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Ketogenic diet activates protective γδ T cell responses against influenza virus infection

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Science Immunology  15 Nov 2019:
Vol. 4, Issue 41, eaav2026
DOI: 10.1126/sciimmunol.aav2026
  • Fig. 1 γδ T cells are required for improved survival of KD-fed mice during IAV infection.

    (A) Body weight change (pooled from five independent experiments for a total of n = 25 chow and n = 33 KD mice analyzed) and (B) survival after infection with 108 PFU of A/PR8 IAV in chow (n = 7) versus KD (n = 10). Survival differences are representative of three independent experiments and were calculated by log-rank test. (C) Blood oxygen saturation kinetics after sublethal infection with 2.5 × 107 PFU of A/PR8 IAV. n = 5 mice per group. (D) BAL viral titers on day 3 after infection with 108 PFU of A/PR8 IAV. Pooled from two independent experiments (chow, n = 8; KD, n = 10). (E) Heat map of RNA-seq top 10 differentially expressed genes (day 3 after infection, based on adjusted P < 0.05) in IAV-infected whole lung tissue of KD-fed versus chow-fed mice. (F) Representative FACS plot of lung γδ T cells on day 3 after infection. (G) Percentage and (H) absolute number of γδ T cells in the lungs 3 days after IAV infection. n = 5 mice per group. (F to H) Data are representative of more than five independent experiments. (I) Body weight change after infection of Mx1 (n = 21) versus Tcrd−/− Mx1 mice fed KD (n = 20) with 108 PFU of IAV. Pooled from three independent experiments. (J) Survival after infection of Mx1 (n = 15) versus Tcrd−/− Mx1 mice fed KD (n = 17) with 108 PFU of IAV. Survival differences were calculated by the Gehan-Breslow-Wilcoxon test. (I) For all graphs, each symbol represents an individual mouse, and data are expressed as means ± SEM. Statistical differences were calculated by (A, C, and I) paired two-way ANOVA, and (D, G, and H) unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2 High-fat content of KD is not sufficient to induce protective γδ T cells.

    (A) Body weight change of chow-fed (n = 5), KD-fed (n = 7), and HFD-fed (n = 9) mice after infection with 108 PFU of IAV. (B) Lung γδ T cell abundance 3 days after IAV infection in chow-fed (n = 3), KD-fed (n = 5), and HFD-fed (n = 5) mice. (C) Frequency of γδ T cells from the lungs of chow-fed (n = 4), KD-fed (n = 6), and HFD-fed (n = 5) mice that produce IL-17 after PMA + ionomycin stimulation ex vivo. (D) Bar graph of significantly regulated Tcrd and Tcrg gene segments. FPKM, fragments per kilobase per million mapped reads. (E) Venn diagram of numbers of differentially expressed (DE) genes (FDR, 5%) in chow, KD, and HFD sorted lung γδ T cells on day 3 after infection. (F) IPA of genes selectively induced by KD compared with chow. (A to C) Data are representative of three independent experiments. For all graphs, each symbol represents an individual mouse. Data are expressed as means ± SEM. Statistical differences were calculated by (A) paired two-way ANOVA, (B) two-way ANOVA with Tukey’s correction for multiple comparisons, and (C) one-way ANOVA with Tukey’s correction for multiple comparisons. **P < 0.01, ****P < 0.0001.

  • Fig. 3 Protective γδ T cell expansion requires metabolic adaptation to KD.

    (A) Blood BHB and lung γδ T cells on day 3 after IAV infection in mice fed chow (n = 5) versus KD (n = 5) versus BD (n = 5) beginning 1 week before infection. Statistical differences were calculated by one-way ANOVA with Tukey’s correction for multiple comparisons. ns, not significant. (B) Body weights of IAV-infected mice fed chow (n = 5), KD (n = 5), or BD (n = 5). Statistical differences were calculated by paired two-way ANOVA with Tukey’s correction for multiple comparisons. (A and B) Data are representative of at least two independent repeats. (C) Western blot of mitochondrial oxidative metabolism proteins in whole lung tissue 3 days after IAV infection in chow- and KD-fed mice. Each lane represents an individual mouse. (D) RNA-seq expression data of ketone metabolism genes from sorted γδ T cells 3 days after infection. For all graphs, each symbol represents an individual mouse. Data are expressed as means ± SEM. **P < 0.01, ****P < 0.0001.

  • Fig. 4 KD-induced protective γδ T cells enhance lung barrier function.

    (A) Heat map of significantly regulated genes (based on adjusted P < 0.05) in both comparisons of Mx1 chow versus Mx1 KD, and Mx1 KD versus Mx1 Tcrd−/− KD. Genes are ranked from highest to lowest expression. (B to F) Representative images of lung airway PAS staining. Arrowheads indicate PAS-positive cells. Scale bars, 100 μm. (G) Quantification of PAS staining images. Statistical differences were calculated by one-way ANOVA with Tukey’s correction for multiple comparisons. Data are represented as means ± SEM and n = 4 mice per group. (H) Correlation analysis of lung Scgb1a1 expression with BAL viral titers collected on day 3 after IAV infection. Statistics were calculated by linear regression pooled from chow (n = 5), KD (n = 5), and Mx1 Tcrd−/− KD (n = 4). For all graphs, each symbol represents an individual mouse. *P < 0.05.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/41/eaav2026/DC1

    Fig. S1. Bioinformatics workflow for whole lung RNA-seq analysis.

    Fig. S2. Quantification and phenotype of lung γδ T cells 3 days after IAV infection.

    Fig. S3. RNA-seq bioinformatics of sorted lung γδ T cells 3 days after IAV infection.

    Fig. S4. Transcriptomic signature of KD-specific genes in lung γδ T cells 3 days after IAV infection.

    Fig. S5. γδ T cells expansion precedes IAV infection.

    Fig. S6. Inflammatory myeloid cell infiltration is not affected by KD or HFD.

    Fig. S7. Proliferation and recruitment drive KD-mediated γδ T cell expansion.

    Fig. S8. Bioinformatics workflow for whole lung RNA-seq analysis in Mx1 Tcrd−/− mice.

    Fig. S9. γδ T cell–dependent regulation of KD-induced transcriptome changes.

    Table S1. Significantly regulated pathways in whole lungs of chow versus KD mice 3 days after IAV infection.

    Table S2. Gene set enrichment analysis of sorted lung γδ T cells from HFD-fed versus KD-fed mice.

    Table S3. Gene set enrichment analysis of sorted lung γδ T cells from HFD-fed versus chow-fed mice.

    Table S4. Gene set enrichment analysis of sorted lung γδ T cells from KD-fed versus chow-fed mice.

    Table S5. KD-specific gene signature of lung γδ T cells.

    Table S6. Significantly regulated pathways in whole lungs of Mx1 KD versus Mx1 Tcrd−/− KD mice 3 days after IAV infection.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Bioinformatics workflow for whole lung RNA-seq analysis.
    • Fig. S2. Quantification and phenotype of lung γδ T cells 3 days after IAV infection.
    • Fig. S3. RNA-seq bioinformatics of sorted lung γδ T cells 3 days after IAV infection.
    • Fig. S4. Transcriptomic signature of KD-specific genes in lung γδ T cells 3 days after IAV infection.
    • Fig. S5. γδ T cells expansion precedes IAV infection.
    • Fig. S6. Inflammatory myeloid cell infiltration is not affected by KD or HFD.
    • Fig. S7. Proliferation and recruitment drive KD-mediated γδ T cell expansion.
    • Fig. S8. Bioinformatics workflow for whole lung RNA-seq analysis in Mx1 Tcrd−/− mice.
    • Fig. S9. γδ T cell–dependent regulation of KD-induced transcriptome changes.
    • Table S1. Significantly regulated pathways in whole lungs of chow versus KD mice 3 days after IAV infection.
    • Table S2. Gene set enrichment analysis of sorted lung γδ T cells from HFD-fed versus KD-fed mice.
    • Table S3. Gene set enrichment analysis of sorted lung γδ T cells from HFD-fed versus chow-fed mice.
    • Table S4. Gene set enrichment analysis of sorted lung γδ T cells from KD-fed versus chow-fed mice.
    • Table S5. KD-specific gene signature of lung γδ T cells.
    • Table S6. Significantly regulated pathways in whole lungs of Mx1 KD versus Mx1 Tcrd−/− KD mice 3 days after IAV infection.

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