Elsevier

Bone

Volume 134, May 2020, 115269
Bone

Full Length Article
Post-antibiotic gut dysbiosis-induced trabecular bone loss is dependent on lymphocytes

https://doi.org/10.1016/j.bone.2020.115269Get rights and content

Highlights

  • Antibiotic-induced dysbiosis causes bone loss in multiple strains of mice.

  • Dysbiosis-induced bone loss is dependent on T and B lymphocytes in mice.

  • Bacterial repopulation following antibiotic treatment is different between wild type and lymphocyte deficient mice.

  • L. reuteri supplementation prevents dysbiosis-induced bone loss in mice.

Abstract

Recent studies in mouse models have shown that gut microbiota significantly influences bone health. We demonstrated that 2-week oral treatment with broad spectrum antibiotics followed by 4 weeks of recovery of the gut microbiota results in dysbiosis (microbiota imbalance)-induced bone loss in mice. Because gut microbiota is critical for the development of the immune system and since both microbiota and the immune system can regulate bone health, in this study, we tested the role of the immune system in mediating post-antibiotic dysbiosis-induced bone loss. For this, we treated wild-type (WT) and lymphocyte deficient Rag2 knockout (KO) mice with ampicillin/neomycin cocktail in water for 2 weeks followed by 4 weeks of water without antibiotics. This led to a significant bone loss (31% decrease from control) in WT mice. Interestingly, no bone loss was observed in the KO mice suggesting that lymphocytes are required for dysbiosis-induced bone loss. Bray-Curtis diversity metrics showed similar microbiota changes in both the WT and KO post-antibiotic treated groups. However, several operational taxonomic units (OTUs) classified as Lactobacillales were significantly higher in the repopulated KO when compared to the WT mice, suggesting that these bacteria might play a protective role in preventing bone loss in the KO mice after antibiotic treatment. The effect of dysbiosis on bone was therefore examined in the WT mice in the presence or absence of oral Lactobacillus reuteri treatment for 4 weeks (post-ABX treatment). As hypothesized, mice treated with L. reuteri did not display bone loss, suggesting a bone protective role for this group of bacteria. Taken together, our studies elucidate an important role for lymphocytes in regulating post-antibiotic dysbiosis-induced bone loss.

Introduction

Osteoporosis is characterized by low bone mass and altered bone architecture [1]. Worldwide, this disease is estimated to affect >200 million people [2]. In the US, osteoporosis accounts for over 2 million bone fractures with an estimated $17 billion treatment cost [3]. By 2025, the annual costs associated with osteoporosis are estimated to increase by ~50% [4]. Osteoporosis increases the risk for bone fractures that can lead to morbidity, mortality, loss of independence and decreased quality of life. Around 30% of people with hip fractures die during the first year and 50% have permanent disability [4]. Many factors such as diet, age, sex, and medications can affect bone remodeling and lead to osteoporosis [[5], [6], [7], [8], [9]]. Despite the development of new treatments against osteoporosis, patients are still concerned about drug effectiveness and unwanted side effects.

During the last decade, gut microbiota has emerged as an important regulator of host physiology including bone health. The gut microbiota comprises of ~1000 bacterial species of which the main phyla represented are Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Verrucomicrobia [10]. The intestinal microbiota is composed of both beneficial and harmful bacteria and thus, gut bacteria can be beneficial to the host as well as contribute to disease. For example, beneficial bacteria such as probiotics increase bone density [[11], [12], [13], [14], [15], [16], [17], [18]] while pathogenic bacteria induce bone loss in animal models [19]. Recent clinical studies further highlight the beneficial skeletal effects of ingesting probiotic bacteria in human subjects [[20], [21], [22]]. Studies have also shown that imbalance in gut microbiota (dysbiosis) is involved in the pathogenesis of several diseases such as diabetes and obesity [23,24].

Although early studies in germ free mice implicated an important role for microbiota in bone health, subsequent studies demonstrated that the effect of microbiota on bone health could be influenced by age, sex, and strain of mice used in the studies. Another important variable that can significantly influence the effect of microbiota on bone health in germ free mouse studies is the composition of the gut microbiota used to conventionalize the germ-free mice. The composition of the mouse gut microbiota can be different between facilities, age, and mouse strain and thus can affect host functions differentially [14,[25], [26], [27], [28]]. More importantly, studies in germ free mice are further complicated due to the developmental defects in the immune system [25,29]. To better understand the role of the gut microbiota on bone density, our lab and others have used antibiotics to deplete commensal microbiota. Our lab recently demonstrated that administration of the broad spectrum antibiotics (ampicillin and neomycin) followed by natural gut microbiota repopulation for four weeks leads to dysbiosis and reduced trabecular femoral bone density [30]. This experimental approach is especially useful to understand gut dysbiosis effects on bone density and is distinct from studies that have utilized either germ-free mice or have focused on chronic antibiotic treatment [26,[31], [32], [33]].

The gut-bone signaling mechanisms that account for microbiota regulation of bone density are not fully understood but are thought to involve: improvement of intestinal barrier function [30], alteration of metabolic hormone levels [26], changes in nutrient absorption [34], regulation of the immune system [15,17,35,36], and microbial byproduct regulation of host cell functions [37]. Our lab and others have shown that oral treatment with probiotics can influence the immune system as well as increase bone density in several different mouse models [14,15,17,36,38]. In fact, recent studies have shown that microbiota metabolites can enhance bone health via regulation of T-regulatory lymphocytes [36]. These data suggest a critical role for the immune system in gut microbiota regulation of bone density. In this study, we aimed to identify the requirement of T and B lymphocytes in dysbiosis-induced bone loss in the antibiotic-induced dysbiosis model in mice. Using mice deficient in mature T and B lymphocytes (Rag2 knockout), we demonstrate an important link between the gut microbiota and the lymphocytes in the modulation of bone density following antibiotic-induced dysbiosis.

Section snippets

Animals and experimental design

Wild-type (C57BL/6) and Rag2 knockout (Rag2tm1Mom, C57BL/6 background) male mice were originally purchased from The Jackson Laboratory (Bar Harbour, Maine) and bred in-house. Both strains of mice were housed in the same room and on the same rack to ensure adaptation to identical housing environment and to prevent cage effect. At 12 weeks of age male mice were randomly divided into 4 experimental groups: 1) WT and 2) KO control groups that received water and 3) WT and 4) KO antibiotic groups

Two week-broad spectrum antibiotic treatment depletes fecal microbiota in mice

Oral treatment of mice with the broad-spectrum antibiotics (ABX) ampicillin and neomycin has been shown to deplete gut microbiota [30,39,40,[47], [48], [49]]. We used these two specific antibiotics because they are poorly absorbed in the mouse gut, thereby limiting extra-intestinal effects [[50], [51], [52]]. Twelve-week-old wild type (WT) and Rag2 knockout (KO) male mice were treated for two weeks with ABX (ampicillin: 144 mg/kg/day and neomycin: 72.46 mg/kg/day) in the drinking water (Fig. 1A

Discussion

Antibiotics are widely prescribed for the treatment and prevention of bacterial infections. Several studies have shown that antibiotics can also deplete the commensal flora in the host, leading to bacterial disturbances and long-term changes that can have sustained negative impact on the host [[54], [55], [56], [57], [58], [59]]. We recently demonstrated that gut microbiota repopulation for 4 weeks following 2-weeks of antibiotic treatment (post-ABX) causes microbial dysbiosis and significantly

Acknowledgements

The authors thank the staff of Campus Animal Resources for the excellent care of our animals. The work presented in this study was funded in part by the National Institutes of Health, grants AT007695 (to LRM, RAB and NP), and DK101050 (to LRM and NP); NDR-A was supported by the William Townsend Porter Pre-doctoral Fellowship from the American Physiological Society.

Funding

The work presented in this study was funded in part by NIH AT007695 (to RAB, LRM and NP), and DK101050 (to LRM and NP); NDR-A was supported by the William Townsend Porter Pre-doctoral Fellowship from the American Physiological Society.

Author contribution statement

NDR-A devised and performed experiments, analyzed data, prepared figures and drafted manuscript. JS, AD, LS, CD-S, JG, RB, performed experiments and revised manuscript. NP, LM devised experiments, analyzed data, prepared figures and drafted and edited manuscript.

Declaration of competing interest

None.

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