*Purpose: The stimulator of interferon genes (STING) pathway has been recognized as a crucial signaling cascade of the innate immune system. Activation of the Hedgehog signaling regulates cell differentiation and immune function. Our previous studies have demonstrated that mesenchymal stem cells (MSCs) can regulate macrophage-mediated inflammation in liver ischemia/reperfusion injury (IRI). However, it remains unknown whether MSCs control STING-mediated innate immune responses in orthotopic liver transplantation (OLT). This study tested the mechanistic role of MSC-mediated Hedgehog signaling on STING function in IR-stressed OLT.

*Methods: Using an established mouse model of ex vivo cold preservation (UW solution at 4°C for 16h) followed by syngeneic OLT, the myeloid-specific SMO knockout (SMO^M-KO) and wild-type (WT) mice were used. The OLT recipients were injected with bone marrow-derived MSCs (1×10⁶ cells in PBS/mouse i.v.) 24h prior to liver transplant. In Group 1, donor livers were transplanted from WT to WT mice (WT→WT). In Group 2, WT→ SMO^M-KO. In Group 3, WT→WT (without cell injection). The recipients were sacrificed at 6h and 24h post-OLT. Liver and serum samples were harvested (n=4-5/group). In parallel in vitro study, bone marrow-derived macrophages (BMMs) were transfected with CRISPR/Cas9-mediated Gli1 knockout (KO) or control vector and then co-cultured with MSCs followed by LPS (100 ng/ml) stimulation.

*Results: Adoptive transfer of MSCs ameliorated IR stressed-induced injury in OLT. MSC treatment attenuated sinusoidal congestion, edema/vacuolization, and hepatocyte necrosis in OLT. However, myeloid SMO deficiency in MSC-treated OLT displayed exacerbated IR-induced hepatocellular damage, with increased serum ALT levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators. MSC treatment augmented SMO and Gli1 yet inhibited STING, TBK1, IRF3, and NF-κB activation, whereas disruption of myeloid SMO diminished Gli1 and enhanced STING/TBK1 activity in MSC-treated OLT. Using MSC/macrophage co-culture system, we found that MSC upregulated macrophage SMO and Gli1 expression whereas disruption of SMO inactivated Gli1 and enhanced STING, TBK1, IRF3, and NF-κB activities after LPS stimulation. Interestingly, CRISPR/Cas9-mediated Gli1 KO enhanced STING activity and augmented M1 macrophage iNOS but reduced M2 macrophage Arg1 expression after co-culture with MSCs.

*Conclusions: Adoptive transfer of MSCs activates the Hedgehog/SMO/Gli1 pathway, which controls STING function and induces macrophage differentiation towards an anti-inflammatory M2 phenotype. Our findings provide novel potential therapeutic targets in MSC-based immunotherapy in transplant recipients.

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