Non-classical monocytes as mediators of tissue destruction in arthritis

Abstract

Objectives: Bone destruction in rheumatoid arthritis is mediated by osteoclasts (OC), which are derived from precursor cells of the myeloid lineage. The role of the two monocyte subsets, classical monocytes (expressing CD115, Ly6C and CCR2) and non-classical monocytes (which are CD115 positive, but low in Ly6C and CCR2), in serving as precursors for OC in arthritis is still elusive.

Methods: We investigated CCR2^-/- mice, which lack circulating classical monocytes, crossed into hTNFtg mice for the extent of joint damage. We analysed monocyte subsets in hTNFtg and K/BxN serum transfer arthritis by flow cytometry. We sorted monocyte subsets and analysed their potential to differentiate into OC and their transcriptional response in response to RANKL by RNA sequencing. With these data, we performed a gene ontology enrichment analysis and gene set enrichment analysis.

Results: We show that in hTNFtg arthritis local bone erosion and OC generation are even enhanced in the absence of CCR2. We further show the numbers of non-classical monocytes in blood are elevated and are significantly correlated with histological signs of joint destruction. Sorted non-classical monocytes display an increased capacity to differentiate into OCs. This is associated with an increased expression of signal transduction components of RANK, most importantly TRAF6, leading to an increased responsiveness to RANKL.

Conclusion: Therefore, non-classical monocytes are pivotal cells in arthritis tissue damage and a possible target for therapeutically intervention for the prevention of inflammatory joint damage.

Keywords: arthritis; cytokines; inflammation; synovitis; tnf-alpha.

Figure 1

hTNFtg/CCR2^−/−-deficient mice showed enhanced local bone destruction and osteoclast formation. CCR2^−/− mice were crossed into hTNFtg mice and histological analysis was performed. (A) Quantitative analysis of histological parameters of arthritis (hTNFtg, n=5; hTNFtg/CCR2^−/−, n=9)). Results are shown as mean±SEM (B) Representative histological sections of the hind paws of hTNFtg/CCR2^+/+ and hTNFtg/CCR2^−/− mice stained with H&E and TRAP. Osteoclasts are displayed as purple-stained cells. *P<0.05. (C), Analysis of the percentage of Ly6C⁺ cells (left panel), CD115⁺ cells (middle panel) and Gr1⁺ cells (right panel) shown as per cent of total cells in the synovial membrane of hTNFtg/CCR2^+/+ and hTNFtg/CCR2^−/− mice. TRAP, tartrate-resistant acid phosphatase.

Figure 2

Monocytes in blood and spleen in hTNFtg arthritis. (A) Gating of flow cytometric analysis of myeloid populations in blood obtained from WT and hTNFtg mice. (B,C) Bar graph summarising frequencies of myeloid populations in blood (B) and spleen (C) of WT mice and hTNFtg mice (n=at least 10 per group). Results are shown as mean±SEM. **P<0.01; ***P<0.001. WT, wild type.

Figure 3

Development of arthritis is accompanied by accumulation of circulating mononuclear cells. (A) Clinical assessment of paw swelling and grip strength in WT mice (n=10) and hTNFtg mice (n=9). (B) Characterisation of monocytes under steady-state conditions and during hTNF driven arthritis. Blood from WT mice and hTNFtg were analysed using flow cytometric analysis over time (n=at least 12 per group and time point). Results are shown as mean±SEM. *P<0.05; **P<0.01; ***P<0.001. WT, wild type.

Figure 4

Characterisation of monocytes during hTNF driven arthritis. The number of circulating non-classical monocytes (A) and classical monocytes (B) was correlated with histological markers of joint destruction such as the number of osteoclasts, the area of erosion and inflammation. (C) H&E staining of a hind paw of a hTNFtg mouse (scale bars=1 mm) and immunohistochemical staining for monocyte markers CD115 and Ly6C in an erosion (scale bars=50 µm). Arrows indicate CD115 positive osteoclasts.

Figure 5

Identification of cell populations with osteoclastogenic potential from peripheral blood cells. (A) In vitro OC assay of sorted Ly6C⁺ classical and Ly6C^– non-classical monocytes of WT and hTNFtg mice stimulated with MCSF (4d) and RANKL (3d). Graph shows the number of TRAP positive multinucleated cells (data shown are mean values of five independent experiments±SEM). (B) Volcano plot representation of differential expression analysis of genes in RNA sequencing of WT Ly6C⁺ classical vs Ly6C^– non-classical monocytes after stimulation with MCSF (4d) and RANKL (1d) (n=3 each). The x-axis shows log2 FC in expression, the negative log10 of the p value is plotted on the y-axis. Each gene is represented by one point on the graph. (C) Number of differentially regulated genes (FC≥2, p≤0.05) in RNA sequencing in Ly6C⁺ classical vs Ly6C^– non-classical monocytes. (D) GO enrichment analysis of the transcriptome of Ly6C⁺ classical vs Ly6C^– non-classical monocytes: x-axis shows FC of significantly enriched GO terms in differentially regulated genes in RNA-sequencing of Ly6C⁺ classical vs Ly6C^– non-classical monocytes. The number in the columns represents the –log10 p value of the indicated GO term. **P<0.01; ***P<0.001. FC, fold change; GO, Gene Ontology; OC, osteoclasts; TRAP, tartrate-resistant acid phosphatase; WT, wild type.

Figure 6

Increased expression of RANK-associated signal transduction molecules in non-classical monocytes. (A) Expression levels (FPKM values) of Gab2 and TRAF6 in RNA sequencing dataset of sorted Ly6C⁺ classical and Ly6C^– non-classical monocytes stimulated with MCSF (4d) and RANKL (1d) (n=3 each). Results are shown as mean±SEM (B) Western blot for the presence of TRAF6 in Ly6C⁺ classical and Ly6C^– non-classical monocytes after stimulation with MCSF for 3 days. Actin was used for loading control. Results are representative of three independent experiments. (C) Heat map of osteoclast-related genes of RNA sequencing of classical vs non-classical monocytes stimulated with MCSF (4d) and RANKL (1d) (n=3 each). (D) Western blot for the presence of DC-STAMP in Ly6C⁺ classical and Ly6C^– non-classical monocytes after stimulation with MCSF (4d) and RANKL (1d). (E) Quantification of nuclei/OC of classical and non-classical monocytes derived OCs. Results are shown as mean±SEM. (F) Representative pictures of TRAP staining to detect OCs generated from classical and non-classical monocytes. *P<0.05; **P<0.01; ***P<0.001. OCs, osteoclasts; TRAP, tartrate-resistant acid phosphatase.