In Vitro Immunoregulatory Network Induced By Mycobacterium Leprae Polypeptide ML-LBP21

Objective: We aim to explore the immunity of Mycobacterium leprae polypeptide ML-LBP21 against leprosy,and study the cytokines,chemokines respectively.At the same time,the changes of immune cell subsets after stimulation with ML-LBP21 polypeptide for 12 hours were also studied.Methods:(1)Peripheral blood mononuclear cells(PBMC)were extracted from 15-20 ml peripheral blood of 3 healthy people.Each case was divided into two groups :peptide stimulation for 12 hours group and PBS control group for transcriptome sequencing analysis.The key genes and differential genes expressed in PBMC of human monocytes stimulated by Mycobacterium leprae polypeptide ML-LBP21 in leprosy immunity were selected to explore the possible signaling pathways induced by Mycobacterium leprae polypeptide ML-LBP21.(2)Quantitative polymerase chain reaction(q PCR)was used to detect the m RNA expression levels of IL-1β,IL-6,TNF-α,CXCL1,CXCL2,CXCL3,CXCL8,CCL2,CCL3,CCL7 and CCL8 differential genes,and to explore the role of ML-LBP21 polypeptide in the immune mechanism of leprosy.(3)In order to study the effect of ML-LBP21 peptide on immune cells,the number of monocytes,MAIT cells,NK cells,B cells,Th1,Th2,Th17 and Treg cells before and after stimulation was detected by flow cytometry.Results:(1)By combining qpcr results and KEGG enrichment analysis,it can be concluded that ML-LBP21 may induce the activation of IL-17 and TNF-αinflammatory signaling pathways.(2)The m RNA expression of chemokines and cytokines was up-regulated after stimulation of human peripheral blood mononuclear cells by Mycobacterium leprae polypeptide ML-LBP21;the m RNA expression of CSTL,TLR2 and MMP9 genes in the peptide stimulation group was higher than that in the PBS control group(P < 0.05).The expression level of CCL2 m RNA after 12 hours of peptide treatment was not statistically significant compared with the PBS control group.The m RNA expression levels of CCL3,CCL7 and CCL8 increased after 12 hours of peptide treatment,and the difference was statistically significant(P< 0.05).After 12 hours of polypeptide treatment,CXCL1,CXCL2 and CXCL3 showed an increasing trend,and the difference was statistically significant(P < 0.05).The difference of CXCL8 was not statistically significant(P > 0.05).There was no significant difference in the gene expression of TNF-α,IL-1β and IL-6 after 12 hours of peptide treatment(P > 0.05).(3)Mycobacterium leprae polypeptide ML-LBP21 can increase the number of non-classical mononuclear cells,P<0.05;the number of MAIT cells decreased,and the difference was statistically significant(P<0.05),not statistically significant.Conclusion:(1)ML-LBP21 may induce the activation of IL-17 and TNF-αinflammatory signaling pathways.(2)After ML-LBP21 acted on human mononuclear cells,cytokines and chemokines were up-regulated and the immune microenvironment was changed.(3)ML-LBP21 can increase the number of nonclassical monocytes and decrease the number of MAIT cells.late the increase of nonclassical monocyte cells and the decrease of MAIT cells.