Among the symbiotic bacteria contained in the probiotics lac

Among the symbiotic bacteria contained in the probiotics, lactic-acid-producing bacteria (LAB) have been suggested to confer broad spectrums of health benefits, such as activation of mucosal and systemic immunity and resistance to infectious illnesses (Schiffrin et al., 1995, Wells and Mercenier, 2008). Daily feeding of LAB containing Lactobacillus rhamnosus, Lactobacillus acidophilus, and Bifidobacterium lactis resulted in significant enhancement of innate and acquired immunity (Gill et al., 2000). Lactate produced by the probiotic Lactobacillus casei was critical in modulating inflammation in a model of indomethacin-induced gut damage with decreased neutrophil infiltration and cytokine expression (Watanabe et al., 2009). Although there is strong evidence for the beneficial roles of symbionts, neither the exact role of the differentiation of Lgr5+ ISCs nor the relevance of regulation of the intestinal environments is clear.
Results
Discussion In this study, we demonstrate that oral feeding of human-used symbionts containing LAB, such as Bifidobacterium and Lactobacillus spp., promotes significant acceleration of Lgr5+ ISC proliferation and epithelial development in vivo and ex vivo. Among several metabolites from LAB-type symbionts, lactate is associated with ISC-mediated epithelial development. Paneth devd and intestinal stromal cells highly express Gpr81, a known lactate receptor, and lactate treatment promotes ISC-mediated epithelial regeneration in a Gpr81-dependent manner. Furthermore, feeding of LAB-type symbionts or lactate protects mice from severe gut injury induced by radiation exposure and chemotherapy drug treatment. Thus, we propose that lactate and the receptor Gpr81 promote ISC-mediated epithelial regeneration by stimulation of the Wnt/β-catenin signal pathway in Paneth and intestinal stromal cells. An earlier study demonstrated that supplemental Wnt agonist R-spondin-1 can maintain ISCs in a self-renewing state that are able to fully differentiate epithelial cells in SI organoids (Sato et al., 2009). A subsequent study revealed that Paneth cells, which constitute the crypt niche, support a growth of Lgr5 stem cells by providing Wnt3 (Sato et al., 2011). Those researchers found that Paneth cell-null mice have concomitant loss of Lgr5 stem cells. In contrast, others reported that depletion of Paneth cells or knockout of the Wnt3 gene in the intestinal epithelium did not lead to obvious phenotype changes in vivo, suggesting the possibility of another functional cell niche (Durand et al., 2012, Farin et al., 2012). If so, intestinal myofibroblast-enriched stromal cells may play a functional role in ISC maintenance and differentiation (Kabiri et al., 2014). Intestinal stromal cells express diverse cytokines related to Wnt signaling (Gregorieff et al., 2005) and thus can support the growth of intestinal epithelium in culture (Lahar et al., 2011). We found that expression levels of Wnt/β-catenin pathway-related genes were significantly enhanced in SI organoids in the presence of culture from symbionts or lactate (Figure 1E), and those expression levels were more activated when organoids were co-cultured with intestinal stromal cells (Figure 2B). In parallel, Wnt3 protein was highly expressed in intestinal stromal cells by stimulation with lactate (Figure 2D). As reported by others (Sato et al., 2011), we also confirmed that Paneth cells express high levels of Wnt3 protein (Figure S2). Those results were confirmed in the SI crypts by in situ hybridization staining (Figure S7). We therefore conclude that lactate derived from LAB-type symbionts stimulate both Paneth cells and intestinal stromal cells to secrete Wnt3 and support ISC maintenance and differentiation. Although lactate has been regarded as an intermediate of carbon metabolism contributing to anti-microbial effects of fermented food, recent accumulated evidence suggests that it has unique bioactive properties, such as regulation of immune responses and tissue regeneration in the gut mucosa (Garrote et al., 2015). Others demonstrated that lactate produced by microbial fermentation reduced inflammatory responses in intestinal epithelial cells and myeloid cells by abrogation of TLR and IL-1β-dependent activation (Iraporda et al., 2015). Furthermore, transient starvation enhanced colon epithelial cell turnover on refeeding in the presence of Lactobacillus murinus and its metabolite, lactate (Okada et al., 2013). Orally administered L. rhamnosus GG probiotic and probiotic-derived products protected the murine SI from radiation injury (Ciorba et al., 2012). In addition, treatment with probiotics containing L. acidophilus and Bifidobacterium bifidum resulted in mild diarrhea in cancer patients receiving radiation therapy (Chitapanarux et al., 2010). Taken together with our results, these data indicate that administration of LAB-type symbionts or lactate may be a useful prophylactic strategy to limit gut injury to humans during radiation therapy and chemotherapy.