ATF3 Mediates the Immunoregulatory Role of the Tim‐3 Pathway in Primary Biliary Cholangitis

OBJECTIVE: The immune checkpoint Tim-3 has been implicated in the pathogenesis of primary biliary cholangitis (PBC), but its precise role in modulating macrophage polarization and immune dysregulation remains unclear. This study aimed to define the expression patterns of Tim-3 in PBC patients and elucidate its functional impact on macrophage behavior and disease progression. METHODS: Bulk RNA sequencing was performed using peripheral blood mononuclear cells (PBMCs) derived from PBC patients. The expression of Tim-3 and Toll-like receptor (TLR) 2 was assessed using flow cytometry. A murine model of autoimmune cholangitis was established using syngeneic bile duct protein. The Tim-3/Galectin-9 pathway was blocked using α-lactose both in vivo and in vitro. Short hairpin RNA (shRNA)-mediated silencing of ATF3 expression was conducted in THP-1 cells to explore its role in macrophage regulation. RESULTS: Our results revealed a significant pro-inflammatory innate immune response in the periphery of PBC patients. Advanced PBC patients exhibited reduced M2-like Kupffer cells and decreased expression of Tim-3 and ATF3, whereas Galectin-9 expression was upregulated. TLR2 activation downregulated Tim-3 and ATF3 expression in macrophages. Blockade of the Tim-3/Galectin-9 pathway with α-lactose exacerbated experimental autoimmune cholangitis, reduced M2 Kupffer cells and ATF3 expression, and increased CXCL10 levels in the liver. Furthermore, silencing ATF3 impaired the suppressive effects of the Tim-3/Galectin-9 pathway on TLR2-activated macrophages and primary murine Kupffer cells. CONCLUSION: These findings highlight the prominent pro-inflammatory innate immune response in PBC patients and demonstrate that ATF3 partially mediates the immunomodulatory effects of the Tim-3/Galectin-9 pathway on macrophages.