*Purpose: Liver ischemia-reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and predisposes to rejection crises in orthotopic liver transplantation (OLT). We and others have reported on the regulation of hepatic macrophage activation by Notch1, a highly conserved transmembrane receptor involved in cell fate decision, proliferation and regeneration, in warm hepatic IRI model. However, little is known as to its function in the pathogenesis of liver IRI in OLT. We produced myeloid-specific Notch1-deficent (mNotch1-KO) mice (C57/BL6) to study putative role of Notch1 myeloid cells in IR-stressed OLT recipients.

*Methods: WT (C57/BL6) livers subjected to extended (18 hr) cold storage were transplanted into groups of WT and mNotch1-KO syngeneic recipients; OLT and blood samples were collected at 6h after reperfusion. HMGB1/Histone-enriched liver flush (LF) was collected after infusing cold-stored WT livers with physiological saline. Bone marrow-derived neutrophil cultures from WT and mNotch1-KO mice were stimulated with LPS/LF while mouse primary hepatocyte cultures (WT) were treated with or without IL22.

*Results: In contrast to WT>WT OLT, recipient myeloid Notch1 deficiency (WT>mNotch1-KO) alleviated IRI, as evidenced by: 1/ decreased sALT (2462±1415 vs 5065±2654 IU/L, n=6/6, p=0.020) and sAST (1720±1093 vs 2853±797 IU/L, n=6/6, p=0.030) levels at 6h post-reperfusion; 2/ depressed Suzuki’s histological IRI grading (p<0.05); 3/ lower frequency of TUNEL+ cells (p<0.05); 4/ inhibited macrophage/neutrophil infiltration in OLT (IHC, p<0.05); 5/ decreased mRNA levels coding for CXCL1/CXCL2/CXCL10/TNFα/CHOP (p<0.05). LPS/LF-activated bone marrow-derived neutrophils from mNotch1-KO mice showed decreased mRNA levels coding for CXCL1/CXCL2/TNFα/IL1β/CXCR2 but enhanced mRNA levels for Ym1, as compared to neutrophils from WT mice, indicating disruption of Notch1 signaling shifted neutrophil activation towards anti-inflammatory (N2) phenotype. In addition, neutrophil Notch1 disruption increased the expression of IL22, an anti-inflammatory and cytoprotective cytokine. Immunohistochemistry and flow cytometry analyses revealed IL22 was predominantly expressed by Ly6G-positive neutrophils in OLTs. Addition of IL22 increased p-Stat3 while decreasing CHOP levels in primary mouse hepatocyte cultures (Western Blots).

*Conclusions: Disruption of Notch1 signaling pathway in recipient-derived myeloid cells exerts hepatoprotection in IR-stressed OLT, at least in part by shifting host neutrophils into IL22 driven anti-inflammatory phenotype. By demonstrating pathogenic role of Notch1 in recipient myeloid cells, our findings add new insights to the complex function of Notch1 in OLT recipients.

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