Duodenal mucosa of untreated celiac disease patients has altered expression of the GAS6 and PROS1 and the negative regulator tyrosine kinase TAM receptors subfamily.

Celiac disease (CD) is an immune-driven disease characterized by tissue damage in the small intestine of genetically-susceptible individuals. We evaluated here a crucial immune regulatory pathway involving TYRO3, AXL, and MERTK (TAM) receptors and their ligands PROS1 and GAS6 in duodenal biopsies of controls and CD patients. We found increased GAS6 expression associated with downregulation of PROS1 and variable TAM receptors levels in duodenum tissue of CD patients. Interestingly, CD3+ lymphocytes, CD68+, CD11c+ myeloid and epithelial cells, showed differential expressions of TAM components comparing CD vs controls. Principal component analysis revealed a clear segregation of two groups of CD patients based on TAM components and IFN signaling. In vitro validation demonstrated that monocytes, T lymphocytes and epithelial cells upregulated TAM components in response to IFN stimulation. Our findings highlight a dysregulated TAM axis in CD related to IFN signaling and contribute to a deeper understanding of the pathophysiology of CD.

Differential localization and limited cytotoxic potential of duodenal CD8+ T cells.

The duodenum is a major site of HIV persistence during suppressive antiretroviral therapy despite harboring abundant tissue-resident memory (Trm) CD8+ T cells. The role of duodenal Trm CD8+ T cells in viral control is still not well defined. We examined the spatial localization, phenotype, and function of CD8+ T cells in the human duodenal tissue from people living with HIV (PLHIV) and healthy controls. We found that Trm (CD69+CD103hi) cells were the predominant CD8+ T cell population in the duodenum. Immunofluorescence imaging of the duodenal tissue revealed that CD103+CD8+ T cells were localized in the intraepithelial region, while CD103-CD8+ T cells and CD4+ T cells were mostly localized in the lamina propria (LP). Furthermore, HIV-specific CD8+ T cells were enriched in the CD69+CD103-/lo population. However, the duodenal HIV-specific CD8+ Trm cells rarely expressed canonical molecules for potent cytolytic function (perforin and granzyme B) but were more polyfunctional than those from peripheral blood. Taken together, our results show that duodenal CD8+ Trm cells possess limited perforin-mediated cytolytic potential and are spatially separated from HIV-susceptible LP CD4+ T cells. This could contribute to HIV persistence in the duodenum and provides critical information for the design of cure therapies.

Administration of Human Derived Upper gut Commensal Prevotella histicola delays the onset of type 1 diabetes in NOD mice.

BACKGROUND: Type 1 diabetes (T1D) is an autoimmune disease that is increasing in prevalence worldwide. One of the contributing factors to the pathogenesis of T1D is the composition of the intestinal microbiota, as has been demonstrated. in T1D patients, with some studies demonstrating a deficiency in their levels of Prevotella. We have isolated a strain of Prevotella histicola from a duodenal biopsy that has anti-inflammatory properties, and in addition, alters the development of autoimmune diseases in mouse models. Therefore, our hypothesis is that the oral administration of P. histicola might delay the development of T1D in the non-obese diabetic (NOD) mice. To assess this, we used the following materials and methods. Female NOD mice (ages 5-8 weeks) were administered every other day P. histicola that was cultured in-house. Blood glucose levels were measured every other week. Mice were sacrificed at various time points for histopathological analysis of the pancreas. Modulation of immune response by the commensal was tested by analyzing regulatory T-cells and NKp46+ cells using flow cytometry and intestinal cytokine mRNA transcript levels using quantitative RT-PCR. For microbial composition, 16 s rRNA gene analysis was conducted on stool samples collected at various time points. RESULTS: Administration of P. histicola in NOD mice delayed the onset of T1D. Beta diversity in the fecal microbiomes demonstrated that the microbial composition of the mice administered P. histicola was different from those that were not treated. Treatment with P. histicola led to a significant increase in regulatory T cells with a concomitant decrease in NKp46+ cells in the pancreatic lymph nodes as compared to the untreated group after 5 weeks of treatment. CONCLUSIONS: These observations suggest that P. histicola treatment delayed onset of diabetes by increasing the levels of regulatory T cells in the pancreatic lymph nodes. This preliminary work supports the rationale that enteral exposure to a non pathogenic commensal P. histicola be tested as a future therapy for T1D.

Frontiers in Celiac Disease: Where Autoimmunity and Environment Meet.

Celiac disease is a chronic, immune-mediated enteropathy driven by dietary gluten found in genetically susceptible hosts. It has a worldwide distribution, is one of the most common autoimmune disorders globally, and is the only autoimmune condition for which the trigger is known. Despite advances in characterizing mechanisms of disease, gaps in understanding of celiac disease pathogenesis remain. A "frontier" concept is considering what moves an HLA-DQ2 or DQ8-positive individual from asymptomatic gluten tolerance to celiac disease manifestation. In this arena, environmental triggers, including age at the time of initial gluten exposure, the occurrence of usual childhood viral infections, and microbiome alterations have emerged as key events in triggering the symptomatic disease. Pathologists play a major role in frontier aspects of celiac disease. This includes the discovery that duodenal mucosal histology in follow-up biopsies does not correlate with ongoing patient symptoms, antitissue transglutaminase antibody titers and diet adherence in celiac disease patients. Further, in light of recent evidence that the detection of monoclonal T-cell populations in formalin-fixed biopsies is not specific for type II refractory celiac disease, pathologists should resist performing such analyses until common causes of "apparent" refractoriness are excluded. The promise of therapies in celiac disease has led to clinical trials targeting many steps in the inflammatory cascade, which depend upon a pathologist's confirmation of the initial diagnosis and evaluation of responses to therapies. As pathologists continue to be active participants in celiac disease research, partnering with other stakeholders, we will continue to impact this important autoimmune disease.

Regulation of intestinal immunity and tissue repair by enteric glia.

Tissue maintenance and repair depend on the integrated activity of multiple cell types1. Whereas the contributions of epithelial2,3, immune4,5 and stromal cells6,7 in intestinal tissue integrity are well understood, the role of intrinsic neuroglia networks remains largely unknown. Here we uncover important roles of enteric glial cells (EGCs) in intestinal homeostasis, immunity and tissue repair. We demonstrate that infection of mice with Heligmosomoides polygyrus leads to enteric gliosis and the upregulation of an interferon gamma (IFNγ) gene signature. IFNγ-dependent gene modules were also induced in EGCs from patients with inflammatory bowel disease8. Single-cell transcriptomics analysis of the tunica muscularis showed that glia-specific abrogation of IFNγ signalling leads to tissue-wide activation of pro-inflammatory transcriptional programs. Furthermore, disruption of the IFNγ-EGC signalling axis enhanced the inflammatory and granulomatous response of the tunica muscularis to helminths. Mechanistically, we show that the upregulation of Cxcl10 is an early immediate response of EGCs to IFNγ signalling and provide evidence that this chemokine and the downstream amplification of IFNγ signalling in the tunica muscularis are required for a measured inflammatory response to helminths and resolution of the granulomatous pathology. Our study demonstrates that IFNγ signalling in enteric glia is central to intestinal homeostasis and reveals critical roles of the IFNγ-EGC-CXCL10 axis in immune response and tissue repair after infectious challenge.

Diagnosing coeliac disease: A literature review.

Coeliac disease (CD) is an autoimmune gastroenteropathy triggered by gliadin and gliadin-tissue transglutaminase (tTG) complexes. CD is one of the few autoimmune diseases with an accurate, non-invasive serological test. Anti-endomysial, anti-tTG and anti-deaminated gliadin peptides (DGP) antibodies are currently used for serological tests with tTG ELISAs being the superior test. Duodenal biopsy, although invasive, is the gold standard for CD diagnosis. HLA genotyping and flow cytometry can also be used as supplementary tests. The incidence of CD is rising globally although the reasons for this remain unclear. In addition, the true incidence of coeliac disease in African populations remains unknown although recent work suggests that South African populations express the alleles associated with this disease. This review examines the pathogenesis and diagnosis of coeliac disease and considers novel and innovative biomarkers in its diagnosis specifically in an African population.

A Randomized Trial of a Transglutaminase 2 Inhibitor for Celiac Disease.

BACKGROUND: In celiac disease, small intestinal transglutaminase 2 causes deamidation of glutamine residues in gluten peptides, which enhances stimulation of T cells and leads to mucosal injury. Inhibition of transglutaminase 2 is a potential treatment for celiac disease. METHODS: In a proof-of-concept trial, we assessed the efficacy and safety of a 6-week treatment with ZED1227, a selective oral transglutaminase 2 inhibitor, at three dose levels as compared with placebo, in adults with well-controlled celiac disease who underwent a daily gluten challenge. The primary end point was the attenuation of gluten-induced mucosal damage, as measured by the ratio of villus height to crypt depth. Secondary end points included intraepithelial lymphocyte density, the Celiac Symptom Index score, and the Celiac Disease Questionnaire score (for assessment of health-related quality of life). RESULTS: Of the 41 patients assigned to the 10-mg ZED1227 group, the 41 assigned to the 50-mg group, the 41 assigned to the 100-mg group, and the 40 assigned to the placebo group, 35, 39, 38, and 30 patients, respectively, had adequate duodenal-biopsy samples for the assessment of the primary end point. Treatment with ZED1227 at all three dose levels attenuated gluten-induced duodenal mucosal injury. The estimated difference from placebo in the change in the mean ratio of villus height to crypt depth from baseline to week 6 was 0.44 (95% confidence interval [CI], 0.15 to 0.73) in the 10-mg group (P = 0.001), 0.49 (95% CI, 0.20 to 0.77) in the 50-mg group (P<0.001), and 0.48 (95% CI, 0.20 to 0.77) in the 100-mg group (P<0.001). The estimated differences from placebo in the change in intraepithelial lymphocyte density were -2.7 cells per 100 epithelial cells (95% CI, -7.6 to 2.2) in the 10-mg group, -4.2 cells per 100 epithelial cells (95% CI, -8.9 to 0.6) in the 50-mg group, and -9.6 cells per 100 epithelial cells (95% CI, -14.4 to -4.8) in the 100-mg group. Use of the 100-mg dose may have improved symptom and quality-of-life scores. The most common adverse events, the incidences of which were similar across all groups, were headache, nausea, diarrhea, vomiting, and abdominal pain. Rash developed in 3 of 40 patients (8%) in the 100-mg group. CONCLUSIONS: In this preliminary trial, treatment with ZED1227 attenuated gluten-induced duodenal mucosal damage in patients with celiac disease. (Funded by Dr. Falk Pharma; CEC-3 EudraCT number, 2017-002241-30.).

Disruption of the pro-inflammatory, anti-inflammatory cytokines and tight junction proteins expression, associated with changes of the composition of the gut microbiota in patients with irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) is a pathologic condition characterized by changes in gut microbiome composition, low-grade inflammation, and disruption of intestinal wall permeability. The interaction between the gut microbiome and the disease manifestation remains unclear. The changing of tight junction proteins and cytokines expression throughout the gastrointestinal tract in IBS patients has not been studied yet. AIM OF THE STUDY: To assess the changes of gut microbiome composition, tight junction proteins, and cytokines expression of intestinal mucosa from the duodenum to the distal part of the colon in IBS patients and healthy volunteers. METHODS: In 31 IBS patients (16 patients with IBS-D; 15 patients with IBS-C) and 10 healthy volunteers the expression of CLD-2, CLD-3, CLD-5, IL-2, IL-10, and TNF-α in mucosal biopsy specimens was determined by morphological and immune-histochemical methods. The qualitative and quantitative composition of the intestinal microbiota was assessed based on 16S rRNA gene sequencing in both groups of patients. RESULTS: The expression of IL-2 and TNF-α was significantly increased in IBS patients compared with the controls (p<0.001), with a gradual increase from the duodenum to the sigmoid colon. The expression of IL-10, CLD-3, and CLD-5 in mucosal biopsy specimens of these patients was lower than in the control group (p<0.001). Increased ratios of Bacteroidetes and decreased ratios of Firmicutes were noted in IBS patients compared to healthy volunteers (p<0.05). CONCLUSION: IBS patients have impaired gut permeability and persisting low-grade inflammation throughout the gastrointestinal tract. Changes in the gut microbiota may support or exacerbate these changes.

Gastric Mucosal Immune Profiling and Dysregulation in Idiopathic Gastroparesis.

INTRODUCTION: It is unclear how immune perturbations may influence the pathogenesis of idiopathic gastroparesis, a prevalent functional disorder of the stomach which lacks animal models. Several studies have noted altered immune characteristics in the deep gastric muscle layer associated with gastroparesis, but data are lacking for the mucosal layer, which is endoscopically accessible. We hypothesized that immune dysregulation is present in the gastroduodenal mucosa in idiopathic gastroparesis and that specific immune profiles are associated with gastroparesis clinical parameters. METHODS: In this cross-sectional prospective case-control study, routine endoscopic biopsies were used for comprehensive immune profiling by flow cytometry, multicytokine array, and gene expression in 3 segments of the stomach and the duodenal bulb. Associations of immune endpoints with clinical parameters of gastroparesis were also explored. RESULTS: The gastric mucosa displayed large regional variation of distinct immune profiles. Furthermore, several-fold increases in innate and adaptive immune cells were found in gastroparesis. Various immune cell types showed positive correlations with duration of disease, proton pump inhibitor dosing, and delayed gastric emptying. DISCUSSION: This initial observational study showed immune compartmentalization of the human stomach mucosa and significant immune dysregulation at the level of leukocyte infiltration in idiopathic gastroparesis patients that extends to the duodenum. Select immune cells, such as macrophages, may correlate with clinicopathological traits of gastroparesis. This work supports further mucosal studies to advance our understanding of gastroparesis pathophysiology.

The Effects of Helicobacter pylori Infection on Microbiota Associated With Gastric Mucosa and Immune Factors in Children.

Background: Helicobacter pylori infection is the main cause of chronic gastritis in children. Little is known about the effect of Helicobacter pylori on microbiota and immunity. This study was aimed at characterizing stomach microbiota and immune-regulatory properties of children with Helicobacter pylori colonization. Methods: We studied 122 children who had undergone gastric endoscopy due to gastrointestinal symptoms, 57 were diagnosed with Helicobacter pylori infection. Endoscopic mucosal biopsy samples were obtained for DNA and RNA extraction. Microbiomes were analyzed by 16S rRNA profiling, with the differentially expressed genes analyzed using RNA sequencing. The RNA-sequencing results of selected genes were validated by qRT-PCR. Results: Bacterial diversity of Helicobacter pylori-positive gastric specimens were lower than those of negative, and both groups were clearly separated according to beta diversity. Helicobacter pylori-positive group significantly reduced proportions of six phyla and eight genera; only Helicobacter taxa were more abundant in Helicobacter pylori-negative group. Gastric tissues RNA sequencing showed increased expression of multiple immune response genes in Helicobacter pylori -infection. Helicobacter pylori -infected children with restructured gastric microbiota had higher levels of FOXP3, IL-10, TGF-ß1 and IL-17A expressions, which were consistent with increased CD4+T cell and macrophagocyte, compared with non-infected children. Conclusions: Presence of Helicobacter pylori significantly influences gastric microbiota and results in lower abundance of multiple taxonomic levels in children. Meanwhile, it affects gastric immune environment and promotes the occurrence of gastritis. Clinical Trial Registration: [http://www.chictr.org.cn], identifier [ChiCTR1800015190].

The role of inflammatory cytokines in anemia and gastrointestinal mucosal injury induced by foot electric stimulation.

Foot electrical stimulation (FES) has been considered as a classic stressor that can disturb homeostasis. Acute anemia was observed in the model induced by FES. The aim of this study was to explore the role of inflammatory cytokines underlying the acute anemia and gastrointestinal (GI) mucosal injury in the FES. Twenty-four male Kunming mice (20 ± 2 g) were randomly divided into control group and experimental group. The mice were placed in a footshock chamber that can generate 0.5 mA electrical impulse periodically for 0.5 h. After the process, red blood cell count, hemoglobin concentration and hematocrit, the levels of corticotropin releasing hormone (CRH) in serum and hypothalamus, and adrenocorticotropic hormone (ACTH) in serum and pituitary were detected separately. In addition, we investigated the expressions of inflammatory cytokines (IL-1, IL-6, TNF-α, iNOS, and IL-10) in the hypothalamus and duodenum by Polymerase Chain Reaction (PCR). Results showed that this FES model induced anemia, increased CRH and ACTH activity in the serum after the FES. Moreover, the expressions of IL-1ß, IL-6, TNF-α, and iNOS were significantly increased following the process, while IL-10 was not activated. These findings suggest that anemia, the inflammatory cytokines in the hypothalamus and duodenum of the mice in the model induced by FES is closely related to GI mucosal injury/bleeding. Taken together, these results underscore the importance of anemia, GI mucosal injury/bleeding and stress, future studies would be needed to translate these findings into the benefit of affected patients.

Immune Checkpoint Inhibitor-Induced Upper Gastrointestinal Tract Inflammation Shows Morphologic Similarities to, but Is Immunologically Distinct From, Helicobacter pylori Gastritis and Celiac Disease.

CONTEXT.­: Immune checkpoint inhibitor (CPI) therapies are associated with multi-organ immune-related adverse events. Although colonic mucosal changes have been described, inflammatory changes incited by CPIs in the upper gastrointestinal tract have not been well characterized. OBJECTIVE.­: To investigate morphologic and immunologic changes incited by CPI therapy in the upper gastrointestinal tract. DESIGN.­: We compared the morphology and immune cell phenotype of gastric and duodenal biopsies from patients treated with anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4) or anti-programmed death receptor-1/programmed death ligand-1 (PD-1/PD-L1) antibodies with biopsies from patients with Helicobacter pylori gastritis, patients with celiac disease, and normal controls. RESULTS.­: Gastric biopsies from patients on CPIs showed chronic gastritis mimicking H pylori gastritis. However, CPI gastritis demonstrated greater numbers of CD8+ intraepithelial lymphocytes, less lamina propria inflammation, fewer plasma cells and CD20+ B cells, fewer lymphoid aggregates, and reduced CD4:CD8 ratio in both the lamina propria and the epithelial layer. There were no differences between anti-CTLA-4 and anti-PD-1/PD-L1 gastritis, except for more lymphoid aggregates in anti-PD-1/PD-L1 gastritis. Duodenal biopsies from patients on CPIs revealed chronic duodenitis with villous blunting, mimicking celiac disease. Compared with celiac disease, CPI duodenitis demonstrated higher prevalence of neutrophilic infiltrates and erosions, increased lamina propria CD3 and CD8 T cells, and reduced CD4:CD8 ratio. Upper gastrointestinal biopsies were more inflamed than concomitant colonic biopsies in the majority of patients. CONCLUSIONS.­: The morphologic and immunophenotypic distinctions between CPI-associated upper gastrointestinal injuries and common infectious and autoimmune diseases may provide useful discriminators when clinicians are confronted with gastric and duodenal inflammatory changes in patients receiving CPI therapy.

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.

Impaired Duodenal Palmitoylethanolamide Release Underlies Acid-Induced Mast Cell Activation in Functional Dyspepsia.

BACKGROUND & AIMS: Acid hypersensitivity is claimed to be a symptomatic trigger in functional dyspepsia (FD); however, the neuroimmune pathway(s) and the mediators involved in this process have not been investigated systematically. Palmitoylethanolamide (PEA) is an endogenous compound, able to modulate nociception and inflammation, but its role in FD has not been assessed. METHODS: Duodenal biopsy specimens from FD and control subjects, and peroxisome proliferator-activated receptor-α (PPARα) null mice were cultured at a pH of 3.0 and 7.4. Mast cell (MC) number, the release of their mediators, and the expression of transient receptor potential vanilloid receptor (TRPV)1 and TRPV4, were evaluated. All measurements also were performed in the presence of a selective blocker of neuronal action potential (tetradotoxin). FD and control biopsy specimens in acidified medium also were incubated in the presence of different PEA concentrations, alone or combined with a selective PPARα or PPAR-γ antagonist. RESULTS: An acid-induced increase in MC density and the release of their mediators were observed in both dyspeptic patients and controls; however, this response was amplified significantly in FD. This effect was mediated by submucosal nerve fibers and up-regulation of TRPV1 and TRPV4 receptors because pretreatment with tetradotoxin significantly reduced MC infiltration. The acid-induced endogenous release of PEA was impaired in FD and its exogenous administration counteracts MC activation and TRPV up-regulation. CONCLUSIONS: Duodenal acid exposure initiates a cascade of neuronal-mediated events culminating in MC activation and TRPV overexpression. These phenomena are consequences of an impaired release of endogenous PEA. PEA might be regarded as an attractive therapeutic strategy for the treatment of FD.

Genome-Wide Transcriptomic Analysis of Intestinal Mucosa in Celiac Disease Patients on a Gluten-Free Diet and Postgluten Challenge.

BACKGROUND & AIMS: Gluten challenge studies are instrumental in understanding the pathophysiology of celiac disease. Our aims in this study were to reveal early gluten-induced transcriptomic changes in duodenal biopsies and to find tools for clinics. METHODS: Duodenal biopsies were collected from 15 celiac disease patients on a strict long-term gluten-free diet (GFD) prior to and post a gluten challenge (PGC) and from 6 healthy control individuals (DC). Biopsy RNA was subjected to genome-wide 3' RNA-Seq. Sequencing data was used to determine the differences between the three groups and was compared to sequencing data from the public repositories. The biopsies underwent morphometric analyses. RESULTS: In DC vs. GFD group comparisons, 167 differentially expressed genes were identified with 117 genes downregulated and 50 genes upregulated. In PGC vs. GFD group comparisons, 417 differentially expressed genes were identified with 195 genes downregulated and 222 genes upregulated. Celiac disease patients on a GFD were not "healthy". In particular, genes encoding proteins for transporting small molecules were expressed less. In addition to the activation of immune response genes, a gluten challenge induced hyperactive intestinal wnt-signaling and consequent immature crypt gene expression resulting in less differentiated epithelium. Biopsy gene expression in response to a gluten challenge correlated with the extent of the histological damage. Regression models using only four gene transcripts described 97.2% of the mucosal morphology and 98.0% of the inflammatory changes observed. CONCLUSIONS: Our gluten challenge trial design provided an opportunity to study the transition from health to disease. The results show that even on a strict GFD, despite being deemed healthy, patients reveal patterns of ongoing disease. Here, a transcriptomic regression model estimating the extent of gluten-induced duodenal mucosal injury is presented.

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).

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.

mTOR sustains inflammatory response in celiac disease.

Celiac disease (CD) is an enteropathy triggered by the ingestion of gluten proteins in genetically predisposed individuals and characterized by excessive activation of effector immune cells and enhanced production of inflammatory cytokines. However, factors/mechanisms that amplify the ongoing mucosal inflammation in CD are not fully understood. In this study, we assessed whether mammalian target of Rapamycin (mTOR), a pathway that combines intra- and extra-cellular signals and acts as a central regulator for the metabolism, growth, and function of immune and non-immune cells, sustains CD-associated immune response. Our findings indicate that expression of phosphorylated (p)/active form of mTOR is increased in protein lysates of duodenal biopsy samples taken from patients with active CD (ACD) as compared to normal controls. In ACD, activation of mTOR occurs mainly in the epithelial compartment and associates with enhanced expression of p-4EBP, a downstream target of mTOR complex (mTORC)1, while expression of p-Rictor, a component of mTORC2, is not increased. Stimulation of mucosal explants of inactive CD patients with pepsin-trypsin-digested (PT)-gliadin or IFN-γ/IL-21, two cytokines produced in CD by gluten-specific T cells, increases p-4EBP expression. Consistently, blockade of such cytokines in cultures of ACD mucosal explants reduces p-4EBP. Finally, we show that inhibition of mTORC1 with rapamycin in ACD mucosal explants reduces p-4EBP and production of IL-15, a master cytokine produced by epithelial cells in this disorder. Our data suggest that ACD inflammation is marked by activation of mTORC1 in the epithelial compartment.

Aggregative Adherence Fimbriae II of Enteroaggregative Escherichia coli Are Required for Adherence and Barrier Disruption during Infection of Human Colonoids.

Symptomatic and asymptomatic infection with the diarrheal pathogen enteroaggregative Escherichia coli (EAEC) is associated with growth faltering in children in developing settings. The mechanism of this association is unknown, emphasizing a need for better understanding of the interactions between EAEC and the human gastrointestinal mucosa. In this study, we investigated the role of the aggregative adherence fimbriae II (AAF/II) in EAEC adherence and pathogenesis using human colonoids and duodenal enteroids. We found that a null mutant in aafA, the major subunit of AAF/II, adhered significantly less than wild-type (WT) EAEC strain 042, and adherence was restored in a complemented strain. Immunofluorescence confocal microscopy of differentiated colonoids, which produce an intact mucus layer comprised of the secreted mucin MUC2, revealed bacteria at the epithelial surface and within the MUC2 layer. The WT strain adhered to the epithelial surface, whereas the aafA deletion strain remained within the MUC2 layer, suggesting that the presence or absence of AAF/II determines both the abundance and location of EAEC adherence. In order to determine the consequences of EAEC adherence on epithelial barrier integrity, colonoid monolayers were exposed to EAEC constructs expressing or lacking aafA Colonoids infected with WT EAEC had significantly decreased epithelial resistance, an effect that required AAF/II, suggesting that binding of EAEC to the epithelium is necessary to impair barrier function. In summary, we show that production of AAF/II is critical for adherence and barrier disruption in human colonoids, suggesting a role for this virulence factor in EAEC colonization of the gastrointestinal mucosa.