1. Epidemiology and Global Health
  2. Immunology and Inflammation
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Contrasting effects of Western vs Mediterranean diets on monocyte inflammatory gene expression and social behavior in a primate model

  1. Corbin SC Johnson
  2. Carol Shively
  3. Kristofer T Michalson
  4. Amanda J Lea
  5. Ryne J DeBo
  6. Timothy D Howard
  7. Gregory A Hawkins
  8. Susan E Appt
  9. Yongmei Liu
  10. Charles E McCall
  11. David M Herrington
  12. Edward H Ip
  13. Thomas C Register  Is a corresponding author
  14. Noah Snyder-Mackler  Is a corresponding author
  1. Department of Psychology, University of Washington, United States
  2. Department of Pathology, Section on Comparative Medicine, Wake Forest School of Medicine, United States
  3. Lewis-Sigler Institute for Integrative Genomics, Princeton University, United States
  4. Department of Ecology and Evolutionary Biology, Princeton University, United States
  5. Department of Biochemistry, Wake Forest School of Medicine, United States
  6. Division of Cardiology, Duke University School of Medicine, United States
  7. Department of Internal Medicine, Section of Molecular Medicine, Wake Forest School of Medicine, United States
  8. Department of Internal Medicine, Section on Cardiovascular Medicine, Wake Forest School of Medicine, United States
  9. Department of Biostatistics and Data Science, Wake Forest School of Medicine, United States
  10. Center for Studies in Demography and Ecology, University of Washington, United States
  11. Department of Biology, University of Washington, United States
  12. School of Life Sciences, Arizona State University, United States
  13. Center for Evolution & Medicine, Arizona State University, United States
Research Article
Cite this article as: eLife 2021;10:e68293 doi: 10.7554/eLife.68293
8 figures, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements
Diet effects on monocyte gene expression.

(A) Diet was significantly associated with the first principal component of gene expression (59% variance explained, t(25.1) = 4.4, p = 1.72 x 10−4). (B) The average effect size of diet on Western genes was 60% stronger than the effect size of diet on Mediterranean genes (Mann-Whitney U = 4.1 x 106, p = 6.1 x 10−117). (C) Western genes (orange) contained more M1 genes than expected by chance, indicating that the Western diet induced a shift toward a proinflammatory monocyte phenotype. Western genes were enriched for proinflammatory (M1-like) genes (fold-enrichment = 1.27, 95% CI = 1.09, 1.46), while Mediterranean genes (blue) were depleted of these same M1-like genes (fold-enrichment = 0.74, 95% CI = 0.61, 0.88). Regulatory (M2-like) genes were also under-represented in Mediterranean genes (fold-enrichment = 0.67, 95% CI = 0.40, 0.97), but not in Western genes (fold-enrichment = 0.95, 95% CI = 0.60, 1.35). Figure 1—figure supplement 1. The sets of Western and Mediterranean genes were compared to genes implicated in 103 complex human diseases and traits (Zhang et al., 2020). Fisher’s Exact Tests were used to calculate the enrichment of trait-associated genes in Western genes that are depicted here (FDR < 0.02, 95% CI) and no traits were enriched in Mediterranean genes. Figure 1—figure supplement 2. Western-diet fed animals exhibited significantly higher expression of pro-inflammatory genes involved in the conserved transcriptional response to adversity (CTRA Cole et al., 2015; Mann-Whitney U = 222, p = 0.016) and lower expression of antiviral- and antibody-related CTRA genes (Mann-Whitney U = 82, p = 0.023), both consistent with the CTRA. See Supplementary file 1A for CTRA categories.

Figure 1—figure supplement 1
Genes with significantly higher expression in monkeys fed the Western diet (‘Western genes’) were enriched for genes associated with numerous complex human diseases and traits.
Figure 1—figure supplement 2
Expression of genes in the conserved transcriptional response to adversity (CTRA Cole et al., 2015) indicate inflammatory effects of a Western diet that parallel the effects of social adversity.
Transcription factor (TF) binding motifs correlated with diet effects on gene expression.

The log-odds ratio of TF binding motif enrichment in Western genes (orange) or Mediterranean genes (blue) are depicted on the x-axis. The y-axis shows the effect size of diet on the expression of the gene that encodes for the TF. Only TFs with binding motifs significantly enriched in either gene set and that were detectably expressed in our samples are shown, with those significantly affected by diet (FDR < 0.05) outlined and labeled.

Diet altered monocyte gene co-expression.

(A) The Pearson correlation between each pair of genes within each of the experimental diets. Gene pairs that were significantly differently correlated between diets are highlighted in blue (n = 445 significant pairs, FDR < 0.2). (B) Of the genes involved in significant pairs, some were paired with more genes than expected by chance, called ‘hub’ genes (n = 16 hub genes; dotted black line is the maximum number of significant pairs expected by chance). The strongest hub gene was the non-coding RNA RF00283. (C) Residual normalized expression of RF00283 is significantly greater in Western- than Mediterranean-fed monkeys (βdiet = 0.51, FDR = 2.3 x 10−6). (D) Example of a differential correlation involving RF00283. Residual normalized expression of RF00283 is plotted against expression of KLF11, a differentially-expressed transcription factor that regulates insulin and has been associated with type II diabetes in humans (Neve et al., 2005). The two genes were more highly expressed in Western monocytes, were positively correlated with one another in Western-fed monkeys (Pearson’s r = 0.61, p = 4.2 x 10−3), were negatively correlated in Mediterranean-fed monkeys (Pearson’s r = −0.63, p = 0.011), and were differentially correlated between the two diets (p = 4.1 x 10−5, FDR = 0.11).

Figure 4 with 2 supplements
Diet alters behavioral phenotype.

(A) Three behaviors were significantly different between the two diet groups. Monkeys fed the Mediterranean diet spent more time in body contact (Holm-Bonferroni adjusted p (pHB) = 1.1 x 10−5) and resting (pHB = 1.6 x 10−3) than Western-fed monkeys. Monkeys eating the Western diet spent more time alone than Mediterranean-fed monkeys (pHB = 4.9 x 10−3). (B) Principal component 2 (PC2) explained 19% of the variance in behavior and was the only PC significantly correlated with diet. (C) PC2 represents a composite measure of diet-altered behavior, as individual loadings onto PC2 (‘DAB scores’; 19% of all variance in behavior) were significantly higher in Mediterranean diet compared to Western diet animals (t(26.8) = 4.13, p = 3.2 x 10−4). (D) Six of the 20 behaviors observed are significantly correlated with DAB score (pHB < 0.05). Here, significant correlations with DAB score in which behaviors are more frequent in Mediterranean diet or Western diet monkeys are indicated with blue or orange points, respectively. Figure 4—figure supplement 1. There were no differences between the Western- and Mediterranean-fed groups in the rates (A) or duration (B) of behaviors during the baseline phase, prior to diet manipulation. The boxplots depict the per-group medians and interquartile ranges for each behavior. Animals fed the Western diet are colored orange, and those fed the Mediterranean diet colored blue. Significant differences between the diet groups in the rates (C) or duration (D) of behaviors during the experimental phase are indicated (Mann-Whitney U test, Holm-Bonferroni adjusted p < 0.05 *, p < 0.01 **, p < 0.001 ***). Figure 4—figure supplement 2. The first axis of variance in behavior—which explained 31% of the overall variance—was significantly positively correlated with dominance rank across diets (Pearson’s r = 0.84, p = 3.9 x 10−10). All monkeys are assigned a rank between 0 and 1 based on the outcomes of dyadic interactions, where a higher rank indicates more dominant social status.

Figure 4—figure supplement 1
Diet manipulation altered behavior.
Figure 4—figure supplement 2
The first PC of all behavioral data captures dominance rank.
Behavior partially mediates the effect of diet on gene expression for 24% of diet-associated genes.

(A) Diet-altered behavior (DAB) mediated the effect of diet on gene expression for 24% (n = 1199) of genes for which diet had an effect (differentially expressed genes or DEGs). For 18% of DEGs, gene expression mediated the effect of diet on DAB score. (B) DAB score mediated 23–97% of the total effect of diet on gene expression in 1199 genes (n = 712 Western genes, orange; n = 487 Mediterranean genes, blue). DAB score mediated a greater number of Western genes than Mediterranean genes (p = 1.5 x 10−21) and accounted for a greater portion of the effect size of diet (p = 6.4 x 10−25) in Western genes. (C) In gene-by-gene models of DAB score as a function of diet + gene expression, gene expression mediated 17–51% of the total effect of diet on DAB in 898 genes (n = 523 Western genes; n = 375 Mediterranean genes). Gene expression mediated a greater number of Western genes than Mediterranean genes (p = 4.6 x 10−14), although expression of these genes did not account for more of the effect of diet on DAB score than Mediterranean genes (Mann-Whitney U = 1.0 x 105, p = 0.55).

Appendix 1—figure 1
RNA Integrity was correlated with both uncorrected gene expression and relative rank.

(A) RNA integrity (RIN) was correlated with PC1 of gene expression (62% of overall variance in gene expression) prior to correction for batch effects (Pearson’s r = 0.41, p = 0.020). Because of this, RIN was included as a batch effect prior to downstream analysis. (B) RIN was also correlated with relative dominance rank (Pearson’s r = 0.49, p = 5.1 x 10−3). Points are colored to indicate Western (orange) or Mediterranean (blue) diet to show that diet did not have an interactive effect in either case.

Appendix 2—figure 1
Greater phenotypic variability in Western diet fed monkeys does not show consistency in individual responsiveness across phenotypes.

(A) Monkeys fed the Western diet showed more variability than monkeys fed the Mediterranean diet in both diet-altered behavior (DAB) and change in body weight. However, the two phenotypes were not correlated within monkeys fed the Western diet (Pearson’s rWestern = −0.26, p = 0.28). (B) Western fed monkeys also showed more variability in the first principal component (PC1) of gene expression than Mediterranean fed monkeys. DAB and PC1 of gene expression were not significantly correlated in Western fed monkeys (Pearson’s rWestern = −0.13, p = 0.60). (C) PC1 of gene expression and change in body weight were not significantly correlated in Western fed monkeys (Pearson’s rWestern = −0.22, p = 0.36).

Appendix 2—figure 2
Quality control of cell purity by CD14 and CD3 expression levels: three samples were excluded due to lower CD14 and high CD3 – possible T cell contamination.

Normalized expression (reads per kilobase million) of CD14 and CD3 are plotted as markers of monocytes and T cells, respectively. Three samples were excluded as outliers due to possible T cell contamination.

Tables

Table 1
Comparison of nutritional contents of diet patterns in human with nonhuman primate diets used in the current study.
Diet CompositionHumanNonhuman Primate
WesternMediterraneanWestern*Mediterranean*Chow
% of Calories
Protein15§1716§1618
Carbohydrate51§5154§5469
Fat33§3231§3113
% of Total fats
Saturated33§2136§2126
Monounsaturated36§5636§5728
Polyunsaturated24§1526§2032
Other nutrients
ω6:ω3 Fatty Acids15:1††2.1-3:1‡‡14.8:1††2.9:1‡‡12:01
Cholesterol mg/Cal0.13§0.160.16§0.15trace
Fiber g/Cal0.01§0.03§§0.02§0.04§§0.01
Sodium mg/Cal1.7§,¶¶1.3¶,§§1.7§,¶¶1.1¶,§§0.25
  1. * Developed and prepared at Wake Forest School of Medicine.

    LabDiet Chemical Composition Diet 5037/8. Type of fat known in 86% of total fat. Omega-6 from corn and pork fat.

  2. Human carbohydrate calories include alcohol.

    §(Centers for Disease Control and Prevention (CDC), 2014).

  3. ¶¶ (Powles et al., 2013).

    Reprinted from Shively et al., 2019, Obesity with permission (Shively et al., 2019).

Additional files

Supplementary file 1

Supplementary tables 1-6.

Suppl.tab 1A Effect of diet on gene expression. Suppl.tab 1B Effects of diet on gene expression (FDR < 0.05 β sort). Suppl.tab 2A Biological processes enriched in western genes compared to other measured genes Suppl.tab 2B. Biological processes enriched in mediterranean genes compared to other measured genes. Suppl.tab 3. Transcription factor binding site motif enrichment. Suppl.tab 4A. Diet correlation and gene set enrichment of weighted gene co-expression network analysis (WGCNA) co-expression modules. Suppl.tab 4B. Biological processes enriched in WGCNA co-expression modules. Suppl.tab 5A. Gene pair correlations across and within diet groups. Suppl.tab 5B. Differentially Correlated Genes. Suppl.tab 6A. Biological processes enriched in behavior-mediated differentially-expressed genes (DEGs) Suppl.tab 6B. Biological processes enriched in behavior-mediated western genes Suppl.tab 6C. Biological processes enriched in behavior-mediated mediterranean genes.

https://cdn.elifesciences.org/articles/68293/elife-68293-supp1-v2.xlsx
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https://cdn.elifesciences.org/articles/68293/elife-68293-transrepform-v2.docx

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