The ST2⁺ Treg/amphiregulin axis protects from immune-mediated hepatitis

Introduction: The alarmin IL-33 has been implicated in the pathology of immune-mediated liver diseases. IL-33 activates regulatory T cells (Tregs) and type 2 innate lymphoid cells (ILC2s) expressing the IL-33 receptor ST2. We have previously shown that endogenous IL-33/ST2 signaling activates ILC2s that aggravate liver injury in murine immune-mediated hepatitis. However, treatment of mice with exogenous IL-33 before induction of hepatitis ameliorated disease severity. Since IL-33 induces expression of amphiregulin (AREG) crucial for Treg function, we investigated the immunoregulatory role of the ST2(+) Treg/AREG axis in immune-mediated hepatitis. Methods: C57BL/6, ST2-deficient (Il1rl1(-/-)) and Areg(-/-) mice received concanavalin A to induce immune-mediated hepatitis. Foxp3Cre(+) x ST2fl/fl mice were pre-treated with IL-33 before induction of immune-mediated hepatitis. Treg function was assessed by adoptive transfer experiments and suppression assays. The effects of AREG and IL-33 on ST2(+) Tregs and ILC2s were investigated in vitro. Immune cell phenotype was analyzed by flow cytometry. Results and discussion: We identified IL-33-responsive ST2(+) Tregs as an effector Treg subset in the murine liver, which was highly activated in immune-mediated hepatitis. Lack of endogenous IL-33 signaling in Il1rl1(-/-) mice aggravated disease pathology. This was associated with reduced Treg activation. Adoptive transfer of exogenous IL-33-activated ST2(+) Tregs before induction of hepatitis suppressed inflammatory T-cell responses and ameliorated disease pathology. We further showed increased expression of AREG by hepatic ST2(+) Tregs and ILC2s in immune-mediated hepatitis. Areg(-/-) mice developed more severe liver injury, which was associated with enhanced ILC2 activation and less ST2(+) Tregs in the inflamed liver. Exogenous AREG suppressed ILC2 cytokine expression and enhanced ST2(+) Treg activation in vitro. In addition, Tregs from Areg(-/-) mice were impaired in their capacity to suppress CD4(+) T-cell activation in vitro. Moreover, application of exogenous IL-33 before disease induction did not protect Foxp3Cre(+) x ST2fl/fl mice lacking ST2(+) Tregs from immune-mediated hepatitis. In summary, we describe an immunoregulatory role of the ST2(+) Treg/AREG axis in immune-mediated hepatitis, in which AREG suppresses the activation of hepatic ILC2s while maintaining ST2(+) Tregs and reinforcing their immunosuppressive capacity in liver inflammation.