Medicine, Dentistry & Health Sciences Collected Works - Research Publications
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ItemNo Preview AvailableMUC13 Cell Surface Mucin Limits Salmonella Typhimurium Infection by Protecting the Mucosal Epithelial Barrier(Elsevier, 2023)BACKGROUND & AIMS: MUC13 cell surface mucin is highly expressed on the mucosal surface throughout the intestine, yet its role against bacterial infection is unknown. We investigated how MUC13 impacts Salmonella typhimurium (S Tm) infection and elucidated its mechanisms of action. METHODS: Muc13-/- and wild-type littermate mice were gavaged with 2 isogenic strains of S Tm after pre-conditioning with streptomycin. We assessed clinical parameters, cecal histology, local and systemic bacterial load, and proinflammatory cytokines after infection. Cecal enteroids and epithelial cell lines were used to evaluate the mechanism of MUC13 activity after infection. The interaction between bacterial SiiE and MUC13 was assessed by using siiE-deficient Salmonella. RESULTS: S Tm-infected Muc13-/- mice had increased disease activity, histologic damage, and higher local and systemic bacterial loads. Mechanistically, we found that S Tm binds to MUC13 through its giant SiiE adhesin and that MUC13 acts as a pathogen-binding decoy shed from the epithelial cell surface after pathogen engagement, limiting bacterial invasion. In addition, MUC13 reduces epithelial cell death and intestinal barrier breakdown by enhancing nuclear factor kappa B signaling during infection, independent of its decoy function. CONCLUSIONS: We show for the first time that MUC13 plays a critical role in antimicrobial defense against pathogenic S Tm at the intestinal mucosal surface by both acting as a releasable decoy limiting bacterial invasion and reducing pathogen-induced cell death. This further implicates the cell surface mucin family in mucosal defense from bacterial infection.
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ItemMUC1-mediated Macrophage Activation Promotes Colitis-associated Colorectal Cancer via Activating the Interleukin-6/Signal Transducer and Activator of Transcription 3 Axis(ELSEVIER INC, 2022)BACKGROUND & AIMS: MUC1 is abnormally expressed in colorectal cancer, including colitis-associated colorectal cancer (CAC), but its role in tumorigenesis is unclear. This study investigated MUC1's effects in murine models of colitis and CAC and elucidated mechanisms of action. METHODS: Colitis and CAC were induced in mice by exposure to dextran sodium sulfate or azoxymethane plus dextran sodium sulphate. Clinical parameters, immune cell infiltration, and tumor development were monitored throughout disease progression. Experiments in knockout mice and bone marrow chimeras were combined with an exploration of immune cell abundance and function. RESULTS: Deficiency of Muc1 suppressed inflammation, inhibited tumor progression, increased abundance of CD8+ T lymphocytes, and reduced abundance of macrophages in colon tumors. Bone marrow chimeras showed promotion of CAC was primarily mediated by Muc1-expressing hematopoietic cells, and that MUC1 promoted a pro-tumoral immunosuppressive macrophage phenotype within tumors. Mechanistic studies revealed that Muc1 deficiency remarkably reduced interleukin-6 levels in the colonic tissues and tumors that was mainly produced by infiltrating macrophages at day 21, 42, and 85. In bone marrow-derived macrophages, MUC1 promoted responsiveness to chemoattractant and promoted activation into a phenotype with high Il6 and Ido1 expression, secreting factors which inhibited CD8+ T cell proliferation. MUC1 potently drives macrophages to produce interleukin-6, which in turn drives a pro-tumorigenic activation of signal transducer and activator of transcription 3 in colon epithelial tumor and stromal cells, ultimately increasing the occurrence and development of CAC. CONCLUSIONS: Our findings provide cellular and molecular mechanisms for the pro-tumorigenic functions of MUC1 in the inflamed colon. Therapeutic strategies to inhibit MUC1 signal transduction warrant consideration for the prevention or therapy of CAC.
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ItemSecreted NF-κB suppressive microbial metabolites modulate gut inflammation(CELL PRESS, 2022-04-12)Emerging evidence suggests that microbiome-host crosstalk regulates intestinal immune activity and predisposition to inflammatory bowel disease (IBD). NF-κB is a master regulator of immune function and a validated target for the treatment of IBD. Here, we identify five Clostridium strains that suppress immune-mediated NF-κB activation in epithelial cell lines, PBMCs, and gut epithelial organoids from healthy human subjects and patients with IBD. Cell-free culture supernatant from Clostridium bolteae AHG0001 strain, but not the reference C. bolteae BAA-613 strain, suppresses inflammatory responses and endoplasmic reticulum stress in gut epithelial organoids derived from Winnie mice. The in vivo responses to Clostridium bolteae AHG0001 and BAA-613 mirror the in vitro activity. Thus, using our in vitro screening of bacteria capable of suppressing NF-κB in the context of IBD and using an ex vivo organoid-based approach, we identify a strain capable of alleviating colitis in a relevant pre-clinical animal model of IBD.
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ItemMUC13 overexpression in renal cell carcinoma plays a central role in tumor progression and drug resistance(WILEY, 2017-05-15)Metastatic renal cell carcinoma is a largely incurable disease, and existing treatments targeting angiogenesis and tyrosine kinase receptors are only partially effective. Here we reveal that MUC13, a cell surface mucin glycoprotein, is aberrantly expressed by most renal cell carcinomas, with increasing expression positively correlating with tumor grade. Importantly, we demonstrated that high MUC13 expression was a statistically significant independent predictor of poor survival in two independent cohorts, particularly in stage 1 cancers. In cultured renal cell carcinoma cells MUC13 promoted proliferation and induced the cell cycle regulator, cyclin D1, and inhibited apoptosis by inducing the anti-apoptotic proteins, BCL-xL and survivin. Silencing of MUC13 expression inhibited migration and invasion, and sensitized renal cancer cells to killing by the multi-kinase inhibitors used clinically, sorafenib and sunitinib, and reversed acquired resistance to these drugs. Furthermore, we demonstrated that MUC13 promotion of renal cancer cell growth and survival is mediated by activation of nuclear factor κB, a transcription factor known to regulate the expression of genes that play key roles in the development and progression of cancer. These results show that MUC13 has potential as a prognostic marker for aggressive early stage renal cell cancer and is a plausible target to sensitize these tumors to therapy.
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ItemGut microbiota shape the inflammatory response in mice with an epithelial defect(TAYLOR & FRANCIS INC, 2021-01-01)Intestinal epithelial cell endoplasmic reticulum (ER) stress has been implicated in intestinal inflammation. It remains unclear whether ER stress is an initiator of or a response to inflammation. Winnie mice, carrying a Muc2 gene mutation resulting in intestinal goblet cell ER stress, develop spontaneous colitis with a depleted mucus barrier and increased bacterial translocation. This study aims to determine whether the microbiota was required for the development of Winnie colitis, and whether protein misfolding itself can initiate inflammation directly in absence of the microbiota. To assess the role of microbiota in driving Winnie colitis, WT and Winnie mice on the same background were rederived into the germ-free facility and housed in the Trexler-type soft-sided isolators. The colitis phenotype of these mice was assessed and compared to WT and Winnie mice housed within a specific pathogen-free facility. We found that Winnie colitis was substantially reduced but not abolished under germ-free conditions. Expression of inflammatory cytokine genes was reduced but several chemokines remained elevated in absence of microbiota. Concomitantly, ER stress was also diminished, although mucin misfolding persisted. RNA-Seq revealed that Winnie differentiated colon organoids have decreased expression of the negative regulators of the inflammatory response compared to WT. This data along with the increase in Mip2a chemokine expression, suggests that the epithelial cells in the Winnie mice are more responsive to stimuli. Moreover, the data demonstrate that intestinal epithelial intrinsic protein misfolding can prime an inflammatory response without initiating the unfolded protein response in the absence of the microbiota. However, the microbiota is necessary for the amplification of colitis in Winnie mice. Genetic predisposition to mucin misfolding in secretory cells initiates mild inflammatory signals. However, the inflammatory signal sets a forward-feeding cycle establishing progressive inflammation in the presence of microbiota.Abbreviations: Endoplasmic Reticulum: ER; Mucin-2: Muc-2; GF: Germ-Free; Inflammatory Bowel Disease: IBD.
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ItemMucoadhesive functionality of cell wall structures from fruits and grains: Electrostatic and polymer network interactions mediated by soluble dietary polysaccharides(NATURE PORTFOLIO, 2017-11-17)We demonstrate the enhancement of intestinal mucin (Muc2) binding to plant cell wall structures from fruit (parenchymal apple tissue) and grain (wheat endosperm) mediated by soluble dietary fibers embedded within cellulose networks. Mucin binding occurs through two distinct mechanisms; for pectin polysaccharides characteristic of fruits and vegetables, it is governed by molecular mucoadhesive interactions, while for neutral polysaccharides, arabinoxylan and β-glucan characteristic of cereal grains, the interaction stems from the properties of their polymer network. Based on microrheological and microscopic measurements, we show that neutral dietary fiber polysaccharides do not adhere to intestinal mucin, but are capable of disrupting the mucin network, which facilitates interpenetration of mucin molecules into the polysaccharide mesh. This effect becomes significant in the context of 'whole foods', where soluble fibers are incorporated within the gel-like matrix of cellulose-reinforced plant cell wall structures. The result of mucoadhesion assay and analysis of microscopy images points to the critical role of entanglements between mucin and polysaccharides as a lock-in mechanism preventing larger mucin from escaping out of plant cell wall structures. These results provide the first indication that non-pectin soluble dietary fiber may influence mucosal interactions, mucus barrier properties, and transmucosal transport of nutrients.
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ItemGlucocorticoids alleviate intestinal ER stress by enhancing protein folding and degradation of misfolded proteins(ROCKEFELLER UNIV PRESS, 2013-06-03)Endoplasmic reticulum (ER) stress in intestinal secretory cells has been linked with colitis in mice and inflammatory bowel disease (IBD). Endogenous intestinal glucocorticoids are important for homeostasis and glucocorticoid drugs are efficacious in IBD. In Winnie mice with intestinal ER stress caused by misfolding of the Muc2 mucin, the glucocorticoid dexamethasone (DEX) suppressed ER stress and activation of the unfolded protein response (UPR), substantially restoring goblet cell Muc2 production. In mice lacking inflammation, a glucocorticoid receptor antagonist increased ER stress, and DEX suppressed ER stress induced by the N-glycosylation inhibitor, tunicamycin (Tm). In cultured human intestinal secretory cells, in a glucocorticoid receptor-dependent manner, DEX suppressed ER stress and UPR activation induced by blocking N-glycosylation, reducing ER Ca(2+) or depleting glucose. DEX up-regulated genes encoding chaperones and elements of ER-associated degradation (ERAD), including EDEM1. Silencing EDEM1 partially inhibited DEX's suppression of misfolding-induced ER stress, showing that DEX enhances ERAD. DEX inhibited Tm-induced MUC2 precursor accumulation, promoted production of mature mucin, and restored ER exit and secretion of Winnie mutant recombinant Muc2 domains, consistent with enhanced protein folding. In IBD, glucocorticoids are likely to ameliorate ER stress by promoting correct folding of secreted proteins and enhancing removal of misfolded proteins from the ER.
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ItemMucin 1 (MUC1) is a novel partner for MAL2 in breast carcinoma cells(BMC, 2009-01-28)BACKGROUND: The MAL2 gene, encoding a four-transmembrane protein of the MAL family, is amplified and overexpressed in breast and other cancers, yet the significance of this is unknown. MAL-like proteins have trafficking functions, but their molecular roles are largely obscure, partly due to a lack of known binding partners. METHODS: Yeast two-hybrid screening of a breast carcinoma cDNA expression library was performed using a full-length MAL2 bait, and subsequent deletion mapping experiments were performed. MAL2 interactions were confirmed by co-immunoprecipitation analyses and confocal microscopy was employed to compare protein sub-cellular distributions. Sucrose density gradient centrifugation of membranes extracted in cold Triton X-100 was employed to compare protein distributions between Triton X-100-soluble and -insoluble fractions. RESULTS: The tumor-associated protein mucin 1 (MUC1) was identified as a potential MAL2 partner, with MAL2/MUC1 interactions being confirmed in myc-tagged MAL2-expressing MCF-10A cells using co-immunoprecipitation assays. Deletion mapping experiments demonstrated a requirement for the first MAL2 transmembrane domain for MUC1 binding, whereas the MAL2 N-terminal domain was required to bind D52-like proteins. Confocal microscopy identified cytoplasmic co-localisation of MUC1 and MAL2 in breast cell lines, and centrifugation of cell lysates to equilibrium in sucrose density gradients demonstrated that MAL2 and MUC1 proteins were co-distributed between Triton X-100-soluble and -insoluble fractions. However co-immunoprecipitation analyses detected MAL2/MUC1 interactions in Triton X-100-soluble fractions only. Myc-MAL2 expression in MCF-10A cells was associated with both increased MUC1 detection within Triton X-100-soluble and -insoluble fractions, and increased MUC1 detection at the cell surface. CONCLUSION: These results identify MUC1 as a novel MAL2 partner, and suggest a role for MAL2 in regulating MUC1 expression and/or localisation.
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ItemNo Preview AvailableInfection's Sweet Tooth: How Glycans Mediate Infection and Disease Susceptibility(ELSEVIER SCI LTD, 2018-02)Glycans form a highly variable constituent of our mucosal surfaces and profoundly affect our susceptibility to infection and disease. The diversity and importance of these surface glycans can be seen in individuals who lack a functional copy of the fucosyltransferase gene, FUT2. Representing around one-fifth of the population, these individuals have an altered susceptibility to many bacterial and viral infections and diseases. The mediation of host-pathogen interactions by mucosal glycans, such as those added by FUT2, is poorly understood. We highlight, with specific examples, important mechanisms by which host glycans influence infection dynamics, including by: acting as pathogen receptors (or receptor-decoys), promoting microbial stability, altering the physical characteristics of mucus, and acting as immunological markers. We argue that the effect glycans have on infection dynamics has profound implications for many aspects of healthcare and policy, including clinical management, outbreak control, and vaccination policy.
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ItemNo Preview AvailableEpithelial Sel1L is required for the maintenance of intestinal homeostasis(AMER SOC CELL BIOLOGY, 2016-02-01)Inflammatory bowel disease (IBD) is an incurable chronic idiopathic disease that drastically decreases quality of life. Endoplasmic reticulum (ER)-associated degradation (ERAD) is responsible for the clearance of misfolded proteins; however, its role in disease pathogenesis remains largely unexplored. Here we show that the expression of SEL1L and HRD1, the most conserved branch of mammalian ERAD, is significantly reduced in ileal Crohn's disease (CD). Consistent with this observation, laboratory mice with enterocyte-specific Sel1L deficiency (Sel1L(ΔIEC)) develop spontaneous enteritis and have increased susceptibility to Toxoplasma gondii-induced ileitis. This is associated with profound defects in Paneth cells and a disproportionate increase of Ruminococcus gnavus, a mucolytic bacterium with known association with CD. Surprisingly, whereas both ER stress sensor IRE1α and effector CHOP are activated in the small intestine of Sel1L(ΔIEC) mice, they are not solely responsible for ERAD deficiency-associated lesions seen in the small intestine. Thus our study points to a constitutive role of Sel1L-Hrd1 ERAD in epithelial cell biology and the pathogenesis of intestinal inflammation in CD.