Background & objectives Alcoholic hepatitis (AH) is a common but life-threatening disease with limited treatment options. It is thought to result from hepatocellular damage, but the presence of cholestasis worsens prognosis, so we examined whether bile ducts participate in the pathogenesis of this disease.
Design Cholangiocytes derived from human bile ducts were co-cultured with neutrophils from patients with AH or controls. Loss of type 3 inositol 1,4,5-trisphosphate receptor (ITPR3), an apical intracellular calcium channel necessary for cholangiocyte secretion, was used to reflect cholestatic changes. Neutrophils in contact with bile ducts were quantified in liver biopsies from patients with AH and controls and correlated with clinical and pathological findings.
Results Liver biopsies from patients with AH revealed neutrophils in contact with bile ducts, which correlated with biochemical and histological parameters of cholestasis. Cholangiocytes co-cultured with neutrophils lost ITPR3, and neutrophils from patients with AH were more potent than control neutrophils. Biochemical and histological findings were recapitulated in an AH animal model. Loss of ITPR3 was attenuated by neutrophils in which surface membrane proteins were removed. RNA-seq analysis implicated integrin β1 (ITGB1) in neutrophil-cholangiocyte interactions and interference with ITGB1 on cholangiocytes blocked the ability of neutrophils to reduce cholangiocyte ITPR3 expression. Cell adhesion molecules on neutrophils interacted with ITGB1 to trigger RAC1-induced JNK activation, causing a c-Jun-mediated decrease in ITPR3 in cholangiocytes.
Conclusions Neutrophils bind to ITGB1 on cholangiocytes to contribute to cholestasis in AH. This previously unrecognised role for cholangiocytes in this disease alters our understanding of its pathogenesis and identifies new therapeutic targets.
- alcoholic liver disease
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MHN and JW are joint senior authors.
Contributors MT performed the majority of experiments, analysed the data and wrote the first draft of the manuscript. PTV participated in histological analysis of biopsy specimens. MTG helped with imaging of NETs and super-resolution imaging. MH contributed to clinical data collection for each patient group. MER provided access to relevant human liver biopsy specimens and contributed to clinical data of all patients in which liver biopsies were obtained. MO-M contributed to the clinical study of alcoholic hepatitis and cirrhosis. SA assisted with RNA-seq analysis. TK assisted with cell culture studies. RK assisted with neutrophil calcium signalling. EK assisted with NFκB silencing and TLR4 inhibitors. RRR obtained clinical specimens, including all blood samples. JMB provided assistance related to the NHC cell line. MHN conceived of the idea, designed the research project, supervised the study and edited the manuscript. JW designed the research, planned and conducted experiments, analysed the data, supervised the study, generated figures and edited the manuscript.
Funding This work was supported by the Gladys Phillips Crofoot Professorship and grants from the National Institutes of Health (P01-DK57751, P30-DK34989, R01-DK114041, R01-DK112797 and S10-OD023598).
Competing interests None declared.
Patient consent for publication Not required.
Ethics approval This study was conducted under the auspices of protocols approved by the Institutional Review Board on the Protection of the Rights of Human Subjects (Yale University). The Human Investigation Committee protocol numbers are HIC-2000025846 and HIC-1304011763. Written informed consent was obtained from all participants. The study protocol conformed to the ethical guidelines of the Helsinki Declaration. Animal studies were approved by Yale University’s Institutional Animal Care and Use Committee (IACUC).
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement The RNA-seq data have been deposited in the Gene Expression Omnibus under the accession number GSE146899.
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