Background/Purpose: Monocytes and Macrophages play critical roles in immune homeostasis and host defense. Recent studies identified that macrophages are highly heterogeneous in terms of location, origin and function. However, the sources of macrophages in the synovium and their functions are not yet clear. We performed BrdU incorporation and RNAseq profiling of blood monocytes and synovial macrophages from normal C57BL/6 mice under homeostatic conditions to identify functional differences.

Methods:

Five distinct macrophage populations in synovium were determined by flow cytometry, based on subsetting CD45⁺CD64⁺CD11b⁺ cells by their expressions of Ly6C, MHCII and F4/80. Three F4/80^int (FI) subsets defined as Ly6C+MHCII- (FI1), Ly6C+MHCII+ (FI2) and Ly6C-MHCII+ (FI3); and 2 subsets of F4/80^hi (FH) cells defined as MHCII^– (FH1) and MHCII⁺ (FH2) were identified. BrdU was used to identify proliferating cells. Populations of synovial macrophages and classical (CM) and non-classical (NCM) circulating monocytes were sorted by flow cytometry. High quality mRNAs from each of the cell populations was processed for RNAseq. The gene expression profiles and pathways were analyzed by Genee software and GOrilla database.

Results:

BrdU+ CM and FI1 and FI2 macrophages that originated from bone marrow monocyte progenitors were detected within 24 hours. NCM and FI3 macrophages were BrdU+ on day 3. The FH1 and FH2 subsets were not renewed by circulating monocytes over 5 days, but were slowly proliferating. The distinct gene expression patterns were identified for each cell type by principal component analysis and pairwise correlation among cell groups. Genes that were highly expressed in CM were enriched in immune system processes, leukocyte activation, and signal transduction pathways such as Irf8, Stat1, Syk and Lyn. The majority of genes (65%) expressed in FH1 macrophages were maintained compared with CMs. This ratio of maintained genes gradually decreased going from the FI3, FH2 to FH1 subsets (60-54%). Further, comparing each macrophage subset to CMs, the ratio of differentially expressed genes (DEGs) increased from 14% (FI1), 23% (FI3), 27% (FH2) to 30% in FH1 macrophages. Of interest, host defense genes were highly represented in the FI1 macrophages compared with CMs. Expression of genes in the FH1 subset was highly correlated with those expressed in microglia and alveolar macrophages.

Conclusion:

CM rapidly enter synovial tissue under homeostatic conditions but differentiate no further the FI3 population. FH macrophages are long-lived tissue resident macrophages of the joints. These observations suggest that under homeostatic conditions the FH1 subset provides a niche that prevents the further differentiation of FI1 macrophages to tissue resident macrophages. Further studies will address the mechanisms contributing to this niche.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/tissue-resident-macrophages-establish-a-niche-that-limits-monocyte-to-macrophage-differentiation-in-synovial-tissue-during-homeostasis/