The online version of this article (https://doi.org/10.1186/s13075-018-1704-y) contains supplementary material, which is available to authorized users.
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.
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.
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.
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.
Additional file 1: Figure S1. The effect of different sizes of MSU crystals on MLO-Y4 cell viability. The alamarBlue® assay was used to determine the viability of MLO-Y4 cells cultured with different sizes of MSU crystals for 24 h. Viability was assessed 24 and 48 h after the addition of MSU crystals. Data shown are pooled from three biological repeats and are presented as mean (SEM), two-way ANOVA: PInteraction = 0.86; PMSU crystal size = 0.96; and PMSU crystal concentration = 0.0001 for the 24 h time point; and PInteraction = 0.13; PMSU crystal size = 0.21; and PMSU crystal concentration < 0.0001 for the 48 h time point. (JPG 154 kb)13075_2018_1704_MOESM1_ESM.jpg
Additional file 2: Figure S2. Indirect effects of MSU crystal-stimulated RAW264.7 macrophage conditioned medium on MLO-Y4 cell viability. RAW264.7 macrophages were cultured with or without 0.5 mg/mL 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 at different concentrations (5%, 20%, and 40% final concentration in a well) for 24 h. The alamarBlue® assay was used to determine MLO-Y4 cell viability 24 h and 48 h after the addition of conditioned medium. Data shown are pooled from three biological repeats and are presented as mean (SEM), two-way ANOVA: PInteraction = 0.16; PTime = 0.74; and PConditioned media concentration = 0.17. (JPG 152 kb)13075_2018_1704_MOESM2_ESM.jpg
Additional file 3: Figure S3. RAW264.7 macrophage expression of TNF-α and PGE2 in response to MSU crystals. RAW264.7 macrophages were cultured with or without 0.5 mg/mL MSU crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. The concentration of TNF-α, PGE2, IL-1β, IL-6, RANKL, and OPG in conditioned medium samples were measured by ELISA. IL-6 and RANKL were undetected in all samples. Data shown are pooled from three biological repeats and are presented as mean (SEM), two-tailed paired t test as indicated between groups. (JPG 156 kb)13075_2018_1704_MOESM3_ESM.jpg
Additional file 4: Figure S4. The effect of neutralizing TNF-α on MLO-Y4 cell inflammation induced by MSU crystal-stimulated RAW264.7 macrophages. RAW264.7 macrophages were cultured with or without 0.5 mg/mL MSU crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium and either 5 μg/mL neutralizing TNF-α antibody or 5 μg/mL IgG isotype control were added to MLO-Y4 cells for 24 h and MLO-Y4 cells were then harvested and mRNA extracted for analysis of gene expression by real-time PCR. Data shown are pooled from four biological repeats and are presented as mean (SEM), one-way ANOVA with post-hoc Sidak’s test between groups as indicated. NS no significant difference. (JPG 203 kb)13075_2018_1704_MOESM4_ESM.jpg
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- Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state
Karen E. Callon
Mei Lin Tay
- BioMed Central
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