Search

Background & Aims In colorectal cancer, approximately 95% of patients are refractory to immunotherapy because of low antitumor immune responses. Therefore, there is an exigent need to develop treatments that increase antitumor immune responses and decrease tumor burden to enhance immunotherapy. Methods The gut microbiome has been described as a master modulator of immune responses. We administered the human commensal, Lactobacillus rhamnosus GG (LGG), to mice and characterized the changes in the gut immune landscape. Because the presence of lactobacilli in the gut microbiome has been linked with decreased tumor burden and antitumor immune responses, we also supplemented a genetic and a chemical model of murine intestinal cancer with LGG. For clinical relevance, we therapeutically administered LGG after tumors had formed. We also tested for the requirement of CD8 T cells in LGG-mediated modulation of gut tumor burden. Results We detected increased colonic CD8 T-cell responses specifically in LGG-supplemented mice. The CD8 T-cell induction was dependent on dendritic cell activation mediated via Toll-like receptor-2, thereby describing a novel mechanism in which a member of the human microbiome induces an intestinal CD8 T-cell response. We also show that LGG decreased tumor burden in the murine gut cancer models by a CD8 T-cell–dependent manner. Conclusions These data support the potential use of LGG to augment antitumor immune responses in colorectal cancer patients and ultimately for increasing the breadth and efficacy of immunotherapy., Graphical abstract

Parathyroid hormone (PTH) is a critical regulator of skeletal development that promotes both bone formation and bone resorption. Using microbiota depletion by wide-spectrum antibiotics and germ-free (GF) female mice, we showed that the microbiota was required for PTH to stimulate bone formation and increase bone mass. Microbiota depletion lowered butyrate levels, a metabolite responsible for gut-bone communication, while reestablishment of physiologic levels of butyrate restored PTH-induced anabolism. The permissive activity of butyrate was mediated by GPR43 signaling in dendritic cells and by GPR43-independent signaling in T cells. Butyrate was required for PTH to increase the number of bone marrow (BM) regulatory T cells (Tregs). Tregs stimulated production of the osteogenic Wnt ligand Wnt10b by BM CD8+ T cells, which activated Wnt-dependent bone formation. Together, these data highlight the role that butyrate produced by gut luminal microbiota plays in triggering regulatory pathways, which are critical for the anabolic action of PTH in bone.

The use of beneficial bacteria to promote gastrointestinal heath is widely practiced, however, the mechanisms whereby many of these microbes elicit their beneficial effects remain elusive. Previously, we conducted a screen for the discovery of novel beneficial microbes and identified the potent cytoprotective effects of a strain of Lactococcus lactis subsp. cremoris. Here, we show that dietary supplementation with L. lactis subsp. cremoris induced transcript enrichment of a set of genes within the colon whose functions are associated with host cell and microbe interactions. Specifically, L. lactis subsp. cremoris induced the expression of tlr2, which we show was required for L. lactis subsp. cremoris to elicit its beneficial effects on the intestine. L. lactis subsp. cremoris did not confer beneficial effects in mice deficient in TLR-2, or deficient in its adaptor protein Myd88 in chronic gut injury models. In addition to cytoprotection, culture supernatant from L. lactis subsp. cremoris accelerated epithelial migration in a cultured epithelial cell scratch wound assay; and effect that was abrogated by a TLR-2 antagonist. Furthermore, L. lactis subsp. cremoris accelerated epithelial tissue restitution following the infliction of a colonic wound biopsy in a TLR-2 and Myd88-dependent manner. Within colonic wounds, L. lactis subsp. cremoris induced the activation of signaling pathways that function in tissue restitution following injury, including the ERK signaling pathway, and of focal adhesion complex (FAC) proteins. Together, these data demonstrate that L. lactis subsp. cremoris signals via the TLR2/MyD88-axis to confer cytoprotection and accelarated tissue restituion in the gut epithelium. These data point to evolving adaptations where beneficial gut microbes moduate innate immune signaling to excert positive influnces on host physiology.

The gut microbiome is a key regulator of bone health that affects postnatal skeletal development and skeletal involution. Alterations in microbiota composition and host responses to the microbiota contribute to pathological bone loss, while changes in microbiota composition that prevent, or reverse, bone loss may be achieved by nutritional supplements with prebiotics and probiotics. One mechanism whereby microbes influence organs of the body is through the production of metabolites that diffuse from the gut into the systemic circulation. Recently, short-chain fatty acids (SCFAs), which are generated by fermentation of complex carbohydrates, have emerged as key regulatory metabolites produced by the gut microbiota. This Review will focus on the effects of SCFAs on the musculoskeletal system and discuss the mechanisms whereby SCFAs regulate bone cells.

Purpose: Determine whether optimizing the antenatal diet, with either direct or indirect butyrate supplementation, reduces the severity of intestinal injury in offspring with colitis. Butyrate is a short-chain fatty acid that has been shown to enhance intestinal barrier function, regulate intestinal mucosal immunity and reduce intestinal inflammation. Butyrate levels can be increased by ingestion of butyrate, a HF diet or certain probiotics. Therefore, we hypothesize that consuming a diet that increases butyrate levels prenatally will reduce the severity of intestinal injury in offspring with colitis. Methods: Mating pairs of C57BL/6 mice were fed either 1% butyrate, high fiber (HF) diet or probiotics with or without a …

Genetic factors account for the majority of the variance of human bone mass, but the contribution of non-genetic factors remains largely unknown. By utilizing maternal/offspring transmission, cohabitation, or fecal material transplantation (FMT) studies, we investigated the influence of the gut microbiome on skeletal maturation. We show that the gut microbiome is a communicable regulator of bone structure and turnover in mice. In addition, we found that the acquisition of a specific bacterial strain, segmented filamentous bacteria (SFB), a gut microbe that induces intestinal Th17 cell expansion, was sufficient to negatively impact skeletal maturation. These findings have significant translational implications, as the identification of methods or timing of microbiome transfer may lead to the development of bacteriotherapeutic interventions to optimize skeletal maturation in humans. Moreover, the transfer of SFB-like microbes capable of triggering the expansion of human Th17 cells during therapeutic FMT procedures could lead to significant bone loss in fecal material recipients.

Varieties and cultivars of the cruciferous vegetable Brassica oleracea are widely presumed to elicit positive influences on mammalian health and disease, particularly related to their indole and sulforaphane content. However, there is a considerable gap in knowledge regarding the mechanisms whereby these plant-derived molecules elicit their beneficial effects on the host. In this study, we examined the chemical variation between B. oleracea varieties and evaluated their capacity to both activate Nrf2 in the Drosophila intestine and elicit cytoprotection. Ten types of edible B. oleracea were purchased and B. macrocarpa was wild collected. Fresh material was dried, extracted by double maceration and green kale was also subjected to anaerobic fermentation before processing. Untargeted metabolomics was used to perform Principal Component Analysis. Targeted mass spectral analysis determined the presence of six indole species and quantified indole. Extracts were tested for their capacity to activate Nrf2 in the Drosophila intestine in third instar Drosophila larvae. Cytoprotective effects were evaluated using a paraquat-induced oxidative stress gut injury model. A “Smurf” assay was used to determine protective capacity against a chemically induced leaky gut. Extracts of Brussels sprouts and broccoli activated Nrf2 and protected against paraquat-induced damage and leaky gut. Lacto-fermented kale showed a cytoprotective effect, increasing survival by 20% over the non-fermented extract, but did not protect against leaky gut. The protective effects observed do not directly correlate with indole content, suggesting involvement of multiple compounds and a synergistic mechanism.

Cardiometabolic syndrome encompasses intertwined risk factors such as hypertension, dyslipidemia, elevated triglycerides, abdominal obesity, and other maladaptive metabolic and inflammatory aberrations. As the molecular mechanisms linking cardiovascular disease and metabolic disorders are investigated, endocannabinoids have emerged as molecules of interest. The endocannabinoid system (ECS) of biologically active lipids has been implicated in several conditions, including chronic liver disease, osteoporosis, and more recently in cardiovascular diseases. The gut microbiome is a major regulator of inflammatory and metabolic signaling in the host, and if disrupted, has the potential to drive metabolic and cardiovascular diseases. Extensive studies have unraveled the impact of the gut microbiome on host physiology, with recent reports showing that gut microbes exquisitely control the ECS, with significant influences on host metabolic and cardiac health. In this review, we outline how modulation of the gut microbiome affects host metabolism and cardiovascular health via the ECS, and how these findings could be exploited as novel therapeutic targets for various metabolic and cardiac diseases.

Gut mucosal immune cells play an essential role in health due to their ability to orchestrate host signaling events in response to exogenous antigens. These antigens may originate from microorganisms including viruses, commensal or pathogenic bacteria, or single-celled eukaryotes, as well as from dietary foodstuff-derived proteins or products. A critical technological capacity to understand host responses to antigens is the ability to efficiently isolate and functionally characterize immune cells from intestinal tissues. Additionally, after characterization, it is of paramount importance to understand the exact functions of these immune cells under different disease states or genetic variables. Here, we outline methods for immune cell isolation from murine small and large intestines with the goal of undertaking a functional analysis of isolated cell types using antibody array platforms.

Gut mucosal immune cells play an essential role in health due to their ability to orchestrate host signaling events in response to exogenous antigens. These antigens may originate from microorganisms including viruses, commensal or pathogenic bacteria, or single-celled eukaryotes, as well as from dietary foodstuff-derived proteins or products. A critical technological capacity to understand host responses to antigens is the ability to efficiently isolate and functionally characterize immune cells from intestinal tissues. Additionally, after characterization, it is of paramount importance to understand the exact functions of these immune cells under different disease states or genetic variables. Here, we outline methods for immune cell isolation from murine small and large intestines with the goal of undertaking a functional analysis of isolated cell types using antibody array platforms.

Disease states are often linked to large scale changes in microbial community structure that obscure the contributions of individual microbes to disease. Establishing a mechanistic understanding of how microbial community structure contribute to certain diseases, however, remains elusive thereby limiting our ability to develop successful microbiome-based therapeutics. Human microbiota-associated (HMA) mice have emerged as a powerful approach for directly testing the influence of microbial communities on host health and disease, with the transfer of disease phenotypes from humans to germ-free recipient mice widely reported. We developed a HMA mouse model of the human vaginal microbiota to interrogate the effects of Bacterial Vaginosis (BV) on pregnancy outcomes. We collected vaginal swabs from 19 pregnant African American women with and without BV (diagnosed per Nugent score) to colonize female germ-free mice and measure its impact on birth outcomes. There was considerable variability in the microbes that colonized each mouse, with no association to the BV status of the microbiota donor. Although some of the women in the study had adverse birth outcomes, the vaginal microbiota was not predictive of adverse birth outcomes in mice. However, elevated levels of pro-inflammatory cytokines in the uterus of HMA mice were detected during pregnancy. Together, these data outline the potential uses and limitations of HMA mice to elucidate the influence of the vaginal microbiota on health and disease.

The intestinal epithelium is a highly dynamic structure that rejuvenates in response to acute stressors and can undergo alterations in cellular composition as animals age. The microbiota, acting via secreted factors related to indole, appear to regulate the sensitivity of the epithelium to stressors and promote epithelial repair via IL-22 and type I IFN signaling. As animals age, the cellular composition of the intestinal epithelium changes, resulting in a decreased proportion of goblet cells in the colon. We show that colonization of young or geriatric mice with bacteria that secrete indoles and various derivatives or administration of the indole derivative indole-3 aldehyde increases proliferation of epithelial cells and promotes goblet cell differentiation, reversing an effect of aging. To induce goblet cell differentiation, indole acts via the xenobiotic aryl hydrocarbon receptor to increase expression of the cytokine IL-10. However, the effects of indoles on goblet cells do not depend on type I IFN or on IL-22 signaling, pathways responsible for protection against acute stressors. Thus, indoles derived from the commensal microbiota regulate intestinal homeostasis, especially during aging, via mechanisms distinct from those used during responses to acute stressors. Indoles may have utility as an intervention to limit the decline of barrier integrity and the resulting systemic inflammation that occurs with aging.

Increasingly, the s-galactoside binding lectins, termed galectins, are being recognized as critical regulators of cell function and organismal homeostasis. Within the context of the mucosal surface, galectins are established regulators of innate and adaptive immune responses, microbial populations, and several critical epithelial functions, including cell migration, proliferation, and response to injury. However, given their complex tissue distribution and expression patterns, the role within specific processes remains poorly understood. We took a genetic approach to understand the role of endogenous galectin-9 (Gal-9), a mucosal galectin that has been linked to inflammatory bowel disease, within the context of the murine intestine. We found that Gal-9–deficient (Gal9−/−) animals show increased sensitivity to chemically induced colitis and impaired proliferation in the setting of acute injury. Moreover, our results show that Gal9−/−-derived enteroids show impaired growth ex vivo. Consistent with a model in which endogenous Gal-9 controls epithelial growth and repair, we found that Gal9−/− animals showed increased sensitivity to intestinal challenge in multiple models of epithelial injury, including acute irradiation injury and ectopic wound biopsies. Finally, our results show that regenerating crypts from patient biopsy specimens show increased expression of Gal-9, indicating these processes may be conserved in humans. Taken together, these studies implicate Gal-9 in the regulation of cellular proliferation and epithelial restitution after intestinal epithelial injury.

Obesity and obesity-related metabolic disorders are linked to the intestinal microbiome. However, the causality of changes in the microbiome-host interaction affecting energy metabolism remains controversial. Here, we show the microbiome-derived metabolite δ-valerobetaine (VB) is a diet-dependent obesogen that is increased with phenotypic obesity and is correlated with visceral adipose tissue mass in humans. VB is absent in germ-free mice and their mitochondria but present in ex-germ-free conventionalized mice and their mitochondria. Mechanistic studies in vivo and in vitro show VB is produced by diverse bacterial species and inhibits mitochondrial fatty acid oxidation through decreasing cellular carnitine and mitochondrial long-chain acyl-coenzyme As. VB administration to germ-free and conventional mice increases visceral fat mass and exacerbates hepatic steatosis with a western diet but not control diet. Thus, VB provides a molecular target to understand and potentially manage microbiome-host symbiosis or dysbiosis in diet-dependent obesity.

Bone loss is a frequent but not universal complication of hyperparathyroidism. Using antibiotic-treated or germ-free mice, we show that parathyroid hormone (PTH) only caused bone loss in mice whose microbiota was enriched by the Th17 cell-inducing taxa segmented filamentous bacteria (SFB). SFB+ microbiota enabled PTH to expand intestinal TNF+ T and Th17 cells and increase their S1P-receptor-1 mediated egress from the intestine and recruitment to the bone marrow (BM) that causes bone loss. CXCR3-mediated TNF+ T cell homing to the BM upregulated the Th17 chemoattractant CCL20, which recruited Th17 cells to the BM. This study reveals mechanisms for microbiota-mediated gut–bone crosstalk in mice models of hyperparathyroidism that may help predict its clinical course. Targeting the gut microbiota or T cell migration may represent therapeutic strategies for hyperparathyroidism. T cells are involved in the bone loss induced by parathyroid hormone (PTH), but their origin is unknown. Here, the authors show that the intestinal microbiota is required for PTH to induce bone loss and describes mechanisms for microbiota-mediated gut–bone crosstalk in mouse models of hyperparathyroidism.

Intestinal homeostasis is regulated in-part by reactive oxygen species (ROS) that are generated in the colonic mucosa following contact with certain lactobacilli. Mechanistically, ROS can modulate protein function through the oxidation of cysteine residues within proteins. Recent advances in cysteine labeling by the Isotope Coded Affinity Tags (ICATs) technique has facilitated the identification of cysteine thiol modifications in response to stimuli. Here, we used ICATs to map the redox protein network oxidized upon initial contact of the colonic mucosa with Lactobacillus rhamnosus GG (LGG). We detected significant LGG-specific redox changes in over 450 proteins, many of which are implicated to function in cellular processes such as endosomal trafficking, epithelial cell junctions, barrier integrity, and cytoskeleton maintenance and formation. We particularly noted the LGG-specific oxidation of Rac1, which is a pleiotropic regulator of many cellular processes. Together, these data reveal new insights into lactobacilli-induced and redox-dependent networks involved in intestinal homeostasis.

Background & Aims: Ileal bile acid absorption is mediated by uptake via the apical sodium-dependent bile acid transporter (ASBT), and export via the basolateral heteromeric organic solute transporter α-β (OSTα-OSTβ). In this study, we investigated the cytotoxic effects of enterocyte bile acid stasis in Ostα-/- mice, including the temporal relationship between intestinal injury and initiation of the enterohepatic circulation of bile acids. Methods: Ileal tissue morphometry, histology, markers of cell proliferation, gene, and protein expression were analyzed in male and female wild-type and Ostα-/- mice at postnatal days 5, 10, 15, 20, and 30. Ostα-/-Asbt-/- mice were generated and analyzed. Bile acid activation of intestinal Nrf2-activated pathways was investigated in Drosophila. Results: As early as day 5, Ostα-/- mice showed significantly increased ileal weight per length, decreased villus height, and increased epithelial cell proliferation. This correlated with premature expression of the Asbt and induction of bile acid–activated farnesoid X receptor target genes in neonatal Ostα-/- mice. Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase-1 and Nrf2–anti-oxidant responsive genes were increased significantly in neonatal Ostα-/- mice at these postnatal time points. Bile acids also activated Nrf2 in Drosophila enterocytes and enterocyte-specific knockdown of Nrf2 increased sensitivity of flies to bile acid–induced toxicity. Inactivation of the Asbt prevented the changes in ileal morphology and induction of anti-oxidant response genes in Ostα-/- mice. Conclusions: Early in postnatal development, loss of Ostα leads to bile acid accumulation, oxidative stress, and a restitution response in ileum. In addition to its essential role in maintaining bile acid homeostasis, Ostα-Ostβ functions to protect the ileal epithelium against bile acid–induced injury. NCBI Gene Expression Omnibus: GSE99579. Keywords: Ileum, Reactive Oxygen Species, Nuclear Factor Erythroid-Derived 2-Like 2, Neonate, Drosophila

OBJECTIVES: Fracture is a traumatic event that can initiate systemic bone loss and lead to increased future risk of fracture, especially in the elderly. We sought to determine whether modulation of the gut microbiome using a daily probiotic supplement would dampen systemic inflammatory responses to fracture to accelerate bone repair and preserve distant bone sites. METHODS: Twelve-week-old male C57BL/6 J mice were randomly assigned to receive the probiotic Bifidobacterium adolescentis (1 × 10(8) CFU) via a daily oral gavage or PBS vehicle control. After two weeks, experimental mid-diaphyseal femoral fractures were created. Bones were analyzed using micro-CT at 10 and 18-days post-fracture. Midsagittal histological sections of fractured femora were stained with TRAP to quantify osteoclast number within the callus. RNA isolated from the small intestines was analyzed for gene expression of tight junction proteins. Serum levels of 20 cytokines were measured and analyzed using principal components analysis (PCA). RESULTS: Micro-CT of fractured limbs revealed a significant increase in callus bone volume (+28%) at day 18 post-fracture in mice receiving the B. adolescentis supplement. The total number of osteoclasts within the fracture callus at day 18 was also significantly increased (+158%) in the probiotic group suggesting more advanced remodeling of the callus. Micro-CT analyses of the L3 vertebral body, a distant bone site, revealed significantly higher trabecular bone volume at day 10 (+ 22%) and 18 (+11%) post-fracture in the probiotic group. PCA of serum cytokines revealed distinct clustering of the B. adolescentis and PBS groups indicating an anti-inflammatory effect by B. adolescentis. In the small intestines, probiotic supplementation significantly increased gene expression of tight junction proteins occludin, Jam3, ZO-1, and claudin-15 at both day 10 and day 18 post-fracture. CONCLUSIONS: Daily B. adolescentis supplementation not only accelerated fracture healing but also protected against vertebral body bone loss following fracture. These effects are likely a consequence of tightened intestinal barrier integrity, thus preventing systemic inflammation stemming from fracture-induced gut permeability. FUNDING SOURCES: Funding provided by Emory University and the Burroughs Wellcome Fund.

Background & Aims Identifying the functional elements that mediate efficient gut epithelial growth and homeostasis is essential for understanding intestinal health and disease. Many of these processes involve the Lactobacillus-induced generation of reactive oxygen species by NADPH oxidase (Nox1). However, the downstream signaling pathways that respond to Nox1-generated reactive oxygen species and mediate these events have not been described. Methods Wild-type and knockout mice were fed Lactobacillus rhamnosus GG and the transcriptional and cell signaling pathway responses in the colon measured. Corroboration of data generated in mice was done using in organoid tissue culture and in vivo gut injury models. Results Ingestion of L rhamnosus GG induces elevated levels of leptin in the gut epithelia, which as well as functioning in the context of metabolism, has pleiotropic activity as a chemokine that triggers cell proliferation. Consistently, using gut epithelial-specific knockout mice, we show that L rhamnosus GG–induced elevated levels of leptin is dependent on a functional Nox1 protein in the colonic epithelium, and that L rhamnosus GG–induced cell proliferation is dependent on Nox1, leptin, and leptin receptor. We also show that L rhamnosus GG induces the JAK-STAT signaling pathway in the gut in a Nox1, leptin, and leptin receptor–dependent manner. Conclusions These results demonstrate a novel role for leptin in the response to colonization by lactobacilli, where leptin functions in the transduction of signals from symbiotic bacteria to subepithelial compartments, where it modulates intestinal growth and homeostasis., Graphical abstract

Recent evidence has demonstrated that reactive oxygen (eg, hydrogen peroxide) can activate host cell signaling pathways that function in repair. We show that mice deficient in their capacity to generate reactive oxygen by the NADPH oxidase 2 holoenzyme, an enzyme complex highly expressed in neutrophils and macrophages, have disrupted capacity to orchestrate signaling events that function in mucosal repair. Similar observations were made for mice after neutrophil depletion, pinpointing this cell type as the source of the reactive oxygen driving oxidation-reduction protein signaling in the epithelium. To simulate epithelial exposure to high levels of reactive oxygen produced by neutrophils and gain new insight into this oxidation-reduction signaling, epithelial cells were treated with hydrogen peroxide, biochemical experiments were conducted, and a proteome-wide screen was performed using isotope-coded affinity tags to detect proteins oxidized after exposure. This analysis implicated signaling pathways regulating focal adhesions, cell junctions, and maintenance of the cytoskeleton. These pathways are also known to act via coordinated phosphorylation events within proteins that constitute the focal adhesion complex, including focal adhesion kinase and Crk-associated substrate. We identified the Rho family small GTP-binding protein Ras-related C3 botulinum toxin substrate 1 and p21 activated kinases 2 as operational in these signaling and localization pathways. These data support the hypothesis that reactive oxygen species from neutrophils can orchestrate epithelial cell-signaling events functioning in intestinal repair.

Background While very low birth weight (VLBW) infants often require multiple red blood cell transfusions, efforts to minimize transfusion-associated risks have resulted in more restrictive neonatal transfusion practices. However, whether restrictive transfusion strategies limit transfusions without increasing morbidity and mortality in this population remains unclear. Recent epidemiologic studies suggest that severe anemia may be an important risk factor for the development of necrotizing enterocolitis (NEC). However, the mechanism whereby anemia may lead to NEC remains unknown. Study design and methods The potential impact of anemia on neonatal inflammation and intestinal barrier disruption, two well-characterized predisposing features of NEC, was defined by correlation of hemoglobin values to cytokine levels in premature infants and by direct evaluation of intestinal hypoxia, inflammation and gut barrier disruption using a pre-clinical neonatal murine model of phlebotomy-induced anemia (PIA). Results Increasing severity of anemia in the preterm infant correlated with the level of IFN-gamma, a key pro-inflammatory cytokine that may predispose an infant to NEC. Gradual induction of PIA in a pre-clinical model resulted in significant hypoxia throughout the intestinal mucosa, including areas where intestinal macrophages reside. PIA-induced hypoxia significantly increased macrophage pro-inflammatory cytokine levels, while reducing tight junction protein ZO-1 expression and increasing intestinal barrier permeability. Macrophage depletion reversed the impact of anemia on intestinal ZO-1 expression and barrier function. Conclusions Taken together, these results suggest that anemia can increase intestinal inflammation and barrier disruption likely through altered macrophage function, leading to the type of predisposing intestinal injury that may increase the risk for NEC.

Animal models have played a key role in providing an understanding of the mechanisms that govern the pathophysiology of intestinal diseases. To expand on the repertoire of organisms available to study enteric diseases, we report on the use of the Drosophila melanogaster model to identify a novel function of an effector protein secreted by Vibrio parahaemolyticus, which is an enteric pathogen found in contaminated seafood. During pathogenesis, V. parahaemolyticus secretes effector proteins that usurp the host’s innate immune signaling pathways, thus allowing the bacterium to evade detection by the innate immune system. One secreted effector protein, VopA, has potent inhibitory effects on mitogen-activated protein kinase (MAPK) signaling pathways via the acetylation of critical residues within the catalytic loops of mitogen-activated protein kinase kinases (MAPKKs). Using the Drosophila model and cultured mammalian cells, we show that VopA also has potent modulating activity on focal adhesion complex (FAC) proteins, where VopA markedly reduced the levels of focal adhesion kinase (FAK) phosphorylation at Ser910, whereas the phosphorylation levels of FAK at Tyr397 and Tyr861 were markedly increased. Cultured cells expressing VopA were also impaired in their ability to migrate and repopulate areas subjected to a scratch wound. Consistently, expression of VopA in Drosophila midgut enterocytes disrupted the normal enterocyte arrangement. Finally, VopA inhibited apoptosis in both Drosophila tissues and mammalian cultured cells. Together, our data show that VopA can alter normal intestinal homeostatic processes to facilitate opportunities for V. parahaemolyticus to prolong infection within the host.

The gut microbiome is a dynamic community of microorganisms within the intestinal lumen. Many studies have suggested a role for gut-resident microbes in modulating host health, however the mechanisms by which these communities impact systemic physiology remain largely unknown. In this study, metabolomic and transcriptional profiling of germ-free and conventionalized mouse liver revealed an upregulation of the Nrf2 antioxidant and xenobiotic response in microbiome-replete animals. Using a Drosophila-based screening assay, we identified members of the genus Lactobacillus capable of stimulating Nrf2. Indeed, in both flies and mice, the human commensal Lactobacillus rhamnosus GG (LGG) potently activated Nrf2 in the fat body and liver, respectively, and protected against acetaminophen hepatotoxicity. MS/MS characterization of the portal circulation of LGG-treated mice identified a small molecule activator of Nrf2, 5-methoxyindoleacetic acid (5-MIAA), produced by LGG. Taken together, these data demonstrate a mechanism by which intestinal microbes modulate hepatic homeostasis and susceptibility to oxidative injury.

A distinct taxon of the Drosophila microbiota, Lactobacillus plantarum, is capable of stimulating the generation of reactive oxygen species (ROS) within cells, and inducing epithelial cell proliferation. Here we show microbial-induced ROS generation within Drosophila larval stem cell compartments exhibits a distinct spatial distribution. Lactobacilli-induced ROS is strictly excluded from defined midgut compartments that harbor adult midgut progenitor (AMP) cells, forming a functional “ROS sheltered zone” (RSZ). The RSZ is undiscernible in germ-free larvae, but forms following mono-colonization with L. plantarum. L. plantarum is a strong activator of the ROS-sensitive CncC/Nrf2 signaling pathway within enterocytes. Enterocyte-specific activation of CncC stimulated the proliferation of AMPs, demonstrating that pro-proliferative signals are transduced from enterocytes to AMPs. Mechanistically, we show that the cytokine Upd2 is expressed in the gut following L. plantarum colonization in a CncC dependent fashion, and may function in lactobacilli-induced AMP proliferation and intestinal tissue growth and development.

A eubiotic microbiota influences many physiological processes in the metazoan host, including development and intestinal homeostasis. Here, we have shown that the intestinal microbiota modulates inflammatory responses caused by sex steroid deficiency, leading to trabecular bone loss. In murine models, sex steroid deficiency increased gut permeability, expanded Th17 cells, and upregulated the osteoclastogenic cytokines TNFα (TNF), RANKL, and IL-17 in the small intestine and the BM. In germ-free (GF) mice, sex steroid deficiency failed to increase osteoclastogenic cytokine production, stimulate bone resorption, and cause trabecular bone loss, demonstrating that the gut microbiota is central in sex steroid deficiency-induced trabecular bone loss. Furthermore, we demonstrated that twice-weekly treatment of sex steroid-deficient mice with the probiotics Lactobacillus rhamnosus GG (LGG) or the commercially available probiotic supplement VSL#3 reduces gut permeability, dampens intestinal and BM inflammation, and completely protects against bone loss. In contrast, supplementation with a nonprobiotic strain of E. coli or a mutant LGG was not protective. Together, these data highlight the role that the gut luminal microbiota and increased gut permeability play in triggering inflammatory pathways that are critical for inducing bone loss in sex steroid-deficient mice. Our data further suggest that probiotics that decrease gut permeability have potential as a therapeutic strategy for postmenopausal osteoporosis.