Background:Corneal transparency may be compromised by viral infections causing corneal scarring, edema, and neovascularization. Ocular injury results from collateral damage induced by exacerbated immune response in corneal stroma. Myofibroblasts play a key role in this process by producing a disorganized extracellular matrix and inflammatory mediators. However, the immune response profile of myofibroblasts during viral infections is still under study. The aim of this work was to analyze the cytokine profile of human limbal myfibroblasts (HLMs) stimulated with the double-stranded RNA analog polyinosinic:polycytidylic acid (poly I:C) and to identify their signaling pathways. Methods:HLMs were isolated from cadaveric sclera-corneal rims and stimulated with poly I:C (10 μg/ml) for 12 h. The secretion of 36 cytokines was measured using the Human Cytokine Array Panel A. The secretion of IFN-β was quantified by ELISA. The expression of pattern recognition receptors (PRRs) such as TLR3, RIG-1 and MDA5 were analyzed by western blot assays. Furthermore, translocation of the nuclear factors NF-κB, IRF3, and IRF7 was assessed by fluorescence staining. In addition, the differentially expressed cytokines were analyzed using the Core Analysis Tool of the Ingenuity Pathway Analysis IPA software. Results:HLMs stimulated with poly I:C increased (fold change > 2) the secretion of G-CSF, sTREM-1, CXCL1, CCL1, CXCL8, CXCL10, CXCL11, CCL2, CCL5, IL-13, IL-6, IL-1ra, and IFN-β compared with HLMs under basal conditions. Poly I:C stimulation also induced the expression of RIG-1 (p < 0.001), but the expression of TLR3 and MDA5 was unmodified. Finally, HLMs increased nuclear translocation of NF-κB, IRF3, and IRF7 after poly I:C stimulation. Bioinformatic analysis identified canonical signaling pathways associated with cell adhesion and diapedesis, chemokine signaling, and activation of IRFs by cytosolic pattern recognition receptors. Conclusions:These results demonstrate that HLMs secrete cytokines involved in immune cell activation and chemotaxis. The data suggest a key role for HLMs during viral infections in cornea and extend our knowledge about the signaling pathways they trigger.Keywords:Antiviral response; Cornea; Inflammation; Innate immunity; Myofibroblats.

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