Infection and antibiotic-associated changes in the fecal microbiota of C. rodentium ϕstx2dact-infected C57BL/6 mice.

Enterohemorrhagic Escherichia coli causes watery to bloody diarrhea, which may progress to hemorrhagic colitis and hemolytic-uremic syndrome. While early studies suggested that antibiotic treatment may worsen the pathology of an enterohemorrhagic Escherichia coli (EHEC) infection, recent work has shown that certain non-Shiga toxin-inducing antibiotics avert disease progression. Unfortunately, both intestinal bacterial infections and antibiotic treatment are associated with dysbiosis. This can alleviate colonization resistance, facilitate secondary infections, and potentially lead to more severe illness. To address the consequences in the context of an EHEC infection, we used the established mouse infection model organism Citrobacter rodentium ϕstx2dact and monitored changes in fecal microbiota composition during infection and antibiotic treatment. C. rodentium ϕstx2dact infection resulted in minor changes compared to antibiotic treatment. The infection caused clear alterations in the microbial community, leading mainly to a reduction of Muribaculaceae and a transient increase in Enterobacteriaceae distinct from Citrobacter. Antibiotic treatments of the infection resulted in marked and distinct variations in microbiota composition, diversity, and dispersion. Enrofloxacin and trimethoprim/sulfamethoxazole, which did not prevent Shiga toxin-mediated organ damage, had the least disruptive effects on the intestinal microbiota, while kanamycin and tetracycline, which rapidly cleared the infection without causing organ damage, caused a severe reduction in diversity. Kanamycin treatment resulted in the depletion of all but Bacteroidetes genera, whereas tetracycline effects on Clostridia were less severe. Together, these data highlight the need to address the impact of individual antibiotics in the clinical care of life-threatening infections and consider microbiota-regenerating therapies.IMPORTANCEUnderstanding the impact of antibiotic treatment on EHEC infections is crucial for appropriate clinical care. While discouraged by early studies, recent findings suggest certain antibiotics can impede disease progression. Here, we investigated the impact of individual antibiotics on the fecal microbiota in the context of an established EHEC mouse model using C. rodentium ϕstx2dact. The infection caused significant variations in the microbiota, leading to a transient increase in Enterobacteriaceae distinct from Citrobacter. However, these effects were minor compared to those observed for antibiotic treatments. Indeed, antibiotics that most efficiently cleared the infection also had the most detrimental effect on the fecal microbiota, causing a substantial reduction in microbial diversity. Conversely, antibiotics showing adverse effects or incomplete bacterial clearance had a reduced impact on microbiota composition and diversity. Taken together, our findings emphasize the delicate balance required to weigh the harmful effects of infection and antibiosis in treatment.

Dietary κ-carrageenan facilitates gut microbiota-mediated intestinal inflammation.

The inflammatory effects of carrageenan (CGN), a ubiquitous food additive, remains controversial. Gut microbiota and intestinal homeostasis may be a breakthrough in resolving this controversy. Here we show that, κ-CGN did not cause significant inflammatory symptoms, but it did cause reduced bacteria-derived short-chain fatty acids (SCFAs) and decreased thickness of the mucus layer by altering microbiota composition. Administration of the pathogenic bacterium Citrobacter rodentium, further aggravated the inflammation and mucosal damage in the presence of κ-CGN. Mucus layer degradation and altered SCFA levels could be reproduced by fecal transplantation from κ-CGN-fed mice, but not from germ-free κ-CGN-fed mice. These symptoms could be partially repaired by administering probiotics. Our results suggest that κ-CGN may not be directly inflammatory, but it creates an environment that favors inflammation by perturbation of gut microbiota composition and then facilitates expansion of pathogens, and this effect may be partially reversed by the introduction of probiotics.

Protective and Anti-Inflammatory Effects of Protegrin-1 on Citrobacter rodentium Intestinal Infection in Mice.

Infectious intestinal colitis, manifesting as intestinal inflammation, diarrhea, and epithelial barrier disruption, affects millions of humans worldwide and, without effective treatment, can result in death. In addition to this, the significant rise in antibiotic-resistant bacteria poses an urgent need for alternative anti-infection therapies for the treatment of intestinal disorders. Antimicrobial peptides (AMPs) are potential therapies that have broad-spectrum antimicrobial activity due to their (1) unique mode of action, (2) broad-spectrum antimicrobial activity, and (3) protective role in GI tract maintenance. Protegrin-1 (PG-1) is an AMP of pig origin that was previously shown to reduce the pathological effects of chemically induced digestive tract inflammation (colitis) and to modulate immune responses and tissue repair. This study aimed to extend these findings by investigating the protective effects of PG-1 on pathogen-induced colitis in an infection study over a 10-day experimental period. The oral administration of PG-1 reduced Citrobacter rodentium intestinal infection in mice as evidenced by reduced histopathologic change in the colon, prevention of body weight loss, milder clinical signs of disease, and more effective clearance of bacterial infection relative to challenged phosphate-buffered saline (PBS)-treated mice. Additionally, PG-1 treatment altered the expression of various inflammatory mediators during infection, which may act to resolve inflammation and re-establish intestinal homeostasis. PG-1 administered in its mature form was more effective relative to the pro-form (ProPG-1). To our knowledge, this is the first study demonstrating the protective effects of PG-1 on infectious colitis.

Reversal of Pathogen-Induced Barrier Defects in Intestinal Epithelial Cells by Contra-pathogenicity Agents.

BACKGROUND: Environmental enteropathy (EE) is associated with stunting, impairment of responses to oral vaccines, and other adverse health consequences in young children throughout the developing world. EE is characterized by chronic low-grade intestinal inflammation and disrupted epithelial barrier integrity, partly resulting from dysregulation of tight junction proteins, observed in other enteropathies such as celiac disease. During EE, this dysregulation of tight junction expression amplifies translocation of pathogenic bacteria across the intestinal mucosa. AIMS: The aim was to determine whether enteropathogen-mediated epithelial barrier failure can be ameliorated using contra-pathogenicity therapies. METHODS: Intestinal epithelial barrier damage was assessed in Caco-2 cells incubated with three important enteropathogens identified in EE patients: Enteropathogenic Escherichia coli (EPEC), Citrobacter rodentium (C. rodentium), and Cryptosporidium parvum (C. parvum). Potential therapeutic molecules were tested to detect effects on transepithelial resistance (TER), bacterial translocation (BT), claudin-4 expression, and regulation of the inflammatory cytokine response. RESULTS: All three enteropathogens compared to uninfected cells, reduced TER (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0007), reduced claudin-4 expression, and permitted BT (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0003) through the monolayer. Zinc, colostrum, epidermal growth factor, trefoil factor 3, resistin-like molecule-ß, hydrocortisone, and the myosin light chain kinase inhibitor ML7 (Hexahydro-1-[(5-iodo-1-naphthalenyl)sulfonyl]-1H-1,4-diazepine hydrochloride); ML7) improved TER (up to 70%) and decreased BT (as much as 96%). Only zinc demonstrated modest antimicrobial activity. CONCLUSION: The enteropathogens impaired intestinal-epithelial barrier integrity with dysregulation of claudin-4 and increased bacterial translocation. Enteropathogen-mediated damage was reduced using contra-pathogenicity agents which mitigated the effects of pathogens without direct antimicrobial activity.

STING controls intestinal homeostasis through promoting antimicrobial peptide expression in epithelial cells.

Stimulator of interferon genes (STING) has been shown to play a critical role in orchestrating immune responses to various pathogens through sensing cyclic dinucleotides. However, how STING regulates intestinal homeostasis is still not completely understood. In this study, we found that STING-/- mice were more susceptible to enteric infection with Citrobacter rodentium compared to wild-type (WT) mice evidenced by more severe intestinal inflammation and impaired bacterial clearance. STING-/- mice demonstrated lower expression of REG3γ but not ß-defensins and Cramp in IECs. Consistently, STING-/- IECs showed reduced capacity to inhibit bacterial growth. STING agonists, both 10-carboxymethyl-9-acridanone (CMA) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA), promoted REG3γ expression IECs. Furthermore, STING agonists promoted WT but not REG3γ-deficient IEC bacterial killing. Mechanistically, STING agonists activated STAT3 and promoted glycolysis in IECs. Inhibition of STAT3 pathway and glycolysis suppressed STING-induced REG3γ production in IECs, and abrogated STING-mediated IEC killing of C. rodentium. Additionally, treatment with the STING ligand, 2,3-cGAMP, inhibited C. rodentium-induced colitis in vivo. Overall, STING promotes IEC REG3γ expression to inhibit enteric infection and intestinal inflammation, thus, maintaining the intestinal homeostasis.

Indole-3-Carbinol Inhibits Citrobacter rodentium Infection through Multiple Pathways Including Reduction of Bacterial Adhesion and Enhancement of Cytotoxic T Cell Activity.

Intestinal inflammation is associated with an increased risk of developing colorectal cancer and may result from dysregulated responses to commensal bacteria or exposure to bacterial pathogens. Dietary modulation of intestinal inflammation may protect against development of colon cancer. However, the precise diet-derived components and underlying mechanisms remain elusive. Citrobacter rodentium (Cr) induces acute intestinal inflammation and has been used to study the role of inflammation in the susceptibility to colon cancer. Here we examine the effects of indole-3-carbinol (I3C), a dietary compound with anticarcinogenic properties, on intestinal immune and inflammatory responses to Cr infection and adhesion to colonic cells in vitro. C57BL/6J mice were fed a diet with/without 1 µmol/g I3C and infected with Cr. Compared to infected mice fed with a control diet, consumption of a 1 µmol I3C/g diet significantly reduced fecal excretion of Cr, Cr colonization of the colon, and reduced colon crypt hyperplasia. Furthermore, expression of Cr-induced inflammatory markers such as IL-17A, IL-6, and IL1ß were attenuated in infected mice fed with the I3C diet, compared to mice fed a control diet. The expression of cytotoxic T cell markers CD8 and FasL mRNA were increased in I3C-fed infected mice. In-vitro, I3C inhibited Cr growth and adhesion to Caco-2 cells. I3C alleviates Cr-induced murine colitis through multiple mechanisms including inhibition of Cr growth and adhesion to colonic cells in vitro and enhancement of cytotoxic T cell activity.

Opioid use potentiates the virulence of hospital-acquired infection, increases systemic bacterial dissemination and exacerbates gut dysbiosis in a murine model of Citrobacter rodentium infection.

Opioid analgesics are frequently prescribed in the United States and worldwide. However, serious side effects such as addiction, immunosuppression and gastrointestinal symptoms limit their use. It was recently demonstrated that morphine treatment results in a significant disruption in gut barrier function, leading to an increased translocation of gut commensal bacteria. Further studies have indicated distinct alterations in the gut microbiome and metabolome following morphine treatment, contributing to the negative consequences that are associated with opioid use. However, it is unclear how opioids modulate gut homeostasis in the context of a hospital-acquired bacterial infection. Citrobacter rodentium is an ideal murine model of human infections with enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). In the current study, a mouse model of C. rodentium infection was used to investigate the role of morphine in the modulation of gut homeostasis in the context of a hospital-acquired bacterial infection. Morphine treatment resulted in 1) the promotion of C. rodentium systemic dissemination, 2) an increase in the expression of the virulence factors of C. rodentium colonization in intestinal contents, 3) altered gut microbiome, 4) damaged integrity of gut epithelial barrier function, 5) inhibition of the C. rodentium-induced increase in goblet cells, and 6) dysregulated IL-17A immune response. This study demonstrates and further validates a positive correlation between opioid drug use/abuse and an increased risk of infections, suggesting that the overprescription of opioids may increase the susceptibility to hospital-acquired infection.

Twin-Arginine Translocation System Is Involved in Citrobacter rodentium Fitness in the Intestinal Tract.

The twin-arginine translocation (Tat) system is involved in not only a wide array of cellular processes but also pathogenesis in many bacterial pathogens; thus, this system is expected to become a novel therapeutic target to treat infections. To the best of our knowledge, involvement of the Tat system has not been reported in the gut infection caused by Citrobacter rodentium Here, we studied the role of Tat in C. rodentium gut infection, which resembles human infection with enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). A C. rodentium Tat loss-of-function mutant displayed prolonged gut colonization, which was explained by reduced inflammatory responses and, particularly, neutrophil infiltration. Further, the Tat mutant had colonization defects upon coinfection with the wild-type strain of C. rodentium The Tat mutant also became hypersensitive to bile acids, and an increase in fecal bile acids fostered C. rodentium clearance from the gut lumen. Finally, we show that the chain form of C. rodentium cells, induced by a Tat-dependent cell division defect, exhibits impaired resistance to bile acids. Our findings indicate that the Tat system is involved in gut colonization by C. rodentium, which is associated with neutrophil infiltration and resistance to bile acids. Interventions that target the Tat system, as well as luminal bile acids, might thus be promising therapeutic strategies to treat human EHEC and EPEC infections.

Long-Term Effects of Early-Life Antibiotic Exposure on Resistance to Subsequent Bacterial Infection.

Early-life antibiotic exposure may provoke long-lasting microbiota perturbation. Since a healthy gut microbiota confers resistance to enteric pathogens, we hypothesized that early-life antibiotic exposure would worsen the effects of a bacterial infection encountered as an adult. To test this hypothesis, C57BL/6 mice received a 5-day course of tylosin (macrolide), amoxicillin (ß-lactam), or neither (control) early in life and were challenged with Citrobacter rodentium up to 80 days thereafter. The early-life antibiotic course led to persistent alterations in the intestinal microbiota and even with pathogen challenge 80 days later worsened the subsequent colitis. Compared to exposure to amoxicillin, exposure to tylosin led to greater disease severity and microbiota perturbation. Transferring the antibiotic-perturbed microbiota to germfree animals led to worsened colitis, indicating that the perturbed microbiota was sufficient for the increased disease susceptibility. These experiments highlight the long-term effects of early-life antibiotic exposure on susceptibility to acquired pathogens.IMPORTANCE The gastrointestinal microbiota protects hosts from enteric infections; while antibiotics, by altering the microbiota, may diminish this protection. We show that after early-life exposure to antibiotics host susceptibility to enhanced Citrobacter rodentium-induced colitis is persistent and that this enhanced disease susceptibility is transferable by the antibiotic-altered microbiota. These results strongly suggest that early-life antibiotics have long-term consequences on the gut microbiota and enteropathogen infection susceptibility.

Ground flaxseed reverses protection of a reduced-fat diet against Citrobacter rodentium-induced colitis.

Flaxseed is high in ω-3 polyunsaturated fatty acids, fiber, and lignans known to lower cholesterol levels. However, its use for prevention or treatment of inflammatory bowel diseases has yielded mixed results, perhaps related to dietary interactions. In this study, we evaluated the impact of ground flaxseed supplementation on the severity of Citrobacter rodentium-induced colitis in the setting of either a high-fat (HF, ~36%kcal) or reduced-fat (RF, ~12%kcal) diet. After weaning, C57BL/6 mice ( n = 8-15/treatment) were fed ground flaxseed (7 g/100 g diet) with either HF (HF Flx) or RF (RF Flx) diets for 4 wk before infection with C. rodentium or sham gavage. Weight changes, mucosal inflammation, pathogen burden, gut microbiota composition, tissue polyunsaturated fatty acids, and cecal short-chain fatty acids were compared over a 14-day infection period. The RF diet protected against C. rodentium-induced colitis, whereas the RF Flx diet increased pathogen burden, exacerbated gut inflammation, and promoted gut dysbiosis. When compared with the RF diet, both HF and HF Flx diets resulted in more severe pathology in response to C. rodentium infection. Our findings demonstrate that although an RF diet protected against C. rodentium-induced colitis and associated gut dysbiosis in mice, beneficial effects were diminished with ground flaxseed supplementation. NEW & NOTEWORTHY Our results demonstrate a strong protective effect of a reduced-fat diet against intestinal inflammation, dysbiosis, and pathogen burden during Citrobacter rodentium-induced colitis. However, ground flaxseed supplementation in the setting of a reduced-fat diet exacerbated colitis despite higher levels of intestinal n-3 polyunsaturated fatty acids and cecal short-chain fatty acids.

Card9 mediates susceptibility to intestinal pathogens through microbiota modulation and control of bacterial virulence.

OBJECTIVE: In association with innate and adaptive immunity, the microbiota controls the colonisation resistance against intestinal pathogens. Caspase recruitment domain 9 (CARD9), a key innate immunity gene, is required to shape a normal gut microbiota. Card9-/- mice are more susceptible to the enteric mouse pathogen Citrobacter rodentium that mimics human infections with enteropathogenic and enterohaemorrhagic Escherichia coli. Here, we examined how CARD9 controls C. rodentium infection susceptibility through microbiota-dependent and microbiota-independent mechanisms. DESIGN: C. rodentium infection was assessed in conventional and germ-free (GF) wild-type (WT) and Card9-/- mice. To explore the impact of Card9-/-microbiota in infection susceptibility, GF WT mice were colonised with WT (WT→GF) or Card9-/- (Card9-/- →GF) microbiota before C. rodentium infection. Microbiota composition was determined by 16S rDNA gene sequencing. Inflammation severity was determined by histology score and lipocalin level. Microbiota-host immune system interactions were assessed by quantitative PCR analysis. RESULTS: CARD9 controls pathogen virulence in a microbiota-independent manner by supporting a specific humoral response. Higher susceptibility to C. rodentium-induced colitis was observed in Card9-/- →GF mice. The microbiota of Card9-/- mice failed to outcompete the monosaccharide-consuming C. rodentium, worsening the infection severity. A polysaccharide-enriched diet counteracted the ecological advantage of C. rodentium and the defective pathogen-specific antibody response in Card9-/- mice. CONCLUSIONS: CARD9 modulates the susceptibility to intestinal infection by controlling the pathogen virulence in a microbiota-dependent and microbiota-independent manner. Genetic susceptibility to intestinal pathogens can be overridden by diet intervention that restores humoural immunity and a competing microbiota.

Galactooligosaccharide supplementation provides protection against Citrobacter rodentium-induced colitis without limiting pathogen burden.

Many enteric pathogens, including Salmonella and enteropathogenic and enterohemorrhagic Escherichia coli, express adhesins that recognize and bind to carbohydrate moieties expressed on epithelial cells. An attractive strategy for inhibiting bacterial adherence employs molecules that mimic these epithelial binding sites. Prebiotic oligosaccharides are non-digestible, fermentable fibres capable of modulating the gut microbiota. Moreover, they may act as molecular decoys that competitively inhibit adherence of pathogens to host cells. In particular, galactooligosaccharides (GOS) and other prebiotic fibres have been shown to inhibit pathogen adherence to epithelial cells in vitro. In the present study, we determined the ability of prophylactic GOS administration to reduce enteric pathogen adherence both in vitro and in vivo as well as protect against intestinal inflammation. GOS supplementation significantly reduced the adherence of the epithelial-adherent murine bacterial pathogen Citrobacter rodentium in a dose-dependent manner to the surface of epithelial cells in vitro. A 1- to 2-log reduction in bacterial adherence was observed at the lowest and highest doses tested, respectively. However, mouse studies revealed that treatment with GOS neither reduced the adherence of C. rodentium to the distal colon nor decreased its dissemination to systemic organs. Despite the absence of adherence inhibition, colonic disease scores for GOS-treated, C. rodentium-infected mice were significantly lower than those of untreated C. rodentium-infected animals (P=0.028). Together, these data suggest that GOS has a direct protective effect in ameliorating disease severity following C. rodentium infection through an anti-adherence-independent mechanism.

Retinoic Acid Mediated Clearance of Citrobacter rodentium in Vitamin A Deficient Mice Requires CD11b+ and T Cells.

Vitamin A deficiency affects over 250 million preschool-age children worldwide and is associated with increased childhood mortality and risk of developing enteric infections. Vitamin A deficient (A-) mice developed chronic Citrobacter rodentium infection. A single oral dose of retinoic acid (RA) at d7 post-infection was sufficient to induce clearance of the pathogen in A- mice. RA treatment of A- mice induced il17 expression in the colon. In A- mice, colonic IL-17 was primarily produced by CD11b+ cells; however, in A+ mice, the major source of colonic IL-17 was CD4+ T cells. To determine the cellular targets of vitamin A required for host resistance to C. rodentium, mice that express a dominant negative (dn) retinoic acid receptor (RAR) in T cells (T-dnRAR) or macrophage/neutrophils (LysM-dnRAR) were used. T-dnRAR mice had T cells that produced a robust intestinal IL-17 response and for 40% of the mice was enough to clear the infection. The remainder of the T-dnRAR mice developed a chronic infection. A- LysM-dnRAR mice developed early lethal infections with surviving mice becoming chronically infected. RA treatment of A- LysM-dnRAR mice was ineffective for inducing colonic IL-17 or clearing C. rodentium. Retinoid signaling is required in T cells and CD11b+ cells for complete elimination of enteric pathogens.

Citrobacter rodentium Relies on Commensals for Colonization of the Colonic Mucosa.

We investigated the role of commensals at the peak of infection with the colonic mouse pathogen Citrobacter rodentium. Bioluminescent and kanamycin (Kan)-resistant C. rodentium persisted avirulently in the cecal lumen of mice continuously treated with Kan. A single Kan treatment was sufficient to displace C. rodentium from the colonic mucosa, a phenomenon not observed following treatment with vancomycin (Van) or metronidazole (Met). Kan, Van, and Met induce distinct dysbiosis, suggesting C. rodentium relies on specific commensals for colonic colonization. Expression of the master virulence regulator ler is induced in germ-free mice, yet C. rodentium is only seen in the cecal lumen. Moreover, in conventional mice, a single Kan treatment was sufficient to displace C. rodentium constitutively expressing Ler from the colonic mucosa. These results show that expression of virulence genes is not sufficient for colonization of the colonic mucosa and that commensals are essential for a physiological infection course.

Bacterial Chat: Intestinal Metabolites and Signals in Host-Microbiota-Pathogen Interactions.

Intestinal bacteria employ microbial metabolites from the microbiota and chemical signaling during cell-to-cell communication to regulate several cellular functions. Pathogenic bacteria are extremely efficient in orchestrating their response to these signals through complex signaling transduction systems. Precise coordination and interpretation of these multiple chemical cues is important within the gastrointestinal (GI) tract. Enteric foodborne pathogens, such as enterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica serovar Typhimurium, or the surrogate murine infection model for EHEC, Citrobacter rodentium, are all examples of microorganisms that modulate the expression of their virulence repertoire in response to signals from the microbiota or the host, such as autoinducer-3 (AI-3), epinephrine (Epi), and norepinephrine (NE). The QseBC and QseEF two-component systems, shared by these pathogens, are involved in sensing these signals. We review how these signaling systems sense and relay these signals to drive bacterial gene expression; specifically, to modulate virulence. We also review how bacteria chat via chemical signals integrated with metabolite recognition and utilization to promote successful associations among enteric pathogens, the microbiota, and the host.

Bile Acid Administration Elicits an Intestinal Antimicrobial Program and Reduces the Bacterial Burden in Two Mouse Models of Enteric Infection.

In addition to their chemical antimicrobial nature, bile acids are thought to have other functions in the homeostatic control of gastrointestinal immunity. However, those functions have remained largely undefined. In this work, we used ileal explants and mouse models of bile acid administration to investigate the role of bile acids in the regulation of the intestinal antimicrobial response. Mice fed on a diet supplemented with 0.1% chenodeoxycholic acid (CDCA) showed an upregulated expression of Paneth cell α-defensins as well as an increased synthesis of the type-C lectins Reg3b and Reg3g by the ileal epithelium. CDCA acted on several epithelial cell types, by a mechanism independent from farnesoid X receptor (FXR) and not involving STAT3 or ß-catenin activation. CDCA feeding did not change the relative abundance of major commensal bacterial groups of the ileum, as shown by 16S analyses. However, administration of CDCA increased the expression of ileal Muc2 and induced a change in the composition of the mucosal immune cell repertoire, decreasing the proportion of Ly6G+ and CD68+ cells, while increasing the relative amount of IgGκ+ B cells. Oral administration of CDCA to mice attenuated infections with the bile-resistant pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium, promoting lower systemic colonization and faster bacteria clearance, respectively. Our results demonstrate that bile acid signaling in the ileum triggers an antimicrobial program that can be potentially used as a therapeutic option against intestinal bacterial infections.

Inflammasome Activation by ATP Enhances Citrobacter rodentium Clearance through ROS Generation.

BACKGROUND: Nod-like receptor family, pyrin domain containing 3 (NLRP3) is an important cytosolic sensor of cellular stress and infection. Once activated, NLRP3 forms a multiprotein complex (inflammasome) that triggers the maturation and secretion of interleukin (IL)-1ß and IL-18. We aimed to define the consequences of NLRP3 induction, utilizing exogenous adenosine triphosphate (ATP) as an inflammasome activator, to determine if inflammasome activation increases macrophage killing of Citrobacter rodentium and define mechanisms. METHODS: Bacterial survival was measured using a gentamicin protection assay. Inflammasome activation or inhibition in mouse J774A.1 macrophages were assessed by measuring IL-1ß; cytokines and reactive oxygen species (ROS) were measured by ELISA and DCFDA, respectively. RESULTS: Activation of the inflammasome increased bacterial killing by macrophages and its inhibition attenuated this effect with no impact on phagocytosis or cell death. Furthermore, inflammasome activation suppressed pro-inflammatory cytokines during infection, possibly due to more effective bacterial killing. While the infection increased ROS production, this effect was reduced by inflammasome inhibitors, indicating that ROS is inflammasome-dependent. ROS inhibitors increased bacterial survival in the presence of ATP, suggesting that inflammasome-induced bacterial killing is mediated, at least in part, by ROS activity. CONCLUSION: Improving inflammasome activity during infection may increase bacterial clearance by macrophages and reduce subsequent microbe-induced inflammation.

Hyaluronan 35kDa treatment protects mice from Citrobacter rodentium infection and induces epithelial tight junction protein ZO-1 in vivo.

Maintaining a healthy intestinal barrier, the primary physical barrier between intestinal microbiota and the underlying lamina propria, is critical for optimal health. Epithelial integrity is essential for the prevention of the entrance of luminal contents, such as bacteria and their products, through the large intestinal barrier. In this study, we investigated the protective functions of biosynthetic, specific sized, hyaluronan around 35kDa (HA35) on intestinal epithelium in healthy mice, as well as mice infected Citrobacter rodentium, an established model that mimics infection with a serious human pathogen, enteropathogenic E. coli (EPEC). Our results reveal that treatment with HA35 protects mice from Citrobacter infection and enhances the epithelial barrier function. In particular, we have found that HA35 induces the expression of tight junction protein zonula occludens (ZO)-1 in both healthy and Citrobacter infected mice, as demonstrated by immunoflurorescence and Western blot analyses. Furthermore, we determined that HA35 treatment enhances ZO-1 expression and reduces intestinal permeability at the early stages of dextran sulfate sodium (DSS)-induced colitis in mice. Together, our data demonstrate that the expression and functionality of tight junctions, are increased by HA35 treatment, suggesting a novel mechanism for the protection from Citrobacter infection.

Model of Host-Pathogen Interaction Dynamics Links In Vivo Optical Imaging and Immune Responses.

Tracking disease progression in vivo is essential for the development of treatments against bacterial infection. Optical imaging has become a central tool for in vivo tracking of bacterial population development and therapeutic response. For a precise understanding of in vivo imaging results in terms of disease mechanisms derived from detailed postmortem observations, however, a link between the two is needed. Here, we develop a model that provides that link for the investigation of Citrobacter rodentium infection, a mouse model for enteropathogenic Escherichia coli (EPEC). We connect in vivo disease progression of C57BL/6 mice infected with bioluminescent bacteria, imaged using optical tomography and X-ray computed tomography, to postmortem measurements of colonic immune cell infiltration. We use the model to explore changes to both the host immune response and the bacteria and to evaluate the response to antibiotic treatment. The developed model serves as a novel tool for the identification and development of new therapeutic interventions.

Antimicrobial growth promoters modulate host responses in mice with a defined intestinal microbiota.

Antibiotics can promote growth in livestock (antimicrobial growth promoters, AGPs), however lack of knowledge regarding mechanisms has hampered the development of effective non-antibiotic alternatives. Antibiotics affect eukaryotic cells at therapeutic concentrations, yet effects of AGPs on host physiology are relatively understudied, partially due to the complexity of host-microorganism interactions within the gastrointestinal tract. To determine the direct effects of AGPs on the host, we generated Altered Schaedler Flora (ASF) mice, and administered chlortetracycline (CTC) and tylosin phosphate (TYL) in feed. Mice were challenged with Citrobacter rodentium to determine how AGPs alter host responses to physiological stress. Although CTC and TYL had inconsistent effects on the ASF taxa, AGPs protected mice from weight loss following C. rodentium inoculation. Mice treated with either CTC or TYL had lower expression of ßd1 and Il17a in the intestine and had a robust induction of Il17a and Il10. Furthermore, AGP administration resulted in a lower hepatic expression of acute phase proteins (Saa1, Hp, and Cp) in liver tissue, and ameliorated C. rodentium-induced reductions in the expression of genes involved in lipogenesis (Hmgcl and Fabp1). Collectively, this indicates that AGPs directly affect host physiology, and highlights important considerations in the development of non-antibiotic alternatives.