Fatima Abji1, Mozhgan Rasti1, Alejandro Gómez-Aristizábal1, Carla Muytjens1, Mahmoud Saifeddine2, Koichiro Mihara2, Majid Motahhari2, Rajiv Gandhi1,3, Sowmya Viswanathan1,4,5, Morley D Hollenberg2,6, Katerina Oikonomopoulou1, Vinod Chandran1,7,8,9,10. 1. Schroeder Arthritis Institute, Krembil Research Institute, University Health Network, Toronto, ON, Canada. 2. Department of Physiology & Pharmacology, University of Calgary Cumming School of Medicine, Calgary, AB, Canada. 3. Division of Orthopaedic Surgery, Department of Surgery, Toronto Western Hospital, Toronto, ON, Canada. 4. Institute of Biomedical Engineering, University of Toronto, Toronto, ON, Canada. 5. Division of Hematology, Department of Medicine, University of Toronto, Toronto, ON, Canada. 6. Department of Medicine, University of Calgary Cumming School of Medicine, Calgary, AB, Canada. 7. Division of Rheumatology, Department of Medicine, University of Toronto, Toronto, ON, Canada. 8. Institute of Medical Science, University of Toronto, Toronto, ON, Canada. 9. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. 10. Department of Medicine, Memorial University of Newfoundland, St. John's, NL, Canada.
Abstract
Objective: Multiple proteinases are present in the synovial fluid (SF) of an arthritic joint. We aimed to identify inflammatory cell populations present in psoriatic arthritis (PsA) SF compared to osteoarthritis (OA) and rheumatoid arthritis (RA), identify their proteinase-activated receptor 2 (PAR2) signaling function and characterize potentially active SF serine proteinases that may be PAR2 activators. Methods: Flow cytometry was used to characterize SF cells from PsA, RA, OA patients; PsA SF cells were further characterized by single cell 3'-RNA-sequencing. Active serine proteinases were identified through cleavage of fluorogenic trypsin- and chymotrypsin-like substrates, activity-based probe analysis and proteomics. Fluo-4 AM was used to monitor intracellular calcium cell signaling. Cytokine expression was evaluated using a multiplex Luminex panel. Results: PsA SF cells were dominated by monocytes/macrophages, which consisted of three populations representing classical, non-classical and intermediate cells. The classical monocytes/macrophages were reduced in PsA compared to OA/RA, whilst the intermediate population was increased. PAR2 was elevated in OA vs. PsA/RA SF monocytes/macrophages, particularly in the intermediate population. PAR2 expression and signaling in primary PsA monocytes/macrophages significantly impacted the production of monocyte chemoattractant protein-1 (MCP-1). Trypsin-like serine proteinase activity was elevated in PsA and RA SF compared to OA, while chymotrypsin-like activity was elevated in RA compared to PsA. Tryptase-6 was identified as an active serine proteinase in SF that could trigger calcium signaling partially via PAR2. Conclusion: PAR2 and its activating proteinases, including tryptase-6, can be important mediators of inflammation in PsA. Components within this proteinase-receptor axis may represent novel therapeutic targets.
Objective: Multiple proteinases are present in the synovial fluid (SF) of an arthritic joint. We aimed to identify inflammatory cell populations present in psoriatic arthritis (PsA) SF compared to osteoarthritis (OA) and rheumatoid arthritis (RA), identify their proteinase-activated receptor 2 (PAR2) signaling function and characterize potentially active SF serine proteinases that may be PAR2 activators. Methods: Flow cytometry was used to characterize SF cells from PsA, RA, OA patients; PsA SF cells were further characterized by single cell 3'-RNA-sequencing. Active serine proteinases were identified through cleavage of fluorogenic trypsin- and chymotrypsin-like substrates, activity-based probe analysis and proteomics. Fluo-4 AM was used to monitor intracellular calcium cell signaling. Cytokine expression was evaluated using a multiplex Luminex panel. Results: PsA SF cells were dominated by monocytes/macrophages, which consisted of three populations representing classical, non-classical and intermediate cells. The classical monocytes/macrophages were reduced in PsA compared to OA/RA, whilst the intermediate population was increased. PAR2 was elevated in OA vs. PsA/RA SF monocytes/macrophages, particularly in the intermediate population. PAR2 expression and signaling in primary PsA monocytes/macrophages significantly impacted the production of monocyte chemoattractant protein-1 (MCP-1). Trypsin-like serine proteinase activity was elevated in PsA and RA SF compared to OA, while chymotrypsin-like activity was elevated in RA compared to PsA. Tryptase-6 was identified as an active serine proteinase in SF that could trigger calcium signaling partially via PAR2. Conclusion: PAR2 and its activating proteinases, including tryptase-6, can be important mediators of inflammation in PsA. Components within this proteinase-receptor axis may represent novel therapeutic targets.
Authors: Hendrik Ungefroren; David Witte; Koichiro Mihara; Bernhard H Rauch; Petra Henklein; Olaf Jöhren; Shirin Bonni; Utz Settmacher; Hendrik Lehnert; Morley D Hollenberg; Roland Kaufmann; Frank Gieseler Journal: Mol Pharmacol Date: 2017-08-25 Impact factor: 4.436
Authors: Deepak R Jadon; Raj Sengupta; Alison Nightingale; Hui Lu; Juliet Dunphy; Amelia Green; James T Elder; Rajan P Nair; Eleanor Korendowych; Mark A Lindsay; Neil J McHugh Journal: Arthritis Res Ther Date: 2017-09-21 Impact factor: 5.156