Epithelial colonization by gut dendritic cells promotes their functional diversification.

Dendritic cells (DCs) patrol tissues and transport antigens to lymph nodes to initiate adaptive immune responses. Within tissues, DCs constitute a complex cell population composed of distinct subsets that can exhibit different activation states and functions. How tissue-specific cues orchestrate DC diversification remains elusive. Here, we show that the small intestine included two pools of cDC2s originating from common pre-DC precursors: (1) lamina propria (LP) CD103+CD11b+ cDC2s that were mature-like proinflammatory cells and (2) intraepithelial cDC2s that exhibited an immature-like phenotype as well as tolerogenic properties. These phenotypes resulted from the action of food-derived retinoic acid (ATRA), which enhanced actomyosin contractility and promoted LP cDC2 transmigration into the epithelium. There, cDC2s were imprinted by environmental cues, including ATRA itself and the mucus component Muc2. Hence, by reaching distinct subtissular niches, DCs can exist as immature and mature cells within the same tissue, revealing an additional mechanism of DC functional diversification.

IL-33 Induces Murine Intestinal Goblet Cell Differentiation Indirectly via Innate Lymphoid Cell IL-13 Secretion.

Regulation of the intestinal mucus layer by goblet cells is important for preventing inflammation and controlling infection. IL-33, a cytokine upregulated in inflammatory bowel disease and helminth infection, induces intestinal goblet cells, but the mechanism remains unclear. Enteroids are three-dimensional structures of primary small intestinal epithelial cells that contain all differentiated intestinal epithelial cell types. We developed an enteroid-immune cell coculture model to determine the mechanism through which IL-33 affects intestinal goblet cell differentiation. We report that IL-33 does not directly induce goblet cell differentiation in murine enteroids; however, IL-13, a cytokine induced by IL-33, markedly induces goblet cells and gene expression consistent with goblet cell differentiation. When enteroids are cocultured with CD90+ mesenteric lymph node cells from IL-33-treated mice, IL-33 then induces IL-13 secretion by group 2 innate lymphoid cells and enteroid gene expression consistent with goblet cell differentiation. In cocultures, IL-33-induced Muc2 expression is dependent on enteroid Il4ra expression, demonstrating a requirement for IL-13 signaling in epithelial cells. In vivo, IL-33-induced intestinal goblet cell hyperplasia is dependent on IL-13. These studies demonstrate that IL-33 induces intestinal goblet cell differentiation not through direct action on epithelial cells but indirectly through IL-13 production by goup 2 innate lymphoid cells.

Impaired O-Glycosylation at Consecutive Threonine TTX Motifs in Mucins Generates Conformationally Restricted Cancer Neoepitopes.

Autoantibody signatures of circulating mucin fragments stem from cancer tissues, and microenvironments are promising biomarkers for cancer diagnosis and therapy. This study highlights dynamic epitopes generated by aberrantly truncated immature O-glycosylation at consecutive threonine motifs (TTX) found in mucins and intrinsically disordered proteins (IDPs). NMR analysis of synthetic mucin models having glycosylated TTX motifs and colonic MUC2 tandem repeats (TRs) containing TTP and TTL moieties unveils a general principle that O-glycosylation at TTX motifs generates a highly extended and rigid conformation in IDPs. We demonstrate that the specific conformation of glycosylated TTX motifs in MUC2 TRs is rationally rearranged by concerted motions of multiple dihedral angles and noncovalent interactions between the carbohydrate and peptide region. Importantly, this canonical conformation of glycosylated TTX motifs minimizes steric crowding of glycans attached to threonine residues, in which O-glycans possess restricted orientations permitting further sugar extension. An antiadhesive microarray displaying synthetic MUC2 derivatives elicited the presence of natural autoantibodies to MUC2 with impaired O-glycosylation at TTX motifs in sera of healthy volunteers and patients diagnosed with early stage colorectal cancer (CRC). Interestingly, autoantibody levels in sera of the late stage CRC patients were distinctly lower than those of early stage CRC and normal individuals, indicating that the anti-MUC2 humoral response to MUC2 neoepitopes correlates inversely with the CRC stage of patients. Our results uncovered the structural basis of the creation of dynamic epitopes by immature O-glycosylation at TTX motifs in mucins that facilitates the identification of high-potential targets for cancer diagnosis and therapy.

Aggregative Adherence Fimbriae II of Enteroaggregative Escherichia coli Are Required for Adherence and Barrier Disruption during Infection of Human Colonoids.

Symptomatic and asymptomatic infection with the diarrheal pathogen enteroaggregative Escherichia coli (EAEC) is associated with growth faltering in children in developing settings. The mechanism of this association is unknown, emphasizing a need for better understanding of the interactions between EAEC and the human gastrointestinal mucosa. In this study, we investigated the role of the aggregative adherence fimbriae II (AAF/II) in EAEC adherence and pathogenesis using human colonoids and duodenal enteroids. We found that a null mutant in aafA, the major subunit of AAF/II, adhered significantly less than wild-type (WT) EAEC strain 042, and adherence was restored in a complemented strain. Immunofluorescence confocal microscopy of differentiated colonoids, which produce an intact mucus layer comprised of the secreted mucin MUC2, revealed bacteria at the epithelial surface and within the MUC2 layer. The WT strain adhered to the epithelial surface, whereas the aafA deletion strain remained within the MUC2 layer, suggesting that the presence or absence of AAF/II determines both the abundance and location of EAEC adherence. In order to determine the consequences of EAEC adherence on epithelial barrier integrity, colonoid monolayers were exposed to EAEC constructs expressing or lacking aafA Colonoids infected with WT EAEC had significantly decreased epithelial resistance, an effect that required AAF/II, suggesting that binding of EAEC to the epithelium is necessary to impair barrier function. In summary, we show that production of AAF/II is critical for adherence and barrier disruption in human colonoids, suggesting a role for this virulence factor in EAEC colonization of the gastrointestinal mucosa.

Defining cooperative roles for colonic microbiota and Muc2 mucin in mediating innate host defense against Entamoeba histolytica.

Amebiasis is caused by the protozoan parasite Entamoeba histolytica (Eh), a potentially fatal disease occurring mainly in developing countries. How Eh interacts with innate host factors in the gut is poorly understood. Eh resides and feed in/on the outer colonic mucus layer and thus share an ecological niche with indigenous microbiota. As gut microbiota regulates innate immune responses, in this study we characterized the cooperative roles that microbiota and the mucus layer play in Eh-induced pro-inflammatory responses in the colon. To study this, we used antibiotics treated and non-treated specific pathogen free Muc2-/- and Muc2+/+ littermates and germ-free mice inoculated with Eh in colonic loops as a short infection model. In antibiotic treated Muc2-/- and Muc2+/+ littermates, Eh elicited robust mucus and water secretions, enhanced pro-inflammatory cytokines and chemokine expression with elevated MPO activity and higher pathology scores as compared to the modest response observed in non-antibiotic treated littermates. Host responses were microbiota specific as mucus secretion and pro-inflammatory responses were attenuated following homologous fecal microbial transplants in antibiotic-treated Muc2+/+ quantified by secretion of 3H-glucosamine newly synthesized mucin, Muc2 mucin immunostaining and immunohistochemistry. Eh-elicited pro-inflammatory responses and suppressed goblet cell transcription factor Math1 as revealed by in vivo imaging of Eh-colonic loops in Math1GFP mice, and in vitro using Eh-stimulated LS174T human colonic goblet cells. Eh in colonic loops increased bacterial translocation of bioluminescent E. coli and indigenous bacteria quantified by FISH and quantitative PCR. In germ-free animals, Eh-induced mucus/water secretory responses, but acute pro-inflammatory responses and MPO activity were severely impaired, allowing the parasite to bind to and disrupt mucosal epithelial cells. These findings have identified key roles for intestinal microbiota and mucus in regulating innate host defenses against Eh, and implicate dysbiosis as a risk factor for amebiasis that leads to exacerbated immune responses to cause life-threatening disease.

Immunoprotective Effect of Chitosan Particles on Hymenolepis nana - Infected Mice.

Hymenolepis nana is the most commonly known intestinal cestode infecting mainly human. This study aimed to investigate the potential effect of chitosan particles (CSP) to enhance the immune system against H. nana infection. Determination of worm burden, egg output, histopathological changes, oxidative stress markers (lipid peroxidation and reduced glutathione), goblet (GCs) and mucosal mast cells (MMCs) counts in intestinal ileum was performed. In addition, levels of intestinal mRNA expression of interleukin (IL)-4, IL-9, stem cell factor (SCF), type I and II interferons (IFN)-α/ γ, tumour necrosis factor (TNF)-α, mucin 2 (MUC2) and inducible nitric oxide synthase (iNOs) were investigated using real-time PCR. The results indicated induced reductions in adult worm and egg counts in infected mice after CSP treatment. This was associated with improvement in tissue morphometric measurements and oxidative stress which were altered after infection. Expression levels of iNOs, IFN-α, IFN-γ, TNF-α and IL-9 were decreased by CSP. Conversely, expression levels of MUC2, IL-4 and SCF increased compared to infected untreated group. In addition, GCs and MMCs counts were normalized by CSP. In conclusion, this study could indicate the immunoprotective effect of CSP against H. nana infection. This was characterized with Th2 anti-inflammatory responses.

Natural anti-intestinal goblet cell autoantibody production from marginal zone B cells.

Expression of a germline VH3609/D/JH2 IgH in mice results in the generation of B1 B cells with anti-thymocyte/Thy-1 glycoprotein autoreactivity by coexpression of Vk21-5/Jk2 L chain leading to production of serum IgM natural autoantibody. In these same mice, the marginal zone (MZ) B cell subset in spleen shows biased usage of a set of Ig L chains different from B1 B cells, with 30% having an identical Vk19-17/Jk1 L chain rearrangement. This VH3609/Vk19-17 IgM is reactive with intestinal goblet cell granules, binding to the intact large polymatrix form of mucin 2 glycoprotein secreted by goblet cells. Analysis of a µκ B cell AgR (BCR) transgenic (Tg) mouse with this anti-goblet cell/mucin2 autoreactive (AGcA) specificity demonstrates that immature B cells expressing the Tg BCR become MZ B cells in spleen by T cell-independent BCR signaling. These Tg B cells produce AGcA as the predominant serum IgM, but without enteropathy. Without the transgene, AGcA autoreactivity is low but detectable in the serum of BALB/c and C.B17 mice, and this autoantibody is specifically produced by the MZ B cell subset. Thus, our findings reveal that AGcA is a natural autoantibody associated with MZ B cells.

Intraductal papillary mucinous neoplasms.

PURPOSE OF REVIEW: Our understanding of intraductal papillary mucinous neoplasm (IPMN) of the pancreas has remarkably grown within the last decade; nonetheless there is still an ongoing controversy if the majority of these potentially malignant neoplasms need to be resected or if observation in a subset is well tolerated. RECENT FINDINGS: Novel cyst fluid biomarkers, like Gnas mutations or mab DAS-1, could play a pivotal role in the distinction of IPMN vs. other cystic lesions, in the sub-classification of IPMN and in the detection of IPMN with high-grade dysplasia or invasive cancer. Other recent studies focused on natural history of minimal- and extensive-mixed IPMN and the safety of the 2012 AIP guidelines. Small series also described that observation can be an option in selected frail patients with MD-IPMN. Further, data from a large European multicenter study analysis indicated that patients with IPMN do not have an increased frequency of extrapancreatic malignancies. SUMMARY: Increasing knowledge about the nature of IPMN and their subtypes has resulted in an individualized approach in diagnosis and treatment. Owing to the availability of accurate diagnostic instruments, timing and indication for pancreatic resection have become more selective, sparing patients with harmless IPMN from major surgery.

The effect of glycosylation on the antibody recognition of a MUC2 mucin epitope.

MUC2 glycoprotein, produced by the epithelium of the colon and built up mainly of repeat units of (1) PTTTPITTTTTVTPTPTPTGTQT(23) , can be overexpressed or underglycosylated in gastrointestinal diseases, e.g. in case of colon carcinoma. We have been studying the epitope structure of the MUC2 by focusing on the repeat unit with the mucin peptide specific MAb 996 monoclonal antibody. This antibody recognizes the (18) PTGTQ(22) sequence as minimal, and (16) PTPTGTQ(22) as optimal epitope within the underglycosylated glycoprotein. In this article, we aim to clarify the effect of glycosylation of the epitope on MAb 996 antibody binding including its correlation with the secondary structure of the modified peptides: glycosylation in the epitope core and in the flank. For this we have prepared the (16) PTPTGTQ(22) peptide glycosylated with N-acetylgalactoseamine (Tn antigen) in position 17, 19, 21, or on all three threonines. The MAb 996 antibody binding properties of the peptides were characterized in competitive ELISA experiments, and their solution secondary structure was studied by circular dichroism spectroscopy in water and in the ordered structure promoting trifluoroethanol. Our results show that glycosylation in position 19 (peptide (16) PTPT(GalNAcα)GTQ(22) ) resulted in enhanced antibody recognition and significantly altered secondary structure, while glycosylation in position 21 completely demolished the binding. These findings could be useful in determining the nature of antigen-antibody interaction, and perhaps designing synthetic peptide vaccines for tumor therapy.

Predictors of lymph node metastasis in T1 colorectal carcinoma: an immunophenotypic analysis of 265 patients.

BACKGROUND: The appropriateness of endoscopic resection in patients with T1 colorectal carcinomas is unclear. Highly precise predictors of lymph node metastasis are required to optimize the outcomes of treatments for T1 colorectal carcinomas. OBJECTIVE: The purpose of this work was to identify predictors of lymph node metastasis by examining the clinicopathologic significance of immunophenotypes found in T1 colorectal carcinomas. DESIGN: This was a retrospective study. SETTINGS: The study was conducted at a university hospital. PATIENTS: Included were 265 patients with T1 colorectal carcinoma who underwent radical surgery. INTERVENTIONS: Patients with T1 colorectal carcinoma were managed. MAIN OUTCOME MEASURES: Immunophenotypes were associated with various clinicopathologic parameters, and CD10 expression was strongly associated with lymph node metastasis. RESULTS: The levels of MUC2, MUC5AC, and CD10 expression were individually significantly associated with tumor location, growth pattern, histologic type, invasive potential, and metastatic potential. The incidence of lymph node metastasis was significantly associated with each of the 5 following parameters: depth of submucosal invasion (p = 0.005), tumor budding (p < 0.001), lymphatic invasion (p < 0.001), MUC2 expression (p = 0.006), and CD10 expression (p < 0.001). Multivariate analysis showed that CD10 expression (OR, 9.2 [95% CI, 2.5-39.8]; p = 0.001) and lymphatic invasion (OR, 6.3 [95% CI, 2.5-17.7]; p < 0.001) were independently associated with lymph node metastasis. LIMITATIONS: This study was limited by its small sample size, intraobserver variation attributed to immunohistochemical staining, and potential selection bias because surgically resected specimens were collected instead of endoscopically resected specimens. CONCLUSIONS: We suggest that CD10 expression is closely associated with lymph node metastasis in T1 colorectal carcinoma.

Loss of TMF/ARA160 protein renders colonic mucus refractory to bacterial colonization and diminishes intestinal susceptibility to acute colitis.

TMF/ARA160 is a Golgi-associated protein with several cellular functions, among them direction of the NF-κB subunit, p65 RelA, to ubiquitination and proteasomal degradation in stressed cells. We sought to investigate the role of TMF/ARA160 under imposed stress conditions in vivo. TMF(-/-) and wild-type (WT) mice were treated with the ulcerative agent dextran sulfate sodium (DSS), and the severity of the inflicted acute colitis was determined. TMF(-/-) mice were found to be significantly less susceptible to DSS-induced colitis, with profoundly less bacterial penetration into the colonic epithelia. Surprisingly, unlike in WT mice, no bacterial colonies were visualized in colons of healthy untreated TMF(-/-) mice, indicating the constitutive resistance of TMF(-/-) colonic mucus to bacterial retention and penetration. Gene expression analysis of colon tissues from unchallenged TMF(-/-) mice revealed 5-fold elevated transcription of the muc2 gene, which encodes the major component of the colonic mucus gel, the MUC2 mucin. Accordingly, the morphology of the colonic mucus in TMF(-/-) mice was found to differ from the mucus structure in WT colons. The NF-κB subunit, p65, a well known transcription inducer of muc2, was up-regulated significantly in TMF(-/-) intestinal epithelial cells. However, this did not cause spontaneous inflammation or increased colonic crypt cell proliferation. Collectively, our findings demonstrate that absence of TMF/ARA160 renders the colonic mucus refractory to bacterial colonization and the large intestine less susceptible to the onset of colitis.

MUC2 expression and prevalence of high-grade dysplasia and invasive carcinoma in mixed-type intraductal papillary mucinous neoplasm of the pancreas.

BACKGROUND/OBJECTIVES: Morphological types and mucin protein expressions classify intraductal papillary mucinous neoplasms (IPMNs). Main duct (MD)-IPMN mostly consists of intestinal type (I-type), which expresses MUC2. Branch duct (BD)-IPMN mostly consists of gastric type (G-type), which does not express MUC2. However, the definition of mixed-type IPMN has yet to be clarified and it contains various histological types. The aim of this study was to investigate the relationship between MUC2 expression and the presence of high-grade dysplasia (HGD) and invasive carcinoma, especially in mixed-type IPMN. METHODS: This retrospective study included 101 consecutive patients with surgically resected IPMNs between April 2001 and October 2012. All patients were morphologically classified into four distinct types (I-type, G-type, PB-type: pancreatobilliary, O-type: oncocytic) and immunohistochemical reactivity of various anti-mucin antibodies were investigated. RESULTS: According to the classification of the 2012 international guidelines, the numbers (and histomorphological types: I/G/PB/O) of MD, mixed-type, and BD-IPMNs were 16 (12/4/0/0), 45 (16/28/1/0), and 40 (0/38/1/1). Prevalence of MUC2 expression in MD, mixed-type, and BD-IPMNs were 75% (12/16), 36% (16/45), and 0% (0/40). In mixed-type IPMN, the prevalence of HGD and/or invasive carcinoma in MUC2-positive IPMN was significantly higher than that of MUC2-negative IPMN (HGD + invasive carcinoma: 88% vs. 38%, p = 0.0017; invasive carcinoma: 50% vs. 21%, p = 0.042). Multivariate analysis showed that MUC2 expression is an independent predictive factor of HGD and invasive carcinoma in mixed IPMN (odds ratio 14.6, 95% CI 2.5-87.4, p = 0.003). CONCLUSIONS: In mixed-type IPMN, MUC2 expression clearly identified HGD and invasive carcinoma and may provide most appropriate surgical indication.

Muc2 protects against lethal infectious colitis by disassociating pathogenic and commensal bacteria from the colonic mucosa.

Despite recent advances in our understanding of the pathogenesis of attaching and effacing (A/E) Escherichia coli infections, the mechanisms by which the host defends against these microbes are unclear. The goal of this study was to determine the role of goblet cell-derived Muc2, the major intestinal secretory mucin and primary component of the mucus layer, in host protection against A/E pathogens. To assess the role of Muc2 during A/E bacterial infections, we inoculated Muc2 deficient (Muc2(-/-)) mice with Citrobacter rodentium, a murine A/E pathogen related to diarrheagenic A/E E. coli. Unlike wildtype (WT) mice, infected Muc2(-/-) mice exhibited rapid weight loss and suffered up to 90% mortality. Stool plating demonstrated 10-100 fold greater C. rodentium burdens in Muc2(-/-) vs. WT mice, most of which were found to be loosely adherent to the colonic mucosa. Histology of Muc2(-/-) mice revealed ulceration in the colon amid focal bacterial microcolonies. Metabolic labeling of secreted mucins in the large intestine demonstrated that mucin secretion was markedly increased in WT mice during infection compared to uninfected controls, suggesting that the host uses increased mucin release to flush pathogens from the mucosal surface. Muc2 also impacted host-commensal interactions during infection, as FISH analysis revealed C. rodentium microcolonies contained numerous commensal microbes, which was not observed in WT mice. Orally administered FITC-Dextran and FISH staining showed significantly worsened intestinal barrier disruption in Muc2(-/-) vs. WT mice, with overt pathogen and commensal translocation into the Muc2(-/-) colonic mucosa. Interestingly, commensal depletion enhanced C. rodentium colonization of Muc2(-/-) mice, although colonic pathology was not significantly altered. In conclusion, Muc2 production is critical for host protection during A/E bacterial infections, by limiting overall pathogen and commensal numbers associated with the colonic mucosal surface. Such actions limit tissue damage and translocation of pathogenic and commensal bacteria across the epithelium.

Effect of core epitope modification on the antibody recognition of a MUC2 mucin peptide.

Identification of protein epitopes via combinatorial chemistry was one of the most important discoveries of the past three decades. Mapping of linear antibody epitopes can be achieved rapidly and cost-effectively by the polymer pin-bound peptide approach. In this article, the determination of the fine epitope structure of MUC2 mucin glycoprotein is described by using specific monoclonal antibody. We have used positional scanning combinatorial approach, and also parallel synthesis. The residues within the MUC2 epitope (18)PTGTQ(22) of MAb 996 were replaced by all other proteinogenic amino acids on pin-bound peptide libraries, and their antibody binding was studied in modified ELISA. Thr(19) was the least important of the residues in antibody recognition; most of the other amino acids could be replaced, except Pro. The other residues cannot be replaced without loss of antibody binding, where both the size and character of the amino acids were important. The significance of the non-chiral Gly(20) residue was further studied by competitive ELISA of parallelly synthesized soluble peptides containing L - or D-Ala instead of Gly residue. However, the D-Ala-containing oligopeptides showed no antibody binding; therefore, the backbone conformation is characteristic of that of L-amino acid containing peptides in this position as well. With the combinatorial approach we obtained relevant information about the contribution of individual amino acid side chains to the MAb 996 antibody binding within the PTGTQ predominant MUC2 mucin epitope. These results could be utilized for the design of synthetic antigens for detection of MUC2 protein core-specific antibodies related to carcinoma(s).

γδ T cells play a protective role during infection with Nippostrongylus brasiliensis by promoting goblet cell function in the small intestine.

The intestinal epithelium is rich in γδ T cells and the gut is a site of residence for a wide variety of pathogens, including nematodes. Although CD4+ T-cell receptor (TCR) -αß+ T helper type 2 T cells are essential for the expulsion of intestinal nematodes, little information is available on the function of γδ T cells in this type of infection. Here, we demonstrate two major functions of γδ T cells as a potently protective T-cell population against Nippostrongylus brasiliensis infection using γδ T-cell-deficient (TCR-δ(-/-) ) mice. First, γδ T cells are required to initiate rapid expulsion of adult worms from the intestine and to limit egg production. Second, γδ T cells prevent the pathological intestinal damage associated with nematode infection, evident by increased clinical disease and more severe microscopic lesions in infected TCR-δ(-/-) mice. γδ T-cell deficiency led to delayed goblet cell hyperplasia in association with reduced expression of phosphorylated STAT6, MUC2, Trefoil factor-3 (TFF3) and T helper type 2 cytokines including interleukin-13 (IL-13). TCR-δ(-/-) mice also produced more interferon-γ than wild-type mice. Within the intraepithelial lymphocyte compartment, γδ T cells produced IL-13. Adoptive transfer of γδ T cells or administration of recombinant IL-13 to TCR-δ(-/-) mice successfully reduced the egg production by N. brasiliensis. Collectively, these data provide strong evidence that γδ T cells play an important role in controlling infection with intestinal nematodes and limiting infection-induced pathology.

Host Protective Mechanisms to Intestinal Amebiasis.

The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis, an infection that manifests as colitis and, in some cases, liver abscess. A better understanding of host protective factors is key to developing an effective remedy. Recently, significant advances have been made in understanding the mechanisms of MUC2 production by goblet cells upon amebic infection, regulation of antimicrobial peptide production by Paneth cells, the interaction of commensal microbiota with immune stimulation, and host genetics in conferring protection from amebiasis. In addition to host pathways that may serve as potential therapeutic targets, significant progress has also been made with respect to development of a vaccine against amebiasis. Here, we aim to highlight the current understanding and knowledge gaps critically.

Bifidobacterium dentium-derived y-glutamylcysteine suppresses ER-mediated goblet cell stress and reduces TNBS-driven colonic inflammation.

Endoplasmic reticulum (ER) stress compromises the secretion of MUC2 from goblet cells and has been linked with inflammatory bowel disease (IBD). Although Bifidobacterium can beneficially modulate mucin production, little work has been done investigating the effects of Bifidobacterium on goblet cell ER stress. We hypothesized that secreted factors from Bifidobacterium dentium downregulate ER stress genes and modulates the unfolded protein response (UPR) to promote MUC2 secretion. We identified by mass spectrometry that B. dentium secretes the antioxidant γ-glutamylcysteine, which we speculate dampens ER stress-mediated ROS and minimizes ER stress phenotypes. B. dentium cell-free supernatant and γ-glutamylcysteine were taken up by human colonic T84 cells, increased glutathione levels, and reduced ROS generated by the ER-stressors thapsigargin and tunicamycin. Moreover, B. dentium supernatant and γ-glutamylcysteine were able to suppress NF-kB activation and IL-8 secretion. We found that B. dentium supernatant, γ-glutamylcysteine, and the positive control IL-10 attenuated the induction of UPR genes GRP78, CHOP, and sXBP1. To examine ER stress in vivo, we first examined mono-association of B. dentium in germ-free mice which increased MUC2 and IL-10 levels compared to germ-free controls. However, no changes were observed in ER stress-related genes, indicating that B. dentium can promote mucus secretion without inducing ER stress. In a TNBS-mediated ER stress model, we observed increased levels of UPR genes and pro-inflammatory cytokines in TNBS treated mice, which were reduced with addition of live B. dentium or γ-glutamylcysteine. We also observed increased colonic and serum levels of IL-10 in B. dentium- and γ-glutamylcysteine-treated mice compared to vehicle control. Immunostaining revealed retention of goblet cells and mucus secretion in both B. dentium- and γ-glutamylcysteine-treated animals. Collectively, these data demonstrate positive modulation of the UPR and MUC2 production by B. dentium-secreted compounds.

MUC2 mucin is a major carrier of the cancer-associated sialyl-Tn antigen in intestinal metaplasia and gastric carcinomas.

Changes in mucin protein expression and in glycosylation are common features in pre-neoplastic lesions and cancer and are therefore used as cancer-associated markers. De novo expression of intestinal mucin MUC2 and cancer-associated sialyl-Tn antigen are frequently observed in intestinal metaplasia (IM) and gastric cancer. However, despite that these antigens often co-localize, MUC2 has not been demonstrated to be a carrier of sialyl-Tn. By using the in situ proximity ligation assay (in situ PLA), we herein could show that MUC2 is a major carrier of the sialyl-Tn antigen in all IM cases and in most gastric carcinoma cases. The requirement by in situ PLA for the presence of both antigens in close proximity increases the selectivity compared to measurement of co-localization, as determined by immunohistochemistry. Identification of the mucin which is the carrier of a carbohydrate structure offers unique advantages for future development of more accurate diagnostic and prognostic markers.

The protective roles of NLRP6 in intestinal epithelial cells.

The evolution of chronic inflammatory diseases is thought to be due to a combination of host genetic variations and environmental factors that include the alteration of intestinal flora, termed "dysbiosis." The intestinal mucosal barrier includes a chemical barrier and physical barrier that have important roles in protecting the intestine against inflammatory injury. The chemical barrier includes antimicrobial peptides (AMPs), and the physical barrier includes a mucous layer, a monolayer of intestinal epithelial cells and cell junctions. The intestinal mucosal barrier is not a static barrier, but rather, it strongly interacts with the gut microbiome and cells of the immune system. Correct expression of AMPs, together with mucus and balanced epithelial cell proliferation, prevents the occurrence of disease. NLRP6, a member of the nucleotide-binding domain, leucine-rich repeat-containing (NLR) innate immune receptor family, participates in the progression of intestinal inflammation and enteric pathogen infections. It has become apparent in recent years that NLRP6 is important in disease pathogenesis, as it responds to internal ligands that lead to the release of AMPs and mucus, thus regulating the regeneration of intestinal epithelial cells. This review summarizes the activation of NLRP6 and its protective role in the intestinal epithelial cell.

The Nlrp6 inflammasome is not required for baseline colonic inner mucus layer formation or function.

The inner mucus layer (IML) is a critical barrier that protects the colonic epithelium from luminal threats and inflammatory bowel disease. Innate immune signaling is thought to regulate IML formation via goblet cell Nlrp6 inflammasome activity that controls secretion of the mucus structural component Muc2. We report that isolated colonic goblet cells express components of several inflammasomes; however, analysis of IML properties in multiple inflammasome-deficient mice, including littermate-controlled Nlrp6-/- , detect a functional IML barrier in all strains. Analysis of mice lacking inflammasome substrate cytokines identifies a defective IML in Il18-/- mice, but this phenotype is ultimately traced to a microbiota-driven, Il18-independent effect. Analysis of phenotypic transfer between IML-deficient and IML-intact mice finds that the Bacteroidales family S24-7 (Muribaculaceae) and genus Adlercrutzia consistently positively covary with IML barrier function. Together, our results demonstrate that baseline IML formation and function is independent of inflammasome activity and highlights the role of the microbiota in determining IML barrier function.