*Purpose: Yes-associated protein (YAP), a downstream effector of the Hippo pathway, tightly regulates cell growth, survival, and tissue inflammation. Activation of heat shock factor 1 (HSF1) has a profound impact on innate immunity during liver inflammatory response. However, it remains unknown as to whether and how the myeloid YAP-HSF1 signaling may control NLRP3 activation in sterile inflammation during liver injury. This study investigated the mechanistic link between YAP and HSF1 in the modulation NLRP3-mediated innate immune response in liver ischemia and reperfusion injury (IRI).

*Methods: Myeloid-specific YAP knockout (YAP^M-KO) and floxed YAP (YAP^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 HSF1 knockout (KO) vector followed by LPS (100 ng/ml) stimulation.

*Results: Ischemia/reperfusion (IR) stress induced YAP and HSF1 activation evidenced by increased nuclear YAP and HSF1 expression in hepatic Kupffer cells. Myeloid YAP deficiency exacerbated IR-induced liver damage, with increased serum ALT levels, macrophage/neutrophil accumulation, and pro-inflammatory cytokine/chemokine production compared to the YAP^FL/FL controls. Unlike in the YAP^FL/FL controls, YAP^M-KO enhanced TXNIP, RIPK3, p-MLKL, NEK7, NLRP3, ASC, cleaved caspase-1 activation in ischemic livers. However, adoptive transfer of lentivirus HSF1-modified macrophages into mice markedly improved liver function with reduced TXNIP, RIPK3, p-MLKL, and NEK7/NLRP3 but augmented TRX1 in IR-challenged livers. Moreover, disruption of RIPK3 in YAP^M-KO mice with an in vivo mannose-mediated RIPK3 siRNA delivery system diminished IR-triggered hepatocyte death. In vitro studies showed that macrophage YAP and HSF1 co-localized in the nucleus whereby HSF1 interacted with YAP in response to LPS stimulation. Disruption of HSF1 with a CRISPR/Cas9 HSF1 KO transfection in YAP^FL/FL macrophage augmented TXNIP and RIPK3 activation leading to enhanced NEK7/NLRP3 function and RIPK3-mediated hepatocyte necroptosis after macrophage/hepatocyte co-culture.

*Conclusions: Myeloid-specific YAP controls NLRP3-triggered liver inflammation and RIPK3-mediated hepatocyte necroptosis through interaction with HSF1. HSF1 is required for the myeloid YAP-mediated immune regulation in liver IRI. Our findings demonstrate the key mechanistic role of myeloid YAP in liver inflammatory injury, and provide novel therapeutic potential in organ IRI and transplant recipients.

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