Akkermansia muciniphila-Derived Extracellular Vesicles as a Mucosal Delivery Vector for Amelioration of Obesity in Mice

Abstract

Recent evidence suggests that probiotics can restore the mucosal barrier integrity, ameliorate inflammation, and promote homeostasis required for metabolism in obesity by affecting the gut microbiota composition. In this study, we investigated the effect of Akkermansia muciniphila and its extracellular vesicles (EVs) on obesity-related genes in microarray datasets and evaluated the cell line and C57BL/6 mice by conducting RT-PCR and ELISA assays. A. muciniphila-derived EVs caused a more significant loss in body and fat weight of high-fat diet (HFD)-fed mice, compared with the bacterium itself. Moreover, treatment with A. muciniphila and EVs had significant effects on lipid metabolism and expression of inflammatory markers in adipose tissues. Both treatments improved the intestinal barrier integrity, inflammation, energy balance, and blood parameters (i.e., lipid profile and glucose level). Our findings showed that A. muciniphila-derived EVs contain various biomolecules, which can have a positive impact on obesity by affecting the involved genes. Also, our results showed that A. muciniphila and its EVs had a significant relationship with intestinal homeostasis, which highlights their positive role in obesity treatment. In conclusion, A. muciniphila-derived EVs can be used as new therapeutic strategies to ameliorate HFD-induced obesity by affecting various mechanisms.

Keywords: Akkermansia muciniphila; Angplt4; extracellular vesicles; gut microbiota; obesity; peroxisome proliferator-activated receptors; tight junction; toll-like receptors.

FIGURE 1

Morphologic characterization of EVs and impact of Akkermansia muciniphila and its EV administration on body and adipose weight, food intake, and blood parameters in both ND- and HFD-fed mice after 5 weeks (n = 5 for each group). (A) Illustration showing obesity induction and amelioration after treatment. (B) Scanning electron micrograph image of A. muciniphila-derived EVs. (C) Body weight gain per mouse. (D) Average daily food intake. (E) Epidydimal adipose weight (EAT). (F) Histopathology image. Scale bar is 50 μm. (G) Total cholesterol levels. (H) Triglyceride levels and (I) glucose levels. ^∗P < 0.05 and ^∗∗P < 0.01 were considered statistically significant, respectively. ND, normal diet + PBS; NA.m, normal diet + A. muciniphila (10⁹ CFU); NEV, normal diet + EVs (10 μg protein); HFD, high-fat diet + PBS; HA.m, high-fat diet + A. muciniphila (10⁹ CFU); HEV, high-fat diet + EVs (10 μg protein).

FIGURE 2

Heatmap and PCA correlation show different genes involved in inflammation and FA oxidation in the epididymal adipose tissue between HFD and ND mice. (A) Heatmap plot revealed inflammatory genes (TNF-α, IL-6, and TLR-4) and TGF-β were expressed at significantly higher levels in the obese than in the normal group. (B) Regulator genes involved in FA oxidation and inflammation (PPAR-γ and PPAR-α) were downregulated in HFD- compared to ND-fed mice. PCA plot showed that the ND group was clustered relatively from the HFD group. The HFD and ND groups are indicated by orange and blue colors, respectively.

FIGURE 3

The effect of A. muciniphila and its EVs on mRNA expression of genes in epidydimal adipose tissue in C57bl/6 mice. Relative mRNA expression of (A) PPAR-γ, (B) PPAR-α, (C) TGF-β, (D) TLR-4, (E) TNF-α, and (F) IL-6. Data are normalized using HPRT as control gene. ^∗P < 0.05 and ^∗∗P < 0.01 were considered statistically significant, respectively. ND, normal diet + PBS; NA.m, normal diet + A. muciniphila (10⁹ CFU); NEV, normal diet + EVs (10 μg protein); HFD, high-fat diet + PBS; HA.m, high-fat diet + A. muciniphila (10⁹ CFU); HEV, high-fat diet + EVs (10 μg protein).

FIGURE 4

The assessment of A. muciniphila and its EVs effect on obesity-related genes in the colon of HFD- and ND-fed mice. Both mice administrated to this bacterium and EVs for 5 weeks. (A) Histopathology image. M, tunica muscularis; S, submucosa layer. Crypt depth (arrows). Mucous thickness (arrowheads). Scale bar is 200 μm. Expression of (B) ZO-1, (C) OCLDN, (D) CLDN-1, (E) CLDN-2, (F) TLR-4, (G) TLR-2, (H) TNF-α, (I) IL-10, and (J) ANGPTL4. Data are normalized using RPL-19 as control gene. ^∗P < 0.05 and ^∗∗P < 0.01 were considered statistically significant, respectively. ND, normal diet + PBS; NA.m, normal diet + A. muciniphila (10⁹ CFU); NEV, normal diet + EVs (10 μg protein); HFD, high-fat diet + PBS; HA.m, high-fat diet + A. muciniphila (10⁹ CFU); HEV, high fat-diet + EVs (10 μg protein).

FIGURE 5

The effect of A. muciniphila and its EVs on the study’s genes and cytokine secretion in the Caco-2 cell line. Caco-2 monolayers were treated with A. muciniphila (MOI₁₀₀) and EV concentration (10 μg) for 24 h. The expression of genes: (A) ZO-1, OCLDN, and CLDN-1; (B) TLR-2 and TLR-4; and (C) ANGPTL4. The levels of cytokines: (D) IL-6 and IL-8; (E) TNF-α and IL-10; and (F) IFN-γ and IL-4. Expression data are normalized using GAPDH as control gene. ^∗P < 0.05 and ^∗∗P < 0.01 were considered statistically significant, respectively.

FIGURE 6

Administration of A. muciniphila and its EVs improves the intestinal and metabolic homeostasis in obese mice. Obesity is associated with the disruption of the intestinal barrier integrity, inflammation, and fat mass gain (left). Oral administration of A. muciniphila or its EVs increase the expression of tight junction proteins and TLR-2 and reduce the expression of TLR-4 and pro-inflammatory cytokines in the colon of obese mice (right). Also, treatment with A. muciniphila and its EVs affects FA oxidation, local inflammation genes, and fat-mass loss in the EAT of obese mice. HFD, high-fat diet; EVs, extracellular vesicles; TLR, toll-like receptor; LPS, lipopolysaccharide; PPAR, peroxisome proliferator-activated receptor; Zo-1, zonula occludens-1; Ocldn, occludin; Cldn, claudin; Angptl4, angiopoietin-like 4; TNF-α, tumor necrosis factor-α; IL-10, interleukin-10; TGF-β, transforming growth factor-β; and Glc, glucose.