Effects Of Exosomes Mediated Peste Des Petits Ruminants Virus On The Expression Of Immune Related Cytokines In Goat Cells

Peste des petits ruminants(PPR),commonly known as sheep plague,is an acute viral infectious disease of goats,sheep and other small ruminants.PPR caused by Peste des petits ruminants virus(PPRV)belongs to the Morbolivirus of Paramyxoviridae.The disease is characterized by acute gastritis,bronchopneumonia,enteritis and abortion of pregnant ewes.At present,the disease has become an endemic disease in many countries,which directly restricts the development of sheep industry.PPRV has a strong phagocytic effect on lymphocytes and epithelial cells of infected host.The main pathogenic feature of PPRV is immunosuppression.Exosomes are membrane vesicles secreted by eukaryotic cells under both pathological and physiological conditions.They reach the surrounding target cells or remote target cells through paracrine and biological circulatory system,and transmit biological information to the target cells.Recent studies have shown that exosomes mediated virus escape or suppression of host immune system may be a key way of virus pathogenesis.Virus infection can induce host cells to secrete a large number of exosomes containing viral RNA or/ and cell-derived RNA and other proteins.These exosomes can be transferred to nearby or remote cells with blood circulation,etc.,and then regulate the gene expression level of these recipient cells.Peripheral blood mononuclear cells(PBMCs)play an important role in pathogen recognition and early innate immune response initiation.Goat PBMCs are the main cell models for studying the interaction between PPRV and infected host.Our previous studies showed that PPRV infection increased the exosomes release level of goats PBMCs,suggesting that exosomes are involved in the pathogenesis of PPRV in goats.The purpose of this study was to investigate the composition of exosomes from PBMCs infected with PPRV and the way of exosomes entering into goat maternal-fetal cells,and to further study the effect of co-culture of PBMCs derived exosomes infected with PPRV and recipient cells on the expression of immune related factors in cells.The research contents are as follows:1.Analysis of the components of exosomes derived from PPRV-infected goat PBMCs.The components of exosomes derived from PPRV-infected goat PBMCs were analyzed by deep sequencing technology and liquid chromatography tandem mass spectrometry.The deep sequencing data showed that,compared with the exosomes of the uninfected group,the mi RNA expression level has changed significantly,including 31 mi RNAs expression was significantly up-regulated,and the expression of 290 mi RNAs was significantly downregulated.KEGG analysis showed that the significantly enriched pathways include metabolic pathway(ko01100),cancer pathway(ko05200),PI3K-Akt signaling pathway(ko04151),endocytosis pathway(ko04144),MAPK signaling pathway(ko04010),Ras signaling pathway(ko04014),Rap1 signaling pathway(ko04015),focal adhesion(ko04510),actin cytoskeleton regulation(ko04810),c AMP signaling pathway(ko04024).Protein expression level analysis showed that the expression of 118 proteins was up-regulated and the expression of 33 proteins was down-regulated in the exosomes of the infected group,compared with the exosomes of the uninfected group.KEGG analysis showed that there was a total of 18 significantly enriched signaling pathways,of which the 6 most significantly enriched pathways included Th17 cell differentiation(chx04659),Th1 and Th2 cell differentiation(chx04658),endoplasmic reticulum protein processing(chx04141),Estrogen signaling pathway(chx04915),IL-17 signaling pathway(chx04657),necrotizing prolapses(chx04217).In addition,based on proteomics analysis and Western blot analysis,it is shown that PPRV-infected PBMCs-derived exosomes contain high levels of PPRV H protein,N protein and V protein.2.Analysis of the pathway of exosomes entering goat maternal-fetal cells.In order to determine the entry pathway of exosomes into goat cells,the exosomes from goat PBMCs were labeled with PKH-26 and co-cultured with goat maternal-fetal cells for different time(0h,6h,12 h,24h,48h).The results of laser confocal microscopy showed that the exosomes could enter into goat PBMCs,goat endometrial epithelial cells(EECs)and goat fetal fibroblasts(GEFs).In order to further study the specific pathway of exosomes entering into cells,we pretreated different cells with relevant entry pathway inhitibor drugs.Then detected the exosomes from PBMCs by IF and FC.The results showed that exosomes entered PBMCs mainly through the CME and CDE pathway;exosomes entered EECs mainly through the CME、CDE pathway and dynamin dependent pathway,and were also significantly affected by PPRV receptor protein Nectin-4;exosomes entered GEFs mainly through CDE pathway and dynamin dependent pathway;exosomes entering PBMCs,EECs and GEFs are all accompanied by the regulation of macropinocytosis by PI3 K.3.The effect of exosomes on the expression of immune related cytokines in the recipient cells.In order to determine the effect of exosomes derived from PPRV infected cells on the expression of immune related cytokines in goat recipient cells.The exosomes of PPRV infected goat PBMCs were extracted and co-cultured with PBMCs.The results of q PCR and Western blot data showed that the IFN-γ、TNF-α and IL-10 expression levels in goat PBMCs co-cultured with exosomes increased,while the IFN-α expression level decreased.Combined with our previous results that PPRV H protein can stimulate SLAM expression and the role of SLAM in regulating cytokine expression,this research further detected the effect of exosomes derived from PPRV infected cells on SLAM expression in goat PBMCs.The results showed that exosomes derived from PPRV infected cells loaded with high level of PPRV H protein could significantly increase SLAM expression in goat PBMCs.In addition,PPRV infection with PBMCs derived exosomes also cause IL-4、IL-8、IL-10 and TNF-α increased in EECs and GEFs,and IFN-β decreased.In conclusion,the mi RNA and protein expression profiles of exosomes derived from PPRV-infected PBMCs were significantly different from those from uninfected cells,and they contained high levels of PPRV N protein,H protein,and V protein.Exosomes could enter different types of goat cells through a variety of ways,and infection exosomes can affect the expression levels of immune related cytokines in the recipient cells.This research lays a foundation for future research of exosomes in PPRV-induced immunosuppression of infected goats,and in particular provides new ideas for the further study of the mechanism of PPRV causing miscarriage in pregnant ewes.