Intestinal Mucosal Tissue Research Articles

LncRNACNN3-206 activates intestinal epithelial cell apoptosis and invasion by sponging miR-212, an implication for Crohn's disease

BACKGROUNDStatistics indicate that the incidence of Crohn’s disease (CD) is rising in many countries. The poor understanding on the pathological mechanism has limited the development of effective therapy against this disease. Previous studies showed that long noncoding RNAs (lncRNAs) could be involved in autoimmune diseases including CD, but the detailed molecular mechanisms remain unclear.AIMTo identify the differentially expressed lncRNAs in the intestinal mucosa associated with CD, and to characterize their pathogenic role(s) and related mechanisms.METHODSThe differential expression of lncRNAs was screened by high-throughput RNA sequencing, and the top candidate genes were validated in an expanded cohort by real-time PCR. The regulatory network was predicted by bioinformatic software and competitive endogenous RNA analysis, and was characterized in Caco-2 and HT-29 cell culture using methods of cell transfection, real-time PCR, Western blotting analysis, flow cytometry, and cell migration and invasion assays. Finally, these findings were confirmed in vivo using a CD animal model.RESULTSThe 3' end of lncRNACNN3-206 and the 3’ UTR of Caspase10 contain high-affinity miR212 binding sites. lncRNACNN3-206 expression was found to be significantly increased in intestinal lesions of CD patients. Activation of the lncRNACNN3-206-miR-212-Caspase10 regulatory network led to increased apoptosis, migration and invasion in intestinal epithelial cells. Knockdown of lncRNACNN3-206 expression alleviated intestinal mucosal inflammation and tissue damage in the CD mouse model.CONCLUSIONlncRNACNN3-206 may play a key role in CD pathogenesis. lncRNACNN3-206 could be a therapeutic target for CD treatment.

DOP64 Discovery of highly selective small-molecule oral inhibitors of integrin α4β7 for the treatment of inflammatory bowel diseases

Abstract Background Integrins play a key role in facilitating immune cell trafficking throughout the body and represent an important receptor family for therapeutic intervention. In particular, the α4β7 integrin is a clinically validated target for the treatment of inflammatory bowel diseases (IBD), as exemplified by the humanised monoclonal antibody vedolizumab, which blocks the interactions between α4β7-expressing lymphocytes and its ligand MAdCAM-1. This blockade leads to the inhibition of these circulating lymphocytes from exiting the bloodstream and entering intestinal mucosal tissues resulting in a decrease in mucosal inflammation in patients. While oral inhibitors of the α4β7 integrin are advantageous over biologics, the efforts have been impeded by challenges to achieve desired selectivity and optimal DMPK properties. The aim of this study was to develop and characterise orally bioavailable small-molecule inhibitors of the α4β7 integrin and to determine their therapeutic potential. Methods Oral small-molecule inhibitors targeting the α4β7 integrin were discovered using Morphic Integrin Technology (MInT) platform. These small-molecule inhibitors were tested for potency and selectivity against a broad panel of integrin family members in multiple biochemical and cell-based functional assays in a ligand-competitive fashion. An acute PD assay with CFSE-labelled lymphocytes was developed to evaluate the activity of the small-molecule compounds in blocking lymphocyte trafficking to gut-associated lymphoid tissues in mice. The in vivo activity was also examined through changes in circulating α4β7+ CD4+ T memory cells in a relevant non-human primate model. Results Key drug candidate small molecules demonstrated over 1000-fold selectivity in vitro against a broad panel of integrin family members, including the α4β1 integrin. These compounds effectively blocked lymphocyte trafficking to mesenteric lymph nodes and Peyer’s patches in the gut in a dose-dependent manner, similar to an α4β7-specific antibody, in an acute gut-homing assay in mice. Additionally, these inhibitors also demonstrated effective occlusion of immune trafficking in a relevant non-human primate model. The lead compound has favourable DMPK properties, good oral bioavailability and is projected to have sufficient exposure in humans to effectively block α4β7-expressing immune cells in circulation. Conclusion Potent, selective, oral small-molecule inhibitors of α4β7 integrin have been discovered that demonstrate on-target, mechanistic efficacy in two animal models relevant to human IBD. It has the potential to be an effective and safe therapeutic in monotherapy as well as serving as a backbone for combination with other IBD drugs.

Gut Microbiota and Cancer of the Host: Colliding Interests.

Cancer develops in multicellular organisms from cells that ignore the rules of cooperation and escape the mechanisms of anti-cancer surveillance. Tumorigenesis is jointly encountered by the host and microbiota, a vast collection of microorganisms that live on the external and internal epithelial surfaces of the body. The largest community of human microbiota resides in the gastrointestinal tract where commensal, symbiotic and pathogenic microorganisms interact with the intestinal barrier and gut mucosal lymphoid tissue, creating a tumor microenvironment in which cancer cells thrive or perish. Aberrant composition and function of the gut microbiota (dysbiosis) has been associated with tumorigenesis by inducing inflammation, promoting cell growth and proliferation, weakening immunosurveillance, and altering food and drug metabolism or other biochemical functions of the host. However, recent research has also identified several mechanisms through which gut microbiota support the host in the fight against cancer. These mechanisms include the use of antigenic mimicry, biotransformation of chemotherapeutic agents, and other mechanisms to boost anti-cancer immune responses and improve the efficacy of cancer immunotherapy. Further research in this rapidly advancing field is expected to identify additional microbial metabolites with tumor suppressing properties, map the complex interactions of host-microbe 'transkingdom network' with cancer cells, and elucidate cellular and molecular pathways underlying the impact of specific intestinal microbial configurations on immune checkpoint inhibitor therapy.

Clinical features of Epstein-Barr virus in the intestinal mucosa and blood of patients with inflammatory bowel disease.

Background:The role of Epstein–Barr virus (EBV) in inflammatory bowel disease (IBD) remains to be elucidated. The aim of this study was to investigate the presence of EBV in the blood and intestinal mucosa of patients with IBD and evaluate the association between EBV positivity and IBD.Methods:Patients with IBD, hospitalized between January 2015 and April 2018, were enrolled. The EBV-DNA load in blood samples from each subject was analyzed by quantitative real-time polymerase chain reaction. EBV-encoded small-RNA 1 (EBER-1) was detected by in-situ hybridization in intestinal mucosa tissue sections of patients with IBD.Result:EBV-DNA was detected in 48 out of 568 patients with IBD (8.4%), and EBER-1 positivity was detected in 27 of these patients (56.3%). Refractory IBD and severe mucosal inflammation were more common in patients with detectable levels of EBER-1 than in those without; the number of EBER-1-positive cells positively correlated with mucosal inflammation (P value < 0.05). Age (≥60 years old) and use of azathioprine were risk factors for EBV infection. There was no significant difference in clinical remission rate and surgical rate between the EBER-1 positive group and EBER-1 negative group, antiviral group and the non-antiviral group, among IBD patients who tested positive for EBV-DNA.Conclusion:Elderly patients with IBD, treated with azathioprine, are more susceptible to EBV positivity. Further, EBV mucosal detection correlated with the severity of mucosal damage and refractoriness, but not prognosis.

Long Noncoding RNA H19 Impairs the Intestinal Barrier by Suppressing Autophagy and Lowering Paneth and Goblet Cell Function.

Background & AimsThe protective intestinal mucosal barrier consists of multiple elements including mucus and epithelial layers and immune defense; nonetheless, barrier dysfunction is common in various disorders. The imprinted and developmentally regulated long noncoding RNA H19 is involved in many cell processes and diseases. Here, we investigated the role of H19 in regulating Paneth and goblet cells and autophagy, and its impact on intestinal barrier dysfunction induced by septic stress.MethodsStudies were conducted in H19-deficient (H19-/-) mice, mucosal tissues from patients with sepsis, primary enterocytes, and Caco-2 cells. Septic stress was induced by cecal ligation and puncture (CLP), and gut permeability was detected by tracer fluorescein isothiocyanate–dextran assays. The function of Paneth and goblet cells was examined by immunostaining for lysozyme and mucin 2, respectively, and autophagy was examined by microtubule-associated proteins 1A/1B light chain 3 II immunostaining and Western blot analysis. Intestinal organoids were isolated from H19-/- and control littermate mice and treated with lipopolysaccharide (LPS).ResultsIntestinal mucosal tissues in mice 24 hours after exposure to CLP and in patients with sepsis showed high H19 levels, associated with intestinal barrier dysfunction. Targeted deletion of the H19 gene in mice enhanced the function of Paneth and goblet cells and promoted autophagy in the small intestinal mucosa. Knockout of H19 protected Paneth and goblet cells against septic stress, preserved autophagy activation, and promoted gut barrier function after exposure to CLP. Compared with organoids from control littermate mice, intestinal organoids isolated from H19-/- mice had increased numbers of lysozyme- and mucin 2–positive cells and showed increased tolerance to LPS. Conversely, ectopic overexpression of H19 in cultured intestinal epithelial cells prevented rapamycin-induced autophagy and abolished the rapamycin-induced protection of the epithelial barrier against LPS.ConclusionsIn investigations of mice, human tissues, primary organoids, and intestinal epithelial cells, we found that increased H19 inhibited the function of Paneth and goblet cells and suppressed autophagy, thus potentially contributing to barrier dysfunction in intestinal pathologies.

Protective effect of extracorporeal membrane oxygenation on intestinal mucosal injury after cardiopulmonary resuscitation in pigs.

The present study aimed to explore the protective effects of extracorporeal membrane oxygenation (ECMO) on intestinal mucosal injury following cardiopulmonary resuscitation (CPR), and to assess the potential mechanisms involved. A total of 24 healthy adult domestic pigs were selected as the study subjects. A ventricular fibrillation model was induced through programmed electric stimulation. Subsequently, the animals were randomly divided into conventional CPR and CPR+ECMO groups (n=12 per group). The mortality and hemodynamic parameters of the two groups were compared. The expression levels of inflammatory cytokines in the serum and intestinal mucosa were detected by ELISAs. The intestinal mucosa was subjected to hematoxylin and eosin, and immunohistochemical staining, followed by electron microscopy, to assess the degree of apoptosis and necrosis. The animals in both groups recovered from the programmed ventricular fibrillation. In the CPR group, two animals died at 2 h and two more animals died a further 2 h later, resulting in a 33.3% mortality rate, whereas no cases of mortality were observed in the CPR+ECMO group. Compared with the animals in the CPR group, the hemodynamic parameters of the animals in the CPR+ECMO group revealed significantly improved outcomes. Multiple inflammatory factors (tumor necrosis factor α, interleukin-1 and interleukin-6), myeloperoxidase and malondialdehyde levels were decreased, whereas Na/Ca-ATPase and superoxide dismutase levels were elevated in the intestinal mucosa of animals in the CPR+ECMO group compared with those in the CPR group. Additionally, pathological staining demonstrated that the intestinal mucosa tissue in the CPR+ECMO group exhibited less apoptosis, necrosis and inflammatory cell infiltration, which was further supported by a decrease in Bax expression and an increase in Bcl-2 expression. Overall, ECMO after CPR reduced the intestinal mucosal barrier injury after spontaneous circulation recovery, and the mechanism involved decreased inflammation and apoptosis.

Cyclophosphamide Treatment Mimics Sub-Lethal Infections With Encephalitozoon intestinalis in Immunocompromised Individuals.

Microsporidia, including Encephalitozoon intestinalis, are emerging pathogens which cause opportunistic infections in immunocompromised patients, such as those with AIDS, cancer, the elderly and people on immunosuppressive drugs. Intestinal mucosa (IM) is crucial for developing an efficient adaptive immune response against pathogenic micro-organisms, thereby preventing their colonization and subsequent infection. As immunosuppressive drugs affect the intestinal immune response is little known. In the present study, we investigated the immune response to E. intestinalis infection in the IM and gut-associated lymphoid tissue (GALT) in cyclophosphamide (Cy) immunosuppressed mice, to mimic an immunocompromised condition. Histopathology revealed lymphoplasmacytic enteritis at 7 and 14 days-post-infection (dpi) in all infected groups, however, inflammation diminished at 21 and 28 dpi. Cy treatment also led to a higher number of E. intestinalis spores and lesions, which reduced at 28 dpi. In addition, flow cytometry analysis demonstrated CD4+ and CD8+ T cells to be predominant immune cells, with up-regulation in both Th1 and Th2 cytokines at 7 and 14 dpi, as demonstrated by histopathology. In conclusion, Cy treatment reduced GALT (Peyer’s plaques and mesenteric lymph nodes) and peritoneum populations but increased the T-cell population in the intestinal mucosa and the production of pro-and anti-inflammatory cytokines, which were able to eliminate this opportunistic fungus and reduced the E. intestinalis infection.

DESIGN, DEVELOPMENT, AND CHARACTERIZATION OF OCIMUM BASILICUM MUCILAGE-BASED, MODIFIED RELEASE MUCOADHESIVE GASTROSPHERES OF CARVEDILOL

Objective: The present investigations aim to develop mucoadhesive gastrospheres of carvedilol using sodium alginate and Ocimum basilicum seed mucilage combination blend oral use.&#x0D; Methods: The gastrospheres were prepared by ionotropic gelation method using 32 factorial designs, the concentration of sodium alginate and O. basilicum mucilage was independent variables while % drug content (DC), % entrapment efficiency, and % drug release at 12 h were dependent variables. The gastrospheres were evaluated for other parameters such as Fourier-transform infrared (FTIR), powder X-ray diffraction, differential scanning calorimetric, and in vitro mucoadhesion studies.&#x0D; Results: In optimization studies from statistical second-order complete model equation among the polymers used O. basilicum mucilage had a more profound effect on DC and % encapsulation efficiency as compared to sodium alginate. The mean particle size of gastrospheres when measured by optical microscopy technique ranged from 774 to 882 μm. The percentage of gastrosphere adhering to goat intestinal mucosal tissue varied from 10% to 65% over 8 h in 0.1N HCl, whereas this was varied from 40% to 60% in phosphate buffer pH 6.8 with provided 12 h of controlled release following zero-order release pattern.&#x0D; Conclusion: Studies conclude that mucilage of O. basilicum can be used as controlled release mucoadhesive material in the formulation of gastrospheres.

Hsa-miR-375 promotes the progression of inflammatory bowel disease by upregulating TLR4.

To elucidate the biological function of hsa-miR-375 in the progression of inflammatory bowel disease (IBD) and the potential mechanism. Intestinal mucosa tissues of 26 IBD patients and 30 healthy volunteers who underwent colonoscopy were harvested for determining hsa-miR-375 level by quantitative Real-time polymerase chain reaction (qRT-PCR). Binding of hsa-miR-375 to toll-like receptor 4 (TLR4) was verified by the dual-luciferase reporter gene assay. Changes in the viability and apoptosis in Caco-2 cells influenced by hsa-miR-375 were examined by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. The regulatory effect of hsa-miR-375 on the intestinal epithelial barrier was examined by detecting transepithelial electrical resistance (TEER) and lucifer yellow flux. Relative levels of TLR4, nuclear factor-kappa B (NF-κB), zonula occludens-1 (ZO-1), occludin and inflammatory factors in Caco-2 cells were detected by qRT-PCR, Western blot and enzyme-linked immunosorbent assay (ELISA). Hsa-miR-375 was downregulated in intestinal mucosa tissues of patients with Crohn's disease (CD) and ulcerative colitis (UC). Knockdown of hsa-miR-375 decreased viability and TEER, but elevated apoptotic rate and lucifer yellow flux. Overexpression of hsa-miR-375 achieved the opposite trends. TLR4 was the direct downstream of hsa-miR-375, and its level was negatively mediated by hsa-miR-375. In addition, TLR4 level in Caco-2 cells was upregulated after LPS induction, while hsa-miR-375 level was unchangeable. Knockdown of hsa-miR-375 upregulated NF-κB and pro-inflammatory factors TNF-α, IL-1β, IL-6 and IL-8, and downregulated ZO-1, occludin and anti-inflammatory factor IL-10. Hsa-miR-375 is involved in the pathogenesis of IBD by upregulating TLR4 and inducing NF-κB activation.

Significance of intestinal fatty acid binding protein in evaluation of intestinal barrier dysfunction of mice at early stage of severe burn injury

Objective: To investigate the significance of intestinal fatty acid binding protein (IFABP) in the evaluation of intestinal barrier dysfunction of mice at the early stage of severe burn injury. Methods: Thirty-six 8-week-old C57BL/6 male mice were collected and divided into normal control group (n=6) and scald group (n=30) according to random number table. Back of each mouse in scald group was placed into hot water of 90 ℃ for 10 s, causing full-thickness scald (hereinafter refer to as burn) of 30% total body surface area, while mice in normal control group were not inflicted with burns. Six mice in normal control group were taken, and 6 mice in scald group at 1, 2, 6, 12, and 24 h post injury were taken respectively. The portal vein blood of each mouse was extracted and the plasma was separated to measure intestinal permeability with fluorescin isothiocyanate-dextran fluorescence probe tracing method and plasma IFABP content by enzyme-linked immunosorbent assay. The distal ileum tissue of mice in normal control group and scald group at each time point post injury was collected to observe the morphology of the intestinal mucosa tissue by hematoxylin-eosin staining. Data were processed with one-way analysis of variance and Student-Newman-Keuls test, and pearson correlation test was used to analyze the correlation between intestinal permeability and plasma IFABP content of burned mice. Results: (1) At 1, 2, 6, 12, and 24 h post injury, the intestinal permeability of mice in scald group was 2.7±0.8, 5.4±2.5, 7.3±4.2, 12.4±6.1, 1.4±0.7, respectively, obviously higher than 1.0±0.4 of normal control group (P<0.05 or P<0.01). The intestinal permeability of mice in scald group showed an increasing trend post injury, reaching the peak at 12 h post injury, and rapidly falling back at 24 h post injury. (2) At 1, 2, 6, 12, and 24 h post injury, the plasma IFABP content of mice in scald group was (64±11), (59±12), (76±18), (111±22), and (66±10) ng/mL, obviously higher than (35±8) ng/mL in normal control group (P<0.05 or P<0.01). The plasma IFABP content of mice in scald group showed an increasing trend post injury, reaching the peak at 12 h post injury, and rapidly decreasing at 24 h post injury. (3) Uniform thickness of mucosa, intact epithelia, regularly arranged villi, and no inflammatory cell infiltration were observed in ileum of mice in normal control group. In ileum of mice in scald group, shortened villi of mucosa with different degrees, edema of lamina propria, and infiltration of neutrophils were observed at 1 and 2 h post injury; obviously damaged and partially exfoliated ileal mucosa, disorderly arranged and broken villi, degenerated and necrotic epithelial cells, dilated central lacteal, and infiltration of lymphocytes and neutrophils were observed at 6 and 12 h post injury; the damage of ileal mucosa was alleviated, and basically intact epithelia, dilated central lacteal, and infiltration of inflammatory cells were observed at 24 h post injury. (4) There was a significantly positive correlation between the intestinal permeability and the plasma IFABP content of burned mice (r=0.841, P<0.05). Conclusions: The plasma IFABP can be used as a good biological indicator for the evaluation of intestinal barrier dysfunction of mice at the early stage of severe burn injury.

MLKL attenuates colon inflammation and colitis-tumorigenesis via suppression of inflammatory responses.

The mixed lineage kinase domain-like protein (MLKL) has emerged as a critical mediator of necroptosis, which results in the release of cellular damage-associated molecular patterns (DAMPs). However, its physiological role in regulating inflammation is not fully understood. We herein showed that Mlkl-/- mice were highly susceptible to colitis and colitis-associated tumorigenesis (CAT), which was associated with massive leukocyte infiltration and increased inflammatory responses. Moreover, we used bone marrow transplantation to reveal that MLKL in inflammatory cells is crucial for its role on colitis. Intestinal mucosal tissue and polyps isolated from Mlkl-/- mice exhibited increased ERK activation and elevated expression of genes associated with inflammation and cancer. Mechanistically, enhanced inflammation in Mlkl-/- mice was due to MEK/ERK activation particularly in dendritic cells (DCs). Our results demonstrate the role of MLKL in maintaining intestinal homeostasis and protecting against colitis and tumorigenesis.

RNA-Binding Protein HuR Regulates Paneth Cell Function by Altering Membrane Localization of TLR2 via Post-transcriptional Control of CNPY3

Paneth cells secrete antimicrobial proteins including lysozyme via secretory autophagy as part of the mucosal protective response. The ELAV like RNA-binding protein 1 (ELAVL1, also called HuR) regulates stability and translation of messenger RNAs (mRNAs) and many aspects of mucosal physiology. We studied the posttranscriptional mechanisms by which HuR regulates Paneth cell function. Intestinal mucosal tissues were collected from mice with intestinal epithelium (IE)-specific disruption of HuR (IE-HuR-/-), HuRfl/fl-Cre- mice (controls), and patients with inflammatory bowel diseases and analyzed by histology and immunohistochemistry. Paneth cell functions were determined by lysozyme-immunostaining assays. We isolated primary enterocytes from IE-HuR-/- and control mice and derived intestinal organoids. HuR and the chaperone CNPY3 were overexpressed from transgenes in intestinal epithelial cells (IECs) or knocked down with small interfering RNAs. We performed RNA pulldown assays to investigate interactions between HuR and its target mRNAs. Intestinal tissues from IE-HuR-/- mice had reduced numbers of Paneth cells, and Paneth cells had fewer lysozyme granules per cell, compared with tissues from control mice, but there were no effects on Goblet cells or enterocytes. Intestinal mucosa from patients with inflammatory bowel diseases had reduced levels of HuR and fewer Paneth cells. IE-HuR-/- mice did not have the apical distribution of TLR2 in the intestinal mucosa as observed in control mice. IECs from IE-HuR-/- mice expressed lower levels of CNPY3. Intestinal organoids from IE-HuR-/- mice were smaller and contained fewer buds compared with those generated from controls, and had fewer lysozyme-positive cells. In IECs, knockdown of HuR decreased levels of the autophagy proteins LC3-I and LC3-II, compared with control cells, and prevented rapamycin-induced autophagy. We found HuR to interact directly with the Cnpy3 mRNA coding region and increase levels of CNPY3 by increasing the stability and translation of Cnpy3 mRNA. CNPY3 bound TLR2, and cells with knockdown of CNPY3 or HuR lost membrane localization of TLR2, but increased cytoplasmic levels of TLR2. In studies of mice, IECs, and human tissues, we found HuR to increase expression of CNPY3 at the posttranscriptional level. CNPY3 is required for membrane localization of TLR2 and Paneth cell function.

The effects and mechanisms of myeloid differentiation protein 2 on intestinal mucosal permeability in mice with chronic colitis.

The present study was designed to investigate the mechanism of myeloid differentiation protein 2 (MD2) on intestinal mucosa destruction in mice with chronic colitis. Briefly, a chronic colitis mouse model was established by the administration of dextran sulfate sodium (DSS) in transgenic mice of MD2 overexpression (Transgenic, MD2-Tg) and C57BL/6 wild-type mice (MD2-WT). In addition, Caco-2 cells were cultured to form a monolayer cell model in vitro. The small interfering RNA was utilized to silence the MD2 gene in Caco-2 cells, and tumor necrosis factor-α (TNF-α) was used to establish the model of intestinal mucosal inflammation. After DSS induction, the intestinal mucosal tissue inflammation was more severe in MD2-Tg mice than MD2-WT. In addition, the intestinal mucosa was severely damaged, the intestinal mucosal permeability was increased, bacterial translocation was obvious, and the expression levels of MD2, MyD88, Toll-like receptor 4 (TLR4), and HMGB1 in mucosal tissues were significantly increased, while the expression levels of tight junction proteins, occludin, and claudin-1 were significantly lower in MD2-Tg mice compared with those in MD2-WT mice. TNF-α could induce inflammatory apoptosis in Caco-2 cell models. After MD2 silencing, the apoptotic level was decreased, the value of transepithelial electrical resistance was increased, the permeability of intestinal mucosa was decreased, the cellular expression levels of MD2, MyD88, TLR4, and HMGB1 were decreased, while the expression levels of tight junction proteins, occludin and claudin-1 were increased. MD2 could aggravate the destruction of intestinal mucosa in chronic colitis through the HMGB1-TLR4-MyD88 pathway.

Molecular mechanisms of interleukin-38 inhibiting inflammatory bowel disease in children by regulating nuclear factor-κB and signal transduction and activator of transcription 3 pathway

Objective To explore the role of interleukin (IL)-38 in inhibiting inflammatory bowel disease (IBD) in children and to investigate the potential molecular mechanisms. Methods From January 2014 to October 2017, 67 patients with ulcerative colitis (UC) and 115 patients with Crohn′s disease (CD) admitted to Wuhan Children′s Hospital were recruited, and 40 individuals with normal endoscopic findings were selected as control. Serum levels of IL-38 of IBD patients and healthy control were determined by enzyme-linked immunosorbent assay (ELISA). Immunohistochemical staining (IHC) was used to detect the expression level of IL-38, nuclear factor κB (NF-κB), phosphorylated signal transduction and activator of transcription 3 (p-STAT3), C-reaction protein (CRP) and erythrocyte sedimentation rate (ESR) in the intestinal mucosa of IBD patients and healthy controls. The extent of disease, therapeutic agents and disease activity scores (Mayo score system for UC patients, Crohn′s disease activity index (CDAI) for CD patients) were evaluated. IL-38-C57BL/6 transgenic mice model was established, and dextran sulfate sodium was used to induce IBD mice model. The intestinal inflammation levels were compared between the wild type IBD mice and IL-38 transgenic IBD mice. The levels of IL-38, NF-κB and p-STAT3 in intestinal mucosa of mice of different groups were determined by IHC. The ratio of CD4+ IL-17+ T helper (Th) 17 cells in peripheral blood of mice of different groups was detected by flow cytometry. Independent sample t test, chi square test and Pearson correlation were performed for statistical analysis. Results The results of ELISA showed that the serum levels of IL-38 of UC and CD patients were (6.1±1.9) ng/L and (9.8±2.1) ng/L, respectively, which both were lower than that of healthy controls ((16.4±2.7) ng/L), and the differences were statistically significant (t=23.107 and 15.853, both P<0.05). The results of IHC indicated that the levels of IL-38 in the intestinal mucosal tissues of UC and CD patients were 0.04±0.01 and 0.03±0.01, respectively, which were both lower than that of healthy controls (0.18±0.02), and the differences were statistically significant (t=48.186 and 69.443, both P<0.05). The levels of NF-κB and p-STAT3 of UC and CD patients were 0.150±0.030, 0.160±0.040 and 0.130±0.030, 0.110±0.010, which were all higher than those of healthy controls (0.020±0.003 and 0.010±0.002), and the differences were statistically significant (tUC=27.273 and 23.078, tCD=23.657 and 62.684; all P<0.05). The number of patients with disease at active phase, CRP level, ESR and disease activity scores of UC and CD patients with low IL-38 expression were all significantly higher than those of patients with high IL-38 expression (χUC2=11.552, tUC=7.118, 8.991 and 7.086; χCD2=5.675, tCD=9.559, 9.358 and 11.268; all P<0.05). The results of Pearson correlation analysis demonstrated that the level of IL-38 in the intestinal mucosal tissue of UC patients was negatively correlated with CRP, ESR and Mayo scores (r=-0.291, -0.672 and -0.639; all P<0.05). And the level of IL-38 in the intestinal mucosal tissue of CD patients was negatively correlated with CRP, ESR and CDAI (r=-0.559, -0.471 and -0.353; all P<0.05). The IHC results showed that the levels of NF-κB and p-STAT3 of IL-38 transgenic IBD mice were lower than those of wild type IBD mice (0.14±0.02 vs. 0.32±0.06, 0.12±0.02 vs. 0.44±0.07), and the differences were statistically significant (t=6.971 and 10.767, both P<0.05). The results of flow cytometry showed that the ratio of CD4+ IL-17+ Th17 cells in the peripheral blood of IL-38 transgenic IBD mice was lower than that of wild type IBD mice (0.030±0.006 vs. 0.280±0.050), and the difference was statistically significant (t=12.160, P<0.05). Conclusions The expression level of IL-38 significantly decreases in the intestinal mucosal tissues of IBD patients, while the level of NF-κB and p-STAT3 significantly increases. IL-38 may inhibit IBD by regulating NF-κB and p-STAT3 signaling pathway to alleviate intestinal immune reaction. Key words: Inflammatory bowel diseases; NF-kappa B; interleukin-38; STAT3

Clock Gene Disruption Is an Initial Manifestation of Inflammatory Bowel Diseases

Sleep disruption modifies the immune system and can trigger flares of inflammatory bowel diseases (IBD). Changes in expression of clock genes have been reported in patients with IBD. We investigated whether a change in the circadian clock is an early event in development of IBD. We performed a prospective study of patients younger than 21 years old who underwent diagnostic endoscopies at the pediatric and adult gastroenterology units at the Tel Aviv Sourasky Medical Center from August 2016 through August 2017. Questionnaires were completed by 32 patients with IBD (8-21 years old) and 18 healthy individuals (controls) that provided data on demographics, sleep, disease activity scores. We also obtained data on endoscopic scores, anthropometric parameters, blood level of C-reactive protein (CRP), and fecal level of calprotectin. Peripheral blood and intestinal mucosa samples were analyzed for expression levels of clock gene (CLOCK, BMAL1, CRY1, CRY2, PER1, and PER2). Levels of CRP and fecal calprotectin were significantly higher in patients with IBD compared with controls (P<.05). Expression levels of clock genes (CLOCK, CRY1, CRY2, PER1, and PER2) were significantly lower in inflamed intestinal mucosa from patients compared with intestinal mucosa from controls (P<.05). Expression levels of all clock genes except for PER2, were also significantly lower in non-inflamed intestinal mucosal tissues from patients compared with controls (P<.05). Expression levels of clock genes (CLOCK, BMAL1, CRY1, CRY2, PER1 and PER2) were lower in white blood cells from patients with IBD compared with controls. This reduction was greater in white blood cells from patients with ulcerative colitis than in patients with Crohn's disease. Young, newly diagnosed, untreated patients with IBD have reduced expression of clock genes in inflamed and non-inflamed intestinal mucosal samples, and also in blood cells, compared with healthy individuals. Alterations in expression of clock genes might be an early event in IBD pathogenesis. ClinicalTrials.gov Identifier: NCT03662646.

Alterations of the Mice Gut Microbiome via Schistosoma japonicum Ova-Induced Granuloma.

Schistosomiasis, also called bilharziasis, is a neglected tropical disease induced by Schistosoma spp. that causes hundreds of millions of infections. Although Schistosoma ova-induced granulomas commonly cause inflammation, hyperplasia, ulceration, micro abscess formation, and polyposis, the role of the egg granuloma on the gut microbiome remains unclear. To explore the role, gut microbial communities in mice infected with Schistosoma japonicum were surveyed. Female C57BL/6 and BALB/c mice were exposed to cercariae of S. japonicum for 45 and 65 days and then sacrificed. Intestinal contents and feces were collected, DNA was extracted, and high-throughput 16S rRNA gene-based pyrosequencing was used to provide a comparative analysis of gut microbial diversity. The intestinal mucosal tissues were also examined. Histopathologic analysis demonstrated that the basic structure of the colonic mucosa was damaged by ova-induced granuloma. Regarding the gut microbiome, 2,578,303 good-quality sequences were studied and assigned to 25,278 Operational Taxonomic Units (OTUs) at a threshold of 97% similarity. The average number of OTUs for C57BL/6 and BALB/c were 545 and 530, respectively. At the phylum level, intestinal microbial communities were dominated by Firmicutes, Bacteroidetes, Proteobacteria, and Verrucomicrobia. Infection with S. japonicum modified bacterial richness in the fecal associated microbiota. Exposure significantly modified bacterial community composition among different groups. At the phylogenetic levels, LEfSe analysis revealed that several bacterial taxa were significantly associated with the S. japonicum-infected mice. The present results suggest that egg granulomas in the intestine influence differentiation of the gut microbial community under pathophysiological conditions. This result suggests that intestinal microbiome-based strategies should be considered for early diagnosis, clinical treatment, and prognosis evaluation of schistosomiasis.

Comparing DNA methylation profiles in saliva and intestinal mucosa

BackgroundAltered epigenetic profiles are a feature of intestinal diseases, including ulcerative colitis and Crohn’s disease. DNA methylation studies in these diseases have utilised intestinal mucosal tissue or blood which can be difficult to collect, particularly for large-scale research studies. Saliva is an attractive alternative for epigenetic studies as it is easy to collect and provides high quality methylation profiles. The aim of the study was to determine the utility of saliva as an alternative for DNA methylation studies of intestinal disorders.ResultsDNA methylation in saliva and intestinal mucosa samples were compared in individuals (n = 10) undergoing endoscopies using the Illumina Infinium Methylation 450 K Beadchip array. We found that DNA methylation was correlated between tissue types within an individual (Pearson correlation co-efficient r = 0.92 to 0.95, p < 0.001). Of the 48,541 probes (approximately 11% of CpG sites) that were differentially methylated between saliva and intestinal mucosa (adjusted p < 0.001, |Δβ| ≥ 20%), these mapped to genes involved in tissue-specific pathways, including the apelin signalling and oxytocin pathways which are important in gastrointestinal cytoprotection and motility.ConclusionsThis study suggests that saliva has the potential to be used as an alternate DNA source to invasive intestinal mucosa for DNA methylation research into intestinal conditions.

Expression and clinical significance of 5-HT and 5-HT3R in the intestinal mucosa of patient with diarrhea-type irritable bowel syndrome.

Expression levels and clinical significance of 5-HT and 5-HT3R in the intestinal mucosa tissue of patients with diarrhea-type irritable bowel syndrome (D-IBS) were investigated. A retrospective analysis was performed on 46 tissue specimens (observation group) of the intestinal mucosa of patients with D-IBS, who were diagnosed in the Tongde Hospital of Zhejiang Province and received colonoscopy from March 2016 to December 2017, and 18 tissue specimens (control group) of the intestinal mucosa of healthy subjects who received physical examinations. The expression levels of 5-HT and 5-HT3R in the intestinal mucosa tissue of patients in the observation and control group were detected by ELISA, and the relationship between 5-HT, 5-HT3R and the clinicopathological parameters of patients with D-IBS was analyzed. Pearson's correlation analysis was used to analyze the correlation of 5-HT and 5-HT3R in the intestinal mucosa tissue of patients with D-IBS. The expression levels of 5-HT and 5-HT3R in the intestinal mucosa tissue of patients in the observation group were significantly higher than those of the patients in the control group (344.86±67.52 ng/ml and 13.04±8.34 pg/ml) (P<0.001). There was a positive correlation between the expression level of 5-HT and the expression level of 5-HT3R in the intestinal mucosa tissue of patients with D-IBS (r=0.725, P<0.001). The expression levels of 5-HT and 5-HT3R in the intestinal mucosa tissue of patients with D-IBS were both significantly higher than those of the healthy subjects. The expression levels of 5-HT and 5-HT3R in patients with D-IBS were correlated with age, sex and the history of gastrointestinal infection. 5-HT and 5-HT3R may be involved in the pathogenesis of D-IBS, and potentially used for clinical treatment.

Age-associated Impairment of the Mucus Barrier Function is Associated with Profound Changes in Microbiota and Immunity

Aging significantly increases the vulnerability to gastrointestinal (GI) disorders but there are few studies investigating the key factors in aging that affect the GI tract. To address this knowledge gap, we used 10-week- and 19-month-old litter-mate mice to investigate microbiota and host gene expression changes in association with ageing. In aged mice the thickness of the colonic mucus layer was reduced about 6-fold relative to young mice, and more easily penetrable by luminal bacteria. This was linked to increased apoptosis of goblet cells in the upper part of the crypts. The barrier function of the small intestinal mucus was also compromised and the microbiota were frequently observed in contact with the villus epithelium. Antimicrobial Paneth cell factors Ang4 and lysozyme were expressed in significantly reduced amounts. These barrier defects were accompanied by major changes in the faecal microbiota and significantly decreased abundance of Akkermansia muciniphila which is strongly and negatively affected by old age in humans. Transcriptomics revealed age-associated decreases in the expression of immunity and other genes in intestinal mucosal tissue, including decreased T cell-specific transcripts and T cell signalling pathways. The physiological and immunological changes we observed in the intestine in old age, could have major consequences beyond the gut.

Effects of N-Acetyl-Cysteine Supplementation through Drinking Water on the Glutathione Redox Status during the Weaning Transition of Piglets.

This study investigated the effect of N-acetyl-cysteine (NAC) supplementation through drinking water on animal performance and the glutathione (GSH) redox system in weaned piglets, particularly in relation to the immediate post-weaning feed intake. To this end, 168 piglets were weaned and either fed ad libitum or fasted the first two days, and either or not administered 200 mg/L NAC via the drinking water until d14 post-weaning. Next to animal performance until day 42 (d42), the GSH redox system was measured in erythrocytes, small intestinal mucosa, liver, lung, and kidney tissue at d0, d2, and d14 post-weaning. Animal performance and GSH levels were not affected by NAC, nor by fasting. Irrespective of treatment, a significant drop in GSH at d2 post-weaning was found as compared to d0, in particular in liver (−69%), distal jejunal mucosa (−72%), and lung tissue (−80%). Post-weaning changes of the GSH redox status were strongly tissue-dependent. To conclude, this research indicates that GSH redox homeostasis was largely affected in multiple organs during the weaning transition. NAC supplementation did not increase GSH levels in any tissue, not even in fasted animals, questioning the fact if cysteine is the first or only limiting factor determining the rate of GSH synthesis in the early post-weaning phase.