Goblet Cell Function Research Articles

Randomised double-masked placebo-controlled trial of the cumulative treatment efficacy profile of intense pulsed light therapy for meibomian gland dysfunction

PurposeTo assess long-term cumulative treatment effects of intense pulsed light (IPL) therapy in meibomian gland dysfunction (MGD). MethodsEighty-seven symptomatic participants (58 female, mean ± SD age, 53 ± 16 years) with clinical signs of MGD were enrolled in a prospective, double-masked, parallel-group, randomised, placebo-controlled trial. Participants were randomised to receive either four or five homogeneously sequenced light pulses or placebo treatment to both eyes, (E-Eye Intense Regulated Pulsed Light, E-Swin, France). Visual acuity, dry eye symptomology, tear film parameters, and ocular surface characteristics were assessed immediately before treatment on days 0, 15, 45, 75, and four weeks after treatment course completion on day 105. Inflammatory and goblet cell function marker expression, and eyelid swab microbiology cultures were evaluated at baseline and day 105. ResultsSignificant decreases in OSDI, SPEED, and SANDE symptomology scores, and meibomian gland capping, accompanied by increased tear film lipid layer thickness, and inhibited Corynebacterium macginleyi growth were observed in both treatment groups (all p < 0.05). Sustained clinical improvements occurred in both treatment groups from day 75, although significant changes from day 45, in lipid layer quality, meibomian gland capping, OSDI and SANDE symptomology, were limited to the five-flash group (all p < 0.05). ConclusionsIPL therapy effected significant improvements in dry eye symptomology, tear film lipid layer thickness, and meibomian gland capping in MGD patients. Five-flash IPL treatment showed superior clinical efficacy to four-flash, and an initial course of at least four treatments is suggested to allow for establishment of sustained cumulative therapeutic benefits prior to evaluation of overall treatment efficacy. Trial registration numberACTRN12616000667415.

Effects of Short-Peptide-Based Enteral Nutrition on the Intestinal Microcirculation and Mucosal Barrier in Mice with Severe Acute Pancreatitis.

Short-peptide-based enteral nutrition (SPEN) is absorbed more efficiently in patients with severe acute pancreatitis (SAP). More importantly, SPEN decreases SAP-induced enterogenous infection risk. This study aims to investigate whether SPEN alleviates intestinal bacterial translocation in mice with SAP, and the underlying mechanisms. The SAP model is established after pre-treatment with SPEN or intact-protein-based enteral nutrition. Although there is no improvement in pancreas injury, as evaluated through Hematoxylin-Eosin staining or serum amylase, SPEN obviously attenuates intestinal bacterial translocation after SAP. To unveil the mechanisms, it is found that the intestinal mechanical barrier destroyed by SAP is significantly relieved by SPEN, which presents with recovered ZO-1 expression, mucus layer, and goblet cell function. Additionally, SPEN alleviates local CCR6/CCL20 induced CD11c+ dendritic cell infiltration, systemic immunosuppression, and inhibits the secretion of luminal secretory immunoglobulin A. Possibly responsible for SAP-induced mucosal dysfunctions, destroyed intestinal mucosal microcirculation and local hypoxia are largely improved in SAP+SPEN group. SPEN can improve downregulated intestinal mucosal microcirculation secondary to SAP, which may be responsible for mucosal inflammation relief, maintenance of the mechanical barrier and mucosal immunity, the correction of systemic immunosuppression, and play a protective role in defending commensal bacterial translocation after SAP.

P071 MODULATION OF THE MUCUS LAYER BY BIFIDOBACTERIUM DENTIUM PROVIDES PROTECTION IN A MODEL OF COLITIS

Abstract Background The intestinal mucus layer serves as a critical interface between the environment and the host. Patients with inflammatory bowel disease (IBD), particularly ulcerative colitis, exhibit reduced synthesis and secretion of the mucus protein MUC2 and decreased mucus thickness. This in turn promotes immune activation and inflammation. The clinical relevance of the mucus layer emphasizes the need to address strategies to modulate this barrier. Although bifidobacteria represent only 3–6% of the healthy adult fecal microbiota, their presence has been associated with numerous health benefits, including bolstering mucus production. However, the molecular mechanisms that underlie these positive effects appear to be strain-specific and are not well defined. We hypothesized that the human-derived Bifidobacterium dentium would increase intestinal mucus synthesis and expulsion via specific metabolites. We also speculated that modulation of goblet cells would be beneficial during colitis. Methods &amp; Results In silico genome analysis revealed that B. dentium lacked the enzymatic repertoire required for degradation of mucin glycans. Consistent with these findings, we found that B. dentium could not use mucin glycans as a primary carbon source in vitro. To examine mucus modulation in vivo, germ-free mice were mono-associated with live or heat-killed B. dentium. Live B. dentium mono-associated mice exhibited increased colonic expression of goblet cell markers Krüppel-Like Factor 4 (Klf4), Relmβ, trefoil factor 3 (Tff3), Muc2, and several mucin glycosyltransferases compared to both heat-killed B. dentium and germ-free counterparts. Likewise, live B. dentium mono-associated colon had increased acidic mucin-filled goblet cells as denoted by MUC2 and PAS-AB staining. In vitro, B. dentium secreted products, including acetate, were able to increase MUC2 levels in T84 cells, mouse colonoids and human colonoids. We also identified that B. dentium secreted products, such as GABA, stimulated autophagy-mediated calcium signaling and MUC2 release. To identify whether B. dentium could enhance MUC2 production in mice harboring a complete microbiota, specific pathogen free mice were treated with live B. dentium by oral gavage. Administration of B. dentium increased the inner mucus layer compared to controls. Moreover, in a TNBS model of colitis, B. dentium treated mice had increased goblet cell numbers and MUC2 mRNA. Mirroring these findings, B. dentium treated mice lost less weight, had improved histology and had decreased levels of TNF, KC (IL-8), and IL-6. Conclusions This work illustrates that B. dentium enhances the intestinal mucus layer and goblet cell function via upregulation of gene expression and autophagy signaling pathways with a net increase in mucin production. Ultimately, these pathways may be targeted for the development of novel therapeutics.

Impacts of Amino Acids on the Intestinal Defensive System.

The intestine interacts with a diverse community of antigens and bacteria. To keep its homeostasis, the gut has evolved with a complex defense system, including intestinal microbiota, epithelial layer and lamina propria. Various factors (e.g., nutrients) affect the intestinal defensive system and progression of intestinal diseases. This review highlights the current understanding about the role of amino acids (AAs) in protecting the intestine from harm. Amino acids (e.g., arginine, glutamine and tryptophan) are essential for the function of intestinal microbiota, epithelial cells, tight junction, goblet cells, Paneth cells and immune cells (e.g., macrophages, B cells and T cells). Through the modulation of the intestinal defensive system, AAs maintain the integrity and function of the intestinal mucosa and inhibit the progression of various intestinal diseases (e.g., intestinal infection and intestinal colitis). Thus, adequate intake of functional AAs is crucial for intestinal and whole-body health in humansand other animals.

Regulatory network analysis of Paneth cell and goblet cell enriched gut organoids using transcriptomics approaches.

The epithelial lining of the small intestine consists of multiple cell types, including Paneth cells and goblet cells, that work in cohort to maintain gut health. 3D in vitro cultures of human primary epithelial cells, called organoids, have become a key model to study the functions of Paneth cells and goblet cells in normal and diseased conditions. Advances in these models include the ability to skew differentiation to particular lineages, providing a useful tool to study cell type specific function/dysfunction in the context of the epithelium. Here, we use comprehensive profiling of mRNA, microRNA and long non-coding RNA expression to confirm that Paneth cell and goblet cell enrichment of murine small intestinal organoids (enteroids) establishes a physiologically accurate model. We employ network analysis to infer the regulatory landscape altered by skewing differentiation, and using knowledge of cell type specific markers, we predict key regulators of cell type specific functions: Cebpa, Jun, Nr1d1 and Rxra specific to Paneth cells, Gfi1b and Myc specific for goblet cells and Ets1, Nr3c1 and Vdr shared between them. Links identified between these regulators and cellular phenotypes of inflammatory bowel disease (IBD) suggest that global regulatory rewiring during or after differentiation of Paneth cells and goblet cells could contribute to IBD aetiology. Future application of cell type enriched enteroids combined with the presented computational workflow can be used to disentangle multifactorial mechanisms of these cell types and propose regulators whose pharmacological targeting could be advantageous in treating IBD patients with Crohn's disease or ulcerative colitis.

The impact of oligosaccharide content, glycosidic linkages and lactose content of galacto-oligosaccharides (GOS) on the expression of mucus-related genes in goblet cells.

Galacto-oligosaccharides (GOS) have been reported to modulate the function of intestinal goblet cells and to improve mucus barrier function. However, GOS is available in many structurally different compositions and it is unknown how GOS structural diversity impacts this modulation of goblet cells. This study aims to investigate the effects of oligosaccharide content and glycosidic linkages of GOS on expression of genes associated with the secretory function of goblet cells. To investigate the effect of oligosaccharide content, LS174T cells were incubated with (β1 → 4)GOS of variable transgalactosylated oligosaccharides and lactose (Lac) composition. To investigate the effect of glycosidic linkages, we compared the effects of (β1 → 4)GOS with (β1 → 3)GOS, and with a mixture of α-linked oligosaccharides (lactose-derived oligosaccharides-LDO). The changes in mRNA expression of mucus-related genes were assessed by RT-PCR. GOS containing Lac significantly enhanced the expression of MUC2, TFF3 and RETNLB but not of Golgi sulfotransferases genes. In contrast, GOS without Lac did not impact these genes. Lac alone significantly enhanced MUC2, TFF3, RETNLB, CHST5, and GAL3ST2 genes suggesting that Lac might be responsible for goblet cell modulation in (β1 → 4)GOS preparations. (β1 → 3)GOS induced the expression of MUC2 and TFF3, and downregulated the RETNLB gene. Compared with the (β1 → 3) and GOS (β1 → 4)GOS, the α-linked LDO significantly upregulated the expression MUC2, TFF3, RETNLB and the Golgi sulfotransferases genes. We identify structural features of GOS that contribute to enhanced mucus integrity. Our study might lead to better GOS formulations for foods to prevent or treat different types of intestinal disorders.

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

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

Human Milk Oligosaccharides Differently Modulate Goblet Cells Under Homeostatic, Proinflammatory Conditions and ER Stress.

ScopeHuman milk oligosaccharides (hMOs) have beneficial effects on intestinal barrier function, but the mechanisms of action are not well understood. Here, the effects of hMOs on goblet cells, which indicate that some hMOs may enhance mucus barrier function through direct modulation of goblet cell function, are studied.Methods and resultsThe modulatory effects of 2′‐fucosyllactose (2′‐FL), 3‐fucosyllactose (3‐FL), lacto‐N‐triaose II (LNT2), and galacto‐oligosaccharides (GOS) on the expression of goblet cell secretory related genes MUC2, TFF3, and RETNLB, and the Golgi‐sulfotransferase genes CHST5 and GAL3ST2 of LS174T are determined by real‐time quantitative RT‐PCR. 3‐FL, LNT2, and GOS‐modulated LS174T gene expression profiles in a dose‐ and time‐dependent manner. In addition, the upregulation of MUC2 is confirmed by immunofluorescence staining. Effects of 2′‐FL, 3‐FL, LNT2, and GOS on gene transcription of LS174T are also assessed during exposure to TNF‐α, IL‐13, or tunicamycin. During TNF‐α challenge, 3‐FL and LNT2 enhance MUC2 and TFF3 gene expression. After IL‐13 exposure, 2′‐FL, 3‐FL, and LNT2 all show upregulating effects on MUC2; 3‐FL and LNT2 also enhance TFF3 expression. LNT2 significantly reverses Tm‐induced downregulation of TFF3, RETNLB, and CHST5.ConclusionThe findings indicate that hMOs may enhance mucus barrier function through direct modulation of intestinal goblet cells. Effects are structure‐ and stressor‐dependent.

Goblet cell associated antigen passages support the induction and maintenance of oral tolerance.

Tolerance to innocuous antigens from the diet and the commensal microbiota is a fundamental process essential to health. Why tolerance is efficiently induced to substances arising from the hostile environment of the gut lumen is incompletely understood but may be related to how these antigens are encountered by the immune system. We observed that goblet cell associated antigen passages (GAPs), but not other pathways of luminal antigen capture, correlated with the acquisition of luminal substances by lamina propria (LP) antigen presenting cells (APCs) and with the sites of tolerance induction to luminal antigens. Strikingly this role extended beyond antigen delivery. The GAP function of goblet cells facilitated maintenance of pre-existing LP T regulatory cells (Tregs), imprinting LP-dendritic cells with tolerogenic properties, and facilitating LP macrophages to produce the immunomodulatory cytokine IL-10. Moreover, tolerance to dietary antigen was impaired in the absence of GAPs. Thus, by delivering luminal antigens, maintaining pre-existing LP Tregs, and imprinting tolerogenic properties on LP-APCs GAPs support tolerance to substances encountered in the hostile environment of the gut lumen.

The assembly of integrated rat intestinal-hepatocyte cultures.

The jejunum is the segment of the small intestine responsible for several metabolism and biotransformation functions. In this report, we have cultured rat jejunum explants in vitro and integrated them with hepatocyte cultures. We have also investigated the changes in jejunum function at different locations since spatial variations in intestinal functions have been reported previously. We divided the length of the rat jejunum into three distinct regions of approximately 9 cm each. We defined the regions as proximal (adjacent to the duodenum), medial, and distal (adjacent to the ileum). Spatiotemporal variations in functions were observed between these regions within the jejunum. Alkaline phosphatase activity (a marker of enterocyte function), decreased twofold between the proximal and distal regions at 4 hr. Lysozyme activity (a marker of Paneth cell function) increased from the proximal to the distal jejunum by 40% at 24 hr. Mucin‐covered areas, a marker of goblet cell function, increased by twofold between the proximal and distal segments of the jejunum at 24 hr. When hepatocytes were integrated with proximal jejunum explants, statistically higher urea (~2.4‐fold) and mucin (57%) production were observed in the jejunum explants. The integrated intestine‐liver cultures can be used as a platform for future investigations.

A BAC Transgene Expressing Human CFTR under Control of Its Regulatory Elements Rescues Cftr Knockout Mice

Small-molecule modulators of cystic fibrosis transmembrane conductance regulator (CFTR) biology show promise in the treatment of cystic fibrosis (CF). A Cftr knockout (Cftr KO) mouse expressing mutants of human CFTR would advance in vivo testing of new modulators. A bacterial artificial chromosome (BAC) carrying the complete hCFTR gene including regulatory elements within 40.1 kb of DNA 5′ and 25 kb of DNA 3′ to the gene was used to generate founder mice expressing hCFTR. Whole genome sequencing indicated a single integration site on mouse chromosome 8 (8qB2) with ~6 gene copies. hCFTR+ offspring were bred to murine Cftr KO mice, producing hCFTR+/mCftr− (H+/m−) mice, which had normal survival, growth and goblet cell function as compared to wild-type (WT) mice. Expression studies showed hCFTR protein and transcripts in tissues typically expressing mCftr. Functionally, nasal potential difference and large intestinal short-circuit (Isc) responses to cAMP stimulation were similar in magnitude to WT mice, whereas small intestinal cAMP ΔIsc responses were reduced. A BAC transgenic mouse with functional hCFTR under control of its regulatory elements has been developed to enable the generation of mouse models of hCFTR mutations by gene editing for in vivo testing of new CF therapies.

The protective effect of icariin and phosphorylated icariin against LPS-induced intestinal goblet cell dysfunction

In this study, we used LS174T cells as a model to investigate the protective effects of icariin and phosphorylated icariin on LPS-induced goblet cell dysfunction. Our results indicated that icariin and phosphorylated icariin increased the cell viability and decreased lactate dehydrogenase activity in LPS-treated LS174T cells. Icariin and phosphorylated icariin attenuated LPS-induced changes in mucin 2 synthesis and secretion. Besides, Icariin and phosphorylated icariin reduced the levels of ROS, MDA, and H2O2 and increased the activity of SOD, GPx, CAT, and T-AOC in LPS-treated LS174T cells. Moreover, the levels of IL-1β, IL-6, IL-8, and TNF-α were reduced in the Icariin and phosphorylated icariin group. Furthermore, Icariin and phosphorylated icariin decreased gene abundance or enzyme activity of Bip, XBP1, GRP78, CHOP, caspase-3, and caspase-4 in LPS-treated LS174T cells. Our data suggest that Icariin and phosphorylated icariin effectively attenuate LPS-induced intestinal goblet cell function damage through regulating oxidative stress, inflammation, apoptosis, and mucin expression.

Bifidobacterium dentium Fortifies the Intestinal Mucus Layer via Autophagy and Calcium Signaling Pathways.

Much remains unknown about how the intestinal microbiome interfaces with the protective intestinal mucus layer. Bifidobacterium species colonize the intestinal mucus layer and can modulate mucus production by goblet cells. However, select Bifidobacterium strains can also degrade protective glycans on mucin proteins. We hypothesized that the human-derived species Bifidobacterium dentium would increase intestinal mucus synthesis and expulsion, without extensive degradation of mucin glycans. In silico data revealed that B. dentium lacked the enzymes necessary to extensively degrade mucin glycans. This finding was confirmed by demonstrating that B. dentium could not use naive mucin glycans as primary carbon sources in vitro To examine B. dentium mucus modulation in vivo, Swiss Webster germfree mice were monoassociated with live or heat-killed B. dentium Live B. dentium-monoassociated mice exhibited increased colonic expression of goblet cell markers Krüppel-like factor 4 (Klf4), Trefoil factor 3 (Tff3), Relm-β, Muc2, and several glycosyltransferases compared to both heat-killed B. dentium and germfree counterparts. Likewise, live B. dentium-monoassociated colon had increased acidic mucin-filled goblet cells, as denoted by Periodic Acid-Schiff-Alcian Blue (PAS-AB) staining and MUC2 immunostaining. In vitro, B. dentium-secreted products, including acetate, were able to increase MUC2 levels in T84 cells. We also identified that B. dentium-secreted products, such as γ-aminobutyric acid (GABA), stimulated autophagy-mediated calcium signaling and MUC2 release. This work illustrates that B. dentium is capable of enhancing the intestinal mucus layer and goblet cell function via upregulation of gene expression and autophagy signaling pathways, with a net increase in mucin production.IMPORTANCE Microbe-host interactions in the intestine occur along the mucus-covered epithelium. In the gastrointestinal tract, mucus is composed of glycan-covered proteins, or mucins, which are secreted by goblet cells to form a protective gel-like structure above the epithelium. Low levels of mucin or alterations in mucin glycans are associated with inflammation and colitis in mice and humans. Although current literature links microbes to the modulation of goblet cells and mucins, the molecular pathways involved are not yet fully understood. Using a combination of gnotobiotic mice and mucus-secreting cell lines, we have identified a human-derived microbe, Bifidobacterium dentium, which adheres to intestinal mucus and secretes metabolites that upregulate the major mucin MUC2 and modulate goblet cell function. Unlike other Bifidobacterium species, B. dentium does not extensively degrade mucin glycans and cannot grow on mucin alone. This work points to the potential of using B. dentium and similar mucin-friendly microbes as therapeutic agents for intestinal disorders with disruptions in the mucus barrier.

Dual roles of IL-18 in colitis through regulation of the function and quantity of goblet cells.

The main aim of the present study was to investigate the dual roles and mechanism of interleukin (IL)-18 in dextran sulfate sodium (DSS)-induced colitis. Firstly, meta-analysis was used to explore whether the levels of IL-18 were different in patients with colon cancer or inflammatory bowel disease. The results demonstrated that IL-18 (rs187238, -137G/C) increased the incidence rate of colon cancer in patients, while IL-18 (rs187238, -137G/C) decreased the incidence rate of ulcerative colitis or Crohn's disease in patients. Therefore, IL-18 (rs187238, -137G/C) may have a dual function in colitis. Next, the functional role of IL-18 in colitis was further investigated, by use of a DSS-induced colitis mouse model. Pre-treatment of the mice with IL-18 increased body weight, augmented colon length, reduced inflammatory infiltration, promoted mucin (Muc)-2 expression, increased the function and quantity of goblet cells and increased the mRNA levels of resistin-like molecule (RELM) β and trefoil factor family (TFF) 3 in mice with DSS-induced colitis, through the IL-22/STAT3 pathway. By contrast, treatment with IL-18 at later stages of the disease reduced body weight, decreased colon length, enhanced inflammatory infiltration and reduced Muc-2 expression, decreased the function and quantity of goblet cells and inhibited the mRNA levels of RELMβ and TFF3 in mice with DSS-induced colitis. In conclusion, IL-18 served a dual function in colitis by regulating the function of goblet cells. The anti-inflammatory effects of IL-18 were observed in the early stage of colitis-induced inflammation, while the pro-inflammatory effects were observed in the later stages of the disease.

Constitutive Ptpn2‐Loss in Mice Decreases Number and Compromises Function of Intestinal Goblet and Paneth cells

Among the ~200 susceptibility loci associated with Inflammatory Bowel Disease (IBD), are loss‐of‐function single nucleotide polymorphisms (SNPs) in the locus (18p11) containing the Protein Tyrosine Phosphatase non‐receptor Type 2 (PTPN2) gene. PTPN2 encodes the T Cell Protein Tyrosine Phosphatase (TCPTP) that functions as a negative regulator of diverse intracellular pathways repressing inflammatory signals. In Ptpn2‐deficient mice, we observed altered intestinal homeostasis in the form of intestinal epithelial barrier defects and altered intestinal microbiome composition. Intestinal epithelial homeostasis is maintained by active‐cycling and slow cycling stem cells confined within the crypt‐based niche and subsequent differentiation into secretory and absorptive epithelial lineages as cells migrate up the crypt‐villus axis. The goal of this study was to determine whether Ptpn2‐loss affects intestinal epithelial cell populations and function that could contribute to altered host‐bacterial interactions.MethodsIntestinal tissues (ileum, cecum, proximal and distal colon) from 21 day old wild type (WT), heterozygous (HET) and constitutive Ptpn2 knockout (KO) Balb/C mice were harvested and processed for imaging to detect proliferation (Ki‐67), apoptosis (TUNEL), Paneth cell (lysozyme), and goblet cell (periodic‐acid Shiff) numbers via staining of specific markers. cDNA was generated by reverse transcriptase, and mRNA expression of key genes associated with Paneth and goblet cells function were measured by PCR.ResultsProximal and distal colon of Ptpn2‐KO mice showed clusters of apoptotic cells at the crypt base in comparison to wildtype. In addition, the number of proliferative cells was higher in the crypts of proximal and distal colon of Ptpn2‐KO mice compared to WT (p=0.0032 and p=0.0005 respectively; n=3). Lysozyme‐positive Paneth cells were significantly reduced in HET mice (p=0.0452 vs. WT; n=2–5) and further reduced in KO mice (p=0.0040; n=4). Moreover, KO mice were almost completely devoid of both lysozyme positive and negative Paneth cells, the latter characterized by large cytosolic granules at the crypt base. mRNA expression of the Paneth cell antimicrobial peptide, Lyz1, was reduced in Ptpn2‐KO mice compared to WT (p=0.017; n=2), whereas expression of antimicrobial peptides Defa5 and Defa6 were not altered. Goblet cell number was slightly reduced in the ileum and distal colon of KO mice (n=2). There was also a preliminary trend towards reduced expression of goblet cell secreted mucin proteins, Muc2, a major mucus layer constituent, and Muc3, a mucin protein homologous to MUC17 in humans shown to be protective against enteroinvasive E. coli, in the cecum of Ptpn2‐KO mice compared to wildtype (n=2).ConclusionPtpn2‐deficiency compromises intestinal epithelial cell differentiation leading to altered numbers and function of Paneth and goblet cells consistent with increased susceptibility to bacterial infection.Support or Funding InformationSupported by NIH 2R01DK091281 (DFM), the Crohn's and Colitis Foundation (DFM), and Science Without Borders Fellowship (VC)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bisphenol A inhibits mucin 2 secretion in intestinal goblet cells through mitochondrial dysfunction and oxidative stress

AimsBisphenol A (BPA) can induce intestinal epithelial cell barrier dysfunction; however, its effects on the intestinal mucus barrier remain unclear. We used LS174T cells as a model to investigate the effects of BPA on the functions of intestinal goblet cells. Main methodsThis study used CCK-8, flow cytometry, ELISA and real-time PCR to investigate the effects of BPA on mitochondrial dynamics, oxidative stress and apoptosis in goblet cells. In addition, mucin synthesis and secretion were evaluated using PAS staining and a PAS assay, respectively. Key findingsOur results indicate that BPA reduced cell viability in a time- and concentration-dependent manner. BPA induced mitochondrial dysfunction, as indicated by the depolarization of the mitochondrial membrane potential, inhibition of mitochondrial respiratory chain complex enzyme activity and reduction of ATP production. Moreover, BPA caused oxidative stress by significantly increasing the accumulation of ROS, as well as oxidative stress products, and reducing the antioxidant capacity. Furthermore, BPA induced intestinal goblet cell apoptosis, accompanied by increased DNA fragmentation, caspase-3, -8, -9,-10 gene expression and enzyme activity. Additionally, BPA inhibited the synthesis and secretion of mucin 2. SignificanceOur data suggest that BPA affected the secretory function of intestinal goblet cells by inducing mitochondrial dysfunction, oxidative stress, and apoptosis.

Interleukin 4 induces rapid mucin transport, increases mucus thickness and quality and decreases colitis and Citrobacter rodentium in contact with epithelial cells

ABSTRACTCitrobacter rodentium infection is a murine model for pathogenic intestinal Escherichia coli infection. C. rodentium infection causes an initial decrease in mucus layer thickness, followed by an increase during clearance. We aimed to identify the cause of these changes and to utilize this naturally occurring mucus stimulus to decrease pathogen impact and inflammation. We identified that mucin production and speed of transport from Golgi to secretory vesicles at the apical surface increased concomitantly with increased mucus thickness. Of the cytokines differentially expressed during increased mucus thickness, IFN-γ and TNF-α decreased the mucin production and transport speed, whereas IL-4, IL-13, C. rodentium and E. coli enhanced these aspects. IFN-γ and TNF-α treatment in combination with C. rodentium and pathogenic E. coli infection negatively affected mucus parameters in vitro, which was relieved by IL-4 treatment. The effect of IL-4 was more pronounced than that of IL-13, and in wild type mice, only IL-4 was present. Increased expression of Il-4, Il-4-receptor α, Stat6 and Spdef during clearance indicate that this pathway contributes to the increase in mucin production. In vivo IL-4 administration initiated 10 days after infection increased mucus thickness and quality and decreased colitis and pathogen contact with the epithelium. Thus, during clearance of infection, the concomitant increase in IL-4 protects and maintains goblet cell function against the increasing levels of TNF-α and IFN-γ. Furthermore, IL-4 affects intestinal mucus production, pathogen contact with the epithelium and colitis. IL-4 treatment may thus have therapeutic benefits for mucosal healing.

TMEM16A is indispensable for basal mucus secretion in airways and intestine.

Transmembrane member 16A (TMEM16A) is the Ca2+-activated chloride channel in airways and intestine. It has been associated with goblet cell metaplasia, as expression of TMEM16A is strongly up-regulated in cystic fibrosis and asthma during mucus hypersecretion. However, the possible role of TMEM16A for mucus production or mucus secretion remains obscure, and whether TMEM16A controls the function of intestinal goblet cells is entirely unknown. Basal mucus secretion in lungs occurs through low levels of ATP in the airway surface liquid. Here, we report for the first time that TMEM16A is essential for basal secretion of mucus in airways and intestine. Airway-ciliated and intestinal epithelial-specific knockout of TMEM16A ( TMEM16Aflox/floxFoxJ1, TMEM16Aflox/floxVil1) leads to accumulation of mucus in airway club (Clara) cells and intestinal goblet cells, respectively. Acute ATP-induced mucus secretion by airway club cells is inhibited when TMEM16A is knocked out in ciliated cells, possibly as a result of compromised release of prosecretory cytokines. Knockdown or inhibition of TMEM16A in human Calu3 airway epithelial cells indicates compromised IL-8 release. In intestinal goblet cells lacking expression of TMEM16A, mucus accumulates as a result of compromised ATP-induced secretion. In contrast, cholinergic mucus secretion by compound exocytosis is independent of TMEM16A. The data demonstrate a previously unrecognized role of TMEM16A for membrane exocytosis and describe a novel, ATP-driven pathway for intestinal mucus secretion. We conclude that ATP-dependent mucus secretion in both airways and intestine requires TMEM16A. The present results may form the basis for a novel, therapeutic approach for the treatment of mucus hypersecretion in inflammatory airway and intestinal disease.-Benedetto, R., Cabrita, I., Schreiber, R., Kunzelmann, K. TMEM16A is indispensable for basal mucus secretion in airways and intestine.

Effects of Epithelial IL-13Rα2 Expression in Inflammatory Bowel Disease.

Background: Mucosal IL-13 Receptor alpha 2 (IL13RA2) mRNA expression is one of the best predictive markers for primary non-responsiveness to infliximab therapy in patients with inflammatory bowel disease (IBD). The objective of this study was to understand how IL-13Rα2, a negative regulator of IL-13 signaling, can contribute to IBD pathology.Methods: IL13RA2 knockout (KO) and wild type (WT) mice were exposed to dextran sodium sulfate (DSS) in drinking water to induce colitis. Furthermore, mucosal biopsies and resection specimen of healthy individuals and IBD patients before the start of anti-tumor necrosis factor (anti-TNF) therapy were obtained for immunohistochemistry and gene expression analysis.Results: After induction of DSS colitis, IL13RA2 KO mice had similar disease severity, but recovered more rapidly than WT animals. Goblet cell numbers and mucosal architecture were also more rapidly restored in IL13RA2 KO mice. In mucosal biopsies of active IBD patients, immunohistochemistry revealed that IL-13Rα2 protein was highly expressed in epithelial cells, while expression was restricted to goblet cells in healthy controls. Mucosal IL13RA2 mRNA negatively correlated with mRNA of several goblet cell-specific and barrier genes, and with goblet cell numbers.Conclusions: The data suggest that IL-13Rα2 on epithelial cells contributes to IBD pathology by negatively influencing goblet cell recovery, goblet cell function and epithelial restoration after injury. Therefore, blocking IL-13Rα2 could be a promising target for restoration of the epithelial barrier in IBD.

The influence of basic treatment with tiotropium bromide of patients with chronic obstructive pulmonary disease stage II on the morpho-functional state of bronchial mucosa and the level of type IV collagen in bronchoalveolar lavage fluid.

The objective of the research was to evaluate the influence of basic treatment of patients with chronic obstructive pulmonary disease with tiotropium bromide on the processes of morphological rearrangement and local barrier defence mechanisms in the bronchial mucosa.&#x0D; Stage II chronic obstructive pulmonary disease is associated with the damage to the bronchi with proliferation of the connective tissue in its proper plate, clear identification of the basal membrane alteration, the presence of fibroblasts, the activation of fibroblasts/myofibroblasts and mucous glands, which is accompanied by the significant increase of type ІV collagen levels by 6.19 times (p&lt;0.05) in bronchoalveolar lavage fluid as compared to the control group indices.&#x0D; The elimination of stage II chronic obstructive pulmonary disease exacerbation and the use of tiotropium bromide within a month was accompanied only by partial improvement of morpho-functional state in relation to both cells of bronchial epithelial lining and adjacent connective tissue of mucosal plate. The prolongation of tiotropium bromide administration from 2 to 6 months, provided positive dynamics of structural morphological changes of the bronchial mucosa (the restoration of the ciliary apparatus of epithelial cells, the normalization of the secretory function of goblet cells, the inactivation of fibroblasts, the initial degeneration of myofibroblasts), thus leading to complete absence of morphological signs of edema or epithelial cell dystrophy.&#x0D; Conclusions. In patients with stage II chronic obstructive pulmonary disease, complete absence of morphological signs of edema or dystrophy of epithelial cells, against the background of collagenolysis in the connective tissue of the proper mucous plate of the bronchi and the highest possible decrease in the number of myofibroblasts, with near-complete levels of type IV collagen normalization in the bronchoalveolar lavage fluid, were identified only within a 6-month treatment with tiotropium bromide.