Parental dietary fat intake alters offspring microbiome and immunity

Abstract

Mechanisms underlying modern increases in prevalence of human inflammatory diseases remain unclear. The hygiene hypothesis postulates that decreased microbial exposure has, in part, driven this immune dysregulation. However, dietary fatty acids also influence immunity, partially through modulation of responses to microbes. Prior reports have described the direct effects of high-fat diets on the gut microbiome and inflammation, and some have additionally shown metabolic consequences for offspring. Our study sought to expand on these previous observations to identify the effects of parental diet on offspring immunity using mouse models to provide insights into challenging aspects of human health. To test the hypothesis that parental dietary fat consumption during gestation and lactation influences offspring immunity, we compared pups of mice fed either a Western diet (WD) fatty acid profile or a standard low-fat diet. All pups were weaned onto the control diet to specifically test the effects of early developmental fat exposure on immune development. Pups from WD breeders were not obese or diabetic, but still had worse outcomes in models of infection, autoimmunity, and allergic sensitization. They had heightened colonic inflammatory responses, with increased circulating bacterial LPS and muted systemic LPS responsiveness. These deleterious impacts of the WD were associated with alterations of the offspring gut microbiome. These results indicate that parental fat consumption can leave a "lard legacy" impacting offspring immunity and suggest inheritable microbiota may contribute to the modern patterns of human health and disease.

Figure 1. Diagrammatic Presentation of Study Design

(a) For experiments evaluating the effects of parental diet, littermate mice were placed on either Low Fat or Western Diet formulations one day prior to being placed in breeding cages. Breeder mice were maintained on the different diets throughout gestation and nursing. When the pups were three weeks post-partum, they were weaned to new cages. All pups were weaned onto the Low Fat control diet. Two to four weeks after weaning, the mice were evaluated in the described models. (b) For evaluation of the effects of active diet consumption, the converse experiment was performed. Pups from breeders on the Low Fat control diet were weaned into new cages and placed on either the Western Diet or Low Fat control diet. (c) For experiments involving co-housing of mice, pups from both Low Fat and Western Diet breeders were weaned into the same cage, and both placed on the Low Fat control diet.

Figure 2. Pups from Western Diet breeders had altered disease susceptibility

(a) Survival after infection with E. coli K1018 in BALB/c mice (n=10-19). (b-d) Staphylococcus aureus (MRSA USA300) skin infection in male BALB/c mice. Lesion sizes (b), day 6 bacterial counts from homogenized skin (c), and mRNA expression in skin abscess tissue normalized against LF controls (dotted line) (d) (n=5-6). (e) Disease free survival after induction of experimental autoimmune encephalitis (EAE) in female BALB/c mice (n=6). (f) EAE scores in C57BL/6 mice (n=7-12). (g-h) Weaned male BALB/c pups were gavaged with peanut protein and cholera toxin weekly for 4-8 weeks before challenge. Temperature drop (g) and symptom scores (h) after challenge (n=5). LF, Low Fat; WD, Western Diet. Results are representative of 3 or more (b, e, f, g, h) or combined from 2-4 (a, c, d) independent experiments and displayed as mean + s.e.m. Significance determined by t test (a-c, e-h) or ANOVA with Bonferroni’s correction (d). All experiments were repeated with similar results in both genders. Gender is indicated when representative experiments are shown; otherwise, data reflects both male and female mice with matched ratios within experiments. Each symbol designates one mouse unless otherwise specified. n designates mouse number per group.

Figure 3. Pups from Western Diet breeders had increased colonic and decreased systemic LPS responses

(a-c) Cytokine production from female excised colons stimulated with LPS for 24-72 hours. (d-e) Representative plots of FoxP3 and CD25 expression of pooled female colonic CD45+, CD4+ cells analyzed by flow cytometry (d) and values from 3 replicate experiments (e) (n=3-5). (f-h) Cytokine production from male splenocytes stimulated with LPS for 24-72 hours. (i-j) Representative plots of FoxP3 and CD25 expression of pooled female splenic CD4+ cells analyzed by flow cytometry (i) and values from 3 replicate experiments (j) (n=3-5). (k) Liver LPS content in homogenized livers. (l) mRNA expression for TLR4 and LPS binding protein (LBP) from thioglycolate-elicited macrophages (n=3-6). LF, Low Fat; WD, Western Diet. Results are representative of 3 or more (a-d, f-i) or combined from (e, j-l) 2-3 independent experiments in BALB/c mice and displayed as mean + s.e.m. Significance determined by t test. All experiments were repeated with similar results in both genders. Gender is indicated when representative experiments are shown; otherwise, data reflects both male and female mice with matched ratios within experiments. Each symbol designates one mouse unless otherwise specified. n designates number of mice per experiment.

Figure 4. Post-weaning exposure to Western Diet did not recapitulate the phenotype of mice exposed during development

Pups from breeders on a standard diet were placed at 3 weeks of age on LF or WD for 2 weeks prior to challenge. (a) Survival after injection with E. coli K1018. (b) Lesion size in male mice and (c) mRNA expression in skin abscess tissue normalized against LF controls (dotted line) following injection with MRSA. (d) Disease free survival in female mice after induction of EAE. (e-f) Temperature change and symptom scores for orally sensitized male mice after challenge with peanut protein. (g-i) Colonic cytokine induction by LPS in female mice. (j) Representative plots of FoxP3 and CD25 expression of pooled female colonic CD45+, CD4+ cells analyzed by flow cytometry (n=5). (k) Liver LPS content. (l-n) Splenic cytokine induction by LPS in male mice. (o) Representative plots of FoxP3 and CD25 expression of female splenic CD4+ cells analyzed by flow cytometry (n=5). LF, Low Fat; WD, Western Diet. Significance determined by t test. Results are representative of 2-3 independent experiments, 5-10 gender- and age-matched BALB/c mice/group unless designated as individual symbol, and displayed as mean + s.e.m.

Figure 5. Co-habitation rescued WD pups from immune alterations

(a-b) Quantitative PCR of selected genes after anti-H3K9Me3 immunoprecipitation of splenic DNA from breeders and both male and female offspring, displayed as the ratio of input DNA and normalized to isotype antibody control. (c) 16S ribosomal RNA genes in cecal stool samples of female mice. Pups from indicated breeder diets were all weaned to LF diet. Female pups were placed in cages with their littermates or co-housed for 4 weeks with pups from breeders fed opposing diet. Each bar represents one mouse; further breakdown of composition within each phylum can be found in Table S1. (d-f) Colonic cytokine induction by LPS in co-housed mice. (g) LPS content in homogenized liver tissue from co-housed mice. (h) Representative plots of FoxP3 and CD25 expression of pooled colonic CD45+, CD4+ cells from co-housed mice (n=3-5) analyzed by flow cytometry. (i-k) LPS induction of cytokines from splenocytes in co-housed mice. (l) Representative plots of FoxP3 and CD25 expression of splenic CD4+ cells from co-housed mice analyzed by flow cytometry (n=3-5). (m) mRNA levels of TLR4 and LBP from thioglycolate-elicited macrophages in co-housed mice relative to LF control (n=3-4). (n) Survival after infection with E. coli K1018 (n=10). LF, Low Fat; WD, Western Diet; M, Male; F, Female. Significance determined by t test (d-n) or ANOVA with Bonferroni’s correction (a-b). Results are representative of 2-3 independent experiments in BALB/c mice and displayed as mean + s.e.m. Each symbol designates one mouse. n designates number of mice per experiment.