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. 2019 May;33(5):6456-6469.
doi: 10.1096/fj.201802477R. Epub 2019 Feb 15.

Obesogenic diet in aging mice disrupts gut microbe composition and alters neutrophil:lymphocyte ratio, leading to inflamed milieu in acute heart failure

Vasundhara Kain  1 , William Van Der Pol  2 , Nithya Mariappan  3 , Aftab Ahmad  3 , Peter Eipers  4 , Deanna L Gibson  5 , Cecile Gladine  6 , Claire Vigor  7 , Thierry Durand  7 , Casey Morrow  4 , Ganesh V Halade  1
Affiliations

Obesogenic diet in aging mice disrupts gut microbe composition and alters neutrophil:lymphocyte ratio, leading to inflamed milieu in acute heart failure

Vasundhara Kain et al. FASEB J. 2019 May.

Abstract

Calorie-dense obesogenic diet (OBD) is a prime risk factor for cardiovascular disease in aging. However, increasing age coupled with changes in the diet can affect the interaction of intestinal microbiota influencing the immune system, which can lead to chronic inflammation. How age and calorie-enriched OBD interact with microbial flora and impact leukocyte profiling is currently under investigated. Here, we tested the interorgan hypothesis to determine whether OBD in young and aging mice alters the gut microbe composition and the splenic leukocyte profile in acute heart failure (HF). Young (2-mo-old) and aging (18-mo-old) mice were supplemented with standard diet (STD, ∼4% safflower oil diet) and OBD (10% safflower oil) for 2 mo and then subjected to coronary artery ligation to induce myocardial infarction. Fecal samples were collected pre- and post-diet intervention, and the microbial flora were analyzed using 16S variable region 4 rRNA gene DNA sequencing and Quantitative Insights Into Microbial Ecology informatics. The STD and OBD in aging mice resulted in an expansion of the genus Allobaculum in the fecal microbiota. However, we found a pathologic change in the neutrophil:lymphocyte ratio in aging mice in comparison with their young counterparts. Thus, calorie-enriched OBD dysregulated splenic leukocytes by decreasing immune-responsive F4/80+ and CD169+ macrophages in aging mice. OBD programmed neutrophil swarming with an increase in isoprostanoid levels, with dysregulation of lipoxygenases, cytokines, and metabolite-sensing receptor expression. In summary, calorie-dense OBD in aging mice disrupted the composition of the gut microbiome, which correlates with the development of integrative and system-wide nonresolving inflammation in acute HF.-Kain, V., Van Der Pol, W., Mariappan, N., Ahmad, A., Eipers, P., Gibson, D. L., Gladine, C., Vigor, C., Durand, T., Morrow, C., Halade, G. V. Obesogenic diet in aging mice disrupts gut microbe composition and alters neutrophil:lymphocyte ratio, leading to inflamed milieu in acute heart failure.

Keywords: inflammation; leukocytes; myocardial infarction; nonresolving inflammation; resolution of inflammation.

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Conflict of interest statement

The authors acknowledge support from U.S. National Institutes of Health (NIH) National Center for Complementary and Integrative Health (NCCIH) (formerly known as NCCAM) Grants AT006704 and HL132989, a University of Alabama at Birmingham (UAB) Pittman Scholar Award (to G.V.H.), and an American Heart Association postdoctoral fellowship (POST31000008 to V.K.). The authors also acknowledge the support of the Microbiome Resource, Comprehensive Cancer Center (P30AR050948), Center for Clinical Translational Science (UL1TR001417), University-Wide Institutional Core, Heflin Center for Genomic Sciences, and Microbiome Center at the University of Alabama at Birmingham. The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Impact of OBD on gut microbiota during aging. A) Study design sketch that delineates C57BL/6 mice age, diet intervention protocol, and parameters studied before and after MI in acute HF. B) PCoA plot of Bray-Curtis (n = 3–8 mice/group).
Figure 2
Figure 2
Impact of OBD and aging on distribution of microbiota taxa. A) Relative abundance by Kruskal-Wallis (KW) mean of the top bacterial phyla. B) Heatmap showing the abundance of significant OTUs. Represented bacterial taxa information (genus, family, and phylum) of these OTUs is also shown (n = 3–8 mice/group).
Figure 3
Figure 3
Aging and OBD impacted on hematology profile post-MI. A) Pie chart displaying white blood cell differential of in young and aging mice fed STD and OBD for 2 mo. Bar graphs represent % of B. B–F) Neutrophil (B), lymphocyte (C), monocyte (D), eosinophil (E), and Basophil (F) population in blood of young and aging mice fed STD and OBD for 2 mo post-MI. BA, basophil; EO, eosinophil; LY, lymphocyte; MO, monocyte; NEU, neutrophil. Values are means ± sem (n = 8–10 mice/group). *P < 0.05 vs. young-STD.
Figure 4
Figure 4
OBD decreased CD169+ macrophages in young and aging mice post-MI. A–D) Immunofluroscence of post-MI spleen sections from young (A, B) and aging (C, D) mice fed normal diet (A, C) and OBD (B, D), presenting 3-color image staining; CD169 (red), F4/80 (green), and nuclei (blue). OBD cleared CD169+ cells in WP and MZ area post-MI, with expansion of F4/80+ in both young and aging mice. Images are representative of 4–5 sections, n = 4/group. Original magnification, ×20. Scale bars, 100 μm.
Figure 5
Figure 5
Aging diminished splenic leukocyte kinetics independent of OBD post-MI. A) Representative flow cytometry dot plots depicting CD45+/CD11b+ population in spleen isolated from young and aging mice fed STD and OBD for 2 mo pre- and post-MI. B) Representative flow cytometry dot plots showing lower CD11b+/F4/80+ population in spleen isolated from young and aging mice fed STD and OBD for 2 mo pre- and post-MI. C) Bar graphs representing percentage of CD11b+ population in spleen at d 1 post-MI. D) Bar graphs representing percentage of F4/80+ population in spleen at d 1 post-MI. Values are mean ± sem (n = 3–5 mice/group/time point for flow cytometry analysis). *P < 0.05 vs. young-STD, $P < 0.05 STD vs. OBD.
Figure 6
Figure 6
Aging diminishes F480+ and Ly6Chi and Ly6G+ cells post-MI irrespective having excess intake of fatty acids. A) Representative flow cytometry dot plots depicting Ly6Chi and Ly6Clo population in spleen isolated from young and aging mice fed STD and OBD for 2 mo pre- and post-MI. B) Representative flow cytometry dot plots showing LY6G population in spleen isolated from young and aging mice fed STD and OBD for 2 mo pre- and post-MI. C) Bar graphs representing % of Ly6G population in spleen at d 1 post-MI. D) Bar graphs representing % of Ly6Clo population in spleen at d 1 post-MI. E) Bar graphs representing % of Ly6G population in spleen at d 1 post-MI. F) Histogram representing change in Ly6G expression in aging mice fed STD and OBD for 2 mo post-MI. Values are means ± sem (n = 3–5 mice/group/time point for flow cytometry analysis). *P < 0.05 vs. young-STD, $P < 0.05 STD vs. OBD.
Figure 7
Figure 7
OBD modulates splenic LOXs, cytokines, and metabolite-sensing receptor expression in young and aging mice post-MI. Bar-graph representing mRNA expression of ALOX-12, ALOX-15, and ALOX-5 (A); IL-1b, TNF-α, and CCL2 (B); MRC-1, Arg-1, and YM-1 (C); and FPR2, GPR40, and GPR120 (D). Gene Expressions are normalized to hypoxanthine phosphoribosyltransferase 1 (HPRT-1) expression. Values are means ± sem (n = 4/group). #P < 0.05 vs. young-STD no MI, *P < 0.05 vs. young-STD post-MI, $P < 0.05 STD vs. OBD.
Figure 8
Figure 8
OBD modulates plasma level of isoprostanes. A–C) Bar graphs representing plasma level of 15-F2t-lsoP (A), 15-epi-15-F2t-lsoP (B), and 5(RS)-5-F2t-lsoP (C). D) The study outcome illustrating impact of OBD on gut microbiome and its impact on systemic inflammation.
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