9788469480243.pdf

TITULO: Patogenia del síndrome reproductivo y respiratorio porcino: evaluación de
la expresión de citoquinas y fenómenos de apoptosis en órganos linfoides y su papel
en la respuesta inmune.
AUTOR: Inmaculada Barranco Cabezudo
© Edita: Servicio de Publicaciones de la Universidad de Córdoba. 2011
Campus de Rabanales
Ctra. Nacional IV, Km. 396 A
14071 Córdoba
www.uco.es/publicaciones
publicaciones@uco.es
ISBN-13: 978-84-694-8024-3
UNIVERSIDAD DE CÓRDOBA
FACULTAD DE VETERINARIA
PATOGENIA DEL SÍNDROME REPRODUCTIVO Y
RESPIRATORIO PORCINO: EVALUACIÓN DE LA EXPRESIÓN DE
CITOQUINAS Y FENÓMENOS DE APOPTOSIS EN ÓRGANOS
LINFOIDES Y SU PAPEL EN LA RESPUESTA INMUNE.
PATHOGENESIS OF PORCINE REPRODUCTIVE AND
RESPIRATORY SYNDROME: EVALUATION OF THE
EXPRESSION OF CYTOKINES AND APOPTOSIS PHENOMENA IN
LYMPHOID ORGANS AND THEIR ROLE IN THE IMMUNE
RESPONSE.
Trabajo presentado por la Licenciada en Veterinaria Dª
Inmaculada Barranco Cabezudo para optar al grado de Doctor
por la Universidad de Córdoba.
Departamento
Comparadas
Córdoba,
de
Anatomía
6
y
Anatomía
Mayo
Patológica
2011
LIBRADO CARRASCO OTERO, CATEDRÁTICO DEL
DEPARTAMENTO
DE
ANATOMÍA
Y
ANATOMÍA
PATOLÓGICA COMPARADAS DE LA FACULTAD DE
VETERINARIA DE LA UNIVERSIDAD DE CÓRDOBA
INFORMA:
Que Dª Inmaculada Barranco Cabezudo, Licenciada en
Veterinaria, ha realizado bajo mi dirección y asesoramiento el
presente trabajo titulado “PATOGENIA DEL SÍNDROME
REPRODUCTIVO Y RESPIRATORIO PORCINO: EVALUACIÓN
DE LA EXPRESIÓN DE CITOQUINAS Y FENÓMENOS DE
APOPTOSIS EN ÓRGANOS LINFOIDES Y SU PAPEL EN LA
RESPUESTA INMUNE” que considero reúne las condiciones y
calidad científica necesarias para optar al grado de Doctor en
Veterinaria.
Y para que conste y surta los efectos oportunos, firmo el
presente informe
en Córdoba a 6 de Mayo de 2011
JAIME
GÓMEZ
LAGUNA,
DOCTOR
EN
VETERINARIA E INVESTIGADOR EN EL CENTRO
TECNOLÓGICO DE INVESTIGACIÓN ALIMENTARIA
CICAP, POZOBLANCO, CÓRDOBA.
INFORMA:
Que Dª Inmaculada Barranco Cabezudo, Licenciada en
Veterinaria, ha realizado bajo mi dirección y asesoramiento el
presente trabajo titulado “PATOGENIA DEL SÍNDROME
REPRODUCTIVO Y RESPIRATORIO PORCINO: EVALUACIÓN
DE LA EXPRESIÓN DE CITOQUINAS Y FENÓMENOS DE
APOPTOSIS EN ÓRGANOS LINFOIDES Y SU PAPEL EN LA
RESPUESTA INMUNE” que considero reúne las condiciones y
calidad científica necesarias para optar al grado de Doctor en
Veterinaria.
Y para que conste y surta los efectos oportunos, firmo el
presente informe
en Córdoba a 6 de Mayo de 2011
A mi familia,
especialmente a mis
Abuelos
“Lo esencial es invisible a los ojos”
(Antoine de Saint-Exupéry, “El Principito”)
TABLE OF CONTENTS
Lista de Abreviaturas/List of Abbreviations
Introducción/ Introduction
Introducción
2
Introduction
5
Resumen/ Summary
Resumen
9
Summary
16
Chapter 1. REVISIÓN BIBLIOGRÁFICA
1.1. SÍNDROME REPRODUCTIVO Y RESPIRATORIO
PORCINO:
1.1.1. Introducción
24
1.1.2. Virus
25
1.1.3. Epidemiología
32
1.1.4. Patogenia
36
1.1.5. Signos clínicos y lesiones
41
1.1.6. Respuesta inmune y vacunación
45
1.2. PAPEL DE LOS ÓRGANOS LINFOIDES EN EL
SÍNDROME
REPRODUCTIVO
Y
RESPIRATORIO
PORCINO.
1.2.1. Estructura y funciones biológicas de los órganos linfoides en
el cerdo.
50
1.2.2. Importancia de los órganos linfoides en el PRRS
54
1.3. PAPEL DE LAS CITOQUINAS EN EL SÍNDROME
REPRODUCTIVO Y RESPIRATORIO PORCINO
1.3.1. Interferones (IFNα, IFNγ)
60
1.3.2. Citoquinas proinflamatorias (IL-1, IL-6, TNFα)
62
1.3.3. Otras citoquinas (IL-10, TGF-β, IL-12).
66
1.4. APOPTOSIS EN PRRS
1.4.1. Conceptos generales
1.4.2. Actualización de los fenómenos de apoptosis en el PRRS
73
77
Chapter 2. OBJETIVOS DE LA TESIS/AIMS OF THE
THESIS
Objetivos de la tesis
84
Aims of the thesis
86
Chapter 3. EXPERIMENTAL STUDIES
3.1. Common experimental design.
3.2.
Differential
expression
89
of
proinflammatory
cytokines in the lymphoid organs of porcine reproductive and
respiratory syndrome virus-infected pigs.
3.3.
IL-10,
IL-12,
91
IFN-α
and
IFN-γ
immunohistochemical expression in lymphoid organs of porcine
reproductive and respiratory syndrome virus-infected pigs. 110
3.4. Apoptosis in lymphoid tissues of PRRSV infected
pigs
detected
by
immunohistochemistry.
tunel
and
cleaved
caspase-3
130
3.5. Immunohistochemical detection of extrinsic and
intrinsic mediators of apoptosis in porcine paraffin-embedded
tissues.
153
Chapter
4.
DISCUSIÓN
GENERAL/
GENERAL
DISCUSSION
Discusión General
176
General Discussion
185
Chapter 5. CONCLUSIONES/ CONCLUSIONS
Conclusiones
195
Conclusions
197
Chapter 7. REFERENCES
200
Currículum Vitae
242
Agradecimientos
246
Patogenia del PRRS/Pathogenesis of PRRS
LISTA DE ABREVIATURAS/LIST OF ABREVIATIONS
Patogenia del PRRS/Pathogenesis of PRRS
LISTA DE ABREVIATURAS/ LIST OF ABBREVIATIONS
ABC: Avidin-Biotin-peroxidase Complex
ANs: Anticuerpos Neutralizantes
ANOVA: Analysis Of VAriance
ADN: Ácido DesoxirriboNucleico
ARN: Ácido RiboNucleico
ASFv: African Swine Fever virus
BALCs: BronchoAlveolar Lavage Cells
BALFs: BronchoAlveolar Lavage Fluids
Bcl: B-cell lymphoma
CISA: Centro de Investigación en Sanidad Animal
CCasp: Cleaved Caspase
CD: Cluster Differentiation
DC: Células Dendríticas/Dendritic Cells
dpi: días post-inoculación/days post-inoculation
E: Envelope
EU: EUropean
EAV: Equine Arteritis Virus
FasL: Fas Ligand
GP: Glicoproteína/Glicoproteine
h: hour
IFN: InterFeróN/ InterFeroN
Ig: Immunoglobulins
IL: InterLeuquina/InterLeukin
iNOS: inducible Nitric Oxide Synthase
Patogenia del PRRS/Pathogenesis of PRRS
Kb: Kilobases
LDEV: Lactate Dehydrogenase-Elevating Virus
LV: Lelistad Virus
M: Membrana/Membrane
mAb: monoclonal Antibody
MHC: Major Histocompatibility Complex
min: minutes
N: Nucleocápside
NK: Natural Killer
OIE: Oficina Internacional de Epizootías
ORF: Open Reading Frame
P: Protein
PAMs: Porcine Alveolar Macrophages
PBMCs: Peripheral Blood Mononuclear Cells
PBS: Phosphate-Buffered Saline
PCV2: Porcine CircoVirus type 2
PRCV: Porcine Respiratory Corona Virus
PRRS: Porcine Reproductive and Respiratory Syndrome
PRRSV: Porcine Reproductive and Respiratory Syndrome
Virus
RPMI medium: Roswell Park Memorial Institute médium
RT: Room Temperature
RT-PCR: Reverse Transcriptase-Polymerase Chain Reaction
SC: Secreting Cells
SD: Standard Deviation
SHFV: Simian Haemorrhagic Fever Virus
Patogenia del PRRS/Pathogenesis of PRRS
SIV: Swine Influenza Virus
SNC: Sistema Nervioso Central
TCID50: 50% Tissue Culture Infectious Dose
TNF: Tumor Necrosis Factor
TNF-R: Tumor Necrosis Factor-Receptors
TUNEL: Terminal deoxynucleotidyl transferase-mediated
dUtp Nick-End Labelling
wpi: weeks post-infection
ZSF: Zinc salts fixative
Patogenia del PRRS/Pathogenesis of PRRS
INTRODUCCIÓN/ INTRODUCTION
1
Patogenia del PRRS/Pathogenesis of PRRS
INTRODUCCIÓN
El Síndrome Reproductivo y Respiratorio Porcino (PRRS,
del inglés Porcine Reproductive and Respiratory Syndrome) es
una enfermedad vírica caracterizada por inducir una respuesta
inmune errática en el hospedador y es considerada como una de
las enfermedades más importantes en la industria del porcino
debido a las importantes pérdidas económicas que ocasiona. A
pesar de que varios estudios se han realizado con el objetivo de
elucidar la respuesta inmune provocada frente al virus del PRRS
(PRRSV, del inglés PRRS virus) todavía quedan muchos
aspectos por esclarecer.
El PRRSV se replica, principalmente, en los macrófagos
alveolares porcinos (MAPs) y, en menor medida, en macrófagos
de otros órganos y en células dendríticas, describiéndose la
replicación del virus tanto en pulmón como en órganos linfoides
de cerdos infectados con el PRRSV, lo que sugiere que estos
órganos desempeñarían un papel importante en la patogenia de
esta enfermedad. Sin embargo, la mayoría de los estudios
centrados en analizar la respuesta inmune provocada tras una
2
Patogenia del PRRS/Pathogenesis of PRRS
infección con el PRRSV se han realizado principalmente sobre
muestras de suero y de pulmón, existiendo escasos datos en lo
que se refiere a la respuesta inmune en los órganos linfoides.
Una característica importante de esta enfermedad es la
persistencia del PRRSV en los animales infectados, lo que es
considerado como indicativo de que tanto la respuesta inmune
humoral como la celular no son capaces de eliminar
completamente el virus. Asimismo, el PRRS se caracteriza por
la instauración de un estado de inmunosupresión en el que
podrían estar implicados los fenómenos de apoptosis, de
linfocitos y macrófagos, inducidos por el PRRSV. Fenómenos
de apoptosis que han sido descritos en esta enfermedad tanto
asociados a la replicación del virus como a la expresión de
diferentes citoquinas.
El principal objetivo de esta tesis fue evaluar la expresión
de citoquinas y de los fenómenos de apoptosis en los órganos
linfoides de cerdos infectados experimentalmente con una cepa
europea del PRRSV y su papel en la respuesta inmune.
3
Patogenia del PRRS/Pathogenesis of PRRS
Los estudios experimentales llevados a cabo en esta tesis
doctoral han sido financiados por el Ministerio de Educación y
Ciencia, mediante los proyectos de investigación
“El papel
del monocito-macrófago en la patogenia del Síndrome
Reproductivo
y Respiratorio
Porcino
y otros
procesos
respiratorios víricos porcinos” (AGL 2006-04146/GAN) y “El
papel de los linfocitos T reguladores (TREG) en el Síndrome
Reproductivo y Respiratorio Porcino: correlación con la
expresión de citoquinas inmunomoduladoras” (AGL200912438/GAN) y un proyecto europeo de la fundación NADIR
(FP7). La doctoranda, Inmaculada Barranco, desarrolló sus
estudios de doctorado gracias a la beca de referencia BES-200714928 del Ministerio de Educación y Ciencia (actual Ministerio
de Ciencia e Innovación).
4
Patogenia del PRRS/Pathogenesis of PRRS
INTRODUCTION
Porcine Reproductive and Respiratory Syndrome (PRRS)
is a viral disease characterized by inducing an erratic host
immune response and considered as one of the most significant
diseases in the swine industry due to causing significant
economis losses. Althoug several studies have been carried out
to elucidate the host immune response evoked against PRRS
virus (PRRSV), there is a lot of aspect wich still remain unclear.
PRRSV replication targets porcine alveolar macrophages
(PAMs), macrophages in other tissues and in minor extent
dendritic cells. In this sense, viral replication has been reported
in both lung and lymphoid organs of PRRSV-infected pigs,
which suggest a role of these organs in the pathogenesis of the
disease. However, the majority of the studies focused on the
immune response evoked after PRRSV infection have been
performed in serum and lung samples and in a lesser extent in
lymphoid tissues.
An important charasteristic of the disease is the
persistence of PRRSV in infected pigs pointing to both humoral
5
Patogenia del PRRS/Pathogenesis of PRRS
and cellular immune response are not able to completely
eliminate the virus, and also the establishment of an
immunosuppression state that could be due to the apoptosis of
lymphocytes and macrophages induced by PRRSV.
Apoptosis phenomena have been reported during PRRS
being linked to both viral particles and the expression of several
cytokines.
Therefore, the main aim of this thesis was to evaluate
the expression of cyokines and apoptosis phenomena in
lymphoid organs of pigs infected with an european PRRSV
strain and their role in the immune response.
The experimental studies carried out in this thesis have
been founded by the Spanish Ministry of Education and Science
through research projects “The role of monocyte-macrophage in
the pathogenesis of Porcine Reproductive and Respiratory
Syndrome
and
other
swine
viral
respiratory
diseases”
(AGL2006-04146/GAN) and “The role of regulatory T cells
(Treg) in the Porcine Reproductive and Respiratory Syndrome:
correlation
6
with
the
expression
of
immunomodulatory
Patogenia del PRRS/Pathogenesis of PRRS
cytokines” (AGL2009-12438/GAN) and a European project of
NADIR foundation (FP7). The PhD student, Inmaculada
Barranco, carried out her doctoral studies supported by a grant
from the Spanish Ministry of Education and Science (now,
Spanish Ministry of Science and Innovation” (BES-200714928).
7
Patogenia del PRRS/Pathogenesis of PRRS
RESUMEN/ SUMMARY
8
Patogenia del PRRS/Pathogenesis of PRRS
RESUMEN
El Síndrome Reproductivo y Respiratorio Porcino (PRRS,
del inglés Porcine Reproductive and Respiratory Syndrome) es
una enfermedad vírica caracterizada por inducir una respuesta
inmune errática en el hospedador y es considerada como una de
las enfermedades más importantes en la industria del porcino
debido a las importantes pérdidas económicas que provoca. A
pesar de que varios estudios se han realizado con el objetivo de
elucidar la respuesta inmune provocada frente al virus del PRRS
(PRRSV, del inglés PRRS virus) todavía quedan muchos
aspectos por entender.
El PRRSV se replica, principalmente, en los macrófagos
alveolares porcinos (MAPs) y, en menor medida, en macrófagos
de otros órganos y en las células dendríticas, describiéndose la
replicación del virus tanto en pulmón como en los órganos
linfoides de cerdos infectados con el PRRSV, y sugiriéndose
que estos órganos tendrían un importante papel en la patogenia
de esta enfermedad. Sin embargo, la mayoría de los estudios
centrados en analizar la respuesta inmune provocada tras una
9
Patogenia del PRRS/Pathogenesis of PRRS
infección con el PRRSV se han realizado principalmente sobre
muestras de suero y de pulmón.
El principal objetivo de esta tesis fue evaluar el papel de la
expresión de citoquinas y los fenómenos de apoptosis en los
órganos linfoides en la patogenia del PRRS.
Para llevar a cabo este estudio, se utilizaron veintiocho
lechones, de cinco semanas de edad, que fueron inoculados con
el aislado 2982 del PRRSV, y sacrificados a 3, 7, 10, 14, 17, 21 y
24 días post-inoculación (dpi). Cuatro lechones, de las mismas
características, fueron usados como control, inoculándolos con
medio estéril, y sacrificándolos al final del experimento. De todos
los animales se tomaron muestras de pulmón, tonsila y nódulos
linfáticos que fueron fijadas en formol tamponado al 10 %, en
solución de sales de Zinc, y en solución de Bouin, para el estudio
histopatológico e inmunohistoquímico.
En nuestro estudio evaluamos la expresión de IL-1α, IL-6 y
TNF-α en la tonsila, y en los nódulos linfáticos retrofaríngeo y
mediastínico de cerdos infectados con el PRRSV, encontrando
una expresión diferente de cada citoquina en función del órgano
10
Patogenia del PRRS/Pathogenesis of PRRS
analizado, lo que muestra un comportamiento diferente del
PRRSV en los órganos linfoides. Este hallazgo podría estar
relacionado con la falta de una respuesta inmune eficaz del
hospedador frente al PRRSV. Así, mientras que en la tonsila la
expresión de estas citoquinas proinflamatorias fue muy pobre,
en el nódulo linfático mediastínico se observó un incremento de
la expresión de TNF-α e IL-1α, siendo la IL-6 la citoquina más
expresada en el nódulo linfático retrofaríngeo El antígeno del
PRRSV se observó fundamentalmente en la médula o
paracorteza de los órganos linfoides analizados, encontrando
tanto en la tonsila como en el nódulo linfático retrofaríngeo una
expresión bimodal, con un primer pico a los 3 dpi y un segundo
a los 14 dpi, mientras que en el nódulo linfático mediastínico
observamos un pico de expresión al comienzo del experimento
disminuyendo posteriormente hasta el final del mismo. Las
células inmunomarcadas tanto para el PRRSV como para las
citoquinas proinflamatorias fueron principalmente macrófagos.
Todas las citoquinas reguladoras estudiadas (IL-10, IL-12,
IFN-α, IFN-γ) fueron expresadas fundamentalmente en las áreas
linforeticulares de la tonsila y en la paracorteza de los nódulos
11
Patogenia del PRRS/Pathogenesis of PRRS
linfáticos retrofaríngeo y mediastínico, principalmente en el
citoplasma de los macrófagos. En la tonsila la expresión de IL10 presentó una curva con un incremento a los 3 , 14 y 24 dpi,
siendo sólo estadísticamente significativo con respecto al grupo
control a los 14 dpi debido a la variabilidad individual. La
expresión de IFN-α, IFN-γ e IL-12 siguieron una tendencia
similar en la tonsila con un pico de expresión estadísticamente
significativo a los 3 dpi. En el nódulo linfático mediastínico,
todas las citoquinas reguladoras mostraron picos de expresión
estadísticamente significativos a los 7, 17 y 24 dpi (IL-12 e IFNγ) o 3, 14 y 24 dpi (IL-10 e IFN-α). Sin embargo, debido a la
variabilidad individual existente la expresión de IL-10 no fue
estadísticamente
significativa.
En
el
nódulo
linfático
retrofaríngeo, todas las citoquinas reguladoras siguieron una
tendencia similar con una expresión máxima a los 3 dpi (IFN-α,
IFN-γ) presentando otros picos de expresión a los 14 dpi así
como al final del estudio (IFN-α, IL-10). En ambos nódulos
linfáticos, la IL-10 fue la citoquina que mostró los niveles más
bajos de expresión, mientras que IFN-γ fue la citoquina más
expresada.
12
Patogenia del PRRS/Pathogenesis of PRRS
La expresión de IFNs en órganos linfoides de cerdos
infectados con el PRRSV junto con la persistencia del virus
encontrada al final del experimento, indica que la vía de
señalización
de
los
IFNs
pued
no
estar
funcionando
adecuadamente.
La presencia de cuerpos apoptóticos y células picnóticas
mostraron un incremento gradual en el nódulo linfático
mediastínico desde el principio del estudio (3 dpi) hasta el final
del mismo. Sin embargo, en la tonsila este incremento fue
menor comparándolo con el grupo control, siendo sólo
estadísticamente significativo al final del experimento (21 y 24
dpi). Por otro lado, la expresión de TUNEL como de Caspasa 3
activada fue similar en ambos órganos analizados con un ligero
aumento al final del experimento. Aunque no se encontró
correlación entre los fenómenos de apoptosis y la expresión del
PRRSV, el comienzo de la expresión de los fenómenos de
apoptosis coincidió con la detección del PRRSV en el nódulo
linfático mediastínico. Sin embargo, en la tonsila se observó un
incremento de los fenómenos de apoptosis sólo al final del
experimento, aunque dichos fenómenos no coincidieron a nivel
13
Patogenia del PRRS/Pathogenesis of PRRS
individual con una mayor expresión de Caspasa 3 activa y/o
TUNEL. Asimismo, en nuestro estudio, encontramos mayor
evidencia de fenómenos de apoptosis en el nódulo linfático
mediastínico que en la tonsila, dónde la expresión de citoquinas
pro-apoptóticas fue muy pobre. Tanto en tonsila como en nódulo
linfático se detectó la presencia de fenómenos de apoptosis de
manera independiente a la expresión de Caspasa 3, sugiriendo
que la inducción de la apoptosis en nuestra infección
experimental con la cepa europea 2982 del PRRSV podría
inducirse por vías independientes de la Caspasa 3.
Finalmente llevamos a cabo un estudio para la puesta a
punto de mediadores de la apoptosis. A partir de dicho estudio,
concluimos que para la detección inmunohistoquímica de los
mediadores de la apoptosis implicados en la vía extrínseca
(caspasa 8 y Fas) el fijador de elección fue la solución de sales
de Zinc y el desenmascaramiento antigénico con tampón citrato,
mientras que para los mediadores implicados en la vía intrínseca
fueron los siguientes: para caspasa 9, fijación en solución de
sales de Zinc con Tween 20 de desenmascaramiento antigénico,
para Bcl-2 e iNOS el fijador de elección fue formol tamponado
14
Patogenia del PRRS/Pathogenesis of PRRS
al 10 % con Proteinasa K y Citrato microondas como
desenmascaramiento antitético respectivamente. Los buenos
resultados obtenidos en la detección, mediante técnicas
inmunohistoquímicas, de diferentes mediadores de la apoptosis
en muestras de tejidos porcinos incluidas en parafina abren la
posibilidad del potencial uso de estos anticuerpos en futuros
estudios.
15
Patogenia del PRRS/Pathogenesis of PRRS
SUMMARY
Porcine Reproductive and Respiratory Syndrome (PRRS)
is a viral disease characterized by inducing an erratic host
immune response and considered as one of the most significant
diseases in the swine industry due to causing significant
economis losses. Althoug several studies have been carried out
to elucidate the host immune response evoked against PRRS
virus (PRRSV), there is a lot of aspect wich still remain unclear.
PRRSV replication targets porcine alveolar macrophages
(PAMs), macrophages in other tissues and in minor extent
dendritic cells. In this sense, viral replication has been reported
in both lung and lymphoid organs of PRRSV-infected pigs,
which suggest a role of these organs in the pathogenesis of the
disease. However, the majority of the studies focused on the
immune response evoked after PRRSV infection have been
performed in serum and lung samples and in a lesser extent in
lymphoid organs.Therefore, the main aim of this thesis was to
evaluate the role of the expression of cytokines and apoptotic
phenomena in lymphoid organs in the pathogenesis of PRRS.
16
Patogenia del PRRS/Pathogenesis of PRRS
To carried out this study twenty eight specific pathogen
free, five weeks old pigs from a PRRSV seronegative farm were
randomly distributed in batches of four and inoculated with
PRRSV field isolate 2982 and humanely killed at 3, 7, 10, 14,
17, 21 and 24 days post-inoculation (dpi). Other four pigs, were
used as controls, inoculated with 1ml of sterile medium and
humanely killed at the end of the study (24dpi). Samples from
the lung, tonsil and lymph nodes were fixed in 10% buffered
formaldehyde, zinc salt fixative and in Bouin solution for
histopathological and immunohistochemical studies.
The expression of IL-1α, IL-6 and TNF-α was examined in
our study in the tonsil, retropharyngeal and mediastinal lymph
nodes of PRRSV-infected pigs. Interestingly, the expression of
each cytokine was different depending on the body compartment
examined. This finding point to a differential behaviour of
PRRSV in the lymphoid organs, which may be related with the
lack of a robust host immune response evoked against the virus.
Whereas the expression of proinflammatory cytokines
observed in the tonsil was very poor, an enhancement was
17
Patogenia del PRRS/Pathogenesis of PRRS
observed on TNF-α and IL-1α levels in the mediastinal lymph
node and IL-6 was the proinflammatory cytokine higher
expressed in the retropharyngeal lymph node. PRRSV antigen
was observed mainly in the medulla and/or in the paracortex of
the different lymphoid tissues analyzed. PRRSV antigen
displayed a bimodal expression in the tonsil and retropharyngeal
lymph node with a first peak of expression at 3 dpi and a second
one at 14 dpi, whereas the mediastinal lymph node had just a
peak of expression at the beginning decreasing onwards until the
end of the experiment. Cells immunollabelled against PRRSV
and proinflammatory cytokines were mainly macrophages.
All regulatory cytokines studied (IL-10, IL-12, IFN-α, IFNγ) were mostly expressed in the lymphoreticular areas of the
tonsil and in the paracortex of retropharyngeal lymph node and
mediastinal lymph node, mainly in the cytoplasm of
macrophages
In the tonsil, the number of IL-10 positive cells displayed a
curve with an increase of its expression at 3 dpi, 14 dpi and
24dpi, being statistically significant only at 14 dpi due to
individual variability. The expression of IFN-α, IFN-γ and IL-12
18
Patogenia del PRRS/Pathogenesis of PRRS
showed a similar trend among them in the tonsil, with a
statistically significant peak of expression at 3 dpi. In the
mediastinal lymph node, all regulatory cytokines showed
statistically significant peaks of expression at 7, 17 and 24 dpi
(IL-12 and IFN-γ) or 3, 14 and 24 dpi (IL-10 and IFN-α).
Nonetheless, the expression of IL-10 was not statistically
significant due to individual variability. In the retropharyngeal
lymph node all regulatory cytokines followed a similar trend
with a maximum expression of all of them at 3 dpi (IFN-α, IFNγ) and another peaks of expression at 14 dpi and at the end of
the study (IFN-α, IL-10). In both lymph nodes IL-10 was the
cytokine which showed the lowest expression whereas IFN-γ
was the one with the highest expression.
The expression of IFNs in the lymphoid organs of PRRSV
infected pigs together to the viral persistence at the end of the
study, indicates that the IFN signaling cascade may not be
working properly.
The presence of apoptotic bodies and cell picnosis, increased
gradually in the mediastinal lymph node from the beginning of
the study (3 dpi) onwards. Nevertheless, in the tonsil the
19
Patogenia del PRRS/Pathogenesis of PRRS
increase of apoptotic bodies and cells picnosis was slighter
compared with the control group, being this increase statistically
significant only at the end of the experiment (21 and 24 dpi). On
the other hand, the expression of TUNEL and cleaved caspase 3
(CCasp3) followed a similar trend in both organs analyzed with
a slight increase at the end of the study. Although no correlation
was observed between apoptotic phenomena and the expression
of PRRSV, the beginning of apoptotic phenomena coincided
with the first detection of PRRSV antigen in the mediastinal
lymph node. However, in the tonsil an enhancement of apoptotic
bodies and cell picnosis was only observed at the end of the
experiment wich did not coincide individually with a higher
expression of CCasp3 and/or TUNEL. Moreover, in our study
there were more evidences of apoptosis in mediastinal lymph
node than in the tonsil, in wich the expression of pro-apoptotic
cytokines was very poor. In both, tonsil and mediastinal lymph
node, apoptotic phenomena were detected independently of
CCasp 3 expression, suggesting that the induction of apoptosis
in our experimental infection with the european strain 2982
20
Patogenia del PRRS/Pathogenesis of PRRS
isolate of PRRSV could be triggered by a caspase 3 independent
pathway.
Finally a study to determine the best fixative and antigen
retrieval method in porcine paraffin embedded tissues for the
immunohistochemical detection of apoptosis mediators was
carried out. According with this study, the best fixative and
antigen retrieval method for the antibodies against caspase 8
and Fas (extrinsic pathway of apoptosis) were Zinc salt
fixative and Citrate microwave unmasking technique, while to
determine the intrinsic pathway the ideal fixative and antigen
retrieval method for each antibody was as follows: for caspase
9, fixation in Zinc salt solution and antigen retrieval with
Tween 20; for Bcl-2 and iNOS the optimal immunolabelling
was observed in 10 % neutral buffered formalin fixed samples
and with Proteinase K and Citrate microwave as antigen
retrieval methods, respectively. The satisfactory results
obtained in our study for a specific immunolabelling in
porcine tissues point to a potential use of these antibodies in
future studies.
21
Patogenia del PRRS/Pathogenesis of PRRS
1. REVISIÓN BIBLIOGRÁFICA
1.1. SÍNDROME REPRODUCTIVO Y RESPIRATORIO
PORCINO:
1.1.1. Introducción
1.1.2. Virus
1.1.3. Epidemiología
1.1.4. Patogenia
1.1.5. Signos clínicos y lesiones
1.1.6. Respuesta inmune y vacunación
1.2. PAPEL DE LOS ÓRGANOS LINFOIDES EN EL PRRS.
1.2.1. Estructura y funciones biológicas de los órganos linfoides
en el cerdo.
1.2.2. Importancia de los órganos linfoides en el PRRS
1.3. PAPEL DE LAS CITOQUINAS EN EL PRRS
1.3.1. Interferones (IFNα, IFNγ)
1.3.2. Citoquinas proinflamatorias (IL-1, IL-6, TNFα)
1.3.3. Otras citoquinas (IL-10, IL-12).
22
Patogenia del PRRS/Pathogenesis of PRRS
1.4. APOPTOSIS EN EL PRRS
1.4.1. Conceptos generales
1.4.2. Actualización de los fenómenos de apoptosis en el PRRS
23
Patogenia del PRRS/Pathogenesis of PRRS
1.1 SÍNDROME REPRODUCTIVO Y RESPIRATORIO
PORCINO.
1.1.1. Introducción.
El Síndrome Reproductivo y Respiratorio Porcino
(PRRS del inglés Porcine Reproductive and Respiratory
Syndrome) es una enfermedad infecciosa del cerdo, descrita por
primera vez en América del Norte en 1987 y que posteriormente
se diagnóstico en Europa (Collins et al., 1992; Edwards et al.,
1992) denominada inicialmente como la “enfermedad misteriosa
del cerdo” o “enfermedad de las orejas azules” (Paton et al.,
1991; Wensvoort et al., 1991). Actualmente se considera que
esta enfermedad tiene una distribución mundial, produciendo
pérdidas económicas significativas en la producción porcina ya
que su capacidad de producir fallos reproductivos en hembras y
problemas respiratorios en lechones y cerdos en crecimientocebo lleva a una inevitable y significativa pérdida. Así se ha
estimado que esta enfermedad origina pérdidas de alrededor de
560 millones de dólares al año en Estados Unidos (Neumann et
24
Patogenia del PRRS/Pathogenesis of PRRS
al., 2005). La dificultad en el control de esta enfermedad es
debida, principalmente, a los múltiples factores variables
implicados en la patogenia, en el genotipo del virus y en la
susceptibilidad del hospedador frente al PRRS (Darwich et al.,
2010). La aparición en 2006 de una cepa del virus del PRRS
altamente virulenta tuvo un efecto devastador en la industria del
porcino en China, y demostró la habilidad del virus de mutar y
evolucionar rápidamente (Tian et al., 2007). Con el fin de
prevenir futuros efectos devastadores de larga duración en la
industria porcina, es necesario profundizar en el conocimiento
de la patogenia del PRRS y de los mecanismos de la respuesta
inmune del hospedador frente a este virus, ya que estos nos
permitirán establecer métodos eficientes para el control de esta
enfermedad.
1.1.2. Virus.
El agente causal del PRRS fue identificado por primera
vez en 1991 por Wensvoort et al. (1991) como virus Lelystad,
siendo ahora más comúnmente denominado como PRRSV (del
25
Patogenia del PRRS/Pathogenesis of PRRS
inglés PRRS virus). El PRRSV es un virus ARN perteneciente
al orden Nidovirales, familia Arteriviridae, género Arterivirus.
Otros miembros de la familia Arteriviridae incluyen el virus de
la arteritis equina (EAV del inglés Equine Arteritis Virus), virus
elevador de lactato deshidrogenasa (LDEV del inglés Lactate
Dehydrogenase-Elevating
Virus),
y
virus
de
la
fiebre
hemorrágica de simios (SHFV del inglés Simian Haemorrhagic
Fever Virus). El PRRSV fue clasificado como un miembro de la
familia Arteriviridae cuando Meulenberg et al. (1993)
reconocieron un número de características físicas y genómicas
similares entre el PRRSV, el EAV y el LDV, como ser virus
ARN de cadena positiva y que tienen la habilidad de replicarse
en macrófagos, sugiriendo que estos tres virus estaban
relacionados evolutivamente.
El PRRSV es un virus pequeño, esférico, envuelto,
formado por una cadena simple de ARN de 13-15 Kb (Benfield
et al., 1992; Morrison et al., 1992). El virión tiene un diámetro
de 50-60 nm con dos fragmentos de lectura abierta (ORF del
inglés Open reading frame) largos (ORF1a y ORF1b) que
26
Patogenia del PRRS/Pathogenesis of PRRS
codifican las proteínas no estructurales y 7 cortos (ORF2-ORF7)
que codifican las proteínas estructurales del virus (Nielsen et al.,
2001; Stadejek et al., 2002). Las proteínas no estructurales y la
secuencia líder 5´ 3´ del virus juegan un papel importante en la
replicación del virus (Fang et al., 2010; Sun et al., 2010).
Considerándose que el modo de replicación del virus lo hace
susceptible a tener
elevados índices de mutación y
recombinación.
Las proteínas estructurales mayores constan de la
nucleocápside (N), la proteína de membrana (M), y la
glicoproteína de envoltura mayor (GP5) (Dea et al., 2000). Las
proteínas estructurales menores incluyen GP2a, GP2b, GP3 y
GP4 (Wu et al., 2001; Wu et al., 2005). (Fig.1).
27
Patogenia del PRRS/Pathogenesis of PRRS
Figura 1. Representación esquemática de PRRSV, mostrando la estructura
icosaédrica del virión, los fragmentos de lectura abierta, así como las
diferentes glicoproteínas y envoltura. Imagen modificada de www.porcilisprrs.com
El PRRSV posee una elevada variabilidad genética y
antigénica, describiéndose dos genotipos principales; el genotipo
Europeo o genotipo I y el genotipo Norteamericano o genotipo
II (Snijder et al., 2004). El genotipo Europeo está representado
por el virus Lelistad (LV) mientras que la cepa ATCC VR 2332
es el prototipo del genotipo Norteamericano. Estos genotipos
tienen importantes diferencias patogénicas y antigénicas
28
Patogenia del PRRS/Pathogenesis of PRRS
presentando sólo un 55-65% de similitud genética, existiendo
además una alta diversidad genética dentro de un mismo
genotipo (Wensvoort et al., 1992; Mardassi et al., 1994; Meng et
al., 1995a, 1995b; Murtaugh et al., 1995; Suárez et al., 1996b;
Drew et al., 1997; Gagnon and Dea, 1998; Dea et al., 2000;
Forsberg et al., 2002; Goldberg et al., 2003; Mateu et al., 2003;
Stadejek et al., 2006) (Fig. 2).
Las proteínas estructurales M y P2b son las más
conservadas (74-81%), mientras que sólo un 51-58% de la
glicoproteína GP5 se conserva entre diferentes aislados del virus
(Meng et al., 1994; Kapur et al., 1996; Wu et al., 2001). En este
sentido, se ha descrito una marcada variabilidad en la
patogenicidad dependiendo del genotipo del virus (Halbur et al.,
1994; Shimizu et al., 1996; Drew, 2000; Thanawongnuwech et
al., 2003). Diferencias significativas entre aislados dentro de un
mismo genotipo también han sido descritas, aunque estas no son
tan marcadas como las que se presentan entre los dos genotipos.
Además, se ha descrito que la vacunación con un genotipo no
protege frente a la infección con otro genotipo diferente
29
Patogenia del PRRS/Pathogenesis of PRRS
(Labarque et al., 2003b; van Woensel et al., 1998).
Inicialmente, las diferencias entre aislados fueron descritas para
el genotipo Norteamericano (Meng et al., 1995a; Andreyev et
al., 1997; Gagnon and Dea, 1998; Allende et al., 1999; Dea et
al., 2000; Goldberg et al., 2000a). Sin embargo, recientemente
se ha demostrado que entre aislados del genotipo Europeo de
pueden existir diferencias de más del 18%, identificándose 4
subtipos diferentes (Drew et al., 1997; Indik et al., 2000;
Forsberg et al., 2001, 2002; Bignotti et al., 2002; Stadejek et al.,
2002, 2006) (Fig.2). En este sentido, Forsberg et al. (2002)
demuestra que la variabilidad entre aislados del genotipo
Europeo era mayor que la variabilidad encontrada entre aislados
del
genotipo
Norteamericano,
probablemente
incremento del número de aislados vacunales.
30
debido
al
Patogenia del PRRS/Pathogenesis of PRRS
Figura 2. Genotipo I (Europeo) y Genotipo II (Norteamericano). Los
diferentes subtipos muestran la diversidad del Genotipo Europeo. Modificado
de Stadejek et al., 2006.
En esta enfermedad se han descrito diferencias entre
razas de cerdos con respecto al título de anticuerpos en suero,
ganancia media diaria y susceptibilidad a las lesiones inducidas
por el PRRSV en pulmón, cerebro y corazón (Halbur et al.,
1997). Todos estos datos sugieren que existe una variación
31
Patogenia del PRRS/Pathogenesis of PRRS
alélica en los genes en la respuesta a la enfermedad producida
por el PRRSV.
La elevada diversidad genética y el elevado número de
cepas de PRRSV junto con la susceptibilidad variable del virus
entre hospedadores hacen muy complicada la tarea de controlar
esta enfermedad. La diferencia entre los dos genotipos sugiere la
necesidad de desarrollar diferentes medidas de control frente al
PRRS dependiendo del genotipo con el que nos encontremos.
Un mayor conocimiento de la relación genética entre aislados
puede ayudar a explicar los mecanismos por los que emergen
nuevos aislados.
1.1.3. Epidemiología.
Actualmente, se considera que el PRRSV es endémico en
la mayor parte de las zonas de producción de ganado porcino del
mundo, considerándose como zonas libres de esta enfermedad
(Fig. 3) a Suecia (Elvander et al., 1997), Noruega (OIE, 1997),
Finlandia (Bøtner 1994), Suiza (Canon et al., 1998), Nueva
Caledonia (OIE, 1996), Nueva Zelanda (Motha et al., 1997),
32
Patogenia del PRRS/Pathogenesis of PRRS
Australia (Garner et al., 1997), Argentina (Perfumo and
Sanguinetti, 2003), Brasil (Ciacci-Zanella et al., 2004) y las islas
caribeñas (Alfonso and Frias-Lepoureau, 2003)
Figura 3. Países considerados libres del virus del PRRS.
El PRRSV tiene la habilidad de transmitirse rápidamente
dentro de las poblaciones y entre poblaciones, lo que explica su
rápida distribución mundial. La principal ruta de transmisión de
33
Patogenia del PRRS/Pathogenesis of PRRS
PRRSV es por contacto directo entre cerdos (Albina, 1997),
aunque los aerosoles y los fómites juegan también un papel
importante en la transmisión horizontal de la enfermedad (De
Jong et al., 1991; Le Potier et al., 1995; Albina, 1997;
Torremorell et al., 1997; Lager and Mengeling, 2000; Kristensen
et al., 2002; Otake et al., 2002b). El PRRSV ha sido aislado de
la mayoría de las secreciones porcinas, incluyendo semen,
sangre, saliva, heces, aerosoles, leche y calostro (Swenson et al.,
1994;
Wagstrom
et
al.,
1997;
Wills
et
al.,
1997a),
demostrándose que los verracos infectados con PRRSV pueden
eliminar el virus a través del semen durante intervalos
prolongados (Prieto and Castro, 2005).
Algunas especies de aves, como vectores pasivos,
pueden estar implicadas en la epidemiología del PRRSV. En
este sentido, Zimmerman et al. (1997) demostraron que cerdos
inoculados intranasalmente con un aislado del PRRSV obtenido
desde heces de patos salvajes llegaron a ser virémicos, y
transmitieron
el
virus
a cerdos
centinela.
Las
agujas
contaminadas y los mosquitos también son considerados
34
Patogenia del PRRS/Pathogenesis of PRRS
vectores pasivos de la enfermedad (Otake et al. 2002c, 2002d).
Por el contrario, los roedores no son susceptibles al PRRSV
(Hooper et al., 1994).
Una característica importante de los virus de la familia
Arteriviridae es su capacidad para producir infecciones
persistentes, capacidad que es muy evidente en PRRSV (Allende
et al., 2000; Bilodeau et al., 1994) ya que este virus puede
permanecer en los tejidos durante varios meses una vez que la
fase aguda de la enfermedad ha remitido. Así, se ha aislado el
PRRSV de muestras de sangre de animales infectados hasta los
23 dpi, con cepas del genotipo Europeo (Prieto et al., 2003) y 28
dpi, con cepas del genotipo Norteamericano (Wills et al., 2003),
o bien hasta los 56 dpi utilizando la técnica RT-PCR (del inglés
Reverse Transcriptase-polymerase Chain reaction) (Wills et al.,
2003). En tonsila, el PRRSV puede ser detectado por RT-PCR
hasta los 251 dpi, aunque el aislamiento del virus de este órgano
tan solo ha sido posible hasta los 56 dpi (Wills et al., 2003).
La edad de los cerdos infectados parece ser un factor
determinante, ya que se ha demostrado que es más común una
35
Patogenia del PRRS/Pathogenesis of PRRS
persistencia prolongada en cerdos jóvenes,
probablemente
debido a que el sistema inmune es más inmaduro y hay una
mayor proporción de células susceptibles (Mengeling et al.,
1994). Sin embargo, a pesar de que la edad es un factor
determinante en la duración de la persistencia de la enfermedad,
a cualquier edad los cerdos permanecen infectados al menos
varias semanas, y con frecuencia meses, tiempo durante el cual
los cerdos se convierten en una fuente de infección.
1.1.4. Patogenia.
El virus del PRRS se replica, preferentemente, en los
macrófagos alveolares porcinos (MAPs) y ,en menor medida, en
otros macrófagos, monocitos y células dendríticas (DCs)
(Molitor
et
al.,
1997;
Bautista
and
Molitor,
1999),
describiéndose la persistencia del virus durante varias semanas
en pulmón y en órganos linfoides (Albina et al., 1994; Wills et
al., 1997b; Allende et al., 2000; Lamontagne et al., 2001, 2003).
Además, el pulmón y los órganos linfoides, con la excepción del
bazo, parecen ser los sitios en los que el virus se replica durante
36
Patogenia del PRRS/Pathogenesis of PRRS
la infección aguda, ya que se han detectado cantidades similares
de virus en ambos tejidos (Xiao et al., 2004). Sin embargo, en
las infecciones persistentes la replicación del virus está
restringida fundamentalmente a la tonsila y nódulos linfáticos
(Wills et al., 1997b; Rossow, 1998; Allende et al., 2000; Xiao et
al., 2004).
En condiciones naturales, la vía más frecuente de entrada
del PRRSV es a través del tracto respiratorio, teniendo lugar
rápidamente una viremia y diseminación por todo el organismo
(Duan et al., 1997). Sin embargo, en condiciones experimentales
se ha conseguido reproducir la enfermedad tras inocular a los
animales
por
intramuscular,
intranasal,
intratraqueal,
intrauterina,
intravenosa
oronasal,
o
oral,
intraperitoneal
(Wensvoort et al., 1991; Christianson et al., 1992; Collins et al.,
1992; Christianson et al., 1993; Rossow et al., 1994; Swenson et
al., 1994; Wills et al., 1994; Pol et al., 1997; Van Reeth et al.,
1999; Yoon et al., 1999).
El PRRS se caracteriza por producir fallos reproductivos
en animales de cría y fallos respiratorios en lechones y cerdos en
37
Patogenia del PRRS/Pathogenesis of PRRS
la
fase
de
crecimiento-cebo.
Como
hemos
indicado
anteriormente, las células en las que el PRRSV se replica
preferentemente son los MAPs, jugando éstos un papel
importante tanto en la respuesta inmune innata como en la
adquirida, llevando a cabo una serie de funciones que incluyen
fagocitosis, inactivación de microorganismos, búsqueda de sitios
en los que hay un daño tisular, procesamiento y presentación de
antígenos a los linfocitos y producción de citoquinas. En el
PRRS entre el 50-65% de los MAPs son destruidos durante las
primeras semanas post-infección, dando lugar a una disminución
en la función de estas células (Molitor et al., 1992; Zhou et al.,
1992; Molitor, 1993) y un descenso en la liberación de aniones
superóxido y peróxido de hidrógeno por macrófagos (Molitor et
al., 1992; Zhou et al., 1992; Thanawongnuwech et al., 1997;
Chiou et al., 2000; López-Fuertes et al., 2000), lo que impide
que se produzca una buena respuesta inmune respiratoria
(Fig.4). No obstante los MAPs recuperan sus funciones a las
cuatro semanas tras la infección con PRRSV (Molitor, 1993;
Done and Paton, 1995).
38
Patogenia del PRRS/Pathogenesis of PRRS
Figura 4. Patogenia de la Forma Respiratoria y la Forma Reproductiva del
PRRS.
En
verracos,
la
infección
por
el
PRRSV
es
multisistémica, siendo posible el aislamiento del virus de
diferentes órganos desde los 2 a los 30 dpi (Prieto et al., 2003).
Tras la infección, el PRRSV se disemina por todo el organismo,
infectando el semen mediante la replicación en órganos del
tracto reproductor o bien por la invasión de monocitos y
macrófagos infectados desde el torrente sanguíneo a los órganos
reproductores, sin la necesidad de que haya una replicación del
39
Patogenia del PRRS/Pathogenesis of PRRS
virus en los órganos reproductores (Prieto et al., 2003) (Fig. 4).
En los testículos el PRRSV puede ser aislado sólo hasta los 8
dpi, lo que indica que éste no es un lugar principal de
replicación del virus (Prieto et al., 2003).
En cerdas reproductoras, la infección por el PRRSV es
más significativa cuando la gestación está más avanzada, ya que
el PRRSV no tiene efecto hasta la implantación del embrión
(Prieto et al., 1997). Cuando las reproductoras son infectadas
intranasalmente el porcentaje de embriones o fetos infectados en
los dos primeros tercios de la gestación es prácticamente nulo
(Mengeling et al., 1994; Prieto et al., 1996) siendo necesario
infectar directamente a los fetos para que se desarrolle la
infección en éstos (Christianson et al., 1993; Lager and
Mengeling, 1995). Mengeling et al.(1994) y Lager et al. (1996)
describieron un 100% de infección en fetos cuando la infección
tiene lugar en el útero a los 90 días de gestación (Fig. 4)
apareciendo nacidos muertos, nacidos débiles que generalmente
mueren antes del destete, incremento en la ratio de muertes antes
40
Patogenia del PRRS/Pathogenesis of PRRS
del destete o bien lechones que sobreviven pero con retraso en el
crecimiento.
1.1.5. Signos clínicos y lesiones.
Las infecciones por el PRRSV pueden variar desde una
infección leve hasta
una infección altamente patógena, que
puede originar una elevada mortalidad en las explotaciones. Los
signos clínicos de la enfermedad dependen tanto de la
condición, raza, edad de los animales infectados como de la
cepa del PRRSV que cause la infección. Así mismo, el PRRS
puede presentarse en dos formas diferenciadas, reproductiva y
respiratoria.
La forma reproductiva se presenta en las cerdas adultas y
se caracteriza por abortos, partos prematuros o retrasados,
mortinatalidad y expulsión de fetos momificados y nacidos
débiles (Plana et al., 1992). En el caso de los machos, el curso
suele ser subclínico pero se producen alteraciones importantes
en la calidad seminal (Prieto et al., 1996). Estas manifestaciones,
tanto del macho como de la hembra no son exclusivas del PRRS
41
Patogenia del PRRS/Pathogenesis of PRRS
y pueden encontrarse en mayor o menor grado en otras
enfermedades. Así, dentro de las enfermedades de tipo
infeccioso, pueden producirse cuadros abortivos que guarden
alguna similitud con el PRRS en la enfermedad de Aujeszky, la
influenza, el mal rojo y la infección con el circovirus porcino
tipo 2 (PCV2 del inglés Porcine Circovirus type 2). Además,
otras enfermedades, como la parvovirosis, pueden dar lugar a
nacidos muertos y momificados.
La forma respiratoria se presenta fundamentalmente en
los lechones y es más difícil de valorar clínicamente ya que el
cuadro, aunque puede ser grave es bastante inespecífico. Los
animales presentan tos y disnea, acompañada de fiebre y pérdida
de apetito, lo que conlleva a una reducción en el crecimiento. La
mortalidad es elevada en cerdos de engorde, de entre las 4 y 10
semanas de edad, y en cerdos neonatales puede ser del 100%
(Rossow, 1998).
Las principales lesiones macroscópicas que se observan
en el PRRS consisten en unos pulmones con un parcheado
oscuro, que no colapsan y con una consistencia gomosa,
42
Patogenia del PRRS/Pathogenesis of PRRS
especialmente en el área ventral de los lóbulos medial y
accesorio, y una hiperplasia de los nódulos linfáticos (Pol et al.,
1991; Halbur et al., 1995a, 1995b; Vézina et al., 1996). Cuando
el virus afecta a las cerdas gestantes, podemos encontrar en los
lechones nacidos débiles o muertos hidrotórax, ascitis y
hemorragias subcutáneas (Plana et al., 1992; Scruggs and
Sorden, 2001). Además, en las camadas infectadas pueden
aparecer fetos momificados y/o macerados, así como edema y
hemorragias en el cordón umbilical (Lager and Halbur, 1996).
El principal hallazgo microscópico en el PRRS es la
presencia de una neumonía intersticial proliferativa multifocal,
caracterizada por una hipertrofia e hiperplasia de los neumocitos
tipo II, infiltración de células mononucleares en los septos
alveolares y presencia de restos celulares y células inflamatorias
en los espacios alveolares (Halbur et al., 1994; Rossow et al.,
1994, 1995). Histopatológicamente también podemos encontrar
una rinitis, caracterizada por una vacuolización de las células
epiteliales, pérdida de los cilios y descamación de la superficie
epitelial (Pol et al., 1991; Collins et al., 1992).
43
Patogenia del PRRS/Pathogenesis of PRRS
La asociación de la infección del virus PRRS con otros
patógenos (Rossow, 1998) y la disminución de las funciones de
los MAPs cuando han sufrido la infección por este virus, ha
sugerido que este tendría un papel inmunosupresor, facilitando
de esta forma la aparición de infecciones concomitantes o
secundarias. Así, se ha demostrado una predisposición a la
infección por Streptococcus suis tanto en animales de engorde
(Galina et al., 1994; Halbur et al., 2000; Thanawongnuwech et
al., 2000) como en lechones infectados en el útero (Feng et al.,
2001). Además, las infecciones concomitantes de PRRS y PCV2
hacen que aumente tanto el rango de mortalidad de lechones
como la gravedad de las lesiones encontradas (Allan et al., 2000;
Harms et al., 2001). Durante la infección con el PRRSV también
se ha descrito un incremento en la susceptibilidad a la infección
con Salmonella choleraesuis (Wills et al., 2000), Bordetella
bronchiseptica
(Brockmeier
et
al.,
2000),
Mycoplasma
hyopneumoniae (Thacker et al., 1999), virus de la gripe porcina
(SIV del inglés Swine Influenza Virus) y coronavirus porcino
(PRCV del inglés Porcine Respiratory Corona Virus) (Van
Reeth et al., 1996). En condiciones de campo, infecciones
44
Patogenia del PRRS/Pathogenesis of PRRS
concomitantes entre el PRRS y Streptococcus suis, Mycoplasma
hyopneumoniae o PCV2 están presentes con elevada frecuencia
(Segalés et al., 2002). Otro de los factores complejos del
PRRSV es que la susceptibilidad a infecciones secundarias varía
entre animales (Rossow, 1998).
1.1.6. Respuesta inmune y vacunación.
Como hemos descrito anteriormente, la persistencia es
una característica del PRRSV, lo que demuestra que la respuesta
inmune de los cerdos frente a este virus es relativamente incapaz
de eliminarlo de la circulación y de los tejidos linfoides.
Los sistemas inmunes innato y adquirido son los
responsables de las principales respuestas defensivas frente a los
microbios y sus antígenos, utilizando diferentes mecanismos
para controlar los mecanismos invasores, por lo que, se pensaba
que los dos funcionaban independientemente. Sin embargo, se
ha hecho cada vez más evidente que ambos interactúan
estrechamente. El sistema inmune innato es inmediato, carece de
especificidad para reconocer a los antígenos y su nivel de
45
Patogenia del PRRS/Pathogenesis of PRRS
respuesta no aumenta con la exposición repetida. Por el
contrario, el sistema inmune adaptativo ofrece respuestas
específicas contra antígenos y patógenos particulares, sus
principales células efectoras son los linfocitos, tiene memoria y
la potencia de su acción aumenta con cada exposición a un
mismo antígeno (Kumar et al., 2008).
La inmunidad frente al PRRSV comienza con una
respuesta inmune antiviral innata, caracterizada por una
producción mínima de interferón tipo I (IFNα/β), por los
macrófagos en el lugar de la infección (Murtaugh et al., 2002).
Al demostrarse que no existe una producción significativa de
IFNα durante la infección por el PRRSV (Albina et al., 1998a;
Buddaert et al., 1998; Van Reeth et al., 1999; Murtaugh et al.,
2002) se considera que la respuesta inmune innata no eficaz en
esta enfermedad (Fig. 5).
La inmunidad humoral antígeno específica aparece
temprano tras la infección (5-7 dpi), aunque los anticuerpos
neutralizantes (ANs) son detectados en suero más tarde
(Loemba et al., 1996; Eichhorn and Frost, 1997; Albina et al.,
46
Patogenia del PRRS/Pathogenesis of PRRS
1998b; Meier et al., 2003). La eficiencia de estos ANs en la
eliminación del PRRSV no está aún clara, mostrando diferentes
autores resultados contradictorios (Yoon et al., 1995; Murtaugh
et al., 2002; Batista et al., 2004) (Fig. 5). La transferencia de una
inmunidad maternal pasiva a los lechones por el calostro es
capaz de protegerlos frente al desarrollo de una sintomatología
clínica y a una reducción de la viremia (Murtaugh et al., 2002).
Figura 5. Esquema de la respuesta inmune en el PRRS.
La respuesta inmune celular mediada, moderada por una
producción de antígenos específicos, es inducida de manera
transitoria a las 4-8 semanas tras la infección por el PRRSV y
47
Patogenia del PRRS/Pathogenesis of PRRS
reestimulada en un periodo de 2-4 semanas tras la reinfección
(López-Fuertes et al., 1999). La respuesta inmune celular está
regulada por diferentes subtipos de linfocitos que dan lugar a la
producción de IFN-γ (Trinchieri, 1995). Sin embargo, la
existencia de una persitencia del PRRSV sugiere que esta
inmunidad celular mediada tampoco es potente y que la
producción de IFN-γ es pobre o inefectiva en los primeros
periodos de la infección (Murtaugh et al., 2002) (Fig 5).
Por otro lado, la exposición frente al PRRSV induce una
inmunidad homóloga, protegiendo eficazmente frente a las
reinfecciones con la misma cepa (Lager et al., 1997).
Es evidente que la interacción entre el virus y la
respuesta inmune del hospedador es compleja, ya que la
infección puede tanto inducir la activación como la subversión
de la respuesta inmune (Murtaugh et al., 2002). La
inmunosupresión inducida por el virus es capaz de prolongar la
persistencia del PRRSV así como de incrementar la gravedad de
otras infecciones secundarias (Mateu and Diaz, 2008).
48
Patogenia del PRRS/Pathogenesis of PRRS
Actualmente el PRRS se está controlando con una
combinación de vacunas y unas buenas prácticas de manejo y
cría en las granjas. En el mercado están disponibles varias
vacunas, incluyendo atenuadas e inactivadas, que permiten
reducir los fallos reproductivos en cerdas y reducir la viremia y
los problemas respiratorios en animales jóvenes. Sin embargo, la
complejidad de la respuesta inmune del hospedador junto con el
amplio número de cepas de PRRSV, hace que las vacunas no
sean siempre eficaces. Además, la protección muchas veces es
sólo parcial, confiriendo protección sólo frente aquellas cepas
antigénicamente homólogas a la cepa de la vacuna (Mateu and
Diaz, 2008). Sin embargo, debido a la elevada variabilidad
genética del PRRSV, es difícil desarrollar una vacuna que
confiera protección suficiente frente a todos los aislados del
PRRSV.
49
Patogenia del PRRS/Pathogenesis of PRRS
1.2. EL PAPEL DE LOS TEJIDOS LINFOIDES EN EL
PRRS.
Como se ha señalado anteriormente la replicación del
PRRSV se produce principalmente en el pulmón y en los en
órganos linfoides. Además, tras la resolución de la fase clínica
de la enfermedad, el PRRSV persiste dentro del tejido linfoide
por un largo periodo de tiempo (Rossow et al., 1996; Wills et
al., 1997b; Horter et al., 2002), sugiriendo que los órganos
linfoides desempeñan un papel importante en la patogenia de la
enfermedad.
1.2.1. Estructura y funciones biológicas de los órganos
linfoides en el cerdo.
Los linfocitos porcinos se producen en los órganos
linfoides primarios en grandes proporciones. En la médula ósea
se producen los linfocitos B, responsables de la respuesta
humoral y en el timo los linfocitos T, responsables de la
50
Patogenia del PRRS/Pathogenesis of PRRS
respuesta celular. Parte de estos linfocitos emigran a los órganos
linfoides secundarios para formar las zonas T o B dependientes.
Estas células son las responsables de las 3 principales
características del sistema inmune: diferenciación de lo propio,
especificidad y memoria (Kumar et al., 2008).
Los órganos linfoides primarios están formados por la
médula ósea y el timo, y su función es la de producir y regular la
producción y diferenciación de los distintos linfocitos. Los
órganos linfoides secundarios están formados a nivel sistémico
por los nódulos linfáticos y el bazo, y a nivel de las mucosas por
el denominado tejido linfoide asociado a mucosas (tonsilas y
placas de Peyer).
En el cerdo, las tonsilas del paladar blando comprenden
la mayor parte del tejido tonsilar (Trautmann and Fiebiger,
1957). Histológicamente, las tonsilas son tejidos linfoepiteliales
compuestos por criptas, linfoepitelio, folículos linfoides,
regiones parafoliculares, tejido conectivo, células linfoides
(linfocitos T y B), células dendríticas y macrófagos. Todos estos
51
Patogenia del PRRS/Pathogenesis of PRRS
componentes proporcionan al cerdo, inmunidad innata, celular y
humoral tanto a nivel local como a nivel sistémico.
Los linfocitos presentes en las áreas foliculares y
parafoliculares de las tonsilas, constituyen la mayor parte de las
células inmunes que se encuentran en la tonsila del cerdo
(Boeker et al., 1999; Salles and Middleton, 2000; Terzic et al.,
2002). Aproximadamente el 75% de los linfocitos de la tonsila
son linfocitos T y un 25% linfocitos B. De los linfocitos T,
aproximadamente el 92% son CD4+ (2%), CD8+ (47%), CD4+
CD8+ (43%) y el 8% restante son células T γδ, cuya función
específica es desconocida pero que puede estar implicada en la
inmunidad mediada por células y en la inmunidad antiviral
(Boeker et al., 1999; Salles and Middleton, 2000).
Tanto la localización física de la tonsila como su
organización histológica, hace que este tejido juegue un papel
importante en la respuesta inmune del hospedador. A pesar de
esto, hay microorganismos capaces de evadir las defensas
inmunológicas y utilizar las tonsilas como una puerta de entrada
en el organismo, un sitio de replicación primaria e incluso un
52
Patogenia del PRRS/Pathogenesis of PRRS
lugar de persistencia. Ejemplos de patógenos porcinos que son
capaces de colonizar las tonsilas son Streptococcus suis tipo 2
(Williams et al., 1973; Davies and Ossowicz, 1991), Salmonella
spp. (Gray et al., 1995), el virus de la peste porcina clásica
(Colgrove et al., 1969; Ressang 1973; Cheville and Mengeling,
1969) y el PRRSV (Rossow et al., 1996; Wills et al., 1997a;
Horter et al., 2002).
La función de los nódulos linfáticos es la de retener los
antígenos que puedan llegar a través de la linfa y proceder a su
presentación
y
procesamiento
antigénico mediante
la
colaboración de los macrófagos y los linfocitos que lo
componen. El cerdo presenta una circulación linfática y
disposición celular diferente a la de otros mamíferos, llegándose
a considerar como invertida (Rothkotter, 2009). Histológica y
funcionalmente se observan dos zonas bien diferenciadas en el
parénquima ganglionar: el tejido cortical, constituido por
los folículos linfoides y el tejido linfoide difuso formado por
linfocitos B, linfocitos T, macrófagos y células dendríticas, y
el tejido medular, constituido principalmente por elementos
53
Patogenia del PRRS/Pathogenesis of PRRS
celulares fijos formados por fibras reticulares y colágeno es
pobre en linfocitos, aunque sí se observan macrófagos y células
dendríticas (Rothkotter, 2009).
1.2.2. Importancia de los órganos linfoides en el PRRS
La infección por el PRRSV induce una leucopenia y
linfopenia transitoria en las primeras semanas de la enfermedad
que se resuelve a los 8-10 días (Christianson et al., 1993;
Nielsen and Botner, 1997; Feng et al., 2002; Lamontagne et al.,
2003). Los nódulos linfáticos presentan lesiones desde los 4
hasta los 28 dpi (Halbur et al. 1995b; Rossow et al. 1994, 1995).
En la mayoría de los cerdos infectados. los nódulos linfáticos
están aumentados entre 2-10 veces, presentando una hiperplasia
nodular linfoide (Lamontagne et al., 2003). Microscópicamente
las lesiones se encuentran predominantemente en los centros
germinales, los cuales están necróticos y depleccionados durante
los primeros días trás la infección. Posteriormente, los centros
germinales aumentan y están compuestos por linfocitos tipo
blastocitos (Lamontagne et al., 2003).
54
Patogenia del PRRS/Pathogenesis of PRRS
En tonsila se ha descrito la persistencia del PRRSV hasta
los 151 dpi (Wills et al., 2003), jugando este órgano un papel
fundamental como barrera primaria en la replicación y
diseminación del virus (Albina et al., 1994; Wills et al., 1997b;
Allende et al., 2000; Lamontagne et al., 2001; Horter et al.,
2002).
La información que existe sobre el efecto del virus del
PRRS sobre las poblaciones de linfocitos T en los tejidos
linfoides es escasa y controvertida, ya que los estudios se han
centrado en el estudio de estas células procedentes de muestras
de sangre, donde sólo se encuentran un 2% del total de linfocitos
T que hay en el organismo (Westermann and Pabst, 1992) y
donde no se ha demostrado ni replicación vírica ni presentación
antigénica. Por lo tanto, es posible que la principal respuesta
dependiente de linfocitos T en el PRRSV ocurra en los órganos
linfoides al igual que ocurre con la respuesta de los linfocitos T
en la inmunodeficiencia de los simios y con el virus de la
inmunodeficiencia humana (VIH) (Kuster et al., 2000; Sopper et
al.,
2003).
Así,
se ha demostrado,
mediante técnicas
55
Patogenia del PRRS/Pathogenesis of PRRS
inmunohistoquímicas (Kawashima et al., 1999) o por citometría
de flujo (Lamontagne et al., 2003), un incremento significativo
de células T CD2+CD8high en el bazo entre los 10 y 45 dpi, lo
que se ha correlacionado con una rápida eliminación del virus de
la sangre y del bazo (Lamontagne et al., 2003). Sin embargo, se
observó un descenso, o cambios no significativos, de las células
T CD8high en el resto de órganos linfoides estudiados
(Kawashima et al., 1999; Lamontagne et al., 2003), lo que
podría estar relacionado con la persistencia del virus en dichos
órganos.
El
incremento
en
el
porcentaje
de
células
T
CD2+CD8high en la infección por el virus del PRRS se reflejó
en un descenso de la ratio CD4/CD8high en el bazo y de la ratio
CD8low/CD8high en el bazo y nódulo linfático mediastínico,
mientras que en la tonsila no se detectaron cambios
significativos (Lamontagne et al., 2003).
Las células T
CD2+CD8low sólo se incrementaron
transitoriamente a los 3 dpi en tonsila. Las células asesinas (NK
del
56
inglés
Natural
Killer)
caracterizadas
como
Patogenia del PRRS/Pathogenesis of PRRS
CD2+CD8lowMIL4+, no presentan variaciones significativas en
el transcurso de la infección por el PRRSV, indicando una falta
de estimulación de la respuesta inmune innata en órganos
linfoides periféricos (Lamontagne et al., 2003) o que células NK
recién producidas fueron reclutadas en los pulmones (Samson et
al., 2000), facilitando una persistencia vírica en órganos
linfoides.
Las
poblaciones
de
células
de
memoria
CD2+CD8lowMIL4- sólo mostraron un descenso significativo
en el bazo a los 3 dpi (Lamontagne et al., 2003). En cambio,
Xiao et al. (2004) describen un descenso de la población de
células T γδ en todos los tejidos examinados, especialmente en
pulmón y nódulos linfáticos. La disminución de células T γδ
puede contribuir a la falta de una adecuada respuesta celular
mediada frente al PRRSV (Murtaugh et al., 2002) y puede
deberse a una baja producción de citoquinas proinflamatorias
(Van Reeth et al., 1999).
Aunque Lamontagne et al. (2003) inicialmente sugirieron
un posible papel de las células CD8high en la eliminación del
virus debido a las coincidencias entre el incremento de esta
57
Patogenia del PRRS/Pathogenesis of PRRS
población y el descenso del título del virus en suero y bazo,
posteriormente observaron la persistencia de ARN vírico en la
sangre y en el bazo, lo que consideraron como un indicador de
un fallo en la eliminación vírica de tipo inmuno mediada.
Además, la persistencia del virus en tonsila y en nódulo linfático
mediastínico es considerado como un indicativo de la ausencia
de una estimulación inmune de células T, lo que estaría
relacionado con los bajos niveles de células CD8high en dichos
órganos o con una rápida muerte de las células linfoides
activadas (Lamontagne et al., 2003).
Kawashima et al. (1999) describen un incremento en el
número de células B en tonsila, placas de Peyer, tejido linfoide
presente en la unión ileocecal, y en los nódulos linfáticos
inguinal superficial, mandibular y traqueobronquial durante una
infección por el PRRSV, mientras que en el timo observan un
descenso de células B.
58
Patogenia del PRRS/Pathogenesis of PRRS
1.3. EL PAPEL DE LAS CITOQUINAS EN EL PRRS:
INTERFERONES, CITOQUINAS PROINFLAMATORIAS
Y OTRAS CITOQUINAS
Las citoquinas son productos polipeptídicos de muchos
tipos celulares, pero principalmente de linfocitos activados y
macrófagos, que funcionan como mediadores de las respuestas
inflamatorias e inmunitarias (Kumar et al., 2008). Numerosos
autores han determinado la expresión de citoquinas en suero y
su relación con la patogenia del PRRS (Van Reeth and
Nauwynck, 2000; Liu et al., 2010; Borghetti et al., 2011) o bien
en pulmón (Gómez-Laguna et al., 2010a). Lunney et al. (2010)
determinaron la expresión génica de citoquinas en órganos
linfoides y su papel en la eliminación del PRRSV. Sin embargo,
no hay estudios que determinen la expresión de citoquinas in
situ en los órganos linfoides y su papel en la patogenia del
PRRS.
59
Patogenia del PRRS/Pathogenesis of PRRS
1.3.1. Interferones (IFNα, IFNγ)
Los
Interferones
(IFNs)
son
glucoproteínas
termoestables, ligeramente básicas y resistentes a las variaciones
de pH, que pertenecen al grupo de las linfoquinas producidas
espontáneamente en pequeñas cantidades por todas las células
animales o humanas como respuesta a una infección viral
inhibiendo de forma inespecífica la replicación viral dentro de
las células del huésped (Tressguerres, 1999; Roitt et al., 2001;
Abbas et al., 2001; Guyton et al., 2001; Vander et al., 2001).
A pesar de que potencialmente todas las células tienen la
capacidad de producir IFNs, lo secretan en mayores cantidades
los leucocitos, fibroblastos y macrófagos (Kumar et al., 2008).
Existiendo dos tipos fundamentales de IFNs, los denominados
Tipo I y Tipo II
IFN tipo I: son una superfamilia que incluyen 7 subfamilias:
IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, IFN-δ, e IFN-τ (Pestka et
al., 2004). Los subtipos IFN-α e IFN-β están muy relacionados,
comparten un receptor común y tienen efectos similares (Van
60
Patogenia del PRRS/Pathogenesis of PRRS
Reeth and Nauwynck, 2000), constituyendo uno de los dos
mecanismos implicados en la respuesta inmune mediada por
citoquinas e induciendo un estado antiviral en las células diana
(Van Reeth and Nauwynck, 2000; Biron and Sen, 2001).
IFN tipo II: incluye al IFN-γ o inmune, está producido
exclusivamente por células del sistema inmune (Boehm et al.,
1997) a diferencia de los IFN tipo I, y representa un importante
regulador de la respuesta inmune adquirida.
Las principales células implicadas en la expresión de
IFN-α e IFN-γ, así como sus funciones biológicas generales y el
papel que juegan en el PRRS se encuentran resumidas en la
tabla 1.
El PRRSV induce niveles mucho más bajos de IFN-α en
los pulmones si lo comparamos con otras enfermedades víricas
como son el SIV o el PRCV (Van Reeth et al., 1999; Van Reeth
et al., 2002). Además, el PRRSV es capaz de inhibir la respuesta
61
Patogenia del PRRS/Pathogenesis of PRRS
de IFN-α (Albina et al., 1998a; Buddaert et al., 1998; Chung et
al., 2004), aunque los mecanismos que utiliza el virus no se
conocen todavía.
Con respecto a IFN-γ, Meier et al. (2003) describen un
incremento inicial en la infección con el PRRSV que facilitaría
la diferenciación de las células T en células secretoras de IFN-γ
(IFN-γ-SC), dando lugar a un incremento eventual de la
respuesta inmune. Sin embargo, otros autores, han descrito que
no se producen cambios en la expresión de IFN-γ tras la
vacunación con una vacuna viva modificada de una cepa
europea del PRRS (Sipos et al., 2003).
1.3.2. Citoquinas proinflamatorias (IL-1, IL-6, TNFα)
Las
citoquinas con actividad proinflamatoria son
producidas por los monocitos y macrófagos activados durante la
respuesta inmune innata, aunque también pueden ser producidas
por linfocitos activados (Th1 o citotóxicos), y otras células no
pertenecientes al sistema inmune. Las principales citoquinas que
participan en los acontecimientos celulares y moleculares
62
Patogenia del PRRS/Pathogenesis of PRRS
asociados con los fenómenos inflamatorios son: IL-1, IL-6,
TNF-α (Kumar et al., 2008).
IL-1, Junto con IL-6 y TNF-α, constituyen una vía alternativa
implicada en la respuesta inmune innata (Biron and Sen, 2001).
Existen dos formas, la IL-1α y la IL-1β que, aunque solamente
tienen un 25 % de homología en su secuencia aminoacídica,
comparten el mismo receptor y ejercen efectos biológicos
similares (Murtaugh et al., 1996; Biron and Sen, 2001). Parte de
sus efectos proinflamatorios se deben a que induce la liberación
de histamina en los mastocitos, generando vasodilatación y
aumento de la permeabilidad vascular en el lugar de la
inflamación. Es el principal pirógeno endógeno, induciendo
fiebre a través de la producción de prostaglandinas. También
promueve la síntesis de proteínas de fase aguda por los
hepatocitos y actúa sobre el SNC induciendo sueño y anorexia,
signos típicamente asociados con los procesos infecciosos
(Kumar et al., 2008).
63
Patogenia del PRRS/Pathogenesis of PRRS
IL-6 Es una citoquina multifuncional que juega un papel
importante en la defensa del hospedador, reacciones de fase
aguda y respuesta inmune. Además de su efecto en la
inflamación, se ha observado que promueve la diferenciación de
linfocitos B hacia células plasmáticas, induciendo la producción
de inmunoglobulinas (Kishimoto et al., 1994). También puede
aumentar la producción de IL-2 y el desarrollo de los
precursores hematopoyéticos dependientes de la IL-3 (Ihle et al.,
1995).
TNF.
Se
han
descrito
dos
moléculas
estrechamente
relacionadas, el TNF-α y el TNF-β, con elevada homología en
su secuencia aminoacídica (Callard and Gearing, 1994). Los
factores de necrosis tumoral fueron descritos inicialmente por
su capacidad de causar necrosis en algunos tumores (Gruss
and Dower, 1995). Con posterioridad, sin embargo, ganaron
protagonismo por las numerosas funciones que ejercen sobre las
respuestas inmunes. Junto con la IL-1 está implicado en los
procesos inflamatorios derivados de los procesos infecciosos,
64
Patogenia del PRRS/Pathogenesis of PRRS
elevando la temperatura corporal y produciendo caquexia y
sueño al actuar sobre el SNC (Kumar et al., 2008). Por otra
parte, induce la expresión de moléculas de adhesión y estimula
la producción de IL-8 por células del endotelio vascular, lo que
contribuye a la extravasación de linfocitos, neutrófilos y
monocitos (Kishimoto et al., 1994). El TNF-β, o linfotoxina, se
une a los mismos receptores que el TNF-α e induce funciones
similares (Van Reeth and Nauwynck, 2000)
Los cambios en la expresión de estas citoquinas se han
relacionado con la modulación de la respuesta inmune frente a
varios virus porcinos (Van Reeth et al., 1999; Van Reeth and
Nauwynck, 2000; Carrasco et al., 2002; Salguero et al., 2002).
El PRRSV ha sido descrito como un pobre inductor de
citoquinas (Van Reeth et al., 1999), sin embargo, GómezLaguna et al., (2010a) describieron un incremento significativo
en la síntesis de citoquinas proinflamatorias en el pulmón de
cerdos infectados con el PRRSV, asociándolo a una síntesis
paracrina de dichas citoquinas (Gómez-Laguna et al., 2010b).
65
Patogenia del PRRS/Pathogenesis of PRRS
Las principales células implicadas en la expresión de las
citoquinas proinflamatorias, así como sus funciones biológicas
generales y el papel que juegan en el PRRS se encuentran
resumidas en la tabla 2.
1.3.3. Otras citoquinas (IL-10, IL-12).
IL-10. Es producida por monocitos/macrófagos, linfocitos T
reguladores, y con menor frecuencia por linfocitos B (Biron and
Sen, 2001; Moore et al., 2001). Es la citoquina inmunosupresora
por excelencia, inhibiendo la síntesis de muchas otras, entre las
que se encuentran IFN-γ, TNF-α, IL-2, IL-12, la expresión de
MHC-II y las moléculas de adhesión en monocitos (Kishimoto
et al., 1994), teniendo también efectos antiproliferativos sobre
muchos tipos celulares. Sin embargo, la IL-10 ejerce también
múltiples actividades inmunomoduladoras, ya que es un cofactor
para el
crecimiento
de líneas
y colonias
de células
mastocíticas in vitro y regula las funciones mediadas por
linfocitos B, induciendo la síntesis de IgG, y linfocitos T,
influyendo en el desarrollo de timocitos y células T. Esta
66
Patogenia del PRRS/Pathogenesis of PRRS
citoquina
también
ejerce
efectos
reguladores
sobre
la
angiogénesis (Callard et al., 1994).
Patton et al. (2009) describen un aumento de la expresión
del CD 163, receptor necesario para la internalización del
PRRSV al incubar in vitro monocitos CD14+ con IL-10
relacionándose con un incremento en la expresión del PRRSV.
En este sentido varios autores señalan que la IL-10 jugaría un
papel importante en la inmunopatogenia del virus del PRRS
(Suradhat et al, 2003; Charerntantanakul et al., 2006; SilvaCampa et al., 2009; Gómez-Laguna et al., 2010a). Así, GómezLaguna et al. (2010a) observaron que la expresión de IL-10 en
el pulmón estaba correlacionada significativamente con la
replicación del virus del PRRS, sugiriendo que PRRSV podría
inducir la expresión de esta citoquina. En este sentido Mitchell
and Kumar, (2004) describen que la IL-10 podría reducir los
niveles de citoquinas implicadas en la eliminación del virus
como IFNs e IL-12. Sin embargo, este papel es controvertido ya
que Díaz et al. (2006) señalan que diferentes cepas de este virus
son capaces de inducir respuestas de IL-10 diferentes en PBMC
67
Patogenia del PRRS/Pathogenesis of PRRS
(del inglés Periferal Blood Mononuclear Cells) de cerdos
infectados con el PRRSV. En este sentido Gimeno et al. (2011)
describen la expresión diferencial de citoquinas utilizando 39
aislados diferentes del genotipo europeo del PRRSV, mostrando
que diferentes aislados son capaces de producir diferentes
patrones de IL-10 y TNF-α in vitro.
IL-12, Es un heterodímero constituido por dos subunidades,
p35, expresada constitutivamente y p40, que se expresa en
respuesta a una amenaza (Biron and Sen, 2001). La síntesis de
ambas subunidades está regulada diferencialmente, siendo
ambas necesarias para la actividad funcional del heterodímero
(Biron and Sen, 2001).
Las principales células implicadas en la expresión de IL10 e IL-12, así como sus funciones biológicas generales y el
papel que juegan en el PRRS se encuentran resumidas en la
tabla 3.
68
Patogenia del PRRS/Pathogenesis of PRRS
Tabla 1. Respuesta inmune, células productoras, funciones biológicas generales y papel de los interferones en el PRRS.
Citoquina
Respuesta
inmune
Células
Productoras
Funciones Biológicas generales
Papel en el PRRS
IFN-α
Innata
Macrófagos
Linfocitos
Fibroblastos
• Antiviral en células diana
• Inhibición de la expresión de IL-12.
• Activación de la citotoxicidad de macrófagos y
células NK.
• In vivo: inducción de la proliferación de
células T CD8
• Diferenciación de células T en IFNγ-SCs en
infecciones víricas
• Correlación entre IFNα-SCs específico de virus e IFNγ-SCs
específico de virus (Royaee et al., 2004)
• Baja respuesta de IFNα por MAPs (Albina et al., 1998a;
Buddaert et al., 1998; Van Reeth et al., 1999) o PBMCs
(Albina et al., 1998a; Chung et al., 2004; Van Gucht et al.,
2004)
• Diferentes aislados de el PRRSV desarrollan diferentes
habilidades para inducir o inhibir IFNα (Lee et al., 2004)
(Cousens et al., 1999; Cella et al., 2000; Kadowaki et
al., 2000; Van Reeth and Nauwynck, 2000; Biron and
Sen, 2001; Tizard, 2008)
IFN-γ
Adaptativa
Células NK
Células T
(CD4+CD8low,
CD4-CD8high y
células T γδ)
• Antiviral en células diana
• Promueve la respuesta inmune Th1 e inhibe la
Th2
•Estimula a los monocitos a un mecanismo de
defensa antimicrobiano mediante la inducción de
óxido nítrico sintetasa.
•Incrementa
la
respuesta
inflamatoria,
incrementando la expresión de los receptores de
TNF
•Aumenta la produccion de citoquinas como IL12 por macrófagos y DC
•Expresión de IFNγ por citoquinas como IL-12,
especialmente en combinación con IL-18
(Abbas et al., 1996; Biron and Sen, 2001, 2007;
Domeika et al., 2002; Rodríguez-Carreño et al.,
2002;Braciale et al., 2007)
•Aumento en la vacunación de el PRRSV utilizando IL-12 o
IFNα como adyuvantes (Foss et al., 2002; Meier et al., 2004)
•Efecto tiempo dosis relacionado con el requerimiento de la
activación macrofágica (Bautista and Molitor, 1999)
•Bloquea la replicación de el PRRSV en cultivos celulares
(Bautista and Molitor, 1999), inhibiendo la síntesis de ARN
vía PKR (Rowland et al., 2001)
•Retrasa la expresión de el PRRS (Meier et al., 2003; Xiao et
al., 2004; Díaz et al., 2005, 2006; Olin et al., 2005)
•IFNγ-SC: correlacionado con viremia en cerdos infectados y
vacunados (Díaz et al., 2005; 2006),protege frente a fallos
reproductivos (Lowe et al., 2005)
•Diferente expresión de IFNγ dependiento de la cepa de el
PRRSV(Thanawongnuwech et al., 2003)
69
Patogenia del PRRS/Pathogenesis of PRRS
Tabla 2. Respuesta inmune, células productoras, funciones biológicas generales y papel de citoquinas proinflamatorias en el PRRS.
Citoquina
Respuesta
inmune
Células
Productoras
Funciones Biológicas generales
Papel en el PRRS
IL-1
Innata
Respuesta
proinflamatoria
Macrófagos
Monocitos
Neutrófilos
Fibroblastos
Células
endoteliales
•Incrementa la respuesta de IFN-γ
•Induce adherencia de leucocitos a células endoteliales.
•Incrementa la permeabilidad microvascular.
•Induce broncoconstricción.
•Pirógeno endógeno.
•Contribuye a la liberación de IL-6
(Murtaugh et al., 1996; Van Reeth and Nauwynck 2000;
Biron and Sen, 2001).
•In Vitro:Ligero incremento en la expresión de IL1α e IL-1β (Thanawongnuwech 2001, 2004)
• In Vivo: incremento en IL-1α e IL-1β en BALF
desde 1 a 52 dpi (Labarque et al., 2003a; Van
Gucht et al., 2003, 2004; Thanawongnuwech et al.,
2004) aunque en suero IL-1β no aumenta hasta los
42 dpi (Thanawongnuwech et al., 2004)
•No cambios en ARNm de IL-1α tras vacunación
con una cepa europea (Sipos et al., 2003).
•El PRRSV disminuye IL-1 y TNF-α en MAPs
infectados in Vitro (López-Fuentes et al., 2000).
IL-6
Innata
Respuesta
proinflamatoria
PBMCs
PAMs
PIMs
Fibroblastos
Células
endoteliales
•Disminuye IL-1 y TNF-α a través de receptores
antagonistas de IL-1 y receptores solubles de TNF- α
•Activación de hepatocitos induciendo la síntesis de
proteínas de fase aguda durante la respuesta de fase
aguda.
•Promociona el crecimiento y diferenciación de células
B en células plasmáticas secretoras de inmunoglobulinas,
jugando un importante papel en la respuesta humoral.
•Respuesta antiinflamatoria inhibiendo la liberación de
IL-1 y TNF- α y reduciendo el flujo de células
inflamatorias al lugar de la inflamación.
(Murtaugh et al., 1996; Scamurra et al., 1996 ; Biron and
Sen, 2001 ; Van Gucht et al., 2003).
•Bajos niveles tras infección con el PRRSV desde
3 a 21 dpi (Asai et al., 1999; Van Gucht et al.,
2003).
•Resultados contradictorios en amplificación de
ARNm de IL-6, siendo detectada desde 0 a 28 dpi
(Feng et al., 2003; Thanawongnuwech et al., 2004)
•Cuando los cerdos son vacunados con una cepa
europea de el PRRSV, IL-6 aumenta a los 22 y 44
dpi (Sipos et al., 2003). La baja respuesta de IL-6
tras inoculación o vacunación muestra que
probablemente esta citoquina no tenga un papel
significante en el PRRS.
70
Patogenia del PRRS/Pathogenesis of PRRS
Citoquina
Respuesta
inmune
Células
Productoras
Funciones Biológicas generales
Papel en el PRRS
TNF-α
Innata
Respuesta
proinflamatoria
Monocitos
Macrófagos
Células NK
•Incremento de la permeabilidad vascular.
•Propiedades de adhesión a las células endoteliales.
•Muerte celular
•Activación de monocitos y neutrófilos.
•Inducción de respuesta IFNγ
•Migración de células dendríticas.
•Aumento de la expresión de MHC clase I.
•Activación de estados antivirales
•Inducción de fiebre, aletargamiento, pérdida de apetito y
proteínas de fase aguda en el hígado.
(Murtaugh et al., 1996; Van Reeth and Nauwynck, 2000;
Biron and Sen, 2001)
•TNFα es expresada in vitro (Thanawongnuwech
et al., 2001, 2004)
•Débil o nula expresión in vivo (Asai et al., 1999;
Choi et al., 2002; Johnsen et al., 2002; Labarque et
al., 2003a; Van Gucht et al., 2003;
Thanawongnuwech et al., 2004)
• Pobre expresión tras vacunación de el PRRSV
(Sipos et al., 2003)
•Reducción de la replicación viral en PAMs tras
adición de TNFα recombinante porcino (LópezFuertes et al., 2000).
•Diferentes aislados de el PRRSV desarrollan
diferentes habilidades para inducir o inhibir TNF-α
(Gimeno et al., 2011)
71
Patogenia del PRRS/Pathogenesis of PRRS
Tabla 3. Respuesta inmune, células productoras, funciones biológicas generales y papel de IL-10 e IL-12 en el PRRS.
Citoquina
Respuesta
inmune
IL-10
Adaptativa
IL-12
72
Innata
Adaptativa
Inmunidad
de las
mucosas
Células
Productoras
Funciones Biológicas generales
Monocitos
Macrófagos
Tregs
Células B
•Inhibición de síntesis de citoquinas: IL-1α, IL-1β,
IL-6, IL-12, IFNγ y TNFα
•Supresión de quemoquinas y síntesis de PGE2.
•Inhibición de la presentación antigénica de las
MHC clase II.
•Inhibición de la expresión de TLR4.
•Disminución de la respuesta Th1 y Th2.
•Inhibición de la proliferación de células T CD4+
•Estimula a las NK y células T CD8+ e induce su
reclutamiento, citotoxicidad y proliferación.
(Cavaillon, 1994; Biron and Sen, 2001; Moore et al.,
2001; Pestka et al., 2004)
DCs activadas
Macrófagos
•Producción tras un desafío de células NK e IFNγ
• Promoción de la diferenciación citolítica de las
células T CD8
•Activación de células T CD4 hacia células T CD4
efectoras.
•Promoción de la inmunidad tipo I contra patógenos
intracelulares.
(Chan et al., 1992; Cesano et al., 1993 ; Hsieh et al.,
1993 ; Macatonia et al., 1995 ; Biron and Sen, 2001,
2007 ; Braciale et al., 2007).
Papel en el PRRS
• In vitro e in vivo: infecciones con el PRRSV muestran
un incremento en los niveles protéicos y genómicos de
IL-10 (Johnsen et al., 2002; Chung and Chae, 2003;
Feng et al., 2003; Labarque et al., 2003a; Suradhat and
Thanawongnuwech, 2003; Suradhat et al., 2003;
Thanawongnuwech
and
Thacker,
2003;
Thanawongnuwech et al., 2004; Díaz et al., 2005, 2006 ;
Gómez-Laguna et al., 2010a).
•Otros autores describen que no hay cambios en la
expresión de IL-10 tras la infección o vacunación con el
PRRSV (López-Fuertes et al., 1999; Sipos et al., 2003).
•IL-10 coincide con bajos niveles de IFNγ-SCs en
PBMCs de cerdos infectados o vacunados con el
PRRSV (Díaz et al., 2005; 2006).
•Diferentes aislados de el PRRSV desarrollan diferentes
habilidades para inducir o inhibir IL-10 (Gimeno et al.,
2011)
•No expresión de ARNm IL-12 por PAMs infectados in
Vitro (Thanawongnuwech et al., 2001).
•Expresión leve-moderada de ARNm IL-12 en PAMs,
BALCs y/o PBMCs (Johnsen et al., 2002; Feng et al.,
2003; Thanawongnuwech and Thacker, 2003).
•Incremento en la expresión de ARNm de IL-12 de 1 a 7
dpi en el pulmón (Chung and Chae, 2003).
•Incremento en la ratio IL-10/IL-12 (Feng et al., 2003).
•Si se usa como adyuvante da lugar a un marcado
incremento de la expresión de IFN-γ (Foss et al., 2002;
Carter and Curiel, 2005) pero no a una mejora en los
títulos de anticuerpos (Foss et al., 2002).
Patogenia del PRRS/Pathogenesis of PRRS
1.4. APOPTOSIS EN EL PRRS
1.4.1. Conceptos generales
La palabra apoptosis proviene del griego, άποπτϖσις,
recordando a las hojas que caen en otoño desde los árboles o a
los pétalos que caen desde las flores (Kerr et al., 1972). La
muerte celular se puede producir de dos maneras diferentes: por
necrosis y por apoptosis (Wyllie et al., 1980). Mientras que la
necrosis es la muerte celular pasiva que sigue a una agresión
celular con daño grave de las membranas (Majno and Joris,
1995), la apoptosis es un proceso activo en respuesta a una
variedad de estímulos fisiológicos y patológicos, en el que la
célula participa de su propia destrucción siguiendo unos pasos
prefijados (Kerr et al., 1972). En la figura 6 se muestran las
principales diferencias que existen entre la necrosis y la
apoptosis.
73
Patogenia del PRRS/Pathogenesis of PRRS
Figura 6. Diferencias principales entre la Necrosis y la Apoptosis
El daño o la modificación del ADN nuclear serían los
principales inductores de la apoptosis. El proceso de apoptosis
puede ser iniciado por diferentes estímulos, fisiológicos y
patológicos, incluyendo la ausencia de factores de crecimiento,
la exposición a diferentes agentes (biológicos, físicos y/o
químicos) y el reconocimiento de modificaciones genéticas
(Bennett et al., 1984; Albright et al., 1997; Clutton, 1997).
Asimismo, cuando los genes que controlan el proceso apoptótico
74
Patogenia del PRRS/Pathogenesis of PRRS
mutan, o se infra o se sobreexpresan, la apoptosis origina
diversas patologías. En este sentido se han identificado
diferentes genes en el proceso apoptótico, ya sea promoviendo o
inhibiendo la apoptosis (oncogenes y genes supresores).
Además, el óxido nítrico podría estimular o inhibir las apoptosis.
Así, mientras que la estimularía en neuronas, en macrófagos, en
las células β pancreáticas y participaría
en el rechazos de
injertos cardíacos en ratas, la inhibiría en ciertas hepatopatías
(Szabolcs M., 1996)
Actualmente se describen dos vías diferentes de
desencadenamiento de la apoptosis, la vía intrínseca y la vía
extrínseca (Roy and Nicholson, 2000) (Fig. 7). La vía intrínseca
es iniciada como respuesta a un estrés celular, como daño en el
ADN, tóxicos y de la replicación de ADN no programada (Roy
and Nicholson, 2000). La proteína p53 es activada tras un daño
en el ADN, desencadenando la apoptosis a través de la
producción de un desequilibrio en la ratio Bax/Bcl-2 (Oltvai et
al., 1993; Miyashita et al., 1994). En células sanas, el Bcl-2 está
presente en la superficie de la membrana mitocondrial
75
Patogenia del PRRS/Pathogenesis of PRRS
previniendo la inducción de la apoptosis mediante el bloqueo de
la acción del Bax y de las proteínas relacionadas. Este
desequilibrio en la ratio Bax/Bcl-2 a favor del Bax y las
proteínas asociadas hace que se produzca una disrupción de la
membrana mitocondrial, dando lugar a la liberación del
citocromo C al citoplasma (Kluck et al., 1997). El citocromo C
liberado forma heterodímeros con la proteínas Apaf-1, formando
el apoptosoma (Zou et al., 1997). La formación del apoptosoma
dará lugar a la activación de la caspasa 9, que a su vez activará
la caspasa 3, dando lugar a una cascada de caspasas que
provocarán la digestión de las proteínas estructurales en el
citoplasma, la degradación del ADN y la fagocitosis de la célula.
La vía extrínseca de la apoptosis tiene lugar cuando uno
o varios receptores de muerte de superficie celular, como los
receptores de la familia del factor de necrosis tumoral (TNF-R)
y los receptores del Fas, se unen a su ligando apropiado como
TNF-α o FasLigand (FasL), (Nagata, 1997; Ashkenazi and
Dixit, 1998). Esta unión activará a la caspasa 8 que a su vez
activará la caspasa 3 (Budihardjo et al., 1999; Earnshaw et al.,
76
Patogenia del PRRS/Pathogenesis of PRRS
1999; Ashkenazi, 2002; Mitchell and Cotran, 2007), originando
una cascada de caspasas comunes a la vía intrínseca que llevarán
a la muerte celular.
Figura 7. Vías Intrínseca y Extrínseca de la apoptosis.
1.4.2. Actualización de los fenómenos de apoptosis en el PRRS
Diferentes virus han desarrollado estrategias para inhibir
la apoptosis de la célula hospedadora en las fases tempranas de
la infección y así asegurarse la replicación vírica y/o inducir la
apoptosis de la célula hospedadora en las fases finales de la
77
Patogenia del PRRS/Pathogenesis of PRRS
infección asegurándose así la diseminación del virus. Así
mientras que el virus de la peste porcina africana (ASFV del
inglés African swine fever virus) codifica proteínas que inhiben
la apoptosis mediante la inactivación del gen p53 o inactivando
la molécula Bax, produciendo un desequilibrio en la ratio
Bax/Bcl-2 a favor de Bcl-2 (Neilan et al., 1993; Afonso et al.,
1996; Brun et al., 1996; Revilla et al., 1997; Young et al., 1997)
y los Baculovirus, disminuyen los fenómenos de apoptosis al
expresar inhibidores de las caspasas (Manji and Friesen, 2001),
el de virus de la arteritis equina (EAV) induce la apoptosis
mediante
una
fragmentación
del
ADN
en
olígomeros
nucleosomales y una activación de las caspasas, estando esta
inducción de los fenómenos de apoptosis correlacionado con el
efecto citopático que produce este virus (Archambault and StLaurent, 2000).
En el transcurso de la la infección por el PRRSV se ha
demostrado el incremento de los fenómenso de apoptosis
(Suárez et al., 1996a; Sur et al., 1997; Fernández et al., 2002;
Gagnon et al., 2003). Sin embargo, aún no está del todo
78
Patogenia del PRRS/Pathogenesis of PRRS
elucidado que mecanismo utiliza el virus del PRRS para inducir
los fenómenos de apoptosis. Así, mientras que algunos estudios
describen la apoptosis como una consecuencia de la replicación
del virus en la célula (apoptosis directa) (Suárez et al., 1996a;
Kim et al, 2002; Coster et al., 2008) y en la que estaría
involucrada la glicoproteína viral GP5 (Suárez et al., 1996a).
Otros estudios describen que el virus induciría, al principio de
la enfermedad,
una inhibición de la apoptosis en las células
infectadas, pero que esta inhibición se tornaría en una inducción
de la apoptosis de las células infectadas al final de la misma
(Coster et al., 2008). Sin embargo, numerosos estudios señalan
que los fenómenos de apoptosis serían inducidos principalmente
en células no infectadas (apoptosis indirecta) (Sur et al., 1998;
Sirinarumitr et al., 1998; Choi and Chae, 2002; Feng et al.,
2002; Labarque et al., 2003a).
Entre los posibles mecanismos por el cual el PRRSV
desencadenaría la apoptosis en las células no infectadas se
considera a la unión del Fas/FasL, ya que Chang et al. (2007)
describen, en un estudio in vitro, que el PRRSV es capaz de
79
Patogenia del PRRS/Pathogenesis of PRRS
inducir la apoptosis de los linfocitos por la vía extrínseca, lo que
para estos autores podría ser la causa de la caracterísitica
inmunosupresión que se produce en el PRRS. Así mismo, se ha
demostrado que algunas citoquinas, como el TNF-α (Larrick and
Wright, 1990), la IL-1 (Dunger et al., 1996; Castigli et al., 2000)
y la IL-10 (Estaquier et al., 1997; Liu et al., 2001) pueden
inducir la apoptosis.
Posiblemente el papel del TNF-α en el mecanismo de la
inducción de la apoptosis en el PRRS no sea importante, ya que
esta citoquina se expresa a niveles muy bajos durante la
infección con este virus (Van Reeth and Nauwynck, 2000;
Gómez-Laguna et al., 2010a). Sin embargo, se han demostrado,
en los pulmones de cerdos infectados con el PRRSV, altos
niveles de IL-1 e IL-10 (Van Reeth and Nauwynck, 2000;
Thanawongnuwech, 2000; Gómez-Laguna et al., 2010a), dos
citoquinas que pueden desencadenar los fenómenos de
apoptosis. Lo que coincide con los estudios de Labarque et al.
(2003a) que describen un pico en la expresión de apoptosis en
pulmones de cerdos infectados con el PRRSV a los 14 dpi,
80
Patogenia del PRRS/Pathogenesis of PRRS
precedido por un pico de producción de IL-1 e IL-10 a los 9 dpi,
sugiriendo un posible papel de estas citoquinas en la inducción
de la apoptosis de los monocitos/macrófagos intersticiales no
infectados con el PRRSV. Sin embargo, esta hipótesis no pudo
ser ratificada mediante un estudio in vitro al no observar
apoptosis en monocitos ni en MAPs tratados previamente con
IL-1 o IL-10 recombinante porcina.
Como hemos comentado anteriormente los órganos
linfoides juegan un papel muy importante en la patogenia del
PRRS, así como la expresión de citoquinas como estrategia del
virus para evadir la respuesta inmune o la inducción por parte
del virus de los fenómenos de apoptosis en macrófagos y
linfocitos, dando lugar a una inmunosupresión e impidiendo el
desarrollo de una correcta respuesta inmune. Sin embargo, son
muy escasos los estudios que han abordado el estudio in situ de
los fenómenos de apoptosis en los órganos linfoides (Sur et al.,
1998) no habiéndose realizado en los órganos linfoides, hasta
la fecha, estudios in vivo que determinen la expresión in situ de
81
Patogenia del PRRS/Pathogenesis of PRRS
citoquinas y el papel que estas y los fenómenos de apoptosis
desempeñarían en la patogenia del PRRS.
82
Patogenia del PRRS/Pathogenesis of PRRS
OBJETIVOS DE LA TESIS/ AIMS OF THE THESIS
83
Patogenia del PRRS/Pathogenesis of PRRS
OBJETIVOS
El objetivo general de esta tesis doctoral fue evaluar el papel de
la expresión de citoquinas y de los fenómenos de apoptosis en
diferentes órganos linfoides en la patogenia del Síndrome
Reproductivo y Respiratorio Porcino. Para alcanzar este objetivo
general planteamos los siguientes objetivos específicos:
1. Evaluar la expresión de citoquinas proinflamatorias y del
antígeno vírico en órganos linfoides (tonsila y nódulos
linfáticos mediastínico y retrofaríngeo) de cerdos
infectados experimentalmente con un aislado europeo
del PRRSV.
2. Determinar el papel que juega la expresión de citoquina
reguladoras (IL-10, IL-12, IFN-γ e IFN-α) en los órganos
linfoides en el transcurso de una infección experimental
con un aislado europeo del PRRSV.
3. Valorar el desarrollo de los fenómenos de apoptosis en
tonsila y nódulo linfático mediastínico de cerdos
inoculados experimentalmente con el aislado 2982 del
PRRSV, así como su correlación con la expresión del
84
Patogenia del PRRS/Pathogenesis of PRRS
antígeno vírico, caspasa 3, TUNEL y citoquinas proapoptóticas.
4. Determinar
el
desenmascaramiento
fijador
ideal
y
el
para
método
la
de
detección
inmunohistoquímica de los mediadores de apoptosis en
muestras de cerdo incluidas en parafina.
85
Patogenia del PRRS/Pathogenesis of PRRS
AIMS
The general aim of the present thesis was to evaluate the role of
the expression of cytokines and apoptosis phenomena in
lymphoid organs in the pathogenesis of Porcine Reproductive
and Respiratory Syndrome. To achieve this general aim, we
carried out the following specific aims:
1. To evaluate the expression of proinflammatory cytokines
and viral antigen in lymphoid organs (mediastinal and
retropharyngeal lymph nodes and tonsil) from pigs
experimentally infected with a European PRRSV strain.
2. To determine the role of the expression of regulatory
cytokines (IL-10, IL-12, IFN-γ and IFN-α) in lymphoid
organs during
an experimental infection with a
European PRRSV strain..
3. To assess the development of apoptosis phenomena in
tonsil and mediastinal lymph node of pig experimentally
inoculated with the isolate 2982 of PRRSV, as well as
their correlation with the expression of viral antigen,
caspasa 3, TUNEL and pro-apoptotic cytokines.
86
Patogenia del PRRS/Pathogenesis of PRRS
4. To determine the ideal fixative and antigen retrieval
method for the immunohistochemical detection of
apoptosis mediators in porcine paraffin embedded
tissues.
87
Patogenia del PRRS/Pathogenesis of PRRS
EXPERIMENTAL STUDIES
3.1. COMMON EXPERIMENTAL DESIGN.
3.2.
DIFFERENTIAL
EXPRESSION
OF
PROINFLAMMATORY CYTOKINES IN THE LYMPHOID
ORGANS
OF
PORCINE
REPRODUCTIVE
AND
RESPIRATORY SYNDROME VIRUS-INFECTED PIGS.
3.3.
IL-10,
IL-12,
IFN-α
AND
IFN-γ
IMMUNOHISTOCHEMICAL EXPRESSION IN LYMPHOID
ORGANS
OF
PORCINE
REPRODUCTIVE
AND
RESPIRATORY SYNDROME VIRUS-INFECTED PIGS.
3.4. APOPTOSIS IN LYMPHOID TISSUES OF PRRSV
INFECTED PIGS DETECTED BY TUNEL AND CLEAVED
CASPASE-3 IMMUNOHISTOCHEMISTRY.
3.5.
IMMUNOHISTOCHEMICAL
EXTRINSIC
AND
APOPTOSIS
IN
TISSUES.
88
INTRINSIC
PORCINE
DETECTION
OF
MEDIATORS
OF
PARAFFIN-EMBEDDED
Patogenia del PRRS/Pathogenesis of PRRS
3.1. COMMON EXPERIMENTAL DESIGN.
Virus
The third passage of the PRRSV field isolate 2982 (kindly
provided by Dr. E. Mateu) was used in this study. The virus was
initially isolated in porcine alveolar macrophages (PAMs) from
serum of a naturally infected piglet during an outbreak of PRRS
affecting a Spanish farm where piglets displayed respiratory
signs. Viral stock was adjusted to a titre of 103.0 TCID50/ml as
determined by means of an immunoperoxidase monolayer assay
(IPMA) (Weensvoort et al., 1991) in PAMs. PRRSV strain 2982
belonged to EU sub-genotype 1 and shared a 93 % similarity to
LV based on ORF5 sequences. The viral stock was free of
aerobic and anaerobic bacterial contamination as determine after
bacteriological culture.
Animals and experimental design
A total of thirty-two, male, five-week-old piglets from a highhealthy farm historically seronegative for PRRSV were used for
the experimental infection. Pigs were clinically healthy and were
housed in biocontainment level III animal facilities at “Centro
de Investigación en Sanidad Animal” (CISA-INIA, Valdeolmos,
89
Patogenia del PRRS/Pathogenesis of PRRS
Madrid, Spain). Pigs were allowed to stay housed in the
biocontainment level III facilities 10 days prior to challenge.
Twenty eight pigs were randomly distributed in batches of four
and inoculated by the intramuscular route, behind the right ear in
the neck with 1 ml the viral inoculum. The four animals of each
batch were killed at 3, 7, 10, 14, 17, 21 and 24 days postinoculation (dpi), respectively. The four remaining pigs, used as
controls, were inoculated with 1 ml of sterile RPMI 1640
medium (BioWhitaker) following the same procedure and
humanely killed at the end of the study (24 dpi). Euthanasia was
performed by initial anesthesia with tiletamine-zolazepam
(ZOLETIL, Virbac) followed by a lethal dose of 5 % sodium
thiopental (THIOVET, Vet Limited). Tissue samples were
analysed by in situ hybridization and were proved as negative to
PCV2. This experiment was carried out under the guidelines of
the European Union (Directive 86/609/EEC) and was approved
by Cordoba University and CISA-INIA Ethical Review
Committees.
90
Patogenia del PRRS/Pathogenesis of PRRS
3.2.
DIFFERENTIAL
PROINFLAMMATORY
EXPRESSION
CYTOKINES
IN
OF
THE
LYMPHOID ORGANS OF PORCINE REPRODUCTIVE
AND RESPIRATORY SYNDROME VIRUS-INFECTED
PIGS.
Porcine Reproductive and Respiratory Sydrome (PRRS) is an
economically significant disease of the modern swine industry
(Neumann et al., 2005). This syndrome is caused by a RNA
virus which belongs to Arteriviridae family, named as PRRS
virus (PRRSV) (Fauquet et al., 2005). PRRSV replication
targets porcine alveolar macrophages (PAMs), macrophages in
other tissues and in minor extent dendritic cells (Molitor et al.,
1997; Bautista and Molitor, 1999).
In this sense, viral
replication has been reported in both lung and lymphoid organs
of PRRSV-infected pigs (Xiao et al., 2004), which suggest a role
of these organs in the pathogenesis of the disease. Moreover,
PRRSV is characterised by inducing an equivocal host immune
response (Darwich et al., 2010) which reinforce the interest on
the study of the lymphoid organs in PRRS.
91
Patogenia del PRRS/Pathogenesis of PRRS
The respiratory form of the disease, morphologically
characterized by interstitial pneumonia, can be induced by
experimental infection of piglets and growing and fattening pigs
(Pol et al., 1991; Collins et al., 1992; Fichtner et al., 1993;
Halbur et al., 1993; Rossow et al., 1994; Gómez-Laguna et al.,
2010b). PRRSV is known to persist during several weeks in
lungs and lymphoid organs and up to 105 days postinfection
(dpi) in tonsils, which play a key role as a first immune barrier
in the viral replication and spread (Albina et al., 1994; Wills et
al., 1997; Allende et al., 2000; Lamontagne et al., 2001; Horter
et al., 2002).
On the other hand, cytokines are mediators of the
immune response and changes in their expression have been
involved in the modulation of the immune response against
several porcine viruses (Van Reeth et al., 1999; Van Reeth and
Nauwynck, 2000; Carrasco et al., 2002; Salguero et al., 2002).
Specifically, proinflammatory cytokines are synthesied during
the early innate immune response, and act as link to the onset of
an adequate adaptative immune response (Kumar et al., 2008).
PRRSV is reported as a poor inducer of cytokines (Van Reeth et
92
Patogenia del PRRS/Pathogenesis of PRRS
al., 1999; Gómez-Laguna et al., 2010b), however, a paracrine
synthesis of proinflammatory cytokines has been reported in the
lung of PRRSV-infected pigs (Gómez-Laguna et al., 2010a).
Recently, our group has studied the acute phase response
in PRRS as well as the expression of proinflammatory cytokines
in the lung of PRRSV infected pigs (Gómez-Laguna et al.,
2010a, 2010b). In the present study, samples from those
previous experiments were used to determine the in situ
expression of proinflammatory cytokines in the lymphoid organs
of PRRSV-infected pigs and to determine their correlation with
the expression of PRRSV antigen.
Material and Methods
Virus, Animals and Experimental Design
The inoculum, animals and experimental design used in
this experiment has been described above in the section “Common
experimental design”.
Clinical Signs, Gross Pathology and Histopathology
The pigs were monitored daily for clinical signs, i.e. rectal
temperature and a clinical respiratory score, as described
93
Patogenia del PRRS/Pathogenesis of PRRS
previously (Halbur et al., 1995b). At the post mortem,
macroscopic lesions of lung and lymphoid tissues were
evaluated by visual inspection following the scoring system
described by Halbur et al. (1995b). Samples from tonsils,
retropharyngeal lymph node and mediastinal lymph node were
collected and fixed in 10% neutral buffered formalin and in
Bouin’s
solution
for
the
histopathological
and
immunohistochemical studies, respectively. Fixed samples were
routinely processed and embedded in paraffin-wax.
Immunohistochemical study
The avidin-biotin-peroxidase complex technique (ABC)
was used for the detection of PRRSV, and cytokine proteins as
described previously (Gómez-Laguna et al., 2010a). Briefly, the
sections were dewaxed and rehydrated through graded ethanol
and the endogenous peroxidase activity was quenched in H2O2
3% in methanol for 30 min. The sections were washed with
phosphate buffered saline (PBS; pH 7.4, 0.01 M) and incubated
for 30 min at room temperature with 100 µl per slide of blocking
solution in a humid chamber. Table 1 summarizes the primary
antibodies and antigen retrieval methods applied. Primary
94
Patogenia del PRRS/Pathogenesis of PRRS
antibodies were incubated overnight at 4 ºC in a humid chamber.
In each case, the corresponding biotinylated secondary antibody
was incubated for 30 min at room temperature. An avidinperoxidase
complex
(Vector
Laboratories,
Burlingame,
California) was applied for 1 h at room temperature. Labelling
was “visualized” by application of the NovaREDTM substrate kit
(Vector Laboratories). Sections were counterstained with
Mayer’s haematoxylin, dehydrated and mounted. For negative
controls, the primary antibody was replaced by blocking
solution, normal serum and isotype-matched reagents of
irrelevant specificity.
Cell counts
The number of positive labelled cells against PRRSV, IL1α, IL-6 and TNF-α antibodies in tonsil, and retropharyngeal
and mediastinal lymph nodes were counted using a method
previously
described
immunolabelled
were
(Salguero
counted
et
in
al.,
25
2005).
non
Cells
overlapping
consecutive selected, high magnification fields of 0.20 mm2
(paracortex and medulla of lymph nodes, and lymphoreticular
areas of tonsils) or 25 non overlapping consecutive selected
95
Patogenia del PRRS/Pathogenesis of PRRS
structures (lymphoid follicles of lymph nodes and tonsils) for
each animal. Results are expressed as the number of cells per
mm2.
Statistical Analysis
The numbers of PRRSV positive cells and cytokineexpressing cells were expressed as a mean ± SD. These values
were evaluated for approximate normality of distribution by the
Kolmogorov-Smirnov test. Differences between the means of
control and inoculated animals were assessed by the MannWhitney-U non-parametric test (GraphPad Instat 3.05, San
Diego, California). Correlation between the expression of virus
and cytokines was assessed by the Spearman test (GraphPad
Instat 3.05). P < 0.05 represented a statistically significant
difference between inoculated and control animals.
96
Patogenia del PRRS/Pathogenesis of PRRS
Table 1. Antibodies source and immunohistochemical techniques used for the immunocharacterisation of PRRSV, macrophages
and cytokines antigens expression.
Type of
Commercial
Specificity
Source
antibody
Anti-PRRSV (clone SDOW-17/SR-30)
Antigen
Fixative
Dilution
origin
Mouse myeloma
Rural
cells
Technologies Inc.
mAb
retrieval
Bouin
1:1.000
HTAR
Anti-human IL-1α
pAb
Rabbit serum
Endogen
Bouin
1:100
Tween 20 0.01 %
Anti-pig IL-6
pAb
Rabbit serum
Endogen
Bouin
1:10
Tween 20 0.01 %
Anti-human TNFα (clone 68B6A3)
mAb
Biosource
Bouin
1:25
Tween 20 0.01 %
NSO Mouse
myeloma cell line
mAb: Monoclonal Antibody. pAb: Polyclonal Antibody. HTAR: High Temperature Antigen Retrieval with citrate buffer ph 6.0.
Tween 20 0.01 %: Tween 20 diluted 0.01 % in PBS during 10’
97
Patogenia del PRRS/Pathogenesis of PRRS
Results
Clinical Signs, Gross Pathology and Histopathology
Control animals displayed neither clinical signs nor gross
lesions throughout the study. Inoculated animals only presented
mild dullness and weight loss from 3 dpi onwards, and a mild
elevation of the rectal temperature at 3 and 10 dpi, but always
within the physiological ranges. Gross lung lesions displayed a
significant increase from 7 dpi onwards, showing a mottled tan
and
rubbery
parenchyma
which
failed
to
collapse.
Retropharyngeal and mediastinal lymph nodes showed a mild to
moderate enlargement from 10 dpi onwards. Microscopically
just a mild hypertrophy of germinal centres and apoptotic bodies
were observed in the lymphoid follicles of lymph nodes of
inoculated animals, and in lesser extent in the tonsil, from 7 dpi
onwards, together with some figures of cell picnosis, mitosis and
necrosis.
Tissue expression of PRRSV antigen in tonsil and lymph nodes
The number of PRRSV antigen labelled cells showed a
mild increase at 3 dpi in the tonsil of PRRSV-infected pigs,
98
Patogenia del PRRS/Pathogenesis of PRRS
being the positive reaction detected mainly within the cytoplasm
of macrophages in the lymphoreticular areas of the tonsil (Fig.
1A). In addition, the viral antigen detection displayed maximum
values at 14 dpi (P<0.05), decreasing onwards (Fig. 2A).
In the retropharyngeal and mediastinal lymph nodes the
detection of PRRSV was mainly observed in the cytoplasm of
macrophages (Fig. 1B). Furthermore, viral antigen was mostly
observed in the paracortex of the retropharyngeal lymph node
and in the medulla of mediastinal lymph node (Fig 1B). The
detection of PRRSV antigen in retropharyngeal and mediastinal
lymph nodes yielded a first peak at 3 and 7dpi (P <0.05),
respectively, followed by a second peak at 14 dpi in the
retropharyngeal lymph node and decreasing onwards (Figs. 3A
and 4A,).
Tissue expression of proinflammatory cytokines in the tonsil
All proinflammatory cytokines studied were mostly
expressed in the lymphoreticular areas of the tonsil, mainly in
the cytoplasm of macrophages and secondly in the cytoplasm of
neutrophils (IL-1α) or lymphocytes (TNF-α, IL-6) (Fig 1F).
Random scarce macrophages and lymphocytes were also
99
Patogenia del PRRS/Pathogenesis of PRRS
immunolabelled against TNF-α and/or IL-6 in the lymphoid
follicles of the tonsil.
None of the proinflammatory cytokines analyzed
displayed significant changes with respect to the control group
in the tonsil (Fig 2 B,C,D).
Tissue expression of proinflammatory cytokines in the lymph
nodes
IL-1α and IL-6 were mainly expressed in the paracortex
of the retropharyngeal lymph node but in the medulla of the
mediastinal lymph node, whereas the expression of TNF-α was
mainly observed in the medulla of both lymphoid organs. The
expression of all these three cytokines was mainly observed in
the cytoplasm of macrophages and in a lesser extent in the
cytoplasm of lymphocytes (TNF-α, IL-6) (Figs. 1D and 1E) and
neutrophils (IL-1α) (Fig. 1C). However, a significant expression
of IL-1α by neutrophils was observed in the mediastinal lymph
node (Fig. 4B).
100
Patogenia del PRRS/Pathogenesis of PRRS
Figure 1. (A) Tonsil of a pig killed at 17 dpi showing macrophages labelled for
expresión of PRRSV. IHC. Bar, 100 µm. Inset: Detail of the cytoplasmic
immunolabelling against PRRSV in the macrophages of the tonsil from the same
animal. IHC. Bar, 50 µm (B) Macrophages immunostained for PRRSV expression in
the mediastinal lymph node of a pig killed at 7 dpi. IHC. Bar, 15 µm. (C) Some
macrophages and a neutrophil (arrow) immunolabelled for IL-1α expression in the
paracortex of the retropharyngeal lymph node of a pig killed at the end of the
experiment. IHC. Bar, 20 µm. (D) Detail of the medulla of the mediastinal lymph
node of a pig killed at 7 dpi with macrophages and lymphocytes (asterisks)
immunolabelled for TNF-α expression. IHC. Bar, 20 µm. (E) Retropharyngeal lymph
node of a pig killed at 3 dpi showing macrophages for expression of IL-6. Notice the
high expression of IL-6 in the medulla of the retropharyngeal lymph node compare
with this expression in the paracortex. IHC. Bar, 100 µm. (F) Lymphoreticular area of
the tonsil of a pig killed at the end of the experiment showing macrophages and
lymphocytes (asterisks) labelled for the expression of IL-6. IHC. Bar, 20 µm.
101
Patogenia del PRRS/Pathogenesis of PRRS
Proinflammatory cytokines displayed different patterns
of expression in retropharyngeal and mediastinal lymph nodes.
Whereas IL-6 was the highest cytokine expressed in the
retropharyngeal lymph node, IL-1α was the one most expressed
in the mediastinal lymph node, with a significant contribution of
IL-1α-expressing neutrophils (Figs. 3B and 4B). On the other
hand, whereas IL-1α and TNF-α were slightly expressed in the
retropharyngeal lymph node, both displayed a higher expression
in mediastinal lymph node following a similar trend with a
statistically significant peak of expression at 7 dpi (Figs 3 and 4)
Figure 2 (A-D) Counts for cells expressing PRRSV antigen, IL-1α, TNF-α and IL-6
respectively in the tonsil of pigs infected with PRRSV. * Indicates statistically
significant differences (P<0.05) between the inoculated group and controls.
102
Patogenia del PRRS/Pathogenesis of PRRS
Figure 3 (A-D) Counts for cells expressing PRRSV antigen, IL-1α, TNF-α
and IL-6 respectively in the retropharyngeal lymph node of pigs infected with
PRRSV. * Indicates statistically significant differences (P<0.05) between the
inoculated group and controls.
Figure 4 (A-D) Counts for cells expressing PRRSV antigen, IL-1α, TNF-α
and IL-6 respectively in the mediastinal lymph node of pigs infected with
PRRSV. * Indicates statistically significant differences (P<0.05) between the
inoculated group and controls.
103
Patogenia del PRRS/Pathogenesis of PRRS
Correlation
between
the
expression
of
PRRSV
and
proinflammatory cytokine antigens
A significant correlation was observed between the
detection of PRRSV and IL-1α (r = 0.76; P<0.05) and also
between the expression of IL-1α and TNF-α (r = 0.83; P<0.05)
in the mediastinal lymph node. On the other hand, no correlation
was observed between the expression of PRRSV and
proinflammatory cytokines neither in the tonsil nor in the
retropharyngeal lymph node (P>0.05), but the maximum of the
expression of proinflammatory cytokines coincided or was
detected just after the peak of viral expression. Furthermore, a
high correlation was observed between the viral expression in
retropharyngeal lymph node and tonsil (r = 0.93; P<0.05).
Additionally, a significant correlation was observed between the
expression of IL-1α (r = 0.79; P<0.05), IL-6 (r = 0.79; P<0.05)
and TNF-α (r = 0.88; P<0.05) in mediastinal lymph node when
compared with the expression of these cytokines in the lung of
PRRSV-infected pigs reported previously in a parallel study
carried out by our group (Gómez-Laguna et al., 2010a).
104
Patogenia del PRRS/Pathogenesis of PRRS
Discussion
PRRSV is known to replicate mainly in porcine alveolar
macrophages (PAMs), macrophages in other tissues and in
minor extent in dendritic cells (Molitor et al., 1997; Bautista and
Molitor, 1999). However, the majority of the studies focused on
the immune response evoked after PRRSV infection have been
performed in serum and lung samples (Batista et al., 2004;
Xibao et al., 2010; Gómez-Laguna et al., 2010a) and in a lesser
extent in the lymphoid tissues in situ (Rossow et al., 1996;
Beyer et al., 2000). Thus, in the present study the in situ
expression of proinflammatory cytokines was examined in the
lymphoid organs of PRRSV-infected pigs in order to put light in
the immune response evoked against PRRSV.
Although oronasal route is considered as the main entry
route of PRRSV (Beyer et al., 2000), in the present study
intramuscular route was selected for the inoculation of the
animals, in order to assess an efficient infection of inoculated
animals. Recently Hermann et al. (2005) compared oral, nasal
and intramuscular routes of exposure to PRRSV, showing that
pigs were the most susceptible by parenteral route, being
105
Patogenia del PRRS/Pathogenesis of PRRS
infected all animals exposed to PRRSV by intramuscular route.
In our study the clinical signs, gross and micro lesions as well as
the detection of PRRSV in lymphoid organs throughout the
study confirm the efficient infection of inoculated animals.
PRRSV antigen was observed mainly in the medulla and/or
in the paracortex of the different lymphoid tissues analyzed. The
expression of PRRSV displayed a bimodal expression in the
tonsil and retropharyngeal lymph node with a first peak of
expression at 3 dpi and a second one at 14 dpi, whereas the
mediastinal lymph node had just a peak of expression at the
beginning decreasing onwards until the end of the experiment.
The tonsil is considered as an immune barrier in PRRSV
replication and spread (Albina et al., 1994; Wills et al., 1997;
Allende et al., 2000; Lamontagne et al., 2001; Horter et al.,
2002). However, despite the expression of PRRSV antigen in
the tonsil a lack of proinflammatory cytokines response was
observed throughout the present study. This lack of response
observed in the tonsil may be related with the PRRSV
persistence in the tonsil of infected pigs (Beyer et al., 2000;
Wills et al., 2003), as well as with the increased susceptibility to
106
Patogenia del PRRS/Pathogenesis of PRRS
secondary pathogens (Wills et al., 2000; Thanawongnuwech et
al., 2000, 2004) reported by other authors.
The expression of IL-1α, IL-6 and TNF-α was examined in
our study in the tonsil, retropharyngeal and mediastinal lymph
nodes of PRRSV-infected pigs. Interestingly, the expression of
each cytokine was different depending on the body compartment
examined. Wheras no expression of proinflammatory cytokines
was observed in the tonsil, an enhancement was observed on
TNF-α and IL-1α levels in the mediastinal lymph node and IL-6
expression in the retropharyngeal lymph node. This finding
point to a differential behaviour of PRRSV in the lymphoid
organs, which may be related with the lack of a robust host
immune response evoked against the virus.
In our study the different response against PRRSV in each
body
compartment
was
reflected
by
the
kinetics
of
proinflammatory cytokines in each lymph node. In this sense,
the expression of proinflammatory cytokines in lymph nodes
draining different areas of the organism represent a useful tool
for the study of the immunopathogenesis of PRRS. This fact is
supported by the correlations observed in our study between the
107
Patogenia del PRRS/Pathogenesis of PRRS
retropharyngeal lymph node and tonsil and between the
mediastinal lymph node and the results observed in the lung
from a previous parallel study of our group (Gómez-Laguna et
al., 2010a).
Proinflammatory cytokines are able to modulate the
expression of CD163, a hemoglobin scavenger receptor which
acts as a PRRSV receptor and is involved in viral uncoating
(Van Gorp et al., 2008). Whereas IL-6 is able to upregulate the
expression of CD163, TNF-α induces a downregulation of this
receptor (Buechler et al., 2000), inhibiting somehow PRRSV
replication. Thus, the imbalance between these cytokines in
lymphoid organs may play a role in the susceptibility to PRRSV
replication.
In conclusion, lymphoid organs and proinflammatory
cytokines were shown to represent an important target of study
for clarifying the immunopathogenesis of PRRS. In our study,
the lack of homogeneity in the immune response observed in the
lymphoid organs of the two body cavities point to another aspect
of the erratic immune response observed in PRRS. Moreover,
the absence of proinflammatory cytokines expression in the
108
Patogenia del PRRS/Pathogenesis of PRRS
tonsil may be of significance both in PRRSV persistence and
susceptibility to concomitant infections.
109
Patogenia del PRRS/Pathogenesis of PRRS
3.3.
IL-10,
IL-12,
IMMUNOHISTOCHEMICAL
IFN-α
AND
EXPRESSION
IFN-γ
IN
LYMPHOID ORGANS OF PORCINE REPRODUCTIVE
AND RESPIRATORY SYNDROME VIRUS-INFECTED
PIGS.
Porcine Reproductive and Respiratory Sydrome (PRRS) is one
of the most economically significant disease of the swine
industry (Neumann et al., 2005), which is caused by PRRS virus
(PRRSV) (Fauquet et al., 2005). PRRSV induces an impairment
of the host immune response favouring a prolonged viraemia
and viral replication (Darwich et al., 2010); however, the exact
mechanism involved in the modulation of the immune response
still remains unclear. PRRSV replication has been reported in
both lung and lymphoid organs of PRRSV-infected pigs (Xiao et
al., 2004), which suggest a role of these organs in the
pathogenesis of the disease.
The production of cytokines is one of the tools used by
macrophages and also by several other immune or non-immune
cells in the defense against pathogens (Kumar et al., 2008). IFN110
Patogenia del PRRS/Pathogenesis of PRRS
γ and IL-12 are classically involved in the subtype of immune
response mediated by Th1 lymphocytes, working both cytokines
in parallel (Biron and Sen, 2001). As well as proinflammatory
cytokines do, IFN-α participates in the innate response,
triggering an antiviral activity by means the differentiation of
naïve T cells into IFN-γ secreting cells and the down-regulation
of IL-12 expression (Biron and Sen, 2001; Tizard, 2008). In
contrast, IL-10 is considered to be an immunosuppressive
cytokine as it down-regulates the expression of several other
cytokines including IL-1α, TNF-α, IL-6, IL-10 itself, IL-12 and
IFN-γ (Biron and Sen, 2001; Moore et al., 2001). Several
studies have examined the role of cytokines in the pathogenesis
of PRRS (Van Reeth and Nauwynck, 2000); however, it is not
clear how the cytokines regulate the onset of the immune
response against the virus. Albina et al. (1994) suggested that
downregulation of IFN-α production may play an important role
in enabling PRRSV replication, likewise, Bautista and Molitor
(1999) reported a significant role of IFN-γ in the protection of
host cells against viral replication. In this sense, another study of
a PRRSV modified-live vaccine showed that upregulation of IL111
Patogenia del PRRS/Pathogenesis of PRRS
10 expression was associated with a lower number of IFN-γ
secreting cells amongst peripheral blood mononuclear cells
(PBMCs) and a lower protection rate after challenging (Díaz et
al., 2006).
Recently, our group has studied the protein expression of
different cytokines in the lung (Gómez-Laguna et al., 2010a)
and serum (Gómez-Laguna et al., 2010b) of PRRSV infected
pigs and it has also been observed in the experimental study
explained before a lack of homogeneity in the expression of
proinflammatory cytokines in the lymphoid organs of PRRSVinfected pigs pointing to a contribution to the erratic immune
response observed in PRRS. In the present study, samples from
those previous experiments were used to determine the in situ
expression of regulatory cytokines in the lymphoid organs of
PRRSV-infected pigs and to determine their correlation with the
expression of PRRSV antigen.
Material and Methods
Virus, Animals and Experimental Design
112
Patogenia del PRRS/Pathogenesis of PRRS
The inoculum, animals and experimental design used in
this experiment have been described above in the section
“Common experimental design”.
Clinical Signs, Gross Pathology and Histopathology
The pigs were monitored daily for clinical signs as
previously described Halbur et al. (1995b). At the post mortem
study, macroscopic lesions were evaluated by following the
scoring system described by Halbur et al. (1995b). Samples from
tonsils, retropharyngeal lymph node and mediastinal lymph node
were collected and fixed in 10% neutral buffered formalin and in
Bouin’s
solution
for
the
histopathological
and
immunohistochemical studies, respectively. Fixed samples were
routinely processed and embedded in paraffin-wax.
Immunohistochemical study
The avidin-biotin-peroxidase complex technique (ABC)
was used for the detection of PRRSV, and cytokine proteins as
described previously (Gómez-Laguna et al., 2010a). The
primary antibodies used were monoclonal anti-PRRSV, clone
SDOW-17/SR-30, diluted 1 in 1000; polyclonal anti-pig IL-10
diluted 1 in 20; polyclonal anti-pig IL-12 diluted 1 in 20;
113
Patogenia del PRRS/Pathogenesis of PRRS
polyclonal anti-pig IFN-γ diluted 1 in 20 and monoclonal antipig IFN-α (clone F17) diluted 1 in 300 . The antigen retrieval
method used for all the cytokines studied was Tween 20
diluted 0.01 % in PBS during 10 min but for monoclonal antiPRRSV, clone SDOW-17/SR-30, a High Temperature Antigen
Retrieval with citrate buffer ph 6.0 was used. Primary
antibodies were incubated overnight at 4 ºC in a humid
chamber. In each case, the corresponding biotinylated
secondary antibody was incubated for 30 min at room
temperature.
An
avidin-peroxidase
complex
(Vector
Laboratories, Burlingame, California) was applied for 1 h at
room temperature. Labelling was “visualized” by application
of the NovaREDTM substrate kit (Vector Laboratories). For
negative controls, the primary antibody was replaced by
blocking solution, normal serum and isotype-matched reagents
of irrelevant specificity. Sections were counterstained with
Mayer’s haematoxylin, dehydrated and mounted.
Cell counts
The number of positive labelled cells against PRRSV,
IL-10, IL-12, IFN-α and IFN-γ antibodies in tonsil, and
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Patogenia del PRRS/Pathogenesis of PRRS
retropharyngeal and mediastinal lymph nodes were counted
using a method previously described (Salguero et al., 2005).
Cells immunolabelled were counted in 25 non overlapping
consecutive selected, high magnification fields of 0.20 mm2
(paracortex and medulla of lymph nodes, and lymphoreticular
areas of tonsils) or 25 non overlapping consecutive selected
structures (lymphoid follicles of lymph nodes and tonsils) for
each animal. Results are expressed as the number of cells per
mm2.
Statistical Analysis
The numbers of PRRSV positive cells and cytokineexpressing cells were expressed as a mean ± SD. These values
were evaluated for approximate normality of distribution by the
Kolmogorov-Smirnov test. Differences between the means of
control and inoculated animals were assessed by the MannWhitney-U non-parametric test (GraphPad Instat 3.05, San
Diego, California). Correlation between the expression of virus
and cytokines was assessed by the Spearman test (GraphPad
Instat 3.05), P < 0.05 represented a statistically significant
difference between inoculated and control animals.
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Patogenia del PRRS/Pathogenesis of PRRS
Results
Clinical Signs, Gross Pathology and Histopathology
The clinical signs, gross and microscopic lesions were
previously described in page.....
Tissue expression of PRRSV antigen in tonsil and lymph nodes
Viral antigen was mostly observed in the cytoplasm of
macrophages in the paracortex of the retropharyngeal lymph
node (Fig. 1A), in the lymphoreticular areas of the tonsil (Fig.
1B) and in the medulla of mediastinal lymph node (Barranco et
al., submitted manuscript). The tissue expression of PRRSV
antigen in tonsil and lymph nodes is represented in figure 2 (Fig.
2A,B,D) together with the expression of PRRSV antigen in lung
described by Gómez-Laguna et. al., (2010a) (Fig. 2C). Briefly,
in the tonsil the viral antigen detection displayed maximum
values at 14 dpi (P < 0.05) decreasing onwards (Fig. 2A). In
retropharyngeal and mediastinal lymph nodes the detection of
PRRSV antigen yielyed a first peak at 3 and 7 dpi (P < 0.05)
respectively followed by a second peak at 14 dpi in the
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Patogenia del PRRS/Pathogenesis of PRRS
retropharyngeal lymph node and decreasing onwards (Fig.
2A,D).
Tissue expression of regulatory cytokines in the tonsil
All regulatory cytokines studied were mostly expressed
in the lymphoreticular areas of the tonsil, mainly in the
cytoplasm of macrophages (Fig. 1C,D).
The number of IL-10 positive cells displayed a curve
with an increase of its expression at 3 dpi, 14 dpi and also at the
end of the experiment (24dpi) , however due to individual
variability, this increase in the expression of IL-10 was
statistically significant only at 14 dpi (Fig 3B). The expression
of IFN-α, IFN-γ and IL-12 showed a similar trend among them
with a statistically significant peak of expression at 3 dpi (Fig
3A,C,D). After 3 dpi, the expression of IFN-α decreased sharply
and remained low until the end of the study (Fig. 3C), whereas
the expression of IFN-γ decreased slightly and remained at
moderate levels until the end of the study with a statistically
significant enhancement at 10 dpi, 14 dpi, 17 dpi and 24 dpi
(Fig. 3D). The expression of IL-12 showed another two peaks at
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Patogenia del PRRS/Pathogenesis of PRRS
14 dpi and 24 dpi, however due to individual variability, this
enhancement was not statistically significant (Fig 3A).
Tissue expression of regulatory cytokines in the lymph nodes
Regulatory cytokines displayed similar patterns of
expression in retropharyngeal and mediastinal lymph nodes
(Figs. 4 and 5). All the regulatory cytokines studied were mainly
expressed in the paracortex of both lymphoid organs (Fig. 1E,F).
The expression of IL-10, IL-12, IFN-α and IFN-γ was mainly
observed in the cytoplasm of macrophages and in a lesser extent
in the cytoplasm of lymphocytes.
In the mediastinal lymph node, all regulatory cytokines
showed statistically significant peaks of expression at 7, 17 and
24 dpi (IL-12 and IFN-γ) (Fig. 4A,D) or 3, 14 and 24 dpi (IL-10
and IFN-α) (Fig. 4B,C). Nonetheless, the expression of IL-10
was not statistically significant due to individual variability. In
the retropharyngeal lymph node all regulatory cytokines
followed a similar trend with a maximum expression of all of
them at 3 dpi (IFN-α, P < 0.05; IFN-γ, P < 0.05) and another
peaks of expression at 14 dpi and at the end of the study (IFN-α,
P < 0.05; IL-10, P < 0.05) (Fig. 5). In both lymph nodes IL-10
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Patogenia del PRRS/Pathogenesis of PRRS
was the cytokine which showed the lowest expression whereas
IFN-γ was the one with the highest expression.
Figure 1. (A) Retropharyngeal lymph node of a pig killed at 17 dpi showing
macrophages labelled for expresión of PRRSV. IHC. Bar, 15 µm. (B) Macrophages of
the tonsil from the same animal immunostained for PRRSV. IHC. Bar, 10 µm. (C)
Some macrophages immunolabelled for IL-10 expression in the tonsil of a pig killed
at 7 dpi. IHC. Bar, 20 µm. (D) Several macrophages from the lymphoreticular area of
the tonsil of a pig killed at 3 dpi immunolabelled for IL-12 expression. IHC. Bar, 50
µm. (E) Retropharyngeal lymph node of a pig killed at 3 dpi showing macrophages
for expression of IFN-α. Notice the high expression of IFN-α in the paracortex of the
retropharyngeal lymph node compare with this expression in the medulla. IHC. Bar,
50 µm. (F) Mediastinal lymph node of a pig killed at 7 dpi showing macrophages and
lymphocytes (asterisks) labelled for the expression of IFN-γ. IHC. Bar, 50 µm.
119
Patogenia del PRRS/Pathogenesis of PRRS
Figure 2 (A-D) Counts for cells expressing PRRSV antigen, in the tonsil,
retropharyngeal lymph node, lung (data from a previous study, GomezLaguna et al., 2010a) and mediastinal lymph node of pigs infected with
PRRSV. * Indicates statistically significant differences (P < 0.05) between
the inoculated group and controls.
Figure 3 (A-D) Counts for cells expressing IL-12, IL-10, IFN-α and IFN-γ
respectively in the tonsil of pigs infected with PRRSV. * Indicates
statistically significant differences (P < 0.05) between the inoculated group
and controls.
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Patogenia del PRRS/Pathogenesis of PRRS
Figure 4 (A-D) Counts for cells expressing IL-12, IL-10, IFN-α and IFN-γ
respectively in the retropharyngeal lymph node of pigs infected with PRRSV.
* Indicates statistically significant differences (P < 0.05) between the
inoculated group and controls.
Figure 5 (A-D) Counts for cells expressing IL-12, IL-10, IFN-α and IFN-γ
respectively in the mediastinal lymph node of pigs infected with PRRSV. *
Indicates statistically significant differences (P < 0.05) between the
inoculated group and controls.
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Patogenia del PRRS/Pathogenesis of PRRS
Correlation between the expression of PRRSV and regulatory
cytokine antigens
In the retropharyngeal lymph node there was a
significant correlation between the detection of PRRSV and
IFN-γ (r = 0.83; P < 0.05) and also between the expression of
IFN-α and IL-10 (r = 0.74; P < 0.05). In the mediastinal lymph
node, a significant correlation was observed between the
detection of PRRSV and IFN-α (r = 0.86; P < 0.05) and between
PRRSV and IL-12 (r = 0.74; P < 0.05). A significant correlation
was observed in all the lymphoid organs examined between the
expression of IL-12 and IFN-α (Table 1). In addition, a
correlation was shown between the expression of IL-12 and
IFN-γ (r = 0.74; P < 0.05). Furthermore, when the expression of
the different antigens in each lymphoid organs was compared
among them a high correlation was observed between PRRSV
expression in retropharyngeal lymph node and tonsil (r = 0.93; P
< 0.01), as well as between the expression of IL-12 (r = 0.76; P
< 0.05) and IFN-α (r = 0.81; P < 0.05) in retropharyngeal lymph
node and tonsil. Tables 1 and 2 summarize the correlation
observed.
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Patogenia del PRRS/Pathogenesis of PRRS
Table 1. Correlation observed between PRRSV, IFN-α, IFN-γ, IL-10 and IL-12 p40 antigens in the different lymphoid organs studied
from PRRSV-infected pigs.
Tonsil
Retropharyngeal Lymph Node
IL-12
PRRSV
IFN-α
PRRSV
IFN-α
IFN-γ
IL-10
p40
-
0.33
0.13
0.31
0.21
-
0.71
0.48
0.90**
-
0.33
0.67
-
0.57
IFN-γ
IL-10
IL-12 p40
-
Mediastinal Lymph Node
IL-12
PRRSV
IFN-α
IFN-γ
IL-10
p40
-
0.55
0.83*
0.38
0.67
-
0.48
0.74*
0.84*
-
0.31
0.69
-
0.62
-
IL-12
PRRSV
-
IFN-α
IFN-γ
IL-10
p40
0.86*
0.45
0.45
0.74*
-
0.38
0.67
0.74*
-
0.6190
0.74*
-
0.50
-
**P < 0.01; *P < 0.05
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Patogenia del PRRS/Pathogenesis of PRRS
Table 2. Correlation observed between Tonsil, Retropharyngeal Lymph Node and Mediastinal Lymph Node expression of the
different regulatory cytokines studied from PRRSV-infected pigs.
IFN-α
Tonsil
R.L.N.
M.L.N
IFN-γ
IL-10
Tonsil
R.L.N
M.L.N
Tonsil
R.L.N
-
0.81*
0.83*
-
0.55
0.19
-
-0.33
-
0.71
-
M.L..
-
IL-12
Tonsil
R.L.N.
M.L.N.
Tonsil
R.L.N
M.L.N
Tonsil
R.L.N
M.L.N
-
0.24
0.81*
-
0.76*
0.45
-
0.93**
0.14
-
0.57
-
0.52
-
0.33
-
R.L.N.: Retropharyngeal Lymph Node; M.L.N.: Mediastinal Lymph Node; **P < 0.01; *P < 0.05
124
PRRSV
-
-
Patogenia del PRRS/Pathogenesis of PRRS
Discussion
The immune system protects host cells from viral
infection, and viruses have evolved to escape from this response
to achieve an efficient proliferation in the host. Host cells
produce cytokines in response to viral infection as a mechanism
of defence. PRRSV replication targets porcine alveolar
macrophages (PAMs), macrophages in other tissues and in
minor extent dendritic cells (Molitor et al., 1997; Bautista and
Molitor, 1999), being characterized by the induction of an
equivocal host immune response (Darwich et al., 2010). There
are several studies focused on the immune response evoked after
PRRSV infection most of which have been performed in serum
and lung samples (Batista et al., 2004; Xibao et al., 2010;
Gómez-Laguna et al., 2010a) and in a lesser extent in the
lymphoid tissues in situ (Rossow et al., 1996; Beyer et al.,
2000). We analyzed in the experimental study explained before
the in situ expression of proinflammatory cytokines in the
lymphoid organs of PRRSV-infected pigs finding a different
expression of these cytokines depending on the examined body
compartment, wich may be related to a differential behaviour of
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Patogenia del PRRS/Pathogenesis of PRRS
PRRSV in the lymphoid organs. In the present study we
analyzed the in situ expression of IL-10, IL-12, IFN-γ and IFNα, which are cytokines involved in the immune response against
viruses, in order to help in the arduous task of deciphering the
immune response evoked against PRRSV.
The expression of IL-10, IL-12, IFN-γ and IFN-α in the
tonsil, retropharyngeal and mediastinal lymph nodes of PRRSVinfected pigs displayed a two-peak curve. Although the
expression of proinflammatory cytokines described before was
different depending on the body compartment examined, the
expression of each cytokine analyzed in the present study was
quite similar in all the lymphoid organs studied, despite the
amount of PRRSV detected was different in each one. This
finding suggests that the adaptative immune response evoked
against PRRSV in this study is more homogeneous than the
proinflammatory response previously observed.
IL-10 is a pleiotropic cytokine with regulatory properties
that is thought to play an important role in PRRSV
immunopathogenesis (Suradhat et al, 2003; Charerntantanakul
et al., 2006; Silva-Campa et al., 2009; Gómez-Laguna et al.,
126
Patogenia del PRRS/Pathogenesis of PRRS
2010a). However, the role of this cytokine in PRRSV is
controversial. According to Díaz et al. (2006) different viral
strains are able to induce different IL-10 responses in PBMC of
PRRSV-naïve pigs. In a parallel study Gómez-Laguna et al.
(2010a) observed that the expression of IL-10 in the lung was
significantly correlated with PRRSV replication, indicating that
PRRSV may induce the expression of IL-10. Interestingly, in the
present study we did no find correlation between IL-10 and
PRRSV in none of the lymphoid tissues analyzed, suggesting
that the expression of IL-10 could be different depending on the
tissue examined.
On the other hand, the IFN family of cytokines is
recognized as a key component of the innate immune response
and the first line of defense against viral infection (Borden et al.,
2007). IFN-α participates in the innate immune response
through their antiviral activity, by inducing the differentiation of
naïve T cells into IFN-γ secreting cells and by down-regulating
the expression of IL-12 (Biron and Sen, 2001; Tizard, 2008). In
addition, IFN-γ and IL-12 are classically involved in the Th1
subtype of immune response, working both cytokines in parallel
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Patogenia del PRRS/Pathogenesis of PRRS
(Biron and Sen, 2001). In our study the expression of IFN-α in
the tonsil and lymph nodes was correlated between them, wich
may be related to the onset of a host innate immune response.
Moreover, the expression of IFN-γ and IL-12 followed also a
similar trend in all the lymphoid organs examined. Despite the
expression of these cytokines, PRRSV antigen was still detected
in moderate amount at the end of the study, which points to an
inefficient viral clearance.
Although IFNs and IL-12 have an antiviral activity, IL10 may reduce the levels of these cytokines involved in viral
clearance (Mitchell and Kumar, 2004). In our study the amount
of IL-10 expressed was mild to moderate being observed a
higher expression of antiviral cytokines. Contrary, the IL10/IFN-γ ratio previously described in the lung by GómezLaguna et al. (2010a) was proportionally higher, which was
related to the viral persistence in the lung of PRRSV infected
pigs.
The expression of IFNs in the lymphoid organs of
PRRSV infected pigs together to the viral persistence at the end
of the study, indicates that the IFN signaling cascade may not be
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Patogenia del PRRS/Pathogenesis of PRRS
working properly. In this sense, non structural protein 2 (Nsp2)
of PRRSV has been shown to inhibit the induction of IFN
regulatory factor 3 (Li et al., 2010), but no role of PRRSV has
been reported on the IFN signaling pathway. Thus, further
studies are being conducted to determine the role of PRRS on
IFN signaling cascade.
In conclusion, considering the different expression of
IFNs, IL-10 and IL-12 in lymphoid organs with respect to the
one described previously in the lung by our group, PRRSV may
be able to impair the host immune response in the lymphoid
organs by a different mechanism than the one described in the
lung.
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Patogenia del PRRS/Pathogenesis of PRRS
3.4. APOPTOSIS IN LYMPHOID TISSUES OF PRRSV
INFECTED PIGS DETECTED BY TUNEL AND CLEAVED
CASPASE-3 IMMUNOHISTOCHEMISTRY.
Apoptosis or “programmed cellular death” is a highly
regulated process modulated by both pro-apoptotic and
antiapoptotic cellular factors and activated by various stimuli
that disturb cell metabolism and physiology. Morphologically,
apoptosis is characterized by peripheral condensation of the
chromatin and cellular shrinkage leading to the formation of
apoptotic bodies, without the onset of an inflammatory response
(Kerr et al., 1972, 1995). Currently, two distinct pathways of
apoptosis have been described, extrinsic and intrinsic pathways
(Roy and Nicholson, 2000; Mitchell and Cotran, 2007). Both
pathways of apoptosis converge in the activation of caspase 3
and continue a common pathway of cell death (Mitchell and
Cotran, 2007).
Caspases or cysteine-aspartic proteases are a family of
cysteine proteases that play essential roles in apoptosis and are
synthesized as inactive precursors that require proteolytic
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Patogenia del PRRS/Pathogenesis of PRRS
conversion to become active caspases (Zhivotovsky et al., 1999;
Hengartner, 2000). Caspase 3 is one of the known effector
caspases wich, once activated, irreversibly executes cell death
through degradation of vital cell proteins and activation of
endonucleases. Consecuently, activation of caspase 3 is
considered a characteristic sign of apoptosis (Huppertz et al.,
1999).
Contributions to histochemical characterization of apoptosis
have been done mainly by studying the final stage of apoptotic
death with the terminal deoxynucleotidyl transferase-mediated
dUTP nick-end labelling (TUNEL) method (Huppertz et al.,
1999). Despite the easy detection of apoptosis by this assay, its
specificity and sensitivity have been strongly criticized (LabatMoleur et al., 1998; Stahelin et al., 1998). However, activated
caspase
3
labelling
is
considered
to
be
a
reliable
immunohistochemical approach to detect and quantify apoptotic
cells in human lymphoid tissue (Dukers et al., 2002).
Nevertheless, the presence of apoptotic cells could be
possible instead a lack of cleaved caspase 3 (CCasp 3) labelling,
since there are authors who described a caspase 3-independent
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Patogenia del PRRS/Pathogenesis of PRRS
apoptosis pathway (Kuida et al., 1996; Piwocka et al., 2002;
Alvarez et al., 2011)
Viral infections may modulate apoptosis inducing either an
up-regulation or a down-regulation of such process as a
mechanism to evade the immune response (Thomson, 2001;
Costers et al., 2008). In this sense, an enhancement of apoptosis
phenomena has been observed in lymphoid organs of pigs
infected with African Swine Fever virus (ASFv) (Salguero et al.,
2005; Fernández de Marco et al., 2007), Porcine Circovirus type
2 (PCV2) (Resendes et al., 2004b) or Porcine reproductive and
respiratory syndrome virus (PRRSV) (Suárez et al., 1996a; Choi
and Chae, 2002; Costers et al., 2008). The apoptosis phenomena
observed in these diseases have been linked directly to viral
replication (Suárez et al., 1996a; Costers et al., 2008) or
indirectly to an increased expression of mediators, such as
cytokines (Choi and Chae, 2002; Salguero et al., 2005;
Fernández de Marco et al., 2007).
Porcine Reproductive and Respiratory Syndrome (PRRS)
constitute one of the most significant diseases in the swine
industry, which is caused by an arterivirus that is considered
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Patogenia del PRRS/Pathogenesis of PRRS
able to modulate the immune response, making easier the
infection with secondary infectious agents. PRRSV can be
detected from 1dpi to 251 dpi in tonsils (Wills et al., 2003),
pointing this persistence to both humoral and cellular immune
response inefficiency to completely eliminate the virus.
Moreover, persistence of the virus in tonsils and mediastinal
lymph nodes indicates either the absence of T cell immune
stimulation, or a fast death of activated lymphoid cells
(Lamontagne et al., 2003). In addition, the apoptosis of
lymphoid cells has been hypothesized to justify the lack of an
efficient cell mediated immune response in PRRS (Lamontagne
et al., 2001; 2003).
However, despite the published studies examining PRRSV
infection and apoptosis, there is still conflicting evidence and
views as to whether PRRSV induces apoptosis directly (within
infected cells) (Suárez et al., 1996a; Kim et al., 2002; Costers et
al., 2008) or indirectly (within bystander cells) (Sur et al., 1997;
Sirinarumitr et al., 1998; Labarque et al., 2003; Choi and Chae,
2002).
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Patogenia del PRRS/Pathogenesis of PRRS
During the first and second experimental study, we
evaluated the expression of PRRSV and cytokines antigens in
the lymphoid organs of PRRSV infected pigs. The main aim of
this study, was to evaluate the apoptosis phenomena by
microscopic examination and to correlate it with PRRSV and
CCasp3 immunohistochemistry and TUNEL method in tonsil
and mediastinal lymph node of PRRSV-infected pigs, as well as
with the expression of pro-apoptotic cytokines.
1. Materials and methods
2.1.Virus, Animals and Experimental Design
The inoculum, animals and experimental design used in
this experiment has been described above in the section “Common
experimental design”.
2.2. Clinical Signs, Gross Pathology and Histopathology
The animals were monitored daily for clinical signs, i.e. rectal
temperature and a clinical respiratory score, as described
previously (Halbur et al., 1995b). At the post mortem,
macroscopic lesions of lung and lymphoid tissues were
evaluated by visual inspection following the scoring system
134
Patogenia del PRRS/Pathogenesis of PRRS
described by Halbur et al. (1995b). Samples from tonsil and
mediastinal lymph node were collected and fixed in 10% neutral
buffered
formalin
for
the
histopathological
and
immunohistochemical studies. Fixed samples were routinely
processed and embedded in paraffin-wax.
Four µm tissue sections from tonsils and mediastinal
lymph node were stained with the routine Mayer´s hematoxylin
and eosin staining for microscopic examination. Samples from
all the animals were analyzed following the score summarize in
table 1.
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Patogenia del PRRS/Pathogenesis of PRRS
Table 1: Score used for tonsil and mediastinal lymph node microscopic examination
LN SIZE
DEPLETION OF
GERMINAL
APOPTOTIC
CELL
AREAS OF
MITOTIC
FC
CENTRES
BODIES
PICNOSIS
NECROSIS
FIGURES
Small: 0
No depletion: 0
No active: 0
None: 0
None: 0
None: 0
None: 0
Nomal: 1
Depletion: 1
Active: 1
Mild: 1
Mild: 1
Mild: 1
Mild: 1
Intense: 2
Intense: 2 Intense: 2
Enlarged: 2
LN: Lymph Node; FC: Follicular Centres
136
Intense: 2
Patogenia del PRRS/Pathogenesis of PRRS
2.3. Immunohistochemical study
The avidin-biotin-peroxidase complex technique (ABC)
was used for the detection of PRRSV and CCasp3 as described
previously (Gómez-Laguna et al., 2010a). The primary
antibodies used were monoclonal anti-PRRSV, clone SDOW17/SR-30, diluted 1 in 1000 and Signal Stain-Cleaved Caspase 3
Asp175 (Cell Signaling, Danvers, MA, USA). Four µm sections
were dewaxed and rehydrated through graded ethanol and a
High Temperature Antigen Retrieval with citrate buffer ph 6.0
was used. Endogenous peroxidase activity was quenched in
H2O2 3% in methanol for 10 min. The sections were washed
with phosphate buffered saline (PBS; pH 7.4, 0.01 M) and
incubated for 1h at room temperature (RT) with 100 µl per slide
of blocking solution in a humid chamber. The primary
antibodies were incubated overnight at 4ºC in a humid chamber.
The corresponding biotinylated secondary antibody was
incubated for 30 min at RT. An avidin-peroxidase complex
(Vector Laboratories, Burlingame, California, USA) was applied
for 30 min at RT. Labelling was “visualized” by application of
the NovaREDTM substrate kit (Vector Laboratories, Burlingame,
137
Patogenia del PRRS/Pathogenesis of PRRS
California, USA). Sections were counterstained with Mayer’s
haematoxylin, dehydrated and mounted. Negative controls
consisted of replacement of the primary antibody by blocking
solution, normal serum and isotype-matched reagents of
irrelevant specificity.
TUNEL
immunolabelling
was
carried
out
with
a
commercial kit (In situ cell death detection, POD, Roche,
Manheim,
Germany,
respectively)
and
following
the
manufacturer’s instructions. Briefly, sequential 4 µm tissue
sections were adhered to silane-coated slides and allowed to dry
at RT. Subsequently, sections were deparaffinized and
rehydrated. Protein digestion was performed by incubating
tissue sections in 20 mg/ml proteinase K recombinant PCR
Grade (Roche Diagnostics, Indianapolis, USA) for 15 min at
37ºC in an humid chamber. Endogenous peroxidase was
inactivated with 2% H2O2 in distilled water (dH2O) for 35 min,
at RT. The labelling mixture was added to sections and
incubated at 37ºC in a humid chamber for 1 h. After stopping
the enzymatic reaction, sections were rinsed with PBS, covered
with Converter-POD (Anti-fluorescein antibody, Fab fragment
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Patogenia del PRRS/Pathogenesis of PRRS
from sheep, conjugated with horse-radish peroxidase) and
incubated for 30 min at 37ºC in a humid chamber. Labelling was
“visualized” by application of the NovaREDTM substrate kit
(Vector Laboratories). Sections were counterstained with
Mayer’s haematoxylin, dehydrated and mounted.
During the first and second experimental studies we
analyzed the expression of several cytokines involved in
apoptotic phenomena such us IL-1, IL-6, TNF-α and IL-10 by
ABC technique in both tonsil and mediastinal lymph node.
2.4. Cell counts
The number of positive cells labelled with antibodies
against PRRSV, CCasp3 and TUNEL in tonsil and mediastinal
lymph node was counted as previously described (Salguero et
al., 2005). Cells immunolabelled were counted in 25 non
overlapping consecutive selected, high magnification fields of
0.20 mm2 (lymphoid follicles, paracortex and medulla of lymph
nodes) or 25 non overlapping consecutive selected structures
(follicles, parafollicular, and lymphoreticular areas of tonsils)
for each animal. Results are expressed as the number of cells per
mm2.
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Patogenia del PRRS/Pathogenesis of PRRS
2.5. Statistical Analysis
The number of PRRSV, CCasp 3 and TUNEL positive
cells was expressed as a mean ± SD. These values were
evaluated for approximate normality of distribution by the
Kolmogorov-Smirnov test. Differences between the means of
control and inoculated animals were assessed by the KruskalWallis test followed by the Mann-Whitney-U non-parametric
test (GraphPad Instat 3.05, San Diego, California). Correlation
between the expression of PRRSV, CCasp3, TUNEL, apoptotic
bodies and picnotic cells was assessed by the Spearman test
(GraphPad Instat 3.05). In addition, the correlation between all
those parameters and the expression of pro-apoptotic cytokines
was performed following the same methodology. P < 0.05
represented a statistically significant difference between
inoculated and control animals.
2. Results
2.1. Clinical Sign, Gross Pathology and Histopathology
The clinical signs, gross and microscopic lesions were
described in the previous experimental studies. Briefly, mild
dullness and weight loss was observed in inoculated animals
140
Patogenia del PRRS/Pathogenesis of PRRS
from the beginning of the study, which showed a non-collapsed,
mottled tan and rubbery lung parenchyma from 7 dpi onwards,
and a mild to moderate enlargement of mediastinal lymph node.
Microscopically the size of lymphoid follicles in most of the
animals in tonsil and mediastinal lymph node were normal
presenting all of them depletion of follicular center and most of
them without an active germinal centre. No necrotic areas were
observed in the tonsil neither in the mediastinal lymph node.
Apoptotic bodies and cell picnosis were presented in both organs
(Fig 1).
Fig. 1. (A) Presence of apoptotic bodies (arrow) and cell picnosis (asterisk)
in the mediastinal lymph node of a 3dpi PRRSV infected pig. H-E Bar =
50µm. (B) Presence of apoptotic bodies (arrow) and cell picnosis (asterisk) in
the tonsil of a 21 dpi PRRSV infected pig. H-E Bar = 40µm
141
Patogenia del PRRS/Pathogenesis of PRRS
In the mediastinal lymph node the presence of apoptotic bodies
and cell picnosis increased gradually from the beginning of the
study (3 dpi) onwards with statistical significant differences with
respect to the control group (Fig 2A). Nevertheless, in the tonsil
the presence of apoptotic bodies and cells picnosis was slighter
with a statistically significant increase only at 21 and 24 dpi
compare with the control group (Fig 2B).
Fig. 2..(A) Apoptotic bodies and cell picnosis expression in mediastinal
lymph node of pigs infected with PRRSV (B) Apoptotic bodies and cell
picnosis expression in tonsil of pigs infected with PRRSV * Indicates
statistically significant differences (P<0.05) between the inoculated group
and controls.
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Patogenia del PRRS/Pathogenesis of PRRS
2.2.Tissue expression of PRRSV antigen in tonsil and lymph
nodes
PRRSV was detected from 3 dpi until the end of the study,
displaying maximum values at 14 dpi in tonsil and at 7 dpi in
mediastinal lymph node, and being observed mainly within the
cytoplasm of macrophages. Table 2 shows PRRSV positive
cells immunolabelled in the tonsil and in the mediastinal lymph
node of control and inoculated animals.
CON
3 dpi
7 dpi
10 dpi
14 dpi
17 dpi
21 dpi
24 dpi
TONSIL
Macrophages Mean ± SD
0.00 ± 0.00
47.40 ± 59.79
7.40 ± 6.02*
0.43 ± 0.30
114.43 ± 25.75*
114.43 ± 25.75*
49.5 ± 19.05*
16.6 ± 18.76*
MEDIASTINAL LYMPH NODE
Macrophages Mean ± SD
8,5±5
99,5±94,04
171,25±100,42*
19,75±15,65
40,25±52,73
8,5±17
23,5±41,74
62,5±67,78
Table 2. PRRSV positive cells immunolabelled in the tonsil and in the
mediastinal lymph node of control and inoculated animals. Data expressed as
mean ± SD of cells/mm2
2.3. Tissue expression of Cleaved Caspase 3 and TUNEL
CCasp3 was mostly expressed in the lymphoreticular area
of the tonsil, mainly by lymphocytes (Fig 3A), whereas TUNEL
reaction was chiefly expressed in the lymphoid follicles by
lymphocytes (Fig 3B).
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Patogenia del PRRS/Pathogenesis of PRRS
In the mediastinal lymph node CCasp3 and TUNEL
reaction were mostly expressed in the paracortex and in the
lymphoid follicles and in a lesser extend in the medulla. CCasp3
expression was observed mainly in macrophages (Fig 3C),
whereas TUNEL reaction was expressed mainly by lymphocytes
(Fig 3D).
Fig. 3.. (A) CCasp 3 immunostaining in the lymphoreticular areas of the
tonsil of a 24 dpi PRRSV infected pig. IHC Bar = 50µm. (B) TUNEL
immunostaining in the follicular areas of the tonsil of a 24 dpi PRRSV
infected pig IHC Bar = 50µm. (C) Macrophages (arrow) immunostained with
CCasp 3 in the mediastinal lymph node of a 24 dpi PRRSV infected pig IHC
Bar = 40µm. (D) Lymphocytes (arrow) immunostained with TUNEL in the
mediastinal lymph node of a 24 dpi PRRSV infected pig IHC Bar = 50µm.
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Patogenia del PRRS/Pathogenesis of PRRS
The expression of CCasp3 in the mediastinal lymph node
was poor throughout the study. Moreover, the expression of
CCasp3 in the tonsil and the TUNEL reaction observed in both
tissues analyzed displayed a similar trend with a slight increase
at the end of the study (Fig 4).
Fig. 4..(A) Counts for cells expressing CCasp3 antigen in the tonsil of pigs
infected with PRRSV. (B) Counts for cells expressing CCasp3 antigen in the
mediastinal lymph node of pigs infected with PRRSV. (C) Counts for cells
expressing TUNEL in the tonsil of pigs infected with PRRSV. (D) Counts for
cells expressing TUNEL in the mediastinal lymph node of pigs infected with
PRRSV.
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Patogenia del PRRS/Pathogenesis of PRRS
2.4. Tissue
expression
of
pro-apoptotic
cytokines
and
correlation with apoptosis
The expression of pro-apoptotic cytokines in both
lymphoid organs were analyzed in the experimental studies
described
before.
Briefly,
in
the
tonsil
none
of
the
proinflammatory cytokines analyzed displayed significant
changes with respect to the control group. Moreover, IL-10 was
also poorly expressed but with a moderate increase at 14 dpi. No
correlation was observed in the tonsil neither between the
expression of pro-apoptotic cytokines and apoptotic phenomena
nor between apoptotic phenomena and PRRSV expression.IL-1α
was the proinflammatory cytokine with the highest expression
followed by TNF-α wich had two peaks of expression at 7 and
14 dpi, whereas IL-10 was increased at 3 dpi and at the end of
the experiment. Despite the expression of IL-1α, TNF-α , IL-10
and PRRSV were not statistically significant correlated with
apoptotic bodies nor cell picnosis, all of them followed a similar
trend throughout the experiment.
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Patogenia del PRRS/Pathogenesis of PRRS
3. Discussion
PRRSV induces an impairment of the host immune
response favouring a prolonged viraemia and viral replication
(Darwich et al., 2010); however, the exact mechanism involved
in the modulation of the immune response still remains unclear.
Lamontagne et al. (2001, 2003) reported a non efficient cell
immune response against PRRSV, which hypothesized to be
related with the apoptosis of lymphoid cells by the direct action
of the virus. Despite numerous studies examine the possible
induction of apoptosis in PRRSV infected cells, it remains
unclear if PRRSV infection results in the direct induction of
apoptosis by viral particles. In this sense, Suárez et al. (1996a)
demonstrated a direct induction of apoptosis in PRRSV infected
cells by the gene product of open reading frame 5 (p25). Other
authors have reported both direct and indirect induction of
apoptosis in PRRSV-infected cells and in bystander cells,
respectively (Sur et al., 1997; 1998), being observed always a
higher number of apoptotic than PRRSV-infected cells (Sur et
al., 1998; Choi and Chae, 2002). Recently, an intrinsic ability of
PRRSV to modulate apoptosis in infected cells has been
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Patogenia del PRRS/Pathogenesis of PRRS
reported, which is independent on the cell type (Costers et al.,
2008). Moreover, these authors described that early in infection,
the balance tends to anti-apoptosis, whereas late in infection, the
balance is driven towards pro-apoptosis (Costers et al., 2008)
In our study a significant increase in the amount of
apoptotic bodies and cell picnosis was observed in pigs
inoculated with PRRSV throughout the experiment in the
mediastinal lymph node and at the end of the study in the tonsil.
Although no correlation was observed between apoptotic
phenomena and the expression of PRRSV, the beginning of
apoptotic phenomena coincided with the first detection of
PRRSV antigen in the mediastinal lymph node. In addition, in
the tonsil a significant enhancement of apoptotic bodies and cell
picnosis was only observed at the end of the experiment
coinciding with a decrease in PRRSV. These results remark that
although PRRSV may be partially involved in a direct induction
of apoptosis, PRRSV induced pro-apoptotic mediators may play
an important role in the onset of apoptosis phenomena.
Apoptosis phenomena have been reported during PRRS
being linked to both viral particles (Suárez et al., 1996a; Costers
148
Patogenia del PRRS/Pathogenesis of PRRS
et al., 2008) and the expression of cytokines (Choi et al., 2002).
TNF-α participates in both the extrinsic and intrinsic pathway of
apoptosis, whereas IL-1 and IL-6 plays a role in the intrinsic
pathway acting as pro- and anti- apoptotic mediators,
respectively (Pollock et al., 2003; Garcia-Tunon et al., 2005;
Alvarez et al., 2011). IL-10 is also considered a pro-apoptotic
mediator (Estaquier et al., 1997; Liu et al., 2001). In a previous
parallel study we observed a lack of homogeneity in the
expression of proinflammatory cytokines in lymphoid organs of
PRRSV-infected pigs. Since all the proinflammatory cytokines
analyzed are involved in apoptosis phenomena it is also
expected to find different expression of apoptotic phenomena in
the lymphoid organs analyzed in the present study. Interestingly,
in our study apoptosis phenomena were more evident in the
mediastinal lymph node than in the tonsil, in wich the
expression of pro-apoptotic cytokines was poor. Furthermore,
the expression of both TNF-α (r = 0.60; P > 0.05) and IL-10 (r =
0.61; P > 0.05) tended to correlate with the presence of
apoptotic bodies and cell pyknosis in the mediastinal lymph
node.
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Patogenia del PRRS/Pathogenesis of PRRS
On the other hand, in the tonsil we found a significant
increase of apoptotic bodies and cell picnosis at the end of the
experiment coinciding with the expression of CCasp3 and
TUNEL. The expression in the tonsil of CCasp3 and TUNEL
was mainly observed in lymphocytes, cells in which the
expression of PRRSV was not observed which support the
hypothesis that PRRSV may induce apoptosis by an indirect
mechanism. Moreover, those animals with higher apoptotic
scores did not correlate with those with higher CCasp3 or
TUNEL counts, which points to an induction of apoptosis by a
caspase 3-independent pathway. This postulate agree with our
results in the mediastinal lymph node, where apoptotic bodies
and cell picnosis were observed throughout the experiment
without an expression of CCasp 3 and TUNEL.
Caspase 3-independent apoptotic pathway have been
described by several authors in human cells in vitro (Untergasser
et al., 2001; Piwocka et al., 2002; Thayyullathul et al., 2008;
Zilkova et al., 2011; Zhang et al., 2011; Alvarez et al., 2011).
Among caspase 3-independent pathways, the generation of
reactive oxygen species (ROS) (Thayyullathi et al., 2008), the
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Patogenia del PRRS/Pathogenesis of PRRS
imbalance of the Bcl-2/Bax ratio (Untergasser et al., 2001) and
the upregulation of FasL (Alvarez et al., 2011), have been
reported as possible mechanisms for the induction of apoptosis.
Specifically, Alvarez et al. (2011) reported that TNF-α induced
an enhancement of FasL and cell death by the release of
cytochrome c from the mitochondria, leading to caspase 9
activation and a caspase 3-independent apoptotic pathway.
Moreover, Chang et al. (2007) described an increase of FasL
capable
of
inducing
Fas/FasL
mediated
apoptosis
in
lymphocytes in pigs with PRRSV infection. In this regard,
taking into account our results we hypothesized that PRRSV
may induce apoptosis through a caspase 3-independent pathway
by Fas/FasL interaction.
In conclusion, different expression of apoptotic phenomena
was observed in the lymphoid organs analyzed which could be
related with the lack of homogeneity in the expression of
proinflammatory cytokines. Furthermore, caspase 3-independent
apoptosis pathways could be present in PRRSV infection, since
no expression of caspase 3 together with morphological
evidence of apoptosis was observed. The study of different
151
Patogenia del PRRS/Pathogenesis of PRRS
apoptosis mediators is encouraged in order to determine the
mechanism used by PRRSV to induce the apoptosis phenomena.
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Patogenia del PRRS/Pathogenesis of PRRS
3.5.
IMMUNOHISTOCHEMICAL
EXTRINSIC
AND
APOPTOSIS
IN
INTRINSIC
PORCINE
DETECTION
OF
MEDIATORS
OF
PARAFFIN-EMBEDDED
TISSUES.
Apoptosis or “programmed cellular death”, both under
physiological and pathological conditions, is characterized by
peripheral condensation of the chromatin and cellular shrinkage
leading to the formation of apoptotic bodies, without the onset
of an inflammatory response (Kerr et al., 1972, 1995). Several
mechanisms may trigger off apoptosis depending on the
expression of specific molecules. Thus, an extrinsic and an
intrinsic pathway have been reported (Roy and Nicholson, 2000;
Mitchell and Cotran, 2007). The extrinsic pathway is started
when an appropiate ligand, such as TNFα or FasLigand (FasL),
is bound to one of several cell surface death-receptors (known as
Tumor Necrosis Factor Receptor family, TNF-R) (Nagata, 1997;
Ashkenazi and Dixit, 1998). These mediators activate caspase 8,
which subsequently activates the executioner caspase 3
(Budihardjo et al., 1999; Earnshaw et al., 1999; Ashkenazi,
2002; Mitchell and Cotran, 2007). On the other hand, the
153
Patogenia del PRRS/Pathogenesis of PRRS
intrinsic pathway is considered a mitochondrial dependent
pathway (Roy and Nicholson, 2000). The Bcl family covers a
group of molecules involved in the mitochondrial permeability,
being differentiated anti-apoptotic (Bcl-2 or Bcl-x) and proapoptotic (Bax, Bak, Bim) molecules (Oltvai et al., 1993;
Miyashita et al., 1994; Reed, 1994; Sedlak et al., 1995; Cory et
al., 2003). The unbalance between these molecules increases the
permeability of mitochondrial membrane, leading to the release
of proteins, just as cytochrome c, which activates caspases. The
release of cytochrome c induces the activation and cleavage of
caspase 9, and the subsequent activation of the executioner
caspase 3 (Zou et al., 1997; Chu et al., 2001; Waterhouse et al.,
2002). Moreover, an upregulation of inducible nitrogen oxide
synthase (iNOS) has been also related to an increase of the
mitochondrial permeability and release of cytochrome c (Kim et
al., 2002; Atre et al., 76 2006). Both extrinsic and intrinsic
pathways of apoptosis converge in the activation of caspase 3
and continue a common pathway of cell death (Mitchell and
Cotran, 2007). Viral infections may modulate apoptosis
inducing either an up-regulation or a down-regulation of such
154
Patogenia del PRRS/Pathogenesis of PRRS
process as a mechanism to evade the immune response
(Thomson, 2001; Costers et al., 2008). In this sense, an
enhancement of apoptosis phenomena has been observed in
lymphoid organs of pigs infected with African Swine Fever
virus (ASFv) (Salguero et al., 2005; Fernández de Marco et al.,
2007), Porcine Circovirus type 2 (PCV2) (Resendes et al., 2004)
or Porcine Reproductive and Respiratory Syndrome virus
(PRRSv) (Suárez et al., 1995a; Choi and Chae, 2002; Costers et
al., 2008). The apoptosis phenomena observed in these diseases
have been linked directly to the viral replication (Suárez et al.,
1996a; Costers et al., 2008) or indirectly to an increased
expression of mediators, such as cytokines (Choi and Chae,
2002; Salguero et al., 2005; Fernández de Marco et al., 2007).
Preservation of antigenicity and structure is essential to carry out
immunohistochemical studies on tissue samples, playing the
fixation a key role to reach this goal. Formaldehyde is the
routine fixative in histopathological studies since is able to
preserve properly the tissue structure. However, aldehyde
fixatives are known to induce cross-links between tissue
proteins inducing the masking of tissue antigens (Ramos-Vara et
155
Patogenia del PRRS/Pathogenesis of PRRS
al., 2008). Thus, recently many efforts have been carried out to
determine which one is the optimal fixative for each antibody as
a previous step to study the pathogenesis of several diseases
(Salguero et al., 2001; Hicks et al., 2006). The main goal of this
study
was
to
standardize
techniques
for
the
immunohistochemical detection of mediators from both
extrinsic (caspase 8, Fas) and intrinsic pathways (caspase 9, Bcl2, iNOS) of apoptosis using different antigen retrieval methods
and fixatives in porcine paraffin embedded tissues.
Materials and methods
Animals and experimental design
To standardize the expression of apoptosis mediator antigens
five 5-week-old pigs from a previous experiment (GómezLaguna et al., 2009) were used. These animals were PRRSvinoculated pigs, with clinical signs and lesions of the disease,
and were selected on the basis of their positive immunolabelling
against caspase 3 and TUNEL (Figs. 1A and 1B).
156
Patogenia del PRRS/Pathogenesis of PRRS
Fig. 1. (A) Anti-caspase 3 immunolabelling in numerous lymphocytes and
macrophages from the lymphoreticular tissue and some lymphocytes in the
lymphoid follicle of the tonsil. IHC. Bar = 100_m. (B) TUNEL labelled cells
and apoptotic bodies (arrow) in the lymphoid follicle of the retropharyngeal
lymph node. IHC. Bar = 25_m.
At the necropsy samples from tonsil, retropharyngeal lymph
node and lung were fixed in various fixatives: 10% neutral
buffered formalin buffered with phosphate-buffered saline
(PBS) pH 7.2 for 18 h; Bouin’s solution (15:5:1 of 10%
formalin, glacial acetic acid, picric acid saturated solution in
distilled water) for 18 h; and, Zinc salts fixative (ZSF) (Tris
buffer pH 7.4 with 0.5% of calcium acetate, 5% of zinc acetate
and 5% of zinc chloride) for 18 h. Samples fixed with the Bouin
solution were rinsed 7 times in ethanol 50%, 30 min each time,
and stored (20-30 min) in ethanol 70% until embedding. Finally,
all the samples fixed with each fixative were routinely processed
157
Patogenia del PRRS/Pathogenesis of PRRS
and embedded in paraffin-wax. Sections (4 µm) of fixed tissue
were used for the immunohistochemical study. Caspase 3 and
TUNEL immunolabelling was carried out with commercial kits
120 (Signal Stain-Cleaved Caspase 3 Asp175, Cell Signaling,
Danvers, MA, USA; In situ cell death detection, POD, Roche,
Manheim,
Germany,
respectively)
and
following
the
manufacturer’s instructions. Briefly, for TUNEL technique,
serial 4 µm tissue sections were adhered to silane-coated slides
and allowed to dry at room temperature (RT). Subsequently,
sections were deparaffinized, rehydrated and incubated with 20
mg/ml proteinase K (Roche Diagnostics, Indianapolis, 125
USA) for 15 min at 37 ºC in an humidity chamber. Endogenous
peroxidase was quenched with 2% H2O2 in distilled water for
35 min, at RT. The labelling mixture was added to sections and
incubated at 37 ºC in an humidity chamber for 1 h. After
stopping the enzymatic reaction, sections were rinsed with PBS,
covered with Converter-POD (Anti-fluorescein antibody, Fab
fragment from sheep, conjugated with horse-radish peroxidase)
and incubated for 30 min at 37 ºC in an humified chamber.
Labelling was “visualized” by application of the NovaREDTM
158
Patogenia del PRRS/Pathogenesis of PRRS
substrate kit (Vector Laboratories). Sections were counterstained
with Mayer’s haematoxylin, dehydrated and mounted.
Immunohistochemical study
The avidin-biotin-peroxidase complex technique (ABC) (Hsu et
al., 1981) was used for the detection of caspases 8 and 9, Fas,
Bcl-2 and iNOS. Briefly, tissue sections were dewaxed and
dehydrated through graded ethanol and the endogenous
peroxidase activity was quenched in H2O2 3% in methanol for
45 min. Different antigen retrieval or permeabilisation methods
were used to test the samples in all different fixations: no
pretreatment, when no antigen retrieval method was performed;
0.01% Tween 20 (Merck & Co, Inc., WhiteHouse Station, NJ,
USA) in PBS pH 7.2 (12 min in a stirrer at room temperature);
microwave in pH 6.0 citrate buffer (5 min at 450W followed by
6 min at 150W); or protease type XIV (Sigma Aldrich Chemie
GmbH, Steinheim, Germany) at 37 ºC for 30 minutes. After
pretreatment, samples were rinsed three times in PBS pH 7.2 for
5 min each and incubated for 30 min at room temperature with
100 µl per slide of blocking solution in a humid chamber, before
incubation overnight at 4 ºC with primary antibody. The clones
159
Patogenia del PRRS/Pathogenesis of PRRS
and sources of primary antibodies are summarized in Table 1. In
each case, the corresponding biotinylated secondary antibody
was incubated for 30 min at room temperature. An avidinbiotin-peroxidase complex (Vector Laboratories; Burlingame,
CA, USA) was 150 applied for 1 h at room temperature.
Labelling was “visualized” by application of the NovaREDTM
substrate kit (Vector Laboratories; Burlingame, CA, USA).
Sections were counterstained with Mayer’s haematoxylin,
dehydrated and mounted. Negative controls consisted of
replacement of the primary antibody by blocking solution,
normal serum and isotype-matched reagents of irrelevant
specificity.
Cell counts
The number of positive cells labelled with antibodies against
caspase 8, caspase 9, Fas, Bcl-2 and iNOS in tonsil,
retropharyngeal lymph node and lung were counted as
previously
described
immunolabelled
were
(Salguero
counted
et
in
al.,
25
2005).
non
Cells
overlapping
consecutive selected, high magnification fields of 0.20 mm2 160
(lymphoid follicles, paracortex and medulla of lymph nodes) or
160
Patogenia del PRRS/Pathogenesis of PRRS
25 non overlapping consecutive selected structures (follicles,
parafollicular, and lymphoreticular areas of tonsils) for each
animal. In the lung sections, it was determined if the
immunolabelled cells were located in the alveoli or in the septa.
Results (number of cells per mm2164 ) were recorded as
follows: +, < 2; ++, 2-5 and +++, > 5 cells/mm2.
161
Patogenia del PRRS/Pathogenesis of PRRS
Table 1
Clones, sources and dilutions of the primary antibodies used for the immunohistochemical detection of extrinsic
and intrinsic pathways of apoptosis.
Antibody
Clone
Source
Dilution
Rabbit Anti-human caspase 8
Clone Asp391
Cell Signaling; Danvers, MA, USA
1:25
Rabbit Anti-human caspase 9
Clone Asp330
Cell Signaling; Danvers, MA, USA
1:25
Santa Cruz Biotech.; Santa Cruz, CA, USA
1:1000
Rabbit Anti-mouse Fas (X- Polyclonal
20)
Mouse Anti-human Bcl-2
Clone 124
Dako Denmark; Glostrup, Denmark
1:50
Rabbit Anti-mouse iNOS
Polyclonal
NeoMarkers; Fremont, CA, USA
1:200
162
Patogenia del PRRS/Pathogenesis of PRRS
Results
Immunohistochemical markers for the extrinsic pathway of
apoptosis
Markers of the extrinsic pathway of apoptosis showed the most
satisfactory immunohistochemical labelling in porcine ZSF
tissues (Table 2). Both antibodies against caspase 8 and Fas
displayed no immunostaining in 10% neutral buffered formalin
and Bouin fixed samples (tonsil, retropharyngeal lymph node,
and lung), but positive immunolabelling with hard to light
background was observed when citrate buffered or protease was
used as antigen retrieval technique with both fixatives (Table 2).
Porcine ZSF tissues s 175 howed positive immunostained cells
for both antibodies against caspase 8 and Fas with light or
without background when Tween 20 or citrate unmasking
methods were respectively used (Table 2).
Immunohistochemical markers for the intrinsic pathway of
apoptosis
The fixation with 10% neutral buffered formalin and Bouin
solution yielded negative results or hard background for
antibody against caspase 9 with any of the antigen retrieval
163
Patogenia del PRRS/Pathogenesis of PRRS
treatments carried out. However, immunopositive cells without
background were observed in ZSF tissue samples when Tween
20 treatment was used (Table 2). No reaction or intense
background was observed with any other combination of
fixative and antigen retrieval. Both Bcl-2 and iNOS antibodies
developed positive immunostaining with light or without
background when citrate antigen retrieval or protease techniques
were used in formalin fixed tissues (Table 2). Bouin and ZSF
samples showed also immunolabelled cells for these antibodies
but with light to hard background (Table 2).
Cell counts and location of immunolabelled cells
Cell counts were performed in the ideal fixative and antigen
retrieval for each antibody: caspase 8 and Fas antibodies in ZSF
tissues and treated with citrate; caspase 9 antibody in ZSF
tissues treated with Tween 20; and Bcl-2 and iNOS antibodies in
10% neutral buffered formalin fixed samples and unmasked
with protease and citrate treatment, respectively. The number of
positive cells against each antibody is expressed as means of all
the animals examined in the study (Table 3). Caspase 3 and
TUNEL techniques immunostained both apoptotic bodies and
164
Patogenia del PRRS/Pathogenesis of PRRS
viable
cells
that
corresponded
with
macrophages
and
lymphocytes from T- and B-cells areas (Figs. 1A and 1B). The
rest of antibodies (caspase 8, caspase 9, Bcl-2, Fas, iNOS)
immunolabelled viable cells corresponding mainly with
lymphocytes from T-cell areas of the examined lymphoid
tissues, lymphoreticular areas 200 of tonsil and paracortex of
201 lymph nodes (Figs. 2A–E). In addition, macrophage-like
cells from the medulla of lymph nodes and from the septum of
the lung were also immunostained (Figs. 2F–G). Few
lymphocytes from B-cell areas (lymphoid follicles) of tonsils
and lymph nodes were positively labelled. Table 3 summarizes
the main structures showing immunolabelled cells for each
organ and antibody. The total number of positive cells against
caspase 3 and TUNEL was equivalent to the one observed for
most of the other mediators of apoptosis evaluated in this study.
Nonetheless, in the retropharyngeal lymph node a higher
number of immunolabelled cells against caspase 3 and TUNEL
was observed in the lymphoid follicles. Contrary, in the medulla
the immunoreactive cells against the intrinsic and extrinsic
mediators of apoptosis were more numerous than caspase 3 and
165
Patogenia del PRRS/Pathogenesis of PRRS
TUNEL (Table 3). Particularly, few epithelial cells from tonsil
crypts were immunolabelled with antibody against caspase 8, as
well as, some neutrophils from the lung or lymphoid tissues with
antibodies against caspase 9 and/or iNOS (Figs. 2F–G).
Fig. 2. (A) Numerous macrophages (arrow) and lymphocytes (arrowhead) immunolabelled
against cleaved caspase 8 (ZSF, Citrate) in the paracortex of the retropharyngeal lymph node.
IHC. Bar = 30µm. (B) Anti-cleaved caspase 9 immunostaining (ZSF, Tween 20) in the
paracortex of a retropharyngeal lymph node. IHC. Bar = 100µm. (C) Abundant lymphocytes
immunostained against Bcl-2 (Formol, Protease) in the paracortex of a retropharyngeal lymph
node. Note the negative immunolabelling of the lymphoid follicle. IHC. Bar = 30µm. (D) AntiFas immunostaining (ZSF, Citrate) in macrophages and lymphocytes in the paracortex of the
tonsil and intraepithelial within a crypt. IHC. Bar = 30µm. (E) Numerous macrophages and some
lymphocytes immunolabelled against iNOS (Formol, Citrate) in the paracortex of the tonsil.
IHC. Bar = 75µm. (F) Anti-iNOS (Formol, Citrate) immunolabelling of two interstitial
macrophages (arrow) in the alveolar septa of the lung. IHC. Bar = 25µm. (G) Two interstitial
macrophages (asterisk), a lymphocyte (arrow), and a neutrophil (arrowhead) immunostained
against cleaved caspase 9 (ZSF, Tween 20) in the alveolar septa of the lung. IHC. Bar = 25µm.
166
Patogenia del PRRS/Pathogenesis of PRRS
Table 2
Treatments and antibodies used for detection of caspases 8 and 9, Fas and iNOS in tonsil, retropharyngeal
lymph node and lung of pigs.
Caspase 8
Caspase 9
Fas
Bcl-2
iNOS
Formol
No pretreatment
Tween 20
Citrate microwave
+
+++
+++
Protease
+
+
+
+++
++
Bouin
No pretreatment
++
Tween 20
+
++
Citrate microwave
+
-/+
++
++
++
Protease
Zinc salts
No pretreatment
++
+
++
+
+
Tween 20
+
+++
Citrate microwave
+++
+
+++
++
+
Protease
- : No reaction; +: Positive reaction and hard background; ++: Positive reaction and light background; +++:
Positive reaction with no background
167
Patogenia del PRRS/Pathogenesis of PRRS
Table 3
Positive cells for caspases 8 and 9, Fas and iNOS in the tonsil, retropharyngeal lymph node and lung of pigs.
Fixative
Antigen retrieval method
Tonsil
Follicles
Caspase 3
Formol
Citrate
TUNEL
Formol
Protease
Caspase 8
ZSF
Citrate
Caspase 9
ZSF
Tween 20
Fas
ZSF
Citrate
Bcl-2
Formol
Protease
iNOS
Formol
Citrate
++
++
++
+
++
+
+
LRT
Retropharyngeal LN
+++
++
+++
++
+++
+++
+++
Follicles
Paracortex
++
+++
++
++
+
+++
+
++
+
+++
+
+++
+
+++
Medulla
Lung
Alveoli
+
+
+++
+
+++
+++
++
+
+++
+
+
+
+
+
Septum
++
+++
++
+
+++
++
2
++
2
ZSF: Zinc Salts Fixative; LRT: LymphoReticular Tissue; +: < 2 cells/0.2mm field; ++: 2-5 cells/0.2mm field;
+++: > 5 cells/0.2mm2 field.
168
Patogenia del PRRS/Pathogenesis of PRRS
Discussion
Immunohistochemical
labelling
represents
nowadays
a
significantly useful tool in research since enables the detection
of antigens by means specific antibodies amplifying the antigenantibody reaction making it easily visible, as well as, to localize
a specific antigen within a lesion contributing to the
understanding of the pathogenesis of several diseases (RamosVara et al., 2008). This technique also allows determining the
spatial correlation between the expression of antigens and
mediators of the immune response, just as cytokines or
apoptosis mediators. However, the fixation process required to
carry out immunohistochemical staining is one of the greatest
inconveniences of this technique. It is well known that aldehyde
fixatives,
routine fixatives in histopathology examinations,
cause cross-linkages masking the antigens present in tissue
sections (Leong and Leong, 2007). Thus, several fixatives and
antigen retrieval methods are proposed in order to obtain the
optimal preservation for each antigen (Salguero et al., 2001). In
the present study a panel of antibodies was used to determine the
extrinsic and intrinsic pathways of apoptosis in porcine fixed
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Patogenia del PRRS/Pathogenesis of PRRS
tissues. To study the extrinsic pathway the antibodies against
caspase 8, and Fas were used, whereas, antibodies against
caspase 9, Bcl-2 and iNOS were use to examine the intrinsic
pathway of apoptosis. All the antibodies were selected on the
basis
of
the
manufacturer
specifications
for
the
immunohistochemical detection of their human, mouse and/or
rat counterparts. As the development and production of new
monoclonal antibodies is both time consuming and expensive, it
seems reasonable to investigate whether existing antibodies
against human or murine antigens can be used successfully in
heterologous species of interest (Pedersen et al., 2002). In this
sense, a considerable homology has been reported between
porcine Fas-associated death domain (FADD) and porcine
procaspase 8 with their human and murine counterparts (Inoue
et al, 2007). Moreover, a marked genomic and antigenic
correlation has been reported between human and porcine
cytokines (Smith, 1990), being observed an 86% of homology
between human and porcine TNFα (Pauli, 1995). Previous
studies have also reported cross reactivity for some of these
antibodies in porcine cell cultures or in porcine frozen tissues
170
Patogenia del PRRS/Pathogenesis of PRRS
(Bai et al., 2004; Ananiadou et al., 2007). These considerations
support the use of human and murine antibodies for
immunohistochemical examination of paraffin wax-embedded
samples with porcine tissues in the present study. In addition,
the positive immunolabelling of these antibodies in our study
confirm their cross-reaction with porcine fixed paraffin
embedded tissues. Previous reports have reported benefits of
fixation with Bouin solution in the immunolabelling of
cytokines and in the study of the pathogenesis of viral diseases
(Salguero et al., 2001; Gómez-Laguna et al., 2010). In the
present study, formalin fixed samples displayed successful
results for Bcl-2 and iNOS, but antibodies against caspase 8,
caspase 9 and Fas required alternative fixatives to get a
satisfactory immunostaining. Bouin solution only enhanced
lightly the detection of these antigens, but with a moderate to
high background. On the other hand, ZSF allowed obtaining a
positive staining without background when citrate microwave
(caspase 8 and Fas) or Tween 20 (caspase 9) unmasking
methods were used. The satisfactory results obtained for a
specific immunolabelling in porcine tissues point to a potential
171
Patogenia del PRRS/Pathogenesis of PRRS
use of these antibodies in future studies. Interestingly, Hicks and
co-authors (2006) found similar results using ZSF for the
immunohistochemical labelling of murine immune system cells.
Theoretically the apoptosis phenomenon is divided into the two
pathways named above, however, it is well known that both
extrinsic and intrinsic pathways interconnect with each other.
Thus, TNFα (a ligand of the extrinsic pathway of apoptosis) has
been suggested to trigger iNOS-induced apoptosis (intrinsic
pathway) in porcine vascular smooth muscle cells (Idel et al.,
2002). On the other hand, the nitric oxide, derived from iNOS
activity, has been reported to inhibit TNFα- and/or Fas-mediated
apoptosis phenomena (Hatano, 2007). Moreover, Fas signaling
has been reported to activate Bid, a proapototic member of the
Bcl family, which increases the mitochondrial permeability
(Myers and McGavin, 2007). Therefore, our results may
increase the knowledge in the interplay between both pathways
of apoptosis in several biological processes. In addition, the
present study allows detecting cells immunolabelled in different
structures from both lymphoid and non-lymphoid organs.
Immunolabelled cells were morphologically identified as
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Patogenia del PRRS/Pathogenesis of PRRS
lymphocytes, macrophages, neutrophils or epithelial cells.
Previous studies have reported apoptosis also in these cells
(Satake et al., 2000; Salguero et al., 2005; Fernández de Marco
et al., 2007). All the antibodies used in this study displayed
similar results than immunolabelled cells against caspase 3 and
TUNEL in the tonsil and in the lung. The discrepancy observed
between these parameters in the lymphoid follicles and medulla
of the retropharyngeal lymph node may be related to the
detection of different stages of the apoptosis phenomenon. Thus,
the higher count of caspase 3 and TUNEL in the lymphoid
follicles of the retropharyngeal lymph node may point to a more
advanced stage of apoptosis, whereas, the higher count of any of
the other mediators, just as caspase 8, in the medulla of the
lymph node may point to a earlier detection of apoptosis. This
finding was also supported by the morphological characteristics
of immunolabelled cells against extrinsic and intrinsic apoptosis
mediators, as viable cells. Therefore, the use of these markers
may be useful also in the detection of different stages of the
apoptosis phenomenon. The results presented in this study
enable the detection of both extrinsic- and intrinsic-apoptosis
173
Patogenia del PRRS/Pathogenesis of PRRS
mediators in specific structures and cells in porcine fixed,
paraffin embedded tissues, representing a worthy tool to study
the pathogenesis of swine diseases, as well as, in xenotransplant
and biomedicine studies.
174
Patogenia del PRRS/Pathogenesis of PRRS
DISCUSIÓN
GENERAL/
GENERAL
DISCUSSION
175
Patogenia del PRRS/Pathogenesis of PRRS
DISCUSIÓN GENERAL
El Síndrome Reproductivo y Respiratorio (PRRS del
ingés Porcine Reproductive and Respiratory Syndrome) es
considerado una de las enfermedades más importantes de la
industria del porcino. A pesar de que varios estudios han sido
llevado a cabo para elucidar respuesta inmune llevada a cabo
por el hospedador frente al virus del PRRS (PRRSV del inglés
PRRS virus) (Yoon et al., 1995; Loemba et al., 1996; Shimizu et
al., 1996; Bautista and Molitor, 1997; Wills et al., 1997a, 1997b;
Albina et al., 1998a, 1998b; Kawashima et al., 1999; LópezFuentes et al., 1999; Allende et al., 2000; Samson et al., 2000;
Lamontagne et al., 2001, 2003; Meier et al., 2003; Xiao et al.,
2004; Díaz et al., 2005), todavía quedan aspectos que sin
esclarecer.
El virus del PRRS se replica, principalmente, en los
macrófagos alveolares porcinos (MAPs) y, en menor medida, en
macrófagos de otros órganos y en las células dendríticas
(Molitor et al., 1997; Bautista and Molitor, 1999). Sin embargo,
la mayoría de los estudios centrados en analizar la respuesta
176
Patogenia del PRRS/Pathogenesis of PRRS
inmune provocada tras una infección con el PRRSV se han
realizado principalmente sobre muestras de suero y de pulmón
(Batista et al., 2004; Xibao et al., 2010; Gómez-Laguna et al.,
2010a) y en menor medida en los órganos linfoides (Rossow et
al., 1996; Beyer et al., 2000).
La producción de citoquinas, es una de las herramientas
utilizadas por los macrófagos así como por otras células del
sistema inmunes o no pertenecientes al sistema inmune, en la
defensa contra patógenos (Kumar et al., 2008). En este sentido,
analizamos la expresión de citoquinas proinflamatorias y de
citoquinas implicadas en la respuesta inmune como IL-10, IL12, IFN-α e IFN-γ en órganos linfoides de cerdos infectados con
el PRRSV para determinar su correlación con la expresión del
antígeno vírico en órganos linfoides, así como para evaluar su
papel en la patogenia de la enfermedad. Así mismo, el PRRSV
desarrolló una expresión bimodal en la tonsila y en el nódulo
linfático retrofaríngeo con un primer pico de expresión a los 3
dpi y un segundo a los 14 dpi, mientras que en el nódulo
linfático mediastínico sólo encontramos un pico de expresión al
177
Patogenia del PRRS/Pathogenesis of PRRS
comienzo de la infección disminuyendo posteriormente hasta el
final del experimento. Interesantemente, la expresión de cada
citoquina estudiada fue diferente dependiendo de la cavidad
analizada. Así, en la tonsila se observó una pobre expresión de
citoquinas proinflamatorias, lo que podría estar relacionado
tanto con la persistencia del PRRSV en tonsila de cerdos
infectados (Beyer et al., 2000; Wills et al., 2003), como con una
mayor susceptibilidad a las infecciones secundarias descritas por
otros autores (Wills et al., 2000; Thanawongnuwech et al., 2000,
2004). Mientras que en el nódulo linfático mediastínico se
observó un incremento en la expresión de TNF-α e IL-1α ,
siendo la IL-6 la citoquina más expresada en el nódulo linfático
retrofaríngeo. Estos hallazgos, señalan un comportamiento
diferente del PRRSV en los órganos linfoides, que podría estar
relacionado con la falta de una respuesta inmune robusta frente
al virus. Adicionalmente, las citoquinas proinflamatorias son
capaces de modular la expresión de CD163, un receptor
neutralizante de la hemoglobina, que actúa como receptor del
PRRSV y está implicado en la internalización del virus (Van
Gorp et al., 2008). Por lo tanto, un desequilibrio entre estas
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Patogenia del PRRS/Pathogenesis of PRRS
citoquinas puede jugar un papel en la capacidad del PRRSV
para replicarse.
Además de las citoquinas proinflamatorias, otras
citoquinas juegan un papel significativo en la patogenia del
PRRS. Se ha hipotetizado que el PRRSV es capaz de evadir la
respuesta inmune induciendo una expresión de IL-10, que podría
a su vez, reducir los niveles de otras citoquinas implicadas en la
eliminación del virus como IFN-α, IFN-γ, IL-12p40 y TNF-α
(Gomez-Laguna et al., 2010a). A pesar de que la expresión de
las citoquinas proinflamatorias fue diferente dependiendo del
órgano examinado, la expresión de IL-10, IL-12p40, IFN-α e
IFN-γ, fue prácticamente similar en todos los órganos linfoides
estudiados, a pesar de que la cantidad de PRRSV detectada fue
diferente. Este hallazgo, sugiere que la respuesta inmune
adaptativa frente al PRRSV es más homogénea que la respuesta
proinflamatoria. A pesar de la actividad antiviral que poseen los
IFNs y la IL-12, la IL-10 es capaz de reducir los niveles de estas
citoquinas implicadas en la eliminación vírica (Mitchell and
Kumar, 2004). En nuestro estudio, la cantidad de IL-10
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Patogenia del PRRS/Pathogenesis of PRRS
expresada fue de leve a moderada, observándose una mayor
expresión de citoquinas con capacidad antiviral. Contrariamente,
la ratio IL-10/IFN-γ previamente descrita en el pulmón por
Gómez-Laguna et al. (2010a) fue proporcionalmente mayor,
relacionándose con la persistencia del PRRSV en pulmón de
cerdos infectados, por lo que la diferente expresión de IFNs, IL10 e IL-12 en los órganos linfoides con respecto al pulmón, nos
indica que el PRRSV es capaz de modular la respuesta inmune
del hospedador en los órganos linfoides de manera diferente a la
previamente descrita en el pulmón. La expresión de IFNs en los
órganos linfoides, junto con la persistencia viral encontrada a
final del estudio, indican que la vía de señalización del IFN
puede no estar funcionando adecuadamente. En este sentido, se
ha demostrado que la proteína 2 no estructural (Nsp2 del inglés
non structural protein 2) del PRRSV es capaz de inhibir la
inducción del factor 3 regulador de IFN (Li et al., 2010), sin
embargo no ha sido descrito en el PRRSV ningún papel en la vía
de señalización del IFN, por lo que futuros estudios están siendo
dirigidos hacia la determinación del papel del PRRS en la vía de
señalización del IFN.
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Patogenia del PRRS/Pathogenesis of PRRS
Por otro lado, las infecciones víricas son capaces de
modular los fenómenos de apoptosis induciendo un aumento o
una disminución de los mismos como mecanismos de evasión de
la respuesta inmune del hospedador (Thomson, 2001; Costers et
al., 2008). La apoptosis es un mecanismo de muerte celular
estrictamente regulado que envuelve a una compleja red de vías
bioquímicas. El que una célula entre en apoptosis o no depende
de un delicado balance de estímulos anti- y pro- apoptóticos.
Los fenómenos de apoptosis han sido descritos en el PRRS
ligados tanto a las partículas víricas (Suárez et al., 1996a;
Costers et al., 2008) como a la expresión de citoquinas (Choi et
al., 2002). Todas las citoquinas proinflamatorias analizadas en
nuestro estudio pueden estar implicadas en los fenómenos de
apoptosis. El TNF-α participa en la vía extrínseca de la
apoptosis, mientras que la IL-1 e IL-6 juegan un papel en la vía
intríseca de la apoptosis actuando como mediadores pro- y antiapoptóticos respectivamente (Pollock et al., 2003; Garcia-Tunon
et al., 2005). Además, algunos autores han descrito un papel
pro-apoptótico para la IL-10 (Estaquier et al., 1997; Liu et al.,
2001). En este sentido, en nuestro estudio, evaluamos los
181
Patogenia del PRRS/Pathogenesis of PRRS
fenómenos de apoptósis mediante examen histopatológico, así
como mediante la expresión de caspasa 3 activada y la técnica
TUNEL en órganos linfoides, encontrando más evidencias de
fenómenos de apoptosis en el nódulo linfático mediastínico que
en la tonsila, donde encontramos una expresión de citoquinas
pro-apoptóticas muy pobre. En ambos órganos estudiados,
encontramos fenómenos de apoptosis a lo largo de todo el
estudio sin que estuviesen correlacionados estos fenómenos con
la expresión de caspasa 3 activada, sugiriendo que el PRRSV en
nuestro estudio induce apoptosis por una vía independiente de la
caspasa 3. La generación de especies reactivas de oxígeno
(ROS, del inglés reactive oxygen species) (Thayyullathi et al.,
2008), el desequilibrio en la ratio Bcl-2/Bax (Untergasser et al.,
2001) así como el incremento de FasL (Alvarez et al., 2011),
han sido descritos como posibles mecanismos de inducción de la
apoptosis
por
vías
independientes
a
la
caspasa
3.
Concretamente, Alvarez et al. (2011) describieron que el TNF-α
induce un incremento del FasL y la muerte celular por la
liberación del citocromo c de la mitocondria, activando la
caspasa 9 y llevando una vía de apoptosis independiente de la
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Patogenia del PRRS/Pathogenesis of PRRS
caspasa3. Además, Chang et al. (2007) describieron un
incremento del FasL capaz de inducir apoptosis mediada por
Fas/FasL en linfocitos de cerdos infectados con el PRRSV. Por
lo tanto, futuros estudios son necesarios para determinar los
mediadores de la apoptosis que utiliza el PRRSV para inducir la
apoptosis por una vía independiente de la caspasa 3, para lo
cual, determinamos el fijador así como el desenmascaramiento
antigénico ideal para la detección inmunohistoquímica de
diferentes mediadores de la apoptosis tanto de la vía extrínseca
como de la vía intrínseca en tejidos de cerdos incluidos en
parafina.
El mejor fijador y desenmascaramiento antigénico
frente a la caspasa 8 y el Fas (mediadores de la vía extrínseca de
la apoptosis) fueron las sales de Zinc y el tampón citrato. Para
determinar la vía intrínseca de la apoptosis los fijadores ideales
así como los desenmascaramientos antigénicos de elección
fueron: Para la caspasa 9, sales de Zinc como fijador y Tween
20 de desenmascaramiento antigéncio, para Bcl-2 e iNOS fue
formol al 10% con proteinasa K tipo XIV y tampón citrato
respectivamente. Los buenos resultados obtenidos para la
inmunotinción de mediadores de apoptosis en tejidos de cerdos
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Patogenia del PRRS/Pathogenesis of PRRS
señalan hacia un potencial uso de estos anticuerpos en futuros
estudios.
En conclusión, el PRRSV en nuestro modelo experimental
induce una expresión diferente de citoquinas y de los fenómenos
de apoptosis en los órganos linfoides, lo que constituye una
herramienta útil para profundizar en el estudio de la patogenia
del PRRSV. Asimismo, nuestros resultados sugieren que el
PRRSV puede utilizar vías independientes a la Caspasa 3 para la
inducción de la apoptosis, lo que abre nuevos campos de
actuación para elucidar el mecanismo empleado por el virus para
inducir dichos fenómenos.
184
Patogenia del PRRS/Pathogenesis of PRRS
GENERAL DISCUSSION
Porcine Reproductive and Respiratory Syndrome (PRRS)
is considered one of the most important diseases in the swine
industry. Although several studies have been carried out to
elucidate the host immune response evoked against PRRS virus
(PRRSV) (Yoon et al., 1995; Loemba et al., 1996; Shimizu et
al., 1996; Bautista and Molitor, 1997; Wills et al., 1997a, 1997b;
Albina et al., 1998a, 1998b; Kawashima et al., 1999; LópezFuentes et al., 1999; Allende et al., 2000; Samson et al., 2000;
Lamontagne et al., 2001, 2003; Meier et al., 2003; Xiao et al.,
2004; Díaz et al., 2005), there are several aspects which still
remain unclear.
PRRSV is known to replicate mainly in porcine alveolar
macrophages (PAMs), macrophages in other tissues and in
minor extent in dendritic cells (Molitor et al., 1997; Bautista and
Molitor, 1999). However, the majority of the studies focused on
the immune response evoked after PRRSV infection have been
performed in serum and lung samples (Batista et al., 2004;
Xibao et al., 2010; Gómez-Laguna et al., 2010a) and in a lesser
185
Patogenia del PRRS/Pathogenesis of PRRS
extent in the lymphoid tissues (Rossow et al., 1996; Beyer et al.,
2000).
The production of cytokines is one of the tools used by
macrophages, and also by several other immune or non-immune
cells, in the defense against pathogens (Kumar et al., 2008). In
this
sense,
we
analyzed
the
in
situ
expression
of
proinflammatory cytokines and cytokines involved in the
immune response like IL-10, IL-12, IFN-α and IFN-γ in the
lymphoid organs of PRRSV-infected pigs to determine their
correlation with the expression of PRRSV antigen in lymphoid
organs and to evaluate their role in the pathogenesis of the
disease. Thus, PRRSV displayed a bimodal expression in the
tonsil and retropharyngeal lymph node with a first peak of
expression at 3 dpi and a second one at 14 dpi, whereas the
mediastinal lymph node had just a peak of expression at the
beginning decreasing onwards until the end of the experiment.
Interestingly, the expression of each cytokine studied was
different depending on the body compartment examined.
Wheras no expression of proinflammatory cytokines was
186
Patogenia del PRRS/Pathogenesis of PRRS
observed in the tonsil, wich may be related with the PRRSV
persistence in the tonsil of infected pigs (Beyer et al., 2000;
Wills et al., 2003), as well as with the increased susceptibility to
secondary pathogens reported by other authors (Wills et al.,
2000; Thanawongnuwech et al., 2000, 2004). Moreover, an
enhancement was observed on TNF-α and IL-1α levels in the
mediastinal
lymph
node
and
IL-6
expression
in
the
retropharyngeal lymph node. This finding point to a differential
behaviour of PRRSV in the lymphoid organs, which may be
related with the lack of a robust host immune response evoked
against the virus. Additionally, proinflammatory cytokines are
able to modulate the expression of CD163, a hemoglobin
scavenger receptor which acts as a PRRSV receptor and is
involved in viral uncoating (Van Gorp et al., 2008). Thus, the
imbalance between these cytokines in lymphoid organs may
play a role in the susceptibility to PRRSV replication.
Besides proinflammatory cytokines, other cytokines have
been reported to play a significant role in the pathogenesis of
PRRS. PRRSV has been hypothesized to evade the local
187
Patogenia del PRRS/Pathogenesis of PRRS
immune response by inducing the expression of IL-10, which
may in turn reduce the levels of cytokines involved in viral
clearance such as IFN-α, IFN-γ, IL-12p40 and TNF-α (GomezLaguna
et
al.,
2010a).
Although
the
expression
of
proinflammatory cytokines was different depending on the body
compartment examined, the expression of IL-10, IL-12p40,
IFN-α and IFN-γ, was quite similar in all the lymphoid organs
studied, despite the amount of PRRSV detected was different in
each one. This finding suggests that the adaptative immune
response evoked against PRRSV is more homogeneous than the
proinflammatory response. Although IFNs and IL-12 have an
antiviral activity, IL-10 may reduce the levels of these cytokines
involved in viral clearance (Mitchell and Kumar, 2004). In our
study the amount of IL-10 expressed was mild to moderate
being observed a higher expression of antiviral cytokines.
Contrary, the IL-10/IFN-γ ratio previously described in the lung
by Gómez-Laguna et al. (2010a) was proportionally higher,
which was related to the viral persistence in the lung of PRRSV
infected pigs, considering the different expression of IFNs, IL10 and IL-12 in lymphoid organs as indicative that the PRRSV
188
Patogenia del PRRS/Pathogenesis of PRRS
may be able to impair the host immune response in the lymphoid
organs by a different mechanism than the one described in the
lung. The expression of IFNs in the lymphoid organs of PRRSV
infected pigs together to the viral persistence at the end of the
study, indicates that the IFN signaling cascade may not be
working properly. In this sense, non structural protein 2 (Nsp2)
of PRRSV has been shown to inhibit the induction of IFN
regulatory factor 3 (Li et al., 2010), but no role of PRRSV has
been reported on the IFN signaling pathway. Thus, further
studies are being conducted to determine the role of PRRS on
IFN signaling cascade.
On the other front, viral infections are known to modulate
apoptosis phenomena inducing either an up-regulation or a
down-regulation of such process as a mechanism of evasion of
the host immune response (Thomson, 2001; Costers et al.,
2008). Apoptosis is a strictly regulated mechanism of cell death
that involves a complex network of biochemical pathways.
Whether a cell undergoes apoptosis or not depends on a delicate
balance
of
anti-
and
pro-apoptotic
stimuli.
Apoptosis
189
Patogenia del PRRS/Pathogenesis of PRRS
phenomena have been reported during PRRS being linked to
both viral particles (Suárez et al., 1996a; Costers et al., 2008)
and the expression of cytokines (Choi et al., 2002). All
proinflammatory cytokines analyzed in our study may be
involved in the apoptotis phenomena. TNF-α participates in the
extrinsic pathway of apoptosis, whereas IL-1 and IL-6 plays a
role in the intrinsic pathway acting as pro- and anti- apoptotic
mediators, respectively (Pollock et al., 2003; Garcia-Tunon et
al., 2005). Additionally, some authors described IL-10 as proapoptotic mediator (Estaquier et al., 1997; Liu et al., 2001). In
this sense, we evaluated apoptosis phenomena by microscopic
examination and by cleaved caspase 3 expression and the
TUNEL technique in lymphoid tissues, finding more evidences
of apoptotic phenomena in the mediastinal lymph node than in
the tonsil, where the expression of pro-apoptotic cytokines was
very poor. In addition, in both, tonsil and mediastinal lymph
node, apoptotic phenomena were described after microscopic
examination throughout the study, without a correlation with the
expression of cleaved caspase 3, suggesting that PRRSV in our
study induces apoptosis by a caspase 3 independent pathway.
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Patogenia del PRRS/Pathogenesis of PRRS
Among caspase 3-independent pathways, the generation of
reactive oxygen species (ROS) (Thayyullathi et al., 2008), the
imbalance of the Bcl-2/Bax ratio (Untergasser et al., 2001) and
the upregulation of FasL (Alvarez et al., 2011), have been
reported as possible mechanisms for the induction of apoptosis.
Specifically, Alvarez et al. (2011) reported that TNF-α induced
an enhancement of FasL and cell death by the release of
cytochrome c from the mitochondria, leading to caspase 9
activation and a caspase 3-independent apoptotic pathway.
Moreover, Chang et al. (2007) described an increase of FasL
capable
of
inducing
Fas/FasL
mediated
apoptosis
in
lymphocytes in pigs with PRRSV infection. Therefore, further
studies should be needed to determine the apoptosis mediators
used by PRRSV to induce apoptosis by a caspase 3 independent
pathway.
In this sense, we determine the ideal fixative and antigen
retrieval method in porcine paraffin embedded tissues for the
immunohistochemical detection of apoptosis mediators, from
both extrinsic and intrinsic pathways. The best fixative and
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Patogenia del PRRS/Pathogenesis of PRRS
antigen retrieval method for the antibodies against caspase 8 and
Fas (extrinsic pathway of apoptosis) were Zinc salt fixative and
Citrate microwave unmasking technique. To determine the
intrinsic pathway the ideal fixative and antigen retrieval method
for each antibody was as follows: for caspase 9, fixation in Zinc
salt solution and antigen retrieval with Tween 20; for Bcl-2 and
iNOS the optimal immunolabelling was observed in 10 %
neutral buffered formalin fixed samples and with Proteinase K
and
Citrate
microwave
as
antigen
retrieval
methods,
respectively. The satisfactory results obtained for a specific
immunolabelling in porcine tissues point to a potential use of
these antibodies in future studies.
In conclusion, PRRSV in our experimental study induces a
different expression of cytokines and apoptotic phenomena in
lymphoid organs, wich constitute an useful tool to deep in the
knowledge of the pathogenesis of PRRSV. Furthemore, our
results suggest that PRRSV could follow a caspase 3
independent pathway to induce apoptosis in lymphoid organs,
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Patogenia del PRRS/Pathogenesis of PRRS
wich give new fields of action to elucidate the mechanism used
by
the
virus
to
induce
these
phenomena.
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Patogenia del PRRS/Pathogenesis of PRRS
CONCLUSIONES/ CONCLUSIONS
194
Patogenia del PRRS/Pathogenesis of PRRS
CONCLUSIONES
1. La cepa 2982 (genotipo europeo) del virus del Síndrome
Reproductivo y Respiratorio Porcino es capaz de evadir la
respuesta inmune induciendo un desequilibrio en la
expresión de citoquinas proinflamatorias a nivel de los
órganos linfoides.
2. La ausencia de expresión de citoquinas proinflamatorias en
la tonsila de cerdos infectados con la cepa 2982 del virus
del
Síndrome
Reproductivo
y
Respiratorio
Porcino
desempeña un papel importante en la persistencia del virus.
3. La cepa 2982 del virus del Síndrome Reproductivo y
Respiratorio Porcino modula la respuesta de IFNs, IL-10 e
IL-12 en los órganos linfoides de manera distinta a la
descrita previamente en el pulmón de cerdos infectados con
esta misma cepa.
4. La expresión de IFNs junto con niveles elevados del virus
del Síndrome Reproductivo y Respiratorio Porcino en
órganos linfoides de cerdos infectados experimentalmente
con la cepa 2982 indican que la vía de señalización de los
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Patogenia del PRRS/Pathogenesis of PRRS
IFNs no se desarrolla de manera eficaz en nuestro modelo
experimental.
5. El desarrollo de los fenómenos de apoptosis desarrollados a
lo largo de la infección con la cepa 2982 del virus del
Síndrome Reproductivo y Respiratorio Porcino coincidió
con una mayor expresión de citoquinas pro-apoptóticas.
6. En la infección con la cepa 2982 del virus del Síndrome
Reproductivo
y
Respiratorio
Porcino
se
producen
fenómenos de apoptosis, en los órganos linfoides, por vías
independientes de la caspasa 3.
7. El formol tamponado al 10% y las sales de Zinc, como
fijadores, y la utilización de Tween 20, tampón citrato y
proteasa tipo XIV, como métodos de desenmascaramiento
antigénico, representaron las combinaciones de elección
para el estudio de los fenómenos de apoptosis en muestras
de tejidos porcinos incluidas en parafina.
196
Patogenia del PRRS/Pathogenesis of PRRS
CONCLUSIONS
1.- The strain 2982 (European genotype) of Porcine
Reproductive and Respiratory Syndrome virus is able to evade
the immune response by inducing an imbalance in the
expression of proinflammatory cytokines in lymphoid organs.
2.- The absence of proinflammatory cytokines expression in
the tonsil from pigs infected with the strain 2982 of Porcine
Reproductive and Respiratory Syndrome virus plays an
important role in the persistence of the virus.
3.- The strain 2982 of
Porcine Reproductive and
Respiratory Syndrome virus modulates the response of IFNs, IL10 and IL-12 in lymphoid organs by a different mechanism than
the one described in the lung of pigs infected with the same
strain.
4.- The expression of IFNs, together with the persistence of
Porcine Reproductive and Respiratory Syndrome virus in the
lymphoid organs of pigs experimentally infected with the strain
2982, indicates that the IFN signaling cascade may not be
working properly in our experimental model.
197
Patogenia del PRRS/Pathogenesis of PRRS
5.- The development of apoptotis phenomena throughout an
infection with the strain 2982 of Porcine Reproductive and
Respiratory
Syndrome
virus
coincided
with
increased
expression of pro-apoptotic cytokines.
6.- During an infection with the strain 2982 of Porcine
Reproductive and Respiratory Syndrome virus the apoptotic
phenomena in lymphoid organs are produced by caspase 3
independent pathways.
7.- Zinc salts and 10 % neutral buffered formalin, as
fixatives, and the use of Tween 20, citrate microwave and
protease type XIV, as antigen retrieval method, represented the
combination of election to study the apoptosis phenomena in
porcine paraffin embedded tissues.
198
Patogenia del PRRS/Pathogenesis of PRRS
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Patogenia del PRRS/Pathogenesis of PRRS
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CURRICULUM VITAE
241
Patogenia del PRRS/Pathogenesis of PRRS
El último estudio experimental de esta tesis, ha sido publicado
este año en la revista científica “Veterinary immunology and
immunopathology” volumen 139, páginas 210-216, estando
actualmente siendo revisados el resto de los estudios
experimentales en revistas indexadas pertenecientes al campo de
las ciencias veterinarias. Los resultados obtenidos de estos
trabajos han sido presentados como comunicaciones en
congresos nacionales e internacionales tal y como se indica a
continuación:
• Barranco I, Gómez-Laguna J, Rodríguez-Gómez IM, Salguero
FJ, Pallarés FJ, Carrasco L, Bernabé A. Role of cytokines as
mediators of the local immune response in lung and tonsil of
pigs inoculated with a european PRRSV field isolate.
Presentado como comunicación tipo oral, en el 28 Meeting of
the European Society of Veterinary Pathology and European
Collage of Veterinary Pathologists, celebrado en Belgrado,
Serbia, del 8 al 11 de Septiembre de 2010.
• Rodríguez-Gómez IM, Gómez-Laguna J, Barranco I, Salguero
FJ, Pallarés FJ, Carrasco L, Ramis G. The role of antigen
presenting cells and T lymphocytes in the tonsil of Porcine
Reproductive and Respiratory Syndrome virus-infected
pigs. Presentado como comunicación tipo oral, en el 28 Meeting
of the European Society of Veterinary Pathology and European
Collage of Veterinary Pathologists, celebrado en Belgrado,
Serbia, del 8 al 11 de Septiembre de 2010.
242
Patogenia del PRRS/Pathogenesis of PRRS
• Gómez-Laguna J, Rodríguez-Gómez IM, Barranco I, Pallarés
FJ, Salguero FJ, Bernabé A, Carrasco L. IL-10 and TGF-β
expresión in the lung and tonsil of pigs experimentally
infected with porcine reproductive and respiratory síndrome
virus. Presentado como comunicación tipo oral, en el XXI
Internacional Pig Veterinary Society (IPVS) Congress,
celebrado en Vancouver, Canadá, del 18 al 21 de Julio de 2010.
• Barranco I, Gómez-Laguna J, Rodríguez-Gómez IM, Salguero
FJ, Pallarés FJ, Bernabé A, Carrasco L. Useful markers to
study extrinsic and intrinsic pathways of apoptosis in
porcine paraffin-embedded tissues. Presentado como
comunicación tipo póster, en el XXI Internacional Pig
Veterinary Society (IPVS) Congress, celebrado en Vancouver,
Canadá, del 18 al 21 de Julio de 2010.
• Rodríguez-Gómez IM, Gómez-Laguna J, Barranco I, Salguero
FJ, Pallarés FJ, Ramis G, Carrasco L. Estudio de las
poblaciones celulares inmunocompetentes en la tonsila de
cerdos infectados con el virus del Síndrome Reproductivo y
Respiratorio Porcino. Presentado como comunicación oral, en
la XXII Reunión de la Sociedad Española de Anatomía
Patológica Veterinaria, celebrada en Valencia, del 16 al 18 de
Junio del 2010.
• Barranco I, Gómez-Laguna J, Rodríguez-Gómez IM, Salguero
FJ, Pallarés FJ, Quereda JJ, Carrasco L. Expresión de
citoquinas inmunomoduladoras en tonsila de cerdos
infectados con el virus del Síndrome Reproductivo y
Respiratorio Porcino. Presentado como comunicación oral, en
la XXII Reunión de la Sociedad Española de Anatomía
Patológica Veterinaria, celebrada en Valencia, del 16 al 18 de
Junio del 2010.
• Barranco I, Gómez-Laguna J, Rodríguez-Gómez IM, Salguero
FJ, Pallarés FJ, Bernabé A, Carrasco L. Determinación del
fijador y desenmascaramiento antigénico ideal para la
detección inmunohistoquímica de diferentes marcadores de
243
Patogenia del PRRS/Pathogenesis of PRRS
las vías extrínseca e intrínseca de la apoptosis. Presentado
como comunicación tipo póster, en la XXII Reunión de la
Sociedad Española de Anatomía Patológica Veterinaria,
celebrada en Valencia, del 16 al 18 de Junio del 2010.
• Barranco I, Gómez-Laguna J, Rodríguez-Gómez IM, Salguero
FJ, Pallarés FJ, Bernabé A, Carrasco L. Mecanismos de
inducción de apoptosis por el virus del Síndrome
Reproductivo y Respiratorio Porcino. Presentado como
comuniación tipo Oral en I Congreso Científico de
Investigadores en Formación, Celebrado en Córdoba, España del
5 al 16 de Octubre del 2009
• Barranco I, Gómez-Laguna J, Rodríguez-Gómez IM, Salguero
FJ, Pallarés FJ, Bernabé A, Carrasco L. Evaluation of the
apoptotic phenomenon and expression of proinflammatory
cytokines in the tonsil of pigs infected with a european
PRRSV field isolate. Presentado como comuniación tipo Oral
en el XXVII Meeting of the European Society of Veterinary
Pathology and European Collage of Veterinary Pathologists,
Celebrado en Olsztyn-kraków, Poland del 9 al 12 de Septiembre
del 2009
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Patogenia del PRRS/Pathogenesis of PRRS
AGRADECIMIENTOS
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Expreso mi agradecimiento a todos los miembros del
Departamento de Anatomía y Anatomía Patológica Comparadas,
que han hecho posible la realización de esta Tesis Doctoral y,
en especial, a los directores de este trabajo, el Dr. D. Librado
Carrasco Otero y el Dr. D. Jaime Gómez-Laguna, por su
rigurosidad científica, su experiencia investigadora, apoyo
constante y mucho más que una inestimable ayuda y confianza
en el desarrollo de la tesis.
A todas aquellas personas que de una u otra manera han
contribuido a que este trabajo se haya realizado.
Y por su puesto a mi familia y amigos, por su paciencia,
apoyo constante y saber estar ahí en los buenos y malos
momentos
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Patogenia del PRRS/Pathogenesis of PRRS
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