Super enhancer regulation of cytokine-induced chemokine production in alcoholic hepatitis

Alcoholic hepatitis (AH) is associated with liver neutrophil infiltration through activated cytokine pathways leading to elevated chemokine expression. Super-enhancers are expansive regulatory elements driving augmented gene expression. Here, we explore the mechanistic role of super-enhancers linking cytokine TNFα with chemokine amplification in AH. RNA-seq and histone modification ChIP-seq of human liver explants show upregulation of multiple CXCL chemokines in AH. Liver sinusoidal endothelial cells (LSEC) are identified as an important source of CXCL expression in human liver, regulated by TNFα/NF-κB signaling. A super-enhancer is identified for multiple CXCL genes by multiple approaches. dCas9-KRAB-mediated epigenome editing or pharmacologic inhibition of Bromodomain and Extraterminal (BET) proteins, transcriptional regulators vital to super-enhancer function, decreases chemokine expression in vitro and decreases neutrophil infiltration in murine models of AH. Our findings highlight the role of super-enhancer in propagating inflammatory signaling by inducing chemokine expression and the therapeutic potential of BET inhibition in AH treatment.


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Mycoplasma contamination -No restrictions on data availability -JASPAR database accessed from http://jaspar.genereg.net/ 4 healthy and 5 AH individuals were used in ChIP-seq and 4 healthy and 6 AH individuals were included in bulk RNA-seq. No sample size calculation was performed. Sample size was sufficient for this exploratory assessment of AH gene expression and histone modification.
No data were excluded from the study.
We generated genome-wide maps for 4 histone modifications including H3K4me1, H3K4me3, H3K27ac, and H3K27me3 in 4 healthy and 5 AH individuals. We generated RNA-seq data in 4 healthy and 6 AH individuals. The different subjects within healthy and AH groups were treated as replicates, respectively, in differential binding analysis and differential gene expression analysis between the two groups. Whole genome sequencing data was not performed without technical replicates but quality control assessment was performed as described. Low quality samples were not used in subsequent analysis.
Randomization is not applicable given exploratory nature of the experiment.
Blinding is not applicable given exploratory nature of the experiment.
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Genome browser session None were used in the study Wildtype C57BL/6 mice from Envigo Laboratories were used in the study. Mice were aged 10-12 weeks. All mice used in experiment were female to minimize heterogeneity of alcohol feeding. Mice were kept in controlled environment, with 12 hours of light/dark (6PM-6AM), ambient temperature at 68-79°F (~20-26°C) with 30-70% humidity.
No wild animals were used.
Study did not involve any samples collected from the field.
All animal work was performed under Mayo Institutional Animal Care and Use Committee oversight, in AAALAC-accredited facilities.
Liver samples were obtained from patients with alcoholic hepatitis at the time of liver transplant or patients with no known chronic liver disease who underwent liver resection for another cause (such as resection of benign liver lesions). Both male and female subjects were included in both groups, age range for patients with AH was 32-60, and control was 58-71. Patients in the AH group were diagnosed with severe AH, and patients in control groups had no known liver disease. Further clinical characteristics are summarized in Supp Fig 1. Alcoholic hepatitis patients were recruited around the time of liver transplant for permission to use explant liver tissue for research studies at University of Lille, France. Control patients were recruited around the time of liver resection surgery for permission to use resected liver tissue for research studies at Mayo Clinic, Rochester, MN. No expect biases, including selfselection bias was expected from patient recruitment. Recruitment and participation in the trial has no impact on patient's clinical treatment course or prognosis. IRB protocols were approved by the Ethics Committee of the University of Lille, France for alcoholic hepatitis samples, and Mayo Clinic for control patients. Studies were performed with informed consent and in accordance with the Declaration of Helsinki.
All RNA-seq and ChIP-seq data generated in this publication will be available publication of this manuscript on the GEO database (GSE155926 and166564).
All RNA-Seq and ChIP-Seq files used in this study No longer applicable 4 normal and 5 AH samples were included in ChIP-seq. 51 bp paired-end reads. Between 23.8 and 56.7 million pairs of raw reads were generated per sample.