Impact of supplementation with a food-derived microbial community on obesity-associated inflammation and gut microbiota composition

Abstract

Background: Obesity is a complex pathology associated with dysbiosis, metabolic alterations, and low-grade chronic inflammation promoted by immune cells, infiltrating and populating the adipose tissue. Probiotic supplementation was suggested to be capable of counteracting obesity-associated immune and microbial alterations, based on its proven immunomodulatory activity and positive effect on gut microbial balance. Traditional fermented foods represent a natural source of live microbes, including environmental strains with probiotic features, which could transiently colonise the gut. The aim of our work was to evaluate the impact of supplementation with a complex foodborne bacterial consortium on obesity-associated inflammation and gut microbiota composition in a mouse model.

Methods: C57BL/6J mice fed a 45% high fat diet (HFD) for 90 days were supplemented with a mixture of foodborne lactic acid bacteria derived from the traditional fermented dairy product "Mozzarella di Bufala Campana" (MBC) or with the commercial probiotic GG strain of Lactobacillus rhamnosus (LGG). Inflammation was assessed in epididymal white adipose tissue (WAT) following HFD. Faecal microbiota composition was studied by next-generation sequencing.

Results: Significant reduction of epididymal WAT weight was observed in MBC-treated, as compared to LGG and control, animals. Serum metabolic profiling showed correspondingly reduced levels of triglycerides and higher levels of HDL cholesterol, as well as a trend toward reduction of LDL-cholesterol levels. Analysis of the principal leucocyte subpopulations in epididymal WAT revealed increased regulatory T cells and CD4⁺ cells in MBC microbiota-supplemented mice, as well as decreased macrophage and CD8⁺ cell numbers, suggesting anti-inflammatory effects. These results were associated with lower levels of pro-inflammatory cytokines and chemokines in WAT explants. Faecal bacterial profiling demonstrated increased Firmicutes/Bacteroidetes ratio in all mice groups following HFD.

Conclusions: Taken together, these results indicate a protective effect of MBC microbiota supplementation toward HFD-induced fat accumulation and triglyceride and cholesterol levels, as well as inflammation, suggesting a stronger effect of a mixed microbial consortium vs single-strain probiotic supplementation. The immunomodulatory activity exerted by the MBC microbiota could be due to synergistic interactions within the microbial consortium, highlighting the important role of dietary microbes with yet uncharacterised probiotic effect.

Keywords: Chronic inflammation; Fermented dairy; Foodborne microbiota; High fat diet; White adipose tissue.

Fig. 1

Experimental design. Six-week-old C57BL/6J male mice were randomly assigned to three experimental groups (four or five animals per group). The mice were fed a standard diet and orally supplemented daily with MBC microbiota, LGG, or PBS (CTRL). After 15 days, the mice were shifted to HFD while continuing bacterial or PBS supplementation for 90 additional days. At the end of the experimental period, blood and epididymal WAT were collected. Faeces were sampled for gut microbiota analysis at the indicated time points: t0, t15, and t105. The experiment was replicated once, and the number of mice in each group for each of the two repetitions is indicated

Fig. 2

Leukocyte subpopulations in epididymal WAT. The effect of bacterial supplementation on the frequency of WAT leukocyte subpopulations was analysed by flow cytometry. The percentage of CD25⁺Foxp3⁺ Treg cells was calculated on T lymphocyte gate (CD4+, a), CD4⁺ and CD8⁺ cell subsets were calculated on lymphocyte gate (CD3⁺, b), whereas CD11b⁺ and F4/80⁺ cells were calculated on leukocyte gate (CD45⁺, c). Black columns: MBC-supplemented mice; grey columns: LGG-supplemented; white columns: CTRL. Each column represents the mean ± SD of nine mice. Means without a common letter significantly differ

Fig. 3

Cytokine and chemokine secretion in epididymal WAT explants. WAT explants were cultured in complete DMEM for 24 h in the presence of ionomycin (1 ng/ml) and PMA (5 ng/ml). Cytokine and chemokine levels were analysed by Luminex assay or by ELISA (see the “Methods” section). Each column represents the mean ± SD of nine mice. Means without a common letter significantly differ

Fig. 4

PCA plot from epididymal WAT immunological profiles. PC1/PC2 score plot showing the distribution of samples in reduced PC1/PC2 space. The percentage variation explained by the plotted principal components is indicated. Symbols refer to individual mice. Red crosses: MBC-supplemented mice; blue squares: LGG-supplemented; black dots: CTRL

Fig. 5

Relative abundance of gut bacterial phyla obtained by NGS of faecal samples. Each bar refers to a single faecal sample and depicts the proportion of OTUs per sample, expressed as percentage. Colour coding of bacterial phyla is shown on the right side. “Others” includes unidentified microorganisms of Bacteria kingdom or of Eukaryota kingdom and unclassified microorganisms

Fig. 6

PCoA plots of unweighted UniFrac distance matrix. PC1/PC2 score plot showing the distribution of samples. The same plots are shown in each panel, with symbols referring to individual samples, but colour coding of each sample refers to time points in a (t0 = red triangles, t15 = blue squares, t105 = orange circles) or treatment type in b (CTRL = red triangles, LGG = blue squares, MBC = orange circles). The percentage variation explained by the plotted principal coordinates is indicated in the axis legend. Score values shown along the axes represent the proportion of dissimilarities captured by each axis