Prostaglandin D2 receptor 2 downstream signaling and modulation of type 2 innate lymphoid cells from patients with asthma

Abstract

Increased production of Prostaglandin D₂ (PGD₂) is linked to development and progression of asthma and allergy. PGD₂ is rapidly degraded to its metabolites, which initiate type 2 innate lymphoid cells (ILC2) migration and IL-5/IL-13 cytokine secretion in a PGD₂ receptor 2 (DP₂)-dependent manner. Blockade of DP₂ has shown therapeutic benefit in subsets of asthma patients. Cellular mechanisms of ILC2 activity in response to PGD₂ and its metabolites are still unclear. We hypothesized that ILC2 respond non-uniformly to PGD₂ metabolites. ILC2s were isolated from peripheral blood of patients with atopic asthma. ILC2s were stimulated with PGD₂ and four PGD₂ metabolites (Δ¹²-PGJ₂, Δ¹²-PGD₂, 15-deoxyΔ^12,14-PGD₂, 9α,11β-PGF₂) with or without the selective DP₂ antagonist fevipiprant. Total RNA was sequenced, and differentially expressed genes (DEG) were identified by DeSeq2. Differential gene expression analysis revealed an upregulation of pro-inflammatory DEGs in ILC2s stimulated with PGD₂ (14 DEGs), Δ¹²-PGD₂ (27 DEGs), 15-deoxyΔ^12,14-PGD₂ (56 DEGs) and Δ¹²-PGJ₂ (136 DEGs), but not with 9α,11β-PGF₂. Common upregulated DEGs were i.e. ARG2, SLC43A2, LAYN, IGFLR1, or EPHX2. Inhibition of DP₂ via fevipiprant mainly resulted in downregulation of pro-inflammatory genes such as DUSP4, SPRED2, DUSP6, ETV1, ASB2, CD38, ADGRG1, DDIT4, TRPM2, or CD69. DEGs were related to migration and various immune response-relevant pathways such as “chemokine (C-C motif) ligand 4 production”, “cell migration”, “interleukin-13 production”, “regulation of receptor signaling pathway via JAK-STAT”, or “lymphocyte apoptotic process”, underlining the pro-inflammatory effects of PGD₂ metabolite-induced immune responses in ILC2s as well as the anti-inflammatory effects of DP₂ inhibition via fevipiprant. Furthermore, PGD₂ and metabolites showed distinct profiles in ILC2 activation. Overall, these results expand our understanding of DP₂ initiated ILC2 activity.

Introduction

Approximately 262 million people worldwide were affected by asthma in 2019, leading to about 450,000 deaths per year with increasing tendency [1]. Asthma causes various clinical symptoms such as shortness of breath, chest tightness, wheezing and coughing [1]. There is still an enormous need of new drugs to treat particularly severe, uncontrolled asthmatic patients not responding sufficiently to current available treatment options [2,3]. The discovery of type 2 innate lymphoid cells (ILC2), which were found to be increased in the airways of severe asthmatics [4,5], led to a reassessment of the pathogenesis of allergic asthma. In addition, development and progression of asthma and allergy is linked to increased production of the ILC2 activating hormone prostaglandin D₂ (PGD₂) [6–9]. PGD₂ is mainly secreted by epithelial cells as well as mast cells and ILC2s, which thereby binds to the PGD₂ receptors 1 (DP₁) and 2 (DP₂, synonym: CRTH2) presumably expressed by Th2 cells, eosinophils and ILC2s [10–12]. While DP₁ signaling is predominantly associated with anti-inflammatory effects such as neuronal protection, inhibition of immune cell function and reduced cell migration [13], activation of DP₂ results in pro-inflammatory outcomes such as increased migration and type 2 cytokine secretion [11,14,15]. However, some mechanisms require cooperative signaling between DP₁ and DP₂, i.e., PGD₂ induced leukotriene C₄ synthesis in eosinophils [16]. Furthermore, thromboxane (TP) receptors [17,18] and peroxisome proliferator activated receptor gamma (PPAR-γ) [19] are also activated by PGD₂. Activation of TP results in smooth muscle contraction [18,20], while signaling via PPAR-γ promotes type 2 allergic responses in mice [21,22] and is involved in modulation of immune and inflammatory responses [23–25]. PPAR-γ receptors are expressed on ILC2s and are further increased following interleukin-33 (IL-33) stimulation [22,26], but there is no clear evidence to date showing the expression of TP receptors on ILC2s.

Within 120 minutes, more than 92% of PGD₂ is transformed either enzymatically or spontaneously to several PGD₂ metabolites such as Δ¹²-PGD₂, Δ¹²-PGJ₂, 15-deoxy-Δ^12,14-PGD₂, 15-deoxy-Δ^12,14-PGJ₂ and 9α,11β-PGF₂ [27,28]. While most of the PGD₂ metabolites have mainly pro-inflammatory effects [15,29], for 15-deoxy-Δ^12,14-PGJ₂ also anti-inflammatory properties were reported [30,31]. These PGD₂ metabolites have selective binding affinity to DP₂ over DP₁ and thus may play a role in inflammatory immune responses via DP₂ signaling [13,32]. We have previously investigated the effects of PGD₂ and its metabolites 13,14-dihydro-15-keto-PGD₂, Δ¹²-PGD₂, 15-deoxy-Δ^12,14-PGD₂, 9α,11β-PGF₂, PGJ_2, Δ¹²-PGJ₂ and 15-deoxy-Δ^12,14-PGJ₂ on ILC2 activity in presence or absence of the selective DP₂ antagonist fevipiprant demonstrating that all selected PGD₂ metabolites except 9α,11β-PGF₂ induced ILC2 migration in a DP₂-dependent manner with EC₅₀ values ranging from 17.4 to 91.7 nM [15]. Compared to PGD₂, ILC2 migration was enhanced in the presence of 15-deoxy-Δ^12,14-PGD₂, Δ¹²-PGJ₂ and 15-deoxy-Δ^12,14-PGJ₂. Additionally, we found that PGD₂ metabolites induce cytokine secretion of IL-5 and IL-13 by ILC2s in a DP₂-dependent manner, whereby 9α,11β-PGF₂ showed reduced potency compared to the other metabolites (IL5 range: 108.1 to 526.9 nM, IL-13 range: 125.2 to 788.3 nM). ILC2s stimulated with Δ¹²-PGD₂ or 15-deoxy-Δ^12,14-PGD₂ secreted numerical elevated levels of the type 2 cytokines IL-5 and IL-13 compared to PGD₂ stimulated ILC2s [15]. As the PGD₂ metabolites showed different potencies to activate ILC2s, these may lead to diverse biological consequences.

Furthermore, inhibition of the DP₂ signaling pathway may be a potential drug target for severe, uncontrolled asthmatic patients [3,33]. Fevipiprant is a potent and selective DP₂ antagonist, which as such is well-tolerated and has shown anti-inflammatory effects such as reduced eosinophilic airway inflammation and declined airway smooth muscle mass in phase 2 clinical trials with patients suffering from severe, uncontrolled asthma [34–36]. In none of the phase 3 studies conducted so far, fevipiprant treatment of patients with severe asthma resulted in statistically significant improved outcomes [37–39]. However, in two phase 3 clinical trials modest reductions of asthma exacerbation rates in the high fevipiprant dose group were observed [38]. In vitro experiments showed that fevipiprant inhibited cell aggregation, migration and cytokine secretion by PGD₂ or PGD₂ metabolites activated human ILC2s [15,40]. However, underlying cellular mechanisms of ILC2 activity via the DP₂ receptor in response to PGD₂ and PGD₂ metabolites are still unclear. We hypothesized that ILC2 respond non-uniformly to PGD₂ metabolites.

Therefore, transcriptomics of ILC2s isolated from blood of asthma patients and incubated with PGD₂ and four PGD₂ metabolites (Δ¹²-PGJ₂, Δ¹²-PGD₂, 15-deoxyΔ^12,14-PGD₂, 9α,11β-PGF₂) in presence or absence of the selective DP₂ antagonist fevipiprant were analyzed to further our understanding of DP₂ initiated ILC2 activity in asthma. These experiments follow and add to previously published in vitro experiments using the same patient material, which have shown that PGD₂ metabolites induce ILC2 migration and IL-5/IL-13 cytokine secretion in a DP₂-dependent manner [15].

Materials and methods

Study design

Four volunteers with mild intermittent atopic asthma (three male/ one female; average age: 28.3 ± 4.0 years; BMI: 19–32 kg/m², with a clinically manifest allergy against house dust mite, no corticosteroids for > four weeks) were enrolled into the study between 9^th November 2020 and 18^th February 2021. Whole blood (500 ml) was collected in 3.8% trisodium citrate and was processed within one hour after blood withdrawal. ILC2s were isolated from the peripheral blood, cultured and expanded. On the one hand, ILC2s were used for migration experiments towards PGD₂ and its metabolites with or without the selective DP₂ antagonist fevipiprant [15], and on the other hand, ILC2s were stimulated with PGD₂ and its metabolites with or without fevipiprant within the culture plates. Total RNA from the stimulated cells within the culture plates was sequenced for transcriptomic analysis (focus of this manuscript), and IL-5/ IL-13 cytokine concentrations were measured in the supernatant [15].

Ethics approval and consent to participate

The protocol was approved by the Ethics Committee of Hannover Medical School, Hannover, Germany under reference 839–2010. The study was conducted at the Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany in accordance with the Declaration of Helsinki and the International Council for Harmonisation (ICH) Harmonised Tripartite Guideline for Good Clinical Practice. Written informed consent was obtained from all participants after they were fully informed about all study aspects before any study-related procedures.

Reagents

PGD₂, Δ¹²-PGJ₂, Δ¹²-PGD₂, 15-deoxy-Δ^12,14-PGJ₂, 9α,11β-PGF₂ were purchased from Cayman Chemicals (Biomol GmBH, Hamburg, Germany). Fevipiprant (GST0000013789) was provided by Novartis Pharma AG (Basel, Switzerland). Reagents were dissolved in sterile-filtered Hybri-Max dimethylsulfoxide (DMSO, Sigma-Aldrich, Taufkirchen, Germany).

Type 2 innate lymphoid cell isolation and cell culture

Details of ILC2 isolation and cell culture as well as reagent information were previously described in detail elsewhere [15]. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from 500 mL peripheral blood of patients with atopic asthma (n = 4) using Ficoll density gradient centrifugation. ILC2 were enriched by depletion of T cells, B cells and monocytes using CD3-, CD14- and CD19-MACS separation beads (Miltenyi Biotech, Bergisch Gladbach, Germany), respectively. Cells were stained for 15 min at RT with a PerCP-Cy5.5-labeled lineage cocktail 1 (CD4, CD8, CD14, CD16, CD19, CD34, CD123, FcεRI), a FITC-labeled lineage cocktail 2 (CD11b, CD56), CD3-BV510, CD127-BV421, CD45-Alexa Fluor 700 and CD294-PE. For more information on flow cytometric antibodies view supplements of our previous publication [15]. Afterwards, ILC2 cells (CD45⁺, CD4^-, CD8^-, CD14^-, CD16^-, CD19^-, CD34^-, CD123^-, FcεRI^-, CD11b^-, CD56^-, CD3^-, CD127⁺, CD294⁺) were sorted with an FACS ARIA Fusion (BD Bioscience) into 96 U bottom well plates (100 cells/ well). The corresponding ILC2 gating strategy is shown in S1 Fig. Sorted cells were expanded with human feeder PBMCs (100,000/ well; 37°C, 5% CO₂) in culture medium (RPMI 1640 Glutamax medium, 1% Pen/Strep, 10% h.i. human AB serum, 25 mM HEPES, 100 U/ ml rh-IL-2, 25 ng/ ml rh-IL-4, and 5 μg/ ml phytohemagglutinin-M (PHA-M)) for three to five weeks.

Type 2 innate lymphoid metabolite incubation and sample processing

Cells (3–15 x10⁴/ well) were incubated with 1 μM fevipiprant for 1 h and afterwards with the EC₇₀ concentration of PGD₂ (EC₇₀ = 348.0 nM) or the selected PGD₂ metabolites Δ¹²-PGD₂ (EC₇₀ = 364.0 nM), Δ¹²-PGJ₂ (EC₇₀ = 759.0 nM), 15-deoxy-Δ^12,14-PGD₂ (EC₇₀ = 446.0 nM) or 9α,11β-PGF₂ (EC₇₀ = 520.6 nM) for 24 h (U-bottom 96-well plates, 37°C, 5% CO₂) in culture medium without IL-2, IL-4 and PHA-M. EC₇₀ concentrations of PGD₂ and metabolites were calculated based on preliminary titration experiments, where IL-5/IL-13 cytokine secretion of ILC2s in the presence of ascending PGD₂ and metabolite concentrations was investigated (results are shown in Fig 4 of our previous publication [15]). For fevipiprant a concentration of 1 μM was chosen, because metabolite-induced cytokine secretion was completely abolished at this concentration (results are shown in Fig 5 of our previous publication [15]). Afterwards, cell samples were centrifuged (5 min, 300 x g, RT), stabilized in RNAprotect Cell Reagent (Qiagen, Venlo, The Netherlands) and stored at -80°C until further handling. Total RNA was isolated from ILC2 using the Rneasy Plus Mini Kit (Qiagen, Venlo, The Netherlands) following the manufacturer’s protocol. Concentration and purity were determined in the isolated RNA solution via absorbance measurement using a NanoDrop device. Eluted RNA was stored at -80°C. For RNA-Sequencing (RNA-Seq) library preparation was performed using the NEBNext Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, Frankfurt am Main, Germany) following the manufacturer’s instructions, and all samples were sequenced paired-end and strand-specific on the NovaSeq 6000 (Illumina, San Diego, USA) with a sequencing-depth of 100 million reads per sample.

Processing of RNA-Seq raw data

Sequenced data were analyzed using the Galaxy web platform (usegalaxy.eu) [41]. Default settings were used for the tool applications, unless otherwise mentioned. For quality control FastQC Galaxy Version 0.72 was applied to the raw data and reports were checked for “per base sequence quality”, “overrepresented sequences” and “adapter content” [42]. Data with poor quality in “per base sequence quality” or “adapter content” were excluded from further analysis. Data with “overrepresented sequences” were trimmed via fastp Galaxy Version 0.20.1 [43] and checked again for quality using FastQC. Reads were mapped to the human GRCh38 reference genome (https://www.gencodegenes.org/human/releases.html) using the Gencode main annotation file via RNA Star Galaxy Version 2.7.8a [44]. From the output with mapped sequences, the number of reads per annotated genes were determined using FeatureCounts Galaxy Version 2.0.1 [45]. Differential expressed gene (DEG) analysis was performed using R (Version R 4.3.1). To remove unwanted variation the control gene method RUVSeq Version 1.34.0 was applied to raw read counts [46]. Using DESeq2 Version 1.40.2 counts were normalized, PCA-Plots were created, and differential expression was calculated using paired sample analysis [47]. Adjusted (adj.) p-values were calculated for multiple testing by DESeq2 using the Benjamini-Hochberg procedure which controls false discovery rate (FDR). Gene names were determined in the manuscript based on ENSEMBL release 103 (February 2021) [48]. DEGs were defined by the following criteria: adjusted p-value ≤ 0.05, BaseMean ≥ 5, and Log2 fold change (|Log2FC|) ≥ 0.58 for deregulated genes.

In addition, genes associated with the PPAR-γ signaling pathway according to the KEGG database (https://www.genome.jp/pathway/hsa03320) were examined for DEGs that were induced by ILC2 stimulation with PGD₂ or its metabolites compared to unstimulated ILC2s (DEGs were defined as described above; results are shown in S1 Table).

Visualization of processed RNA-Seq data

Data were visualized using GraphPad Prism 9.0.1. Gene set enrichment analysis was performed with differentially expressed genes using g:Profiler (Version 2019) to determine enriched pathways (KEGG_pathways), and biological processes (GOTERM_BP_DIRECT) [49]. Adj. p-values were calculated for multiple testing by g:Profiler using the Benjamini-Hochberg procedure which controls FDR. Significantly enriched pathways showed an adj. p-value ≤ 0.05. Selected immune response-relevant enriched pathways were visualized using the R package ggplot2 Version 3.4.4 (entire dataset is given in the supplements).

Results

Type 2 immune response- as well as migration-related genes were upregulated in ILC2s stimulated with PGD₂, Δ¹²PGD₂, Δ¹²PGJ₂ and 15-deoxyΔ^12,14-PGD₂, but not in 9α,11β-PGF₂ stimulated ILC2s

Differential gene expression analysis resulted in 14 differentially expressed genes (DEGs) for PGD₂-, 27 DEGs for Δ¹²-PGD₂-, 136 DEGs for Δ¹²-PGJ₂- and 56 DEGs for 15-deoxy-Δ^12,14-PGD₂-, and none DEGs for 9α,11β-PGF₂-stimulated cells, when compared to unstimulated cells (Fig 1). Common upregulated genes were ARG2, SLC43A2, LAYN, IGFLR1 and EPHX2. Inhibition of the DP₂ receptor via fevipiprant and following stimulation with PGD₂ or metabolites resulted in 97 DEGs for PGD₂-, 109 DEGs for Δ¹²-PGD₂-, 116 DEGs for Δ¹²-PGJ₂-, 46 DEGs for 15-deoxy-Δ^12,14-PGD₂-, and 256 DEGs for 9α,11β-PGF₂-stimulaed ILC2s compared to stimulated ILC2 without fevipiprant (Fig 2). Genes were mainly downregulated including common genes such as DUSP4, SPRED2, DUSP6, ETV1, ASB2, CD38, ADGRG1, DDIT4, TRPM2 and CD69. Detailed results for each gene of all samples are provided in S2 and S3 Tables, including calculated log2 Fold change (log2FC) with corresponding adjusted p-values. As PGD₂ and its metabolites 15-deoxy-Δ^12,14-PGJ₂ and Δ¹²-PGJ₂ from the J-series can bind and activate the PPAR-γ receptor [19,21,50], we have additionally investigated the expression of genes that are related to the PPAR-γ signaling pathway. However, none of the PPAR-γ related genes were significantly differentially expressed in PGD₂ or metabolite stimulated ILC2s compared with unstimulated ILC2s (S1 Table).

[Figure omitted. See PDF.]

DEG analysis of ILC2s stimulated with (a) PGD₂, (b) Δ¹²-PGD₂, (c) Δ¹²-PGJ₂, (d) 15-deoxy-Δ^12,14-PGD₂ and (e) 9α,11β-PGF₂ versus unstimulated ILC2s. DEGs are highlighted in red for upregulated genes (adj. p-value ≤ 0.05, log2FC ≥ 0.58, BaseMean ≥ 5) and in blue for downregulated genes (adj. p-value ≤ 0.05, log2FC ≤ -0.58, BaseMean ≥ 5).

[Figure omitted. See PDF.]

DEG analysis of ILC2s incubated with fevipiprant versus ILC2s stimulated with (a) PGD₂, (b) Δ¹²-PGD₂, (c) Δ¹²-PGJ₂, (d) 15-deoxy-Δ^12,14-PGD₂ and (e) 9α,11β-PGF₂. DEGs are highlighted in red for upregulated genes (adj. p-value ≤ 0.05, log2FC ≥ 0.58, BaseMean ≥ 5) and in blue for downregulated genes (adj. p-value ≤ 0.05, log2FC ≤ -0.58, BaseMean ≥ 5).

PGD₂ and its metabolites show distinct profiles in ILC2 activation

Gene set enrichment analysis with identified DEGs (|log2FC| ≥ 0.58, adj. p-value ≤ 0.05) revealed multiple pathways that play a role in immune responses (main results in Fig 3, all results in S4 and S5 Tables). Unexpectedly, “cell migration” and “cell motility” pathways were only enhanced in Δ¹²-PGJ₂ stimulated ILC2. “Chemokine production” including C-C Motif Chemokine Ligand 4 (CCL4), CCL5, IL-13 as well as IL-17 production was elevated in ILC2s stimulated with in particular PGD₂ and 15-deoxy-Δ^12,14-PGD₂. ILC2s activated with Δ¹²-PGD₂ showed an upregulation of genes associated with the “regulation of receptor signaling pathway via JAK-STAT” and “regulation of eosinophil differentiation”. In addition, identified DEGs of PGD₂, Δ¹²-PGD₂, and 15-deoxy-Δ^12,14-PGD₂, but not Δ¹²-PGJ₂ or 9α,11β-PGF₂ stimulated cells contributed to regulation of lymphocyte and mononuclear cell proliferation. Furthermore, gene set enrichment analysis revealed upregulation of apoptosis-relevant pathways in ILCs stimulated with PGD₂, Δ¹²-PGD₂, Δ¹²-PGJ₂ and 15-deoxy-Δ^12,14-PGD₂, but not with 9α,11β-PGF₂. Only Δ¹²-PGJ₂ stimulated ILC2 showed an upregulation of the “adenylate cyclase-inhibiting adrenergic receptor signaling pathway” as well as ferroptosis related pathways. None DEGs were identified in 9α,11β-PGF₂ stimulated cells, and therefore no 9α,11β-PGF₂ regulated pathways could be found (Fig 3). As expected based on in vitro experiments [15], pre-incubation of ILC2 with the selective DP₂ antagonist fevipiprant followed by stimulation with PGD₂, Δ¹²-PGD₂, Δ¹²-PGJ₂ or 15-deoxy-Δ^12,14-PGD₂ attenuated the “immune response” (Fig 4). This included various pathways regarding “cell migration”, “cytokine production” (e.g., IL-13, CCL4, CCL5), signaling cascades (e.g., “MAPK cascade”, “ERK1 and ERK2 cascade”), as well as regulation of cell activation, proliferation, differentiation, phagocytosis, and death (Fig 4). In contrast, ILC2 incubation with fevipiprant prior to 9α,11β-PGF₂ stimulation resulted in downregulation of only some general immune response-related pathways such as “cell communication” while “cell migration/ adhesion” were upregulated in these cells.

[Figure omitted. See PDF.]

[Figure omitted. See PDF.]

In addition to DEG and gene set enrichment analysis, principal component analysis (PCA) was performed using all ILC2 samples (unstimulated ILC2s, ILC2 ± PGD₂/metabolites ± fevipiprant). Unexpectedly, the samples were not clustered by groups (unstimulated vs. metabolites vs. fevipiprant), but by participants (S2 Fig). The top three genes of the principal component 1 (PC1) were TRDV3, ITGAD and ERAP2, while PC2 was dominated by expression of the top three genes TRDV2, RPS4Y1 and SELL. However, principal component analysis of ILC2s sorted by the four individual participants resulted in group-specific clustering (S3 Fig).

Discussion

We have previously shown that PGD₂ metabolites induce eosinophil shape change, ILC2 cell migration and type 2 cytokine secretion via the DP₂ receptor [15]. However, the corresponding underlying cellular mechanisms of DP₂ and potential differences between PGD₂- and PGD₂ metabolites-induced ILC2 activity are still unclear.

As expected, and in accordance with our previous findings [15], ILC2 stimulation with PGD₂ or PGD₂ metabolites (except 9α,11β-PGF₂) resulted in upregulation of pro-inflammatory genes. ILC2 stimulation resulted in different numbers of DEG depending on the used metabolite, which could indicate that PGD₂ and its metabolites may have different biological purposes in ILC2 activation. In contrast to the other examined metabolites, stimulation with 9α,11β-PGF₂ did not induce DEGs. Therefore, it is possible that 9α,11β-PGF₂ is a degradation product. On the other hand, the used 9α,11β-PGF₂ concentrations may not have been sufficient to compensate the 130 fold reduced DP₂ binding affinity of 9α,11β-PGF₂ compared to PGD₂ [32]. Compared with the other metabolites, stimulation with Δ¹²-PGJ₂ led to the highest DEG number (136 DEGs) followed by 15-deoxy-Δ^12,14-PGD₂ (56 DEG), Δ¹²-PGD₂ (27 DEGs) and then PGD₂ (14 DEGs). Common upregulated genes were e.g. important for T cell immunobiology (ARG2 [51], SLC43A2 [52], IGFLR1 [53]), involved in cell migration (LAYN [54]) or known to be involved in cigarette smoke-induced pulmonary inflammation and autophagy in mice (EPHX2 [55]). Some upregulated genes of this study were previously identified as potential targets for cancer therapeutics (LAYN, IGFLR1 [56]). As LAYN and IGFLR1 appear to play a role in the immune response of asthmatic patient-derived ILC2s, these genes could also have the potential to be valuable targets for the treatment of asthma. DP₂ inhibition via fevipiprant led to downregulation of in particular migration-related genes (DUSP6 [57], ETV1 [58], ASB2 [59,60], CD38 [61], ADGRG1 [62], DDIT4 [63], TRPM2 [64]). Furthermore, genes that are involved in the MAPK- and ERK signaling pathway (DUSP4 [65], SPRED2 [66]) or known to play a role in pathogenesis of allergic airway inflammation (CD69 [67]) were downregulated by DP₂ inhibition.

The prominent pro-inflammatory effects of PGD₂ metabolite stimulation as well as anti-inflammatory effects of the selective DP₂ antagonist fevipiprant were also mirrored by gene set enrichment analysis. With the exception of stimulation with 9α,11β-PGF₂, immune response-related pathways were generally upregulated in PGD₂ or metabolite stimulated ILC2s, while DP₂ inhibition via fevipiprant led to downregulation of these pathways. However, PGD₂ and its metabolites showed variabilities in their potential to activate pro-inflammatory pathways. Metabolites of the D-series (PGD₂, Δ¹²-PGD₂, 15-deoxy-Δ^12,14-PGD₂) have shown a comparable activation pattern in ILC2s including production of different cytokines, regulation of cell proliferation and apoptotic processes. Thereby 15-deoxy-Δ^12,14-PGD₂ induced an enhanced, while Δ¹²-PGD₂ induced an attenuated immune response in ILC2s compared to the origin molecule PGD₂. In contrast, ILC2 stimulation with Δ¹²-PGJ₂ primarily induced “cell motility/migration” and “Ferroptosis”-related pathways such as “iron ion transport”, “NRF2 pathway” or “KEAP-NFE2L2 pathway”. The reason why only Δ¹²-PGJ₂ was able to induce ferroptosis-related pathways in ILC2s, although all metabolites bind to the same receptor DP_2, remains speculative.

The different biological responses of ILC2s to stimulation with PGD₂ or its metabolites could be attributed to additional interactions with other relevant receptors. PGD₂ has been shown to activate DP₁ [13] and TP receptors [17,18]. However, as DP₁ is only expressed at low levels [12] and TP has not yet been clearly reported to be expressed on ILC2 cells, it is unlikely that these signaling pathways are involved in ILC2 activation. Another potentially relevant receptor is PPAR-γ, which can be activated by PGD₂ [19] or its metabolites Δ¹²-PGJ₂ and 15-deoxy-Δ^12,14-PGJ₂ from the J-series [19,21,50]. To the best of our knowledge, there is no literature elucidating effects of PGD₂ metabolites from the D-series on the PPAR-γ receptor. Therefore, the different activation pattern of Δ¹²-PGJ₂ compared to the metabolites from the D- and F-series, may at least be partly attributed to signaling via PPAR-γ. However, genes that are related to the PPAR-γ signaling pathway were not differentially expressed in ILC2s stimulated with Δ¹²-PGJ₂ or other metabolites compared to unstimulated ILC2s (S1 Table). To fully clarify whether the PPAR-γ signaling pathway is involved in ILC2 activation in response to PGD₂ or its metabolites, experiments with a PPAR-γ antagonist such as GW9662 would need to be performed. However, as inhibition of DP₂ via fevipiprant showed powerful inhibitory effects, we assume that ILC2 activation via DP₂ is the most prominent signaling pathway.

Overall, the observations described in this publication are consistent with our previously described in vitro experiments from the same study, where cell migration was reported to be strongest for the J-series metabolites, cytokine secretion was strongest for the D-series metabolites and 9α,11β-PGF₂ only showed barely effects [15]. In addition to the previously performed experiments, transcriptomic analysis revealed new relevant pathways which need to be confirmed in vitro and in vivo.

Interestingly, the inhibitory effects of fevipiprant seemed to be more prominent in cells activated with metabolites from the D-series (PGD₂, Δ¹²-PGD₂, 15-deoxy-Δ^12,14-PGD₂). Since ILC2 stimulation with 9α,11β-PGF₂ did not induce changes in the transcriptomic profile compared to unstimulated cells, the lack of down-regulated pathways after fevipiprant incubation was expected. On the contrary, pathways such as “cell communication, “cell migration” or “cell adhesion” were upregulated in 9α,11β-PGF₂ stimulated cells that were preincubated with fevipiprant. The reason for this remains unclear.

PGD₂ and metabolites bind DP₂ with higher affinity than DP₁ and in the following rank order of potency: PGD₂ (K_i = 2.4) > Δ¹²-PGJ₂ (K_i = 6.8) > Δ¹²-PGD₂ (K_i = 7.63) >>> 9α,11β-PGF₂ (K_i = 315.0) (unknown K_i for 15-deoxy-Δ^12,14-PGD₂) [32,68]. As outlined above, the low DP₂ affinity of 9α,11β-PGF₂ could therefore explain the reduced ability of this metabolite to activate ILC2s. However, the binding affinity of the other metabolites is not in line with the potency to induce DEGs in ILC2s, where the rank order was Δ¹²-PGJ₂ > 15-deoxy-Δ^12,14-PGD₂ > Δ¹²-PGD₂ > PGD₂ > 9α,11β-PGF₂, and therefore does not explain the different ILC2 activation patterns. Another reason for the different potencies of PGD₂ and its metabolites to activate ILC2s could be their local distributions in human tissue. However, the current knowledge about PGD₂ metabolism in human tissue is only present to a limited extend. In plasma, PGD₂ is presumably degraded into Δ¹²-PGJ₂ and Δ¹²-PGD₂, while only small amounts of 15-deoxy-Δ^12,14-PGD₂ are formed [27]. Increased 9α,11β-PGF₂ could be measured in plasma and urine of asthmatic patients following allergen challenge [69]. PGD₂ was found in BAL [9] and in induced sputum [70], but up to date there is no additional information related to its metabolites in these compartments. However, it is known that activated ILC2s migrate from blood into the airways of asthmatics following allergen challenge [5], and secrete endogenous PGD₂ [12]. Due to the known presence of ILC2s and PGD₂ in the airways of asthmatics, it can be hypothesized that PGD₂ metabolites should also be present and therefore may contribute to activation and recruiting of ILC2s via DP₂ signaling.

Unexpectedly, principal component analysis of all samples resulted in participant-specific rather than treatment group-specific clustering. Additional analysis revealed that these clusters are dominated by gene expression of the T Cell Receptor Delta Variable 2 and 3 (TRDV2/ TRDV3), which are both used by T cells for gene rearrangement in order to generate a highly diverse T cell receptor (TCR) repertoire for broad antigen recognition [71]. Interestingly, rearranged TCR genes in ILC2s are aberrant and nonfunctional, leading to the theory that ILC2s arise from failed T cell development [72,73]. Our data emphasize the close relationship of ILC2s to T cells and suggest that there may be subsets of ILC2s based on the expression profile of TRDV genes. Further characteristic PC genes were ITGAD, RPS4Y1 and SELL, which are interestingly all involved in cell migration [74–76]. This could indicate that ILC2 subgroups exist that differ in their migration behaviour.

This study carries limitations. First, we have analyzed biological samples from only four participants. This is not uncommon when analyzing sequencing data [77], but might not be sufficient to draw generalized conclusions about DP₂ dependent ILC2 activity especially because principal component analysis revealed high subject-related dominance. However, with four biological replicates for each experimental group, a sequencing depth of 100 million reads per sample and the described processing of the RNA-Seq raw data, our experimental design is in line with general recommendations for RNA-Seq [78]. Second, ILC2s were isolated from blood from asthmatic patients. Because there are no data with ILC2s obtained from healthy participants, it remains unclear whether the described effects of the metabolites and DP₂ inhibition are specific for asthmatic derived ILC2s or characteristic for ILC2s in general. Regardless, since ILC2s were reported to be increased in the airways of asthmatics [4,5] and are known to be recruited from the blood into the human airways upon allergen challenge [5], we believe that our findings are relevant for a deeper understanding of this disease. Finally, it should be mentioned that fevipiprant is no longer being developed for the treatment of asthma. However, blockade with fevipiprant in the experiments shown here confirmed the DP₂ dependence of ILC2 activities and provides a deeper understanding of the DP₂ signaling pathway. Although fevipiprant is no longer studied in clinical trials, DP₂ antagonism is still a valid treatment strategy, so our findings could be of relevance to other DP₂ antagonists [79,80].

Conclusions

ILC2 metabolite stimulation led to up-, while DP₂ inhibition led to downregulation of migration-related genes, which expression may contribute to ILC2 migration. Other DEGs and corresponding pathway analysis were related to e.g., T-cell immunobiology, pulmonary inflammation or pro-inflammatory signaling pathways and therefore could promote inflammation. Overall, our results expand our understanding of DP₂ initiated ILC2 activity.

Supporting information

S1 Fig. Gating strategy for ILC2 sorting.

After depletion of CD3-, CD14- and CD19-positive cells from PBMCs via magnetic activated cell separation (MACS), cells were stained with a PerCP-Cy5.5-labeled lineage cocktail 1 (CD4, CD8, CD14, CD16, CD19, CD34, CD123, FcεRI), a FITC-labeled lineage cocktail 2 (CD11b, CD56), CD3-BV510, CD127-BV421, CRTH2-PE and CD45-Alexa Fluor 700. (a) Lymphoid cells were gated by cell size (FSC-A) and cell granularity (SSC-A), and (b) discriminated from doublets (clotted cells) by determination of the time of flight that each cells needed to pass the filter (SSC-W). For further gating, (d, f, g, i, j) gates were set according to FMO controls. (c) CD45^high, lineage1^-, (e) lineage2^-, CD3^-, (h) CD127⁺ and CRTH2⁺ cells were defined as ILC2 and sorted into 96 U buttom well plates containing 100 μL supplemented medium. Gate boundaries were not altered between participants.

https://doi.org/10.1371/journal.pone.0307750.s001

(TIF)

S2 Fig. Principal component analysis of sequenced ILC2s (unstimulated = blue, metabolites = green, fevipiprant = red).

https://doi.org/10.1371/journal.pone.0307750.s002

(TIF)

S3 Fig. Principal component analysis, depicting clustering of sequenced ILC2s (unstimulated = blue, metabolites = green, fevipiprant = red) sorted by the four ILC2 donors (a-d = subject 1–4).

https://doi.org/10.1371/journal.pone.0307750.s003

(TIF)

S1 Table. Differential gene expression analysis of genes that are related to the PPAR-γ signaling pathway using DESeq2 in PGD2 or PGD2 metabolite stimulated ILC2s.

https://doi.org/10.1371/journal.pone.0307750.s004

(XLSX)

S2 Table. Differential gene expression analysis using DESeq2 in ILC2s stimulated with PGD2 or PGD2 metabolites versus unstimulated ILC2s.

https://doi.org/10.1371/journal.pone.0307750.s005

(XLSX)

S3 Table. Differential gene expression analysis using DESeq2 in ILC2s incubated with fevipiprant versus ILC2s stimulated with PGD2 or PGD2 metabolites.

https://doi.org/10.1371/journal.pone.0307750.s006

(XLSX)

S4 Table. Gene set enrichment analysis with identified DEGs (|log2FC| ≥ 0.58, adj. p-value ≤ 0.05) of metabolite activated ILC2s versus unstimulated ILC2s using g:Profiler.

https://doi.org/10.1371/journal.pone.0307750.s007

(XLSX)

S5 Table. Gene set enrichment analysis with identified DEGs (|log2FC| ≥ 0.58, adj. p-value ≤ 0.05) of ILC2s incubated with fevipiprant versus ILC2s stimulated with PGD2 or PGD2 metabolites using g:Profiler.

https://doi.org/10.1371/journal.pone.0307750.s008

(XLSX)

Acknowledgments

The authors would like to thank the clinical staff of Fraunhofer ITEM for subject recruitment and are grateful to the study participants for their involvement in the study. Furthermore, the authors would like to thank the Helmholtz Centre for Infection Research for carrying out the sequencing.

Citation: Gress C, Fuchs M, Carstensen-Aurèche S, Müller M, Hohlfeld JM (2024) Prostaglandin D2 receptor 2 downstream signaling and modulation of type 2 innate lymphoid cells from patients with asthma. PLoS ONE 19(7): e0307750. https://doi.org/10.1371/journal.pone.0307750

About the Authors:

Christina Gress

Roles: Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing

Affiliations: Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany, German Center for Lung Research (DZL-BREATH), Hannover, Germany

Maximilian Fuchs

Roles: Formal analysis, Visualization

Affiliation: Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany

Saskia Carstensen-Aurèche

Roles: Investigation, Writing – review & editing

Affiliations: Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany, German Center for Lung Research (DZL-BREATH), Hannover, Germany

Meike Müller

Contributed equally to this work with: Meike Müller, Jens M. Hohlfeld

Roles: Conceptualization, Supervision, Writing – review & editing

E-mail: [email protected] (JMH); [email protected] (MM)

Affiliations: Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany, German Center for Lung Research (DZL-BREATH), Hannover, Germany

Jens M. Hohlfeld

Contributed equally to this work with: Meike Müller, Jens M. Hohlfeld

Roles: Conceptualization, Funding acquisition, Supervision

E-mail: [email protected] (JMH); [email protected] (MM)

Affiliations: Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany, German Center for Lung Research (DZL-BREATH), Hannover, Germany, Department of Respiratory Medicine and Infectious Disease, Hannover Medical School, Hannover, Germany

ORICD: https://orcid.org/0000-0003-2646-6186

References

1. The Global Asthma Network. The Global Asthma Report 2022. Int J Tuberc Lung Dis. 2022;26:1–104. pmid:36303302

2. Peters SP, Ferguson G, Deniz Y, Reisner C. Uncontrolled asthma: a review of the prevalence, disease burden and options for treatment. Respir Med. 2006;100:1139–51. pmid:16713224

3. Corren J. New Targeted Therapies for Uncontrolled Asthma. J Allergy Clin Immunol Pract. 2019;7:1394–403. pmid:31076057

4. Smith SG, Chen R, Kjarsgaard M, Huang C, Oliveria J-P, O’Byrne PM, et al. Increased numbers of activated group 2 innate lymphoid cells in the airways of patients with severe asthma and persistent airway eosinophilia. J Allergy Clin Immunol. 2016;137:75–86.e8. pmid:26194544

5. Winkler C, Hochdörfer T, Israelsson E, Hasselberg A, Cavallin A, Thörn K, et al. Activation of group 2 innate lymphoid cells after allergen challenge in asthmatic patients. J Allergy Clin Immunol. 2019;144:61–69.e7. pmid:30731124

6. Kupczyk M, Kuna P. Targeting the PGD2/CRTH2/DP1 Signaling Pathway in Asthma and Allergic Disease: Current Status and Future Perspectives. Drugs. 2017;77:1281–94. pmid:28612233

7. Domingo C, Palomares O, Sandham DA, Erpenbeck VJ, Altman P. The prostaglandin D2 receptor 2 pathway in asthma: a key player in airway inflammation. Respir Res. 2018;19:189. pmid:30268119

8. Liu MC, Hubbard WC, Proud D, Stealey BA, Galli SJ, Kagey-Sobotka A, et al. Immediate and late inflammatory responses to ragweed antigen challenge of the peripheral airways in allergic asthmatics. Cellular, mediator, and permeability changes. Am Rev Respir Dis. 1991;144:51–8. pmid:2064141

9. Fajt ML, Balzar S, Trudeau JB, Westcott JY, Wenzel SE. Elevated Prostaglandin D2 (PGD2) Levels in Bronchoalveolar Lavage Fluid (BALF) in Severe Asthma. Journal of Allergy and Clinical Immunology. 2010;125:AB43.

10. Athari SS. Targeting cell signaling in allergic asthma. Signal Transduct Target Ther. 2019;4:45. pmid:31637021

11. Mjösberg JM, Trifari S, Crellin NK, Peters CP, van Drunen CM, Piet B, et al. Human IL-25- and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161. Nat Immunol. 2011;12:1055–62. pmid:21909091

12. Maric J, Ravindran A, Mazzurana L, van Acker A, Rao A, Kokkinou E, et al. Cytokine-induced endogenous production of prostaglandin D2 is essential for human group 2 innate lymphoid cell activation. J Allergy Clin Immunol. 2019;143:2202–2214.e5. pmid:30578872

13. Kostenis E, Ulven T. Emerging roles of DP and CRTH2 in allergic inflammation. Trends Mol Med. 2006;12:148–58. pmid:16545607

14. Hirai H, Tanaka K, Yoshie O, Ogawa K, Kenmotsu K, Takamori Y, et al. Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2. J Exp Med. 2001;193:255–61. pmid:11208866

15. Carstensen S, Gress C, Erpenbeck VJ, Kazani SD, Hohlfeld JM, Sandham DA, et al. Prostaglandin D2 metabolites activate asthmatic patient-derived type 2 innate lymphoid cells and eosinophils via the DP2 receptor. Respir Res. 2021;22:262. pmid:34620168

16. Mesquita-Santos FP, Bakker-Abreu I, Luna-Gomes T, Bozza PT, Diaz BL, Bandeira-Melo C. Co-operative signalling through DP(1) and DP(2) prostanoid receptors is required to enhance leukotriene C(4) synthesis induced by prostaglandin D(2) in eosinophils. Br J Pharmacol. 2011;162:1674–85. pmid:20973774

17. Maher SA, Birrell MA, Adcock JJ, Wortley MA, Dubuis ED, Bonvini SJ, et al. Prostaglandin D2 and the role of the DP1, DP2 and TP receptors in the control of airway reflex events. Eur Respir J. 2015;45:1108–18. pmid:25323233

18. Beasley RC, Featherstone RL, Church MK, Rafferty P, Varley JG, Harris A, et al. Effect of a thromboxane receptor antagonist on PGD2- and allergen-induced bronchoconstriction. J Appl Physiol (1985). 1989;66:1685–93. pmid:2525122

19. Harris SG, Phipps RP. Prostaglandin D(2), its metabolite 15-d-PGJ(2), and peroxisome proliferator activated receptor-gamma agonists induce apoptosis in transformed, but not normal, human T lineage cells. Immunology. 2002;105:23–34. pmid:11849312

20. al Jarad N, Hui KP, Barnes N. Effects of a thromboxane receptor antagonist on prostaglandin D2 and histamine induced bronchoconstriction in man. Br J Clin Pharmacol. 1994;37:97–100. pmid:8148229

21. Chen T, Tibbitt CA, Feng X, Stark JM, Rohrbeck L, Rausch L, et al. PPAR-γ promotes type 2 immune responses in allergy and nematode infection. Sci Immunol. 2017;2:5196. pmid:28783701

22. Xiao Q, He J, Lei A, Xu H, Zhang L, Zhou P, et al. PPARγ enhances ILC2 function during allergic airway inflammation via transcription regulation of ST2. Mucosal Immunol. 2021;14:468–78. pmid:32811992

23. Zhang X, Young HA. PPAR and immune system—what do we know? Int Immunopharmacol. 2002;2:1029–44. pmid:12349941

24. Kobayashi Y, Ueki S, Mahemuti G, Chiba T, Oyamada H, Saito N, et al. Physiological levels of 15-deoxy-Delta12,14-prostaglandin J2 prime eotaxin-induced chemotaxis on human eosinophils through peroxisome proliferator-activated receptor-gamma ligation. J Immunol. 2005;175:5744–50. pmid:16237065

25. Ercolano G, Gomez-Cadena A, Dumauthioz N, Vanoni G, Kreutzfeldt M, Wyss T, et al. PPARɣ drives IL-33-dependent ILC2 pro-tumoral functions. Nat Commun. 2021;12:2538. pmid:33953160

26. McSorley HJ, Arthur JSC. The devil’s in the detail: cell-specific role of PPARγ in ILC2 activation by IL-33. Mucosal Immunol. 2021;14:544–6. pmid:33328594

27. Schuligoi R, Schmidt R, Geisslinger G, Kollroser M, Peskar BA, Heinemann A. PGD2 metabolism in plasma: kinetics and relationship with bioactivity on DP1 and CRTH2 receptors. Biochem Pharmacol. 2007;74:107–17. pmid:17452035

28. Pettipher R, Hansel TT. Antagonists of the prostaglandin D2 receptor CRTH2. Drug News Perspect. 2008;21:317–22. pmid:18836589

29. Sandig H, Andrew D, Barnes AA, Sabroe I, Pease J. 9alpha,11beta-PGF2 and its stereoisomer PGF2alpha are novel agonists of the chemoattractant receptor, CRTH2. FEBS Lett. 2006;580:373–9. pmid:16378605

30. Coutinho DS, Anjos-Valotta EA, do Nascimento CVMF, Pires ALA, Napimoga MH, Carvalho VF, et al. 15-Deoxy-Delta-12,14-Prostaglandin J2 Inhibits Lung Inflammation and Remodeling in Distinct Murine Models of Asthma. Front Immunol. 2017;8:740. pmid:28713373

31. Scher JU, Pillinger MH. 15d-PGJ2: the anti-inflammatory prostaglandin? Clin Immunol. 2005;114:100–9. pmid:15639643

32. Sawyer N, Cauchon E, Chateauneuf A, Cruz RPG, Nicholson DW, Metters KM, et al. Molecular pharmacology of the human prostaglandin D2 receptor, CRTH2. Br J Pharmacol. 2002;137:1163–72. pmid:12466225

33. Singh D, Ravi A, Southworth T. CRTH2 antagonists in asthma: current perspectives. Clin Pharmacol. 2017;9:165–73. pmid:29276415

34. Pelaia C, Crimi C, Vatrella A, Busceti MT, Gaudio A, Garofalo E, et al. New treatments for asthma: From the pathogenic role of prostaglandin D2 to the therapeutic effects of fevipiprant. Pharmacol Res. 2020;155:104490. pmid:31682916

35. Gonem S, Berair R, Singapuri A, Hartley R, Laurencin MFM, Bacher G, et al. Fevipiprant, a prostaglandin D2 receptor 2 antagonist, in patients with persistent eosinophilic asthma: a single-centre, randomised, double-blind, parallel-group, placebo-controlled trial. Lancet Respir Med. 2016;4:699–707. pmid:27503237

36. Saunders R, Kaul H, Berair R, Gonem S, Singapuri A, Sutcliffe AJ, et al. DP2 antagonism reduces airway smooth muscle mass in asthma by decreasing eosinophilia and myofibroblast recruitment. Sci Transl Med 2019. pmid:30760581

37. Gevaert P, Bachert C, Maspero JF, Cuevas M, Steele D, Acharya S, et al. Phase 3b randomized controlled trial of fevipiprant in patients with nasal polyposis with asthma (THUNDER). J Allergy Clin Immunol. 2022;149:1675–1682.e3. pmid:35094848

38. Brightling CE, Gaga M, Inoue H, Li J, Maspero J, Wenzel S, et al. Effectiveness of fevipiprant in reducing exacerbations in patients with severe asthma (LUSTER-1 and LUSTER-2): two phase 3 randomised controlled trials. Lancet Respir Med. 2021;9:43–56. pmid:32979986

39. Castro M, Kerwin E, Miller D, Pedinoff A, Sher L, Cardenas P, et al. Efficacy and safety of fevipiprant in patients with uncontrolled asthma: Two replicate, phase 3, randomised, double-blind, placebo-controlled trials (ZEAL-1 and ZEAL-2). EClinicalMedicine. 2021;35:100847. pmid:33997741

40. Hardman C, Chen W, Luo J, Batty P, Chen Y-L, Nahler J, et al. Fevipiprant, a selective prostaglandin D2 receptor 2 antagonist, inhibits human group 2 innate lymphoid cell aggregation and function. J Allergy Clin Immunol. 2019;143:2329–33. pmid:30825466

41. Afgan E, Baker D, Batut B, van den Beek M, Bouvier D, Cech M, et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 2018;46:W537–W544. pmid:29790989

42. Andrews S. FastQC: a quality control tool for high throughput sequence data. 2010. Available from: http://www.bioinformaticsbabrahamacuk/. Accessed 21 Jul 2022.

43. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884–i890. pmid:30423086

44. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. pmid:23104886

45. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30:923–30. pmid:24227677

46. Risso D, Ngai J, Speed TP, Dudoit S. Normalization of RNA-seq data using factor analysis of control genes or samples. Nat Biotechnol. 2014;32:896–902. pmid:25150836

47. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550. pmid:25516281

48. Martin FJ, Amode MR, Aneja A, Austine-Orimoloye O, Azov AG, Barnes I, et al. Ensembl 2023. Nucleic Acids Res. 2023;51:D933–D941. pmid:36318249

49. Raudvere U, Kolberg L, Kuzmin I, Arak T, Adler P, Peterson H, et al. g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res. 2019;47:W191–W198. pmid:31066453

50. Fujimori K, Maruyama T, Kamauchi S, Urade Y. Activation of adipogenesis by lipocalin-type prostaglandin D synthase-generated Δ12-PGJ₂ acting through PPARγ-dependent and independent pathways. Gene. 2012;505:46–52. pmid:22664386

51. Martí I Líndez A-A, Reith W. Arginine-dependent immune responses. Cell Mol Life Sci. 2021;78:5303–24. pmid:34037806

52. Wang W, Zou W. Amino Acids and Their Transporters in T Cell Immunity and Cancer Therapy. Mol Cell. 2020;80:384–95. pmid:32997964

53. Lobito AA, Ramani SR, Tom I, Bazan JF, Luis E, Fairbrother WJ, et al. Murine insulin growth factor-like (IGFL) and human IGFL1 proteins are induced in inflammatory skin conditions and bind to a novel tumor necrosis factor receptor family member, IGFLR1. J Biol Chem. 2011;286:18969–81. pmid:21454693

54. Chen Z, Zhuo W, Wang Y, Ao X, An J. Down-regulation of layilin, a novel hyaluronan receptor, via RNA interference, inhibits invasion and lymphatic metastasis of human lung A549 cells. Biotechnol Appl Biochem. 2008;50:89–96. pmid:17822380

55. Li Y, Yu G, Yuan S, Tan C, Lian P, Fu L, et al. Cigarette Smoke-Induced Pulmonary Inflammation and Autophagy Are Attenuated in Ephx2-Deficient Mice. Inflammation. 2017;40:497–510. pmid:28028752

56. Ren X, Zhang Z. Understanding tumor-infiltrating lymphocytes by single cell RNA sequencing. Adv Immunol. 2019;144:217–45. pmid:31699218

57. Hsu S-F, Lee Y-B, Lee Y-C, Chung A-L, Apaya MK, Shyur L-F, et al. Dual specificity phosphatase DUSP6 promotes endothelial inflammation through inducible expression of ICAM-1. FEBS J. 2018;285:1593–610. pmid:29493888

58. Weng H, Feng W, Li F, Huang D, Lin L, Wang Z. Transcription factor ETV1-induced lncRNA MAFG-AS1 promotes migration, invasion, and epithelial-mesenchymal transition of pancreatic cancer cells by recruiting IGF2BP2 to stabilize ETV1 expression. Growth Factors. 2023;41:152–64. pmid:37428861

59. Shin JH, Moreno-Nieves UY, Zhang LH, Chen C, Dixon AL, Linde MH, et al. AHR Regulates NK Cell Migration via ASB2-Mediated Ubiquitination of Filamin A. Front Immunol. 2021;12:624284. pmid:33717133

60. Lamsoul I, Métais A, Gouot E, Heuzé ML, Lennon-Duménil A-M, Moog-Lutz C, et al. ASB2α regulates migration of immature dendritic cells. Blood. 2013;122:533–41. pmid:23632887

61. Frasca L, Fedele G, Deaglio S, Capuano C, Palazzo R, Vaisitti T, et al. CD38 orchestrates migration, survival, and Th1 immune response of human mature dendritic cells. Blood. 2006;107:2392–9. pmid:16293598

62. Einspahr JM, Tilley DG. Pathophysiological impact of the adhesion G protein-coupled receptor family. Am J Physiol Cell Physiol. 2022;323:C640–C647. pmid:35848619

63. Song X, Liu B, Zhao G, Pu X, Liu B, Ding M, et al. Streptococcus pneumoniae promotes migration and invasion of A549 cells in vitro by activating mTORC2/AKT through up-regulation of DDIT4 expression. Front Microbiol. 2022;13:1046226. pmid:36601406

64. Wang G, Cao L, Liu X, Sieracki NA, Di A, Wen X, et al. Oxidant Sensing by TRPM2 Inhibits Neutrophil Migration and Mitigates Inflammation. Dev Cell. 2016;38:453–62. pmid:27569419

65. Neamatallah T, Jabbar S, Tate R, Schroeder J, Shweash M, Alexander J, et al. Whole Genome Microarray Analysis of DUSP4-Deletion Reveals A Novel Role for MAP Kinase Phosphatase-2 (MKP-2) in Macrophage Gene Expression and Function. Int J Mol Sci 2019. pmid:31336892

66. Wakioka T, Sasaki A, Kato R, Shouda T, Matsumoto A, Miyoshi K, et al. Spred is a Sprouty-related suppressor of Ras signalling. Nature. 2001;412:647–51. pmid:11493923

67. Miki-Hosokawa T, Hasegawa A, Iwamura C, Shinoda K, Tofukuji S, Watanabe Y, et al. CD69 controls the pathogenesis of allergic airway inflammation. J Immunol. 2009;183:8203–15. pmid:19923457

68. Gazi L, Gyles S, Rose J, Lees S, Allan C, Xue L, et al. Delta12-prostaglandin D2 is a potent and selective CRTH2 receptor agonist and causes activation of human eosinophils and Th2 lymphocytes. Prostaglandins Other Lipid Mediat. 2005;75:153–67. pmid:15789622

69. Bochenek G, Nizankowska E, Gielicz A, Swierczyńska M, Szczeklik A. Plasma 9alpha,11beta-PGF2, a PGD2 metabolite, as a sensitive marker of mast cell activation by allergen in bronchial asthma. Thorax. 2004;59:459–64. pmid:15170023

70. Mastalerz L, Celejewska-Wójcik N, Wójcik K, Gielicz A, Januszek R, Cholewa A, et al. Induced sputum eicosanoids during aspirin bronchial challenge of asthmatic patients with aspirin hypersensitivity. Allergy. 2014;69:1550–9. pmid:25123806

71. Papadopoulou M, Sanchez Sanchez G, Vermijlen D. Innate and adaptive γδ T cells: How, when, and why. Immunol Rev. 2020;298:99–116. pmid:33146423

72. Kogame T, Egawa G, Nomura T, Kabashima K. Waves of layered immunity over innate lymphoid cells. Front Immunol. 2022;13:957711. pmid:36268032

73. Shin SB, Lo BC, Ghaedi M, Scott RW, Li Y, Messing M, et al. Abortive γδTCR rearrangements suggest ILC2s are derived from T-cell precursors. Blood Adv. 2020;4:5362–72. pmid:33137203

74. Blythe EN, Weaver LC, Brown A, Dekaban GA. β2 Integrin CD11d/CD18: From Expression to an Emerging Role in Staged Leukocyte Migration. Front Immunol. 2021;12:775447. pmid:34858434

75. Chen X, Tong C, Li H, Peng W, Li R, Luo X, et al. Dysregulated Expression of RPS4Y1 (Ribosomal Protein S4, Y-Linked 1) Impairs STAT3 (Signal Transducer and Activator of Transcription 3) Signaling to Suppress Trophoblast Cell Migration and Invasion in Preeclampsia. Hypertension. 2018;71:481–90. pmid:29378854

76. Ivetic A, Hoskins Green HL, Hart SJ. L-selectin: A Major Regulator of Leukocyte Adhesion, Migration and Signaling. Front Immunol. 2019;10:1068. pmid:31139190

77. Allan DSJ, Cerdeira AS, Ranjan A, Kirkham CL, Aguilar OA, Tanaka M, et al. Transcriptome analysis reveals similarities between human blood CD3- CD56bright cells and mouse CD127+ innate lymphoid cells. Sci Rep. 2017;7:3501. pmid:28615725

78. Williams AG, Thomas S, Wyman SK, Holloway AK. RNA-seq Data: Challenges in and Recommendations for Experimental Design and Analysis. Curr Protoc Hum Genet. 2014;83:11.13.1–20. pmid:25271838

79. Asano K, Sagara H, Ichinose M, Hirata M, Nakajima A, Ortega H, et al. A Phase 2a Study of DP2 Antagonist GB001 for Asthma. J Allergy Clin Immunol Pract. 2020;8:1275–1283.e1. pmid:31778823

80. Schmidt JA, Bell FM, Akam E, Marshall C, Dainty IA, Heinemann A, et al. Biochemical and pharmacological characterization of AZD1981, an orally available selective DP2 antagonist in clinical development for asthma. Br J Pharmacol. 2013;168:1626–38. pmid:23146091