Research ArticleAllergy

Diacylglycerol kinase ζ promotes allergic airway inflammation and airway hyperresponsiveness through distinct mechanisms

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Science Signaling  03 Sep 2019:
Vol. 12, Issue 597, eaax3332
DOI: 10.1126/scisignal.aax3332
  • Fig. 1 DGKζ KO mice are protected from OVA-induced allergic airway inflammation and AHR.

    (A to C) Flow cytometry analysis of cytokine production and GATA3 abundance in WT and DGKζ KO CD4+ T cells activated in vitro with antibodies against CD3 and CD28 for 5 days. Dot plots (A and C) are representative of five independent experiments. The frequency of cytokine-producing cells (B) and the frequency of GATA3+ cells (C) are means ± SEM of 10 mice per group pooled from all experiments. (D) Cytospin analysis of total number of eosinophils (Eos), macrophages (Mac), lymphocytes (Lym), and neutrophils (Neu) in BAL fluid from unchallenged or OVA-challenged WT and DGKζ KO mice, as indicated. Data are means ± SEM of 6 mice per group (unchallenged) and 19 to 22 mice per group (OVA-challenged) from three independent experiments. (E and F) ELISA analysis of the amount of TH1 and TH2 cytokines in BAL fluid (E) and OVA-specific IgG1 and IgG2a antibody in the serum (F) from OVA-challenged WT and DGKζ KO mice. Data are means ± SEM of 14 to 16 mice per group from two independent experiments. (G) H&E staining of lung tissue (left) and compiled histopathological scores (right) from unchallenged and OVA-challenged WT and DGKζ KO mice. Images (left) are representative of two independent experiments. Scale bars, 200 μm. Quantified scores (right) are means ± SEM of four to five mice per group pooled from all experiments. (H) FlexiVent analysis of airway resistance after methacholine treatment of unchallenged or OVA-challenged WT and DGKζ KO mice, as indicated. Data are means ± SEM of six to nine mice per group from two independent experiments. (I and J) Flow cytometry analysis of total immune cells (I) and ELISA for cytokine amounts (J) in BAL fluid from HDM-challenged WT and DGKζ KO mice. Data are means ± SEM of nine mice per group from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant by two-sided unpaired Student’s t test (B to G and I), two-way analysis of variance (ANOVA) with Bonferroni’s posttest (H), or Mann-Whitney U test (J).

  • Fig. 2 Hematopoietic and nonhematopoietic cells differentially contribute to OVA-induced airway inflammation and AHR in the absence of DGKζ.

    (A to C) FlexiVent analysis of airway resistance after methacholine treatment (A), cytospin analysis of total immune cells in BAL fluid (B), and ELISA for cytokine abundance in BAL fluid (C) from OVA-challenged WT and DGKζ KO BM chimeras. Data are means ± SEM of seven to nine mice per group from three independent experiments. (D to F) FlexiVent analysis of airway resistance after methacholine treatment (D), cytospin analysis of total immune cells in BAL fluid (E), and ELISA for cytokine abundance in BAL fluid (F) from OVA-challenged Vav-Cre DGKζfl/fl mice and Vav-Cre controls. Lung resistance values (D) are means ± SEM of seven mice per group from two independent experiments. BAL cell numbers (E) and cytokine abundance (F) are means ± SEM of 13 to 14 mice per group from four independent experiments. (G and H) Cytospin analysis of total immune cells (G) and ELISA for cytokine amounts (H) in BAL fluid from OVA-challenged CD4-Cre DGKζfl/fl mice and CD4-Cre controls. Data are means ± SEM of 12 to 17 mice per group from at least two independent experiments. (I) Flow cytometry analysis of the ratio of adoptively transferred cytokine-producing WT OT-II and DGKζ KO OT-II CD4+ T cells in the spleen of OVA-sensitized congenic WT hosts after ex vivo restimulation with PMA/ionomycin. Data are means ± SEM of 10 mice per group from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant by two-way ANOVA with Bonferroni’s posttest (A and D), two-sided unpaired Student’s t test (B and E to H), Mann-Whitney U test (C), or one-sided Student’s t test (I).

  • Fig. 3 The loss of DGKζ in airway smooth muscle cells protects against AHR.

    (A) FlexiVent analysis of airway resistance after methacholine treatment from OVA-challenged Pirt-Cre controls and Pirt-Cre DGKζfl/fl mice. Data are means ± SEM of 8 to 11 mice per group from two independent experiments. (B to D) FlexiVent analysis of airway resistance after methacholine treatment (A), flow cytometry analysis of total immune cells in BAL fluid (B), and ELISA for cytokine abundance in BAL fluid (C) from OVA-challenged DGKfl/fl controls and Myh11-Cre DGKζfl/fl mice. Lung resistance values (B) are means ± SEM of 9 to 14 mice per group from four independent experiments. BAL cell numbers (C) and cytokine abundance (D) are means ± SEM of 18 to 21 mice per group from six independent experiments. (E) Myograph analysis of the contractile forces generated after methacholine treatment ex vivo of tracheal rings isolated from WT and DGKζ KO mice. Data are means ± SEM of 8 to 11 mice per group from three independent experiments. (F) Myograph analysis of the contractile forces generated after methacholine treatment ex vivo of tracheal rings isolated from DGKfl/fl controls and Myh11-Cre DGKζfl/fl mice. Data are means ± SEM of eight mice per group from two independent experiments. *P < 0.05, **P < 0.01; NS, not significant by two-way ANOVA with Bonferroni’s posttest (A, B, E, and F) or two-sided unpaired Student’s t test (C and D).

  • Fig. 4 Enhancement of ERK signaling in T cells is sufficient to protect from OVA-induced allergic airway inflammation but insufficient to protect from OVA-induced AHR.

    (A) The frequency of cytokine-producing WT and DGKζ KO CD4+ T cells after pretreatment with either vehicle control or U0126 followed by in vitro activation with antibodies against CD3 and CD28 for 5 days. Data are means ± SEM of five mice per group from two independent experiments. (B to D) Flow cytometry analysis of cytokine production and GATA3 abundance in WT and ERKSEM CD4+ T cells after in vitro activation with antibodies against CD3 and CD28 for 5 days. Dot plots (B and D) are representative of three independent experiments. The frequency of cytokine-producing cells (C) and the frequency of GATA3-expressing cells (D) are means ± SEM of six mice per group from three independent experiments. (E to G) Flow cytometry analysis of total immune cells in BAL fluid (E), ELISA for cytokine abundance in BAL fluid (F), and flexiVent analysis of airway resistance after methacholine treatment (G) from OVA-challenged WT and ERKSEM mice. BAL cell numbers (E) and cytokine abundance (F) are means ± SEM of 21 to 26 mice per group from five independent experiments. Lung resistance values (G) are means ± SEM of eight to nine mice per group from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant by two-sided unpaired Student’s t test (A to F) or two-way ANOVA with Bonferroni’s posttest (G).

  • Fig. 5 Pharmacological inhibition of DGK protects against OVA-induced allergic airway inflammation and AHR.

    (A to C) FlexiVent analysis of airway resistance after methacholine treatment (A), flow cytometry analysis of total immune cells in BAL fluid (B), and ELISA for cytokine abundance in BAL fluid (C) from OVA-challenged WT mice treated with either vehicle control or R59949 during the late sensitization and airway challenge phases of the OVA-induced asthma model. Lung resistance values (A) are means ± SEM of seven to eight mice per group from three independent experiments. BAL cell numbers (B) are means ± SEM of 15 to 17 mice per group from four independent experiments. Cytokine amounts (C) are means ± SEM of 10 to 11 mice per group from two independent experiments. (D to F) FlexiVent analysis of airway resistance after methacholine treatment (D), flow cytometry analysis of total immune cells in BAL fluid (E), and ELISA for cytokine abundance in BAL fluid (F) from OVA-challenged WT mice treated with either vehicle control or R59949 only during the airway challenge phase of the OVA-induced asthma model. Data are means ± SEM of 9 to 10 mice per group from two independent experiments. (G) Western blot for pMLC, pAkt, and pMYPT1 in lysates of carbachol-stimulated HASM cells pretreated with vehicle versus R59949. Blots (left) are representative of three independent experiments. Normalized band densities of pMLC (right) are means ± SEM of three donors per condition pooled from all experiments. (H) Analysis of bronchoconstriction (left) and area under curve values (right) after carbachol treatment of human PCLS pretreated with vehicle versus R59949. Data are means ± SEM of 9 to 23 slices per condition from three to seven donors per condition. *P < 0.05, **P < 0.01, ****P < 0.0001; NS, not significant by two-way ANOVA with Bonferroni’s posttest (A and D) or two-sided unpaired Student’s t test (B, C, and E to H).

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/597/eaax3332/DC1

    Fig. S1. DGKζ KO T cells display impaired TH2 differentiation in vitro.

    Fig. S2. DGKζ KO T cells display attenuated TH2 differentiation at early time points.

    Fig. S3. Addition of exogenous IL-4 restores TH2 differentiation in DGKζ KO T cells.

    Fig. S4. DGKα KO mice are partially protected from OVA-induced allergic airway inflammation and AHR.

    Fig. S5. Loss of DGKζ selectively impairs the ability of T cells to produce IL-4 in a T cell–intrinsic manner during TH2 differentiation in vivo.

    Fig. S6. Myh11-Cre is not a driver of the reduction in tension of Myh11-Cre DGKfl/fl tracheal rings in response to methacholine.

    Fig. S7. Inhibition of ERK signaling is sufficient to restore TH2 differentiation in DGKζ KO T cells.

    Table S1. Flow cytometry antibodies.

    Table S2. Immunoblotting antibodies.

  • This PDF file includes:

    • Fig. S1. DGKζ KO T cells display impaired TH2 differentiation in vitro.
    • Fig. S2. DGKζ KO T cells display attenuated TH2 differentiation at early time points.
    • Fig. S3. Addition of exogenous IL-4 restores TH2 differentiation in DGKζ KO T cells.
    • Fig. S4. DGKα KO mice are partially protected from OVA-induced allergic airway inflammation and AHR.
    • Fig. S5. Loss of DGKζ selectively impairs the ability of T cells to produce IL-4 in a T cell–intrinsic manner during TH2 differentiation in vivo.
    • Fig. S6. Myh11-Cre is not a driver of the reduction in tension of Myh11-Cre DGKfl/fl tracheal rings in response to methacholine.
    • Fig. S7. Inhibition of ERK signaling is sufficient to restore TH2 differentiation in DGKζ KO T cells.
    • Table S1. Flow cytometry antibodies.
    • Table S2. Immunoblotting antibodies.

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