The Cervicovaginal Mucus Barrier.

Preterm births are a global health priority that affects 15 million babies every year worldwide. There are no effective prognostic and therapeutic strategies relating to preterm delivery, but uterine infections appear to be a major cause. The vaginal epithelium is covered by the cervicovaginal mucus, which is essential to health because of its direct involvement in reproduction and functions as a selective barrier by sheltering the beneficial lactobacilli while helping to clear pathogens. During pregnancy, the cervical canal is sealed with a cervical mucus plug that prevents the vaginal flora from ascending toward the uterine compartment, which protects the fetus from pathogens. Abnormalities of the cervical mucus plug and bacterial vaginosis are associated with a higher risk of preterm delivery. This review addresses the current understanding of the cervicovaginal mucus and the cervical mucus plug and their interactions with the microbial communities in both the physiological state and bacterial vaginosis, with a focus on gel-forming mucins. We also review the current state of knowledge of gel-forming mucins contained in mouse cervicovaginal mucus and the mouse models used to study bacterial vaginosis.

Muc5b is mainly expressed and sialylated in the nasal olfactory epithelium whereas Muc5ac is exclusively expressed and fucosylated in the nasal respiratory epithelium.

The nose is a complex organ that filters and warms breathing airflow. The nasal epithelium is the first barrier between the host and the external environment and is covered by a mucus gel that is poorly documented. Mucins are large, heavily O-glycosylated polymeric molecules secreted in the nose lumen by specialized cells, and they are responsible for the biochemical properties of the mucus gel. The mucus traps particles and clears them, and it also bathes microbiota, host molecules, and receptors that are all essential for odor perception in the olfactory epithelium. We used histology and immunohistochemistry to study the expression of the two main airway polymeric mucins, Muc5ac and Muc5b, in wild-type, green fluorescent protein-reporter Muc5b, and in genetically Muc5b-deficient mice. We report that Muc5ac is produced by goblet cells at the cell surface in the respiratory epithelium but is not expressed in the olfactory epithelium, whereas Muc5b is secreted by Bowman's glands situated in the lamina propria beneath the olfactory epithelium and also by goblet cells in the distal part of the respiratory epithelium. We also observed that Muc5b-deficient mice exhibited depletion of Bowman's glands. Using lectins, we found that terminally O-glycosylated chains of Muc5b were sialylated but not fucosylated, whereas Muc5ac was fucosylated but not sialylated. Specific localization and specific terminal glycosylation of the two mucins suggest different functions of the mucins.

Ocular mucins in dry eye disease.

Dry eye disease is a common and multifactorial disease with a high prevalence worldwide. Water loss, reduced expression of glycocalyx mucins, and loss of goblet cells secreting gel-forming mucins are hallmarks of dry eye disease. Mucins are large and complex heavily glycosylated proteins. Their organization in the tear film remains unclear, but they play a key role to protect and maintain integrity of the ocular surface. Mice have been extremely valuable mammalian models with which to study ocular physiology and disease, and to evaluate eye therapies. Genetically modified mice and spontaneously occurring mutants with eye defects have proven to be powerful tools for the pharmaceutical industry, clinicians, and basic researchers investigating dry eye disease. However, ocular mucins remain relatively under-studied and inadequately characterized. This review aims to summarize current knowledge about mucin production at the ocular surface in healthy individuals and in dry eye disease, and to compile an overview of mouse models available for the study of mucins in dry eye disease.

Systematic gene overexpression in Candida albicans identifies a regulator of early adaptation to the mammalian gut.

Candida albicans is part of the human gastrointestinal (GI) microbiota. To better understand how C. albicans efficiently establishes GI colonisation, we competitively challenged growth of 572 signature-tagged strains (~10% genome coverage), each conditionally overexpressing a single gene, in the murine gut. We identified CRZ2, a transcription factor whose overexpression and deletion respectively increased and decreased early GI colonisation. Using clues from genome-wide expression and gene-set enrichment analyses, we found that the optimal activity of Crz2p occurs under hypoxia at 37°C, as evidenced by both phenotypic and transcriptomic analyses following CRZ2 genetic perturbation. Consistent with early colonisation of the GI tract, we show that CRZ2 overexpression confers resistance to acidic pH and bile salts, suggesting an adaptation to the upper sections of the gut. Genome-wide location analyses revealed that Crz2p directly modulates the expression of many mannosyltransferase- and cell-wall protein-encoding genes, suggesting a link with cell-wall function. We show that CRZ2 overexpression alters cell-wall phosphomannan abundance and increases sensitivity to tunicamycin, suggesting a role in protein glycosylation. Our study reflects the powerful use of gene overexpression as a complementary approach to gene deletion to identify relevant biological pathways involved in C. albicans interaction with the host environment.

Non-C-mannosylable mucin CYS domains hindered proper folding and secretion of mucin.

The CYS domain occurs in multiple copies in many gel-forming mucins. It is believed that CYS domains can interact with each other in a reversible manner, suggesting a key role of the domain in gel formation. This domain always contains in its amino-terminal sequence the C-mannosylation motif WXXW, but whether the CYS domain is C-mannosylated is debated, and the putative role of C-mannosylation of the domain is unclear. We prepared recombinant CYS domains of the human mucin MUC5B with (WXXW→AXXW) and without a single amino acid mutation and mini-5B mucins made of a large Ser/Thr/Pro region flanked by two CYS domains with the WXXW motif or with the mutated AXXW motif on the first, second or both CYS domains. We found that the single CYS domain and the two CYS domains of mini-5B mucin must be C-mannosylable for the efficient maturation and secretion of the recombinant molecules; otherwise, they are retained in the cell and co-localized with a resident enzyme of the endoplasmic reticulum.

Biological modeling of mucus to modulate mucus barriers.

A recent study using a transgenic mouse, whose intestinal mucus contains a molecule made of 12 copies of a domain found in many gelling mucins, demonstrates that it is possible to strengthen mucus properties in situ, leading to promising new treatment strategies in diseases in which the mucosal barrier is impaired.

[Human milk oligosaccharides play major roles in child development and future health]. / Les oligosaccharides du lait maternel : des rôles majeurs pour le développement de l'enfant et sa santé future.

Human milk oligosaccharides (HMO) represent the third largest component of human breast milk (BM). The BM level is comprised between 5 to 20 g per liter and they have a great structural complexity with more than 150 HMO characterized to date. In this review, we present a summary of the main experimental and clinical data that have demonstrated their multiple biological roles in infants such as for gut development, microbiota, immune protection and neurodevelopment. Some HMO-enriched infant formulas are available yet, even if their benefits on the infant health remain to be confirmed. Further researches could allow therapeutic use in preterm newborns or in infants with intestinal diseases. Experimental data suggest that they could also be used in the prevention of some chronic diseases with immunometabolic or neurodevelopmental components.

Title: Les oligosaccharides du lait maternel : des rôles majeurs pour le développement de l'enfant et sa santé future. Abstract: En raison de sa capacité à fournir des apports nutritionnels optimaux ainsi que de nombreux facteurs bioactifs, tels que des oligosaccharides, le lait maternel est considéré comme le régime alimentaire optimal pour les nouveau-nés. Les oligosaccharides du lait humain (HMO) constituent le troisième composant du lait maternel. Plus de 150 HMO ont été caractérisés, leur concentration variant de 5 à 20 g/L. Certaines préparations infantiles enrichies en HMO sont désormais disponibles, même si leurs effets sur la santé restent à démontrer. La poursuite des recherches pourrait permettre d'envisager leur utilisation chez les enfants prématurés ou présentant des maladies inflammatoires digestives. Des données expérimentales suggèrent en effet que les HMO pourraient prévenir certaines maladies chroniques à composantes immuno-métaboliques ou neurodéveloppementales. Dans cette revue, nous présentons une synthèse des dernières données montrant les effets biologiques de ces oligosaccharides.

A porous cervical mucus plug leads to preterm birth induced by experimental vaginal infection in mice.

During gestation, the cervical mucus plug (CMP) acts to seal the cervical canal. Pilot studies in humans have suggested that a porous CMP may increase the risk of uterine infection and preterm birth. We examined the gel-forming content of the mouse vagina and the CMP. We experimentally infected pregnant mice by intravaginal administration of pathogens related to preterm birth in humans. We found that the epithelium in both the vagina and cervical canal of pregnant mice produced the two gel-forming mucins Muc5b and Muc5ac. The CMP was porous in Muc5b-deficient mice for which intravaginal administration of Escherichia coli O 55 led to the activation of an inflammatory response in the uterus and 100% preterm births. The pathogen was found in the mucus plug and uterus. This study shows that Muc5b is essential for the in vivo barrier function and the prevention of uterine infections during gestation.

[Idiopathic pulmonary fibrosis]. / La fibrose pulmonaire idiopathique.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal lung disease of unknown origin. It is characterized by aberrant scarring of the alveolar epithelium leading to an accumulation of extracellular matrix (ECM). Fibroblastic foci, consisting of fibroblasts and myofibroblasts, are responsible for the excessive production of ECM. The two therapeutic molecules available on the market to date only allow to slow down the evolution of the disease. In this review, we present the mechanisms involved in the progression of the disease, its treatments and the study models.

Title: La fibrose pulmonaire idiopathique. Abstract: La fibrose pulmonaire idiopathique (FPI) est une maladie pulmonaire chronique, évolutive et mortelle dont l'origine est inconnue. Elle se caractérise par une cicatrisation aberrante de l'épithélium alvéolaire aboutissant à une accumulation de matrice extracellulaire (MEC). Les foyers fibroblastiques, constitués de fibroblastes et de myofibroblastes, sont responsables de la production excessive de MEC. Les deux seules molécules thérapeutiques disponibles sur le marché permettent seulement de ralentir l'évolution de la maladie. Dans cette revue, nous présentons les mécanismes impliqués dans la progression de la maladie, ses traitements et les modèles d'étude.

Abnormal expression of Muc5b in Cftr-null mice and in mammary tumors of MMTV-ras mice.

Gel-forming mucins are large, high molecular weight, and heavily O-glycosylated proteins that are responsible for the rheological properties of mucus gel. Among them, the mucin MUC5B has been implicated in breast cancer and cystic fibrosis. We obtained a new polyclonal serum, named CP1, which was isolated from a rabbit immunized with a mouse Muc5b peptide. The immunoprofile of Muc5b was determined on paraffin-embedded and frozen mouse tissue sections and showed a similar expression pattern in mouse to that in the human. The "nonmammary" mucin Muc5b was detected in all mammary tumors analyzed from MMTV-ras mice, suggesting that the CP1 antibody is a valuable tool for investigating the involvement of this mucin in mammary cancer. We also found that uninfected Cftr( -/- ) mice harbored more Clara cells, which were Muc5b-positive, than did their wild-type control littermates. The number of Muc5b-positive cells increased in Cftr( -/- ) mice infected experimentally with Pseudomonas aeruginosa, and the mice developed mucus plugs in their bronchi and bronchioles with a high frequency of Muc5b content (87%, Cohen's kappa = 0.82; p < 0.0001). These findings suggest that mice genetically deficient in the Cftr gene are predisposed to develop mucus plugs and that MUC5B may provide a valuable target for decreasing mucus viscosity in cystic fibrosis.

The characterization of the first anti-mouse Muc6 antibody shows an increased expression of the mucin in pancreatic tissue of Cftr-knockout mice.

Gel-forming mucins are large high-molecular weight secreted O-glycoproteins responsible for the gel-properties of the mucus blanket. Five orthologous gel-forming mucins have been cloned in human and mouse. Among them, the mucin MUC6 has been less studied, particularly in rodents and no anti rodent-Muc6 antibody has been reported yet. In order to further study Muc6 in mice, our aims were to obtain a specific Muc6 antibody, to validate it and to test it in Cftr deficient mice. A polyclonal serum named CP4 was isolated from a rabbit immunized by a mouse Muc6 peptide. In Western blot experiments, the antibody detected a high-molecular weight molecule secreted by the gastric tissue. Using immunohistochemistry, we showed that the antibody reacted strongly with deep glands of duodenum and ileum and mucous neck cells of gastric body. CP4 also recognized Muc6 protein secreted at the surface of the stomach and renal collecting tubules. The centroacinar cells of pancreatic tissue also reacted with the antibody. Cftr-/- mice showed a higher expression of Muc6 at both protein and RNA levels compared with their control Cftr+/+ littermates suggesting that as in the human disease, Muc6 may contribute to the formation of materials that block pancreatic acini and ducts in mouse models of cystic fibrosis. The rabbit anti-mouse Muc6 polyclonal antibody seems highly specific to the mouse mucin and will be useful to study pancreatic pathology in cystic fibrosis.

Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells.

We have previously reported that 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAc alpha-O-bn), an inhibitor of glycosylation, perturbed apical biosynthetic trafficking in polarized HT-29 cells suggesting an involvement of a lectin-based mechanism. Here, we have identified galectin-4 as one of the major components of detergent-resistant membranes (DRMs) isolated from HT-29 5M12 cells. Galectin-4 was also found in post-Golgi carrier vesicles. The functional role of galectin-4 in polarized trafficking in HT-29 5M12 cells was studied by using a retrovirus-mediated RNA interference. In galectin-4-depleted HT-29 5M12 cells apical membrane markers accumulated intracellularly. In contrast, basolateral membrane markers were not affected. Moreover, galectin-4 depletion altered the DRM association characteristics of apical proteins. Sulfatides with long chain-hydroxylated fatty acids, which were also enriched in DRMs, were identified as high-affinity ligands for galectin-4. Together, our data propose that interaction between galectin-4 and sulfatides plays a functional role in the clustering of lipid rafts for apical delivery.

Mucin CYS domain stiffens the mucus gel hindering bacteria and spermatozoa.

Mucus is the first biological barrier encountered by particles and pathogenic bacteria at the surface of secretory epithelia. The viscoelasticity of mucus is governed in part by low energy interactions that are difficult to assess. The CYS domain is a good candidate to support low energy interactions between GFMs and/or mucus constituents. Our aim was to stiffen the mucus from HT29-MTX cell cocultures and the colon of mice through the delivery of a recombinant protein made of hydrophobic CYS domains and found in multiple copies in polymeric mucins. The ability of the delivery of a poly-CYS molecule to stiffen mucus gels was assessed by probing cellular motility and particle diffusion. We demonstrated that poly-CYS enrichment decreases mucus permeability and hinders displacement of pathogenic flagellated bacteria and spermatozoa. Particle tracking microrheology showed a decrease of mucus diffusivity. The empirical obstruction scaling model evidenced a decrease of mesh size for mouse mucus enriched with poly-CYS molecules. Our data bring evidence that enrichment with a protein made of CYS domains stiffens the mucin network to provide a more impermeable and protective mucus barrier than mucus without such enrichment.

Early life nutrition influences susceptibility to chronic inflammatory colitis in later life.

The first thousand days of life are a critical time of development in humans during which the risk profile for diseases in later life can be modified. Nevertheless, long-term consequences of early environment on susceptibility to intestinal diseases have not yet been assessed. Using a mouse model of postnatal growth restriction (PNGR), we showed that early life nutrition influences intestinal maturation and gut health in later life. PNGR induced an alteration of the intestinal barrier in pups at weaning, resulting in increased intestinal permeability, and affected gut bacterial colonization. Specifically, pups with PNGR harbored a decreased bacterial diversity, higher Enterococcus spp., Staphylococcus spp., and Escherichia-Shigella spp., and lower Odoribacter spp. and several members of the Lachnospiraceae family. The lack of an efficient intestinal barrier in early life and the dysbiosis induced by PNGR were associated with a higher susceptibility to chronic colitis in adulthood.

Muc5b-deficient mice develop early histological lung abnormalities.

Gel-forming mucins are the main organic component responsible for physical properties of the mucus hydrogels. While numerous biological functions of these mucins are well documented, specific physiological functions of each mucin are largely unknown. To investigate in vivo functions of the gel-forming mucin Muc5b, which is one of the major secreted airway mucins, along with Muc5ac, we generated mice in which Muc5b was disrupted and maintained in the absence of environmental stress. Adult Muc5b-deficient mice displayed bronchial hyperplasia and metaplasia, interstitial thickening, alveolar collapse, immune cell infiltrates, fragmented and disorganized elastin fibers and collagen deposits that were, for approximately one-fifth of the mice, associated with altered pulmonary function leading to respiratory failure. These lung abnormalities start early in life, as demonstrated in one-quarter of 2-day-old Muc5b-deficient pups. Thus, the mouse mucin Muc5b is essential for maintaining normal lung function.

Architecture of the large membrane-bound mucins.

Epithelial membrane-bound mucins are high molecular mass glycoproteins that may be also secreted or released into the extracellular environment. The genomic and multi-domain organizations of human large epithelial membrane-bound mucins are reviewed here with the purpose to clarify the literature on the subject with the help of mouse sequences. This family of complex molecules contains at least MUC3A, MUC12, MUC17, all organized in a cluster of genes, MUC4 and likely MUC16. In addition, we discuss the splicing events reported for these mucins with an emphasis on the human mucin MUC4.

E-cadherin and beta-catenin mRNA levels throughout colon cancer progression.

BACKGROUND: Although E-cadherin and beta-catenin are key regulators in tumor invasion and proliferation, few studies have been undertaken on the expression of these genes at the messenger ribonucleic acid (mRNA) level in relation to the progression of colon cancer. PATIENTS AND METHODS: In this study, tissue samples from colectomy (n = 37) or hepatectomy (n = 23) were collected in both tumor and adjacent normal tissues. Real-time quantitative reverse transcriptase-polymerase chain reaction was used to quantify E-cadherin and beta-catenin mRNAs in reference to 18S RNA. RESULTS: E-cadherin and beta-catenin levels in colon carcinomas were not statistically different compared with adjacent normal mucosa and were not correlated with tumor, nodes, and metastases (TNM) stage. Conversely, E-cadherin and beta-catenin levels were significantly higher in liver metastases than in adjacent normal tissue. Interestingly, we found that E-cadherin level in liver metastases was correlated to the TNM stage of the related primary tumor: a higher E-cadherin level was found for State I-II TNM. In addition, a high expression of E-cadherin in liver metastases was associated with a lower occurrence of extra-hepatic metastases after resection of liver metastases. CONCLUSION: Taken together, these data show that E-cadherin and beta-catenin expressions are regulated throughout colon cancer progression.

Transgenic Mouse Reporter to Study Muc5b In Vivo.

Dysregulation of gel-forming mucins is associated with many airway diseases. Better knowledge of the pathophysiological mechanisms linking mucins and respiratory diseases will advance the understanding of their pathogenesis and should provide opportunities to develop new therapeutic compounds for treatment. MUC5B and MUC5AC are the two main gel-forming mucins in the respiratory tract. The organization in domains and the expression profile of mouse Muc5b are very similar to those in humans, which makes the mouse a relevant model for studies of the translational activities of human mucins. To assess the in vivo biological functions of Muc5b, a mouse reporter tagged in frame with the green fluorescent protein marker has been engineered by homologous recombination. The proof of concept that this reporter model may be informative for translational studies was confirmed by the finding that interleukin-13 administration in living mice upregulated Muc5b production.

Gel-forming mucin interactome drives mucus viscoelasticity.

Mucus is a hydrogel that constitutes the first innate defense in all mammals. The main organic component of mucus, gel-forming mucins, forms a complex network through both reversible and irreversible interactions that drive mucus gel formation. Significant advances in the understanding of irreversible gel-forming mucins assembly have been made using recombinant protein approaches. However, little is known about the reversible interactions that may finely modulate mucus viscoelasticity, which can be characterized using rheology. This approach can be used to investigate both the nature of gel-forming mucins interactions and factors that influence hydrogel formation. This knowledge is directly relevant to the development of new drugs to modulate mucus viscoelasticity and to restore normal mucus functions in diseases such as in cystic fibrosis. The aim of the present review is to summarize the current knowledge about the relationship between the mucus protein matrix and its functions, with emphasis on mucus viscoelasticity.

[Gel-forming mucins structure governs mucus gels viscoelasticity]. / La structure des mucines conditionne les propriétés viscoélastiques des gels de mucus.

Mucus is the first line of innate mucosal defense in all mammals. Gel­forming mucins control the rheological properties of mucus hydrogels by forming a network in which hydrophilic and hydrophobic regions coexist, and it has been revealed that the network is formed through both covalent links and reversible links such as hydrophobic interactions in order to modulate the structure as a function of the physiological necessities. Here, we review the structure and functions of the mucus in terms of the gel-forming mucins protein-protein interactions, also called interactome. Since it is difficult to characterize the low energy reversible interactions due to their dependence on physico-chemical environment, their role is not well understood. Still, they constitute a promising target to counteract mucus abnormalities observed in mucus-associated diseases.