Vasoactive intestinal peptide promotes host defense against enteric pathogens by modulating the recruitment of group 3 innate lymphoid cells.

Group 3 innate lymphoid cells (ILC3s) control the formation of intestinal lymphoid tissues and play key roles in intestinal defense. They express neuropeptide vasoactive intestinal peptide (VIP) receptor 2 (VPAC2), through which VIP modulates their function, but whether VIP exerts other effects on ILC3 remains unclear. We show that VIP promotes ILC3 recruitment to the intestine through VPAC1 independent of the microbiota or adaptive immunity. VIP is also required for postnatal formation of lymphoid tissues as well as the maintenance of local populations of retinoic acid (RA)-producing dendritic cells, with RA up-regulating gut-homing receptor CCR9 expression by ILC3s. Correspondingly, mice deficient in VIP or VPAC1 suffer a paucity of intestinal ILC3s along with impaired production of the cytokine IL-22, rendering them highly susceptible to the enteric pathogen Citrobacter rodentium This heightened susceptibility to C. rodentium infection was ameliorated by RA supplementation, adoptive transfer of ILC3s, or by recombinant IL-22. Thus, VIP regulates the recruitment of intestinal ILC3s and formation of postnatal intestinal lymphoid tissues, offering protection against enteric pathogens.

Fasting increases microbiome-based colonization resistance and reduces host inflammatory responses during an enteric bacterial infection.

Reducing food intake is a common host response to infection, yet it remains unclear whether fasting is detrimental or beneficial to an infected host. Despite the gastrointestinal tract being the primary site of nutrient uptake and a common route for infection, studies have yet to examine how fasting alters the host's response to an enteric infection. To test this, mice were fasted before and during oral infection with the invasive bacterium Salmonella enterica serovar Typhimurium. Fasting dramatically interrupted infection and subsequent gastroenteritis by suppressing Salmonella's SPI-1 virulence program, preventing invasion of the gut epithelium. Virulence suppression depended on the gut microbiota, as Salmonella's invasion of the epithelium proceeded in fasting gnotobiotic mice. Despite Salmonella's restored virulence within the intestines of gnotobiotic mice, fasting downregulated pro-inflammatory signaling, greatly reducing intestinal pathology. Our study highlights how food intake controls the complex relationship between host, pathogen and gut microbiota during an enteric infection.

Inflammatory bowel disease and immunonutrition: novel therapeutic approaches through modulation of diet and the gut microbiome.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract, thought to at least in part reflect an aberrant immune response to gut bacteria. IBD is increasing in incidence, particularly in populations that have recently immigrated to western countries. This suggests that environmental factors are involved in its pathogenesis. We hypothesize that the increase in IBD rates might reflect the consumption of an unhealthy Western diet, containing excess calories and lacking in key nutritional factors, such as fibre and vitamin D. Several recent studies have determined that dietary factors can dramatically influence the activation of immune cells and the mediators they release through a process called immunonutrition. Moreover, dietary changes can profoundly affect the balance of beneficial versus pathogenic bacteria in the gut. This microbial imbalance can alter levels of microbiota-derived metabolites that in turn can influence innate and adaptive intestinal immune responses. If the diet-gut microbiome disease axis does indeed underpin much of the 'western' influence on the onset and progression of IBD, then tremendous opportunity exists for therapeutic changes in lifestyle, to modulate the gut microbiome and to correct immune imbalances in individuals with IBD. This review highlights four such therapeutic strategies - probiotics, prebiotics, vitamin D and caloric restriction - that have the potential to improve and add to current IBD treatment regimens.

Direct measurement of oxidative and nitrosative stress dynamics in Salmonella inside macrophages.

Many significant bacterial pathogens have evolved virulence mechanisms to evade degradation and exposure to reactive oxygen (ROS) and reactive nitrogen species (RNS), allowing them to survive and replicate inside their hosts. Due to the highly reactive and short-lived nature of ROS and RNS, combined with limitations of conventional detection agents, the mechanisms underlying these evasion strategies remain poorly understood. In this study, we describe a system that uses redox-sensitive GFP to nondisruptively measure real-time fluctuations in the intrabacterial redox environment. Using this system coupled with high-throughput microscopy, we report the intrabacterial redox dynamics of Salmonella enterica Typhimurium (S. Typhimurium) residing inside macrophages. We found that the bacterial SPI-2 type III secretion system is required for ROS evasion strategies and this evasion relies on an intact Salmonella-containing vacuole (SCV) within which the bacteria reside during infection. Additionally, we found that cytosolic bacteria that escape the SCV experience increased redox stress in human and murine macrophages. These results highlight the existence of specialized evasion strategies used by intracellular pathogens that either reside inside a vacuole or "escape" into the cytosol. Taken together, the use of redox-sensitive GFP inside Salmonella significantly advances our understanding of ROS and RNS evasion strategies during infection. This technology can also be applied to measuring bacterial oxidative and nitrosative stress dynamics under different conditions in a wide variety of bacteria.

Dietary vitamin D3 deficiency alters intestinal mucosal defense and increases susceptibility to Citrobacter rodentium-induced colitis.

Vitamin D deficiency affects more that 1 billion people worldwide. Although thought to increase risk of bacterial infections, the importance of vitamin D on host defense against intestinal bacterial pathogens is currently unclear since injection of the active form of vitamin D, 1,25(OH)2D3, increased susceptibility to the enteric bacterial pathogen Citrobacter rodentium by suppressing key immune/inflammatory factors. To further characterize the role of vitamin D during bacteria-induced colitis, we fed weanling mice either vitamin D3-deficient or vitamin D3-sufficient diets for 5 wk and then challenged them with C. rodentium. Vitamin D3-deficient mice lost significantly more body weight, carried higher C. rodentium burdens, and developed worsened histological damage. Vitamin D3-deficient mice also suffered greater bacterial translocation to extra-intestinal tissues, including mesenteric lymph nodes, spleen, and liver. Intestinal tissues of infected vitamin D3-deficient mice displayed increased inflammatory cell infiltrates as well as significantly higher gene transcript levels of inflammatory mediators TNF-α, IL-1ß, IL-6, TGF-ß, IL-17A, and IL-17F as well as the antimicrobial peptide REG3γ. Notably, these exaggerated inflammatory responses accelerated the loss of commensal microbes and were associated with an impaired ability to detoxify bacterial lipopolysaccharide. Overall, these studies show that dietary-induced vitamin D deficiency exacerbates intestinal inflammatory responses to infection, also impairing host defense.

Predictive utility of ANCA positivity and antigen specificity in the assessment of kidney disease in paediatric-onset small vessel vasculitis.

OBJECTIVES: The objective of this study is to evaluate whether anti-neutrophil cytoplasmic antibody (ANCA) seropositivity and antigen specificity at diagnosis have predictive utility in paediatric-onset small vessel vasculitis. METHODS: Children and adolescents with small vessel vasculitis (n=406) stratified according to the absence (n=41) or presence of ANCA for myeloperoxidase (MPO) (n=129) and proteinase-3 (PR3) (n=236) were compared for overall and kidney-specific disease activity at diagnosis and outcomes between 1 and 2 years using retrospective clinical data from the ARChiVe/Paediatric Vasculitis Initiative registry to fit generalised linear models. RESULTS: Overall disease activity at diagnosis was higher in PR3-ANCA and MPO-ANCA-seropositive individuals compared with ANCA-negative vasculitis. By 1 year, there were no significant differences, based on ANCA positivity or specificity, in the likelihood of achieving inactive disease (~68%), experiencing improvement (≥87%) or acquiring damage (~58%). Similarly, and in contrast to adult-onset ANCA-associated vasculitis, there were no significant differences in the likelihood of having a relapse (~11%) between 1 and 2 years after diagnosis. Relative to PR3-ANCA, MPO-ANCA seropositivity was associated with a higher likelihood of kidney involvement (OR 2.4, 95% CI 1.3 to 4.7, p=0.008) and severe kidney dysfunction (Kidney Disease Improving Global Outcomes (KDIGO) stages 4-5; OR 6.04, 95% CI 2.77 to 13.57, p<0.001) at onset. Nonetheless, MPO-ANCA seropositive individuals were more likely to demonstrate improvement in kidney function (improved KDIGO category) within 1 year of diagnosis than PR3-ANCA seropositive individuals with similarly severe kidney disease at onset (p<0.001). CONCLUSIONS: The results of this study suggest important paediatric-specific differences in the predictive value of ANCA compared with adult patients that should be considered when making treatment decisions in this population.

SARS-CoV-2 cross-sectional seroprevalence study among public school staff in Metro Vancouver after the first Omicron wave in British Columbia, Canada.

OBJECTIVE: To determine the SARS-CoV-2 seroprevalence among school workers within the Greater Vancouver area, British Columbia, Canada, after the first Omicron wave. DESIGN: Cross-sectional study by online questionnaire, with blood serology testing. SETTING: Three main school districts (Vancouver, Richmond and Delta) in the Vancouver metropolitan area. PARTICIPANTS: Active school staff enrolled from January to April 2022, with serology testing between 27 January and 8 April 2022. Seroprevalence estimates were compared with data obtained from Canadian blood donors weighted over the same sampling period, age, sex and postal code distribution. PRIMARY AND SECONDARY OUTCOMES: SARS-CoV-2 nucleocapsid antibody testing results adjusted for test sensitivity and specificity, and regional variation across school districts using Bayesian models. RESULTS: Of 1850 school staff enrolled, 65.8% (1214/1845) reported close contact with a COVID-19 case outside the household. Of those close contacts, 51.5% (625/1214) were a student and 54.9% (666/1214) were a coworker. Cumulative incidence of COVID-19 positive testing by self-reported nucleic acid or rapid antigen testing since the beginning of the pandemic was 15.8% (291/1845). In a representative sample of 1620 school staff who completed serology testing (87.6%), the adjusted seroprevalence was 26.5% (95% CrI 23.9% to 29.3%), compared with 32.4% (95% CrI 30.6% to 34.5%) among 7164 blood donors. CONCLUSION: Despite frequent COVID-19 exposures reported, SARS-CoV-2 seroprevalence among school staff in this setting remained no greater than the community reference group. Results are consistent with the premise that many infections were acquired outside the school setting, even with Omicron.

SARS-CoV-2 seroprevalence among Vancouver public school staff in British Columbia, Canada: a cross-sectional study.

OBJECTIVES: Few studies reported COVID-19 cases in schools during the 2020/21 academic year in a setting of uninterrupted in-person schooling. The main objective was to determine the SARS-CoV-2 seroprevalence among school staff in Vancouver public schools. DESIGN: Cumulative incident COVID-19 cases among all students and school staff based on public health data, with an embedded cross-sectional serosurvey among a school staff sample that was compared to period, age, sex and geographical location-weighted data from blood donors. SETTING: Vancouver School District (British Columbia, Canada) from kindergarten to grade 12. PARTICIPANTS: Active school staff enrolled from 3 February to 23 April 2021 with serology testing from 10 February to 15 May 2021. MAIN OUTCOME MEASURES: SARS-CoV-2 seroprevalence among school staff, based on spike (S)-based (unvaccinated staff) or N-based serology testing (vaccinated staff). RESULTS: Public health data showed the cumulative incidence of COVID-19 among students attending in-person was 9.8 per 1000 students (n=47 280), and 13 per 1000 among school staff (n=7071). In a representative sample of 1689 school staff, 78.2% had classroom responsibilities, and spent a median of 17.6 hours in class per week (IQR: 5.0-25 hours). Although 21.5% (363/1686) of surveyed staff self-reported close contact with a COVID-19 case outside of their household (16.5% contacts were school-based), 5 cases likely acquired the infection at school based on viral testing. Sensitivity/Specificity-adjusted seroprevalence in 1556/1689 staff (92.1%) was 2.3% (95% CI: 1.6% to 3.2%), comparable to a sex, age, date and residency area-weighted seroprevalence of 2.6% (95% CI: 2.2% to 3.1%) among 5417 blood donors. CONCLUSION: Seroprevalence among staff was comparable to a reference group of blood donors from the same community. These data show that in-person schooling could be safely maintained during the 2020/21 school year with mitigation measures, in a large school district in Vancouver, Canada.

Ulcerative Colitis-associated E. coli pathobionts potentiate colitis in susceptible hosts.

Ulcerative colitis (UC) is a chronic inflammatory condition linked to intestinal microbial dysbiosis, including the expansion of E. coli strains related to extra-intestinal pathogenic E. coli. These "pathobionts" exhibit pathogenic properties, but their potential to promote UC is unclear due to the lack of relevant animal models. Here, we established a mouse model using a representative UC pathobiont strain (p19A), and mice lacking single immunoglobulin and toll-interleukin 1 receptor domain (SIGIRR), a deficiency increasing susceptibility to gut infections. Strain p19A was found to adhere to the cecal mucosa of Sigirr -/- mice, causing modest inflammation. Moreover, it dramatically worsened dextran sodium sulfate-induced colitis. This potentiation was attenuated using a p19A strain lacking α-hemolysin genes, or when we targeted pathobiont adherence using a p19A strain lacking the adhesin FimH, or following treatment with FimH antagonists. Thus, UC pathobionts adhere to the intestinal mucosa, and worsen the course of colitis in susceptible hosts.

Skin Exposure to Narrow Band Ultraviolet (UVB) Light Modulates the Human Intestinal Microbiome.

The recent worldwide rise in idiopathic immune and inflammatory diseases such as multiple sclerosis (MS) and inflammatory bowel diseases (IBD) has been linked to Western society-based changes in lifestyle and environment. These include decreased exposure to sunlight/UVB light and subsequent impairment in the production of vitamin D, as well as dysbiotic changes in the makeup of the gut microbiome. Despite their association, it is unclear if there are any direct links between UVB light and the gut microbiome. In this study we investigated whether exposing the skin to Narrow Band Ultraviolet B (NB-UVB) light to increase serum vitamin D levels would also modulate the makeup of the human intestinal microbiota. The effects of NB-UVB light were studied in a clinical pilot study using a healthy human female cohort (n = 21). Participants were divided into those that took vitamin D supplements throughout the winter prior to the start of the study (VDS+) and those who did not (VDS-). After three NB-UVB light exposures within the same week, the serum 25(OH)D levels of participants increased on average 7.3 nmol/L. The serum response was negatively correlated to the starting 25-hydroxy vitamin D [25(OH)D] serum concentration. Fecal microbiota composition analysis using 16S rRNA sequencing showed that exposure to NB-UVB significantly increased alpha and beta diversity in the VDS- group whereas there were no changes in the VDS+ group. Bacteria from several families were enriched in the VDS- group after the UVB exposures according to a Linear Discriminant Analysis (LDA) prediction, including Lachnospiracheae, Rikenellaceae, Desulfobacteraceae, Clostridiales vadinBB60 group, Clostridia Family XIII, Coriobacteriaceae, Marinifilaceae, and Ruminococcus. The serum 25(OH)D concentrations showed a correlation with the relative abundance of the Lachnospiraceae, specifically members of the Lachnopsira and Fusicatenibacter genera. This is the first study to show that humans with low 25(OH)D serum levels display overt changes in their intestinal microbiome in response to NB-UVB skin exposure and increases in 25(OH)D levels, suggesting the existence of a novel skin-gut axis that could be used to promote intestinal homeostasis and health. Clinical Trial Registration: clinicaltrials.gov, NCT03962673. Registered 23 May 2019 - Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT03962673?term=NCT03962673&rank=1.

Milk Fat Globule Membrane Supplementation in Formula Modulates the Neonatal Gut Microbiome and Normalizes Intestinal Development.

Breast milk has many beneficial properties and unusual characteristics including a unique fat component, termed milk fat globule membrane (MFGM). While breast milk yields important developmental benefits, there are situations where it is unavailable resulting in a need for formula feeding. Most formulas do not contain MFGM, but derive their lipids from vegetable sources, which differ greatly in size and composition. Here we tested the effects of MFGM supplementation on intestinal development and the microbiome as well as its potential to protect against Clostridium difficile induced colitis. The pup-in-a-cup model was used to deliver either control or MFGM supplemented formula to rats from 5 to 15 days of age; with mother's milk (MM) reared animals used as controls. While CTL formula yielded significant deficits in intestinal development as compared to MM littermates, addition of MFGM to formula restored intestinal growth, Paneth and goblet cell numbers, and tight junction protein patterns to that of MM pups. Moreover, the gut microbiota of MFGM and MM pups displayed greater similarities than CTL, and proved protective against C. difficile toxin induced inflammation. Our study thus demonstrates that addition of MFGM to formula promotes development of the intestinal epithelium and microbiome and protects against inflammation.

Investigation of Host and Pathogen Contributions to Infectious Colitis Using the Citrobacter rodentium Mouse Model of Infection.

Citrobacter rodentium is used as a model organism to study enteric bacterial infections in mice. Infection occurs via the oral-fecal route and results in the pathogen forming attaching and effacing lesions on infected epithelial cells. Moreover, infection leads to a subsequent host-mediated form of colitis. C. rodentium infection is thus an excellent model to study infectious colitis in vivo, while the ability to genetically manipulate C. rodentium virulence genes provides the opportunity to develop clear insights into the pathogenesis of this and related infectious microbes. This chapter outlines the basic techniques involved in setting up a C. rodentium infection in mice and several different methodologies to assess the severity of the infection.