Colonic Goblet Cells Research Articles

TRIM34 attenuates colon inflammation and tumorigenesis by sustaining barrier integrity.

Loss of the colonic inner mucus layer leads to spontaneously severe colitis and colorectal cancer. However, key host factors that may control the generation of the inner mucus layer are rarely reported. Here, we identify a novel function of TRIM34 in goblet cells (GCs) in controlling inner mucus layer generation. Upon DSS treatment, TRIM34 deficiency led to a reduction in Muc2 secretion by GCs and subsequent defects in the inner mucus layer. This outcome rendered TRIM34-deficient mice more susceptible to DSS-induced colitis and colitis-associated colorectal cancer. Mechanistic experiments demonstrated that TRIM34 controlled TLR signaling-induced Nox/Duox-dependent ROS synthesis, thereby promoting the compound exocytosis of Muc2 by colonic GCs that were exposed to bacterial TLR ligands. Clinical analysis revealed that TRIM34 levels in patient samples were correlated with the outcome of ulcerative colitis (UC) and the prognosis of rectal adenocarcinoma. This study indicates that TRIM34 expression in GCs plays an essential role in generating the inner mucus layer and preventing excessive colon inflammation and tumorigenesis.

Bovine Colostrum Before or After Formula Feeding Improves Systemic Immune Protection and Gut Function in Newborn Preterm Pigs

Objectives: Maternal milk is often absent or in limited supply just after preterm birth. Many preterm infants are therefore fed infant formula as their first enteral feed despite an increased risk of feeding intolerance, necrotizing enterocolitis (NEC), and infection. Using preterm pigs as a model for preterm infants, we hypothesized that bovine colostrum given before or after formula feeding would alleviate formula-induced detrimental effects during the first days after preterm birth.Methods: A total of 74 preterm pigs received gradually increasing volumes of formula (F) or bovine colostrum (C) until day 5, when they were euthanized or transitioned to either C or F for another 4 days, resulting in six groups: C or F until day 5 (C5, F5, n = 11 each), C or F until day 9 (CC, FF n = 12–13 each), C followed by F (CF, n = 14), and F followed by C (FC, n = 13).Results: Systemically, colostrum feeding stimulated circulating neutrophil recruitment on day 5 (C5 vs. F5, P < 0.05). Relative to initial formula feeding, initial colostrum feeding promoted the development of systemic immune protection as indicated by a decreased T-helper cell population and an increased regulatory T-cell population (CC + CF vs. FC + FF, P < 0.01). In the gut, colostrum feeding improved intestinal parameters such as villus heights, enzymes, hexose absorption, colonic goblet cell density, and decreased the incidence of severe NEC (27 vs. 64%), diarrhea (16 vs. 49%), and gut permeability on day 5, coupled with lowered expression of LBP, MYD88, IL8, HIF1A, and CASP3 (C5 vs. F5, all P < 0.05). On day 9, the incidence of severe NEC was similarly low across groups (15–21%), but diarrhea resistance and intestinal parameters were further improved by colostrum feeding, relative to exclusive formula feeding (CC, CF, or FC vs. FF, respectively, all P < 0.05). The expression of MYD88 and CASP3 remained downregulated by exclusive colostrum feeding (CC vs. FF, P < 0.01) and colostrum before or after formula feeding down regulated HIF1A and CASP3 expression marginally.Conclusion: Colostrum feeding ameliorated detrimental effects of formula feeding on systemic immunity and gut health in preterm newborns, especially when given immediately after birth.

Gallic acid prevents 1, 2-Dimethylhydrazine induced colon inflammation, toxicity, mucin depletion, and goblet cell disintegration.

1,2-Dimethylhydrazine (DMH), an environmental toxicant specifically targets the colon. The present study was aimed to evaluate the efficacy of gallic acid (GA) against colon toxicity induced by DMH in Wistar rats. GA, a phenolic acid has numerous beneficial properties, which include antiviral, antifungal and antioxidant properties which help cells to overcome oxidative stress and balance the redox homeostasis. GA was administered orally at two doses (25 and 50 mg/kg body weight) once daily for 14 days and a single dose (40 mg/kg body weight) of DMH was administered subcutaneously on 14th day. Animals were sacrificed on the 15th day and we could find that GA at both the doses significantly ameliorates DMH-induced increased toxicity markers and also substantially increases the glutathione content level and activities of detoxifying enzymes. It also ameliorates the expression of proliferation, inflammation, apoptosis, goblet cell disintegration, and mucin depletion in the colon that was elevated upon administration of DMH. Histological alterations provide further confirmation of the protective role of GA against DMH-induced colon toxicity. The results of this study clearly indicate supplementation of GA is beneficial in ameliorating DMH-induced oxidative stress, inflammation, proliferation, apoptosis, mucin depletion, and goblet cell disintegration in colon of Wistar rats.

Rhubarb-Evoke Mucus Secretion through Aggregation and Degranulation of Mast Cell in the Colon of Rat: In vivo and ex vivo studies

Rhubarb is commonly used to treat constipation in China for its function of promoting intestinal movement and optimum water content in feces. However, its mechanism of mucus secretion is vague. The aim of the study is to investigate the role of mast cells and enteric neurons in rhubarb extract (RE)-induced mucus secretion in the rat colon. Immunofluorescence was used to detect histamine receptors. Western blotting and 3,3′-diaminobenzidine (DAB) were applied to explore the content changes of mast cells activation. The changes in colonic goblet cells (GCs) were determined by means of PAS/AB staining. An intestinal perfusion system with a Bradford protein assay kit was directly to estimate in vitro secretion. And the cytokines were investigated with ELISA. The longitudinal aspect of this study indicate that the number and water content of faecal pellets were enhanced after the administration of different doses of RE accompanied by mast cells accumulated and increased the content of interferon (IFN) -γ or decreased the levels of interleukin (IL) −10 at doses of 3 and 6 g/kg. Pretreatment with ketotifen, mast cell stabilizer, had partially inhibited on RE-induced mucus secretion. Furthermore, RE induced the release of acetylcholine and mucin-2 in the colonic tissue and the histamine levels from the faeces. The results suggest that RE induced colonic mucus secretion involves mast cell activation and some cytokine.

Dietary intervention using (1,3)/(1,6)-β-glucan, a fungus-derived soluble prebiotic ameliorates high-fat diet-induced metabolic distress and alters beneficially the gut microbiota in mice model.

Western diet, rich in carbohydrates and fat, is said to be a major factor underlying metabolic syndrome. Interventions with prebiotics, the key modulators of the gut microbiota, have paramount impact on host-associated metabolic disorders. Herein, we investigated the effect of fungus-derived (1,3)/(1,6)-β-glucan, a highly soluble dietary fiber, on high-fat diet (HFD)-induced metabolic distress. Male C57BL/6J mice were fed with different diet groups (n = 11): control diet, HFD, 3g/kg or 5g/kg of β-glucan-incorporated HFD. At the end of experimental study period (12th week), body weight, feces weight and fecal moisture content were observed. Further, colonic motility was measured using activated charcoal meal study. Proteins extracted from liver and intestine tissues were subjected to western blot technique. Paraffin-embedded intestinal tissues were sectioned for histochemical [Periodic acid-Schiff (PAS) and Alcian blue (AB) staining] analysis. Fecal microbiota analysis was performed using MOTHUR bioinformatic software. β-glucan consumption exhibited anti-obesity property in mice groups fed with HFD. In addition, β-glucan ameliorated HFD-induced hepatic stress, colonic motility and intestinal atrophy (reduction in colon length, goblet cells, and mucosal layer thickness). Further, β-glucan incorporation shifted bacterial community by increasing butyrate-producing bacteria such as Anaerostipes, Coprobacillus, and Roseburia and decreasing reportedly obesity-associated bacteria such as Parabacteroides and Lactococcus. Altogether, the outcomes of this present pre-clinical animal study show β-glucan to be a promising therapeutic candidate in the treatment of HFD-induced metabolic distress. Further comprehensive research has to be conducted to brace its clinical relevance, reproducibility and efficacy for aiding human health.

VAMP8-mediated MUC2 mucin exocytosis from colonic goblet cells maintains innate intestinal homeostasis

The mucus layer is the first line of innate host defense in the gut that protects the epithelium by spatially separating commensal bacteria. MUC2 mucin is produced and stored by goblet cells that is constitutively exocytosed or hyper secreted upon sensing a threat. How coordinated mucus exocytosis maintains homeostasis in the intestinal epithelium and modulates the immunological landscape remains elusive. Here we describe how the vesicle SNARE protein VAMP8 coordinates mucin exocytosis from goblet cells. Vamp8−/− exhibit a mild pro-inflammatory state basally due to an altered mucus layer and increased encounters with microbial antigens. Microbial diversity shifts to a detrimental microbiota with an increase abundance of pathogenic and mucolytic bacteria. To alleviate the heavy microbial burden and inflammatory state basally, Vamp8−/− skews towards tolerance. Despite this, Vamp8−/− is highly susceptible to both chemical and infectious colitis demonstrating the fragility of the intestinal mucosa without proper mucus exocytosis mechanisms.

Docosahexaenoic and Eicosapentaenoic Acids Prevent Altered-Muc2 Secretion Induced by Palmitic Acid by Alleviating Endoplasmic Reticulum Stress in LS174T Goblet Cells.

Diets high in saturated fatty acids (FA) represent a risk factor for the development of obesity and associated metabolic disorders, partly through their impact on the epithelial cell barrier integrity. We hypothesized that unsaturated FA could alleviate saturated FA-induced endoplasmic reticulum (ER) stress occurring in intestinal secretory goblet cells, and consequently the reduced synthesis and secretion of mucins that form the protective mucus barrier. To investigate this hypothesis, we treated well-differentiated human colonic LS174T goblet cells with palmitic acid (PAL)—the most commonly used inducer of lipotoxicity in in vitro systems—or n-9, n-6, or n-3 unsaturated fatty acids alone or in co-treatment with PAL, and measured the impact of such treatments on ER stress and Muc2 production. Our results showed that only eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids protect goblet cells against ER stress-mediated altered Muc2 secretion induced by PAL, whereas neither linolenic acid nor n-9 and n-6 FA are able to provide such protection. We conclude that EPA and DHA could represent potential therapeutic nutrients against the detrimental lipotoxicity of saturated fatty acids, associated with type 2 diabetes and obesity or inflammatory bowel disease. These in vitro data remain to be explored in vivo in a context of dietary obesity.

Identification of Key Pro-Survival Proteins in Isolated Colonic Goblet Cells of Winnie, a Murine Model of Spontaneous Colitis.

Accumulating evidence suggests that the goblet cell-derived mucin-2 (Muc2) is a major component of the immune system and that perturbations in Muc2 lead to an ulcerative colitis-like phenotype. The animal model Winnie carries a missense mutation in Muc2 that causes Muc2 misfolding, accumulation in goblet cells, and ER stress. Excessive ER stress is a hallmark of many diseases, including ulcerative colitis, cancer, diabetes and Parkinson's disease. However, rather than committing to cell death, which is the typical outcome of unresolved ER stress, Winnie goblet cells are characterized by hyperproliferation, suggesting additional regulation of this cellular stress response. To elucidate the molecular mechanisms underlying ulcerative colitis in the Winnie model, we isolated goblet cells from Winnie and wild-type mice and used label-free quantitative proteomics and bioinformatics to understand the functional consequences of Muc2 misfolding and accumulation. A large number of changes were identified that highlight a dramatic reprogramming of energy production, including enhanced utilization of butyrate, a key energy source of colonic cells. A major finding was the marked upregulation of the coiled-coil-helix-coiled-coil-helix domain proteins Chchd2, Chchd3, and Chchd6. In particular, we identified and confirmed the upregulation and nuclear translocation of Chchd2, a protein known to inhibit oxidative stress induced apoptosis. This study is the first to apply proteome-level analysis to the preclinical Winnie model of ulcerative colitis. Identification of proteins and pathways affected in isolated Winnie goblet cells provides evidence for novel adaptive mechanisms underlying cell survival under conditions of chronic ER stress.

The Nlrp6 inflammasome is not required for baseline colonic inner mucus layer formation or function.

The inner mucus layer (IML) is a critical barrier that protects the colonic epithelium from luminal threats and inflammatory bowel disease. Innate immune signaling is thought to regulate IML formation via goblet cell Nlrp6 inflammasome activity that controls secretion of the mucus structural component Muc2. We report that isolated colonic goblet cells express components of several inflammasomes; however, analysis of IML properties in multiple inflammasome-deficient mice, including littermate-controlled Nlrp6-/- , detect a functional IML barrier in all strains. Analysis of mice lacking inflammasome substrate cytokines identifies a defective IML in Il18-/- mice, but this phenotype is ultimately traced to a microbiota-driven, Il18-independent effect. Analysis of phenotypic transfer between IML-deficient and IML-intact mice finds that the Bacteroidales family S24-7 (Muribaculaceae) and genus Adlercrutzia consistently positively covary with IML barrier function. Together, our results demonstrate that baseline IML formation and function is independent of inflammasome activity and highlights the role of the microbiota in determining IML barrier function.

Treatment with a Zinc Metalloprotease Purified from Bothrops moojeni Snake Venom (BmooMP-Alpha-I) Reduces the Inflammation in an Experimental Model of Dextran Sulfate Sodium-Induced Colitis.

It has been described that the metalloprotease BmooMP-alpha-I purified from Bothrops moojeni snake venom is able to hydrolyze the TNF molecule. However, this observation has been based mainly on in vitro investigation, in addition to molecular modeling and docking approaches. Considering that there is no in vivo study to demonstrate the biological effects of this enzyme, the major aim to the present work was to investigate whether the BmooMP-alpha-I has any anti-inflammatory efficacy by setting up a murine experimental design of colitis induced by dextran sulfate sodium (DSS). For this purpose, C57BL/6 mice were divided into six groups, as follows: (i) animals without intestinal inflammation, (ii) animals without intestinal inflammation treated with BmooMP-alpha-I (50 μg/animal/day), and (iii) animals with intestinal inflammation induced by 3% of DSS, (iv) mice with intestinal inflammation induced by DSS and treated with BmooMP-alpha-I enzyme at the 50, 25, or 12.5 μg/animal/day dosages by intraperitoneal route. Clinical signs of colitis were observed daily for calculating the morbidity scores, cytokine measurements, and histological features. We observed that the animals treated with different doses of the enzyme presented a remarkable improvement of colitis signs, as confirmed by a significant increase of the intestine length in comparison to the DSS group. Also, no difference was observed between the groups treated with the enzyme or vehicle, as the colon length of these animals was slightly lower than that of the group of healthy animals, without induction of intestinal inflammation. The cytokine quantification in supernatants of intestinal tissue homogenates showed a significant reduction of 38% in IFN-gamma levels, when the animals were treated with 50 μg of the BmooMP-alpha-I compared to the animals receiving DSS only. A significant reduction of 39% in TNF levels was also observed in all doses of treatment with BmooMP-alpha-I, in addition to a significant reduction of 35% in the amount of IL-12p40. Histological examinations revealed that the BmooMP-alpha-I 50 μg treated group preserved colon architecture and goblet cells and reduced the ulcer area, when compared with DSS mice, which showed typical inflammatory changes in tissue architecture, such as ulceration, crypt dilation, loss of tissue architecture, and goblet cell depletion, accompanied by a significant cell infiltration. In conclusion, our results suggest that the improvement of clinical scores and histological findings related to BmooMP-alpha-I treatment in this experimental model could be attributed to the metalloprotease ability to modulate cytokine production locally at the inflamed intestine. These findings highlight the potential anti-inflammatory role and effectiveness of this enzyme as a therapeutic alternative in this type of immunopathological condition.

Gut Microbiota Facilitates the Preventive Effects of Barley Leaf Against Colitis by Improving Pparγ-Mediated Mucosal Barrier Function

Background: Dietary changes associated with westernization have resulted in an increased prevalence of many chronic inflammatory diseases such as inflammatory bowel disease (IBD). Barley leaf (BL) has a long history of use in traditional Chinese medicine with potential health-promoting properties, while its beneficial effects on IBD prevention have not been examined. Methods: By using 16S rDNA gene-based microbiota analysis, electronic microscopy, and RNA sequencing, we examined the mechanisms underlying the preventive effects of BL on colonic inflammation in a dextran sulfate sodium (DSS)-induced colitis mouse model. Findings: BL alleviated DSS-induced colitis severity, as evidenced by reduced body weight loss, disease activity index, and colon shortening. This was companied by inhibited expansion of Enterobacteriaceae in the colonic contents of DSS-treated mice. Furthermore, the beneficial effects of BL against colitis were largely due to its preventive activities before DSS induction. The gut microbiota contributed to the beneficial effects of BL since transfer of BL-cultured fecal microbiota protected against DSS-induced colitis and mucosal barrier damage. Mechanically, BL caused mucus hyperproduction in colonic goblet cells, which was significantly inhibited by treatment with the peroxisome proliferator-activated receptor (PPAR)γ antagonist GW9662. Antibiotic treatment revealed that the effects of BL on mucus hyperproduction and DSS-induced colitis was lost in the absence of gut microbiota. Interpretation: Our results showed that BL protected against DSS-induced colitis by promoting PPARγ-mediated mucus hyperproduction, a mechanism that relied on regulation of the gut microbiota. Funding: This work was supported by Beijing Municipal Science and Technology Project Fund [No. D161100005416001] and Chinese Universities Scientific Fund [No. 2016SP003]. Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: The mouse experiments were performed in accordance with the guidelines of the local ethics committee.

Sprouty2 restricts colonic tuft and goblet cell numbers by repressing epithelial IL‐33 expression

OBJECTIVETuft and goblet cells are secretory cell types of the intestinal epithelium that serve key roles in sensing and responding to inflammation. However, the intracellular mechanisms controlling their numbers and activity are incompletely defined. Here, we investigated the role of Sprouty2 (Spry2), an intracellular modulator of PI3K and MAPK signaling, in tuft and goblet cell development. Both PI3K and MAPK are implicated in control of differentiation in the intestine. Furthermore, both pathways have been linked to expression of IL‐33, a cytokine that indirectly influences secretory differentiation by stimulating IL‐13 release from immune cell populations. We tested the hypothesis that Spry2 controls IL‐33 production in the colon, thus altering intestinal secretory cell development.METHODSMucosal scrapings from wild type mice given dextran sodium sulphate (DSS) colitis, and colonoids treated with TNF, were subjected to qPCR analysis for Spry2 expression. Colons from mice with intestinal epithelial‐specific Spry2 deletion (Spryflox/flox;Villin‐Cre, here called Spry2KOIE) and littermate controls were collected at 8 weeks of age. Cytokine levels and epithelial marker expression were determined by immunostaining and qPCR. Signaling pathway alterations were assessed by dot blot array and confirmed by western blot. In vitro, murine colonoids were treated with IL‐33 or IL‐13 +/− signaling pathway inhibitors (LY294002, PI3K; CHIR99021, GSK3β) to determine the effects on cytokine and epithelial marker expression.RESULTSSpry2 levels were decreased in inflamed colons (59% decrease, p&lt;0.05) and TNF‐treated colonoids (45% decrease, p&lt;0.05), demonstrating regulation by inflammation. Spry2KOIE mice displayed increased colonic IL‐33 expression (80% increase, p&lt;0.05), more mesenchymal IL‐13+ cells, more Dclk1+ tuft cells (2.2‐fold, p&lt;0.01), and more Muc2+ goblet cells (1.57‐fold, p&lt;0.01). These mice also had elevated GSK3β phosphorylation (88% increase, p&lt;0.01) on Ser9 (an inhibitory site phosphorylated by Akt), and increased phospho‐Akt (p&lt;0.01). In vitro, inactivation of GSK3β in murine colonoids with CHIR99021 mimicked the increased IL‐33 observed in vivo, but interestingly did not alter Dclk1 or Muc2 expression. This effect was attenuated by PI3K inhibition. IL‐13, but not IL‐33, induced Dclk1 and Muc2 in colonoids, suggesting an epithelial‐mesenchymal crosstalk circuit drives alterations in tuft and goblet cells following Spry2 loss.CONCLUSIONInflammation‐driven loss of colonic epithelial Spry2 de‐represses PI3K/Akt signals that lead to elevated IL‐33 expression. IL‐33 may act, in turn, to promote mesenchymal IL‐13 release that drives a protective expansion of tuft and goblet cells in the epithelium.Support or Funding InformationNIH R01DK095004 (Frey); Crohn's and Colitis Foundation Career Development Award (Schumacher)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of a probiotic on the morphology and mucin composition of pig intestine.

Although the use of probiotics in human and animal medicine is growing, their mode of action remains poorly understood. This study examined the effects of a multi-strain probiotic (SLAB51™) on the morphology and carbohydrate composition of mucins secreted by goblet cells of intestinal crypts in growing-finishing pigs. Sections of duodenum, caecum and colon from pigs fed for 12 weeks with an orally administered control basal diet (No-Pro) or one with a probiotic blend (Pro) were processed for microscopic analysis and stained with (1) haematoxylin-eosin for structural and morphometrical investigation; (2) conventional histochemistry (periodic acid-Schiff, Alcian Blue pH 2.5, high iron diamine staining) for neutral, acidic non-sulphated, and sulphated mucin analysis; and (3) FITC-labelled MAA-II and SNA lectins for α2,3- and α2,6-sialomucin identification. Compared with No-Pro samples, Pro samples displayed (1) increased goblet cell numbers in all investigated tract crypts; (2) an increase in acidic non-sulphomucins but a decrease in neutral, sulphated and α2,6-sialomucin-secreting goblet cells in the duodenum; (3) decreased crypt depth, an increase in α2,6-sialomucin secretory goblet cells, and a loss of goblet cell-secreting α2,3-sialomucins, which appeared on the apical surface of crypt fundus epithelial cells in the caecum; and (4) an increase in α2,6-sialomucin-producing goblet cells in the colon. Results suggest that treatment with SLAB51™ induces region-specific changes in the morphology and carbohydrate composition of mucins secreted along intestinal tracts of growing-finishing pigs. These changes could ameliorate the health status of the animals, which displayed higher growth performance and meat quality than controls (Tufarelli et al., 2017).

P153 LOSS OF NEUTROPHIL EXTRACELLULAR TRAPS VIA PEPTIDYL ARGININE DEIMINASE-4 DEFICIENCY AGGRAVATES CITROBACTER RODENTIUM- INDUCED GUT INFLAMMATION

Citrobacter rodentium (CR) is an attaching-effacing enteropathogen which infects mice and causes intestinal inflammation. The CR infection model is appropriate for investigating host-pathogen interactions in the gut and understanding the pathogenesis of human inflammatory bowel diseases. Peptidyl arginine deiminase-4 (PAD4) is an enzyme required for the generation of neutrophil extracellular traps (NETs), which can provide antimicrobial effects during the host innate immune response; however, the role of PAD4 against gastrointestinal (GI) infection is largely unknown. In this study, we challenged PAD4-deficient (Pad4-/-) mice and wild type (WT) littermates against CR, and investigated bacteria clearance and gut pathology. Luminal colonization of CR in Pad4-/- mice peaked between 11-14 days post-infection (p.i.), whereas WT mice suppressed the infection by 14 days. Loss of PAD4 substantially increased CR dissemination to the spleen and mesenteric lymph nodes, which prolonged and aggravated CR-associated inflammation as indicated by splenomegaly, colomegaly and elevated systemic and colonic pro-inflammatory markers (lipocalin 2 and serum amyloid A). Likewise, the colonic antimicrobial cytokines, IL-22 and IL-17A, were more upregulated in Pad4-/- than WT mice in response to infection. Histological analysis revealed that transmissible colonic hyperplasia and goblet cell depletion were more pronounced in Pad4-/- than WT mice. Using a bioluminescent strain of CR (ICC180), we observed that the transit of CR through the gastrointestinal tract occurs more rapidly in Pad4-/- mice. Treating WT mice with deoxyribonuclease I (DNase I; disrupts NETs generation), recapitulated the exacerbated CR infection and gut pathology as in PAD4 deficient mice. Administration of the PAD4 inhibitor, Cl-amidine, also aggravated CR infection, but to a lesser extent than those observed in either Pad4-/- or DNase I-treated mice. Taken together, our findings highlight the importance of PAD4 in the mucosal clearance of CR and in resolving the gut-associated inflammation and colitis. In light of the ongoing efforts to target PAD4 or NETs to treat other inflammatory disorders, our findings caution that their inhibition may not be favorable against GI infection.

P153 LOSS OF NEUTROPHIL EXTRACELLULAR TRAPS VIA PEPTIDYL ARGININE DEIMINASE-4 DEFICIENCY AGGRAVATES CITROBACTER RODENTIUM- INDUCED GUT INFLAMMATION

Citrobacter rodentium (CR) is an attaching-effacing enteropathogen which infects mice and causes intestinal inflammation. The CR infection model is appropriate for investigating host-pathogen interactions in the gut and understanding the pathogenesis of human inflammatory bowel diseases. Peptidyl arginine deiminase-4 (PAD4) is an enzyme required for the generation of neutrophil extracellular traps (NETs), which can provide antimicrobial effects during the host innate immune response; however, the role of PAD4 against gastrointestinal (GI) infection is largely unknown. In this study, we challenged PAD4-deficient (Pad4-/-) mice and wild type (WT) littermates against CR, and investigated bacteria clearance and gut pathology. Luminal colonization of CR in Pad4-/- mice peaked between 11-14 days post-infection (p.i.), whereas WT mice suppressed the infection by 14 days. Loss of PAD4 substantially increased CR dissemination to the spleen and mesenteric lymph nodes, which prolonged and aggravated CR-associated inflammation as indicated by splenomegaly, colomegaly and elevated systemic and colonic pro-inflammatory markers (lipocalin 2 and serum amyloid A). Likewise, the colonic antimicrobial cytokines, IL-22 and IL-17A, were more upregulated in Pad4-/- than WT mice in response to infection. Histological analysis revealed that transmissible colonic hyperplasia and goblet cell depletion were more pronounced in Pad4-/- than WT mice. Using a bioluminescent strain of CR (ICC180), we observed that the transit of CR through the gastrointestinal tract occurs more rapidly in Pad4-/- mice. Treating WT mice with deoxyribonuclease I (DNase I; disrupts NETs generation), recapitulated the exacerbated CR infection and gut pathology as in PAD4 deficient mice. Administration of the PAD4 inhibitor, Cl-amidine, also aggravated CR infection, but to a lesser extent than those observed in either Pad4-/- or DNase I-treated mice. Taken together, our findings highlight the importance of PAD4 in the mucosal clearance of CR and in resolving the gut-associated inflammation and colitis. In light of the ongoing efforts to target PAD4 or NETs to treat other inflammatory disorders, our findings caution that their inhibition may not be favorable against GI infection.

Dopamine promotes colonic mucus secretion through dopamine D5 receptor in rats.

Dopamine regulates gastrointestinal mucosal barrier. Mucus plays important roles in the protection of intestinal mucosa. Here, the regulatory effect of dopamine on rat colonic mucus secretion was investigated. RT-PCR, immunofluorescence, Periodic Acid-Schiff reagent assay, Alcian blue-Periodic Acid-Schiff staining, and enzyme-linked immunosorbent assay were used to observe the expression of dopamine receptor and the direct effect of dopamine on the colonic mucus. Mice injected intraperitoneally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) destroying enteric dopamine (DA) neurons, rats microinjected with 6-hydroxydopamine (6-OHDA) into the bilateral substantia nigra damaging central dopaminergic neurons, and dopamine D5 receptor-downregulated transgenic mice were used to detect the effect of endogenous enteric dopamine or dopamine receptors on distal colonic mucus. Our results indicated that D5 immunoreactivity was widely distributed on the colonic goblet cells. Dopamine dose-dependently increased rat distal colonic mucus secretion in vitro. D1-like receptor antagonist SCH23390 inhibited dopamine (1 μΜ)-induced distal colonic mucus secretion. D1-like receptor agonist SKF38393 promoted mucin 2 (MUC2) secretion and increased the intracellular cAMP level of colonic mucosa. D5 receptor-downregulated transgenic mice showed a decreased colonic MUC2 content. MPTP-treated mice exhibited lower colonic dopamine content and decreased colonic mucus content. 6-OHDA rats had an increase in the dopamine content in colonic mucosa but decreases in the protein levels of D1 and D5 receptors and MUC2 content in the colonic mucosa. These findings reveal that dopamine is able to promote distal colonic mucus secretion through the D5 receptor, which provides important evidence to better understand the possible role of dopamine in the colonic mucosal barrier.

Milk phospholipids ameliorate mouse colitis associated with colonic goblet cell depletion via the Notch pathway.

As natural emulsifiers used in food products, phospholipids have been gaining increasing attention, whereas their intestinal health impacts are still unclear. This work aims to investigate the effect of milk phospholipids (MP) on dextran sodium sulfate (DSS)-induced colitis of C57BL/6 mice, compared with soybean phospholipids (SP), as well as the related mechanism. We found that unlike SP, MP supplementation (25 mg per kg BW) had a unique beneficial effect on the 2.5% DSS induced colitis (p < 0.05) and ameliorated the injury status of goblet cells, indicated by the increased number (p < 0.05) and size (p < 0.05) of the goblet cells and more acid mucins and antimicrobial peptides (p < 0.05). Meanwhile, MP supplementation restored the over-activated Notch pathway through balancing the level of Notch pathway ligand Dll4 in the colon subepithelial layer (p < 0.05), thus leading to the increase in the Math1 expression (p < 0.05), and consequently enhanced goblet cell restitution (p < 0.05). Furthermore, MP induced more goblet cells in the colonoid and colonic myofibroblast co-culture system (p < 0.05), which highlighted the indispensable role of colonic myofibroblasts, as an intestinal stem cell niche factor, in the goblet cell modulation effect of MP. These findings indicated that phospholipids from milk instead of soybean attenuated the severity of DSS-induced mouse colitis and prevented the depletion of colonic goblet cells through balancing the over-activated Notch pathway mediated by colonic myofibroblasts.

Defining cooperative roles for colonic microbiota and Muc2 mucin in mediating innate host defense against Entamoeba histolytica.

Amebiasis is caused by the protozoan parasite Entamoeba histolytica (Eh), a potentially fatal disease occurring mainly in developing countries. How Eh interacts with innate host factors in the gut is poorly understood. Eh resides and feed in/on the outer colonic mucus layer and thus share an ecological niche with indigenous microbiota. As gut microbiota regulates innate immune responses, in this study we characterized the cooperative roles that microbiota and the mucus layer play in Eh-induced pro-inflammatory responses in the colon. To study this, we used antibiotics treated and non-treated specific pathogen free Muc2-/- and Muc2+/+ littermates and germ-free mice inoculated with Eh in colonic loops as a short infection model. In antibiotic treated Muc2-/- and Muc2+/+ littermates, Eh elicited robust mucus and water secretions, enhanced pro-inflammatory cytokines and chemokine expression with elevated MPO activity and higher pathology scores as compared to the modest response observed in non-antibiotic treated littermates. Host responses were microbiota specific as mucus secretion and pro-inflammatory responses were attenuated following homologous fecal microbial transplants in antibiotic-treated Muc2+/+ quantified by secretion of 3H-glucosamine newly synthesized mucin, Muc2 mucin immunostaining and immunohistochemistry. Eh-elicited pro-inflammatory responses and suppressed goblet cell transcription factor Math1 as revealed by in vivo imaging of Eh-colonic loops in Math1GFP mice, and in vitro using Eh-stimulated LS174T human colonic goblet cells. Eh in colonic loops increased bacterial translocation of bioluminescent E. coli and indigenous bacteria quantified by FISH and quantitative PCR. In germ-free animals, Eh-induced mucus/water secretory responses, but acute pro-inflammatory responses and MPO activity were severely impaired, allowing the parasite to bind to and disrupt mucosal epithelial cells. These findings have identified key roles for intestinal microbiota and mucus in regulating innate host defenses against Eh, and implicate dysbiosis as a risk factor for amebiasis that leads to exacerbated immune responses to cause life-threatening disease.

Single cell RNA-seq reveals profound transcriptional similarity between Barrett\u2019s oesophagus and oesophageal submucosal glands

Barrett’s oesophagus is a precursor of oesophageal adenocarcinoma. In this common condition, squamous epithelium in the oesophagus is replaced by columnar epithelium in response to acid reflux. Barrett’s oesophagus is highly heterogeneous and its relationships to normal tissues are unclear. Here we investigate the cellular complexity of Barrett’s oesophagus and the upper gastrointestinal tract using RNA-sequencing of single cells from multiple biopsies from six patients with Barrett’s oesophagus and two patients without oesophageal pathology. We find that cell populations in Barrett’s oesophagus, marked by LEFTY1 and OLFM4, exhibit a profound transcriptional overlap with oesophageal submucosal gland cells, but not with gastric or duodenal cells. Additionally, SPINK4 and ITLN1 mark cells that precede morphologically identifiable goblet cells in colon and Barrett’s oesophagus, potentially aiding the identification of metaplasia. Our findings reveal striking transcriptional relationships between normal tissue populations and cells in a premalignant condition, with implications for clinical practice.

Role of metabolic changes of mucosal layer in the intestinal barrier dysfunction following trauma/hemorrhagic shock

BackgroundThe mucosal layer plays an important role in regulating the intestinal barrier function. However, the underlying mechanisms of intestinal barrier dysfunction caused by trauma-hemorrhagic shock (THS) are still unknown. MethodsIn this study, we examined the barrier damages, inflammatory responses as well as the metabolic changes of the mucosal layer of the colon in a THS rat model. ResultsThe results showed that compared to the rats treated with trauma only, THS induced marked failure of intestinal barrier characterized by increased intestinal permeability, inflammatory cell infiltration and decreased expression of genes involved in epithelial integrity. Moreover, decreased colonic mucus content and goblet cell numbers indicated that the mucosal layer was also impaired in response to THS. This was companied by the anomalous inflammatory responses in the tissue. Finally, microdialysis catheter examination showed that metabolites including glycerol, glucose, lactate and pyruvate, glutamate and glutamine were also altered by THS. ConclusionOur results provide evidence that mucus layer-associated metabolic changes may contribute to the THS-induced intestinal barrier dysfunction.