Metabolic regulation by prostaglandin E2 impairs lung group 2 innate lymphoid cell responses

Abstract Background Group 2 innate lymphoid cells (ILC2s) play a critical role in asthma pathogenesis. Non‐steroidal anti‐inflammatory drug (NSAID)‐exacerbated respiratory disease (NERD) is associated with reduced signaling via EP2, a receptor for prostaglandin E2 (PGE2). However, the respective roles for the PGE2 receptors EP2 and EP4 (both share same downstream signaling) in the regulation of lung ILC2 responses has yet been deciphered. Methods The roles of PGE2 receptors EP2 and EP4 on ILC2‐mediated lung inflammation were investigated using genetically modified mouse lines and pharmacological approaches in IL‐33‐induced lung allergy model. The effects of PGE2 receptors and downstream signals on ILC2 metabolic activation and effector function were examined using in vitro cell cultures. Results Deficiency of EP2 rather than EP4 augments IL‐33‐induced mouse lung ILC2 responses and eosinophilic inflammation in vivo. In contrast, exogenous agonism of EP4 and EP2 or inhibition of phosphodiesterase markedly restricts IL‐33‐induced lung ILC2 responses. Mechanistically, PGE2 directly suppresses IL‐33‐dependent ILC2 activation through the EP2/EP4‐cAMP pathway, which downregulates STAT5 and MYC pathway gene expression and ILC2 energy metabolism. Blocking glycolysis diminishes IL‐33‐dependent ILC2 responses in mice where endogenous PG synthesis or EP2 signaling is blocked but not in mice with intact PGE2‐EP2 signaling. Conclusion We have defined a mechanism for optimal suppression of mouse lung ILC2 responses by endogenous PGE2‐EP2 signaling which underpins the clinical findings of defective EP2 signaling in patients with NERD. Our findings also indicate that exogenously targeting the PGE2‐EP4‐cAMP and energy metabolic pathways may provide novel opportunities for treating the ILC2‐initiated lung inflammation in asthma and NERD.

share same downstream signaling) in the regulation of lung ILC2 responses has yet been deciphered.

Methods:
The roles of PGE 2 receptors EP2 and EP4 on ILC2-mediated lung inflammation were investigated using genetically modified mouse lines and pharmacological approaches in IL-33-induced lung allergy model. The effects of PGE 2 receptors and downstream signals on ILC2 metabolic activation and effector function were examined using in vitro cell cultures.
Results: Deficiency of EP2 rather than EP4 augments IL-33-induced mouse lung ILC2 responses and eosinophilic inflammation in vivo. In contrast, exogenous agonism of EP4 and EP2 or inhibition of phosphodiesterase markedly restricts IL-33-induced lung ILC2 responses. Mechanistically, PGE 2 directly suppresses IL-33-dependent ILC2 activation through the EP2/EP4-cAMP pathway, which downregulates STAT5 and MYC pathway gene expression and ILC2 energy metabolism. Blocking glycolysis diminishes IL-33-dependent ILC2 responses in mice where endogenous PG synthesis or EP2 signaling is blocked but not in mice with intact PGE 2 -EP2 signaling.

Conclusion:
We have defined a mechanism for optimal suppression of mouse lung ILC2 responses by endogenous PGE 2 -EP2 signaling which underpins the clinical findings of defective EP2 signaling in patients with NERD. Our findings also indicate that exogenously targeting the PGE 2 -EP4-cAMP and energy metabolic pathways

| INTRODUC TI ON
Asthma is a chronic inflammatory lung disease characterized by bronchoconstriction and airway hyperresponsiveness. Upon contact with allergens, irritants (e.g., pollen, air pollutants) or infections, the damaged lung epithelial cells release pro-allergic cytokines such as interleukin (IL)-33, which rapidly activates many immune cells including group 2 innate lymphoid cells (ILC2s). ILC2s are innate lymphocytes without antigen-specific receptors, but they highly express type 2 helper (Th2) transcription factors such as GATA3 and epithelial cytokine receptors including ST2, a receptor for IL-33. 1 In response to stimuli by epithelial cytokines, ILC2s produce large amounts of type 2 cytokines (e.g., IL-5, IL-13), which initiate the early onset of innate allergic inflammation. 1 Lack or reduction of ILC2s leads to not only decline of type 2 inflammation during diseases, as found in asthma, metabolic diseases and cancer, but also alteration of type 2 immunity following parasite infections. 1,2 ILC2s also contribute to Th2 cell activation, sustaining chronic allergic inflammation. 3,4 ILC2s have been reported to present in human lungs and their cytokine production is associated with disease severity, 5-7 but a possibility that those ILC2s found in human lungs may be contaminated from peripheral blood cannot be ruled out. Furthermore, ILC2s and their cytokine production (e.g., IL-13) are increased in inflamed sinonasal mucosa from chronic rhinosinusitis with nasal polyps, 8 a condition that is associated with hypersensitivity to aspirin.
Eicosanoids are bioactive lipid mediators that play critical roles in regulation of type 2 immune responses and allergic diseases. 9 Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, and indomethacin are widely used for anti-pyretic and analgesic purposes during acute and chronic inflammation through blocking biosynthesis of prostaglandins (PGs) including PGE 2 . 10 However, hypersensitivity reactions to NSAIDs can occur, causing NSAID-exacerbated respiratory disease (NERD), a chronic type 2 immune mediated respiratory disease linked to asthma and nasal polyposis. 11 Increased ILC2 numbers have been observed in the nasal mucosa of patients with NERD, 12 indicating a role for ILC2s in NERD pathogenesis. 13 NERD patients exhibit imbalanced arachidonic acid metabolism with overproduction of PGD 2 and leukotrienes (e.g., LTD 4 and LTB 4 ) but reduction of PGE 2 . 14 PGD 2 and leukotrienes promote ILC2 recruitment to the lung and cytokine production. 15,16 Prostacyclin (also called PGI 2 ) has been reported to restrict ILC2 activation and effector function. 17  may provide novel opportunities for treating the ILC2-initiated lung inflammation in asthma and NERD.

K E Y W O R D S
cellular metabolism, group 2 innate lymphoid cell (ILC2), lung allergy, NSAID-exacerbated respiratory disease, prostaglandin E 2
have reduced expression of the PTGES gene (encoding the key PGE 2 synthase, mPGES1) due to hypermethylation. 18 Genetic studies have also suggested that polymorphisms in the PTGER2 gene (which encodes PGE 2 receptor EP2) are specifically associated with aspirin-intolerant asthma. 19 Moreover, immune cells from the NERD patients display reduced EP2 expression. 14,20 These clinical studies suggest that lessening of PGE 2 -EP2 signaling may be a causative factor in the development of NERD, but the underlying mechanisms remain to be elucidated. PGE 2 plays distinct roles during the sensitization and challenge stages of T cell-mediated allergic inflammation, possibly via both EP2 and EP4. 21 PGE 2 through EP4 suppresses neutrophilic lung inflammation in ex vivo human cell culture systems and various animal models. [22][23][24] Lung allergic responses were increased in mice deficient in PGE 2 synthases such as COX2. 25 Recently, it was reported that PGE 2 suppressed human ILC2 function in vitro through its receptors EP2 and EP4. 26

| Murine lung and BAL single cell suspensions
The murine lungs were digested in Liberase TL (Roche) and DNase I (Sigma) at 37°C for 35 min. Digested tissue was passed through a 100 μm cell strainer and red blood cells lysed using ACK lysing buffer. Viable and dead lung cell counts were determined using 0.4% Trypan Blue solution and a TC10™️ Automated Cell Counter (BioRad).
BAL samples were centrifuged and supernatants from the first BAL retrieval harvested, snap frozen on dry ice and stored at −80°C for subsequent cytokine detection using mouse IL-5/IL-13 ELISA kits according to manufacturer's instructions. Red blood cells were lysed from BAL cell pellets using ACK lysing buffer and cell counts performed as before. Live Lineage(CD11c/CD11b/NK1.1) − CD45 + CD90.2 + ST2 + ILC2s were sorted using a BD FACS Aria II and then cultured in complete RPMI containing 50 μM β-mercaptoethanol with IL-2, IL-7, and IL-33 with or without PGE 2 in a round bottom 96-well plate at 37°C in 5% CO 2 for 3 days.

| Seahorse assays
The Seahorse XFe96-well metabolic analyser (Agilent) was used to investigate the effect PGE 2 may have upon ILC2 glycolysis in vitro.

| RNA-sequencing data analysis
To explore the effect of cAMP on ILC2 gene expression, we first downloaded raw sequence data in FASTQ format from the GEO database (accession number: GSE131996). FastQC was used to assess the quality of FASTQ files (https://www.bioin forma tics.babra ham.ac.uk/proje cts/fastq c/). Sequence reads were mapped to mouse genome using STAR 2.7. 34

| Statistical analyses
All data were expressed as mean ± SD (in vivo) or SEM (in vitro).
For certain experiments where both sexes of animals were used, absolute cell numbers were normalised by sex and data were presented as fold changes. Statistical significance between two groups was examined using an unpaired, 2-tailed Student's ttest. One-way and two-way ANOVA with post-hoc Holm-Sidak's multiple comparisons tests were used to evaluate statistical significance between multiple groups. Statistical analyses were performed using Prism 8 software (Graphpad) and significance was accepted at p < .05.

| Inhibition of endogenous PG synthesis augments IL-33-dependent lung ILC2 responses
At the steady state, PGE 2 is expressed in most tissues including the lung and its levels are elevated by inflammatory stimuli. We thus investigated whether endogenous PGE 2 suppressed lung ILC2 immune responses. We administered intratracheally IL-33 into the lungs of wild-type (WT) C57BL/6 mice. Mice also received indomethacin, an NSAID that blocks the biosynthesis of all PGs including PGE 2 , or vehicle control in drinking water ( Figure 1A). In agreement with a previous report, 38 indomethacin significantly increased IL-33-mediated accumulation of CD45 + Lineage(Lin) − ST2 + ILC2s to the lung and enhanced type 2 cytokine (i.e., IL-5 and IL-13) production from ILC2s ( Figure 1B,C). However, indomethacin treatment had no effect on the accumulation of eosinophils in the lung (data not shown), possibly because indomethacin inhibits all PG synthesis and some PGs have direct actions on eosinophils.
These results suggest that endogenous PGs suppress lung ILC2 responses.

| EP2 deficiency enhances IL-33-induced lung ILC2 responses
As indomethacin inhibits biosynthesis of all PGs, we examined whether PGE 2 is involved in the regulation of lung ILC2 responses, and if yes, through which receptor(s). We examined published datasets 39 and found that ILC2s isolated from various tissues including lung, bone marrow, skin, and intestine have considerably higher gene expression of EP4, followed by EP2 and EP1, and that EP3 has much lower expression levels in ILC2s ( Figure S1).
Given that down-regulation of EP2 is associated with NERD, 14 we first asked whether blockade of endogenous PGE 2 -EP2 signaling influenced lung ILC2 responses. We injected IL-33 into the lungs of EP2-deficient 29 or control mice and measured lung ILC2 activation ( Figure 1D). As expected, EP2-deficiency increased lung ILC2 accumulation and production of type 2 cytokines (IL-5 and IL-13) in response to IL-33 ( Figure 1E,F), although naïve EP2 deficient mice had similar lung ILC2s compared to naive WT mice ( Figure S2A,B). EP2 (Ptger2) gene expression was reduced by half in EP2-heterozygous mice ( Figure S2C), which led to increased IL-5-producing ILC2s but did not affect ST2 + total ILC2s or IL-13producing ILC2s ( Figure 1F), indicating that EP2 expression levels may differently control distinct ILC2 subpopulations. EP2 deficiency also increased lung Lin + CD90.2 + ST2 + Th2 cells, but type 2 cytokine production from T cells was not significantly affected by the loss of EP2 ( Figure 1E,F). In agreement with the increase in ILC2s, EP2-deficient mice had more eosinophils after IL-33 treatment ( Figure 1G and Figure S3). Our data suggest that endogenous PGE 2 -EP2 signaling restricts IL-33-dependent lung ILC2 responses.

| EP4 deficiency does not affect IL-33-induced lung ILC2 responses
To test whether blocking PGE 2 -EP4 signaling modulates lung ILC2 responses, we administered IL-33 to global EP4-deficient 30 and control mice (Figure 2A). Unexpectedly, EP4 deficiency did not enhance ILC2 accumulation in the lung but reduced type 2 cytokine production compared to WT and heterozygous mice ( Figure 2B,C). Global EP4 deficiency also did not increase eosinophils in the lung ( Figure 2D). We then investigated whether specific deletion of EP4 in immune cells including ILC2s enhances lung ILC2 responses. As there were no mouse lines that selectively target only ILC2s, we generated Vav Cre EP4 fl/fl mice by crossing EP4-floxed mice 33 to Vav-cre mice 31 which drives ablation of EP4 in all hematopoietic-derived immune cells (including ILC2s) ( Figure S4A). Naïve Vav Cre EP4 fl/fl mice had similar lung ILC2s to naïve control EP4 fl/fl mice ( Figure S4B,C). A previously published report showed that Vav-dependent EP4 deficiency enhanced lung ILC2 function and eosinophilic inflammation. 27 However, we found that mice with conditional EP4 deficiency in all immune cells had comparable lung ILC2 responses after IL-33 administration, albeit with a moderate reduction of lung ST2 + ILC2 accumulation, compared to control EP4 fl/fl and Vav Cre EP4 fl/+ mice ( Figure 2E,F).
Homozygous Vav Cre EP4 fl/fl mice had a trend to reduce eosinophils ( Figure 2G), which was likely due to a direct effect of EP4

| EP2 and EP4 agonism inhibit IL-33-induced lung ILC2 responses
To examine the effects of exogenous activation of EP2 and EP4 on ILC2 responses in vivo, we intratracheally administered IL-33 to- Engagement of EP2 and EP4 activates the intracellular cAMP pathway. To test if the elevation of cAMP contributes PGE 2 suppression of lung ILC2 responses, we administered 3-isobutyl-1-methylxanthine (IBMX), a non-selective inhibitor of cyclic nucleotide phosphodiesterases (PDEs) that increases the intracellular cAMP level through blocking cAMP degradation. Like EP2 or EP4 agonists, IBMX significantly reduced lung ILC2 numbers and type 2 cytokine production ( Figure 3E,G,I). It is worth to note that although the EP4 agonist markedly suppressed IL-5 production from both IL-5 + IL-13 − and IL-5 + IL-13 + ILC2s in response to IL-33 administration, neither the EP2 agonist nor IBMX reduced IL-5 production from lung ILC2s ( Figure 3H,I). This may be the cause for unchanged eosinophils by EP2 agonist and IBMX as eosinophil activation is driven by IL-5. In addition, the recent studies suggested that platelet activation and platelet-adherent leukocytes were associated with NERD, 40,41 which might link to disruption of eicosanoid balance such as overproduction of cysteinyl leukotriene and reduction of PGE 2 . 14,41 As platelet adherent promotes lung ILC2 effector function, 42,43 we examined whether PGE 2 -EP2-cAMP signaling had an effect on platelet-adherent (i.e., CD41-positive) ILC2s using flow cytometry. IL-33 treatment indeed increased the numbers of CD41 + ILC2s in the lung, and this was abrogated by EP2 agonist and IBMX, although neither IL-33 nor EP2 agonist or IBMX affected CD41 adherent at the single cell level ( Figure S6). Thus, exogenously activated the PGE 2 -EP2/EP4-cAMP pathway suppresses lung ILC2 responses.

| PGE 2 -EP2/EP4-cAMP signaling directly inhibits ILC2 activation
We next examined the underlying mechanisms for PGE 2 Figure 4E). Other cells such as eosinophils and basophils can also produce IL-5, which may be the reason for PGE 2 suppression of IL-5 production in whole Rag2 −/− lung cell culture but not in purified ILC2 cultures. ILC2s have been reported produce the regulatory cytokine IL-10, [45][46][47] and PGE 2 promotes IL-10 production from multiple cell types such as Th2 or macrophages. 10 We thus examined if PGE 2 impacted on IL-10 production from ILC2s.

| cAMP regulates IL-33-dependent and -independent ILC2 gene expression
To understand the underlying mechanisms for PGE 2 suppression of ILC2 responses, we examined a published dataset 44 and analysed the effects of cAMP on ILC2 gene expression. Gene set enrichment analysis showed that, in addition to restricting cell cycle progression as reported previously, 44 cAMP markedly enhanced apoptosisassociated pathways ( Figure 4G). Expression of pro-apoptosis associated genes (e.g., Bad, Bak1, caspases, Pdcd1) were upregulated by cAMP independently of IL-33 ( Figure 4H). In contrast, IL-33induced expression of anti-apoptotic genes (e.g., Bcl2l1, Bcl3, Trp53, Ptk2) were down-regulated by cAMP ( Figure 4H). This differential cAMP-altered expression of pro-and anti-apoptotic genes contributes to overall reduced cell survival. Especially, in consistent to down-regulation of the hallmark c-Myc pathway gene expression by cAMP ( Figure 4G), our data show that PGE 2 -cAMP significantly reduced c-Myc protein expression in ILC2s ( Figure 4I).

| PGE 2 restricts ILC2 cellular metabolism
In this study, we have established that PGE 2 reduces ILC2 survival, cell size, proliferation, and c-Myc expression (Figure 4), all are hallmark events of cellular metabolism. Similarly, in vivo activation of EP4 and, possibly EP2, by their respective agonists reduced Ki-67-expressing ST2 + ILC2s in lungs of mice treated with IL-33 ( Figure 5A,B). In contrast, EP2 deficiency increased Ki-67 + ST2 + ILC2s in the lung ( Figure 5C,D).
Furthermore, IL-33 stimulation induced phosphorylation of the ribosomal protein S6 (pS6), a key signature of cellular metabolic activation. This is again reverted by PGE 2 and cAMP ( Figure 5E,F). These results may imply changes in energy metabolism of ILC2s by PGE 2 -cAMP signaling. To examine if PGE 2 directly regulates ILC2 metabolism, we measured metabolic changes in cultured lung ILC2s using the Seahorse assays. Indeed, PGE 2 -reduced oxygen consumption rate (OCR, an F I G U R E 5 PGE 2 represses ILC2 cellular metabolism, and blockade of glycolysis attenuates EP2 inhibition-augmented lung ILC2 responses. (A and B) Representative flow cytometric dot plots (A) and collective percentages and numbers (B) of lung proliferating Ki-67 + ILC2s in C57BL/6 mice administered intratracheally with IL-33 with PBS (n = 3) or IL-33 with vehicle (n = 4), an EP2 agonist (butaprost, n = 3), an EP4 agonist (L-902688, n = 3), or both the EP2 and EP4 agonists (n = 3) for 3 consecutive days as shown in Figure 3. (C and D) Representative flow cytometric dot plots (C) and collective percentages and numbers (D) of lung proliferating Ki-67 + ILC2s in EP2 +/+ (n = 4), EP2 +/− (n = 2) or EP2 −/− (n = 5) mice administered with IL-33 for 3 consecutive days as shown in Figure 1. (E and F) Representative flow cytometric dot plots (E) and collective expression of pS6 (F) in ILC2s expanded and cultured with IL-33, IL-2 and IL-7 for 3 days in the presence or absence of indicated reagents. (G-I) The oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and proton efflux rate (glycoPER) of ILC2s that were sorted from lungs of Rag2 −/− mice and then cultured with IL-33 with or without PGE 2 for 3 days. (J) Experimental design. Female C57BL/6 mice were administered intratracheally with IL-33 with or without 2-DG for 3 consecutive days and sacrificed 24 h after the last IL-33 challenge. Mice were also treated with indomethacin, an EP2 antagonist (PF-04418948) or vehicle control in drinking water from the day before first IL-33 administration and throughout the experiments. indicator of mitochondrial phosphorylation), extracellular acidification rate (ECAR, an indicator of aerobic glycolysis) and glycolytic proton efflux rate (PER, a real-time indicator of glycolysis rate) of ILC2s ( Figure 5G,H). Furthermore, PGE 2 suppressed not only basal glycolysis (i.e., physiological mitochondrial respiration) but also compensatory glycolysis when mitochondrial respiration is inhibited ( Figure 5I). Thus, our results suggest that PGE 2 directly inhibits IL-33-dependent ILC2 energy metabolism in vitro.
To determine whether PG signaling controls ILC2 responses through inhibiting ILC2 metabolism in vivo, we treated WT C57BL/6 mice with IL-33 and indomethacin ( Figure 5J). Some mice received 2-deoxy-D-glucose (2-DG), a glucose analogue that inhibits glycolysis through blocking glucose hexokinase. 2-DG has no impact on IL-33or helminth infection-induced ILC2 activation, 58,59 but it reduces ILC2 responses when ILC2s are over-activated such as in PD-1-deficient mice. 59 In agreement with these previous reports, 58 Accordingly, overall lung inflammation was also decreased by 2-DG in indomethacin-treated mice as evidenced by reduction of both eosinophils and neutrophils in the BAL fluid ( Figure 5M). 2-DG did not affect lung ILC2 numbers nor cytokine production in both indomethacin-or vehicle-treated mice except the reduction of lung eosinophils in mice treated with indomethacin but not vehicle control ( Figure S11). As indomethacin inhibits production of all PGs, we wondered if PGE 2 , especially the EP2 receptor, inhibits lung ILC2 responses through inhibiting ILC2 metabolism. To address this, we treated mice with PF-04418948, a highly selective EP2 antagonist, 60 with or without 2-DG ( Figure 5J).
In agreement with our observations in EP2KO mice, the EP2 antagonist also significantly increased numbers of ILC2s, Th2 cells, eosinophils and neutrophils in the BAL fluid ( Figure 5N,O). Similar to findings in mice treated with indomethacin, augmented ILC2 responses in EP2 antagonist-treated mice were also markedly reduced by 2-DG although it remained no effects on ILC2 responses in mice treated with vehicle control ( Figure 5N,O). Taken together, these results suggest that PGE 2 impedes ILC2 energy metabolism and constrains IL-33-induced lung type 2 immune responses.

| DISCUSS ION
Here, we report that PGE 2 -EP2/EP4 signaling limits type 2 lung inflammation through negative regulation of ILC2 responses. We have shown that deficiency of EP2, but not EP4, enhanced lung ILC2 responses, suggesting that endogenous ligands (such as PGE 2 and others such as PGE 1 ) more preferentially bind to EP2 than EP4. Although both EP2 and EP4 activate the same downstream cAMP-PKA signaling pathway, they can have redundant or additive functions in vivo.
For example, EP2 and EP4 collaboratively promotes T cell-mediated psoriasis. 61 Under certain circumstances, the in vivo actions of PGE 2 may be dominantly mediated by one receptor, leaving the other dispensable. For example, the effects of PGE 2 on intestinal homeostasis are largely mediated by EP4, and EP2 exerts few effects. 62 Our results may also indicate that in the lung, EP2 may have already been endogenously activated at the optimal level for repressing ILC2 responses. Thus, further activation of EP2 requires stronger stimuli, for example using an agonist with higher EP2 affinity like butaprost (free acid) at relatively higher dosages to achieve satisfactory efficiency in vivo. On the other side, EP4 signaling does not sound to be properly activated by endogenous ligands, thus activation of this receptor by an exogenous agonist results in considerable effects. In addition, the discrepancy in effects between EP4 agonism and EP4 deficiency on regulation of lung ILC2 responses may also indicate that EP4 expression on different lung cell types has distinct effects during type 2 inflammation. For example, PGE 2 may affect T cell functions that counteract PGE 2 inhibition of ILC2s.
Moreover, the strength (e.g., the amounts of local lung resident PGE 2 ) and the timing of endogenous versus exogenous EP4 activation in the course of lung allergic inflammation may also influence the overall effects of EP4 on ILC2 responses. Further studies are needed to finely examine these possibilities. PGE 2 has been indicated to be associated with various lung conditions including neutrophilic inflammation, 22 infections (e.g., mycobacterium tuberculosis 20 ) and COPD. 24 Blocking synthesis of PGE 2 and other PGs by NSAIDs like indomethacin may be beneficial for treating these conditions. It is worth to note that indomethacin was reported to bind to mouse CRTH2, 68 a receptor for prostaglandin D 2 that mediates ILC2 migration. 15,69 It was suggested that CRTH2 mediates ILC2 recruitment to the lung when IL-33 is administered systemically (e.g., via intraperitoneal injection), but it has few effects on ILC2 migration to the lung if IL-33 is administered directly to the lung. 16 In our studies, we intratracheally administered IL-33 into the lung, thus the enhancement of lung ILC2 responses by indomethacin is most likely through inhibition of endogenous PGs. Furthermore, our findings that EP2 rather than EP4 is the dominant mediator of endogenous PGE 2 effects on the regulation of lung ILC2 responses specifically reflect the findings from clinical studies. Indeed, PTGER2 gene polymorphisms are associated with asthma especially NERD and patients with NERD have decreased EP2 expression. 14,19,20 Our results suggest that the down-regulation or alteration of EP2 signaling in NERD patients may contribute to augmented ILC2 responses and inflammation. Thus, pharmacological activation of EP4 may be a promising therapeutic approach to limit augmented ILC2 responses in asthma patients with low EP2 expression or function.
We have also reported that PGE 2 through activation of cAMP suppresses ILC2 energy metabolism, which controls cell survival, proliferation, activation and effector function. Besides PGE 2 , many molecules that can activate the cAMP pathway such as PGI 2, 17 β2-adrenergic receptor agonist (e.g., norepinephrine), 70 and calcaencoding calcitonin gene-related peptide 44,71,72 have all been found to suppress ILC2 responses and allergic lung inflammation. Our findings of regulation of ILC2 gene expression by the PGE 2 -cAMP pathway via IL-33-dependent and -independent mechanisms suggest that PGE 2 may have broad impacts on ILC2-mediated type 2 immune responses. It is worth noting that although cAMP represses ILC2 activation and IL-13 production, it does not inhibit IL-5 expression, which was in line with the findings of other studies. 44 Indeed, cAMP was reported to mediate elevation of IL-5 production from homeostatic ILC2s. 73 Actually, PGE 2 -cAMP signaling suppresses IL-5 production from IL-5/IL-13-co-expressed ILC2s. Thus, it may be worth investigating for the future if PGE 2 -cAMP activates distinct downstream pathways in different ILC2 subsets, for example taking advantage of single cell RNA-sequencing.
Activation of the energy metabolic programs is essential for ILC2 survival, proliferation and effector function as blockade of mTORC1 signaling by rapamycin inhibits IL-33-induced ILC2 responses and airway inflammation. 74 Our observation that blocking glycolysis by 2-DG has no effect on IL-33-or parasite-mediated ILC2 activation at the 'normal' level has also been reported by other groups, 58,59 but it impairs excessive ILC2 responses when ILC2s are 'overactivated', for example, by removing 'endogenous inhibitors' such as PD-1, 59 PGE 2 or EP2. This is probably because normally activated ILC2 had lowgene expression of glycolysis-related enzymes, 75  This current research has several limitations. First, our results clearly indicated that PGE2-EP2 signaling directly suppresses lung ILC2 responses in viitro and in vivo using global EP2-deficient mice.
However, due to the lack of ILC2-specific EP2-deficient mouse lines, we could not address whether ILC2-specific EP2 signalling inhibits lung ILC2 responses and, if yes, whether this is dependent of its inhibition of ILC2 cellular metabolism in vivo. Second, as limited access to key resources (especially for an appropriate seahorse machine), we could not directly check if PGE 2 inhibits ILC2 oxygen consumption and glycolysis through EP2 and EP4. Third, we did not examine if PGE 2 suppression of lung ILC2 responses contributes to human asthma and NERD, which is desired to investigate following this current research.
Our current work has significantly improved our understanding of control of lung allergic responses. First, we have demonstrated that blockade or deficiency of endogenous EP2 signaling augments lung ILC2 responses and eosinophilic inflammation. This is in agreement with published findings from humans that down-regulation of the PGE 2 -EP2 pathway is a feature of NRED. 14,19,20 Second, our results using global and ILC2/Th2-conditional knockout mouse lines clearly showed that while endogenous EP4 signaling is dispensable for control of lung ILC2 responses, EP4 agonism markedly reduced IL-33-dependent lung ILC2 responses. Thus, exogenous activation of EP4 could therapeutically attenuate ILC2-dependent lung allergic reactions, especially in asthma patients where PGE 2 -EP2 signaling is down-regulated. Third, our work suggests that PGE 2 curtails ILC2 cellular metabolism through activating the EP2/EP4/cAMP signalling. More importantly, our findings that in vivo inhibition of glycolysis successfully reduces lung ILC2 responses augmented by diminishing PGE 2 -EP2 signaling suggest a potential strategy for treatment of NERD that has down-regulated EP2 signaling and enhanced ILC2 responses.
Current treatments do not provide a cure for asthma and in the case of NERD patients, such treatments may not be effective. Our work highlights the EP4-cAMP pathway as a potential therapeutic target for asthma and in particular for NERD, where small molecules that harness activation of EP4 and/or elevation of intracellular cAMP levels could be of clinical benefit. Our results show that use of phosphodiesterase inhibitor effectively inhibits ILC2 responses in vitro and in vivo, supporting the development of phosphodiesterase inhibitors as considered for treatment of asthma and other lung diseases. 76,77 Furthermore, we propose that targeting ILC2 energy metabolic pathways (e.g., glycolysis) may be beneficial in the control of NSAID-dependent augmentation of type 2 lung inflammation in patients with NERD.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.