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.

Dietary pectin-derived acidic oligosaccharides improve the pulmonary bacterial clearance of Pseudomonas aeruginosa lung infection in mice by modulating intestinal microbiota and immunity.

BACKGROUND: A predominantly T-helper type 2 (Th2) immune response is critical in the prognosis of pulmonary Pseudomonas aeruginosa infection. But the mucosal and systemic immune responses can be influenced by the intestinal microbiota. METHODS: We assessed the effect of microbiota compositional changes induced by a diet enriched in 5% acidic oligosaccharides derived from pectin (pAOS) on the immune response and outcome of chronic pulmonary P. aeruginosa infection in mice. RESULTS: pAOS promoted Th1 polarization by increasing interferon γ release, upregulating t-bet gene expression, decreasing interleukin 4 secretion, and downregulating gata3 gene expression. pAOS also sustained the release of keratinocyte chemoattractant, recruited polynuclear leukocytes and macrophages, stimulated M1 macrophage activation and interleukin 10 release, and decreased tumor necrosis factor α release in the lung. These effects led to increased bacterial clearance after the first and second P. aeruginosa infections. pAOS modified the intestinal microbiota by stimulating the growth of species involved in immunity development, such as Bifidobacterium species, Sutturella wadsworthia, and Clostridium cluster XIVa organisms, and at the same time increased the production of butyrate and propionate. CONCLUSION: These results suggest that pAOS may have beneficial effects by limiting the number and severity of pulmonary exacerbations in patients chronically infected with P. aeruginosa, such as individuals with cystic fibrosis.

Assembly of the respiratory mucin MUC5B: a new model for a gel-forming mucin.

Mucins are essential components in mucus gels that form protective barriers at all epithelial surfaces, but much remains unknown about their assembly, intragranular organization, and post-secretion unfurling to form mucus. MUC5B is a major polymeric mucin expressed by respiratory epithelia, and we investigated the molecular mechanisms involved during its assembly. Studies of intact polymeric MUC5B revealed a single high affinity calcium-binding site, distinct from multiple low affinity sites on each MUC5B monomer. Self-diffusion studies with intact MUC5B showed that calcium binding at the protein site catalyzed reversible cross-links between MUC5B chains to form networks. The site of cross-linking was identified in the MUC5B D3-domain as it was specifically blocked by D3 peptide antibodies. Biophysical analysis and single particle EM of recombinant MUC5B N terminus (D1D2D'D3; NT5B) and subdomains (D1, D1-D2, D2-D'-D3, and D3) generated structural models of monomers and disulfide-linked dimers and suggested that MUC5B multimerizes by disulfide linkage between D3-domains to form linear polymer chains. Moreover, these analyses revealed reversible homotypic interactions of NT5B at low pH and in high calcium, between disulfide-linked NT5B dimers, but not monomers. These results enable a model of MUC5B to be derived, which predicts mechanisms of mucin intracellular assembly and storage, which may be common to the other major gel-forming polymeric mucins.

Impact of fish oils on the outcomes of a mouse model of acute Pseudomonas aeruginosa pulmonary infection.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that causes pneumonia in immunocompromised humans and severe pulmonary damage in patients with cystic fibrosis. Imbalanced fatty acid incorporation in membranes, including increased arachidonic acid and decreased DHA concentrations, is known to play a critical role in chronic inflammation associated with bacterial infection. Other lipids, such as EPA and alkylglycerols, are also known to play a role in inflammation, particularly by stimulating the immune system, decreasing inflammation and inhibiting bacterial growth. In this context, the goal of the present study was to assess the effect of dietary DHA/EPA, in a 2:1 ratio, and alkylglycerols, as natural compounds extracted from oils of rays and chimeras, respectively, on the inflammatory reaction induced by P. aeruginosa pulmonary infection in mice. To this end, mice were fed with a control diet or isolipidic, isoenergetic diets prepared with oils enriched in DHA/EPA (2:1) or alkylglycerols for 5 weeks before the induction of acute P. aeruginosa lung infection by endotracheal instillation. In our model, DHA/EPA (2:1) significantly improved the survival of mice after infection, which was associated with the acceleration of bacterial clearance and the resolution of inflammation leading to the improvement of pulmonary injuries. By contrast, alkylglycerols did not affect the outcomes of P. aeruginosa infection. Our findings suggest that supplementation with ray oil enriched in DHA/EPA (2:1) can be considered as a preventive treatment for patients at risk for P. aeruginosa infection.

[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.

(n-3) long-chain PUFA differentially affect resistance to Pseudomonas aeruginosa infection of male and female cftr-/- mice.

The aim of this study was to determine whether oral supplementation with EPA/DHA (10.5 and 5.1% of fat, respectively) could improve the outcome of pulmonary P. aeruginosa infection in cftr(-/-) mice compared with wild-type (Wt) mice similarly treated. Because gender could influence the susceptibility of cftr-deficient mice, results were analyzed by gender. Wt and (-/-) mice were randomized for 6 wk to consume a control or EPA/DHA diet, infected with endotracheal injection of 5 × 10(7) CFU/mouse of P. aeruginosa, and killed 24 h later. Cftr(-/-) mice were more susceptible to infection than were Wt mice; (-/-) males had more neutrophils (P < 0.01) and a higher keratinocyte-derived chemokine (KC) level (P < 0.05), and (-/-) females had greater lung injury and mortality (P < 0.05). Female (-/-) mice were more susceptible than (-/-) males with a higher mortality and lung injury (P < 0.05). The EPA/DHA diet reduced neutrophil numbers and KC and IL-6 levels (P < 0.05) in (-/-) males and reduced mortality rate (P < 0.001), lung permeability, and IL-6 level (P < 0.05) in (-/-) females compared with (-/-) mice fed the control diet. These results were associated with a reduction in the pulmonary bacterial load (P < 0.05), an increase in the EPA/DHA concentration in cell membranes of (-/-) males and females (P < 0.01), and an increased weight gain only in males compared with (-/-) mice fed the control diet (P < 0.01). In conclusion, EPA/DHA improves the host resistance of (-/-) mice, although the beneficial effect differed in males and females.

A lipoxygenase with dual positional specificity is expressed in olives (Olea europaea L.) during ripening.

Plant lipoxygenases (LOXs) are a class of widespread dioxygenases catalysing the hydroperoxidation of polyunsaturated fatty acids. Although multiple isoforms of LOX have been detected in a wide range of plants, their physiological roles remain to be clarified. With the aim to clarify the occurrence of LOXs in olives and their contribution to the elaboration of the olive oil aroma, we cloned and characterized the first cDNA of the LOX isoform which is expressed during olive development. The open reading frame encodes a polypeptide of 864 amino acids. This olive LOX is a type-1 LOX which shows a high degree of identity at the peptide level towards hazelnut (77.3%), tobacco (76.3%) and almond (75.5%) LOXs. The recombinant enzyme shows a dual positional specificity, as it forms both 9- and 13-hydroperoxide of linoleic acid in a 2:1 ratio, and would be defined as 9/13-LOX. Although a LOX activity was detected throughout the olive development, the 9/13-LOX is mainly expressed at late developmental stages. Our data suggest that there are at least two Lox genes expressed in black olives, and that the 9/13-LOX is associated with the ripening and senescence processes. However, due to its dual positional specificity and its expression pattern, its contribution to the elaboration of the olive oil aroma might be considered.

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.

Importance of the Phospholipid Core for Mucin Hydrogel Penetration and Mucosal Cell Uptake of Maltodextrin Nanoparticles.

Nanoparticles (NPs) used as mucosal antigen delivery systems are a promising way to vaccinate. However, NPs have to cross the mucus gel and penetrate into mucosal cells to deliver antigens, and a mismatch exists between mucopenetrating NPs, rarely able to interact with cells, and NPs designed to deliver antigens into cells, but often described as mucoadhesives. Here, we compared the ability of cationic maltodextrin-based NPs, either without (NP+) or with an anionic phospholipid core (NPL), to interact with mucins and airway epithelial cells. We showed that their lipid core increased the NPL's mobility in mucin hydrogel by reducing interactions with mucins. Similarly, the uptake and protein delivery by NPLs into airway epithelial cells were not hindered by mucins. This highlights the importance of anionic lipids in the NPL, which are more efficient in crossing the mucin hydrogel while retaining the ability to interact with epithelial cells, an intermediate behavior between mucoadhesive and mucopenetrating NPs.

Impact and consequences of intensive chemotherapy on intestinal barrier and microbiota in acute myeloid leukemia: the role of mucosal strengthening.

Induction chemotherapy (7 + 3 regimen) remains the gold standard for patients with acute myeloid leukemia (AML) but is responsible for gut damage leading to several complications such as bloodstream infection (BSI). We aimed to investigate the impact of induction chemotherapy on the intestinal barrier of patients with AML and in wild-type mice. Next, we assessed the potential benefit of strengthening the mucosal barrier in transgenic mice releasing a recombinant protein able to reinforce the mucus layer (Tg222). In patients, we observed a decrease of plasma citrulline, which is a marker of the functional enterocyte mass, of short-chain fatty acids and of fecal bacterial load, except for Escherichia coli and Enterococcus spp., which became dominant. Both the α and ß-diversities of fecal microbiota decreased. In wild-type mice, citrulline levels decreased under chemotherapy along with an increase of E. coli and Enterococcus spp load associated with concomitant histologic impairment. By comparison with wild-type mice, Tg222 mice, 3 days after completing chemotherapy, had higher citrulline levels, a faster healing epithelium, and preserved α-diversity of their intestinal microbiota. This was associated with reduced bacterial translocations. Our results highlight the intestinal damage and the dysbiosis induced by the 7 + 3 regimen. As a proof of concept, our transgenic model suggests that strengthening the intestinal barrier is a promising approach to limit BSI and improve AML patients' outcome.

Mucin CYS domains are ancient and highly conserved modules that evolved in concert.

BACKGROUND: Many large secreted gel-forming mucins contain multiple copies of a "naked" cysteine-enriched domain (CYS domain) that interrupt or are adjacent to mucin domains (Ser/Thr/Pro regions). There is less similarity of homologous CYS sequences between species than there is across CYS repeats within species. These CYS domains are likely implicated in reversible mucin-mucin interactions that play a central role in mucus properties. RESULTS: The use of multiple genome sequences in a comparative analysis of CYS sequences identified new mucins containing the CYS domain in various species. Furthermore, analysis showed that this domain is at least 650 million-year old and that CYS sequences evolved under strong selective pressure in concerted fashion in all species examined by two mechanisms that never coexist within a given gene: either multiplication of exons encoding CYS modules or successive intra-exonic duplications. CONCLUSION: The CYS sequences appear to have undergone concerted evolution with a high selective pressure to conserve cysteine and other amino acid residues. This domain is the best-conserved domain in secreted mucins pointing toward an important common function in many different organisms.

Dietary (n-3) polyunsaturated fatty acids affect the kinetics of pro- and antiinflammatory responses in mice with Pseudomonas aeruginosa lung infection.

The underlying mechanisms by which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) affect host resistance to Pseudomonas aeruginosa are unclear. The aim of this study was to determine their role on the kinetic of pro- and antiinflammatory response in lung infection. Mice fed either a control diet or a diet enriched with EPA and DHA were infected intratracheally and we studied lung expression of proinflammatory markers [CXCL1, interleukin (IL)-6, tumor necrosis factor-alpha], antiinflammatory markers (IL-10, A20, and IkappaB alpha), and PPARalpha and PPARgamma. The inflammatory response was assessed using recruitment of neutrophils and macrophages into bronchoalveolar lavage fluid, bacterial clearance from the lung, pulmonary injury, and 7-d survival rate. Compared with the control group, EPA and DHA delayed the expression of proinflammatory markers during the first 2 h (P < 0.05), upregulated proinflammatory marker expression (P < 0.05), and induced overexpression of antiinflammatory markers at 8 h (P < 0.05), enhanced recruitment of neutrophils at 16 h (P < 0.05), and induced PPARalpha and PPARgamma overexpression at 4 and 8 h (P < 0.01), respectively. Pulmonary bacterial load decreased and pulmonary injury and mortality were reduced during the first 24 h (P < 0.05). In conclusion, EPA and DHA modulate the balance between pro- and antiinflammatory cytokines, alter the early response of the host to P. aeruginosa infection, and affect the early outcome of infection.