Bitter Melon Polysaccharides Enhance Goblet Cell Function and Mucosal Integrity in DSS‐Induced Colitis Through Microbiota Remodeling

BackgroundUlcerative colitis (UC) is a chronic and acute inflammatory disorder of the colon linked to dysregulated gut mucosal immune response and compromised colonic epithelial barrier function and integrity. Chromogranin-A (CHGA), a pro-peptide secreted by enteroendocrine cells, is highly expressed in colonic tissues of patients with UC. Elevated CHGA has been shown to correlate with UC disease activity and severity. Moreover, complete deletion of CHGA was shown to result in a diminution of pro-inflammatory markers known to disrupt the colonic epithelial barrier function and gut mucosal healing process. However, little is known about the effect of the absence of CHGA on colonic epithelial barrier function and gut mucosal integrity.AimsHere, we characterized the impact of the lack of CHGA on the colonic mucosa and epithelial barrier structure and function using CHGA knockout (CHGA-/-) mice treated with dextran sulfate sodium (DSS) to induce colitis.Methods13–17 weeks old male C57BL/6 wild-type (CHGA+/+) and C57BL/6 CHGA-/- mice were treated for 5 days with 5% DSS to induced acute colitis, control mice received regular water. CHGA mRNA expression, disease activity index (DAI), and macroscopic score (MS) were analyzed. Distal colonic tissues were isolated, and mRNA expression of markers associated with regenerative stem cells (fast-cycling stem cells [Lgr5+] and reserve stem cells [HOPX+] and [LY6a+], Goblet cells functions mucus barrier mucin 2 [MUC2], resistin-like molecule β [RELMβ], WAP 4-disulphide core domain 2 [WFDC2]) and trefoil factor 3 (TFF3) was evaluated by qRT-PCR.ResultsWe validated a beneficial effect of the Lack of CHGA on colitis severity, associated with significantly lower DAI and MS. In colitic CHGA+/+ and CHGA-/- mice, Lgr5+ and HOPX+ were both highly down-regulated, although, compared to CHGA+/+, CHGA-/- mice presented a 10.5fold higher expression of HOPX+. Compared to non-colitic states, Ly6a+ expression was significantly elevated in both colitic CHGA+/+ and CHGA-/- mice, however, no differences in Lgr5+ and Ly6a+ expression were noted between CHGA+/+ and CHGA-/- mice in all conditions. In CHGA+/+ mice, inflammatory conditions led to higher MUC2 and RELMB expression, although, compared to CHGA+/+, these markers were significantly lower in CHGA-/- mice. In colitic conditions, compared to CHGA+/+, CHGA-/- had a significant increase of WFDC2. In non-colitic conditions, mRNA expressions of all markers evaluated between CHGA+/+ and CHGA-/- in this study were unaltered. Finally, no differences were observed in TFF3 gene expression.ConclusionsThese results indicate in the absence of CHGA, the colonic epithelial barrier integrity and function are maintained through the modulation of goblet cells functions and elevated gut mucosa regenerative potential, thus enhancing the mucosal protection to colitis damage.Funding AgenciesCIHR

During aging, the protective function of mucus barrier is significantly reduced among which changes in colonic mucus barrier function received the most attention. Additionally, the incidence of colon-related diseases increases significantly in adulthood, posing a threat to the health of the elderly. However, the specific changes in colonic mucus barrier with aging and the underlying mechanisms have not been fully elucidated. To understand the effects of aging on the colonic mucus barrier, changes in the colonic mucus layer were evaluated in mice aged 2, 12, 18, and 24 months. Microbial invasion, thickness, and structure of colonic mucus in mice at different months of age were analyzed by in situ hybridization fluorescence staining, AB/PAS staining, and cryo-scanning electron microscopy. Results showed that the aged colon exhibited intestinal mucus barrier dys-function and altered mucus properties. During aging, microorganisms invaded the mucus layer to reach epithelial cells. Compared with young mice, the thickness of mucus layer in aged mice in-creased by 11.66 μm. And the contents of the main components and glycosylation structure of colon changed. Among them, the proportion of goblet cells decreased significantly in older mice, and the expression of spdef genes that regulate goblet cell differentiation decreased. Further, the expression of key enzymes involved in mucin core structure formation and glycan modification also changed with aging. The expression of core 1 β1,3-galactosyltransferase (C1GalT1) which is the key enzyme forming the main core structure increased by one time, while core 2 β1,6 N-acetylglucosaminyltransferase (C2GnT) and core 3 β1,3 N-acetylglucosaminyltransferase (C3GnT) decreased 2 to 6- and 2-fold, respectively. Also, the expression of sialyltransferase, one of the mucin-glycan modifying enzymes, was decreased by 1-fold. Overall, our results indicate that the goblet cells/glycosyltransferase/O-glycan axis plays an important role in maintaining the physicochemical properties of colonic mucus and the stability of intestinal environment.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder for which dietary interventions can be a useful treatment. In recent years, the low-FODMAP approach is gaining traction in this regard. The fermentation of these non-absorbed carbohydrates by the gut microbiota can generate toxic glycating metabolites, such as methylglyoxal. These metabolites can have harmful effects by their role in the generation of advanced glycation end products (AGEs), which activates Receptor for AGEs (AGER). Mast cells can be stimulated by AGEs and play a role in IBS. We have treated mice with lactose or fructo-oligosaccharides (FOS), with or without co-administration of pyridoxamine and investigated the colonic mucus barrier. We have found that an increased intake of lactose and fructo-oligosaccharides induces a dysregulation of the colonic mucus barrier, increasing mucus discharge in empty colon, while increasing variability and decreasing average thickness mucus layer covering the fecal pellet. Changes were correlated with increased mast cell counts, pointing to a role for the crosstalk between these and goblet cells. Additionally, AGE levels in colonic epithelium were increased by treatment with the selected fermentable carbohydrates. Observed effects were prevented by co-treatment with anti-glycation agent pyridoxamine, implicating glycation processes in the negative impact of fermentable carbohydrate ingestion. This study shows that excessive intake of fermentable carbohydrates can cause colonic mucus barrier dysregulation in mice, by a process that involves glycating agents and increased mucosal mast cell counts.

Momordica Charantia L. polysaccharides ameliorate colonic mucus barrier damage induced by high fat diet through regulation of gut microbiota and MUC2 expression.

BackgroundThe relationship between disturbances of the gut microbiota and 1,25(OH)2D3 deficiency has been established both in humans and animal models with a vitamin D poor diet or a lack of sun exposure. Our prior study has demonstrated that Cyp27b1−/− (Cyp27b1 knockout) mice that could not produce 1,25(OH)2D3 had significant colon inflammation phenotypes. However, whether and how 1,25(OH)2D3 deficiency due to the genetic deletion controls the gut homeostasis and modulates the composition of the gut microbiota remains to be explored.Results1,25(OH)2D3 deficiency impair the composition of the gut microbiota and metabolite in Cyp27b1−/− mice, including Akkermansia muciniphila, Solitalea Canadensis, Bacteroides-acidifaciens, Bacteroides plebeius and SCFA production. 1,25(OH)2D3 deficiency cause the thinner colonic mucus layer and increase the translocation of the bacteria to the mesenteric lymph nodes. We also found 1,25(OH)2D3 supplement significantly decreased Akkermansia muciniphila abundance in fecal samples of Cyp27b1−/− mice.ConclusionDeficiency in 1,25(OH)2D3 impairs the composition of gut microbiota leading to disruption of intestinal epithelial barrier homeostasis and induction of colonic inflammation. This study highlights the association between 1,25(OH)2D3 status, the gut microbiota and the colonic mucus barrier that is regarded as a primary defense against enteric pathogens.

The colonic mucus layer serves as an important barrier and prevents colonic bacteria from invading the mucosa and cause inflammation. The regulation of colonic mucus secretion is poorly understood. The aim of this study was to investigate the role of the mucus barrier in induction of colitis. Furthermore, regulation of mucus secretion by luminal bacterial products was studied. The colon of anesthetized Muc2(-/-), Muc1(-/-), wild-type (wt), and germ-free mice was exteriorized, the mucosal surface was visualized, and mucus thickness was measured with micropipettes. Colitis was induced by DSS (dextran sodium sulfate, 3%, in drinking water), and disease activity index (DAI) was assessed daily. The colonic mucosa of germ-free and conventionally housed mice was exposed to the bacterial products LPS (lipopolysaccharide) and PGN (peptidoglycan). After DSS induction of colitis, the thickness of the firmly adherent mucus layer was significantly thinner after 5 days and onward, which paralleled the increment of DAI. Muc2(-/-) mice, which lacked firmly adherent mucus, were predisposed to colitis, whereas Muc1(-/-) mice were protected with significantly lower DAI by DSS compared with wt mice. The mucus barrier increased in Muc1(-/-) mice in response to DSS, whereas significantly fewer T cells were recruited to the inflamed colon. Mice housed under germ-free conditions had an extremely thin adherent colonic mucus layer, but when exposed to bacterial products (PGN or LPS) the thickness of the adherent mucus layer was quickly restored to levels observed in conventionally housed mice. This study demonstrates a correlation between decreasing mucus barrier and increasing clinical symptoms during onset of colitis. Mice lacking colonic mucus (Muc2(-/-)) were hypersensitive to DSS-induced colitis, whereas Muc1(-/-) were protected, probably through the ability to increase the mucus barrier but also by decreased T cell recruitment to the afflicted site. Furthermore, the ability of bacteria to regulate the thickness of the colonic mucus was demonstrated.

Chorioamnionitis is a risk factor for necrotizing enterocolitis (NEC). Ureaplasma parvum (UP) is clinically the most isolated microorganism in chorioamnionitis, but its pathogenicity remains debated. Chorioamnionitis is associated with ileal barrier changes, but colonic barrier alterations, including those of the mucus barrier, remain under-investigated, despite their importance in NEC pathophysiology. Therefore, in this study, the hypothesis that antenatal UP exposure disturbs colonic mucus barrier integrity, thereby potentially contributing to NEC pathogenesis, was investigated. In an established ovine chorioamnionitis model, lambs were intra-amniotically exposed to UP or saline for 7 d from 122 to 129 d gestational age. Thereafter, colonic mucus layer thickness and functional integrity, underlying mechanisms, including endoplasmic reticulum (ER) stress and redox status, and cellular morphology by transmission electron microscopy were studied. The clinical significance of the experimental findings was verified by examining colon samples from NEC patients and controls. UP-exposed lambs have a thicker but dysfunctional colonic mucus layer in which bacteria-sized beads reach the intestinal epithelium, indicating undesired bacterial contact with the epithelium. This is paralleled by disturbed goblet cell MUC2 folding, pro-apoptotic ER stress and signs of mitochondrial dysfunction in the colonic epithelium. Importantly, the colonic epithelium from human NEC patients showed comparable mitochondrial aberrations, indicating that NEC-associated intestinal barrier injury already occurs during chorioamnionitis. This study underlines the pathogenic potential of UP during pregnancy; it demonstrates that antenatal UP infection leads to severe colonic mucus barrier deficits, providing a mechanistic link between antenatal infections and postnatal NEC development.

Somatostatin stimulates colonic MUC2 expression through SSTR5-Notch-Hes1 signaling pathway

The VSL# 3 Probiotic Mixture Modifies Microflora but Does Not Heal Chronic Dextran-Sodium Sulfate–Induced Colitis or Reinforce the Mucus Barrier in Mice

Background & AimsThe interorgan crosstalk between the liver and the intestine has been the focus of intense research. Key in this crosstalk are bile acids, which are secreted from the liver into the intestine, interact with the microbiome, and upon absorption reach back to the liver. The bile acid-activated farnesoid X receptor (Fxr) is involved in the gut-to-liver axis. However, liver-to-gut communication and the roles of bile acids and Fxr remain elusive. Herein, we aim to get a better understanding of Fxr-mediated liver-to-gut communication, particularly in colon functioning.MethodsFxr floxed/floxed mice were crossed with cre-expressing mice to yield Fxr ablation in the intestine (Fxr-intKO), liver (Fxr-livKO), or total body (Fxr-totKO). The effects on colonic gene expression (RNA sequencing), the microbiome (16S sequencing), and mucus barrier function by ex vivo imaging were analysed.ResultsDespite relatively small changes in biliary bile acid concentration and composition, more genes were differentially expressed in the colons of Fxr-livKO mice than in those of Fxr-intKO and Fxr-totKO mice (3272, 731, and 1824, respectively). The colons of Fxr-livKO showed increased expression of antimicrobial genes, Toll-like receptors, inflammasome-related genes and genes belonging to the ‘Mucin-type O-glycan biosynthesis’ pathway. Fxr-livKO mice have a microbiome profile favourable for the protective capacity of the mucus barrier. The thickness of the inner sterile mucus layer was increased and colitis symptoms reduced in Fxr-livKO mice.ConclusionsTargeting of FXR is at the forefront in the battle against metabolic diseases. We show that ablation of Fxr in the liver greatly impacts colonic gene expression and increased the colonic mucus barrier. Increasing the mucus barrier is of utmost importance to battle intestinal diseases such as inflammatory bowel disease, and we show that this might be done by antagonising FXR in the liver.Lay summaryThis study shows that the communication of the liver to the intestine is crucial for intestinal health. Bile acids are key players in this liver-to-gut communication, and when Fxr, the master regulator of bile acid homoeostasis, is ablated in the liver, colonic gene expression is largely affected, and the protective capacity of the mucus barrier is increased.

A Dietary Fiber-Deprived Gut Microbiota Degrades the Colonic Mucus Barrier and Enhances Pathogen Susceptibility

Ulcerative colitis (UC) is a type of inflammatory bowel disease associated with immune system dysfunction caused by gut dysbiosis. This study aimed to investigate the alleviating effect of Lactobacillus acidophilus LA85 on colitis and its underlying mechanism using mouse models of dextran sulfate sodium (DSS)-induced UC. The UC mouse models were established by treating C57BL/6J male mice with 2.5% (w/v) DSS in drinking water for 7 days. These mice received supplementation with either L. acidophilus LA85 (1 × 109 colony-forming units/day) or 200 µL of sterile water once daily (LA85-treated and UC model mice, respectively). The disease activity index (DAI), colon length, and histological changes in the colons of mice were then analyzed at Day 21, and the effects of L. acidophilus LA85 on the gut microbiota and serum inflammatory cytokines were also investigated. Compared with the UC model mice, L. acidophilus LA85-treated UC mice showed significant reductions in a variety of colitis symptoms, including weight loss, the DAI score, colon shortening, and colon tissue damage. Lactobacillus acidophilus LA85 supplementation also significantly decreased the serum concentrations of tumor necrosis factor α and interleukin-6 while increasing the serum concentration of IL-10. Furthermore, LA85 supplementation improved the diversity and composition of the gut microbiota, both of which had been decreased by DSS. In particular, L. acidophilus LA85-treated UC mice showed higher relative abundances of Akkermansia and Romboutsia than the UC model mice. These results demonstrate that L. acidophilus LA85 can alleviate inflammatory diseases of the intestine, such as inflammatory bowel disease, by regulating immune responses and restoring the gut microbiota. PRACTICAL APPLICATION: Ulcerative colitis is a type of inflammatory bowel disease caused by imbalance of gut microbiota. This study showed that L. acidophilus LA85 can alleviate DSS-induced colitis in mice through regulation of inflammatory cytokines, protection of intestinal barrier, and regulation of specific gut microbiota. L. acidophilus LA85 is a promising probiotic candidate for the treatment of UC.

Protection of Sophocarpine on Colonic Barrier in DSS-induced Acute Colitis in Mice by Increasing Expression of HNF4α

BackgroundIncreased apoptosis and loss of epithelial barrier function are hallmark features in the pathogenesis of inflammatory bowel disease. While bile acids are classically known for their roles in facilitating digestion and absorption of fats, they have more recently become appreciated as a family of enterocrine hormones that regulate many aspects of intestinal function. However, even though changes in luminal bile acids are closely associated with IBD pathogenesis, their roles in regulating epithelial barrier function are still largely unknown. Lithocholic acid (LCA) is a secondary colonic bile acid whose luminal levels are decreased and which we have recently shown to be protective against the onset of intestinal inflammation. The aim of the current study was to investigate the effects of LCA on colonic epithelial barrier function and apoptosis.MethodsC57BL/6 mice were administered dextran sodium sulphate (DSS, 2.5%) in drinking water for 5 days to induce epithelial damage and intestinal inflammation and disease severity was measured as disease activity index (DAI). Mice received PBS or Na+‐lithocholate (LCA) via IP injection each day. Epithelial permeability was assessed by measuring the flux of orally‐gavaged FITC‐dextran from the lumen into the blood. Levels of cleaved PARP, measured by Western blotting, were used as a marker of apoptosis. To examine the effects of LCA on cytokine‐induced barrier dysfunction in vitro, monolayers of T84 colonic epithelial cells, treated INF‐ɣ and TNF‐α, were employed.ResultsAfter 5 days, animals treated with LCA had a significantly lower DAI score (5.12 ± 1.0) than those of mice treated with DSS alone (9.0 ± 0.5) (n = 12; p ≤ 0.001). DSS treatment increased the flux of FITC‐dextran from the lumen into the blood by 2.3 ± 0.7 fold and this was reduced to 1.3 ± 0.2 fold in LCA‐treated animals (n = 12). The apoptotic marker, cleaved‐PARP, was increased 19.9 ± 10.9 fold in mucosal tissues isolated from DSS treated mice and this was also reduced to 3.9 ± 1.1 fold by LCA treatment (n = 3 – 4; p ≤ 0.01). In monolayers of cultured T84 colonic epithelial cells, treatment with a combination of the proinflammatory cytokines, INF‐ɣ (40 ng/ml) and TNF‐α (20 ng/ml) increased FITC‐dextran flux from the apical to basolateral domain by 4.9 ± 0.7 fold of that in control cells, while treatment with LCA (10 μM) practically abolished this response to 1.3 ± 0.5 fold (n = 8; p ≤ 0.001). Levels of cleaved‐PARP were increased by 4.4 ± 1.8 fold in IFN/TNF treated cells but this was reduced to 1.9 ±0.1 fold in LCA treated cells (n = 4). Similarly, GW4064 (5 μM), an agonist of the nuclear bile acid receptor, FXR, also prevented IFN/TNF‐induced FITC‐dextran flux and PARP cleavage in cultured epithelial cells.ConclusionsBy virtue of its capacity to prevent cytokine‐induced epithelial apoptosis and disruption of barrier function, these data imply a protective role for LCA in conditions of colonic inflammation. Thus, LCA and its associated signalling pathways present promising targets for the development of new approaches to promote colonic barrier function in the treatment of IBD.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Vagus Nerve: A Tonic Inhibitory Influence Associated With Inflammatory Bowel Disease in a Murine Model