TLR3-mediated NET formation and macrophage activation in acute liver failure: Targeting IL-36 signaling to attenuate inflammatory drivers

Abstract

Background: Acute liver failure (ALF), characterized by inflammation-mediated hepatocyte dysfunction, poses a significant global health burden. Although neutrophils and macrophages are central to driving inflammatory responses in ALF, the molecular mechanisms governing IL-36-driven inflammation in toll-like receptor 3 (TLR3)-mediated NETosis and macrophage activation remain poorly understood. This study systematically delineates wherein TLR3 activation induces IL-36 hyperactivation and NETosis, which cooperatively trigger macrophage activation to accelerate ALF progression.

Methods: First, the role of IL-36 signaling was evaluated in the TLR3-provoked mouse peritoneal macrophages (MPMs) and murine bone-derived macrophages (BMDMs) in vitro. Further, the murine model of ALF was established with polyinosinic-polycytidylic acid (Poly(I:C))/D-GalN and acute ethanol gavage in Il1rl2-deficient mice generated by lentivirus-mediated shRNA, and the released NETs were evaluated with immunoblotting and immunofluorescence staining.

Results: We observed Poly(I:C), the specific ligand of TLR3, when combined with extracellular ATP, provoked the IL-36 signaling and the release of IL-1β in macrophages. In contrast, Il36g deficiency reduced the secretion of proinflammatory cytokines, such as IL-1β and HMGB1, upon macrophage activation. While Poly(I:C)/D-GalN or binge ETOH-induced ALF in mice was accompanied by the enhanced level of IL-36γ and NET formation, IL-36R knockdown reversed the liver damage by decreasing the expressions of proinflammatory cytokines and chemokines, reducing the lipid accumulation and preventing the NET formation as well.

Conclusion: Our study demonstrates that TLR3-mediated NET formation was enhanced in ALF, and this process was tightly regulated by IL-36 signaling. These findings suggest that targeting IL-36 represents a potential therapeutic target for modulating inflammation in ALF.

Keywords: Acute liver failure; IL-36 signaling; Macrophage activation; NETosis; TLR3 pathway.