Characterizing how probiotic <i>Lactobacillus reuteri</i> 6475 and lactobacillic acid mediate suppression of osteoclast differentiation.

Abstract

Osteoporosis is a disease that impacts over 200 million people worldwide. Taking into consideration the side effects stemming from medications used to treat this illness, investigators have increased their efforts to develop novel therapeutics for osteoporosis. In a previous study, we demonstrated that ovariectomy-induced bone loss in mice was prevented by treatment with the probiotic bacterium <i>Lactobacillus reuteri</i> 6475 (<i>L. reuteri)</i>, an effect that correlated with reduced osteoclastogenesis in the bone marrow of <i>L. reuteri</i> treated mice. We also demonstrated that <i>L. reuteri</i> directly inhibited osteoclastogenesis <i>in vitro</i>. To better understand how <i>L. reuteri</i> impacts osteoclast formation, we used additional <i>in vitro</i> analyses to identify that conditioned supernatant from <i>L. reuteri</i> inhibited osteoclastogenesis at the intermediate stage of fused polykaryons. To elucidate the effect of <i>L. reuteri</i> treatment on host cell physiology, we performed RNAseq at multiple time points during <i>in vitro</i> osteoclastogenesis and established that <i>L. reuteri</i> downregulated several KEGG pathways including osteoclast differentiation as well as TNF-α, NF-κB, and MAP kinase signaling. These results were consistent with Western Blot data demonstrating that NF-κB and p38 activation were decreased by <i>L. reuteri</i> treatment. We further identified that lactobacillic acid (LA), a cyclopropane fatty acid produced by <i>L. reuteri</i>, contributed significantly to the suppression of osteoclastogenesis. Additionally, we demonstrated that <i>L. reuteri</i> is signaling through the long chain fatty acid receptor, GPR120, to impact osteoclastogenesis. Overall, these studies provide both bacterial and host mechanisms by which <i>L. reuteri</i> impacts osteoclastogenesis and suggest that long chain fatty acid receptors could be targets for preventing osteoclastogenesis.