Infant milk formula, produced by membrane filtration, promotes mucus production in the upper small intestine of young pigs.

Human breast milk promotes maturation of the infant gastrointestinal barrier, including the promotion of mucus production. In the quest to produce next generation infant milk formula (IMF), we have produced IMF by membrane filtration (MEM-IMF). With a higher quantity of native whey protein, MEM-IMF more closely mimics human breast milk than IMF produced using conventional heat treatment (HT-IMF). After a 4-week dietary intervention in young pigs, animals fed a MEM-IMF diet had a higher number of goblet cells, acidic mucus and mucin-2 in the jejunum compared to pigs fed HT-IMF (P < 0.05). In the duodenum, MEM-IMF fed pigs had increased trypsin activity in the gut lumen, increased mRNA transcript levels of claudin 1 in the mucosal scrapings and increased lactase activity in brush border membrane vesicles than those pigs fed HT-IMF (P < 0.05). In conclusion, MEM-IMF is superior to HT-IMF in the promotion of mucus production in the young gut.

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.

Differentiation of intestinal stem cells toward goblet cells under systemic iron overload stress are associated with inhibition of Notch signaling pathway and ferroptosis.

Iron overload can lead to oxidative stress and intestinal damage and happens frequently during blood transfusions and iron supplementation. However, how iron overload influences intestinal mucosa remains unknown. Here, the aim of current study was to investigate the effects of iron overload on the proliferation and differentiation of intestinal stem cells (ISCs). An iron overload mouse model was established by intraperitoneal injection of 120 mg/kg body weight iron dextran once a fortnight for a duration of 12 weeks, and an iron overload enteroid model was produced by treatment with 3 mM or 10 mM of ferric ammonium citrate for 24 h. We found that iron overload caused damage to intestinal morphology with a 64 % reduction in villus height/crypt depth ratio, and microvilli injury in the duodenum. Iron overload mediated epithelial function by inhibiting the expression of nutrient transporters and enhancing the expression of secretory factors in the duodenum. Meanwhile, iron overload inhibited the proliferation of ISCs and regulated their differentiation into secretory mature cells, such as goblet cells, through inhibiting Notch signaling pathway both in mice and enteroid. Furthermore, iron overload caused oxidative stress and ferroptosis in intestinal epithelial cells. In addition, ferroptosis could also inhibit Notch signaling pathway, and affected the proliferation and differentiation of ISCs. These findings reveal the regulatory role of iron overload on the proliferation and differentiation of ISCs, providing a new insight into the internal mechanism of iron overload affecting intestinal health, and offering important theoretical basis for the scientific application of iron nutrition regulation.

Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP4 pathway activation in human colon organoids.

Understanding the mechanisms involved in colonic epithelial differentiation is key to unraveling the alterations causing inflammatory conditions and cancer. Organoid cultures provide an unique tool to address these questions but studies are scarce. We report a differentiation system toward enterocytes and goblet cells, the two major colonic epithelial cell lineages, using colon organoids generated from healthy tissue of colorectal cancer patients. Culture of these organoids in medium lacking stemness agents resulted in a modest ultrastructural differentiation phenotype with low-level expression of enterocyte (KLF4, KRT20, CA1, FABP2) and goblet cell (TFF2, TFF3, AGR2) lineage markers. BMP pathway activation through depletion of Noggin and addition of BMP4 resulted in enterocyte-biased differentiation. Contrarily, blockade of the Notch pathway using the γ-secretase inhibitor dibenzazepine (DBZ) favored goblet cell differentiation. Combination treatment with BMP4 and DBZ caused a balanced strong induction of both lineages. In contrast, colon tumor organoids responded poorly to BMP4 showing only weak signals of cell differentiation, and were unresponsive to DBZ. We also investigated the effects of 1α,25-dihydroxyvitamin D3 (calcitriol) on differentiation. Calcitriol attenuated the effects of BMP4 and DBZ on colon normal organoids, with reduced expression of differentiation genes and phenotype. Consistently, in normal organoids, calcitriol inhibited early signaling by BMP4 as assessed by reduction of the level of phospho-SMAD1/5/8. Our results show that BMP and Notch signaling play key roles in human colon stem cell differentiation to the enterocytic and goblet cell lineages and that calcitriol modulates these processes favoring stemness features.

Seihaito, a Kampo medicine, attenuates IL-13-induced mucus production and goblet cell metaplasia.

Goblet cell hyperplasia and increased mucus production are features of airway diseases, including asthma, and excess airway mucus often worsens these conditions. Even steroids are not uniformly effective in mucus production in severe asthma, and new therapeutic options are needed. Seihaito is a Japanese traditional medicine that is used clinically as an antitussive and expectorant. In the present study, we examined the effect of Seihaito on goblet cell differentiation and mucus production. In in vitro studies, using air-liquid interface culture of guinea-pig tracheal epithelial cells, Seihaito inhibited IL-13-induced proliferation of goblet cells and MUC5AC, a major component of mucus production. Seihaito suppressed goblet cell-specific gene expression, without changing ciliary cell-specific genes, suggesting that it inhibits goblet cell differentiation. In addition, Seihaito suppressed MUC5AC expression in cells transfected with SPDEF, a transcription factor activated by IL-13. Furthermore, Seihaito attenuated in vivo goblet cell proliferation and MUC5AC mRNA expression in IL-13-treated mouse lungs. Collectively, these findings demonstrated that Seihaito has an inhibitory effect on goblet cell differentiation and mucus production, which is at least partly due to the inhibition of SPDEF.

Coarse-grained modeling and dynamics tracking of nanoparticles diffusion in human gut mucus.

The gastrointestinal (GI) tract's mucus layer serves as a critical barrier and a mediator in drug nanoparticle delivery. The mucus layer's diverse molecular structures and spatial complexity complicates the mechanistic study of the diffusion dynamics of particulate materials. In response, we developed a bi-component coarse-grained mucus model, specifically tailored for the colorectal cancer environment, that contained the two most abundant glycoproteins in GI mucus: Muc2 and Muc5AC. This model demonstrated the effects of molecular composition and concentration on mucus pore size, a key determinant in the permeability of nanoparticles. Using this computational model, we investigated the diffusion rate of polyethylene glycol (PEG) coated nanoparticles, a widely used muco-penetrating nanoparticle. We validated our model with experimentally characterized mucus pore sizes and the diffusional coefficients of PEG-coated nanoparticles in the mucus collected from cultured human colorectal goblet cells. Machine learning fingerprints were then employed to provide a mechanistic understanding of nanoparticle diffusional behavior. We found that larger nanoparticles tended to be trapped in mucus over longer durations but exhibited more ballistic diffusion over shorter time spans. Through these discoveries, our model provides a promising platform to study pharmacokinetics in the GI mucus layer.

Intergenerational protective anti-gut commensal immunoglobulin G originates in early life.

Maternal immunoglobulins of the class G (IgGs) protect offspring from enteric infection, but when, where, and how these antibodies are physiologically generated and confer protection remains enigmatic. We found that circulating IgGs in adult mice preferentially bind early-life gut commensal bacteria over their own adult gut commensal bacteria. IgG-secreting plasma cells specific for early-life gut bacteria appear in the intestine soon after weaning, where they remain into adulthood. Manipulating exposure to gut bacteria or plasma cell development before, but not after, weaning reduced IgG-secreting plasma cells targeting early-life gut bacteria throughout life. Further, the development of this anti-gut commensal IgG response coincides with the early-life interval in which goblet cell-associated antigen passages (GAPs) are present in the colon. Offspring of dams "perturbed" by B cell ablation or reduced bacterial exposure in early life were more susceptible to enteric pathogen challenge. In contrast to current concepts, protective maternal IgGs targeted translocating gut commensals in the offspring, not the enteric pathogen. These early-life events affecting anti-commensal IgG production have intergenerational effects for protection of the offspring.

Sex drives colonic mucin sialylation in wild mice.

Mucin protein glycosylation is important in determining biological properties of mucus gels, which form protective barriers at mucosal surfaces of the body such as the intestine. Ecological factors including: age, sex, and diet can change mucus barrier properties by modulating mucin glycosylation. However, as our understanding stems from controlled laboratory studies in house mice, the combined influence of ecological factors on mucin glycosylation in real-world contexts remains limited. In this study, we used histological staining with 'Alcian Blue, Periodic Acid, Schiff's' and 'High-Iron diamine' to assess the acidic nature of mucins stored within goblet cells of the intestine, in a wild mouse population (Mus musculus). Using statistical models, we identified sex as among the most influential ecological factors determining the acidity of intestinal mucin glycans in wild mice. Our data from wild mice and experiments using laboratory mice suggest estrogen signalling associates with an increase in the relative abundance of sialylated mucins. Thus, estrogen signalling may underpin sex differences observed in the colonic mucus of wild and laboratory mice. These findings highlight the significant influence of ecological parameters on mucosal barrier sites and the complementary role of wild populations in augmenting standard laboratory studies in the advancement of mucus biology.

Evaluation of Ki-67, goblet cell and MUC2 mucin RNA expression in dogs with lymphoplasmacytic and granulomatous colitis.

Intestinal mucus barrier disruption may occur with chronic inflammatory enteropathies. The lack of studies evaluating mucus health in dogs with chronic colitis arises from inherent challenges with assessment of the intestinal mucus layer. It is therefore unknown if reduced goblet cell (GBC) numbers and/or mucin 2 (MUC2) expression, which are responsible for mucus production and secretion, correlate with inflammation severity in dogs with granulomatous colitis (GC) or lymphocytic-plasmacytic colitis (LPC). It is undetermined if Ki-67 immunoreactivity, which has been evaluated in dogs with small intestinal inflammation, similarly correlates to histologic severity in GC and LPC. Study objectives included comparing Ki-67 immunoreactivity, GBC population and MUC2 expression in dogs with GC, LPC and non-inflamed colon; and exploring the use of ribonucleic acid (RNAscope®) in-situ hybridization (ISH) to evaluate MUC2 expression in canine colon. Formalin-fixed endoscopic colonic biopsies were obtained from 48 dogs over an eight-year period. A blinded pathologist reviewed all biopsies. Dogs were classified into the GC (n=19), LPC (n=19) or no colitis (NC) (n=10) group based on final histopathological diagnosis. Ki-67 immunohistochemistry, Alcian-Blue/PAS staining to highlight GBCs, and RNAscope® ISH using customized canine MUC2-targeted probes were performed. At least five microscopic fields per dog were selected to measure Ki-67 labelling index (KI67%), GBC staining percentage (GBC%) and MUC2 expression (MUC2%) using image analysis software. Spearman's correlation coefficients were used to determine associations between World Small Animal Veterinary Association histologic score (WHS) and measured variables. Linear regression models were used to compare relationships between WHS with KI67%, GBC%, and MUC2%; and between GBC% and MUC2%. Median WHS was highest in dogs with GC. Median KI67% normalised to WHS was highest in the NC group (6.69%; range, 1.70-23.60%). Median GBC% did not correlate with colonic inflammation overall. Median MUC2% normalised to WHS in the NC group (10.02%; range, 3.05-39.09%) was two- and three-fold higher than in the GC and LPC groups respectively. With increased colonic inflammation, despite minimal changes in GBC% overall, MUC2 expression markedly declined in the LPC group (-27.4%; 95%-CI, -49.8, 5.9%) and mildly declined in the GC and NC groups. Granulomatous colitis and LPC likely involve different pathways regulating MUC2 expression. Decreased MUC2 gene expression is observed in dogs with chronic colitis compared to dogs without colonic signs. Changes in MUC2 expression appear influenced by GBC activity rather than quantity in GC and LPC.

GLP-1R signaling modulates colonic energy metabolism, goblet cell number and survival in the absence of gut microbiota.

OBJECTIVES: Gut microbiota increases energy availability through fermentation of dietary fibers to short-chain fatty acids in conventionally raised mice. Energy deficiency in germ-free (GF) mice increases glucagon-like peptide-1 (GLP-1) levels, which slows intestinal transit. To further analyze the role of GLP-1-mediated signaling in this model of energy deficiency, we re-derived mice lacking GLP-1 receptor (GLP-1R KO) as GF. METHODS: GLP-1R KO mice were rederived as GF through hysterectomy and monitored for 30 weeks. Mice were subjected to rescue experiments either through feeding an energy-rich diet or colonization with a normal cecal microbiota. Histology and intestinal function were assessed at different ages. Intestinal organoids were assessed to investigate stemness. RESULTS: Unexpectedly, 25% of GF GLP-1R KO mice died before 20 weeks of age, associated with enlarged ceca, increased cecal water content, increased colonic expression of apical ion transporters, reduced number of goblet cells and loss of colonic epithelial integrity. Colonocytes from GLP-1R KO mice were energy-deprived and exhibited increased ER-stress; mitochondrial fragmentation, increased oxygen levels and loss of stemness. Restoring colonic energy levels either by feeding a Western-style diet or colonization with a normal gut microbiota normalized gut phenotypes and prevented lethality. CONCLUSIONS: Our findings reveal a heretofore unrecognized role for GLP-1R signaling in the maintenance of colonic physiology and survival during energy deprivation.

REGγ Mitigates Radiation-Induced Enteritis by Preserving Mucin Secretion and Sustaining Microbiome Homeostasis.

Radiation-induced enteritis, a significant concern in abdominal radiation therapy, is associated closely with gut microbiota dysbiosis. The mucus layer plays a pivotal role in preventing the translocation of commensal and pathogenic microbes. Although significant expression of REGγ in intestinal epithelial cells is well established, its role in modulating the mucus layer and gut microbiota remains unknown. The current study revealed notable changes in gut microorganisms and metabolites in irradiated mice lacking REGγ, as compared to wild-type mice. Concomitant with gut microbiota dysbiosis, REGγ deficiency facilitated the infiltration of neutrophils and macrophages, thereby exacerbating intestinal inflammation after irradiation. Furthermore, fluorescence in situ hybridization assays unveiled an augmented proximity of bacteria to intestinal epithelial cells in REGγ knockout mice after irradiation. Mechanistically, deficiency of REGγ led to diminished goblet cell populations and reduced expression of key goblet cell markers, Muc2 and Tff3, observed in both murine models, minigut organoid systems and human intestinal goblet cells, indicating the intrinsic role of REGγ within goblet cells. Interestingly, although administration of broad-spectrum antibiotics did not alter the goblet cell numbers or mucin 2 (MUC2) secretion, it effectively attenuated inflammation levels in the ileum of irradiated REGγ absent mice, bringing them down to the wild-type levels. Collectively, these findings highlight the contribution of REGγ in counteracting radiation-triggered microbial imbalances and cell-autonomous regulation of mucin secretion.

Surfactant protein D prevents mucin overproduction in airway goblet cells via SIRPα.

Mucin overproduction is a common feature of chronic airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), and exacerbates their underlying respiratory condition. Surfactant protein D (SP-D) protects against airway diseases through modulation of immune reactions, but whether it also exerts direct effects on airway epithelial cells has remained unclear. Therefore, we sought to investigate the inhibitory role of SP-D on mucin production in airway epithelial cells. We prepared air-liquid interface (ALI) cultures of human primary bronchial epithelial cells (HBECs), which recapitulated a well-differentiated human airway epithelium. Benzo(a)pyrene (BaP), a key toxicant in cigarette smoke, induced mucin 5AC (MUC5AC) production in ALI-cultured HBECs, airway secretory cell lines, and airway epithelia of mice. Then, the protective effects of SP-D against the BaP-induced mucin overproduction were examined. BaP increased MUC5AC production in ALI cultures of HBECs, and this effect was attenuated by SP-D. SP-D also suppressed the BaP-induced phosphorylation of extracellular signal-regulated kinase (ERK) and MUC5AC expression in NCI-H292 goblet-like cells, but not in NCI-H441 club-like cells. Signal regulatory protein α (SIRPα) was found to be expressed in HBECs and NCI-H292 cells but absent in NCI-H441 cells. In NCI-H292 cells, SP-D activated SH2 domain-containing tyrosine phosphatase-1 (SHP-1), downstream of SIRPα, and knockdown of SIRPα abolished the suppressive effects of SP-D on BaP-induced ERK phosphorylation and MUC5AC production. Consistent with these in vitro findings, intratracheal instillation of SP-D prevented the BaP-induced phosphorylation of ERK and Muc5ac expression in airway epithelial cells in a mouse model. SP-D acts directly on airway epithelial cells to inhibit mucin secretion through ligation of SIRPα and SHP-1-mediated dephosphorylation of ERK. Targeting of SIRPα is therefore a potential new therapeutic approach to suppression of mucin hypersecretion in chronic airway diseases such as COPD and asthma.

Human apical-out nasal organoids reveal an essential role of matrix metalloproteinases in airway epithelial differentiation.

Extracellular matrix (ECM) assembly/disassembly is a critical regulator for airway epithelial development and remodeling. Airway organoid is widely used in respiratory research, yet there is limited study to indicate the roles and mechanisms of ECM organization in epithelial growth and differentiation by using in vitro organoid system. Moreover, most of current Matrigel-based airway organoids are in basal-out orientation where accessing the apical surface is challenging. We present a human apical-out airway organoid using a biochemically defined hybrid hydrogel system. During human nasal epithelial progenitor cells (hNEPCs) differentiation, the gel gradually degrade, leading to the organoid apical surfaces facing outward. The expression and activity of ECM-degrading enzymes, matrix metalloproteinases (MMP7, MMP9, MMP10 and MMP13) increases during organoid differentiation, where inhibition of MMPs significantly suppresses the normal ciliation, resulting in increased goblet cell proportion. Moreover, a decrease of MMPs is found in goblet cell hyperplastic epithelium in inflammatory mucosa. This system reveals essential roles of epithelial-derived MMPs on epithelial cell fate determination, and provides an applicable platform enabling further study for ECM in regulating airway development in health and diseases.

Activation of goblet-cell stress sensor IRE1ß is controlled by the mucin chaperone AGR2.

Intestinal goblet cells are secretory cells specialized in the production of mucins, and as such are challenged by the need for efficient protein folding. Goblet cells express Inositol-Requiring Enzyme-1ß (IRE1ß), a unique sensor in the unfolded protein response (UPR), which is part of an adaptive mechanism that regulates the demands of mucin production and secretion. However, how IRE1ß activity is tuned to mucus folding load remains unknown. We identified the disulfide isomerase and mucin chaperone AGR2 as a goblet cell-specific protein that crucially regulates IRE1ß-, but not IRE1α-mediated signaling. AGR2 binding to IRE1ß disrupts IRE1ß oligomerization, thereby blocking its downstream endonuclease activity. Depletion of endogenous AGR2 from goblet cells induces spontaneous IRE1ß activation, suggesting that alterations in AGR2 availability in the endoplasmic reticulum set the threshold for IRE1ß activation. We found that AGR2 mutants lacking their catalytic cysteine, or displaying the disease-associated mutation H117Y, were no longer able to dampen IRE1ß activity. Collectively, these results demonstrate that AGR2 is a central chaperone regulating the goblet cell UPR by acting as a rheostat of IRE1ß endonuclease activity.

The IRE1ß-mediated unfolded protein response is repressed by the chaperone AGR2 in mucin producing cells.

Effector mechanisms of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) are well-characterised, but how ER proteostasis is sensed is less well understood. Here, we exploited the beta isoform of the UPR transducer IRE1, that is specific to mucin-producing cells in order to gauge the relative regulatory roles of activating ligands and repressing chaperones of the specialised ER of goblet cells. Replacement of the stress-sensing luminal domain of endogenous IRE1α in CHO cells (normally expressing neither mucin nor IRE1ß) with the luminal domain of IRE1ß deregulated basal IRE1 activity. The mucin-specific chaperone AGR2 repressed IRE1 activity in cells expressing the domain-swapped IRE1ß/α chimera, but had no effect on IRE1α. Introduction of the goblet cell-specific client MUC2 reversed AGR2-mediated repression of the IRE1ß/α chimera. In vitro, AGR2 actively de-stabilised the IRE1ß luminal domain dimer and formed a reversible complex with the inactive monomer. These features of the IRE1ß-AGR2 couple suggest that active repression of IRE1ß by a specialised mucin chaperone subordinates IRE1 activity to a proteostatic challenge unique to goblet cells, a challenge that is otherwise poorly recognised by the pervasive UPR transducers.

Sphingosine kinase 1-specific inhibitor PF543 reduces goblet cell metaplasia of bronchial epithelium in an acute asthma model.

Sphingosine kinase 1 (SPHK1) has been shown to play a key role in the pathogenesis of asthma where SPHK1-generated sphingosine-1-phosphate (S1P) is known to mediate innate and adaptive immunity while promoting mast cell degranulation. Goblet cell metaplasia (GCM) contributes to airway obstruction in asthma and has been demonstrated in animal models. We investigated the role of PF543, a SPHK1-specific inhibitor, in preventing the pathogenesis of GCM using a murine (C57BL/6) model of allergen-induced acute asthma. Treatment with PF543 before triple allergen exposure (DRA: House dust mite, Ragweed pollen, and Aspergillus) reduced inflammation, eosinophilic response, and GCM followed by reduced airway hyperreactivity to intravenous methacholine. Furthermore, DRA exposure was associated with increased expression of SPHK1 in the airway epithelium which was reduced by PF543. DRA-induced reduction of acetylated α-tubulin in airway epithelium was associated with an increased expression of NOTCH2 and SPDEF which was prevented by PF543. In vitro studies using human primary airway epithelial cells showed that inhibition of SPHK1 using PF543 prevented an allergen-induced increase of both NOTCH2 and SPDEF. siRNA silencing of SPHK1 prevented the allergen-induced increase of both NOTCH2 and SPDEF. NOTCH2 silencing was associated with a reduction of SPDEF but not that of SPHK1 upon allergen exposure. Our studies demonstrate that inhibition of SPHK1 protected allergen-challenged airways by preventing GCM and airway hyperreactivity, associated with downregulation of the NOTCH2-SPDEF signaling pathway. This suggests a potential novel link between SPHK1, GCM, and airway remodeling in asthma.NEW & NOTEWORTHY The role of SPHK1-specific inhibitor, PF543, in preventing goblet cell metaplasia (GCM) and airway hyperreactivity (AHR) is established in an allergen-induced mouse model. This protection was associated with the downregulation of NOTCH2-SPDEF signaling pathway, suggesting a novel link between SPHK1, GCM, and AHR.

Human conjunctiva organoids to study ocular surface homeostasis and disease.

The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.

Effect of Nano-selenium on Biological Mechanism of Goblet Cells of the Small Intestine Within Laying Hen.

Dietary selenium intake within the normal physiological range is critical for various supporting biological functions. However, the effect of nano-selenium on biological mechanism of goblet cells associated with autophagy is largely unknown.The purpose of this study was to investigate the effect of nano-selenium on the mucosal immune-defense mechanism of goblet cells (GCs) in the small intestine of laying hens.The autophagy was determined by using specific markers. Nano-selenium-treated group of immunohistochemistry (IHC), immunofluorescence (IF), and western blotting (WB) results indicated the strong positive immune signaling of microtubule-associated light chain (LC3) within the mucosal surface of the small intestine. However, weak expression of LC3 was observed in the 3-methyladenine autophagy inhibitor (3-MA) group. IHC and IF staining results showed the opposite tendency for LC3 of sequestosome 1 (P62/SQSTM1). P62/SQSTM1 showed strong positive immune signaling within the mucosal surface of the small intestine of the 3-MAgroup, and weak immune signaling of P62/SQSTM1 in the nano-selenium-treated group. Moreover, pinpointing autophagy was involved in the mucosal production and enrichment of mucosal immunity of the GCs. The morphology and ultrastructure evidence showed that the mucus secretion of GCs was significantly increased after nano-selenium treatment confirmed by light and transmission electron microscopy. Besides that, immunostaining of IHC, IF and WB showed that autophagy enhanced the secretion of Mucin2 (Muc2) protein in nano-selenium-treated group. This work illustrates that the nano-selenium particle might enhance the mucosal immune-defense mechanism via the protective role of GCs for intestinal homeostasis through autophagy.

Reduced miR-513a-5p expression in COPD may regulate airway mucous cell hyperplasia through TFR1-dependent signaling.

Airway mucous cell metaplasia and mucous hypersecretion is one of the key characteristic pathophysiological status of chronic obstructive pulmonary disease (COPD). micro(mi)RNAs are acknowledged as non-encoding RNA molecules playing important roles in gene expression regulation. In this study, we searched the Gene Expression Omnibus (GEO) database for the differentially expressed miRNAs between COPD and non-COPD controls with bioinformatics analysis. Finally, we focused on miR-513a-5p and investigated the potential mechanism by which miR-513a-5p regulates airway mucous hypersecretion and goblet cell metaplasia. A dual-luciferase reporter assay was then showing that miR-513a-5p targeted the 3'-UTR of TFR1 and inhibited its expression in vitro. In vivo transfection demonstrated that TFR1 downregulation partially blocked MUC5AC hypersecretion and goblet cell hyperplasia in COPD model rats. In vitro study, CSE increased the intracellular expression and secretion of MUC5AC by BEAS-2B branchial epithelial cells in the BEAS-2B cell and THP-1 cell coculture system. Coculture with either miR-513a-5p mimic-pretreated or TFR1-deficient THP-1 cells attenuated intracellular MUC5AC expression in BEAS-2B cells exposed to CSE. ELISA demonstrated that transfection of TFR1 siRNA or pretreatment with miR-513a-5p mimic reduced the secretion of inflammatory factors that are responsible for airway goblet cell hyperplasia, such as IL-1ß, IL-13, and IL-17, by THP-1 cells after CSE stimulation. Our findings supported that miR-513a-5p/TFR1 signaling axis might activate macrophages as well as promote airway inflammation and airway mucous cell hyperplasia in COPD.

Sleep Deprivation Impairs Intestinal Mucosal Barrier by Activating Endoplasmic Reticulum Stress in Goblet Cells.

Sleep deficiency is associated with intestinal inflammatory conditions and is increasingly recognized as a public health concern worldwide. However, the effects of sleep deficiency on intestinal goblet cells (GCs), which play a major role in intestinal barrier formation, remain elusive. Herein, the effects of sleep deprivation on intestinal GCs were determined using a sleep-deprivation mouse model. Sleep deprivation impaired the intestinal mucosal barrier and decreased the expression of tight junction proteins. According to single-cell RNA sequencing and histologic assessments, sleep deprivation significantly reduced GC numbers and mucin protein levels in intestinal tissues. Furthermore, sleep deprivation initiated endoplasmic reticulum stress by activating transcription factor 6 and binding Ig protein. Treatment with melatonin, an endoplasmic reticulum stress regulator, significantly alleviated endoplasmic reticulum stress responses in intestinal GCs. In addition, melatonin increased the villus length, reduced the crypt depth, and restored intestinal barrier function in mice with sleep deprivation. Overall, the findings revealed that sleep deprivation could impair intestinal mucosal barrier integrity and GC function. Targeting endoplasmic reticulum stress could represent an ideal strategy for treating sleep deficiency-induced gastrointestinal disorders.