key: cord-0259720-p198bpd5 authors: Pott, Fabian; Postmus, Dylan; Brown, Richard J. P.; Wyler, Emanuel; Neumann, Elena; Landthaler, Markus; Goffinet, Christine title: Single-cell transcriptomic analysis of antiviral responses and viral antagonism in Chikungunya virus-infected synovial fibroblasts date: 2021-06-11 journal: bioRxiv DOI: 10.1101/2020.06.07.138610 sha: ef2317f7c41f999fca298e758cd2333658315250 doc_id: 259720 cord_uid: p198bpd5 In recent years, (re-)emerging arboviruses including Chikungunya virus (CHIKV) and Mayaro virus (MAYV) have caused growing concern due to expansion of insect vector ranges. No protective vaccine or specific antiviral strategies are currently available. Long-term morbidity after CHIKV infection includes debilitating chronic joint pain, which has associated health and economic impact. Here, we analyzed the early cell-intrinsic response to CHIKV and MAYV infection in primary human synovial fibroblasts. This interferon-competent cell type represents a potential source of polyarthralgia induced by CHIKV infection. Synovial fibroblasts from healthy and osteoarthritic donors were similarly permissive to CHIKV and MAYV infection ex vivo. Using RNA-seq, we defined a CHIKV infection-induced transcriptional profile with several hundred interferon-stimulated and arthralgia-mediating genes upregulated. Type I interferon was both secreted by infected fibroblasts and protective when administered exogenously. IL-6 secretion, which mediates chronic synovitis, however, was not boosted by infection. Single-cell RNA-seq and flow cytometric analyses uncovered an inverse correlation of activation of innate immunity and productive infection at the level of individual cells. In summary, primary human synovial fibroblasts serve as bona-fide ex vivo primary cell model of CHIKV infection and provide a valuable platform for studies of joint tissue-associated aspects of CHIKV immunopathogenesis. after CHIKV infection includes debilitating chronic joint pain, which has associated health and 23 economic impact. Here, we analyzed the early cell-intrinsic response to CHIKV and MAYV 24 infection in primary human synovial fibroblasts. This interferon-competent cell type represents Whitney-U-tests when comparing between cell lines or between cell lines and primary cells. For treatment analysis, ratio paired t-tests were applied. For IC50 calculation, nonlinear fit 198 curves with variable slopes were calculated. FDR correction was applied for RNA-seq analysis 199 and Bonferroni correction for Gene Ontology analysis. P values <0.05 were considered 200 significant (*), <0.01 very significant (**), <0.001 highly significant (***); < 0.0001 extremely 201 significant, n.s. = not significant (≥0.05). 202 Osteoarthritic fibroblasts are susceptible and permissive to CHIKV and MAYV infection 204 First, we examined the ability of primary human synovial fibroblasts to support the entire 205 CHIKV and MAYV replication cycle. Therefore, we infected synovial fibroblasts obtained Susceptibility of cells to CHIKV infection is enhanced by the attachment factor MXRA8 [12] and the cytosolic protein FHL-1 is essential for CHIKV genome replication [13] . We 220 confirmed expression of these two cellular cofactors in OASF and HSF by immunoblotting 221 and/or immunofluorescence (Fig. 1C) . We assessed the functional relevance of the MXRA8 222 attachment factor using a soluble MXRA8-Fc fusion protein, which blocks the binding site on 223 the E1-E2 glycoprotein complex on the virus surface [12, 38] . At a low MOI, MXRA8-Fc-224 preincubated CHIKV was 50 % less infectious to synovial fibroblasts, and this inhibition was 225 abolished when saturating amounts of infectious virus particles were used (Fig. 1D ), indicating 226 that endogenous MXRA8 contributes, at least partially, to CHIKV entry in OASF. Subsequently, we investigated whether IL-1β-mediated activation of synovial 228 fibroblasts, a hallmark of rheumatoid arthritis [39] [40] [41] , modulates their susceptibility to CHIKV 229 infection. Treatment with IL-1β, did not alter the percentage of EGFP-positive cells upon 230 CHIKV challenge (Fig. 1E) , while readily inducing IL-6 secretion (Fig. 1F) . Conversely, CHIKV infection only very mildly, if at all, enhanced IL-1β-induced IL-6 secretion (Fig. 1F ). These data suggest that CHIKV infection of synovial fibroblasts neither induces nor modulates 233 IL-6 secretion, arguing against their activation. To determine the importance of IFN-mediated antiviral immunity in this primary cell (ISGs) was induced at the protein level in a C9 treatment-sensitive manner, including IFITM3, 254 ISG15, and MX2. As expected, production of the viral E1-E2 and capsid proteins was 255 detectable specifically in CHIKV-infected, but not in cells exposed to C9-pretreated virus ( (Fig. 2F, right panel) . mRNAs for all IFN receptors were detectable and stable with 281 exception of IFNLR1, whose expression was upregulated upon CHIKV infection (Fig. S1E ). Established host factors for CHIKV as well as fibroblast marker genes and cellular 294 We noticed very little inter-donor variation regarding the distribution of identified viral reads 295 along the viral genome. The 5´ region of the genome, encoding the non-structural CHIKV 296 proteins, was replicated to a lower extent than the 3´, 26S subgenomic promoter-driven, 297 structural protein-encoding genomic region. Interestingly, this differential abundance of 5´and 298 3´ reads was also detected in cultures inoculated with C9-neutralized virus, suggesting infection 299 in a small number of cells (Fig. 3A) . Overall, the 26S subgenomic viral RNA was 5.3-fold more IFN-α and -λ induced expression of IFIT1 and MX2 was higher in U2OS cells than in HFF-1. 330 We next investigated the sensitivity of CHIKV infection to IFN when applied four hours post- Single-cell (sc) RNA-seq of the very same cells showed very little inter-donor 362 variability, and we merged data from both donors throughout the rest of the analysis (Fig. 5C ). In order to identify potential correlations of viral RNA abundance and the cellular transcription 364 profile, we compared the expression of CHIKV RNA to expression of 203 IFN signaling genes 365 listed in the REACTOME database (identifier R-HSA-913531, Table 1 ). For each cell, the 366 expression of this collection of genes was summarized using Seurat's AddModuleScore It is tempting to speculate that the synovial fibroblast-specific hyperreactivity is linked 535 to the long-term arthralgia observed in vivo in chronic CHIKV patients, and that 536 pharmacological interference with hyperinflammation represents a feasible intervention 537 approach towards the alleviation of long-term arthralgia. In rheumatoid arthritis, hyperactivated 538 synovial fibroblasts invade the joint matrix, destroying/disrupting the cartilage and causing 539 long-term inflammation [28, 72] . 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