Effect Of E.maxima Th1 Cytokine Inhibitory Molecules On Chicken Th1/Th2 Cell Differentiation Signaling Pathways

Avain coccidiosis is a global epidemic parasitic disease caused by single or mixed infection of Eimeria in the poultry industry.When avain coccidia infects the host,cellular immunity plays a significant role in anti-coccidiosis immunity,and Th1 cytokines play a crucial role in stimulating cellular immunity.E.maxima is one of the most prevalent species with moderate pathogenicity.Infection of E.maxima reduces the conversion efficiency of feed,carcass quality and egg production of infected chickens,even results in death due to severe infection.E.maxima infection can inhibit the expression of Th1 cytokines and escape immune attack from the host.In our previous study,we screened four Th1 cytokine inhibitory molecules(EmSAG,EmHPSP-1,EmHPSP-2 and EmHPSP-3)from the E.maxima sporozoite cDNA expression library.Meanwhile,we have documented that the above inhibitory molecules exerted its inhibitory effect by binding to the receptors on chicken peripheral blood mononuclear cells.However,related immune cell signaling pathway induced by them on chicken peripheral blood mononuclear cells and innate immune cells need to be further determined.In order to clarify these issues,the following research is carried out in present study.In order to investigate the effects of Th1 cytokine inhibitory molecules on the transcription level of related cytokines in chicken peripheral blood mononuclear cells.The above four Th1 cytokines inhibitory molecules were expressed and purified,respectively(rEmSAG,rEmHPSP-1,rEmHPSP-2 and rEmHPSP-3).The transcription levels of transcription factors(T-bet and GATA3)and related cytokines(IFN-γ,IL-2,IL-4 and IL10)on peripheral blood mononuclear cells induced by Th1 cytokine inhibitory molecules were detected by real-time quantitative PCR.The results showed that the inhibitory molecules EmSAG,EmHPSP-1,EmHPSP-2 and EmHPSP-3 could inhibit the transcription level of T-bet and IL-2 and IFN-γ significantly.In order to study the proteomics of chicken peripheral blood mononuclear cells stimulated by Th1 cytokine inhibitory molecules,Th1 cytokine inhibitory molecules and ConA were co-incubated with chicken peripheral blood mononuclear cells and found 46 differential protein spots were screened by 2-DE.By MALDI-TOF technology and database search,17 differential proteins were identified and analyzed by GO annotation and KEGG pathway.It was found that Pyruvate kinase PKM as a common specific differential protein exists in chicken peripheral blood mononuclear cells induced by four inhibitory molecules.PDZ and LIM domain protein 1 isoform X2 are specific differential proteins produced by chicken peripheral blood mononuclear cells induced by rEmHPSP-1 or rEmHPSP-3.Proteasome C1 subunit is a specific differential protein produced by chicken peripheral blood mononuclear cells induced by rEmSAG.Plastin-2 and Nucleophosmin are unique differential proteins produced by chicken peripheral blood mononuclear cells induced by rEmHPSP-1.The specific differential proteins induced by rEmHPSP-2 in chicken peripheral blood mononuclear cells are Myosin regulatory light chain 2,SH3 domain-binding glutamic acidrich-like protein and L-lactate dehydrogenase A chain.The results showed that these proteins are mostly involved in metabolic processes and signaling pathways.In order to study the effect of E.maxima Th1 cytokine inhibitory molecules on signaling pathways related to Th1/Th2 cell differentiation,Th1 cytokine inhibitory molecules and ConA were used to costimulate chicken peripheral blood mononuclear cells.The proliferation effect of the Th1 cytokine inhibitory molecules on the peripheral blood lymphocytes was detected by CCK8 Kit.Real-time quantitative PCR was used to detect the changes of Th1/Th2 cell differentiation signaling pathway molecules at the transcription level.Western blot was used to detect the changes of key molecular proteins and phosphorylated proteins.Finally,RNA interference was used to verify the effects of key molecules.It was found that the inhibitory molecules of EmSAG,EmHPSP-1,EmHPSP-2 and EmHPSP-3 can inhibit the proliferation of chicken peripheral blood mononuclear cells.And qPCR showed that STAT1,STAT4 and STAT6 are key molecules of Th1/Th2 cell differentiation signal pathway.Western blot showed that the four inhibitory molecules down-regulated the expression levels of STAT1 and STAT4 in chicken peripheral blood mononuclear cells,and up-regulated the expression levels of STAT6 protein in chicken peripheral blood mononuclear cells.After the interference of STAT6,a key molecule in the signaling pathway of Th2 cell differentiation,the inhibition ability of the four inhibitory molecules on Thl cytokines was decreased,and the promotion effect of Th2 cytokines was blocked.In this study,we found that Th1 cytokine inhibitory molecules can inhibit Th1/Th2 cell differentiation signal transduction pathway by inhibiting the expression of STAT1 and STAT4 phosphorylated proteins,which are key molecules in the Thl cell differentiation pathway.In addition,the signal transduction of Th2 cell differentiation pathway was promoted by promoting the expression of STAT6 phosphorylated protein,a key molecule in Th2 cell differentiation pathway.This study reveals the mechanism by which Th1 cytokine inhibitory molecules inhibit the expression of chicken immune cell signaling pathways,which also has important reference value to elucidate the immune evasion mechanism of avain coccidia.