Pathogenesis Of Chicken Immunological Tolerance Induced By Avian Leukosis Virus Subgroup J

Avian leukosis virus subgroup J(ALV-J)is a tumorigenic retrovirus that is clinically induced to cause myeloid leukemia and other various types of neoplastic diseases in broilers and laying hens.Since the virus was discovered in 1991,ALV-J has been widely found in various flocks around the world and has been experiencing intermittent outbreaks.It has caused a devastating blow to the chicken industry worldwide.At present,ALV-J infection in various breeds of chickens in China is quite common,which has brought huge economic losses to the poultry breeding industry.In addition to causing a decline in the performance of the chicken and inducing tumors,a more serious but easily overlooked hazard is that ALV-J infection can cause severe immunological tolerance in infected chickens.Moreover,immunological tolerance is a necessary condition for tumor formation and opportunistic infections.Regrettably,little is known about the underlying causes and molecular mechanism of ALV-J-induced immunological tolerance.Therefore,elucidating the pathogenesis of ALV-J inducing host immunological tolerance is of practical and important significance for the prevention and control of ALV-J.This study established an experimental model of ALV-J indused immunological tolerance,from the perspective of developmental,cytological,immunological and molecular biology to determine the immune status of ALV-J infected chicken and its internal relationship with immunological tolerance.Using the cutting-edge technology of modern life sciences to detect and analyze the differential expression of related proteins in ALV-J infected cells,to clarify its role in B cell development and function-related signaling pathways,and to analyze the molecular mechanism of ALV-J indused immunological tolerance.In order to study the difference of immunological tolerance induced by congenital infection and acquired infection,this study established an experimental model by intra-injection of ALV-J to simulate congenital infection and chick injection of ALV-J to simulate acquired infection.The study found that ALV-J intraembryonic infection can cause persistent viremia in infected chickens but no antibody response(ie,immunological tolerance),while only a part of chickens infected with ALV-J showed immunological tolerance.ELISA results showed that the levels of total immunoglobulin(Ig M and Ig G)in the blood of immune-tolerant chickens were significantly reduced.The number and developmental dynamics of Ig M+ and Ig G+ B cells in immune organs of immune-tolerant chickens were further detected by immunohistochemistry.The activation and proliferation of B cells from infected chicken spleen after stimulation by thymus independent antigen lipopolysaccharide(LPS)were detected and evaluated.The results showed that the ability of B cells to develop and differentiate into Ig M+ and Ig G+ antibody-producing cells(especially Ig G-type cells)was severely inhibited in immune-tolerant chickens,the activation and expansion of B cells in splenic germinal center were also inhibited after mitogen stimulation.In order to understand the difference of immune response to specific antigen stimulation between chickens infected with ALV-J congenitally and acquired,the titers of Ig M-type and Ig G-type antibodies produced by the host responding to the stimulation of sheep red blood cells(SRBC)and attenuated Marek's disease virus(MDV)type 1 vaccine were further tested.The results showed that the titers of Ig M-type and Ig G-type antigen-specific antibodies produced by congenitally infected chickens decreased significantly.However,although the titers of Ig G-type antigen-specific antibodies in acquired infected chickens decreased significantly,the titers of Ig M-type antigen-specific antibodies were basically normal.These data suggested that the acquired infection of ALV-J had little effect on the early development of B cells in chickens.In conclusion,ALV-J congenital infection inhibits the development and maturation of B cells,and inhibits the production of immunoglobulin and the ability of immunoglobulin class switch recombination.To further explore the cytological mechanism of ALV-J congenital infection-induced immunological tolerance,the developmental dynamics of bursa B cells from chicken embryo to chicken were examined.The results of cell morphology showed that the bursa of Fabricius was badly developed and the lymphoid follicle could not form obvious medulla and cortex in chickens infected with ALV-J in embryonic stage,which suggested that these lymphoid follicles were in a naive state.However,in chickens infected with ALV-J after hatching,most of the lymphoid follicles can form the cortex although the development of bursa of Fabricius were also inhibited.These results demonstrated that ALV-J congenital infection has seriously impactd the development of B cells in bursa of Fabricius.Flow cytometry analysis further found that ALV-J intra-embryonic infection resulted in higher B cell infection rate(about 27%),and the result of immunohistochemistry showed that the volume of gp85 antigen-positive B cell and the ratio of nuclear to cytoplasm were large,and these cells were mostly in early development stage.The above results indicated that the congenital infection of ALV-J leads to the late developmental inhibition of embryonic B cells,and thus impaired the ability of B cells to produce immunoglobulin.Flow cytometry analysis confirmed that ALV-J blocked the differentiation of CD117+ progenitor B cells in bursa of Fabricius.The data from vitro experiments further confirmed that ALV-J inhibited the proliferation,immunoglobulin gene class switch recombination of CD117+ B cells in vitro after stimulated by LPS and IL-4.Comprehensive analysis of the above-mentioned in vitro and in vivo experimental data suggested that ALV-J-induced immunological tolerance is closely related to B cell developmental resistance and dysfunction,and the key reason for the occurrence of this "anergy" state of B cells is that ALV-J selectively acted the early development of B cells and their further inhibited the development and maturation.Related proteomic analysis for cells infected with ALV-J showed that ALV-J altered the protein expression level of tyrosine kinase Lyn in DF-1 cells.Lyn is a core factor in the B cell signaling pathway of B cells,which is involved in biological processes related to B cell proliferation,differentiation,maturation or tolerance.Therefore,to clarify the molecular pathogenesis of B cell anergy induced by ALV-J,this study further analyzed the effect of ALV-J on the expression of Lyn in B cells and its effect on BCR signaling pathway.The results of western-blot and immunohistochemistry showed that the expression level of Lyn in bursal cells have increased in the chickens infected with ALV-J in embryonic stage.In vivo experiments showed that the expression of Lyn in B cells infected by ALV-J was abnormal.This study further investigated the effect of ALV-J on Lyn in BCR signal transduction in vitro.By laser confocal microscopy,it was found that the gp85 protein was mainly distributed in the cytoplasm after ALV-J infection of B cells,and some of the B cells had positive signals of gp85 in the cytoplasm and nucleus.The results of q RT-PCR and Western-blot were consistent with the results of in vivo experiments,that is,ALV-J infection resulted in up-regulation of Lyn m RNA level and protein level in B cells.Lyn has both positive and negative regulation in BCR signal transduction,which depends on the activation level of different phosphorylation sites.To clarify the dominant role of Lyn in B cells of ALV-J infected group,the phosphorylation level of Lyn protein and its downstream direct substrate Syk protein after BCR signal activation were further detected.Flow cytometry analysis showed that when BCR signal was activated,the phosphorylation levels of Tyr397(activated positive regulation)and Tyr507(activated negative regulation)in infected B cells were increased to different extents.At this time,the phosphorylation level of Syk,the core kinase of BCR signal transduction and the direct substrate of Lyn,is particularly important.Flow cytometry analysis showed that the phosphorylation level of Syk decreased significantly at this time,suggesting that Lyn played a major role in inhibiting BCR signal transduction in B cells of infected group.Tto confirm this effect,we further interfered with Lyn in B cells by sh RNA and detected the phosphorylation level of related proteins.The results showed that the phosphorylation level of downstream protein Syk increased significantly after Lyn sh RNA interference on B cells of infected group.The above experimental data further indicateed that Lyn plays a inhibitory role in BCR signaling in infected B cells,resulting in the blockage of B cell signaling cascade in B cells.In summary,the immunological tolerance induced by ALV-J were associated with B-cell anergy.The underlying cause is that ALV-J blocked the development and differentiation of CD117+ progenitor B cells,the molecular pathogenesis of B-cell anergy was ALV-J activates the inhibitory effect of Lyn in BCR signaling.In conclusion,the pathogenesis of ALV-J inducing immunological tolerance was comprehensively analyzed from both cellular and molecular dimensions,which provided a theoretical basis for the prevention and control of avian leukemia and for revealing the mechanism of related viruses inducing immunological tolerance.