Transcriptional profiling of murine macrophages stimulated with cartilage fragments revealed a strategy for treatment of progressive osteoarthritis

Abstract

Accumulating evidence suggests that synovitis is associated with osteoarthritic process. Macrophages play principal role in development of synovitis. Our earlier study suggests that interaction between cartilage fragments and macrophages exacerbates osteoarthritic process. However, molecular mechanisms by which cartilage fragments trigger cellular responses remain to be investigated. Therefore, the current study aims at analyzing molecular response of macrophages to cartilage fragments. To this end, we analyzed the transcriptional profiling of murine macrophages exposed to cartilage fragments by RNA sequencing. A total 153 genes were differentially upregulated, and 105 genes were down-regulated in response to cartilage fragments. Bioinformatic analysis revealed that the most significantly enriched terms of the upregulated genes included scavenger receptor activity, integrin binding activity, TNF signaling, and toll-like receptor signaling. To further confirm our results, immunohistochemical staining was performed to detected regulated molecules in synovial tissues of OA patients. In consistence with RNA-seq results, MARCO, TLR2 and ITGα5 were mainly detected in the intima lining layer of synovial tissues. Moreover, blockade of TLR2 or ITGα5 but not Marco using specific antibody significantly reduced production of TNF-α in stimulated macrophages by cartilage fragments. Our data suggested that blocking TLR2 or ITGα5 might be promising therapeutic strategy for treating progressive osteoarthritis.

Flightless I is a catabolic factor of chondrocytes that promotes hypertrophy and cartilage degeneration in osteoarthritis

“…In fact, the presence of inflammatory cytokines in the synovium provokes cartilage breakage and the resulting fragments/tears can then cause further pathological changes ( Macfarlane et al, 2019 ; Hamasaki et al, 2019 ). Consistent with these findings, our earlier studies demonstrated that cartilage fragments elucidate a robust inflammatory response in macrophages that are associated with increased catabolism in cartilages ( Hamasaki et al, 2019 , Hamasaki et al, 2020 ). Therefore, investigating the intercellular communication within the joint microenvironment namely between cells that reside in the synovium and cartilage would be expected to provide clues for development of a novel treatment.…”

“…Under this condition, the production of proinflammatory cytokines in the synovium alter the chondrogenic phenotype and induce differentiation, hypertrophy and apoptosis leading to cartilage degeneration ( Abramson et al., 2006 , De Lange-Brokaar et al, 2012 ). In fact, our earlier findings, using cocultured chondrocytes model, demonstrated that cartilage fragments activate NF-κB signaling in macrophages resulting in the production of inflammatory cytokines that mediate the production of catabolic factors in chondrocytes ( Hamasaki et al., 2019 , 2020 ). Therefore, it is very important to study the molecular changes in chondrocytes that are cocultured with cartilage fragment-stimulated macrophages as a step toward developing a deeper understanding of synovium-cartilage crosstalk in the OA process.…”

“…575102 Critical commercial assays Human Flightless I Homolog ELISA Kit MyBioSource, Huissen, Netherlands catalog No. MBS459349 Deposited data Raw and Analysis data This paper GSE171420 Raw and Analysis data of articular cartilage Soul et al, 2018 E-MTAB-6266 Raw and Analysis data of articular cartilage Ramos et al, 2014 GSE57218 Raw and Analysis data of articular cartilage Dunn et al, 2016 E-MTAB-4304 Raw and Analysis data of human chondrocyte stimulated with IL-1β Comblain et al, 2016 GSE75181 Raw and Analysis data of mouse chondrocyte stimulated with IL-1β Son et al, 2017 GSE104793 Raw and Analysis data of bovine chondrocyte stimulated with IL-1β Lv et al, 2019 https://www.nature.com/articles/s41598-018-36500-2 Experimental models: Organisms/strains Mouse C57BL/6J The Jackson Laboratory RRID: IMSR_JAX:000664 Software and algorithms Image J Schneider et al., 2012 https://imagej.nih.gov/ij/ Cufflinks and Cuffdiff Hamasaki et al, 2020 http://cole-trapnell-lab.github.io/cufflinks/cuffdiff/index.html …”

“…Macrophages, chondrocytes and cartilage fragments were isolated from C57BL/6 mice and cultured as earlier described ( Hamasaki et al, 2019 , Hamasaki et al, 2020 ). Briefly, bone marrow cells isolated using MACS beads (Monocyte isolation kit BM, Miltenyi Biotec) were differentiated into macrophages by stimulation with 50 ng/mL mouse recombinant macrophage colony-stimulating factor (MCSF; Peprotech, Tokyo, Japan) for 7 days.…”

“…Only high-quality libraries were subjected to Illumina HiSeq 2500 (Illumina sequencing platforms). The paired-end reads (100 bp) were trimmed by fastp, aligned, normalized, and then annotated using Cufflinks and Cuffdiff ( Hamasaki et al, 2020 ). Significantly expressed genes with p-value < 0.05 were subjected to gene ontology (GO) enrichment and pathway enrichment analyses of KEGG (Kyoto Encyclopedia of Genes and Genome).…”