Despite the heterogeneity of FLS, most total cells still retain fibroblast characteristics
We established the integrity of the total cell population. FLS are pluripotent cells. To strengthen the evidence that these cells are all fibroblasts at initial input before analysis and have true heterogeneity between subtypes and subpopulations of cells, we overlayed universal markers using Loupe Cell Browser and calculated the percentages of total cells that express a group of fibroblast markers. Muhl et al. established a group of markers based on literature as no single transcript serves as a universal fibroblast marker [27]. As a group, these markers can identify fibroblasts within total cell populations (LUM, LOXL1, COL1A1, COL5A1, PDFGRA, COL1A2, FBLN1, FBLN2, and CD34). Transcripts chosen from several markers are needed to robustly distinguish fibroblasts from other subpopulations.
We determined that 92% of oligoarticular (oligo) JIA FLS express fibroblast markers, 90% of prior to extension (ETB) JIA FLS express fibroblast markers, and 94% of poly JIA FLS express fibroblast markers (Fig. 1A). Heatmaps provide visualizations of the expression of the individual fibroblast markers and their expression pattern in individual cell clusters (Fig. 1B). It is important to note that FLS are identified by both upregulation and downregulation of these specific genes and we confirm that no single marker can be used to identify a FLS but a combination of these markers can distinguish this cell type (Fig. 1B). This data confirms that total JIA FLS are indeed fibroblast-like synoviocytes. All cell clusters had the expression of fibroblast markers. While we establish that our initial total cell input has fibroblast origins, the following scRNA-seq analysis will reveal that these cells are heterogeneous when we compare subtypes of JIA. Additionally, within each subtype, there are conserved subpopulations of cells, and those subpopulations provide another layer of heterogeneity to the genetic fingerprint of JIA FLS.
Despite a single cell type used for initial input for all JIA subtypes, there is heterogeneity between the subtypes of disease
CellRanger aggregate was performed on a single JIA subtype to aggregate datasets into a single output for multi-sample analysis. Figure 2A provides a flow chart for data input and downstream analysis performed. First, we analyzed the overall differences between JIA subtypes, oligoarticular JIA (oligo), extend-to-be JIA (ETB), and polyarticular JIA (poly). Heatmaps reveal the 30 top variable genes which show heterogeneity within subtypes between single cells (Fig. 2B). These genes have the greatest differential expression and account for the cell dispersion between single cells within each JIA subtype. When comparing specific genes, there are 16 unique differentially expressed genes within cells from oligo, 22 unique differentially expressed genes within cells from ETB, and 15 unique differentially expressed genes within cells from poly (Fig. 2C). When comparing subtypes, there are 4 genes that overlap between oligo and ETB (Fig. 2C). Of note, FBLN1 is differentially expressed. Of the 11 genes that overlap between oligo and poly, S100A4 is differentially expressed, as well as COMP and TIMP1 (Fig. 2C). There were 6 genes that overlap between ETB and poly. From this list, COL3A1, HAPLN1, and SFRP4 are differentially expressed within single cells from each subtype (Fig. 2C).
The most prominent cluster subpopulations of FLS co-localize together but cluster dispersion increases as the disease course becomes more severe
Each group of replicates was used as separate inputs in the Seurat data analysis package from Bioconductor. Regardless of the JIA subtype, FLS separate into distinguishable communities of cell subpopulations that vary in their gene expression. Despite a single cell type, FLS, used for initial input, the individual cells separate into various clusters of subpopulations of cells that vary in their gene expression patterns (Fig. 3A). While cells within a cluster have similar expression patterns, there are groups of cells that have different expression patterns from their neighboring clusters, demonstrating that FLS are heterogenous.
For oligoarticular JIA FLS samples, all communities co-localize together with mild separation of cluster number 4 (Fig. 3A). ETB JIA FLS samples have more separation of clusters 4, 5, and 6 while polyarticular JIA FLS samples have the most separation among their cell communities (Fig. 3A). Increases in the heterogeneity of FLS correlate to dispersion between cell clusters and this separation corresponds to progression to more severe disease courses.
Cell clusters were identified as various subpopulations of known cell types
We used unbiased clustering since there is little known about marker genes in FLS subtypes. For validation, cells were automatically annotated using the SingleR data package from Bioconductor. Seurat single analysis was done to analyze the JIA subtypes and identify shared populations across data sets and downstream comparative analysis. Among these shared populations were fibroblast-like cells, chondrocyte-like cells, and smooth muscle cell-like cells (Fig. 3B). Other, smaller subpopulations included osteoblasts and mesenchymal stem cells (Fig. 3B).
Identifying the top genes that account for dispersion of cells between subtypes signifies transcriptome differences between subpopulations of cells within JIA subtypes
Seurat single analysis identified the top genes of each projected cell type for each subtype. Dot plots provide visualization of the average expression of each gene in each cell type and the percentage of cells that express that gene (Fig. 3C). These genes are the highest contributors to variation among the cells. Genes that contribute the most variation among cells are as follows: oligo — COL1A1, MT-ND1, TUBA1B, ACAN, and IFI27; ETB — MTRNR2LB, SERPINE1, TOP2A, MEG3, COL3A1, and VCAM; and poly — ACP5, SPP1, TNFAIP6, IGFBP5, AKAP12, MFAP5, and S100A4 (Fig. 3C). Several of these genes encode surface and secreted proteins that have been associated with musculoskeletal and developmental diseases.
Cells that were identified as chondrocytes are the majority cell-type subpopulation with polyarticular JIA FLS having the largest percentage of chondrocytes
While many cell subpopulations were identified, the three most populated cell-type subpopulations across JIA subtypes were identified as chondrocyte-like, fibroblast-like, and smooth muscle cell-like cells (Fig. 4A). As the cells evolve toward more chondrocyte-like characteristics, their fibroblast-like cell subpopulation and smooth muscle cell-like subpopulation decrease as the disease course becomes more severe, particularly between oligoarticular JIA FLS and polyarticular JIA FLS samples (Fig. 4B).
The heterogeneity of JIA FLS persists in differential gene expression within cell subpopulations of JIA subtypes
The most prominent subpopulations between JIA subtypes were the same: chondrocyte-like cells, fibroblast-like cells, and smooth muscle cell-like cells; however, transcript analysis revealed the differential expression of genes for these subpopulations between JIA subtypes. Seurat single analysis identified the top differential genes of each projected cluster or cell subpopulation for each JIA subtype (Fig. 5A). Further analysis revealed overlap among the chondrocyte-like subpopulation gene lists between JIA subtypes. By removing the overlapping genes, we identified unique genes that distinguish the chondrocyte-like subpopulations between JIA subtypes (Supplemental table 1).
When analyzing gene expression of the subpopulation of fibroblast-like cells and smooth muscle cell-like cells within the JIA subtypes, we identified unique genes that distinguish the fibroblast-like subpopulations and the smooth muscle cell-like subpopulations between JIA subtypes (Fig. 5B, C; Supplemental table 2).
Considering the chondrocyte-like cell cluster of JIA FLS is the predominant subpopulation across JIA subtypes, the expression of unique genes in these cells could distinguish between JIA subtypes. While many genes overlap, the unique genetic fingerprint of this subpopulation shows that even in a single uniform cluster of chondrocyte-like cells, there is heterogeneity between JIA subtypes. This prompted us to closely examine the transcriptome of the chondrocyte-like subpopulation within each JIA subtype.
Further analysis of the chondrocyte-like subpopulation reveals biologically relevant evidence that contributes to the pathology of JIA
Seurat integrated analysis allows for the comparison between JIA subtypes. This analysis identifies the top differential genes of each projected cell subpopulation for each JIA subtype and allows us to determine genes that not just distinguish between subpopulations of JIA subtypes but between the JIA subtypes themselves. Positive differentially expressed genes are also calculated for each subpopulation between subtypes. This allows for the identification of differentially expressed markers across JIA subtypes.
When comparing the chondrocyte-like subpopulation from ETB JIA FLS to the chondrocyte-like subpopulation from oligo JIA FLS, there were 211 differentially expressed genes (Supplemental table 3). Of these genes, 82 are upregulated in the oligo when compared to ETB and 129 are upregulated in ETB when compared to oligo. Ingenuity pathway analysis (IPA) revealed the top diseases and disorders associated with this gene list: cancer, organismal injury and abnormality, gastrointestinal disease, and immunological disease. These diseases and disorders are associated with cell movement, cell death, cell proliferation, and cell function. The top 3 differentially expressed genes within this subpopulation were LRRC15, MTRNR2L1, and GREM2 (Fig. 6). For this comparison, LRRC15 is expressed in 35.8% of chondrocyte-like cells in oligo JIA FLS compared to just 6.1% of chondrocyte-like cells in ETB JIA FLS. 67.3% of chondrocyte-like cells in oligo JIA FLS express GREM1 compared to 34.8% of chondrocyte-like cells in ETB JIA FLS. GREM2 is expressed in 45.8% of the chondrocyte-like cells in oligo JIA FLS compared to 11.5% of chondrocyte-like cells in ETB JIA FLS.
When comparing the chondrocyte-like subpopulation from ETB JIA FLS to the chondrocyte-like subpopulation from poly JIA FLS, there were 440 differentially expressed genes (Supplemental table 4). Of these genes, 167 are upregulated in poly when compared to ETB and 273 are upregulated in ETB when compared to poly. IPA revealed the top diseases and disorders associated with this gene list: osteoarthritis, connective tissue disorders, inflammatory disease, and skeletal and muscular disorders. These diseases and disorders are associated with cell movement, cell death, cell proliferation, and cell function. The top 3 differentially expressed genes within this subpopulation were S100A4, TIMP3, and NBL1 (Fig. 6). For this comparison, S100A4 is expressed in 89.6% of chondrocyte-like cells in ETB JIA FLS compared to 39% of chondrocyte-like cells in poly JIA FLS. 75.8% of chondrocyte-like cells in ETB JIA FLS express TIMP3 compared to 48.2% of chondrocyte-like cells in poly JIA FLS. NBL1 is expressed in 72.4% of the chondrocyte-like cells in ETB JIA FLS compared to 40.7% of chondrocyte-like cells in poly JIA FLS.
When comparing the chondrocyte-like subpopulation from oligo JIA FLS to the chondrocyte-like subpopulation from poly JIA FLS, there were 266 differentially expressed genes (Supplemental table 5). Of these genes, 124 are upregulated in poly when compared to oligo and 142 are upregulated in oligo when compared to poly. IPA revealed the top diseases and disorders associated with this gene list: cancer, organismal injury and abnormality, gastrointestinal disease, and connective tissue disorders. These diseases and disorders are associated with cell movement, cell death, cell proliferation, and cell function. The top 3 differentially expressed genes within this subpopulation were CRLF1, MFAP5, and TNXB (Fig. 6). For this comparison, CRLF1 is expressed in 63.1% of chondrocyte-like cells in oligo JIA FLS compared to 29.2% of chondrocyte-like cells in poly JIA FLS. 58.7% of chondrocyte-like cells in oligo JIA FLS express MFAP5 compared to 19.7% of chondrocyte-like cells in poly JIA FLS. TNXB is expressed in 44.6% of the chondrocyte-like cells in oligo JIA FLS compared to 11.6% of chondrocyte-like cells in poly JIA FLS. Unique genetic fingerprints within the chondrocyte-like subpopulation can be used to further distinguish between JIA subtypes.