Bone mass protective potential mediated by bovine milk basic protein requires normal calcium homeostasis in mice.

OBJECTIVES: Milk provide protective effects against bone loss caused by an impaired calcium balance. Although the effects of some elements have previously been confirmed, the involvement of milk basic protein (MBP) in bone mineral metabolism remains poorly characterized. Moreover, the importance of mineral nutrition sufficiency to establish the effect of MBP must be evaluated. METHODS: First, to evaluate the physiological conditions required for MBP activity, we examined the bone and mineral phenotypes of mice that suffer from insufficient calcium absorption due to a lack of intestinal vitamin D signaling. Second, to determine whether vitamin D signaling affects the effect of MBP on bone resorption, in vitro osteoclastogenesis were assessed using bone marrow cells. RESULTS: In mice with systemic vitamin D receptor (Vdr) inactivation, dietary MBP supplementation was unable to normalize hypercalcemia and hyperparathyroidism and failed to rescue bone mineralization impairments. In contrast, calcium and bone homeostasis responded to MBP supplementation when Vdr inactivation was restricted to the intestines. Hyperparathyroidism in intestine-specific Vdr knockout mice was also improved by MBP supplementation, along with a decrease in bone resorption in response to the level of serum tartrate-resistant acid phosphatase 5b. These results corresponded with a reduction in tartrate-resistant acid phosphatase-stained osteoclast numbers and the eroded surface on the tibia. MBP treatment dose-dependently suppressed osteoclastogenesis in cultured bone marrow macrophages regardless of vitamin D activity. These effects of MBP were blunted when parathyroid hormone was added to the culture medium, which is in line with the in vivo phenotype observed with systemic Vdr inactivation and suggests that severe hyperparathyroidism limits MBP activity in the bone. CONCLUSIONS: Therefore, adaptive calcium homeostasis is an essential requirement when MBP exerts protective effects through the inhibition of bone resorption.

Effector memory CD4+T cells in mesenteric lymph nodes mediate bone loss in food-allergic enteropathy model mice, creating IL-4 dominance.

Intestinal inflammation can be accompanied by osteoporosis, but their relationship, mediated by immune responses, remains unclear. Here, we investigated a non-IgE-mediated food-allergic enteropathy model of ovalbumin (OVA) 23-3 mice expressing OVA-specific T-cell-receptor transgenes. Mesenteric lymph nodes (MLNs) and their pathogenic CD4+T cells were important to enteropathy occurrence and exacerbation when the mice were fed an egg-white (EW) diet. EW-fed OVA23-3 mice also developed bone loss and increased CD44hiCD62LloCD4+T cells in the MLNs and bone marrow (BM); these changes were attenuated by MLN, but not spleen, resection. We fed an EW diet to F1 cross offspring from OVA23-3 mice and a mouse line expressing the photoconvertible protein KikGR to track MLN CD4+T cells. Photoconverted MLN CD44hiCD62LloCD4+T cells migrated predominantly to the BM; pit formation assay proved their ability to promote bone damage via osteoclasts. Significantly greater expression of IL-4 mRNA in MLN CD44hiCD62LloCD4+T cells and bone was observed in EW-fed OVA23-3 mice. Anti-IL-4 monoclonal antibody injection canceled bone loss in the primary inflammation phase in EW-fed mice, but less so in the chronic phase. This novel report shows the specific inflammatory relationship, via Th2-dominant-OVA-specific T cells and IL-4 production, between MLNs and bone, a distant organ, in food-allergic enteropathy.

Milk basic protein supplementation exerts an anti-inflammatory effect in a food-allergic enteropathy model mouse.

To examine novel functions of milk basic protein (MBP) in T-cell-related inflammatory diseases, such as autoimmune diseases and allergies, we evaluated the effects of MBP on the causative responses of ovalbumin (OVA)-specific T cells in a food-allergic enteropathy model, OVA23-3 mice, which express an OVA-specific T-cell receptor gene. The OVA-specific CD4+ T cells of the mesenteric lymph nodes (MLN) from OVA23-3 mice were cultured with CD11c+ dendritic cells of MLN from BALB/cA mice in the absence or presence of MBP following stimulation with OVA; then the levels of CD69 expression and the levels of cytokine production by CD4+ T cells were measured to evaluate activation. The effects of MBP supplementation of OVA 23-3 mice were assessed by feeding a diet containing OVA (OVA diet) with or without MBP for 28 d. Intestinal inflammation, together with activation and cytokine production of CD4+ T cells by MLN, as well as femoral bone mineral density, were measured. In in vitro culture, MBP inhibited excess activation and IL-4 production by CD4+ T cells. The supplementation of MBP to the OVA diet attenuated OVA-specific IgE production in OVA-diet-fed OVA23-3 mice and slightly resolved developing enteropathy caused by excess IL-4 production by CD4+ T cells. Feeding OVA diet to OVA23-3 mice exhibited bone loss accompanied with enteropathy, whereas MBP supplementation prevented bone loss and increased osteoprotegerin, an osteoclastogenesis inhibitory factor, in the mice. The inhibition of T-cell-activation in both MLN and bone marrow by MBP supplementation may help prevent increased IgE levels caused by excessive IL-4 production and bone loss accompanied by enteropathy. Our findings show that MBP may help attenuate both T-cell-related inflammation and bone loss.

Milk Basic Protein Facilitates Increased Bone Mass in Growing Mice.

Milk basic protein (MBP) comprises a group of basic whey proteins and is effective in preventing bone loss by promoting bone deposition (bone formation) and suppressing withdrawn (bone resorption). We previously revealed the bone protective effects of MBP during life phases involving excessive bone resorption, such as in adults and postmenopausal women, and in animal models (ovariectomized rats and mice). However, it was unclear whether MBP increases bone mass during the growth stage, when there is more bone formation than resorption. We therefore investigated the effect of MBP supplementation on bone mass in 6-wk-old mice provided water supplemented with MBP [0.01%, 0.1%, 1.0% (w/w)] or deionized water (control) ad libitum for 10 wk. Analysis by micro-computerized tomography showed that MBP significantly increased tibia cortical bone mineral density and femur trabecular bone volume to tissue volume compared with mice provided deionized water. Next, the function of MBP in bone remodeling (bone formation and resorption) was evaluated using an in vitro system and the results demonstrated that MBP directly promoted osteoblast proliferation and inhibited osteoclastogenesis. Moreover, the plasma level of insulin-like growth factor-1 was increased by MBP supplementation, suggesting that MBP indirectly promoted osteoblast proliferation/differentiation. These effects enhance bone formation and/or inhibit bone resorption, resulting in increased bone mass in growing mice.

Milk basic protein increases ghrelin secretion and bone mineral density in rodents.

OBJECTIVES: Milk basic protein (MBP), a mixture of proteins isolated from bovine milk, is known to increase bone formation. Ghrelin, a stomach-derived peptide hormone, also has been reported to stimulate osteoblast formation. The aim of this study was to determine whether MBP-induced bone formation is mediated via ghrelin. METHODS: MBP was chronically administered to mice in their drinking water for 3 wk, and body weight, water intake, and bone mineral density were measured. Additionally, plasma bone-specific alkaline phosphatase, tartrate-resistant acid phosphatase isoform 5b, and ghrelin concentrations were determined by enzyme-linked immunosorbent assay. To examine the direct effect of MBP on ghrelin secretion, gastric tissue culture and primary mucosal cells were stimulated by MBP. RESULTS: The in vivo study of young, growing mice showed that chronic MBP intake for 3 wk increased the plasma ghrelin concentration and bone mineral density of the hind limb tibia. In vitro studies using minced rat gastric mucosa tissues and primary murine isolated gastric mucosal cells revealed that MBP stimulated ghrelin release in a dose-dependent manner. Moreover, MBP-induced ghrelin secretion was partly inhibited by adrenergic blockers. CONCLUSIONS: These findings suggest a novel mechanism by which MBP directly acts on ghrelin secretion. Additionally, the elevated ghrelin level induced by MBP may act as a mediator for bone formation.

Lactobacillus gasseri SBT2055 induces TGF-ß expression in dendritic cells and activates TLR2 signal to produce IgA in the small intestine.

Probiotic bacteria provide benefits in enhancing host immune responses and protecting against infection. Induction of IgA production by oral administration of probiotic bacteria in the intestine has been considered to be one reason for this beneficial effect, but the mechanisms of the effect are poorly understood. Lactobacillus gasseri SBT2055 (LG2055) is a probiotic bacterium with properties such as bile tolerance, ability to improve the intestinal environment, and it has preventive effects related to abdominal adiposity. In this study, we have found that oral administration of LG2055 induced IgA production and increased the rate of IgA(+) cell population in Peyer's patch and in the lamina propria of the mouse small intestine. The LG2055 markedly increased the amount of IgA in a co-culture of B cells and bone marrow derived dendritic cells (BMDC), and TLR2 signal is critical for it. In addition, it is demonstrated that LG2055 stimulates BMDC to promote the production of TGF-ß, BAFF, IL-6, and IL-10, all critical for IgA production from B cells. Combined stimulation of B cells with BAFF and LG2055 enhanced the induction of IgA production. Further, TGF-ß signal was shown to be critical for LG2055-induced IgA production in the B cell and BMDC co-culture system, but TGF-ß did not induce IgA production in a culture of only B cells stimulated with LG2055. Furthermore, TGF-ß was critical for the production of BAFF, IL-6, IL-10, and TGF-ß itself from LG2055-stimulated BMDC. These results demonstrate that TGF-ß was produced by BMDC stimulated with LG2055 and it has an autocrine/paracrine function essential for BMDC to induce the production of BAFF, IL-6, and IL-10.