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. 2018 Feb 23;50(2):e450.
doi: 10.1038/emm.2017.282.

Akkermansia muciniphila-derived extracellular vesicles influence gut permeability through the regulation of tight junctions

Chaithanya Chelakkot  1 , Youngwoo Choi  2 , Dae-Kyum Kim  1 , Hyun T Park  2 , Jaewang Ghim  3 , Yonghoon Kwon  2 , Jinseong Jeon  2 , Min-Seon Kim  4 , Young-Koo Jee  5 , Yong S Gho  2 , Hae-Sim Park  6 , Yoon-Keun Kim  7 , Sung H Ryu  1   2
Affiliations

Akkermansia muciniphila-derived extracellular vesicles influence gut permeability through the regulation of tight junctions

Chaithanya Chelakkot et al. Exp Mol Med. .

Abstract

The gut microbiota has an important role in the gut barrier, inflammation and metabolic functions. Studies have identified a close association between the intestinal barrier and metabolic diseases, including obesity and type 2 diabetes (T2D). Recently, Akkermansia muciniphila has been reported as a beneficial bacterium that reduces gut barrier disruption and insulin resistance. Here we evaluated the role of A. muciniphila-derived extracellular vesicles (AmEVs) in the regulation of gut permeability. We found that there are more AmEVs in the fecal samples of healthy controls compared with those of patients with T2D. In addition, AmEV administration enhanced tight junction function, reduced body weight gain and improved glucose tolerance in high-fat diet (HFD)-induced diabetic mice. To test the direct effect of AmEVs on human epithelial cells, cultured Caco-2 cells were treated with these vesicles. AmEVs decreased the gut permeability of lipopolysaccharide-treated Caco-2 cells, whereas Escherichia coli-derived EVs had no significant effect. Interestingly, the expression of occludin was increased by AmEV treatment. Overall, these results imply that AmEVs may act as a functional moiety for controlling gut permeability and that the regulation of intestinal barrier integrity can improve metabolic functions in HFD-fed mice.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Microbial diversity and relative abundances of EVs in human feces. Metagenomic data showing microbial diversity. (a) Microbial diversity in the feces of type 2 diabetic patients and healthy controls. (b) Microbial diversity in the fecal EVs of type 2 diabetic patients and healthy controls. (c) Heat map of normalized EV contents. Only species that showed significant differences were used to draw this heat map (P<0.05). The order of species was sorted by the content ratio (type 2 diabetes patients over healthy controls), showing that A. muciniphila might be a candidate EV-secreting bacterium that suppresses type 2 diabetes.
Figure 2
Figure 2
In vivo distribution of Cy7-labeled A. muciniphila-derived EVs in mice. Cy7-labeled AmEVs (50 μg of total protein) were orally administered to C57BL/6 mice. (a) In vivo fluorescent whole-body images of AmEVs in mice were acquired with an IVIS spectrum. (b) Ex vivo image of various tissues, including liver, fat, muscle, pancreas, spleen and large intestine, after 6 h of EV feeding. (c) Radiant efficiency was measured using the Living Image 3.1 software. All data are presented as the mean±s.e.m. of 3 experiments, with n=5–7 animals per group for each experiment. *P<0.05.
Figure 3
Figure 3
A. muciniphila-derived EVs improve gut permeability in high-fat diet (HFD)-fed mice. Mice were fed a normal chow diet (NCD) or a HFD for 12 weeks, followed by oral administration of AmEVs (10 μg per mouse) every other day for 2 weeks. (a) Body weight changes were measured at the indicated time points. The graph shows body weight change from the day that EV feeding started. (b) In vivo intestinal permeability assay in NCD, NCD+AmEV, HFD and HFD+AmEV mice conducted after 4 h of treatment with FITC-dextran. The serum FITC concentration was measured using blood collected by retro-orbital bleeding, and fluorescence was measured using a spectrofluorometer. Data are shown as the mean±s.e.m. (c) Gross imaging of the colons dissected from NCD-, NCD+AmEV-, HFD- and HFD+AmEV-fed mice. (d) The colon length of NCD-, NCD+AmEV-, HFD- and HFD+AmEV-fed mice. (e) Hematoxylin and eosin staining of colon sections from NCD-, NCD+AmEV-, HFD- and HFD+AmEV-fed mice. Red arrows indicate the recruitment of immune cells. Scale bar is 50 μm. (f) Immunohistochemical images showing occludin expression (occludin—red, nucleus—blue) in NCD-, NCD+AmEV-, HFD- and HFD+AmEV-fed mice. Scale bar is 50 μm. All data are presented as the mean±s.e.m. of 3 experiments; n=5–7 per group; *P<0.05, **P<0.01.
Figure 4
Figure 4
A. muciniphila-derived EVs reduce LPS-induced intestinal permeability through AMPK phosphorylation. (a) Trans-epithelial electrical resistance in Caco-2 cells 4 h after treatment with LPS (5 μg ml−1), LPS (5 μg ml−1)+EcEVs (1 μg ml−1) and LPS (5 μg ml−1)+AmEVs (1 μg ml−1). (b) In vitro permeability assay in Caco-2 cells 4 h after LPS (5 μg ml−1), LPS (5 μg ml−1)+EcEV (1 μg ml−1) and LPS (5 μg ml−1)+AmEV (1 μg ml−1) treatment. FITC-dextran was added to the upper chamber in the Caco-2 transwell chamber assay, and the transport of FITC-labeled dextran across the Caco-2 monolayer culture was measured by fluorescence spectrofluorometry. (c) Expression of tight junction proteins in Caco-2 cells after LPS, LPS+EcEV and LPS+AmEV treatments. A graph showing the relative occludin expression (normalized to actin) is also shown (*P<0.05). (d) AmEV treatment increased occludin expression and phosphorylation of AMPKα2 thr-172 in a dose-dependent manner. Caco-2 cells were treated with 0.1, 1 and 10 μg ml−1 AmEVs, and the expression of occludin, p-AMPK, total AMPK and actin was analyzed after 4 h by immunoblotting. A graph showing the relative protein expression normalized to actin is shown (occludin expression in NT vs the specified treatment group ##P<0.01, ###P<0.001; p-AMPK expression in NT vs the specified treatment group **P<0.01). (e) Time course of AmEV-induced occludin and p-AMPK expression in Caco-2 cells. Caco-2 cells were treated with 1 μg ml−1 AmEVs, and the expression of occludin, p-AMPK, total AMPK and actin was analyzed after 1, 2, 4 and 8 h by immunoblotting. The graph shows the relative protein expression after normalization to actin (occludin expression in NT vs the specified time point #P<0.05; p-AMPK expression in NT vs the specified time point *P<0.05, **P<0.01). (f) Western blot showing the effect of the AMPK inhibitor on AmEV-treated Caco-2 cells. Caco-2 cells were treated with the AMPK-specific inhibitor compound C for 30 min, after which 1 μg ml−1 AmEVs was applied. Protein expression of occludin and p-AMPK was observed after 4 h. The graph shows the relative occludin expression after normalization to actin (occludin expression in NT vs the specified time point #P<0.05). All data are presented as the mean±s.e.m. of three experiments performed in triplicate. NS, not significant.
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