Mechanistic Study Of Antibacterial Effect Of Heterophil Based On H/L Directional Selection Chicken Line

Heterophils have phagocytosis and pathogens killing functions,which play an indispensable role in the immune defense in the host.The ratio of heterophils to lymphocytes(H/L ratio)can reflect a readiness to cope with infection through injury(via heterophils)rather than with a communicable disease(via lymphocytes).Previous studies have shown that low ratio of H/L significantly enhanced the ability of Salmonella resistance and heterophils function in chickens.In this study,the chickens came from different H/L selection lines,which have the difference in Salmonella resistance and heterophils functions.To further elucidate how the H/L selection influencing the Salmonella resistance as well as the molecular mechanism of heterophils functions,genome-wide association study(GWAS)and selective sweep analysis were performed.The results provide a theoretical support for the chicken antibacterial breeding.After more than 9 generations selection,the H/L ratio of H/L selection line significantly decreased(from 0.5 to 0.25).The population structure and the selective sweep analysis of random individuals between the H/L selection line(41chickens)and the control line(31 chickens)were performed based on the 55 K SNP genotyping chip.The results suggested that continuous selection of H/L caused genome differentiation.We further sequenced 368 chickens from the H/L selection line,control line,and the F2 population crossed by the two lines.By using the selective sweep analysis(5th generation of H/L selection line,n=43;9th generation of H/L selection line,n=92;non-selection line,n=92),we found that TIAM1,INPP5 D,CLDN8,TJP1,MYO1 E,EIF4G3,THBS2,CORIN,and ST6GALNAC3 genes were selected,which related to immune and disease resistance.The GWAS on H/L ratio(5th generation of H/L selection line,n=35;9th generation of H/L selection line,n=73;F2 population,n=141)showed the SNPs were located on a 900 Kb region of chromosome 5(chr5: 12.4Mb-13,3Mb)significantly correlated to the H/L ratio.We identified that the PTPRJ and its downstream INPP5 D gene are the candidate genes,which influence the heterophils functions.Differential expression analysis of individuals with high(n=8)and low(n=8)PTPRJ gene expression in heterophils transcriptomes showed that the expression of LYN,SHP1,and SHIP1 genes,which are in the downstream of PTPRJ related to immunosuppression,significantly reduced in the low PTPRJ gene expression group.KEGG analysis results showed that the classical TLR4-My D88 signaling pathway,oxidative burst,phagocytosis,and chemokine production pathways related to heterophils function were enriched.These results suggested that PTPRJ may be involved in the regulation of chicken heterophil function.Association analysis was carried out between Jinlinghua chicken(n=1,199)with different H/L ratio and significant SNP loci on PTPRJ gene.The results showed that the SNP(rs736799474)in PTPRJ downstream was significantly correlated to H/L ratio(P < 0.05),i.e.,the H/L ratio of CC genotype individuals was significantly lower than that of TT genotype individuals.The double luciferase assay and EMSA for this site showed that rs736799474 located in the enhancer region.The enhancer effect is weakened and the expression of PTPRJ is down-regulated after T > C mutation.In order to effectively supplement the results of functional gene identification based on specific breeding lines(H/L directional breeding lines),GWAS analysis was carried out on white-feathered broilers(n=1,650)and yellow-feathered broilers(n=1,199).Genes,such as C1 QBP in PTPRJ regulatory network,were identified as candidate genes related to H/L.The sulfur relay system pathway and calcium signaling pathway were candidate pathways affecting H/L.In conclusion,this study identified that PTPRJ gene is the key regulatory gene for Salmonella resistance and heterophil function improvement in H/L directional breeding line.It is found that H/L directional breeding may affect the immune function of chickens.H/L directional breeding decreased PTPRJ enhancer activity and down-regulated PTPRJ expression,which may further down-regulate the immunosuppressive effect of downstream genes(LYN,SHP1,and SHIP1).Thereby the heterophils function and the body's resistance to Salmonella improved.