A Potential Probiotic for Diarrhea: Clostridium tyrobutyricum Protects Against LPS-Induced Epithelial Dysfunction via IL-22 Produced By Th17 Cells in the Ileum.

Probiotics are clinically used for diarrhea and inflammatory bowel diseases in both humans and animals. Previous studies have shown that Clostridium tyrobutyricum (Ct) protects against intestinal dysfunction, while its regulatory function in the gut needs further investigation and the related mechanisms are still not fully elucidated. This study aims to further verify the protective function of Ct and reveal its underlying mechanisms in alleviating diarrhea and intestinal inflammation. Ct inhibited LPS-induced diarrhea and intestinal inflammation in the ileum. IL-22 was identified and the protective role of Ct in the ileum presented an IL-22-dependent manner according to the transcriptomic analysis and in vivo interference mice experiments. The flow cytometric analysis of immune cells in the ileum showed that Ct enhanced the proportions of Th17 cells in response to LPS. The results of in situ hybridization further verified that Ct triggered Th17 cells to produce IL-22, which combined with IL-22RA1 expressed in the epithelial cells. Moreover, Ct was unable to enhance the levels of short-chain fatty acids (SCFAs) in the ileum, suggesting that the protective role of Ct in the ileum was independent of SCFAs. This study uncovered the role of Ct in alleviating diarrhea and inflammation with the mechanism of stimulating Th17 cells in the lamina propria to produce IL-22, highlighting its potential application as a probiotic for diarrhea and inflammation in the ileum.

Psychological stress impairs IL22-driven protective gut mucosal immunity against colonising pathobionts.

Crohn's disease is an inflammatory disease of the gastrointestinal tract characterized by an aberrant response to microbial and environmental triggers. This includes an altered microbiome dominated by Enterobacteriaceae and in particular adherent-invasive E. coli (AIEC). Clinical evidence implicates periods of psychological stress in Crohn's disease exacerbation, and disturbances in the gut microbiome might contribute to the pathogenic mechanism. Here we show that stress-exposed mice develop ileal dysbiosis, dominated by the expansion of Enterobacteriaceae. In an AIEC colonisation model, stress-induced glucocorticoids promote apoptosis of CD45+CD90+ cells that normally produce IL-22, a cytokine that is essential for the maintenance of ileal mucosal barrier integrity. Blockade of glucocorticoid signaling or administration of recombinant IL-22 restores mucosal immunity, prevents ileal dysbiosis, and blocks AIEC expansion. We conclude that psychological stress impairs IL-22-driven protective immunity in the gut, which creates a favorable niche for the expansion of pathobionts that have been implicated in Crohn's disease. Importantly, this work also shows that immunomodulation can counteract the negative effects of psychological stress on gut immunity and hence disease-associated dysbiosis.

Ileitis-associated tertiary lymphoid organs arise at lymphatic valves and impede mesenteric lymph flow in response to tumor necrosis factor.

Lymphangitis and the formation of tertiary lymphoid organs (TLOs) in the mesentery are features of Crohn's disease. Here, we examined the genesis of these TLOs and their impact on disease progression. Whole-mount and intravital imaging of the ileum and ileum-draining collecting lymphatic vessels (CLVs) draining to mesenteric lymph nodes from TNFΔARE mice, a model of ileitis, revealed TLO formation at valves of CLVs. TLOs obstructed cellular and molecular outflow from the gut and were sites of lymph leakage and backflow. Tumor necrosis factor (TNF) neutralization begun at early stages of TLO formation restored lymph transport. However, robustly developed, chronic TLOs resisted regression and restoration of flow after TNF neutralization. TNF stimulation of cultured lymphatic endothelial cells reprogrammed responses to oscillatory shear stress, preventing the induction of valve-associated genes. Disrupted transport of immune cells, driven by loss of valve integrity and TLO formation, may contribute to the pathology of Crohn's disease.

Epithelial-myeloid exchange of MHC class II constrains immunity and microbiota composition.

Intestinal epithelial cells (IECs) have long been understood to express high levels of major histocompatibility complex class II (MHC class II) molecules but are not considered canonical antigen-presenting cells, and the impact of IEC-MHC class II signaling on gut homeostasis remains enigmatic. As IECs serve as the primary barrier between underlying host immune cells, we reasoned that IEC-intrinsic antigen presentation may play a role in responses toward the microbiota. Mice with an IEC-intrinsic deletion of MHC class II (IECΔMHC class II) are healthy but have fewer microbial-bound IgA, regulatory T cells (Tregs), and immune repertoire selection. This was associated with increased interindividual microbiota variation and altered proportions of two taxa in the ileum where MHC class II on IECs is highest. Intestinal mononuclear phagocytes (MNPs) have similar MHC class II transcription but less surface MHC class II and are capable of acquiring MHC class II from IECs. Thus, epithelial-myeloid interactions mediate development of adaptive responses to microbial antigens within the gastrointestinal tract.

Heat stress inhibits expression of the cytokines, and NF-κB-NLRP3 signaling pathway in broiler chickens infected with salmonella typhimurium.

High ambient temperature has potential influence on oxidative stress, or systemic inflammation affecting poultry production and immune status of chickens. Heat stress (HS) induces intestinal inflammation and increases susceptibility of harmful pathogens, such as Salmonella and Escherichia coli. Intestinal inflammation is a common result of body immune dysfunction. Therefore, we designed an experiment to analyze the effects of 35 ± 2 °C HS on salmonella infection in chickens through regulation of the immune responses. 40 broiler chickens were randomly divided into 4 groups: control group, heat stress (HS) group, salmonella typhimurium (ST) group and model group (heat stress + salmonella typhimurium, HS + ST). Birds in HS and model group were treated with 35 ± 2 °C heat stress 6 h a day and for 14 continuous days. Then, ST and model group birds were orally administrated with 1 mL ST inoculum (109 cfu/mL). Chickens were sacrificed at the 4th day after ST administration and ileum tissues were measured. We observed that heat stress decreased ileum TNF-α and IL-1ß protein expressions. Concomitantly heat stress decreased NLRP3 and Caspase-1 protein levels. The protein expressions of p-NF-κB-p65 and p-IκB-α in ileum. Heat stress also inhibited IFN-α, p-IRF3 and p-TBK1, showing a deficiency in the HS + ST group birds. Together, the present data suggested that heat stress suppressed intestinal immune activity in chickens infected by salmonella typhimurium, as observed by the decrease of immune cytokines levels, which regulated by NF-κB-NLRP3 signaling pathway.

Effects of a Low Dose of T-2 Toxin on the Percentage of T and B Lymphocytes and Cytokine Secretion in the Porcine Ileal Wall.

Plant materials used in the production of pig feed are frequently contaminated with mycotoxins. T-2 toxin is a secondary metabolite of selected Fusarium species, and it can exert a harmful influence on living organisms. Most mycotoxins enter the body via the gastrointestinal tract, and they can modulate the gut-associated lymphoid tissue (GALT) function. However, little is known about the influence of low T-2 toxin doses on GALT. Therefore, the aim of this study was to evaluate the effect of T-2 toxin administered at 50% of the lowest-observed-adverse-effect level (LOAEL) on the percentage of CD2+ T cells, CD4+ T helper cells, CD8+ cytotoxic T cells, CD4+CD8+ double-positive T cells, TCRγδ+ cells, CD5+CD8- B1 cells, and CD21+ B2 cells, and the secretion of proinflammatory (IFN-γ, IL-1ß, IL-2, IL-12/23p40, IL-17A), anti-inflammatory, and regulatory (IL-4, IL-10, TGF-ß) cytokines in the porcine ileal wall. The results of the study revealed that T-2 toxin disrupts the development of tolerance to food antigens by enhancing the secretion of proinflammatory and regulatory cytokines and decreasing the production of anti-inflammatory TGF-ß. T-2 toxin triggered the cellular response, which was manifested by an increase in the percentage of CD8+ T cells and a decrease in the percentage of B2 and Tγδ lymphocytes.

Fecal Dipeptidyl Peptidase-4: An Emergent Biomarker in Inflammatory Bowel Disease.

INTRODUCTION: Dipeptidyl peptidase-4 (DPP-4) is a membrane-bound glycoprotein that acts as a receptor but also exists in a soluble form. It has been recognized as a mediator of inflammation and considered a biomarker in inflammatory bowel disease (IBD). METHODS: We evaluated a prospectively recruited cohort, consisting of 101 patients with IBD, using validated clinical indexes; 22 patients with ulcerative colitis (UC) underwent endoscopic evaluation. Fecal DPP-4 (fDPP-4) levels were analyzed and correlated with clinical scores, Mayo endoscopic score (in UC patients), serum DPP-4, C-reactive protein, and fecal calprotectin. Immunohistochemical staining for DPP-4 in intestinal biopsies was also performed. RESULTS: When compared with remitters, median fDPP-4 levels were higher in patients with ileal Crohn's disease (CD) (7,584 [1,464-7,816] vs 2,104 [630-2,676] ng/mL, P = 0.015) and lower in patients with UC exhibiting clinical activity (1,213 [559-1,682] vs 7,814 [2,555-7,985] ng/mL, P < 0.001). Patients with UC presenting endoscopic activity also had lower levels than remitters (939 [559-1,420] vs 7,544 [4,531-7,940] ng/mL, P = 0.006). Fecal DPP-4 discriminated clinical activity from remission with areas under the curve of 0.76 (95% confidence interval [CI] 0.58-0.94, P = 0.015) and 0.80 (95% CI 0.68-0.93, P < 0.001) in CD and UC, respectively; it allowed to differentiate endoscopic activity in patients with UC, with areas under the curve of 0.84 (95% CI 0.63-1.00, P = 0.009). Immunohistochemical analysis revealed higher DPP-4 apical expression in UC remitters, but no statistically significant differences were revealed between patients with ileal CD. DISCUSSION: Our results suggest that fDPP-4 can be used as a biomarker of IBD activity, particularly in UC. The expression profiles in intestinal tissue might represent a functional compartmentalization of DPP-4 expression.

C-reactive protein/abumin ratio is a useful biomarker for predicting the mucosal healing in the Crohn disease: A retrospective study.

ABSTRACT: Ileocolonoscopy is currently recognized as the gold standard for evaluating mucosal healing in patients with Crohn disease (CD). However, the ideal noninvasive marker to assess mucosal healing instead of invasive ileocolonoscopy is not available. This study aimed to determine the correlations between the mucosal healing and serological optimizing markers in CD.This retrospective study consecutively included 62 CD patients with 137 hospitalizations between March 2014 and March 2020. On the basis of the Simple Endoscopic Score for Crohn's disease (SES-CD), the CD patients were divided into mucosal healing group (SES-CD ≤ 2) and nonmucosal healing group (SES-CD > 2). We collected the results of ileocolonoscopy examination and inflammatory markers and then serological optimizing markers, including C-reactive protein/albumin ratio (CRP/ALB), platelet/albumin ratio (PLT/ALB), neutrophil-lymphocyte ratio (NLR), and platelet-lymphocyte ratio (PLR) were calculated. The control group consisted of 50 healthy volunteers in the corresponding period.We found that CRP/ALB, PLT/ALB, NLR, and PLR were correlated with the mucosal healing of CD, and the correlation of CRP/ALB with the mucosal healing was the highest (r = -0.64). Receiver operating characteristic (ROC) analysis showed that the area under the curve (AUC) of CRP/ALB (0.87) was higher than NLR (0.69), PLR (0.72), and PLT/ALB (0.81). In the efficacy of assessing the mucosal healing in CD, the sensitivity of CRP/ALB, NLR, PLR, and PLT/ALB were 91.1%, 83.9%, 73.2%, and 73.2%, respectively, and the specificity was 76.5%, 46.9%, 64.2%, and 75.3%, respectively.CRP/ALB was the most appropriate marker to assess CD mucosal healing among the serological optimizing markers.

Molecular mechanism of Escherichia coli H10407 induced diarrhoea and its control through immunomodulatory action of bioactives from Simarouba amara (Aubl.).

Enterotoxigenic Escherichia coli (ETEC) infection is a major cause of death in children under the age of five in developing countries. ETEC (O78:H11:CFA/I:LT+:ST+) mechanism has been studied in detail with either heat labile (LT) or heat stable (ST) toxins using in vitro and in vivo models. However, there is no adequate information on ETEC pathogenesis producing both the toxins (LT, ST) in BALB/c mice model. In this study, female mice have been employed to understand ETEC H10407 infection induced changes in physiology, biochemical and immunological patterns up to seven days post-infection and the antidiarrhoeal effect of Simarouba amara (Aubl.) bark aqueous extract (SAAE) has also been looked into. The results indicate that BALB/c is sensitive to ETEC infection resulting in altered jejunum and ileum histomorphology. Withal, ETEC influenced cAMP, PGE2, and NO production resulting in fluid accumulation with varied Na+, K+, Cl-, and Ca2+ levels. Meanwhile, ETEC subverted expression of IL-1ß, intestine alkaline phosphatase (IAP), and myeloperoxidase (MPO) in jejunum and ileum. Our data also indicate the severity of pathogenesis reduction which might be due to attainment of equilibrium after reaching optimum rate of infection. Nevertheless, degree of pathogenesis was highly significant (p < 0.01) in all the studied parameters. Besides that, SAAE was successful in reducing the infectious diarrhoea by inhibiting ETEC H10407 in intestine (jejunum and ileum), and shedding in feces. SAAE decreased cAMP, PGE2, and fluid accumulation effectively and boosted the functional activity of immune system in jejunum and ileum IAP, MPO, IL-1ß, and nitric oxide.

Inflammasome Activation in Ankylosing Spondylitis Is Associated With Gut Dysbiosis.

OBJECTIVE: We undertook this study to evaluate the activation and functional relevance of inflammasome pathways in ankylosing spondylitis (AS) patients and rodent models and their relationship to dysbiosis. METHODS: An inflammasome pathway was evaluated in the gut and peripheral blood from 40 AS patients using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), flow cytometry, and confocal microscopy, and was compared to that of 20 healthy controls and 10 patients with Crohn's disease. Bacteria was visualized using silver stain in human samples, and antibiotics were administered to HLA-B27-transgenic rats. The NLRP3 inhibitor MCC950 was administered to SKG mice, and ileal and joint tissues were assessed by IHC analysis and real-time qRT-PCR. The role of inflammasome in modulating the interleukin-23 (IL-23)/IL-17 axis was studied ex vivo. RESULTS: Expression levels of Nlrp3, Nlrc4, and Aim2 were increased in the gut of HLA-B27-transgenic rats and reduced by antibiotic treatment (P < 0.05). In curdlan-treated SKG mice, NLRP3 blockade prevented ileitis and delayed arthritis onset (P < 0.05). Compared to healthy controls, AS patients demonstrated overexpression of NLRP3 (fold induction 2.33 versus 22.2; P < 0.001), NLRC4 (fold induction 1.90 versus 6.47; P < 0.001), AIM2 (fold induction 2.40 versus 20.8; P < 0.001), CASP1 (fold induction 2.53 versus 24.8; P < 0.001), IL1B (fold induction 1.07 versus 10.93; P < 0.001), and IL18 (fold induction 2.56 versus 15.67; P < 0.001) in the ileum, and caspase 1 activity was increased (P < 0.01). The score of adherent and invasive mucosa-associated bacteria was higher in AS (P < 0.01) and correlated with the expression of inflammasome components in peripheral blood mononuclear cells (P < 0.001). NLRP3 expression was associated with disease activity (the Ankylosing Spondylitis Disease Activity Score using the C-reactive protein level) (r2 = 0.28, P < 0.01) and with IL23A expression (r2 = 0.34, P < 0.001). In vitro, inflammasome activation in AS monocytes was paralleled by increased serum levels of IL-1ß and IL-18. Induction of IL23A, IL17A, and IL22 was IL-1ß-dependent. CONCLUSION: Inflammasome activation occurs in rodent models of AS and in AS patients, is associated with dysbiosis, and is involved in triggering ileitis in SKG mice. Inflammasomes drive type III cytokine production with an IL-1ß-dependent mechanism in AS patients.

Lost in Translation: Lack of CD4 Expression due to a Novel Genetic Defect.

CD4 expression identifies a subset of mature T cells primarily assisting the germinal center reaction and contributing to CD8+ T-cell and B-cell activation, functions, and longevity. Herein, we present a family in which a novel variant disrupting the translation-initiation codon of the CD4 gene resulted in complete loss of membrane and plasma soluble CD4 in peripheral blood, lymph node, bone marrow, skin, and ileum of a homozygous proband. This inherited CD4 knockout disease illustrates the clinical and immunological features of a complete deficiency of any functional component of CD4 and its similarities and differences with other clinical models of primary or acquired loss of CD4+ T cells. The first inherited loss of any functional component of CD4, including soluble CD4, is clinically distinct from any other congenital or acquired CD4 T-cell defect and characterized by compensatory changes in T-cell subsets and functional impairment of B cells, monocytes, and natural killer cells.

Giardia Is Often Overlooked on Histopathologic Examination: A High-Volume, Single-Institution Experience.

AIMS. GIARDIA: is sometimes missed by the pathologist, and we sought to determine how often this occurs at our institution-a large tertiary care center with a subspecialty gastrointestinal pathology service and what certain clinical and histologic clues can be used to flag cases with a higher likelihood of infection, targeting them for greater scrutiny. METHODS AND RESULTS: We identified a set of patients who tested positive for Giardia with a stool-based test, and who also received a small bowel biopsy at a similar time-point. These biopsies were retrospectively reviewed for Giardia, finding 8 positive cases. The organism was prospectively detected in 4 cases (50%) but overlooked in the remaining 4 cases (50%). Three of the 4 cases missed cases showed only rare organisms. The detected cases tended to more frequently have prominent lymphoid aggregates (3 detected cases, 0 overlooked cases) and intraepithelial lymphocytosis (3 detected cases, 0 overlooked cases). Certain clinical and histologic clues can be used to flag cases with a higher likelihood of infection. Specifically, we found abnormalities of the mucosa (active inflammation, intraepithelial lymphocytosis, villous expansion, prominent lymphoid aggregates) in each case, and 4 of 8 cases were from immunocompromised patients. Finally, 2 of 8 cases were terminal ileum biopsies. CONCLUSIONS: Biopsies with a histologic abnormality or those from immunocompromised patients should receive greater attention. Routinely looking for Giardia at that terminal ileum is necessary.

Uric acid drives intestinal barrier dysfunction through TSPO-mediated NLRP3 inflammasome activation.

BACKGROUND AND AIM: Intestinal epithelial dysfunction is the foundation of various intestinal and extra-intestinal diseases, while the effects and mechanism of uric acid on the intestinal barrier are little known. TSPO has been shown to be related to the generation of ROS and is involved in regulating inflammation, whether uric acid drives intestinal epithelial dysfunction through TSPO-mediated NLRP3 inflammasome activation is unknown. METHODS: UOX gene knockout mouse (UOX-/-) were used for models of hyperuricemia. Fluorescein isothiocyanate (FITC)-labeled dextran was used to assess in vivo intestinal permeability. Serum lipopolysaccharide (LPS) and culture supernatants IL-1ß were measured using ELISA Kit. IEC-6 exposed to different concentrations of uric acid was used for in vitro experiment. Protein content and mRNA were assessed using Western blotting and Q-PCR, respectively. Intracellular ROS was determined using flow cytometry and fluorescence microscope. Mitochondrial membrane potential was detected on an immunofluorescence. Small interfering RNA transfection was used to assess the interaction between translocator protein (TSPO) and NLRP3 inflammasome. N-acetyl-L-cysteine (NAC) was used as ROS scavenger. RESULTS: Our results showed that hyperuricemia mice were characteristic by increased intestinal permeability. Hyperuricemia upregulated TSPO, increased production of ROS and activated NLRP3 inflammasome, which resulted in lower expression of occludin and claudin-1. In vitro, we showed that soluble uric acid alone increased the expression of TSPO, depolarized mitochondrial membrane potential, increased ROS release and activated NLRP3 inflammasome, which further reduced the expression of occludin and claudin-1. Silencing TSPO suppressed NLRP3 inflammasome activation and increased expression of claudin-1 and occludin, which was accompanied by lower levels of ROS. Scavenging ROS also significantly inhibited NLRP3 inflammasome activation without change of TSPO, indicating that TSPO-mediated NLRP3 inflammasome activation was dependent on ROS. CONCLUSIONS: In conclusion, uric acid drives intestinal barrier dysfunction through TSPO-mediated NLRP3 inflammasome.

Faecal Biomarkers in Inflammatory Bowel Diseases: Calprotectin Versus Lipocalin-2-a Comparative Study.

BACKGROUND AND AIMS: Faecal biomarkers, particularly calprotectin [FCAL], have become important diagnostic and monitoring tools in inflammatory bowel diseases [IBD]. As FCAL is mainly produced by neutrophils, we hypothesised that faecal lipocalin-2 [FLCN2], also expressed by intestinal epithelial cells [IEC], could be beneficial in specific clinical situations. METHODS: We compared clinical and endoscopic activity-related correlations between FCAL and FLCN2, assayed from the same sample, in a cohort of 132 patients (72 Crohn's disease [CD]) and 40 controls. A detailed analysis of cellular origins was done by confocal microscopy and flow cytometry. To evaluate the potential to detect low-grade inflammation, we studied faecal and tissue concentrations in a cohort with clinical, endoscopic, and histological remission. RESULTS: There was an excellent correlation between FCAL and FLCN2 [rS = 0.87, p <0.001] and comparable sensitivity and specificity to predict clinical and endoscopic disease activity, with optimal thresholds for endoscopic activity of 73.4 and 1.98 µg/g in ulcerative colitis [UC] and 78.4 and 0.56 µg/g in Crohn's disease for FCAL and FLCN2, respectively. Strong co-expression of both proteins was observed in granulocytes and macrophages. IECs expressed LCN2 but not CAL. In our IBD cohort in deep remission neither FCAL nor FLCN2 was different from controls; yet mucosal LCN2 but not CAL expressions remained elevated in the rectum of UC and the ileum of CD patients. CONCLUSIONS: This study corroborates the diagnostic equivalence of FLCN2 and FCAL in IBD. In remission, persistent mucosal overexpression renders LCN2 an attractive candidate for molecular inflammation warranting further investigation.

Intestinal Receptor of SARS-CoV-2 in Inflamed IBD Tissue Seems Downregulated by HNF4A in Ileum and Upregulated by Interferon Regulating Factors in Colon.

BACKGROUND: Patients with inflammatory bowel disease [IBD] are considered immunosuppressed, but do not seem more vulnerable for COVID-19. Nevertheless, intestinal inflammation has shown to be an important risk factor for SARS-CoV-2 infection and prognosis. Therefore, we investigated the role of intestinal inflammation on the viral intestinal entry mechanisms, including ACE2, in IBD. METHODS: We collected inflamed and uninflamed mucosal biopsies from Crohn's disease [CD] [n = 193] and ulcerative colitis [UC] [n = 158] patients, and from 51 matched non-IBD controls for RNA sequencing, differential gene expression, and co-expression analysis. Organoids from UC patients were subjected to an inflammatory mix and processed for RNA sequencing. Transmural ileal biopsies were processed for single-cell [sc] sequencing. Publicly available colonic sc-RNA sequencing data, and microarrays from tissue pre/post anti-tumour necrosis factor [TNF] therapy, were analysed. RESULTS: In inflamed CD ileum, ACE2 was significantly decreased compared with control ileum [p = 4.6E-07], whereas colonic ACE2 was higher in inflamed colon of CD/UC compared with control [p = 8.3E-03; p = 1.9E-03]. Sc-RNA sequencing confirmed this ACE2 dysregulation and exclusive epithelial ACE2 expression. Network analyses highlighted HNF4A as key regulator of ileal ACE2, and pro-inflammatory cytokines and interferon regulating factors regulated colonic ACE2. Inflammatory stimuli upregulated ACE2 in UC organoids [p = 1.7E-02], but not in non-IBD controls [p = 9.1E-01]. Anti-TNF therapy restored colonic ACE2 regulation in responders. CONCLUSIONS: Intestinal inflammation alters SARS-CoV-2 coreceptors in the intestine, with opposing dysregulations in ileum and colon. HNF4A, an IBD susceptibility gene, seems an important upstream regulator of ACE2 in ileum, whereas interferon signalling might dominate in colon.

Effect of Crohn's Disease on Villous Length and CYP3A4 Expression in the Pediatric Small Intestine.

Changes in absorptive capacity and first-pass metabolism in the small intestine affect oral drug bioavailability. Characterization of such changes as a consequence of inflammation is important for developing physiologically-based pharmacokinetic (PBPK) models for inflammatory bowel disease. We sought to elucidate the impact of small intestinal Crohn's disease (CD) on villous length and CYP3A4 expression in children. Freshly frozen duodenal and terminal ileum (TI) biopsies from 107 children (1-19 years) with and without CD were evaluated for active inflammation. Villous length and CYP3A4 mRNA/protein expression were compared among regions of active and inactive inflammation in CD and controls. A twofold reduction in villous length was observed in inflamed duodena and ilia of children with CD, but in the absence of regional inflammation, villi in CD were comparable in length to controls. Expression of CYP3A4 mRNA correlated significantly with villous length in the TI (P = 0.0003), with a trend observed in the duodenum that did not reach statistical significance. In the presence of active inflammation, a significant decrease in CYP3A protein expression was confirmed in the duodenum, where protein expression also correlated significantly with villous length across diagnoses (P < 0.0001). Our findings suggest that previous observations of decreased CYP3A4 expression and function in inflamed intestine may not be due solely to downregulation by inflammatory cytokines, but also to villous blunting and subsequent loss of surface area for protein expression. This information is relevant for PBPK model development and could aid with dose adjustment decisions for oral CYP3A4 substrates administered during CD flare (e.g., budesonide).

Paneth Cell Alertness to Pathogens Maintained by Vitamin D Receptors.

BACKGROUND AND AIMS: Vitamin D exerts a regulatory role over mucosal immunity via the vitamin D receptor (VDR). Although Paneth cells and their products are known to regulate the commensal and pathogenic microbiota, the role that VDRs in Paneth cells play in these responses is unknown. METHODS: We identified the decreased intestinal VDR significantly correlated with reduction of an inflammatory bowel disease risk gene ATG16L1 and Paneth cell lysozymes in patients with Crohn's disease. We generated Paneth cell-specific VDR knockout (VDRΔPC) mice to investigate the molecular mechanisms. RESULTS: Lysozymes in the Paneth cells were significantly decreased in the VDRΔPC mice. Isolated VDRΔPC Paneth cells exhibited weakened inhibition of pathogenic bacterial growth and displayed reduced autophagic responses. VDRΔPC mice had significantly higher inflammation after Salmonella infections. VDRΔPC mice also showed high susceptibility to small intestinal injury induced by indomethacin, a nonsteroidal anti-inflammatory drug. Co-housing of VDRΔPC and VDRlox mice made the VDRΔPC less vulnerable to dextran sulfate sodium colitis, suggesting the transmission of protective bacterial from the VDRlox mice. Thus, a lack of VDR in Paneth cells leads to impaired antibacterial activities and consequently increased inflammatory responses. Genetically and environmentally regulated VDRs in the Paneth cells may set the threshold for the development of chronic inflammation, as observed in inflammatory bowel diseases. CONCLUSIONS: We provide new insights into the tissue-specific functions of VDRs in maintaining Paneth cell alertness to pathogens in intestinal disorders. Targeting the VDR affects multiple downstream events within Paneth cells that inhibit intestinal inflammation and establish host defense against enteropathogens.

Macrophage migration inhibitory factor (MIF) and pregnancy may impact the balance of intestinal cytokines and the development of intestinal pathology caused by Toxoplasma gondii infection.

Toxoplasma gondii (T. gondii) is an intracellular parasite responsible for causing toxoplasmosis. When infection occurs during pregnancy, it can produce severe congenital infection with ocular and neurologic damage to the infant. From the oral infection parasite reaches the intestine, causing inflammatory response, damage in tissue architecture and systemic dissemination. Macrophage migration inhibition factor (MIF) is a cytokine secreted from both immune and non-immune cells, including gut epithelial cells. MIF is described to promote inflammatory responses, to be associated in colitis pathogenesis and also to play role in maintaining the intestinal barrier. The aim of the present study was to evaluate the influence of the pregnancy and MIF deficiency on T. gondii infection in the intestinal microenvironment and to address how these factors can impact on the intestinal architecture and local cytokine profile. For this purpose, small intestine of pregnant and non-pregnant C57BL/6 MIF deficient mice (MIF-/-) and Wild-type (WT) orally infected with 5 cysts of ME-49 strain of T. gondii were collected on day 8th of infection. Intestines were processed for morphological and morphometric analyses, parasite quantification and for cytokines mensuration. Our results showed that the absence of MIF and pregnancy caused an increase in T. gondii infection index. T. gondii immunolocalization demonstrated that segments preferentially infected with T. gondii were duodenum and ileum. The infection caused a reduction in the size of the intestinal villi, whereas, infection associated with pregnancy caused an increase in villi size due to edema caused by the infection. Also, the goblet cell number was increased in the ileum of MIF-/- mice, when compared to the corresponding WT group. Analyses of cytokine production in the small intestine showed that MIF was up regulated in the gut of pregnant WT mice due to infection. Also, infection provoked an intense Th1 response that was more exacerbated in pregnant MIF-/- mice. We also detected that the Th2/Treg response was more pronounced in MIF-/- mice. Altogether, our results demonstrated that pregnancy and MIF deficiency interferes in the balance of the intestinal cytokines and favors a Th1-immflamatory profile, which in turn, impact in the development of pathology caused by T. gondii infection in the intestinal microenvironment.