*Purpose: Innate immune-driven local inflammation induced by ischemia/reperfusion injury (IRI) causes hepatic dysfunction and failure following liver transplantation. Although mesenchymal stem cell (MSC)-based therapy has been successfully applied in various human diseases, a number of clinical phase III trials of MSC immunotherapy failed to meet the primary endpoints because of the low efficacy of engrafted cells. CD47/SIRPα signaling has been implicated in regulating innate immunity during inflammatory response. This study was designed to enhance the immunosuppressive ability of MSC by dissecting the functional roles and molecular mechanisms of the CD47/SIRPα signaling in MSC-mediated immune regulation during liver IRI.

*Methods: Using a mouse liver IR injury model, the myeloid-specific β-catenin knockout (β-catenin^M-KO) and β-catenin-proficient (β-catenin^FL/FL) mice were i.v. injected with MSCs (1×10⁶) 24h prior to liver ischemia. Mice were sacrificed after 6h of reperfusion. For in vitro study, MSCs were transfected with CRISPR/Cas9-mediated CD47 knockout (KO) or control vector, and then co-cultured with bone marrow-derived macrophages (BMMs) followed by LPS stimulation.

*Results: Adoptive transfer of MSCs ameliorated IR-induced liver damage as evidenced by reduced sALT levels and histological Suzuki’s scores, diminished macrophage/neutrophil accumulation and inflammatory mediators. MSC treatment augmented CD47, signal regulatory protein alpha (SIRPα), and β-catenin but reduced Foxo1 and TLR4 expression in ischemic livers. However, disruption of myeloid β-catenin in MSC-transferred mice exacerbated liver IRI with augmented Foxo1 and TLR4 activity. Using a MSC/macrophage co-culture system, increased MSC CD47 and macrophage SIRPα expression were found after LPS stimulation. MSC increased SIRPα, p-Akt, and β-catenin expression in LPS-stimulated macrophages. However, CD47 KO in MSC reduced macrophage p-Akt and β-catenin but enhanced Foxo1 and TLR4 activity after co-culture. Moreover, disruption of macrophage β-catenin reduced M2 macrophage Arg1 and augmented M1 macrophage iNOS with increased proinflammatory TNF-α, IL-β, and MCP-1 expression even co-cultured with MSC.

*Conclusions: MSC promotes CD47/SIRPα interaction, which in turn modulates the Akt-β-catenin axis leading to diminished Foxo1/TLR4 activity in IR-triggered liver inflammation. Our findings may provide novel therapeutic strategies to treat transplant recipients using genetically modified MSC approaches or pharmacological interventions targeting the CD47/SIRPα signaling in the donor or in the graft before implantation or reperfusion.

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