Dopamine receptor D2 confers colonization resistance via microbial metabolites.

The gut microbiome has major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens1-3, including enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea and acute renal failure4,5 (haemolytic uremic syndrome). Although gut microorganisms can provide colonization resistance by outcompeting some pathogens or modulating host defence provided by the gut barrier and intestinal immune cells6,7, this phenomenon remains poorly understood. Here, we show that activation of the neurotransmitter receptor dopamine receptor D2 (DRD2) in the intestinal epithelium by gut microbial metabolites produced upon dietary supplementation with the essential amino acid L-tryptophan protects the host against Citrobacter rodentium, a mouse AE pathogen that is widely used as a model for EHEC infection8,9. We further find that DRD2 activation by these tryptophan-derived metabolites decreases expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Our results reveal a noncanonical colonization resistance pathway against AE pathogens that features an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization in the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches targeting DRD2 with dietary or pharmacological interventions to improve gut health and treat gastrointestinal infections, which afflict millions globally.

Pectins that Structurally Differ in the Distribution of Methyl-Esters Attenuate Citrobacter rodentium-Induced Colitis.

INTRODUCTION: Pectins have anti-inflammatory properties on intestinal immunity through direct interactions on Toll-like receptors (TLRs) in the small intestine or via stimulating microbiota-dependent effects in the large intestine. Both the degree of methyl-esterification (DM) and the distribution of methyl-esters (degree of blockiness; DB) of pectins contribute to this influence on immunity, but whether and how the DB impacts immunity through microbiota-dependent effects in the large intestine is unknown. Therefore, this study tests pectins that structurally differ in DB in a mouse model with Citrobacter rodentium induced colitis and studies the impact on the intestinal microbiota composition and associated attenuation of inflammation. METHODS AND RESULTS: Both low and high DB pectins induce a more rich and diverse microbiota composition. These pectins also lower the bacterial load of C. rodentium in cecal digesta. Through these effects, both low and high DB pectins attenuate C. rodentium induced colitis resulting in reduced intestinal damage, reduced numbers of Th1-cells, which are increased in case of C. rodentium induced colitis, and reduced levels of GATA3+ Tregs, which are related to tissue inflammation. CONCLUSION: Pectins prevent C. rodentium induced colonic inflammation by lowering the C. rodentium load in the caecum independently of the DB.

Glycine Attenuates Citrobacter rodentium-Induced Colitis by Regulating ATF6-Mediated Endoplasmic Reticulum Stress in Mice.

SCOPE: Inflammatory bowel disease (IBD) is an inflammatory gastrointestinal disorder in which endoplasmic reticulum (ER) stress and dysbiosis of the intestinal microbiota are implicated. Glycine supplementation is reported to reduce inflammatory responses in experimental colitis. However, the underlying mechanisms responsible for the beneficial effects remain unclear. METHODS AND RESULTS: Female C57BL/6 mice are orally administered with glycine (3.5 or 5.2 g kg-1 body weight) for 14 continuous days. On day 8 post-glycine supplementation, the mice are orally inoculated with 2 × 109 CFU Citrobacter rodentium (C. rodentium). The results show that glycine alleviates C. rodentium-induced body weight loss, increased disease activity index and spleen weight, colon length shortening, and colonic hyperplasia. Glycine suppresses the activation and infiltration of inflammatory cells, and secretion of pro-inflammatory cytokines in the colon tissues. The apoptosis of colon epithelial cells is also abrogated by glycine, which is associated with the inactivation of activating transcription factor 6α (ATF6α)-C/EBP homologous protein (CHOP) signaling. In addition, glycine administration increases α diversity, restores ß diversity, and abolishes the reduction in Lactobacillus, Bifidobacterium, Alistipes, Turicibacter, and Alloprevotella in the colon. CONCLUSIONS: Glycine supplementation is a nutritional strategy that may ameliorate C. rodentium-induced colitis by regulating ATF6α-CHOP-mediated ER stress and enhancing the abundance of Lactobacillus.

Plant- and Fish-Derived n-3 PUFAs Suppress Citrobacter Rodentium-Induced Colonic Inflammation.

SCOPE: Marine-derived n-3 PUFAs may ameliorate inflammation associated with inflammatory bowel diseases. Plant-derived n-3 PUFAs are thought to be inferior owing to shorter chain lengths. The aim of this study is to compare the impact of plant- and fish-derived PUFAs on murine colitis. METHODS AND RESULTS: C57BL/6 mice are fed high fat (36% kcal) diets with either 2.5% w/w sunflower oil (SO), flaxseed oil (FSO), ahiflower oil (AO), or fish oil (FO). After 4 weeks, mice are orogastrically challenged with Citrobacter rodentium (108 CFU) or sham gavaged. Fecal shedding is assayed at 2, 7, 10, and 14 days post infection (PI), and fecal microbiota at 14 days PI. Colonic inflammation and lipid mediators are measured. Supplementation regulates intestinal inflammation with crypt lengths being 66, 73, and 62 ±17 µm shorter (compared to SO) for FSO, AO, and FO respectively, p < 0.01. FSO blunts pathogen shedding at the peak of infection and FSO and AO both enhance fecal microbial diversity. FO attenuates levels of lipoxin and leukotriene B4 while plant oils increase pro-resolving mediator concentrations including D, E, and T-series resolvins. CONCLUSION: Plant and fish n-3 PUFAs attenuate colitis-induced inflammation while exhibiting characteristic pro-resolving lipid mediator metabolomes. Plant oils additionally promote microbial diversity.

Diet-derived galacturonic acid regulates virulence and intestinal colonization in enterohaemorrhagic Escherichia coli and Citrobacter rodentium.

Enteric pathogens sense the complex chemistry within the gastrointestinal tract to efficiently compete with the resident microbiota and establish a colonization niche. Here, we show that enterohaemorrhagic Escherichia coli and Citrobacter rodentium, its surrogate in a mouse infection model, sense galacturonic acid to initiate a multi-layered program towards successful mammalian infection. Galacturonic acid utilization as a carbon source aids the initial pathogen expansion. The main source of galacturonic acid is dietary pectin, which is converted to galacturonic acid by the prominent member of the microbiota, Bacteroides thetaiotamicron. This is regulated by the ExuR transcription factor. However, galacturonic acid is also sensed as a signal through ExuR to modulate the expression of the genes encoding a molecular syringe known as a type III secretion system, leading to infectious colitis and inflammation. Galacturonic acid acts as both a nutrient and a signal directing the exquisite microbiota-pathogen relationships within the gastrointestinal tract. This work highlights that differential dietary sugar availability influences the relationship between the microbiota and enteric pathogens, as well as disease outcomes.

Pea polyphenolics and hydrolysis processing alter microbial community structure and early pathogen colonization in mice.

Health benefits associated with pea consumption have been attributed to the fiber and polyphenolic content concentrated within the pea seed coat. However, the amount of pea polyphenols can vary between cultivars, and it has yet to be studied whether pea polyphenols impact the intestinal microbiota. We hypothesized that pea polyphenols promote a healthy microbiome that supports intestinal integrity and pathogen colonization resistance. To investigate the effects of pea polyphenols, pea cultivars rich and poor in proanthocyanidins were supplemented in raw or acid hydrolyzed form to an isocaloric diet in mice. Acid hydrolysis increases the absorption of pea polyphenols by cleaving polymeric proanthocyanidins to their readily absorbable anthocyanidin monomers. After 3 weeks of diet, mice were challenged with Citrobacter rodentium and pathogen colonization and inflammation were assessed. Counter to our hypothesis, pea seed coat fraction supplementation, especially the non-hydrolyzed proanthocyanidin-rich fraction diet adversely increased C. rodentium pathogen load and inflammation. Ileal, cecal and colon microbial communities were notably distinct between pea seed cultivar and hydrolysis processing. The consumption of intact proanthocyanidins decreased microbial diversity indicating that proanthocyanidins have antimicrobial properties. Together our results indicate supplementation of raw pea seed coat rich in proanthocyanidins adversely affect intestinal integrity. However, acid hydrolysis processing restored community structure and colonization resistance, and the anthocyanidin-rich fractions reduced weight gain on a high fat diet. Establishing a clear understanding of the effects of pea fiber and polyphenolic form on health will help to develop research-based pea products and dietary recommendations.

Colonic levels of vasoactive intestinal peptide decrease during infection and exogenous VIP protects epithelial mitochondria against the negative effects of IFNγ and TNFα induced during Citrobacter rodentium infection.

Citrobacter rodentium infection is a model for infection with attaching and effacing pathogens, such as enteropathogenic Escherichia coli. The vasoactive intestinal peptide (VIP) has emerged as an anti-inflammatory agent, documented to inhibit Th1 immune responses and successfully treat animal models of inflammation. VIP is also a mucus secretagogue. Here, we found that colonic levels of VIP decrease during murine C. rodentium infection with a similar time dependency as measurements reflecting mitochondrial function and epithelial integrity. The decrease in VIP appears mainly driven by changes in the cytokine environment, as no changes in VIP levels were detected in infected mice lacking interferon gamma (IFNγ). VIP supplementation alleviated the reduction of activity and levels of mitochondrial respiratory complexes I and IV, mitochondrial phosphorylation capacity, transmembrane potential and ATP generation caused by IFNγ, TNFα and C. rodentium infection, in an in vitro mucosal surface. Similarly, VIP treatment regimens that included the day 5-10 post infection period alleviated decreases in enzyme complexes I and IV, phosphorylation capacity, mitochondrial transmembrane potential and ATP generation as well as increased apoptosis levels during murine infection with C. rodentium. However, VIP treatment failed to alleviate colitis, although there was a tendency to decreased pathogen density in contact with the epithelium and in the spleen. Both in vivo and in vitro, NO generation increased during C. rodentium infection, which was alleviated by VIP. Thus, therapeutic VIP administration to restore the decreased levels during infection had beneficial effects on epithelial cells and their mitochondria, but not on the overall infection outcome.

Cooperative Metabolic Adaptations in the Host Can Favor Asymptomatic Infection and Select for Attenuated Virulence in an Enteric Pathogen.

Pathogen virulence exists on a continuum. The strategies that drive symptomatic or asymptomatic infections remain largely unknown. We took advantage of the concept of lethal dose 50 (LD50) to ask which component of individual non-genetic variation between hosts defines whether they survive or succumb to infection. Using the enteric pathogen Citrobacter, we found no difference in pathogen burdens between healthy and symptomatic populations. Iron metabolism-related genes were induced in asymptomatic hosts compared to symptomatic or naive mice. Dietary iron conferred complete protection without influencing pathogen burdens, even at 1000× the lethal dose of Citrobacter. Dietary iron induced insulin resistance, increasing glucose levels in the intestine that were necessary and sufficient to suppress pathogen virulence. A short course of dietary iron drove the selection of attenuated Citrobacter strains that can transmit and asymptomatically colonize naive hosts, demonstrating that environmental factors and cooperative metabolic strategies can drive conversion of pathogens toward commensalism.

Phytonutrient diet supplementation promotes beneficial Clostridia species and intestinal mucus secretion resulting in protection against enteric infection.

Plant extracts, or phytonutrients, are used in traditional medicine practices as supplements to enhance the immune system and gain resistance to various infectious diseases and are used in animal production as health promoting feed additives. To date, there are no studies that have assessed their mechanism of action and ability to alter mucosal immune responses in the intestine. We characterized the immunomodulatory function of six phytonutrients: anethol, carvacrol, cinnamaldehyde, eugenol, capsicum oleoresin and garlic extract. Mice were treated with each phytonutrient to assess changes to colonic gene expression and mucus production. All six phytonutrients showed variable changes in expression of innate immune genes in the colon. However only eugenol stimulated production of the inner mucus layer, a key mucosal barrier to microbes. The mechanism by which eugenol causes mucus layer thickening likely involves microbial stimulation as analysis of the intestinal microbiota composition showed eugenol treatment led to an increase in abundance of specific families within the Clostridiales order. Further, eugenol treatment confers colonization resistance to the enteric pathogen Citrobacter rodentium. These results suggest that eugenol acts to strengthen the mucosal barrier by increasing the thickness of the inner mucus layer, which protects against invading pathogens and disease.

Long-term selenium deficiency increases the pathogenicity of a Citrobacter rodentium infection in mice.

Citrobacter rodentium is a mouse pathogen that causes infectious colitis and shares characteristics with human enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli, including the ability to cause attaching and effacing lesions in the colon and serves as a useful model to study the pathogenicity of these bacteria. In this study, mice were fed a selenium-deficient diet for 5 or 20 weeks and then infected with C. rodentium. Colonization of the colon by C. rodentium was similar in mice fed adequate or selenium-deficient diets, but total bacterial colonization of the spleen was elevated in mice fed selenium-deficient diet for 20 weeks. Infection-induced changes to the colon included inflammatory cell infiltration, gross changes in crypt architecture, and ulceration and denuding of the epithelial layer that were greatest in mice fed a selenium-deficient diet for 20 weeks. Expression of pro-inflammatory genes was significantly higher 12-days post-infection in mice fed the selenium-deficient diet for 20 weeks compared to mice fed a selenium-adequate diet or selenium-deficient diet for 5 weeks. Diarrhea was prevalent in mice fed the selenium-deficient diet for 20 weeks but not 5 weeks, and this was associated with decreased expression of solute carrier family 26a3 and carbonic anhydrase IV, genes involved in ion transport. These results indicated that selenium played an important role in resistance to the pathological effects of a C. rodentium infection, and therefore, selenium status may be important in the expression of human disease caused by common food-borne bacteria.

The pathogenicity of an enteric Citrobacter rodentium Infection is enhanced by deficiencies in the antioxidants selenium and vitamin E.

The pathogenesis of a Citrobacter rodentium infection was evaluated in mice fed diets with a single deficiency in either selenium or vitamin E or with a double deficiency in both selenium and vitamin E compared to mice on nutritionally adequate diets. Mice fed the selenium- and vitamin E-deficient diet for 6 weeks had increased loads of C. rodentium in the colon and spleen, which were not observed in mice fed either of the singly deficient diets or the adequate diet. Infected mice fed the doubly deficient diet had increased colon crypt hyperplasia and an influx of infiltrating cells along with gross changes to crypt architecture, including ulceration and denuding of the epithelial layer. Cytokine and chemokine mRNA levels in the colon were measured by real-time PCR. Expression of proinflammatory cytokines and chemokines was upregulated on day 12 after infection with C. rodentium in mice fed the doubly deficient diet compared to mice fed the control diet. Heme oxygenase 1, an enzyme upregulated by oxidative stress, also was more highly induced in infected mice fed the doubly deficient diet. Production of C. rodentium antigen-specific IgM and IgG antibodies was not affected by feeding the doubly deficient diet. The results indicated that selenium and vitamin E play an important role in host resistance and in the pathology induced by C. rodentium, an infection that mimics disease caused by common food-borne bacterial pathogens in humans.