Transcriptomic identification of TMIGD1 and its relationship with the ileal epithelial cell differentiation in Crohn's disease.

Crohn's disease (CD) is a complex and multifactorial illness. There are still considerable gaps in our knowledge regarding its pathophysiology. A transcriptomic approach could shed some light on little-known biological alterations of the disease. We therefore aimed to explore the ileal transcriptome to gain knowledge about CD. We performed whole transcriptome gene expression analysis on ileocecal resections from CD patients and inflammatory bowel disease-free controls, as well as on a CD-independent cohort to replicate selected results. Normalized data were hierarchically clustered, and gene ontology and the molecular network were studied. Cell cultures and molecular methods were used for further evaluations. Genome-wide expression data analysis identified a robust transmembrane immunoglobulin domain-containing 1 (TMIGD1) gene underexpression in CD tissue, which was even more marked in inflamed ileum, and which was replicated in the validation cohort. Immunofluorescence showed TMIGD1 to be located in the apical microvilli of well-differentiated enterocytes but not in intestinal crypt. This apical TMIGD1 was lower in the noninflamed tissue and almost disappeared in the inflamed mucosa of surgical resections. In vitro studies showed hypoxic-dependent TMIGD1 decreased its expression in enterocyte-like cells. The gene enrichment analysis linked TMIGD1 with cell recovery and tissue remodeling in CD settings, involving guanylate cyclase activities. Transcriptomics may be useful for finding new targets that facilitate studies of the CD pathology. This is how TMIGD1 was identified in CD patients, which was related to multiciliate ileal epithelial cell differentiation.NEW & NOTEWORTHY This is a single-center translational research study that aimed to look for key targets involved in Crohn's disease and define molecular pathways through different functional analysis strategies. With this approach, we have identified and described a novel target, the almost unknown TMIGD1 gene, which may be key in the recovery of injured mucosa involving intestinal epithelial cell differentiation.

Immunological Differences between Lymphocytic and Collagenous Colitis.

BACKGROUND: Lymphocytic (LC) and collagenous (CC) colitis are the two major forms of microscopic colitis (MC). The aim of this study was to identify similarities and differences in their mucosal immune characteristics. METHODS: Colonic biopsies from 15 CC, 8 LC, and 10 healthy controls were collected. Mucosal lymphocytes were assessed by flow cytometry. Tissue gene expression and protein levels were determined by real-time PCR and ELISA, respectively. RESULTS: LC patients had lower numbers of CD4(+) and double-positive CD4(+)CD8(+)mucosal T lymphocytes, and higher numbers of CD8(+) and CD4(+)TCRγδ(+) mucosal T cells, compared with controls and CC patients. Regulatory Treg (CD4(+)CD25(+)FOXP3(+)) and double-negative (CD3(+)CD4(-)CD8(-)) T cell percentages were higher in both CC and LC compared with controls, coupled with higher levels of the anti-inflammatory IL-10, both at mRNA and protein levels. By contrast, Th1 and Th17 cells were lower in both CC and LC, although gene expression of Th1/Th17 cytokines was higher in both. CONCLUSION: CC and LC share some regulatory and effector mechanisms, but not others. Higher IL-10 levels and higher Treg and double-negative T cell percentages, found in both CC and LC, could be responsible for the lack of progression of structural damage and the blockade of proinflammatory cytokine production. However, CC and LC are revealed as separate, independent entities, as they show clearly different mucosal lymphocyte profiles, which could be caused by different luminal triggers of the two diseases. Hence, CC and LC are two closely related but independent intestinal disorders.

Regional Specialisation of T Cell Subsets and Apoptosis in the Human Gut Mucosa: Differences Between Ileum and Colon in Healthy Intestine and Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: There is very limited information regarding region-specific immunological response in human intestine. We aimed to determine differences in immune compartmentalisation between ileum and colon in healthy and inflamed mucosa. METHODS: T cell profile and its apoptosis were measured by flow cytometry, Th1, Th17, Treg [CD4(+)CD25(+)FOXP3(+)], double positive [DP, CD3(+)CD4(+)CD8(+)] and double negative T cells [DN, CD3(+)CD4(-)CD8(-)], immunohistochemistry [FOXP3, caspase-3], and real-time polymerase chain reaction [PCR] [IFN-γ, IL-17-A, and FOXP3] on biopsies from different regions of healthy intestine and of intestine in inflammatory bowel diseases. RESULTS: Healthy colon showed higher percentages of Treg, Th17, and DN, and lower numbers of DP T cells compared with ileum [p < 0.05]. Some but not all region-specific differences were lost in inflammatory conditions. Disease-specific patterns were found: a Th1/Th17 pattern and a Th17 pattern in Crohn's disease and ulcerative colitis respectively, whereas a reduction in Th1/Th17 was found in microscopic colitis. In colonic Crohn's disease and microscopic colitis, DN T cells had a pattern inverse to that of Th1/Th17 (increase in microscopic colitis [p < 0.05] and decrease in Crohn's disease [p < 0.005]). Higher levels of lymphocyte apoptosis were found in healthy colon compared with the ileal counterparts [p = 0.001]. All forms of colonic inflammation presented a dramatic decrease in apoptosis compared with healthy colon. By contrast ileal Crohn's disease showed higher levels of cleaved-Caspase(+) CD3(+) cells. CONCLUSIONS: Immunological differences exist in healthy gastrointestinal tract. Inflammatory processes overwhelm some location-specific differences, whereas others are maintained. Care has to be taken when analysing immune response in intestinal inflammation, as location-specific differences may be relevant.

Interleukin-10 Enhances the Intestinal Epithelial Barrier in the Presence of Corticosteroids through p38 MAPK Activity in Caco-2 Monolayers: A Possible Mechanism for Steroid Responsiveness in Ulcerative Colitis.

Glucocorticosteroids are the first line therapy for moderate-severe flare-ups of ulcerative colitis. Despite that, up to 60% of patients do not respond adequately to steroid treatment. Previously, we reported that low IL-10 mRNA levels in intestine are associated with a poor response to glucocorticoids in active Crohn's disease. Here, we test whether IL-10 can favour the response to glucocorticoids by improving the TNFα-induced intestinal barrier damage (assessed by transepithelial electrical resistance) in Caco-2 monolayers, and their possible implications on glucocorticoid responsiveness in active ulcerative colitis. We show that the association of IL-10 and glucocorticoids improves the integrity of TNFα-treated Caco-2 cells and that p38 MAPK plays a key role. In vitro, IL-10 facilitates the nuclear translocation of p38 MAPK-phosphorylated thereby modulating glucocorticoids-receptor-α, IL-10-receptor-α and desmoglein-2 expression. In glucocorticoids-refractory patients, p38 MAPK phosphorylation and membrane desmoglein-2 expression are reduced in colonic epithelial cells. These results suggest that p38 MAPK-mediated synergism between IL-10 and glucocorticoids improves desmosome straightness contributing to the recovery of intestinal epithelium and reducing luminal antigens contact with lamina propria in ulcerative colitis. This study highlights the link between the intestinal epithelium in glucocorticoids-response in ulcerative colitis.

Comparison of lymphocyte isolation methods for endoscopic biopsy specimens from the colonic mucosa.

An ideal method of immune cell isolation should provide maximum cell yield without disturbing functional properties. Intestinal endoscopic biopsies, in contrast to surgical samples, allow the study of all disease stages but have the drawback of a minimum amount of tissue available, making protocol optimization mandatory. We compared for the first time two methods of separation of colonic epithelium and five methods of lamina propria cell isolation for colonic biopsy specimens (mechanical, enzymatic and organ culture protocols). Lymphocyte number, viability and phenotype (CD45+, CD103+, CD3+, CD4+, CD8+, CD19+, CD16-56+) were analyzed by flow cytometry. Neither of the two epithelial detachment protocols achieved proper epithelial separation, though the high intensity ion chelation method was more accurate. Maximum cell yield of lamina propria lymphocytes without phenotypic modification was obtained with overnight smooth enzymatic digestion. High dose collagenase incubation caused a marked decrease in CD4+ lymphocytes of the lamina propria as compared to low enzymatic method (p=0.004). Mechanical and biopsy culture are not advisable methods because of the low cell yield, and phenotypic alterations and high contamination rate, respectively.

Bacteria and spontaneous experimental colitis: immunological changes.

BACKGROUND: Intestinal commensal flora seems to be a requisite for both human and experimental intestinal inflammation. Our aim was to assess the immunological changes in the colon of IL-10(-/-) mice depending on the environmental conditions. MATERIALS AND METHODS: Twelve wild-type (WT) and 24 IL-10(-/-) 4-week-old mice were kept under specific pathogen-free (SPF) conditions for 4 weeks. Half of them were transferred to a conventional environment. Mice were sacrificed at 12 weeks of age, and the incidence and severity of colitis was assessed. Intraepithelial (IEL) and lamina propria (LPL) lymphocytes were assessed for phenotype and apoptosis by flow cytometry. Toll-like receptors 2 (TLR2) and TLR9 expression was assessed by real-time PCR. Immunohistochemical analyses for cell apoptosis, TLR2 and MyD88 were also performed. RESULTS: IL-10(-/-) mice shifted to conventional conditions showed a greater incidence (66% vs. 50%) and severity of colitis than animals kept under SPF conditions (P = 0·009). The number of CD3+ IEL was higher and their apoptosis rate lower in IL-10(-/-) than in their WT counterparts, regardless of the environment. In LPL, however, these differences were only observed in mice shifted to conventional conditions. TLR2 expression was significantly increased in SPF-housed IL-10(-/-) mice when compared to WT controls. Immunohistochemistry demonstrated the loss of TLR2 and MyD88 in damaged areas. CONCLUSIONS: In SPF conditions, IL-10 deficiency appears to be compensated by an increased epithelial TLR2 expression, thus resulting in a milder colonic damage. However, in conventional conditions, this compensatory mechanism would be exceeded inducing a more severe colonic damage with activation of LPL immune cells.

Therapeutic effect of antisecretory factor-rich egg yolk on the late phases of 2,4,6-trinitrobenzenesulphonic acid colitis in mice.

Antisecretory factor (AF) is expressed in all tissues of mammals, inhibits intestinal hypersecretion and has anti-inflammatory properties as well. Endogenous AF synthesis may be stimulated by feeding hydrothermally processed cereals. Alternatively, freeze-dried egg yolk can be used as a source of exogenous AF. Several reports have suggested that AF from freeze-dried egg yolk may be useful in inflammatory bowel disease. We assessed the effect of freeze-dried, AF-rich egg yolk intake on 2,4,6-trinitrobenzenesulphonic acid (TNBS) colitis. Balb/c mice were randomised to receive (1) AF in sterile drinking-water (4 g/l, n 38) and (2) sterile drinking-water alone (vehicle, n 38) from TNBS or saline administration onwards. Different subsets of mice were killed at weeks 1-3 after TNBS or saline administration. Macroscopic and microscopic damage was assessed in colonic specimens. Eicosanoid and cytokine production was evaluated in supernatants of 24 h-incubated colonic explants. Myeloperoxidase activity was measured in frozen colonic samples, while apoptosis was assessed in paraffined samples by the in situ oligoligation method. AF-treated mice showed a milder colonic damage compared with the vehicle group, which became statistically significant at week 3. This was accompanied by decreased IL-2, IL-1 and leukotriene B4 production at weeks 2 and 3, as well as increased interferon-γ at week 1, in AF-treated mice compared with vehicle-treated mice. AF-treated mice had significantly increased counts of apoptotic cells in the lamina propria at weeks 1 and 2 post-TNBS. In conclusion, the administration of AF-rich egg yolk has a therapeutic effect in the late phases of TNBS colitis in Balb/c mice.

Apoptosis resistance of mucosal lymphocytes and IL-10 deficiency in patients with steroid-refractory Crohn's disease.

BACKGROUND: Apoptosis resistance of T-cells is considered an abnormality of immune pathways in Crohn's disease (CD). It has been previously shown that corticosteroids induce apoptosis of cells involved in inflammation. Thus, our aim was to assess the apoptosis of mononuclear cells and pro/antiinflammatory cytokines in the intestinal mucosa of patients with active CD, related to steroid response, and identify cellular and molecular factors that may predict this response to therapy. METHODS: Patients with CD (n = 26), ulcerative colitis (UC) (n = 32), and controls (n = 10) were prospectively studied with mucosal biopsies before and 7-10 days after corticosteroid treatment. Immunophenotype and apoptosis of T and B lymphocytes, plasma cells, and macrophages were assessed by flow cytometry, immunohistochemistry, and immunofluorescence. The cytokine expression pattern was evaluated by quantitative polymerase chain reaction (PCR). RESULTS: Apoptosis resistance of T and B lymphocytes was observed only in steroid-refractory and -dependent CD patients as compared to responsive patients (P = 0.032; P = 0.004, respectively), being evident after steroid treatment. Interleukin (IL)-10 was markedly increased at baseline in steroid-responsive patients compared to the nonresponders (P = 0.006; sensitivity: 88.8%; specificity: 66.6% to predict steroid response). CONCLUSIONS: Apoptosis resistance of mucosal T and B cells in steroid-refractory and -dependent CD patients appears during the evolution of the acute phase, limiting its clinical application as a predictor marker. In contrast, increased expression of IL-10 at an early stage of active steroid-sensitive CD patients supports its usefulness at predicting a good steroid response. Steroid-dependent and -refractory CD patients share similar molecular and cellular pathophysiological mechanisms.

Lactobacillus fermentum CECT 5716 prevents and reverts intestinal damage on TNBS-induced colitis in mice.

BACKGROUND: Probiotics attenuate gut inflammation when administered before experimental colitis, but data on their effect after colitis induction are scarce. We aimed to evaluate the effects of Lactobacillus fermentum CECT 5716 on gut injury when administered either before or after trinitrobencene sulfonic acid (TNBS) colitis in Balb/c mice. METHODS: In a preventive study, probiotic or vehicle was administered for 2 weeks before colitis. Then mice were allocated to: probiotic + TNBS, probiotic + sham, vehicle + TNBS, or vehicle + sham, and sacrificed 72 hours later. In a therapeutic study, mice were allocated into the same groups as before. Probiotic or vehicle were administered for 3 weeks. Mice were sacrificed at weeks 1, 2, and 3 after TNBS. Histological score, myeloperoxidase activity, and eicosanoid and cytokine production in colonic explant cultures were measured. Immunohistochemistry for nitrotyrosine and MyD88 was also performed. RESULTS: In the preventive study, colitis was milder with probiotic than with vehicle (P = 0.041). This was associated with increased PGE(2), IL-2, and IL-4 production, as well as attenuated nitrotyrosine staining in the former. In the therapeutic study, histological score at week 1 post-TNBS was higher in probiotic than in vehicle fed mice (P = 0.018). However, at weeks 2 and 3 the histological score was significantly lower-with decreased IL-6 production and increased MyD88 staining-in mice receiving the probiotic. CONCLUSIONS: Pretreatment with L. fermentum CECT 5716 attenuates TNBS colitis, an effect that seems to be due to its antioxidant abilities. When administered after TNBS, this probiotic is also effective in accelerating colitis recovery, and this is associated with an enhanced Toll-like receptor function.

Partial replacement of dietary (n-6) fatty acids with medium-chain triglycerides decreases the incidence of spontaneous colitis in interleukin-10-deficient mice.

Enteral nutrition has a primary therapeutic effect in active Crohn's disease. It is unknown which nutrient(s) account for this action, but a role for both the amount and type of dietary fat has been postulated. Some clinical and experimental data suggest that medium-chain triglycerides (MCT) may reduce intestinal inflammation. We aimed to assess the effect of replacing part of the dietary fat with MCT on the incidence and severity of colitis in interleukin (IL)-10(-/-) mice under specific pathogen-free conditions. Twenty-four IL-10(-/-) 4-wk-old mice were randomized to receive a control diet based on sunflower oil [(n-6) fatty acids (FA)] and an experimental isocaloric, isonitrogenous diet with 50% sunflower and 50% coconut oil (MCT diet). When the mice were 12 wk old, they were killed and the colon was examined for the presence of colitis, lymphocyte subpopulations and apoptosis, ex vivo cytokine production in supernatant of colon explants, toll-like receptor (TLR)-2 and TLR-9 mRNA, and FA profile in colonic tissue homogenates. Colitis incidence was lower in the IL-10(-/-) mice fed the MCT diet (1/12) than in the mice fed the control diet (8/12; P = 0.03). The histological damage score was also lower in the former (P < 0.0005). Feeding the MCT diet resulted in fewer total and apoptotic intraepithelial CD3+ and lamina propria CD3+CD4+ lymphocytes, as well as downregulated production of IL-6 and interferon-gamma, and reduced TLR-9 mRNA. We conclude that partial replacement of dietary (n-6) FA with MCT decreases the incidence of colitis in a model of spontaneous intestinal inflammation and provide experimental arguments for a possible primary therapeutic effect of MCT in human Crohn's disease.

Macronutrients and bioactive molecules: is there a specific role in the management of inflammatory bowel disease?

The effect of bioactive nutrient molecules on inflammatory response has an archetype in Inflammatory Bowel Disease. The exacerbated inflammatory response in such conditions can be nutritionally modified by 2 ways: changing the response of the host, or changing the composition of the intestinal ecosystem. Host response can be modified by changing the cell structure and function which is nutrient dependent. Nutrient deprivation will lead to a situation where there is not enough building material for cell replacement and the synthesis of mediators (enzymes, hormones, etc). However, this may occur even in a situation where there is no quantitative nutrient deprivation but only qualitative changes. In Inflammatory Bowel Disease, changes in the sources of some nutrients such as lipids or carbohydrates (CHO) can modify the inflammatory response. Lipids, by changing cell membrane composition, may modify the pattern of eicosanoid synthesis, intracellular signal transmission and activation of nuclear transcription factors, which modify the expression of some genes-that is, changing the host response. On the other hand, certain sources of carbohydrates, by undergoing anaerobic bacterial fermentation, drop the pH in the intestinal lumen favoring the growth of certain strains of bacteria which act favorably in maintaining tolerance in the bowel. In addition, as a consequence of CHO fermentation, short-chain fatty acids are produced which, especially butyrate, may act in 2 ways: by providing energy to the epithelial cells, but also as anti-inflammatory substrate-that is, modifying at least 1 of the mechanisms triggering the inflammatory response enhancement. However, it should not be forgotten that the cellular response to dietary modifications will depend on the individual genome, as has been recently observed. This may explain why some individuals do and others do not show a similar response to dietary interventions.