Microarray of PBMC from patients with decompensated cirrhosis (AD) and ACLF stimulated with CpG-DNA and treated with human serum albumin (HSA)

Human serum albumin (HSA) is an emerging treatment for preventing excessive systemic inflammation and organ failure(s) in patients with acutely decompensated (AD) cirrhosis. Here, we investigated the molecular mechanisms underlying the immunomodulatory properties of HSA. Administration of HSA to patients with AD cirrhosis with elevated circulating bacterial DNA (CpG-DNA) was associated with reduced plasma cytokine levels. In isolated leukocytes, HSA abolished CpG-DNA-induced cytokine expression and release independently of its oncotic and scavenging properties. Similar anti-inflammatory effects were observed with recombinant human albumin. HSA exerted widespread changes on the immune cell transcriptome, specifically in genes related to the endosomal compartment involved in cytosolic DNA sensing and type I interferon responses. Flow cytometry and confocal microscopy analyses revealed that HSA was taken up by leukocytes and internalized in vesicles positively stained with EEA1, a marker of early endosomes. Indeed, HSA and CpG-DNA colocalized in endosomes, the compartment where CpG-DNA binds to TLR9, its cognate receptor. Furthermore, HSA also inhibited poly-(I:C)- and LPS-induced IRF3 phosphorylation and TRIF-mediated responses, which are exclusive of endosomal TLR3 and TLR4 signaling, respectively. The immunomodulatory actions of HSA did not compromise leukocyte defensive mechanisms such as phagocytosis, efferocytosis and intracellular ROS production. The in vitro immunomodulatory effects of HSA were confirmed in vivo in analbuminemic humanized FcRn transgenic mice. In conclusion, these findings indicate that HSA internalizes in immune cells and modulates their responses through interaction with endosomal TLR signaling, thus providing a mechanism for the benefits of HSA infusions in patients with cirrhosis. Microarray of PBMC from patients with decompensated cirrhosis (AD) and ACLF stimulated with CpG-DNA and treated with human serum albumin (HSA). PBMC were isolated from 20 ml of EDTA blood using a ficoll-Hypaque density gradient separation method. PBMC were seeded at a density of 1.0x10e6 cells/mL and incubated with HSA (15 mg/ml) for 24 h in the presence of CpG-DNA (4 µM) or vehicle for the last 4 hours. Cells were collected and RNA was isolated using TRIzol reagent and quantified with nanodrop. RNA integrity was re-checked in the Bioanalyzer 2100 before performing microarray hybridization. The average RIN value of total RNAs was 9.05 (from 8 to 9.5 range). Processing of RNA samples, fragmentation and labelling of single-stranded-cDNA were prepared according to Affymetrix WT PLUS Reagent Kit user guide in an automated system (Beckman FX robot). Following fragmentation and terminal labeling, single-stranded-cDNAs were hybridized for 17 hours at 45ºC on the GeneChip Human gene 2.1ST 24 - array plates, using the automated GeneTitan System, which includes the hybridization oven, the fluidic station and the scanner.