The study shows that soluble mediators secreted from macrophages in response to stimulation by MSU crystals affect gene expression and secretion of factors from osteoblastic cells. An experimental system based on mouse cell lines was used to model osteoblast differentiation in the presence of soluble factors released from MSU crystal-stimulated macrophages. Conditioned medium from MSU-stimulated RAW264.7 macrophages potently inhibited the osteoblastic differentiation of MC3T3-E1 cells as demonstrated by the reduced expression of osteoblast marker genes and inhibition of matrix mineralization. A second system, based on the human THP-1 monocytic cell line and primary HOBs, was used to model the effects of MSU crystals within the tophus on differentiated osteoblasts. Inhibition of osteoblast marker genes was less consistent in this system. In both experimental systems, conditioned medium from MSU stimulated macrophages/ monocytes increased the expression and secretion of proinflammatory factors from the osteoblastic cells.
For the preparation of the conditioned medium used in our study, macrophage/ monocytic cell lines were incubated with MSU crystals alone, without additional stimulators of inflammation. Activation of the NLRP3 inflammasome and the subsequent release of IL-1β are critical steps in the initiation of the acute inflammatory response to MSU crystals during a gout flare [9, 10]. For this reason, when constructing in vitro systems that model gout flares, MSU crystals are added to macrophages primed with either lipopolysaccharide or phorbol 12-myristate 13-acetate [11, 12]. However, in the current study, we aimed to model the extracellular environment of the osteoblast in tophaceous gout rather than during acute flares. Tophi are not acutely inflamed, and studies mostly find persistent, low-grade inflammation in asymptomatic tophaceous gout [9, 13]. Therefore, we believe that our experimental system represents a relevant in vitro model to examine the indirect, macrophage-mediated effects of MSU crystals on osteoblasts in tophaceous gout.
Conditioned medium from RAW264.7 cells stimulated with 0.3 or 0.5 mg/mL MSU crystals inhibited the expression of Runx2, Sp7, and Col1a and blocked the characteristic increase in the expression of the late osteogenic markers Dmp1, Bglap, and Ibsp in MC3T3-E1 cells. The inhibitory effect of the conditioned medium was also demonstrated in assays of matrix mineralization by MC3T3-E1 cells. The inhibition of matrix mineralization was generally consistent with the gene expression profile, although a conditioned medium generated in RAW264.7 cells with 0.1 mg/mL MSU, which did not affect gene expression, effectively inhibited matrix mineralization. It is possible that under these culture conditions, additional factors required for matrix mineralization, which were not measured in this study, were not produced adequately. A previous study investigating the direct effect of MSU crystals on MC3T3-E1 cells found a similar inhibition of osteoblast marker genes and matrix mineralization . In the current study, we also determined the expression of COL1A1, IBSP, and BGLAP in HOBs following a short incubation with a conditioned medium from MSU crystal-stimulated THP-1 monocytes. In this experimental system, only BGLAP expression was lower in the presence of MSU crystal-stimulated conditioned medium than in the control. The direct effect of MSU crystals on HOBs was studied by Bouchard et al. . Analysis of proteins secreted from HOBs in response to 0.3 mg/mL MSU crystals found that alkaline phosphatase and osteocalcin concentrations were reduced to 72% and 58%, respectively, of their concentrations in control cultures. Taken together, these results indicate that in joints affected by tophaceous gout, MSU crystals negatively affect osteoblast function through a combination of direct and indirect mechanisms.
We studied the indirect effects of MSU crystals on OPG and RANKL, two proteins expressed in cells of the osteoblast lineage, which are key regulators of osteoclast formation and bone resorption. The binding of RANKL to RANK, a receptor expressed on osteoclast precursor cells, drives osteoclast differentiation, whereas OPG is a decoy receptor that binds RANKL and inhibits osteoclast differentiation . Studies that determined the circulating concentrations of OPG and RANKL in patients with gout found an imbalance between the two regulator proteins that may be associated with bone erosion in tophaceous gout [16,17,18]. In our study, incubation with a conditioned medium from MSU crystal-stimulated macrophages/ monocytes reduced OPG gene expression and concentration of secreted protein in long-term cultures of MC3T3-E1 cells, but increased the concentration of secreted OPG in short-term HOB cultures. The different OPG responses in the two culture systems could be related to the nature of the osteoblastic cells used: the first system used a cell line of early osteoblasts induced to differentiate in vitro, and the second used mature osteoblasts obtained from human trabecular bone. Reduced concentration of OPG results in increased bone resorption that could be relevant to bone erosion in gout-affected joints, whereas an increase in OPG concentration could potentially contribute to the formation of the typical boney structures or represent a compensatory mechanism aimed at restoring normal bone metabolism in an environment of excessive bone resorption. Consistent with previous reports, RANKL was not expressed in MC3T3-E1 cells . RANKL was only detected in gene expression analysis in HOBs, where its expression was twofold higher in cells incubated with a conditioned medium from MSU crystal-stimulated THP-1 monocytes than in control cells. In a previous study, an indirect effect of MSU crystals on RANKL was determined in the MLO-Y4 osteocytic cell line, where incubation with a conditioned medium from RAW264.7 cells stimulated with 0.5 mg/mL MSU crystals increased RANKL gene expression by approximately sixfold . In the MLO-Y4 cells, OPG gene expression was only inhibited transiently, whereas OPG protein secretion remained unchanged. Although findings from in vitro studies and correlation analyses suggest a potential role for the disruption of RANKL/OPG balance in the development of bone erosion in gout, so far, there is no direct clinical evidence to support this role.
Imaging and pathology studies have established that tophi are closely associated with bone erosion in the gout-affected joint [3, 20]. A recent study found that the sizes of the two tophus components, the urate crystal core and surrounding inflammatory soft tissue, were independently associated with bone erosion . These findings suggest that a better understanding of the inflammatory environment in and around the tophus could help identify mechanisms that underlie bone erosion in gout. In cells of the monocyte lineage used in our study, MSU crystals directly stimulated PGE2 and TNF-α secretion from RAW264.7 cells, and IL-1β and TNF-α secretion from THP-1 cells. Although not determined in the current study, previous studies suggest that the signaling pathways most likely to mediate the increased production of these inflammatory factors in monocyte/macrophages are NF-kappa B, C/EBP beta (CCAAT/enhancer binding protein beta), and c-Jun [22, 23]. COX-2 is also activated by IL-1α and IL-1β, a mechanism that can contribute to the increased production of PGE2 [24,25,26]. We found that MSU crystals increased IL-1β expression in THP-1 cells but not in RAW264.7 cells. IL-1α concentrations were not determined in the current study, and we cannot exclude a potential role for IL-1α in mediating the indirect effect of MSU crystals in osteoblastic cells. Secretion of PGE2 and TNF-α from RAW264.7 cells and human peripheral blood mononuclear cells in response to MSU crystals has been previously described [5, 27]. In vivo, characterization of the cellular component of the tophus indicated that macrophages are continuously recruited into the tophi and that mono- and multinucleated macrophages are present in the soft tissue surrounding the MSU crystal core [1, 6]. A large number of cells in the tophus express the COX-2 enzyme  and the inflammatory cytokines IL-1β  and TNF-α . Bone cells are another potential source of inflammatory mediators in the gout-affected joint. In our study, a conditioned medium from MSU crystal-stimulated monocytes/ macrophages induced the expression of genes encoding COX-2 and IL-6 and the secretion of PGE2 and IL-6 from MC3T3-E1 cells and HOBs. A similar, indirect response to MSU crystals has been demonstrated in the osteocytic cell line MLO-Y4. In these cells, a conditioned medium from MSU crystal-stimulated RAW264.7 macrophages induced the secretion of PGE2, TNF-α, and IL-6 . Studies of the direct response of cells of the osteoblast lineage to MSU crystals found the stimulation of PGE2, IL-6, and IL-8 in HOBs , whereas in MLO-Y4 cells, MSU had no direct effect on the expression of inflammatory genes . The proinflammatory mediators that were stimulated directly in monocytic and indirectly in osteoblastic cells in our study are all known to affect both osteoclasts and osteoblasts. TNF-α, IL-6, and PGE2 stimulate osteoclast formation and bone resorption, particularly in pathological contexts [28,29,30]. PGE2 and IL-6 also stimulate osteoblast differentiation and bone formation [31, 32], whereas TNF-α inhibits these processes [33, 34]. Increased concentrations of the proinflammatory mediators in the extracellular environment around the tophus could contribute to the imbalanced bone remodeling that leads to bone erosion and aberrant bone formation.
We used the COX-2 selective inhibitor SC-236 to examine whether PGE2, which is secreted from HOBs in response to a conditioned medium from MSU crystal-stimulated THP-1 cells, acts in an autocrine fashion to induce the changes seen in HOBs. We chose to study the role of COX-2 activation because we had previously shown that the indirect effects of MSU crystals in the osteocytic cell line MLO-Y4 were reduced by COX-2 inhibition, whereas TNF-α inhibition was without effect . As expected, SC-236 inhibited PGE2 secretion from HOBs. However, it only partially suppressed the activation of IL6 gene expression and had no effect on IL-6 secretion, suggesting that COX-2 activation is not a central mechanism mediating this response. COX-2 activation might play a more important role in osteocytes, as a similar experiment in MLO-Y4 cells found that COX-2 inhibition strongly suppressed the induced secretion of IL-6 .
Our study had several limitations. We did not perform an analysis of the specific contribution of all the secreted factors identified in monocyte/ macrophage conditioned medium to the osteoblast effects. In future studies, TNF-α inhibitors, antagonists of the EP4 receptor—the main receptor that mediates PGE2 activity in osteoblasts , and the IL-1 receptor antagonist anakinra could be used to determine the role of each: TNF-α, PGE2, and IL-1 in MSU crystal macrophage-mediated effects in osteoblasts. The fact that this was an in vitro study could be considered another limitation relating to the clinical relevance of our findings. However, the research question addressed in this study originated from observations made in clinical samples of people with erosive gout. Specifically, histological analysis of joints affected by gout demonstrating the presence of MSU crystals, macrophage-rich tophus tissue, and areas of bone erosion provides the clinical context for our study.