Obesity Reshapes the Microbial Population Structure along the Gut-Liver-Lung Axis in Mice

Abstract

The microbiome is emerging as a major player in tissue homeostasis in health and disease. Gut microbiome dysbiosis correlates with several autoimmune and metabolic diseases, while high-fat diets and ensuing obesity are known to affect the complexity and diversity of the microbiome, thus modulating pathophysiology. Moreover, the existence of a gut-liver microbial axis has been proposed, which may extend to the lung. In this context, we systematically compared the microbiomes of the gut, liver, and lung of mice fed a high-fat diet to those of littermates fed a matched control diet. We carried out deep sequencing of seven hypervariable regions of the 16S rRNA microbial gene to examine microbial diversity in the tissues of interest. Comparison of the local microbiomes indicated that lung tissue has the least diverse microbiome under healthy conditions, while microbial diversity in the healthy liver clustered closer to the gut. Obesity increased microbial complexity in all three tissues, with lung microbial diversity being the most modified. Obesity promoted the expansion of Firmicutes along the gut-liver-lung axis, highlighting staphylococcus as a possible pathologic link between obesity and systemic pathophysiology, especially in the lungs.

Keywords: 16S rRNA; comparative analysis; firmicutes; gut; high-fat diet; liver; lung; microbiome; obesity; staphylococcus.

Figure 1

Mice fed with a high-fat diet (HFD) developed obesity and non-alcoholic liver fatty liver disease (NAFLD). Mice fed with HFD for 16 weeks presented with (A) statistically significant higher body weight from the tenth week of HFD onwards, (B) elevated ALT levels, and (C) decreased AST/ALT ratio in plasma after 16 weeks of HFD. (D) Representative images from the histopathology (hematoxylin & eosin staining) of gut, liver, and lung tissues, illustrating lipid deposition in the liver and gut after 16 weeks of HFD. Hematoxylin stains cell nuclei (purple) and eosin stains the extracellulal matrix (pink). The “bubbles” appearing in HDF liver and gut samples are lipid droplets. Statistical significance was assessed through the Friedman test followed by pairwise Mann-Whitney tests (in A) and Mann-Whitney tests (in B and C); * p < 0.05 was considered significant.

Figure 2

HFD-driven obesity triggers changes in the microbiome composition of all tissues examined. (A) HFD-driven obesity results in a greater number of detected Amplicon Sequence Variants (ASVs) in all tissues. (B) α-diversity per sample and diet. Shannon’s index was used to evaluate sample biodiversity per tissue and diet. (C) Similarity of samples per tissue and diet as described by β-diversity. Aitchison distance was used to account for the compositional nature of 16S rRNA sequencing data. (D) Venn diagrams of common phyla or (E) families upon CD or HFD in gut, liver, and lung.

Figure 3

Relative abundance of phyla detected in gut, liver, and lung of control and HFD-fed mice. Heatmap and respective dendrogram of non-zero abundance phyla under CD (A) and HFD (B). (C) Relative abundance of all detected phyla. Relative abundance calculations for all panels were based on GCN values. For panels A and B, only phyla with an abundance greater than zero after value rounding to two decimal places were considered. Heatmaps are scaled per phylum. Manhattan distance was used to perform hierarchical clustering of the tissues with complete linkage in panels A and B.

Figure 4

HFD consistently affects specific microbial families and genera. (A) Heatmap and tissue dendrogram of HFD-to-CD differences in family relative abundance (B) Heatmap of HFD-to-CD difference in genera relative abundance. For both panels, only those taxa with HFD-to-CD relative abundance differences other than zero in at least one tissue after rounding to two decimal places were considered. Heatmaps are scaled per taxon. Manhattan distance was used to perform hierarchical clustering of tissues with complete linkage. Relative abundance was calculated based on GCN abundance values. Families and respective genera are coded with the same color in both panels.