Intestinal Goblet Cells Research Articles

Differential influence of YAP1 and TAZ on differentiation of intestinal epithelial cell: A review.

Intestinal cell stemness, proliferation and differentiation are complex processes all occurring in distinct compartments of the crypt that need to be closely regulated to ensure proper epithelial renewal. The involvement of the Hippo pathway in intestinal epithelial proliferation and regeneration after injury via the regulation of its effectors YAP1 and TAZ has been well-documented over the last decade. The implication of YAP1 and TAZ on intestinal epithelial cell differentiation is less clear. Using intestinal cell models in which the expression of YAP1 and TAZ can be modulated, our group showed that YAP1 inhibits differentiation of the two main intestinal epithelial cell types, goblet and absorptive cells through a specific mechanism involving the repression of prodifferentiation transcription factor CDX2 expression. Further analysis provided evidence that the repressive effect of YAP1 on intestinal differentiation is mediated by regulation of the Hippo pathway by Src family kinase activity. Interestingly, the TAZ paralog does not seem to be involved in this process, which provides another example of the lack of perfect complementarity of the two main Hippo effectors.

Fenretinide Reduces Intestinal Mucin-2-Positive Goblet Cells in Chronic Alcohol Abuse

Introduction: Alcohol-induced thickening of the gut mucosal layer and increased expression of goblet cell gel-forming mucins, such as mucin-2 (MUC2) are associated with disruptions to the gut barrier in alcoholic liver disease (ALD). Interest in drugs that can target gut mucins in ALD has grown; however to date, no studies have examined the properties of drugs on expression of gut mucins in models of ALD. We previously demonstrated that at 10 mg/kg/day, the drug fenretinide (N-[4-hydroxyphenyl] retinamide [Fen]), a synthetic retinoid, mitigates alcohol-associated damage to the gut barrier and liver injury in a murine model of ALD. Methods: In this study, we specifically sought to examine the effects of Fen on gut goblet cells, and expression of mucins, including MUC2 using a 25-day Lieber-DeCarli model of chronic alcohol intake. Results: Our results show that chronic alcohol intake increased gut-mucosal thickening, goblet cell numbers, and mRNA and protein expression of MUC2 in both the ileum and colon. Alcohol intake was associated with marked decreases in ileal and colonic Notch signaling, levels of Notch ligands Dll1 and Dll4, and increases in the expression of Notch-associated genes indispensable for goblet cell specification, including Math1 and Spdef. Interestingly, ileal and colonic expression of KLF4, which is involved in terminal differentiation of goblet cells, was reduced in mice chronically fed alcohol. Coadministration of alcohol with Fen at 10 mg/kg/day significantly reduced alcohol-associated increases in ileal and colonic mucosal thickening, ileal Muc2, colonic Muc2, Muc5ac and Muc6 mRNAs, and goblet cell numbers. We also found that Fen strongly prevented alcohol-mediated suppression of the Notch ligand Dll1, Notch signaling, and alcohol-induced increases in expression of Notch-associated goblet cell specification genes in both the ileum and colon. In the absence of alcohol, Fen treatments alone at 10 mg/kg/day had no effects on any of the goblet cell-related endpoints. Conclusion: These data show for the first time that the drug Fen possesses mucosal layer-modulating properties in response to chronic alcohol abuse. These data warrant further preclinical examination of Fen given the need for anti-ALD drugs and emerging evidence of a role for intestinal goblet cell mucins in the progression of ALD.

Effects of dietary Saccharomyces cerevisiae on growth, intestinal and liver health, intestinal microbiota and disease resistance of channel catfish (Ictalurus punctatus)

Probiotics are an eco-friendly alternative to antibiotics and play an important role in ensuring the sustainability of aquaculture. The aim of the present study was to investigate the effects of Saccharomyces cerevisiae on the growth, intestinal and liver health, intestinal microbiota, and disease resistance of channel catfish (Ictalurus punctatus). Channel catfish were randomly allotted into two treatments, control group (basal diet) and SCC group (basal diet with 2% Saccharomyces cerevisiae culture), and fed for 12 weeks. The results showed that the weight gain of SCC group was significantly increased (P < 0.05) and FCR was significantly decreased (P < 0.05) when compared with the control group. SCC diet significantly improved the intestinal structural integrity of channel catfish and increased the number of intestinal goblet cell. The expression of intestinal gene HIF1α was increased in SCC group, while the expressions of NF-κB in the intestine and liver were significantly decreased (P < 0.05). The relative abundance of Firmicutes tended to increase, while Proteobacteria had a tendency to decrease (0.20 > P > 0.05). In addition, the survival rate of channel catfish was significantly increased in SCC group after being challenged with mixture of A. veronii HM091 and A. hydrophila NJ-1. In conclusion, the supplementation of Saccharomyces cerevisiae culture improved the growth, intestinal and liver health and disease resistance of channel catfish.

Lambs fed diets containing by-product from coconut processing: histomorphometry characteristics in the digestive and renal systems.

This study aimed to evaluate the histological characteristics in the digestive and renal systems of lambs fed diets containing coconut by-product (CB). A total of 35 male lambs with an initial weight of 16.9 ± 2.93kg were distributed in a completely randomized design with five levels of CB in the diet (0; 4.8; 9.6; 14.4 and 19.2% in total dry matter). Samples of the liver, kidney, rumen, and intestine were histomorphometrically evaluated, and the data were submitted to regression analysis, at a 5% error probability. The inclusion of CB linearly decreased the dry matter intake and caused a quadratic effect for the height of ruminal papillae, absorption area, epithelium thickness, as well as for average daily gain. The inclusion of CB linearly increased the mucous layer and reduced the submucosal layer, as well as promoted a decrease in goblet cells in the small intestine. The inclusion of CB did not influence hepatic glycogen; additionally, the histopathological examination did not reveal liver damage or congestion, vacuolization, and necrosis of the renal tissue. Therefore, our results indicate that CB can be included in lambs diet up to the level of 7.2% without causing changes in the histomorphometry characteristics of the gastrointestinal tract and changes in liver and kidney tissue that compromise animal performance.

Loss of Myosin Vb leads to dysregulation of colonic goblet cell structure and function

BackgroundIntestinal goblet cells secrete a protective mucus layer preventing bacteria and harmful ingested substances from contacting the columnar epithelial cells that line the intestine. Single cell RNAseq data has revealed that goblet cells have high expression of Myosin 5b (Myo5b), a molecular motor that regulates vesicle trafficking in epithelial cells. Myo5b is most widely studied in the small intestine, but it’s role in the colonic epithelium and particularly goblet cells is unknown. A recent study found that Myo5b expression was reduced in inflammatory bowel disease (IBD), a condition that is characterized by dysfunctional mucus; potentially providing a link between Myo5b and goblet cell function. We hypothesized that loss of Myo5b would perturb goblet cell mucus production.MethodsAdult control and intestinal specific tamoxifen inducible VillinCreERT2;Myo5bflox/flox(Myo5b KO) mice were injected intraperitoneally with 2 mg of tamoxifen to deplete intestinal Myo5b. After four days colonic tissue was collected and analyzed.ResultsWe found that loss of Myo5b in vivo results in reduced number of goblet cells in the colon by H&amp;E staining. Muc2 immunostaining and alcian blue staining similarly demonstrated a reduction of mucin within goblet cells. Mucin proteins are heavily o‐glycosylated which makes up 80% of the molecular weight. Normally, mucin structures are terminated by sialic acid which provides a negative charge that protects the mucus protein from bacterial degradation. Staining for sialic acid with the lectin SNA revealed a decrease in sialic acid levels in the mucus of Myo5b KO mice compared to controls. Once sialic acid is removed from mucins, the underlying glycan structures are exposed. Using the lectin WGA to identify the internal glycan oligosaccharide n‐acetyl glucosamine, we observed increased exposure of n‐acetyl glucosamine in Myo5b KO mice compared to control mice; which was consistent with our sialic acid findings. Intestinal goblet cells also secrete wound healing factors including the peptide Trefoil factor 3 (TFF3). Similar to the pattern we observed for Muc2 and sialic acid content, we found decreased levels of TFF3 in the mucosa of Myo5b KO mice.ConclusionsOur data shows that loss of functional Myo5b in the colonic epithelium results in decreased goblet cell numbers and alterations in mucin composition. These data suggest that Myo5b may play an important role in goblet cell mucus production and that mutations affecting Myo5b function may contribute to IBD.

Interleukin-10 regulates goblet cell numbers through Notch signaling in the developing zebrafish intestine

Cytokines are immunomodulatory proteins that orchestrate cellular networks in health and disease. Among these, interleukin (IL)-10 is critical for the establishment of intestinal homeostasis, as mutations in components of the IL-10 signaling pathway result in spontaneous colitis. Whether IL-10 plays other than immunomodulatory roles in the intestines is poorly understood. Here, we report that il10, il10ra, and il10rb are expressed in the zebrafish developing intestine as early as 3 days post fertilization. CRISPR/Cas9-generated il10-deficient zebrafish larvae showed an increased expression of pro-inflammatory genes and an increased number of intestinal goblet cells compared to WT larvae. Mechanistically, Il10 promotes Notch signaling in zebrafish intestinal epithelial cells, which in turn restricts goblet cell expansion. Using murine organoids, we showed that IL-10 modulates goblet cell frequencies in mammals, suggesting conservation across species. This study demonstrates a previously unappreciated IL-10-Notch axis regulating goblet cell homeostasis in the developing zebrafish intestine and may help explain the disease severity of IL-10 deficiency in the intestines of mammals.

Disordered Expression of Tight Junction Proteins Is Involved in the Mo-induced Intestinal Microenvironment Dysbiosis in Sheep.

To evaluate the molybdenum (Mo)-induced changes of intestinal morphology and the relationship of intestinal tight junction (TJ) proteins expression and intestinal barrier function, a total of 20 healthy sheep were randomly divided into five groups of four: 0, 5, 10, 20, and 50mg/kg BW/day Na2MoO4·2H2O were administrated in five groups named control group, Mo 5 group, Mo 10 group, Mo 20 group, and Mo 50 group, respectively. After 28days of Mo treatment, the duodenum, the jejunum, and the ileum tissue were collected. The histopathology and the developmental parameters were evaluated by hematoxylin-eosin staining. The intestinal epithelial cell DNA damage was detected by TdT-mediated dUTP nick end labeling assay. The intestinal glycoprotein and the goblet cells were analyzed by Alcian Blue-Periodic Acid-Schiff (AB-PAS) staining and PAS staining, respectively. TJ proteins were determined by immunofluorescence technology. Results showed that excessive Mo significantly decreased the small intestinal villus height (VH), crypt depth (CD), VH/CD, and mucosal thickness (P < 0.05 or P < 0.01) while induced the damage of DNA in small intestinal epithelial cells. Moreover, excessive Mo injured intestinal barrier function by decreasing the percent of glycoprotein distribution area (P < 0.05) and the relative density of intestinal goblet cells (P < 0.05). Mo treatment induced decreased (P < 0.01) expression of Zonula Occludens-1, Occludin, and Claudin-1. In conclusion, excessive Mo interfered with the expression of TJ proteins, inhibited intestinal epithelial development, and further aggravated the intestinal barrier function damage, leading to disturbing the small intestinal microenvironment balance.

Lactobacillus casei Improve Anti-Tuberculosis Drugs-Induced Intestinal Adverse Reactions in Rat by Modulating Gut Microbiota and Short-Chain Fatty Acids.

The adverse effects of anti-tuberculosis (TB) drugs in the intestines were related to alteration of the intestinal microbiota. However, there was less information about microbial metabolism on the adverse reactions. This study aimed to explore whether Lactobacillus casei could regulate gut microbiota or short-chain fatty acids (SCFAs) disorders to protect intestinal adverse reactions induced by isoniazid (H) and rifampicin (R). Male Wistar rats were given low and high doses of Lactobacillus casei two hours before daily administration of anti-TB drugs. After 42 days, colon tissue and blood were collected for analysis. The feces at two-week and six-week were collected to analyze the microbial composition and the content of SCFAs in colon contents was determined. Supplementation of Lactobacillus casei increased the proportion of intestinal goblet cells induced by H and R (p < 0.05). In addition, HR also reduced the level of mucin-2 (p < 0.05), and supplementation of Lactobacillus casei restored. After two weeks of HR intervention, a decrease in OTUs, diversity index, the abundance of Bacteroides, Akkermansia, and Blautia, and an increase of the abundance of Lacetospiraceae NK4A136 group and Rumencoccus UCG-005, were observed compared with the control group (p all < 0.05). These indices in Lactobacillus casei intervention groups were similar to the HR group. Six-week intervention resulted in a dramatic reduction of Lacetospiraceae NK4A136 group, butyric acid, valeric acid and hexanoic acid, while an increase of Bacteroides and Blautia (p all < 0.05). Pretreatment with Lactobacillus casei significantly increased the content of hexanoic acid compared with HR group (p < 0.05). Lactobacillus casei might prevent intestinal injury induced by anti-tuberculosis drugs by regulating gut microbiota and SCFAs metabolism.

The gastrointestinal tract histology of Eliomys quercinus and Glis glis

The Garden Dormouse (Eliomys quercinus) and the Edible Dormouse (Glis glis) differ in their omnivorous diet, with the first being more carnivorous and readily consuming small vertebrates and even poisonous invertebrates, while the latter consumes more plant-based foods. These differences could lead to histological differences in their gastrointestinal tract, which we wanted to test. Six animals of each species were caught in nature and fed the same diet for two weeks to diminish the impact of the short-term external factors. Tissue samples were taken of middle oesophagus, glandular stomach, middle small intestines and large intestines. They were stained with H&amp;amp;E for measuring the thickness of layers in oesophagus gastric mucosa and epithelial cell height in the intestines. Alcian blue - PAS stain was used for goblet cell count and type identification in the intestines, differentiating blue (acidic), lilac (neutral) and purple (mixed) cells. Results showed Eliomys quercinus having greater variance in oesophageal layer thicknesses, significantly thicker muscularis mucosae and thinner tunica muscularis in the oesophagus, and thicker gastric mucosa compared to Glis glis. Eliomys quercinus also had a significantly higher total goblet cell count in both intestines and a higher count of each type in the large intestine, although Glis glis had far more mixed and neutral goblet cells in the small intestine. Both showed intriguingly low numbers of neutral goblet cells in the small intestine. These results indicate interesting differences in gastrointestinal histology and goblet cell distribution in comparison with their distinct diets.

Effects of dietary galactooligosaccharide on growth, antioxidants, immunity, intestinal morphology and disease resistance against Aeromons hydrophila in juvenile hybrid sturgeon (Acipenser baerii♀ × A. schrenckii♂)

The present study investigated the effects of dietary galactooligosaccharide (GOS) supplementation on growth, antioxidant capacity, immunity, intestinal morphology and resistance to Aeromons hydrophila of hybrid sturgeon (Acipenser baerii♀ × A. schrenckii♂). A total number of 360 fish were randomly distributed into 12 aquaria and fed diets supplemented with GOS at 0%, 1%, 3% and 5% concentrations (designated as control, GOS1, GOS3 and GOS5) for 8 weeks. Then 30 fish from each group were intraperitoneally challenged with A. hydrophila, and mortality was recorded for the next 15 days. The results showed that there were significant differences of final body weight (FBW), weight gain (WG), specific growth rate (SGR) and feed conversion ratio (FCR) in sturgeon fed GOS diets. The lysozyme, ACP, AKP and MPO activities in sturgeon serum fed GOS diets were higher than those with control diet. Significant increase of SOD, CAT and GSH-PX capacity and remarkably decrease of MDA content were observed in liver of fish fed GOS diets. Remarkable enhancement of muscular thickness, villus and microvilli height, and goblet cells in mid intestine was observed in GOS supplemented groups. In spiral valve, supplementation of any quantities of GOS promoted an evident increase in the muscular thickness, villus height and goblet cells, while the villus width and crypt depth were relatively invariant. The IL-1β and IL-8 mRNA levels were decreased and TNF-α gene expression was increased by dietary GOS. The challenge test with A. hydrophila resulted in lower mortalities by GOS administration in a dose-dependent manner and the highest relative percent survival (36.67 ± 1.92%) was observed in fish fed GOS5 diet. The study concludes that GOS inclusion at the rate of 3% is recommended to increase the growth performance, antioxidant capability and immunity as well as regulate intestinal morphology and promote resistance against A. hydrophila in hybrid sturgeon.

Comparative microanatomical and histochemical biodistribution profiles of different types of mucins in the intestinal mucosa of some tetrapod representatives.

The microanatomical features of the intestinal tract mucosa layer in different species of tetrapoda vary according to the type of species, tissue, and function of the targeted cells. In the present study, we have evaluated the histological and histochemical variations of the intestinal tract in four species representing superclass tetrapoda. Bufo regularis (toad), Trachylepis quinquetaeniata (lizard), Columba livia domestica (pigeon) and Mus musculus (mouse) were used as representatives for amphibians, reptilians, avians and mammalians respectively. Histologically, the ileum's mucosal layer of the lower tetrapods (toad and lizard) was almost similar and consists of elongated finger-like shape villi lined with simple columnar epithelium and goblet cells. Similarly, the microanatomical features in ileum of higher tetrapod representatives (pigeon and mouse) were characterized by the presence of villi lined with simple columnar epithelium and scattered goblet cells as well as intestinal glands (crypts of Lieberkühn) at the bases of the intestinal villi. In the toad rectum, the mucosal layer was similar to that of the ileum but with shorter villi and more numerous goblet cells. However, the mucosal layer of the rectum in the lizard had low numbers of absorptive columnar epithelial cells with abundant goblet basal cells. Comparatively, the pigeon's rectal mucosa had almost a similar structure to that of ileum but in leaf-like shaped villi. Finally, the rectum of the mouse has narrow rectal pits, instead of villi, lined with goblet cells and absorptive epithelial cells. Histochemically, the ileum in the four studied tetrapod representatives showed varying biodistribution profiles of neutral, sulfated and carboxylated mucins. There are variations encountered in the intestinal brush border and goblet cells of villi in all species as well as the crypts of Lieberkühn in higher tetrapods. Also, the rectum of all tetrapod species showed weak to strong positive signals for the three types of mucins in the brush border and goblet cells of villi in all species and crypts of Lieberkühn in higher tetrapods as well. In addition, the brush border of toad's rectum was lacking sulfated mucins and that of the lizard did not have any type of mucins. The data of this study will contribute to understand the relationship between the microanatomical features and mucins biodistribution profiles in the mucosal layer of tetrapod intestinal tract and their functions.

Heat Stress-Induced Dysbiosis of Porcine Colon Microbiota Plays a Role in Intestinal Damage: A Fecal Microbiota Profile.

The pathological mechanisms of gastrointestinal disorders, including inflammatory bowel disease (IBD), in pigs are poorly understood. We report the induction of intestinal inflammation in heat-stressed (HS) pigs, fecal microbiota transplantation from pigs to mice, and explain the role of microorganisms in IBD. 24 adult pigs were subjected to HS (34 ± 1 °C; 75–85% relative humidity for 24h) while 24 control pigs (CP) were kept at 25 ± 3°C and the same humidity. Pigs were sacrificed on days 1, 7, 14, 21. Colonic content microbiome analyses were conducted. Pseudo-germ-free mice were fed by gavage with fecal microbiota from HS-pigs and CP to induce pig-like responses in mice. From 7 d, HS-pigs exhibited fever and diarrhea, and significantly lower colonic mucosal thickness, crypt depth/width, and goblet cell number. Compared with each control group, the concentration of cortisol in the peripheral blood of HS pigs gradually increased, significantly so on days 7, 14, and 21 (P < 0.01). While the concentration of LPS in HS pigs' peripheral blood was significantly higher on days 7, 14 (P < 0.01), and 21 (P < 0.05) compared with that of the control group. The colonic microbiome composition of HS-pigs was different to that of CP. By day 14, opportunistic pathogens (e.g., Campylobacterales) had increased in HS-pigs. The composition of the colonic microbiome in mice administered feces from HS-pigs was different from those receiving CP feces. Bacteroides were significantly diminished, Akkermansia were significantly increased, and intestinal damage and goblet cell numbers were higher in mice that received HS-pig feces. Moreover, we verified the relevance of differences in the microbiota of the colon among treatments. Heat stress promotes changes in gut microbiome composition, which can affect the colonic microbial structure of mice through fecal microbiota transplantation; the molecular mechanisms require further investigation. This study enhanced our understanding of stress-induced inflammation in the colon and the increase in diarrhea in mammals subjected to prolonged HS. Our results provide useful information for preventing or ameliorating deficits in pig production caused by prolonged exposure to high temperatures.

Early Antibiotic Exposure Alters Intestinal Development and Increases Susceptibility to Necrotizing Enterocolitis: A Mechanistic Study.

Increasing evidence suggests that prolonged antibiotic therapy in preterm infants is associated with increased mortality and morbidities, such as necrotizing enterocolitis (NEC), a devastating gastrointestinal pathology characterized by intestinal inflammation and necrosis. While a clinical correlation exists between antibiotic use and the development of NEC, the potential causality of antibiotics in NEC development has not yet been demonstrated. Here, we tested the effects of systemic standard-of-care antibiotic therapy for ten days on intestinal development in neonatal mice. Systemic antibiotic treatment impaired the intestinal development by reducing intestinal cell proliferation, villi height, crypt depth, and goblet and Paneth cell numbers. Oral bacterial challenge in pups who received antibiotics resulted in NEC-like intestinal injury in more than half the pups, likely due to a reduction in mucous-producing cells affecting microbial–epithelial interactions. These data support a novel mechanism that could explain why preterm infants exposed to prolonged antibiotics after birth have a higher incidence of NEC and other gastrointestinal disorders.

A9 SIALIC ACID METABOLISM PLAYS A KEY ROLE IN INTESTINAL FITNESS AND VIRULENCE OF AN ATTACHING/ EFFACING BACTERIAL PATHOGEN

BackgroundThe gastrointestinal (GI) mucus barrier acts as an important interface between the host and luminal gut microbes, beyond its role in limiting direct contact between noxious luminal agents and the underlying intestinal epithelium. Mucus is comprised of highly glycosylated mucin proteins, modified by O-glycan side chains, formed by five sugar monomers, including sialic acid. Sialic acid frequently occupies the terminal position of O-glycans and can be cleaved by microbial sialidases. We hypothesize that upon entering their hosts, enteric pathogens, such as the attaching and effacing (A/E) family (EHEC, EPEC and Citrobacter rodentium) metabolically adapt to their intestinal environment, and express key virulence factors by sensing and metabolizing mucin sugars, such as sialic acid.AimsInvestigate the role of sialic acid in regulating the fitness and virulence strategies of the A/E pathogen C. rodentium within the GI environment.MethodsC57Bl/6 mice were orally infected with either wildtype (WT) or mutant strains of C. rodentium to study bacterial pathogenicity in vivo. Sialic acid was localized in mouse colonic tissue sections through lectin staining, and quantified in mouse feces and mucus scrapings using a commercial kit. Protein secretion by C. rodentium, in the presence or absence of sialic acid was analyzed by SDS-PAGE and mass spectrometry. Intestinal epithelial cell lines were infected with enteropathogenic E. coli (EPEC) or C. rodentium to examine bacterial adherence.ResultsSialic acid was expressed widely in the GI tracts of mice, primarily in the colonic mucus layer and by intestinal goblet cells. Both EPEC and C. rodentium were found to take up and metabolize sialic acid through the transporter NanT. A C. rodentium strain deficient in sialic acid uptake ( ΔnanT) was dramatically impaired in colonizing the intestines of mice and was rapidly cleared. Sialic acid also impacted C. rodentium’s virulence by inducing the secretion of two key virulence factors, which significantly enhanced the pathogen’s adhesion to intestinal epithelial cells. Moreover, sialic acid increased C. rodentium’s ability to degrade mucus, due to the increased production of these two secreted virulence factors.ConclusionsWe demonstrate that sialic acid, a mucin-derived sugar, is an essential nutrient for A/E pathogens to thrive and expand within their host’s intestines. Moreover, sialic acid enhances pathogen virulence by inducing secretion of two important virulence factors, which increase adhesion to the epithelium and promote the degradation of mucus.Funding AgenciesCCC, CIHRCH.I.L.D. Fdn

Spray-Dried Porcine Plasma Promotes the Association Between Metabolic and Immunological Processes at Transcriptional Level in Gilthead Sea Bream (Sparus aurata) Gut

The spray-dried porcine plasma (SDPP) is an abattoir by-product used in animal nutrition with beneficial effects reported in livestock and commercial aquatic species. Previous results have found that the dietary inclusion of SDPP in gilthead sea bream (Sparus aurata) increased the density of intestinal goblet cells, and it did not result in significant changes in the autochthonous microbiota. However, there is no comprehensive data on the mechanisms that could take place on the intestine of gilthead sea bream fed with an SDPP-supplemented diet. For this reason, this study aimed to unveil the biological mechanisms modulated in response to the dietary administration of SDPP in the gilthead sea bream gut. To achieve this goal, we made a microarrays-based transcriptomic approach in gut samples from gilthead sea bream fed with an SDPP-supplemented diet for 95 days. As control diet, we used a protein-rich commercial feed (51% crude protein, 17% crude fat, and 20.6 MJ/kg gross energy) which was supplemented with 3% SDPP at the expense of LT70 fishmeal. The microarray analyses showed a total of 803 (468 up- and 335 down-regulated) differential expressed genes (DEGs). The functional network analysis revealed that dietary inclusion of SDPP induced sustained changes in 120 biological processes, grouped in 12-clusters. Among them, the metabolic-related process (cellular catabolic process, organic substance catabolic process, protein metabolism process), protein transport, and leukocyte mediated immunity interacted in the leading interactome network. This evidence confirms the previous evidence of the enhancement of the mucosal health status in response to the dietary administration of SDPP and provides further understanding of the mode of action of this ingredient in aquafeeds.

Acanthopanax senticosus total flavonoids alleviate lipopolysaccharide-induced intestinal inflammation and modulate the gut microbiota in mice

Here, we study the therapeutic effect of Acanthopanax senticosus total flavonoids (ASTFs) using a mouse intestinal inflammation model. The inflammation model used in the present study was developed through lipopolysaccharide (LPS) treatment of mice. The experimental mice were divided into a control group, model group (10 mg/kg LPS), dexamethasone group (1 mg/kg DEX) and ASTF low-, medium- and high-dosage groups (200, 400 and 800 mg/kg, respectively). The morphological and structural changes in the ileum, jejunum and duodenum were observed using HE staining. The number of intestinal goblet cells (GCs) was calculated based on PAS staining. The contents of interleukin (IL)-1β, IL-6, prostaglandin E2 (PGE2) and tumor necrosis factor α (TNF-α) were determined using enzyme-linked immunosorbent assay (ELISA) and the related mRNA expression level were measured by RT-PCR. The protein expression levels of Toll-like receptor 4 (TLR4), MyD88, p65 and p-p65 were measured using Western blotting. In addition, the 16S rRNA sequences of bacterial taxa were amplified and analyzed to assess changes in the intestinal microbes of LPS-induced mice and also in response to regulation by ASTF. Following intervention with ASTF, different therapeutic effects were shown according to the various dosages tested, all of which resulted in improved intestinal morphology and an increased number of intestinal GCs, while the contents of IL-1β, IL-6, PGE2 and TNF-α and the related mRNA expression level were significantly reduced. The TLR4, MyD88 and p-p65/p-65 protein expression levels were also significantly reduced. In addition, 16S rRNA sequencing results show that LPS disrupts the structure of mouse gut microbes, though we observed that normal microbial status can be restored through ASTF intervention.

WR-GLP2, a glucagon-like peptide 2 from hybrid crucian carp that protects intestinal mucosal barrier and inhibits bacterial infection

Glucagon-like peptide 2 (GLP2) is a proglucagon-derived peptide produced by intestinal enteroendocrine L-cells. The main biological actions of GLP2 in mammals are related to regulating energy absorption and maintaining the morphology, integrity of intestinal mucosa. However, the in vivo function of fish GLP2 in intestinal barrier and immune defense is essentially unknown. With an aim to elucidate the antimicrobial mechanism of GLP2 in fish, we in this study examined the function of GLP2 from hybrid crucian carp. Hybrid crucian carp GLP2 (WR-GLP2) possesses the conserved glucagon like hormones 2 domain. WR-GLP2 is mainly expressed in the intestine and is significantly upregulated after Aeromonas hydrophila infection. AB-PAS staining analysis showed WR-GLP2 significantly increased the number of goblet cells in intestine. WR-GLP2 induced significant inductions in the expression of the antimicrobial molecules (MUC2, Lyzl-1, Hepcidin-1 and LEAP-2) and tight junctions (ZO-1, Occludin and Claudin-4). In addition, WR-GLP2 significantly alleviated the intestinal apoptosis, thereby enhancing host's resistance against Aeromonas hydrophila infection. Together these results indicate that WR-GLP2 is involved in intestinal mucosal barrier and immune defense against pathogen infection.

Giardia duodenalis cysteine proteases cleave proteinase-activated receptor-2 to regulate intestinal goblet cell mucin gene expression

Giardia duodenalis cysteine proteases have been identified as key virulence factors and have been implicated in alterations to intestinal goblet cell activity and mucus production during Giardia infection. The present findings demonstrate a novel mechanism by which Giardia cysteine proteases modulate goblet cell activity via cleavage and activation of protease-activated receptor 2. Giardia duodenalis (assemblage A) increased MUC2 mucin gene expression in human colonic epithelial cells in a manner dependent upon both protease-activated receptor 2 activation and Giardia cysteine protease activity. Protease-activated receptor 2 cleavage within the N-terminal activation domain by Giardia proteases was confirmed using a nano-luciferase tagged recombinant protease-activated receptor 2. In keeping with these observations, the synthetic protease-activated receptor 2-activating peptide 2fLIGRLO-amide increased Muc2 gene expression in a time-dependent manner. Calcium chelation and inhibition of the ERK1/2 mitogen activated protein kinase pathway inhibited Muc2 upregulation during Giardia infection, consistent with canonical protease-activated receptor 2 signaling pathways. Giardia cysteine proteases cleaved both recombinant protease-activated receptor 1 and protease-activated receptor 2 within their extracellular activation domains with isolate-dependent efficiency that correlated with the production of cysteine protease activity. Protease-activated receptors represent a novel target for Giardia cysteine proteases, and these findings demonstrate that protease-activated receptor 2 can regulate mucin gene expression in intestinal goblet cells.

METHYLTRANSFERASE SMYD2 MODULATES THERAPEUTIC EFFECTS OF EPIDERMAL GROWTH FACTOR IN INFLAMMATORY BOWEL DISEASE

Abstract BACKGROUND Inflammatory bowel disease (IBD) is a chronic and remitting inflammatory disorder of the gastrointestinal tract. A prior clinical trial demonstrated the ability of epidermal growth factor receptor (EGFR) ligand EGF to induce and maintain remission in ulcerative colitis. As a protein lysine methyltransferase, SET and MYND domain-containing protein 2 (SMYD2) regulates various cellular functions, including chromatin remodeling and tumorigenesis. A bioinformatic study has shown that EGFR might be a candidate for SMYD2-mediated methylation. In this research, we aimed to investigate whether SMYD2 depletion could synergize with EGFR-activating treatment to promote intestinal goblet cell regeneration in IBD. METHODS SMYD2 flox C57BL/6 (SMYD2fl/fl) and intestine epithelial cell-specific conditional knockout of SMYD2 (SMYD2ΔIEC) male mice (8 to10-week old) were given 3 cycles of DSS treatment, each cycle including 5 days of 2% DSS in drinking water followed by 16 days of normal drinking water. EGF (50 μg/kg/day) was administered via intraperitoneal injection once per day for 6 days during the third cycle of DSS treatment (from day 42 to 47 along with DSS administration). The disease activity index (DAI), including body weight loss, stool consistency and colorectal bleeding, was monitored and calculated daily. Histologic scoring was used to evaluate the extent of histopathologic change in middle colon tissue. Goblet cell differentiation and maturation was analyzed via Alcian Blue/periodic acid-Schiff (AB/PAS) staining and immunostaining. RESULTS EGF treatment significantly increased the number of goblet cells (AB/PAS staining positive cells) in both SMYD2fl/fl and SMYD2ΔIEC mice. However, in SMYD2ΔIEC mice, more mature goblet cells were observed. In contrast, in SMYD2fl/fl mice, most goblet cells were accumulated at the lower half of colon crypts with small mucin particles. As compared with SMYD2fl/fl mice receiving EGF treatment, SMYD2 depletion combined with EGF injection (6 days) failed to significantly improve body weight loss and DAI scoring during DSS treatment, as well as histologic scoring (including inflammatory cell infiltration and epithelial tissue damage). Immunostaining of goblet cell differentiation markers (including ATOH1, SPDEF and KLF4) revealed no significant difference between SMYD2fl/fl and SMYD2ΔIEC mice. CONCLUSION SMYD2 depletion combined with EGF treatment could promote goblet cell expansion and maturation in mouse DSS-induced chronic colitis. However, the mechanism by which SMYD2 depletion facilitates goblet cell maturation needs further investigation.

P007 ORMDL proteins shape homeostasis in the intestinal epithelium by regulating endoplasmic reticulum architecture and autophagy

Abstract Background ORM1-like protein 3 (ORMDL3) is an endoplasmic reticulum (ER)-resident protein that has been implicated in ER homeostasis and sphingolipid biosynthesis. Genome-wide association studies identified susceptibility loci around the ORMDL3 gene for inflammatory diseases, including asthma and IBD. Preliminary studies suggested a role of ORMDL proteins within the ER stress-induced unfolded protein response (UPR). Here, we set out to investigate the role of ORMDL proteins in intestinal inflammation. Methods Mice with constitutive and conditional knockouts (KO) of Ormdl1, Ormdl2 and Ormdl3 in intestinal epithelial cells were generated. From these, intestinal organoids were generated to perform functional assessment of ER stress, autophagy, and inflammatory responses. We performed 3’-single cell RNA sequencing (scRNAseq) of small intestine tissue of animals with triple conditional knockout of Ormdl1/Ormdl2/Ormdl3 (“Ormdl123-VilCre”) vs. wildtype to identify cell type-specific effects of epithelial ablation of ORMDL proteins. 16S rDNA sequencing was performed on stool samples from these animals. Results Mice deficient for ORMDL displayed reduced body weight dependent on the number of knockouts. Ormdl1/2/3-KO mice are embryonically lethal while Ormdl1/2/3-VilCre animals were viable without signs of spontaneous inflammation. Ormdl1- and Ormdl3-KO animals displayed reduced number of Paneth and goblet cells in the small intestine, accompanied by massive dilation/ballooning of the ER. This phenotype was strongly pronounced in Ormdl1/3-KO and Ormdl1/2/3-VilCre animals. ORMDL3 co-localises and co-precipitates with LC3, indicating a direct interaction with autophagy proteins, while loss of ORMDL proteins impaired autophagic flux. Interestingly, UPR and Cxcl1 expression were insufficiently induced in intestinal organoids from Ormdl1/2/3-VilCre animals treated with tunicamycin. scRNAseq analysis of small intestine tissue surprisingly revealed compensatory induction of antimicrobial peptides in non-Paneth cells and vice versa downregulation of apoptosis regulating gene in Ormdl1/2/3-VilCre mice. We observed an expansion of myeloid and plasma cells in Ormdl1/2/3-VilCre mice, while no changes in epithelial cell proportions can be identified. These findings were accompanied by dysbiosis of the stool microbiome (β-diversity, Bray-Curtis dissimilarity) of Ormdl1/2/3-VilCre animals. Conclusion Our data suggest a critical regulatory role of the IBD risk gene ORMDL3 in the ER-autophagy axis in intestinal epithelial cells, shaping the mucosa homeostasis on cellular and microbiome level. Functional assessment of inflammation in Ormdl1/2/3-VilCre animals (e.g., DSS colitis model) will be performed to understand the role of ORMDL in intestinal inflammation.