Prostaglandin D2 activates group 2 innate lymphoid cells through chemoattractant receptor-homologous molecule expressed on TH2 cells

Abstract

Background: Activation of the group 2 innate lymphoid cell (ILC2) population leads to production of the classical type 2 cytokines, thus promoting type 2 immunity. Chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2), a receptor for prostaglandin D₂ (PGD₂), is expressed by human ILC2s. However, the function of CRTH2 in these cells is unclear.

Objectives: We sought to determine the role of PGD₂ and CRTH2 in human ILC2s and compare it with that of the established ILC2 activators IL-25 and IL-33.

Methods: The effects of PGD₂, IL-25, and IL-33 on the cell migration, cytokine production, gene regulation, and receptor expression of ILC2s were measured with chemotaxis, ELISA, Luminex, flow cytometry, quantitative RT-PCR, and QuantiGene assays. The effects of PGD₂ under physiologic conditions were evaluated by using the supernatant from activated mast cells.

Results: PGD₂ binding to CRTH2 induced ILC2 migration and production of type 2 cytokines and many other cytokines. ILC2 activation through CRTH2 also upregulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA). The effects of PGD₂ on ILC2s could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist.

Conclusions: PGD₂ is an important and potent activator of ILC2s through CRTH2 mediating strong proallergic inflammatory responses. Through IgE-mediated mast cell degranulation, these innate cells can also contribute to adaptive type 2 immunity; thus CRTH2 bridges the innate and adaptive pathways in human ILC2s.

Keywords: Group 2 innate lymphoid cell; IL-25; IL-33; PGD(2); adaptive type 2 immunity; chemoattractant receptor-homologous molecule expressed on T(H)2 cells; innate type 2 immunity.

Fig E1

Isotype controls for ILC2 isolation (Fig 1).

Fig E2

Comparison of Lin⁻CD127⁺CRTH2⁻ and Lin⁻CD127⁺CRTH2⁺ cells from human skin. A, Expression of ST2 (blue line) on CRTH2⁻ cells was much lower than expression on CRTH2⁺ cells. The red line shows unstained cells. B, Lin⁻CD127⁺CRTH2⁺(white columns) but not Lin⁻CD127⁺CRTH2^- cells (black columns) responded to IL-33 stimulation by IL-13 production (n = 2).

Fig E3

Expression of ST2, IL-17RA, and CRTH2 in ILC2s (skin) is regulated by PGD₂ in a dose-dependent manner. The mRNA level of ST2, CRTH2, IL-17RA, and IL-17RB in the cell pellets of ILC2s after stimulation with various concentrations of PGD₂ is shown. The mRNA levels in the cells treated with 1 nmol/L PGD₂ were treated as 1-fold (n = 2).

Fig E4

CRTH2 mediates proinflammatory cytokine production in ILC2s (skin) in response to supernatants from activated mast cells. Concentrations of IL-3, IL-8, IL-9, IL-21, GM-CSF, and CSF-1 in supernatants after ILC2 incubation with 1:1.5 diluted supernatants of mast cells treated with medium (white bars) or IgE/anti-IgE antibody with (black bars) or without (gray bars) diclofenac in the presence or absence of TM30089 for 3 hours. *P < .05 (n = 2).

Fig E5

Cytokine production by ILC2s (skin) in response to supernatants from activated mast cells is inhibited by CysLT₁ antagonist partially but not by D prostanoid receptor antagonist. The effects of TM30089, BWA868C, montelukast, and their combination on the production of IL-13 (protein), IL-3, and GM-CSF (mRNA) in ILC2s treated with the supernatant from IgE/anti-IgE-activated mast cells (gray bars) were examined with ELISA or quantitative RT-PCR (n = 1).

Fig 1

ILC2 isolation. ILC2s isolated from human skin were lineage marker–negative (CD3, CD4, CD8, CD14, CD19, CD56, CD11c, CD11 b, FcεRI, T-cell receptor γδ, T-cell receptor αβ, and CD123), CD45^high, IL-7Rα⁺, and CRTH2⁺. Isotype controls are shown in Fig E1.

Fig 2

Migration of ILC2s (A and B, skin; C, blood) to PGD₂ is mediated by CRTH2. Fig 2, A, Migration after stimulation with IL-25, IL-33, or PGD₂. Fig 2, B, Migration after exposure to PGD₂ in the absence or presence of TM30089. Fig 2, C, Migration in response to PGD₂, IL-33, or IL-25 alone or in combination with or without TM30089. *P < .05 (n = 3).

Fig 3

CRTH2 mediates type 2 cytokine production in ILC2s (skin) in response to PGD₂. A, mRNA levels of cytokines in cells (mRNA) and cytokine concentrations in supernatants (protein) after incubation with various concentration of PGD₂. B, Concentrations of cytokines released after treatments are as indicated. *P < .05 between control and other treatments and **P < .05 between PGD₂ and indicated treatments (n = 3).

Fig 4

Activation of CRTH2 evokes proinflammatory cytokine production in ILC2s (skin). A, Cytokine concentrations after stimulation with PGD₂. B, mRNA levels of cytokines (mRNA) and concentrations of cytokines (protein) after treatments, as indicated. C, mRNA level of IFN-γ after treatments. *P < .05 between control and other treatments and **P < .05 between PGD₂ and indicated treatments (n = 3).

Fig 5

Activation of CRTH2 modulates receptor expression in ILC2s (A, blood; B and C, skin). Fig 5, A, mRNA levels of receptor genes after treatments (n = 3). The expression of ST2 (Fig 5, B) and CRTH2 (Fig 5, C) in ILC2s after incubation with medium or PGD₂ with or without TM30089 for 4 (Fig 5, B) or 6 (Fig 5, C) hours, as determined by using fluorescence-activated cell sorting, is shown.

Fig 6

Effect of mast cell supernatants on activation of ILC2s (skin) is mediated by CRTH2. A, Levels of PGD₂ and IL-13 in supernatants of mast cells treated with medium (white bars) or IgE/anti-IgE antibody with (black bars) or without (gray bars) diclofenac. Supernatants were assigned as supernatants 1 to 3. B, ILC2 migration after exposure to supernatants with or without TM30089. C and D, mRNA and protein levels of cytokines (Fig 6, C) and mRNA levels of receptors (Fig 6, D) in ILCs after incubation with supernatants with or without TM30089 for 3 hours. *P < .05 (n = 2).