TNFa has Differential Effects on the Transcriptome Profile of Selected Populations in Murine Cartilage

To further our understanding of the role of tumor necrosis factor (TNFα) on the inflammatory response in cartilage, we explored its effects on the transcriptome profile of murine epiphyseal chondrocytes at the total and single cell (sc) resolution. Gene set enrichment analysis of total RNA-Seq from TNFα-treated chondrocytes revealed enhanced response to biotic stimulus, defense and immune response and cytokine mediated signaling and suppressed cartilage and skeletal morphogenesis and development. scRNA-Seq analyzed 14,239 cells and 24,320 genes and distinguished 16 cell clusters. The more prevalent ones were constituted by cells of the limb bud, chondrogenic cells and fibroblasts comprising ~73% of the cell population. Genes expressed by joint fibroblasts were detected in 5 clusters comprising ~45% of the cells isolated. Pseudotime trajectory finding revealed an association between fibroblast and chondrogenic clusters which was not modified by TNFα. TNFα decreased the total cells recovered by 18.5% and the chondrogenic, limb bud and mesenchymal clusters by 32%, 27% and 7%, respectively. TNFα had profound effects on the insulin-like growth factor (IGF) axis decreasing Igf1, Igf2 and Igfbp4 and inducing Igfbp3 and Igfbp5, explaining an inhibition of collagen biosynthesis, cartilage and skeletal morphogenesis. Ingenuity Pathway Analysis of scRNA-Seq data revealed that TNFα enhanced the osteoarthritis, rheumatoid arthritis, pathogen induced cytokine storm and interleukin 6 signaling pathways and suppressed fibroblast growth factor signaling. In conclusion, epiphyseal chondrocytes are constituted by diverse cell populations distinctly regulated by TNFα to promote inflammation and suppression of matrix biosynthesis and growth. Chondrocyte-enriched cells were isolated from the epiphyses of long bones of the hind and fore limbs from 3- to 4-day-old C57BL/6 mice.b Cultured in the presence and absence of TNFa.