Background/Purpose: Macrophages are critical to the pathogenesis of rheumatoid arthritis(RA), but several distinct macrophage subpopulations co-exist in the synovium of joints. In steady-state, tissue-resident macrophages maintain joint integrity and are required for tissue homeostasis. On the other hand, macrophages contribute to inflammation through the production of cytokines, release of degradative enzymes, and recruitment of other immune cells. However, we currently lack specific targets to regulate macrophage function at the molecular level in the context of developing and relapsing disease.

Methods: We utilized functional genomic analysis of murine and human synovium including single cell-CITE and ATAC seq.

Results: Based on our analysis of gene expression, we characterized the 4 synovial macrophage populations as follows: MA=CX3CR1+MHCII- tissue-resident synovial lining, MB=CX3CR1+MHCII+ newly infiltrating cells; MC=CX3CR1-MHCII- tissue-resident interstitial, MD=CX3CR1-MHCII+ monocyte-derived interstitial. We then characterized the 4 subpopulations of synovial macrophages at days 0, 3, 7, 13 and 21 post serum transfer induced arthritis (STIA). We clustered the genes that were differentially expressed over the course of STIA in any subpopulation into 6 clusters (STIA I-VI). STIA I-III represent an increase in expression of inflammation and immune response genes that is most prominent in infiltrating MB. In contrast, STIA IV-VI represent a decrease in expression of homeostatic (largely interstitial MC and MD), cell cycle (MB), and tissue-resident genes (preferentially MA), respectively. To achieve better resolution on the transcriptional profile of synovial macrophages, we performed bulk RNA-seq on each subpopulation as well as classical and non-classical monocytes. We defined 5 clusters of genes that vary in expression across macrophages: c1=MA-specific; c2=tissue-resident-specific (MA and MC), c3=monocyte-derived-specific (MB and MD), c4=tissue-resident and monocyte; c5=MB and monocyte. To identify TFs that regulate each cluster, we performed motif-finding using the HOMER package. We identified that the MAFB sequence motif was associated with c3 and increased in expression from monocytes to tissue-resident macrophages supporting its role in monocyte-macrophage transition. On the other hand, the MEF2C sequence motif and gene expression suggested it played a role in tissue-residency. By performing a similar analysis on STIA time course clusters, we found that MAFB is associated with increased expression of inflammatory genes, whereas MEF2C is linked with decreased expression of homeostatic and tissue-resident genes. We also determined that macrophage sub-populations identified in mice were recapitulated in human patients from the synovial tissue of 7 RA patients. We found that patients exhibited varying macrophage composition, specifically, the proportion of MA and MC tended to be negatively correlated with disease severity, while MB and MD exhibited the opposite trend.

Conclusion: These studies established the dynamics and transcriptional regulation of monocyte-derived macrophages in inflammatory arthritis from mice and human synovium.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/transcriptional-factor-profiling-denotes-specific-synovial-macrophage-heterogeneity/