Background/Purpose: Rheumatoid arthritis (RA) is an autoimmune disease of the joints associated with accelerated aging and increased mortality. Further, RA is linked with a number of co-morbidities including cardiovascular disease, which accounts for 40% of RA mortality. Synovial macrophages are a key effector cell in joint inflammation, and a reduction in sublining synovial macrophages is the only current reproducible biomarker for successful response to therapy. Thus, characterization of synovial macrophages during disease could provide insight into progression of RA and increased mortality upon aging. To that end, the aim of this study was to use KRN Ag⁷mice that develop spontaneous arthritis and atherosclerosis to monitor changes in the cellular composition of arthritic synovium in parallel with non-diseased C57Bl/6 controls.

Methods: KRN Ag⁷ and C57Bl/6 mice were bred in house and euthanized at desired timepoints (1, 3, 6, 9, 12, and 18 months) when ankles were collected for extraction of synovium. Single cell suspensions were prepared from synovial tissue and stained with an antibody cocktail designed to identify monocyte and macrophage populations, which were sorted from single cell suspensions using BD FACSAria 4-Laser. Statistical analysis was carried out in Flowjo v9 and Prism7. Statistical significance was defined P ≤ 0.05.

Results: KRN Ag⁷ mice displayed arthropathy from birth, and 20% increased mortality compared to C57Bl/6 at 12 months. Flow cytometry analysis of synovial tissue showed a trend towards an overall reduction in CD11b⁺ leukocytes in KRN Ag⁷ mice, which became statistically significant from 6 months. Further analysis of KRN Ag⁷ synovium identified two phases of disease, an inflammatory phase from months 1-3 and an attempted resolution phase from 6 months. The inflammatory phase was characterized by significant increases in eosinophils and neutrophils and a decrease in dendritic cells compared to C57Bl/6. During resolution phase levels of these three populations trended towards C57Bl/6 levels, but remained significantly different. Levels of neutrophils in KRN Ag⁷ mice also correlated with Ly6c^lo monocytes, which are required for neutrophil recruitment. Levels of CD11b⁺CD64⁺Ly6c^lo macrophages in KRN Ag⁷ synovium peaked in inflammation phase but remained lower than those of C57Bl/6 at all time points. Subdivision of macrophages into 4 populations based on expression of MHCII and CX3CR1 identified populations associated with both phases of disease. MHCII⁺CX3CR1^– and MHCII^–CX3CR1⁺ macrophages were significantly higher and lower respectively than C57Bl/6 during inflammation, but during initial resolution at 6 months reached comparable levels to those of C57Bl/6. However this was not maintained, and levels of both populations returned to baselines by 18 months. Neither MHCII⁺CX3CR1⁺ nor MHCII^–CX3CR1^– macrophages significantly changed.

Conclusion: Using KRN Ag⁷ mice, that best recapitulate human disease, we have characterized the cellular dynamics in arthritis upon aging, which are distinct from those observed in non-arthritic mice. By identifying the critical populations, and further characterization of their transcriptional profile, we can provide insight into RA progression with age.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mapping-changes-in-monocyte-and-macrophage-populations-in-the-synovium-an-aging-study-in-arthritic-krn-ag7-mice/