Mucin dynamics and enteric pathogens McGuckin, Michael A; Lindén, Sara K; Sutton, Philip ...
Nature reviews. Microbiology,
04/2011, Volume:
9, Issue:
4
Journal Article
Peer reviewed
Open access
The extracellular secreted mucus and the cell surface glycocalyx prevent infection by the vast numbers of microorganisms that live in the healthy gut. Mucin glycoproteins are the major component of ...these barriers. In this Review, we describe the components of the secreted and cell surface mucosal barriers and the evidence that they form an effective barricade against potential pathogens. However, successful enteric pathogens have evolved strategies to circumvent these barriers. We discuss the interactions between enteric pathogens and mucins, and the mechanisms that these pathogens use to disrupt and avoid mucosal barriers. In addition, we describe dynamic alterations in the mucin barrier that are driven by host innate and adaptive immune responses to infection.
The airways mucus gel performs a critical function in defending the respiratory tract against pathogenic and environmental challenges. In normal physiology, the secreted mucins, in particular the ...polymeric mucins MUC5AC and MUC5B, provide the organizing framework of the airways mucus gel and are major contributors to its rheological properties. However, overproduction of mucins is an important factor in the morbidity and mortality of chronic airways disease (e.g., asthma, cystic fibrosis, and chronic obstructive pulmonary disease). The roles of these enormous, multifunctional, O-linked glycoproteins in health and disease are discussed.
Mucin glycoproteins are secreted in large quantities by mucosal epithelia and cell surface mucins are a prominent feature of the glycocalyx of all mucosal epithelia. Currently, studies investigating ...the gastrointestinal mucosal barrier use either animal experiments or non-in vivo like cell cultures. Many pathogens cause different pathology in mice compared to humans and the in vitro cell cultures used are suboptimal because they are very different from an in vivo mucosal surface, are often not polarized, lack important components of the glycocalyx, and often lack the mucus layer. Although gastrointestinal cell lines exist that produce mucins or polarize, human cell line models that reproducibly create the combination of a polarized epithelial cell layer, functional tight junctions and an adherent mucus layer have been missing until now. We trialed a range of treatments to induce polarization, 3D-organization, tight junctions, mucin production, mucus secretion, and formation of an adherent mucus layer that can be carried out using standard equipment. These treatments were tested on cell lines of intestinal (Caco-2, LS513, HT29, T84, LS174T, HT29 MTX-P8 and HT29 MTX-E12) and gastric (MKN7, MKN45, AGS, NCI-N87 and its hTERT Clone5 and Clone6) origins using Ussing chamber methodology and (immuno)histology. Semi-wet interface culture in combination with mechanical stimulation and DAPT caused HT29 MTX-P8, HT29 MTX-E12 and LS513 cells to polarize, form functional tight junctions, a three-dimensional architecture resembling colonic crypts, and produce an adherent mucus layer. Caco-2 and T84 cells also polarized, formed functional tight junctions and produced a thin adherent mucus layer after this treatment, but with less consistency. In conclusion, culture methods affect cell lines differently, and testing a matrix of methods vs. cell lines may be important to develop better in vitro models. The methods developed herein create in vitro mucosal surfaces suitable for studies of host-pathogen interactions at the mucosal surface.
The bacterium Helicobacter pylori can cause peptic ulcer disease, gastric adenocarcinoma and MALT lymphoma. The cell-surface mucin MUC1 is a large glycoprotein which is highly expressed on the ...mucosal surface and limits the density of H. pylori in a murine infection model. We now demonstrate that by using the BabA and SabA adhesins, H. pylori bind MUC1 isolated from human gastric cells and MUC1 shed into gastric juice. Both H. pylori carrying these adhesins, and beads coated with MUC1 antibodies, induced shedding of MUC1 from MKN7 human gastric epithelial cells, and shed MUC1 was found bound to H. pylori. Shedding of MUC1 from non-infected cells was not mediated by the known MUC1 sheddases ADAM17 and MMP-14. However, knockdown of MMP-14 partially affected MUC1 release early in infection, whereas ADAM17 had no effect. Thus, it is likely that shedding is mediated both by proteases and by disassociation of the non-covalent interaction between the alpha- and beta-subunits. H. pylori bound more readily to MUC1 depleted cells even when the bacteria lacked the BabA and SabA adhesins, showing that MUC1 inhibits attachment even when bacteria cannot bind to the mucin. Bacteria lacking both the BabA and SabA adhesins caused less apoptosis in MKN7 cells than wild-type bacteria, having a greater effect than deletion of the CagA pathogenicity gene. Deficiency of MUC1/Muc1 resulted in increased epithelial cell apoptosis, both in MKN7 cells in vitro, and in H. pylori infected mice. Thus, MUC1 protects the epithelium from non-MUC1 binding bacteria by inhibiting adhesion to the cell surface by steric hindrance, and from MUC1-binding bacteria by acting as a releasable decoy.
The inability of pancreatic β-cells to make sufficient insulin to control blood sugar is a central feature of the aetiology of most forms of diabetes. In this review we focus on the deleterious ...effects of oxidative stress and endoplasmic reticulum (ER) stress on β-cell insulin biosynthesis and secretion and on inflammatory signalling and apoptosis with a particular emphasis on type 2 diabetes (T2D). We argue that oxidative stress and ER stress are closely entwined phenomena fundamentally involved in β-cell dysfunction by direct effects on insulin biosynthesis and due to consequences of the ER stress-induced unfolded protein response. We summarise evidence that, although these phenomenon can be driven by intrinsic β-cell defects in rare forms of diabetes, in T2D β-cell stress is driven by a range of local environmental factors including increased drivers of insulin biosynthesis, glucolipotoxicity and inflammatory cytokines. We describe our recent findings that a range of inflammatory cytokines contribute to β-cell stress in diabetes and our discovery that interleukin 22 protects β-cells from oxidative stress regardless of the environmental triggers and can correct much of diabetes pathophysiology in animal models. Finally we summarise evidence that β-cell dysfunction is reversible in T2D and discuss therapeutic opportunities for relieving oxidative and ER stress and restoring glycaemic control.
Abstract
Mucus is characterized by multiple levels of assembly at different length scales which result in a unique set of rheological (flow) and mechanical properties. These physical properties ...determine its biological function as a highly selective barrier for transport of water and nutrients, while blocking penetration of pathogens and foreign particles. Altered integrity of the mucus layer in the small intestine has been associated with a number of gastrointestinal tract pathologies such as Crohn’s disease and cystic fibrosis. In this work, we uncover an intricate hierarchy of intestinal mucin (Muc2) assembly and show how complex rheological properties emerge from synergistic interactions between mucin glycoproteins, non-mucin proteins, and Ca
2+
. Using a novel method of mucus purification, we demonstrate the mechanism of assembly of Muc2 oligomers into viscoelastic microscale domains formed via hydrogen bonding and Ca
2+
-mediated links, which require the joint presence of Ca
2+
ions and non-mucin proteins. These microscale domains aggregate to form a heterogeneous yield stress gel-like fluid, the macroscopic rheological properties of which are virtually identical to that of native intestinal mucus. Through proteomic analysis, we short-list potential protein candidates implicated in mucin assembly, thus paving the way for identifying the molecules responsible for the physiologically critical biophysical properties of mucus.
Endoplasmic reticulum (ER) stress may be both a trigger and consequence of chronic inflammation. Chronic inflammation is often associated with diseases that arise because of primary misfolding ...mutations and ER stress. Similarly, ER stress and activation of the unfolded protein response (UPR) is a feature of many chronic inflammatory and autoimmune diseases. In this review, we describe how protein misfolding and the UPR trigger inflammation, how environmental ER stressors affect antigen presenting cells and immune effector cells, and present evidence that inflammatory factors exacerbate protein misfolding and ER stress. Examples from both animal models of disease and human diseases are used to illustrate the complex interactions between ER stress and inflammation, and opportunities for therapeutic targeting are discussed. Finally, recommendations are made for future research with respect to the interaction of ER stress and inflammation.
Mucosa-associated bacteria are increased in inflammatory bowel disease (IBD), which suggests the possibility of an increased source of digestible endogenous mucus substrate. We hypothesized that ...mucolytic bacteria are increased in IBD, providing increased substrate to sustain nonmucolytic mucosa-associated bacteria.
Mucolytic bacteria were characterized by the ability to degrade human secretory mucin (MUC2) in pure and mixed anaerobic cultures. Real-time PCR was used to enumerate mucosa-associated mucolytic bacteria in 46 IBD and 20 control patients. Bacterial mucolytic activity was tested in vitro using purified human MUC2.
We confirm increased total mucosa-associated bacteria 16S rRNA gene in macroscopically and histologically normal intestinal epithelium of both Crohn's disease (CD) (mean 1.9-fold) and ulcerative colitis (UC) (mean 1.3-fold). We found a disproportionate increase in some mucolytic bacteria. Mean Ruminococcus gnavus were increased >4-fold and Ruminococcus torques ∼100-fold in macroscopically and histologically normal intestinal epithelium of both CD and UC. The most abundantly detected mucolytic bacterium in controls, Akkermansia muciniphila, was reduced many fold in CD and in UC. Coculture of A. muciniphila with MUC2 as the sole carbon source led to reduction in its abundance while it augmented growth of other bacteria.
Mucolytic bacteria are present in healthy humans, where they are an integral part of the mucosa-associated bacterial consortium. The disproportionate increase in R. gnavus and R. torques could explain increased total mucosa-associated bacteria in IBD.
Objective
The spondyloarthritides share genetic susceptibility, interleukin‐23 (IL‐23) dependence, and the involvement of microbiota. The aim of the current study was to elucidate how host genetics ...influence gut microbiota and the relationship between microbiota and organ inflammation in spondyloarthritides.
Methods
BALB/c ZAP‐70W163C–mutant (SKG) mice, Toll‐like receptor 4 (TLR‐4)–deficient SKG mice, and wild‐type BALB/c mice were housed under specific pathogen–free conditions. SKG and wild‐type BALB/c mice were maintained under germ‐free conditions, and some of these mice were recolonized with altered Schaedler flora. All of the mice were injected intraperitoneally with microbial β‐1,3‐glucan (curdlan). Arthritis, spondylitis, and ileitis were assessed histologically. Microbiome composition was analyzed in serial fecal samples obtained from mice that were co‐housed beginning at the time of weaning, using 454 pyrosequencing. Infiltrating cells and cytokines in the peritoneal cavity were measured by flow cytometry and enzyme‐linked immunosorbent assay. Cytokine, endoplasmic reticulum (ER) stress marker, and tight junction protein transcription was measured by quantitative real‐time polymerase chain reaction.
Results
Microbiota content and response to curdlan varied according to whether T cell receptor signal strength was normal or was impaired due to the ZAP‐70W163C mutation. Curdlan triggered acute inflammation regardless of the presence of the SKG allele or microbiota. However, no or limited microbiota content attenuated the severity of arthritis. In contrast, ileal IL‐23 expression, ER stress, lymph node IL‐17A production, goblet cell loss, and ileitis development were microbiota‐dependent. Ileitis but not arthritis was suppressed by microbiota transfer upon co‐housing SKG mice with wild‐type BALB/c mice, as well as by TLR‐4 deficiency.
Conclusion
The interaction between immunogenetic background and host microbiota leads to an IL‐23–dependent loss of mucosal function, triggering ileitis in response to curdlan.
Background & Aims Protein misfolding and endoplasmic reticulum (ER) stress have been observed in intestinal secretory cells from patients with inflammatory bowel diseases and induce intestinal ...inflammation in mice. However, it is not clear how immune factors affect ER stress and therefore disease symptoms. Methods We analyzed the effects of interleukin (IL)-10 on ER stress in intestinal tissues in wild-type C57BL/6, Winnie , IL-10 −/− , and Winnie × IL-10 +/− mice. In Winnie mice, misfolding of the intestinal mucin Muc2 initiates ER stress and inflammation. We also analyzed the effects of different inhibitors of IL-10 signaling and the N -glycosylation inhibitor tunicamycin in cultured human LS174T goblet cells. Results Administration of neutralizing antibodies against IL-10 or its receptor (IL-10R1) to Winnie mice rapidly exacerbated ER stress and intestinal inflammation compared with mice given vehicle (controls). Antibodies against IL-10 also increased accumulation of misfolded Muc2 in the ER of goblet cells of Winnie mice and increased T-cell production of inflammatory cytokines. Winnie × IL-10 +/− mice and IL-10 −/− mice with a single Winnie allele each developed more severe inflammation than Winnie mice or IL-10 −/− mice. Administration of tunicamycin to wild-type mice caused intestinal ER stress, which increased when IL-10R1 was blocked. In LS174T cells, induction of ER stress with tunicamycin and misfolding of MUC2 were reduced by administration of IL-10; this reduction required STAT1 and STAT3. In LS174T cells incubated with tunicamycin, IL-10 up-regulated genes involved in MUC2 folding and in ER-associated degradation and maintained correct folding of MUC2, its transport from the ER, and its O -glycosylation and secretion. Conclusions IL-10 prevents protein misfolding and ER stress by maintaining mucin production in goblet cells and helps the intestine preserve the mucus barrier.
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