Supplementary materials to Beneficial Microbes: Bacillus subtilis DE111 partially reverses endothelial dysfunction in western-diet fed mice

Imbalances in the gut microbiome have emerged as an important factor in endothelial dysfunction, a significant risk factor for cardiovascular disease. Thus, interventions targeting the microbiome may prove helpful in preventing or reversing this impairment. We previously reported that a spore-forming Bacillus subtilis DE111 improved endothelial function in a cohort of healthy, non-obese humans after a four-week intervention. Building on these promising results, the present study sought to investigate whether administering B. subtilis DE111 could reverse endothelial dysfunction in mice with diet-induced obesity. Male C57BL/6J mice were fed a Western diet (WD; n=24) or standard diet (SD; n=24) for ten weeks to induce endothelial dysfunction, after which half of the animals in each group (n=12) were allocated to receive B. subtilis DE111 (hereafter, PB) formulated into the diets for an additional eight weeks. Outcomes included endothelial-dependent arterial dilation, glucose tolerance, body weight, microbiota profiles, and assessments of intestinal permeability and mucosal immunity markers. Furthermore, a cell culture model of gut barrier function was used to further assess the effects of PB on gut barrier integrity. PB treatment significantly attenuated WD-induced mesenteric endothelial-dependent arterial dilation, independent of changes in other cardiometabolic parameters or changes in gut barrier function. In vitro trans-epithelial electrical resistance of the Caco-2 cell culture confirmed that neither PB-conditioned media nor faecal waters from B. subtilis-treated human stool resulted in gut barrier improvements, nor did they protect against inflammation-associated barrier disruptions. Microbiome analysis revealed significant differences in Shannon’s alpha diversity of WD-fed animals. These data suggest that while PB consumption significantly attenuated WD diet-induced endothelial dysfunction, the underlying mechanisms of this protection were not identified. These results appear to be independent of PB-mediated improvements in gut barrier function or the other investigated parameters, and thus further studies should explore B. subtilis-mediated immune responses or metabolite production as mechanisms underlying these endothelial protective effects.