Goblet Cell Expansion Research Articles

Loss of Elp3 blocks intestinal tuft cell differentiation via an mTORC1-Atf4 axis

Intestinal tuft cells are critical for anti-helminth parasite immunity because they produce IL-25, which triggers IL-13 secretion by activated group 2 innate lymphoid cells (ILC2s) to expand both goblet and tuft cells. We show that epithelial Elp3, a tRNA-modifying enzyme, promotes tuft cell differentiation and is consequently critical for IL-25 production, ILC2 activation, goblet cell expansion and control of Nippostrongylus brasiliensis helminth infection in mice. Elp3 is essential for the generation of intestinal immature tuft cells and for the IL-13-dependent induction of glycolytic enzymes such as Hexokinase 1 and Aldolase A. Importantly, loss of epithelial Elp3 in the intestine blocks the codon-dependent translation of the Gator1 subunit Nprl2, an mTORC1 inhibitor, which consequently enhances mTORC1 activation and stabilizes Atf4 in progenitor cells. Likewise, Atf4 overexpression in mouse intestinal epithelium blocks tuft cell differentiation in response to intestinal helminth infection. Collectively, our data define Atf4 as a negative regulator of tuft cells and provide insights into promotion of intestinal type 2 immune response to parasites through tRNA modifications.

Club cell CREB regulates the goblet cell transcriptional network and pro-mucin effects of IL-1B.

Introduction: Club cells are precursors for mucus-producing goblet cells. Interleukin 1β (IL-1B) is an inflammatory mediator with pro-mucin activities that increases the number of mucus-producing goblet cells. IL-1B-mediated mucin production in alveolar adenocarcinoma cells requires activation of the cAMP response element-binding protein (CREB). Whether the pro-mucin activities of IL-1B require club cell CREB is unknown. Methods: We challenged male mice with conditional loss of club cell Creb1 and wild type littermates with intra-airway IL-1B or vehicle. Secondarily, we studied human "club cell-like" H322 cells. Results: IL-1B increased whole lung mRNA of secreted (Mucin 5ac, Mucin 5b) and tethered (Mucin 1, Mucin 4) mucins independent of genotype. However, loss of club cell Creb1 increased whole lung mRNA of member RAS oncogene family (Rab3D), decreased mRNA of the muscarinic receptor 3 (M3R) and prevented IL-1B mediated increases in purinergic receptor P2Y, (P2ry2) mRNA. IL-1B increased the density of goblet cells containing neutral mucins in wildtype mice but not in mice with loss of club cell Creb1. These findings suggested that club cell Creb1 regulated mucin secretion. Loss of club cell Creb1 also prevented IL-1B-mediated impairments in airway mechanics. Four days of pharmacologic CREB inhibition in H322 cells increased mRNA abundance of forkhead box A2 (FOXA2), a repressor of goblet cell expansion, and decreased mRNA expression of SAM pointed domain containing ETS transcription factor (SPDEF), a driver of goblet cell expansion. Chromatin immunoprecipitation demonstrated that CREB directly bound to the promoter region of FOXA2, but not to the promoter region of SPDEF. Treatment of H322 cells with IL-1B increased cAMP levels, providing a direct link between IL-1B and CREB signaling. Conclusion: Our findings suggest that club cell Creb1 regulates the pro-mucin properties of IL-1B through pathways likely involving FOXA2.

Activating transcription factor 5 (ATF5) controls intestinal tuft and goblet cell expansion upon succinate-induced type 2 immune responses in mice.

Intestinal tuft cells, a chemosensory cell type in mucosal epithelia that secrete interleukin (IL)-25, play a pivotal role in type 2 immune responses triggered by parasitic infections. Tuft cell-derived IL-25 activates type 2 innate lymphoid cells (ILC2) to secrete IL-13, which, in turn, acts on intestinal stem or transient amplifying cells to expand tuft cells themselves and mucus-secreting goblet cells. However, the molecular mechanisms of tuft cell differentiation under type 2 immune responses remain unclear. The present study investigated the effects of the deletion of activating transcription factor 5 (ATF5) on the type 2 immune response triggered by succinate (a metabolite of parasites) in mice. ATF5 mRNAs were expressed in the small intestine, and the loss of the ATF5 gene did not affect the gross morphology of the tissue or the basal differentiation of epithelial cell subtypes. Succinate induced marked increases in tuft and goblet cell numbers in the ATF5-deficient ileum. Tuft cells in the ATF5-deficient ileum are assumed to be a subtype of intestinal tuft cells (Tuft-2 cells) marked by the transcription factor Spib. Exogenous IL-25 induced similar increases in tuft and goblet cell numbers in wild-type and ATF5-deficient ilea. IL-13 at a submaximal dose enhanced tuft cell differentiation more in ATF5-deficient than in wild-type intestinal organoids. These results indicate that the loss of ATF5 enhanced the tuft cell-ILC2 type 2 immune response circuit by promoting tuft cell differentiation in the small intestine, suggesting its novel regulatory role in immune responses against parasitic infections.

Experimental study of the mechanism of induction of conjunctival goblet cell hyperexpression using CHIR-99021 in vitro

A component of the tear film, mucin is produced by conjunctival goblet cells and is crucial to preserving the tear film's stability. Severe thermal burns, chemical burns, and severe ocular surface diseases can cause extensive damage to the conjunctiva, destroy the secretory function of goblet cells, and affect the stability of the tear film and integrity of the ocular surface. Currently, the expansion efficiency of goblet cells in vitro is low. In this study, we observed that rabbit conjunctival epithelial cells exhibited dense colony morphology after stimulation with the Wnt/β-catenin signaling pathway activator CHIR-99021 and promoted the differentiation of conjunctival goblet cells and the expression of its specific marker Muc5ac, among which the best induction effect was observed after 72 h in vitro culture with 5 μmol/L CHIR-99021. Under optimal culture conditions, CHIR-99021 increased the expression levels of the Wnt/β-catenin signaling pathway factors Frzb, β-catenin, SAM pointed domain containing ETS transcription factor, and glycogen synthase kinase-3β and the levels of the Notch signaling pathway factors Notch1 and Krüppel-like factor 4 while decreasing the expression levels of Jagged-1 and Hes1. The expression level of ABCG2, a marker of epithelial stem cells, was raised to keep rabbit conjunctival epithelial cells from self-renewing. Our study showed that CHIR-99021 stimulation successfully activated the Wnt/β-catenin signaling pathway and conjunctival goblet cell differentiation was stimulated, in which the Notch signaling pathway played a combined role. Those results provide a novel idea for the expansion of goblet cells in vitro.

Dynamic states of cervical epithelia during pregnancy and epithelial barrier disruption

The cervical epithelium undergoes changes in proliferation, differentiation, and function that are critical to ensure fertility and maintain pregnancy. Here, we identify cervical epithelial subtypes in non-pregnant, pregnant, and in labor mice using single-cell transcriptome and spatial analysis. We identify heterogeneous subpopulations of epithelia displaying spatial and temporal specificity. Notably in pregnancy, two goblet cell subtypes are present in the most luminal layers with one goblet population expanding earlier in pregnancy than the other goblet population. The goblet populations express novel protective factors and distinct mucosal networks. Single-cell analysis in a model of cervical epithelial barrier disruption indicates untimely basal cell proliferation precedes the expansion of goblet cells with diminished mucosal integrity. These data demonstrate how the cervical epithelium undergoes continuous remodeling to maintain dynamic states of homeostasis in pregnancy and labor, and provide a framework to understand perturbations in epithelial health that increase the risk of premature birth.

Intestinal Epithelial Digestive, Transport, and Barrier Protein Expression Is Increased in Environmental Enteric Dysfunction

Environmental enteric dysfunction (EED) is characterized by malabsorption and diarrhea that result in irreversible deficits in physical and intellectual growth. We sought to define the expression of transport and tight junction proteins by quantitative analysis of duodenal biopsies from patients with EED. Biopsies from Pakistani children with confirmed EED diagnoses were compared to those from age-matched North American healthy controls, patients with celiac disease, and patients with nonceliac disease with villous atrophy or intraepithelial lymphocytosis. Expression of brush border digestive and transport proteins and paracellular (tight junction) proteins was assessed by quantitative multiplex immunofluorescence microscopy. EED was characterized by partial villous atrophy and marked intraepithelial lymphocytosis. Epithelial proliferation and enteroendocrine, tuft, and Paneth cell numbers were unchanged, but there was significant goblet cell expansion in EED biopsies. Expression of proteins involved in nutrient and water absorption and that of the basolateral Cl− transport protein NKCC1 were also increased in EED. Finally, the barrier–forming tight junction protein claudin-4 (CLDN4) was significantly upregulated in EED, particularly within villous enterocytes. In contrast, expression of CFTR, CLDN2, CLDN15, JAM-A, occludin, ZO-1, and E-cadherin was unchanged. Upregulation of a barrier–forming tight junction protein and brush border and basolateral membrane proteins that support nutrient and water transport in EED is paradoxical, as their increased expression would be expected to be correlated with increased intestinal barrier function and enhanced absorption, respectively. These data suggest that EED activates adaptive intestinal epithelial responses to enhance nutrient absorption but that these changes are insufficient to restore health.

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

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

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

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

Up-regulation of gasdermin C in mouse small intestine is associated with lytic cell death in enterocytes in worm-induced type 2 immunity

"Taste-like" tuft cells in the intestine trigger type 2 immunity in response to worm infection. The secretion of interleukin-13 (IL-13) from type 2 innate lymphoid cells (ILC2) represents a key step in the tuft cell-ILC2 cell-intestinal epithelial cell circuit that drives the clearance of worms from the gut via type 2 immune responses. Hallmark features of type 2 responses include tissue remodeling, such as tuft and goblet cell expansion, and villus atrophy, yet it remains unclear if additional molecular changes in the gut epithelium facilitate the clearance of worms from the gut. Using gut organoids, we demonstrated that IL-4 and IL-13, two type 2 cytokines with similar functions, not only induced the classical type 2 responses (e.g., tuft cell expansion) but also drastically up-regulated the expression of gasdermin C genes (Gsdmcs). Using an in vivo worm-induced type 2 immunity model, we confirmed the up-regulation of Gsdmcs in Nippostrongylus brasiliensis-infected wild-type C57BL/6 mice. Consistent with gasdermin family members being principal effectors of pyroptosis, overexpression of Gsdmc2 in human embryonic kidney 293 (HEK293) cells triggered pyroptosis and lytic cell death. Moreover, in intestinal organoids treated with IL-4 or IL-13, or in wild-type mice infected with N. brasiliensis, lytic cell death increased, which may account for villus atrophy observed in worm-infected mice. Thus, we propose that the up-regulated Gsdmc family may be major effectors for type 2 responses in the gut and that Gsdmc-mediated pyroptosis may provide a conduit for the release of antiparasitic factors from enterocytes to facilitate the clearance of worms.

Tnfaip8 Regulates Intestinal Stemness and Terminal Differentiation of Secretory Progenitors

The intestine is a dynamic environment that relies on constant self-renewal of the intestinal epithelium to maintain homeostasis. Maintenance of stem cell populations as well as coordinated cell fate determination is necessary for coordinating intestinal function and regeneration. Tumor necrosis factor-alpha-induced protein 8 (TIPE0) is a regulator of phosphoinositide 3-kinase (PI3K)-mediated signaling through the sequestration of membranous PIP2. The PI3K/protein kinase B (Akt)/ꞵ-catenin pathway regulates proliferation in the intestine in conjunction with Wnt signalling. We recently found that loss of Tipe0 leads to hyperactivation of microbiome-mediated Akt/ꞵ-catenin signaling in the intestine and a single-cell RNA sequencing survey of Tipe0-/- small intestine found that Tipe0-/- intestinal cells were shifted towards an undifferentiated state, with the notable exception of goblet cells, which were expanded.Here, we performed more in depth analysis of our existing sc-RNASeq data set to better characterize the changes in intestinal differentiation seen with loss of TIPE0, using pseudo-time based approaches. We hypothesized that various transcription factors/epigenetic regulators would be differentially expressed in the Tipe0-/- intestine, underlying the expansion of goblet cells we had previously seen. In these studies, we confirmed that loss of Tipe0 causes a shift towards an undifferentiated state with an increase in Lgr5+ stem cells and transitional enterocytes and a consequent decrease in terminally differentiated enterocytes with the exception of goblet cells which were also enriched. Additionally, in this process we identified three possible novel regulators of terminal cell fate decisions in the intestinal secretory lineage: Nupr1, Kdm4a, and Gatad1. We propose that these novel regulators drive epigenetic changes involved in goblet cell (Nupr1) or tuft cell (Kdm4a and Gatad1) fate commitment and that TIPE0 may play a role in orchestrating terminal differentiation, likely through its regulation of homeostatic Akt/ꞵ-catenin signaling.

Sprouty2 limits intestinal tuft and goblet cell numbers through GSK3\u03b2-mediated restriction of epithelial IL-33

Dynamic regulation of intestinal cell differentiation is crucial for both homeostasis and the response to injury or inflammation. Sprouty2, an intracellular signaling regulator, controls pathways including PI3K and MAPKs that are implicated in differentiation and are dysregulated in inflammatory bowel disease. Here, we ask whether Sprouty2 controls secretory cell differentiation and the response to colitis. We report that colonic epithelial Sprouty2 deletion leads to expanded tuft and goblet cell populations. Sprouty2 loss induces PI3K/Akt signaling, leading to GSK3β inhibition and epithelial interleukin (IL)-33 expression. In vivo, this results in increased stromal IL-13+ cells. IL-13 in turn induces tuft and goblet cell expansion in vitro and in vivo. Sprouty2 is downregulated by acute inflammation; this appears to be a protective response, as VillinCre;Sprouty2F/F mice are resistant to DSS colitis. In contrast, Sprouty2 is elevated in chronic colitis and in colons of inflammatory bowel disease patients, suggesting that this protective epithelial-stromal signaling mechanism is lost in disease.

Lactobacillus Protects Against S. Typhimurium-Induced Intestinal Inflammation by Determining the Fate of Epithelial Proliferation and Differentiation.

The influence of the intestinal microbiota, such as Lactobacillus, on the intestinal mucosa, particularly intestinal stem cells, remains incompletely understood. In this study, mice and intestinal organoids are used to explore the regulatory effect of Lactobacillus on the proliferation and differentiation of intestinal epithelial cells. This study demonstrates that S. typhimurium causes intestinal epithelial damage and affected growth of intestinal organoids. S. typhimurium also colonizes the intestine and then causes pathological changes to the intestinal epithelium, intestinal inflammation, and even death. However, L. acidophilus alleviates damage to intestinal organoids, increases the survival ratio of mice infected with S. typhimurium, and reduces tumor necrosis factor-α (TNF-α) secretion. Moreover, L. acidophilus affects the differentiation of epithelial cells through inhibition of the excessive expansion of goblet cells and Paneth cells induced by S. typhimurium to avoid over-exhaustion. Finally, it is also demonstrated that L. acidophilus ameliorates overactivation of Wnt/β-catenin pathway by Salmonella, depending on the contact with toll-like receptor 2 (TLR2), to affect the proliferation of the intestinal epithelium. This study demonstrates that L. acidophilus protects the intestinal mucosa against S. typhimurium infection through not only the inhibition of pathogen invasion but also determination of the fate of the intestinal epithelium.

Lactobacillus Modulates Intestinal Epithelial Proliferation to Alleviate S. Typhimurium Induced Mucosal Barrier Damage Through Wnt/β-Catenin Pathway

The influence of intestinal microbiota, such as Lactobacillus, on intestinal mucosa, especially in the proliferation of intestinal stem cells, are still incompletely understood. In this study, intestinal organoids and mice were used to explore proliferation effect of Lactobacillus on intestinal epithelial through modulation of Wnt/βcatenin pathway. The study demonstrated that L. acidophilus increased survival ratio of mice infected with S. typhimurium, as well as reduced TNF-α secretion and S. typhimurium colonization in feces. S. typhimurium infection caused crypt hyperplasia with enhancement of PCNA cells and crypt length remarkably with over-expression of β-catenin, which was also remitted by L. acidophilus. Similar protective effects of L. acidophilus on inhibiting intestinal inflammation and crypt hyperplasia induced by S. typhimurium was also observed in intestinal organoids model by alleviating TNF-α secretion and β-catenin over-expression. S. typhimurium infection induced hyperplasia of UEA-1 goblet cells and Lysozyme Paneth cells both in mice and organoids to defend against pathogen invasion through exploiting TLR2 and TLR4. However, L. acidophilus ameliorated over-activation of TLRs and Wnt/β-catenin pathway to impair excessive expansion of goblet cells and Paneth cells induced by S. typhimurium. Our study implied that L. acidophilus protected intestinal mucosa against S. typhimurium infection by moderate modulating Wnt/β-catenin pathways to affect intestinal epithelial proliferation. Funding Statement: This study was supported by the National Natural Science Foundation of China (31502024), National Key Research and Development Program of China (2018YFD0500600), Jiangsu Agriculture Science and Technology Innovation Fund (JASTIF, CX[15]1066) and PAPD. Declaration of Interests: There are no conflicts of interest (financial, professional or personal) related to this manuscript. Ethics Approval Statement: All animal experiments were carried out in strict accordance with the regulations in the Guide for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People's Republic of China. All efforts were made to minimize suffering. The animal protocol was approved by the University of Nanjing Agriculture University Committee on Animal Resources Committee.

Dietary fiber sources and non-starch polysaccharide-degrading enzymes modify mucin expression and the immune profile of the swine ileum.

Due to their complex chemical and physical properties, the effects and mechanisms of action of natural sources of dietary fiber on the intestine are unclear. Pigs are commonly fed high-fiber diets to reduce production costs and non-starch polysaccharide (NSP)-degrading enzymes have been used to increase fiber digestibility. We evaluated the expression of mucin 2 (MUC2), presence of goblet cells, and ileal immune profile of pigs housed individually for 28 days and fed either a low fiber diet based on corn-soybean meal (CSB, n = 9), or two high fiber diets formulated adding 40% corn distillers’ dried grains with solubles (DDGS, n = 9) or 30% wheat middlings (WM, n = 9) to CSB-based diet. Pigs were also fed those diets supplemented with a NSP enzymes mix (E) of xylanase, β-glucanase, mannanase, and galactosidase (n = 8, 10, and 9 for CSB+E, DDGS+E and WM+E, respectively). Feeding DDGS and WM diets increased ileal MUC2 expression compared with CSB diet, and this effect was reversed by the addition of enzymes. There were no differences in abundance of goblet cells among treatments. In general, enzyme supplementation increased gene expression and concentrations of IL-1β, and reduced the concentrations of IL-4, IL-17A and IL-11. The effects of diet-induced cytokines on modulating intestinal MUC2 were assessed in vitro by treating mouse and swine enteroids with 1 ng/ml of IL-4 and IL-1β. In accordance with previous studies, treatment with Il-4 induced Muc2 and expansion of goblet cells in mouse enteroids. However, swine enteroids did not change MUC2 expression or number of goblet cells when treated with IL-4 or IL-1β. Our results suggest that mucin and immune profile are regulated by diet in the swine intestine, but by mechanisms different to mouse, emphasizing the need for using appropriate models to study responses to dietary fiber in swine.

Dual-specificity phosphatase 6 deletion protects the colonic epithelium against inflammation and promotes both proliferation and tumorigenesis.

The Ras/mitogen‐activated protein kinase (MAPK) pathway controls fundamental cellular processes such as proliferation, differentiation, and apoptosis. The dual‐specificity phosphatase 6 (DUSP6) regulates cytoplasmic MAPK signaling by dephosphorylating and inactivating extracellular signal‐regulated kinase (ERK1/2) MAPK. To determine the role of DUSP6 in the maintenance of intestinal homeostasis, we characterized the intestinal epithelial phenotype of Dusp6 knockout (KO) mice under normal, oncogenic, and proinflammatory conditions. Our results show that loss of Dusp6 increased crypt depth and epithelial cell proliferation without altering colonic architecture. Crypt regeneration capacity was also enhanced, as revealed by ex vivo Dusp6 KO organoid cultures. Additionally, loss of Dusp6 induced goblet cell expansion without affecting enteroendocrine and absorptive cell differentiation. Our data also demonstrate that Dusp6 KO mice were protected from acute dextran sulfate sodium‐induced colitis, as opposed to wild‐type mice. In addition, Dusp6 gene deletion markedly enhanced tumor load in Apc Min/+ mice. Decreased DUSP6 expression by RNA interference in HT29 colorectal cancer cells enhanced ERK1/2 activation levels and promoted both anchorage‐independent growth in soft agar as well as invasion through Matrigel. Finally, DUSP6 mRNA expression in human colorectal tumors was decreased in advanced stage tumors compared with paired normal tissues. These results demonstrate that DUSP6 phosphatase, by controlling ERK1/2 activation, regulates colonic inflammatory responses, and protects the intestinal epithelium against oncogenic stress.

Mechanistic de-risking using 3D organoids: addressing human relevance of rat goblet cell expansion

Mechanistic de-risking using 3D organoids: addressing human relevance of rat goblet cell expansion

Activation of intestinal tuft cell-expressed Sucnr1 triggers type 2 immunity in the mouse small intestine

The hallmark features of type 2 mucosal immunity include intestinal tuft and goblet cell expansion initiated by tuft cell activation. How infectious agents that induce type 2 mucosal immunity are detected by tuft cells is unknown. Published microarray analysis suggested that succinate receptor 1 (Sucnr1) is specifically expressed in tuft cells. Thus, we hypothesized that the succinate-Sucnr1 axis may be utilized by tuft cells to detect certain infectious agents. Here we confirmed that Sucnr1 is specifically expressed in intestinal tuft cells but not in other types of intestinal epithelial cells, and demonstrated that dietary succinate induces tuft and goblet cell hyperplasia via Sucnr1 and the tuft cell-expressed chemosensory signaling elements gustducin and Trpm5. Conventional mice with a genetic Sucnr1 deficiency (Sucnr1-/-) showed diminished immune responses to treatment with polyethylene glycol and streptomycin, which are known to enhance microbiota-derived succinate, but responded normally to inoculation with the parasitic worm Nippostrongylus brasiliensis that also produces succinate. Thus, Sucnr1 is required for microbiota-induced but not for a generalized worm-induced type 2 immunity.

Histopathological study of common carp (Cyprinus carpio) fed aflatoxin-contaminated diets

This study aimed to evaluate the effects of aflatoxin-contaminated diet on histopathological alterations of the gill, liver, kidney and intestine tissues in common carp. Fish were randomly distributed into 15 tanks, i.e. in five experimental groups; (I) control fed with normal diet without solvent and aflatoxin, (II) positive control received feed with only solvent, and (III-V) fed on diets containing 0.5, 0.7 and 1.4 mg kg -1 of aflatoxin, respectively. After 21-days, 12 fish per treatment were randomly caught, anesthetized and euthanized. Then, histological sections of the tissues were prepared. The main aflatoxicosis symptoms in the gills were fusion and disorganisation of the secondary gill lamellae, shortening of the secondary lamellae, inflammation of mucous membranes, and exfoliation of the gill epithelium. Liver of the infected fish indicated cloudy swelling of hepatocytes, cellular hypertrophy, formation of vacuoles in the cytoplasm, and necrosis of liver parenchyma. Expansion of Bowman’s space, necrosis of urinary tract, exfoliation and degeneration of the urinary tract epithelium, expansion of the urinary lumen and dilation of the urinary space were observed symptoms in the kidney. Changes in the intestine of the aflatoxin-treated fish were; expansion of goblet cells, necrosis of mucous layers, exfoliation of the mucous epithelium, and bleeding in the intestinal wall. The results indicates that feeding common carp with diets contaminated with aflatoxin, even in low concentrations (≤ 1.4 mg kg -1 feed) can cause histopathological damages and disturb their physiological balance.

Artificial Polymeric Scaffolds as Extracellular Matrix Substitutes for Autologous Conjunctival Goblet Cell Expansion.

PurposeWe fabricated and investigated polymeric scaffolds that can substitute for the conjunctival extracellular matrix to provide a substrate for autologous expansion of human conjunctival goblet cells in culture.MethodsWe fabricated two hydrogels and two silk films: (1) recombinant human collagen (RHC) hydrogel, (2) recombinant human collagen 2-methacryloylxyethyl phosphorylcholine (RHC-MPC) hydrogel, (3) arginine-glycine-aspartic acid (RGD) modified silk, and (4) poly-D-lysine (PDL) coated silk, and four electrospun scaffolds: (1) collagen, (2) poly(acrylic acid) (PAA), (3) poly(caprolactone) (PCL), and (4) poly(vinyl alcohol) (PVA). Coverslips and polyethylene terephthalate (PET) were used for comparison. Human conjunctival explants were cultured on scaffolds for 9 to 15 days. Cell viability, outgrowth area, and the percentage of cells expressing markers for stratified squamous epithelial cells (cytokeratin 4) and goblet cells (cytokeratin 7) were determined.ResultsMost of cells grown on all scaffolds were viable except for PCL in which only 3.6 ± 2.2% of the cells were viable. No cells attached to PVA scaffold. The outgrowth was greatest on PDL-silk and PET. Outgrowth was smallest on PCL. All cells were CK7-positive on RHC-MPC while 84.7 ± 6.9% of cells expressed CK7 on PDL-silk. For PCL, 87.10 ± 3.17% of cells were CK7-positive compared to PET where 67.10 ± 12.08% of cells were CK7-positive cells.ConclusionsBiopolymer substrates in the form of hydrogels and silk films provided for better adherence, proliferation, and differentiation than the electrospun scaffolds and could be used for conjunctival goblet cell expansion for eventual transplantation once undifferentiated and stratified squamous cells are included. Useful polymer scaffold design characteristics have emerged from this study.

Type 3 muscarinic receptors contribute to intestinal mucosal homeostasis and clearance of Nippostrongylus brasiliensis through induction of TH2 cytokines.

Despite increased appreciation for the role of nicotinic receptors in the modulation of and response to inflammation, the contribution of muscarinic receptors to mucosal homeostasis, clearance of enteric pathogens, and modulation of immune cell function remains relatively undefined. Uninfected and Nippostrongylus brasiliensis-infected wild-type and type 3 muscarinic receptor (M3R)-deficient (Chrm3(-/-)) mice were studied to determine the contribution of M3R to mucosal homeostasis as well as host defense against the TH2-eliciting enteric nematode N. brasiliensis Intestinal permeability and expression of TH1/TH17 cytokines were increased in uninfected Chrm3(-/-) small intestine. Notably, in Chrm3(-/-) mice infected with N. brasiliensis, small intestinal upregulation of TH2 cytokines was attenuated and nematode clearance was delayed. In Chrm3(-/-) mice, TH2-dependent changes in small intestinal function including smooth muscle hypercontractility, increased epithelial permeability, decreased epithelial secretion and absorption, and goblet cell expansion were absent despite N. brasiliensis infection. These findings identify an important role for M3R in host defense and clearance of N. brasiliensis, and support the expanding role of cholinergic muscarinic receptors in maintaining mucosal homeostasis.