Interleukin-34 Reprograms Glycolytic and Osteoclastic Rheumatoid Arthritis Macrophages via Syndecan 1 and Macrophage Colony-Stimulating Factor Receptor

Abstract

Objective: In rheumatoid arthritis (RA), elevated serum interleukin-34 (IL-34) levels are linked with increased disease severity. IL-34 binds to 2 receptors, macrophage colony-stimulating factor receptor (M-CSFR) and syndecan 1, which are coexpressed in RA macrophages. Expression of both IL-34 and syndecan 1 is strikingly elevated in the RA synovium, yet their mechanisms of action remain undefined. This study was undertaken to investigate the mechanism of action of IL-34 in RA.

Methods: To characterize the significance of IL-34 in immunometabolism, its mechanism of action was elucidated in joint macrophages, fibroblasts, and T effector cells using RA and preclinical models.

Results: Intriguingly, syndecan 1 activated IL-34-induced M-CSFR phosphorylation and reprogrammed RA naive cells into distinctive CD14+CD86+GLUT1+ M34 macrophages that expressed elevated levels of IL-1β, CXCL8, and CCL2. In murine M34 macrophages, the inflammatory phenotype was accompanied by potentiated glycolytic activity, exhibited by transcriptional up-regulation of GLUT1, c-Myc, and hypoxia-inducible factor 1α (HIF-1α) and amplified pyruvate and l-lactate secretion. Local expression of IL-34 provoked arthritis by expanding the glycolytic F4/80-positive, inducible nitric oxide synthase (iNOS)-positive macrophage population, which in turn attracted fibroblasts and polarized Th1/Th17 cells. The cross-talk between murine M34 macrophages and Th1/Th17 cells broadened the inflammatory and metabolic phenotypes, resulting in the expansion of IL-34 pathogenicity. Consequently, IL-34-instigated joint inflammation was alleviated in RAG^-/- mice compared to wild-type mice. Syndecan 1 deficiency attenuated IL-34-induced arthritis by interfering with joint glycolytic M34 macrophage and osteoclast remodeling. Similarly, inhibition of glycolysis by 2-deoxy-d-glucose reversed the joint swelling and metabolic rewiring triggered by IL-34 via HIF-1α and c-Myc induction.

Conclusion: IL-34 is a novel endogenous factor that remodels hypermetabolic M34 macrophages and facilitates their cross-regulation with T effector cells to advance inflammatory bone destruction in RA.

Figure 1.. Expression of IL-34 and its receptors, M-CSFR and SDC-1, in NL, OA and RA specimens.

(A) GM-CSF, M-CSF and IL-34 protein concentrations were determined in plasma and SF samples collected from NL (plasma: n=39), OA (plasma: n=10; SF: n=32) and RA (plasma: n=39; SF: n=45) donors. NL, OA and RA synovial tissues were stained for IL-34 (B), M-CSFR (C) and SDC-1 (E) and scored on a 0–5 scale in suppl-Fig. S1A–C (for IL-34, NL: n=9, OA: n=11, RA: n=9; for M-CSFR, NL: n=9, OA: n=11, RA: n=8; for SDC-1, NL: n=8, OA: n=10, RA: n=10). (D) A biotinylated anti-IL-34 Ab was used to compare the amount of IL-34 retained on a recombinant SDC-1-coated plate versus a PBS-coated plate, both incubated with various doses of IL-34 (0.24 to 1000 ng/ml). F) Western blot analysis was used to evaluate the expression of SDC-1 (32 kDa) and M-CSFR (full-length: 140 kDa; intracellular domain: 52 kDa) in RA PB MΦ, T cells and RA FLS. Equal loading was confirmed by quantifying Actin (42 kDa). The data are shown as mean ± SEM. * p<0.05 ** p<0.01 *** p<0.001.

Figure 2.. Joint inflammation was induced by local IL-34 expression.

WT mice were i.a. injected 1x/week with Ad-Ctrl and Ad-IL-34. (A) Joint circumference was monitored over 16 days (n=16). 16 days post-onset, mice were sacrificed and joints were processed for IHC, qRT-PCR and protein estimation. (B) Tissue sections were stained with H&E and scored for synovial lining thickness, inflammation and bone erosion on a 0–5 scale in the suppl-Fig. S2A (n=5). (C) Tissue sections were stained for MΦ markers F4/80, iNOS, Arg1 and staining was scored on a 0–5 scale in the suppl-Fig. S2B (n=5). By qRT-PCR, joint RNA levels of various monokines (D-E) or osteoclastic factors (F) were determined following Ad-IL-34 or control treatment (n=8). The data are shown as mean ± SEM or as minimum-maximum box-and-whisker plots (centerline = median). * p<0.05 ** p<0.01 *** p<0.001.

Figure 3.. M34 MΦ signaling and signature is dependent on SDC-1 ligation.

(A) Cells were pre-incubated with buffer, anti-SDC-1 Ab (1:100) or anti-M-CSFR Ab (10 μg/ml) for 2h before IL-34 (100 ng/ml) stimulation for 5 or 10 minutes and lysates were used for WB quantification of pM-CSFR and pERK. WB shown is one representative out of 4 independent experiments. (B) In vitro differentiated MΦs derived from negatively selected RA monocytes were control-treated or stimulated with IL-34 (300 ng/ml) for 24h and then stained with Ab against CD14, CD86 and CD206 for flow cytometry. (B) The contour plot of CD14⁺ CD86⁺ cells for one representative experiment out of 5 is shown. Among CD14⁺ gated cells, the MFI of CD86 and CD206 staining was determined (n=5) in the suppl-Fig. S3A. (C) The qRT-PCR analysis was performed to determine the expression of M1 and M2 cytokines by RA in vitro differentiated MΦs stimulated with IL-34 (300 ng/ml) for 8h, expressed as a fold change compared to control treatment (n=4). (D-F) Prior to IL-34 stimulation, RA cells were control-treated or pre-incubated with anti-SDC-1 (1:100) or anti-M-CSFR Ab (10 μg/ml) (CCL2: n=8; TNF: n=6; IL-6: n=8). Protein induction was compared to control treatment and was analyzed using the Wilcoxon signed-rank test. The effect of neutralizing Abs compared to sole IL-34 stimulation was analyzed using a one-tailed Wilcoxon matched-pairs signed-rank test. The data are shown as mean ± SEM or as minimum-maximum box-and-whisker plots (centerline = median). * p<0.05 ** p<0.01 *** p<0.001.

Figure 4.. IL-34-induced metabolic changes promote progressive inflammation.

(A) In vitro differentiated MΦs, derived from negatively selected monocytes, were control-treated or stimulated with IL-34 (300 ng/ml) for 24h. A representative contour plot and the mean percentage of CD14⁺GLUT1⁺ cells (n=5) are shown. Significance was evaluated using a paired t-test. (B) The Seahorse cell energy phenotype assay was employed to study the glycolytic (ECAR) capacity of PBS- versus IL-34-pre-treated RAW 264.7 cells (n=5). (C) The concentration of L-lactate and pyruvate in the conditioned media of murine MΦs (24h) was determined colorimetrically (n=4). (D) Under hypoglycemic conditions, murine BM-derived MΦs were untreated or stimulated with IL-34 (1 μg/ml) for 6h and expression of metabolic genes was examined by qRT-PCR (n=4). WT mice were i.a. injected 1x/week with Ad-Ctrl and Ad-IL-34. (E) The mice that received Ad-IL-34 were either i.p. treated with 2-DG or placebo and joint circumference was monitored over 16 days (n=10). (F) Tissue sections were stained for C-MYC and HIF1α or H&E stained and staining was scored on a 0–5 scale in the suppl-Figs. S3I–J (n=4). The data are shown as mean ± SEM or as minimum-maximum box-and-whisker plots (centerline = median). * p<0.05 ** p<0.01 *** p<0.001.

Figure 5.. T cells contribute to IL-34-induced joint inflammation.

(A) Supernatant levels of IL-17 were measured in the BMMΦ-splenocyte co-cultures (5 days) treated with control or IL-34 (1 μg/ml) in the presence or absence of 2-DG (5 mM) (n=3). As a positive control (+) co-cultures were stimulated with TGFβ (1ng/ml) + IL-6 (20 ng/ml). (B) Joint CD3+ T cells were stained in WT mice that received Ad-Ctrl and Ad-IL-34 on days 0, 7, 14, and were sacrificed on day 16. Immunostaining was scored on a 0–5 scale in the suppl-Fig. S5A (n=5). (C) Joint circumference was monitored in WT and RAG−/− mice that were i.a. injected with Ad-Ctrl or Ad-IL-34 (1x/week) and were sacrificed on day 17 (n=10). (D) Ankle joints were stained for H&E or F4/80, iNOS and Arg1 (n=4). H&E and positive MΦ immunostaining were scored on 0–5 scale in the suppl-Fig. S5D–E. (E) Joint expression of monokines (Ad-Ctrl (WT): n=7; Ad-IL-34 (WT): n=5; Ad-IL-34 (RAG−/−): n=6) and osteoclastic factors (F; n=6) were determined by qRT-PCR. The data are shown as mean ± SEM. * p<0.05 ** p<0.01 *** p<0.001.

Figure 6.. SDC-1 modulates IL-34-driven joint inflammation, glycolysis and osteoclastogenesis.

WT and SDC-1−/− mice were i.a. injected with Ad-Ctrl or Ad-IL-34 (1x/week). (A) Joint circumference was monitored over 15 days, thereafter mice were sacrificed and differences between treatment groups were evaluated using the non-parametric Mann-Whitney test (Ad-Ctrl (WT): n=7; other groups: n=8). (B) Transcriptional regulation of joint inflammatory mediators was evaluated by qRT-PCR (n=4). (C) Under hypoglycemic conditions, BMMΦs from WT and SDC-1−/− mice were untreated or stimulated with IL-34 (1 μg/ml) for 6h and expression of glycolytic genes was examined by qRT-PCR (n=4). (D) Transcription levels of joint osteoclastic factors were determined by qRT-PCR in WT and SDC-1−/− mice that received control or Ad-IL-34 local injection (n=4). (E) To assess osteoclastogenesis in vitro, BM-derived pre-osteoclasts from WT and SDC-1−/− mice were untreated (-Ctrl), cultured in suboptimal conditions (15 ng/ml M-CSF and RANKL; 15/15) or exposed to 15/15 + IL-34 (300 ng/ml). Following 14 days of differentiation (fresh stimuli 2x/week), TRAP staining was performed and TRAP⁺ multinuclear osteoclasts were counted in the suppl-Fig. S5G (n=4). (F) Illustrates that IL-34 binding to M-CSFR/SDC-1 cultivates RA inflammatory and glycolytic M34 MΦs that are predisposed to osteoclastogenesis. The data are shown as mean ± SEM, * p<0.05 ** p<0.01 *** p<0.001.