Macrophage Modulated By Tim-3/Galectin-9 Pathway In Primary Biliary Cholangitis

Objective:Our previous works have found that abnormal expression of T cell immunoglobulin and mucin domain 3(Tim-3)and Galectin-9 in a murine model of autoimmune cholangitis.The dysfunction of T cell immunoglobulin and mucin domain 3(Tim-3)/Galectin-9 pathway is involved in diverse autoimmune diseases.Therefore,further Research on immune checkpoints including Tim-3 and other inhibitory genes expressed on peripheral mononuclear cells(PBMCs)from patients with primary cholangitis(PBC),especially monocyte subsets,by using transcriptomic analysis.Risk factors involved in PBC and the potential regulatory role should also be analyzed.The primary aim of this study was to investigate the function and modulation of Tim-3/Gal ectin-9 pathway on target cells in PBC.Methods:Differentially expressed genes containing proinflammatory and inhibitory genes between PBC and HC were screened by using transcriptomic analysis.Gene Set Enrichment analysis and Protein-Protein Interaction Networks(PPI)were applied to analyze risk factors and hub gene involved in PBC and the potential regulatory role.According to the results of transcriptomic analysis,the expression of Tim-3 and Toll-like receptor(TLR2)on monocyte subsets including classical monocytes(cMO),intermediate monocytes(iMO)and nonclassical monocytes(ncMO)was determined by flow cytometry.Enzyme-linked immunosorbent assay(ELISA)was applied to detect serum levels of soluble Tim-3(sTim-3),Galectin-9 and cytokines/chemokines Tumor Necrosis Factor-alpha(TNF-α),Interferon gamma(IFN-γ),Interleukin(IL)-1β,IL-2,IL-6,IL-8,IL-10 and IL-12,CC-chemokine ligand 3,CCL3,CCL4,CCL19,CCL20,C-X-C motif chemokine 9(CXCL9),CXCL10,CXCL11,CXCL16 and C-X3-C motif chemokine 1(CX3CL1).The expression of Tim-3,Galectin-9 and Activating transcription factor 3(ATF3),and polarization of hepatic macrophages of PBC patients were observed by using immunohistochemistry(IHC).The relationship between sTim-3 or Galectin-9 and cytokines/chemokines and clinical features was analyzed.THP-1 cells were differentiated into macrophages,then activation of TLR2 pathway was performed.The expression of Tim-3,Galectin-9,ATF3 and cytokines/chemokines were detected by Western Blot or ELISA.The Tim-3/Galectin-9 pathway was blocked to observe the polarization of macrophages,the expression of ATF3 and the secretion of cytokines/chemokines.At the same time,the potential biological role of stim-3 was also investigated.A murine model of autoimmune cholangitis was induced,and Tim-3/Galectin-9 pathway was blocked in vivo.Histologically,liver inflammation and polarization of liver macrophages was observed,the expression of ATF3 and CXCL10 in mouse liver was detected by IHC.The modulation of Tim-3/Galectin-9 pathway on TLR2-activated macrophages after ATF3 silence.Results:Transcriptomics suggested that the up-regulated genes were predominantly pro-inflammatory in PBC patients.However,there were no significant difference in immune checkpoints including Tim-3 and most inhibitory genes.Gene Set Enrichment analysis indicated that antigen recognition,infection,oxidative stress,cytokine pathway and Toll-like receptor signaling pathway were involved in PBC.Addtionally,endogenous ligands of Toll-like receptors were upregulated in PBMCs of PBC patients.Up-regulation of TLR2 and ATF3 in PBMCs of patients with PBC were further confirmed by Real-Time Quantitative Reverse Transcription PCR.ICH showed that the expression of ATF3 was decreased in the liver of advanced PBC patients.The proportion of peripheral monocyte in patients with PBC was increased,especially advanced stage.In addition,monocyte-to-lymphocyte ratio was an independent predictor of prognosis in patients with PBC.Flow cytometry revealed that the peripheral iMO and ncMO were significantly increased in PBC patients,and reduced hepatic M2-like macrophages of advanced PBC patients were found by using immunofluorescence.Compared to HC,circulated Tim-3+CD14+and TLR2+CD16+MO were elevated in PBC patients.In advanced PBC patients,Tim-3 expression was reduced in the liver,whereas Galectin-9 expression was upregulated.High serum sTim-3 and low Galectin-9 was detected in PBC patients,but there was no significant correlation with cytokines or clinical features.THP-1 cells were classically polarized into macrophages,the expression of Tim-3 was declined in M1-like macrophages,and ATF3 was significantly increased in M2-like macrophages.Both Pam3csk4 and lipopolysaccharide(LPS),the TLR2 and TLR4 agonist respectively,raised the expression of Tim-3 and ATF3 in THP-1 cells,while declined in macrophages.α-lactose was used block Tim-3/Galectin-9 pathway,which promoted the secretion of Galectin-9,IL-6 and CXCL10 from M0 macrophages.Except for increased ATF3 expression,recombinant human Tim-3 Fc Chimera Protein(rhTim-3 Fc)had a similar biofunction with α-lactose.Recombinant human Galectin-9(rhGalectin-9)up-regulated the expression Tim-3 and ATF3,while down-regulated the secretion of IL-6 and CXCL10 of Pam3csk4-treated macrophages.But there was no significant change in these macrophages pre-treated with α-lactose or rhTim-3 Fc.Intraperitoneal injection of α-lactose did not aggravate liver inflammation or promote the process of liver fibrosis in autoimmune cholangitis mice,but it induced lymphocyte infiltration in the portal area of the liver in mice.α-lactose reduced mice liver M2 macrophages,an increase of CXCL10 and decreased ATF3 expression in liver also were observed in autoimmune cholangitis mice treat with α-lactose.Silencing ATF3 inhibited THP-1 cells proliferation,but no effect on expression of Tim-3 and Galectin-9 in macrophages.After silencing ATF3,the inhibitory ability of rhGalectin-9 on cytokine secretion of Pam3csk4-stimulated macrophages was lower than shNC group.Conclusion:Predominant innate immune response and pro-inflammatory polarization of monocyte/macrophage in PBC patients.Almost up-regulated genes in PBC patients are mainly pro-inflammatory,cytokines/chemokines containing CXCL9-10 participate in the development of PBC.In addition,TLR2 and ATF3 are the key genes involved in the development of PBC.PBC patients have pro-inflammatory monocytes/macrophages and increased percentage of TLR2+CD16+monocytes.PBC patients have an increase of serum cytokines/chemokines and the dysfunction of Tim-3/Galectin-9 pathway.Activation of TLR2 pathway downregulates the expression of Tim-3 and ATF3 on macrophages.The modulation of Tim-3/Galectin-9 pathway on macrophages is involved in the development of PBC.Furthermore,ATF3 partially mediates the negative modulation of Tim-3/Galectin-9 pathway on macrophages in PBC.