BACKGROUND & AIMS:Clostridioides difficile is a toxin-secreting bacteria that is an urgent antimicrobial resistance threat, with approximately 25% of patients developing recurrent infections. Inflammatory bowel disease (IBD) patients are at increased risk of severe, recurrent C. difficile infection. METHODS:To investigate a role for C. difficile infection in IBD pathogenesis, we collected peripheral blood and stool from 20 each of ulcerative colitis patients, Crohn's disease patients, and healthy control subjects. We used a flow cytometric activation induced marker assay to quantify C. difficile toxin-specific CD4+ T cells and 16S ribosomal RNA sequencing to study microbiome diversity. RESULTS:We found IBD patients had significantly increased levels of C. difficile toxin B-specific CD4+ T cells, but not immunoglobulin G or immunoglobulin A, compared with healthy control subjects. Within antigen-specific CD4+ T cells, T helper type 17 cells and cells expressing the gut homing receptor integrin β7 were reduced compared with healthy control subjects, similar to our previous study of non-IBD patients with recurrent C. difficile infection. Stool microbiome analysis revealed that gut homing, toxin-specific CD4+ T cells negatively associated with microbial diversity and, along with T helper type 17 cells, positively associated with bacteria enriched in healthy control subjects. CONCLUSIONS:These data suggest that IBD patients, potentially due to underlying intestinal dysbiosis, experience undiagnosed C. difficile infections that result in impaired toxin-specific immunity. This may contribute to the development of inflammatory T cell responses toward commensal bacteria and provide a rationale for C. difficile testing in IBD patients.

The offspring of women in the poorest socio-economic groups in Western societies have an increased risk of developing non-communicable disease in adult life. Deprivation is closely related to the consumption of a diet with an excess of energy (sugar and fat), salt and a shortage of key vitamins. To test the hypothesis that this diet adversely affects the development and long-term health of the offspring, we have formulated two rodent diets, one with a nutrient profile corresponding to the diet of pregnant women in the poorest socio-economic group (DEP) and a second that incorporated current UK recommendations for the diet in pregnancy (REC). Female rats were fed the experimental diets for the duration of gestation and lactation and the offspring compared with those from a reference group fed the AIN-93G diet. The growth trajectory of DEP and REC offspring was reduced compared with the AIN-93G. The REC offspring diet had a transient increase in adipose reserves at weaning, but by 30 weeks of age the body composition of all three groups was similar. The maternal diet had no effect on the homoeostatic model assessment index or the insulin tolerance of the offspring. Changes in hepatic gene expression in the adult REC offspring were consistent with an increased hepatic utilisation of fatty acids and a reduction in de novo lipogenesis. These results show that despite changes in growth and adiposity maternal metabolic adaptation minimises the adverse consequences of the imbalanced maternal diet on the metabolism of the offspring.

Regulatory T cell (Treg) therapy is a promising strategy to treat inflammatory bowel disease (IBD). Data from animal models has shown that Tregs specific for intestinal antigens are more potent than polyclonal Tregs at inhibiting colitis. Flagellins, the major structural proteins of bacterial flagella, are immunogenic antigens frequently targeted in IBD subjects, leading to the hypothesis that flagellin-specific Tregs could be an effective cell therapy for IBD. We developed a novel chimeric antigen receptor (CAR) specific for flagellin derived from Escherichia coli H18 (FliC). We used this CAR to confer FliC-specificity to human Tregs and investigated their therapeutic potential. FliC-CAR Tregs were activated by recombinant FliC protein but not a control flagellin protein, demonstrating CAR specificity and functionality. In a humanized mouse model, expression of the FliC-CAR drove preferential migration to the colon and expression of the activation marker PD1. In the presence of recombinant FliC protein in vitro, FliC-CAR Tregs were significantly more suppressive than control Tregs and promoted the establishment of colon-derived epithelial cell monolayers. These results demonstrate the potential of FliC-CAR Tregs to treat IBD and more broadly show the therapeutic potential of CARs targeting microbial-derived antigens.

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging variants of concern 1,2 . Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern 3,4 . Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle 5,6 . Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 10 6 in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids 7 . In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern.

Acute damage to the intestinal epithelium can be repaired via de-differentiation of mature intestinal epithelial cells (IECs) to a stem-like state, but there is a lack of knowledge on how intestinal stem cells function after chronic injury, such as in inflammatory bowel disease (IBD). We developed a chronic-injury model in human colonoid monolayers by repeated rounds of air-liquid interface and submerged culture. We use this model to understand how chronic intestinal damage affects the ability of IECs to (1) respond to microbial stimulation, using the Toll-like receptor 5 (TLR5) agonist FliC and (2) regenerate and protect the epithelium from further damage. Repeated rounds of damage impair the ability of IECs to regrow and respond to TLR stimulation. We also identify mRNA expression and DNA methylation changes in genes associated with IBD and colon cancer. This methodology results in a human model of recurrent IEC injury like that which occurs in IBD.

proteolytic µ Proteolytic of relative to vehicle-treated cells (screen at 10 in fluorescence (IC 50 50 were after generating a nonlinear regression analysis from a log([Compound]) versus a proteolytic activity GraphPad

Clostridioides difficile infection (CDI) is the leading cause of gastroenteritis-associated death in North America.1 The first-line treatment is typically vancomycin, which kills vegetative forms and eliminates production of the 2 pathogenic toxins: TcdA and TcdB.2 However, 25%–35% of patients experience recurrent disease.1 Fecal microbiota transplant (FMT) is an effective second-line therapy for recurrent CDI,3 but it remains difficult to access and carries infectious risk if not done carefully; in addition, multiple treatments may be needed.

Summary The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced against emerging variants of concern (VOCs) 1,2 . Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against VOCs 3,4 . Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs), such as TMPRSS2, whose essential role in the virus lifecycle is responsible for the cleavage and priming of the viral spike protein 5–7 . Here, we identify and characterize a small-molecule compound, N-0385, as the most potent inhibitor of TMPRSS2 reported to date. N-0385 exhibited low nanomolar potency and a selectivity index of >10 6 at inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids 8 . Importantly, N-0385 acted as a broad-spectrum coronavirus inhibitor of two SARS-CoV-2 VOCs, B.1.1.7 and B.1.351. Strikingly, single daily intranasal administration of N-0385 early in infection significantly improved weight loss and clinical outcomes, and yielded 100% survival in the severe K18-human ACE2 transgenic mouse model of SARS-CoV-2 disease. This demonstrates that TTSP-mediated proteolytic maturation of spike is critical for SARS-CoV-2 infection in vivo and suggests that N-0385 provides a novel effective early treatment option against COVID-19 and emerging SARS-CoV-2 VOCs.

Intestinal epithelial cells provide a front line of defense by establishing a barrier against food Ags, pathogens, and commensal microorganisms. This defense includes the establishment of a tolerogenic environment in the gastrointestinal (GI) tract. The intestinal epithelium replenishes itself by cell turnover every 4-5 days, and this process is facilitated by various pathways of communication between the intestinal epithelial cells (IECs), the underlying stromal cell network, and professional immune cells, which together help establish a proper intestinal stem cell (ISC) niche in the crypt. However, during a state of inflammation, such as in inflammatory bowel diseases (IBD), these communication pathways can be altered, and this can lead to the development of inflammatory IECs within the crypt that further drive inflammation. Here, we review the current literature looking at crosstalk between immune cells, stromal cells, and IECs: how does the immune system potentially alter the ISC niche, and how do IECs influence intestinal immunity? We discuss the latest research using single cell RNA sequencing and intestinal organoid cultures to help answer these questions. A better understanding of this complex crosstalk can help lead to a better understanding of intestinal biology in general, and more efficient therapeutic approaches to treat IBD.

Clostridioides difficile is a Gram-positive, toxin-producing bacteria that is a major cause of hospital-acquired infection.1Clements A.C. et al.Lancet Infect Dis. 2010; 10: 395-404Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar During C difficile infection (CDI), the bacterial toxins Clostridioides difficile toxin A (TcdA) and B (TcdB) are produced, which cause intestinal epithelial injury and inflammation and symptoms including diarrhea and abdominal pain.2Aktories K. et al.Annu Rev Microbiol. 2017; 71: 281-307Crossref PubMed Scopus (85) Google Scholar First-line CDI treatment is typically vancomycin or metronidazole, antibiotics that kill vegetative forms and eliminate toxin production. However, in 25%–35% of patients, commensals fail to effectively repopulate the colon, allowing antibiotic-resistant C difficile spores to germinate and cause recurrent disease.1Clements A.C. et al.Lancet Infect Dis. 2010; 10: 395-404Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar Despite the prevalence of CDI, the immune response to this pathogen is poorly characterized. Most previous studies have focused on analysis of antitoxin antibodies, which do not correlate with protection from CDI.3Wullt M. et al.Clin Vaccine Immunol. 2012; 19: 1552-1554Crossref PubMed Scopus (22) Google Scholar We hypothesized that the CD4+ T-cell response to TcdA and TcdB after natural infection with C difficile may be a new way to characterize how pathogen-specific immunity shapes disease susceptibility and severity. We specifically aimed to identify distinguishing features of CD4+ T-cell immunity in patients with primary versus recurrent CDI compared to healthy control individuals with no clinical history of CDI. Study protocols were approved by the clinical research ethics boards of the University of British Columbia (H15-01682, H09-01238, and H18-02553) and the Vancouver Coastal Health Authority (V15-01682). All participants were ≥18 years old and provided written informed consent. Cohort demographics are shown in Supplementary Table 1, and inclusion/exclusion criteria are provided in the Supplementary Methods. Patients with new-onset disease were those experiencing their first known CDI episode; those with recurrent disease had experienced >2 recurrences. The OX40 assay was performed as described.4Cook L. et al.Cell Mol Gastroenterol Hepatol. 2020; 9: 485-506Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar The antibodies used were CD3-BV785, CD8-APCFire750, CD39-FITC, CXCR3-BV421, CCR6-APC, and CCR4-BV605 (BioLegend) and CD4-AlexaFluor700, CD25-PECy7, CD134(OX40)-PE, and Integrin β7-PECy5 (Becton, Dickinson and Company). A 4-laser LSRII FortessaX20 flow cytometer (Becton, Dickinson and Company) was used for data acquisition, using application settings. Assay cutoffs were >0.02% (mean ± 3 standard deviations of unstimulated wells) of CD4+ T cells and at least 20 cells in the gate. Analysis was performed by using FlowJo software, version 10.6. (Treestar Inc). See the Supplementary Methods for additional details. To quantify toxin-specific CD4+ T cells, we used the OX40 assay,4Cook L. et al.Cell Mol Gastroenterol Hepatol. 2020; 9: 485-506Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar which detects induced coexpression of CD25 and OX40 after 44 hours of incubation with antigen. Pediacel (Sanofi Pasteur Ltd), a childhood pentavalent vaccine, was a positive control antigen (Figure 1A). To confirm antigen-specific enrichment within the CD25+OX40+ versus CD25–OX40– T-cell population, CD4+ T cells were sorted from TcdB-stimulated assays, with only CD25+OX40+ cells proliferating in response to TcdB restimulation (Figure 1B). The majority of healthy control individuals had detectable TcdB-, but not TcdA-, specific CD4+ T cells. All patients with CDI tended to have higher frequencies of TcdB- and TcdA-specific CD4+ T cells, with this increase significant for patients with recurrent CDI. There were no differences in the frequencies of Pediacel-specific CD4+ T cells between groups (Figure 1C), indicating that the increase in TcdA- and TcdB-specific CD4+ T cells is antigen- and disease-specific. We found no correlation between toxin-specific CD4+ T cells and Age, Treatment with Systemic antibiotics, Leukocyte count, serum Albumin, Serum creatinine as a measure of renal function (ATLAS) score in the new-onset cohort (Figure 1D). However, in the recurrent CDI cohort, the frequency of TcdA-, and, to a greater extent, TcdB-specific CD4+ T cells was significantly higher in patients with moderate/severe disease compared to mild disease (Figure 1E). To identify T-cell epitopes, a peptide pool of 15mers overlapping by 12 amino acids from the C-terminal combined repetitive oligopeptides (CROPs) domain of TcdB (TcdBCROPS) was used for tetramer-guided mapping in patients with HLA-DRB1∗07:01–recurrent CDI. CD4+ T cells specific for 3 peptides (5′-3′): peptide 37, VGFVTINDKVFYFSDSGIIE (amino acids [aa] 2119–2138); peptide 38, KVFYFSDSGIIESGVQNIDD (aa2127–2146); and peptide 64, FTDEYIAATGSVIIDGEEYY (aa2335–2354) were detected in n = 2 patients (Supplementary Figure 1C) and may represent immunogenic regions of TcdB. Enzyme-linked immunosorbent assays were used to measure IgG and IgA antibodies specific for TcdA, TcdB, and TcdBCROPS, a target of neutralizing antibodies.5Chen P. et al.Nat Struct Mol Biol. 2019; 26: 712-719Crossref PubMed Scopus (12) Google Scholar We found no differences in IgG levels, but anti-TcdB IgA levels were significantly higher in both CDI patient groups compared to healthy control individuals (Supplementary Figure 1A). Antibody levels did not correlate with disease severity in the new-onset (data not shown) or recurrent groups (Supplementary Figure 1B). We next quantified the proportions of T helper (Th) Th1, Th2, Th17, and Th17.1 cells within TcdB-specific CD4+ T cells (Supplementary Figure 1D). TcdB-specific CD4+ T cells were enriched for Th17 cells, and the proportion of Th17 cells was significantly reduced in patients with recurrent, compared to new-onset, CDI (Figure 1F). This was unique to TcdB as no differences were observed for Pediacel-specific Th17 cells. To confirm the Th17 cell phenotype of TcdB-specific CD4+ T cells, CD25+OX40+CD4+ T cells were restimulated with phorbol myristate acetate/ionomycin, showing significantly higher levels of the Th17 cell–associated cytokines interleukin (IL) 10, IL17A, IL17F, and IL22 compared to CD25–OX40–CD4+ T cells (Supplementary Figure 1E). Similarly, after TcdB-stimulated in vitro expansion, TcdB-specific CD4+ T cells expressed the Th17 cell marker CD161 and secreted Th17 cell cytokines (Supplementary Figure 1F). We report here that the T-cell, but not antibody, responses to TcdB distinguished patients with CDI from healthy control individuals and positively correlated with disease severity in recurrent CDI. The finding that TcdB-specific CD4+ T cells were enriched for Th17 cells echoes what has been shown for Th cell responses to other intestinal microbes, both pathogenic and commensal.4Cook L. et al.Cell Mol Gastroenterol Hepatol. 2020; 9: 485-506Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar,6Becattini S. et al.Science. 2015; 347: 400-406Crossref PubMed Scopus (202) Google Scholar,7Gaffen S.L. Moutsopoulos N.M. Sci Immunol. 2020; 5: eaau4594Crossref PubMed Scopus (22) Google Scholar The importance of IL17 in the gut for protection against infections has been well-described,7Gaffen S.L. Moutsopoulos N.M. Sci Immunol. 2020; 5: eaau4594Crossref PubMed Scopus (22) Google Scholar although there are also pathogenic roles described for Th17 cells in CDI,8Saleh M.M. et al.Cell Host Microbe. 2019; 25: 756-765Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar highlighting a need for further work into the protective versus pathogenic effects of toxin-specific Th17 cells. Although TcdB-specific CD4+ T cells were present in most individuals, their frequency was significantly increased with repeated and more severe C difficile infection. Thus, toxin-specific CD4+ T cells rather than antibodies are potentially a useful biomarker of disease severity. In conclusion, this study provides new mechanistic insight into how immunity to C difficile develops after natural infection or colonization with C difficile and suggests that effective T-cell immunity to C difficile requires the development of Th17 cells. The authors wish to thank Drs Rebecca Gomez, I-Ting Chow, and Eddie A. James at the Benaroya Research Institute at Virginia Mason, Seattle, for their assistance with tetramer-guided epitope mapping experiments; Xiaojiao Wang, Hannah Peters, Laura Oliveira, Torey Lau, and Regan Mah at the University of British Columbia, Vancouver, Canada, for their assistance with experiments, patient recruitment, and critical revision of manuscript; and Dr. Simon Hirota at the University of Calgary for assistance with protocols for growing C difficile. Laura Cook, PhD (Conceptualization: Supporting; Formal analysis: Lead; Funding acquisition: Supporting; Investigation: Lead; Methodology: Lead; Writing – original draft: Lead); William D. Rees, MSc (Formal analysis: Supporting; Investigation: Supporting; Writing – review & editing: Supporting); May Q. Wong, BSc (Formal analysis: Supporting; Investigation: Supporting; Methodology: Supporting; Writing – review & editing: Supporting); William W. Kwok, PhD (Conceptualization: Supporting; Data curation: Supporting; Methodology: Supporting; Writing – review & editing: Supporting); Megan K. Levings, PhD (Conceptualization: Equal; Funding acquisition: Equal; Supervision: Equal; Writing – review & editing: Equal); Theodore S. Steiner, MD (Conceptualization: Equal; Funding acquisition: Lead; Resources: Equal; Supervision: Equal; Writing – review & editing: Equal). Inclusion criteria was a laboratory-confirmed CDI diagnosed by a positive tcdB PCR assay result with the presence of diarrhea (≥3 loose or watery bowel movements per day for ≥2 consecutive days), need for antibiotic treatment, and lack of spontaneous resolution. Exclusion criteria for all cohorts were HIV infection, receipt of intravenous immunoglobulin or rituximab within 6 months, hemorrhagic disorder precluding phlebotomy, or pregnancy.•New-onset CDI cohort: patients with a primary incidence of CDI receiving physician-prescribed metronidazole or vancomycin, samples collected within 5 days of diagnosis.•Recurrent CDI cohort: patients receiving assessment and/or treatment for recurrent CDI with ≥1 episode of CDI and ≥1 unprovoked recurrence of CDI (return of diarrhea within 2 months after successful treatment for CDI, without additional antibiotics).•Healthy control group: no previous diagnosis of CDI, immune compromise, or chronic intestinal diseases/conditions. A medical history was obtained from all study participants. Patients with CDI received phone follow-ups every 14 days for 45 days to assess for recurrent CDI (initial cessation of diarrhea, followed by recurrence within 45 days from completion of therapy and a positive stool test result for C difficile). For disease severity classification, individuals with new-onset CDI were given an ATLAS score using published criteria.1Hernandez-Garcia R. Garza-Gonzalez E. Miller M. et al.Application of the ATLAS score for evaluating the severity of Clostridium difficile infection in teaching hospitals in Mexico.Braz J Infect Dis. 2015; 19: 399-402Crossref PubMed Scopus (11) Google Scholar Individuals with recurrent CDI were classified as having mild disease if they never required hospitalization. If they required hospitalization or administration of intravenous fluids during a CDI episode, they were defined as having moderate disease, and if they had multiple recurrent hospitalizations, they were classified as having severe disease. Peripheral blood was collected into 9-mL sodium heparin vacutainers (Becton, Dickinson and Company [BD]) and transported at ambient temperature. OX40 assays were set up by using whole blood within 4 hours of collection, and plasma aliquots were stored at –80°C. Staphylococcal enterotoxin B (Sigma-Aldrich) was used at 1 μg/mL. Pediacel, a pentavalent vaccine containing components of pertussis vaccine, diphtheria and tetanus toxoids, inactivated poliomyelitis vaccine, and Haemophilus influenzae B conjugate vaccine (Sanofi Pasteur Ltd, Lyon, France) was used at 1/40 dilution. C difficile TcdA and TcdB toxoids (formaldehyde inactivated, List Biological Laboratories Inc) were used at 10 μg/mL and 5 μg/mL, respectively. Soluble anti-CD3 (clone OKT3, UBC Antibody Lab) was used at 0.25 μg/mL. Tetanus toxoid was used at 2 μg/mL (Enzo Life Sciences, Inc). C. difficile ATCC strain 9689 was grown on YT agar plates anaerobically for 48–72 hours at 37°C; then colonies were grown in brain heart infusion broth at 37°C in an anaerobic chamber for 24–48 hours. Genomic DNA was extracted as previously described,2Sim J.H. Anikst V. Lohith A. et al.Optimized protocol for simple extraction of high-quality genomic DNA from Clostridium difficile for whole-genome sequencing.J Clin Microbiol. 2015; 53: 2329-2331Crossref PubMed Scopus (10) Google Scholar and TcdBCROPS was amplified by using the following primers: 5′-GGTTCGAACTATTCACTAATCACTAATTGAGC-3′ and 5′-CTCGGATCCTGAAGAAAATAAGGTGTCACAAG-3′. TcdBCROPS was expressed with a 6xHis tag in Escherichia coli BL21(DE3)PLysS and purified by metal affinity chromatography as previously described.3Donnelly M.A. Steiner T.S. Two nonadjacent regions in enteroaggregative Escherichia coli flagellin are required for activation of toll-like receptor 5.J Biol Chem. 2002; 277: 40456-40461Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar Quantification of anti-TcdA, TcdB IgG, and IgA levels in plasma was performed by enzyme-linked immunosorbent assay (ELISA), as previously described,4Cook L. Lisko D.J. Wong M.Q. et al.Analysis of flagellin-specific adaptive immunity reveals links to dysbiosis in patients with inflammatory bowel disease.Cell Mol Gastroenterol Hepatol. 2020; 9: 485-506Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar using plates coated with 1 μg/mL of TcdA or TcdB toxoids from List Biological Laboratories Inc (Campbell) or 1/100 dilution of Pediacel. Levels of anti-TcdBCROPS IgG and IgA were measured by ELISA using 1 μg/mL. A standard made from pooled plasma samples from participants in our cohorts with high-titer antibodies was included with each ELISA and used to generate relative arbitrary units for cross-assay standardization. Cell supernatants were collected after 5 hours of stimulation with 10 ng/mL phorbol 12-myristate 13-acetate (PMA) and 500 ng/mL ionomycin. Concentrations of secreted cytokines were measured by using a 13-plex Th cytokine bead array kit (BioLegend) according to manufacturer's directions. Cytokine bead array data were acquired on a 3-laser Cytoflex cytometer (Beckman Coulter) and analyzed with FCAP array v3 software (BD). Approximately 30 mL of peripheral blood was centrifuged at 500g for 15 minutes, and 5 mL of the buffy coat layer was collected and mixed with 3 mL Iscove's modified Dulbecco's medium (Thermo Fisher Scientific), with 4 mL plated per well in a 6-well tissue culture plate (BD) and stimulated with 5 μg/mL TcdB for 40–48 hours. CD4+ T cells were isolated by using the CD4+ RosetteSep enrichment cocktail (STEMCELL Technologies) and TcdB-specific live CD4+CD25+OX40+ were isolated by using a 4-laser FACSAria IIu cell sorter (BD) to >95% purity. Sorted cells were expanded with autologous antigen-presenting cells and antigen (or soluble anti-CD3) in the presence of 200 U/mL IL2 (Proleukin). Antigen-presenting cells were obtained from peripheral blood mononuclear cells by negative isolation with CD3+ EasySep kit (Miltenyi Biotec), irradiated at 25 Gy, and plated at 1 × 105 cells/well in U-bottom 96-well plates; 20,000 sorted CD4+ T cells added, and cells were incubated at 37°C (5% CO2). T cells were restimulated every 14 days. Culture media was X-VIVO 15 (Lonza) supplemented with 5% heat-inactivated human serum (NorthBio Inc), 1% Glutamax (Invitrogen), and 1% penicillin-streptomycin (Invitrogen). Cell proliferation was measured by staining with 5 μmol/L Cell Proliferation Dye eF450 (Invitrogen). Epitope mapping was performed as previously described5Archila L.L. Kwok W.W. Tetramer-guided epitope mapping: A rapid approach to identify HLA-restricted T-cell epitopes from composite allergens.Methods Mol Biol. 2017; 1592: 199-209Crossref PubMed Scopus (9) Google Scholar to identify HLA-DRB–restricted peptide epitopes within the CROPS domain of C difficile TcdB (UniProt identifier: P18177, aa1831–2366) using a peptide library of 20mers overlapping by 12 amino acids (total of 66 peptides, Mimotopes). MP63, an influenza (flu) epitope, was used as a positive control tetramer.6Ge X. Tan V. Bollyky P.L. et al.Assessment of seasonal influenza A virus-specific CD4 T-cell responses to 2009 pandemic H1N1 swine-origin influenza A virus.J Virol. 2010; 84: 3312-3319Crossref PubMed Scopus (91) Google Scholar Briefly, peripheral blood mononuclear cells were stimulated with pools of 5 consecutive and overlapping peptides per well. After 14 days, cells were stained with patient-specific HLA-DRB1 tetramers loaded with a mix of peptides from each pool. Cultures with tetramer staining were further analyzed with tetramers loaded with individual peptides. Statistical analyses between 2 groups used the Mann-Whitney U test or, for paired samples, Wilcoxon signed rank test. Analysis of ≥3 groups used Kruskal-Wallis 1-way analysis of variance (ANOVA) or, for paired samples, a Friedman 1-way ANOVA with Dunn multiple comparison posttest. P values were considered significant when <.05. Prism, version 8 (GraphPad Software Inc) was used for all statistical analyses. Error bars represent median ± interquartile range; ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗P ≤ .0001; ns indicates not significant. NA, not applicable.

Intestinal epithelial cells provide a front line of defense by establishing a barrier against food Ags, pathogens, and commensal microorganisms. This defense includes the establishment of a tolerogenic environment in the gastrointestinal (GI) tract. The intestinal epithelium replenishes itself by cell turnover every 4-5 days, and this process is facilitated by various pathways of communication between the intestinal epithelial cells (IECs), the underlying stromal cell network, and professional immune cells, which together help establish a proper intestinal stem cell (ISC) niche in the crypt. However, during a state of inflammation, such as in inflammatory bowel diseases (IBD), these communication pathways can be altered, and this can lead to the development of inflammatory IECs within the crypt that further drive inflammation. Here, we review the current literature looking at crosstalk between immune cells, stromal cells, and IECs: how does the immune system potentially alter the ISC niche, and how do IECs influence intestinal immunity? We discuss the latest research using single cell RNA sequencing and intestinal organoid cultures to help answer these questions. A better understanding of this complex crosstalk can help lead to a better understanding of intestinal biology in general, and more efficient therapeutic approaches to treat IBD.

Abstract Intestinal epithelial cells provide a front line of defense by establishing a barrier against food Ags, pathogens, and commensal microorganisms. This defense includes the establishment of a tolerogenic environment in the gastrointestinal (GI) tract. The intestinal epithelium replenishes itself by cell turnover every 4–5 days, and this process is facilitated by various pathways of communication between the intestinal epithelial cells (IECs), the underlying stromal cell network, and professional immune cells, which together help establish a proper intestinal stem cell (ISC) niche in the crypt. However, during a state of inflammation, such as in inflammatory bowel diseases (IBD), these communication pathways can be altered, and this can lead to the development of inflammatory IECs within the crypt that further drive inflammation. Here, we review the current literature looking at crosstalk between immune cells, stromal cells, and IECs: how does the immune system potentially alter the ISC niche, and how do IECs influence intestinal immunity? We discuss the latest research using single cell RNA sequencing and intestinal organoid cultures to help answer these questions. A better understanding of this complex crosstalk can help lead to a better understanding of intestinal biology in general, and more efficient therapeutic approaches to treat IBD.

ABSTRACT Background & Aims Clostridioides difficile is a leading cause of infectious diarrhea and an urgent antimicrobial resistant threat. Symptoms are caused by its toxins, TcdA and TcdB, with many patients developing recurrent C. difficile infection (CDI), requiring fecal microbiota transplant (FMT). Antibody levels have not been useful in predicting patient outcomes, which is an unmet need. We aimed to characterize T cell-mediated immunity to C. difficile toxins and assess how these responses were affected by FMT. Methods We obtained blood samples from patients with newly acquired CDI, recurrent CDI (with a subset receiving FMT), inflammatory bowel disease with no history of CDI, and healthy individuals (controls). Toxin-specific CD4 + T cell responses were analysed using a whole blood flow cytometry antigen-induced marker assay. Serum antibodies were measured by ELISA. Tetramer guided mapping was used to identify HLA-II-restricted TcdB epitopes and DNA was extracted from TcdB-specific CD4 + T cells for TCR repertoire analysis by Sanger sequencing. Results CD4 + T cell responses to C. difficile toxins were functionally diverse. Compared to controls, individuals with CDI, or inflammatory bowel disease had significantly higher frequencies of TcdB-specific CD4 + T cells. Subjects with recurrent CDI had reduced proportions of TcdB-specific CD4 + Th17 cells, FMT reversed this deficit and increased toxin-specific antibody production. Conclusions These data suggest that effective T cell immunity to C. difficile requires the development of Th17 cells. In addition, they show that an unknown aspect of the therapeutic effect of FMT may be enhanced T and B cell-mediated immunity to TcdB. GRAPHICAL ABSTRACT

The intestinal epithelium is replenished every 3-4 days through an orderly process that maintains important secretory and absorptive functions while preserving a continuous mucosal barrier. Intestinal epithelial cells (IECs) derive from a stable population of intestinal stem cells (ISCs) that reside in the basal crypts. When intestinal injury reaches the crypts and damages IECs, a mechanism to replace them is needed. Recent research has highlighted the existence of distinct populations of acute and chronic damage-associated ISCs and their roles in maintaining homeostasis in several intestinal perturbation models. What remains unknown is how the damage-associated regenerative ISC population functions in the setting of chronic inflammation, as opposed to acute injury. What long-term consequences result from persistent inflammation and other cellular insults to the ISC niche? What particular "regenerative" cell types provide the most efficacious restorative properties? Which differentiated IECs maintain the ability to de-differentiate and restore the ISC niche? This review will cover the latest research on damage-associated regenerative ISCs and epigenetic factors that determine ISC fate, as well as provide opinions on future studies that need to be undertaken to understand the repercussions of the emergence of these cells, their contribution to relapses in inflammatory bowel disease, and their potential use in therapeutics for chronic intestinal diseases.

BACKGROUND:Mycobacterium avium subspecies paratuberculosis (MAP), a member of the mycobacteriaceae family, causes Johne's disease in ruminants, which resembles Crohn's disease (CD) in humans. MAP was proposed to be one of the causes of human CD, but the evidence remains elusive. Macrophages were reported to be the only cell where MAP proliferates in ruminants and humans and is likely the major producer of TNFα-associated inflammation. However, whether human dendritic cells (DCs), another major antigen-presenting cell (APC), have the ability to harbor MAP and disseminate infection, remains unknown.METHODS:Human monocyte-derived dendritic cells (moDCs) were infected with MAP and phagocytosis and intracellular survival were quantified by immunofluorescence (IF) and colony counts, respectively. MoDC cytokine expression was measured via ELISA and their activation state was measured via flow cytometry.RESULTS:We showed that MAP can infect and replicate in human moDCs as means to evade the immune system for successful infection, through inhibition of the phago-lysosome fusion via the secretion of protein tyrosine phosphatase PtpA. This mechanism initially led to a state of tolerance in moDCs and then subsequently caused a pro-inflammatory response as infection persisted, characterized by the upregulation of IL-6 and TNFα, and downregulation of IL-10. Moreover, we showed that moDCs have the ability to phagocytose up to 18% of MAP, when exposed at a multiplicity of infection of 1:1.CONCLUSION:Infection and subsequent proliferation of MAP within moDCs could provide a unique means for the dissemination of MAP to lymphoid tissue, while altering immune responses to facilitate the persistence of infection of host tissues in CD.

Background: The increased incidence of diseases, including metabolic syndrome and infertility, may be related to exposure to the mixture of chemicals, which are ubiquitous in the modern environment (environmental chemicals, ECs). Xeno-detoxification occurs within the liver which is also the source of many plasma proteins and growth factors and plays an important role in the regulation of homeostasis. Objectives: The objective of this study was to investigate the effects of ECs on aspects of liver function, in a well characterized ovine model of exposure to a real-life EC mixture. Methods: Four groups of sheep (n = 10-12/sex/treatment) were maintained long-term on control or sewage sludge-fertilized pastures: from conception to culling at 19 months of age in females and from conception to 7 months of age and thereafter in control plots until culling at 19 months of age in males. Environmental chemicals were measured in sheep livers and RNA and protein extracts were assessed for exposure markers. Liver proteins were resolved using 2D differential in-gel electrophoresis and differentially expressed protein spots were identified by liquid chromatography/tandem mass spectroscopy. Results: Higher levels of polycyclic aromatic hydrocarbons (PAHs) and lower levels of polychlorinated biphenyls (PCBs) in the livers of control males compared to control females indicated sexually dimorphic EC body burdens. Increased levels of the PAHs Benzo[a] anthracene and chrysene and reduced levels of PCB 153 and PCB 180 were observed in the livers of continuously exposed females. EC exposure affected xenobiotic and detoxification responses and the liver proteome in both sexes and included major plasma-secreted and blood proteins, and metabolic enzymes whose pathway analysis predicted dysregulation of cancer-related pathways and altered lipid dynamics. The latter were confirmed by a reduction in total lipids in female livers and up-regulation of cancerrelated transcript markers in male livers respectively by sewage sludge exposure. Conclusions: Our results demonstrate that chronic exposure to ECs causes major physiological changes in the liver, likely to affect multiple systems in the body and which may predispose individuals to increased disease risks.

Iron deficiency is common in pregnant and lactating women and is associated with reduced cognitive development of the offspring. Since iron affects lipid metabolism, the availability of fatty acids, particularly the polyunsaturated fatty acids required for early neural development, was investigated in the offspring of female rats fed iron-deficient diets during gestation and lactation. Subsequent to the dams giving birth, one group of iron-deficient dams was recuperated by feeding an iron-replete diet. Dams and neonates were killed on postnatal days 1, 3 and 10, and the fatty acid composition of brain and stomach contents was assessed by gas chromatography. Changes in the fatty acid profile on day 3 became more pronounced on day 10 with a decrease in the proportion of saturated fatty acids and a compensatory increase in monounsaturated fatty acids. Long-chain polyunsaturated fatty acids in the n-6 family were reduced, but there was no change in the n-3 family. The fatty acid profiles of neonatal brain and stomach contents were similar, suggesting that the change in milk composition may be related to the changes in the neonatal brain. When the dams were fed an iron-sufficient diet at birth, the effects of iron deficiency on the fatty acid composition of lipids in both dam's milk and neonates' brains were reduced. This study showed an interaction between maternal iron status and fatty acid composition of the offspring's brain and suggests that these effects can be reduced by iron repletion of the dam's diet at birth.

Background and Aims: Endoplasmic reticulum [ER] stress in intestinal epithelial cells [IECs] contributes to the pathogenesis of inflammatory bowel disease [IBD]. We hypothesized that ER stress changes innate signalling in human IECs, augmenting toll-like receptor [TLR] responses and inducing pro-inflammatory changes in underlying dendritic cells [DCs]. Methods: Caco-2 cells and primary human colon-derived enteroid monolayers were exposed to ATP [control stressor] or thapsigargin [Tg] [ER stress inducer], and were stimulated with the TLR5 agonist flagellin. Cytokine release was measured by an enzyme immunoassay. ER stress markers CHOP, GRP78 and XBP1s/u were measured via quantitative PCR and Western blot. Monocyte-derived DCs [moDCs] were cultured with the IEC supernatants and their activation state was measured. Responses from enteroids derived from IBD patients and healthy control participants were compared. Results: ER stress enhanced flagellin-induced IL-8 release from Caco-2 cells and enteroids. Moreover, conditioned media activated DCs to become pro-inflammatory, with increased expression of CD80, CD86, MHCII, IL-6, IL-15 and IL-12p70 and decreased expression of CD103 and IL-10. Flagellin-induced IL-8 production correlated with DC activation, suggesting a common stress pathway. Moreover, there were distinct differences in cytokine expression and basal ER stress between IBD and healthy subject-derived enteroid monolayers, suggesting a dysregulated ER stress pathway in IBD-derived enteroids. Conclusions: Cellular stress enhances TLR5 responses in IECs, leading to increased DC activation, indicating a previously unknown mechanistic link between epithelial ER stress and immune activation in IBD. Furthermore, dysregulated ER stress may be propagated from the intestinal epithelial stem cell niche in IBD patients.

Nutritional science has traditionally used the reductionist approach to understand the roles of individual nutrients in growth and development. The macronutrient dense but micronutrient poor diets consumed by many in the Western world may not result in an overt deficiency; however, there may be situations where multiple mild deficiencies combine with excess energy to alter cellular metabolism. These interactions are especially important in pregnancy as changes in early development modify the risk of developing non-communicable diseases later in life. Nutrient interactions affect all stages of fetal development, influencing endocrine programming, organ development and the epigenetic programming of gene expression. The rapidly developing field of stem cell metabolism reveals new links between cellular metabolism and differentiation. This review will consider the interactions between nutrients in the maternal diet and their influence on fetal development, with particular reference to energy metabolism, amino acids and the vitamins in the B group.