Clinical and Molecular Analyses of Intestinal Goblet Cell Acidomucins and Cysteine Metabolism

Goblet cells are key contributors to intestinal mucosal barrier function through their role in production of mucins and other important secretory products, which include trefoil factor 3 (TFF3) and resistin like molecule ?? (RETNLB). Goblet cell mucins show great structural heterogeneity but can be broadly classified into neutral and acidic subtypes (chemotypes), which can be further classified into sulfomucins or sialomucins based on the presence of terminal sulfate or sialic acid groups on the oligosaccharide chains. Sulfomucins and sialomucins vary regionally throughout the gastrointestinal tract and changes in their expression have been observed in diseases such as inflammatory bowel disease and colorectal cancer. It is likely that there is variation in these chemotypes among individuals and that both regional and interindividual variation in sulfo- and sialomucins may influence microbial colonization. However, quantitative data and a description of interindividual variation in sulfo- and sialomucin content in the human colon are lacking. There is an absence of data describing the relationship between microbial species and these mucin chemotypes in the human colon. In Chapter 2, we begin to fill these gaps in the literature via a pilot study examining sulfo- and sialomucins in the healthy human colon and their relationship with sulfate reducing bacteria (SRB). Quantitative differences in mucin abundance among specific regions of the colon and individuals are described. In addition, we observe that the relationship between these mucin chemotypes and SRB is not influenced by location in the colon, but rather by the host. In Chapter 3, we begin to identify factors that may contribute to changes in sulfomucin in disease and possibly interindividual variation, using human adenocarcinoma-derived LS174T cells, which have a goblet cell-like phenotype and produce both sulfo- and sialomucins. We specifically focused on the effects of bacterial flagellin, IL-13, and TNF?? on the expression of genes encoding the major secretory mucin, MUC2, and Golgi sulfotransfereases, CHST5 and GAL3ST2. In addition, expression of sulfomucin and Sulfo Lea antigen, which is synthesized in part by GAL3ST2, was examined. Overall, the results indicated that both host and microbial factors influence sulfomucin expression, possibly via modulation of CHST5 and GAL3ST2 expression. Because goblet cells synthesize and secrete cysteine-rich products, including mucins, TFF3, and RETNLB, they may have a high cysteine requirement. Furthermore, they may require additional cysteine, which can be catabolized to sulfate, for mucin sulfation. Cysteine is also used in the production of other proteins as well as essential molecules including glutathione, taurine, and pyruvate. However, high cysteine levels are cytotoxic. Mammals regulate cysteine metabolism to maintain levels within a range that is sufficient for synthesis of essential molecules but below the level of cytotoxicity through the use of cysteine dioxygenase (CDO), which catalyzes the first step in cysteine catabolism. In Chapter 4, CDO expression and localization in mouse small and large intestine is examined using immunohistochemical and immunofluorescence staining techniques. We observed that CDO is expressed in goblet cells as well as Paneth and enteroendocrine cells, which are also members of the secretory lineage, while expression was absent in absorptive enterocytes. We postulate that this striking difference in CDO expression between the absorptive and secretory cell lineages is due to higher cysteine catabolism requirements in goblet, Paneth, and enteroendocrine cells relative to absorptive epithelial cells, either to synthesize additional taurine and sulfate or to metabolize excess cysteine when cells are not actively synthesizing cysteine-rich secretory products. In Chapter 5, we examine the possibility that goblet cells are using CDO to synthesize taurine and determine whether inflammatory stimuli alter taurine biosynthesis, using the LS174T cell line as a model. There are two pathways for taurine synthesis. One pathway involves the oxidation of cysteine via CDO to cysteinesulfinate, which is decarboxylated by cysteinesulfinic acid decarboxylase (CSAD) to hypotaurine. The other pathway involves the conversion of cysteine to Coenzyme A, which releases cysteamine during turnover. Cysteamine is then oxidized to hypotaurine via cysteamine dioxygenase (ADO). In both pathways, hypotaurine is then oxidized to taurine. We confirm that LS174T cells synthesize taurine via the ADO pathway and possibly the CDO/CSAD pathway. We also demonstrate that certain inflammatory triggers can enhance taurine biosynthesis and/or export, which may provide taurine for other cell types in an in vivo setting. The anti-inflammatory effects of taurine in the intestine have been described, so these results may represent a novel mechanism by which goblet cells attenuate inflammation. Overall, the findings described in this dissertation provide novel information regarding the role of intestinal goblet cells in mucosal barrier function

Protective effect of stromal Dickkopf-3 in prostate cancer: opposing roles for TGFBI and ECM-1

Aberrant transforming growth factor–β (TGF-β) signaling is a hallmark of the stromal microenvironment in cancer. Dickkopf-3 (Dkk-3), shown to inhibit TGF-β signaling, is downregulated in prostate cancer and upregulated in the stroma in benign prostatic hyperplasia, but the function of stromal Dkk-3 is unclear. Here we show that DKK3 silencing in WPMY-1 prostate stromal cells increases TGF-β signaling activity and that stromal cellconditioned media inhibit prostate cancer cell invasion in a Dkk-3-dependent manner. DKK3 silencing increased the level of the cell-adhesion regulator TGF-β–induced protein (TGFBI) in stromal and epithelial cell-conditioned media, and recombinant TGFBI increased prostate cancer cell invasion. Reduced expression of Dkk-3 in patient tumors was associated with increased expression of TGFBI. DKK3 silencing reduced the level of extracellular matrix protein-1 (ECM-1) in prostate stromal cell-conditioned media but increased it in epithelial cell-conditioned media, and recombinant ECM-1 inhibited TGFBI-induced prostate cancer cell invasion. Increased ECM1 and DKK3 mRNA expression in prostate tumors was associated with increased relapse-free survival. These observations are consistent with a model in which the loss of Dkk-3 in prostate cancer leads to increased secretion of TGFBI and ECM-1, which have tumor-promoting and tumor-protective roles, respectively. Determining how the balance between the opposing roles of extracellular factors influences prostate carcinogenesis will be key to developing therapies that target the tumor microenvironment

Pyrosequencing-Based Analysis of the Mucosal Microbiota in Healthy Individuals Reveals Ubiquitous Bacterial Groups and Micro-Heterogeneity

This study used 16S rRNA-based pyrosequencing to examine the microbial community that is closely associated with the colonic mucosa of five healthy individuals. Spatial heterogeneity in microbiota was measured at right colon, left colon and rectum, and between biopsy duplicates spaced 1 cm apart. The data demonstrate that mucosal-associated microbiota is comprised of Firmicutes (50.9%±21.3%), Bacteroidetes (40.2%±23.8%) and Proteobacteria (8.6%±4.7%), and that interindividual differences were apparent. Among the genera, Bacteroides, Leuconostoc and Weissella were present at high abundance (4.6% to 41.2%) in more than 90% of the studied biopsy samples. Lactococcus, Streptococcus, Acidovorax, Acinetobacter, Blautia, Faecalibacterium, Veillonella, and several unclassified bacterial groups were also ubiquitously present at an abundance <7.0% of total microbial community. With the exception of one individual, the mucosal-associated microbiota was relatively homogeneous along the colon (average 61% Bray-Curtis similarity). However, micro-heterogeneity was observed in biopsy duplicates within defined colonic sites for three of the individuals. A weak but significant Mantel correlation of 0.13 was observed between the abundance of acidomucins and mucosal-associated microbiota (P-value  =  0.04), indicating that the localized biochemical differences may contribute in part to the micro-heterogeneity. This study provided a detailed insight to the baseline mucosal microbiota along the colon, and revealed the existence of micro-heterogeneity within defined colonic sites for certain individuals

On the Relationship between Sialomucin and Sulfomucin Expression and Hydrogenotrophic Microbes in the Human Colonic Mucosa

The colonic mucus layer is comprised primarily of acidomucins, which provide viscous properties and can be broadly classified into sialomucins or sulfomucins based on the presence of terminating sialic acid or sulfate groups. Differences in acidomucin chemotypes have been observed in diseases such as colorectal cancer and inflammatory bowel disease, and variation in sialo- and sulfomucin content may influence microbial colonization. For example, sulfate derived from sulfomucin degradation may promote the colonization of sulfate-reducing bacteria (SRB), which through sulfate respiration generate the genotoxic gas hydrogen sulfide. Here, paired biopsies from right colon, left colon, and rectum of 20 subjects undergoing routine screening colonoscopies were collected to enable parallel histochemical and microbiological studies. Goblet cell sialo- and sulfomucins in each biopsy were distinguished histochemically and quantified. Quantitative PCR and multivariate analyses were used to examine the abundance of hydrogenotrophic microbial groups and SRB genera relative to acidomucin profiles. Regional variation was observed in sialomucins and sulfomucins with the greatest abundance of each found in the rectum. Mucin composition did not appear to influence the abundance of SRB or other hydrogenotrophic microbiota but correlated with the composition of different SRB genera. A higher sulfomucin proportion correlated with higher quantities of Desulfobacter, Desulfobulbus and Desulfotomaculum, relative to the predominant Desulfovibrio genus. Thus, acidomucin composition may influence bacterial sulfate respiration in the human colon, which may in turn impact mucosal homeostasis. These results stress the need to consider mucus characteristics in the context of studies of the microbiome that target intestinal diseases

The Border in Ireland and Trade, Déjà vu time again!

During December’s general election campaign, PM Johnson promised that he had an “oven ready” withdrawal deal he’d agreed with the EU. As he claimed incessantly, the only impediment to its implementation was Parliament’s refusal to fulfil the will of the people. Support for the Conservatives, he faithfully assured potential voters, would “GetBrexit done”

Data from: Anthrax toxin receptor 1 is essential for arteriogenesis in a mouse model of hindlimb ischemia

Anthrax toxin receptor 1/tumor endothelial marker 8 (Antxr1 or TEM8) is up-regulated in tumor vasculature and serves as a receptor for anthrax toxin, but its physiologic function is unclear. The objective of this study was to evaluate the role of Antxr1 in arteriogenesis. The role of Antxr1 in arteriogenesis was tested by measuring gene expression and immunohistochemistry in a mouse model of hindlimb ischemia using wild-type and ANTXR1-/- mice. Additional tests were performed by measuring gene expression in in vitro models of fluid shear stress and hypoxia, as well as in human muscle tissues obtained from patients having peripheral artery disease. We observed that Antxr1 expression transiently increased in ischemic tissues following femoral artery ligation and that its expression was necessary for arteriogenesis. In the absence of Antxr1, the mean arterial lumen area in ischemic tissues decreased. Antxr1 mRNA and protein expression was positively regulated by fluid shear stress, but not by hypoxia. Furthermore, Antxr1 expression was elevated in human peripheral artery disease requiring lower extremity bypass surgery. These findings demonstrate an essential physiologic role for Antxr1 in arteriogenesis and peripheral artery disease, with important implications for managing ischemia and other arteriogenesis-dependent vascular diseases

Assessment of the volume of SWCNT aggregates in the lung tissue sections from w/t and MPO k/o mice.

<p>a. Representative images of the lung tissue sections. Insert - higher magnifigation (2.5× zoom) of a field illustrating the presence of SWCNT (green punctuate spots pointed by white arrows). b. Quantitation of SWCNT aggregates (SWCNT volume/total lung volume) using their specific absorbance (750–850 nm), * p<0.05, <i>vs</i> w/t 1 day post exposure, # p<0.05, <i>vs</i> w/t 28 days post exposure. c. Assessment of SWCNT aggregates - number per microscopic field - using an automated IN Cell Analyser 1000 microscope, * p<0.05, <i>vs</i> w/t 1 day post exposure, # p<0.05, <i>vs</i> w/t 28 days post exposure.</p

Characterization of pulmonary inflammatory responses to SWCNT in w/t and MPO k/o mice at day 1 after pharyngeal aspiration exposure.

<p>a–c. Levels of pro-inflammatory cytokines (a - TNF-α; b - IL-6; c – MCP-1) in BAL fluid of w/t and MPO k/o mice. Mean ± SEM (n = 6 mice/group). *p<0.05, <i>vs</i> control PBS-exposed mice. d. Content of PMNs in BAL fluid of w/t and MPO k/o mice. Mean ± SEM (n = 6 mice/group). *p<0.05, <i>vs</i> control PBS-exposed mice. e. Typical microscopic images of inflammatory cells in BAL fluid with SWCNT inclusions (red arrows). f. Content of PMNs with engulfed SWCNT in BAL fluid of w/t and MPO k/o mice. Mean ± SEM (n = 6 mice/group). *p<0.05, <i>vs</i> w/t mice.</p