Distinct fibroblast subsets drive inflammation and damage in arthritis

Abstract

The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune-mediated inflammatory diseases (IMIDs)^1,2. However, it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue-driven processes observed in IMIDs, such as inflammation and damage^3-5. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of fibroblast activation protein-α (FAPα)⁺ fibroblasts suppressed both inflammation and bone erosions in mouse models of resolving and persistent arthritis. Single-cell transcriptional analysis identified two distinct fibroblast subsets within the FAPα⁺ population: FAPα⁺THY1⁺ immune effector fibroblasts located in the synovial sub-lining, and FAPα⁺THY1^- destructive fibroblasts restricted to the synovial lining layer. When adoptively transferred into the joint, FAPα⁺THY1^- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation, whereas transfer of FAPα⁺ THY1⁺ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell-based therapies aimed at modulating inflammation and tissue damage.

Extended data 1. Mouse synovial FAPα expression.

(a) Expression of PDPN and FAPα by immunohistochemistry in ankle joints (representative of n=8 mice). Flow cytometry of digested synovia: (b) gating strategy for SFs in digested synovia, (c) representative expression of PDPN and FAPα during STIA, (d) corresponding absolute numbers of FAPα⁺ cells (n=10 mice per group), (e) plot of FAPα expression in THY1^- (LL, blue) or THY1⁺ (SL, red) PDPN⁺ cells and PDPN^- THY1⁺ pericytes (black) in day 9 STIA synovia (each plot representative of n=12 mice, numbers=percentage of cells). (f) Quantification of Fap transcript expression in sort purified cells (day 9 STIA synovia, n=12 mice). (g) Immunofluorescence staining for PDPN, FAPα and THY1 expression in day 9 STIA ankle joints (representative of n=12 mice). Spearman’s correlation analysis between the total (black), LL (blue) and SL (red) FAPα expressing cells quantified by flow cytometry and (h) the change in ankle joint thickness, (i) cartilage destruction and (j) bone erosion (both by histology) (n=40 mice). (k) Representative bioluminescence of in vivo imaging of FAPα-DTR⁺ mice treated with DTx or Vehicle and (l) quantification of bioluminescence (n=8 mice per group). (m) Quantification of synovial FAPα⁺ cells following administration of either DTx or Vehicle (n=8 mice per group). (n) Immunohistochemistry staining of FAPα (red) expression in ankle joints following DTx (representative of n=8 mice). (o) Total number of CD45^- CD31^- cells by flow cytometry in day 9 STIA synovia compared to non-arthritic (control) mice following DTx (n=7 mice per group). (p) H&E staining and quantification of cellularity following DTx treatment. Arrow indicates SM. Data are expressed as the average number of cells per quantified per histological section (n=8 mice per group). Statistics: 2-way ANOVA with Tukey’s post hoc, (f,n,p). Data represented as Mean±S.D., except f,n which are shown as box plots (centre line, median; box limits, upper and lower quartiles; whiskers, maximum and minimum values.

Extended data 2. Effects of FAPα cell deletion.

Change in wrist and ankle joint thickness during STIA with AUC analysis following FAPα cell deletion at (a) days 5 and 7 and (b) days 10 and 12 or (c) prophylactically prior to the induction of STIA (n=8, mice per group). (d) Representative time course analysis of structural joint damage assessed by micro-CT following FAPα cell deletion at days 3 and 5 following induction of STIA, with (e) quantification of bone erosion and new bone formation (n=8 mice per group), combined for front and hind paws. (f) Histological examination of ankle joint tissue sections at day 12 STIA with quantification of bone erosion, pannus formation and bone formation (all by H&E) and cartilage destruction (by Safranin O staining) (n=12 mice per group). (g) Representative images of cathepsin k immunohistochemical staining of osteoclasts (brown) in the ankle joints of day 12 STIA mice. (h) Number of osteoclasts (cathepsin k positive) per tissue section in DTR^- versus DTR⁺ mice at day 12 STIA compared to non-arthritic control mice (n=12 mice per group). (i) Expression of bone turnover markers including osteoclast and osteoblast markers in whole paw tissue analysed by RT-PCR (n=8 mice per group, data are expressed as mean fold change in expression compared to expression in non-arthritic mice). Statistics: Mann Whitney test, a-c, 2-way ANOVA with Tukey’s post hoc, e, 1-way ANOVA with Tukey’s post hoc, f,h. Data represented as Mean±S.D., except AUC analysis in a-c which are shown as box plots (centre line, median; box limits, upper and lower quartiles; whiskers, maximum and minimum values.

Extended data 3. Effect of FAPα cell deletion on leucocyte infiltration.

(a) Flow cytometry plot of peripheral blood monocytes (numbers=percentage of positive cells) with quantification at day 9 STIA following DTx at day 3 and day 5 (n=6 mice per group). (b) Flow cytometry gating strategy for leucocyte populations in digested synovia. (c) Numbers of leucocytes and percentage of MHC Class II expressing F4/80 cells (M1) in hind limb joints of day 28 persistent STIA mice analysed by flow cytometry (n=13 mice per group). (d) Representative immunohistochemical staining of macrophages (F4/80⁺, brown; nuclei blue) in the ankle joint tissue sections at day 12 STIA mice following DTx at day 3 and 5 (representative of n=6 mice). (e) Percentage of F4/80⁺ macrophages staining positive for MHC Class II as detected by flow cytometry in day 12 STIA digested synovia from DTR^+/- mice following DTx at day 3 and 5 (n=13 mice per group). (f) Expression of functional macrophage markers detected by RT-PCR in sort purified macrophages (CD45⁺ CD11b⁺ F4/80⁺) isolated from the synovia of day 12 STIA mice following DTx at day 3 and 5 (n=7 mice per group). (g) Number of FAPα expressing cells quantified by flow cytometry from digested synovia (n=8 mice), popliteal (draining) and mesenteric (non-draining) lymph nodes (n=6 mice) following IA administration of DTx into the ankle joint during STIA (harvested day 14) and (h) daily change in weight from baseline (expressed as percentage of original body weight) in STIA mice compared to non-arthritic mice (n=6 mice). (i) Effect of local deletion of synovial FAPα expressing cells (by IA injection of DTx); on ankle joint thickness in the resolving model of STIA model when compared to the wrist joints on the same mouse (non-deleted limbs) (n=8 mice) and to non-arthritic DTR+ and DTR- injected mice (n=6 mice per group) with AUC analysis. (j) Representative micro-CT images of day 14 STIA and non-arthritic control mice following IA injection of DTx and quantification of bone erosion and new bone formation (STIA DTR^- n=10, STIA DTR⁺ n=13, DTR^- and DTR⁺ n=8). (k) Quantification of the number of fibroblasts and leucocytes in digested synovia of day 9 STIA mice analysed by flow cytometry following IA administration of DTx (n=8 mice). Statistics: 2-way ANOVA with Tukey’s post hoc a,c,e,h,k, two-tailed paired Student’s t-test, f,g, 1-way ANOVA with Tukey’s multiple comparison tests, I,j. Data represented as Mean±S.D., except a,c,e,f,g,k and AUC analysis in I, which are shown as box plots (centre line, median; box limits, upper and lower quartiles; whiskers, maximum and minimum values.

Extended data 4. 10x Chromium single cell RNAseq (droplet based single cell) analysis of CD45^- cell populations from inflamed synovium.

(a) t-SNE projection of non-hematopoietic stromal cells from the inflamed mouse joint (n=3 biological replicates, day 9 STIA) showing the initial automatic cluster assignments from Seurat (projection is identical to that shown in Fig 3a). (b) The same t-SNE plot coloured for biological replicate. (c) The barplot shows the number of cells in each cluster stratified by replicate. (d) Cluster cell identification: the five panels of violin plots show expression (normalised, log-transformed counts of the cells from all of the n=3 biological replicates, y axes) of known cell type marker genes (for fibroblasts, lining layer fibroblasts, osteoblasts, chondrocytes, and vascular cells) in each of the automatically assigned clusters (x axes). The colours of violin plots correspond to those shown in Extended data Data 4a.

Extended data 5. continued cluster identification analysis.

The four panels of violin plots show expression (normalised, log-transformed counts of the cells from all of the n=3 biological replicates, y axes) of known cell type marker genes (for pericytes, muscle cells, erythrocytes and the cell cycle) in each of the automatically assigned clusters (x axes). The colours of violin plots correspond to those shown in Extended data 4a.

Extended data 6. Differential gene expression between fibroblast clusters.

The heat map shows the (row-scaled) expression of the top 20 (by p-value) discovered significant, conserved marker genes for each cluster (BH adjusted p-value <0.1 in separate tests of cells from each of the n=3 biological replicate samples, two-sided Wilcoxon tests). Each column represents a single fibroblast and each row the given gene. The cluster identification is indicated for each column. LL: lining layer fibroblasts correspond to F5 and are PDPN⁺ THY1^- and SL: sub-lining layer fibroblasts correspond to F1-F4 fibroblast subsets and are PDPN⁺ THY1^-.

Extended data 7. Differential gene expression in specific fibroblast clusters.

(a) A set of violin plots showing gene expression (normalised, log-transformed counts of the cells from all of the n=3 biological replicates, x axes) of additional fibroblast markers in each of the F1-F5 fibroblast clusters (y axes) (corresponds to Fig 3b). (b) t-SNE projection of fibroblasts from the inflamed mouse joint coloured by replicate (corresponds to Fig 3b). (c) A set of violin plots showing gene expression (normalised, log-transformed counts of the cells from all of the n=3 biological replicates, x axes) of known markers for chemokines in each of the F1-F5 fibroblast clusters (y axes) (corresponds to Fig 3B). (d) Number of cells in each cluster stratified by replicate.

Extended data 8. Trajectory analysis and identification of fibroblast subpopulations from human RA patients.

(a) The Heatmap shows genes most strongly up- or downregulated across the inferred F1-F2-F5 trajectory in the mouse fibroblasts from the STIA model (as determined by Slingshot). (b-e) Re-analysis of CEL-Seq2 single-cell RNA-sequencing dataset from 20 RA patients. (b) t-SNE projection of the RA patient fibroblasts indicating the automatic cluster assignments from Seurat. (c) The sets of violin plots show expression (normalised, log-transformed counts, cells from all n=20 RA patients, x axes) of cluster marker genes in each of the RA patient fibroblast clusters (y axes). The violin plots are grouped into six sets comprising of ‘other markers’ (known markers or markers reported by Zhang et al) or of markers characteristic of each of the Hs RA F1-F5 clusters as indicated. (d) The same t-SNE plot as in (a) coloured by patient ID. (e) The barplot shows the number of patients represented in each assigned cluster.

Extended data 9. Bulk RNA sequencing of sort purified FAPα expressing LL and SL cell populations from the inflamed hind limb joints of day 9 STIA mice.

(a) Gating strategy for flow cytometry based cell sorting from day 9 STIA digested synovia gated on CD45^- CD31^- live cells. Coloured gates correspond to each sort purified population and the percentage gated cell population is indicated. (b) Principal components analysis (PCA) reveals each population clusters according to either a SL phenotype or a LL phenotype. Each dot presents a single biological replicate sample and is coloured according to the gating strategy outlined in (a). (c) The heat map shows the differential expression of the 50 most significant genes (by p value) for each population (BH adjusted p-value <0.1) and reveals distinct transcriptional profiles between THY1⁺ and THY1^- cell populations. (d) Expression of specific genes RNA sequencing in PDPN⁺ FAPα⁺ THY1^- versus PDPN⁺ FAPα⁺ THY1⁺ sort purified cells. For each heatmap a column represents a single biological replicate and coloured according to the gating strategy in (a). Biological replicates represent cells isolated and purified from the digestion of synovia from the hind limbs of a single mouse (n=10 THY1^- FAPα⁺, n=5 THY1^- FAPα^-, n=6 THY1⁺ FAPα^- and n=12 THY1⁺ FAPα⁺ samples).

Extended data 10. Effect of IA injection of fibroblast subsets.

(a) Effect on ankle joint thickness of IA injection of 500,000 sort purified PDPN⁺ FAPα⁺ THY1^- (blue) or PDPN⁺ FAPα⁺ THY1⁺ (red) cells into the ankle joint of CIA mice at the first sign of joint inflammation (day 0) compared to contralateral sham injected joints (n=8 mice per group). (b) Representative images of micro-CT analysis and quantification of bone erosion and new bone formation (n=8 mice per group). (c) Flow cytometric analysis of leucocytes in the digested synovia of injected joints, 7 days post injection (n=8 mice per group). (d) Representative confocal microscopy of ankle joint tissue of mice injected with red Tomato expressing PDPN⁺ FAPα⁺ THY1^- or PDPN⁺ FAPα⁺ THY1⁺ isolated from day 9 STIA cells and injected into day 3 STIA recipient mice (representative images from n=6 mice, 14 days post injection). (e) Flow cytometric analysis of digested synovia, 14 days post injection of cell trace labelled cells (isolated from day 9 STIA mice and injected into the ankle joint of day 3 STIA recipient mice), gated on the CD45^- CD31^- PDPN⁺ cell fraction. Percentage of THY1⁺ (red) or THY1^- (blue) cell in this gated cell fraction is indicated (representative of n=6 per group). (f) Percentage of engraftment and viability of injected cells 14 days post injection into the ankle joints of STIA mice (n=8 per treatment group, two tailed Student’s t-test). Engraftment is expressed as the percentage recovery of the original injected cell number. Statistics: 1-way ANOVA with Bonferroni post hoc test, b,c and AUC analysis a. Paired two-tailed Student’s t-test f. Data represented as Mean±S.D., except c,e and AUC analysis in a, which are shown as shown as box plots (centre line, median; box limits, upper and lower quartiles; whiskers, maximum and minimum values.

Fig. 1. FAPα⁺ SFs accumulate in the arthritic joint.

(a) Representative images of FAPα expression in synovial tissue and (b) quantification, with (c) matched Fap transcript expression in SFs expanded in vitro (n=8 control, 9 resolving and 11 RA, patient samples). (d) CyToF viSNE plots of CD45^- cells and (e) confocal microscopy of RA synovium (both representative of n=8, RA patient samples). (f) Serial measurements of bioluminescence signal in FAPα-luciferase mice and (g) quantification during STIA (n=8 mice). (h) Spearman’s correlation between bioluminescence and joint thickness (n=30 mice). (i) Representative image of FAPα (red) expression in hind limb joints of day 9 STIA mice, arrows indicate FAPα expression and (j) quantification (n=10 mice per group). (k) Fap transcript expression in sort purified synovial CD45^- CD31^- cells during STIA (n=8 mice, per time point). (l) Fold change in mRNA expression of stromal markers in the synovia of day 9 STIA compared to control mice (n=8 mice). (m) Spearman’s correlation between combined expression of Pdpn, Thy1 and Fap, and ankle joint thickness (n=44 mice). (n) Change in absolute numbers and percentage of Ki67⁺ and BrdU⁺ cells during STIA (n=6 mice). Statistics: Kruskal-Wallis with Dunn’s post-hoc, b,c, 1-way ANOVA with Dunnett’s post hoc, compared to day 0, g or day 3 k, two-tailed Mann-Whitney test j, 2-way ANOVA with Tukey’s post hoc, l,n. Data represented as Mean±S.D., except g,k,l, which are shown as box plots (centre line, median; box limits, upper and lower quartiles; whiskers, maximum and minimum values).

Fig. 2. Deletion of FAPα expressing cells attenuates synovial inflammation.

(a) Joint inflammation in deleted (STIA DTR⁺, n=13) versus non-deleted (STIA DTR^-, n=9) STIA mice compared to non-arthritic deleted (DTR⁺, n=6) and non-deleted (DTR^-, n=6) mice, with AUC analysis. (b) Effect of sustained FAPα cell deletion on joint inflammation in persistent STIA (STIA, DTR⁺/DTR^- n=8 mice, and non-arthritic DTR⁺/DTR^-, n=4 mice), with AUC analysis. (c) Representative micro-CT and histological images of day 28 persistent STIA mice with (d) quantification of micro-CT data, osteoclast number and histomorphometric analysis of bone erosion, pannus formation and cartilage damage, arrows indicate areas of interest (all: n=16 mice per group). (e) Representative histology at day 9 STIA and quantification (n=14 mice per group), arrow shows leucocyte infiltration and SM=synovial membrane. (f) Spearman’s correlation between the percentage of FAPα cell deletion achieved at day 9 and change in ankle joint thickness (n=45 mice). (g) Quantification of the number of fibroblasts and pericytes (n=8 mice) and (h) leucocyte subsets (n=13 mice) in day 9 STIA synovia analysed by flow cytometry. (i) Expression of selected mRNA transcripts and proteins (by luminex analysis following stimulation ex vivo with TNFα) in the synovia of DTR^- and DTR⁺ day 9 STIA mice following DTx treatment, expressed as fold change in expression compared to non-arthritic mice (n=8 mice per group for mRNA and n=6 mice per group for protein analysis). Statistics: 1-way ANOVA with Dunnett’s multiple comparison test a,b, two-tailed paired Student’s t-test (d,e), 2-way ANOVA with Tukey’s multiple comparison test (f,g). Data represented as Mean±S.D., except AUC analysis (in a and b) and f,g, which are shown as box plots (centre line, median; box limits, upper and lower quartiles; whiskers, maximum and minimum values).

Fig. 3. Single-cell RNA-sequencing reveals distinct fibroblast subsets.

(a) t-SNE projection showing 2814 non-hematopoietic (CD45^- CD31^- cells) stromal cells from the inflamed synovium of day 9 STIA mice (n=3 biological replicates). Cells are coloured according to major cell type based on expression of known marker genes (Extended data 4 and 5). (b) Unsupervised graph-based clustering of fibroblasts (n=1725 cells) reveals five subsets (F1-F5) in the inflamed STIA joints. For (c) and (d) conserved cluster marker genes were first identified as those with a maximum BH adjusted p value < 0.1 in separate tests of cells from each of the n=3 biological replicates (two-sided Wilcoxon tests, supplementary table 1). (c) Over-representation analysis of Gene Ontology (GO) categories suggests different functions for the five STIA fibroblast subsets. Significant enrichments amongst cluster markers (BH adjusted p < 0.05, one-sided Fisher’s exact tests, supplementary table 2) are shown in colour. (d) Expression of marker genes (x axes) in the identified STIA fibroblast clusters (y axes). The first panels show expression of known pan-fibroblast markers. The remaining sets of panels show examples of identified conserved makers genes for each fibroblast cluster F1-F5. (e) Embedding of the STIA fibroblasts in the first three components of a diffusion reveals possible relationships between the clusters. (f) Comparison of the fibroblasts clusters from the mouse (Mm) STIA model with those from human (Hs) RA patients. Hierarchical clustering of orthologous (one-to-one) cluster markers identified three sets of homologous subpopulations. (g) Selected markers commonly expressed between homologous human and mouse clusters.

Fig. 4. Fibroblast subsets are responsible for different aspects of disease pathology.

(a) Luminex analysis of stimulated FAPα⁺ THY1^- and FAPα⁺ THY1⁺ cells isolated from day 9 STIA synovia, (b) secretion of RANKL and OPG (n=6 mice per group) and (c) quantification of the synovial RANKL⁺ THY1^-/+ cells by flow cytometry (numbers=percentage of cells, n=8 mice per group). (d) Effect of FAPα⁺ THY1^- and FAPα⁺ THY1⁺ cells on the number of osteoclasts, N. Oc, (n=4 mice per group) and matrix degradation in vitro (n=8 mice per group). (e) Effect of IA injection of PDPN⁺ FAPα⁺ THY1^- or PDPN⁺ FAPα⁺ THY1⁺ cells into the ankle joints of day 3 STIA mice compared to contra-lateral sham injected joints, with AUC analysis (n=8 mice per group). (f) Flow cytometric analysis of absolute number of leucocyte subsets isolated from the digested synovia of injected joints at day 12 (n=8 mice per group). (g) Representative images at day 12 and (h) quantification of micro-CT data, and histomorphometric analysis of osteoclast number, cartilage damage, bone erosion and synovial pannus (n=16 mice per group). (i) Mass cytometry (CyToF) analysis of PDPN⁺ FAPα⁺ and THY1^+/- cells in synovium of patients with OA and RA (n=15 OA, n=8 RA patient samples). (j) Spearman’s correlation between THY1⁺ FAPα⁺ cells in RA and serum ESR level and synovial Krenn score (n=8 RA patient samples). Statistics: two-tailed paired Student’s t-test (a-d,l), 1-way ANOVA with Dunnett’s post hoc test compared to sham injected (e,f), two-tailed paired Student’s t-test (AUC, e and f,h). Data represented as Mean±S.D., except c,d,f,l and AUC analysis in e, which are shown as box plots (centre line, median; box limits, upper and lower quartiles; whiskers, maximum and minimum values).