Polyunsaturated fatty acids and lipid mediators controlling chronic inflammation in asthma

Introduction

Asthma is a common chronic heterogeneous inflammatory respiratory disease that affects about 300 million people worldwide and is characterized by wheezing, coughing, shortness of breath, reduced expiratory airflow, hyperresponsiveness, airway remodeling, and mucus overproduction [1, 2, 3].

Asthma includes phenotypes and endotypes that differ in trigger factors, the degree of control over the disease, the nature of the immune response [4, 5]. The asthma phenotypes reflect clinical and physiological signs and trigger factors, and allow predicting the efficacy of treatment and controlling the dynamics of the disease development [6]. There are several asthma phenotypes: allergic asthma, non-allergic asthma, late-onset asthma, asthma with fixed airflow obstruction, and asthma in obese patients [7]. The asthma endotypes are distinguished based on specific pathobiological markers [8]. The Th2 (T helper 2 cells) asthma endotype is associated with the recruitment of type 2 innate lymphoid cells (ILC2), IgE-secreting B cells, and eosinophils [9]. This endotype may include subendotypes characterized by high levels of interleukins (IL)-5, IL-13 or IgE. The Th2 endotype includes early-onset asthma in children and adolescents, eosinophilic asthma in adults, aspirin-induced asthma, asthma-COPD overlap syndrome (ACOS), etc. The patients without atopy and allergy symptoms have the non-Th2 endotype [10]. The activation of bronchial epithelial cells, along with a production of IL-33, IL-25, IL-17 and thymic stromal lymphopoietin (TSLP), are observed in this type of inflammation. The Th17 endotype is diagnosed in steroid-resistant asthma [11]. The mixed Th2/Th17 endotype is known as well [12]. Non-Th2 asthma endotype includes exercise-induced asthma, asthma associated with obesity, late-onset asthma, asthma in smokers, or neutrophilic asthma in non-smokers, to name a few.

However, even the detection of asthma phenotypes and endotypes aimed at choosing effective therapeutic strategies for relieving the symptoms of the disease does not allow neutralizing chronic inflammation in the bronchopulmonary system [13]. The number of asthma patients worldwide will be approaching 400 million people during next five years [14]. The high prevalence of this pathology is associated with reduced life quality in patients, difficulties in controlling the disease symptoms, patient resistance to glucocorticosteroids (GCS), and economic burden on the health care system [7]. Asthma treatment involves inhaled β2-adrenergic receptor (β2-AR) agonists, inhaled and systemic GCSs, and leukotriene synthesis inhibitors (LTSIs) in combination with leukotriene receptor antagonists (LTRAs) [15]. Most asthma patients respond well to GCS monotherapy or its combination with long-acting β2-agonists and/or LTRAs, which leads to the achievement and maintenance of the disease control. At the same time, such therapy is not effective enough in 5-10% of cases [16]. Besides, extended GCS administration results in numerous side effects, which reduces the efficacy of treatment [17, 18]. Hence, further studies of the regulatory mechanisms of inflammation and ways of its resolution in asthma are essential.

Impaired inflammation resolution mechanisms play an important role in the development of persistent low-grade bronchopulmonary inflammation [13, 19, 20]. The functioning of immunocompetent cells involved in the resolution process depends on the composition of fatty acids (FAs) in their cell membranes, dietary intake of FAs, and the capability to metabolize polyunsaturated fatty acids (PUFAs) [21]. Therefore, the modification of FA composition can lead to disruption of regulatory mechanisms of the inflammatory process [22, 23]. It is well known that ω-3 and ω-6 PUFAs are precursors for the synthesis of proinflammatory eicosanoids, specialized pro-resolving mediators (SPMs) [24, 25], and endocannabinoids [26]. Endocannabinoids are a group of endogenous lipids that influence the resolution of inflammation, but can also increase the production of proinflammatory mediators [27]. The resolution of acute inflammation consists in switching the synthesis of proinflammatory lipid mediators to the formation of SPMs [28]. The impairment of this process underlies the development of chronic inflammation [29, 30, 31, 32, 33]. Therefore, the study of the role of eicosanoids, SPM and endocannabinoids in the regulation of inflammatory responses in bronchial asthma allows us to expand our understanding of the mechanisms of impaired resolution of inflammation and find new targets for asthma treatment. [30, 31, 34].

It is noteworthy that ω-3 PUFAs and SPMs are agonists of nuclear peroxisome proliferator-activated receptors (PPARs) and glucocorticoid receptor (GR) that exhibit anti-inflammatory effects [35]. The activation of PPARs and GR leads to the initiation of the signaling pathways blocking the expression of inflammatory nuclear transcription factors, such as nuclear factor-kappa B (NF-kB) and activating protein 1 (AP-1) [36]. On the contrary, ω-6 PUFAs and their proinflammatory derivates enhance the expression of NF-kB triggering the synthesis of inflammatory cytokines, nitric oxide, etc. [37]. There is evidence that ω-6 PUFAs and their oxidized forms are responsible for the development of resistance to GCSs in some asthma patients via increasing the NF-kB expression and decreasing GR expression [38]. Endocannabinoids are also agonists for several subtypes of PPARs (α/γ/δ) [39, 40, 41]. Therefore, maintaining a balance between ω-3 and ω-6 PUFAs is an important factor determining the inhibition of inflammation by stimulating the formation of SPMs. The results of contemporary studies established that SPMs are potentially the most effective therapeutic targets in asthma and allergic diseases [42]. These results created fundamentally new opportunities for developing an effective strategy for the treatment of chronic inflammatory diseases of the bronchopulmonary system based on correcting the processes of inflammation resolution.

The effectiveness of ω-3 PUFAs intake to correct the lipid metabolism disorders and immune imbalance in asthma is under investigation [43, 44, 45]. Numerous epidemiological studies of the effect of consuming oily fish or supplements containing ω-3 PUFAs on the development of allergic reactions and asthma during pregnancy, breastfeeding, and during different age periods were carried out [46, 47, 48]. These results confirm the protective properties of ω-3 PUFAs and the relationship between a reduction in fish oil intake and elevated number of people suffering from asthma or other allergic diseases. At the same time, there are many conflicting data on the effectiveness of ω-3 PUFAs in chronic inflammation [49, 50]. Therefore, despite the prospects and great interest in the therapeutic potential of ω-3 PUFAs, the mechanisms of their anti-inflammatory action require clarification.

This review systematizes general knowledge on the biosynthesis and metabolic transformations of long-chain ω-3 and ω-6 PUFAs, their functions as precursors for the formation of proinflammatory and specialized pro-resolving lipid mediators. A contemporary view of the role of lipid mediators in the pathogenesis of chronic inflammation in asthma is highlighted. Some clinical aspects of the anti-inflammatory and pro-resolving action of ω-3 PUFAs are described. Prospects for the use of ω-3 PUFAs aimed at the resolution of chronic bronchopulmonary inflammation are shown.

The role of ω3 and ω6 polyunsaturated fatty acids

FAs are carboxylic acids with a carbon chain containing carboxyl and methyl groups at the ends. FAs are classified into saturated (with no double bonds) and unsaturated. The latter are further classified into monounsaturated (possessing one double bond) and polyunsaturated (containing from two to six double bonds). PUFAs are classified with respect to the position of the first double bond into ω-9, ω-7, ω-6, and ω-3 FAs [51, 52]. According to the length of the carbon chain (C), FAs can be divided into long-chain (C12-C22), medium-chain (C7-C12) and short-chain (C2-C6). Medium- and short-chain FAs are synthesized de novo or are a result of fat consumption. Short-chain FAs are synthesized in the intestine and then enter the circulatory system. Since some ω-3 and ω-6 PUFAs are essential (α-linolenic and linoleic acids), their sufficient intake with food is a prerequisite for the normal functioning of the body [53, 54].

The stages of the synthesis of ω-6 PUFAs from linoleic acid and ω-3 PUFAs from α-linolenic acid, as well as enzymes involved in PUFA biosynthesis, are presented in Table 1.

Table 1. Biosynthesis of ω-3 and ω-6 PUFAs

The biosynthesis and metabolic transformations of ω-6 and ω-3 PUFAs are mediated by the same enzymes responsible for elongation and desaturation: elongase 2 (ELOVL2), elongase 5 (ELOVL5), Δ5 desaturase (delta-5 desaturase, or D5D, also known as FADS1), and Δ6 desaturase (delta-6 desaturase, or D6D, also known as FADS2) [55]. Elongases are responsible for the lengthening of the PUFA hydrocarbon chain. Desaturases catalyze the transformation of a single bond between carbon atoms into the double unsaturated bond (C=C) [56]. Since the enzymes involved in the synthesis of 20-22 PUFAs are universal for both ω-3 PUFA and ω-6 PUFAs, these FAs compete for the enzyme system. The mechanism underlying the development of a certain metabolic pathway mediated by elongases and desaturases is still not fully understood.

Subsequently, the metabolic transformations of PUFAs follow the enzymatic oxidation pathways for eicosanoid (proinflammatory mediators) and SPM production (pro-resolving mediators) [30]. PUFAs are released from membrane phospholipids with the participation of phospholipase A2 (PLA2). The latter can be of three different types: cytosolic calcium-dependent PLA2 (cPLA2), cytosolic calcium-independent PLA2 (iPLA2), and secreted PLA2 (sPLA2)) [57]. The synthesis of eicosanoids and SPMs are catalyzed by cyclooxygenases (COX1 and COX2), lipoxygenases (5-LOX, 8-LOX, 12-LOX, 15-LOX), cytochrome P450 (CYP 450) and leukotriene A4 hydrolase (LTA4H) [58, 59].

The role of eicosanoids in asthma pathogenesis

Eicosanoids include leukotrienes (LTs), prostanoids (prostaglandins – PGs, cyclopentenone PGs, thromboxanes – TXs), and metabolites of hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acids (DHETs) [60-68].

LTs are classified into LTB4, LTB5 (the less potent LTB4 analog), as well as LTC4, LTD4, LTE4, LTF4 that belong to the group of cysteinyl leukotrienes (CysLT) and are formed after the conversion of arachidonic acid (AA) to LT A4 [61, 62, 69-72].

Prostanoids include PGs (prostaglandin I2 [PGI2 or prostacyclin], PGD2, PGE2, PGF2α), cyclopentenone PGs (15-deoxy-∆-12,14-prostaglandin J2 [15d-PGJ2], Δ12-PGJ2, PGJ2, PGC2, PGA1, PGA2), and TXs (thromboxane А2 [TxА2], TXB2) [73-90].

Eicosanoids are involved in the regulation of various processes occurring in the cell [58, 67-94] (Table 2).

Table 2. Eicosanoids and their functions

Leukotrienes

LTB4 plays an important role in airway inflammation [61]. LTB4 mediates plasma exudation, thereby causing the mucosal edema. This LT is a neutrophil chemoattractant: it stimulates macrophages to produce interferon-alpha (IFN-alpha), IL-17, IL-6, and IL-1-beta. LTB4 and LTB5 bind to the LTB4R1 and LTB4R2 receptors (also known as BLT1 and BLT2, respectively).

CysLTs are mainly secreted by mast cells and leukocytes [70] and act via three major G protein-coupled receptors (CysLT1R, CysLT2R, and CysLT3R) [61]. The biosynthesis of LTC4 in mast cells is catalyzed by cPLA2 α, 5-LOX, 5-lipoxygenase-activating protein (FLAP) and LTC4 synthase (LTC4S). The enzyme cPLA2α induces AA synthesis; 5-LOX and FLAP oxidize AA to produce LTA4, and LTC4S conjugates LTA4 with glutathione to synthesize LTC4. Cellular proteases metabolize LTC4 to generate more stable products (LTD4 and LTE4) [71]. Then, all three LTs bind to CysLTR1 and CysLTR2 receptors. It is well known that CysLTs are proinflammatory mediators, powerful bronchoconstrictors; they cause hyperresponsiveness and edema of the bronchi, and are involved in airway remodeling. The role of CysLTs in the development of allergies and asthma was extensively studied in this regard [61, 70]. LTD4 binds to CysLT1R and is the most potent bronchoconstrictor [71]. Many studies established that CysLT1R antagonists, such as montelukast, have a beneficial effect on asthmatics, especially GCS-resistant asthma patients [61]. LTD4 can induce the inflammatory reaction mediated by airway epithelial cells and promote airway remodeling in both allergic and non-allergic asthma [72]. 

Рrostanoids

Prostanoids are a large group of inflammatory mediators (PGs and TXs) derived from AA [73]. Prostaglandins are powerful vasodilators, inhibit platelet aggregation, and regulate smooth muscle cell contraction. Most PGs have a proinflammatory action (for example, PGE2 and PGD2), but cyclopentenone PG (PGA2 and 15d-PGJ2) is characterized by anti-inflammatory and antioxidant effects. Structural differences between PGs and the variety of their receptors (G protein-coupled receptors) determine different biological activities of PGs and their opposite effects in different tissues.

The interaction between PGI2 and the prostacyclin receptor (the prostaglandin I2 receptor or IP) leads to activating vasodilation and myocardial protection suppressing platelet aggregation and inhibiting smooth muscle proliferation [73, 74].

PGE2 is among the most abundant proinflammatory PGs. It causes vasodilation and elevated permeability of lung microvessels, which results in accumulating inflammatory cells at the site of infection. PGE2 increases mucociliary transport and liquid volume of the airway surface, and provokes type 2 T helper cell (Th2) responses. However, PGE2 can also perform pro-resolving functions. The multifunctionality of this PG is associated with the presence of a different type of its receptors [75]. The prostaglandin E2 (PGE2) receptors include the following isoforms: prostaglandin E2 receptor 1 (EP1), prostaglandin E2 receptor 2 (EP2), prostaglandin E2 receptor 3 (EP3), prostaglandin E2 receptor 4 (EP4) [76]. EP1 receptor mediates hyperalgesia, suppresses pain, stimulates colon cancer development in mice, induces hypertension in diabetic mice, and enhances the differentiation of uncommitted T cell lymphocytes to the Th1 cell phenotype differentially involved in etiology of acute brain injuries. The activation of EP2 promotes the development of inflammation by regulating B cell immunoglobulin class, and maturation of T lymphocyte CD4−CD8− cells to CD4+CD8+ cells. However, EP2 activation also suppresses inflammation by affecting immune cells (neutrophils, monocytes, macrophages, dendritic cells, NK cells, fibroblasts) [77]. EP3, like EP4, have different functions; at the same time many of them were found only in animals, but not in humans. EP3 play an important role in reducing allergic reactivity (at least in mice). H. Gao et al. demonstrated that cough induced by PGE2 and depolarization of the vagus nerve in vitro were inhibited by the administration of EP3 receptor [78].

PGF2α is a potent bronchoconstrictor and inflammatory mediator [79]. This prostaglandin is involved in the regulation of lipopolysaccharide-induced systemic inflammation [80]. Additionally, PGF2α concentration in blood plasma is a marker of oxidative stress in asthma [81].

PGD2 is expressed on eosinophils, basophils, Th2 cells, ILC2, macrophages, as well as mast, epithelial and smooth muscle cells [74, 82]. Prostanoid PGD2 causes bronchospasm, vasodilation and regulates functioning of immune cells through binding receptors DP1 (receptor 1 PGD2) and DP2 (receptor 2 PGD2). DP1 is expressed on platelets, eosinophils, basophils, T cells, macrophages, and endothelial cells [83]. It mediates vasodilatation, induces smooth muscle relaxation and eosinophil apoptosis. DP2 receptor has a wide range of ligands: PGD2 and its metabolites, including Δ12PGD2, Δ12PGJ2, 15-deoxy-Δ12,14PGD2, 15-deoxy12,14PGJ2, and 9α11βPGF2 [84]. PGD2, just as PGF2α, is a bronchoconstrictor and mediator of airway inflammation [79]. It was shown that PGF2α and PGD2 levels positively correlated with the severity of airflow obstruction in asthma, while PGE2 was not associated with lung functioning [85]. Concentrations of PGD2 and CysLT in exhaled air condensate and sputum were remarkably higher in asthma patients than in healthy subjects, and these parameters varied depending on the severity of asthma [86].

The analysis of metabolites of PGs, CysLTs, and isoprostanes provides the basis for a new, noninvasive approach to molecular phenotyping of asthma in adults and adolescents [62]. Concentrations of these metabolites in healthy subjects (except for PGE2) are not associated with age, body mass index, or sex. High levels of LTE4 and PGD2 are related to lower lung function, increased concentration of nitric oxide and increased amounts of eosinophils in the blood, sputum, and urine in adolescents with asthma [62].

TxA2 is produced by platelets, neutrophils, monocytes, macrophages, and cells of the lung parenchyma [87, 88]. It is known that PG can be converted to TxA2 by the TxA2 synthase; the latter is expressed in the lungs, kidneys, liver, monocytes and megakaryocytes. TxA2 transmits signal through the TP receptor (thromboxane receptor) (TPα and TPβ). TPα activates adenylate cyclase that leads to an increase in cAMP levels and the induction of intracellular signaling, while TPβ inhibits it. TxA2 is activated during tissue damage and inflammation, causing platelet aggregation and vasoconstriction. At the same time, PGI2 inhibits platelet aggregation and smooth muscle proliferation and induces vasodilation. S. Braune et al. described the effect of prostanoids on platelets, which are key participants in thrombosis and hemostasis. [89]. Since TxA2 and PGI2 have antagonistic effects on thrombosis and atherogenesis, their balance is necessary for maintaining vascular homeostasis. The activation of TP affects platelet aggregation and airway smooth muscle contraction; therefore, increased TxA2 activity may play a role in the pathogenesis of asthma. Interestingly, mast cells release PGD2 under in vitro conditions, but inhibition of this process by using hematopoietic prostaglandin-D-synthase (hPGDS) results in PGE2 and TXA2 secretion [90]. Contrarywise, if thromboxane synthase activity is suppressed, PGD2 level increases. The authors emphasized that it is necessary to prove the presence of these changes in vivo and their clinical significance.

TXA₂ is an extremely unstable metabolite that is hydrolyzed to stable but physiologically inert TxB₂ in the absence of the enzyme [91]. TxB₂ correlates with the severity of airflow obstruction in asthma [85, 92-94].

Thus, prostanoids are involved in many physiological and pathophysiological processes. Further study of the role of eicosanoids in asthma pathogenesis is necessary for developing personalized treatment of this pathology.

Pro-resolving lipid mediators in asthma pathogenesis

SPMs include resolvins (Rvs), lipoxins (LXs), protectins (PDs), maresins (MaRs), and 15-epi-lipoxin A4 [34, 95-101]. SPMs act on receptors of various cells of the immune system: lymphocytes, neutrophils, macrophages, endothelial and epithelial cells [20]. They limit the production of proinflammatory mediators, including PGs, LTs, and selective cytokines; stimulate bacterial phagocytosis and efferocytosis of apoptotic cells, thereby ensuring the restoration of homeostasis (Table 3).

Table 3. Pro-resolving lipid mediators and their functions

Decreased SPM synthesis is observed in asthma, which contributes to the chronicity of the inflammatory process in the bronchopulmonary system [20, 34, 96]. Therefore, the administration of SPMs can be an effective way to treat asthma. However, insufficient understanding of the SPM signaling pathways and rapid inactivation of SMPs limit their translational potential. Stable low-molecular-weight SPM mimetics and agonists of their receptor became new potential medicinal drugs. Recently, preclinical studies demonstrated that they can effectively reduce asthma manifestations by the resolution of inflammation [42].

Maresins

MaR biosynthesis is initiated in macrophages. The 12-LOX enzyme converts docosahexaenoic acid to 13S, 14S-epoxymaresin; then hydrolase (or epoxide hydrolase) stimulates the formation of MaR1 and MaR2 from this metabolite. Recently discovered SPM sulfido-conjugates (SPM-SCs) are also synthesized from 13S, 14S-epoxymaresin [31, 34, 98]. The SPM group includes maresin conjugates in tissue regeneration (MCTRs), protectin conjugates in tissue regeneration (PCTRs), and resolvin conjugates in tissue regeneration (RCTRs) [99]. MCTR1 is formed from 13S, 14S-epoxymeresin (under the influence of glutathione-s-transferase mu4 [GSTM4] and/or leukotriene-C4-synthase [LTC4S]). Then gamma-glutamyltransferase (GGT) converts MCTR1 to MCTR2, which is followed by conversion into MCTR3 via dipeptidase (DPEP). MaRs are believed to act as potent mediators of the inflammatory activity of macrophages, thereby contributing to the resolution of acute inflammation and tissue regeneration. MaR1 exhibits powerful resorption, regenerative, and antiplatelet properties [100]. MaR2 modulates an adaptive immune response in human peripheral blood lymphocytes. These data provide new possibilities for developing approaches to the modulation of chronic inflammation via affecting MaR2 [101]. MCTRs are the most common CysLTs in the lungs of healthy people, while other CysLTs are widely expressed in patients with asthma [31, 98]. MCTRs act as regenerating agents with anti-inflammatory properties [34]. The decreased MCTR1 level and the increased MCTR3 level in asthma indicate the enzymatic conversion of MCTR1 to MCTR3. However, this process is slower than the conversion of LTC4 to LTD4 and LTE4. In patients with severe asthma, CysLT production is elevated, while LX synthesis is reduced. Particularly, MCTRs was shown to block the LTD4-induced contraction of the respiratory muscles and facilitate the resolution of airway allergic response in mice [99].

Lipoxins

LXs (LXA4 and LXB4) are derivatives of AA, but unlike eicosanoids, they exhibit pronounced anti-inflammatory properties and activate the processes of resolution and recovery [102]. LXs can be formed in several ways. For example, LXA4 and LXB4 are synthesized by neutrophils through 5-LOX pathway; and by eosinophils, neutrophils and macrophages via 5-LOX and 15-LOX; while LXA4 is generated by platelets via 12-LOX. LXs suppress IL-8 secretion by leukocytes and epithelial cells and also stimulate macrophage efferocytosis [103]. LXA4 and LXB4 are derivatives of LOX-mediated metabolism and important pro-resolving mediators in asthma [104]. The therapeutic potential of 15-epi-LXA4 for the resolution of acute lung inflammation has been demonstrated [97]. It was noted that LXs, PGE2 and 15d-PGJ2, are capable of inducing reprogramming of macrophages to the M2 phenotype in vitro and in vivo. Although PGE2 and LXA4 exhibited beneficial effects in patients with asthma, further investigation of their mechanisms of action is required, since, according to the obtained results, PGE2 caused side effects and LXA4 had low stability [104]. Taking into account the discovered disorders in LX production in asthma, these mediators may be of significant interest as a therapeutic target.

Resolvins

Resolvins are biosynthesized from ω-3 PUFAs: eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [105]. Rv E-series (RvE1, RvE2) are derived from EPA, and Rv D-series (RvD1, RvD2, RvD3, RvD4, RvD5, RvD6) are formed from DHA [106, 107].

The anti-inflammatory effect of RvE1 is associated with its interaction with PPARs and blocking of NF-kB expression [108]. The RvE1 receptors are LT B4 receptor 1 (BLT1) and G-protein coupled receptor (Chemerin Receptor 23 – ChemR23). RvE1 has an antagonistic effect on BLT1 and blocks biological effects of proinflammatory LTs. At the same time, RvE1 exhibits a synergistic effect on ChemR23, inhibits the activation of NF-kB and enhances phagocytosis [109]. RvE2 exerts a biological effect similar to RvE1, regulates neutrophil chemotaxis, and activates phagocytosis and synthesis of anti-inflammatory cytokines. RvD1 acts through LX A4 receptor (ALX) and G-protein-coupled receptor 32 (GPR32). Resolvin E4 (RvE4: 5S, 15S-dihydroxy-eicosapentaenoic acid) is a recently discovered resolvin that increases the efferocytosis of apoptotic cells by macrophages [110, 111].

RvD1 and RvD2 arrest the inflammatory process by modulating the adaptive immune response of human peripheral blood lymphocytes [107]. These lipid mediators reduce cytokine production by activating CD8+, Th1 and Th17 cells. Furthermore, they prevent the differentiation of CD4+ T cells into Th1 and Th17 by suppressing their transcription factors. In particular, RvD1 attenuates local and systemic inflammatory responses in sepsis-induced cardiac injury [112].

The mechanism of action of RvD and RvE in bronchopulmonary pathology is under investigation [31, 113, 114, 115]. RvE1 contributes to the resolution of airway inflammation by inhibiting eosinophil and lymphocyte recruitment and the production of IL-6, IL-13 and IL-23. RvE1 reduces airway sensitivity and inflammation in asthmatic mice [114]. Additionally, 17-epi-RvD1 is effective for the resolution of acute lung inflammation [97]. MaR1 and RvD1 inhibit the synthesis of cytokines by ILC2s and thereby participate in the control of ILC2-mediated eosinophilic airway inflammation and diseases [116]. Therefore, these lipid mediators may be the basis for developing a therapeutic strategy to alleviate airway inflammation.

Protectins

Protectins are formed from DHA and are synthesized by a number of cells, including brain cells, monocytes, and CD4⁺ lymphocytes [117]. PD1 (the main member of the protectin family) exhibits potent anti-inflammatory and neuroprotective actions through interacting with PPARs, blocking NF-κB expression, decreasing COX-2 expression and PG production. PD1 is a regulator of the protein synthesis of the B-cell lymphoma 2 (BCL2) family that has a pronounced antiapoptotic effect.

A decrease in PD1 level was revealed in severe and uncontrolled asthma. Recently, PD1 was identified in exhaled breath condensate in asthma patients during exacerbation of the disease. In addition, PD1 reduces the level of PGD2 involved in airway hyperresponsiveness. The ability of PD1 to inhibit 15-LOX expression and, therefore, to suppress LT biosynthesis was shown as well [98]. It was demonstrated that intravenous injection of PD1 into allergen-sensitized mice prior to aerosol allergen administration prevented the development of airway hyperresponsiveness, as well as eosinophilic and T-cell-mediated inflammation in these animals [98]. Protectin is able to reduce the inflammatory response in lung tissue after sepsis by activating PPARγ and inhibiting phosphorylation and activation of NF-κB p65 [117]. Protectin can modulate the epithelial cell repair, and activate the proliferation of primary rat lung fibroblasts and the differentiation of myofibroblasts [118]. Overall, the anti-inflammatory activity of PD1 indicates its therapeutic potential in asthma.

Endocannabinoids in asthma pathogenesis

The endocannabinoid system performs important functions: from regulating energy balance, metabolism of carbohydrates and lipids to participating in the immune response [119]. Although the greatest number of studies on the biology of endocannabinoid system was carried out in the field of neurology and psychiatry, accumulated data have demonstrated the importance of this system in various diseases such as cancer, Alzheimer’s disease, multiple sclerosis, cardiovascular disease, chronic pain, obesity, and asthma [120-122].

This system includes the endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), cannabinoid receptors and enzymatic systems involved in their synthesis, transport, metabolism, and decomposition [27]. Anandamide is biosynthesized from N-arachidonoyl phosphatidylethanolamine (NAPE) in humans. In turn, NAPE arises by the transfer of AA from lecithin to the free amine of cephalin via the enzyme N-acyltransferase [123]. Anandamide is synthesized from NAPE by several pathways that involve enzymes such as phospholipase A2, phospholipase C, and N-acetylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD); 2-arachidonoylglycerol is derived from diacylglycerol (DAG) containing AA. The subsequent metabolism of endocannabinoids by the CYP (cytochrome P450)-mediated mechanism leads to the formation of many highly active eicosanoids with different physiological effects [28].

Cannabinoids bind mainly to CB1 and CB2 receptors [124]. CB1 receptors are expressed in areas of the brain responsible for motor and cognitive functions, memory, learning, positive emotions, regulation of homeostasis and reproduction, body temperature, sleep and wakefulness, as well as in white and brown adipose tissues, liver, skeletal muscles, pancreas, and bronchi [125]. CB2 receptors are localized in lymphoid organs, on B cells, monocytes/macrophages, and eosinophils [126]. The activation of this receptor type on mast cells has a direct anti-inflammatory effect. Current research data indicate the important role of mast cells in the pathophysiology of asthma and reveal the mechanism of their effect on respiratory function.

There are other types of cannabinoid-activated receptors – G-protein coupled receptors. They are located in the central nervous system, intestines, liver, bones, skeletal muscles, and adipose tissue (for example, GPR18, GPR55, GPR119, etc.) [124]. In addition to CB receptors, endocannabinoids can bind and activate ion channels with a transient receptor potential (TRP) [40, 127]. Moreover, 2-AG is an agonist for several subtypes of PPARs [39, 40]. A. Lago-Fernandez et al. analyzed the relationships between endocannabinoids and PPARs in different pathologies, and also the analgesic, antitumor, and neuroprotective properties of various phytogenic, endogenous, and synthetic cannabinoids [128].

Particular attention should be paid to the participation of the endocannabinoid system in the immune response. The mechanism of immunosuppression, mediated by cannabinoids, were studied in vitro and in vivo models of many diseases, such as multiple sclerosis, diabetes, septic shock, rheumatoid arthritis, and allergic asthma [129-132]. The recent review authored by N.E. Kaminski et al. summarized key research on the mechanisms of action of cannabinoids in animals and humans (including meta-analyses and randomized clinical trials), along with the results of genetic analysis of single nucleotide polymorphisms in the CNR1 and CNR2 genes located on CB1 and CB2 receptors, which indicates the important role of these receptors in immune-mediated diseases [129]. Z.P. Espinosa-Riquer et al. demonstrated that 2-AG and its CB2 receptors play an important role in the inhibition of innate immune reactions in vivo [130].

The contribution of the endocannabinoid system in asthma pathogenesis was extensively studied in recent years. It was shown that receptors interacting directly with endocannabinoids, specifically, PPAR-α, GRP 55, TRPV1, CB1, and CB2 [39], were localized in the bronchi of mice [41]. It is known that TRPV, TRPA1, TRPM8, PPARs are involved in asthma pathogenesis [39, 40, 127]. A number of innate immune cells, including airway epithelial cells, dendritic cells, macrophages, natural killer (NK) cells, and group 2 (ILC2) are important in the development of asthma. M.E. Ferrini et al. demonstrated the contribution of allergen-induced CB2 activation in the regulation of the activity of NK cells in chronic lung inflammation [133]. Mice lacking CB2 receptors had an increased number of NK cells in the lungs, along with a reduced amount of ILC2. M. Ferrini et al. proposed that NK cells limit ILC2 response in allergic airway inflammation [133]. It is worth noting that NK cells are regulated by a number of endogenously produced eicosanoids, including prostaglandins (PGD2, PGE2), and CB2 [133]. Some authors investigated in vitro the effect of 2-AG on the production of cytokines by lung NK cells. It was revealed that this cannabinoid inhibits the synthesis of IFN-γ by studied cells, which indicates that endocannabinoids play a key role in the suppression of cytokine production by NK cells in the lungs via interacting with CB2 receptors. Increased activity of endocannabinoids is observed under conditions of prostaglandin deficiency. Further studies of the effect of CB2 activation on markers of airway inflammation are needed [134]. Future research of the therapeutic potential of endocannabinoids as PPAR agonists in asthma is of great interest.

Cannabinoids or cannabinoid-based medicinal drugs are promising as anti-inflammatory or immunosuppressive agents [129]. However, currently, there is some controversy regarding the advisability of therapeutic use of these compounds [132]. The conflicting effects of endocannabinoids observed in clinical trials are related to high heterogeneity of their receptors and complexity of signaling.

The relationship between eicosanoids, pro-resolving lipid mediators, endocannabinoids and nuclear transcription factors in asthma

Peroxisome proliferator-activated receptors

PPARs are involved in the regulation of inflammatory responses and lipid metabolism [36]. These receptors belong to the superfamily of hormonal nuclear transcription factors with anti-inflammatory properties (48 members) [135]. Three isoforms of PPARs are known: PPARα (NR1C1), PPARβ/δ (NR1C2), and PPARγ (NR1C3). Natural ligands for all PPAR isoforms are PUFAs, endocannabinoids, eicosanoids and their products: 12(S)-HETE, 15(S)-HETE, 13-hydroxyoctadeca-dienoic acid (13-HODE). Other natural ligands for PPARs are SPMs (Rv, PD, MaR, LX, 15-epi-LXA₄) (Table 4).

Table 4. Peroxisome proliferator-activated receptors and their natural ligands

PPARα is produced in the liver, kidney, heart, skeletal muscle, brown adipose tissue, as well as by epithelial cells, macrophages, lymphocytes, and dendritic cells of the respiratory tract mucosa [136]. This receptor isoform stimulates the expression of genes of enzymes involved in β-oxidation and regulates the metabolism of lipids, carbohydrates and amino acids. PPARα reduces the synthesis of proinflammatory mediators and modulates the production of adhesive and chemotactic molecules [36].

PPARβ/δ is expressed in all organs and tissues; however, their synthesis is most pronounced in the brain, liver, skin, adipose tissue, and skeletal muscles [137]. PPARβ/δ is involved in fatty acid oxidation; it regulates plasma lipid and blood glucose levels, increases the sensitivity of cells to insulin, and participates in wound healing [135].

There are three types of PPARγ: PPARγ1 is expressed in all tissues and cells, PPARγ2 is mainly produced in adipose tissue, and PPARγ3 is synthesized in colon, spleen, white adipose tissue and by macrophages. This receptor is an important regulator of cellular homeostasis and energy metabolism [138].

AA metabolites (PGA2 and 15d-PGJ2) modulate PPARγ activity, while 5(S)-HETE, 8(S)-HETE, 8S-HPETE, 15-HETE, and LTB4 interact with PPARα [139]. PPARβ/δ can be activated by prostacyclin and 15-HETE [140]. Nevertheless, 5(S)-HETE, 8(S)-HETE, 15-HETE, and LTB4 are weaker activators [141]. It is well known that the production of PGE2 and PGD2, which are transformed into anti-inflammatory PGA2 and 15d-PGJ2, is increased in inflammation [141].

All PPAR isoforms are expressed in the lung epithelium and endothelium, by dendritic cells, eosinophils, fibroblasts, and macrophages. PPARs modulate lipid homeostasis and are involved in the control of genes responsible for the oxidative metabolism of lipids: carnitine palmitoyltransferase I (CPT1), pyruvate dehydrogenase lipoamide kinase isozyme 4 mitochondrial (PDK4), CYP4A, and acyl-CoA oxidase 1 (ACOX 141) [142, 143]. Wide expression of PPARs in the bronchopulmonary system, their anti-inflammatory effect and their ability to regulate lipid metabolism attracted close attention of scientists as potential therapeutic targets in asthma [35].

Glucocorticoid receptor

GR is ubiquitously expressed and acts as a ligand-dependent transcription factor [144]. In the absence of the ligand, GR is located in the cytoplasm and is part of the complex that includes heat shock proteins (hsp90, hsp70, and hsp56), immunophilins, and p23. Ligand binding to GR results in the translocation of the receptor into the nucleus, where it interacts with DNA [145]. It is assumed that direct binding of GR to DNA activates transcription, while binding of GR to proinflammatory transcription factors suppresses the process [146]. An important coregulator is the peroxisome proliferator-activated γ-coactivator 1-α (PGC1α) that interacts with GR to modulate gene expression. PGC1α forms a stable complex with the ancestral variant of the GR (AncGR2) LBD [147].

The GR receptor has several isoforms, the most studied of which are GRα and GRβ [144]. The GRα isoform stimulates transcriptional activity of GR, while GRβ is involved in disrupting GRα-mediated GR activity. GRβ also regulates many genes associated with the inflammatory process in a GRα-independent manner [148]. The GR receptor modulates the transcription of proinflammatory and anti-inflammatory genes through multiple isoforms initiating various mechanisms.

Glucocorticoids (GCs) used in asthma therapy act via the GR receptor [149]. Inhaled GCs are effective for most asthma patients; however, some patients exhibit poor asthma control even after taking high doses of local or systemic GCs. Targeting GR-associated pathways can overcome GC resistance. GCs have activating and inhibitory effects on the immune system; therefore, they are able to regulate the immune balance between the response to antigens and excessive inflammation [150].

NF-κB

The proinflammatory role of NF-κB is well understood [151]. The NF-κB signalling is involved in the progression of cancer, in particular, in lung tumorigenesis. The p53 activity is related to the induction of apoptosis, while the stimulation of NF-κB promotes resistance to apoptosis [152]. Various NF-κB antagonists are currently developed to suppress the beta-activity of IκB kinase (IKK) in lung cancer [153]. Accumulated evidence indicates the involvement of NF-κB (the p65 subunit of NF-κB) in acute lung injury [154]. The NF-κB pathway contributes to the pathogenesis of asthma. The pathway plays an important role in mucus overproduction and airway remodeling processes [108]. NF-kB activation implicates TLR signaling cascades that control the synthesis of proinflammatory cytokines and pro-allergic mediators (TSLP, IL-25, IL-33), enhancing the Th2-mediated immune response [155]. NF-κB expression is significantly increased in patients with severe asthma; therefore, many drugs used in asthma therapy inhibit inflammatory reactions in the airways through inactivating the NF-κB pathway [156, 157].

Activating protein 1

Activating protein 1 (AP-1) is a key transcription factor that regulates a wide range of cellular processes, including apoptosis, differentiation, migration, and transformation of cells, and is also involved in the expression of cytokine genes [158]. Four subtypes of the protein were identified: Jun (v-Jun, c-Jun, JunB, and JunD), Fos (v-Fos, c-Fos, FosB, Fral, and Fra2), ATF (ATF2, ATF3/LRF1, B-ATF, JDP1, and JDP2) and MAF (c-Maf, MafB, MafA, MafG/F/K, and Nrl). These subtypes are capable of forming dimers. Despite high degree of structural homology, dimers differ in their capability to activate or suppress gene expression, which suggests their specific regulatory functions. Various methyltransferases regulate the activity of AP-1 in different ways [158]. AP-1 activity is induced by physiological and environmental stimuli (growth factors, neurotransmitters, polypeptide hormones, cytokines, UV exposure, bacterial and viral infections). Increased AP-1 expression was detected in the airways of asthma patients. Fos-associated antigen 2 (Fra-2) belongs to the AP-1 family and is involved in different aspects of cell functioning. The surge of Fra-2 expression was described in several chronic lung diseases, in particular, asthma. High levels of Fra-2 expression trigger inflammation associated with remodeling of the parenchyma and vasculature, which leads to fibrosis and pulmonary hypertension [159].

The relationships between eicosanoids, pro-resolving lipid mediators, endocannabinoids and nuclear transcription factors (PPARs, GR, AP-1, NF-kB) in asthma

The imbalance between anti-inflammatory and proinflammatory nuclear transcription factors plays an important role in the response to treatment [35, 143]. As mentioned above, asthma therapy includes inhaled β2-AR agonists, inhaled and systemic GCs, LTSIs and LTRAs [15]. Resistance to GCs in patients with severe asthma remains a serious clinical problem [16-18, 160]. GC resistance depends on the expression and affinity of GR, as well as many intracellular mediators controlling the signal transduction pathway [149, 161]. The anti-inflammatory effect of GCs is implemented via GR that suppresses the activity of proinflammatory NF-kB and AP-1 (this mechanism is known as transrepression) [162]. This results in the inhibition of the inflammatory response in the bronchopulmonary system. It should be noted that different way of implementing the anti-inflammatory action of GCs is PPAR activation [163]. Despite efforts of the pharmaceutical and academic communities, the search for selective GR modulators with potent anti-inflammatory or anticancer properties, but with fewer side effects, has had so far a somewhat limited success [164]. Several selective GR agonists are currently in preclinical development, but only three of them (GW870086X, AZD5423, AZD7594) have potential therapeutic value for asthma treatment and are in clinical trials [165].

Asthma is characterized by the imbalance between anti-inflammatory (GR, PPARs) and proinflammatory (AP-1, NF-κB) nuclear transcription factors [35, 143]. The resolution of acute inflammation is the process of switching the synthesis of proinflammatory lipid mediators to the formation of SPMs [28]. It is known that proinflammatory stimuli activate NF-κB, AP-1 and promote the synthesis of eicosanoids (PGD2 and PGE2), triggering PPARs (PPARα and PPARγ). Activated PPARs, in turn, inhibit NF-κB activity, which leads to the suppression of inflammation. Possible mechanisms of NF-kB inactivation include not only direct binding of NF-kB to PPARs, but also the indirect effect of PPARs via stimulating the production of antioxidant enzymes and reducing the concentration of reactive oxygen species. The endocannabinoid system is also involved in this process since endocannabinoids are agonists for several subtypes of PPARs [39, 40]. Pro-resolving lipid mediators also exert anti-inflammatory and cytoprotective effects through their interaction with PPARs, GRs and blockade of NF-κB [58, 25]. According to S. Muralikumar et al., the highest activity of FAs and their derivatives as PPARγ agonists is characteristic in a decreasing order for RvE1, PD1, DHA, EPA, and LXA4 [166].

The pathways of the balance regulation between proinflammatory and anti-inflammatory nuclear transcription factors by lipid metabolites of ω-3 and ω-6 PUFAs are currently actively studied [19, 24, 25]. These receptors are expressed by cells of the bronchopulmonary system and play an important role in its immunometabolic homeostasis.

It is known that ω-3 and ω-6 PUFAs are precursors for the synthesis of SPMs [24, 25] and endocannabinoids [26]. There is evidence that ω-6 PUFAs and their oxidized form can lead to the development of resistance to GCSs in asthma patients through the increase in NF-kB expression and the decrease in GR activity [38]. At the same time, the metabolite of linoleic acid also causes resistance to GCSs [38]. Therefore, maintaining a balance between ω-3 and ω-6 PUFAs allows reducing inflammation in the bronchopulmonary system by stimulating the production of SPMs. Hence, this mechanism can be one of the goals in overcoming the resistance to GCSs in asthma [42]. PUFAs are of considerable interest as modulators of chronic inflammation in asthma. The study of the effect of PUFA-containing supplements on the production of SPMs, which are agonists of PPARs, opens up new opportunities in the development of an effective therapeutic strategy for asthma via correcting the resolution of inflammation rather than suppressing its onset and development [29].

Epidemiological and clinical studies on the effectiveness of using ω-3 PUFAs in asthma

Recent changes in the diet leading to a low ratio of ω-3 and ω-6 PUFAs contribute to the exacerbation of asthma [46, 167]. Therefore, ω-3 PUFA supplementation (primarily, supplements containing pure EPA, DHA, or their combination) is proposed as the supportive treatment for this pathology [44]. It is believed that ω-3 PUFAs play an anti-inflammatory role by competing with the arachidonic acid cascade to attenuate the action of proinflammatory eicosanoids (PGs and LTs) or by its converting to potent SPMs [47]. Eosinophils play an essential role in allergic inflammatory diseases and asthma due to their ability to produce CysLT. Its biosynthesis is important as a proallergic mechanism. However, eosinophils synthesize SPMs from DHA and AA, which implied their regulatory role in inflammation [168]. Therefore, the impairment of PUFA metabolism in eosinophils may be an important component in asthma pathogenesis.

Intake of ω-3 PUFAs is more effective for reducing IL-17A in asthma patients than sublingual immunotherapy, while both methods are effective according to indicators of asthma control test (ACT), peak expiratory flow rate (PEFR), and forced expiratory volume in the first second (FEV1) [45]. It was shown that the EPA/DHA ratio is higher in controlled or partially controlled asthma, compared with uncontrolled asthma [169]. Patients with a high ratio required inhaled corticosteroids less than patients with a low ratio. This study suggests that a higher level of ω-3 PUFAs plays an important role in the response of asthma patients to the treatment [169]. The EPA intake is associated with a decrease in the number of eosinophils in the airways and the downregulation of the expression of inflammatory mediators in the lungs. Intravenous administration of 12-hydroxy-17,18-epoxyeicosatetraenoic acid (12-OH-17,18-EpETE), an EPA metabolite, attenuate eosinophilic airway inflammation [170]. At the same time, DHA is more effective for modulating cardiometabolic risk factors [171]. In contrast to the use of EPA, the combination of EPA and DHA significantly improves the clinical course of asthma (decreased airway hyperresponsiveness, eosinophilic inflammation, and levels of inflammatory cytokines (IL-5, IFN-γ, and IL-6). The combined use of EPA and DHA was confirmed to reduce an airway inflammation by promoting the production of pro-resolving lipid mediators RvD1 and RvD4 [172]. When using particular FAs as therapeutic agents in asthma, their different effects should be taken into account.

Despite positive results of using ω-3 PUFAs in asthma therapy, the EPIC (European Prospective Investigation into Cancer and Nutrition Heidelberg cohort) study did not reveal their therapeutic effect [173]. The combined ω-3 and ω-6 PUFA supplementation at a dose of 1.19 g/day did not cause a bronchoprotective and anti-inflammatory effect in patients with exercise-induced asthma [174].

There is limited evidence for the benefits of ω-3 PUFAs in reducing the risk of allergic diseases and asthma in childhood [175]. It is thought that atopic sensitization can be prevented by eating fish during pregnancy, infancy, and childhood [176]. In children with the common variant of FADS, the high intake of EPA and DHA from fish diet during childhood is closely related to the low risk of asthma incidence before the middle adolescence [48]. According to other data, the consumption of fish and FAs during pregnancy is associated with the development of asthma in their children [177]. In a recent review, Y. Zhang et al. analyzed data on the effect of ω-3 PUFAs on the course of allergic diseases in childhood, using the PubMed, EMBASE, and international trial registers of the Cochrane Central Register of Controlled Trials (ClinicalTrials.gov and ISRCTN Registry) up to 2018. The evidence from meta-analysis-based studies demonstrated that ω-3 PUFAs did not significantly alter the risk of atopy and eczema [49]. Another study presented the results of randomized controlled trials evaluating the effect of ω-3 PUFA intake during pregnancy on the incidence of asthma in children, based on publications from the PubMed, Embase and Central databases up to 2017 [50]. Interestingly, the consumption of ω-3 PUFAs during pregnancy reduces the incidence of asthma in children, but not the incidence of asthma in pregnant women. I. Trambusti et al. summarized data on the potential role of prenatal and perinatal dietary and nutritional interventions in the primary prevention of asthma in children [43]. The authors suggested that further research is needed to elucidate the strategies of primary prevention of childhood asthma.

Consumption of ω-3 PUFAs is associated with an anti-inflammatory effect [178, 179]. G. Piazzi et al. discovered that the dietary fats (eicosapentaenoic acid-free fatty acid) led to a switch from ω-6 to ω-3 PUFAs and a reduction in PGE2 production [180]. However, PUFAs are susceptible to oxidation, which is accompanied by the formation of proinflammatory mediators [44]. A.M. Corteselli et al. studied the effect of tropospheric ozone (O₃) on the development of oxidative stress in human airway epithelial cells when taking ω-3 PUFAs [44]. The increased formation of lipid hydroperoxides as a result of the reaction of O₃ with PUFAs was confirmed [44]. Given the proinflammatory activity of lipid hydroperoxides, further study of the potential role of ω-3 PUFAs in increasing human susceptibility to adverse health effects of O₃ is needed.

The lack of standardized doses significantly limits the use of ω-3 PUFAs as well [21]. Remarkably, both high (6.2 g/day [3.7 g EPA and 2.5 g DHA]) and low doses (3.1 g/day [1.8 g EPA and 1.3 g DHA]) of ω-3 PUFAs equally enfeebled bronchospasm and reduced the levels of airway inflammation markers [178].

The effect of SPMs in vivo is just beginning to be studied. A number of studies proposed a promising approach to personalized therapy of inflammation-related diseases by using ω-3 PUFA derivates [96]. RvE1 is known to reduce airway sensitivity and inflammation in asthmatic mice. Although ω-3 PUFAs are precursors of RvE1, it was demonstrated that they have no effect on RvE1 levels in airway inflammation and reactive airway disease. Therefore, ω-3 PUFA supplementation insignificantly affects airway inflammation and reactive airway disease [114]. The inconsistency of the available data suggests the prospects for further study of this issue.

Conclusion

Inflammation is an acute response of tissues and systems to traumatic or stressful influences. The resolution of inflammation is known as a controlled termination program of the inflammatory response. It is assumed that chronic inflammation in the lungs may be the result of a defect in the resolution of the inflammatory process, viz., the functional imbalance between eicosanoids, endogenous specialized pro-resolving lipid mediators and endocannabinoids. The precursors for the formation of lipid mediators are PUFAs. Resolvins, protectins and maresins are synthesized from ω-3 PUFAs; eicosanoids, lipoxins, endocannabinoids are derived from ω-6 PUFAs. All lipid mediators directly (through membrane receptors, intracellular nuclear transcription factors) or indirectly interact with each other, thereby forming a single metabolic network that controls the inflammatory response. It has been established that SPMs are PPARs and GR agonists. The anti-inflammatory effect of these receptors is to suppress the activity of proinflammatory transcriptional nuclear factors, NF-kB and AP-1.

Conflict of interest

We declare that we have no conflicts of interest.

Funding

Our project was funded from the federal budget of the Russian Federation.