*Purpose: Neutrophil extracellular trap (NET) formation relates to various diseases, including sterile inflammation. Although neutrophils express proficient CEACAM1 (CC1, also known as CD66a) long isoform, its role in the regulation of NET and orthotopic liver transplantation (OLT) remains unknown. Here, we analyzed mouse and human OLT biopsies (Bx) to determine how neutrophil CC1L isoform may regulate NET formation and contribute to OLT outcomes.

*Methods: In the experimental arm, donor WT (C57/BL6) livers were transplanted, after cold storage (4^oC/18h), to groups of syngeneic WT or CC1-deficient (CC1-KO) mouse recipients, and sampled at 6h post-OLT. In parallel in vitro study, neutrophils isolated from WT or CC1-KO mice were stimulated by S1P (sphingosine 1 phosphate) with or without Bafilomycin A1 (BafA1; lysosome inhibitor). In the clinical arm, human hepatic biopsies obtained at 2h post-OLT Bx (n=55) were screened for CC1L expression (Western blots) and for pro-inflammatory phenotype (RT-PCR).

*Results: Recipient CC1 null mutation augmented IRI-OLT (WT>CC1-KO) as evidenced by increased sAST/sALT levels (p<0.05) as compared with WT recipients. Western blot analysis of OLT samples identified enhanced expression of activated caspase-11 and citrullinated histone H3 (cit H3; neutrophil extracellular trap marker) in CC1-KO recipients. In vitro, CC1-deficient neutrophils showed enhanced cleaved caspase 11, Gasdermin D and cathepsin B expression, and subsequent higher NET formation against the S1P challenge. Blockade of cathepsin B by BafA1 increased NET formation, while inhibition of S1P signaling suppressed caspase 11 activation and increased cathepsin B expression, and ultimately inhibited NET formation. This suggested the critical role of caspase 11/lysosome pathway in the regulation of NET formation. Consistent with the murine data, CC1L expression in human liver Bx correlated negatively with serum transaminase release at postoperative day 1. When differentially screened for low (n=28) vs. high (n=27) CC1L expression Bx groups, CC1L low human OLTs showed enhanced innate/adaptive immune activation (RT-PCR), and higher incidence of early allograft dysfunction (EAD; 28.6% vs 11.1%).

*Conclusions: As a checkpoint regulator of IR-stress and sterile hepatic inflammation, neutrophil CC1L may serve as a novel regulator of NET formation and a target for therapeutic modulation in OLT recipients.

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