Macrophage Notch1 inhibits TAK1 function and RIPK3-mediated hepatocyte necroptosis through activation of β-catenin signaling in liver ischemia and reperfusion injury

Background: Notch signaling is highly conserved and critically involved in cell differentiation, immunity, and survival. Activation of the Notch pathway modulates immune cell functions during the inflammatory response. However, it remains unknown whether and how the macrophage Notch1 may control the innate immune signaling TAK1, and RIPK3-mediated hepatocyte necroptosis in liver ischemia and reperfusion injury (IRI). This study investigated the molecular mechanisms of macrophage Notch1 in modulating TAK1-mediated innate immune responses and RIPK3 functions in liver IRI. Methods: Myeloid-specific Notch1 knockout (Notch1M-KO) and floxed Notch1 (Notch1FL/FL) mice (n= 6/group) were subjected to 90 min partial liver warm ischemia followed by 6 h of reperfusion. In a parallel in vitro study, bone marrow-derived macrophages (BMMs) were isolated from these conditional knockout mice and transfected with CRISPR/Cas9-mediated beta-catenin knockout (KO) vector followed by LPS (100 ng/ml) stimulation. Results: IR stress-induced Notch1 activation evidenced by increased nuclear Notch intracellular domain (NICD) expression in liver macrophages. Myeloid Notch1 deficiency exacerbated IR-induced liver damage, with increased serum ALT levels, macrophage/neutrophil accumulation, and proinflammatory cytokines/chemokines production compared to the Notch1FL/FL controls. Unlike in the Notch1FL/FL controls, Notch1M-KO enhanced TRAF6, TAK1, NF-kB, RIPK3, and MLKL but reduced beta-catenin activation in ischemic livers. However, adoptive transfer of lentivirus beta-catenin-modified macrophages markedly improved liver function with reduced TRAF6, p-TAK1, RIPK3 and p-MLKL in IR-challenged livers. Moreover, disruption of RIPK3 in Notch1M-KO mice with an in vivo mannose-mediated RIPK3 siRNA delivery system diminished IR-triggered hepatocyte death. In vitro studies showed that macrophage NICD and beta-catenin co-localized in the nucleus, whereby beta-catenin interacted with NICD in response to LPS stimulation. Disruption of beta-catenin with a CRISPR/Cas9-mediated beta-catenin KO in Notch1FL/FL macrophage augmented TRAF6 activation leading to enhanced TAK1 function. While CRISPR/Cas9-mediated TRAF6 KO in Notch1M-KO macrophage inhibited RIPK3-mediated hepatocyte necroptosis after co-culture with primary hepatocytes. Conclusions: Macrophage Notch1 controls TAK1-mediated innate immune responses and RIPK3-mediated hepatocyte necroptosis through activation of beta-catenin. beta-catenin is required for the macrophage Notch1-mediated immune regulation in liver IRI. Our findings demonstrate that the macrophage Notch1-beta-catenin axis is a crucial regulatory mechanism in IR-triggered liver inflammation and provide novel therapeutic potential in organ IRI and transplant recipients.

Automated summary

This study demonstrated that macrophage Notch1 controls TAK1-mediated innate immune responses and RIPK3-mediated hepatocyte necroptosis through activation of beta-catenin. Beta-catenin is essential for macrophage Notch1-mediated immune regulation in liver ischemia and reperfusion injury (IRI), indicating its potential as a therapeutic target. The macrophage Notch1-beta-catenin axis is a crucial regulatory mechanism in IR-triggered liver inflammation, offering novel therapeutic possibilities in organ IRI and transplant recipients.