Introduction
Platelet-Activating Factor Receptor (Ptafr /PAF-R) is hypomethylated in experimental cirrhosis. Its ligand, the Platelet Activating Factor (PAF) is a proinflammatory phospholipid with multiple biological activities, as aggregation of platelets and modulation of blood pressure1,2. PAF/PAF-R interaction has been shown to be relevant in pulmonary3 and renal4 fibrosis progression. However, the contribution of this pathway to chronic liver disease (CLD) pathophysiology remains unknown.
Aims & Methods
To characterize the contribution of the PAF/PAF-R pathway in CLD progression. A methylation array was performed in hepatic macrophages sorted from control and cirrhotic mice by intragastric administration of CCl4. Ptafr gene (qPCR) and protein (WB, IHC) expression was evaluated in the livers of cirrhotic and control mice and in primary non-parenchymal liver cells by flow cytometry; as well as in immortalized Kupffer cells (imKCs) in response to different proinflammatory stimuli by qPCR and WB. The presence of the PAF ligand was evaluated in serum, and the supernatant of immortalized hepatic sinusoidal endothelial cells (imLSECs) treated with proinflammatory stimuli by ELISA. The expression of enzymes involved in PAF synthesis and degradation was studied in liver samples. The consequences of treating imKCs with PAF in vitro were evaluated by qPCR and WB. The effect of PAF/PAF-R pathway hampering was tested both in vitro and in vivo by use of a PAF antagonist BN-52021.
Results
The Pafr promoter is hypomethylated in primary hepatic macrophages isolated from cirrhotic mice. We detected a significant increase in PAFR gene and protein expression in the liver of these animals. Liver cells that express PAFR during experimental cirrhosis are primarily hepatic macrophages. In vitro treatment of imKCs with the inflammatory ligands CpG and TNFα induces PAFR expression. Furthermore, stimulation of imKCs with PAF induces a polarization of imKCs toward a proinflammatory profile, increasing gene expression of proinflammatory cytokines (TNF, p≤0.01; IL1β, p≤0.01; TGFβ1, p≤0.01) and chemokines (CCL3, p≤0.05; CCL20, p≤0.0001; CXCL15, p≤0.05; and CXCL16, p≤0.01;), as well as reducing the expression of anti-inflammatory cytokines (IL4, p≤0.01; IL10, p≤.0.0001; and IL-13, p≤0.01 ). This polarization was blocked by the administration of BN-52021 to cell culture. We observed an increase in gene expression of Lpcat1, the enzyme responsible for PAF synthesis, and a decrease in Pafah2, the enzyme that degrades PAF, in liver samples from cirrhotic mice compared to controls, which explains the increase in PAF detected in the serum of cirrhotic mice. All inflammatory stimuli used (IL1b, TNFa, CpG, and LPS) induced PAF secretion by imLSECs in vitro. Cirrhotic mice treatment with BN-52102 resulted in fibrosis and liver damage amelioration (measured by sirius red, aSMA and Vimentin immunostaining), improvement of biochemical markers (hepatic transferases and bile acids) and a reduction of gene expression of proinflammatory and profibrogenic markers (Timp1, Acta2, Col1a1, IL-4, 13, 1b, Tnf, Tgfb).
Conclusion
We have detected the activation of the PAF/PAF-R pathway in experimental cirrhosis. PAF signalling through its receptor induces proinflammatory polarization of hepatic macrophages in vitro. The blockade of receptor activation with a ligand antagonist BN-52021 reduces liver damage and proinflammatory gene expression in cirrhotic mice. These data suggest the value of blocking the PAF/PAF-R pathway to reduce the progression of experimental cirrhosis and its potential application as a therapeutic target.
References
1- Yin et al. Diabetes Metab Syndr Obes. 2022 Jul 8:15:2003-30.
2- Yang et al. Gut. 2004 Jun; 53(6): 877-83.
3- Shahzad et al. J Clin Med. 2024 Apr 6;13(7):2126.
4- Jiang et al. Ren Fail. 2022; 44(1): 660-71.
