Background/Purpose: Macrophages fulfill critical functions in maintaining tissue homeostasis in steady-state, as well as in inflammation and immune response.  In the joint synovium, we have previously shown using single-cell RNA-seq that four distinct populations of macrophage co-exist in steady state: synovial lining, interstitial, antigen-presenting, and infiltrating.  Our goal is to understand the role of these different macrophages in the inflammatory phase that follows joint trauma.  Synovial inflammation has been shown to key in the development of long term cartilage damage and osteoarthritis (OA): thus, macrophages may provide a therapeutic target for prevention and treatment.

Methods: We used a mouse model of post-traumatic OA (CIOA) where mice develop cartilage damage a few weeks after intra-articular injection of collagenase into the synovial space of the knee.  We euthanized male mice (aged 8-12 weeks) at days 0, 3, 7, 11, and 28 post-CIOA.  Knees were dissected, extraneous muscle tissue resected, and joints processed for Fluorescence-Activated Cell Sorting (FACS).  Macrophages were identified as CD45+CD11B+Ly6G-Ly6C-CD64+ cells and further subdivided into four populations by expression of MHCII and CX3CR1.  RNA was extracted from these cells and RNA-seq was performed using full-length SMART-seq v4 Ultra Low Input Kit.  After sequencing on Illumina Nextseq, bcl files were demultiplexed and resulting fastq files were trimmed with Trimmomatic, aligned to mm10 with tophat, and mapped to genes with HTseq to generate a table of gene expression for analysis.

Results: All macrophage populations were increased in number during peak inflammation on day 3 post-CIOA and returned to approximately steady-state levels by day 28.  We found that each macrophage sub-population exhibited distinct gene expression at day 0 but their transcriptional profiles appeared to converge on day 3.  By resolution of inflammation on day 28, the transcriptional profile of each sub-population resembled steady-state.  The transcriptional signatures over time suggested two dominant trends drove macrophage response: genes that were differentially expressed primarily in the synovial lining population and genes that were differentially expression in all four sub-populations in the same pattern.  Synovial lining macrophages up-regulated of cell cycle and monocyte genes suggesting this population may expand through a combination of proliferation and differentiating monocytes.  This population down-regulated genes associated with its specific synovial lining phenotype, which was confirmed by overlap with genes from human data.  The expression patterns observed in all macrophages included the up-regulation of genes associated with IL-1B/IL-6 inflammation and down-regulation of homeostatic genes.

Conclusion: Understanding the function of macrophages following joint trauma is likely to provide insight into the resulting joint instability that leads to the development of cartilage damage and OA.  By investigating the role of specific macrophage subsets, we may elucidate potential targets for the prevention or attenuation of OA-associated cartilage damage.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-dynamics-of-macrophage-sub-populations-in-the-inflammatory-phase-following-joint-trauma/