Molecular reframing of fibroblasts during resolution of arthritis [scRNA 2]

Fibroblasts are key orchestrators of inflammation. Little is known whether these cells change phenotype during resolution of inflammation. We adopted a method to visualise fibroblast activation during inflammation in humans in vivo, which is based on a fibroblast activation protein (FAP) tracer detected by positron emission tomography (PET). While tracer accumulation was high in active arthritis, it decreased significantly after TNF- and IL-17A inhibition. Biopsy-based scRNA-seq analyses in experimental arthritis demonstrated that FAP signal reduction reflected a phenotypic switch from pro-inflammatory MMP3+/IL6+ fibroblasts (high FAP internalisation) to pro-resolving CD200+DKK3+ fibroblasts (low FAP internalisation). Spatial transcriptomics of human joints revealed that pro-resolving niches of CD200+DKK3+ fibroblasts clustered with innate lymphoid cells (ILC)2, whereas MMP3+/IL6+ fibroblasts were co-localised with inflammatory immune cells. CD200+DKK3+ fibroblasts stabilised the ILC2 phenotype and induced resolution of arthritis via CD200/CD200R1 pathway. Taken together, these data suggest a dynamic molecular regulation of the mesenchymal compartment during resolution of inflammation. Overall design: Cells from synovial joints were separated by fluorescence-activated cell sorting to isolate viable CD45- GR-1 cells or viable CD45- FAP+ Pdpn+ and processed for single-cell RNA sequencing using 10x Genomics Chromium Kits. Three libraries were generated for CD45- cells without enrichment for FAP+ Pdpn+ cells, each containing cells from four individual animals separated by antibody hashes directed against CD45 and B2m. Each of these three libraries contained a healthy wild-type reference (#4) and 3 arthritic hTNFtg197+ animals treated with Il23mc for 9 days (#1-3). Arthritic animals were either untreated or treated with TNF- or IL-17-inhibiting antibodies (one condition per library). An additional library was generated with viable CD45- FAP+ Pdpn+ cells containing five individually hashtag-tagged arthritic hTNFtg197+ animals treated with Il23mc for 9 days.