Thus, it is possible that effects on the release of biochemical bone markers from cortical bone to serum might be confounded by abundant release from trabecular bone. Therefore, further analyses of other more specific cortical bone parameters, such as osteoclast number in cortical bone, are required to confirm that the protective effect of probiotics on ovx-induced cortical bone loss is mediated via altered cortical bone resorption. Mechanistic studies of the bone sparing effect of probiotic treatment in ovx mice revealed that the expression of several osteolytic cytokines such as TNFa and IL-1b as well as the RANKL/OPG ratio in cortical bone were suppressed by probiotic treatment. TNFa promotes osteoclastogenesis indirectly by stimulating RANKL expression by marrow stromal cells and osteoblasts and by direct stimulation of OCL precursors exposed to permissive levels of RANKL. IL-1 is a downstream regulator of the effects of TNFa on osteoclastogenesis. The inhibitory effect of probiotics on the RANKL/OPG ratio in the present study was mainly due to an increased expression of OPG in cortical bone. OPG directly inhibits OCL differentiation at a late stage in a dose dependant manner. Together, these findings indicate that probiotic treatment suppress osteoclastogenesis, resulting in reduced OCL-mediated bone resorption. Treg cells are critical for maintaining self-tolerance and negatively regulate immune responses. In an earlier study, Lavasani et al demonstrated that the probiotic strains used in the present study induce Treg cells. Several probiotic L. strains have been described to have a therapeutic effect in experimental mouse models of inflammatory bowel disease, atopic dermatitis, and rheumatoid arthritis associated with enrichment of Treg cells in the inflamed regions. This inhibitory effect of probiotic L. strains was recently shown to depend on suppressive motifs in the DNA enriched in these strains that potently prevented dendritic cell activation and maintained Treg cell conversion during inflammation. TGFb is crucial for the induction and activity of Treg cells. Interestingly, ovx decreased Treg cells in bone marrow in veh but not probiotic treated mice in the present study. Furthermore, the expression of TGFb1 was increased by probiotic compared to veh treatment after ovx, suggesting that probiotic treatment prevents down regulation of Treg cells via an induction of TGFb1. In vitro studies have shown that Treg cells directly inhibit OCL differentiation and function and that this effect of Treg cells is stimulated by estrogen and Dasatinib dependent on expression of TGFb1. In addition, adoptive transfer of Treg cells decreases the number of OCLs and limits bone loss in ovx mice. Collectively these findings may suggest that the suppressive effect of probiotic treatment on inflammatory cytokines and bone resorption involves effects by Treg cells. In conclusion, treatment with L. para or the L. mix prevents ovx-induced cortical bone loss.