Abstract
Background. Osteoarthritis (OA) is no longer considered only a disease of the cartilage, with disruption of synovial tissue homeostasis a recognised feature. The aim of this study was to characterise the OA synovial secretome (including soluble inflammatory mediators and exosome content) and uptake of isolated exosomes by primary OA chondrocytes, thereby interrogating potential tissue communication pathways in OA.
Methodology. Human OA synovial explants were cultured in the presence/absence of IL-1β or protease activated receptor (PAR)2 agonist peptide (SLIGKV-NH2) or reverse peptide (RP). Conditioned medium (CM) was harvested after 48h (n=13). Dead cells and debris were eliminated by successive centrifugations at increasing speeds, with exosomes isolated by ultracentrifuge. Western blot analysis was used to confirm the presence of exosome markers (CD9, CD81, HSP70 and CD63), with scanning electron microscopy (SEM) used to verify exosome size. Isolated exosomes were labelled with Exo-Red (stains RNA) or Exo-Green (stains protein) and uptake by primary human OA chondrocytes evaluated using immunofluorescence imaging. Cytokine levels in CM were evaluated using ELISA.
Results. Exosome isolation success was determined by the presence of associated markers, with a size range of 30-150 nm. Stimulation with IL-1β or SLIGKV-NH2 did not significantly alter the protein concentration of the preparations. Exosomes carried protein and RNA cargo (as evidenced by specific staining) and were taken up by primary chondrocytes after 4h exposure. Levels of IL-6, TNFα and MMP-3 in CM were significantly increased by both IL-1β (p<0.001). Interestingly, only SLIGKV-NH2 significantly increased IL-8 (p<0.001), demonstrating differential regulation by PAR2.
Conclusion. OA synovial tissue has the potential to impact chondrocyte behaviour through the release of soluble mediators and exosomes. Future studies exploring both chondrocyte gene expression post exosome uptake, and characterisation of cargo, will provide insight into the role of the synovium in OA cartilage pathology.Sponsored by: University of the West of Scotland PhD Studentship, Arthritis Research UK (reference no. 4988) & Tenovus Scotland (reference no. S16/16)
Methodology. Human OA synovial explants were cultured in the presence/absence of IL-1β or protease activated receptor (PAR)2 agonist peptide (SLIGKV-NH2) or reverse peptide (RP). Conditioned medium (CM) was harvested after 48h (n=13). Dead cells and debris were eliminated by successive centrifugations at increasing speeds, with exosomes isolated by ultracentrifuge. Western blot analysis was used to confirm the presence of exosome markers (CD9, CD81, HSP70 and CD63), with scanning electron microscopy (SEM) used to verify exosome size. Isolated exosomes were labelled with Exo-Red (stains RNA) or Exo-Green (stains protein) and uptake by primary human OA chondrocytes evaluated using immunofluorescence imaging. Cytokine levels in CM were evaluated using ELISA.
Results. Exosome isolation success was determined by the presence of associated markers, with a size range of 30-150 nm. Stimulation with IL-1β or SLIGKV-NH2 did not significantly alter the protein concentration of the preparations. Exosomes carried protein and RNA cargo (as evidenced by specific staining) and were taken up by primary chondrocytes after 4h exposure. Levels of IL-6, TNFα and MMP-3 in CM were significantly increased by both IL-1β (p<0.001). Interestingly, only SLIGKV-NH2 significantly increased IL-8 (p<0.001), demonstrating differential regulation by PAR2.
Conclusion. OA synovial tissue has the potential to impact chondrocyte behaviour through the release of soluble mediators and exosomes. Future studies exploring both chondrocyte gene expression post exosome uptake, and characterisation of cargo, will provide insight into the role of the synovium in OA cartilage pathology.Sponsored by: University of the West of Scotland PhD Studentship, Arthritis Research UK (reference no. 4988) & Tenovus Scotland (reference no. S16/16)
| Original language | English |
|---|---|
| Publication status | Accepted/In press - 5 Dec 2017 |
| Event | 2017 British Society for Immunology Congress - Brighton, United Kingdom Duration: 4 Dec 2017 → 7 Dec 2017 http://www.bsicongress.com/full-programme/ |
Conference
| Conference | 2017 British Society for Immunology Congress |
|---|---|
| Abbreviated title | BSI |
| Country/Territory | United Kingdom |
| City | Brighton |
| Period | 4/12/17 → 7/12/17 |
| Internet address |
Keywords
- Exosomes
- Extracellular vesicles
- Inflammation
- Osteoarthritis
- Cartilage, Articular
- Synovial Membrane
- Synovitis
