Distinct roles for dietary lipids and Porphyromonas gingivalis infection on atherosclerosis progression and the gut microbiota

Abstract

Mounting evidence in humans supports an etiological role for the microbiota in inflammatory atherosclerosis. Atherosclerosis is a progressive disease characterized by accumulation of inflammatory cells and lipids in vascular tissue. While retention of lipoprotein into the sub-endothelial vascular layer is believed to be the initiating stimulus leading to the development of atherosclerosis, activation of multiple pathways related to vascular inflammation and endothelial dysfunction sustain the process by stimulating recruitment of leukocytes and immune cells into the sub-endothelial layer. The Gram-negative oral pathogen Porphyromonas gingivalis has been associated with the development and acceleration of atherosclerosis in humans and these observations have been validated in animal models. It has been proposed that common mechanisms of immune signaling link stimulation by lipids and pathogens to vascular inflammation. Despite the common outcome of P. gingivalis and lipid feeding on atherosclerosis progression, we established that these pro-atherogenic stimuli induced distinct gene signatures in the ApoE^-/- mouse model of atherosclerosis. In this study, we further defined the distinct roles of dietary lipids and P. gingivalis infection on atherosclerosis progression and the gut microbiota. We demonstrate that diet-induced lipid lowering resulted in less atherosclerotic plaque in ApoE^-/- mice compared to ApoE^-/- mice continuously fed a Western diet. However, the effect of diet-induced lipid lowering on plaque accumulation was blunted by P. gingivalis infection. Using principal component analysis and hierarchical clustering, we demonstrate that dietary intervention as well as P. gingivalis infection result in distinct bacterial communities in fecal and cecal samples of ApoE^-/- mice as compared to ApoE^-/- mice continuously fed either a Western diet or a normal chow diet. Collectively, we identified distinct microbiota changes accompanying atherosclerotic plaque, suggesting a future avenue for investigation on the impact of the gut microbiota, diet, and P. gingivalis infection on atherosclerosis.

Keywords: Atherosclerosis; Gut microbiota; Immunity; Inflammation; Lipids; Oral microbiota.

Figure 1. Experimental protocol

Four treatment groups were studied: NC=normal chow for 22 weeks (n=6), WD=Western diet for 22 weeks (n=6), WD-NC=Western diet for 11 weeks followed by normal chow for 11 weeks (n=7), WD-NC+Pg=Western diet for 11 weeks followed by normal chow for 11 weeks and P. gingivalis oral infection at midpoint (n=15). All mice were sacrificed at 22 weeks; analysis of plaque index in the aorta and its main braches was performed and fecal and cecal samples were collected at this time.

Figure 2. Atherosclerotic plaque development in the aorta and its main branches

A. Representative images showing differential plaque accumulation among the four treatment groups. white arrow=innominate artery, green arrow=left common carotid artery, red arrow=left subclavian artery B. Quantitative assessment of plaque development among the four treatment groups, shown as plaque index. Plaque formation in the aortic arch, innominate artery, left common carotid artery, and left subclavian artery was quantified. 6 grades were assigned based on the percent plaque area vs. total blood vessel area: 0, no plaque; 1, 1-20%; 2, 21-40%; 3, 41-60%; 4, 61-80%; 5, 81-100%. Numbers from all four locations were summed to generate the plaque index. NC=normal chow for 22 weeks (n=6), WD=Western diet for 22 weeks (n=6), WD-NC=Western diet for 11 weeks followed by normal chow for 11 weeks (n=7), WD-NC+Pg=Western diet for 11 weeks followed by normal chow for 11 weeks and P. gingivalis oral infection at midpoint (n=15). Data were analyzed by one-way ANOVA (p<0.0001) using Bonferroni Post Test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3. Relative abundance of phyla in cecal samples

A. Phylum Bacteroidetes (family S24-7). Data were analyzed by one-way ANOVA (p=0.0128). B. Linear correlation between plaque index and relative abundance of phylum Bacteroidetes (S24-7) in cecal samples. Pearson r = 0.6515, p=0.0006. C. Phylum Firmicutes. Data were analyzed by one-way ANOVA (p=0.0245). D. Phylum Tenericutes. Data were analyzed by one-way ANOVA (p=0.0007). NC=normal chow for 22 weeks (n=5), WD=Western diet for 22 weeks (n=6), WD-NC=Western diet for 11 weeks followed by normal chow for 11 weeks (n=6), WD-NC+Pg=Western diet for 11 weeks followed by normal chow for 11 weeks and P. gingivalis oral infection at midpoint (n=8).

Figure 4. Alpha diversity measures

Rarefaction analysis of fecal and cecal samples. A. Chao1 (richness) and B. Shannon Index (abundance and evenness). Lines represent the mean+/-standard deviation. The analysis was performed on a randomly selected subset of 1,000-32,000 sequences per sample over 10 iterations.

Figure 5. Hierarchical clustering and PCA

A-C. PCA based on treatment group. PC1 (25%), PC2 (11%), PC3 (8%). Log₂ transformed, 577/2406 variables were significant, p<0.01, q<0.04166 D. PCA based on plaque index category. PC1 (35%), PC2 (10%), PC3 (9%). Log₂ transformed, 246/2406 variables were significant, p<0.01, q< 0.096312 E. Heatmap based on treatment group, metadata group memberships (site of sample collection, plaque index, treatment group, original cage) are indicated by colored blocks at the top or bottom of the heatmap. Log₂ transformed, Top 5% (120 variables), p<1.35e-7, q<2.6311e-6. NC=normal chow for 22 weeks (cages 10 and 18), WD=Western diet for 22 weeks (cages 7 and 12), WD-NC=Western diet for 11 weeks followed by normal chow for 11 weeks (cages 11 and 15), WD-NC+Pg=Western diet for 11 weeks followed by normal chow for 11 weeks and P. gingivalis oral infection at midpoint (cages 8 and 9).

Figure 6. Linear discriminant analysis effect size (LEfSe) of cecal samples

A. NC vs. WD-NC vs. WD-NC+Pg. B. WD vs. WD-NC vs. WD-NC+Pg. C. NC vs. WD. D. WD vs. WD-NC. E. WD-NC vs. WD-NC+Pg. Left side= linear discriminant analysis (LDA) plots, listing differentially abundant taxa (α = 0.05) with an LDA score higher than 2.0. The taxon name is preceded by one of the following: p_=phylum, c_=class, o_=o_order, f_=f_family, g_=genus, s_=species. Colored bars indicate LDA score as well as the group in which that taxon was most abundant. White letters within the colored bars correspond to the cladogram, on the right.