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. 2017 Aug 1;199(3):1096-1104.
doi: 10.4049/jimmunol.1601569. Epub 2017 Jun 30.

Leukotriene C4 Potentiates IL-33-Induced Group 2 Innate Lymphoid Cell Activation and Lung Inflammation

Sean J Lund  1 , Alex Portillo  1 , Kellen Cavagnero  1 , Rachel E Baum  1 , Luay H Naji  1 , Jana H Badrani  1 , Amit Mehta  2 , Michael Croft  2 , David H Broide  1 , Taylor A Doherty  3
Affiliations

Leukotriene C4 Potentiates IL-33-Induced Group 2 Innate Lymphoid Cell Activation and Lung Inflammation

Sean J Lund et al. J Immunol. .

Abstract

Asthma is a complex disease that is promoted by dysregulated immunity and the presence of many cytokine and lipid mediators. Despite this, there is a paucity of data demonstrating the combined effects of multiple mediators in asthma pathogenesis. Group 2 innate lymphoid cells (ILC2s) have recently been shown to play important roles in the initiation of allergic inflammation; however, it is unclear whether lipid mediators, such as cysteinyl leukotrienes (CysLTs), which are present in asthma, could further amplify the effects of IL-33 on ILC2 activation and lung inflammation. In this article, we show that airway challenges with the parent CysLT, leukotriene C4 (LTC4), given in combination with low-dose IL-33 to naive wild-type mice, led to synergistic increases in airway Th2 cytokines, eosinophilia, and peribronchial inflammation compared with IL-33 alone. Further, the numbers of proliferating and cytokine-producing lung ILC2s were increased after challenge with both LTC4 and IL-33. Levels of CysLT1R, CysLT2R, and candidate leukotriene E4 receptor P2Y12 mRNAs were increased in ILC2s. The synergistic effect of LTC4 with IL-33 was completely dependent upon CysLT1R, because CysLT1R-/- mice, but not CysLT2R-/- mice, had abrogated responses. Further, CysLTs directly potentiated IL-5 and IL-13 production from purified ILC2s stimulated with IL-33 and resulted in NFAT1 nuclear translocation. Finally, CysLT1R-/- mice had reduced lung eosinophils and ILC2 responses after exposure to the fungal allergen Alternaria alternata Thus, CysLT1R promotes LTC4- and Alternaria-induced ILC2 activation and lung inflammation. These findings suggest that multiple pathways likely exist in asthma to activate ILC2s and propagate inflammatory responses.

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Figures

Figure 1
Figure 1
LTC4 potentiates IL-33 mediated airway inflammation. WT, IL-7R−/− (IL-7RKO), and RAG2−/− (RAG2KO) mice were challenged with either PBS, IL-33, LTC4, or both IL-33 and LTC4 once a day for 3 days. On day 4, the mice were euthanized and BAL and lungs were collected. (A) Total eosinophils in the BAL of wild type mice. (B) IL-5 and (C) IL-13 in the BAL of wild type mice. (D) H&E stained lung sections from PBS, IL-33, LTC4, or both IL-33 and LTC4 challenged wild type mice. Scale bar is 100 μm. (E) Total eosinophils in the BAL (top) and lungs (bottom) of challenged IL-7R−/− and RAG2−/− mice. (F) BAL IL-5 and IL-13 in challenged IL-7R−/− and RAG2−/− mice. Data shown are representative of 2–5 independent experiments with 4 mice per group. *p < 0.05, *** p < 0.005, unpaired t test.
Figure 2
Figure 2
LTC4 potentiates IL-33-induced ILC2 activation and cytokine production. WT mice were challenged with PBS, IL-33, LTC4, or the combination of IL-33 and LTC4 once a day for 3 days. On day 4, mice were euthanized and BAL and lungs were collected. (A) Total numbers of ILC2s, (B) proliferating Ki67+ ILC2s, and (C) IL-5+ ILC2s in the lungs of wild type mice enumerated. (D) Percentages of lung ILC2s within the lymphocyte gate. (E) FACS plots of ILC2 Ki67 percent and (F) ILC2 IL-5 and IL-13. ILC2s were defined as CD45+ lineage-negative Thy1.2+ lymphocytes. FACS plots in (E) and (F) gated on ILC2s. Data shown are representative of 5 independent experiments, with 4 mice per group. * p < 0.05, *** p < 0.005, unpaired t test.
Figure 3
Figure 3
LTC4 potentiation of IL-33-induced ILC2 responses occurs independent of adaptive immunity. IL-7R−/− (IL-7RKO) and RAG2−/− (RAG2KO) were challenged with either PBS, IL-33, LTC4, or both IL-33 and LTC4 once a day for 3 days. On day 4, mice were euthanized and BAL and lungs were collected. (A) Total numbers of ILC2s, (B) proliferating Ki67+ ILC2s, and (C) IL-5+ ILC2s in the lungs of RAG2−/− enumerated. (D) (E), FACS plots of ILC2 Ki67 in RAG2 knockout mice. (D) Percentages of lung ILC2s within the lymphocyte gate. (E) FACS plots of ILC2 Ki67 percent and (F) ILC2 IL-5 and IL-13 shown from RAG2 KO mice. ILC2s were defined as CD45+ lineage-negative Thy1.2+ lymphocytes. FACS plots in (E) and (F) gated on ILC2s. Data shown are representative of 2–3 independent experiments with 2 mice per group. * p < 0.05, *** p < 0.005, unpaired t test.
Figure 4
Figure 4
LTC4 potentiation of IL-33-induced airway inflammation is dependent on CysLT1R. WT, CysLT1R−/− (1R), and CysLT2R−/− (2R) mice were challenged with IL-33, LTC4, or the combination of IL-33 and LTC4 once a day for 3 days. On day 4, mice were euthanized and BAL and lungs were collected. (A) Total eosinophils in the BAL (A) and lungs (B) were enumerated. (C) H&E stained lung sections from from WT, CysLT1R−/− and CysLT2R−/− challenged with IL-33 or IL-33 and LTC4. Images shown are 10×, scale bar is 100 μm. Data shown are representative of 2–4 independent experiments with 2 mice per group. * p < 0.05, *** p < 0.005, unpaired t test.
Figure 5
Figure 5
Potentiation of ILC2 proliferation by LTC4 is dependent on CysLT1R. WT, CysLT1R−/− (1R), and CysLT2R −/− (2R) mice were challenged with IL-33, LTC4, or the combination of IL-33 and LTC4 once a day for 3 days. On day 4, mice were euthanized and BAL and lungs were collected. (A) Percent of ILC2s within lymphocyte gate and (B) Ki-67+ ILCs enumerated. (C) Total numbers of ILC2s and (D) total numbers of Ki67+ ILC2 also shown. Data shown are representative of 2–5 independent experiments with 4 mice per group. * p < 0.05, *** p < 0.005, unpaired t test.
Figure 6
Figure 6
ILC2 cytokine production potentiation by LTC4 is dependent on CysLT1R. WT, CysLT1R−/− (1R), and CysLT2R−/− (2R) mice were challenged with either PBS, IL-33, LTC4, or both IL-33 and LTC4 once a day for 3 days. On day 4, the mice were euthanized and BAL and lungs were collected. (A) Concentration of IL-5 and IL-13 in the BAL of wild type, CysLT1R−/−, and CysLT2R−/− mice. (B) Total numbers of IL-5+ and IL-13+ ILC2 in wild type, CysLT1R−/−, and CysLT2R−/− mice and (C–D) FACS plots of IL-5 and IL-13. Gated on CD45+ Thy1.2+ lineage-negative lymphocytes. Data shown are representative of 5 independent experiments with 3 mice per group. * p < 0.05, *** p < 0.005 unpaired t test.
Figure 7
Figure 7
CysLTs directly potentiate ILC2 activation and NFAT1 translocation. Lung ILC2s were purified and then stimulated with media, IL-33, or IL-33 in combination with each of the leukotrienes for 6 hours. (A) Concentration of IL-5 and (B) IL-13 from culture supernatants. (C) Representative immunofluorescence of ILC2s with NFAT1 staining (red) with nuclear staining (blue). Data shown are representative of 2 independent experiments. * p < 0.05, ** p < 0.01 *** p < 0.005 unpaired t test.
Figure 8
Figure 8
CysLT1R is required for ILC2 responses during allergen challenge with Alternaria alternata. WT and CysLT1R knockout mice were challenged with 30μg Alternaria alternata on day 0 followed by 3μg on days 9 and 12. Lung cells were analyzed on D14. (A) Percent of lung eosinophils in naïve and challenged WT and CysLT1R knockout mice. (B) Total number of ILC2s in naïve and challenged WT and CysLT1R knockout mice. (C) Total number of Ki67+ ILC2 in naïve and challenged WT and CysLT1R knockout mice (left) and representative Ki-67% within the ILC2 population from lungs of challenged WT and CysLT1R knockout mice (right). (D) IL-5+ and (E) IL-13+ ILC2s in the lungs of challenged WT and CysLT1R knockout mice (left) and representative ILC2 IL-5% (D) and IL-13% (E) from lungs of challenged WT and CysLT1R knockout mice. Data shown are representative of 2 independent experiments. * p < 0.05, ** p < 0.01 *** p < 0.005 unpaired t test.
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References

    1. Weiss JW, Drazen JM, McFadden ER, Jr, Weller PF, Corey EJ, Lewis RA, Austen KF. Comparative bronchoconstrictor effects of histamine, leukotriene C, and leukotriene D in normal human volunteers. Trans Assoc Am Physicians. 1982;95:30–35. - PubMed
    1. Weiss JW, Drazen JM, Coles N, McFadden ER, Jr, Weller PF, Corey EJ, Lewis RA, Austen KF. Bronchoconstrictor effects of leukotriene C in humans. Science. 1982;216:196–198. - PubMed
    1. Laitinen LA, Laitinen A, Haahtela T, Vilkka V, Spur BW, Lee TH. Leukotriene E4 and granulocytic infiltration into asthmatic airways. Lancet. 1993;341:989–990. - PubMed
    1. Sims JE, Smith DE. The IL-1 family: regulators of immunity. Nat Rev Immunol. 2010;10:89–102. - PubMed
    1. Moussion C, Ortega N, Girard JP. The IL-1-like cytokine IL-33 is constitutively expressed in the nucleus of endothelial cells and epithelial cells in vivo: a novel ‘alarmin’? PLoS One. 2008;3:e3331. - PMC - PubMed

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