Sex steroid deficiency-associated bone loss is microbiota dependent and prevented by probiotics

Abstract

A eubiotic microbiota influences many physiological processes in the metazoan host, including development and intestinal homeostasis. Here, we have shown that the intestinal microbiota modulates inflammatory responses caused by sex steroid deficiency, leading to trabecular bone loss. In murine models, sex steroid deficiency increased gut permeability, expanded Th17 cells, and upregulated the osteoclastogenic cytokines TNFα (TNF), RANKL, and IL-17 in the small intestine and the BM. In germ-free (GF) mice, sex steroid deficiency failed to increase osteoclastogenic cytokine production, stimulate bone resorption, and cause trabecular bone loss, demonstrating that the gut microbiota is central in sex steroid deficiency-induced trabecular bone loss. Furthermore, we demonstrated that twice-weekly treatment of sex steroid-deficient mice with the probiotics Lactobacillus rhamnosus GG (LGG) or the commercially available probiotic supplement VSL#3 reduces gut permeability, dampens intestinal and BM inflammation, and completely protects against bone loss. In contrast, supplementation with a nonprobiotic strain of E. coli or a mutant LGG was not protective. Together, these data highlight the role that the gut luminal microbiota and increased gut permeability play in triggering inflammatory pathways that are critical for inducing bone loss in sex steroid-deficient mice. Our data further suggest that probiotics that decrease gut permeability have potential as a therapeutic strategy for postmenopausal osteoporosis.

Figure 1. GF mice are protected against the loss of trabecular bone induced by sex steroid deficiency.

Assessment of femoral bone structure by in vitro μCT in Conv.R mice, GF mice, and Col.GF mice following either leuprolide (375 μg/month) or vehicle control treatment for 10 weeks. (A) Images of representative 3-dimensional μCT reconstructions of examined femurs from each group. (B) BV/TV. (C) Ct.V. (D) Ct.Th. (E) Tb.N. (F) Tb.Sp. (G) Tb.Th. n = 10 mice per group in all panels. Data are expressed as mean ± SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and post hoc tests applying the Bonferroni correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the indicated groups.

Figure 2. GF mice are protected against the increase in bone turnover induced by sex steroid deprivation.

(A) The images show tartrate-resistant acid phosphatase–stained (TRAP-stained) sections of the distal femur. Original magnification ×40. (B) Images are representative sections displaying the calcein double-fluorescence labeling. Original magnification ×20. (C) Serum levels of type 1 cross-linked CTX. (D) Serum levels of osteocalcin. n = 10 mice per group in all panels. Data are expressed as mean ±SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and post hoc tests applying the Bonferroni correction for multiple comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the corresponding vehicle group.

Figure 3. The BM and the intestine of GF mice are protected against the increase in osteoclastogenic cytokines induced by sex steroid deprivation.

(A–C) Levels of the osteoclastogenic cytokines TNF, RANKL, and IL-17 in the BM of Conv.R, GF, and Col.GF mice following either leuprolide (375 μg/month) or vehicle control treatment for 10 weeks. (D–F) Relative frequency of BM CD4⁺TNF⁺ cells, of CD8⁺TNF⁺ cells, and of Th17 cells in mice from experimental groups described in A. (G and H) qPCR analysis measuring transcript levels of the Th17 cell–inducing transcription factors Rora and Rorgt in CD4⁺ T cells from the BM of mice in experimental groups described in A. (I–K) qPCR analysis measuring transcript levels of TNF, Rankl, and Il17 in the SI of mice in experimental groups described in A. (L–N) Flow cytometry analysis to measure the relative abundance of cytokine-producing CD4⁺ T cells in the SILP of ovx and sham-operated mice at 4 weeks following surgery. n = 10 mice per group in all panels. Data are expressed as mean ±SEM. All data were normally distributed according to the Shapiro-Wilk normality test. A–K were analyzed by 2-way ANOVA and post hoc tests applying the Bonferroni correction for multiple comparisons. L–N were analyzed by unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the indicated group.

Figure 4. Sex steroid deprivation promotes intestinal permeability.

(A–D) qPCR analysis measuring transcript levels of the tight junction proteins claudin 2, claudin 3, claudin 15, and Jam3 in the SI of Conv.R, and GF mice, and in Col.GF mice, following either leuprolide (375 μg/month) or vehicle control treatment for 10 weeks. (E) Measurement of serum endotoxin levels in mice from experimental groups described in A. (F) Measurement of gut permeability in conventional ovx and sham-operated mice. At 4 weeks after surgery, FITC-dextran was administered orally to each experimental subject. Serum FITC-dextran levels were measured 4 hours later. n = 10 mice per group in all panels. Data are expressed as mean ±SEM. All data were normally distributed according to the Shapiro-Wilk normality test. A–E were analyzed by 2-way ANOVA and post hoc tests applying the Bonferroni correction for multiple comparisons. F was analyzed by unpaired t tests. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the corresponding vehicle or sham group. ND, not detectable. EU, endotoxin units.

Figure 5. Supplementation of the indigenous microbiota with probiotics prevents sex steroid–induced bone loss.

Conventional ovx and sham-operated mice were supplemented twice a week with 1 × 10⁹ CFU of either VSL#3, LGG, LGG-M, E. coli, or vehicle. Data are expressed as mean ±SEM. (A) In vivo prospective measurements of spine BV/TV by μCT scanning at baseline and 2 and 4 weeks after surgery. n = 10–14 mice per group. Data were normally distributed according to the Shapiro-Wilk normality test and analyzed by ANOVA for repeated measures and post hoc tests applying the Bonferroni correction for multiple comparisons. (B and C) In vitro analysis of femoral BV/TV and Ct.V by μCT scanning. n = 10–14 mice per group. (D and E) Serum levels of type 1 cross-linked CTX and serum levels of osteocalcin. n = 10 mice per group randomly selected from a total of 10–14 mice per group. Data in B–E were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and post hoc tests applying the Bonferroni correction for multiple comparisons. ^†P < 0.05, ^††P < 0.01 ^†††P < 0.001, and ^††††P < 0.0001 compared with baseline. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with sham vehicle. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, and ^####P < 0.0001 compared with ovx vehicle.

Figure 6. Supplementation of the indigenous microbiota with probiotics prevents the increase in gut permeability induced by sex steroid depletion.

(A) Measurement of gut permeability in conventional ovx and sham-operated mice. At 4 weeks after surgery, FITC-dextran was administered orally to each experimental subject. Serum FITC-dextran levels were measured 4 hours later. Mice were supplemented twice a week with 1 × 10⁹ CFU total of either VSL#3, LGG, LGG-M, E. coli, or vehicle. (B) Endotoxin levels in the serum of mice from experimental groups described in A. (C–F) qPCR analysis measuring transcript levels of the tight junction proteins claudin 2, claudin 3, claudin 15, and Jam3 in the SI of mice from experimental groups described in A. n = 10 mice per group in all panels randomly selected from a total of 10–14 mice per group. Data are expressed as mean ±SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and post hoc tests applying the Bonferroni correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with sham vehicle. ^##P < 0.01, ^###P < 0.001, and ^####P < 0.0001 compared with ovx vehicle.

Figure 7. Supplementation of the indigenous microbiota with probiotics modulates the generation of SI and BM osteoclastogenic cytokines following sex steroid depletion.

(A–C) qPCR analysis measuring transcript levels of Tnf, Rankl, and Il17 in the SI tissue of conventional ovx and sham-operated mice at 4 weeks after surgery. Mice were supplemented twice a week with 1 × 10⁹ CFU total of either VSL#3, LGG, LGG-M, E. coli, or vehicle. (D–F) qPCR analysis measuring transcript levels of Tnf, Rankl, and Il17 in the BM of mice treated as described in A. n = 10 mice per group in all panels randomly selected from a total of 10–14 mice per group. Data are expressed as mean ±SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and post hoc tests applying the Bonferroni correction for multiple comparisons. **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with sham vehicle. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, and ^####P < 0.0001 compared with ovx vehicle.