Muriel Mercier-Bonin

Stress disrupts intestinal mucus barrier in rats via mucin O-glycosylation shift: prevention by a probiotic treatment.

Despite well-known intestinal epithelial barrier impairment and visceral hypersensitivity in irritable bowel syndrome (IBS) patients and IBS-like models, structural and physical changes in the mucus layer remain poorly understood. Using a water avoidance stress (WAS) model, we aimed at evaluating whether 1) WAS modified gut permeability, visceral sensitivity, mucin expression, biochemical structure of O-glycans, and related mucus physical properties, and 2) whether Lactobacillus farciminis treatment prevented these alterations. Wistar rats received orally L. farciminis or vehicle for 14 days; at day 10, they were submitted to either sham or 4-day WAS. Intestinal paracellular permeability and visceral sensitivity were measured in vivo. The number of goblet cells and Muc2 expression were evaluated by histology and immunohistochemistry, respectively. Mucosal adhesion of L. farciminis was determined ex situ. The mucin O-glycosylation profile was obtained by mass spectrometry. Surface imaging of intestinal mucus was performed at nanoscale by atomic force microscopy. WAS induced gut hyperpermeability and visceral hypersensitivity but did not modify either the number of intestinal goblet cells or Muc2 expression. In contrast, O-glycosylation of mucins was strongly affected, with the appearance of elongated polylactosaminic chain containing O-glycan structures, associated with flattening and loss of the mucus layer cohesive properties. L. farciminis bound to intestinal Muc2 and prevented WAS-induced functional alterations and changes in mucin O-glycosylation and mucus physical properties. WAS-induced functional changes were associated with mucus alterations resulting from a shift in O-glycosylation rather than from changes in mucin expression. L. farciminis treatment prevented these alterations, conferring epithelial and mucus barrier strengthening.

Impact of mycotoxins on the intestine: are mucus and microbiota new targets?

ABSTRACT There is an increasing awareness of the deleterious effects attributed to mycotoxins during their fate within the gut, particularly for deoxynivalenol (DON), zearalenone (ZEN), ochratoxin A (OTA), fumonisin B1 (FB1), aflatoxin B1 (AFB1), and patulin (PAT). Evidence indicates that disruption of the epithelial barrier is well established. However, intestinal barrier function on its luminal side involves two other partners, mucus and microbiota, which have rarely been considered in the context of mycotoxin exposure. The current review aimed at providing a summary of DON, ZEN, OTA, FB1, AFB1, and PAT effects on intestinal barrier function, with special focus on mucus and microbiota. DON, ZEN, OTA, FB1, AFB1, and PAT are known to markedly affect epithelial cell integrity and functions. Regarding mucus, DON is the most documentated mycotoxin. In vivo, toxicological impact of DON generally has only been assessed through goblet cell number. Evaluation of the mycotoxins/mucus interplay considering other indicators such as composition, thickness, and penetrability of mucus, mucin O-glycosylation thus warrants further attention. With respect to microbiota, few short-term studies to date have been reported indicating deleterious effects. However, long-term exposure to mycotoxins may also produce significant changes in microbiota composition and metabolic activity, which requires further experimentation. In conclusion, mucus and microbiota are key targets for dietary mycotoxins although assessment of induced effects is preliminary. A significant research effort is now underway to determine the adverse consequences of mycotoxins on mucus and microbiota considered as individual but also as tightly connected gut players.

Transmission electron microscopy for elucidating the impact of silver-based treatments (ionic silver versus nanosilver-containing coating) on the model yeast Saccharomyces cerevisiae

After exposure to ionic silver or nanosilver-containing plasma coating, the same visual aspect of scanning transmission electron microscopy (STEM) images was observed for the model yeast Saccharomyces cerevisiae. The main common feature was the presence of electron-dense nodules all over the cell. However, high resolution TEM (HRTEM), STEM, energy dispersive x-ray microanalysis spectroscopy (EDS) and electron microdiffraction revealed some striking differences. Regarding ionic silver exposure, the formation of electron-dense nodules was related to the Ag(+) reactivity towards sulfur-containing compounds to form clusters with Ag(2)S-like structures, together with the production of a few silver nanocrystals, mainly at the cell wall periphery. For nanosilver-based treatment, some sulfur-containing silver clusters preferentially located at the cell wall periphery were detected, together with nodules composed of silver, sulfur and phosphorus all over the cell. In both silver-based treatments, nitrogen and silver signals overlapped, confirming the affinity of silver entities for proteinaceous compounds. Moreover, in the case of nanosilver, interactions of silver with phosphorus-containing subcellular structures were indicated.

Adhesion of the genome-sequenced Lactococcus lactis subsp. cremoris IBB477 strain is mediated by specific molecular determinants

Understanding the nature of mucus-microbe interactions will provide important information that can help to elucidate the mechanisms underlying probiotic adhesion. This study focused on the adhesive properties of the Lactococcus lactis subsp. cremoris IBB477 strain, previously shown to persist in the gastrointestinal tract of germ-free rats. The shear flow-induced detachment of L. lactis cells was investigated under laminar flow conditions. Such a dynamic approach demonstrated increased adhesion to bare and mucin-coated polystyrene for IBB477, compared to that observed for the MG1820 control strain. To identify potential genetic determinants giving adhesive properties to IBB477, the improved high-quality draft genome sequence comprising chromosome and five plasmids was obtained and analysed. The number of putative adhesion proteins was determined on the basis of surface/extracellular localisation and/or the presence of adhesion domains. To identify proteins essential for the IBB477 specific adhesion property, nine deletion mutants in chromosomal genes have been constructed and analysed using adhesion tests on bare polystyrene as well as mucin-, fibronectin- or collagen IV-coated polystyrene plates in comparison to the wild-type strain. These experiments demonstrated that gene AJ89_07570 encoding a protein containing DUF285, MucBP and four Big_3 domains is involved in adhesion to bare and mucin-coated polystyrene. To summarise, in the present work, we characterised the adhesion of IBB477 under laminar flow conditions; identified the putative adherence factors present in IBB477, which is the first L. lactis strain exhibiting adhesive and mucoadhesive properties to be sequenced and demonstrated that one of the proteins containing adhesion domains contributes to adhesion.

Mucus and microbiota as emerging players in gut nanotoxicology: The example of dietary silver and titanium dioxide nanoparticles

ABSTRACT Given the growing use of nanotechnology in many common consumer products, including foods, evaluation of the consequences of chronic exposure to nanoparticles in humans has become a major public health issue. The oral route of exposure has been poorly explored, despite the presence of a fraction of nanosized particles in certain food additives/supplements and the incorporation of such particles into packaging in contact with foods. After their ingestion, these nanoparticles pass through the digestive tract, where they may undergo physicochemical transformations, with consequences for the luminal environment, before crossing the epithelial barrier to reach the systemic compartment. In this review, we consider two examples, nanosilver and nanotitanium dioxide. Despite the specific features of these particles and the differences between them, both display a close relationship between physicochemical reactivity and bioavailability/biopersistence in the gastrointestinal tract. Few studies have focused on the interactions of nanoparticles of silver or titanium dioxide with the microbiota and mucus. However, the microbiota and mucus play key roles in intestinal homeostasis and host health and are undoubtedly involved in controlling the distribution of nanoparticles in the systemic compartment.

Renal tubular fluid shear stress facilitates monocyte activation toward inflammatory macrophages

Modified urinary fluid shear stress (FSS) induced by variations of urinary fluid flow and composition is observed in early phases of most kidney diseases. Recently, we reported that renal tubular FSS promotes endothelial cell activation and subsequent adhesion of human monocytes, thereby suggesting that changes in urinary FSS can induce the development of inflammation (Miravète M, Klein J, Besse-Patin A, Gonzalez J, Pecher C, Bascands JL, Mercier-Bonin M, Schanstra JP, Buffin-Meyer B, BBRC 407: 813-817, 2011). Here, we evaluated the influence of tubular FSS on monocytes as they play an important role in the progression of inflammation in nephropathies. Human renal tubular cells (HK-2) were exposed to FSS 0.01 Pa for 30 min or 5 h. Treatment of human THP-1 monocytes with the resulting conditioned medium (FSS-CM) modified the expression of macrophage differentiation markers, suggesting differentiation toward the inflammatory M1-type macrophage. The effect was confirmed in freshly isolated human monocytes. In contrast to endothelial cells, the activation of monocytes by FSS-CM did not require TNF-α. Cytokine array analysis of FSS-CM showed that FSS modified secretion of cytokines by HK-2 cells, particularly by increasing secretion of TGF-β and by decreasing secretion of C-C chemokine ligand 2 (CCL2). Neutralization of TGF-β or CCL2 supplementation attenuated the effect of FSS-CM on macrophage differentiation. Finally, FSS-injured HK-2 cells expressed and secreted early biomarkers of tubular damage such as kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin. In conclusion, changes in urinary FSS should now also be considered as potential insults for tubular cells that initiate/perpetuate interstitial inflammation.

Renal tubular fluid shear stress promotes endothelial cell activation.

Modified urinary fluid shear stress (FSS) induced by variations of urinary fluid flow and composition is observed in early phases of most kidney diseases. In this study, we hypothesized that changes in urinary FSS represent a tubular aggression that contributes to the development of inflammation, a key event in progression of nephropathies. Human renal tubular cells (HK-2) were exposed to FSS for 30 min at 0.01 Pa. Treatment of human endothelial cells (HMEC-1) with the resulting conditioned medium (FSS-CM) increased C-C chemokine ligand 2 (CCL2) and tumor necrosis factor (TNF)-α protein secretion, increased endothelial vascular adhesion molecule-1 (VCAM-1) mRNA expression and stimulated adhesion of human (THP-1) monocytes to the endothelial monolayer. These effects were TNF-α dependent as they were abolished by neutralization of TNF-α. Interestingly, the origin of TNF-α was not epithelial, but resulted from autocrine endothelial production. However, in contrast to short term FSS, long term FSS (5h) induced the release of the key inflammatory proteins CCL2 and TNF-α directly from tubular cells. In conclusion, these results suggest for the first time that urinary FSS can contribute to the inflammatory state involved in initiation/perpetuation of renal diseases.

Evaluation of adhesion force between functionalized microbeads and protein-coated stainless steel using shear-flow-induced detachment.

Spherical microbeads functionalized with two types of chemical groups (NH(2), OH) were chosen as a simplified bacterial model, in order to elucidate the role of macromolecular interactions between specific biopolymers and 316 L stainless steel, in the frame of biofilm formation in the marine environment. NH(2) microbeads were used in their native form or after covalent binding to BSA or different representative poly-amino acids. OH microbeads were used in their native form. Adhesion force between microbeads and bare or BSA-coated stainless steel was quantified at nanoscale. Shear-flow-induced detachment experiments were combined with a simplified version of a theoretical model, based on the balance of hydrodynamic forces and torque exerted on microbeads. A maximal adhesion force of 27.6+/-8.5 nN was obtained for BSA-coated NH(2) microbeads. The high reactivity of OH functional groups was assessed (adhesion force of 15.6+/-4.8 nN for large microbeads). When charge-conducting stainless steel was coated with BSA, adhesion force was significantly lower than the one estimated with the bare surface, probably due to an increase in hydrophilic surface properties or suppression of charge transfer. The mechanism for microbead detachment was established (mainly rolling). The flow chamber and the associated theoretical modelling were demonstrated to be a relevant approach to quantify nanoscale forces between interacting surfaces.

Mucus organisation is shaped by colonic content; a new view

The colonic mucus barrier is commonly described as a continuous double layer covering the epithelium, separating the microbiota from the intestinal tissue. This model is currently considered valid throughout the colon. The colon is characterised by regional anatomo-functional specificities such as presence and consistency of contents and location. In this study, we characterised the organisation of the colonic mucus barrier in proximal and distal colon of rodents by histological and FISH staining, taking into account aforementioned specificities. By using longitudinal sections and imaging extensive areas of tissue with and without colonic contents, we have obtained a spatiotemporal overview of mucus organisation in the colon. We describe for the first time that the colonic mucus layer covers the faeces instead of the epithelium in the distal colon. This faecal mucus layer confines the microbiota to the faeces and prevents it from remaining in empty distal colon. In the proximal colon, the mucus did not form a separating layer between bacteria and epithelium. We conclude that the organisation of colonic mucus is reliant on the presence of the colonic content, and the location within the colon. Our findings reopen the discussion on the nature of the colonic mucus barrier.

Dynamics of detachment of Escherichia coli from plasma-mediated coatings under shear flow.

A series of plasma-mediated coatings, containing silver nanoparticles embedded in an organosilicon or silica-like matrix, were deposited onto stainless steel and chemically characterized. Their anti-adhesive properties were evaluated in vitro towards Escherichia coli by performing shear-flow induced detachment experiments. Increasing the wall shear stress facilitated E. coli cell detachment, irrespective of the coating characteristics. When nanosilver was incorporated, cell detachment was lower, probably due to the affinity of the embedded silver for biological components of the cell wall. The presence of methyl groups in the matrix network could also promote enhanced hydrophobic interactions. Within the population fraction remaining attached to the coating under increasing shear flow, different association phenotypes were observed, viz. progressively lying flat, moving laterally, remaining tethered, or rotating by a single anchoring point, until alignment with the flow direction. This re-orientation phenotype and its relation with detachment were dependent of the coating. The effects of such heterogeneities should be more deeply explored.