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, 140 (4), 1090-1100.e11

Cysteinyl Leukotriene E 4 Activates Human Group 2 Innate Lymphoid Cells and Enhances the Effect of Prostaglandin D 2 and Epithelial Cytokines

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Cysteinyl Leukotriene E 4 Activates Human Group 2 Innate Lymphoid Cells and Enhances the Effect of Prostaglandin D 2 and Epithelial Cytokines

Maryam Salimi et al. J Allergy Clin Immunol.

Abstract

Background: Group 2 innate lymphoid cells (ILC2s) are a potential innate source of type 2 cytokines in the pathogenesis of allergic conditions. Epithelial cytokines (IL-33, IL-25, and thymic stromal lymphopoietin [TSLP]) and mast cell mediators (prostaglandin D2 [PGD2]) are critical activators of ILC2s. Cysteinyl leukotrienes (cysLTs), including leukotriene (LT) C4, LTD4, and LTE4, are metabolites of arachidonic acid and mediate inflammatory responses. Their role in human ILC2s is still poorly understood.

Objectives: We sought to determine the role of cysLTs and their relationship with other ILC2 stimulators in the activation of human ILC2s.

Methods: For ex vivo studies, fresh blood from patients with atopic dermatitis and healthy control subjects was analyzed with flow cytometry. For in vitro studies, ILC2s were isolated and cultured. The effects of cysLTs, PGD2, IL-33, IL-25, TSLP, and IL-2 alone or in combination on ILC2s were defined by using chemotaxis, apoptosis, ELISA, Luminex, quantitative RT-PCR, and flow cytometric assays. The effect of endogenous cysLTs was assessed by using human mast cell supernatants.

Results: Human ILC2s expressed the LT receptor CysLT1, levels of which were increased in atopic subjects. CysLTs, particularly LTE4, induced migration, reduced apoptosis, and promoted cytokine production in human ILC2s in vitro. LTE4 enhanced the effect of PGD2, IL-25, IL-33, and TSLP, resulting in increased production of type 2 and other proinflammatory cytokines. The effect of LTE4 was inhibited by montelukast, a CysLT1 antagonist. Interestingly, addition of IL-2 to LTE4 and epithelial cytokines significantly amplified ILC2 activation and upregulated expression of the receptors for IL-33 and IL-25.

Conclusion: CysLTs, particularly LTE4, are important contributors to the triggering of human ILC2s in inflammatory responses, particularly when combined with other ILC2 activators.

Keywords: Group 2 innate lymphoid cell; IL-2; IL-25; IL-33; atopic dermatitis; leukotriene E(4); prostaglandin D(2); thymic stromal lymphopoietin.

Figures

Fig 1
Fig 1
Increased expression of CysLT1 by ILC2s in patients with atopic dermatitis (AD).A, mRNA for cysLT receptors in cultured ILC2s with quantitative RT-PCR. B, Expression of CysLT1 with flow cytometry. C and D, CysLT1+ ILC2s detected in PBMCs from healthy subjects and patients with AD (Fig 1, C) and cohort mean fluorescence intensity CysLT1 (Fig 1, D). FSC, Forward scatter. E, mRNA for CysLT1 in blood ILC2s. *P < .05 (Fig 1, A, n = 10; Fig 1, D, n = 4-8; Fig 1, E, n = 3).
Fig 2
Fig 2
Migration of cultured ILC2s in response to cysLTs. A, Migration of ILC2s to cysLTs or IL-33 examined with chemotaxis assays. B, Migration of ILC2s in response to LTE4, PGD2, IL-33, IL-25, and TSLP alone or in combination in the presence/absence of montelukast. *P < .05. Data in Fig 2, A, are representative of 4 experiments (Fig 2, B, n = 3).
Fig 3
Fig 3
Effect of cysLTs on apoptosis of cultured ILC2s. A, Annexin-V+ cells detected by using flow cytometry after IL-2 withdrawal in the presence/absence of cysLTs. B and C, Annexin-V+ cells at 18 hours after IL-2 withdrawal in the presence of LTC4(dashed line), LTD4(dotted line), or LTE4 (solid line; Fig 3, B) or LTE4/montelukast (Fig 3, C). *P < .05. Data in Fig 3, A, are representative of 3 experiments (Fig 3, B, n = 4; Fig 3, C, n = 3).
Fig 4
Fig 4
Effect of cysLTs and CysLT1 antagonist on IL-13 production in cultured ILC2s. A, IL-13 concentrations in cell supernatants measured with ELISA after treatment with LTC4(dashed line), LTD4(dotted line), or LTE4(solid line). B, IL-13 production by ILC2s induced by 100 nmol/L LTE4 in the presence of montelukast. Data in Fig 4, A, are representative of 5 experiments (Fig 4, B, n = 1). EC50, Half maximal effective concentration; IC50, half maximal inhibitory concentration.
Fig 5
Fig 5
LTE4 enhancement of effects of PGD2 and epithelial cytokines on cytokine production by cultured ILC2s. Cytokine concentrations after ILC2 treatment with LTE4 and PGD2 alone or in combination (A) or LTE4, IL-33, IL-25, and TSLP alone or in combination (B) in the presence (black bar)/absence (white bars) of montelukast. Cytokines were measured with the multiplex bead array. *P < .05 (n = 3).
Fig 6
Fig 6
Effect of IL-2 and other stimulators on cytokine and receptor expression in cultured ILC2s. Cytokine concentrations in supernatants (A) or mRNA levels of receptors in ILC2s (B) after treatment with combinations of LTE4, PGD2, IL-33, IL-25, and TSLP with or without montelukast in the presence (black bars)/absence (white bars) of IL-2 measured with the multiplex bead array (Fig 6, A) or quantitative RT-PCR (Fig 6, B). *P < .05 (n = 3-5).
Fig 7
Fig 7
Effect of endogenous cysLTs on cytokine production in cultured ILC2s. A, Levels of LTE4 in supernatants of mast cells treated with medium (white bar, ns) or IgE/anti-IgE with (gray bar, IgE+MK) or without (black bar, IgE) MK886. B and C, Protein (Fig 7, B) and mRNA (Fig 7, C) levels of cytokines of ILC2s after incubation with supernatants with or without montelukast for 4 hours. *P < .05 (n = 4).
Fig E1
Fig E1
ILC2 isolation. ILC2s isolated from human blood were lineage marker negative (CD3, CD14, CD19, CD56, CD11c, CD11b, FcεRI, TCRγδ, TCRα,β and CD123), CD45high, IL-7 receptor α–positive, and CRTH2+ cells. FSC, Forward scatter.
Fig E2
Fig E2
GPR99 is not expressed by human ILC2s and TH2 cells. The protein level of GPR99 in cell lysates was detected by using Western blotting. NIH3T3 cells were used as a positive control.
Fig E3
Fig E3
CysLTs do not affect proliferation of ILC2s in culture. Levels of Cell Trace Violet in ILC2s were measured with flow cytometry at day 4 after cell labeling with Cell Trace Violet and then incubation with 100 nmol/L LTC4, LTD4, or LTE4 (n = 3).
Fig E4
Fig E4
LTE4 synergistically enhanced gene transcription of type 2 cytokines induced by PGD2 in cultured human ILC2s. mRNA levels of type 2 cytokines in ILC2s after treatment with LTE4 and PGD2 alone or their combination for 4 hours were determined by using quantitative RT-PCR. *P < .05 (n = 3).
Fig E5
Fig E5
LTE4 and PGD2 enhanced the effects of epithelial cytokines on type 2 cytokine production by ILC2s ex vivo. A, IL-5+ ILC2s detected in fresh PBMCs after stimulation with IL-33 (50 ng/mL), IL-25 (50 ng/mL), and TSLP (50 ng/mL) or a combination of these epithelial cytokines with LTE4 (50 nmol/L) and PGD2 (100 nmol/L) by using intracellular staining. B, Proportion of type 2 cytokine (IL-5 and IL-13)–positive cells in gated LinCD45+ cells after indicated treatments. *P < .05. Data in Fig E5, A, are representative of 3 independent experiments (Fig E5, B, n = 3).
Fig E6
Fig E6
IL-2–enhanced cytokine production in ILC2s in response to LTE4, IL-33, IL-25, and their combinations. IL-13 concentrations in ILC2 culture after incubation with LTE4, IL-33, or IL-25 or their combinations in the presence (black bars) or absence (white bars) of IL-2 were measured with the multiplex bead array. *P < .05 (n = 3).
Fig E7
Fig E7
Effect of combination of IL-2 and other stimulators on gene expression of cytokines in ILC2 cells. mRNA levels of cytokines in cultured ILC2s after treatment with different combinations of LTE4, PGD2, IL-33, IL-25, and TSLP with or without montelukast in the presence (black bars) or absence (white bars) of IL-2 for 4 hours were measured with quantitative RT-PCR. *P < .05 (n = 3).
Fig E8
Fig E8
Relationship between IL1RL1 gene regulation and cytokine productions in ILC2s after various treatments was analyzed by using Spearman analysis.

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