Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s

Abstract

Group 2 innate lymphoid cells (ILC2s) and type 2 helper T cells (Th2 cells) are the primary source of interleukin 5 (IL-5) and IL-13 during type 2 (allergic) inflammation in the lung. In Th2 cells, T cell receptor (TCR) signaling activates the transcription factors nuclear factor of activated T cells (NFAT), nuclear factor κB (NF-κB), and activator protein 1 (AP-1) to induce type 2 cytokines. ILC2s lack a TCR and respond instead to locally produced cytokines such as IL-33. Although IL-33 induces AP-1 and NF-κB, NFAT signaling has not been described in ILC2s. In this study, we report a nonredundant NFAT-dependent role for lipid-derived leukotrienes (LTs) in the activation of lung ILC2s. Using cytokine reporter and LT-deficient mice, we find that complete disruption of LT signaling markedly diminishes ILC2 activation and downstream responses during type 2 inflammation. Type 2 responses are equivalently attenuated in IL-33- and LT-deficient mice, and optimal ILC2 activation reflects potent synergy between these pathways. These findings expand our understanding of ILC2 regulation and may have important implications for the treatment of airways disease.

Figure 1.

LTs are sufficient to rapidly activate lung ILC2s. (A) Gene expression measured by quantitative RT-PCR on cells sorted from lungs (CD4 and ILC2) or blood (eosinophils) of naive mice. (B) Experimental workflow for in vitro ILC2 stimulation assay. (C–F) Frequency (C and E) and MFI (D and F) of Smart13 reporter–positive lung ILC2s sorted from naive wild-type (C and D) or the indicated (E and F) mice and treated in vitro for 6 h with the indicated LTs. (C and D) Four 10-fold dilutions from 100 to 0.1 nM are shown. (E and F) 10 nM of the indicated LTs. (G and H) Frequency of Smart13 reporter–positive ILC2s in lungs (G) and amount of IL-5 protein in BAL (H) of mice treated for 6 h with 200 ng of the indicated LTs. Data are representative of two experiments (A, E, and F) or pooled from at least three experiments (C, D, G, and H). n = 3 technical replicates (A, E, and F) or at least 3 biological replicates (C and D) or biological replicates as shown (G and H). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with PBS by one-way ANOVA). Data are means ± SEM. bg, background.

Figure 2.

Lung ILC2 homeostasis is largely intact in the absence of LTs. (A and B) Number of ILC2s (A) and frequency of Smart13 reporter–positive ILC2s (B) in the lungs of naive mice of the indicated genotypes. (C–F) Frequency (C and E) and MFI (D and F) of Smart13 reporter–positive lung ILC2s sorted from naive mice of the indicated genotypes and treated in vitro for 6 h with 10 nM of the indicated LTs (C and D) or 10 ng/ml IL-33 (E and F). Data are pooled from at least three experiments (A and B) or representative of two experiments (C–F). n = 3 technical replicates (C–F) or biological replicates as shown (A and B). **, P < 0.01 (compared with WT[B6] by one-way ANOVA). One high Ltc4s^−/− outlier was excluded from B by Grubb’s test (P < 0.01). Data are means ± SEM. bg, background.

Figure 3.

LTs are required for lung ILC2 activation during type 2 inflammation. (A) Frequency of Smart13 reporter–positive ILC2s in the lungs of mice of the indicated genotypes treated 8 h with intranasal chitin. (B–E) Mice of the indicated genotypes infected for 7 d with N. brasiliensis (Nb) and then analyzed for number of lung ILC2s (B), frequency of Smart13 reporter–positive ILC2s (C), MFI of Smart13 reporter on ILC2s (D), and number of lung eosinophils (E). (F) BAL from mice of the indicated genotypes infected 7 d with N. brasiliensis. (G) OD420 of BAL in F to quantify red blood cell infiltrate. Data are representative of two experiments (F) or pooled from two (G) or at least three experiments (A–E). n = biological replicates as shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with treated WT[B6] by one-way ANOVA). Data are means ± SEM.

Figure 4.

IL-33 and LTs are nonredundant and signal with different kinetics in lung ILC2s. (A–D) IL-13 (A) or IL-5 (B) protein in the BAL or number of eosinophils (C) or ILC2s (D) in the lungs of the indicated mice infected 7 d with N. brasiliensis. (E–G) Expression of Il33r (E), Cysltr1 (F), or Ltb4r1 (G) measured by quantitative RT-PCR in ILC2s sorted from naive lungs of the indicated mice. (H and I) Frequency (H) or MFI (I) of Smart13 reporter–positive ILC2s sorted from the lungs of Arg1^Yarg/Yarg;Il13^Smart/Smart mice and treated in vitro for 6 h with IL-33 or LTC₄, as indicated. Four 10-fold dilutions from 100 nM (LTC₄) and 100 ng/ml (IL-33) are shown. (J and K) Frequency (J) or MFI (K) of Smart13 reporter–positive ILC2s sorted from lungs of Arg1^Yarg/Yarg;Il13^Smart/Smart mice with or without anti–IL-33R antibody, as indicated. Cells were treated 6 h in vitro with 10 nM LTC₄ or 10 ng/ml IL-33. (L and M) MFI (L) or frequency (M) of the Smart13 reporter on lung ILC2s sorted from IL13^Smart/Smart mice and treated in vitro for the indicated times with 10 nM LTC₄ or 10 ng/ml IL-33. (N) IL-5 protein in the supernatants of cells in L and M. The data shown are pooled from two experiments (A–G) or representative of two experiments (H–N). n = 3 technical replicates (H–N) or biological replicates as shown (A–G). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with WT[B6] by one-way ANOVA). One high WT(B6) outlier was excluded from C by Grubb’s test (P < 0.01). Data are means ± SEM. bg, background.

Figure 5.

NFAT-dependent LTC₄ signaling synergizes with IL-33 for optimal ILC2 activation. (A) MFI of Smart13 reporter on ILC2s sorted from lungs of IL13^Smart/Smart mice and treated 6 h in vitro with 10 nM LTC₄, 10 ng/ml IL-33, 30 ng/ml PMA, and/or 500 ng/ml ionomycin (Iono), as indicated. (B and C) Frequency (B) or MFI (C) of Smart13 reporter–positive ILC2s sorted from lungs of IL13^Smart/Smart mice and treated in vitro for 30 min with 10 µM sotrastaurin, 100 µM caffeic acid phenethyl ester (CAPE), or 100 nM cyclosporine A (Cyclo A) as indicated, followed by 6-h treatment with 10 nM LTC₄ or 10 ng/ml IL-33 in the presence of the indicated inhibitors. (D and E) Fluorescence microscopy of ILC2s sorted from lungs of WT(B6) mice and treated 30 min with cyclosporine A (CSA) as indicated followed by 90-min treatment with 100 nM LTC₄, 100 ng/ml IL-33, 30 ng/ml PMA, and/or 500 ng/ml ionomycin, as indicated. Cells were stained with anti-NFATC1 (D) or anti-NFATC2 (E) and DAPI (blue). White asterisks indicate nuclear localization of NFAT. Bars, 10 µm. (F) Quantification of cells in E with nuclear localization of NFATC2. At least 40 cells were counted for each condition. P + I, PMA + ionomycin. (G) MFI of the Smart13 reporter on ILC2s sorted from the lungs of IL13^Smart/Smart mice and treated in vitro for 6 h with the indicated combinations of IL-33 with LTC₄ or LTD₄. (H–K) Il13^Smart/Smart mice treated 6 h with 50 ng IL-33 or LTC₄ given intranasally alone or in combination and analyzed for frequency (H) or MFI (I) of Smart13 reporter–positive ILC2s in lungs or IL-5 (J) or IL-13 (K) in the BAL. (L–N) Quantitative RT-PCR analysis of the indicated genes in ILC2s (lineage⁻;Thy1.2⁺;IL-33R⁺) or other ILCs (lineage⁻;Thy1.2⁺;IL-33R⁻) sorted from the lungs of naive WT(B6) mice. (O) Expression of Il5 measured by quantitative RT-PCR in ILC2s sorted from naive lungs of the indicated mice. The data shown are pooled from three (A–C and H–K) or two (L–N) experiments or are representative of two (D–F and O) or three (G) experiments. n = 3 (A–C), 2 (F) or 1 (G) technical replicates, or 5–6 (L–N) or biological replicates as shown (F, H–K, and O). #, not detected. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with PBS by one-way ANOVA [H–K and O] or pairwise comparison by Student’s t test [L–N]). Data are means ± SEM. bg, background.