NF-κB Pathway,Lymphocyte Antigens And Other Immune-related Genes Expression In Chicken Erythrocytes Treated With Avian Influenza Virus Subtype H9N2 And E.coli

Chicken erythrocytes in blood vessels are the most abundant circulating cells,which participate in the host’s immune responses.The NF-κB plays a vital role in the inflammatory response following viral infections.Though,the expression of the NF-κB pathway,lymphocyte antigens and other immune-related genes in chicken erythrocytes infected with LPAIV H9N2 and E.coli has not been extensively studied.This in-vivo and in-vitro study determined interaction between Avian Influenza virus subtype H9N2,E.coli and chicken erythrocytes.Furthermore to highlight the role of Nuclear factor кB pathway in chickens,we examined m RNA expressions of important Nuclear factor кB pathway genes,lymphocytes antigen genes and other immune-related genes in H9N2 and E.coli treated chicken erythrocytes post-treatment at certain time periods,which played a vital role.The role of erythrocytes in chicken immunity was also highlighted in this study.This in-vivo and in-vitro study was planned to determine the interaction of LPAIV H9N2 and E.coli with chicken erythrocytes using indirect immunofluorescence,transmission electron and Scanning electron microscopy.Immunohistochemistry(IHC)performed for My D88 immune-related genes.Transcriptome sequencing was done to find out total number of upregulated and downregulated gene expressions,followed by investigating My D88(Myeloid differentiation primary response 88),CCL5(C-C Motif Chemokine Ligand 5),MDA5(melanoma differentiation-associated protein 5)IKBKE(inhibitor of nuclear factor kappa B kinase subunit epsilon),NFKBIA(NF-κB inhibitor alpha),NFKBIE(NF-κB inhibitor epsilon),IFN-α(Interferon Alpha),CSF3(Colony Stimulating Factor 3),TRAF6(Tumor necrosis factor receptor-associated factor 6),Lymphocyte antigen 75,LY6 E,LY86 and LY96 m RNA expression using quantitative real-time PCR(q RT-PCR)both in-vivo and in-vitro at different time intervals 0,2,6,10 h and at day 3rd,7th and 14 th for H9N2 while for E.coli on0,0.5,1,2 h and at day 1st 3rd and 7th post-treatment.The indirect immunofluorescence,transmission electron microscopy and scanning electron microscopy results revealed that there was a significant interaction between erythrocytes and LPAIV H9N2 and E.coli.IHC results confirmed the expressions of My D88 and TRAF6 in infected tissue.The transcriptome sequencing analysis evaluated differentially expressed genes(DEG’s)in H9N2 treated erythrocytes at 2 h(879 DEG’s 412↑ and 467↓)and at 7 day(2,550 DEG’s 1,129↑ and 1,421↓)among control and experiment group.Quantitative analysis revealed significant(P < 0.05)variations of m RNA expression of NF-κB pathway genes(NFKBIA,IKBKE,NFKBIE,IFN-α,My D88,MDA5,CCL5,TRAF6,CSF3 and Lymphocytes antigens(LY6E,LY75,LY86,LY96),)at certain time intervals(0,2,6,10 h and at day 3rd,7th and 14 th post-treatment)in response to LPAIV H9N2 and on 0,0.5,1,2 h and at day 1st 3rd and 14 th in response to E.coli in treated erythrocytes as compared to nontreated control group.This in-vivo and in-vitro study provided the first evidence of erythrocyte-pathogen Interactions,which can constitutively express several NF-κB pathway genes(NFKBIA,IKBKE,NFKBIE),Lymphocytes antigens(LY6E,LY75,LY86,LY96),and other immune regulated genes(IFN-α,My D88,MDA5,CCL5,TRAF6,CSF3,erythrocyte functions in immunity and propose in light of recent-omics research that the nucleated erythrocytes may have a direct role in the immune response.Our findings hold potential to provide a new perspective for poultry health to find therapeutic intervention for infection caused by microbial pathogens by targeting candidate genes from immune response pathways.