"Western Diet"-Induced Adipose Inflammation Requires a Complex Gut Microbiota

Hao Q Tran, Alexis Bretin, Aneseh Adeshirlarijaney, Beng San Yeoh, Matam Vijay-Kumar, Jun Zou, Timothy L Denning, Benoit Chassaing, Andrew T Gewirtz, Hao Q Tran, Alexis Bretin, Aneseh Adeshirlarijaney, Beng San Yeoh, Matam Vijay-Kumar, Jun Zou, Timothy L Denning, Benoit Chassaing, Andrew T Gewirtz

Abstract

Background & aims: Consumption of a low-fiber, high-fat, Western-style diet (WSD) induces adiposity and adipose inflammation characterized by increases in the M1:M2 macrophage ratio and proinflammatory cytokine expression, both of which contribute to WSD-induced metabolic syndrome. WSD-induced adipose inflammation might result from endoplasmic reticulum stress in lipid-overloaded adipocytes and/or dissemination of gut bacterial products, resulting in activation of innate immune signaling. Hence, we aimed to investigate the role of the gut microbiota, and its detection by innate immune signaling pathways, in WSD-induced adipose inflammation.

Methods: Mice were fed grain-based chow or a WSD for 8 weeks, assessed metabolically, and intestinal and adipose tissue were analyzed by flow cytometry and quantitative reverse transcription polymerase chain reaction. Microbiota was ablated via antibiotics and use of gnotobiotic mice that completely lacked microbiota (germ-free mice) or had a low-complexity microbiota (altered Schaedler flora). Innate immune signaling was ablated by genetic deletion of Toll-like receptor signaling adaptor myeloid differentiation primary response 88.

Results: Ablation of microbiota via antibiotic, germ-free, or altered Schaedler flora approaches did not significantly impact WSD-induced adiposity, yet dramatically reduced WSD-induced adipose inflammation as assessed by macrophage populations and cytokine expression. Microbiota ablation also prevented colonic neutrophil and CD103- dendritic cell infiltration. Such reduced indices of inflammation correlated with protection against WSD-induced dysglycemia, hypercholesterolemia, and liver dysfunction. Genetic deletion of myeloid differentiation primary response 88 also prevented WSD-induced adipose inflammation.

Conclusions: These results indicate that adipose inflammation, and some aspects of metabolic syndrome, are not purely a consequence of diet-induced adiposity per se but, rather, may require disturbance of intestine-microbiota interactions and subsequent activation of innate immunity.

Keywords: Altered Schaedler Flora; Antibiotics; Germ-Free; High-Fat Diet; Metabolic Syndrome; Microbiota; MyD88.

Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Characterization of changes in tissue leukocytes in response to diet-induced obesity. Four-week-old male C57BL/6J mice were purchased from The Jackson Laboratory and housed for 2 weeks to favor microbiota stabilization. Subsequently, half of the mice were switched to a Western-style, high-fat diet (60% kcal from fat) or continued on a standard grain-based chow as a control. After 8 weeks on a WSD, mice were killed and biometric measurements are shown for (A) final body weight and (B) epididymal adipose weight. (C) In addition, a 15-hour fasting blood glucose level was measured before euthanasia. Epididymal adipose tissue was analyzed by flow cytometry to quantify the following: (D) macrophages, (E) M1:M2 macrophage ratio, (F) DCs, (G) CD11b+CD103- and CD11b-CD103+ DC subsets, and (H) eosinophils and neutrophils. Colon tissue was used for flow cytometry to quantify as cells per organ: (I) DCs, (J) CD103+/- dendritic subsets, and (K) eosinophils and neutrophils. Data are the means ± SD (N=5). Statistical significance was determined using the t test. P < .05, brackets indicate significance. eAT, epididymal adipose tissue.
Figure 2
Figure 2
Gating scheme for myeloid cells in adipose, small intestine, colon tissue, spleen, and PPs. Data were collected until 100,000 events were reached in the Live/CD45+ gate. All cells were gated for appropriate side scatter area/forward scatter area and singlets. (A) Gating scheme of macrophages from epididymal adipose and intestinal tissue. Live CD45+MHCII+ cells were identified as macrophages if they were F4/80+ (or CD11b+) and CD64+. Of epididymal adipose tissue macrophages, M1 macrophages were identified as CD301-, M2 macrophages were identified as CD301+. (B) Gating scheme of DCs from epididymal adipose and intestinal tissue. Live CD45+MHCII+ cells were identified as DCs if they were CD11c+ but CD64-. DC subsets were identified as either CD11b+CD103- (Q3) or CD11b-CD103+ (Q1) in the adipose or CD103+/- in the small intestine and colon. (C) Gating scheme of eosinophils and neutrophils from epididymal adipose and intestinal tissue. Live CD45+MHCII- cells were identified as eosinophils if they were Siglec-F+Ly6-G- or neutrophils if they were Siglec-F-Ly6-G+, these populations also both are CD11b+. (D) Gating scheme of myeloid cells from splenic tissue. Live CD45+MHCII+ cells were gated to exclude B cells (CD19+) and T cells (CD3+). Of this CD45+MHCII+CD19-CD3- population, CD11chigh cells were classified as DCs, which then were classified as either CD8+/-, while CD11b+CD11c- cells were categorized as either eosinophils (Ly6-CloGr-1-), neutrophils (Gr-1+), or inflammatory monocytes (Ly6-ChighGr-1-). (E) Gating scheme of DCs from PPs. Dendritic cells were identified as live CD11c+ and subdivided as either preDCs or mature DCs based on MHCII expression. Mature MHCII+ DCs were categorized as either myeloid DCs (CD8a-CD11b+) or lymphoid DCs (CD8a+CD11b-).
Figure 3
Figure 3
An obesogenic diet alters myeloid cell populations in the small intestine, and to a lesser extent in the spleen and PPs. (A) After 8 weeks on chow (orange circles) or a WSD (blue squares), mice (N=5) were killed and colon tissue was analyzed for macrophages. Small intestinal tissue was analyzed for the following: (B) DCs, (C) CD103+/- DCs, (D) eosinophils and neutrophils, and (E) macrophages. Splenic tissue was analyzed for (F) eosinophils, neutrophils, inflammatory monocytes, and (G) CD8+/- classical dendritic cells (cDCs). (H–I) PPs were analyzed for total DCs and DC subsets. Data are the means ± SD. Statistical significance was determined using the t test. P < .05, brackets indicate significance.
Figure 4
Figure 4
Microbiota ablation did not mitigate WSD-induced adiposity but ameliorated features of metabolic syndrome. (A) Four-week-old male C57BL/6J conventionally raised mice were purchased from The Jackson Laboratory, 4-week-old male C57BL/6 mice possessing an ASF were obtained from the Georgia State University breeding repository, and 3- to 5-week-old male C57BL/6 GF mice were purchased from Taconic Biosciences (N=5). At day 0, animals were switched to a high-fat diet (60% kcal from fat) for 8 weeks or continued on a standard grain-based chow as a control. An additional group of conventional C57BL/6J mice were started on an antibiotic cocktail 3 days before high-fat diet administration and maintained throughout the experiment. At day 56, mice were euthanized for tissue collection and biometric measurements, data of WSD-treated mice represented here are relative to the chow-fed mice of their corresponding groups: (B) final body weight, (C) epididymal adipose tissue weight, (D) subcutaneous adipose tissue weight, (E) brown adipose tissue weight, (F) 15-hour fasting blood glucose level, (G) ALT concentration, (H) AST concentration, (I) total serum cholesterol, (J) liver weight, (K) colon weight/length ratio, and (L) spleen weight. Data are the means ± SD. Statistical significance was determined using 1-way analysis of variance corrected for multiple comparisons with a Bonferroni test. *P ≤ .05, **P ≤ .01, ***P ≤ .001, and ****P ≤ .0001. ABX, antibiotic; Conv, conventional; ns, nonsignificant.
Figure 5
Figure 5
Absolute values of biometric measurements and tissue/macromolecule quantifications corresponding toFigure 4. Comparison of conventional mice with antibiotic-treated, ASF, and GF mice (n=5-7): (A) final body weight, (B) changes in weight in antibiotic-treated mice, (C) epididymal adipose tissue weight, (D) subcutaneous adipose tissue weight, (E) brown adipose tissue weight, (F) fasting blood glucose level, (G) ALT concentration, (H) AST concentration, (I) total serum cholesterol, (J) liver weight, (K) colon weight/length ratio, and (L) spleen weight. Data are the means ± SD. Statistical significance was determined using 1-way analysis of variance corrected for multiple comparisons with a Bonferroni test. *P ≤ .05, **P ≤ .01, ***P ≤ .001, and ****P ≤ .0001. ABX, antibiotic; Conv, conventional; ns, nonsignificant.
Figure 6
Figure 6
Hepatic steatosis is reduced in antibiotic-treated and ASF mice on a WSD compared with conventional controls. Extended analysis of mice from Figure 2. Four-week-old male C57BL/6J conventionally raised mice were purchased from The Jackson Laboratory, 4-week-old male C57BL/6 mice possessing an ASF were obtained from the Georgia State University breeding repository, and 3- to 5-week-old male C57BL/6 GF mice were purchased from Taconic Biosciences. At day 0, animals were switched to a high-fat diet (60% kcal from fat) for 8 weeks or continued on a standard grain-based chow as a control. An additional group of conventional C57BL/6J mice were started on an antibiotic cocktail 3 days before high-fat diet administration and maintained throughout the experiment. At day 56, mice were killed and liver tissues were collected to assess (A) hepatic steatosis and (B) perform H&E staining. Data are the means ± SD. Statistical significance was determined using 1-way analysis of variance corrected for multiple comparisons with a Bonferroni test. ***P ≤ .001. ABX, antibiotic; Conv, conventional; ns, nonsignificant.
Figure 7
Figure 7
Microbiota ablation reduces macrophage infiltration and M1 macrophage polarization, reducing the M1:M2 ratio. Epididymal adipose tissue from the epididymitis of mice was analyzed using flow cytometry to quantify the following: (A) macrophages and (B) M1 and M2 macrophage subsets. Data from WSD-treated mice represented here are relative to the chow-fed mice of their corresponding groups, as follows: (C) number of macrophages per gram of adipose tissue, (D) M1 macrophages as a percentage of total macrophages, (E) M1 macrophages as the number of cells per gram of adipose tissue, (F) M2 macrophages as a percentage of total macrophages (∗∗P ≤ .01), and (G) M2 macrophages as the number of cells per gram of adipose tissue. (H) M1:M2 macrophage ratios were quantified using the percentage of M1 and M2 macrophages. (I) Epididymal adipose tissue collected at death also was analyzed for cytokines via quantitative reverse-transcription polymerase chain reaction. Data are the means ± SD (N=5-7). Statistical significance was determined using 1-way analysis of variance corrected for multiple comparisons with a Bonferroni test. *P ≤ .05, ***P ≤ .001, and ****P ≤ .0001. #Limited sample quantity prevented the use of statistical analysis. ABX, antibiotic; Conv, conventional; eAT, epididymal adipose tissue; ns, nonsignificant.
Figure 8
Figure 8
Microbiota ablation modestly protects against eosinophil loss during WSD feeding. (A) Epididymal adipose tissue from the epididymitis of mice was analyzed using flow cytometry to quantify eosinophils. (B) Eosinophils of WSD-treated mice represented as the number per gram of adipose tissue is relative to chow-fed mice. Data are the means ± SD (N=5-7). Statistical significance was determined using 1-way analysis of variance corrected for multiple comparisons with a Bonferroni test. *P ≤ .05. ABX, antibiotic; Conv, conventional; eAT, epididymal adipose tissue; ns, nonsignificant.
Figure 9
Figure 9
Microbiota ablation reduces DC alterations and neutrophil cell infiltration in the colon. Colon tissue was analyzed using flow cytometry to quantify the following: (A) DCs, (B) CD103+/- DCs, and (C) neutrophils. Data from WSD-treated mice represented here are relative to the chow-fed mice of their corresponding groups, as follows: (D) number of DCs, (E) number of CD103- DCs, (F) number of CD103+ DCs, and (G) number of neutrophils per colon. Data are the means ± SD (N=5-7). Statistical significance was determined using 1-way analysis of variance corrected for multiple comparisons with a Bonferroni test. *P ≤ .05 and **P ≤ .01. ABX, antibiotic; Conv, conventional; ns, nonsignificant.
Figure 10
Figure 10
Transplant of microbiota from obese mice mildly recapitulates features of obesity and myeloid cell alterations. Four-week-old male C57BL/6 GF mice were purchased from Taconic Biosciences and were administered feces from either chow-(blue boxes) or WSD-(orange circles)fed mice by oral gavage. These animals subsequently were maintained in a sterile environment for 4 weeks before terminal tissue collection and biometric quantification, data are as follows: (A) final body weight; (B) epididymal adipose tissue weight; (C) subcutaneous adipose tissue weight; (D) brown adipose tissue weight; (E) 15-hour fasting blood glucose level; (F) ALT concentration, AST concentration, and total serum cholesterol; (G) liver weight; (H) colon weight/length ratio; and (I) spleen weight. Epididymal adipose tissue was analyzed using flow cytometry to quantify the following: (J) macrophages, (K and L) M1 macrophages, (M and N) M2 macrophages, (O) M1:M2 ratio, and (P) eosinophils. (Q) Epididymal adipose tissue collected at death also was analyzed for cytokines via quantitative reverse-transcription polymerase chain reaction. Colon tissue was analyzed using flow cytometry to quantify the following: (R) DCs, (S) CD103- DCs, (T) CD103+ DCs, and (U) neutrophils. Data are the means ± SD (N=3-4). Statistical significance was determined using the t test. P < .05, brackets indicates significance. eAT, epididymal adipose tissue; FMT, fecal microbiota transplant; mRNA, messenger RNA.
Figure 11
Figure 11
Genetic depletion of TLR signaling adaptor MyD88 phenocopies microbiota ablation in response to WSD. Conventionally raised 3- to 5-week-old male C57BL/6J, wild-type and MyD88 KO mice were purchased from The Jackson Laboratory. Animals were placed on a high-fat diet (60% kcal from fat) for 8 weeks or continued on a standard grain-based chow as a control. At day 56, mice were killed for tissue collection and biometric measurements, data from WSD-treated mice represented here are relative to the chow-fed mice of their corresponding groups, as follows: (A) final body weight; (B) epididymal adipose tissue weight; (C) 15-hour fasting blood glucose level; (D) ALT concentration, AST concentration, and total cholesterol; (E) colon weight/length ratio; and (F) spleen weight. Epididymal adipose tissue from the epididymitis of mice was analyzed using flow cytometry and quantitative reverse-transcription polymerase chain reaction to quantify the following: (G) macrophages, (H and I) M1 macrophages, (J and K) M2 macrophages, (L) M1:M2 ratio, (M) inflammatory cytokines, and (N) eosinophils. Colon tissue was used to quantify the following: (O) DCs, (P) CD103- DCs, (Q) CD103+ DCs, and (R) neutrophils. Data are the means ± SD (N=5). Statistical significance was determined using 1-way analysis of variance corrected for multiple comparisons with a Bonferroni test. *P ≤ .05, **P ≤ .01, ***P ≤ .001, and ****P ≤ .0001. eAT, epididymal adipose tissue; mRNA, messenger RNA; ns, nonsignificant.
Figure 12
Figure 12
Absolute values of biometric measurements and tissue/macromolecule quantifications corresponding toFigure 11: (A) body weight; (B) epididymal adipose tissue weight; (C) 15-hour fasting blood glucose level; (D) colon weight/length ratio; (E) ALT concentration, AST concentration, and total cholesterol; and (F) spleen weight. Data are the means ± SD (N=5). Statistical significance was determined using 1-way analysis of variance corrected for multiple comparisons with a Bonferroni test. *P ≤ .05, **P ≤ .01, ***P ≤ .001, and ****P ≤ .0001. ns, nonsignificant.

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