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. 2016 Apr 8;11(4):e0153180.
doi: 10.1371/journal.pone.0153180. eCollection 2016.

Lactobacillus reuteri 6475 Increases Bone Density in Intact Females Only under an Inflammatory Setting

Fraser L Collins  1 , Regina Irwin  1 , Hayley Bierhalter  1 , Jonathan Schepper  1 , Robert A Britton  2 , Narayanan Parameswaran  1 , Laura R McCabe  1   3   4
Affiliations

Lactobacillus reuteri 6475 Increases Bone Density in Intact Females Only under an Inflammatory Setting

Fraser L Collins et al. PLoS One. .

Abstract

Background & aims: We previously demonstrated that short-term oral administration of the probiotic Lactobacillus reuteri 6475 enhanced bone density in male but not female mice. We also established that L. reuteri 6475 enhanced bone health and prevented bone loss in estrogen-deficient female mice. In this study, we tested whether a mild inflammatory state and/or a long-term treatment with the probiotic was required to promote a positive bone effect in estrogen-sufficient female mice.

Methods: A mild inflammatory state was induced in female mice by dorsal surgical incision (DSI). Following DSI animals were orally supplemented with L. reuteri or vehicle control for a period of 8 weeks. Gene expression was measured in the intestine and bone marrow by qPCR. Distal femoral bone density and architecture was analyzed by micro-CT.

Results: We report that 8 weeks after DSI there is a significant increase in the weight of spleen, thymus and visceral (retroperitoneal) fat pads. Expression of intestinal cytokines and tight junction proteins are also altered 8 weeks post-DSI. Interestingly, L. reuteri treatment was found to display both intestinal region- and inflammation-dependent effects. Unexpectedly we identified that 1) L. reuteri treatment increased bone density in females but only in those that underwent DSI and 2) DSI benefited cortical bone parameters. In the bone marrow, dorsal surgery induced CD4+ T cell numbers, a response that was unaffected by L. reuteri treatment, whereas expression of RANKL, OPG and IL-10 were significantly affected by L. reuteri treatment.

Conclusion: Our data reveals a previously unappreciated effect of a mild surgical procedure causing a long-lasting effect on inflammatory gene expression in the gut and the bone. Additionally, we demonstrate that in intact female mice, the beneficial effect of L. reuteri on bone requires an elevated inflammatory status.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. DSI Increases General Markers of Inflammation.
a) Body Mass, b) spleen c) thymus d) retroperitoneal fat and e) inguinal fat were weighed after an 8 week period in non-surgery and DSI mice ± L. reuteri. DSI significantly increased spleen weight (p<0.0001), thymus weight (p<0.0001) and retroperitoneal fat (p<0.001) but had no significant effect on body mass or inguinal fat. n = 9–18 per group. Data is mean ± SEM. Statistical analysis performed by 1-way ANOVA with Fisher’s LSD post-test.
Fig 2
Fig 2. L. reuteri Significantly Increases Bone Volume in Female Mice 8 Weeks after DSI.
Non-surgery and DSI mice were treated ± L. reuteri for 8 weeks and trabecular bone density analyzed by μCT. i) L. reuteri significantly increased %BVF in the DSI mice compared to DSI control (p<0.05) and non-surgery control (p<0.05). No significant difference was observed between the non-surgery and DSI controls. L. reuteri had no effect in the non-surgery cohort. Representative μCT isosurface images of ii) DSI and iii) DSI + L. reuteri. n = 8–18 per group. Statistical analysis performed by 1-way ANOVA with Fisher’s LSD post-test.
Fig 3
Fig 3. DSI and L. reuteri Significantly Alter Regional Intestinal Gene Expression.
Non-surgery and DSI mice were treated ± L. reuteri for 8 weeks and gene expression analyzed in the jejunum and colon by qPCR. In the jejunum DSI significantly increased expression of TNFα (p<0.001), IL-1β (p<0.01), IFNγ (p<0.05) and TGFβ (p<0.001). Significant decreases in IL-10 (p<0.01) and occludin (p<0.01) were observed. In the non-surgery cohort L. reuteri significantly reduced IL-10 (p<0.01) expression. In the DSI cohort significant reductions were observed for TNFα (p<0.0001), IFNγ (p<0.001) and TGFβ (p<0.0001). In the colon, DSI resulted in a significant increase in TNFα (p<0.05) and a decrease in occludin expression (p<0.05). L. reuteri significantly reduced expression of occludin (p<0.05), IL-1β (p<0.05) and TGFβ (p<0.01) in the non-surgery cohort. In the DSI cohort, L. reuteri significantly increased expression of TNFα (p<0.01) and IL-1β (p<0.01) while increasing expression of IL-10. n = 8–10 per group. * = significant to non-surgery control, ^ = significant to DSI control. Statistical analysis performed by 1-way ANOVA with Fisher’s LSD post-test.
Fig 4
Fig 4. DSI Increases CD4+ T Cell Numbers in Bone Marrow and Significantly Increases Markers of Osteoblast Bone Formation after 8 Weeks.
Non-surgery and DSI mice were treated ± L. reuteri for 8 weeks. Bone marrow was isolated and CD4+ T cell numbers analyzed by flow cytometry and osteoclast and osteoblast bone remodeling markers analyzed. a) Representative flow cytometry scatter plots. CD4+ T cells numbers were significantly increased in DSI mice (p<0.0001) compared to the non-surgery cohort. L. reuteri had no significant effect on CD4+ T cell numbers. b) MAR was measured by calcein incorporation, DSI + L. reuteri significantly increased MAR compared to non-surgery controls (p<0.05). Representative fluorescent images of calcein incorporation are included on the right. c, d) DSI had no effect on serum TRAP5b (c) but significantly increased levels of serum osteocalcin (d) compared to the non-surgery control (p<0.01). L. reuteri had no effect on TRAP5b or osteocalcin levels. n = 7–18 per group. * = significant to non-surgery control, ^ = significant to DSI control. Data is mean ± SEM. Statistical analysis performed by 1-way ANOVA with Tukey post-test or Fisher’s LSD test.
Fig 5
Fig 5. DSI and L. reuteri Alter Bone Marrow Gene Expression at 4 and 8 Weeks.
Bone marrow was isolated from 4 and 8 week Non-surgery and DSI mice ± L. reuteri and gene expression analyzed by qPCR. At 4 weeks L. reuteri significantly reduced RANKL expression in the DSI cohort (p<0.05). L. reuteri additionally increased OPG expression resulting in a significant increase in the OPG:RANKL ratio (p<0.05) compared to the DSI control. At 8 weeks RANKL and OPG expression trended higher in the DSI control compared to the non-surgery control, resulting in an increased OPG:RANKL ratio. IL-10 expression was elevated in the DSI control over the non-surgery control. In the non-surgery cohort L. reuteri increased OPG and IL-10 expression. In the DSI cohort, L. reuteri decreased RANKL while increasing OPG expression resulting in a trend towards an increased OPG:RANKL ratio. n = 6–10 per group. * = significant to non-surgery control, ^ = significant to DSI control. Statistical analysis performed by 1-way ANOVA with Fisher’s LSD test.
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