Peritoneal macrophage heterogeneity in decompensated liver cirrhosis

Peritoneal macrophages (PM) are thought to regulate peritoneal inflammation and control bacterial infections in decompensated liver cirrhosis. The aim of this study was to characterize human PM heterogeneity. Employing CD206 surface expression, we identified subsets of human large (LPM) and small (SPM) PM, which differed in granularity and maturation states. FACS-sorted LPM from patients with decompensated cirrhosis revealed discrete transcriptome clusters, comprising more than 4000 differentially regulated genes involved in cell cycle, metabolism, and immune signaling. Mononuclear cells from patients with decompensated liver cirrhosis (n=4) were purified from ascitic fluid by Ficoll density gradient centrifugation using Lympholyte-H (Cedarlane). Peritoneal macrophages were enriched using positive magnetic assisted cell sorting (Milteny Biotec). PM subsets (SPMs and LPMs) were sorted by flow cytometry on a FACSAria III (BD Biosciences) using monoclonal antibodies against CD14 (PE) and CD206 (APC or FITC). Sorted cells were seeded in cell culture plates at a density of about 100k cell per cm2 (0.75 – 1.0 mio cells per well) and treated with 10 ng/ml E. coli lipopolysaccharide (LPS) for 3h or left untreated. Total RNA was isolated and microarray analysis was performed by a commercial transcriptomics provider (Aros Applied Biotechnology, Eurofins, Aarhus, Denmark) using the Affymetrix Human Clariom S HT 24 array plate and the WT plus kit for labeling. CHP files were created using Transcriptome analysis concole (TAC, ThermoFisher Scientific). GC Correction, SST (Signal Space Transformation), RMA background and quantile normalization were applied. Differential gene expression between SPMs and LPMs was analyzed in steady state or following LPS stimulation. Before extracting differentially expressed genes, patient dependent differences were eliminated using repeated measure option (pairwise analysis or batch effect removal) of TAC software package.