De novo fatty-acid synthesis protects invariant NKT cells from cell death, thereby promoting their homeostasis and pathogenic roles in airway hyperresponsiveness
- Department of Pathology, Seoul National University College of Medicine, Republic of Korea
- Laboratory of Immune Regulation in Department of Biomedical Sciences, Seoul National University College of Medicine, Republic of Korea
- Laboratory of Immunology and Vaccine Innovation, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Republic of Korea
- Department of Internal Medicine, Seoul National University Bundang Hospital, Republic of Korea
- Institute of Allergy and Clinical Immunology, Seoul National University Medical Research Council, Republic of Korea
Figures
Figure 1 with 1 supplement
The fatty-acid synthesis pathway is upregulated in invariant natural-killer T (iNKT) cells during murine allergic asthma.
(A, B) WT mice were subjected to ovalbumin (OVA)-induced asthma. (A) Schematic depiction of the OVA-induced allergic asthma model (left). The airway resistance of naïve, OVA-sensitized, and OVA-sensitized and OVA-challenged WT mice (right). (B) Gene expression of enzymes related to glycolysis, the tricarboxylic acid (TCA) cycle, β-oxidation, and fatty-acid synthesis in lung iNKT cells that were sorted on d0, d7, and d17. The data shown in (A) and (B) are pooled data from four independent experiments (n = 8/group). (C) In vitro gene expression of enzymes related to glycolysis (Hk2 and Pkm2), the TCA cycle (Sdh2b and Idh1a), and fatty-acid synthesis (Acc1 and Fasn) in CD8+ T, CD4+ T, and iNKT cells before and after CD3/CD28 stimulation. Representative results from two independent experiments (n = 4/group) are shown. (D–I) Cd4-CreAccl1fl/fl mice and the Acc1fl/fl control mice were subjected to OVA-induced asthma (D–F) or house-dust mite (HDM)-induced asthma (G–I). (D, G) Airway resistance. (E, H) Numbers and survival status of iNKT cells that were sorted from the lung by flow cytometry. (F, I) Cytokine expression in iNKT cells sorted from the lung. The data shown in (D–I) are pooled data from four independent experiments (n = 8/group). All data are presented as mean ± SEM. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, as determined by unpaired two-tailed t-tests.
Figure 1—figure supplement 1
Cd4-Cre::Acc1fl/fl– mice fail to develop airway resistance in the house-dust mite (HDM)-induced asthma model.
(A) Gene set enrichment analysis (GSEA) in invariant natural-killer T (iNKT) versus CD4+ T cells. Acc1fl/fl and Cd4-Cre::Accl1fl/fl mice were intranasally challenged with ovalbumin (OVA) (B–F) or HDM (G–K), and various parameters were measured. (B, G) Total cell counts from the lungs and bronchoalveolar lavage fluids (BALFs) (C, D, H, I). Estimation of immune cell subsets in the BALFs (C, H) and lungs (D, I) via flow cytometry (E, F, J, K). Expression levels of transcription factors (E, J) and cytokines (F, K) in CD4+ T (excluding iNKTs) cells sorted from lungs. Data were pooled from four independent experiments with n = 8 per group and presented as means ± SEMs. ns, not significant. *p<0.05, **p<0.01, ***p<0.001 as determined by unpaired two-tailed t-tests.
Figure 2
ACC1 deficiency in murine invariant natural-killer T (iNKT) cells associates with failure to develop α-GalCer-induced AHR.
Cd4-Cre::Acc1fl/fl and Acc1fl/fl mice were administered α-GalCer intranasally. (A) Airway resistance. (B) Total immune cell numbers in the lungs and bronchoalveolar lavage fluid (BALF). (C, D) Numbers of immune-cell subsets in the BALF (C) and lungs (D). (E, F) Expression levels of transcription factors (E) and cytokines (F) in the conventional CD4+ T cell population that was sorted from the lungs. (G) Numbers and survival status of iNKT cells sorted from the lungs. (H) Cytokine expression in iNKT cells sorted from the lungs. All data are pooled from five independent experiments (n = 10/group) and presented as mean ± SEM. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, as determined by unpaired two-tailed t-tests.
Figure 3 with 1 supplement
Adoptive transfer of WT, but not ACC1-deficient, invariant natural-killer T (iNKT) cells restores the ability of NKT-knockout mice to develop ovalbumin (OVA)-induced asthma.
iNKT cells sorted from naïve Cd4-Cre::Acc1fl/fl and Acc1fl/fl mice were adoptively transferred into naïve WT and Jα18 KO mice, after which OVA-induced asthma was initiated. (A) Airway resistance. (B) Total inflammatory cell numbers in the lungs and bronchoalveolar lavage fluid (BALF). (C, D) Numbers of immune-cell subsets in BALF (C) and lungs (D). (E, F) Expression levels of transcription factors (E) and cytokines (F) in CD4+ T cells sorted from the lungs. (G) Cytokine expression in iNKT cells sorted from the lungs. (H, I) Numbers (H) and survival status (I) of iNKT cells from the lungs. (J) Representative histological lung section images and histological scores. Scale bar = 200 µM. The data in (A–J) are pooled data from four independent experiments (n = 8/group). All data are presented as mean ± SEM. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, as determined by unpaired two-tailed t-tests.
Figure 3—figure supplement 1
Deficiency of ACC1 in invariant natural-killer T (iNKT) cells perturbed their thymic development and peripheral homeostasis in cell intrinsic manner.
(A) The percentages of iNKT cells in the thymi, spleens, and livers from Cd4-Cre::Acc1fl/fl and Acc1fl/fl mice were measured via flow cytometry. (B) Analysis of CD4+ SP thymocytes, CD8+ SP thymocytes, CD4+CD8+ DP thymocytes, CD4–CD8– DN thymocytes, and CD8+ T, CD4+ T cells from the spleens of Cd4-Cre::Acc1fl/fl and Acc1fl/fl mice. (C) Thymic iNKT cells from thymus of Cd4-Cre::Acc1fl/fl and Acc1fl/fl mice were enriched via magnetic-associated cell sorting (MACS). Percentages of NKT1s, NKT2s, and NKT17s and developmental stages of MACS-purified CD3+-CD1d tetramer+ iNKT cells were measured via flow cytometry. (A–C) Data were pooled from four independent experiments with n = 4 per group. (D) Mixed bone marrow chimeras were generated by adoptive transfer of a mixture of WT CD45.1 bone marrow and CD45.2 Cd4-Cre::Acc1fl/fl bone marrow into irradiated WT mice. To investigate the reconstitution, CD1d tetramer+ iNKT cells were stained with CD45.1 and CD45.2 antibodies via flow cytometry. (E, F) The percentages of iNKT cells in the thymi, spleens, and livers (E) and of CD4+SP, CD8+SP, CD4+CD8+DP, and CD4–CD8–DN thymocytes and splenic CD4+ T, CD8+ T cells, CD3e+ T cells, B cells (F) were measured via flow cytometry. (G, H) iNKT cells sorted from the livers of Cd4-Cre::Acc1fl/fl and Acc1fl/fl mice were stimulated with CD3/CD28 antibodies. (G) The levels of IL-4 and IFN-γ in the culture supernatants were assessed by ELISA. (H) Expression of CD69 was estimated via flow cytometry. (D–H) Data were pooled from three independent experiment with n = 30 per group and presented as means ± SEMs. ns, not significant. *p<0.05, **p<0.01, ***p<0.001 as determined by unpaired two-tailed t-tests.
Figure 4
The high glycolytic capacity of ACC1-deficient invariant natural-killer T (iNKT) cells associates with their enhanced cell death.
iNKT cells were sorted from the liver of naïve Cd4-Cre::Acc1fl/fl and Acc1fl/fl mice and used directly in the experiments. The two iNKT cell preparations were compared. (A) Volcano plot of the gene expression. (B) Signature gene expression, as determined by gene set enrichment analysis (GSEA). (C) Heat map of the fatty-acid synthesis, glycolysis, cell death, and cytokine genes. (D) Metabolome data map of the glycolysis, tricarboxylic acid (TCA) cycle, and fatty-acid synthesis metabolites. (E) Seahorse extracellular flux analysis of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). (F) Expression of glycolysis-related (Hk2, Pkm2) and gluconeogenesis-related (Fbp1, G6pase) genes. (G) 2-NBDG uptake. (H) GLUT1 expression. (I) Correlation between iNKT-cell death and maximal glycolytic activity. (J, K) The iNKT cells were treated with 2-DG to inhibit glycolysis. The effect of 2-DG on cell death (J) and intrinsic cell death-related gene expression (K) was then determined. In (A–D), the iNKT cells were from 8 mice/group. The data in (D) are presented as mean ± SEM. The data in (E–K) are pooled data from five independent experiments (n = 5/group) and are presented as mean ± SEM. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, as determined by unpaired two-tailed t-tests.
Figure 5 with 1 supplement
Low PPARγ expression in ACC1-deficient invariant natural-killer T (iNKT) cells contributes to their enhanced cell death.
iNKT cells sorted from the liver of naïve Cd4-Cre::Acc1fl/fl and Acc1fl/fl mice were either used directly in the experiment (A) or cultured with and/or without anti-CD3/CD28 with and without the indicated additives (C–H). (A) Heat map of the transcription factor genes and RT-PCR confirmation of Pparg expression in the uncultured iNKT-cell preparations. (B) CD3/CD28-stimulated and unstimulated iNKT cells were cultured with and without pioglitazone and Pparg, Cepba, Fbp1, G6pase, Hk2, and Pkm2 expression was examined. (C, D) Unstimulated iNKT cells were cultured with and without pioglitazone and 2-NBDG uptake, GLUT1 expression (C), and cell death (D) were examined. (E) CD3/CD28-stimulated and unstimulated iNKT cells were cultured with and without palmitate and/or FABP inhibitor and Pparg, Cepba, Fbp1, G6pase, Hk2, and Pkm2 expression was examined. (F) Unstimulated iNKT cells were cultured with and without palmitate and Fabp1, Fabp3, and Fabp5 expression was examined. (G, H) Unstimulated iNKT cells were cultured with and without palmitate and/or FABP inhibitor and PPARγ expression (G), 2-NBDG uptake, and cell death (H) were examined. (I) Graphical abstract of ACC1-FABP-PPARγ in iNKT cells are shown. The data in (B–H) are pooled data from four independent experiments (n = 4/group) and are presented as mean ± SEM. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, as determined by unpaired two-tailed t-tests.
Figure 5—figure supplement 1
ACC1 deficiency does not affect epigenetic modification of Pparg in invariant natural-killer T (iNKT) cells.
(A) Schematic structure of the Pparg promoter and identification of the CpG-enriched regions. Pparg methylation was measured via pyrosequencing. (B, C) Expression levels of Pparg, Hk2, Bcl2, Bak (B), and cell death (C) of iNKT cells from Cd4-Cre::Acc1fl/fl or Acc1fl/fl mice in the presence or absence of HDAC inhibitor treatment. Data were pooled from four independent experiments with n = 4 per group and presented as means ± SEMs. ns, not significant. *p<0.05, **p<0.01, ***p<0.001 as determined by unpaired two-tailed t-tests.
Figure 6 with 2 supplements
Pretreatment of ACC1-deficient invariant natural-killer T (iNKT) cells with PPARγ agonist or exogenous fatty acid restores ovalbumin (OVA)-induced asthma.
iNKT cells sorted from naïve Cd4-Cre::Acc1fl/fl and Acc1fl/fl mice were pretreated with and without pioglitazone or palmitate and then adoptively transferred into naïve WT and Jα18 KO mice. OVA-induced asthma was then initiated. (A) Airway resistance. (B) Representative histological images and histological scores of lung sections. Scale bar = 200 µM. (C) Total immune cell numbers in the lungs and bronchoalveolar lavage fluids (BALFs). (D, E) Immune-cell subsets in the BALFs (D) and lungs (E). (F, G) Transcription factor (F) and cytokine (G) expression in the total CD4+ T cell population from the lungs. (H) Cell numbers and survival status of the lung iNKT cells. (I) Cytokine expression in the lung iNKT cells. (J) PPARγ, 2-NBDG, and GLUT1 expression in the lung iNKT cells. The data are pooled from four independent experiments (n = 8/group) and are presented as mean ± SEM. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, as determined by unpaired two-tailed t-tests.
Figure 6—figure supplement 1
Adoptive transfer of Acc1-deficient invariant natural-killer T (iNKT) cells pretreated with exogenous fatty acid or PPAR-γ agonist into iNKT cell-deficient mice (Jα18 KO) restores AHR in the house-dust mite (HDM)-induced asthma model.
(A) Airway resistance was measured in WT, Jα18KO, and Jα18KO mice adoptively transferred with iNKT cells from Acc1fl/fl or Cd4-Cre::Acc1fl/fl mice in the presence or absence of pioglitazone or palmitate treatment upon HDM challenge. (B) Representative histological images of lung sections from the indicated mouse groups challenged with HDM. Scale bar = 200 µM. (C–E) Total cell numbers in the lungs and bronchoalveolar lavage fluids (BALFs) were counted (C), and subsets of immune cells in the BALFs (D) and lungs (E) were estimated via flow cytometry. (F, G) Expression levels of transcription factors (F) and cytokines (G) were measured in CD4+ T cells sorted from the lungs of mice with ovalbumin (OVA)-induced asthma. (H) Expression levels of cytokines were measured in iNKT cells sorted from the lungs of each mouse group. (I) Heat map of Ccr4, Ccr6, Cxcr3, and Cxcr4 expression in WT and ACC1-deficient iNKT cells from RNA-seq data and RT-PCR validation of the chemokine receptor expression levels in WT and ACC1-deficient iNKT cells. Data were pooled from four independent experiments with n = 8 per group and presented as means ± SEMs. n.s, not significant. *p<0.05, **p<0.01, ***p<0.001 as determined by unpaired two-tailed t-tests.
Figure 6—figure supplement 2
Genes for fatty acid metabolism are highly expressed in invariant natural-killer T (iNKT) cells from peripheral blood mononuclear cells (PBMCs) of patients with allergic asthma.
(A, B) The expression levels of ACC1, FASN, PPARG (A), HK2, and FBP1 (B) in CD8+ T, CD4+ T, regulatory T cells, and iNKT cells from peripheral blood samples of healthy donors and patients with non-allergic asthma or allergic asthma (n = 10 per group). (C) The expression levels of IL4, IL13, IFNG, IL10 were measured in iNKT cells from peripheral bloods of healthy donors and patients with non-allergic asthma or allergic asthma. Data are presented as means ± SEMs. ns, not significant. *p<0.05, **p<0.01, ***p<0.001 as determined by Mann–Whitney U test.
Additional files
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Supplementary file 1
List of reagents used for flow cytometry.
- https://cdn.elifesciences.org/articles/87536/elife-87536-supp1-v1.docx
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Supplementary file 2
List of primer sequences used for real-time PCR.
- https://cdn.elifesciences.org/articles/87536/elife-87536-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/87536/elife-87536-mdarchecklist1-v1.docx
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De novo fatty-acid synthesis protects invariant NKT cells from cell death, thereby promoting their homeostasis and pathogenic roles in airway hyperresponsiveness
eLife 12:RP87536.
https://doi.org/10.7554/eLife.87536.4
