Fecal-adherent mucus is a non-invasive source of primary human MUC2 for structural and functional characterization in health and disease.

The O-glycoprotein Mucin-2 (MUC2) forms the protective colon mucus layer. While animal models have demonstrated the importance of Muc2, few studies have explored human MUC2 in similar depth. Recent studies have revealed that secreted MUC2 is bound to human feces. We hypothesized human fecal MUC2 (HF-MUC2) was accessible for purification and downstream structural and functional characterization. We tested this via histologic and quantitative imaging on human fecal sections; extraction from feces for proteomic and O-glycomic characterization; and functional studies via growth and metabolic assays in vitro. Quantitative imaging of solid fecal sections showed a continuous mucus layer of varying thickness along human fecal sections with barrier functions intact. Lectin profiling showed HF-MUC2 bound several lectins but was weak to absent for Ulex europaeus 1 (α1,2 fucose-binding) and Sambucus nigra agglutinin (α2,6 sialic acid-binding), and did not have obvious b1/b2 barrier layers. HF-MUC2 separated by electrophoresis showed high molecular weight glycoprotein bands (∼1-2 MDa). Proteomics and Western analysis confirmed the enrichment of MUC2 and potential MUC2-associated proteins in HF-MUC2 extracts. MUC2 O-glycomics revealed diverse fucosylation, moderate sialylation, and little sulfation versus porcine colonic MUC2 and murine fecal Muc2. O-glycans were functional and supported the growth of Bacteroides thetaiotaomicron (B. theta) and short-chain fatty acid (SCFA) production in vitro. MUC2 could be similarly analyzed from inflammatory bowel disease stools, which displayed an altered glycomic profile and differential growth and SCFA production by B. theta versus healthy samples. These studies describe a new non-invasive platform for human MUC2 characterization in health and disease.

The immune interactions of gut glycans and microbiota in health and disease.

The human digestive system harbors a vast diversity of commensal bacteria and maintains a symbiotic relationship with them. However, imbalances in the gut microbiota accompany various diseases, such as inflammatory bowel diseases (IBDs) and colorectal cancers (CRCs), which significantly impact the well-being of populations globally. Glycosylation of the mucus layer is a crucial factor that plays a critical role in maintaining the homeostatic environment in the gut. This review delves into how the gut microbiota, immune cells, and gut mucus layer work together to establish a balanced gut environment. Specifically, the role of glycosylation in regulating immune cell responses and mucus metabolism in this process is examined.

Age-related decline in goblet cell numbers and mucin content of the human colon: Implications for lower bowel functions in the elderly.

BACKGROUND & AIMS: Older people experience a greater incidence of lower bowel disorders, including constipation. Causes can include factors associated with growing older, such as use of medications or disease, but compounded by degenerative changes within the bowel wall. It has been suggested that the latter is exacerbated by loss of an effective mucosal barrier to luminal contents. In human colon, little is known about the impact of ageing on key components of this barrier, namely the goblet cells and mucin content. METHODS: Changes in the number of goblet cells and density of mucin content were investigated in macroscopically normal human ascending (AC; n = 13) and descending (DC; n = 14) colon from elderly (≥ 67 years) and younger adults (60 years and below). Samples were serially sectioned and stained for haematoxylin and eosin to assess tissue morphology, and alcian blue periodic acid Schiff (ABPAS) and MUC-2 antibody to identify goblet cells producing mucins. New procedures in visualization and identification of goblet cells and mucin contents were employed to ensure unbiased counting and densitometric analysis. RESULTS: Compared with the younger adults, the numbers of goblet cells per crypt were significantly lower in the elderly AC (72 ± 1.2 vs 51 ± 0.5) and DC (75 ± 2.6 vs. 54 ± 1.9), although this reduction did not reach statistical significance when assessed per mucosal area (AC: P = 0.068; DC: P = 0.096). In both regions from the elderly, numerous empty vesicles (normally containing mucins) were observed, and some areas of epithelium were devoid of goblet cells. Thus, the density of mucin content per unit mucosal area were significantly reduced with age. CONCLUSIONS: Ageing could result in a reduced number of goblet cells and development of degenerative changes in mucin production. Together, these have implications for the mucus barrier function in the colon of elderly individuals.

Evaluation the effect of dietary vitamin E, sesamin and thymoquinone bioactive compounds on immunological response, intestinal traits and MUC-2 gene expression in broiler Japanese quails (Coturnix japonica).

The current study was performed to determine the effect of dietary vitamin E, sesamin and thymoquinone bioactive lignans derived from sesame and black seed on immunological response, intestinal traits and Mucin2 gene expression in broiler quails. Three hundred and fifty (one days-old) quails were allotted to seven dietary treatments with five replicates as an experimental randomized design study. Treatments were basal diet as a control, control +100 and +200 mg of vitamin E, sesamin and thymoquinone per each kg of diet respectively. At 35 d of age, two quails from each pen were chosen, weighted, slaughtered, eviscerated and lymphoid organ relative weights were measured. Anti-body titers against Newcastle disease (ND), Sheep red blood cell (SRBC), and infectious bronchitis virus (IBV) and Avian influenza (AI) vaccination were determined. The serum activities of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and serum antioxidant activates such as superoxide dismutase (SOD),glutathione peroxidase(GPX), catalase (CAT) and total antioxidant capacity (TAC) were examined. The cell mediated immunity by dinitrochlorobenzene (DNCB) and phytohemagglutinin (PHA) challenges were assessed. The microflora populations of ileum, morphological traits of jejunum and mucin2 gene expression were analyzed. Data showed that the lymphoid organ (thymus, spleen and Bursa) relative weights and antibody titer against HI, AI, SRBC and IB vaccination were increased compared to the control (p ≤ 0.05). Serum activities of ALP, ALT and AST were decreased under influences of dietary treatments (p ≤ 0.05). The serum antioxidant activates of GPX,SOD,CAT and TAC were increased and Increasing in mean skin thickness after DNCB challenge and decrease wing web swelling response to PHA mitojen injection were observed (p ≤ 0.05). Salmonella enterica, E-coli and Coliforms colonies were decrease and Lactobacillus colonies increased instead (p ≤ 0.05). The villus height and surface, crypt depth and goblet cells density were increased compared to the control (p ≤ 0.05). The expression of MUC2 gene increased under influnces of vitamin E, sesamin and thymoquinone supplemented diets (p ≤ 0.05).

Mucin-Phenotype and Expression of the Protein V-Set and Immunoglobulin Domain Containing 1 (VSIG1): New Insights into Gastric Carcinogenesis.

In gastric cancer (GC), intestinal metaplasia (IM) is a common precursor lesion, but its relationship to the MUC2/MUC5AC/CDX2 axis is not completely understood. Although V-set and immunoglobulin domain containing 1 (VSIG1) is supposed to be a specific marker for gastric mucosa and GC, respectively, no data about its relationship with IM or mucin phenotype have been published. The aim of our study was to explore the possible linkage between IM and these four molecules. The clinicopathological features of 60 randomly selected GCs were examined in association with VSIG1, MUC2, MUC5AC and CDX2. Two online database platforms were also used to establish the transcription factors (TFs) network involved in MUC2/MUC5AC/CDX2 cascade. IM was more frequently encountered in females (11/16 cases) and in patients below 60 years old (10/16 cases). Poorly differentiated (G3) carcinomas tended to show a loss of CDX2 (27/33 cases) but not of MUC2 and MUC5AC. MUC5AC and CDX2 were lost in parallel with the depth of invasion of the pT4 stage (28/35 and 29/35 cases), while an advanced Dukes-MAC-like stage was only correlated with CDX2 and VSIG1 loss (20/37 and 30/37 cases). VSIG1 was directly correlated with MUC5AC (p = 0.04) as an indicator of gastric phenotype. MUC2-negative cases showed a propensity towards lymphatic invasion (37/40 cases) and distant metastases, while CDX2-negative cases tended to associate with hematogenous dissemination (30/40 cases). Regarding the molecular network, only 3 of the 19 TFs involved in this carcinogenic cascade (SP1, RELA, NFKB1) interacted with all targeted genes. In GC, VSIG1 can be considered an indicator of gastric phenotype carcinomas, where carcinogenesis is mainly driven by MUC5AC. Although infrequently encountered in GC, CDX2 positivity might indicate a locally advanced stage and risk for vascular invasion, especially in tumors developed against the background of IM. The loss of VSIG1 indicates a risk for lymph node metastases.

Multivariate Prognostic Models for Patients with Stages I and Ii Colon Carcinoma: a Strobe-Compliant Retrospective Cohort Study.

Background: Colorectal cancer is the most frequent gastrointestinal malignancy worldwide. The value of adjuvant treatment is controversial in Stages I and II. Objective: The aim of this study was to construct post-operative prognostic models applicable to patients with stages I-II colon carcinoma (CC). Methods: This is a retrospective cohort study of patients with Stage I-II CC treated over a 25-year period. Exposure was defined as clinical, histopathological, and immunohistochemical factors (including CDX2 and MUC2 expression). Patients were randomly allocated to either a "modeling set" or a "validation set". Factors associated with recurrence, disease-free survival (DFS), and overall survival (OS) were defined in the "modeling set". Their performances were tested in the "validation set". Results: From a total of 556 recruited patients, 339 (61%) were allocated to the "modeling set" and 217 (39%) to the "validation set". Three models explaining recurrence, DFS, and OS were described. Tumor location in the left colon (Hazards ratio [HR] = 1.57; 95% Confidence interval [CI] 0.99-2.48), lymphocyte (HR = 0.46; 96% CI 0.27-0.88) and monocyte (HR = 0.99; 95% CI 0.99-1) counts, neutrophil/platelet ratio (HR = 1.3; 95% CI 0.74-2.3, and HR = 2.3; 95% CI 1.3-4.1; for second and third category, respectively), albumin/monocyte ratio (HR = 0.43; 95% CI 0.21-0.87), and microscopic residual disease after surgery (HR = 8.7; 95% CI 3.1-24) were independently associated with OS. T classification and expression of CDX2 and/or MUC2 were not independently associated with recurrence or prognosis. Conclusion: These models are simple and readily available, and distinguish the risk and prognosis in patients with CC stages I and II; these models require cheaper processes than the use of more sophisticated molecular biology techniques. They may guide either the need for adjuvant therapy versus post-operative surveillance only, as well as aid in the design of clinical trials.

[Mucins expression in gastric cancer: connection of MUC status with clinical, morphological and prognostic characteristics of the tumor]. / Ekspressiya mutsinov v rake zheludka: svyaz' MUC-statusa s kliniko-morfologicheskimi i prognosticheskimi kharakteristikami opukholi.

OBJECTIVE: Clarification of the prognostic value and relationship of MUC-phenotypes of gastric cancer with clinical and morphological parameters. MATERIAL AND METHODS: Surgical material from 310 patients with a verified diagnosis of gastric cancer was studied. Samples were immunohistochemically stained with antibodies to MUC2, CD10, MUC5AC. The results were compared with clinical and morphological characteristics of gastric cancer and patient survival data. RESULTS: The MUC-null and MUC-mix groups significantly differ in the prevalence of subtotal/total tumors from the MUC-I group (p=0.022 and p=0.007, respectively), where there are significantly fewer such tumors. Tubular tumors were more common in the MUC-null group compared to the MUC-G (p=0.026) and MUC-mix (p=0.006) groups, and there were fewer cases with the presence of "signet-ring" cells in the MUC-null group (p=0.000). When studying the discohesive histological type, the literature data on smaller tumor sizes and a lower frequency of lymph node metastasis for MUC-G status were not confirmed, but a more frequent proximal localization of MUC-I tumors was found (p=0.003). No statistically significant differences in survival were found in the analysis of the total sample. Differences in survival were found only in discohesive cancers, where the best survival was recorded for the MUC-null group, and the worst for the MUC-mix group (p=0.022). MUC status is not an independent predictor of gastric cancer (HR=1.662, p=0.093). CONCLUSION: Between tumors with different MUC statuses, there were differences in localization and belonging to individual histological types. Significant differences in survival were found only for discohesive cancers with MUC-null and MUC-mix statuses. Separation of gastric cancers according to MUC status may have only limited predictive value in selected histological forms of cancer.

Oxidized phospholipids cause changes in jejunum mucus that induce dysbiosis and systemic inflammation.

We previously reported that adding a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to a Western diet (WD) ameliorated systemic inflammation. To determine the mechanism(s) responsible for these observations, Ldlr-/- mice were fed chow, a WD, or WD plus Tg6F. We found that a WD altered the taxonomic composition of bacteria in jejunum mucus. For example, Akkermansia muciniphila virtually disappeared, while overall bacteria numbers and lipopolysaccharide (LPS) levels increased. In addition, gut permeability increased, as did the content of reactive oxygen species and oxidized phospholipids in jejunum mucus in WD-fed mice. Moreover, gene expression in the jejunum decreased for multiple peptides and proteins that are secreted into the mucous layer of the jejunum that act to limit bacteria numbers and their interaction with enterocytes including regenerating islet-derived proteins, defensins, mucin 2, surfactant A, and apoA-I. Following WD, gene expression also decreased for Il36γ, Il23, and Il22, cytokines critical for antimicrobial activity. WD decreased expression of both Atoh1 and Gfi1, genes required for the formation of goblet and Paneth cells, and immunohistochemistry revealed decreased numbers of goblet and Paneth cells. Adding Tg6F ameliorated these WD-mediated changes. Adding oxidized phospholipids ex vivo to the jejunum from mice fed a chow diet reproduced the changes in gene expression in vivo that occurred when the mice were fed WD and were prevented with addition of 6F peptide. We conclude that Tg6F ameliorates the WD-mediated increase in oxidized phospholipids that cause changes in jejunum mucus, which induce dysbiosis and systemic inflammation.

The IgGFc-binding protein FCGBP is secreted with all GDPH sequences cleaved but maintained by interfragment disulfide bonds.

Mucus forms an important protective barrier that minimizes bacterial contact with the colonic epithelium. Intestinal mucus is organized in a complex network with several specific proteins, including the mucin-2 (MUC2) and the abundant IgGFc-binding protein, FCGBP. FCGBP is expressed in all intestinal goblet cells and is secreted into the mucus. It is comprised of repeated von Willebrand D (vWD) domain assemblies, most of which have a GDPH amino acid sequence that can be autocatalytically cleaved, as previously observed in the mucins MUC2 and mucin-5AC. However, the functions of FCGBP in the mucus are not understood. We show that all vWD domains of FCGBP with a GDPH sequence are cleaved and that these cleavages occur early during biosynthesis in the endoplasmic reticulum. All cleaved fragments, however, remain connected via a disulfide bond within each vWD domain. This cleavage generates a C-terminal-reactive Asp-anhydride that could react with other molecules, such as MUC2, but this was not observed. Quantitative analyses by MS showed that FCGBP was mainly soluble in chaotropic solutions, whereas MUC2 was insoluble, and most of the secreted FCGBP was not covalently bound to MUC2. Although FCGBP has been suggested to bind immunoglobulin G, we were unable to reproduce this binding in vitro using purified proteins. In conclusion, while the function of FCGBP is still unknown, our results suggest that it does not contribute to covalent crosslinking in the mucus, nor incorporate immunoglobulin G into mucus, instead the single disulfide bond linking each fragment could mediate controlled dissociation.

Fusobacteriumnucleatum Adheres to Clostridioides difficile via the RadD Adhesin to Enhance Biofilm Formation in Intestinal Mucus.

BACKGROUND & AIMS: Although Clostridioides difficile infection (CDI) is known to involve the disruption of the gut microbiota, little is understood regarding how mucus-associated microbes interact with C difficile. We hypothesized that select mucus-associated bacteria would promote C difficile colonization and biofilm formation. METHODS: To create a model of the human intestinal mucus layer and gut microbiota, we used bioreactors inoculated with healthy human feces, treated with clindamycin and infected with C difficile with the addition of human MUC2-coated coverslips. RESULTS: C difficile was found to colonize and form biofilms on MUC2-coated coverslips, and 16S rRNA sequencing showed a unique biofilm profile with substantial cocolonization with Fusobacterium species. Consistent with our bioreactor data, publicly available data sets and patient stool samples showed that a subset of patients with C difficile infection harbored high levels of Fusobacterium species. We observed colocalization of C difficile and F nucleatum in an aggregation assay using adult patients and stool of pediatric patients with inflammatory bowel disease and in tissue sections of patients with CDI. C difficile strains were found to coaggregate with F nucleatum subspecies in vitro; an effect that was inhibited by blocking or mutating the adhesin RadD on Fusobacterium and removal of flagella on C difficile. Aggregation was shown to be unique between F nucleatum and C difficile, because other gut commensals did not aggregate with C difficile. Addition of F nucleatum also enhanced C difficile biofilm formation and extracellular polysaccharide production. CONCLUSIONS: Collectively, these data show a unique interaction of between pathogenic C difficile and F nucleatum in the intestinal mucus layer.

Zranb1-mutant mice display abnormal colonic mucus production and exacerbation of DSS-induced colitis.

Expression of mucin MUC2, a component of the colonic mucus layer, plays a crucial role in intestinal homeostasis. Here, we describe a new regulator of MUC2 expression, the deubiquitinase ZRANB1 (Trabid). A ZRANB1 mutation changing cysteine to serine in amino acid position 443, affects ubiquitination. To analyze ZRANB1 function in the intestine, we generated Zranb1 C443S mutant knock-in (Zranb1C443S/C443S) mice using the CRISPR/Cas9 system. Zranb1C443S/C443S mice exhibited decreased mRNA expression and MUC2 production. Colonic organoids from Zranb1C443S/C443S mice displayed decreased Muc2 mRNA expression following differentiation into goblet cells. Finally, we analyzed dextran sulfate sodium-induced colitis to understand ZRANB1's role in intestinal inflammation. Zranb1C443S/C443S mice with colitis exhibited significant weight loss, reduced colon length, and worsening clinical and pathological scores, indicating that ZRANB1 contributes to intestinal homeostasis. Together, these results suggest that ZRANB1 regulates MUC2 expression and intestinal inflammation, which may help elucidating the pathogenesis of inflammatory bowel disease and developing new therapeutics targeting ZRANB1.

Oral Corticosteroids Impair Mucin Production and Alter the Posttransplantation Microbiota in the Gut.

INTRODUCTION: Gut microbiota alterations cause inflammation in patients with ulcerative colitis (UC). Fecal microbiota transplantation (FMT) enables manipulating the microbiota's composition, but the mechanisms underlying colonization of the posttransplantation microbiota are poorly understood. METHODS: In this open-label, nonrandomized study, the FMT efficacy and changes in the gut microbiota were evaluated in 8 UC patients with mild-to-moderately active endoscopic colonic lesions. Compositional changes in the fecal and mucosal microbiotas between donors and recipients were examined via 16S rRNA-based sequencing. To investigate the effects of oral corticosteroids on microbiota colonization, FMT was performed in germ-free prednisolone (PSL)-administered mice to examine the factors determining colonization. RESULTS: Four UC patients achieved clinical remission (CR) after FMT, and 3 also achieved endoscopic remission. The fecal microbiotas of the CR patients changed similar to those of the donors after FMT. The mucin-coding gene, MUC2, was less expressed in the colons of the PSL-dependent patients than in the PSL-free patients. In the mice, PSL treatment decreased the fecal mucin production and altered the posttransplantation fecal microbiota composition. Adding either exogenous mucin or the mucin secretagogue, rebamipide, partially alleviated the PSL-induced dysbiosis of the gut microbiota. Administering rebamipide with FMT from healthy donors relieved inflammation in mice with Enterococcus faecium-induced colitis. CONCLUSION: Colonic mucin controlled the gut microbiota composition, and oral corticosteroid treatment modified the gut microbiota partly by reducing the colonic mucin.

Increased Listeria monocytogenes Dissemination and Altered Population Dynamics in Muc2-Deficient Mice.

The mucin Muc2 is a major constituent of the mucus layer that covers the intestinal epithelium and creates a barrier between epithelial cells and luminal commensal or pathogenic microorganisms. The Gram-positive foodborne pathogen Listeria monocytogenes can cause enteritis and also disseminate from the intestine to give rise to systemic disease. L. monocytogenes can bind to intestinal Muc2, but the influence of the Muc2 mucin barrier on L. monocytogenes intestinal colonization and systemic dissemination has not been explored. Here, we used an orogastric L. monocytogenes infection model to investigate the role of Muc2 in host defense against L. monocytogenes Compared to wild-type mice, we found that Muc2-/- mice exhibited heightened susceptibility to orogastric challenge with L. monocytogenes, with higher mortality, elevated colonic pathology, and increased pathogen burdens in both the intestinal tract and distal organs. In contrast, L. monocytogenes burdens were equivalent in wild-type and Muc2-/- animals when the pathogen was administered intraperitoneally, suggesting that systemic immune defects related to Muc2 deficiency do not explain the heightened pathogen dissemination observed in oral infections. Using a barcoded L. monocytogenes library to measure intrahost pathogen population dynamics, we found that Muc2-/- animals had larger pathogen founding population sizes in the intestine and distal sites than observed in wild-type animals. Comparisons of barcode frequencies suggested that the colon becomes the major source for seeding the internal organs in Muc2-/- animals. Together, our findings reveal that Muc2 mucin plays a key role in controlling L. monocytogenes colonization, dissemination, and population dynamics.

DOCK4 stimulates MUC2 production through its effect on goblet cell differentiation.

The intestinal mucosa is in continuous contact with milliard of microorganisms, thus intestinal epithelial barrier is a critical component in the arsenal of defense mechanisms required to prevent infection and inflammation. Mucin 2 (MUC2), which is produced by the goblet cells, forms the skeleton of the intestinal mucus and protects the intestinal tract from self-digestion and numerous microorganisms. Dedicator of cytokinesis 4 (DOCK4) is a member of the DOCK-B subfamily of the DOCK family of guanine nucleotide exchange factors. It is reported that DOCK4 plays a critical role in the repair of the barrier function of the intestinal epithelium after chemical damage. In this study, the role of DOCK4 in the goblet cell differentiation and MUC2 production is explored. Disordered intestinal epithelium and shortage of goblet cells were observed in DOCK4 gene knockout mice. Furthermore, DOCK4 deletion contributed to the low expression of MUC2 and the goblet cell differentiation/maturation factors including growth factor independent 1 (Gfi1) and SAM pointed domain epithelial-specific transcription factor (Spdef) in mouse ileums and colons. Overexpression of DOCK4 caused a marked increase in Gfi1, Spdef, and MUC2, while siRNA knockdown of endogenous DOCK4 significantly decreased Gfi1, Spdef, and MUC2 in HT-29 cells. In addition, MUC2, DOCK4, and the goblet cell differentiation/maturation factors mRNA levels were decreased in colorectal cancer samples compared with normal colons. A significant positive correlation was found between MUC2 and DOCK4. In conclusion, DOCK4 may serve as a critical regulator of goblet cell differentiation and MUC2 production in the intestine.

Prognostic significance of MUC2, CDX2 and SOX2 in stage II colorectal cancer patients.

BACKGROUND: Colorectal cancer (CRC) remains a serious health concern worldwide. Despite advances in diagnosis and treatment, about 15 to 30% of stage II CRC patients subjected to tumor resection with curative intent, develop disease relapse. Moreover, the therapeutic strategy adopted after surgery is not consensual for these patients. This supports the imperative need to find new prognostic and predictive biomarkers for stage II CRC. METHODS: For this purpose, we used a one-hospital series of 227 stage II CRC patient samples to assess the biomarker potential of the immunohistochemical expression of MUC2 mucin and CDX2 and SOX2 transcription factors. The Kaplan-Meier method was used to generate disease-free survival curves that were compared using the log-rank test, in order to determine prognosis of cases with different expression of these proteins, different mismatch repair (MMR) status and administration or not of adjuvant chemotherapy. RESULTS: In this stage II CRC series, none of the studied biomarkers showed prognostic value for patient outcome. However low expression of MUC2, in cases with high expression of CDX2, absence of SOX2 or MMR-proficiency, conferred a significantly worst prognosis. Moreover, cases with low expression of MUC2 showed a significantly clear benefit from treatment with adjuvant chemotherapy. CONCLUSION: In conclusion, we observe that patients with stage II CRC with low expression of MUC2 in the tumor respond better when treated with adjuvant chemotherapy. This observation supports that MUC2 is involved in resistance to fluorouracil-based adjuvant chemotherapy and might be a promising future predictive biomarker in stage II CRC patients.

Effect of Senecio scandens ethanol extract on gut microbiota composition in mice.

The gut microbiota inhabits the animal intestinal tract, and dysbiosis of the gut microbiota may result in disease. Senecio scandens has pharmaceutical antibacterial activities and is regarded as a broad-spectrum antibiotic in traditional Chinese medicine. Extracts of S. scandens are reported to show strong antimicrobial activity, and quercetin significantly decreases some species in the caecal microflora. However, the bactericidal effects of the extracts on the gut microbiota remain obscure. Here, we supplied ethanol extract of S. scandens, which might possibly be used as an alternative for chemical antibiotics, to mice to investigate the state of the intestinal microbiota. Our studies included a control group, low-, moderate-, and high-dose ethanol extract groups, and cefixime capsule group. The ethanol extract groups did not present reduced diversity or differences in the gut microbiota balance. There were significant differences between the ethanol extract and cefixime capsule groups in terms of the gut microbiota. The control and ethanol extract groups contained similar bacteria, which suggested that the ethanol extract has no inhibitory effect on the gut microbiota in vivo. Bifidobacteriales and Lactobacillus acidophilus were significantly increased in the high-dose group. Both secretory immunoglobulin A and mucin 2 concentrations increased as the dose of ethanol extract increased. The functional prediction differences between the control and ethanol extract groups decreased with increasing extract doses, which indicated that the low-dose and high-dose extract treatments might regulate different pathways and functions of the gut microbiota. The results also highlighted the prevention of bacterial drug resistance in the ethanol extract groups.

Alteration of Colonic Mucin Composition and Cytokine Expression in Acute Swine Dysentery.

Swine dysentery (SD) is an enteric disease associated with strongly ß-hemolytic Brachyspira spp. that cause mucohemorrhagic diarrhea primarily in grower-finisher pigs. We characterized alteration of colonic mucin composition and local cytokine expression in the colon of pigs with acute SD after B. hyodysenteriae (Bhyo) infection and fed either a diet containing 30% distillers dried grains with solubles (DDGS) or a control diet. Colonic tissue samples from 9 noninoculated pigs (Control, N = 4; DDGS, N = 5) and 10 inoculated pigs experiencing acute SD (Bhyo, N = 4; Bhyo-DDGS, N = 6) were evaluated. At the apex of the spiral colon, histochemical staining with high-iron diamine-Alcian blue revealed increased sialomucin (P = .008) and decreased sulfomucin (P = .027) in Bhyo pigs relative to controls, with a dietary effect for sulfomucin. Noninoculated pigs fed DDGS had greater expression of sulfomucin (P = .002) compared to pigs fed the control diet. Immunohistochemically, there was de novo expression of mucin 5AC (MUC5AC) in the Bhyo group while mucin 2 (MUC2) expression was not significantly different between groups. RNA in situ hybridization to detect the pro-inflammatory cytokine IL-1ß often showed increased expression in the Bhyo group although without statistical significance, and this was not correlated with MUC5AC or MUC2 expression, suggesting IL-1ß is not a major regulator of their secretion in acute SD. Expression of the anti-inflammatory cytokine TGF-ß1 was significantly suppressed in the Bhyo group compared to controls (P = .005). This study reveals mucin and cytokine alterations in the colon of pigs with experimentally induced SD and related dietary effects of DDGS.

Mucins Dynamics in Physiological and Pathological Conditions.

Maintaining intestinal health requires clear segregation between epithelial cells and luminal microbes. The intestinal mucus layer, produced by goblet cells (GCs), is a key element in maintaining the functional protection of the epithelium. The importance of the gut mucus barrier is highlighted in mice lacking Muc2, the major form of secreted mucins. These mice show closer bacterial residence to epithelial cells, develop spontaneous colitis and became moribund when infected with the attaching and effacing pathogen, Citrobacter rodentium. Furthermore, numerous observations have associated GCs and mucus layer dysfunction to the pathogenesis of inflammatory bowel disease (IBD). However, the molecular mechanisms that regulate the physiology of GCs and the mucus layer remain obscured. In this review, we consider novel findings describing divergent functionality and expression profiles of GCs subtypes within intestinal crypts. We also discuss internal (host) and external (diets and bacteria) factors that modulate different aspects of the mucus layer as well as the contribution of an altered mucus barrier to the onset of IBD.

Sodium channel TRPM4 and sodium/calcium exchangers (NCX) cooperate in the control of Ca2+-induced mucin secretion from goblet cells.

Regulated mucin secretion is essential for the formation of the mucus layer that protects the underlying epithelial cells from foreign particles. Alterations in the quantity or quality of secreted mucins are therefore detrimental to airway and colon physiology. Based on various biochemical assays in several human cell lines, we report here that Na+/Ca2+ exchanger 2 (NCX2) works in conjunction with transient receptor potential cation channel subfamily M member 4 (TRPM4), and perhaps TRPM5, Na+ channels to control Ca2+-mediated secretion of both mucin 2 (MUC2) and MUC5AC from HT29-18N2 colonic cancer cells. Differentiated normal bronchial epithelial (NHBE) cells and tracheal cells from patients with cystic fibrosis (CFT1-LC3) expressed only TRPM4 and all three isoforms of NCXs. Blocking the activity of TRPM4 or NCX proteins abrogated MUC5AC secretion from NHBE and CFT1-LC3 cells. Altogether, our findings reveal that NCX and TRPM4/TRPM5 are both required for mucin secretion. We therefore propose that these two proteins could be potential pharmacological targets to control mucus-related pathologies such as cystic fibrosis.

Calcium-activated chloride channel regulator 1 (CLCA1) forms non-covalent oligomers in colonic mucus and has mucin 2-processing properties.

Calcium-activated chloride channel regulator 1 (CLCA1) is one of the major nonmucin proteins found in intestinal mucus. It is part of a larger family of CLCA proteins that share highly conserved features and domain architectures. The CLCA domain arrangement is similar to proteins belonging to the ADAM (a disintegrin and metalloproteinase) family, known to process extracellular matrix proteins. Therefore, CLCA1 is an interesting candidate in the search for proteases that process intestinal mucus. Here, we investigated CLCA1's biochemical properties both in vitro and in mucus from mouse and human colon biopsy samples. Using immunoblotting with CLCA1-specific antibodies and recombinant proteins, we observed that the CLCA1 C-terminal self-cleavage product forms a disulfide-linked dimer that noncovalently interacts with the N-terminal part of CLCA1, which further interacts to form oligomers. We also characterized a second, more catalytically active, N-terminal product of CLCA1, encompassing the catalytic domain together with its von Willebrand domain type A (VWA). This fragment was unstable but could be identified in freshly prepared mucus. Furthermore, we found that CLCA1 can cleave the N-terminal part of the mucus structural component MUC2. We propose that CLCA1 regulates the structural arrangement of the mucus and thereby takes part in the regulation of mucus processing.