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Oral treatment with probiotic bacteria has been shown to prevent bone loss in multiple models of osteoporosis. In previous studies we demonstrated that oral administration of  Lactobacillus reuteri  in healthy male mice increases bone density. The host and bacterial mechanisms of these effects however are not well understood. The objective of this study was to understand the role of lymphocytes in mediating the beneficial effects of  L. reuteri  on bone health in male mice. We administered  L. reuteri  in drinking water for 4 weeks to wild type or Rag knockout (lack mature T and B lymphocytes) male mice. While  L. reuteri  treatment increased bone density in wild type, no significant increases were seen in Rag knockout mice, suggesting that lymphocytes are critical for mediating the beneficial effects of  L. reuteri  on bone density. To understand the effect of  L. reuteri  on lymphocytes in the intestinal tissues, we isolated mesenteric lymph node (MLN) from naïve wild type mice. In  ex vivo  studies using whole mesenteric lymph node (MLN) as well as CD3 +  T-cells, we demonstrate that live  L. reuteri  and its secreted factors have concentration-dependent effects on the expression of cytokines, including anti-inflammatory cytokine IL-10. Fractionation studies identified that the active component of  L. reuteri  is likely water soluble and small in size (<3 kDa) and its effects on lymphocytes are negatively regulated by a RIP2 inhibitor, suggesting a role for NOD signaling. Finally, we show that T-cells from MLNs treated with  L. reuteri  supernatants, secrete factors that enhance osterix (transcription factor involved in osteoblast differentiation) expression in MC3T3-E1 osteoblasts. Together, these data suggest that  L. reuteri  secreted factors regulate T-lymphocytes which play an important role in mediating the beneficial effects of  L. reuteri  on bone density.

Beneficial effects of Lactobacillus reuteri 6475 on bone density in male mice is dependent on lymphocytes