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. 2015 Nov;8(6):1313-23.
doi: 10.1038/mi.2015.21. Epub 2015 Apr 8.

The prostaglandin D₂ receptor CRTH2 regulates accumulation of group 2 innate lymphoid cells in the inflamed lung

E D Tait Wojno  1   2 , L A Monticelli  1 , S V Tran  1 , T Alenghat  2 , L C Osborne  1 , J J Thome  3 , C Willis  4 , A Budelsky  4 , D L Farber  3   5 , D Artis  1
Affiliations

The prostaglandin D₂ receptor CRTH2 regulates accumulation of group 2 innate lymphoid cells in the inflamed lung

E D Tait Wojno et al. Mucosal Immunol. 2015 Nov.

Abstract

Group 2 innate lymphoid cells (ILC2s) promote type 2 cytokine-dependent immunity, inflammation, and tissue repair. Although epithelial cell-derived cytokines regulate ILC2 effector functions, the pathways that control the in vivo migration of ILC2s into inflamed tissues remain poorly understood. Here, we provide the first demonstration that expression of the prostaglandin D2 (PGD2) receptor CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) regulates the in vivo accumulation of ILC2s in the lung. Although a significant proportion of ILC2s isolated from healthy human peripheral blood expressed CRTH2, a smaller proportion of ILC2s isolated from nondiseased human lung expressed CRTH2, suggesting that dynamic regulation of CRTH2 expression might be associated with the migration of ILC2s into tissues. Consistent with this, murine ILC2s expressed CRTH2, migrated toward PGD2 in vitro, and accumulated in the lung in response to PGD2 in vivo. Furthermore, mice deficient in CRTH2 exhibited reduced ILC2 responses and inflammation in a murine model of helminth-induced pulmonary type 2 inflammation. Critically, adoptive transfer of CRTH2-sufficient ILC2s restored pulmonary inflammation in CRTH2-deficient mice. Together, these data identify a role for the PGD2-CRTH2 pathway in regulating the in vivo accumulation of ILC2s and the development of type 2 inflammation in the lung.

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Figures

Figure 1
Figure 1. Human ILC2s isolated from the peripheral blood are more likely to express CRTH2 than ILC2s isolated from the lung
CRTH2 expression by CD45+ lineage (CD3/CD5/CD14/CD16/CD19/CD56/FcεRIα) negative (lin−) CD127+IL-33R+ ILC2s in (a) PBMCs and (b) lung parenchyma from the same deceased adult organ donor (collected at the time of organ procurement for transplantation). Left plots gated on CD45+lin− cells. FMO, fluorescence minus one negative staining control. (c) Frequency of CRTH2+ cells of human CD45+lin−CD127+IL-33R+ ILC2s from the PBMCs and lung parenchyma isolated from the same deceased adult organ donor. (a–c) Blood n=8; lung n=8. Results are mean ± sem; *, P ≤ 0.5 using (c) a paired two-tailed students t-test.
Figure 2
Figure 2. Murine ILC2s express CRTH2 and accumulate in the lung in response to PGD2
Gpr44 RNA transcript expression by β2m+ lineage (CD3/CD4/ CD19/CD11b/CD11c/NK1.1) negative (lin−) CD90.2+CD127+IL-33R+ ILC2s in (a) PBMCs and (b) lung parenchyma from C57BL/6 WT mice. Left plots gated on β2m+lin− cells. FMO, fluorescence minus one negative (no probe) staining control. (c) Gpr44 and Actβ expression in cells sort-purified from rmIL-33-treated C57BL/6 WT mice. As a positive control and consistent with previous studies, lung eosinophils expressed Gpr44. (d) In vitro migration of spleen and lung ILC2s sort-purified from rmIL-33-treated C57BL/6 WT mice in response to PGD2. (e) Total numbers of ILC2s in the right (R) superior lung lobe of naïve C57BL/6 WT and Gpr44−/− mice treated i.n. with PGD2 for 3 days determined using flow cytometry (ILC2s are CD45+ lineage (CD3/CD4/CD5/CD19/CD11b/CD11c/NK1.1) negative CD90.2+CD25+CD127+IL-33R+). Data are representative of (a,b,e) 2–4 mice per group per experiment and 2 independent experiments or (c,d) ILC2s from 5 pooled mice per experiment and 5 independent experiments. Results are mean ± sem; *, P ≤ 0.05; **, P ≤ 0.01; using (a,b) a two-tailed students t-test or (e) a one-way ANOVA test and Bonferroni post-hoc testing.
Figure 3
Figure 3. CRTH2 mediates accumulation of ILC2s in the murine lung and pulmonary inflammation
C57BL/6 WT and Gpr44−/− mice infected with N. brasiliensis were examined at day 32 following infection (N, naive; Nb, N. brasiliensis). (a) Total numbers of ILC2s in the lung determined using flow cytometry (ILC2s are CD45+ lineage (CD3/CD4/CD5/CD19/CD11b/CD11c/NK1.1) negative CD90.2+CD25+CD127+IL-33R+). (b) H & E staining of lung sections. Scale bars = 1 mm (left) or 100 µm (right). (c) Pathology score based on histology. (d) Periodic acid schiff/Alcian Blue staining of lung sections depicting bronchiolar epithelium. Scale bars = 50 µm. (e) Real-time PCR analysis of gene expression in lung tissue. Total numbers of (f) CD4+ T cells and (g) CD11b+CD11cint macrophages determined using flow cytometry. Data are representative of 1–4 mice per group per experiment and 5 independent experiments. Results are mean ± sem; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 using (a,e–g) a one-way ANOVA test and Bonferroni post-hoc testing or (c) a two-tailed students t-test.
Figure 4
Figure 4. A CRTH2-specific inhibitor prevents accumulation of ILC2s in the murine lung and pulmonary inflammation following helminth infection
C57BL/6 WT mice treated with or without the CRTH2 inhibitor OC00459 infected with N. brasiliensis were examined at day 32 following infection (N, naive; Nb, N. brasiliensis). (a) Total numbers of ILC2s in the lung determined using flow cytometry (ILC2s are CD45+ lineage (CD3/CD4/CD5/CD19/CD11b/CD11c/NK1.1) negative CD90.2+CD25+CD127+IL-33R+). (b) H & E staining of lung sections. Scale bars = 1 mm (left) or 100 µm (right). (c) Pathology score based on histology. (d) Periodic acid schiff/Alcian Blue staining of lung sections depicting bronchiolar epithelium. Scale bars = 50 µm. (e) Real-time PCR analysis of gene expression in lung tissue. Total numbers of (f) CD4+ T cells and (g) CD11b+CD11cint macrophages determined using flow cytometry. Data are representative of 2–4 mice per group per experiment and 2 independent experiments. Results are mean ± sem; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 using (a,e–g) a one-way ANOVA test and Bonferroni post-hoc testing or (c) a two-tailed students t-test.
Figure 5
Figure 5. Hematopoietic expression of CRTH2 mediates ILC2 accumulation in the murine lung and pulmonary inflammation
CD45.1 C57BL/6 WT mice were lethally irradiated and reconstituted with CD45.2 C57BL/6 WT bone marrow (BM) (WT-WT BM mice), Gpr44−/− BM (WT-KO BM mice) or WT and Gpr44−/− BM at a 1:1 ratio (WT-50/50 BM mice). Chimeric mice were infected with N. brasiliensis and examined at day 32 following infection (N, naive; Nb, N. brasiliensis). (a) Total numbers of ILC2s in the lung determined using flow cytometry (ILC2s are CD45.2+ lineage (CD3/CD4/CD5/CD19/CD11b/ CD11c/NK1.1) negative (lin−) CD90.2+CD25+ CD127+ IL-33R+). (b) H & E staining of lung sections. Scale bars = 1 mm (left) or 100 µm (right). (c) Pathology score based on histology. (d) Periodic acid schiff/Alcian Blue staining of lung sections depicting bronchiolar epithelium. Scale bars = 50 µm. (e) Real-time PCR analysis of gene expression in lung tissue. Total numbers of (f) CD4+ T cells and (g) CD11b+CD11cint macrophages as determined using flow cytometry. (h) Representative flow cytometry plot showing the distribution of donor WT (red) and Gpr44−/− (blue) ILC2s (% of ILC2s) and (i) total numbers of donor ILC2s of WT and Gpr44−/− origin in the lung. Plot gated on CD45.2+lin−CD25+CD127+IL-33R+ ILC2s. Data are representative of 2–4 mice per group per experiment and 3 independent experiments. Results are mean ± sem; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 using (a,e) a one-way ANOVA test and Bonferroni post-hoc testing or (c,f–i) a two-tailed students t-test and (i) Welch’s correction for unequal variance.
Figure 6
Figure 6. CRTH2-expressing ILC2s mediate pulmonary inflammation in Gpr44−/− mice
C57BL/6 WT and Gpr44−/− mice infected with N. brasiliensis were examined at day 32 following infection (N, naive; Nb, N. brasiliensis). Some Gpr44−/− mice received pulmonary CD45.1+ Gpr44+/+ ILC2s intra-venously (i.v.) every week starting the day of infection. (a) Representative flow cytometry plot showing sort-purified donor CD45.1+ Gpr44-sufficient ILC2s. Plot gated on CD45.1+ lineage (CD3/CD4/CD5/CD19/CD11b/CD11c/NK1.1) negative (lin−) CD90.2+CD25+ cells, and percentage is ILC2s of CD45.1+ cells. (b) Representative flow cytometry plots showing donor CD45.1+ Gpr44-sufficient ILC2s in the lung of recipient Gpr44−/− mice at day 32 following N. brasiliensis infection. Left: gated on CD45.2−lin− cells; middle: gated on CD45.2−lin−CD45.1+CD90.2+CD25+ cells; right: gated on donor-derived ILC2s. (c) H & E staining of lung sections. Scale bars = 1 mm (left) or 100 µm (right). (d) Pathology score based on histology. (e) Periodic acid schiff/Alcian Blue staining of lung sections depicting bronchiolar epithelium. Scale bars = 50 µm. (f) Real-time PCR analysis of gene expression in lung tissue. Total numbers of (g) CD4+ T cells and (h) CD11b+CD11cint macrophages as determined using flow cytometry. Data are representative of 2–4 mice per group per experiment and 3 independent experiments. Results are mean ± sem; *, P ≤ 0.05; ***, P ≤ 0.001 using (d,f–h) a one-way ANOVA test and Bonferroni post-hoc testing.
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