Antibiotic pretreatment alleviates liver transplant damage in mice and humans
Kojiro Nakamura, … , Ronald W. Busuttil, Jerzy W. Kupiec-Weglinski
Kojiro Nakamura, … , Ronald W. Busuttil, Jerzy W. Kupiec-Weglinski
Published July 22, 2019
Citation Information: J Clin Invest. 2019;129(8):3420-3434. https://doi.org/10.1172/JCI127550.
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Research Article Immunology

Antibiotic pretreatment alleviates liver transplant damage in mice and humans

Abstract

Although modifications of gut microbiota with antibiotics (Abx) influence mouse skin and cardiac allografts, its role in orthotopic liver transplantation (OLT) remains unknown. We aimed to determine whether and how recipient Abx pretreatment may affect hepatic ischemia-reperfusion injury (IRI) and OLT outcomes. Mice (C57BL/6) with or without Abx treatment (10 days) were transplanted with allogeneic (BALB/c) cold-stored (18 hours) livers, followed by liver and blood sampling (6 hours). We divided 264 human OLT recipients on the basis of duration of pre-OLT Abx treatment into control (Abx-free/Abx <10 days; n = 108) and Abx treatment (Abx ≥10days; n = 156) groups; OLT biopsy (Bx) samples were collected 2 hours after OLT (n = 52). Abx in mice mitigated IRI-stressed OLT (IRI-OLT), decreased CCAAT/enhancer-binding protein homologous protein (CHOP) (endoplasmic reticulum [ER] stress), enhanced LC3B (autophagy), and inhibited inflammation, whereas it increased serum prostaglandin E2 (PGE2) and hepatic PGE2 receptor 4 (EP4) expression. PGE2 increased EP4, suppressed CHOP, and induced autophagosome formation in hepatocyte cultures in an EP4-dependent manner. An EP4 antagonist restored CHOP, suppressed LC3B, and recreated IRI-OLT. Remarkably, human recipients of Abx treatment plus OLT (Abx-OLT), despite severe pretransplantation clinical acuity, had higher EP4 and LC3B levels but lower CHOP levels, which coincided with improved hepatocellular function (serum aspartate aminotransferase/serum aspartate aminotransferase [sALT/sAST]) and a decreased incidence of early allograft dysfunction (EAD). Multivariate analysis identified “Abx-free/Abx <10 days” as a predictive factor of EAD. This study documents the benefits of Abx pretreatment in liver transplant recipients, identifies ER stress and autophagy regulation by the PGE2/EP4 axis as a homeostatic underpinning, and points to the microbiome as a therapeutic target in OLT.

Authors

Kojiro Nakamura, Shoichi Kageyama, Takahiro Ito, Hirofumi Hirao, Kentaro Kadono, Antony Aziz, Kenneth J. Dery, Matthew J. Everly, Kojiro Taura, Shinji Uemoto, Douglas G. Farmer, Fady M. Kaldas, Ronald W. Busuttil, Jerzy W. Kupiec-Weglinski

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Figure 3

CHOP expression is inhibited concomitantly with LC3B enhancement via PGE2/EP4 signaling in hepatocytes as well as by recipient Abx pretreatment in OLT.

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CHOP expression is inhibited concomitantly with LC3B enhancement via PGE...
(A) Primary mouse hepatocytes were incubated with DMSO, PGE2 (5 μM), or PGE2 (10 μM) for 60 minutes. Western blot detection and relative intensity ratios of CHOP and p-S6K. VCL expression served as an internal control and was used for normalization (n = 3/group). (B) Primary mouse hepatocytes were incubated with PGE2 (5 μM) for 60 minutes, and the EP4 antagonist (20 nM, ONO-AE3-208) was supplemented 15 minutes prior to PGE2 incubation in some cell cultures. Western blot detection and relative intensity ratios of CHOP and p-S6K with VCL normalization (n = 3/group). (C) Representative immunohistochemical images show detection of LC3B (red) in hepatocytes cultured with DMSO, PGE2, or PGE2 plus the EP4 antagonist (actin: green; DAPI: blue). n = 3. Original magnification, ×400. (D) BALB/c mouse livers subjected to 18 hours of cold storage were transplanted orthotopically into allogeneic C57BL/6 mice pretreated or not with Abx for 10 days, followed by OLT sampling 6 hours after reperfusion. Western blot detection of CHOP, p-S6K, and LC3B and relative quantifications with VCL normalization in OLT (n = 4/group). (E) Representative immunohistochemical detection of LC3B (green) in OLT, with or without recipient Abx pretreatment (DAPI: blue). n = 3. Original magnification, x400. Data indicate the mean ± SEM. *P < 0.05, Student’s t test or 1-way ANOVA followed by Tukey’s HSD test.