Estudio de la respuesta inmune, innata y adquirida, en equinos infectados con el Virus de la Anemia Infecciosa Equina

Abstract

El Virus de la Anemia Infecciosa Equina (VAIE) ocasiona una enfermedad persistente en equinos, que culmina en una prolongada fase asintomática, evidenciando un estado de equilibrio permanente entre la infección viral y el sistema inmune del animal. A fin de analizar la participación de receptores y citoquinas en la respuesta inmune de equinos que cursan esta etapa de la infección, se investigó, mediante transcripción reversa seguida de PCR semicuantitativa, la expresión de receptores y citoquinas en células mononucleares de sangre periférica (PBMC). Por otra parte, mediante inmunohistoquímica se estudió la expresión de dichas moléculas en hígado y bazo, órganos considerados reservorio viral. Se emplearon, además, estudios de estimulación in vitro de células dendríticas para evaluar la capacidad de los receptores de la familia TLRs, de reconocer secuencias conservadas del VAIE y finalmente se estudió la secreción de citoquinas y la expresión de receptores en PBMC, en respuesta al estímulo con secuencias virales conservadas. Los resultados mostraron niveles basales de IFN-alfa disminuidos en PBMC de equinos VAIE(+), mientras que en órganos reservorio presentan niveles disminuidos de IFN-gama, sugiriendo la presencia de mecanismos regulatorios que limitan la expresión de citoquinas antivirales. Los estudios de estimulación in vitro mostraron grandes variaciones en los perfiles de citoquinas entre los equinos asintomáticos. Estos resultados evidencian la complejidad del estudio de infecciones lentivirales en especies no endocriadas y concuerdan con investigaciones realizadas en otras infecciones retrovirales, respecto de los perfiles de citoquinas, la variabilidad entre individuos y su relación con la progresión de la enfermedad.

The Equine Infectious Anemia Virus (EIAV) causes persistent disease in horses that is characterized by lethargy and chronic anemia. Equine Infectious Anemia (EIA) has a cyclical and dynamic evolution, in which the animals remain free of clinical symptoms but become life-long EIAV carriers. A key role in controlling viral replication at this stage of infection was assigned to the immune system. To examine the role of receptors and cytokines in horse's immune response during this stage of infection, reverse transcription followed by semiquantitative PCR was performed in peripheral blood mononuclear cells (PBMC). Moreover, the expression of such molecules in viral reservoir organs such liver and spleen, was studied by immunohistochemistry. On the other hand, in vitro stimulation of dendritic cells was done in order to evaluate the ability of Toll-Like Receptors family, to recognize conserved sequences of EIAV. Finally, cytokines and receptor expression in PBMC in response to specific stimulation with conserved viral sequences was evaluated. The results showed decreased levels of IFN-alpha in PBMC of EIAV(+) horses, while decreased levels of IFN-gamma in reservoir organs were found, suggesting the presence of regulatory mechanisms that limit the expression of antiviral cytokines. PBMC in vitro stimulation studies showed large variations in cytokine profiles among asymptomatic equines. These results show the complexity of the study of lentiviral infections in not inbred species and are consistent with research conducted in other retroviral infections, regarding cytokine profiles, variability between individuals and their relationship to disease progression.The Equine Infectious Anemia Virus (EIAV) causes persistent disease in horses that is characterized by lethargy and chronic anemia. Equine Infectious Anemia (EIA) has a cyclical and dynamic evolution, in which the animals remain free of clinical symptoms but become life-long EIAV carriers. A key role in controlling viral replication at this stage of infection was assigned to the immune system. To examine the role of receptors and cytokines in horse's immune response during this stage of infection, reverse transcription followed by semiquantitative PCR was performed in peripheral blood mononuclear cells (PBMC). Moreover, the expression of such molecules in viral reservoir organs such liver and spleen, was studied by immunohistochemistry. On the other hand, in vitro stimulation of dendritic cells was done in order to evaluate the ability of Toll-Like Receptors family, to recognize conserved sequences of EIAV. Finally, cytokines and receptor expression in PBMC in response to specific stimulation with conserved viral sequences was evaluated. The results showed decreased levels of IFN-alpha in PBMC of EIAV(+) horses, while decreased levels of IFN-gamma in reservoir organs were found, suggesting the presence of regulatory mechanisms that limit the expression of antiviral cytokines. PBMC in vitro stimulation studies showed large variations in cytokine profiles among asymptomatic equines. These results show the complexity of the study of lentiviral infections in not inbred species and are consistent with research conducted in other retroviral infections, regarding cytokine profiles, variability between individuals and their relationship to disease progression.