Crypt Goblet Cells Research Articles

Effect of increasing supplementation of dietary glycine on growth performance, meat quality, liver characteristics, and intestinal health in broiler chickens raised under heat stress conditions

The current study aimed to investigate the effect of increasing supplementation of dietary glycine (Gly) on growth performance, meat quality, liver characteristics, and intestinal health in broiler chickens raised under heat stress (HS) conditions. A total of one thousand six hundred 25-d-old broiler chickens were randomly allotted to 1 of 5 dietary treatments with 8 replicates. Each replicate comprised 20 male and 20 female birds. A negative control (NC) diet was prepared to meet or exceed energy and nutrient requirement estimates, whereas a positive control (PC) diet was formulated to contain increasing concentrations of AMEn by 50 kcal/kg as well as those of digestible amino acids, total Ca, and available P by 10% compared with the respective concentrations in the NC diet. Three additional diets were prepared by supplementing the NC diet with 0.4, 0.8, or 1.6% Gly. All chickens were raised under cyclic HS conditions at 29°C ± 0.89°C for 10 h/d and 23°C ± 1.45°C for the remaining time over an 18-d feeding trial. Results indicated that broiler chickens fed the NC diet had a greater (P < 0.05) FCR than those fed the PC diet under HS conditions. Increasing supplementation of up to 1.6% Gly in diets decreased (linear, P < 0.001) FCR in broiler chickens. Increasing supplementation of dietary Gly tended to increase (linear, P = 0.070) water holding capacity in the breast meat. Increasing supplementation of dietary Gly decreased (linear, P < 0.05) serum aspartate aminotransferase concentrations and tended to decrease blood heterophil:lymphocyte (linear, P = 0.083) and liver malondialdehyde concentrations (quadratic, P = 0.084). A tendency for increased villus height (linear, P = 0.086) and a significant increase in villus height:crypt depth ratio and goblet cell numbers (linear, P < 0.05) were identified following increasing Gly supplementation. In conclusion, increasing supplementation of dietary Gly improved feed efficiency, meat quality, liver health, and intestinal morphology possibly by mitigating oxidative stress and stress response in broiler chickens raised under HS conditions.

Sex differences in intestinal morphology and increase in diencephalic neuropeptide Y gene expression in female but not male Pekin ducks exposed to chronic heat stress.

The impact of heat stress (HS) on production is intricately linked with feed intake. We investigated the effects of HS on intestines and diencephalic genes in Pekin ducks. One hundred and sixty adult ducks were allocated to two treatment rooms. The control room was maintained at 22°C and the HS room at 35°C for the first 10 h of the day then reduced to 29.5°C. After 3 weeks, 10 hens and 5 drakes were euthanized from each room and jejunum and ileum collected for histology. Brains were collected for gene expression analysis using qRT-PCR. Intestinal morphology data were analyzed with two-way ANOVA and diencephalic gene data were analyzed with Kruskal-Wallis test. There was an increase in villi width in the ileum (p = .0136) and jejunum (p = .0019) of HS hens compared to controls. HS drakes showed a higher crypt depth (CD) in the jejunum (p = .0198) compared to controls. There was an increase in crypt goblet cells (GC) count in the ileum (p = .0169) of HS drakes compared to HS hens. There was higher villi GC count (p = .07) in the jejunum of HS drakes compared to controls. There was an increase in the crypt GC density (p = .0054) in the ileum, not jejunum, of HS drakes compared to HS hens. Further, there were no differences in the proopiomelanocortin gene expression in either sex but there was an increase in the expression of neuropeptide Y (NPY) gene in HS hens (p = .031) only and a decrease in the corticotropin releasing hormone gene in the HS drakes (p = .037) compared to controls. These data show that there are sex differences in the effect of HS on gut morphology while the upregulation in NPY gene may suggest a role in mediating response to chronic HS.

Heat shock protein 90 and prolyl hydroxylase 2 co-regulate hypoxia-inducible factor-1α expression in porcine small intestinal epithelial cells under heat stress

Heat stress (HS) poses a substantial threat to animal growth and development, resulting in declining performance and economic losses. The intestinal system is susceptible to HS and undergoes intestinal hyperthermia and pathological hypoxia. Hypoxia-inducible factor-1α (HIF-1α), a key player in cellular hypoxic adaptation, is influenced by prolyl-4-hydroxylase 2 (PHD2) and heat shock protein 90 (HSP90). However, the comprehensive regulation of HIF-1α in the HS intestine remains unclear. This study aims to explore the impact of HS on pig intestinal mucosa and the regulatory mechanism of HIF-1α. Twenty-four Congjiang Xiang pigs were divided into the control and five HS-treated groups (6, 12, 24, 48, and 72 h). Ambient temperature and humidity were maintained in a thermally-neutral state (temperature–humidity index (THI) < 74) in the control group, whereas the HS group experienced moderate HS (78 < THI <84). Histological examination revealed villus exfoliation after 12 h of HS in the duodenum, jejunum, and ileum, with increasing damage as HS duration extended. The villus height to crypt depth ratio (V/C) decreased and goblet cell number increased with prolonged HS. Quantitative real-time PCR, Western blot, and immunohistochemistry analysis indicated increased expression of HIF-1α and HSP90 in the small intestine with prolonged HS, whereas PHD2 expression decreased. Further investigation in IPEC-J2 cells subjected to HS revealed that overexpressing PHD2 increased PHD2 mRNA and protein expression, while it decreases HIF-1α. Conversely, interfering with HSP90 expression substantially decreased both HSP90 and HIF-1α mRNA and protein levels. These results suggest that HS induces intestinal hypoxia with concomitant small intestinal mucosal damage. The expression of HIF-1α in HS-treated intestinal epithelial cells may be co-regulated by HSP90 and PHD2 and is possibly linked to intestinal hyperthermia and hypoxia.

Phosphatidylethanolamine Improves Postnatal Growth Retardation by Regulating Mucus Secretion of Intestinal Goblet Cells in Piglets.

Phosphatidylethanolamine (PE), a multifunctional phospholipid, is necessary for neonate development. This study aimed to explore the impact of the regulation of exogenous PE on postnatal growth retardation (PGR) by improving intestinal barrier function. Thirty-two neonatal pigs were divided into four groups according to their body weight (BW 2.79 ± 0.50 kg or 1.88 ± 0.40 kg) at 7 days old, CON-NBW, PE-NBW, CON-PGR, and PE-PGR. PE was supplemented to NBW piglets and PGR piglets during lactation and post-weaning periods. Compared with the NBW piglets, the growth performance of PGR piglets was lower, while PE improved the poor growth performance. PGR piglets showed injured intestinal morphology, as evidenced by the reduced ratio of villus height to crypt depth (VH/CD) and goblet cell numbers in the jejunum and ileum. PE recovered the intestinal barrier injury by increasing VH/CD and goblet cell numbers. The decreased MUC2 mRNA and protein expressions were observed in the small intestine of PGR piglets, and PE remarkably increased the expression of MUC2. Mechanistically, PE increased the goblet cell differentiation promoting gene spdef mRNA levels and reduced the mRNA expressions involved in endoplasmic reticulum stress in the jejunal and ileal mucosa of PGR piglets. Overall, we found that PE alleviated growth retardation by regulating intestinal health and generalized its application in neonates.

A12 ROLE OF INTESTINAL EPITHELIAL HNF4A IN PROTECTING AGAINST ACUTE INFLAMMATORY STIMULI

Abstract Background Inflammatory bowel disease is a group of chronic resulting from complex interactions between host susceptibility genes, the host microbiome, the immune system, and environmental triggers. The gastrointestinal epithelium acts as a barrier against harmful stimuli, and defects in its integrity are a precursor for the development of IBD. Our laboratory has shown that the conditional deletion of Hnf4a in the mouse intestinal epithelium leads to chronic intestinal inflammation. However, the functional role of HNF4A on epithelial barrier integrity is still controversial. Aims To evaluate the impact of Hnf4a deletion on barrier integrity during acute inflammation. Methods We used a tamoxifen-inducible CreER-loxP system to delete Hnf4a in the adult intestinal epithelium (Hnf4aΔIEC-ind). Intestinal permeability was assessed in Hnf4aΔIEC-ind and control mice in the collected serum after oral gavage of FITC-dextran. Mice were infected with an invasion-attenuated strain of Salmonella Typhimurium (SB103). S. Typhimurium loads were scored in the feces, liver, and spleen tissue. Histological analysis was carried out by various staining. Also, we used a model of T-cell-induced enteropathy triggered by a T-cell activating monoclonal anti-CD3ε Ab (145-2C11). Mice were assessed for clinical signs of enteropathy and sacrificed 1-, 3-, and 5 days post-injection. Gene expression of selected targets was studied by RT-qPCR. Results FITC-dextran assay showed a significant increase of the intestinal permeability in uninfected mutant mice compared to controls. In contrast, an oral infection with an invasion-deficient S. Typhimurium strain did not differentially impact in tissue bacterial loads or epithelial damage between groups. Likewise, the loss of epithelial HNF4A led to increased Muc2 transcripts expression in the ileum of infected mutant mice but not in the colon. Histological examinations at four days post-infection showed an elevated number and size of goblet cells in the ileal crypts of mutant mice. Bacterial localization using the general bacterial probe EUB388-cy3 (red) staining showed that infected mutant mice tended to enhance barrier mucus layer thickness compared to the controls. On the contrary, acute T cell activation with anti-CD3 mAb induced a significant increase of mRNA expression of Cxcl1 in the small intestine of mutant mice. H&amp;E staining showed elongated ileal crypts in mutant mice compared to controls after anti-CD3 injection. Conclusions Altogether, these results support that HNF4A could play a crucial adaptive role in modulating the small intestine epithelial barrier function and controlling the immune system during acute inflammatory stress. Funding Agencies CCC, CIHR

P072 LDN-071, a novel first-in-class amino acid-based PAI-1 inhibitor, significantly reduces the severity of experimental colitis in mice

Abstract Background Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders characterised by alterations of acute relapses and remission. The past decades have brought substantial advances in the pharmacological management of IBD by wide range of medicines. Although, the sustained efficacy of the currently available anti-inflammatory therapies is still far from optimal. Therefore, the development of novel therapies represents a major unmet clinical need in this field. Recently the enrichment of the coagulation pathway genes and among these, Plasminogen activator inhibitor 1 (PAI-1) was described in IBD, whereas PAI-1 emerged as a link between the epithelium and inflammation. Moreover, the genetic deletion of PAI-1 impaired the severity of experimental colitis in mice. Based on these, our aim was to develop and test novel PAI-1 inhibitors for the treatment of IBD. Methods Novel, amino acid-based PAI-1 inhibitors were synthetized by A3SMO® platform. The inhibitory potential and cytotoxicity effects of the peptides were determined by in vitro assays. Based on the in vitro results LDN-071 was selected for further analysis. Experimental colitis was induced in mice with 3% DSS and after the induction of colitis 10 mg/bwkg LDN-071 was administered per os for four consecutive days. The changes of body weight, spleen weight and colon length were measured. Haematoxylin eosin (HE) and Periodic acid-Schiff (PAS) staining were performed for histology. ELISA and qRT-PCR measurements were applied to analyse the mucosal cytokine expressions. In vivo toxicity of LDN-071 was determined in mice. Results LDN-071 significantly inhibited PAI-1 in vitro without decreasing the cell viability in a concentration range of 1µM-1mM. The administration of 10 mg/bwkg LDN-071 in the DSS-treated mice prevented the body weight loss, the development of bloody diarrhoea, the decrease of colon length and increase of the spleen weight, which were displayed by the DSS+vehicle-treated animals. The histological analysis of the colon revealed that DSS treatment induced a severe transmural inflammation, ulceration, loss of crypt structures and goblet cell depletion. Importantly, all of these parameters were significantly impaired by the administration of 10 mg/bwkg LDN-071. Additionally, mucosal gene and protein expressions of TNF-α, IL-1β and IL-6 were significantly impaired by PAI-1 inhibition. Importantly, no signs of toxicity were LDN-071 observed in mice exposed to 100 mg/bwkg LDN-071 for 7 days. Conclusion Our results showed that LDN-071, a novel amino acid-based inhibitor of PAI-1. It significantly reduces the severity of experimental colitis in mice. We propose that the inhibition of PAI-1 could be a potential novel therapeutic strategy in IBD.

Lactobacillus-derived indole derivatives ameliorate intestinal barrier damage in rat pups with complementary food administration.

The consumption of complementary foods can bring about diarrhea and intestinal barrier dysfunction in infants. In this study, three different Lactobacillus strains combined with L-tryptophan (Trp) were administered to rat pups with complementary foods. Complementary food feeding caused inflammatory cell infiltration, crypt structure irregularity and goblet cell reduction in the colon tissues of the rat pups. However, the oral administration of Trp combined with Lactiplantibacillus plantarum DPUL-S164 or Limosilactobacillus reuteri DPUL-M94 significantly restored the pathological changes in the colon tissues and inhibited the expression of pro-inflammatory cytokines in the colon and ileum of the rat pups. M94 or S164 combined with Trp intervention could promote the expression of cell differentiation genes and tight junction proteins, and restore the intestinal barrier damage caused by complementary foods in rat pups by activating the aryl hydrocarbon receptors (AhR) and nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. In addition, the indole-3-lactic acid (ILA), indole-3-propionic acid (IPA), or indole-3-carbaldehyde (I3C) level in the cecal contents of the rat pups increased after intervention of Trp combined with S164 or M94, which may account for the amelioration of intestinal barrier damage in rat pups administered with complementary foods. Furthermore, S164 or M94 combined with Trp intervention up-regulated the relative abundance of f_Lactobacillaceae, f_Akkermansiaceae, g_Lactobacillus, and g_Akkermansia in the intestinal tract of the rat pups. In conclusion, S164 or M94 combined with Trp intervention can ameliorate complementary food-induced intestinal barrier damage and gut flora disorder in rat pups by producing ILA, IPA, or I3C, which are AhR ligands.

Integrated microbiome, metabolome and transcriptome profiling reveals the beneficial effects of fish oil and Bacillus subtilis jzxj-7 on mouse gut ecosystem.

The effects of fish oil (FO) and Bacillus subtilis jzxj-7 (JZXJ-7) on the colonic physiology, bacteria, metabolites, and gene expressions were studied in C57BL/6J mice. Co-administration of FO and JZXJ-7 was more beneficial than individual supplementation, as evidenced by improved growth performance, enhanced colon crypt depth and goblet cell numbers. FO + JZXJ-7 inhibited colonic fibrosis by downregulating fibrosis marker protein expression and upregulating occludin, claudin-2 and claudin-4 gene expressions. FO + JZXJ-7 ameliorated oxidative stress and inflammation by increasing catalase, superoxide dismutase, total anti-oxidation capacity, and reducing colon tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 levels. Mechanistically, FO + JZXJ-7 modulated the colon micro-ecological environment by enriching Roseburia, Lachnospiraceae NK4B4, Faecalibaculum and Lactococcus and its derived short-chain fatty acids, and activating Ppara and Car1 mediated peroxisome proliferators-activated receptor (PPAR) and phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling. Overall, FO + JZXJ-7 may serve as a promising nutraceutical to improve health by boosting the growth of colonic beneficial bacteria, altering metabolic phenotype, and regulating gene expression.

Evaluation the effect of dietary vitamin E, sesamin and thymoquinone bioactive compounds on immunological response, intestinal traits and MUC-2 gene expression in broiler Japanese quails (Coturnix japonica)

The current study was performed to determine the effect of dietary vitamin E, sesamin and thymoquinone bioactive lignans derived from sesame and black seed on immunological response, intestinal traits and Mucin2 gene expression in broiler quails. Three hundred and fifty (one days-old) quails were allotted to seven dietary treatments with five replicates as an experimental randomized design study. Treatments were basal diet as a control, control +100 and +200 mg of vitamin E, sesamin and thymoquinone per each kg of diet respectively. At 35 d of age, two quails from each pen were chosen, weighted, slaughtered, eviscerated and lymphoid organ relative weights were measured. Anti-body titers against Newcastle disease (ND), Sheep red blood cell (SRBC), and infectious bronchitis virus (IBV) and Avian influenza (AI) vaccination were determined. The serum activities of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and serum antioxidant activates such as superoxide dismutase (SOD),glutathione peroxidase(GPX), catalase (CAT) and total antioxidant capacity (TAC) were examined. The cell mediated immunity by dinitrochlorobenzene (DNCB) and phytohemagglutinin (PHA) challenges were assessed. The microflora populations of ileum, morphological traits of jejunum and mucin2 gene expression were analyzed. Data showed that the lymphoid organ (thymus, spleen and Bursa) relative weights and antibody titer against HI, AI, SRBC and IB vaccination were increased compared to the control (p ≤ 0.05). Serum activities of ALP, ALT and AST were decreased under influences of dietary treatments (p ≤ 0.05). The serum antioxidant activates of GPX,SOD,CAT and TAC were increased and Increasing in mean skin thickness after DNCB challenge and decrease wing web swelling response to PHA mitojen injection were observed (p ≤ 0.05). Salmonella enterica, E-coli and Coliforms colonies were decrease and Lactobacillus colonies increased instead (p ≤ 0.05). The villus height and surface, crypt depth and goblet cells density were increased compared to the control (p ≤ 0.05). The expression of MUC2 gene increased under influnces of vitamin E, sesamin and thymoquinone supplemented diets (p ≤ 0.05).

The arginine methyltransferase PRMT5 promotes mucosal defense in the intestine.

PRMT5 is a type II arginine methyltransferase abundantly expressed in the colonic epithelium. It is up-regulated in inflammatory bowel disease and colorectal cancer. However, its role in mucosal defense against enteric infection has not been studied. Here, we report that Prmt5 in the murine colon is up-regulated in response to Citrobacter rodentium infection. Pathogen clearance in mice with haploinsufficient expression of Prmt5 is significantly delayed compared with wildtype littermate controls. Transcriptomic analyses further reveal that PRMT5 regulates the expression of canonical crypt goblet cell genes involved in mucus production, assembly, and anti-microbial responses via methyltransferase activity-dependent and -independent mechanisms. Together, these findings uncover PRMT5 as a novel regulator of mucosal defense and a potential therapeutic target for treating intestinal diseases.

Intestinal permeability and peripheral immune cell composition are altered by pregnancy and adiposity at mid- and late-gestation in the mouse.

It is clear that the gastrointestinal tract influences metabolism and immune function. Most studies to date have used male test subjects, with a focus on effects of obesity and dietary challenges. Despite significant physiological maternal adaptations that occur across gestation, relatively few studies have examined pregnancy-related gut function. Moreover, it remains unknown how pregnancy and diet can interact to alter intestinal barrier function. In this study, we investigated the impacts of pregnancy and adiposity on maternal intestinal epithelium morphology, in vivo intestinal permeability, and peripheral blood immunophenotype, using control (CTL) and high-fat (HF) fed non-pregnant female mice and pregnant mice at mid- (embryonic day (E)14.5) and late (E18.5) gestation. We found that small intestine length increased between non-pregnant mice and dams at late-gestation, but ileum villus length, and ileum and colon crypt depths and goblet cell numbers remained similar. Compared to CTL-fed mice, HF-fed mice had reduced small intestine length, ileum crypt depth and villus length. Goblet cell numbers were only consistently reduced in HF-fed non-pregnant mice. Pregnancy increased in vivo gut permeability, with a greater effect at mid- versus late-gestation. Non-pregnant HF-fed mice had greater gut permeability, and permeability was also increased in HF-fed pregnant dams at mid but not late-gestation. The impaired maternal gut barrier in HF-fed dams at mid-gestation coincided with changes in maternal blood and bone marrow immune cell composition, including an expansion of circulating inflammatory Ly6Chigh monocytes. In summary, pregnancy has temporal effects on maternal intestinal structure and barrier function, and on peripheral immunophenotype, which are further modified by HF diet-induced maternal adiposity. Maternal adaptations in pregnancy are thus vulnerable to excess maternal adiposity, which may both affect maternal and child health.

Highland barley β-glucan boosted intestinal epithelial regeneration via cell cycle and autophagy

Cell cycle progression and autophagy take a crucial part in stimulating the regeneration of gut epithelial which definitely contributes to maintain the integrity of the intestinal barrier. Our previous research demonstrated that highland barley β-glucan (HBBG) significantly mitigated the damaged gastric mucosal and intestinal barrier of colitis mice, whenas the mechanism remains indistinct. In this study, we found that HBBG remitted the increase of disease activity index, the shortening of colon length and the damage of intestinal epithelial structure in mice with dextran sulphate sodium induced colitis. Furthermore, HBBG observably boosted gut epithelial regeneration on account of PCNA+ transit amplifying cells, Ki67+ crypt base columnar cells and goblet cells. Interestingly, HBBG relieved the apoptosis and suppressive proliferation of IEC-6 cells induced by lipopolysaccharide. The results of immunohistochemistry and flow cytometry demonstrated that HBBG promoted the cell cycle progression. Likewise, HBBG increased cell cycle related proteins CyclinD1 and CDK4 in IEC-6 cells. In the meanwhile, HBBG dramatically enhanced the autophagy. Excitingly, HBBG augmented the nuclear translocation of TFEB and phosphorylation of AMPK, which hinted HBBG activated autophagy via AMPK-TFEB signal. Expectantly, the treatment of 3-methyladenine, an autophagy inhibitor, inverted the promoted proliferation in IEC-6 cells caused by HBBG. Thereout, we presumed that HBBG accelerated the regeneration of intestinal epithelial by means of promoting cell cycle progression and activating the autophagy.

Role of Bifidobacterium infantis in the Treatment of Duodenal and Colon Inflammation in Induced Ulcerative Colitis

&#x0D; &#x0D; &#x0D; &#x0D; This study aimed to investigate the potential protective role of Bifidobacterium (B.) infantis in alleviating ‎‎induced duodenal and colon inflammation associated with ulcerative colitis (UC). Female albino Wister rats ‎‎(n=24) ‎were randomized into four experimental groups: Control Negative, acetic acid-induced colitis (AA colitis), ‎B. infantis-treated (Bifido), orally gavaged with 1 mL of reference strain ‎B. infantis at 108 CFU/mL for four weeks prior to the induction of colitis,‎ and Bifido+AA ‎colitis. Colitis was induced via ‎intrarectal administration of 4% AA solution‎. Seven days post-colitis induction, blood samples were ‎obtained to assess protein levels, and histopathological evaluations were conducted on duodenal and ‎colon tissues. ‎Additionally, immunohistochemical assessments for B-cell lymphoma 2‎ (Bcl-2) in colon and ‎myeloperoxidase (MPO) in duodenum ‎sections were performed‎. Results revealed that B. infantis treatment significantly elevated serum albumin and total ‎protein levels in the Bifido and Bifido+AA colitis groups, approximating those in the Control Negative group. Histopathological and morphological changes of duodenum in AA colitis revealed ulceration of the mucosal epithelium, submucosal inflammatory cellular infiltration, tissue depression resulting in villus atrophy, and crypt hyperplasia. Additionally, ‎colonic crypt gland atrophy and goblet cells depletion were observed. Most of these changes were ameliorated in the Bifido and Bifido+AA colitis groups. Immunohistochemical analysis displayed marked immunopositivity ‎ of Bcl-2 in colon and MPO in duodenum sections of the Bifido and Bifido+AA colitis groups, indicating the antiapoptotic and anti-inflammatory roles of B. infantis. This study demonstrates that B. infantis exerts a protective effect against AA-induced UC by normalizing serum protein levels, ameliorating histopathological alterations, and ‎modulating apoptotic and inflammatory markers. These findings underscore B. infantis as a ‎promising therapeutic agent for UC and warrant further research to elucidate the underlying ‎molecular mechanisms.&#x0D; &#x0D; &#x0D; &#x0D;

Regional epithelial cell diversity in the small intestine of pigs.

Understanding regional distribution and specialization of small intestinal epithelial cells is crucial for developing methods to control appetite, stress, and nutrient uptake in swine. To establish a better understanding of specific epithelial cells found across different regions of the small intestine in pigs, we utilized single-cell RNA sequencing (scRNA-seq) to recover and analyze epithelial cells from duodenum, jejunum, and ileum. Cells identified included crypt cells, enterocytes, BEST4 enterocytes, goblet cells, and enteroendocrine (EE) cells. EE cells were divided into two subsets based on the level of expression of the EE lineage commitment gene, NEUROD1. NEUROD1hi EE cells had minimal expression of hormone-encoding genes and were dissimilar to EE cells in humans and mice, indicating a subset of EE cells unique to pigs. Recently discovered BEST4 enterocytes were detected in both crypts and villi throughout the small intestine via in situ staining, unlike in humans, where BEST4 enterocytes are found only in small intestinal villi. Proximal-to-distal gradients of expression were noted for hormone-encoding genes in EE cells and nutrient transport genes in enterocytes via scRNA-seq, demonstrating regional specialization. Regional gene expression in EE cells and enterocytes was validated via quantitative PCR (qPCR) analysis of RNA isolated from epithelial cells of different small intestinal locations. Though many genes had similar patterns of regional expression when assessed by qPCR of total epithelial cells, some regional expression was only detected via scRNA-seq, highlighting advantages of scRNA-seq to deconvolute cell type-specific regional gene expression when compared to analysis of bulk samples. Overall, results provide new information on regional localization and transcriptional profiles of epithelial cells in the pig small intestine.

Clearance of small intestinal crypts involves goblet cell mucus secretion by intracellular granule rupture and enterocyte ion transport.

Goblet cells in the small intestinal crypts contain large numbers of mucin granules that are rapidly discharged to clean bacteria from the crypt. Because acetylcholine released by neuronal and nonneuronal cells controls many aspects of intestinal epithelial function, we used tissue explants and organoids to investigate the response of the small intestinal crypt to cholinergic stimulation. The activation of muscarinic acetylcholine receptors initiated a coordinated and rapid emptying of crypt goblet cells that flushed the crypt contents into the intestinal lumen. Cholinergic stimulation induced an expansion of the granule contents followed by intracellular rupture of the mucin granules. The mucus expanded intracellularly before the rupture of the goblet cell apical membrane and continued to expand after its release into the crypt lumen. The goblet cells recovered from membrane rupture and replenished their stores of mucin granules. Mucus secretion from the goblet cells depended on Ca2+ signaling and the expansion of the mucus in the crypt depended on gap junctions and on ion and water transport by enterocytes adjacent to the goblet cells. This distinctive mode of mucus secretion, which we refer to as "expanding secretion," efficiently cleans the small intestine crypt through coordinated mucus, ion, and fluid secretion by goblet cells and enterocytes.

Expand, push, break, release, repair: A unique way to secrete macromolecules.

Regulated exocytosis is a fundamental process in specialized secretory organs. In this issue of Science Signaling, Dolan et al. describe a distinctive modality of secretion in the goblet cells of the small intestine crypts driven by intracellular cargo expansion.

Evaluation of the Number of Goblet Cells in Crypt of Colonic Mucosa Submitted to Experimental Gastric Restriction

Obesity‐related comorbidity is defined as a condition caused by overweight/obesity or known to contribute to the presence or severity of the condition. These comorbid conditions are expected to improve or go into remission in the presence of effective and sustained weight loss. However, the mechanisms of surgical weight loss and the long‐term consequences of bariatric surgery remain unclear. World Health Organization reports obesity as a rising chronic condition with many devastating health consequences. Gastric reduction procedures are currently employed to treat morbid obesity and there are few reports concerning the consequent effects in large intestine.MethodsExperimental model of gastric restriction was devised using rats. The animals were submitted to surgical gastrostomy and a cylindrical loofah was inserted into the stomach. We studied 30 adult male Wistar rats divided in three groups: the stomach reduction group (R20), the sham group (SH20), which underwent the same procedure except for the loofah insertion and the control group (C20). Animals were fed and kept in separate cages. Two colon samples were collected 2 cm from the cecum and were individually packed into containers. Samples were submitted to routine histological processing for Alcian Blue, Periodic Acid and Reagent of Schiff (PAS) and High Iron Diamine (HID AB). The sections were analyzed for the length of the crypts and mucous cells. Goblet cells were obtained in all groups as mean, median, minimum, and maximum and were compared between groups using generalized estimation equations (GEE) with normal distribution and identity link function supposing exchangeable correlation matrix between fields. Bonferroni's multiple comparison was applied to identify differences between groups. The results are described by the mean value with respective standard deviation.ResultsThe analysis of PAS and AB Goblet cells count on R20 group indicated a statistically significant reduction and no shallower crypts as compared to the C20. The sialomucins and sulphomucins number cells were found with no enhance. There was not difference regarding on R20 compared to C20 and SH20.ConclusionThese findings might be a result of colonic mucosae atrophy secondary to the effects of gastric restriction in this model. Restriction decreases the content of neutral mucins and total acidic mucins, without interfering with the sulfomucin and sialomucin acid mucin subtypes.

Gut microbiota-derived butyrate regulates gut mucus barrier repair by activating the macrophage/WNT/ERK signaling pathway.

Ulcerative colitis (UC) is majorly associated with dysregulation of the dynamic cross-talk among microbial metabolites, intestinal epithelial cells, and macrophages. Several studies have reported the significant role of butyrate in host-microbiota communication. However, whether butyrate provides anti-inflammatory profiles in macrophages, thus contributing to UC intestinal mucus barrier protection, has currently remained elusive. In the current study, we found that butyrate increased mucin production and the proportion of mucin-secreting goblet cells in the colon crypt in a macrophage-dependent manner by using clodronate liposomes. Furthermore, in vivo and in vitro studies were conducted, validating that butyrate facilitates M2 macrophage polarization with the elevated expressions of CD206 and arginase-1 (Arg1). In macrophages/goblet-like LS174T cells co-culture systems, butyrate-primed M2 macrophages significantly enhanced the expression of mucin-2 (MUC2) and SPDEF (goblet cell marker genes) than butyrate alone, while blockade of WNTs secretion or ERK1/2 activation significantly decreased the beneficial effect of butyrate-primed macrophages on goblet cell function. Additionally, the adoptive transfer of butyrate-induced M2 macrophages facilitated the generation of goblet cells and mucus restoration following dextran sulfate sodium (DSS) insult. Taken together, our results revealed a novel mediator of macrophage-goblet cell cross-talk associated with the regulation of epithelial barrier integrity, implying that the microbial metabolite butyrate may serve as a candidate therapeutic target for UC.

Lactobacillus plantarum metabolites reduce deoxynivalenol toxicity on jejunal explants of piglets

The deterioration of food and feed stuffs and toxic intestinal effects due to fungal colonization and concomitant production of mycotoxins is an increasing concern. The development of fungi resistance to many commonly used chemical preservatives adds further alarm. Therefore, effective detoxification methods would be useful in counteracting this problem. Biotransformation/adsorption of mycotoxins by lactic acid bacteria and their metabolites is a promising approach to minimize the deleterious effects of mycotoxins. The objective of the present study was to evaluate the beneficial effects of Lactobacillus plantarum metabolites in reducing deoxynivalenol intestinal toxicity. To achieve this aim, histological, morphometrical and oxidative stress analyses were performed in the intestinal mucosa of piglets exposed to deoxynivalenol alone or associated with two strains (SN1 and SN2) of L. plantarum subsp. plantarum metabolites. Metabolites were obtained after dichloromethane (D) or ethyl acetate (A) extraction. Jejunal explants were exposed to the following treatments for 2 and 4 h a) culture medium (control group); b) deoxynivalenol (DON, 10 μM); c) L. plantarum metabolites DSN1; d) L. plantarum metabolites DSN1+DON; e) L. plantarum metabolites DSN2; f) L. plantarum metabolites DSN2+DON; g) L. plantarum metabolites ASN1; h) L. plantarum metabolites ASN1+DON; i) L. plantarum metabolites ASN2; j) L. plantarum metabolites ASN2+DON. The metabolites were incubated 1 h previously to DON challenge (one and 3 h of exposure). Histological assessment showed DON-treated explants with villi fusion and atrophy, multifocal apical necrosis and cuboid or flattened enterocytes with 2 and 4 h of exposure, while LP metabolites groups individually or associated with DON remained like control. The density of goblet cells in villi and crypts was reduced in DON explants compared to control group with 2 and 4 h of exposure; on the other hand, a significant increase in this parameter was achieved in LP metabolites groups compared to DON. Morphometric evaluation showed no difference in villi height or crypts depth in any treated explants. Overall, oxidative stress response assessments showed that explants exposed to SN1 extracted with dichloromethane and ethyl acetate, and SN2 extracted with dichloromethane reduced superoxide anion production. In conclusion, L. plantarum metabolites induced beneficial effects in intestinal mucosa, reducing the toxic effects of DON on intestinal morphology and oxidative response.

Self-Cross-Linked Hydrogel of Cysteamine-Grafted γ-Polyglutamic Acid Stabilized Tripeptide KPV for Alleviating TNBS-Induced Ulcerative Colitis in Rats.

KPV (Lys-Pro-Val), which is a tripeptide derived from α-MSH (α-melanocyte-stimulating hormone), has an anti-inflammatory effect on colitis. However, KPV solution is very unstable when rectally administered, compromising its therapeutic efficacy. Herein, cysteamine-grafted γ-polyglutamic acid (SH-PGA) was synthesized by conjugating cysteamine with the carboxyl groups of γ-PGA. The synthesized SH-PGA has the thiol grafting amount of 4.5 ± 0.3 mmol/g. Without the use of the cross-linker, the SH-PGA hydrogel with 4% of the polymer was formed by self-cross-linking of thiol groups. Moreover, the formation of the SH-PGA hydrogel was not affected by KPV. The KPV/SH-PGA hydrogel presented higher elastic modulus (G') than the corresponding viscous modulus (G″) at 0.01-10 Hz, exhibiting good mechanical stability. The KPV/SH-PGA hydrogel presented a shear-thinning behavior, which was helpful for rectal administration. Only 30% of KPV was released from the KPV/SH-PGA hydrogel within 20 min, followed by a sustained-release behavior. Importantly, the stability of KPV in the SH-PGA hydrogel was obviously enhanced, which was presented by detecting its anti-inflammatory activity and promoting cell migration potential after 2 h of exposure to 37 °C. The enhanced therapeutic effect of the KPV/SH-PGA hydrogel on colitis was confirmed on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced ulcerative colitis rats. The colitis symptoms including body weight loss and the disease activity index score were obviously attenuated by rectally administering the KPV/SH-PGA hydrogel. Besides, the KPV/SH-PGA hydrogel treatment prevented the colon shortening of TNBS-infused rats and decreased the colonic myeloperoxidase level. The morphology of the colon including the epithelial barrier, crypt, and intact goblet cells was recovered after KPV/SH-PGA hydrogel treatment. Besides, the KPV/SH-PGA hydrogel decreased the expression of proinflammatory cytokines such as tumor necrosis factor α and interleukin 6. Collectively, the KPV/SH-PGA hydrogel may provide a promising strategy for the treatment of ulcerative colitis.