Lipid Mediator Regulation of Group 2 Innate Lymphoid Cells

ILC2s were originally found to be activated by epithelial cell derived cytokines to induce the secretion of Th2 cytokines, IL-5 and IL-13. Recent research has shown that lipid mediators play a large role in the activation and inhibition of ILC2 function. Unlike the traditional epithelial cell derived cytokines IL-33 and IL-25, lipid mediators have been shown to promote ILC2 secretion of not only IL-5 and IL-13, but the secretion of IL-4 as well. Prostaglandin D2 has been shown to be a potent chemoattractant of ILC2s as well as a potent activator of ILC2s to release Th2 cytokines. In addition to prostaglandin D2, cysteinyl leukotrienes also activate ILC2s to secrete Th2 cytokines during inflammation. Notably, lipid mediators have been shown to work in concert with epithelial cell derived cytokines to increase IL-5 and IL-13 secretion from ILC2s. On the other hand, lipid meditators prostaglandin I2 and lipoxin A4 are the first identified lipid mediator inhibitors of ILC2 function, and thus limit ILC2 contribution to Th2 inflammation. ILC2s play a potential significant role in Th2 mediated inflammation in a variety of allergic disease states, such as asthma, atopic dermatitis, and chronic rhinosinusitis. The identification of lipid mediators as activators and inhibitors of ILC2 function provides additional therapeutic targets for altering ILC2 function during disease states.


Introduction
Group 2 innate lymphoid cells (ILC2) represent a group of cells that lack conventional lineage markers associated with well-established immune and inflammatory cells, such as T and B cells, mast cells, basophils, myeloid cells, and erythroid cells. Despite lacking lineage markers, ILC2s have been shown to express CD127 (IL-7Rα), CD25 (IL-2Rα), inducible T-cell costimulatory (ICOS), Sca-1, and the receptors for IL-25 (IL-17RB) and IL-33 (T1/ST2) [1]. One of the defining characteristics of ILC2s is the production of large amounts of Th2 cytokines upon stimulation. ILC2s have been shown to produce IL-5 and IL-13 in response to IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) and produce IL-4 in response to lipid mediators [2][3][4]. Specifically, ILC2s have been shown to have a larger capacity to produce Th2 cytokines compared to other cell types on a per cell basis [5]. Compared to other ILC group members, the secretion of Th2 cytokines is specific to ILC2s compared to Th1 cytokines secreted by ILC1, and Th17 cytokines released by ILC3. ILC2s also secrete other inflammatory mediators including IL-9 which can regulate immune cell survival, and amphiregulin which can regulate tissue repair [6][7][8][9][10].
There are increasing numbers of studies demonstrating an association between ILC2s and allergic inflammation, despite ILC2s not directly responding to allergen exposure, as they do not express antigen recognition receptors. Patients with chronic rhinosinusitis (CRS) and atopic dermatitis show increased levels of ILC2s in nasal polyps and skin lesions respectively [11][12][13]. Recent studies show elevated ILC2 levels in both the sputum and blood of adult severe asthmatics compared to mild asthmatics, as well as in the BAL, induced sputum, and blood of children with severe asthma compared to children without [14,15].
In addition, in mice, ILC2s have been shown to directly influence and contribute to the development of airway hyperresponsiveness (AHR). In the absence of IL-13-producing CD4 T cells, ILC2s are sufficient for induction of AHR and IL-13 production in response to either allergen or glycolipids when wildtype ILC2s are transferred to IL-13 deficient mice [16,17]. Additionally, during influenza infection in mice, ILC2s can induce AHR through the IL-33-IL-13 axis, which is independent of Th2 cells and adaptive immunity [18].
Despite ILC2 involvement in Th2 inflammation, an initial innate stimulation of epithelial cells, mast cells, eosinophils, and/or myeloid cells is required for ILC2 activation. ILC2s do not express antigen recognition receptors and thus do not directly interact with an antigen. However they are activated through either secreted mediators or direct cell contact with activated cells. Initially ILC2s were shown to be activated by IL-25, IL-33, and TSLP, which are epithelial cell-derived cytokines. In the past few years, studies have shown ILC2 activation by tumor necrosis factor (TNF)-like ligand 1A (TL1A) and modulation by prostaglandin and leukotriene lipid mediators [19,20].

Lipid Mediator Activation of ILC2
Several cell types have been known to rapidly produce lipid mediators, such as activated mast cells, macrophages, dendritic cells, and eosinophils [21]. A study by Roediger et al. found that mast cells and ILC2s interact in vivo [22]. This study demonstrated that mouse dermal ILC2s migrated closely to resident mast cells and formed stable interactions between the dermal ILC2s and resident mast cells [22]. In patients with CRS, it has been shown that the percentage of ILC2s found in eosinophilic nasal polyps was significantly higher compared to the percentage of ILC2s found in non-eosinophilic nasal polyps, and that an overall positive correlation existed between the percentages of eosinophils and ILC2s in nasal polyps [23]. This was also confirmed by a recent study by Bal et al., which determined that in patients with CRS, ILC2s were present in the majority of nasal polyps with eosinophils, however in nasal polyps with few or lacking eosinophils ILC2s were not detected [24]. Within the nasal polyps, ILC2s and eosinophils co-localized spatially such that areas with high ILC2 density also had high eosinophil density [24]. In addition, this study showed ILC2 distribution in the nasal polyp was not random but was embedded in areas of the nasal polyp with higher eosinophil density. Together these studies provide evidence of ILC2 interactions with both mast cells and eosinophils in vivo, which suggests crosstalk between the cell types and a mechanism by which these cells can lead to ILC2 activation potentially through lipid mediator release from mast cells and/or eosinophils.
Lipid mediators is a broader term for a class of bioactive lipids that includes eicosanoids, such as prostaglandins (PG) and leukotrienes (LT). Eicosanoids have long since been associated with Th2 inflammatory diseases and share a common origin, from arachidonic acid [25][26][27]. Arachidonic acid can be metabolized into downstream prostaglandins by the cyclooxygenase enzymes, COX-1 and COX-2, and to leukotrienes by the 5-lipoxygenase enzyme. Specifically, prostaglandin D 2 (PGD 2 ) and cysteinyl leukotrienes (CysLTs) have been shown to promote ILC2 responses [4,28], while PGI 2 and LXA 4 have been shown to dampen ILC2 responses [29,30]. This review focuses on the regulation of ILC2s by these lipid mediators.

Prostaglandin D 2 (PGD 2 )
PGD 2 is strongly correlated to a variety of allergic disorders, as it is generated by eosinophils and in large quantities by IgE-activated mast cells [26]. Therefore, it is not surprising that studies have shown the PGD 2 signaling pathway plays a role in allergic rhinitis, asthma [31,32] atopic dermatitis [33,34] CRS and nasal polyposis [35][36][37], and aspirin exacerbated respiratory disease (AERD) [38]. PGD 2 has also been found to be a more potent bronchoconstrictor than histamine in normal and atopic individuals [39]. This long standing strong correlation between PGD 2 and allergic inflammation makes PGD 2 a potential important mediator in ILC2 activation, especially as ILC2s express CRTH2, a receptor for PGD 2 . PGD 2 binds to two receptors DP1 and CRTH2 (also known as DP2) [40]. CRTH2 is highly expressed on the surface of ILC2s and is a commonly used phenotypic ILC2 marker in humans [41]. CRTH2 is a G protein coupled receptor involved in the chemotaxis of cells toward PGD 2 [40]. PGD 2 induces the chemotaxis of human peripheral blood ILC2s from both allergic and nonallergic individuals, however allergic ILC2s showed enhanced chemotaxis compared to nonallergic [42]. In addition, the chemoattractant effect of PGD 2 was dependent on CRTH2, as incubation of human peripheral blood cells with a pharmacologic CRTH2 antagonist inhibited PGD 2 -induced ILC2 chemotaxis [42]. In addition to the chemoattractant effect of PGD 2 , PGD 2 has been shown to induce IL-13 secretion from human peripheral blood ILC2s [30]. Notably, PGD 2 induction of IL-13 secretion from ILC2s was significantly reduced when ILC2s were also treated with a CRTH2 receptor antagonist, however the DP1 antagonist diminished PGD 2 induced ILC2, IL-13 secretion but not enough to achieve statistical significance. In addition, the stimulation of peripheral blood ILC2s with a CRTH2 agonist, but not a DP1 agonist, significantly increased IL-13 secretion [30]. This study suggests that while PGD 2 can signal through both the DP1 and CRTH2 receptors, CRTH2 plays a larger role in PGD 2 induced IL-13 secretion from ILC2s.
ILC2s are enriched in nasal polyps of patients with eosinophilic CRS and systemic corticosteroids are associated with reduced ILC2 levels in nasal polyps [23]. Prednisone like other corticosteroids have been shown to inhibit the prostaglandin metabolic pathway. Therefore, it is possible that administration of corticosteroids could influence PGD 2induced ILC2 responses. A recent study demonstrated that IL-33 and TSLP stimulate mast cells to produce PGD 2 , and that TSLP expression in nasal polyps of AERD patients correlates with PGD 2 -generating enzymes [43]. The potential of both TSLP and PGD 2 to activate ILC2s and TSLP to further induce PGD 2 generation suggests a network of signals that can occur during Th2 inflammation that results in synergistic ILC2 responses.
Similarly to human blood ILC2s, Tait Wojno et al. showed that murine ILC2s also express CRTH2 and exposure to PGD 2 causes ILC2 accumulation in the mouse lung in vivo [28]. Mice infected with N. brasiliensis had increased lung inflammation and ILC2 accumulation [28]. However when utilizing mice lacking CRTH2 or using a specific CRTH2 inhibitor, there was decreased ILC2 levels in the airways and decreased inflammation following N. brasiliensis infection. Therefore, N. brasiliensis-induced lung inflammation and airway ILC2 accumulation is CRTH2 dependent [28]. In addition, another study by Xue et al. showed that CRTH2 mediates the chemotaxis of ILC2s isolated from human skin, as well as peripheral blood, as PGD 2 induced migration of ILC2s was reduced by a CRTH2 specific inhibitor, TM30089 [3]. PGD 2 signaling through CRTH2 induces the activation of human ILC2s and the production of Th2 cytokines IL-4, IL-5, and IL-13 [3]. In addition, stimulation of ILC2s with PGD 2 in combination with IL-33 and IL-25 had increased induction of IL-4, IL-5, IL-13, as well as IL-9, when compared to IL-33 and IL-25 stimulation alone. PGD 2 also upregulates the expression of the IL-33 receptor, while it downregulates CRTH2 expression in human ILC2s [3]. Thus, PGD 2 can potentiate IL-33 and IL-25 responses and increase IL-33/IL-25 induced Th2 cytokine production [3,30]. Together these studies demonstrate the role of PGD 2 not only as a major chemoattractant for ILC2s to the site of inflammation, but also as a potent ILC2 activator to express Th2 cytokines.

Cysteinyl Leukotrienes (CysLTs)
CysLTs comprise of LTC 4 , LTD 4 , and LTE 4 , which are generated by cell types important to allergic inflammation including eosinophils, mast cells, basophils, and macrophages [44]. LTC 4 is the parent LT that is metabolized into LTD 4 and LTE 4 [21]. The main receptors for CysLTs are CysLT 1 R and CysLT 2 R. LTD 4 can bind both receptors, however it binds CysLT 1 R with higher affinity than CysLT 2 R. CysLT 2 R binds both LTC 4 and LTD 4 with equal affinity [45]. CysLTs have a variety of proinflammatory activities which are associated with allergic inflammation during innate immune responses, such as increased eosinophil migration, and activation of mast cells, macrophages, dendritic cells, and neutrophils [46]. Due to the proinflammatory nature of CysLTs, CysLTs have been associated with several allergic diseases such as asthma, allergic rhinitis, and AERD, as well as other diseases such as COPD [45][46][47][48]. Therefore, it is possible that CysLT production activates ILC2s during allergic inflammation, as CysLTs affect a wide array of cells and are strongly correlated to allergic inflammation.
Mouse lung and bone marrow ILC2s have been shown to express CysLT 1 R [4]. Additionally mouse lung ILC2s produce the Th2 cytokines IL-5 and IL-13 after stimulation with LTD 4 . Unlike IL-33 and IL-25, LTD 4 has been shown to also induce the secretion of  [49]. This study demonstrates a potential role for the ILC2-IL-4 axis in Th2 differentiation and provides a previously unidentified role for ILC2s in immunity which may be regulated by leukotrienes.

Prostaglandin I 2 (PGI 2 )
Similarly to other prostaglandins, PGI 2 is generated from arachidonic acid metabolism through the cyclooxygenase enzymes, however it also requires downstream conversion by PGI synthase (PGIS) [26]. PGI 2 is also known as prostacylin, and signals through the prostacylin receptor, IP. PGI 2 functions in both the innate and adaptive immune systems as mainly an immunosuppressive mediator. PGI 2 -IP activation of downstream protein kinase A leads to several antiinflammatory actions, such as reduced cell proliferation, relaxation of smooth muscle, and other cellular inhibitory mechanisms (i.e., IL-10 production) [50]. PGI 2 has a well-established correlation to allergic disorders, as several studies in mice have shown that PGI 2 administration diminishes features of allergic and asthmatic airways including airway eosinophilia, Th2 cytokine production, and AHR, while PGI 2 signaling deficiency leads to exaggerated allergic inflammation [51][52][53][54]. In addition, it has been shown that PGI 2 levels are elevated in the lungs of individuals during allergic inflammation, which demonstrates that PGI 2 is present during allergic inflammation in humans [26]. Therefore, PGI 2 is considered an important player in the regulation and dampening of Th2 inflammation.
A study by Zhou et al. investigated the role of PGI 2 in ILC2 regulation and found an inhibitory effect of PGI 2 on ILC2 function [29]. This study showed that mouse lung ILC2s express the PGI 2 receptor IP. Isolated wildtype mouse bone marrow ILC2s when treated with a PGI 2 analog, cicaprost, had significantly diminished IL-33induced IL-13 secretion in culture. Interestingly, cicaprost also inhibited IL-33 induced ILC2 proliferation. The effect of PGI 2 was specific to the IP receptor, as bone marrow ILC2s from mice deficient in IP stimulated with IL-33 and cicaprost maintained similar levels of IL-13 secretion and proliferation to IL-33 treatment alone. Similarly to mouse ILC2s, human blood ILC2s stimulated in vitro with IL-2 and IL-33 had diminished IL-5 and IL-13 secretion when treated with cicaprost compared to vehicle treated. These findings were expanded to show that mice deficient in IP had increased ILC2 accumulation in the lungs in response to Alternaria extract treatment compared to wildtype mice also treated with Alternaria. In addition, IP deficient mice had increased Th2 cytokine secretion when treated with Alternaria, while the number of CD4+ cells expressing Th2 cytokines was not altered by IP deficiency. Moreover, when wildtype mice were administered cicaprost together with Alternaria, there was diminished airway inflammation and diminished ILC2 secretion of Th2 cytokines. These findings support a role for PGI 2 as a potent inhibitor of ILC2 secretion of Th2 cytokines, as well as an inhibitor of ILC2 proliferation, which suggests that PGI 2 regulates airway inflammation through inhibition of ILC2 function.

Lipoxin A 4 (LXA 4 )
Lipoxins, like leukotrienes and prostaglandins, are derived from arachidonic acid, however they are a product of 5-, 12-, and 15lipoxygenases [55]. The primary lipoxins found in mammals are LXA 4 and its isomer LXB 4 . Lipoxins have a variety of pro-resolution and anti-inflammatory effects on cells. They have been shown to inhibit neutrophil and eosinophil recruitment and activation, stimulate macrophage phagocytosis of apoptotic cells, inhibit TNF secretion from T cells, as well as having effects on dendritic cells and monocytes, and on structural cells, i.e., fibroblasts and epithelial cells [56]. In addition to the active downstream effects of lipoxins, lipoxin generation is inversely related to leukotriene biosynthesis and therefore dampen leukotriene pro-inflammatory effects. Inflammatory mediators, such as IL-4, and IL-13, can enhance 15-lipoxygenase expression and subsequent lipoxin generation [56]. Due to the proresolution nature of lipoxins, the dysregulation of lipoxin synthesis has a role in a variety of diseases, including respiratory inflammation, renal diseases, and cancer [55]. Specifically, LXA 4 has a variety of proresolution actions that inhibit AHR and pulmonary inflammation, and if unregulated can lead to allergic and asthmatic symptoms [57]. Therefore it is not surprising that several studies have shown lower levels of LXA 4 correlate with the degree of airflow obstruction in asthmatics, and diminished LXA 4 levels are found in patients with asthma [58][59][60][61].
A study by Barnig et al. found that human peripheral blood ILC2s from both healthy and asthmatic individuals express the natural receptor for LXA 4 , ALX/FPR2, as well as the receptor for an additional pro-resolving mediator, CMKLR1 [30]. In addition, the authors found that PGD 2 potentiates IL-2/IL-25/IL-33 induced IL-13 secretion from ILC2 which is reduced when pretreated with equimolar amounts of LXA 4 . This reduction in ILC2 IL-13 production by LXA 4 was dependent on ALX/FPR2, as incubation with the receptor antagonist, WRW4, lowered LXA 4 inhibition of ILC2 secretion of IL-13. These findings are in agreement with the well-established role of lipoxins as anti-inflammatory and pro-resolution mediators.

Summary
While ILC2s were originally characterized as innate lymphoid cells that respond to epithelial-derived cytokines, IL-33, TSLP, and IL-25, an increasing amount of studies are demonstrating lipid mediators as significant regulators of ILC2 function. ILC2s are a significant source of Th2 cytokines, including IL-13, IL-5, and IL-4. In addition, PGD 2 has been shown to promote ILC2 recruitment and amplify ILC2 cytokine responses, as well as work with epithelial cell-derived cytokines to promote a more robust activation of ILC2s. Importantly, PGI 2 and LXA 4 have been shown to dampen ILC2 responses, demonstrating the role of lipid mediators as both positive and negative regulators of ILC2 function. Due to the increasing understanding of the role of ILC2 in Th2 inflammation, targeting of lipid mediators, both activating and inhibiting, provide attractive potential targets for a variety of allergic disorders.