Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state

Abstract

Background: Bone erosion is a frequent complication of gout and is strongly associated with tophi, which are lesions comprising inflammatory cells surrounding collections of monosodium urate (MSU) crystals. Osteocytes are important cellular mediators of bone remodeling. The aim of this study was to investigate the direct effects of MSU crystals and indirect effects of MSU crystal-induced inflammation on osteocytes.

Methods: For direct assays, MSU crystals were added to MLO-Y4 osteocyte cell line cultures or primary mouse osteocyte cultures. For indirect assays, the RAW264.7 macrophage cell line was cultured with or without MSU crystals, and conditioned medium from these cultures was added to MLO-Y4 cells. MLO-Y4 cell viability was assessed using alamarBlue® and LIVE/DEAD® assays, and MLO-Y4 cell gene expression and protein expression were assessed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Histological analysis was used to examine the relationship between MSU crystals, inflammatory cells, and osteocytes in human joints affected by tophaceous gout.

Results: In direct assays, MSU crystals reduced MLO-Y4 cell and primary mouse osteocyte viability but did not alter MLO-Y4 cell gene expression. In contrast, conditioned medium from MSU crystal-stimulated RAW264.7 macrophages did not affect MLO-Y4 cell viability but significantly increased MLO-Y4 cell expression of osteocyte-related factors including E11, connexin 43, and RANKL, and inflammatory mediators such as interleukin (IL)-6, IL-11, tumor necrosis factor (TNF)-α and cyclooxygenase-2 (COX-2). Inhibition of COX-2 in MLO-Y4 cells significantly reduced the indirect effects of MSU crystals. In histological analysis, CD68⁺ macrophages and MSU crystals were identified in close proximity to osteocytes within bone. COX-2 expression was also observed in tophaceous joint samples.

Conclusions: MSU crystals directly inhibit osteocyte viability and, through interactions with macrophages, indirectly promote a shift in osteocyte function that favors bone resorption and inflammation. These interactions may contribute to disordered bone remodeling in gout.

Keywords: Bone erosion; Gout; Inflammation; Osteocyte; Urate.

Fig. 1

The direct effects of MSU crystals on osteocyte viability. The alamarBlue® assay was used to determine the viability of a MLO-Y4 cells and primary mouse osteocytes cultured with monosodium urate (MSU) crystals for 24 h, b MLO-Y4 cells cultured with soluble urate for 24 h, and c MLO-Y4 cells cultured with different types of crystals for 24 h. Viability was assessed 24 and 48 h after the addition of crystals or soluble urate. Data shown are pooled from three to four biological repeats and are presented as mean (SEM); by two-way ANOVA a P_Interaction < 0.0001 for MLO-Y4 cells, P_Interaction = 0.026 for primary mouse osteocytes, b P_Interaction = 0.24, and c P_Interaction = 0.057 at 24 h, P_Interaction < 0.0001 at 48 h; with post-hoc Dunnett’s test *p < 0.05, **p < 0.01, and ***p < 0.001 versus control (no crystals or soluble urate) at that time point. d The LIVE/DEAD® assay was used to determine the percentage of dead MLO-Y4 cells within three separate layers of the collagen gel following culture with MSU crystals for 24 h or 48 h. Data shown are pooled from four biological repeats and are presented as mean (SEM); one-way ANOVA p < 0.0001 at 24 h, p = 0.004 at 48 h; with post-hoc Sidak’s test ***p < 0.001 versus control (no MSU crystals) for each layer of the gel. BCP basic calcium phosphate, CPPD calcium pyrophosphate dehydrate

Fig. 2

Direct effects of MSU crystals on MLO-Y4 cell expression of bone-related or inflammatory genes. Real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes in MLO-Y4 cells, following culture with 0.1 mg/mL monosodium urate (MSU) crystals for 0, 1, 6, and 24 h. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P_Interaction > 0.1 for all genes. OPG osteoprotegerin, RANKL receptor activator of nuclear factor kappa-B ligand, TNF tumor necrosis factor

Fig. 3

Indirect effects of MSU crystal-stimulated RAW264.7 macrophage conditioned medium on MLO-Y4 cell gene expression. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 0, 1, 6, and 24 h. MLO-Y4 cells were harvested and real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P_Interaction = 0.007 for Tnfrsf11b, P_Interaction = 0.0005 for Tnfa, P_Interaction < 0.0001 for all other genes; with post-hoc Sidak’s test *p < 0.05, **p < 0.01, and ***p < 0.001 versus control conditioned medium at that time point. OPG osteoprotegerin, RANKL receptor activator of nuclear factor kappa-B ligand, TNF tumor necrosis factor

Fig. 4

Secretion of proinflammatory mediators by MLO-Y4 cells in response to MSU crystal-stimulated RAW264.7 macrophages. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 24 h and supernatants harvested. The concentrations of a tumor necrosis factor (TNF)-α, b prostaglandin E₂ (PGE₂), c interleukin (IL)-6, and d osteoprotegerin (OPG) protein in the RAW264.7 macrophage conditioned medium samples (control and MSU crystal-stimulated) and the MLO-Y4 cell supernatants were measured by ELISA. Data shown are pooled from three biological repeats and are presented as mean (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference

Fig. 5

Effects of COX-2 inhibition on MLO-Y4 cell responses to MSU crystal-stimulated RAW264.7 macrophage conditioned medium. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. A cyclooxygenase-2 (COX-2)-specific inhibitor (SC-236) was added to MLO-Y4 cells for 1 h prior to the addition of 40% conditioned medium for 24 h. MLO-Y4 cells were then harvested for mRNA gene expression analysis and supernatants harvested for protein quantification. a Changes in mRNA expression of inflammatory genes: tumor necrosis factor (TNF)-α, COX-2, interleukin (IL)-6, and IL-11; and bone-related genes: receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG). b Changes in TNF-α, prostaglandin E₂ (PGE₂), and IL-6 protein levels in MLO-Y4 cell supernatants. Data shown are pooled from five biological repeats and are presented as (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference

Fig. 6

Histological analysis of human joint tissue affected by tophaceous gout. a,b Representative photomicrographs of joint samples affected by tophaceous gout, showing both MSU crystals (indicated by asterisks) and associated inflammatory tissue in close proximity to bone (a, toluidine blue staining viewed using light microscopy; b, viewed using polarizing light microscopy with a red compensator). Immunohistochemistry staining for c CD68⁺ cells (macrophages) and d COX-2 expression in human joint tissue affected by tophaceous gout