*Purpose: Although CEACAM1 (CM1; carcinoembryonic antigen-related cell adhesion molecule 1; CD66a) regulates hepatic metabolism, its role in orthotopic liver transplantation (OLT) is unknown. A recent study identified P38 MAPK signaling in the mechanism of cell death. We analyzed mouse OLTs and human OLT biopsies (Bx) to determine as to whether/how hepatocyte CM1-p38 axis may contribute to OLT outcomes.

*Methods: WT or CM1-deficient (CM1-KO) mouse livers (C57/BL6) were transplanted, after cold storage (UW/4℃/18h), to syngeneic WT recipients, and sampled at 6h post-OLT. Separate groups of cold-stored WT or CM1-KO livers were perfused with saline to collect liver flush (LF). Bone marrow-derived macrophage (BMDM) cultures were then stimulated with LF from WT vs. CM1-KO cold-stored livers. In the clinical arm, human cold-stored pre-OLT Bx (n=63) were screened for CM1/phosphor-p38 (pP38) expression by Western blots (WB), while corresponding post-OLT Bx (at 2h post-reperfusion) were screened by RT-PCR.

*Results: Compared with WT>WT mouse OLTs, donor liver CM1 disruption (CM1-KO>WT) aggravated OLT damage, evidenced by: 1/ sALT/sAST levels (n=10/10, p<0.05),; 2/Suzuki's histological hepatic grading (p<0.05); 3/ frequency of TUNEL+ cells (p<0.05); 4/ increased serum HMGB1 levels (ELISA, p<0.05); 5/ enhanced Ly6G+/CD11b+ cell infiltration (IHC, p<0.05); and 5/ raised MCP1/CXCL2/CXCL10/IL1β/IL2 (p<0.05). In primary mouse hepatocyte cultures, CM1 deficiency augmented pP38 expression (WB, p<0.05), evidenced by AST/ALT release into culture medium (p<0.05). Moreover, CM1-deficient cold-stored livers showed enhanced pP38 expression (p<0.05), with LF from CM1-KO grafts enriched in HMGB1/Histone H3 (WB, p<0.05), and readily triggering BMDM activation in vitro, evidenced by MCP1/CXCL2/CXCL10 levels (p<0.05), as compared to LF from WT grafts. In the clinical arm, pre-OLT CM1 levels in human hepatic Bx negatively correlated with pP38 (r=-0.369, p=0.003); sALT (r=-0.315, p=0.012) and sAST(r=-0.269, p=0.033) at POD1. Unlike in pre-OLT "high" CM1 group (n=31), pre-OLT "low" CM1 livers (n=32) were characterized by: 1/ increased sAST (710±299 vs 268±40 IU/L, p=0.014) and sALT (420±49 vs 287±45 IU/L, p=0.021) at POD1; 2/ enhanced TLR4/CD86/CD68 (macrophage markers), Cathepsin G (neutrophil marker), CD4/CD28 (T cell markers) in OLT Bx; and 3/ impaired overall graft survival (3-y: 76.7 vs 90.8%, p=0.1219) and rejection-free graft survival (3-y: 64.7 vs 81.5%, p=0.1383).

*Conclusions: This translational study identifies a novel hepatocyte CM1-P38 signaling pathway, which protects liver grafts against ischemia/surgery stress and controls innate – adaptive immune interface/OLT outcomes. Hence, CM1-P38 axis represents not only a new biomarker of hepatocellular function in cold-stored livers, but may also serve as a target for therapeutic intervention in OLT recipients.

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