Bacterial specificity of the gut microbiome predicts bone density in primary hyperparathyroidism

Abstract

Primary hyperparathyroidism causes mild-to-severe bone loss, but the reason for this heterogeneity is unclear. We investigated the role of the microbiome in 50 primary hyperparathyroidism patients. Microbiome transfers from primary hyperparathyroidism patients with and without osteoporosis to germ-free mice replicated the human bone phenotype and regulated TNF⁺ T cells and Th17 cells in mice. Accordingly, circulating TNF⁺ T cells and Th17 cells and TNF/IL17 production predicted bone density in primary hyperparathyroidism patients. Bifidobacterium longum, TNF⁺ T cells, and Th17 cells were mediators of bone loss in primary hyperparathyroidism patients, while Bifidobacterium longum supplementation caused PTH to expand TNF⁺ T cells and Th17 cells and induce bone loss in mice. Our findings link Bifidobacterium longum-induced TNF⁺ T cells and Th17 cells to bone loss in patients with primary hyperparathyroidism. Bifidobacterium longum abundance may determine the skeletal phenotypes of patients with primary hyperparathyroidism and allow prediction of their risk of bone loss. Microbiome modifications by antibiotics or precision probiotics might offer novel preventive approaches for the skeletal complications of primary hyperparathyroidism.

References

[1] Walker, M. D, Bilezikian, J. P. Primary hyperparathyroidism. In: Feingold KR, Adler RA, Ahmed SF, et al. (eds). Endotext. South Dartmouth (MA): MDText.com, Inc (2000).

[2] Bilezikian JP. Primary hyperparathyroidism. J. Clin. Endocrinol. Metab., 2018, 103: 3993-4004.

[3] Potts, J. Primary hyperparathyroidism. in Metabolic Bone Diseases Vol. 1 (eds. Avioli, L. V. & Krane, S.) 411–442 (Academic Press, 1998).

[4] Silva BC, Bilezikian JP. Skeletal abnormalities in hypoparathyroidism and in primary hyperparathyroidism. Rev. Endocr. Metab. Disord., 2021, 22: 789-802.

[5] Calvi LM, et al.. Activated parathyroid hormone/parathyroid hormone-related protein receptor in osteoblastic cells differentially affects cortical and trabecular bone. J. Clin. Invest., 2001, 107: 277-286.

[6] Qin L, Raggatt LJ, Partridge NC. Parathyroid hormone: a double-edged sword for bone metabolism. Trends Endocrinol. Metab., 2004, 15: 60-65.

[7] Young N, Mikhalkevich N, Yan Y, Chen D, Zheng WP. Differential regulation of osteoblast activity by Th cell subsets mediated by parathyroid hormone and IFN-gamma. J. Immunol., 2005, 175: 8287-8295.

[8] Terauchi M, et al.. T lymphocytes amplify the anabolic activity of parathyroid hormone through Wnt10b signaling. Cell Metab., 2009, 10: 229-240.

[9] Tawfeek H, et al.. Disruption of PTH Receptor 1 in T Cells Protects against PTH-Induced Bone Loss. PLoS One, 2010, 5: e12290.

[10] Bedi B, et al.. Silencing of parathyroid hormone (PTH) receptor 1 in T cells blunts the bone anabolic activity of PTH. Proc. Natl. Acad. Sci. USA, 2012, 109: E725-E733.

[11] Gao Y, et al.. T cells potentiate PTH-induced cortical bone loss through CD40L signaling. Cell Metab., 2008, 8: 132-145.

[12] Li JY, et al.. IL-17A is increased in humans with primary hyperparathyroidism and mediates PTH-induced bone loss in mice. Cell Metab., 2015, 22: 799-810.

[13] Li, J. Y. et al. IL-17 receptor signaling in osteoblasts/osteocytes mediates PTH-induced bone loss and enhances osteocytic RANKL production. J. Bone Miner. Res.34, 349–360 (2019).

[14] Yu M, et al.. PTH induces bone loss via microbial-dependent expansion of intestinal TNF⁺ T cells and Th17 cells. Nat. Commun., 2020, 11. ArticleID: 468

[15] Li JY, et al.. Parathyroid hormone-dependent bone formation requires butyrate production by intestinal microbiota. J. Clin. Investig., 2020, 130: 1767-1781.

[16] Pacifici R. Role of gut microbiota in the skeletal response to PTH. J. Clin. Endocrinol. Metab., 2021, 106: 636-645.

[17] Turner JE, et al.. CCR6 recruits regulatory T cells and Th17 cells to the kidney in glomerulonephritis. J. Am. Soc. Nephrol., 2010, 21: 974-985.

[18] Lam J, et al.. TNF-alpha induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand. J. Clin. Invest., 2000, 106: 1481-1488.

[19] McArthur MA, Chen WH, Magder L, Levine MM, Sztein MB. Impact of CD4+ T cell responses on clinical outcome following oral administration of wild-type enterotoxigenic Escherichia coli in humans. PLoS Negl. Trop. Dis., 2017, 11: e0005291.

[20] Srinivasan A, Foley J, McSorley SJ. Massive number of antigen-specific CD4 T cells during vaccination with live attenuated Salmonella causes interclonal competition. J. Immunol., 2004, 172: 6884-6893.

[21] Mazmanian SK, Round JL, Kasper DL. A microbial symbiosis factor prevents intestinal inflammatory disease. Nature, 2008, 453: 620-625.

[22] Ren D, Wang D, Liu H, Shen M, Yu H. Two strains of probiotic Lactobacillus enhance immune response and promote naive T cell polarization to Th1. Food Agric. Immunol., 2019, 30: 281-295.

[23] Ivanov II, et al.. Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. Cell Host Microbe, 2008, 4: 337-349.

[24] Ivanov II, et al.. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell, 2009, 139: 485-498.

[25] Gaboriau-Routhiau V, et al.. The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses. Immunity, 2009, 31: 677-689.

[26] Atarashi K, et al.. Th17 cell induction by adhesion of microbes to intestinal epithelial cells. Cell, 2015, 163: 367-380.

[27] Tan TG, et al.. Identifying species of symbiont bacteria from the human gut that, alone, can induce intestinal Th17 cells in mice. Proc. Natl. Acad. Sci. USA, 2016, 113: E8141-E8150.

[28] Pal, S. et al. Gender affirming hormone therapy preserves skeletal maturation in young mice via the gut microbiome. J. Clin. Invest.134, e175410 (2024).

[29] Powell WFJr, et al.. Targeted ablation of the PTH/PTHrP receptor in osteocytes impairs bone structure and homeostatic calcemic responses. J. Endocrinol., 2011, 209: 21-32.

[30] Paik J, et al.. Potential for using a hermetically-sealed, positive-pressured isocage system for studies involving germ-free mice outside a flexible-film isolator. Gut Microbes, 2015, 6: 255-265.

[31] Tomura M, et al.. Activated regulatory T cells are the major T cell type emigrating from the skin during a cutaneous immune response in mice. J. Clin. Invest., 2010, 120: 883-893.

[32] Tomura M, et al.. Monitoring cellular movement in vivo with photoconvertible fluorescence protein “Kaede” transgenic mice. Proc. Natl. Acad. Sci. USA, 2008, 105: 10871-10876.

[33] Yu, M. et al. Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF+ T cells and Th17 cells. J. Clin. Invest.131, e143137 (2021).

[34] Tingley D, Yamamoto Y, Hirose K, Keele L, Imai K. mediation: R Package for causal mediation analysis. J. Stat. Softw., 2014, 59: 1-38.

[35] Jovanovic DV, et al.. IL-17 stimulates the production and expression of proinflammatory cytokines, IL-beta and TNF-alpha, by human macrophages. J. Immunol., 1998, 160: 3513-3521.

[36] Sato K, et al.. Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction. J. Exp. Med., 2006, 203: 2673-2682.

[37] Komatsu N, Takayanagi H. Autoimmune arthritis: the interface between the immune system and joints. Adv. Immunol., 2012, 115: 45-71.

[38] Li JY, et al.. Sex steroid deficiency-associated bone loss is microbiota dependent and prevented by probiotics. J. Clin. Invest., 2016, 126: 2049-2063.

[39] Guss JD, et al.. Alterations to the gut microbiome impair bone strength and tissue material properties. J. Bone Miner. Res., 2017, 32: 1343-1353.

[40] Tyagi, A. M. et al. The gut microbiota is a transmissible determinant of skeletal maturation. eLife10, e64237 (2021).

[41] Li, C. et al. Gut microbiota composition and bone mineral loss-epidemiologic evidence from individuals in Wuhan, China. Osteoporos. Int.30, 1003–1013 (2019).

[42] Gray SM, et al.. Mouse adaptation of human inflammatory bowel diseases microbiota enhances colonization efficiency and alters microbiome aggressiveness depending on the recipient colonic inflammatory environment. Microbiome, 2024, 12. ArticleID: 147

[43] Yang, J. C. et al. Biogeographical distribution of gut microbiome composition and function is partially recapitulated by fecal transplantation into germ-free mice. ISME J.19, wrae250 (2025).

[44] Hansdah K, Lui JC. Emerging Insights into the Endocrine Regulation of Bone Homeostasis by Gut Microbiome. J. Endocr. Soc., 2024, 8: bvae117.

[45] Patterson E, et al.. Bifidobacterium longum 1714 improves sleep quality and aspects of well-being in healthy adults: a randomized, double-blind, placebo-controlled clinical trial. Sci. Rep., 2024, 14. ArticleID: 3725

[46] Yao S, Zhao Z, Wang W, Liu X. Bifidobacterium longum: protection against inflammatory bowel disease. J. Immunol. Res., 2021, 2021: 8030297.

[47] Sapra L, et al.. Bifidobacterium longum ameliorates ovariectomy-induced bone loss via enhancing anti-osteoclastogenic and immunomodulatory potential of regulatory B cells (Bregs). Front. Immunol., 2022, 13. ArticleID: 875788

[48] Roberts, J. L. et al. Bifidobacterium longum supplementation improves age-related delays in fracture repair. Aging Cell22, e13786 (2023).

[49] Ruiz L, Delgado S, Ruas-Madiedo P, Sanchez B, Margolles A. Bifidobacteria and their molecular communication with the immune system. Front. Microbiol., 2017, 8: 2345.

[50] Ma B, et al.. Strain-specific alterations in gut microbiome and host immune responses elicited by tolerogenic Bifidobacterium pseudolongum. Sci. Rep., 2023, 13. ArticleID: 1023

[51] Lopez P, Gueimonde M, Margolles A, Suarez A. Distinct Bifidobacterium strains drive different immune responses in vitro. Int. J. Food Microbiol., 2010, 138: 157-165.

[52] Arpaia N, et al.. Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature, 2013, 504: 451-455.

[53] Smith PM, et al.. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science, 2013, 341: 569-573.

[54] Whittier DE, et al.. Guidelines for the assessment of bone density and microarchitecture in vivo using high-resolution peripheral quantitative computed tomography. Osteoporos. Int., 2020, 31: 1607-1627.

[55] Sode M, Burghardt AJ, Pialat JB, Link TM, Majumdar S. Quantitative characterization of subject motion in HR-pQCT images of the distal radius and tibia. Bone, 2011, 48: 1291-1297.

[56] Dar, H. Y. et al. Callus gammadelta T cells and microbe-induced intestinal Th17 cells improve fracture healing in mice. J. Clin. Invest.133, e166577 (2023).

[57] Walter J, Armet AM, Finlay BB, Shanahan F. Establishing or exaggerating causality for the gut microbiome: lessons from human microbiota-associated rodents. Cell, 2020, 180: 221-232.

[58] Britton GJ, et al.. Microbiotas from humans with inflammatory bowel disease alter the balance of gut Th17 and RORgammat(+) regulatory T cells and exacerbate colitis in mice. Immunity, 2019, 50: 212-224 e214.

[59] Goodrich JK, et al.. Human genetics shape the gut microbiome. Cell, 2014, 159: 789-799.

[60] Pal, S. et al. The microbiome restrains melanoma bone growth by promoting intestinal NK and Th1 cells homing to bone. J. Clin. Invest.132, e157340 (2022).

[61] Bouxsein ML, et al.. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J. Bone Miner. Res., 2010, 25: 1468-1486.

Funding

U.S. Department of Health & Human Services | National Institutes of Health (NIH)(DK124821)

RIGHTS & PERMISSIONS

The Author(s)