Avaliação in vitro de alterações biomecânicas, bioquímicas e moleculares de sinoviócitos semelhantes a fibroblastos humanos (HFLS) infectados pelo vírus Chikungunya

Abstract

Chikungunya virus (CHIKV) is an arbovirus transmitted by Aedes sp. mosquitoes that causes Chikungunya fever (CHIKF) and could be progress to Rheumatic Chronic Syndrome (RSC). The synovial joints are affected during this infection, and fibroblastlike synoviocytes (FLS) are the major cell type in the composition of synovial tissue. FLS in pathologies such as rheumatoid arthritis (RA), assumes an aggressive profile, causing severe tissue damage in the inflammatory process. This damage can occur due to changes in cellular biomechanical properties. Thus, the aim of this work was to understand biomechanical, biochemical and molecular changes in human fibroblast-like synoviocytes (HFLS) after CHIKV infection in vitro. Determination of the MOI was performed by MTT assay and viral infection was confirmed by flow cytometry, RT-PCR and infection of Vero E6 cells with HFLS supernatant. The following were evaluated: morphological changes and Young`s modulus by atomic force microscopy (AFM); cytoskeleton changes by fluorescence analysis of F-actin labeling; biochemical changes were determined by Raman spectroscopy; relative gene expression analysis by RT-qPCR. Also, the quantification of chemokines was measured in the supernatant using the CBA methodology. As result, HFLS were infected in vitro since 46.8% of the cells were CHIKV-positive after 48h of infection in the intracellular flow cytometry analysis. In addition, viral RNA in the cell supernatant was detected by RT-PCR. The detection of plaque-forming units (PFUs) in the monolayer of infected Vero E6 cells indicated the presence of viable viral particles in the HFLS supernatant. AFM analysis shows significant changes in cell morphology and nanoindentation analysis detected a 107.46% increase in Young's elastic modulus, suggesting alterations in the cytoskeleton. Fluorescence cytoskeleton staining indicated disruption of F-actin filaments after 12h of infection. Raman spectroscopy and PCA analysis show a total variance of 65% between uninfected and CHIKV-infected HFLS. In addition, the major alterations detected were related to amide I, amide III, β-sheets, lipids and collagen, with signatures in 795, 977, 1031, 1223, 1290, 1444, 1495, 1635, 1660 and 1709 cm-1 . Relative gene expression shows an increase in the expression of MMP1, VEGFA and UGDH. Also, an increase in the IP10 and a reduction in MCP-1 chemokine levels were detected in the supernatant 48 after viral infection. In summary, the results presented in this work help elucidate the cellular and molecular changes involved in the CHIKV-HFLS interaction, thus contributing to developing new therapeutic strategies for this arbovirus.