Mincle-GSDMD-mediated release of IL-1β containing small extracellular vesicles contributes to ethanol-induced liver injury

Background & Aims Macrophage inducible C-type lectin (Mincle) is expressed on Kupffer cells and senses ethanol-induced danger signals released from dying hepatocytes and promotes IL-1β production. However, it remains unclear what and how ethanol-induced Mincle ligands activate downstream signaling events to mediate IL-1β release and contribute to alcohol-associated liver disease (ALD). In this study, we investigated the association of circulating β-glucosylceramide (β-GluCer), an endogenous Mincle ligand, with severity of ALD and examined the mechanism by which β-GluCer engages Mincle on Kupffer cells to release IL-1β in the absence of cell death and exacerbates ALD. Approach and Results Concentrations of β-GluCer were increased in serum of patients with severe AH and correlated with disease severity. Challenge of Kupffer cells with LPS and β-GluCer induced formation of a Mincle and Gsdmd -dependent secretory complex containing chaperoned full-length GSDMD (Hsp90-CDC37-NEDD4) with polyubiquitinated pro-IL-1β and components of the Casp8-NLRP3 inflammasome loaded as cargo in small extracellular vesicles (sEV). Gao-binge ethanol exposure to wild-type, but not Mincle -/- and Gsdmd -/- , mice increased release of IL-1β containing sEVs from liver explant cultures. Myeloid-specific deletion of Gsdmd similarly decreased the formation of sEVs by liver explant cultures and protected mice from ethanol-induced liver injury. sEVs collected from ethanol-fed wild-type, but not Gsdmd -/- , mice promoted injury of cultured hepatocytes and, when injected into wild-type mice, aggravated Gao-binge ethanol-induced liver injury. Conclusion β-GluCer functions as a DAMP activating Mincle-dependent GSDMD-mediated formation and release of IL-1β-containing sEVs, which in turn exacerbate hepatocyte cell death and contribute to the pathogenesis of ALD. ### Competing Interest Statement The authors have declared no competing interest. * Mincle : macrophage inducible c-type lectin GSDMD : gasdermin d IL-1β : interleukin-1β β-GluCer : β-glucosylceramide LPS : lipopolysaccharide ALD : alcohol liver disease sEV : small extracellular vesicles AH : alcohol-associated hepatitis Casp : caspase CDC37 : cell deivision cycle 37 HSP90 : heat shock protein 90 NEDD4 : neural precursor cell expressed developmentally down-regulated protein 4 SAP130 : spliceosome-associated protein 130 NLRP3 : NLR family pyrin domain containing 3 MELD : model for end-stage liver disease TDB : trehalose-6,6-dibehenate ATP : adenosine triphosphate DAMP : Damage-associated molecular pattern HC : Healthy control LDH : Lactate Dehydrogenase imKC : immortalized Kupffer cell SAA1 : Serum Amyloid A1 ALT : alanine aminotransferase AST : aspartate aminotransferase TNFa : tumour necrosis factor alpha WT : wild type EtOH : ethanol.