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ObjectiveThe intestinal immune response could play an important role in obesity-related comorbidities. We aim to study the profile of duodenal cytokines and chemokines in patients with morbid obesity (MO), its relation with insulin resistance (IR) and the intake of metformin, and with the evolution of MO after sleeve gastrectomy (SG).Research design and methodsDuodenal levels of 24 cytokines and 9 chemokines were analyzed in 14 nonobese and in 54 MO who underwent SG: with lower IR (MO-lower-IR), with higher IR (MO-higher-IR), and with type 2 diabetes treated with metformin (MO-metf-T2DM).ResultsMO-lower-IR had higher levels of cytokines related to Th1, Th2, Th9, Th17, Th22, M1 macrophages, and chemokines involved in the recruitment of macrophages and T-lymphocytes (p < 0.05), and total (CD68 expression) and M1 macrophages (ITGAX expression) (p < 0.05) when compared with nonobese patients, but with a decrease in M2 macrophages (MRC1 expression) (p < 0.05). In MO-higher-IR, these chemokines and cytokines decreased and were similar to those found in nonobese patients. In MO-metf-T2DM, only IL-4 (Th2) and IL-22 (Th22) increased their levels with regard to MO-higher-IR (p < 0.05). In MO-higher-IR and MO-metf-T2DM, there was a decrease of CD68 expression (p < 0.05) while ITGAX and MRC1 were similar with regard to MO-lower-IR. We found an association between CXCL8, TNFβ and IL-2 with the evolution of body mass index (BMI) after SG (p < 0.05).ConclusionsThere is an association between a higher IR and a lower duodenal immune response in MO, with a slight increase in those patients with metformin treatment. Intestinal immune response could be involved in the evolution of BMI after SG.

Plant sterols and stanols inhibit intestinal cholesterol absorption and consequently lower serum LDL-cholesterol (LDL-C) concentrations. The underlying mechanisms are not yet known. In vitro and animal studies have suggested that changes in intestinal sterol metabolism are attributed to the LDL-C-lowering effects of plant stanol esters. However, similar studies in human subjects are lacking. Therefore, we examined the effects of an acute intake of plant stanol esters on gene expression profiles of the upper small intestine in healthy volunteers. In a double-blind cross-over design, fourteen healthy subjects (eight female and six male; age 21–55 years), with a BMI ranging from 21 to 29 kg/m2, received in random order a shake with or without plant stanol esters (4 g). At 5 h after consumption of the shake, biopsies were taken from the duodenum (around the papilla of Vater) and from the jejunum (20 cm distal from the papilla of Vater). Microarray analysis showed that the expression profiles of genes involved in sterol metabolism were not altered. Surprisingly, the pathways involved in T-cell functions were down-regulated in the jejunum. Furthermore, immunohistochemical analysis showed that the number of CD3 (cluster of differentiation number 3), CD4 (cluster of differentiation number 4) and Foxp3+ (forkhead box P3-positive) cells was reduced in the plant stanol ester condition compared with the control condition, which is in line with the microarray data. The physiological and functional consequences of the plant stanol ester-induced reduction of intestinal T-cell-based immune activity in healthy subjects deserve further investigation.

Background Identification of adjuvant treatment is necessary for rapid and effective treatment in patients with celiac disease. In a pilot randomized controlled trial, the effect of prednisolone on enterocyte apoptosis and regeneration in celiac disease was investigated. Patients and Methods Thirty-three treatment-naïve patients with celiac disease were randomized to either gluten-free diet (GFD, n  = 17) or GFD + prednisolone (1 mg/kg for 4 weeks, n  = 16). Duodenal biopsies were taken at baseline and at 4 and 8 weeks posttreatment. Six patients with functional dyspepsia were recruited as controls. All these biopsies were stained for markers of intrinsic apoptotic pathway (AIF, H2AX, p53), common apoptotic pathway (CC3, M30), apoptotic inhibitors (XIAP, Bcl2), and epithelial proliferation (Ki-67). Apoptotic (AI) and proliferation indices (PI) were compared. Results At baseline duodenal biopsies, the end apoptotic products H2AX and M30 were significantly increased. In comparison with those treated with GFD alone, after 4 weeks of GFD + prednisolone treatment, some markers of both intrinsic and common apoptotic pathways showed rapid decline. After prednisolone withdrawal, there was overexpression of H2AX, CC3, and p53 in the latter group. In comparison with those treated with only GFD, patients treated with prednisolone showed suppression of mucosal PI, which started rising again after withdrawal of prednisolone. Conclusions Apoptosis takes place in mucosal epithelium in celiac disease. Addition of short course of prednisolone suppresses apoptosis rapidly. However, it also suppresses epithelial regeneration; hence, if used, it should be withdrawn after an initial short course. (Registered at clinicaltrials.gov; NCT01045837)

The parasitic helminth Trichinella spiralis, which poses a serious health risk to animals and humans, can be found worldwide. Recent findings indicate that a rare type of gut epithelial cell, tuft cells, can detect the helminth, triggering type 2 immune responses. However, the underlying molecular mechanisms remain to be fully understood. Here we show that both excretory–secretory products (E–S) and extract of T. spiralis can stimulate the release of the cytokine interleukin 25 (IL-25) from the mouse small intestinal villi and evoke calcium responses from tuft cells in the intestinal organoids, which can be blocked by a bitter-taste receptor inhibitor, allyl isothiocyanate. Heterologously expressed mouse Tas2r bittertaste receptors, the expression of which is augmented during tuftcell hyperplasia, can respond to the E–S and extract as well as to the bitter compound salicin whereas salicin in turn can induce IL-25 release from tuft cells. Furthermore, abolishment of the G-protein γ13 subunit, application of the inhibitors for G-protein αo/i, Gβγ subunits, and phospholipase Cβ2 dramatically reduces the IL-25 release. Finally, tuft cells are found to utilize the inositol triphosphate receptor type 2 (Ip₃r2) to regulate cytosolic calcium and thus Trpm5 activity, while potentiation of Trpm5 by a sweet-tasting compound, stevioside, enhances tuft cell IL-25 release and hyperplasia in vivo. Taken together, T. spiralis infection activates a signaling pathway in intestinal tuft cells similar to that of taste-bud cells, but with some key differences, to initiate type 2 immunity.

The intestinal immune system has the challenging task of tolerating foreign nutrients and the commensal microbiome, while excluding or eliminating ingested pathogens. Failure of this balance leads to conditions such as inflammatory bowel diseases, food allergies and invasive gastrointestinal infections 1 . Multiple immune mechanisms are therefore in place to maintain tissue integrity, including balanced generation of effector T (T H ) cells and FOXP3 + regulatory T (pT reg ) cells, which mediate resistance to pathogens and regulate excessive immune activation, respectively 1 – 4 . The gut-draining lymph nodes (gLNs) are key sites for orchestrating adaptive immunity to luminal perturbations 5 – 7 . However, it is unclear how they simultaneously support tolerogenic and inflammatory reactions. Here we show that gLNs are immunologically specific to the functional gut segment that they drain. Stromal and dendritic cell gene signatures and polarization of T cells against the same luminal antigen differ between gLNs, with the proximal small intestine-draining gLNs preferentially giving rise to tolerogenic responses and the distal gLNs to pro-inflammatory T cell responses. This segregation permitted the targeting of distal gLNs for vaccination and the maintenance of duodenal pT reg cell induction during colonic infection. Conversely, the compartmentalized dichotomy was perturbed by surgical removal of select distal gLNs and duodenal infection, with effects on both lymphoid organ and tissue immune responses. Our findings reveal that the conflict between tolerogenic and inflammatory intestinal responses is in part resolved by discrete gLN drainage, and encourage antigen targeting to specific gut segments for therapeutic immune modulation. Immune responses in the gut and associated draining lymph nodes differ between tolerogenic and inflammatory depending on their anatomical location.

A new evaluation of previously published data suggested to us that the accumulation of mutations might slow, rather than increase, as individuals age. To explain this unexpected finding, we hypothesized that normal stem cell division rates might decrease as we age. To test this hypothesis, we evaluated cell division rates in the epithelium of human colonic, duodenal, esophageal, and posterior ethmoid sinonasal tissues. In all 4 tissues, there was a significant decrease in cell division rates with age. In contrast, cell division rates did not decrease in the colon of aged mice, and only small decreases were observed in their small intestine or esophagus. These results have important implications for understanding the relationship between normal stem cells, aging, and cancer. Moreover, they provide a plausible explanation for the enigmatic age-dependent deceleration in cancer incidence in very old humans but not in mice.

In vitro intestinal epithelial models are being developed for studying gut physiology, barrier function, and drug transport. There are two major in vitro models which are currently used: cell line-derived monolayers (such as Caco-2/HT29 cocultures) and primary cell monolayers derived from human intestinal organoids. To conduct a side by side comparison of organoid-derived and cell line-derived intestinal epithelial monolayers, assessing their morphological features, barrier performance, and secretome under similar culture conditions. Human duodenum-derived organoids and Caco-2/HT29 cocultures (9:1 ratio) were seeded onto Transwell inserts and were harvested at the same time for comparative analysis. Barrier function was assessed by transepithelial electrical resistance (TEER) and Lucifer Yellow permeability. Structural characterization was performed using immunofluorescence microscopy, scanning and transmission electron microscopy. Secretion of growth factors and chemokines was quantified on apical and basal sides via multiplex assays. Both models formed intact epithelial barriers with robust tight junctions and displayed appropriate apical-basolateral polarization, confirmed by the presence of microvilli. However, the organoid-derived model exhibited occasional villus-like protrusions and ultrastructural crypt-like invaginations, homogenous secretion of Mucin 2 over the surface, and release of epithelial growth factor whereas the cell line model exhibited longer and more densely packed microvilli and higher apical secretion of vascular endothelial growth factor. The barrier properties were comparable except that the organoid model had decreased permeability (Lucifer Yellow). This study highlights key structural, functional, and molecular similarities and differences that influence the physiological relevance and application scope of each model. It enables educated decision making when choosing the best model for a particular \"Context of Use\" weighing the pros and cons between experimental convenience and physiological relevance.

The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs) in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS) found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from her6/HES1 that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development and physiology.

New therapeutic molecules for farm animals are needed to address worldwide problems in the food industry, like the rise of resistance among ruminant parasites and pathogenic microbes. Since in vivo testing would involve an excessive number of animals, with consequent ethical and economic issues, the generation of sheep intestinal organoids represents a promising close-to-reality in vitro model for veterinary drug development; however, the characterization and application of such organoids remain limited. In this study, ovine intestinal organoids were generated from adult LGR5+ stem cells from the intestinal crypts of freshly slaughtered lambs, and developed in an in vitro culture system. Morphological analysis via brightfield microscopy and immunocytochemical staining revealed a pseudostratified epithelium with multiple cell types, and distinct apical–basal polarity, while RNA sequencing validated the preservation of the physiological characteristics of the original organ. The development and characterization of a robust and reproducible protocol for culturing sheep duodenum intestinal organoids in a high-throughput screening (HTS) compatible format demonstrated reliability in HTS applications, with Z’-factor tests indicating robust assay performance. Dose–response studies using pre-identified compounds showed comparable pharmacodynamic profiles between mouse and sheep organoids. These findings establish sheep intestinal organoids as an innovative tool for veterinary pharmacology and toxicology, offering a cost-effective and sustainable platform to address challenges such as drug resistance and improve livestock health.

Celiac disease (CD) is an immune-mediated disorder triggered by gluten exposure. The contribution of the adaptive immune response to CD pathogenesis has been extensively studied, but the absence of valid experimental models has hampered our understanding of the early steps leading to loss of gluten tolerance. Using intestinal organoids developed from duodenal biopsies from both non-celiac (NC) and celiac (CD) patients, we explored the contribution of gut epithelium to CD pathogenesis and the role of microbiota-derived molecules in modulating the epithelium’s response to gluten. When compared to NC, RNA sequencing of CD organoids revealed significantly altered expression of genes associated with gut barrier, innate immune response, and stem cell functions. Monolayers derived from CD organoids exposed to gliadin showed increased intestinal permeability and enhanced secretion of pro-inflammatory cytokines compared to NC controls. Microbiota-derived bioproducts butyrate, lactate, and polysaccharide A improved barrier function and reduced gliadin-induced cytokine secretion. We concluded that: (1) patient-derived organoids faithfully express established and newly identified molecular signatures characteristic of CD. (2) microbiota-derived bioproducts can be used to modulate the epithelial response to gluten. Finally, we validated the use of patient-derived organoids monolayers as a novel tool for the study of CD.