RESUMO
To close the gap between ultra-hygienic research mouse models and the much more environmentally exposed conditions of humans, we have established a system where laboratory mice are raised under a full set of environmental factors present in a naturalistic, farmyard-type habitat-a process we have called feralization. In previous studies we have shown that feralized (Fer) mice were protected against colorectal cancer when compared to conventionally reared laboratory mice (Lab). However, the protective mechanisms remain to be elucidated. Disruption of the protective intestinal barrier is an acknowledged player in colorectal carcinogenesis, and in the current study we assessed colonic mucosal barrier properties in healthy, feralized C57BL/6JRj male mice. While we found no effect of feralization on mucus layer properties, higher expression of genes encoding the mucus components Fcgbp and Clca1 still suggested mucus enforcement due to feralization. Genes encoding other proteins known to be involved in bacterial defense (Itln1, Ang1, Retnlb) and inflammatory mechanisms (Zbp1, Gsdmc2) were also higher expressed in feralized mice, further suggesting that the Fer mice have an altered intestinal mucosal barrier. These findings demonstrate that microbial experience conferred by housing in a farmyard-type environment alters the intestinal barrier properties in mice possibly leading to a more robust protection against disease. Future studies to unravel regulatory roles of feralization on intestinal barrier should aim to conduct proteomic analyses and in vivo performance of the feralized mice intestinal barrier.
Assuntos
Animais de Laboratório , Colo , Fazendas , Abrigo para Animais , Mucosa Intestinal , Laboratórios , Animais , Feminino , Masculino , Camundongos , Animais de Laboratório/microbiologia , Animais de Laboratório/fisiologia , Colo/microbiologia , Colo/fisiologia , Microbioma Gastrointestinal , Regulação da Expressão Gênica , Íleo/microbiologia , Íleo/fisiologia , Mucosa Intestinal/anatomia & histologia , Mucosa Intestinal/crescimento & desenvolvimento , Mucosa Intestinal/microbiologia , Mucosa Intestinal/fisiologia , Camundongos Endogâmicos C57BLRESUMO
A key feature in intestinal immunity is the dynamic intestinal barrier, which separates the host from resident and pathogenic microbiota through a mucus gel impregnated with antimicrobial peptides. Using a forward genetic screen, we have found a mutation in Tvp23b, which conferred susceptibility to chemically induced and infectious colitis. Trans-Golgi apparatus membrane protein TVP23 homolog B (TVP23B) is a transmembrane protein conserved from yeast to humans. We found that TVP23B controls the homeostasis of Paneth cells and function of goblet cells, leading to a decrease in antimicrobial peptides and more penetrable mucus layer. TVP23B binds with another Golgi protein, YIPF6, which is similarly critical for intestinal homeostasis. The Golgi proteomes of YIPF6 and TVP23B-deficient colonocytes have a common deficiency of several critical glycosylation enzymes. TVP23B is necessary for the formation of the sterile mucin layer of the intestine and its absence disturbs the balance of host and microbe in vivo.
Assuntos
Mucosa Intestinal , Intestinos , Proteínas de Membrana , Animais , Camundongos , Microbioma Gastrointestinal , Glicosilação , Células Caliciformes/metabolismo , Complexo de Golgi/metabolismo , Homeostase , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Intestinos/metabolismo , Proteínas de Membrana/metabolismo , Muco , Celulas de Paneth/metabolismoRESUMO
The metalloproteases meprin α and meprin ß are highly expressed in the healthy gut but significantly decreased in inflammatory bowel disease, implicating a protective role in mucosal homeostasis. In the colon, meprin α and meprin ß form covalently linked heterodimers tethering meprin α to the plasma membrane, therefore presenting dual proteolytic activity in a unique enzyme complex. To unravel its function, we applied N-terminomics and identified galectin-3 as the major intestinal substrate for meprin α/ß heterodimers. Galectin-3-deficient and meprin α/ß double knockout mice show similar alterations in their microbiome in comparison to wild-type mice. We further demonstrate that meprin α/ß heterodimers differentially process galectin-3 upon bacterial infection, in germ-free, conventionally housed (specific pathogen-free), or wildling mice, which in turn regulates the bacterial agglutination properties of galectin-3. Thus, the constitutive cleavage of galectin-3 by meprin α/ß heterodimers may play a key role in colon host-microbiome homeostasis.
Assuntos
Galectina 3 , Metaloendopeptidases , Camundongos , Animais , Galectina 3/genética , Galectina 3/metabolismo , Metaloproteases/metabolismo , Proteólise , Camundongos Knockout , HomeostaseRESUMO
The intestinal tract is an ecosystem in which the resident microbiota lives in symbiosis with its host. This symbiotic relationship is key to maintaining overall health, with dietary habits of the host representing one of the main external factors shaping the microbiome-host relationship. Diets high in fiber and low in fat and sugars, as opposed to Western and high-fat diets, have been shown to have a beneficial effect on intestinal health by promoting the growth of beneficial bacteria, improve mucus barrier function and immune tolerance, while inhibiting pro-inflammatory responses and their downstream effects. On the contrary, diets low in fiber and high in fat and sugars have been associated with alterations in microbiota composition/functionality and the subsequent development of chronic diseases such as food allergies, inflammatory bowel disease, and metabolic disease. In this review, we provided an updated overview of the current understanding of the connection between diet, microbiota, and health, with a special focus on the role of Western and high-fat diets in shaping intestinal homeostasis by modulating the gut microbiota.
Assuntos
Ecossistema , Microbioma Gastrointestinal , Dieta Hiperlipídica , Fibras na Dieta , Muco , AçúcaresRESUMO
The colonic mucus layer is organized as a two-layered system providing a physical barrier against pathogens and simultaneously harboring the commensal flora. The factors contributing to the organization of this gel network are not well understood. In this study, the impact of transglutaminase activity on this architecture was analyzed. Here, we show that transglutaminase TGM3 is the major transglutaminase-isoform expressed and synthesized in the colon. Furthermore, intrinsic extracellular transglutaminase activity in the secreted mucus was demonstrated in vitro and ex vivo. Absence of this acyl-transferase activity resulted in faster degradation of the major mucus component the MUC2 mucin and changed the biochemical properties of mucus. Finally, TGM3-deficient mice showed an early increased susceptibility to Dextran Sodium Sulfate-induced colitis. Here, we report that natural isopeptide cross-linking by TGM3 is important for mucus homeostasis and protection of the colon from inflammation, reducing the risk of colitis.
Assuntos
Colo/metabolismo , Muco/metabolismo , Transglutaminases/metabolismo , Animais , Colite/etiologia , Colite/metabolismo , Camundongos , Mucina-2/metabolismoRESUMO
The intestinal mucus layer, an important element of epithelial protection, is produced by goblet cells. Intestinal goblet cells are assumed to be a homogeneous cell type. In this study, however, we delineated their specific gene and protein expression profiles and identified several distinct goblet cell populations that form two differentiation trajectories. One distinct subtype, the intercrypt goblet cells (icGCs), located at the colonic luminal surface, produced mucus with properties that differed from the mucus secreted by crypt-residing goblet cells. Mice with defective icGCs had increased sensitivity to chemically induced colitis and manifested spontaneous colitis with age. Furthermore, alterations in mucus and reduced numbers of icGCs were observed in patients with both active and remissive ulcerative colitis, which highlights the importance of icGCs in maintaining functional protection of the epithelium.
Assuntos
Colo/citologia , Células Caliciformes/fisiologia , Mucosa Intestinal/citologia , Muco/fisiologia , Animais , Diferenciação Celular , Colite/induzido quimicamente , Colite/fisiopatologia , Colite Ulcerativa/patologia , Colite Ulcerativa/fisiopatologia , Colo/fisiologia , Células Caliciformes/citologia , Humanos , Mucosa Intestinal/fisiologia , Intestino Delgado/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-ets/genética , TranscriptomaRESUMO
The intestinal mucus layer is a physical barrier separating the tremendous number of gut bacteria from the host epithelium. Defects in the mucus layer have been linked to metabolic diseases, but previous studies predominantly investigated mucus function during high-caloric/low-fiber dietary interventions, thus making it difficult to separate effects mediated directly through diet quality from potential obesity-dependent effects. As such, we decided to examine mucus function in mouse models with metabolic disease to distinguish these factors. Here we show that, in contrast to their lean littermates, genetically obese (ob/ob) mice have a defective inner colonic mucus layer that is characterized by increased penetrability and a reduced mucus growth rate. Exploiting the coprophagic behavior of mice, we next co-housed ob/ob and lean mice to investigate if the gut microbiota contributed to these phenotypes. Co-housing rescued the defect of the mucus growth rate, whereas mucus penetrability displayed an intermediate phenotype in both mouse groups. Of note, non-obese diabetic mice with high blood glucose levels displayed a healthy colonic mucus barrier, indicating that the mucus defect is obesity- rather than glucose-mediated. Thus, our data suggest that the gut microbiota community of obesity-prone mice may regulate obesity-associated defects in the colonic mucosal barrier, even in the presence of dietary fiber.
Assuntos
Microbioma Gastrointestinal , Mucosa Intestinal/metabolismo , Obesidade/patologia , Animais , Glicemia/análise , Colo/metabolismo , Colo/microbiologia , Colo/patologia , Feminino , Glucose/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Obesos , Obesidade/genética , FenótipoRESUMO
Calcium-activated chloride channel regulator 1 (CLCA1) is one of the major nonmucin proteins found in intestinal mucus. It is part of a larger family of CLCA proteins that share highly conserved features and domain architectures. The CLCA domain arrangement is similar to proteins belonging to the ADAM (a disintegrin and metalloproteinase) family, known to process extracellular matrix proteins. Therefore, CLCA1 is an interesting candidate in the search for proteases that process intestinal mucus. Here, we investigated CLCA1's biochemical properties both in vitro and in mucus from mouse and human colon biopsy samples. Using immunoblotting with CLCA1-specific antibodies and recombinant proteins, we observed that the CLCA1 C-terminal self-cleavage product forms a disulfide-linked dimer that noncovalently interacts with the N-terminal part of CLCA1, which further interacts to form oligomers. We also characterized a second, more catalytically active, N-terminal product of CLCA1, encompassing the catalytic domain together with its von Willebrand domain type A (VWA). This fragment was unstable but could be identified in freshly prepared mucus. Furthermore, we found that CLCA1 can cleave the N-terminal part of the mucus structural component MUC2. We propose that CLCA1 regulates the structural arrangement of the mucus and thereby takes part in the regulation of mucus processing.
Assuntos
Canais de Cloreto/química , Canais de Cloreto/metabolismo , Colo/metabolismo , Mucosa Intestinal/metabolismo , Mucina-2/metabolismo , Multimerização Proteica , Proteólise , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Domínios Proteicos , Estrutura Quaternária de ProteínaRESUMO
[This corrects the article DOI: 10.3389/fphys.2019.00694.].
RESUMO
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.
Assuntos
Colo/imunologia , Células Caliciformes/imunologia , Inflamassomos/imunologia , Mucosa Intestinal/imunologia , Muco/imunologia , Receptores de Superfície Celular/imunologia , Animais , Colo/metabolismo , Colo/microbiologia , Microbioma Gastrointestinal/imunologia , Células Caliciformes/metabolismo , Células Caliciformes/microbiologia , Inflamassomos/genética , Inflamassomos/metabolismo , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Interleucina-18/genética , Interleucina-18/imunologia , Interleucina-18/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mucina-2/imunologia , Mucina-2/metabolismo , Muco/metabolismo , Muco/microbiologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/imunologiaRESUMO
Calcium-activated anion secretion is expected to ameliorate cystic fibrosis, a genetic disease that carries an anion secretory defect in exocrine tissues. Human patients and animal models of the disease that present a mild intestinal phenotype have been postulated to bear a compensatory calcium-activated anion secretion in the intestine. TMEM16A is calcium-activated anion channel whose presence in the intestinal epithelium is contradictory. We aim to test the functional expression of TMEM16A using animal models with Cftr and/or Tmem16a intestinal silencing. Expression of TMEM16A was studied in a wild type and intestinal Tmem16a knockout mice by mRNA-seq, mass-spectrometry, q-PCR, Western blotting and immunolocalization. Calcium-activated anion secretion was recorded in the ileum and proximal colon of these animals including intestinal Cftr knockout and double mutants with dual Tmem16a and Cftr intestinal ablation. Mucus homeostasis was studied by immune-analysis of Mucin-2 (Muc2) and survival curves were recorded. Tmem16a transcript was found in intestine. Nevertheless, protein was barely detected in colon samples. Electrophysiological measurements demonstrated that the intestinal deletion of Tmem16a did not change calcium-activated anion secretion induced by carbachol or ATP in ileum and proximal colon. Muc2 architecture was not altered by Tmem16a silencing as was observed when Cftr was deleted from mouse intestine. Tmem16a silencing neither affected animal survival nor modified the lethality observed in the intestinal Cftr-null mouse. Our results demonstrate that TMEM16A function in the murine intestine is not related to electrogenic calcium-activated anion transport and does not affect mucus homeostasis and survival of animals.
RESUMO
Many epithelial surfaces of the body are covered with protective mucus, and disrupted mucus homeostasis is coupled to diseases such as ulcerative colitis, helminth infection, cystic fibrosis, and chronic obstructive lung disease. However, little is known how a balanced mucus system is maintained. By investigating the involvement of proteases in colonic mucus dynamics we identified metalloprotease activity to be a key contributor to mucus expansion. The effect was mediated by calcium-activated chloride channel regulator 1 (CLCA1) as application of recombinant CLCA1 on intestinal mucus in freshly dissected tissue resulted in increased mucus thickness independently of ion and mucus secretion, but dependent on its metallohydrolase activity. Further, CLCA1 modulated mucus dynamics in both human and mouse, and knock-out of CLCA1 in mice was compensated for by cysteine proteases. Our results suggest that CLCA1 is involved in intestinal mucus homeostasis by facilitating processing and removal of mucus to prevent stagnation. In light of our findings, we suggest future studies to investigate if upregulation of CLCA1 in diseases associated with mucus accumulation could facilitate removal of mucus in an attempt to maintain homeostasis.
Assuntos
Canais de Cloreto/metabolismo , Colo/metabolismo , Muco/metabolismo , Animais , Canais de Cloreto/genética , Homeostase , Humanos , Metaloproteases/metabolismo , Camundongos , Camundongos Knockout , Proteólise , Proteoma/químicaRESUMO
Innate immune signaling pathways contribute to the protection of host tissue when bacterially challenged. Colonic goblet cells are responsible for generating the two mucus layers that physically separate the luminal microbiota from the host epithelium. Analysis of colonic tissues from multiple mouse strains allowed us to identify a "sentinel" goblet cell (senGC) localized to the colonic crypt entrance. This cell nonspecifically endocytoses and reacts to the TLR2/1, TLR4, and TLR5 ligands by activating the Nlrp6 inflammasome downstream of TLR- and MyD88-dependent Nox/Duox reactive oxygen species synthesis. This triggers calcium ion-dependent compound exocytosis of Muc2 mucin from the senGC and generates an intercellular gap junction signal; in turn, this signal induces Muc2 secretion from adjacent goblet cells in the upper crypt, which expels bacteria. Thus, senGCs guard and protect the colonic crypt from bacterial intruders that have penetrated the inner mucus layer.
Assuntos
Colo/imunologia , Colo/microbiologia , Microbioma Gastrointestinal/imunologia , Células Caliciformes/imunologia , Mucina-2/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Bactérias/imunologia , Cálcio/metabolismo , Endocitose/imunologia , Imunidade Inata , Inflamassomos/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/genética , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor 5 Toll-Like/metabolismoRESUMO
The secreted, goblet cell-derived protein Clca1 (chloride channel regulator, calcium-activated-1) has been linked to diseases with mucus overproduction, including asthma and cystic fibrosis. In the intestine Clca1 is found in the mucus with an abundance and expression pattern similar to Muc2, the major structural mucus component. We hypothesized that Clca1 is required for the synthesis, structure or barrier function of intestinal mucus and therefore compared wild type and Clca1-deficient mice under naive and at various time points of DSS (dextran sodium sulfate)-challenged conditions. The mucus phenotype in Clca1-deficient compared to wild type mice was systematically characterized by assessment of the mucus protein composition using proteomics, immunofluorescence and expression analysis of selected mucin genes on mRNA level. Mucus barrier integrity was assessed in-vivo by analysis of bacterial penetration into the mucus and translocation into sentinel organs combined analysis of the fecal microbiota and ex-vivo by assessment of mucus penetrability using beads. All of these assays revealed no relevant differences between wild type and Clca1-deficient mice under steady state or DSS-challenged conditions in mouse colon. Clca1 is not required for mucus synthesis, structure and barrier function in the murine colon.
Assuntos
Canais de Cloreto/fisiologia , Colite/metabolismo , Colo/metabolismo , Muco/metabolismo , Animais , Colite/induzido quimicamente , Colite/patologia , Colo/microbiologia , Colo/patologia , Sulfato de Dextrana , Fezes/microbiologia , Expressão Gênica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microbiota , Mucina-2/metabolismo , Permeabilidade , RNA Ribossômico 16S/genéticaRESUMO
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 Peyer's 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.