Sjoerd van der Post

The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system

The gastrointestinal tract is covered by mucus that has different properties in the stomach, small intestine, and colon. The large highly glycosylated gel‐forming mucins MUC2 and MUC5AC are the major components of the mucus in the intestine and stomach, respectively. In the small intestine, mucus limits the number of bacteria that can reach the epithelium and the Peyers patches. In the large intestine, the inner mucus layer separates the commensal bacteria from the host epithelium. The outer colonic mucus layer is the natural habitat for the commensal bacteria. The intestinal goblet cells secrete not only the MUC2 mucin but also a number of typical mucus components: CLCA1, FCGBP, AGR2, ZG16, and TFF3. The goblet cells have recently been shown to have a novel gate‐keeping role for the presentation of oral antigens to the immune system. Goblet cells deliver small intestinal luminal material to the lamina propria dendritic cells of the tolerogenic CD103+ type. In addition to the gel‐forming mucins, the transmembrane mucins MUC3, MUC12, and MUC17 form the enterocyte glycocalyx that can reach about a micrometer out from the brush border. The MUC17 mucin can shuttle from a surface to an intracellular vesicle localization, suggesting that enterocytes might control and report epithelial microbial challenge. There is communication not only from the epithelial cells to the immune system but also in the opposite direction. One example of this is IL10 that can affect and improve the properties of the inner colonic mucus layer. The mucus and epithelial cells of the gastrointestinal tract are the primary gate keepers and controllers of bacterial interactions with the host immune system, but our understanding of this relationship is still in its infancy.

Composition and functional role of the mucus layers in the intestine

In discussions on intestinal protection, the protective capacity of mucus has not been very much considered. The progress in the last years in understanding the molecular nature of mucins, the main building blocks of mucus, has, however, changed this. The intestinal enterocytes have their apical surfaces covered by transmembrane mucins and the whole intestinal surface is further covered by mucus, built around the gel-forming mucin MUC2. The mucus of the small intestine has only one layer, whereas the large intestine has a two-layered mucus where the inner, attached layer has a protective function for the intestine, as it is impermeable to the luminal bacteria.

Function of the CysD domain of the gel-forming MUC2 mucin.

The colonic human MUC2 mucin forms a polymeric gel by covalent disulfide bonds in its N- and C-termini. The middle part of MUC2 is largely composed of two highly O-glycosylated mucin domains that are interrupted by a CysD domain of unknown function. We studied its function as recombinant proteins fused to a removable immunoglobulin Fc domain. Analysis of affinity-purified fusion proteins by native gel electrophoresis and gel filtration showed that they formed oligomeric complexes. Analysis of the individual isolated CysD parts showed that they formed dimers both when flanked by two MUC2 tandem repeats and without these. Cleavages of the two non-reduced CysD fusion proteins and analysis by MS revealed the localization of all five CysD disulfide bonds and that the predicted C-mannosylated site was not glycosylated. All disulfide bonds were within individual peptides showing that the domain was stabilized by intramolecular disulfide bonds and that CysD dimers were of non-covalent nature. These observations suggest that CysD domains act as non-covalent cross-links in the MUC2 gel, thereby determining the pore sizes of the mucus.

Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB)

Background: MUC2 polymers form the mucus layer of colon that separates luminal bacteria from the epithelium. Results: P. gingivalis secrets a protease that cleaves the MUC2 mucin, a cleavage modulated by O-glycosylation. Conclusion: Bacteria can disrupt the MUC2 polymer via proteolytic cleavage. However, O-glycosylation can inhibit this process. Significance: Bacteria can dissolve the protective inner mucus layer, potentially triggering colitis. The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic peptide covering the IRTT sequence. Only GalNAc-T3 was able to glycosylate the second Thr in IRTT, rendering the sequence resistant to cleavage by RgpB. Furthermore, when GalNAc-T3 was expressed in CHO cells expressing the MUC2 C terminus, the second threonine was glycosylated, and the protein became resistant to RgpB cleavage. These findings suggest that bacteria can produce proteases capable of dissolving the inner protective mucus layer by specific cleavages in the MUC2 mucin and that this cleavage can be modulated by site-specific O-glycosylation.

Characterization of human thymic exosomes.

Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytometry, proteomic analysis and microRNA profiling we describe the morphology, size, density, protein composition and microRNA content of human thymic exosomes. The thymic exosomes share characteristics with previously described exosomes such as antigen presentation molecules, but they also exhibit thymus specific features regarding surface markers, protein content and microRNA profile. Interestingly, thymic exosomes carry proteins that have a tissue restricted expression in the periphery which may suggest a role in T cell selection and the induction of central tolerance. We speculate that thymic exosomes may provide the means for intercellular information exchange necessary for negative selection and regulatory T cell formation of the developing thymocytes within the human thymic medulla.

Inhibition of Cyclooxygenase-2 Prevents Chronic and Recurrent Cystitis

The spread of multidrug-resistant microorganisms globally has created an urgent need for novel therapeutic strategies to combat urinary tract infections (UTIs). Immunomodulatory therapy may provide benefit, as treatment of mice with dexamethasone during acute UTI improved outcome by reducing the development of chronic cystitis, which predisposes to recurrent infection. Here we discovered soluble biomarkers engaged in myeloid cell development and chemotaxis that were predictive of future UTI recurrence when elevated in the sera of young women with UTI. Translation of these findings revealed that temperance of the neutrophil response early during UTI, and specifically disruption of bladder epithelial transmigration of neutrophils by inhibition of cyclooxygenase-2, protected mice against chronic and recurrent cystitis. Further, proteomics identified bladder epithelial remodeling consequent to chronic infection that enhances sensitivity to neutrophil damage. Thus, cyclooxygenase-2 expression during acute UTI is a critical molecular trigger determining disease outcome and drugs targeting cyclooxygenase-2 could prevent recurrent UTI.

Altered Expression of Autoimmune Regulator in Infant Down Syndrome Thymus, a Possible Contributor to an Autoimmune Phenotype

Down syndrome (DS), caused by trisomy of chromosome 21, is associated with immunological dysfunctions such as increased frequency of infections and autoimmune diseases. Patients with DS share clinical features, such as autoimmune manifestations and specific autoantibodies, with patients affected by autoimmune polyendocrine syndrome type 1. Autoimmune polyendocrine syndrome type 1 is caused by mutations in the autoimmune regulator (AIRE) gene, located on chromosome 21, which regulates the expression of tissue-restricted Ags (TRAs) in thymic epithelial cells. We investigated the expression of AIRE and TRAs in DS and control thymic tissue using quantitative PCR. AIRE mRNA levels were elevated in thymic tissue from DS patients, and trends toward increased expression of the AIRE-controlled genes INSULIN and CHRNA1 were found. Immunohistochemical stainings showed altered cell composition and architecture of the thymic medulla in DS individuals with increased frequencies of AIRE-positive medullary epithelial cells and CD11c-positive dendritic cells as well as enlarged Hassall’s corpuscles. In addition, we evaluated the proteomic profile of thymic exosomes in DS individuals and controls. DS exosomes carried a broader protein pool and also a larger pool of unique TRAs compared with control exosomes. In conclusion, the increased AIRE gene dose in DS could contribute to an autoimmune phenotype through multiple AIRE-mediated effects on homeostasis and function of thymic epithelial cells that affect thymic selection processes.

Proteomic Study of the Mucin Granulae in an Intestinal Goblet Cell Model

Goblet cells specialize in producing and secreting mucus with its main component, mucins. An inducible goblet-like cell line was used for the purification of the mucus vesicles stored in these cells by density gradient ultracentrifugation, and their proteome was analyzed by nanoLC-MS and MS/MS. Although the density of these vesicles coincides with others, it was possible to reveal a number of proteins that after immunolocalization on colon tissue and functional analyses were likely to be linked to the MUC2 vesicles. Most of the proteins were associated with the vesicle membrane or their outer surface. The ATP6AP2, previously suggested to be associated with vesicular proton pumps, was colocalized with MUC2 without other V-ATPase proteins and, thus, probably has roles in mucin vesicle function yet to be discovered. FAM62B, known to be a calcium-sensitive protein involved in vesicle fusion, also colocalized with the MUC2 vesicles and is probably involved in unknown ways in the later events of the MUC2 vesicles and their secretion.

Membrane Protein Profiling of Human Colon Reveals Distinct Regional Differences

The colonic epithelium is a highly dynamic system important for the regulation of ion and water homeostasis via absorption and secretion and for the maintenance of a protective barrier between the outer milieu and the inside of the body. These processes are known to gradually change along the length of the colon, although a complete characterization at the protein level is lacking. We therefore analyzed the membrane proteome of isolated human (n = 4) colonic epithelial cells from biopsies obtained via routine colonoscopy for four segments along the large intestine: ascending, transverse, descending, and sigmoid colon. Label-free quantitative proteomic analyses using high-resolution mass spectrometry were performed on enriched membrane proteins. The results showed a stable level for the majority of membrane proteins but a distinct decrease in proteins associated with bacterial sensing, cation transport, and O-glycosylation in the proximal to distal regions. In contrast, proteins involved in microbial defense and anion transport showed an opposing gradient and increased toward the distal end. The gradient of ion-transporter proteins could be directly related to previously observed ion transport activities. All individual glycosyltransferases required for the O-glycosylation of the major colonic mucin MUC2 were observed and correlated with the known glycosylation variation along the colon axis. This is the first comprehensive quantitative dataset of membrane protein abundance along the human colon and will add to the knowledge of the physiological function of the different regions of the colonic mucosa. Mass spectrometry data have been deposited to the ProteomeXchange with the identifier PXD000987.

Structural characterization of bacterioferritin from Blastochloris viridis.

Iron storage and elimination of toxic ferrous iron are the responsibility of bacterioferritins in bacterial species. Bacterioferritins are capable of oxidizing iron using molecular oxygen and import iron ions into the large central cavity of the protein, where they are stored in a mineralized form. We isolated, crystallized bacterioferritin from the microaerophilic/anaerobic, purple non-sulfur bacterium Blastochloris viridis and determined its amino acid sequence and X-ray structure. The structure and sequence revealed similarity to other purple bacterial species with substantial differences in the pore regions. Static 3- and 4-fold pores do not allow the passage of iron ions even though structural dynamics may assist the iron gating. On the other hand the B-pore is open to water and larger ions in its native state. In order to study the mechanism of iron import, multiple soaking experiments were performed. Upon Fe(II) and urea treatment the ferroxidase site undergoes reorganization as seen in bacterioferritin from Escherichia coli and Pseudomonas aeruginosa. When soaking with Fe(II) only, a closely bound small molecular ligand is observed close to Fe1 and the coordination of Glu94 to Fe2 changes from bidentate to monodentate. DFT calculations indicate that the bound ligand is most likely a water or a hydroxide molecule representing a product complex. On the other hand the different soaking treatments did not modify the conformation of other pore regions.