Jean-Luc Desseyn

The extraordinarily complex but highly structured organization of intestinal mucus-gel unveiled in multicolor images.

The mucus that coats the gastrointestinal tract of all mammals is a dynamic and sticky gel layer and represents the first protective barrier between the host and the hostile environment. There is, however, a lack of detailed knowledge about the mucus gel organization because of the high water content and the complexity of MUC2, the main gel-forming molecule in the intestine. Histological staining and a multilabel immunofluorescence method were used to examine mucus blankets and Muc2 in mouse colon and ileum samples fixed in Carnoys solution, unveiling an extraordinarily complex but highly structured mucus gel organization. The inner firmly adherent mucus blanket consists of alternating layers. The thicker outer loosely adherent mucus blanket in the colon is made of alternating laminated layers and loose curl-like structures. The layers consist of Muc2 molecules with different fucosylation states and glycoforms remain unmixed in the mucus. Importantly, distinct goblet cell subpopulations throughout the ileum along the crypt-to-villus axis with an alternation of goblet cells secreting fucosylated and non-fucosylated Muc2 are observed. A better understanding of the mucus structure should contribute to improve the efficiency of DNA and drug delivery and will allow for a better understanding and treatment of inflammatory and infectious intestinal diseases.

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

BACKGROUNDnMany 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.nnnRESULTSnThe 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.nnnCONCLUSIONnThe 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.

(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.

Dietary Pectin–Derived Acidic Oligosaccharides Improve the Pulmonary Bacterial Clearance of Pseudomonas aeruginosa Lung Infection in Mice by Modulating Intestinal Microbiota and Immunity

BACKGROUNDnA 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.nnnMETHODSnWe 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.nnnRESULTSnpAOS 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.nnnCONCLUSIONnThese 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.

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.

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 kappaxa0=xa00.82; pxa0<xa00.0001). 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.

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.

Modulation of host defence against bacterial and viral infections by omega-3 polyunsaturated fatty acids.

OBJECTIVESnAlthough n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs) are used widely in the treatment of chronic inflammatory diseases, their effect in infectious disease requires a particular attention.nnnMETHODSnThe present article discusses their anti-inflammatory and immune properties involved in the host defence and presents a systematic review of the effects of their oral administration on the prevention and outcome of experimental and clinical infections.nnnRESULTSnAt a dose corresponding to an human dose of 500xa0mg/day, n-3 LC-PUFAs intake is beneficial against experimental infections caused by extracellular pathogens including Streptococcus pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus by reducing inflammation, and reduces the incidence of pneumococcal infections in the elderly, but at 2-4-fold higher doses as occurs in some human intervention and/or during long-term it becomes detrimental in intestinal infections with Citrobacter rodentium or Helicobacter hepaticus by exacerbating anti-inflammatory response. They are also harmful against infections caused by intracellular pathogens as Mycobacterium tuberculosis, Salmonella, Influenza virus and Herpes simplex virus by affecting the immune cell response.nnnCONCLUSIONnThe effects of n-3-LC-PUFAs on infections depend on the pathogen and the n-3 LC-PUFA dose and timing. Caution should be recommended for high-dose and long-term supplementation in humans.

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.

Preclinical mouse model to monitor live Muc5b-producing conjunctival goblet cell density under pharmacological treatments

Purpose Modification of mucous cell density and gel-forming mucin production are established hallmarks of mucosal diseases. Our aim was to develop and validate a mouse model to study live goblet cell density in pathological situations and under pharmacological treatments. Methods We created a reporter mouse for the gel-forming mucin gene Muc5b. Muc5b-positive goblet cells were studied in the eye conjunctiva by immunohistochemistry and probe-based confocal laser endomicroscopy (pCLE) in living mice. Dry eye syndrome (DES) model was induced by topical application of benzalkonium chloride (BAK) and recombinant interleukine (rIL) 13 was administered to reverse the goblet cell loss in the DES model. Results Almost 50% of the total of conjunctival goblet cells are Muc5b+ in unchallenged mice. The decrease density of Muc5b+ conjunctival goblet cell population in the DES model reflects the whole conjunctival goblet cell loss. Ten days of BAK in one eye followed by 4 days without any treatment induced a −18.3% decrease in conjunctival goblet cell density. A four days of rIL13 application in the DES model restored the normal goblet cell density. Conclusion Muc5b is a biological marker of DES mouse models. We bring the proof of concept that our model is unique and allows a better understanding of the mechanisms that regulate gel-forming mucin production/secretion and mucous cell differentiation in the conjunctiva of living mice and can be used to test treatment compounds in mucosal disease models.