Colonisation of the colonic mucus gel layer with butyrogenic and hydrogenotropic bacteria in health and ulcerative colitis

Butyrate is the primary energy source for colonocytes and is essential for mucosal integrity and repair. Butyrate deficiency as a result of colonic dysbiosis is a putative factor in ulcerative colitis (UC). Commensal microbes are butyrogenic, while others may inhibit butyrate, through hydrogenotropic activity. The aim of this study was to quantify butyrogenic and hydrogenotropic species and determine their relationship with inflammation within the colonic mucus gel layer (MGL). Mucosal brushings were obtained from 20 healthy controls (HC), 20 patients with active colitis (AC) and 14 with quiescent colitis (QUC). Abundance of each species was determined by RT-PCR. Inflammatory scores were available for each patient. Statistical analyses were performed using Mann–Whitney-U and Kruskall-Wallis tests. Butyrogenic R. hominis was more abundant in health than UC (p < 0.005), prior to normalisation against total bacteria. Hydrogenotropic B. wadsworthia was reduced in AC compared to HC and QUC (p < 0.005). An inverse correlation existed between inflammation and R. hominis (ρ − 0.460, p < 0.005) and B. wadsworthia (ρ − 0.646, p < 0.005). Other hydrogenotropic species did not widely colonise the MGL. These data support a role for butyrogenic bacteria in UC. Butyrate deficiency in UC may be related to reduced microbial production, rather than inhibition by microbial by-products.

Effect of snail mucus on angiogenesis during wound healing

Background: Angiogenesis is the process through which new blood vessels are formed from existing ones. This process plays an important role in supplying the oxygen and nutrients needed for cellular metabolism and eliminating cell debris during wound healing. Snail mucus can bind to several factors that stimulate angiogenesis, including vascular endothelial growth factor, platelet-derived growth factor, and fibroblast growth factor. The aim of this study is to observe changes in angiogenesis during the healing of wounds topically applied with snail mucus. Methods: Punch biopsy was performed on the back of male Wistar rats to obtain four wounds, and different concentrations of snail mucus were applied to each of these wounds. The animals were sacrificed on days 2, 4, and 7 to observe the extent of angiogenesis during wound healing by microscopy. Results: Two-way ANOVA showed differences in number of blood vessels formed (p = 0.00) and day of observation (p = 0.00) between groups. Post hoc Tukey’s HSD test showed that 24% snail mucus treatment does not significantly affect wound healing (p = 0.488); by contrast, treatment with 48% and 96% snail mucus demonstrated significant effects on angiogenesis (p = 0.01). Spearman’s test showed interactive effects between snail mucus concentration and day of observation on the extent of angiogenesis (p = 0.001, R = 0.946). Conclusion: Topical application of snail mucus gel can increase angiogenesis during wound healing in Wistar rat skin.

Colonisation of the Colonic Mucus Gel Layer With Butyrogenic and Hydrogenotropic Bacteria in Health and Ulcerative Colitis

Butyrate is the primary energy source for colonocytes and is essential for mucosal integrity and repair. Butyrate deficiency as a result of colonic dysbiosis is a putative factor in ulcerative colitis (UC). Commensal microbes are butyrogenic, while others have an inhibitory effect, through hydrogenotropic activity. The aim of this study was to quantify butyrogenic and hydrogenotropic species and determine their relationship with inflammation within the colonic mucus gel layer (MGL).Mucosal brushings were obtained from 20 patients with active colitis (AC), 20 healthy controls (HC) and 14 with quiescent colitis (QUC). Abundance of each species was determined by RT-PCR. Inflammatory scores were available for each patient. Statistical analyses were performed using Mann-Whitney-U and Kruskall-Wallis tests.Butyrogenic R. hominis was more abundant in health than UC (p<0.005). Hydrogenotropic B. wadsworthia was reduced in AC compared to HC and QUC (p<0.005). An inverse correlation existed between inflammation and R. hominis (ρ -0.460, p >0.005) and B. wadsworthia (ρ -0.646, p >0.005). Other hydrogenotropic species did not widely colonise the MGL. These data support a role for butyrogenic and some species of hydrogenotropic bacteria in UC. Butyrate deficiency in UC may be related to reduced microbial production, rather than inhibition by microbial by-products.

Modeling airway dysfunction in asthma using synthetic mucus biomaterials

As asthma worsens, occlusion of airways with mucus significantly contributes to airflow obstruction and reduced lung function. Recent evidence from clinical studies has shown mucus obtained from adults and children with asthma possesses altered mucin composition. However, how these changes alter the functional properties of the mucus gel is not yet fully understood. To study this, we have engineered a synthetic mucus biomaterial to closely mimic the properties of native mucus in health and disease. We demonstrate this model possesses comparable biophysical and transport properties to native mucus ex vivo collected from human subjects and in vitro isolated from human airway epithelial (HAE) tissue cultures. We found by systematically varying mucin composition that mucus gel viscoelasticity is enhanced when predominantly composed of mucin 5AC (MUC5AC), as is observed in asthma. As a result, asthma-like synthetic mucus gels are more slowly transported on the surface of HAE tissue cultures and at a similar rate to native mucus produced by HAE cultures stimulated with the type 2 cytokine IL-13, known to contribute to airway inflammation and MUC5AC hypersecretion in asthma. We also discovered the barrier function of asthma-like synthetic mucus towards influenza A virus was impaired as evidenced by the increased frequency of infection in MUC5AC-rich hydrogel coated HAE cultures. Together, this work establishes a biomaterial-based approach to understand airway dysfunction in asthma and related muco-obstructive lung diseases.Graphical Abstract

Influenza A virus diffusion through mucus gel networks

ABSTRACTIn this study, influenza A virus (IAV) and nanoparticle diffusion in human airway mucus was quantified using fluorescent video microscopy and multiple particle tracking. In previous work, it was determined that mucin-associated sialic acid acts as a decoy receptor for IAV hemagglutinin binding, and that virus passage through the mucus gel layer is facilitated through the sialic-acid cleaving enzyme, neuraminidase (NA), also present on the IAV envelope. However, our data suggests the mobility of IAV in mucus is significantly influenced by the mesh structure of the gel, as measured by nanoparticle probes, and NA activity is not required to facilitate virus passage through mucus gels. Using newly developed analyses, the binding affinity of IAV to the 3D mucus meshwork was estimated for individual virions with dissociation constants in the mM range, indicative of weak and reversible IAV-mucus interactions. We also found IAV diffusion significantly increased in mucus when treated with a mucolytic agent to break mucin-mucin disulfide bonds. In addition, IAV diffusion was significantly limited in a synthetic mucus model as crosslink density was systematically increased and network pore size was reduced. The results of this work provide important insights on how the balance of adhesive and physical barrier properties of mucus influence the dissemination of IAV within the lung microenvironment.

Less Reactive Thiol Ligands: Key towards Highly Mucoadhesive Drug Delivery Systems

As less reactive s-protected thiomers can likely interpenetrate the mucus gel layer to a higher extent before getting immobilized via disulfide bond formation with mucins, it was the aim of this study to develop a novel type of s-protected thiomer based on the less reactive substructure cysteine-N-acetyl cysteine (Cys-NAC) in order to obtain improved mucoadhesive properties. For this purpose, two types of s-protected thiomers, polyacrylic acid-cysteine-mercaptonicotinic acid (PAA-Cys-MNA) and polyacrylic acid-cysteine-N-acetyl cysteine (PAA-Cys-NAC), were synthesized and characterized by Fourier-transform infrared spectroscopy (FT-IR) and the quantification of attached disulfide ligands. The viscosity of both products was measured in the presence of NAC and mucus. Both thiomers were also evaluated regarding swelling behavior, tensile studies and retention time on the porcine intestinal mucosa. The FT-IR spectra confirmed the successful attachment of Cys-MNA and Cys-NAC ligands to PAA. The number of attached sulfhydryl groups was in the range of 660–683 µmol/g. The viscosity of both s-protected thiomers increased due to the addition of increasing amounts of NAC. The viscosity of the mucus increased in the presence of 1% PAA-Cys-MNA and PAA-Cys-NAC 5.6- and 10.9-fold, respectively, in comparison to only 1% PAA. Both s-protected thiomers showed higher water uptake than unmodified PAA. The maximum detachment force (MDF) and the total work of adhesion (TWA) increased in the case of PAA-Cys-MNA up to 1.4- and 1.6-fold and up to 2.4- and 2.8-fold in the case of PAA-Cys-NAC. The retention of PAA, PAA-Cys-MNA, and PAA-Cys-NAC on porcine intestinal mucosa was 25%, 49%, and 76% within 3 h, respectively. The results of this study provide evidence that less reactive s-protected thiomers exhibit higher mucoadhesive properties than highly reactive s-protected thiomers.

P075 Muc5ac expression protects the colonic barrier in experimental colitis

Background The mucus gel layer (MGL) lining the colon is integral to exclusion of bacteria and maintaining intestinal homeostasis in health and disease. MGL defects allowing bacteria to directly contact the colonic surface are commonly observed in ulcerative colitis (UC). The major macromolecular component of the colonic MGL is the secreted gel-forming mucin, MUC2, whose expression is essential for homeostasis in health. In UC, another gel-forming mucin, MUC5AC is induced. In mice, Muc5ac is protective during intestinal helminth infection. Here, we tested the expression and functional role of MUC5AC/Muc5ac in colitis patient biopsies and murine colitis. Methods We measured MUC5AC/Muc5ac expression in UC patient biopsies and during acute dextran sulphate sodium (DSS) colitis. We performed DSS-colitis in mice deficient in Muc5ac (Muc5ac−/−) to model the potential functional role of Muc5ac in colitis. To assess MGL integrity, we quantified bacterial–epithelial interaction and translocation to mesenteric lymph nodes (MLNs). Antibiotic treatment was performed to directly investigate the role of colonic bacteria in our murine colitis studies. Results Colonic MUC5AC/Muc5ac mRNA expression increased significantly in active UC and murine colitis. Muc5ac−/− mice experienced worsened injury and inflammation in DSS-colitis compared with controls. This was associated with increased bacterial–epithelial contact and translocation to the MLN. Antibiotic treatment normalised colitis severity in Muc5ac−/− mice to that of antibiotic treated controls. Conclusion We demonstrate for the first time that MUC5AC/Muc5ac induction in acute colitis controls injury by reducing bacterial breach of the MGL. Therefore, developing strategies to induce MUC5AC expression may protect the intestinal barrier in UC.

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.

The abundance of Akkermansia muciniphila and its relationship with sulphated colonic mucins in health and ulcerative colitis

Akkermansia muciniphila utilises colonic mucin as its substrate. Abundance is reduced in ulcerative colitis (UC), as is the relative proportion of sulphated mucin in the mucus gel layer (MGL). It is unknown if these phenomena are related, however reduced sulphated mucins could contribute to reduced abundance, owing to a lack of substrate. The aim of this study was to quantify A. muciniphila within the MGL and to relate these findings with markers of inflammation and the relative proportion of sulphomucin present. Colonic biopsies and mucus brushings were obtained from 20 patients with active UC (AC), 14 with quiescent UC (QUC) and 20 healthy controls (HC). A. muciniphila abundance was determined by RT-PCR. High iron diamine alcian-blue staining was performed for histological analysis. Patients with AC had reduced abundance of A. muciniphila compared to HC and QUC. A positive association was found between A. muciniphila abundance and higher percentage of sulphated mucin (ρ 0.546, p = 0.000). Lower abundances of A. muciniphila correlated with higher inflammatory scores (ρ = 0.294 (p = 0.001)). This study confirms an inverse relationship between A. muciniphila and inflammation and a positive association between A. muciniphila abundance and percentage of sulfated mucin in the MGL.