Cholangiopathies, including primary sclerosing cholangitis (PSC) are cholestatic liver diseases that target cholangiocytes. No definitive treatments for PSC exist and curative measures are elusive, thus translational studies are critically needed. PSC is characterized by ductular reaction (DR) encompassing enhanced bile duct mass, biliary senescence, inflammation, peribiliary fibrosis and increased portal mast cell (MC) presence/activation. During liver damage, MCs migrate to the liver and reside near damaged/senescent bile ducts and inhibition of biliary-derived senescent associated secretory phenotypes (SASP) reduces PSC phenotypes in mice. The exact source of MC infiltration/activation or stabilization remains unclear. Melatonin exerts its effects via specific receptors, MT1 and MT2, which are expressed by cholangiocytes and upregulated in cholestasis but have differential effects on liver damage. Knockdown of MT1 decreases cholestatic liver injury, whereas knockdown of MT2 increases cholestatic liver injury. Anti-inflammatory and anti-oxidant properties of melatonin are well-known; however, melatonin can also inhibit HA signaling, suggesting a link between these two molecules that, to date, has remained elusive. One key element between melatonin signaling and MC- histamine (HA) is the alarmin and SASP, IL-33, which is the ligand of the ST2 receptor (expressed on MCs) and IL-33/ST2 are upregulated in cholestasis. Since melatonin modulates IL-33/ST2 signaling which regulates MC activation/stabilization, there is a natural link between these two. Preliminary data demonstrates that (i) IL-33/ST2 signaling increases in human PSC and Mdr2-/- mice that correlates with increased HA content; (ii) Mdr2-/- mice treated with an ST2 inhibitor have reduced DR, inflammation and IL-33 content; and (ii) melatonin therapy decreases MC activation and biliary IL-33 content. These data support our overall hypothesis that, during liver damage, increased systemic and hepatic IL-33 levels induce MC infiltration/activation driving PSC phenotypes, which can be ameliorated via modulation of melatonin/MT-1 signaling. SA1 Hypothesis: Hepatic IL-33/ST2 signaling drives MC infiltration/activation and PSC phenotypes. We will evaluate (i) the cellular source(s) of IL- 33 using in vivo models and in vitro systems; and (ii) effects of IL-33/ST2 modulation on MC migration/activation and PSC phenotypes. SA2 Hypothesis: MCs infiltration increases IL-33 during liver damage and manipulation of MC mediators decreases IL-33/ST2 signaling. We will determine if (i) manipulation of MC-HA signaling directly alters IL-33/ST2 signaling, using cultured systems and 3D human organoids; (ii) changes in MC-HA/HR signaling in vivo mediates IL-33/ST2 outcomes and (iii) if manipulation of MCs in vitro affects PSC phenotypes. SA3 Hypothesis: Exogenous melatonin or manipulation of melatonin signaling decreases liver damage by stabilization of MCs and downregulation of IL-33/ST2 and there is an autoc...