Inflammatory Fibroblast‐Like Synoviocyte‐Derived Exosomes Aggravate Osteoarthritis via Enhancing Macrophage Glycolysis

Abstract The severity of osteoarthritis (OA) and cartilage degeneration is highly associated with synovial inflammation. Although recent investigations have revealed a dysregulated crosstalk between fibroblast‐like synoviocytes (FLSs) and macrophages in the pathogenesis of synovitis, limited knowledge is available regarding the involvement of exosomes. Here, increased exosome secretion is observed in FLSs from OA patients. Notably, internalization of inflammatory FLS‐derived exosomes (inf‐exo) can enhance the M1 polarization of macrophages, which further induces an OA‐like phenotype in co‐cultured chondrocytes. Intra‐articular injection of inf‐exo induces synovitis and exacerbates OA progression in murine models. In addition, it is demonstrated that inf‐exo stimulation triggers the activation of glycolysis. Inhibition of glycolysis using 2‐DG successfully attenuates excessive M1 polarization triggered by inf‐exo. Mechanistically, HIF1A is identified as the determinant transcription factor, inhibition of which, both pharmacologically or genetically, relieves macrophage inflammation triggered by inf‐exo‐induced hyperglycolysis. Furthermore, in vivo administration of an HIF1A inhibitor alleviates experimental OA. The results provide novel insights into the involvement of FLS‐derived exosomes in OA pathogenesis, suggesting that inf‐exo‐induced macrophage dysfunction represents an attractive target for OA therapy.


Figure S4 .
Figure S4.Characterization of ctr-exo and inf-exo.(a) Diameter distribution of ctr-exo and inf-exo determined using Nano Track Analysis (NTA).(b) Western blot confirmation of exosomal markers in ctr-exo and inf-exo.

Figure S5 .
Figure S5.Inflammation is an important pathological feature of synovial lesions in OA.(a) Bar plot for top 10 enriched terms using GO enrichment analysis of differentially expressed genes between human OA and non-OA synovium.The raw transcriptome data was obtained from GSE89408 dataset.(b) and (c) Representative images of IF staining for NLRP3 and iNOS of OA or non-OA synovium specimens from patients and mice.Scale bar, 100 μm.

Figure S6 .
Figure S6.The effects of exosomes derived from human fibroblast-like synoviocytes (FLSs) on THP-1 cells.(a) and (b) Schemes for investigating the effects of exosomes derived from human FLSs on THP-1 cells.Primary human FLSs (passage 0) were isolated from human non-OA or OA synovium.The cell culture media of non-OA FLSs, OA FLSs, and IL-1β stimulated non-OA FLSs were collected for exosome isolation and the cells were collected for RNA and protein analysis.Exosomes from non-OA FLSs, OA FLSs, and IL-1β-stimulated FLSs were named ctr-exo, OA-exo, and inf-exo, respectively.(c), (d), and (e) Western blot and qPCR analysis of matrix degenerative enzymes and cytokines of FLSs.Western blot analysis (f), Transmission electron microscope (g), and Nano track analysis (h) for exosome characterization.(i) Expression of M1-related inflammatory genes in macrophages stimulated by different exosomes.n=3 for each group.** P < 0.01, * P < 0.05, ns, not significant, versus the indicated groups, Student's t-test.

Figure S7 .
Figure S7.Volcano map of differentially expressed genes in macrophages after stimulation of the two exosomes.Volcano map showing 1,461 downregulated and 1,663 upregulated mRNAs in inf-exo treated macrophages compared to ctr-exo treated macrophages, fulfilling the criteria |log2 (fold change)| ≥ 1 and P＜0.05.The volcano map was generated using an online tool (www.omicstudio.cn).

Figure S8 .
Figure S8.Expression of M2-related genes in macrophages stimulated by ctr-exo and inf-exo.n=3 for each group.ns, P > 0.05, versus the indicated groups, Student's t-test.

Figure S9 .
Figure S9.Similar effects of LPS-primed macrophages and inf-exo-primed macrophages on co-cultured chondrocytes.(a) Scheme for co-culture of macrophages and chondrocytes.The macrophages were stimulated with ctr-exo, inf-exo, and LPS (100 ng/mL) for 24 hours prior to co-culture and the chondrocytes were subjected to further western blot analysis.(b) and (c) Western blot analysis and corresponding quantification for chondrocytes after co-culture with macrophages.n=3 for each group, ** P < 0.01, versus the indicated groups, one-way ANOVA, Dunnett's multiple comparisons test.

Figure S10 .
Figure S10.(a) and (b) Pictures of the cell culture media after exosome supplement for 48 hours, top panel with macrophages inoculated, bottom panel with medium only and no cells

Figure S11 .
Figure S11.Involvement of HIF1A in inf-exo-triggered macrophage dysfunction.(a) Characteristic motif for HIF1A binding location (HRE) in the promoter regions.(b) and (c) Western blot analysis and quantification of HIF1A expression in macrophages after si-RNA or scramble sequence transfection.(d) Transcription level of Hif1a expression after si-RNA transfection.(e) and (f) Quantification of the M1-polarization-related protein and glycolysis enzymes in Fig. 6p, q. n=3 for each group.** P < 0.01, * P < 0.05, ns, not significant, versus the indicated groups, Student's t-test.

Figure S12 .
Figure S12.Safety of PX-478 treatment and μ-CT reconstruction of knees.(a) Representative H.E. staining images of important organs after 8-week PX-478 injection.(b) Histogram of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA).n=6 for each group.ns, not significant, one-way ANOVA.(c) Three-dimensional reconstruction of μ-CT scanning of knee joints, with osteophytes labeled with yellow color.Scale bar, 1 mm.(d) The sagittal view of the knee joints, red line indicating the subchondral bone plate.Scale bar, 1 mm.

Table S4 .
Primers for quantitative real-time polymerase chain reaction (qRT-PCR) of human