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 2

Abx treatment increases serum PGE2 and enhances EP4 expression in OLT.

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Abx treatment increases serum PGE2 and enhances EP4 expression in OLT. (...
(A–C) Intestine tissue and serum samples from portal veins of naive and Abx-treated (10 days) C57BL/6 mice were analyzed. (C, F, and G) BALB/c livers subjected to 18 hours of cold storage were transplanted into C57BL/6 mice pretreated or not with Abx for 10 days, followed by serum and liver sampling 6 hours after OLT. (D and E) Primary mouse hepatocytes were incubated with or without PGE2 (5 μM) for the indicted time periods. (A) Western blot detection and the relative intensity ratio of COX2 in intestines of naive and Abx-treated mice. β-Actin expression served as an internal control and was used for normalization (n = 3/group). (B) Representative immunohistochemical staining of COX2 in intestines from naive and Abx-treated mice (n = 3/group). Original magnification, ×100. (C) Serum PGEM levels were measured by ELISA (n = 4–6/group). *P < 0.05 versus peripheral/naive; #P < 0.05 versus portal/naive; §P < 0.05 versus peripheral/OLT. (D) qRT-PCR detection of mRNA coding for EP4 with TBP normalization (n = 4/group). †P < 0.05 versus hepatocytes without PGE2. (E) Western blot detection and relative intensity ratios of EP4 in hepatocytes. VCL expression served as an internal control and was used for normalization (n = 2/group). (F) Western blot detection of EP4 and its relative intensity ratio with VCL normalization in OLT (n = 4/group). (G) Representative immunohistochemical staining of EP4 in OLT with or without recipient Abx treatment (n = 3/group). Original magnification, ×200. Data indicate the mean ± SEM. Statistical significance was determined by Student’s t test or 1-way ANOVA followed by Tukey’s HSD test.