POLY(I:C) INDUCES MORPHOLOGICAL CHANGES AND TYPE I INTERFERON-MEDIATED INFLAMMATORY RESPONSE IN MESOTHELIAL CELLS FROM PERITONEUM

Abstract Background and Aims Peritonitis is a major cause of morbidity and discontinuation of the therapy in peritoneal dialysis (PD) patients. Most of peritonitis episodes during PD may be imputed to bacterial infection, although in about 20% of the cases a viral origin may be hypothesized. Toll-like receptors (TLRs) play a critical role in innate immune responses by specifically recognizing molecular patterns from different microorganisms, including bacteria, fungi, and viruses. Polyinosinic:polycytidylic acid (Poly(I:C)) is a synthetic analogue of double-stranded RNA (dsRNA) that mimics the anti-viral immune response by activating TLR3. Upon activation, TLR3 signaling pathway leads to the induction of NF-ΚB pathway and the interferon response (IRF3 activation). The aim of this work is to study the ex vivo and in vivo effect of Poly(I:C) treatment in primary human mesothelial cells (MCs) and mice peritoneum. Methods MCs were collected from effluent fluids of 8 clinically stable PD patients and amplified for ex vivo experiments. MCs were treated with Poly(I:C) (2 ng/μl) or TGFβ1 (2 ng/ml). After 48 hours of stimulation, samples were collected for RNA subsequent analysis. Poly(I:C) was intraperitoneally delivered to mice daily for 10 days, using two different doses: 30 mg/kg (n = 6) and 90 mg/kg (n = 4) of weight. Non-treated mice were used as control (n = 6). Mice were euthanized and peritoneal tissue was collected for the subsequent RNA, protein, and histological experiments. Gene expression analyses of TLRs, interferon-stimulated genes (ISGs), and other inflammatory markers were performed by qPCR. Protein analysis from cell lysates was performed by western blot and cytokine and chemokine levels from cell supernatants were detected by ELISA assay. Results MCs from PD patients expressed a specific subset of TLRs, which were modulated by stimulation with Poly(I:C), being TLR3 the most induced receptor. Additionally, Poly(I:C) induced a bona fide mesothelial to mesenchymal transition (MMT), characterized by the acquisition of a spindle-like morphology, increased expression of mesenchymal markers such as SNAIL, TGFB1, FN1, MMP9, and MMP14, and decreased expression of the epithelial markers ECAD and CALB2. Moreover, Poly(I:C) increased mRNA and protein levels of several cytokines and chemokines, such as TNFα, IL-6, IL-1β, IFNβ, CXCL8, and CXCL9; and ISGs, including CXCL10, MX1, IFIT1, and IFITM1. In vivo, Poly(I:C) administration in mice induced peritoneal inflammation, characterized by increased gene expression of proinflammatory response-related factors, including Ccl5, Arg1, and the ISGs Cxcl10 and Ifit1, in a dose-dependent manner. Conclusion Treatment with Poly(I:C) is sufficient to induce a profibrotic/proinflammatory response in MCs in ex vivo and in vivo settings. These discoveries highlight the role of viral infections in peritoneum damage and may provide insight for further studies aimed at specifically counteracting the effect of viral infections in peritonitis in PD patients.