The prostaglandin D2 receptor 2 pathway in asthma: a key player in airway inflammation.

Asthma is characterised by chronic airway inflammation, airway obstruction and hyper-responsiveness. The inflammatory cascade in asthma comprises a complex interplay of genetic factors, the airway epithelium, and dysregulation of the immune response.Prostaglandin D2 (PGD2) is a lipid mediator, predominantly released from mast cells, but also by other immune cells such as TH2 cells and dendritic cells, which plays a significant role in the pathophysiology of asthma. PGD2 mainly exerts its biological functions via two G-protein-coupled receptors, the PGD2 receptor 1 (DP1) and 2 (DP2). The DP2 receptor is mainly expressed by the key cells involved in type 2 immune responses, including TH2 cells, type 2 innate lymphoid cells and eosinophils. The DP2 receptor pathway is a novel and important therapeutic target for asthma, because increased PGD2 production induces significant inflammatory cell chemotaxis and degranulation via its interaction with the DP2 receptor. This interaction has serious consequences in the pulmonary milieu, including the release of pro-inflammatory cytokines and harmful cationic proteases, leading to tissue remodelling, mucus production, structural damage, and compromised lung function. This review will discuss the importance of the DP2 receptor pathway and the current understanding of its role in asthma.

Background

Asthma affects approximately 358 million people worldwide [1], and is characterised by chronic airway inflammation, reversible airway obstruction and hyper-responsiveness. The heterogeneous nature of this condition may cause difficulty in predicting response to treatment in a particular patient [2, 3].

Despite the availability of clinical practice guidelines and standard-of-care therapy, a large proportion of asthma patients remain symptomatic and experience poor quality-of-life [4, 5]. There is a high unmet need for novel asthma therapies, especially for patients with severe disease. Effective disease control is dependent in part by treatment adherence [6], which can be influenced by route of administration. Adherence to inhaled therapies, particularly maintenance therapies such as inhaled corticosteroids, is often poor, and is driven by the complexity of the inhaler, as well as errors during device use, such as improper actuation–inhalation coordination [7]. A clinical consequence of poor or non-adherence to inhaled therapies is increase of symptoms and eventually the occurrence of exacerbations [8]. Adherence to oral asthma treatment has been shown to be superior to that of inhaled therapies [9, 10], however oral therapy options for the management of asthma are presently quite limited. Hence, effective new oral therapies may help the management of severe or insufficiently controlled asthma [11, 12], as has been the case with the recent introduction of biological therapies via subcutaneous injection.

A treatment target with a novel mechanism of action that has gained significant interest in recent years and which has promise to be accessible by small molecule-based oral therapies, is the receptor 2 (DP2) of prostaglandin D2 (PGD2). This receptor is also referred to in the literature as the chemoattractant receptor homologous molecule expressed on TH2 cells (CRTH2) [13], and is expressed on the membrane surface of TH2 cells, type 2 innate lymphoid cells (ILC2), mast cells and eosinophils [14-16]. This review aims to discuss the current understanding of the DP2 receptor signalling pathway in asthma.

Allergen-dependant and non-allergen-dependent stimulation

The inflammatory cascade in asthma comprises a complex interplay of factors. In a large proportion of patients, asthma is associated with a type 2 immune response (Type 2-high asthma) [17, 18]. Until recently, only the allergen-dependent immune pathway was considered to be an important target for asthma treatment. However, it is now clear that both the non-allergen- and allergen-dependent immune pathways are involved in the pathophysiological and immunological responses in asthma [19]. As PGD2, a pro-inflammatory lipid mediator, release is stimulated following both non-allergen-dependent (infections, physical stimuli or chemical stimuli) and allergen-dependent immune activation, the DP2 receptor pathway has relevance in both atopic and non-atopic asthma (Fig. 1) [16, 20].

Fig. 1

Overview of the DP2 receptor-mediated response of immune cells in the inflammatory pathway. Proposed schematic providing an overview of the DP2 receptor-mediated response of various immune cells, including mast cells, TH2 cells, ILC2 and eosinophils, and the subsequent effect on inflammation in the asthmatic airways through increased inflammatory cell chemotaxis and cytokine production. Abbreviations, APC: antigen presenting cell; DP2: prostaglandin D2 receptor 2; IgE: immunoglobulin E; IL: interleukin; ILC2: type 2 innate lymphoid cell; PGD2: prostaglandin D2

PGD2 release from immune cells

PGD2 is released following activation of the immune system, which can be either non-allergen- or allergen-dependent (Fig. 1); the non-allergen-dependent pathway comprises indirect activation of mast cells via the processing of physical agents, chemical agents or infections by antigen presenting cells, or direct activation via complement, sphingolipids and others. Through the allergen-dependent pathway, inhaled allergens trigger a cascade of events that provoke the release of PGD2, initiating a signalling cascade through the DP2 receptor in target cells (TH2 cells, ILC2 and eosinophils). Inhaled antigens are presented to CD4+ T lymphocytes by allergen presenting cells. In allergic patients, these T lymphocytes differentiate to acquire a TH2 cell profile, producing significant amounts of IL-4 and IL-13, which promote IgE class-switching in B lymphocytes [21-23]. Mast cells are subsequently activated upon allergen-induced cross-linking of adjacent high-affinity IgE Fc receptor (FcεRI)-bound IgE at the cell surface [24].

PGD2 is primarily released from mast cells through activation of hematopoietic PGD synthase, resulting in nanomolar local concentrations of the mediator [25]. Mast cells are tissue-resident cells that can be activated and degranulated in minutes [26]. They are widely distributed at mucosal surfaces and in tissues throughout the body, and play a central role in the pathophysiology of asthma, not only by mediating immunoglobulin E (IgE)-dependent allergic responses, but also in non-IgE-mediated mechanisms [27, 28]. Mast cell numbers are similarly increased in both allergic and non-allergic asthma, although response to cyclic adenosine monophosphate (cAMP) is higher in allergic than in non-allergic patients [29].

Aside from mast cells, other cell types can also produce PGD2 under certain conditions, including biologically meaningful quantities in TH2 cells [13, 30, 31]. Macrophages [32], and dendritic cells [33, 34] also produce small amounts of PGD2.

PGD2 receptors

PGD2 mainly exerts its biological effect via high affinity interactions with two structurally and pharmacologically distinct receptors (the prostaglandin D2 receptor 1 [DP1] and the DP2 receptor) [13]. At micromolar concentrations, PGD2 can also stimulate the thromboxane receptor [35].

DP1, a 359 amino acid, ~40 kDa G-protein-coupled prostaglandin receptor, was the first PGD2 receptor to be identified [36, 37]. It mediates a range of effects, which are mostly non-inflammatory in nature; vasodilation, inhibition of cell migration, relaxation of smooth muscle, and eosinophil apoptosis [38].

The DP2 receptor is a 395 amino acid, 43 kDa G-protein-coupled prostaglandin receptor. Binding of PGD2 to the DP2 receptor on immune cells induces a myriad of pro-inflammatory downstream effects, which significantly contribute to the recruitment, activation and/or migration of TH2 cells, ILC2, and eosinophils, thereby fuelling the inflammatory cascade in asthma [14, 38–41]. PGD2 metabolites (DK-PGD2, Δ12PGJ2, 15-deoxy- Δ12,14PGD2, and deoxy- Δ12,14PGJ2) also activate the DP2 receptor [42-44].

Cells expressing the DP2 receptor

The DP2 receptor plays a key role in the pathophysiology of asthma: it induces and amplifies the inflammatory cascade [16, 25, 45, 46]. This type of receptor can be found in many cell types, however the key cells of the DP2 receptor pathway include TH2 cells, ILC2 cells and eosinophils, suggesting a homeostatic role for this receptor (Fig. 1) [14–16, 47]. In addition, type 2 cytotoxic T (Tc2) lymphocytes were recently shown to be activated by PGD2 acting via the DP2 receptor, thus contributing to the pathogenesis of eosinophilic asthma [41].

Effects of the DP2 receptor on TH2 cells

PGD2 preferentially upregulates IL-4, IL-5 and IL-13 expression (type 2 cytokines) in TH2 cells in a dose-dependent manner [48] and induces TH2 cell migration [46] via its high affinity interaction with the DP2 receptor (Fig. 1).

DP2 receptor activation has shown a potent effect on TH2 cell migration in vitro, highlighting a key function of this receptor in mediating the chemotaxis of TH2 lymphocytes [49]. As elevated levels of circulating DP2+CD4+ T cells is a hallmark feature of severe asthma [50], this provides a DP2 receptor-rich environment upon which already increased levels of PGD2 levels may act, further perpetuating the inflammatory cascade.

Effects of the DP2 receptor on ILC2 cells

ILC2 is a cell type that may link the non-allergen- and allergen-dependent responses in asthma. ILC2 cell activation is triggered by inflammatory mediators released from epithelial and immune cells (e.g. IL-33 and PGD2), and is associated with increased production of type 2 cytokines [51]. Thus, ILC2 cells facilitate a TH2 immune response that can be independent of the allergen [52].

Secretion of IL-4, IL-5 and IL-13 from ILC2 cells is increased in response to DP2 receptor stimulation in a dose-dependent manner [16].

In response to IL-33, ILC2 cell activation was initially reported to produce high levels of IL-5 and IL-13 in vitro, but very low levels of IL-4. Interestingly, recent studies have shown that when their DP2 receptor is stimulated, ILC2 cells produce higher levels of IL-4 [53].

Meanwhile, DP2 stimulation alone remarkably increases ILC2 cell migration, which is 4.75-fold greater than that of IL-33 [16].

Effects of the DP2 receptor on eosinophils

Eosinophils are involved in airway hyper-responsiveness, mucus hypersecretion, tissue damage and airway remodelling in asthma. Eosinophil activation is also associated with increased cytokine production, which has various downstream immunomodulatory effects [54]. DP2 receptor activation at the eosinophil surface facilitates the trans-endothelial migration and influx of eosinophils, increases eosinophil degranulation and induces eosinophil shape change [40, 55, 56]. Eosinophil shape change in response to DP2 activation [57] is similar to that visualised previously with eotaxin stimulation [58].

Eosinophil influx and activation can cause detrimental effects on the epithelial lining of the lungs of asthma patients. This happens through degranulation and release of harmful mediators such as eosinophil cationic protein, eosinophil peroxidase, eosinophil protein X and cytotoxic major basic protein [19, 59, 60]. Additionally, eosinophils release transforming growth factor (TGF)-ß which induces apoptotic effects upon airway epithelial cells, contributing to airway tissue denudation. Moreover, eosinophils enhance airway smooth muscle cell proliferation, further contributing to structural remodelling of the pulmonary architecture [61]. Charcot-Leyden crystals, a product of activated eosinophils, are detectable in expectorated sputum samples from asthma patients [62]. These crystals are largely comprised of the toxic enzyme lysophospholipase (also known as phospholipase B), and may contribute to eosinophil-driven tissue denudation in the lungs [63].

As mentioned previously, in addition to the direct effects, DP2 receptor activation also has indirect effects on eosinophils by inducing the release of IL-4, IL-5 and IL-13 from TH2 cells and ILC2, which affect eosinophil maturation, apoptosis and migration to the lungs.

Effects of DP2-mediated cytokine release

DP2 receptor activation increases release of cytokines from ILC2 and TH2 cells. These cytokines cause some of the characteristic features of asthma, including airway inflammation, IgE production, mucus metaplasia, airway hyper-reactivity, smooth muscle remodelling and eosinophilia [52, 64]. We will review the effects of the key cytokines released:

IL-4 enhances the migration of eosinophils, which is a key step in the inflammatory cascade. To do this, in synergy with tumour necrosis factor (TNF)-α, IL-4 increases the expression of vascular cell adhesion molecule-1 (VCAM-1) and P selectin on the surface of the vascular endothelium, which facilitates the trans-endothelial passage of eosinophils from the bloodstream into the lung parenchyma [19, 65]. Meanwhile, IL-4 also stimulates the release of eotaxin, a potent and selective eosinophil chemoattractant, from the vascular endothelium (Fig. 1). Eotaxin facilitates eosinophil migration [66, 67]. Differentiation and proliferation of TH2 cells is also promoted by IL-4 [39].

IL-5 is directly involved in the differentiation and maturation of eosinophils in the bone marrow, eosinophil chemotaxis to sites of inflammation, and local eosinophilopoiesis [68, 69]. It also inhibits eosinophil apoptosis, leading to the accumulation of these cells at sites of inflammation, which in turn perpetuates and prolongs the inflammatory cycle [70].

IL-13 is known to induce goblet cell hyperplasia, mucus production, and airway hyper-responsiveness, leading to airway inflammation and tissue remodelling [39, 64]. Furthermore, IL-4 and IL-13 released from TH2 and ILC2 in response to DP2 receptor activation promote immunoglobulin class switching from IgM to IgE antibodies in B cells and plasma cells, which leads to further mast cell recruitment, activation and PGD2 release at sites of inflammation [16, 20, 71, 72]. It also contributes to the release of eotaxin (together with IL-4), which as mentioned above, facilitates eosinophil migration.

Levels of other pro-inflammatory cytokines are also increased upon activation of DP2 receptors, including IL-8, IL-9 and granulocyte–macrophage colony-stimulating factor, which may additionally contribute to excessive immune cell chemotaxis, associated proteases and enhanced airway inflammation in asthma [16].

Results from phase II clinical studies suggest that blocking the activation of the DP2 receptor pathway with DP2 receptor antagonists reduces the symptoms associated with asthma, improves pulmonary function and inhibits eosinophil shape change, while showing indirect signs (sputum eosinophil reduction) of the potential to decrease the number of exacerbations experienced by severe asthma patients [73-80].

Further evidence for DP2 receptor pathway importance in asthma

PGD2 levels are increased in asthma, with increased levels in patients with severe disease [27, 81], and in response to allergen challenge [82, 83]. The number of DP2 receptor-positive cells within the submucosal tissue is also significantly higher in patients with severe asthma compared with healthy controls [84]. Interestingly, an association between a single nucleotide polymorphism in the DP2 receptor (rs533116) and allergic asthma has also been reported [85].

PGD2 protein and DP2 receptor expression levels in bronchoalveolar lavage fluid (BALF) from severe asthmatic patients were shown to be significantly higher than from healthy controls or patients with mild or moderate asthma [27, 81]. Interestingly, Murray et al. [82] demonstrated a 150-fold increase in PGD2 levels in BALF from asthma patients within nine minutes of local antigen (Dermatophagoides pteronyssinus) challenge, demonstrating that allergen-induced PGD2 release is an early and rapid event. Furthermore, a study by Wenzel and colleagues showed that allergen challenge in atopic asthma patients induced a significant increase in BALF PGD2 levels compared with atopic patients without asthma [83].

Of significant interest is the sustained activity of PGD2-derived metabolites despite extensive and rapid PGD2 metabolism. The PGD2-derived metabolites PGJ2 and Δ12-PGJ2, are themselves known to be potent DP2 receptor agonists, thereby demonstrating the sustained and prolonged activity of the DP2 receptor via the metabolites of PGD2 [45]. Despite the short half-life of PGD2 in plasma (~30 min), its biological activity towards the DP2 receptor is maintained through the formation of these metabolites, which are more stable than the parent compound, highlighting their potential role in perpetuating the inflammatory cascade [45].

Blockage of PGD2 via DP2 receptor antagonism inhibits inflammatory cell chemotaxis and also reduces type 2 pro-inflammatory cytokine production, which provides further evidence of the vital role played by PGD2 and its interaction with the DP2 receptor in asthma [46]. Of note, DP2 receptor antagonism has also been shown to decrease airway smooth muscle cell mass and chemotaxis of these cells towards PGD2 [86, 87].

Role of the DP2 receptor pathway in virus-induced asthma

Viruses, such as rhinovirus (RV), influenza A, and respiratory syncytial virus (RSV), are a major cause of asthma exacerbations and can activate the DP2 receptor pathway [88]. These respiratory viruses produce double-stranded RNA (dsRNA) during replication, which activates the non-allergen-dependent immune response and results in increased chemokine synthesis from airway epithelial and innate immune cells [88, 89]. A recent study also suggests the involvement of the DP2 receptor pathway in augmenting virus-mediated airway eosinophilic inflammation [88]. It shows that DP2 receptor stimulation followed by eosinophil recruitment into the airways is a major pathogenic factor in the dsRNA-induced enhancement of airway inflammation and bronchial hyper-responsiveness [88].

PGD2 levels have also been found to be increased after viral challenge in asthma patients, which may act synergistically with IL-33 to further drive type 2 cytokine production [90, 91]. The role of PGD2 in RV16-induced asthma exacerbations was recently investigated in atopic asthma patients [91]. In this study, baseline PGD2 levels were higher in asthmatic patients versus healthy controls. Furthermore, RV16 infection induced a greater PGD2 increase in asthmatic patients compared with the healthy participants. The largest RV16-mediated PGD2 increase was observed in those with severe and poorly-controlled asthma, suggesting a potential role for PGD2 in driving asthma exacerbations [91].

Polyinosinic:polycytidylic acid (poly I:C) is an immunostimulant; it is structurally similar to double-stranded RNA, which is present in some viruses and is a “natural” stimulant of toll-like receptor 3 (TLR3), which is expressed in the membrane of B-cells, macrophages and dendritic cells. Thus, poly I:C can be considered a synthetic analogue of double-stranded RNA and can simulate viral infections. Early evidence from poly I:C murine asthma models suggests that a selective DP2 receptor antagonist may dose-dependently block the aforementioned virus-induced T2 response, and may help to reduce the inflammation caused by virus-mediated asthma exacerbations [92].

Conclusions

The DP2 receptor pathway is known to play a key role in the pathophysiology of asthma via induction and amplification of the inflammatory cascade by exerting direct effects on immune cells, including TH2 cella, ILC2 and eosinophils [16, 46, 55]. IL-4, IL-5 and IL-13 release from DP2 receptor-activated immune cells can have significant effects on immune cell influx, degranulation, tissue remodelling and mucus production in the airways, leading to structural damage, fibrosis and reduced pulmonary function [64]. Additionally, the effect of DP2 receptor activation on eosinophil activation and migration leads to tissue damage, through release of harmful cationic proteins and enhanced proliferation of airway smooth muscle cells [93].

This review highlights the important pro-inflammatory role of the DP2 receptor pathway in asthma. Furthermore, multiple DP2 receptor antagonists are currently under clinical investigation [73–75, 77–80], for asthma therapies. Indeed, in a 12-week study in patients with allergic asthma that was uncontrolled by low-dose ICS, the oral DP2 receptor antagonist fevipiprant (150 mg once daily or 75 mg twice daily) produced significant improvements in pre-dose FEV1 compared with placebo [73]. Further, in patients with moderate to severe eosinophilic asthma, fevipiprant significantly reduced mean sputum eosinophil percentage compared with placebo [80]. Initial positive findings have also been reported with timapiprant (OC00459) [78], BI 671800 [77], setipiprant [94] , MK-1029 and ADC-3680 [95] , but not with AZD1981 [75]. Hence, the clinical outcomes of larger, phase III clinical studies involving DP2 receptor antagonists are eagerly awaited.