*Purpose: The CD47-signal regulatory protein alpha (SIRPα) signaling pathway plays important roles in immune homeostasis and tissue inflammatory response. Gli1, the downstream effector of the Hedgehog pathway has been shown to regulate immune cell activation and inflammatory response. However, it remains unknown as to whether and how the CD47-SIRPα signaling may influence the Hedgehog pathway to control innate immune response in mesenchymal stem cell (MSC)-mediated immune regulation during liver inflammatory injury. This study investigated the roles and molecular mechanisms of MSC CD47-mediated Gli1/Notch1 signaling in ischemia/reperfusion-triggered liver inflammation.

*Methods: Myeloid-specific SMO or Notch1 knockout (SMO^M-KO and Notch1^M-KO) and floxed SMO or Notch1 (SMO^FL/FL and Notch1^FL/FL) mice (n=6/group) were i.v. injected with bone marrow-derived MSCs or genetically modified MSCs (1×10⁶ cells in PBS/mouse) 24h prior to surgical procedure, and then subjected to 90 min partial liver warm ischemia followed by 6 h of reperfusion. In parallel 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 (100 ng/ml) stimulation.

*Results: Adoptive transfer of MSCs increased CD47 expression and ameliorated liver IRI. However, deletion of CD47 in MSCs exacerbated IR-induced hepatocellular damage, with increased serum ALT levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators. MSC treatment augmented SIRPα, Hedgehog/SMO/Gli1, and Notch1 intracellular domain (NICD) whereas CD47-deficient MSC treatment reduced these gene expressions in IR-stressed livers. Moreover, disruption of myeloid SMO or Notch1 increased IR-triggered liver inflammation with diminished Gli1 and NICD but enhanced NEK7 and NLRP3 activation in MSC-transferred mice. Using MSC/macrophage co-culture system, we found that MSC CD47 and macrophage SIRPα expression were increased after LPS stimulation. The CD47-SIRPα interaction increased macrophage Gli1 and NICD nuclear translocation whereby NICD interacted with Gli1 and regulated its target gene Dvl2, which in turn inhibited NEK7/NLRP3 activity.

*Conclusions: The CD47-SIRPα signaling activates the Hedgehog/SMO/Gli1 pathway, which controls NEK7/NLRP3 activity through a direct interaction between Gli1 and NICD. NICD is a novel coactivator of Gli1, and the target gene Dvl2 regulated by the NICD-Gli1 complex is crucial for the modulation of NLRP3-driven inflammatory response in MSC-mediated immune regulation. Our findings provide novel potential therapeutic targets in MSC-based immunotherapy of sterile inflammatory liver injury.

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