Akkermansia muciniphila Adheres to Enterocytes and Strengthens the Integrity of the Epithelial Cell Layer - PubMed (original) (raw)

Akkermansia muciniphila Adheres to Enterocytes and Strengthens the Integrity of the Epithelial Cell Layer

Justus Reunanen et al. Appl Environ Microbiol. 2015 Jun.

Abstract

Akkermansia muciniphila is a Gram-negative mucin-degrading bacterium that resides in the gastrointestinal tracts of humans and animals. A. muciniphila has been linked with intestinal health and improved metabolic status in obese and type 2 diabetic subjects. Specifically, A. muciniphila has been shown to reduce high-fat-diet-induced endotoxemia, which develops as a result of an impaired gut barrier. Despite the accumulating evidence of the health-promoting effects of A. muciniphila, the mechanisms of interaction of the bacterium with the host have received little attention. In this study, we used several in vitro models to investigate the adhesion of A. muciniphila to the intestinal epithelium and its interaction with the host mucosa. We found that A. muciniphila adheres strongly to the Caco-2 and HT-29 human colonic cell lines but not to human colonic mucus. In addition, A. muciniphila showed binding to the extracellular matrix protein laminin but not to collagen I or IV, fibronectin, or fetuin. Importantly, A. muciniphila improved enterocyte monolayer integrity, as shown by a significant increase in the transepithelial electrical resistance (TER) of cocultures of Caco-2 cells with the bacterium. Further, A. muciniphila induced interleukin 8 (IL-8) production by enterocytes at cell concentrations 100-fold higher than those for Escherichia coli, suggesting a very low level of proinflammatory activity in the epithelium. In conclusion, our results demonstrate that A. muciniphila adheres to the intestinal epithelium and strengthens enterocyte monolayer integrity in vitro, suggesting an ability to fortify an impaired gut barrier. These results support earlier associative in vivo studies and provide insights into the interaction of A. muciniphila with the host.

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Figures

FIG 1

FIG 1

Live-dead fluorescence staining of A. muciniphila cells exposed to different atmospheres. A. muciniphila cells were incubated for 1 h under an aerobic, 5% CO2, or anaerobic atmosphere. Relative fluorescence units (RFU) were measured after staining of the cells with DAPI or propidium iodide (PI), which stains only dead or seriously impaired cells. Background fluorescence from nonstained cells has been subtracted from the RFU values obtained. The results shown are means and standard deviations for four parallel samples.

FIG 2

FIG 2

Adhesion of A. muciniphila cells to the Caco-2 and HT29 human epithelial cell lines. Adhesion was carried out under an anaerobic or 5% CO2 atmosphere and was assessed by immunofluorescence microscopy. Enterocyte nuclei were strained with DAPI (blue), and A. muciniphila cells were stained with a polyclonal rabbit antiserum raised against whole A. muciniphila cells and a secondary antibody conjugated with Alexa Fluor 594 (red).

FIG 3

FIG 3

Binding of A. muciniphila to ECM proteins. Metabolically labeled A. muciniphila cells were allowed to bind to different human extracellular matrix proteins. The results shown are means and standard deviations for five parallel wells. The asterisk indicates a level of binding significantly different (P < 0.05) from background binding (to BSA).

FIG 4

FIG 4

Adherence of A. muciniphila to the Caco-2 and HT-29 cell lines and to human intestinal mucus. A. muciniphila and L. rhamnosus GG (positive-control strain) cells were allowed to bind to human enterocytes or immobilized intestinal mucus. Means and standard deviations for five parallel wells are shown.

FIG 5

FIG 5

Adherence of A. muciniphila to Caco-2 and HT-29 cells at different growth stages. Levels of binding to mucus are shown for comparison. Data are means and standard deviations for five parallel wells. The asterisk indicates a significant difference (P < 0.05) in adhesion to the different cell lines at the same growth stage.

FIG 6

FIG 6

Impact of A. muciniphila, B. fragilis, or E. coli on the development of the TER of a Caco-2 monolayer. Means and standard deviations for three parallel wells are shown. Asterisks indicate TER values significantly different (P < 0.05) from that of the control (growth medium without bacteria).

FIG 7

FIG 7

Induction of IL-8 production in HT-29 cells by A. muciniphila, B. fragilis, and E. coli. Means and standard deviations for three parallel wells are shown. Asterisks indicate IL-8 production levels significantly (P < 0.05) above the background level (growth medium without bacteria or LPS). LPS (1 ng ml−1) from E. coli was included as a positive control.

FIG 8

FIG 8

Analysis of LPS and lipid A contents of A. muciniphila. (A) Western blotting of whole-cell lysates of A. muciniphila (lane 2) and E. coli (positive control) (lane 3) using an antiserum against E. coli LPS. Lane 1, molecular mass standard. (B) Electron micrographs of thin-sectioned and immunostained A. muciniphila and E. coli bacteria. The bacteria were immunostained using an antiserum against E. coli lipid A and 10-nm colloidal gold particles conjugated to protein A (pAg). Arrows indicate 10-nm gold particles. Bars, 500 nm.

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