Background/Purpose: Rheumatoid arthritis (RA) manifests in persistent synovial inflammation, cellular infiltration and pro-inflammatory cytokine production, resulting in progressive joint destruction. Macrophages have been implicated in RA progression and persistence through production of degradative enzymes, cytokines, and chemokines. However, the mechanisms underlying these activities are not fully elucidated. We previously demonstrated that naïve mouse joints contain both MHC II⁺ (monocyte-derived) and MHC II^– (tissue-resident) macrophages. Interestingly, we have shown that the monocyte-derived macrophages drive inflammation, while the tissue-resident macrophages are involved in resolution based on an acute model of inflammatory arthritis. Thus, we have optimized a multi-parameter flow cytometry protocol to isolate synovial macrophage subsets to perform subset-specific transcriptomic analysis.

Methods: We used 3 mouse model of arthritis: the acute inducible K/BxN serum transfer-induced arthritis (STIA) model, and chronic inducible collagen induced arthritis (CIA) model, and the spontaneous KRN/Ag7 mice. STIA and CIA was induced in 10-12 week old female C57BL/6 mice. Flow cytometric analysis was employed to delineate macrophage subsets via expression of MHC II and CX3CR1 to obtain 4 distinct macrophage populations. These populations were sorted throughout the course of arthritis by FACS. RNA was extracted from sorted macrophage populations and processed for RNA-seq using Quantseq 3’ mRNA library preparation. The RNA-seq libraries were sequenced on an Illumina NextSeq 500 to an average depth of 5 million reads. The reads were aligned and mapped to genes using STAR and HTseq respectively.

Results: We observe by flow cytometry that the predominance of individual synovial macrophage subsets shift throughout the initiation, progression and resolution phases of arthritis and eventually return to their steady-state phenotype. PCA analysis of macrophage subsets in a naïve mouse show distinct transcriptional profiles. Analysis of the transcriptional profiles over the course of arthritis reveals that each macrophage subset responds differently at each phase of inflammation. K-means clustering of specific synovial macrophage subsets have identified distinct gene clusters and cellular processes that are differentially regulated to dictate their function during arthritis. Synovial macrophage populations from each arthritic model display a similar response to inflammation, suggesting that the transcriptional signatures and function of macrophages are consistent in inflammatory arthritis.

Conclusion: We conclude that fluctuations in the synovial macrophage populations over the course of arthritis coincide with the different phases of joint inflammation. These dynamics indicate that the specific macrophage subsets have different function during the course of disease as evidenced by their distinct transcriptional profiles. These high-throughput genomic approaches applied to macrophage subsets from models of both acute and chronic inflammatory arthritis allows us to comprehensively map their role in disease, thereby providing insight into potentially useful targets for therapy.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dynamics-of-transcriptional-signatures-from-purified-synovial-macrophage-subsets-during-acute-and-chronic-murine-models-of-inflammatory-arthritis/