Mechanisms of gut microbiota-mediated bone remodeling

Abstract

The mechanisms underlying the systemic effects mediated by gut microbiota are under active investigation. In addition to local, direct effects of gut microbiota on the host, metabolic products from microbiota may act peripherally, reaching distal organs through the circulation. In our recent publication we demonstrated that gut microbiota influence bone remodeling distally, promoting both bone resorption and formation. We proposed that these effects are mediated, at least in part, by the induction of insulin like growth factor (IGF-1) by the microbiota metabolite short chain fatty acids (SCFA). Here we explore additional mechanisms by which microbial metabolites could directly or indirectly alter host bone remodeling. We discuss whether SCFA directly modulate bone resorption by their actions on osteoclasts, and test the possibility that serotonin is another gut microbiota derived long-distance mediator of effects on bone remodeling. A detailed understanding of the mechanisms of microbiota effect on bone remodeling could help establish potential therapeutic strategies to promote bone health.

Keywords: GPR109; SCFA; bone; gut microbiota; serotonin.

Figure 1.

Butyrate and proprionate inhibit osteoclast differentiation in vitro. (A) Osteoclasts were differentiated from equivalent numbers of bone marrow derived macrophages (BMDM) in the presence of M-CSF and RANKL. GPR41, GPR109, and GPR43 expression was assessed by real-time PCR in osteoclast cultures before, 24 h, or 48 h after addition of RANKL. Gene expression was normalized to Hprt. (B) Equal numbers of BMDM were treated with acetate, proprionate, and butyrate at the indicated concentrations in the presence of RANKL and M-CSF and TRAP positive multinuclear cell (MNC) were counted. Representative TRAP stained are shown. (C) 200 μM of individual SCFA were added before, 24 h, or 48 h after addition of RANKL and TRAP positive MNC were counted. Data are shown as mean ± SEM. Difference between groups are determined by one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 2.

GPR109 is not required for butyrate inhibition of osteoclast differentiation and deficiency increases trabecular bone mass. (A) An equal number of total bone marrow cells isolated from WT and GPR109 KO mice was differentiated in the presence of M-CSF and RANKL and osteoclast formation was quantified based on number of TRAP positive MNC formed. (B) BMDM from WT and GPR109 KO mice were treated with 100 µM butyrate and RANKL was added simultaneously to induce osteoclast differentiation. (C) Trabecular and (D) cortical bone parameters determined by microCT. The region of interest was thresholded using a global threshold that set the bone/marrow cutoff at 352.3 mg HA/cm³ for trabecular bone and 589.4 mg HA/cm³ for cortical bone. (E) CTX-I and (F) P1NP levels in the serum from WT and GPR109 KO mice. Data are shown as mean ± SEM. Difference between groups are compared by Student's t test in panel A, C, D, E and F; and one-way ANOVA in panel B. * P < 0.05; ** P < 0.01.

Figure 3.

Regulation of serotonin by microbiota and its metabolites. (A) Peripheral Serotonin levels in GF mice and littermates colonized for 1 month starting at 8 weeks of age. Between group differences were compared by Mann-Whitney test; *** p < 0.001. (B) Peripheral Serotonin levels in SPF mice and SPF mice treated with two different antibiotic cocktails with or without SCFA supplementation. Antibiotic cocktail 1 contains 1 mg/mL each of ampicillin, metronidazole and neomycin and 0.5 mg/mL vancomycin. Antibiotic cocktail 2 contains 0.2 mg/mL gentamicin, 0.15 mg/mL ciprofloxacin, 2 mg/mL streptomycin, and 1 mg/mL bacitracin. (C) Expression of Tph1, Chga, Slc6a4 and Maoa in the colon from SPF mice treated with antibiotic cocktail 1 with or without SCFA supplementation. Data are shown as mean ± SEM. Differences between multiple groups were compared by one-way ANOVA followed with Kruskal-Wallis post-hoc test. * p < 0.05; ** p < 0.01; ns, not statistically significant.