Antagonizing epigenetically controlled PAF/PAF-R pathway improves liver function during experimental cirrhosis

Platelet-activating factor (PAF) phospholipid is mainly produced by macrophages and involved in pro-inflammatory responses. We evaluated the regulation of PAF-R gene expression during experimental cirrhosis and whether antagonizing its ligand PAF improves liver function and inflammation. Patients with cirrhosis and CCl 4 -induced cirrhotic C57Bl/6 mice were included in the study. A subgroup of mice was treated with either PAF antagonist BN-52021 or a DNMT inhibitor, Aza, for two weeks before laparotomies. Sorted hepatic macrophages were subjected to a genome-wide DNA methylation study, and Ptafr expression analysed by Western Blot, qPCR, and immunohistochemistry in liver tissue. Immortalized Kupffer cells (imKCs) were stimulated with PAF and antigenic ligands. Cytokine and chemokine expression were measured. Biochemical and hepatic markers of liver damage were assessed. Hepatic PAF-R increased in patients and the CCl 4 cirrhotic model. PAF antagonism reduced hepatic structural damage and improved endothelial function in cirrhotic mice. Also in vivo , PAF-R signalling pathway inhibition rebalanced hepatic cytokine response modifying the Th17-Treg axis in experimental cirrhosis. PAF-R was induced by CpG and TNF-α in vitro , and the PAF-R/PAF pathway stimulation elicited proinflammatory cytokine production in imKCs. PAF-R expression was controlled by Ptafr promoter CpGs demethylation in hepatic macrophages from cirrhotic mice. This mechanism was confirmed by targeting enzymes inhibiting DNMTs in charge of DNA-methylation with Aza in imKCs. PAF antagonist BN-52021 disrupts PAF-R/PAF signaling counteracting PAF-R overexpression, and ameliorates liver injury in cirrhotic mice . Ptafr expression is controlled by promoter DNA demethylation leading to PAFR overexpression in hepatic macrophages. PAF / PAF-R pathway activation was found upregulated in plasma and liver tissue from cirrhotic patients. Analysis of publicly available sc-RNAseq data hinted towards hepatic macrophages as key players in pathway upregulation. The expression of PAF-R is mainly controlled by epigenetic dysregulation (promoter demethylation) in cirrhosis, but it can also by induced by CpG and TNF-⍺. Activation of the PAF/PAF-R pathway in vitro induces the production of proinflammatory cytokines and chemokines on immortalized murine Kupffer cells (imKCs), while administration of a PAF antagonist (BN-52021) prevents the proinflammatory differentiation of imKCs. Interestingly, blocking the PAF/PAF-R pathway in experimental cirrhosis ameliorates liver fibrosis and inflammation, and positively impacts on liver and endothelial function. • PAF/PAF-R signaling pathway is overactivated in patients with cirrhosis. • PAF/PAF-R blockade ameliorates liver injury during experimental cirrhosis. • Antagonizing PAF/PAF-R pathway restores homeostatic Treg/Th17 immune balance. • Promoter DNA methylation controls PAF-R expression in hepatic macrohages.