Influence of Gut Microbiota on Progression to Tuberculosis Generated by High Fat Diet-Induced Obesity in C3HeB/FeJ Mice

Abstract

The administration of a high fat content diet is an accelerating factor for metabolic syndrome, impaired glucose tolerance, and early type 2 diabetes. The present study aims to assess the impact of a high fat diet on tuberculosis progression and microbiota composition in an experimental animal model using a C3HeB/FeJ mouse strain submitted to single or multiple consecutive aerosol infections. These models allowed us to study the protection induced by Bacillus Calmette-Guérin vaccination as well as by the natural immunity induced by chemotherapy after a low dose Mycobacterium tuberculosis infection. Our results show that a high fat diet is able to trigger a pro-inflammatory response, which results in a faster progression toward active tuberculosis and an impaired protective effect of BCG vaccination, which is not the case for natural immunity. This may be related to dysbiosis and a reduction in the Firmicutes/Bacteroidetes ratio in the gut microbiota caused by a decrease in the abundance of the Porphyromonadaceae family and, in particular, the Barnesiella genus. It should also be noted that a high fat diet is also related to an increase in the genera Alistipes, Parasuterella, Mucispirillum, and Akkermansia, which have previously been related to dysbiotic processes. As diabetes mellitus type 2 is a risk factor for developing tuberculosis, these findings may prove useful in the search for new prophylactic strategies for this population subset.

Keywords: BCG; C3HeB/FeJ; comorbidity; gut microbiota; high fat diet; mice; obesity; tuberculosis.

Figure 1

Weight evolution during the experiment in pre- and post-infection status. Weight evolution of the same experiment is represented by comparing ND and HFD (A–F) and Sham and BCG (G–L). Panels represent: pre-infection and sham (A), SI and sham (B), MCI and sham (C), pre-infection and BCG (D), SI and BCG (E), MCI and BCG (F), pre-infection and ND (G), SI and ND (H), MCI and ND (I), pre-infection and HFD (J), SI and HFD (K), MCI and HFD (L). Linear regression are represented together with p-values. Slopes' values are included in Table S1.

Figure 2

Bacillary load progression at different end time points (w3, w4, and w16) are shown as log CFUs/ml. Each panel compares sham and BCG vaccinated groups: ND and SI (A), ND and MCI (B), HFD and SI (C), HFD and MCI (D). Box and whiskers plots show the minimum, first quartile, median, third quartile and maximum values. *p < 0.05, **p < 0.01, ***p < 0.001; Mann–Whitney test.

Figure 3

Progression of lung disease at different end time points (w3, w4, and w16) is shown as the percentage of damaged area. Each panel compares sham and BCG vaccinated groups: ND and SI (A), ND and MCI (B), HFD and SI (C), HFD and MCI (D). Bar plots show median with range. *p < 0.05; Mann–Whitney test.

Figure 4

Quality of the lesions found at week 16 post-infection. The tissue sections show a proliferative (A) and an exudative (B) lesion stained with H/E. (C) Relative percentage of exudative lesions at week 16. Black bars represent ND groups while red bars represent HFD groups. Hatched bars in each type of diet represent vaccination Bar plots show median with range. Asterisk indicates differences between vaccination and infection in each diet; hashtag indicates differences between diets. *p < 0.05, ^#p < 0.05; Mann–Whitney test.

Figure 5

Principal component analysis of inflammatory mediators. (A) Representation of animals according to experimental groups with 95% confidence ellipses. (B) Samples colored by type of infection.

Figure 6

Evolution of survival after Mtb infection for the first experiment. Each panel compares sham and BCG vaccinated groups: ND and SI (A), ND and MCI (B), HFD and SI (C), HFD and MCI (D). Median survival times and p-values are indicated in each panel. Log-rank test.

Figure 7

Evolution of survival after Mtb infection for the second experiment. Each panel compares sham and NI groups: ND (A) and HFD (B). Median survival times and p-values are indicated in each panel. Log-rank test.

Figure 8

Analysis of the microbiota diversity based on 16S rRNA sequencing. In each panel the ND group is colored in black and the HFD group in red. Rarefaction curves at week 16 (A) and week 28 (B) after starting the different diets are represented. (C) NMDS ordination in samples following different diets. (D) Shannon, Simpson and inverse Simpson diversity indexes. **p < 0.01, ***p < 0.001; Mann–Whitney test.

Figure 9

Taxonomic composition of the intestinal microbiota based on 16S rRNA sequencing at week 16 after starting both diets. Analysis of the relative abundance of the most abundant phylum (A), class (B), order (C), family (D), and genus (E) in ND vs. HFD. (F) Bar plot representation of the OTUs of each sample according the taxonomic classification. (G) Regression plot of Firmicutes and Bacteroidetes phyla against animal weight and analysis of the F/B ratio in ND and HFD. *p < 0.05, **p < 0.01, ***p < 0.001; Mann–Whitney test.

Figure 10

Taxonomic composition of the intestinal microbiota based on 16S rRNA sequencing at week 28 after starting both diets. Analysis of the relative abundance of the most abundant phylum (A), class (B), order (C), family (D), and genus (E) in ND vs. HFD. (F) Bar plot representation of the OTUs of each sample according the taxonomic classification. (G) Regression plot of Firmicutes and Bacteroidetes phyla against animal weight and analysis of the F/B ratio in ND and HFD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Mann–Whitney test.

Figure 11

Taxonomic composition of the intestinal microbiota based on 16S rRNA sequencing between different diets and under chemotherapy. Analysis of the relative abundance of the most abundant phylum (A), class (B), order (C), family (D), and genus (E). (F) Bar plot representation of the OTUs of each sample according the taxonomic classification. (G) Analysis of the F/B ratio. *p < 0.05, **p < 0.01; Mann–Whitney test.