Histone acetylation/deacetylation play an essential role in modifying chromatin structure and in regulating cell plasticity in eukaryotic cells. Therefore, histone deacetylase (HDAC) pharmacological inhibitors are promising tools in the therapy of fibrotic diseases and in cancer. Peritoneal fibrosis is a pathological process characterized by many cellular and molecular alterations, including the acquisition of invasive/pro-fibrotic abilities by mesothelial cells (MCs) through induction of mesothelial to mesenchymal transition (MMT). The aim of this study was to characterize the molecular mechanism of the antifibrotic role of HDAC1 inhibition. Specifically, treatment with MS-275, an HDAC1-3 inhibitor previously known to promote MMT reversal, induced the expression of several TGFBRI mRNA-targeting miRNAs. Among them, miR-769-5p ectopic expression was sufficient to promote MMT reversal and to limit MC migration and invasion, whereas miR-769-5p silencing further enhanced mesenchymal gene expression. These results were confirmed by HDAC1 genetic silencing. Interestingly, miR-769-5p silencing maintained mesenchymal features despite HDAC1 inhibition, thus indicating that it is necessary to drive MMT reversal induced by HDAC1 inhibition. Besides TGFBRI, miR-769-5p was demonstrated to target SMAD2/3 and PAI-1 expression directly. When analyzing molecular mechanisms underlying miR-769-5p expression, we found that the transcription factor Wilms’ tumor 1 (WT1), a master gene controlling MC development, binds to the miR-769-5p promoter favoring its expression. Interestingly, both WT1 expression and binding to miR-769-5p promoter were increased by HDAC1 inhibition and attenuated by TGFβ1 treatment. Finally, we explored the significance of these observations in the cell-to-cell communication: we evaluated the ability of miR-769-5p to be loaded into extracellular vesicles (EVs) and to promote MMT reversal in recipient mesenchymal-like MCs. Treatment of fibrotic MCs with EVs isolated from miR-769-5p over-expressing MCs promoted the down-regulation of specific mesenchymal targets and the reacquisition of an epithelial-like morphology. In conclusion, we highlighted an HDAC1-WT1-miR-769-5p axis potentially relevant for therapies aimed at counteracting organ fibrosis.
While blue LED (b-LED) light is increasingly being studied for its cytotoxic activity towards bacteria in therapy of skin-related infections, its effects on eukaryotic cells plasticity are less well characterized. Moreover, since different protocols are often used, comparing the effect of b-LED towards both microorganisms and epithelial surfaces may be difficult. The aim of this study was to analyze, in the same experimental setting, both the bactericidal activity and the effects on human keratinocytes. Exposure to b-LED induced an intense cytocidal activity against Gram-positive (i.e, Staphylococcus aureus) and Gram-negative (i.e., Pseudomonas aeruginosa) bacteria associated with catheter-related infections. Treatment with b-LED of a human keratinocyte cell line induced a transient cell cycle arrest. At the molecular level, exposure to b-LED induced a transient downregulation of Cyclin D1 and an upregulation of p21, but not signs of apoptosis. Interestingly, a transient induction of phosphor-histone γ-H2Ax, which is associated with genotoxic damages, was observed. At the same time, keratinocytes underwent a transient epithelial to mesenchymal transition (EMT)-like phenotype, characterized by E-cadherin downregulation and SNAIL/SLUG induction. As a functional readout of EMT induction, a scratch assay was performed. Surprisingly, b-LED treatment provoked a delay in the scratch closure. In conclusion, we demonstrated that b-LED microbicidal activity is associated with complex responses in keratinocytes that certainly deserve further analysis.
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.
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