Could Arachidonic Acid-Derived Pro-Resolving Mediators Be a New Therapeutic Strategy for Asthma Therapy?

Asthma represents one of the leading chronic diseases worldwide and causes a high global burden of death and disability. In asthmatic patients, the exacerbation and chronification of the inflammatory response are often related to a failure in the resolution phase of inflammation. We reviewed the role of the main arachidonic acid (AA) specialized pro-resolving mediators (SPMs) in the resolution of chronic lung inflammation of asthmatics. AA is metabolized by two classes of enzymes, cyclooxygenases (COX), which produce prostaglandins (PGs) and thromboxanes, and lypoxygenases (LOX), which form leukotrienes and lipoxins (LXs). In asthma, two primary pro-resolving derived mediators from COXs are PGE2 and the cyclopentenone prostaglandin15-Deoxy-Delta-12,14-PGJ2 (15d-PGJ2) while from LOXs are the LXA4 and LXB4. In different models of asthma, PGE2, 15d-PGJ2, and LXs reduced lung inflammation and remodeling. Furthermore, these SPMs inhibited chemotaxis and function of several inflammatory cells involved in asthma pathogenesis, such as eosinophils, and presented an antiremodeling effect in airway epithelial, smooth muscle cells and fibroblasts in vitro. In addition, PGE2, 15d-PGJ2, and LXs are all able to induce macrophage reprogramming to an alternative M2 pro-resolving phenotype in vitro and in vivo. Although PGE2 and LXA4 showed some beneficial effects in asthmatic patients, there are limitations to their clinical use, since PGE2 caused side effects, while LXA4 presented low stability. Therefore, despite the strong evidence that these AA-derived SPMs induce resolution of both inflammatory response and tissue remodeling in asthma, safer and more stable analogs must be developed for further clinical investigation of their application in asthma treatment.


INTRODUCTION
Asthma is a high prevalence chronic inflammatory pulmonary disease, the respiratory symptoms of which include cough, wheezing, shortness of breath, and chest tightness which leads to elevated morbidity, mortality, and disease social and economic costs (1)(2)(3). Pulmonary inflammation is the hallmark of asthma, which is driven by a Th2 immune reponse to inhaled allergens, and associated with infiltration of the bronchial mucosa with eosinophils, CD4+ T cells, macrophages and, in exacerbations and severe cases, neutrophils (4,5). Macrophages are classified into classical (M1) or alternative activation (M2a, M2b, M2c, or M2d subtypes). During allergic asthma, under exposure to Th2 cytokines (IL-4 and IL-13), macrophages are reprogrammed to M2a profile and perform diverse functions ranging between protective and pathogenic roles (6)(7)(8)(9). Airway remodelling is another key feature of asthma pathogenesis and can precede the development of inflammation (10). It is characterized by mucous gland and airway smooth muscle (ASM) cells hyperplasia and/or hypertrophy, deposition of extracellular matrix (EM) proteins, and myofibroblast proliferation, leading to the thickening and occlusions of airways (11). In severe asthmatic patients, a failure in pro-resolving pathways extends the pro-inflammatory mechanisms, resulting in a chronic inflammation, which is associated with a major cause of admission to the intensive care unit and high mortality rates (12,13). Lipid mediators, such as those originated by arachidonic acid (AA), are key factors of the resolution of inflammation, once they orchestrate the clearance of pro-inflammatory cells and signals promoting tissue restoration (13,14). In this review, we discussed the impact of AA-derived specialized pro-resolving mediators (SPMs) in the resolution of inflammation and remodeling in asthma.

RESOLUTION OF INFLAMMATION
The resolution of inflammation is an active and controlled process that reduces inflammation through the elimination of danger signals, leading to the restoration of tissue homeostasis and preventing the progression towards an uncontrolled chronic inflammatory state. Thus, catabolization and antagonization of pro-inflammatory mediators, a decrease in leukocyte numbers at inflammatory sites, and tissue repair are key events in the resolution process (13). It is notewothy that different from classical anti-inflammatory molecules, the SPMs modulate the end of the inflammatory response, without promoting unwanted immunosuppression (15). During the resolution phase, leukocyte apoptosis and metabolization of intracellular inflammatory signals lead to the clearance of inflammatory cells by specialized phagocytes. Together, these events promote the end of the acute inflammatory response and initiate tissue repair and healing (12,16).
Among the SPMs, the lipid mediators activate many aspects of the resolution process (23). These endogenous mediators are biosynthesized in local inflamed tissue microenvironments, and can control the magnitude/duration of the inflammatory response as well as the timing of tissue restoration (17). They primarily come from the metabolism of polyunsaturated fatty acids, such as AA, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and docosapentaenoic acid (DPA) (15). Interestingly, several AAderived mediators have consistently presented pro-resolving and tissue protecting activities in asthma (24,25).

SPMs DERIVED FROM CYCLOOXYGENASES
Cyclooxygenases (COX), especially COX-2 isoform, play a pivotal role in the conversion of AA into different pro-inflammatory lipid mediators, including prostaglandins (PG) and thromboxanes (26). Despite the clear ligation of COX-2 activity with the development of the inflammatory response, it has also been proved that the inhibition of this enzyme impairs leukocyte clearance, indicating that some COX-2 derived mediators possess pro-resolving action. This occurs mainly due to the ability of COX-2 to metabolize EPA into resolvins, which are one of the main classes of SPMs (27). In asthma, beyond resolvins, the COX-2 activity also culminates in the formation of other important SPMs, such as PGE 2 and 15-Deoxy-Delta-12,14-PGJ 2 (15d-PGJ 2 ), a metabolite of PGD 2 (28). PGE 2 is synthesized by three distinct enzymes, microsomal PGE synthase-1 (mPGES-1), mPGES-2, and cytosolic PGES (cPGES), which use PGH 2 as substrate. The actions of PGE 2 are mediated by four distinct 7TM receptors (EP1-EP4) (29). Although PGE 2 is a pro-inflammatory mediator, several works have shown that this lipid presents pro-resolving actions in some contexts (30). So, what determines when PGE 2 presents pro-resolving effects? There are three major factors, not mutually exclusive: i) time: the kinetics of PGE 2 release can separate its pro-inflammatory and pro-resolving effects due to the presence of different targets (31,32); ii) context: eg. PGE 2 can inhibit ERK activation and MMP-1 secretion by gastric epithelial cells in the presence of cytokines, however, in their absence, PGE 2 does the opposite (31,33,34); iii) concentration: eg. very low PGE 2 concentrations inhibit chondrocyte-dependent collagen cleavage in osteoarthritis cartilage, while higher concentrations enhance it (35).
IL-4 and IL-13, essential cytokines in the asthma pathogenesis (4), suppressed PGE 2 production by dendritic cells through reduction of COX-2 and mPGES-1 expression (36). Also, asthmatic patients presented an inverse correlation between the sputum levels of PGE 2 and eosinophil numbers (37,38), suggesting that PGE 2 may reduce airway eosinophilia in these patients. Inhaled PGE 2 markedly inhibits the early and late bronchoconstrictor response to an allergen in asthmatic patients (39); however, these effects may be related only to the PGE 2 -induced ASM relaxation (40). Nevertheless, COX-1 knock out (KO) and EP2KO mice that were ovalbumin (OVA)-sensitized and challenged showed increased eosinophilia and Th2 cytokines levels in the lungs and bronchoalveolar lavage (BAL), respectively, compared to wild-type (WT) mice (41,42). Besides, treatment with PGE 2 inhibited the house dust mite (HDM)-induced lung eosinophilia (43), and OVAprovoked accumulation of eosinophils and Th2 cytokines in the BAL (42), probably because PGE 2 can inhibit b 2 integrin and Lselectin function with a consequent reduction in eosinophil migration (44,45). Furthermore, prior studies of our group showed that PGE 2 derived from eosinophils induced an early resolution of allergic pleural edema (25,46).
Until now, there has been no agreement on the effects of PGE 2 on the differentiation of naïve T cells to Th1, Th2, or Th17 (47); however, type 2 innate lymphoid cells (ILC2), that emerged in the literature as novel Th2 cytokine-producing cells, strongly express both EP2 and EP4. PGE 2 inhibited proliferation, activation, and release of cytokines by ILC2 (48,49). Besides, alveolar macrophages from asthmatics presented a reduction in the EP2 expression (50) and PGE 2 generation, in parallel with decreased efferocytosis of apoptotic cells (51). PGE 2 is a well-known inductor of M2 macrophage reprogramming (52). Furthermore, PGE 2 induced IL-10 production by macrophages in vitro, and the adoptive transfer of those PGE 2 -treated macrophages led to fewer infiltrating eosinophils, macrophages, activated TCD4+, and regulatory T lymphocytes in lungs of HDM-exposed mice (43).
In lung fibroblasts, there is an inverse relationship between COX-2 and mPGES-1 expression and the number of allergen challenges, resulting in a reduction in PGE 2 production by those cells (53). Besides, mPGES-1 KO mice showed an augmentation of allergen-induced vascular smooth muscle cell numbers and thickness of intrapulmonary vessels (54). PGE 2 also inhibited fibroblast migration, proliferation, collagen deposition, and myofibroblast differentiation in the lung (55). In vitro, PGE 2 decreased the expression of tenacin C and fibronectin by human fibroblast and ASM cells (56), reduced the proliferation of ASM cells derived from asthmatic patients (57), and upregulated the expression of the anti-inflammatory protein tristetraprolin in human ASM cells (58). Prior investigations of our group revealed that the instillation of glucagon induced a high production of PGE 2 into the lungs (59). Also, we reported that a non-selective COX inhibitor decreased the inhibitory effect of glucagon on OVAinduced collagen deposition in the lungs (60), suggesting that the anti-remodeling effect of glucagon depends on PGE 2 production. Interestingly, inhaled PGE 2 showed bronchodilator capacity in small clinical trials with asthmatic patients (61, 62) ( Table 1).
Despite the possible benefits of PGE 2 in asthma, non-selective COX inhibitors improved specific airway conductance and airway constriction of asthmatics (63,64), suggesting that COX-derivatives may play a role in the development or worsening of asthma. Nevertheless, patients with mild allergic asthma treated with specific COX-2 inhibitors did not present an effect on lung function and eosinophil accumulation in the sputum (65) ( Table  1). Although PGE 2 acts directly in the resolution of inflammation, it can also drive a pro-inflammatory response in human fibroblast and ASM cells (56). Furthermore, PGE 2 apparently desensitized b2 adrenergic receptors during asthma exacerbation triggered by Etoricoxib

COX-2 inhibitor
Etoricoxib had no effect on allergen-induced airflow obstruction and sputum eosinophils, basal lung function, or methacholine responsiveness in mild asthma patients No side effects were observed Pioglitazone

PPAR-g agonist
Pioglitazone had no effect on symptoms, airflow obstruction and inflammation in patients with severe asthma Peripheral edema and presumptive angioedema  Rhinovirus infection (69). A high dose of PGE 2 can also induce airway contraction in asthmatic patients, probably through activating different receptors (70), and cough by activation of EP3 receptor (71). As the most of pro-resolving actions of PGE 2 are related to the activation of EP2, the development of selective agonists of this receptor can be a good strategy to be consider for treating asthma in the future. 15d-PGJ 2 is formed spontaneously by a series of dehydration of PGD 2 (72), and it is produced abundantly in the inflamed site, making it important in the resolution of the inflammation (73). Most of the pro-resolving actions of 15d-PGJ 2 depend on the peroxisome proliferator-activated receptor-gamma (PPARg) activation, but some of its effects are independent of this receptor (74). In asthmatic patients, there is a reduction in the PPARg expression in BAL cells (75). Furthermore, polymorphism of the PPARG gene may be related to an increased risk of asthma development (76). Activation of PPARg by synthetic agonists reduced the levels of Th2 cytokines and inhibited AHR, the influx of eosinophils and structural changes in the airway wall in murine OVA-challenge models of asthma (77,78). Together, these data indicate that the reduction in PPARg expression by inflammatory cells in asthmatic patients may be one of the mechanisms that contribute to the development of chronic asthma.
In a model of carrageenin-induced pleurisy, 48h after the provocation, when mononuclear cells dominate the reaction up to the resolution, there was an immense increase in COX-2 protein expression and 15d-PGJ 2 levels coincident with inflammatory resolution and associated with minimal exudate PGE 2 levels. In this model, the use of both nonselective or selective COX-2 inhibitors, 24h after carrageenin challenge, increased the number of inflammatory cells and exudate volume in parallel to a reduction in the 15d-PGJ 2 levels. In addition, 15d-PGJ 2 reversed the selective-COX-2 inhibitor-induced rise in cell number and exudate volume, indicating that the production of 15d-PGJ 2 is important to the resolution in this model (79). The pro-resolving effect of 15d-PGJ 2 was related to an induction of apoptosis of inflammatory cells (80). Besides, 15d-PGJ 2 also regulates the balance of cytokines and chemokines that control leukocyte trafficking during acute inflammation, promotes M2 macrophage differentiation, as well as the efflux of macrophage to draining lymphatics, facilitating the resolution of inflammation (81). This pro-resolving effect of 15d-PGJ 2 may be dependent on PPARg, once IL-4-induced PPARg activity becomes indispensible for M2 activation (82,83).
In an OVA-induced asthma model, KO mice for PGD synthase (H-PGDS), an enzyme that catalyzes PGH 2 into PGD 2 , showed accelerated chronic allergic lung eosinophil inflammation in parallel to an increase in the local levels of TNFa and eotaxin-1. Furthermore, the exogenous administration of 15d-PGJ 2 decreased the excessive eosinophilic infiltration and TNFa and eotaxin-1 levels noted in those mice (84). Furthermore, the activation of PPARg reduced OVA-induced eosinophilia and IL-4, IL-5, and IL-6 levels in the lungs of mice (85). We previously showed that interventional treatment with 15d-PGJ 2 inhibited both OVA-and HDM-induced eosinophils accumulation and IL-5 and IL-13 levels in the lungs (86). The pro-resolving effect of 15d-PGJ 2 on lung eosinophilia is probably related to its ability to block the traffic and induce apoptosis of these granulocytes (87). The inhibitory effect of 15d-PGJ 2 on eosinophil migration is possibly dependent on PPARg, once the activation of this receptor by synthetic agonists inhibits chemotaxis of eosinophils (85). However, the pro-apoptotic effect of 15d-PGJ 2 is independent of PPARg (87). 15d-PGJ 2 also inhibited T lymphocyte proliferation in a mechanism probably dependent on PPARg, as it is mimicked by PPARg synthetic agonists (88,89).
We previously demonstrated that interventional treatment with 15d-PGJ 2 reversed structural changes related to airway remodeling, including epithelial thickening, mucus exacerbation, and EM deposition, in both OVA and HDM murine models of asthma (86). These antiremodeling effects of 15d-PGJ 2 may be related to its ability to reduce differentiation of fibroblasts into myofibroblasts, the proliferation of myofibroblasts (90), and fibroblast growth factor-induced human ASM cell proliferation (91). Although PPARg agonists are extremely promising to asthma therapy, unfortunately severe asthmatic patients treated with pioglitazone did not present with an improvement in asthma features and showed significant side effects (66) ( Table 1).
The failure in the generation and action of LXs is associated with more severe airway inflammation (99). Indeed, severely asthmatic patients presented a reduction of LXA 4 levels in BAL fluid, sputum, and whole blood compared to moderately asthmatic individuals. This reduction in LXA 4 concentrations observed in severe asthma was associated with a higher degree of airway obstruction (24). Eosinophils from the blood of asthmatic patients presented a decreased ALXR expression compared to those obtained from healthy individuals (100). Furthermore, transgenic mice that overexpress ALXR showed a reduction in OVA-induced eosinophilia in the BAL and lung tissue (101). We previously showed that 15-epi-LXA 4 analogs inhibited OVA-induced pleural eosinophil influx by reducting local eotaxin and IL-5 generation (102). We also noted that 15-epi-LXA 4 analogs accelerate the drainage of OVA-induced pleural edema (25). In human eosinophils, LXA 4 inhibited chemotaxis toward chemoattractants (103), and granulocyte-macrophage colony-stimulating factor- induced IL-13 and eotaxin release in vitro (104). In spite of inhibiting eosinophil migration, 15-epi-LXA 4 is a potent chemoattractant to monocytes in vitro (105) and restored the balance between M2 and M1 populations into the lungs in a murine model of pulmonary damage induced by bleomycin (106). Furthermore, LXA 4 stimulates macrophage efferocytosis of apoptotic polymorphonuclear cells and cellular debris (107). Among the ILC family, natural killer (NK) cells and ILC2s are important in the control and exacerbation of asthma, respectively. NK cell depletion induced a persistent allergic airway inflammation in association with reduction of the LXA 4 levels in the BAL (108). LXA 4 enhanced activated NK cells-induced eosinophil apoptosis through ALXR activation (109). Meanwhile, the blood and sputum of patients with severe asthma presented elevated numbers of ILC2 compared to mild asthmatics, which was related to persistent airway eosinophilia (110). LXA 4 inhibited both PGD 2 -and IL-25 plus IL-33-induced IL-13 release by ILC2 in vitro in a mechanism dependent on ALXR activation (109).
In vitro, LXA 4 and 15-epi-LXA 4 reduced IL-8 secretion induced by serum amyloid A in a human alveolar A549 cell line (111). Also, activation of ALXR by LXA 4 increased basal proliferation and wound repair of human airway epithelial cells (112). In a murine model of asthma caused by OVA, airway epithelial cells presented with an increased expression of ALXR (101) and LXB 4 reduced mucus production (113). ASM hypertrophy and hyperplasia, as well as accumulation of muscle cells in the subepithelial layer, are some of the changes observed in asthma remodeling. It was shown that LXA 4 reduced both LTE 4 -and IL-13-primed ASM migration toward plateletderived growth factor in vitro (114). Another critical pathological feature of airway remodeling in asthma is the EM deposition in the peribronchiolar area. It is noteworthy that both fibroblasts and myofibroblasts can express ALXR (115). Moreover, LXA 4 inhibited connective tissue growth factor-induced human lung fibroblast proliferation in vitro (115) and blocked TGF-btriggered increase in a-smooth muscle actin expression and collagen release by human myofibroblasts in vitro (116). Besides, treatment with 15-epi-LXA 4 reversed bleomycinpromoted fibrosis and lung damage in mice (106). Altogether, these data suggest a potential role of LXs in the resolution of the airway and peribronchiolar remodeling observed in asthmatics.
Due to the possible therapeutic application of LXA 4 , some clinical trials using this LX, its analogues, or LXA 4 receptor agonist BML-111 were administered in asthmatic patients. The nebulization of LXA 4 reduced LTC4-induced bronchoconstriction (67); however, the rapid inactivation and significant instability to exposure to light and acids of LXA 4 (117) make its clinical use difficult. Furthermore, the inhalation of LXA 4 analog or BML-111, which is more potent and stable than LXA 4 itself (118), improved the lung function (68) ( Table 1). Interestingly, both LXA 4 analog and BML-111 were well tolerated and presented no side effects (68).

CONCLUSION
This mini-review presents several aspects of the pro-resolving effects of COX-and LOX-derivative mediators in asthma (Figure 1), addressing their efficacy and current limitations for clinical use. Nevertheless, the review presents several strong pieces of evidence that support the development of new drugs based on analogs of PGE 2 , 15d-PGJ 2 , and LXs with better physical-chemical properties, allowing greater stability and superior selectivity for specific receptors. Moreover, new analogs of AA-derived SPMs could also improve efficiency and reduce the required dose of glucocorticoid, the latter often leading to adverse effects and steroid-refractoriness, despite being the best asthma treatment so far.

AUTHOR CONTRIBUTIONS
DI, MF, and DC contributed to the conception and design of the study, wrote the manuscript, discussed the content, and contributed to the manuscript revision. MM discussed the content and contributed to the manuscript revision. VC contributed to the conception and design of the study, wrote the manuscript, discussed the content, and contributed to the manuscript revision. All authors reviewed and/or edited the manuscript prior submission. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by PrInt Fiocruz-CAPES Program N°0 1/2020.