Study On Duck Blood-Spleen Barrier And The Mechanism Of DTMUV Invading Duck Spleen

As the primary site for immunocyte proliferation and differentiation,the spleen,which is part of the blood circulatory system,is the largest secondary lymphoid organ in birds and plays an important role in resisting circulating pathogens as a haematopoietic organ,which depends upon the immunological barrier present in the spleen.Immunological barrier as"first line of defense" of the body is a physiological structure that can protect foreign materials from entering the body or parts of the body.The barrier in the spleen,called the blood-spleen barrier(BSB),is a filtration bed located between arterial and venous vessels,which was initially identified through plasmodium research in mammals.The location of the BSB in mammals was found in the splenic marginal zone between white pulp and red pulp and is composed of endothelial cells,macrophages,reticular tissue,and fibers.The avian spleen has no marginal zone,while has periellipsoidal lymphatic sheaths(PELS)in the white pulp,that are thought to be functionally similar to the marginal zone of mammals.The previous research in our lab showed that the BSB of chicken is located in ellipsoid and PELS.However,BSB plays an important role in immune response.The sensitivity and immune response of ducks to various viruses are different from that of chickens.The morphology and immune mechanism of BSB have not been reported in duck as a common waterfowl.In April 2010,an infectious disease of egg-laying ducks outbroke in the region of southeastern coastal provinces of China,which could cause symptoms of retarded growth,the decline of egg production and death.Ovary hemorrhage and follicle rupture in egg-laying ducks are the main pathological changes of this disease that is called the duck hemorrhagic ovaritis.It was found that this disease was caused by a novel type of flavivirus infection,named as duck Tembusu virus(DTMUV).At present,most of the studies on DTMUV are focused on the aetiological research of the virus.However,the research on the pathogenic mechanism of the virus to ducks is still lacking.The molecular pathogenesis is still not entirely clear.The previous studies have reported that DTMUV accumulates firstly into the spleen after invading into body and causes obvious lesions.In this study,the existence and structure feature of the duck BSB were first identified by morphological methods.In addition,XZ-2012 virus strain was used to establish model,and the distribution rule in the spleens of adult egg-laying shelducks was investigated.The pathological injury of spleen and barrier caused by virus was detected by morphological methods.The expression change of the relevant genes and proteins were studied by transcriptome sequencing(RNA-seq)technique to reveal the molecular mechanism of the virus invading the spleen and immune response.The outcome of this study would provide a theoretical basis for avian immunological mechanism and prevention of Flavivirus diseases.Experiment I Identification and structural characteristics of the BSB in ducks To identify the existence and composition of the BSB in ducks,the microanatomical structures of the spleen were investigated by intravenous injection of ink,using microtomy,silver staining,enzymatic histochemistry technique and transmission electron microscopy.The results showed that the white pulp in duck spleen consists of ellipsoids,PELS,splenic nodules and periarterial lymphatic sheaths(PALS),and there was no presence of marginal zone.Splenic nodules are located at the beginning of the central artery,which was surrounded by the PALS.The sheathed capillary had cuboidal-shaped endothelial cells,surrounded by the ellipsoid,which is similar to high endothelial venue(HEV).The splenic ellipsoid was formed by reticular cells.However,ellipsoid-associated cells were not obvious.PELS was showed more than PALS.At the different time point of ink injection(10min,30min,3h,6h,9h,15h,24h,36h,2d,3d,5d),the results showed that carbon particles were restricted around the sheathed capillary at beginning,then were trapped to the PELS.With the time course,the carbon particles were appeared around central artery and splenic nodules.The number of the carbon particles was decreasing.Reticular fibers were densely distributed in basement membrane of the sheathed capillary endothelium and the border between red pulp and PELS.The enzymatic histochemistry results showed that macrophages were mainly distributed at the border between red pulp and PELS.These results suggested that the BSB resisted carbon particles around sheathed capillaries as a mechanical barrier at the beginning of carbon particles invasion,then as a biological barrier of phagocytosis,carbon particles were phagocytosed by macrophages and bedetained at the border of PELS and red pulp,and moved in red pulp and PALS,contributing to antigen information transfer for the specific immunity.In conclusion,the BSB was found in duck spleen for the first time,which was a reticular structure between the arterial and venous vessels,including cubic endothelial cells,reticular cells,ellipsoid-associated macrophages,reticular fibers and protecting the spleen from invasion of circulating pathogens.The research of blood-spleen barrier in ducks provides theoretical foundation for avian immune mechanism and epidemic prevention.Experiment ? Dynamic distribution characteristics of DTMUV invading the duck spleen To observe whether BSB block the invasion of DTMUV,the isolated and purified DTMUV strain XZ-2012 was used as a strain model,to intramuscularly inject the six-month egg-laying shelducks with the infective dose of 104TCID50.The dynamic distribution of the virus in spleen at different time post-infection(pi)was studied using RT-PCR,RT-qPCR,ELISA,immunofluorescence and transmission electron microscopy(TEM).The results showed that the virus occurred in the spleen after 2 hpi and lasted up to 18 dpi.The registered viral load increased from 2 hpi to 3 dpi,and then it diminished from 6 dpi to 18 dpi with a slight rise at 12 dpi.From 2 hpi to 6 dpi the positive reactions were mostly distributed in the PELS of spleen white pulp,few being found in the sheathed capillary.From 9 dpi to 18 dpi,the positive reactions were migrating into PALS around the central artery through the red pulp.Under TEM,the virus particles could be observed mostly in lymphocytes and macrophages.It was suggested that DTMUV invaded the spleen at 2 hpi and replicated significantly from 1 dpi to 3 dpi,being eliminated from 9 dpi to 18 dpi.This is the first study on the dynamic distribution of DTMUV from invasion to elimination in duck spleen conducted by molecular and morphological methods.It could provide theoretical basis for the occurrence,development and detoxification of the virus in the organs of the immune system.Experiment ? Pathological study of BSB and its surrounding tissue caused by DTMUV infecting duck spleen To observe the pathological change of spleen tissue and the change of barrier structure caused by DTMUV invading duck blood-spleen barrier,104 TCIDso dose of DTMUV XZ-2012 strain was intramuscularly injected into 6-month-old egg-laying ducks divided into seven different post-infection(pi)times(2h,12h,1d,3d,6d,9d,18d).The pathological changes of the spleen were observed by HE staining,ink injection,reticular fiber staining and TEM.The infected spleens showed the different degrees of status marmoratus,especially at 12hpi-3dpi.The results of HE staining showed that the normal duck spleen consisted of red pulp and white pulp which included ellipsoid and PELS,PALS and splenic nodules.From 2hpi to 12hpi,there were slight vacuolar degeneration at the border between ellipsoid and PELS and on the wall of central artery in duck spleen.At 1dpi,there were more red blood cells in spleen tissue and more vacuolar degeneration between ellipsoid and PELS.At 3dpi,the inflammatory area around the ellipsoid was enlarged and the inflammation was enhanced.At 6dpi,the number of lymphocytes in the spleen was increased,and the tissue around the ellipsoid began to recover.At 9dpi,there were much hemosiderin around the ellipsoid.At 18dpi,hemosiderin migrated to the periphery of the splenic nodules through the red pulp.Ultrastructural images showed that the endothelial cells of sheathed capillary the spleen were loosely arranged after infection compared with the normal spleen.The cells and their mitochondria,endoplasmic reticulum were obvious swelling.The number of carbon particles blocked by ellipsoid in the infected spleen was decreased significantly in the injected groups.The number of carbon particles in the ellipsoid increased from 3 dpi to 6dpi,and the carbon particles in the PELS were dispersed,then recovered gradually.Reticular fibers correspond to blocking carbon particles.In conclusion,DTMUV might enter into the spleen through endocytosis,cause lymphocyte and macrophage edema and damage of blood-spleen barrier structure.At the later stage of infection,inflammatory reaction was weakened,lymphocyte increase,spleen corpuscle proliferation,ellipsoid-associated macrophages the function of phagocyte and barrier restored.This study provides a theoretical basis for the pathogenicity of flavivirus and the destruction of the immune barrier structure.Experiment ? Expression dynamic of immune-related genes in spleen of DTMUV invading duck spleen based on RNA-seq technology Six-month egg-laying shelducks were intramuscularly injected with DTMUV strain XZ-2012.Using Illumina HiSeq 2500 sequencing,RNA-Seq sequencing and differential expression genes(DEGs)analysis were performed on duck spleen samples of normal and seven groups(2h,12h,1d,3d,6d,9d,18d)at different infected times.The expression patterns of key genes and proteins were verified by RT-qPCR and Western blot.The results showed that a total of 16,127 genes(14,165 known genes and 1,962 new genes)were identified from eight libraries.According to the Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis and weighted co-expression network analysis(WGCNA),17 barrier-related key genes in 5 significant enrichment pathways and 23 immune-related key genes in 6 significant enrichment pathways were screened,respectively.The expression levels of RIG-? like receptors(RLRs),downstream IRF7 and proinflammatory cytokines IL-6 from RIG-I signaling pathway were upregulated inapparently at 2 hpi and apparently at 3 dpi,while the expression of MHC-? and barrier-related factors regulating tight junctions and reticular fibers were obviously downregulated at 2 hpi.The expression levels of downstream antiviral cytokines type-? IFNs,anti-inflammatory cytokines IL-10,cell adhesion molecules(CAMs),chemokines and their receptors associated with lymphocyte homing were significantly upregulated at 3 dpi.The population of lymphocytes was increased at 6 dpi.The immune function of spleen was recovered starting from 9 dpi.The findings of this study suggest that DTMUV invaded into the spleen via RIG-I signaling pathway and enhanced immune evasion by inhibiting MHC-? expression during the early stage of infection.Additionally,DTMUV induced PELS lesions through activating IL-6 expression.Similarly,DTMUV increased the expression levels of RLRs,antiviral type-? IFNs,lymphocyte homing-related genes and proteins as well as the number of lymphocytes in the infected duck spleen.Taken altogether,these findings provide new insights into the study of cellular and molecular mechanisms of DTMUV infection spleen in duck vivo.