It is well established that physiological generation of low levels of ROS act as critical second messengers in multiple signaling pathways. These include the regulatory networks that control growth and differentiation in disparate biological systems, including the gut of many metazoans. However, the molecular mechanism of ROS production within the intestine is unknown. Recent reports have shown that the ROS‐generating enzyme NADPH oxidase 1 (Nox1) is highly expressed by colon epithelia. We report that Lactobacillus spp. are potent inducers of endogenous ROS generation, and of ROS‐dependent cellular proliferation within intestines of two metazoan models, namely the fruitfly Drosophila melanogaster, and the mouse. Moreover, we show that these induced responses are diminished in mice or Drosophila that are selectively deficient for Nox1 within intestinal epithelial cells. Together, these results implicate Nox1 in epithelial cell homeostasis and reveal a novel mechanism for the maintenance of intestinal tissue structure.

Summary An optimal gut microbiota influences many beneficial processes in the metazoan host. However, the molecular mechanisms that mediate and function in symbiont-induced host responses have not yet been fully characterized. Here, we report that cellular ROS enzymatically generated in response to contact with lactobacilli in both mice and Drosophila has salutary effects against exogenous insults to the intestinal epithelium via the activation of Nrf2 responsive cytoprotective genes. These data show that the xenobiotic inducible Nrf2 pathway participates as a signaling conduit between the prokaryotic symbiont and the eukaryotic host. Indeed, our data imply that the capacity of lactobacilli to induce redox signaling in epithelial cells is a highly conserved hormetic adaptation to impel cellular conditioning to exogenous biotic stimuli. These data also highlight the role the microbiota plays in eukaryotic cytoprotective pathways, and may have significant implications in the characterization of a eubiotic microbiota.

The mammalian gut microbiota is essential for normal intestinal development, renewal and repair. Injury to the intestinal mucosa can occur with infection, surgical trauma, and in idiopathic inflammatory bowel disease. Repair of mucosal injury, termed restitution, as well as restoration of intestinal homeostasis involves induced and coordinated proliferation and migration of intestinal epithelial cells. N-formyl peptide receptors (FPRs) are widely expressed pattern recognition receptors that can specifically bind and induce responses to host derived and bacterial peptides and small molecules. Here we report that specific members of the gut microbiota stimulate FPR1 on intestinal epithelial cells to generate reactive oxygen species via enterocyte NADPH oxidase NOX1, causing rapid phosphorylation of Focal Adhesion Kinase (FAK) and ERK MAPK. These events stimulate migration and proliferation of enterocytes adjacent to colonic wounds. Together, these findings identify a novel role of FPR1 as pattern recognition receptors for perceiving the enteric microbiota that promotes repair of mucosal wounds via generation of ROS from the enterocyte NOX1.

Lactobacillus rhamnosus GG is a widely used probiotic, and the strain's salutary effects on the intestine have been extensively documented. We previously reported that strain GG can modulate inflammatory signaling, as well as epithelial migration and proliferation, by activating NADPH oxidase 1-catalyzed generation of reactive oxygen species (ROS). However, how strain GG induces these responses is unknown. Here, we report that strain GG's probiotic benefits are dependent on the bacterial-epithelial interaction mediated by the SpaC pilin subunit. By comparing strain GG to an isogenic mutant that lacks SpaC (strain GG⍀spaC), we establish that SpaC is necessary for strain GG to adhere to gut mucosa, that SpaC contributes to strain GG-induced epithelial generation of ROS, and that SpaC plays a role in strain GG's capacity to stimulate extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling in enterocytes. In addition, we show that SpaC is required for strain GG-mediated stimulation of cell proliferation and protection against radiologically inflicted intestinal injury. The identification of a critical surface protein required for strain GG to mediate its probiotic influence advances our understanding of the molecular basis for the symbiotic relationship between some commensal bacteria of the gut lumen and enterocytes. Further insights into this relationship are critical for the development of novel approaches to treat intestinal diseases.

Objective: Bacillus subtilis probiotics have been shown to influence several aspects of the human gut including motility, epithelial strength, inflammation, etc. that may change bowel movement frequency and/or type. To explore the efficacy of Bacillus subtilis (B. subtilis) DE111 in this regard, 50 people were evaluated by their stool profile, food diary and questionnaire while taking a probiotic or placebo daily over the course of 105-days. Method: The stools were scored based on the Bristol Stool Chart index and safety was assessed using blood markers during the 0, 45 and 105 days. Results: Statistically significant results showed those in the DE111 group moved to a healthier bowel index while those in the Placebo group stayed the same. Conclusion: The study provided evidence that DE111 may improve occasional constipation and/or diarrhea in healthy individuals.

A growing body of data has emerged showing that members of the gut microbiota are essential for the normal physiology and homeostasis of the intestine. However, the molecular basis for these processes are not well understood. In this study, we describe a novel role for the commensal bacterium Lactobacillus in intestinal cytoprotection. We show that this bacterial genus specifically induces cellular reactive oxygen species (ROS) in both Drosophila and murine guts. This ROS induction in turn stimulates the redox‐sensitive Nrf2 pathway and leads to consequent ARE (Antioxidant Response Element)‐dependent cytoprotective gene expression. Nrf2/ARE pathway mediated cytoprotection is well established in many tissues, but its stimulation by gut bacteria is completely unrecognized. Using Nrf2 knockout Drosophila and mice, we demonstrate that Nrf2 is required for protection against a variety of intestinal insults. Together, these data show that gut microbial stimulated ROS generation modulates the Nrf2 signaling pathway thus maintaining intestinal epithelial homeostasis and cytoprotection, and that this effect is evolutionarily conserved.

Lactobacillus rhamnosus GG (LGG) is a widely used probiotic, and the strain’s salutary effects on the intestine have been extensively documented. We have demonstrated this strain can modulate inflammatory signaling, as well as epithelial migration and proliferation via stimulation of Nox1 dependent reactive oxygen species. However, how this strain mediates these effects is largely unknown. Here, we report that LGG’s probiotic benefits are dependent on the SpaC pilin subunit. By comparing LGG to an isogenic mutant that lacks SpaC (LGGΩSpaC) in a variety of in vitro and in vivo assays, we establish that SpaC is necessary for LGG to adhere to gut mucosa, that SpaC contributes to LGG’s ability to induce the epithelial generation of ROS, and that SpaC plays a role in LGG’s capacity to stimulate the phosphorylation of ERK in enterocytes. In addition, we show that SpaC is required for LGG‐mediated stimulation of cell proliferation, and protection against radiologically‐inflicted intestinal injury. The identification of a critical surface protein required for LGG to mediate its probiotic influence furthers our understanding of the molecular basis for the symbiotic relationship between some commensal bacteria of the gut lumen and enterocytes. Further insights into this relationship are critical for the development of novel approaches to treat inflammatory bowel diseases and other intestinal disorders. Grant Funding Source: Supported by NIH RO1 AI 64462

It is well established that physiological generation of low levels of ROS act as critical second messengers in multiple signaling pathways. These include the regulatory networks that control growth and differentiation in disparate biological systems, including the gut of many metazoans. However, the molecular mechanism of ROS production within the intestine is unknown. Recent reports have shown that the ROS‐generating enzyme NADPH oxidase 1 (Nox1) is highly expressed by colon epithelia. We report that Lactobacillus spp. are potent inducers of endogenous ROS generation, and of ROS‐dependent cellular proliferation within intestines of two metazoan models, namely the fruitfly Drosophila melanogaster, and the mouse. Moreover, we show that these induced responses are diminished in mice or Drosophila that are selectively deficient for Nox1 within intestinal epithelial cells. Together, these results implicate Nox1 in epithelial cell homeostasis and reveal a novel mechanism for the maintenance of intestinal tissue structure.