*Purpose: Foxo1 plays important roles in cell metabolism, oxidative stress, inflammation, and apoptosis. Activation of Hedgehog/Gli1 signaling regulates cell growth, differentiation, and immune function. However, it remains unknown whether and how myeloid Foxo1 signaling may modulate the Hedgehog/Gli1 pathway in sterile inflammatory liver injury. This study investigated the functional roles and molecular mechanisms of myeloid Foxo1 signaling in the modulation of NLRP3-mediated innate immune responses during ischemia/reperfusion (IR)-triggered liver inflammation.

*Methods: Myeloid-specific Foxo1 knockout (Foxo1^M-KO) and floxed Foxo1 (Foxo1^FL/FL) mice (n=6/group) were subjected to 90 min partial liver warm ischemia followed by 6 h of reperfusion. In parallel in vitro study, bone marrow-derived macrophages (BMMs) were isolated from these conditional knockout mice and transfected with CRISPR/Cas9-mediated Gli1 or Snail knockout vector followed by LPS (100 ng/ml) stimulation.

*Results: Foxo1^M-KO mice were resistant to IR-induced hepatocellular damage, with reduced serum ALT levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared to the Foxo1^FL/FL controls. Unlike in the Foxo1^FL/FL controls, Foxo1^M-KO enhanced β-catenin, Hedgehog signaling effector Gli1, and Snail but reduced RIPK3, NEK7/NLRP3 activation in ischemic livers. Disruption of Gli1 in Foxo1^M-KO livers deteriorated liver function with diminished Snail while enhancing RIPK3 and NEK7/NLRP3 activation. For the in vitro studies, we found that macrophage Foxo1 and β-catenin co-localized in the nucleus whereby Foxo1 interacts with β-catenin in response to inflammatory stimulation. Importantly, nuclear Foxo1 competed with TCF for interaction with β-catenin. Disruption of the Foxo1-β-catenin axis by Foxo1 deletion enhanced β-catenin/TCF binding, activated Gli1/Snail signaling, leading to inhibited RIPK3 and NEK7/NLRP3. Moreover, macrophage Gli1 or Snail knockout activated RIPK3, which augmented hepatocyte necroptosis after macrophage/hepatocyte co-culture while macrophage RIPK3 knockout diminished NEK7/NLRP3-mediated immune and inflammatory responses.

*Conclusions: Myeloid-specific Foxo1 deficiency promotes the Hedgehog/Gli1 signaling and mitigates IR-induced hepatocellular injury through disruption of nuclear Foxo1-β-catenin interaction, which in turn enhances β-catenin/TCF binding and activates the Hedgehog/Gli1/Snail signaling, resulting in reduced NEK7/NLRP3-driven liver inflammation and RIPK3-mediated hepatocyte necroptosis. Our findings underscore the crucial role of myeloid Foxo1 signaling in the modulation of NLRP3-mediated innate immunity and inflammatory responses during sterile inflammatory liver injury, and imply the therapeutic potential in organ IRI and transplant recipients.

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