Resident Synovial Macrophages in Synovial Fluid: Implications for Immunoregulation in Infectious and Inflammatory Arthritis

Abstract

Objectives: Resident synovial macrophages (RSM) provide immune sequestration of the joint space and are likely involved in initiation and perpetuation of the joint-specific immune response. We sought to identify RSM in synovial fluid (SF) and demonstrate migratory ability, in additional to functional changes that may perpetuate a chronic inflammatory response within joint spaces.

Methods: We recruited human patients presenting with undifferentiated arthritis in multiple clinical settings. We used flow cytometry to identify mononuclear cells in peripheral blood and SF. We used a novel transwell migration assay with human ex-vivo synovium obtained intra-operatively to validate flow cytometry findings. We used single cell RNA-sequencing (scRNA-seq) to further identify macrophage/monocyte subsets. ELISA was used to evaluate the bone-resorption potential of SF.

Results: We were able to identify a rare population of CD14^dim, OPG⁺, ZO-1⁺ cells consistent with RSM in SF via flow cytometry. These cells were relatively enriched in the SF during infectious processes, but absolutely decreased compared to healthy controls. Similar putative RSM were identified using ex vivo migration assays when MCP-1 and LPS were used as migratory stimulus. scRNA-seq revealed a population consistent with RSM transcriptionally related to CD56⁺ cytotoxic dendritic cells and IDO⁺ M2 macrophages.

Conclusion: We identified a rare cell population consistent with RSM, indicating these cells are likely migratory and able to initiate or coordinate both acute (septic) or chronic (autoimmune or inflammatory) arthritis. RSM analysis via scRNA-seq indicated these cells are M2 skewed, capable of antigen presentation, and have consistent functions in both septic and inflammatory arthritis.

Keywords: Type A synoviocyte; inflammatory arthritis; joint space immunoregulation; resident synovial macrophage; septic arthritis.

Figure 1:

Resident Synovial Macrophage-like cells. Live, CD56⁻CD3⁻CD20⁻CD11c⁻ TREM2⁺OPG⁺CD68⁺CD11b⁺HLA-DR⁺CX3CR1⁺ cells were identified with an enrichment of CD14^dim cells in the SF compared to PBMCs (A, n=6 patients for preliminary evaluation for RSM cells). These CD14^dim macrophages were decreased in absolute frequency in inflammatory and septic arthritis compared to controls (B, n=22 patients, 5-12 patients per group) but were relatively enriched in the SF of pathologic joints when SFCs were compared to PBMC, shown by ratio >1 (C, n=22 patients, 5-12 patients per group). Back-gating on the Alive/CD14^dim population to identify M2 macrophages (D, inset), 78.6% were double positive for OPG and ZO-1 (D, representative patient with inflammatory arthritis). Two-way ANOVA with Tukey’s post-hoc correction.

Figure 2:

Transwell synovial cell migration assay. Schematic of the experimental set up (A). Resident synovial macrophage markers TREM-2+, OPG+, CX3CR1+, ZO-1+ and F11R+ were evaluated within the migratory monocyte/macrophage population (Alive/CD56−/CD3−/CD11c−/CD20−/CD14+/CD11b+) in a representative patient treated with 250 ng/mL of MCP-1 (B). tSNE plots of the same patient’s migratory myeloid cells demonstrating ZO-1 has the most delineation from other markers (C).

Figure 3:

ELISA results of SF supernatant protein concentration. OPG, sRANKL, and ratio of OPG:sRANKL (A). TRAP and TGF-β1, (B). Measures of TIMP1, MMP9, or ratio of TIMP1:MMP9. Normal (n=9), inflammatory (n=6), and septic (n=11). Multiple Mann-Whitney tests with FDR rate <0.05.

Figure 4:

Unsupervised Cluster Analysis showing composite of 3 patients with inflammatory arthritis (A). Clusters were manually identified based on top ten expressed genes (B) in addition to classical markers. Analysis performed in R with resolution of 0.4 and dimensions 1:30. N = 3 patients per inflammatory and septic arthritis.

Figure 5:

Differentially Expressed Genes (DEGs) (A). Conserved genes where Log2FC > 1.5 and the change in percent expression in both septic arthritis and inflammatory arthritis was > 0.7 in each cluster compared to all other clusters. Gene names in •bold represent genes that were also in the top 10 expressed genes in Figure 4.

Figure 6:

Top 15 most up or downregulated pathways using DEGs and ReactomeGSA.