Fig 2: LR intake modulates Treg-Th17 cell balance in vivo. Cells from BM, PP, spleen and LN of mice from Sham, Ovx and Ovx+LR groups were harvested and analysed by Flow cytometry for percentage of (A) CD4+Foxp3+Tregs. (B) CD8+Foxp3+Tregs. (C) CD4+Roryt+Th17 cells. (D) CD4+RANKL+T cells. Data are reported as Mean ± SEM. Similar results were obtained in two independent experiments with n = 6. Statistical significance of each parameter was assessed by ANOVA followed by paired group comparison. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with indicated groups.

Fig 3: High dietary salt intake skews expression of osteoclastogenic factors. (A) Serum samples of mice were analyzed for secretion of various proinflammatory and anti-inflammatory cytokines by CBA (B) Average percentage (%) of CD4+RANKL+T cells in bone marrow (BM). (C) Average percentage (%) of total RANKL in bone marrow (BM). The results were evaluated by using ANOVA with subsequent comparisons by Student t test for paired or nonpaired data, as appropriate. Analysis was performed using Sigma plot software (Systat Software, Inc., Germany). Values are reported as mean ± SEM (n = 10) and similar results were obtained in three independent experiments. Statistical significance was defined as p ≤ 0.05 (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001) with respect to low salt diet group.

Fig 4: LR-CM inhibits RANKL stimulated F-actin ring formation: Bone Marrow Macrophages (BMMs) were treated with M-CSF (30 ng/ml) and RANKL (100 ng/ml) with or without Lactobacillus rhamnosus—Conditioned Media (LR-CM) at different ratios of 1:10 and 1:1 for 6 days. F-actin and nuclei were stained with FITC-conjugated phalloidin and DAPI respectively. Images were captured in fluorescence microscope (Imager.Z2 Zeiss microscope) at 10× magnification. (B) Number of F-actin rings. (C) Number of nuclei per osteoclasts. (D) Area of F-actin rings. The above images are indicative of one independent experiment and similar results were obtained in three different independent experiments. Statistical significance was considered as p ≤ 0.05 (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001) with respect to indicated groups.

Fig 5: TLR9−/− mice had low bone mass, increased osteoclastogenesis and systemic inflammation. a Representative 3D μCT images of femurs and L3 vertebrae from TLR9−/− (KO) and wildtype (WT) mice. b Trabecular bone microarchitecture of femurs and L3 vertebrae showing bone mineral density (BMD), bone volume per tissue volume (BV/TV), number of trabeculae (Tb. N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). KO, n = 8; WT, n = 9. c Representative HE (upper panels) and TRAP (lower panels) stained images of distal femurs from each group. d Circulating CTX and P1NP levels. KO, n = 6; WT, n = 8. e Histomorphometric analysis of bone resorption indices. n = 8 per group. f–h In vitro osteoclastogenesis assay. f TRAP-stained osteoclast like cells (OCLs). g Quantification of number and area of TRAP+ multinucleated OCLs. h qPCR analysis of osteoclast signature genes’ expression at the end of assay. i Western blots showing expression of the indicated molecules in early osteoclast precursors. j–k In vitro osteoclastogenesis of wildtype BMNCs in the presence of TLR9 antagonist. j TRAP-stained OCLs. k Quantification of OCL numbers. l Spleens from 8-week-old male TLR9−/− and wildtype mice. m Levels of TNFα, IFNγ, IL1β, and RANKL in serum and bone marrow supernatants. n = 3 to 13 per group. n Splenic T cell populations analyzed by flow cytometry. n = 6–10 per group. o The bone marrow T cell populations. n = 5–10 per group. The numbers in n and o represent the frequencies in total splenic or bone marrow cells. Eight-week-old sex-matched mice were used in d, e and m–o. One-way ANOVA with Turkey’s multiple comparison tests was conducted in k. In other panels, statistical significance was determined using an unpaired two-tailed t-test. Error bars represent the s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1 and ns P > 0.05