Bile acids engage the SIPR-STAT3 signaling axis to modulate regulatory T cell responses in fibrosing cholangiopathies

Abstract

Background & aims: Regulatory T cells (Tregs), a subset of CD4 lymphocytes, protect against inflammatory tissue injury. However, it is currently unknown how retention of bile acids (BA) in fibrosing cholangiopathies like biliary atresia or PSC shape hepatic Treg responses.

Methods: To induce sclerosing cholangitis (SC), mice were fed a diet containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine for 14 days, followed by 28 days on regular chow to assess tissue repair. Serial hepatic cell and nuclear preparations were subjected to single-cell RNA sequencing and ATAC-seq to define gene regulatory networks controlling Tregs under cholestatic conditions. Candidate molecules mediating the effects of tauro- and chenodeoxycholic acid (T/CDCA) on Tregs were validated in vitro, across three murine models of SC, and in liver tissue samples from 130 infants with biliary atresia.

Results: Single-cell analyses revealed that Tregs acquired a Th17-like transcriptional program during cholestasis and upregulated amphiregulin (Areg) during the repair phase. S1P receptors were identified as mediators of T/CDCA effects on Tregs, and this was validated both in vitro and in the Abcb4^-/- model of SC. Deletion of Stat3 in CD4⁺ cells enhanced hepatic Treg responses following bile duct ligation. Pharmacologic reduction of hepatic BA concentrations using an IBAT (ileal bile acid transporter) inhibitor increased hepatic Treg numbers and attenuated liver injury and fibrosis in Abcb4^-/- mice. These protective effects were lost upon Treg depletion or AREG neutralization. Finally, in infants with biliary atresia, a liver transcriptional profile consistent with Treg activation and AREG upregulation at diagnosis was associated with improved 2-year native liver survival.

Conclusion: Bile acids suppress Treg regulatory function by promoting a Th17-like phenotype, thereby limiting their capacity to mitigate immune-mediated cholangiocyte injury. Restoring Treg function and amphiregulin expression may represent a novel therapeutic strategy in fibrosing cholangiopathies.

Impact and implications: In this study, we examined the role of CD4 lymphocytes in controlling bile duct epithelial injury in fibrosing cholangiopathies, with potential implications for developing targeted therapies for BA and PSC. Using single-cell genomics, functional assays, and complementary mouse models of sclerosing cholangitis, we investigated the mechanisms by which chenodeoxycholic acid-derived bile acids determine polarization and suppressor functions of CD4 lymphocytes. Interventions such as reducing hepatic bile acid concentrations with an IBAT (ileal bile acid transporter) inhibitor, antagonizing STAT3 in CD4⁺ cells, or blocking S1P receptors enhanced hepatic regulatory T cell responses and protected against cholestatic liver injury in experimental models. These preclinical findings provide a foundation for future clinical trials in patients with fibrosing cholangiopathies.

Keywords: Amphiregulin; Childhood Liver Disease Research Network; Extrahepatic biliary atresia; Gene regulatory networks; Ileal bile acid transporter; Primary sclerosing cholangitis; Regulatory T cells (Tregs); Sphigosin-1-phosphate receptor; Taurochenodeoxycholic acid; Th17 polarization.