*Purpose: We and others have reported on benefits of endoplasmic reticulum (ER) stress modulation and autophagy enhancement to mitigate ischemia-reperfusion injury (IRI) in orthotopic liver transplantation (OLT). Although gut microbiota may influence skin/cardiac allograft rejection, its role in OLT recipients is unknown. We employed a mouse allo-OLT model and analyzed human OLTs to determine as to whether and how recipient antibiotics pretreatment (Abx Rx) may influence ER stress – autophagy crosstalk and OLT outcomes.

*Methods: In the experimental arm, mouse recipients (C57BL/6) with or without Abx Rx (amoxicillin, x10 days) were transplanted with allogeneic (Balb/c) cold-stored (18h) livers, followed by liver/blood sampling at 6h post-reperfusion. In the clinical arm, 264 human OLT recipients were retrospectively divided into Abx (Abx Rx ≥10 days, n=156) vs control (Abx-free or Abx<10 days, n=108) groups and analyzed by a logistic regression analysis; while OLT biopsies (Bx; n=52) collected at 2h after reperfusion were analyzed by Western blots (WB).

*Results: In mouse allo-OLT recipients, Abx Rx mitigated hepatic IRI, evidenced by: 1/ depressed sALT/sAST, Suzuki’s histological grading, frequency of TUNEL+ cells; 2/ inhibited neutrophil/macrophage infiltration (IHC); 3/ decreased CHOP, pS6K (mTOR indicator) but enhanced LC3B-II (WB) expression; and 4/ suppressed mRNA levels coding for CXCL10/MCP1/CXCL2. Abx Rx increased serum Prostaglandin E2 (PGE2) levels (ELISA), enhanced COX2 (an enzyme for PGE2 synthesis) in the intestine (WB/IHC), and increased hepatic PGE2 receptor 4 (EP4) expression (WB/IHC). In primary mouse hepatocyte cultures, addition of PGE2 increased EP4, suppressed pS6K/CHOP and promoted LC3B, while EP4 antagonist exerted opposite effects. Addition of EP4 antagonist to Abx-treated OLT recipients restored pS6K/CHOP while limiting LC3B-II conversion; and recreated IRI, evidenced by sALT/sAST, Suzuki’s grading and frequency of TUNEL+ cells. In the clinical arm, OLT patients in Abx Rx group (n=156) had decreased sALT (p<0.001), sAST (p=0.044); and experienced lower incidence of early allograft dysfunction (EAD, 21.8 vs 32.4%, p=0.038) as compared to control OLTs (n=108). A multivariate analysis identified cold ischemia time (p=0.001) and Abx<10 days regimen (odds ratio=1.79, p=0.049) as independent EAD risk factors. WB analyses of clinical liver Bx revealed Abx group (n=28) had higher EP4 (p=0.022), LC3B (p=0.038) and LC3B-II (p=0.007); but lower pS6K (p=0.033) and CHOP (p=0.056) as compared to controls (n=24).

*Conclusions: This translational study documents striking benefits of Abx Rx prior to OLT and identifies PGE2/EP4-dependent ER stress/autophagy regulation as important mechanistic underpinnings, implementing gut microbiome as putative target for novel therapeutic intervention in OLT.

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