Fig 2: (A-C) Synovial cellularity, fibrosis and hyperplasia were evaluated in wild-type (WT) mice that received repeated intra-articular injections of recombinant murine NGF (IA 50 ng or 500 ng, 5 μL, 2x/week) or vehicle (5 μL, 2x/week) for 4 weeks; n=5 mice/group. Scores are shown for the medial and the lateral compartments, as well as the total joint scores; (D-F) Representative histological images of medial and lateral tibial synovia showing overview and zoomed in images (in black insets) from knee sections of mice injected with (D) vehicle, (E) 50 ng NGF, or (F) 500 ng NGF mice, respectively. Histological images show all 3 pathological features scored in (A-C). The zoomed in images in black insets show fibrosis and increased cellularity in NGF-injected mice. Black arrows show representative areas used to score hyperplasia. Two-way ANOVA with Sidak’s post-test. Scale bar = 200μm for overview images and 100μm for zoomed in images.

Fig 3: NGF-induced changed in lining fibroblast transcriptome. (A) Heatmap of top gene set enrichment analysis-derived leading-edge genes, demonstrating coordinated upregulation of genes related to collagen fibril organization, smoothened signaling, and ossification; (B) Transcription factor binding motif analysis (RcisTarget) of lining fibroblasts in NGF vs. Veh revealed significantly enriched motif transfac_pre_M01014 for the Sox family of transcription factors, predicted by direct annotation; (C) Expression of predicted transcription factors by lining fibroblasts in Veh and NGF groups, demonstrating highest expression of Sox5 (red arrow); (D) Gene regulatory network plot for Sox5 in lining fibroblasts.

Fig 4: Single cell RNA sequencing of whole synovium response to intra-articular NGF injection. (A) Annotation of distinct synovial cell clusters. Twelve major cell types were identified and annotated (fibroblasts (sublining and lining), endothelial cells (vascular and lymphatic), and macrophages/monocytes comprised the majority, and smaller populations of pericytes, vascular smooth muscle cells (VSMCs), Schwann cells, T cells, perineurial cells, and dendritic cells were also detected; (B) Proportional cell counts of synovial cells in Veh and NGF groups were similar; (C) Expression of cluster-defining genes; (D) Cluster-specific response to NGF treatment, enumerated as # DEGs per cluster, shows the largest number of NGF-induces differences in gene expression occurred in sublining and lining fibroblasts, endothelial cells, and Schwann cells. NGF-induced differential gene expression of these clusters is shown as (E) volcano plots of differential gene expression (cutoffs at FDR < 0.05 and |log2(fold change)| > 0.585, and (F) gene set enrichment (top 10 enriched pathways shown, sorted by normalized enrichment score, NES).

Fig 5: (A) Quantification of neuronal signal in the medial synovium of NaV1.8-tdTomato mice injected with vehicle (5 μL, 2x/week) or recombinant murine NGF (IA 500 ng, 5 μL, 2x/week) for 4 weeks; n=5 mice/group; (B,C) Representative confocal images of the medial compartment of vehicle and NGF treated groups respectively showing NaV1.8 fibers in white arrows; (D) Quantification of neuronal signal in the lateral synovium of NaV1.8-tdTomato mice injected with vehicle (5 μL, 2x/week) or recombinant murine NGF (500 ng, 5 μL, 2x/week) for 4 weeks; n=5 mice/group; (E,F) Representative confocal images of the lateral compartment of vehicle and NGF treated groups, respectively. Scale bar=100μm.