Goblet Cell Expression Research Articles

Rab7-dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal homeostasis

Inflammation in ulcerative colitis is typically restricted to the mucosal layer of distal gut. Disrupted mucus barrier, coupled with microbial dysbiosis, has been reported to occur prior to the onset of inflammation. Here, we show the involvement of vesicular trafficking protein Rab7 in regulating the colonic mucus system. We identified a lowered Rab7 expression in goblet cells of colon during human and murine colitis. In vivo Rab7 knocked down mice (Rab7KD) displayed a compromised mucus layer, increased microbial permeability, and depleted gut microbiota with enhanced susceptibility to dextran sodium-sulfate induced colitis. These abnormalities emerged owing to altered mucus composition, as revealed by mucus proteomics, with increased expression of mucin protease chloride channel accessory 1 (CLCA1). Mechanistically, Rab7 maintained optimal CLCA1 levels by controlling its lysosomal degradation, a process that was dysregulated during colitis. Overall, our work establishes a role for Rab7-dependent control of CLCA1 secretion required for maintaining mucosal homeostasis.

Rab7-dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal homeostasis.

Inflammation in ulcerative colitis is typically restricted to the mucosal layer of distal gut. Disrupted mucus barrier, coupled with microbial dysbiosis, has been reported to occur prior to the onset of inflammation. Here, we show the involvement of vesicular trafficking protein Rab7 in regulating the colonic mucus system. We identified a lowered Rab7 expression in goblet cells of colon during human and murine colitis. In vivo Rab7 knocked down mice (Rab7KD) displayed a compromised mucus layer, increased microbial permeability, and depleted gut microbiota with enhanced susceptibility to dextran sodium-sulfate induced colitis. These abnormalities emerged owing to altered mucus composition, as revealed by mucus proteomics, with increased expression of mucin protease chloride channel accessory 1 (CLCA1). Mechanistically, Rab7 maintained optimal CLCA1 levels by controlling its lysosomal degradation, a process that was dysregulated during colitis. Overall, our work establishes a role for Rab7-dependent control of CLCA1 secretion required for maintaining mucosal homeostasis.

Activation of goblet cell Piezo1 alleviates mucus barrier damage in mice exposed to WAS by inhibiting H3K9me3 modification

BackgroundOur recent studies found that intestinal mechanical signals can regulate mucus synthesis and secretion of intestinal goblet cells through piezo type mechanosensitive ion channel component 1 (Piezo1), but the detailed molecular mechanisms remain to be investigated. Previous studies using a water avoidance stress (WAS) model reported decreased intestinal mucus accompanied by abnormal intestinal motility. It has also been reported that the expression of mucin2 was negatively correlated with histone H3 lysine 9 trimethylation (H3K9me3), a key regulator of histone methylation, and that mechanical stimulation can affect methylation. In this study, we aimed to determine whether and how Piezo1 expressed on goblet cells regulates mucus barrier function through methylation modification.MethodsA murine WAS model was established and treated with Yoda1 (Piezo1 agonist), and specific Piezo1 flox-mucin2 Cre mice were also tested. The mucus layer thickness and mucus secretion rate of mouse colonic mucosa were detected by a homemade horizontal Ussing chamber, intestinal peristaltic contraction was detected by the ink propulsion test and organ bath, goblet cells and mucus layer morphology were assessed by HE and Alcian blue staining, mucus permeability was detected by FISH, and the expression levels of Piezo1, H3K9me3 and related molecules were measured by Western blots and immunofluorescence. LS174T cells were cultured on a shaker board in vitro to simulate mechanical stimulation. Piezo1 and H3K9me3 were inhibited, and changes in mucin2 and methylation-related pathways were detected by ELISAs and Western blots. ChIP-PCR assays were used to detect the binding of H3K9me3 and mucin2 promoters under mechanical stimulation.ResultsCompared with those of the controls, the mucus layer thickness and mucus secretion rate of the mice exposed to WAS were significantly decreased, the mucus permeability increased, the number of goblet cells decreased, and the intestinal contraction and peristalsis were also downregulated and disordered. Intraperitoneal injection of Yoda1 improved mucus barrier function and intestinal contraction. In the colonic mucosa of mice exposed to WAS, Piezo1 was decreased, and histone H3 lysine 9 trimethylation (H3K9me3) and methyltransferase suppressor of variegation 3–9 homolog 1 (SUV39h1) were increased, but activating Piezo1 alleviated these effects of WAS. Piezo1 flox-mucin2 Cre mice showed decreased mucus expression and increased methylation compared to wild-type mice. Cell experiments showed that mechanical stimulation induced the activation of Piezo1, decreased H3K9me3 and SUV39h1, and upregulated mucin2 expression. Inhibition of Piezo1 or H3K9me3 blocked the promoting effect of mechanical stimulation on LS174T mucin2 expression. The binding of H3K9me3 to the mucin2 promoter decreased significantly under mechanical stimulation, but this could be blocked by the Piezo1 inhibitor GsMTx4.ConclusionPiezo1 mediates mechanical stimulation to inhibit SUV39h1, thereby reducing H3K9me3 production and its binding to the mucin2 promoter, ultimately promoting mucin2 expression in goblet cells. This study further confirmed that piezo1 on goblet cells could regulate mucus barrier function through methylation.

Galacto-Oligosaccharides Increase the Abundance of Beneficial Probiotic Bacteria and Improve Gut Architecture and Goblet Cell Expression in Poorly Performing Piglets, but Not Performance.

Poorly performing piglets receiving commercial milk replacers do not benefit from the naturally occurring probiotic galacto-oligosaccharides otherwise found in sow milk. Study objectives were to investigate the effects of complete milk replacer supplemented with galacto-oligosaccharides on the microbiome, gut architecture and immunomodulatory goblet cell expression of poorly performing piglets that could benefit from milk replacement feeding when separated from sows and housed with fit siblings in environmentally controlled pens. The study is novel in that it is one of the first to investigate the effects of supplementing complete milk replacer with galacto-oligosaccharides in poorly performing piglets. Gastrointestinal tract samples were collected from piglets, and the microbiome composition was assessed by 16s ribosomal ribonucleic acid gene sequencing. Gut architectural features, villus/crypt ratio and enumeration of goblet cells in tissues were assessed by histopathological techniques. The most abundant taxa identified at the genus level were Lactobacillus, Streptococcus, Prevotella, Lactococcus and Leuconostoc. Milk replacer plus galacto-oligosaccharides significantly improved gut architectural features and villus/crypt ratio throughout the gastrointestinal tract, increased the number of goblet cells and revealed a differential abundance of beneficial probiotic bacteria, particularly Lactobacillus and Bifidobacterium. In these respects, galacto-oligosaccharide-supplemented milk replacer may be a useful addition to animal husbandry in poorly performing, non-thriving animals when moved to environmentally controlled pens away from sows and fit siblings, thereby modulating the microbiome and gastrointestinal tract performance.

The Milk Active Ingredient, 2'-Fucosyllactose, Inhibits Inflammation and Promotes MUC2 Secretion in LS174T Goblet Cells In Vitro.

In several mice inflammatory models, human milk oligosaccharides (HMOs) were shown to protect the intestinal barrier by promoting mucin secretion and suppressing inflammation. However, the functions of the individual HMOs in enhancing mucin expression in vivo have not been compared, and the related mechanisms are not yet to be clarified. In this study, we investigated the modulatory effects of 2′-fucosyllactose (2′-FL), 3′-sialyllactose (3′-SL), galacto-oligosaccharide (GOS) and lactose (Lac) on goblet cells’ functions in vitro. The appropriate dosage of the four chemicals was assessed in LS174T cells using the CCK-8 method. Then they were supplemented into a homeostasis and inflammatory environment to further investigate their effects under different conditions. Mucin secretion-related genes, including mucin 2 (MUC2), trefoil factor family 3 (TFF3), resistin-like β (RETNLB), carbohydrate sulfotransferase 5 (CHST5) and galactose-3-O-sulfotransferase 2 (GAL3ST2), in LS174T cells were detected using quantitative RT-qPCR. The results showed that 2′-FL (2.5 mg/mL, 72 h) was unable to increase MUC2 secretion in a steady-state condition. Comparatively, it exhibited a greater ability to improve mucin secretion under an inflammatory condition compared with GOS, demonstrated by a significant increase in TFF3 and CHST5 mRNA expression levels (p > 0.05). However, 3′-SL and Lac exhibited no effects on mucin secretion. To further investigate the underlying mechanism via which 2′-FL enhanced goblet cells’ secretion function, the NOD-like receptor family pyrin domain containing 6 (NLRP6) gene, which is closely related to MUC2 secretion, was silenced using the siRNA method. After silencing the NLRP6 gene, the mRNA expression levels of MUC2, TFF3 and CHST5 in the (2′-FL + tumor necrosis factor α (TNF-α) + NLRP6 siRNA) group were significantly decreased compared with the (2′-FL + TNF-α) group (p > 0.05), indicating that NLRP6 was essential for MUC2 expression in goblet cells. We further found that 2′-FL could significantly decrease toll-like receptor 4 (TLR4, p < 0.05), myeloid differential protein-88 (MyD88, p < 0.05) and nuclear factor kappa-B (NF-κB, p < 0.05) levels in LS174T inflammatory cells, even when the NLRP6 was silenced. Altogether, these results indicated that in goblet cells, 2′-FL exerts its function via multiple processes, i.e., by promoting mucin secretion through NLRP6 and suppressing inflammation by inhibiting the TLR4/MyD88/NF-κB pathway.

Gastrointestinal consequences of lipopolysaccharide-induced lung inflammation

BackgroundRespiratory inflammation is the body’s response to lung infection, trauma or hypersensitivity and is often accompanied by comorbidities, including gastrointestinal (GI) symptoms. Why respiratory inflammation is accompanied by GI dysfunction remains unclear. Here, we investigate the effect of lipopolysaccharide (LPS)-induced lung inflammation on intestinal barrier integrity, tight-junctions, enteric neurons and inflammatory marker expression.MethodsFemale C57bl/6 mice (6–8 weeks) were intratracheally administered LPS (5 µg) or sterile saline, and assessed after either 24 or 72 h. Total and differential cell counts in bronchoalveolar lavage fluid (BALF) were used to evaluate lung inflammation. Intestinal barrier integrity was assessed via cross sectional immunohistochemistry of tight junction markers claudin-1, claudin-4 and EpCAM. Changes in the enteric nervous system (ENS) and inflammation in the intestine were quantified immunohistochemically using neuronal markers Hu + and nNOS, glial markers GFAP and S100β and pan leukocyte marker CD45.ResultsIntratracheal LPS significantly increased the number of neutrophils in BALF at 24 and 72 h. These changes were associated with an increase in CD45 + cells in the ileal mucosa at 24 and 72 h, increased goblet cell expression at 24 h, and increased expression of EpCAM at 72 h. LPS had no effect on the expression of GFAP, S100β, nor the number of Hu + neurons or proportion of nNOS neurons in the myenteric plexus.ConclusionsIntratracheal LPS administration induces inflammation in the ileum that is associated with enhanced expression of EpCAM, decreased claudin-4 expression and increased goblet cell density, these changes may contribute to systemic inflammation that is known to accompany many inflammatory diseases of the lung.

Smoking modulates different secretory subpopulations expressing SARS-CoV-2 entry genes in the nasal and bronchial airways

SARS-CoV-2 infection and disease severity are influenced by viral entry (VE) gene expression patterns in the airway epithelium. The similarities and differences of VE gene expression (ACE2, TMPRSS2, and CTSL) across nasal and bronchial compartments have not been fully characterized using matched samples from large cohorts. Gene expression data from 793 nasal and 1673 bronchial brushes obtained from individuals participating in lung cancer screening or diagnostic workup revealed that smoking status (current versus former) was the only clinical factor significantly and reproducibly associated with VE gene expression. The expression of ACE2 and TMPRSS2 was higher in smokers in the bronchus but not in the nose. scRNA-seq of nasal brushings indicated that ACE2 co-expressed genes were highly expressed in club and C15orf48+ secretory cells while TMPRSS2 co-expressed genes were highly expressed in keratinizing epithelial cells. In contrast, these ACE2 and TMPRSS2 modules were highly expressed in goblet cells in scRNA-seq from bronchial brushings. Cell-type deconvolution of the gene expression data confirmed that smoking increased the abundance of several secretory cell populations in the bronchus, but only goblet cells in the nose. The association of ACE2 and TMPRSS2 with smoking in the bronchus is due to their high expression in goblet cells which increase in abundance in current smoker airways. In contrast, in the nose, these genes are not predominantly expressed in cell populations modulated by smoking. In individuals with elevated lung cancer risk, smoking-induced VE gene expression changes in the nose likely have minimal impact on SARS-CoV-2 infection, but in the bronchus, smoking may lead to higher viral loads and more severe disease.

Design of functional decellularized matrix for conjunctival epithelial stem cell maintenance and ocular surface reconstruction

Conjunctival defects severely impair ocular surface homeostasis and vision.However, the existing conjunctival reconstruction methods are still unable to achieve satisfactory functional replacement of conjunctiva. Scaffold materials that provide a more stem-cell-friendly microenvironment are in urgent need of improvement. Herein we reported a functional conjunctival reconstruction scaffold constructed from decellularized matrix derived from rabbit subconjunctival fibroblasts (DM-SCF) loaded with conjunctival epithelial stem cells (CjESCs) for ocular surface repair. The DM-SCF had a thickness and mechanical strength close to that of normal conjunctiva and showed excellent biocompatibility. CjESCs inoculated on DM-SCF maintained good stem cell properties, which showed long-term maintenance of low differentiation and good proliferation activity.DM-SCF transplantation with CjESCs showed satisfactory results in rabbit models of a large conjunctival defect.The conjunctiva was reconstructed with abundant goblet cell expression and regular fiber arrangement. Protein mass spectrometry revealed the active extracellular matrix components of DM-SCF, regulating the activation of Wnt/β-catenin and Notch pathways for stem cell maintenance. Overall, our study developed a promising new strategy for ocular surface reconstruction by optimizing bioactive scaffold and providing functional stem cell niche, so as to achieve long-standing and superior functional repair.

The IL-25-dependent tuft cell circuit driven by intestinal helminths requires macrophage migration inhibitory factor (MIF)

Macrophage migration inhibitory factor (MIF) is a key innate immune mediator with chemokine- and cytokine-like properties in the inflammatory pathway. While its actions on macrophages are well-studied, its effects on other cell types are less understood. Here we report that MIF is required for expansion of intestinal tuft cells during infection with the helminth Nippostrongylus brasiliensis. MIF-deficient mice show defective innate responses following infection, lacking intestinal epithelial tuft cell hyperplasia or upregulation of goblet cell RELMβ, and fail to expand eosinophil, type 2 innate lymphoid cell (ILC2) and macrophage (M2) populations. Similar effects were observed in MIF-sufficient wild-type mice given the MIF inhibitor 4-IPP. MIF had no direct effect on epithelial cells in organoid cultures, and MIF-deficient intestinal stem cells could generate tuft cells in vitro in the presence of type 2 cytokines. In vivo the lack of MIF could be fully compensated by administration of IL-25, restoring tuft cell differentiation and goblet cell expression of RELM-β, demonstrating its requirement upstream of the ILC2-tuft cell circuit. Both ILC2s and macrophages expressed the MIF receptor CXCR4, indicating that MIF may act as an essential co-factor on both cell types to activate responses to IL-25 in helminth infection. [Display omitted]

Mechanical stimulation activates Piezo1 to promote mucin2 expression in goblet cells.

Studies on the regulation of mucin2 expression in intestinal goblet cells by the endocrine system and the immune system have been comprehensive, but the effects of abundant mechanical factors in the intestinal microenvironment on goblet cells are not clear. We constructed mechanical stimulation models in vivo and in vitro to explore the effect of mechanical stimulation on intestinal goblet cells. Piezo1 expression and function were regulated through model mouse and drugs to explored whether Piezo1 mediated mechanical stimulation. The results showed that hydrostatic pressure could promote mucus secretion in the mouse colon, and both traction force and shear force could promote the expression of mucin2 in the LS174T cell line. We further found that the Piezo1 protein, which was abundantly expressed in goblet cells, acted as a mechanoreceptor. Knockout of Piezo1 in the intestinal epithelial cells of mice could reduce the promotion of mucus secretion by pressure stimulation, and the specific downregulation of Piezo1 protein in LS174T cells or Piezo1 inhibitor treatment could significantly reduce the promotion of mucin2 expression in goblet cells by mechanical stimulation; however, treatment with a Piezo1 agonist had the opposite effect. Moreover, we found that Piezo1 regulated mucin2 expression through the downstream Erk/Ca2+ pathway. In short, our study confirmed for the first time that goblet cells are mechanoreceptive cells that can directly sense mechanical stimulation in the intestinal tract and respond back through the Piezo1-Erk/Ca2+ -mucin2 pathway.

18 COVID-19 AND THE GUT IN HUMAN OBESITY: OVEREXPRESSION OF SARS-COV-2 RECEPTORS REVEALED BY DEEP SINGLE CELL RNA-SEQ

IntroductionObesity is a major risk factor for severe coronavirus disease, and clinical evidence now supports the GI tract, in addition to the respiratory system, as a potential route for SARS-CoV-2 infection. Expression of viral entry factors ACE2, TMPRSS2, and CTSL have been detected along the human GI tract including gastric, ileal and colonic mucosa. It is unclear whether obesity confers increased susceptibility to initial SARS-CoV-2 infection, or what gut mechanisms in obesity predispose to vulnerability to SARS-CoV-2. Thus, we aimed to investigate, by single cell RNA-sequencing (scRNA-Seq) of human colonic mucosa, whether patients with obesity may be more susceptible to SARS-CoV-2 infection, by virtue of enhanced expression of SARS-CoV2 entry cofactors followed protein assessment in colon biopsies.MethodsWe studied 19 patients: 10 lean (age 33±3y, BMI 23±1kg/m2, 90% female), and 9 with obesity (age 43±3y, BMI 36±1kg/m2, 89% female). Human colonic biopsies from lean (n=4 scRNA-Seq; n=6 validation) and obesity (n=6 scRNASeq; n=3 validation) participants were obtained by sigmoidoscopy. Biopsies were dissociated, and viable cells were FACS-isolated. Chromium-10X Genomics was used for scRNA-Seq library prep, followed by Illumina HiSeq4000 sequencing. COVID-19 entry factors displaying significant differential expression between lean and obesity were then validated for gene, and protein expression in the validation cohort using Illumina TruSeq, and quantitative immunofluorescence confocal microscopy, respectively.ResultsThe initial dataset analysis revealed sequencing of 59,653 cells, 705 million reads, at 127,000 reads per cell. The human colonic mucosa partitioned into 20 cell subsets (Fig1A,B), and 15 of the 20 clusters displayed detectable expression of at least one of the COVID-19 entry factors: TMPRSS2, CTSL, or ACE2 (Fig1C,D,E). Goblet cell expression of TMPRSS2 was increased 4.6-fold (p<0.05), stromal cell expression of CTSL was increased 1.2-fold (p<0.0001), and ACE2 expression was increased 1.27-fold (p<0.001) in crypt-top (CT) colonocytes of obesity compared to lean controls (Fig2A). Colonic overexpression of TMPRSS2 mRNA (p<0.05) and protein (p<0.05), and CTSL (p<0.05) mRNA, but not ACE2 mRNA, in obesity was further validated in a second validation cohort (Fig2B-F).ConclusionsscRNA-Seq analysis of human colonic epithelium in obesity compared to healthy controls revealed multiple epithelial cell subsets (goblet cell, stromal, and colonocytes) with overexpression of COVID-19 entry factors TMPRSS2, CTSL, and ACE2, confirming the digestive system as a portal for infection by SARS-CoV-2.Furthermore goblet, stromal, and colonocyte-specific overexpression of TMPRSS2, CTSL, and ACE2 in obesity may play a significant role in increased initial susceptibility to COVID-19, and worse disease outcomes in human obesity.Figure 1Single-Cell RNA-Seq Profiling of Human Colonic Epithelium in Obesity. A) t-SNE plot of single-cell RNA-seq profiles of native human colonic epithelial cells, colored by cluster number and identity, listed by largest to smallest population, and annotated by cluster identity, determined by highest ranking gene marker for the colonic cells clustered and profiled between lean and obesity, where dotted blue, red, and green circles represents goblet cells, stromal cells, and CT colonocytes, respectively. B) Proposed cluster identities based on conserved expression of known markers for annotated cell types. Clusters identified displayed expression of at least one of the COVID-19 entry factors: TMPRSS2, CTSL, or ACE2. The average proportion of cells in annotated clusters expressing, C) TMPRSS2, D) CTSL, and E) ACE2 among all studied participants.Figure 2Differential Expression of COVID-19 Entry factors. A) COVID-19 entry factors, TMPRSS2, CTSL, ACE2, displaying differential expression in the single-cell dataset comparing obesity to lean controls in the specified cell clusters. Blue, red, and green bars represent goblet cells, stromal cells, and CT colonocytes, respectively. Light and dark shaded bars represent the lean an obesity groupings, respectively. B) COVID-19 entry factors, TMPRSS2, CTSL, ACE2, displaying differential mRNA expression in the validation cohort dataset comparing obesity to lean in whole colonic mucosal tissue. Quantification of protein expression of C,D) TMPRSS2 and E,F) CTSL in the validation cohort dataset comparing obesity to lean in whole colonic mucosal tissue. Significance testing used a two-tailed student t-test compared between lean and obesity; *p<0.05, **p<0.01, ***p<0.0001. L= Lean, OB= Obesity.

A219 PROTECTIVE EFFECTS OF AKKERMANSIA MUCINIPHILA ON INTESTINAL BARRIER FUNCTION AND INFLAMMATION

Abstract Background Akkermansia muciniphila, an anaerobic gram-negative bacteria, accounts for ~3% of human gut microbiota. Despite its mucolytic nature, A. muciniphila has been shown to stimulate mucin production, enhance anti-inflammatory regulatory T cell proliferation and improve gut barrier integrity. Interestingly, an inverse relationship has been established between A. muciniphila and several disease states including inflammatory bowel disease (IBD) suggesting it may have protective and anti-inflammatory effects. However, the precise role and mechanism of A. muciniphila in the pathogenesis of colitis remains unknown. Thus, we hypothesize that A. muciniphila may induce protective effects on intestinal inflammation by influencing host immune response and epithelial barrier integrity. Aims (1) To investigate the protective role of A. muciniphila in intestinal inflammation in a chemically induced model of IBD and (2) to investigate the protective role of A. muciniphila in intestinal inflammation and host defense in a model of enteric parasitic infection. Methods Colitis was induced in germ-free C57BL/6 mice with 2.5% dextran sulphate sodium (DSS) after treatment with either C57BL/6 wild-type (WT) cecal contents or WT cecal contents supplemented with A. muciniphila. Colitis severity was assessed by disease activity index (DAI), macroscopic and histological scores, myeloperoxidase (MPO) assay and cytokine expression. In addition, colitis was induced by Trichuris muris, an intestinal nematode, following treatment with A. muciniphila. Post-infection, the severity of intestinal inflammation was assessed by worm burden, goblet cell staining, cytokines analysis, MPO activity and Muc2 expression. Microbial composition was assessed by 16s rRNA gene sequencing. Results In preliminary studies, mice treated with A. muciniphila and administered DSS for 5 days yielded a significant decrease in DAI, macroscopic scoring, and MPO values compared with controls. IL-10 was also elevated in mice receiving A. muciniphila. Groups receiving A. muciniphila in the T. muris model trended toward decreased worm burden, IL-4, IL-13, as well as increased levels of IL-10, goblet cell expression, and Muc2 and Muc5ac expression. A significant decrease in MPO activity was also observed in the group receiving the A. muciniphila-supplemented gavage. Microbial analysis indicated that 3 weeks post-gavage Akkermansia levels were significantly elevated in groups receiving the A. muciniphila-supplemented WT cecal contents versus WT alone. This significance was maintained post-T. muris infection. Conclusions These findings suggest that A. muciniphila may have a protective role in the context of intestinal inflammation. This research has the potential to fuel the development of novel treatments by utilizing this protective role in IBD. Funding Agencies CIHR

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.

Bovine milk-derived exosomes enhance goblet cell activity and prevent the development of experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is characterized by intestinal injury and impaired mucin synthesis. We recently showed that breast milk exosomes from rodents promote intestinal cell viability, epithelial proliferation, and stem cell activity, but whether they also affect mucus production is unknown. Therefore, the aim of this study was to investigate the effects of bovine milk-derived exosomes on goblet cell expression in experimental NEC and delineate potential underlying mechanisms of action. Exosomes were isolated from bovine milk by ultracentrifugation and confirmed by Nanoparticle Tracking Analysis and through the detection of exosome membrane markers. To study the effect on mucin production, human colonic LS174T cells were cultured and exposed to exosomes. Compared to control, exosomes promoted goblet cell expression, as demonstrated by increased mucin production and relative expression levels of goblet cell expression markers trefoil factor 3 (TFF3) and mucin 2 (MUC2). In addition, exosome treatment enhanced the expression of glucose-regulated protein 94 (GRP94), the most abundant intraluminal endoplasmic reticulum (ER) chaperone protein that aids in protein synthesis. Furthermore, experimental NEC was induced in mouse pups by hyperosmolar formula feeding, lipopolysaccharide administration and hypoxia exposure on postnatal days 5–9. Milk exosomes were given with each gavage feed. NEC was associated with ileal morphological injury and reduction in MUC2+ goblet cells and GRP94+ cells per villus. Exosome administration to NEC pups prevented these changes. This research highlights the potential novel application of milk-derived exosomes in preventing the development of NEC in high-risk infants when breast milk is not available.

P059 INDOLEAMINE 2,3-DIOXYGENASE 1 EXPRESSION PROMOTES EPITHELIAL SECRETORY CELL DIFFERENTIATION VIA ARYL HYDROCARBON RECEPTOR

In mouse models of IBD, the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) has been shown to limit disease severity. Our studies indicate that intestinal epithelial cells are an important source of IDO1 expression in these conditions; however the mechanism remains unknown. We have identified IDO1 expression in goblet cells as well as colocalization with secreted mucin. As mucin is both a physical barrier against bacterial invasion and an energy substrate for luminal microbes, we hypothesized that epithelial IDO1 may limit colitis severity is through enhancement of mucus barrier function. We generated a novel transgenic mouse model which overexpresses EGFP-tagged IDO1 specifically in the intestinal epithelium (IDO1-TG). Primary epithelial spheroid cultures were utilized to assess growth and gene expression by RT-qPCR and immunofluorescence in vitro. Histology revealed an increase in goblet and Paneth cells in IDO1-TG small intestine tissue. Analysis of gene expression in enteroids revealed higher levels of stem cell markers Lgr5 and Lrig1 in IDO1-TG compared to wildtype (WT). Additionally, the secretory lineage transcription factor Atoh1 and downstream effector Gfi1 were significantly upregulated in IDO1-TG, leading to enhanced secretory lineage markers for Paneth cells, goblet cells, and enteroendocrine cells, and a reduction in absorptive cell markers. Enteroids genetically deficient of Ido1 showed the opposite trend. Inhibition of IDO1 did not reduce expression of secretory cell markers, but incubation with a specific inhibitor of the aryl hydrocarbon receptor restored baseline expression levels. Taken together, these data indicate expression of IDO1 in the intestinal epithelium promotes secretory cell differentiation and mucus production, whereby identifying a novel mechanism by which non-enzymatic activity of IDO1 modulates intestinal homeostasis.

P059 INDOLEAMINE 2,3-DIOXYGENASE 1 EXPRESSION PROMOTES EPITHELIAL SECRETORY CELL DIFFERENTIATION VIA ARYL HYDROCARBON RECEPTOR

In mouse models of IBD, the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) has been shown to limit disease severity. Our studies indicate that intestinal epithelial cells are an important source of IDO1 expression in these conditions; however the mechanism remains unknown. We have identified IDO1 expression in goblet cells as well as colocalization with secreted mucin. As mucin is both a physical barrier against bacterial invasion and an energy substrate for luminal microbes, we hypothesized that epithelial IDO1 may limit colitis severity is through enhancement of mucus barrier function. We generated a novel transgenic mouse model which overexpresses EGFP-tagged IDO1 specifically in the intestinal epithelium (IDO1-TG). Primary epithelial spheroid cultures were utilized to assess growth and gene expression by RT-qPCR and immunofluorescence in vitro. Histology revealed an increase in goblet and Paneth cells in IDO1-TG small intestine tissue. Analysis of gene expression in enteroids revealed higher levels of stem cell markers Lgr5 and Lrig1 in IDO1-TG compared to wildtype (WT). Additionally, the secretory lineage transcription factor Atoh1 and downstream effector Gfi1 were significantly upregulated in IDO1-TG, leading to enhanced secretory lineage markers for Paneth cells, goblet cells, and enteroendocrine cells, and a reduction in absorptive cell markers. Enteroids genetically deficient of Ido1 showed the opposite trend. Inhibition of IDO1 did not reduce expression of secretory cell markers, but incubation with a specific inhibitor of the aryl hydrocarbon receptor restored baseline expression levels. Taken together, these data indicate expression of IDO1 in the intestinal epithelium promotes secretory cell differentiation and mucus production, whereby identifying a novel mechanism by which non-enzymatic activity of IDO1 modulates intestinal homeostasis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Epithelial cell types and their proposed roles in maintaining the mucosal barrier in human chagasic-megacolonic mucosa.

Patients suffering from chagasic megacolon must have an intact mucosal barrier as they survive this chronic disease for decades. A key structure of the mucosal barrier are epithelial cells. Vasoactive-intestinal-peptide (VIP)-positive nerve fibres are involved in influencing, e.g., epithelial cell proliferation, mucus secretion (e.g., mucin 2 and trefoil factor 3 of goblet cells) and inflammation or autoimmunity, all putative and/or known factors altered in chagasic megacolon. We analyzed qualitatively and quantitatively goblet cells, their specific markers, such as mucin 2 (MUC2) and trefoil factor 3 (TFF3) and enterocytes, the relation of VIP-immunoreactive nerve fibres to the epithelia, the distribution of gelsolin, a protein involved in chronic inflammation processes in the epithelia, and the proliferation rate of epithelial cells by combined 4',6-diamidino-2-phenylindole (DAPI) and phosphohistone-H3 (PHH3) staining. Goblet cells were the dominating epithelial cell type. They accounted for 38.4% of all epithelial cells in controls and changed to 58.9% in the megacolonic parts. In contrast to the overall expression in goblet cells of control epithelia, TFF3 was confined to goblet cells at the base of the crypts whereas MUC2 was found only in luminal goblet cells. Gelsolin-positive goblet cells were predominantly recognized within the controls. Finally, the mean value of mitosis increased from 1.5% within the controls up to 2.6% in the anal parts of the chagasic sepcimens. Taken together, increased cell proliferation, preponderance of goblet cells, differential MUC 2, and TFF 3 expression might all be factors maintaining an intact mucosal barrier within chagasic megacolon.

Lactobacillus rhamnosus GG treatment improves intestinal permeability and modulates inflammatory response and homeostasis of spleen and colon in experimental model of Pseudomonas aeruginosa pneumonia

Recent clinical trials and invivo models demonstrate probiotic administration can reduce occurrence and improve outcome of pneumonia and sepsis, both major clinical challenges worldwide. Potential probiotic benefits include maintenance of gut epithelial barrier homeostasis and prevention of downstream organ dysfunction due to systemic inflammation. However, mechanism(s) of probiotic-mediated protection against pneumonia remain poorly understood. This study evaluated potential mechanistic targets in the maintenance of gut barrier homeostasis following Lactobacillus rhamnosus GG (LGG) treatment in a mouse model of pneumonia. Studies were performed in 6-8 week old FVB/N mice treated (o.g.) with or without LGG (109CFU/ml) and intratracheally injected with Pseudomonas aeruginosa or saline. At 4, 12, and 24h post-bacterial treatment spleen and colonic tissue were collected for analysis. Pneumonia significantly increased intestinal permeability and gut claudin-2. LGG significantly attenuated increased gut permeability and claudin-2 following pneumonia back to sham control levels. As mucin expression is key to gut barrier homeostasis we demonstrate that LGG can enhance goblet cell expression and mucin barrier formation versus control pneumonia animals. Further as Muc2 is a key gut mucin, we show LGG corrected deficient Muc2 expression post-pneumonia. Apoptosis increased in both colon and spleen post-pneumonia, and this increase was significantly attenuated by LGG. Concomitantly, LGG corrected pneumonia-mediated loss of cell proliferation in colon and significantly enhanced cell proliferation in spleen. Finally, LGG significantly reduced pro-inflammatory cytokine gene expression in colon and spleen post-pneumonia. These data demonstrate LGG can maintain intestinal barrier homeostasis by enhancing gut mucin expression/barrier formation, reducing apoptosis, and improving cell proliferation. This was accompanied by reduced pro-inflammatory cytokine expression in the gut and in a downstream organ (spleen). These may serve as potential mechanistic targets to explain LGG's protection against pneumonia in the clinical and invivo setting.

Intestinal NHE8 is highly expressed in goblet cells and its expression is subject to TNF-α regulation.

While the intestine plays an important role in digestion and absorption, the mucus lining the epithelium represents a pivotal function in mucosal protection. Goblet cells are scattered in both the crypts and among enterocytes, and they secrete an important component of mucus, mucin. We have reported that sodium/hydrogen exchanger (NHE) 8 is a novel player in mucosal protection, since loss of NHE8 function resulted in reduced mucin production and increased bacterial adhesion. While NHE8 has been shown to be expressed in enterocytes and its expression is reduced during intestinal inflammation, nothing is known about the role of NHE8 in goblet cells. This current study is designed to define the expression of NHE8 and the role of TNF-α in the regulation of NHE8 in goblet cells. Using HT29-MTX cells as an in vitro model, we detected abundant NHE8 mRNA in goblet cells. Immunohistochemical staining localized NHE8 protein on the plasma membrane and in the intracellular compartments in goblet cells. Furthermore, NHE8 expression in goblet cells is regulated by the proinflammatory cytokine TNF-α. The expression of NHE8 in HT29-MTX cells was significantly reduced at both mRNA and protein levels in the presence of TNF-α. This inhibition of NHE8 mRNA expression could be blocked by the transcriptional inhibitor actinomycin D. Promoter reporter assay showed that NHE8 promoter activity was indeed reduced by TNF-α. Mechanistically, TNF-α reduced Sp3 protein binding to the human NHE8 basal promoter region. Therefore, NHE8 is expressed in goblet cells, and the inflammatory cytokine TNF-α downregulates NHE8 expression by a transcriptional mechanism.

Potential role of aquaporin 3 in gastric intestinal metaplasia

Gastric intestinal metaplasia (GIM) is a pre-cancerous condition and a pivotal step in the formation of gastric cancer (GC). Aquaporin 3 (AQP3) has been found to be expressed in goblet cells rather than mucus-secreting glands. To investigate the characteristics of GIM in non-cancerous tissues adjacent to GC, as well as the expression and role of AQP3 in GIM tissues, 16 patients diagnosed with gastric adenocarcinoma of intestinal type located in the lesser curve of the antrum were consecutively enrolled in this study. A new pathological technology called "gastric mucosal sausage roll" was introduced. GIM was determined according to the updated Sydney system, and AQP3 expression in goblet cells was determined by immunohistochemistry. GIM was found in all stomach specimens, and its incidence increased with progression to GC (P < 0.001). GIM prevalence displayed remarkable association with the distance to GC in the anterior gastric wall tissues (P = 0.016) and tissues toward the cardia (P = 0.014), such that GIM was more common in the areas closer to GC (P < 0.001). AQP3 was found to be expressed in 67.71% of parts with GIM, and AQP3 immunoreactivity was identified more frequently in severe GIM areas (P < 0.001). In short, the incidence and severity of GIM correlated with the distance from GC, and AQP3 was differentially expressed in goblet cells, with most AQP3-positive goblet cells presenting in severe GIM. Together, this study suggests that AQP3 may play an important role in gastric carcinogenesis from GIM.