Osteoporosis is a disease characterized by low bone mass, which can subsequently lead to an increased risk of sustaining a bone fracture. With the advancements made in research and medicine in the past century, the average lifespan has increased substantially. However, this has also lead to an increase in the elderly population that is susceptible to age-related diseases such as osteoporosis. It is currently estimated that over 300 million people worldwide are impacted by osteoporosis. Taking into consideration the side effects stemming from medications used to treat this illness, there has been an increase in research efforts to develop novel therapeutics for osteoporosis. One area of research that has garnered recent interest involves investigating the therapeutic potential of the gut microbiota in bone health. As a result, an overarching goal in this area of research revolves around identifying microbes that impact bone health and understanding the mechanisms mediating these responses. In this thesis, I present the beneficial use of the probiotic bacterium Lactobacillus reuteri (L. reuteri) in an in vivo murine model of osteoporosis mediated by estrogen deficiency. Using female Balb/c mice that are rendered estrogen deficient following ovariectomy, we demonstrated that supplementation with L. reuteri was capable of preventing bone loss. In addition to this, we identified that the process of osteoclastogenesis was down-regulated following L. reuteri treatment in these mice suggesting that this could be the mechanism by which L. reuteri confers its benefit on bone health. Osteoclasts are the main cell type responsible for bone resorption. Using an in vitro model of osteoclastogenesis, we demonstrated that cell-free conditioned medium (CCM) from L. reuteri inhibited the maturation of osteoclasts from macrophages. We further characterized this by demonstrating that L. reuteri CCM halted osteoclastogenesis at an intermediate stage characterized by fused polykaryons. Using an antagonist to the G protein coupled receptor GPR120, we decreased the ability of L. reuteri CCM to suppress osteoclastogenesis from 70% to 38% suggesting that L. reuteri is partially signaling through GPR120 to suppress osteoclastogenesis. Taking into account that GPR120 was a receptor for long chain fatty acids, we investigated the impact of lactobacillic acid (LA), a long chain fatty acid produced by L. reuteri, and observed that the suppression of osteoclastogenesis by L. reuteri involved the production of LA. Moreover, purified LA could suppress osteoclast formation in a dose dependent manner. To elucidate the effect of L. reuteri treatment on host cell physiology, we performed RNA sequencing at multiple time points during osteoclastogenesis. An analysis of the transcriptome data identified several pathways that were modulated following L. reuteri treatment. Further investigations indicated that NF-κB and p38 activation were impacted by L. reuteri in RAW264.7 cells. These sets of experiments have led to the identification of a possible effector molecule produced by L. reuteri and mechanism by which it acts to benefit bone health. In the last part of this thesis, I present an in vivo murine model using germ free (GF) mice to study the impact of the gut microbiota on bone health. By colonizing GF mice with microbiotas with different compositions, the goal was to identify specific microbes that could be impacting bone health in either a positive or negative manner. Interestingly enough, we discovered that the microbial communities that were introduced into the different groups of mice in our studies did not impact bone health in comparison to the GF control group. This was in stark contrast to existing literature that reported a deleterious effect in bone health following the introduction of a gut microbiota. This reinforced the fact that our knowledge remains limited in terms of understanding how the gut microbiota impacts bone health. Nevertheless, the discoveries stemming from these studies contribute to the growing body of work in this discipline and will guide future research that aims to uncover novel therapeutic options to combat osteoporosis.
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