Background/Purpose: Rheumatoid Arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints. Macrophages are a key mediator of pro-inflammatory signaling, contributing to inflammation and subsequent joint destruction seen in RA. Recent research has revealed that macrophages in the joint synovium exhibit phenotypically distinct subpopulations, which may diverge in contributing towards either RA pathogenesis or potential remission. There is a need to further understand how synovial macrophage subpopulations are repopulated after disruption to better characterize varying phenotypic trajectories.

Methods: Bone marrow chimeras (BMC) were generated by transplanting cells from CD45.1 mice (donor) into CD45.2 mice (recipient) that were irradiated with shielded ankle joints. Ankle joints were extracted from BMC controls (day 0) and those treated with clodronate-laden liposomes (day 7) to deplete circulating monocytes. CITE-seq was performed on sorted synovial macrophages (CD45+CD11b+CD4-CD8-Ly6G-SiglecF-NK1.1-CD64+ cells) using the 10x Chromium instrument and processed using the CellRanger pipeline. Clustering, differential gene expression analysis, and pathway analysis was performed using Seurat and fgsea for each timepoint separately and combined. Trajectory analysis was performed using Monocle after merging day 0 and day 7 data.

Results: Distinct macrophage subpopulations were seen in both day 0 and day 7 samples. MHCII genes (H2-Eb1, H2-Ab1, H2-Aa) were differentially expressed in CD45.1-enriched clusters at day 0. For both timepoints, clusters of synovial lining macrophages (CX3CR1+) were CD45.2-enriched. Genes associated with toll-like receptor signaling pathways (Tlr2, Tlr7, Tlr8) marked CD45.2-enriched clusters at day 0. Genes coding for Ap1 (Jun), a major transcription factor regulating the pro-inflammatory program of macrophages, were repressed at day 7 compared to day 0. Macrophages at day 7 differentially expressed genes coding for metalloproteases (Mmp12, Mmp14, Mmp19) implicated in extracellular matrix degradation, suggesting heightened migratory capacity. Enrichment for pathways associated with extracellular matrix interactions were confirmed in our trajectory analysis from day 0 to day 7.

Conclusion: These results suggest the existence of transcriptional heterogeneity among macrophages in the synovium, as well as changes over time following disruption. Future experiments will test how macrophage repopulation is altered under inflammatory conditions.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/investigating-macrophage-repopulation-in-the-synovium/