Synovial fluid monocyte/macrophage subsets and their correlation to patient-reported outcomes in osteoarthritic patients: a cohort study

Abstract

Background: Chronic, low-grade inflammation of the synovium (synovitis) is a hallmark of osteoarthritis (OA), thus understanding of OA immunobiology, mediated by immune effectors, is of importance. Specifically, monocytes/macrophages (MΦs) are known to be abundantly present in OA joints and involved in OA progression. However, different subsets of OA MΦs have not been investigated in detail, especially in terms of their relationship with patient-reported outcome measures (PROMs). We hypothesized that levels of synovial fluid (SF) MΦ subsets are indicative of joint function and quality of life in patients with OA, and can therefore serve as biomarkers and therapeutic targets for OA.

Methods: In this cohort study, synovial fluid leukocytes (SFLs, N = 86) and peripheral blood mononuclear cells (n = 53) from patients with knee OA were characterized. Soluble MΦ receptors and chemokine (sCD14, sCD163, CCL2, CX3CL1) levels were detected in SF using immunoassays. Linear models, adjusted for sex, age and body mass index, were used to determine associations between SF MΦs and soluble factors with PROMs (N = 83). Pearson correlation was calculated to determine correlation between MΦ subsets, T cells and soluble factors.

Results: SF MΦs were the most abundant SFLs. Within these, the double-positive CD14⁺CD16⁺-MΦ subset is enriched in knee OA SF compared to the circulation. Importantly, MΦ subset ratios correlated with PROMs, specially stiffness, function and quality of life. Interestingly, the SF CD14⁺CD16⁺-MΦ subset ratio correlated with SF chemokine (C-C motif) ligand 2 (CCL2) levels but not with levels of sCD163 or sCD14; we found no association between PROMs and either SF CCL2, sCD163, sCD14 or CX3CL1 (which was below detection levels). All SF MΦs displayed high levels of HLA-DR, suggesting an activated phenotype. Correlation between OA SF MΦ subsets and activated CD4⁺ T cell subsets suggests modulation of CD4⁺ T cell activation by MΦs.

Conclusion: SF MΦ subsets are associated with knee OA PROMs and display an activated phenotype, which may lead to modulation of CD4⁺ T cell activation. Knee OA SF MΦ subsets could serve as knee OA function biomarkers and as targets of novel therapeutics.

Keywords: Leukocytes; Monocytes/macrophages; Osteoarthritis; PROMs; Synovial fluid.

Fig. 1

Experimental outline. a Venn diagram of samples and data collected: blood, synovial fluids (SFs), Kellgren–Lawrence (KL) grading and patient-reported outcome measures (PROMs). b Experimental outline and methods; OA state: i.e. Early (KL I/II) or Late (KL III/IV). PBMCs, peripheral blood mononuclear cells; MΦ, monocytes/macrophages; SF, synovial fluid; CCL2, chemokine (C-C motif) ligand 2

Fig. 2

Knee osteoarthritis (KOA) synovial fluid (SF) monocytes/macrophages (MΦs) are the most abundant SF leukocytes (SFLs) and have different prevalence to those in the circulation. a Frequency of SFL and peripheral blood mononuclear cell (PBMC) (N = 40) populations. No statistical analysis is shown for neutrophils. b MΦ subset ratios, in PBMC and SF MΦs (N = 40). a and b Wilcoxon signed test (paired) was used. P values shown are corrected for multiple comparisons. Box plots indicate median with interquartile range for box and Tukey-style whiskers. Lines between boxplots indicate significant differences, with adjusted p value shown on top. White dots with white box represent data from PBMCs; black dots with gray box represent data from SFLs. NK, natural killer

Fig. 3

Pro-inflammatory monocytes/macrophages (MΦ) subset correlate with synovial fluid (SF) chemokine (C-C motif) ligand 2 (CCL2) levels. Positive correlation between SF CCL2 levels and CD14⁺ CD16⁺−MΦs (to total SF MΦs) ratio, N = 81. Pearson r and adjusted p value. Dashed line indicates regression resulting from general linear modeling approximation

Fig. 4

Knee osteoarthritis (KOA) synovial fluid (SF) monocytes/macrophages (MΦ) subsets and CD4+ T cells display an activated phenotype and correlate with each other. a Levels of HLA-DR expression in MΦs: mean fluorescence intensity (MFI) on peripheral blood mononuclear cell (PBMC) and SF MΦ subpopulations (SF leukocytes (SFLs), N = 40). b Frequency of activated cells in CD4⁺ T cells: CD69 + (early activation; N = 37), CD25+ (intermediate activation, SFLs, N = 37), HLA-DR+ (late activation, SFLs, N = 15). c Negative correlation between CD14⁺CD16⁺-MΦs (to total SF MΦs) ratio and the frequency of early activated (CD69⁺) CD4⁺ T cells; N = 82. d Negative correlation between CD14⁺CD16^neg-MΦs (to total SF MΦs) ratio and the frequency of late activated (HLA-DR⁺) CD4⁺ T cells; N = 32. a and b Median with interquartile range for box and Tukey-style whiskers. Lines between boxplots indicate significant differences with adjusted p value on top. White dots with white box represent data from PBMCs; black dots with gray box represent data from SFLs. c and d Pearson correlation coefficient (r) and adjusted p value shown