The role of goblet cells and mucus in intestinal homeostasis.

The intestinal tract faces numerous challenges that require several layers of defence. The tight epithelium forms a physical barrier that is further protected by a mucus layer, which provides various site-specific protective functions. Mucus is produced by goblet cells, and as a result of single-cell RNA sequencing identifying novel goblet cell subpopulations, our understanding of their various contributions to intestinal homeostasis has improved. Goblet cells not only produce mucus but also are intimately linked to the immune system. Mucus and goblet cell development is tightly regulated during early life and synchronized with microbial colonization. Dysregulation of the developing mucus systems and goblet cells has been associated with infectious and inflammatory conditions and predisposition to chronic disease later in life. Dysfunctional mucus and altered goblet cell profiles are associated with inflammatory conditions in which some mucus system impairments precede inflammation, indicating a role in pathogenesis. In this Review, we present an overview of the current understanding of the role of goblet cells and the mucus layer in maintaining intestinal health during steady-state and how alterations to these systems contribute to inflammatory and infectious disease.

Epithelial GPR35 protects from Citrobacter rodentium infection by preserving goblet cells and mucosal barrier integrity.

Goblet cells secrete mucin to create a protective mucus layer against invasive bacterial infection and are therefore essential for maintaining intestinal health. However, the molecular pathways that regulate goblet cell function remain largely unknown. Although GPR35 is highly expressed in colonic epithelial cells, its importance in promoting the epithelial barrier is unclear. In this study, we show that epithelial Gpr35 plays a critical role in goblet cell function. In mice, cell-type-specific deletion of Gpr35 in epithelial cells but not in macrophages results in goblet cell depletion and dysbiosis, rendering these animals more susceptible to Citrobacter rodentium infection. Mechanistically, scRNA-seq analysis indicates that signaling of epithelial Gpr35 is essential to maintain normal pyroptosis levels in goblet cells. Our work shows that the epithelial presence of Gpr35 is a critical element for the function of goblet cell-mediated symbiosis between host and microbiota.

Mucus threads from surface goblet cells clear particles from the airways.

BACKGROUND: The mucociliary clearance system driven by beating cilia protects the airways from inhaled microbes and particles. Large particles are cleared by mucus bundles made in submucosal glands by parallel linear polymers of the MUC5B mucins. However, the structural organization and function of the mucus generated in surface goblet cells are poorly understood. METHODS: The origin and characteristics of different mucus structures were studied on live tissue explants from newborn wild-type (WT), cystic fibrosis transmembrane conductance regulator (CFTR) deficient (CF) piglets and weaned pig airways using video microscopy, Airyscan imaging and electron microscopy. Bronchoscopy was performed in juvenile pigs in vivo. RESULTS: We have identified a distinct mucus formation secreted from the surface goblet cells with a diameter less than two micrometer. This type of mucus was named mucus threads. With time mucus threads gathered into larger mucus assemblies, efficiently collecting particles. The previously observed Alcian blue stained mucus bundles were around 10 times thicker than the threads. Together the mucus bundles, mucus assemblies and mucus threads cleared the pig trachea from particles. CONCLUSIONS: These results demonstrate that normal airway mucus is more complex and has a more variable structural organization and function than was previously understood. These observations emphasize the importance of studying young objects to understand the function of a non-compromised lung.

Development and Functional Properties of Intestinal Mucus Layer in Poultry.

Intestinal mucus plays important roles in protecting the epithelial surfaces against pathogens, supporting the colonization with commensal bacteria, maintaining an appropriate environment for digestion, as well as facilitating nutrient transport from the lumen to the underlying epithelium. The mucus layer in the poultry gut is produced and preserved by mucin-secreting goblet cells that rapidly develop and mature after hatch as a response to external stimuli including environmental factors, intestinal microbiota as well as dietary factors. The ontogenetic development of goblet cells affects the mucin composition and secretion, causing an alteration in the physicochemical properties of the mucus layer. The intestinal mucus prevents the invasion of pathogens to the epithelium by its antibacterial properties (e.g. ß-defensin, lysozyme, avidin and IgA) and creates a physical barrier with the ability to protect the epithelium from pathogens. Mucosal barrier is the first line of innate defense in the gastrointestinal tract. This barrier has a selective permeability that allows small particles and nutrients passing through. The structural components and functional properties of mucins have been reviewed extensively in humans and rodents, but it seems to be neglected in poultry. This review discusses the impact of age on development of goblet cells and their mucus production with relevance for the functional characteristics of mucus layer and its protective mechanism in the chicken's intestine. Dietary factors directly and indirectly (through modification of the gut bacteria and their metabolic activities) affect goblet cell proliferation and differentiation and can be used to manipulate mucosal integrity and dynamic. However, the mode of action and mechanisms behind these effects need to be studied further. As mucins resist to digestion processes, the sloughed mucins can be utilized by bacteria in the lower part of the gut and are considered as endogenous loss of protein and energy to animal. Hydrothermal processing of poultry feed may reduce this loss by reduction in mucus shedding into the lumen. Given the significance of this loss and the lack of precise data, this matter needs to be carefully investigated in the future and the nutritional strategies reducing this loss have to be defined better.

Hypoxia and heat stress affect epithelial integrity in a Caco-2/HT-29 co-culture.

Hypoxia and hyperthermia, which can be induced by high environmental temperature or strenuous exercise, are two common stressors that affect intestinal epithelial integrity and lead to multiple clinical symptoms. In this study, we developed an in-vitro intestinal monolayer model using two human colonic epithelial cell lines, Caco-2 and HT-29, co-cultured in Transwell inserts, and investigated the effects of heat treatment and/or hypoxia on the epithelial barrier function. The monolayer with a ratio of 9:1 (Caco-2:HT-29) showed high trans-epithelial electrical resistance (TEER), low Lucifer Yellow permeability and high mucin production. Hyperthermia and/or hypoxia exposure (2 h) triggered heat shock and oxidative stress responses. HSP-70 and HSF-1 protein levels were up-regulated by hyperthermia, which were further enhanced when hyperthermia was combined with hypoxia. Increased HIF-1α protein expression and Nrf2 nuclear translocation was only caused by hypoxia. Hyperthermia and/or hypoxia exposure disrupted the established monolayer by increasing paracellular permeability, decreasing ZO-1, claudin-3 and occludin protein/mRNA expression, while enhancing E-cadherin protein expression. Tight junction protein distribution in the monolayer was also modulated by the hyperthermia and/or hypoxia exposure. In addition, transcription levels of mucin genes, MUC-2 and MUC-5AC, were increased after 2 h of hyperthermia and/or hypoxia exposure. In conclusion, this Caco-2/HT-29 cell model is valid and effective for studying detrimental effects of hyperthermia and/or hypoxia on intestinal barrier function and related heat shock and oxidative stress pathways and can be used to investigate possible interventions to reverse hyperthermia and/or hypoxia-induced intestinal epithelial injury.

An intercrypt subpopulation of goblet cells is essential for colonic mucus barrier function.

The intestinal mucus layer, an important element of epithelial protection, is produced by goblet cells. Intestinal goblet cells are assumed to be a homogeneous cell type. In this study, however, we delineated their specific gene and protein expression profiles and identified several distinct goblet cell populations that form two differentiation trajectories. One distinct subtype, the intercrypt goblet cells (icGCs), located at the colonic luminal surface, produced mucus with properties that differed from the mucus secreted by crypt-residing goblet cells. Mice with defective icGCs had increased sensitivity to chemically induced colitis and manifested spontaneous colitis with age. Furthermore, alterations in mucus and reduced numbers of icGCs were observed in patients with both active and remissive ulcerative colitis, which highlights the importance of icGCs in maintaining functional protection of the epithelium.

Reduced Neurog3 Gene Dosage Shifts Enteroendocrine Progenitor Towards Goblet Cell Lineage in the Mouse Intestine.

BACKGROUND & AIMS: Transient expression of Neurog3 commits intestinal secretory progenitors to become enteroendocrine-biased progenitors and hence drive enteroendocrine differentiation. Loss of Neurog3 in mouse resulted in the depletion of intestinal enteroendocrine cells (EECs) and an increase in goblet cells. Earlier studies in developing mouse pancreas identified a role of Neurog3 gene dosage in endocrine and exocrine cell fate allocation. We aimed to determine whether Neurog3 gene dosage controls fate choice of enteroendocrine progenitors. METHODS: We acquired mutant Neurog3 reporter mice carrying 2, 1, or null Neurog3 alleles to study Neurog3 gene dosage effect by lineage tracing. Cell types arising from Neurog3+ progenitors were determined by immunohistochemistry using antibodies against intestinal lineage-specific markers. RNA sequencing of sorted Neurog3+/+, Neurog3+/-, or bulk intestinal cells were performed and differentially expressed genes were analyzed. RESULTS: We identified 2731 genes enriched in sorted Neurog3+/+-derived cells in the Neurog3+/+EYFP mouse intestine when compared with bulk duodenum epithelial cells. In the intestine of Neurog3+/-EGFP heterozygous mouse, we observed a 63% decrease in EEC numbers. Many Neurog3-derived cells stained for goblet marker Mucin 2. RNA sequencing of sorted Neurog3+/- cells uncovered enriched expression of genes characteristic for both goblet and enteroendocrine cells, indicating the mixed lineages arose from Neurog3+ progenitors. Consistent with this hypothesis, deletion of both Neurog3 alleles resulted in the total absence of EECs. All Neurog3+-derived cells stained for Mucin 2. CONCLUSIONS: We identified that the fate of Neurog3+ enteroendocrine progenitors is dependent on Neurog3 gene dosage. High Neurog3 gene dosage enforces the commitment of secretory progenitors to an EE lineage, while constraining their goblet cell lineage potential. Transcriptome profiling data was deposited to Gene Ontology omnibus, accession number: GSE149203.

Bioelectric Responses of Conjunctival Goblet Cells to Dry Eye: Impact of Ion Channels on Exocytotic Function and Viability.

How ion channels impact the response of the ocular surface to dry eye is only beginning to be explored. Here, we review recent progress and provide new experimental data clarifying the exocytosis-altering actions of ion channels in conjunctival goblet cells whose release of tear-stabilizing mucin is a key adaptive response to the pre-ocular hyperosmolarity that characterizes dry eye. Patch-clamp recordings of goblet cells located in freshly excised rat conjunctiva reveal that these mucin-releasing cells respond to sustained hyperosmolarity by sequentially activating their ATP-sensitive potassium (KATP), nonspecific cation (NSC), voltage-gated calcium (VGCC), and P2X7 channels; each of which modulates exocytosis. Based on these and other new findings, we now identify four stages in the bioelectric response of conjunctival goblet cells to extracellular hyperosmolarity. To better characterize these stages, we report that high-resolution membrane capacitance (Cm) measurements of the exocytotic activity of single goblet cells demonstrate that the replenishment of mucin-filled granules after neural-evoked exocytosis is a multi-hour process, which VGCCs markedly accelerate. Yet, we also discovered that VGCC activation is high-risk since hyperosmotic-induced goblet cell death is boosted. With dry eye treatments being far from optimal, elucidating the physiologic and pathobiologic impact of the KATP/NSC/VGCC/P2X7 pathway provides a new opportunity to identify novel therapeutic strategies.

Mucins in Intestinal Mucosal Defense and Inflammation: Learning From Clinical and Experimental Studies.

Throughout the gastrointestinal (GI) tract, a distinct mucus layer composed of highly glycosylated proteins called mucins plays an essential role in providing lubrication for the passage of food, participating in cell signaling pathways and protecting the host epithelium from commensal microorganisms and invading pathogens, as well as toxins and other environmental irritants. These mucins can be broadly classified into either secreted gel-forming mucins, those that provide the structural backbone for the mucus barrier, or transmembrane mucins, those that form the glycocalyx layer covering the underlying epithelial cells. Goblet cells dispersed among the intestinal epithelial cells are chiefly responsible for the synthesis and secretion of mucins within the gut and are heavily influenced by interactions with the immune system. Evidence from both clinical and animal studies have indicated that several GI conditions, including inflammatory bowel disease (IBD), colorectal cancer, and numerous enteric infections are accompanied by considerable changes in mucin quality and quantity. These changes include, but are not limited to, impaired goblet cell function, synthesis dysregulation, and altered post-translational modifications. The current review aims to highlight the structural and functional features as well as the production and immunological regulation of mucins and the impact these key elements have within the context of barrier function and host defense in intestinal inflammation.

Refined sorghum flours precooked by extrusion enhance the integrity of the colonic mucosa barrier and promote a hepatic antioxidant environment in growing Wistar rats.

The effects of precooked-refined sorghum flour consumption on antioxidant status, lipid profile, and colonic and bone health were evaluated. Twenty-four male Wistar rats were fed with control diet (C), or red or white precooked-refined sorghum based diets (SD) for 60 days. The intake of SD was lower than that of C, but the efficiency of all diets was similar. Rats fed with SD showed lower feces excretion, cecal pH and enzyme activities (ß-glucosidase, ß-glucuronidase and mucinase) than C. White SD improved intestinal architecture, cell proliferation and apoptosis, upregulated ZO1 and occludin tight junction proteins and stimulated goblet cell differentiation, enhancing the integrity of the mucosa barrier in both proximal and distal colonic mucosa in a better way than red SD. Consumption of SD significantly decreased serum triglyceride levels compared with the C diet. The mineral content of the right femur was not different among diets. The liver enzyme activities (superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase) did not show differences among diets. Liver reducing power and reduced glutathione/oxidize glutathione ratio were higher for animals consuming SD than C. It can be concluded that the consumption of precooked refined sorghum flours still has beneficial effects for health, mainly at the colonic level, despite the lower phenolics and fibre contents of refined flours with respect to whole grain flours.

An indisputable role of NHE8 in mucosal protection.

The loss of the intestinal Na+/H+ exchanger isoform 8 (NHE8) results in an ulcerative colitis-like condition with reduction of mucin production and dysbiosis, indicating that NHE8 plays an important role in intestinal mucosal protection. The aim of this study was to investigate the potential rebalance of the altered microbiota community of NHE8-deficient mice via fecal microbiota transplantation (FMT) and feeding probiotic VSL#3. We also aimed to stimulate mucin production by sodium butyrate administration via enema. Data from 16S rRNA sequencing showed that loss of NHE8 contributes to colonic microbial dysbiosis with reduction of butyrate-producing bacteria. FMT increased bacterial adhesion in the colon in NHE8 knockout (NHE8KO) mice. Periodic-acid Schiff reagent (PAS) stain and quantitative PCR showed no changes in mucin production during FMT. In mice treated with the probiotic VSL#3, a reduction of Lactobacillus and segmented filamentous bacteria (SFB) in NHE8KO mouse colon was detected and an increase in goblet cell theca was observed. In NHE8KO mice receiving sodium butyrate (NaB), 1 mM NaB stimulated Muc2 expression without changing goblet cell theca, but 10 mM NaB induced a significant reduction of goblet cell theca without altering Muc2 expression. Furthermore, 5 mM and 10 mM NaB-treated HT29-MTX cells displayed increased apoptosis, while 0.5 mM NaB stimulated Muc2 gene expression. These data showed that loss of NHE8 leads to dysbiosis with reduction of butyrate-producing bacteria and FMT and VSL#3 failed to rebalance the microbiota in NHE8KO mice. Therefore, FMT, VSL#3, and NaB are not able to restore mucin production in the absence of NHE8 in the intestine.NEW & NOTEWORTHY Loss of Na+/H+ exchanger isoform 8 (NHE8), a Slc9 family of exchanger that contributes to sodium uptake, cell volume regulation, and intracellular pH homeostasis, resulted in dysbiosis with reduction of butyrate-producing bacteria and decrease of Muc2 production in the intestine in mice. Introducing fecal microbiota transplantation (FMT) and VSL#3 in NHE8 knockout (NHE8KO) mice failed to rebalance the microbiota in these mice. Furthermore, administration of FMT, VSL#3, and sodium butyrate was unable to restore mucin production in the absence of NHE8 in the intestine.

Calcineurin Inhibitor Voclosporin Preserves Corneal Barrier and Conjunctival Goblet Cells in Experimental Dry Eye.

Objective: The purpose of this study was to evaluate the potential of voclosporin (VOS) in preventing goblet cell (GC) loss and modulating interferon-gamma (IFN-γ) producing CD4+ T cells in the mouse desiccating stress (DS) dry eye model. Methods: Mice were subjected to DS and treated topically with vehicle, VOS, or cyclosporine A as a treatment control. Corneal barrier function was evaluated after 5 and conjunctival GC density after 10 days of desiccation. CD4+ T cells were isolated from ocular surface draining lymph nodes of dry eye donor mice and adoptively transferred into immune deficient RAG1-/- mice from which tears and conjunctiva were collected for the evaluation of inflammatory cytokines/chemokines and GC density. Results: Compared to the vehicle-treated group, VOS was significantly better in preserving corneal barrier function and preventing DS-induced conjunctival GC loss. CD4+ T cells from VOS treated dry eye donors caused less conjunctival GC loss than vehicle and suppressed expression of IFN-γ signature genes to a similar extent and transforming growth factor-beta to a greater extent than cyclosporine in adoptive transfer recipients. Conclusion: These findings suggest that VOS preserves corneal barrier function and conjunctival GCs and suppresses IFN-γ producing CD4+ T cells in experimental dry eye.

Rotavirus infection induces glycan availability to promote ileum-specific changes in the microbiome aiding rotavirus virulence.

Multiple studies have identified changes within the gut microbiome in response to diarrheal-inducing bacterial pathogens. However, examination of the microbiome in response to viral pathogens remains understudied. Compounding this, many studies use fecal samples to assess microbiome composition; which may not accurately mirror changes within the small intestine, the primary site for most enteric virus infections. As a result, the functional significance of small intestinal microbiome shifts during infection is not well defined. To address these gaps, rotavirus-infected neonatal mice were examined for changes in bacterial community dynamics, host gene expression, and tissue recovery during infection. Profiling bacterial communities using 16S rRNA sequencing suggested significant and distinct changes in ileal communities in response to rotavirus infection, with no significant changes for other gastrointestinal (GI) compartments. At 1-d post-infection, we observed a loss in Lactobacillus species from the ileum, but an increase in Bacteroides and Akkermansia, both of which exhibit mucin-digesting capabilities. Concomitant with the bacterial community shifts, we observed a loss of mucin-filled goblet cells in the small intestine at d 1, with recovery occurring by d 3. Rotavirus infection of mucin-producing cell lines and human intestinal enteroids (HIEs) stimulated release of stored mucin granules, similar to in vivo findings. In vitro, incubation of mucins with Bacteroides or Akkermansia members resulted in significant glycan degradation, which altered the binding capacity of rotavirus in silico and in vitro. Taken together, these data suggest that the response to and recovery from rotavirus-diarrhea is unique between sub-compartments of the GI tract and may be influenced by mucin-degrading microbes.

The Immunological Basis of Dry Eye Disease and Current Topical Treatment Options.

Homeostasis of the lacrimal functional unit is needed to ensure a well-regulated ocular immune response comprising innate and adaptive phases. When the ocular immune system is excessively stimulated and/or immunoregulatory mechanisms are disrupted, the balance between innate and adaptive phases is dysregulated and chronic ocular surface inflammation can result, leading to chronic dry eye disease (DED). According to the Tear Film and Ocular Surface Society Dry Eye Workshop II definition, DED is a multifactorial disorder of the ocular surface characterized by impairment and loss of tear homeostasis (hyperosmolarity), ocular discomfort or pain, and neurosensory abnormalities. Dysregulated ocular immune responses result in ocular surface damage, which is a further contributing factor to DED pathology. Several therapeutics are available to break the vicious circle of DED and prevent chronic disease and progression, including immunosuppressive agents (steroids) and immunomodulators (cyclosporine and lifitegrast). Given the chronic inflammatory nature of DED, each of these agents is commonly used in clinical practice. In this study, we review the immunopathology of DED and the molecular and cellular actions of current topical DED therapeutics to inform clinical decision making.

Melatonin controls microbiota in colitis by goblet cell differentiation and antimicrobial peptide production through Toll-like receptor 4 signalling.

Microbial dysbiosis has long been postulated to be associated with the pathogenesis of inflammatory bowel disease (IBD). Although evidence supporting the anti-colitic effects of melatonin have been accumulating, it is not clear how melatonin affects the microbiota. Herein, we investigated the effects of melatonin on the microbiome in colitis and identified involvement of Toll-like receptor (TLR) 4 signalling in the effects. Melatonin improved dextran sulfate sodium (DSS)-induced colitis and reverted microbial dysbiosis in wild-type (WT) mice but not in TLR4 knockout (KO) mice. Induction of goblet cells was observed with melatonin administration, which was accompanied by suppression of Il1b and Il17a and induction of melatonin receptor and Reg3ß, an antimicrobial peptide (AMP) against Gram-negative bacteria. In vitro, melatonin treatment of HT-29 intestinal epithelial cells promotes mucin and wound healing and inhibits growth of Escherichia coli. Herein, we showed that melatonin significantly increases goblet cells, Reg3ß, and the ratio of Firmicutes to Bacteriodetes by suppressing Gram-negative bacteria through TLR4 signalling. Our study suggests that sensing of bacteria through TLR4 and regulation of bacteria through altered goblet cells and AMPs is involved in the anti-colitic effects of melatonin. Melatonin may have use in therapeutics for IBD.

Vasoactive intestinal peptide regulates ileal goblet cell production in mice.

Innervation of the intestinal mucosa has gained more attention with demonstrations of tuft and enteroendocrine cell innervation. However, the role(s) these fibers play in maintaining the epithelial and mucus barriers are still poorly understood. This study therefore examines the proximity of mouse ileal goblet cells to neuronal fibers, and the regulation of goblet cell production by vasoactive intestinal peptide (VIP). An organotypic intestinal slice model that maintains the cellular diversity of the intestinal wall ex vivo was used. An ex vivo copper-free click-reaction to label glycosaminoglycans was used to identify goblet cells. Pharmacological treatment of slices was used to assess the influence of VIP receptor antagonism on goblet cell production and neuronal fiber proximity. Goblet cells were counted and shown to have at least one peripherin immunoreactive fiber within 3 µm of the cell, 51% of the time. Treatment with a VIP receptor type I and II antagonist (VPACa) resulted in an increase in the percentage of goblet cells with peripherin fibers. Pharmacological treatments altered goblet cell counts in intestinal crypts and villi, with tetrodotoxin and VPACa substantially decreasing goblet cell counts. When cultured with 5-Ethynyl-2'-deoxyuridine (EdU) as an indicator of cell proliferation, colocalization of labeled goblet cells and EdU in ileal crypts was decreased by 77% when treated with VPACa. This study demonstrates a close relationship of intestinal goblet cells to neuronal fibers. By using organotypic slices from mouse ileum, vasoactive intestinal peptide receptor regulation of gut wall goblet cell production was revealed.

SIRT2 Contributes to the Regulation of Intestinal Cell Proliferation and Differentiation.

BACKGROUND AND AIMS: Intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. Disruption of this homeostasis is associated with disorders such as inflammatory bowel disease (IBD). We investigated the role of Sirtuin 2 (SIRT2), a NAD-dependent protein deacetylase, in intestinal epithelial cell (IEC) proliferation and differentiation and the mechanism by which SIRT2 contributes to maintenance of intestinal cell homeostasis. METHODS: IECs were collected from SIRT2-deficient mice and patients with IBD. Expression of SIRT2, differentiation markers (mucin2, intestinal alkaline phosphatase, villin, Na,K-ATPase, and lysozyme) and Wnt target genes (EPHB2, AXIN2, and cyclin D1) was determined by western blot, real-time RT-PCR, or immunohistochemical (IHC) staining. IECs were treated with TNF or transfected with siRNA targeting SIRT2. Proliferation was determined by villus height and crypt depth, and Ki67 and cyclin D1 IHC staining. For studies using organoids, intestinal crypts were isolated. RESULTS: Increased SIRT2 expression was localized to the more differentiated region of the intestine. In contrast, SIRT2 deficiency impaired proliferation and differentiation and altered stemness in the small intestinal epithelium ex vivo and in vivo. SIRT2-deficient mice showed decreased intestinal enterocyte and goblet cell differentiation but increased the Paneth cell lineage and increased proliferation of IECs. Moreover, we found that SIRT2 inhibits Wnt/ß-catenin signaling, which critically regulates IEC proliferation and differentiation. Consistent with a distinct role for SIRT2 in maintenance of gut homeostasis, intestinal mucosa from IBD patients exhibited decreased SIRT2 expression. CONCLUSION: We demonstrate that SIRT2, which is decreased in intestinal tissues from IBD patients, regulates Wnt-ß-catenin signaling and is important for maintenance of IEC proliferation and differentiation.

Identification of Key Pro-Survival Proteins in Isolated Colonic Goblet Cells of Winnie, a Murine Model of Spontaneous Colitis.

BACKGROUND: Accumulating evidence suggests that the goblet cell-derived mucin-2 (Muc2) is a major component of the immune system and that perturbations in Muc2 lead to an ulcerative colitis-like phenotype. The animal model Winnie carries a missense mutation in Muc2 that causes Muc2 misfolding, accumulation in goblet cells, and ER stress. Excessive ER stress is a hallmark of many diseases, including ulcerative colitis, cancer, diabetes and Parkinson's disease. However, rather than committing to cell death, which is the typical outcome of unresolved ER stress, Winnie goblet cells are characterized by hyperproliferation, suggesting additional regulation of this cellular stress response. METHODS: To elucidate the molecular mechanisms underlying ulcerative colitis in the Winnie model, we isolated goblet cells from Winnie and wild-type mice and used label-free quantitative proteomics and bioinformatics to understand the functional consequences of Muc2 misfolding and accumulation. RESULTS: A large number of changes were identified that highlight a dramatic reprogramming of energy production, including enhanced utilization of butyrate, a key energy source of colonic cells. A major finding was the marked upregulation of the coiled-coil-helix-coiled-coil-helix domain proteins Chchd2, Chchd3, and Chchd6. In particular, we identified and confirmed the upregulation and nuclear translocation of Chchd2, a protein known to inhibit oxidative stress induced apoptosis. CONCLUSIONS: This study is the first to apply proteome-level analysis to the preclinical Winnie model of ulcerative colitis. Identification of proteins and pathways affected in isolated Winnie goblet cells provides evidence for novel adaptive mechanisms underlying cell survival under conditions of chronic ER stress.

Human Colon-on-a-Chip Enables Continuous In Vitro Analysis of Colon Mucus Layer Accumulation and Physiology.

BACKGROUND & AIMS: The mucus layer in the human colon protects against commensal bacteria and pathogens, and defects in its unique bilayered structure contribute to intestinal disorders, such as ulcerative colitis. However, our understanding of colon physiology is limited by the lack of in vitro models that replicate human colonic mucus layer structure and function. Here, we investigated if combining organ-on-a-chip and organoid technologies can be leveraged to develop a human-relevant in vitro model of colon mucus physiology. METHODS: A human colon-on-a-chip (Colon Chip) microfluidic device lined by primary patient-derived colonic epithelial cells was used to recapitulate mucus bilayer formation, and to visualize mucus accumulation in living cultures noninvasively. RESULTS: The Colon Chip supports spontaneous goblet cell differentiation and accumulation of a mucus bilayer with impenetrable and penetrable layers, and a thickness similar to that observed in the human colon, while maintaining a subpopulation of proliferative epithelial cells. Live imaging of the mucus layer formation on-chip showed that stimulation of the colonic epithelium with prostaglandin E2, which is increased during inflammation, causes rapid mucus volume expansion via an Na-K-Cl cotransporter 1 ion channel-dependent increase in its hydration state, but no increase in de novo mucus secretion. CONCLUSIONS: This study shows the production of colonic mucus with a physiologically relevant bilayer structure in vitro, which can be analyzed in real time noninvasively. The Colon Chip may offer a new preclinical tool to analyze the role of mucus in human intestinal homeostasis as well as diseases, such as ulcerative colitis and cancer.