Introduction

Asthma is a globally important non-communicable disease of a major public health concern due to its significant morbidity, mortality, and clinical severity in both children and adults.¹ Although prevalence, severity, and mortality vary with respect to the geographical location, psychological, and environmental factors, genetic determinants as well as sex also play a prominent role in modulating disease severity.^2,3 As per the evidence, low- and middle-income countries exhibit higher mortality ratios compared to high-income countries, where the disease is most prevalent.⁴ More than 300 million people are affected by asthma in the world and it is expected that a further 100 million will be affected by 2025.⁵ Although the role of key molecular players in the progression of the disease is yet to be understood, the disease is characterized by the persistence of chronic airway inflammation, respiratory tract infection, airway wall remodeling, edema, and mucus hypersecretion, which is significantly associated with prolonged cough, chest pain or chest tightness, episodes of wheezing and shortness of breath.⁶

Studies on in vitro and in vivo techniques include diverse experimental approaches in cell culture and human clinical models represent the basic molecular network involving potent molecular signals and their pathophysiologic scenario instigating cellular activation, oxidative stress stimulating inflammatory milieu leading to the pathogenesis, and severity of the disease.^7–10 Epidemiological evidence indicates significant involvement of multicellular populations and their activation leading to disease progression and pathogenesis. The cellular activation and severity are characterized by the release of potential biochemical and inflammatory mediators such as cytokines, chemokines, eicosanoids, growth factors, oxidative stress, neural mediators, nitric oxides, and abnormal expression of cell adhesion molecules. Accumulation of these factors promotes smooth muscle cell hyperplasia, goblet cell hyperplasia, epithelial/subepithelial shedding and fibrosis, and blood vessel dysfunction ultimately leading to airway remodeling.¹¹ Exposure to environmental stimuli and allergens causes activation and recruitment of of a variety of cellular populations such as macrophages, dendritic cells, airway epithelial cells, neutrophils, eosinophils, and mast cells inducing cellular damage and inflammatory response in patients with asthma.¹²

Eosinophils are in activated state in asthma

Eosinophils are the major inflammatory cells which play a crucial role in the development of allergic and non-allergic asthma phenotypes.^13,14 The cell is a bone marrow-derived granulocyte with bilobed nucleus having characteristics of acidophilic granules and originated from the eosinophil colony, forming a unit of myeloid progenitor cells and regulated by a variety of signaling molecules like chemokines, cytokines, and adhesion molecules. The cells play an important role, particularly in the immune response against allergic inflammation and parasitic reactions. Eosinophil development in the bone marrow is stimulated by interleukin-3 (IL-3) and granulocyte macrophage colony-stimulating factor (GM-CSF), early differentiation factor and interleukin-5 (IL-5), late differentiation factor which is only active in eosinophil and basophils.^15–17 Eosinophilia in patients with asthma–especially sputum and blood–are significantly associated with basement membrane inflammation of airway mucosa in comparison to the non-atopic control subjects.^18–20

Plenty of evidence indicates variations in eosino-phil count with respect to different corticosteroids thus regulating asthma severity.^15,21,22 Airway eosinophilia is significantly associated with exacerbation and plays a predominant role in airway remodeling. Several cell adhesion receptor expressions on activated eosinophils are highly crucial in mediating eosinophil arrest in the vascular region, extravasations into the airway wall through bronchial and epithelial tissue to the airway lumen. It is evident that eosinophils in their activated state are characterized by an increase in inflammatory mediators and granular proteins in the blood, sputum samples, bronchoalveolar lavage fluid (BAL), and bronchial biopsy specimens in patients with asthma.^23–25 Eosinophil infiltration has been observed to cause vasodilatation, microvascular obstruction, and disruption of epithelial cells.²⁶

Both genetic and environmental factors containing allergens activate a variety of immune cells and epithelial cells finally leading to eosinophil activation, recruitment, and trafficking.²⁷ Activated eosinophils are characterized by overexpression of several eosinophil surface adhesion molecules; some receptors also behave as enzymes, receptors for immunoglobulin G (IgG) immunoglobulin A (IgA), cytokines, chemokines and complement proteins which are implicated in cell adhesion, apoptosis, signal transduction, eosinophil trafficking, proliferation, activation and promotion of survival and death.^28,29 The activation state of the eosinophils constitutes several activation markers that play a key role in airway remodeling, eosinophil trafficking, and extravasations.³⁰ Although several cell surface proteins are observed to report the eosinophil activation, the role of integrins is highly crucial. Earlier evidence also indicates that integrins play a predominant role in pulmonary function and airway inflammation. General markers of eosino-phil activation include clusters of differentiation 69 (CD69), CD11b, CD4, CD9, CD35, CD44, CD45RO, CD48, CD58, CD63, CD66e, CD11a, CD69, CD67, CD81, CD11c, CD18, CD16, CD32, CD64, CD23, CD89, CD54, CD123, CD125, CD62L, CD162, CD108, CD29, CD18, CD32, β2, β1, thymic stromal lymph-opoietin receptor (TSLPR), neuropeptide SR, interleukin 17 receptor A (IL-17RA), IL-13Rα1, human leukocyte antigen DR isotype (HLADR), Galectin 3, platelet-derived growth factor B chain (PDGF-B), and stem cell factor (SCF) (Figure 1). It was reported that eosinophils and T lymphocytes are increased in the asthmatic airways with respect to non-asthmatic controls.^31–34 However, the receptors are also expressed by other cells such as neutrophils, basophils, and mast cells. The only receptor specific to the eosinophils is the epidermal growth factor-like module containing mucin-like hormone-like receptor 1 (EMR1).³⁵

Figure 1 Upregulation of eosinophil surface adhesion molecules in asthma. The eosinophils in their activated state are characterized by overexpression of several cell adhesion receptors as shown in the figure. Instigated by several stimuli eosinophil displays the following CAMs and also releases potent toxic signaling molecules. This contributes to a concomitant increase in inflammatory mediators and granular proteins in the blood, sputum samples, BAL, and bronchial biopsy in patients with asthma, culminating in pathological manifestations.

Molecular players leading to eosinophil activation

Accumulation of activated eosinophils in the submucosa region is the prominent indicator of inflammation in asthma. Activated eosinophils are reported to be significantly associated with asthma severity. On activation, eosinophils release several potent toxic signaling molecules such as major basic proteins, eosinophil peroxidase, eosinophilic cationic proteins, and lipid mediators rendering tissue damage and subsequently leading to allergic manifestation.³⁶ The role of cysteinyl leukotriene in the exacerbation of tissue eosinophilia has been previously described. An elevated level of cysteinyl leukotriene in modulating bronchoconstriction, mucus hypersecretion, tissue infiltration, vascular permeability, and inflammation has been reported by earlier studies.^37,38 In addition, the role of leukotriene in inducing eosinophil activation by stimulating lung cells for the production of regulated on activation, normal T cell expressed and secreted (RANTES) has been proposed.³⁹ Furthermore, an elevated level of leukotriene B4 (LTB4) has been reported, this is a lipid mediator with possible chemo attracting property that has been investigated in patients with asthma. The molecule is released by a variety of cell types including mast cells, macrophages, and neutrophils which mediate its action by acting on G-protein coupled receptor (GPCR) and LTB4 receptor 1 (BLT1) expressed by eosinophils. Although the role of leukotriene B4 receptor (LTB4) in human eosinophil activation, chemotaxis, and pathogenesis of asthma is poorly understood, the potent role of LTB4 in the activation of mice eosinophils has been previously described.⁴⁰ The increase in cytokine content and its implications in the activation of asthma have been well studied. Several pro- inflammatory cytokines have been observed to be produced by the activated eosinophils such as interleukin 1 beta chain (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) along with chemokines including IL-8/chemokine (C-X-C motif) ligand 8 (CXCL8), growth-regulated oncogene (GRO)-α/CXCL1, macrophage inflammatory protein-1β (MIP-1β)/ chemokine (C-C motif) ligand 4 (CCL4), membrane cofactor protein (MCP-1)/CCL2, and RANTES/CCL5.^41,42 In addition to this, both TNF-α and interferon-gamma (IFN-γ) can induce the expression and release of type 1 T helper (Th1) and Th2 derived chemokines such as CXCL-9 and CXCL-10, possibly by activating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), signal transducer and activator of transcription (STAT1), and STAT6.⁴³

The response to different allergens, bacterial, fungal, and parasitic infection induces the production of cytokines such as interleukin 3 (IL-3), IL-4, IL-5, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF).^44,45 It was also previously reported that TNF-α plays an important role in the secretion of IL-4, IL-6, and IFN-γ from a variety of immune cells including eosinophils. Earlier evidence suggests the possible role of IL-23, a member of the IL-12 family that plays an important role in asthma severity. IL-23 is expressed by dendritic cells and macrophages in response to microbial stimuli such as peptidoglycan, lipopolysaccha-rides (LPS). The physiological role of IL-17A, IL-17F, and IL-23 has been elucidated in eosinophil activation in asthma and its role in inflammation.^46,47 IL-23 can also stimulate IL-17F production by eosinophils. Synergistically, IL-23 and IL-17F activate eosinophils and produce IL-1β and IL-6, which in turn, in combined treatment, can induce CXCL1, CXCL8, and chemokine 14 (CCl4).⁴⁸ Activation of eosinophils is associated with an intracellular signaling pathway, leading to the secretion of several cytokines transforming growth factor-beta (TGF-β), MIP-1α, (GM-CSF), TNF-α, IL-1α, IL-3, IL-5, IL-6, IL-8 can induce allergic response and tissue damage.⁴⁴

Both IL-1β and IL-6 can induce the expression of transcription factor retinoic-acid-receptor-related orphan nuclear receptor gamma (RORy), which can be further sustained by the action of IL-17A and IL-6, leading to Th17 lymphocyte differentiation.^49,50 The study was further supported by the evidence that involvement of p38 mitogen-activated protein kinases (p38-MAPK) signaling pathway by which IL-23 and IL-17A can potentiate their action, whereas IL-17F possibly stimulates extracellular-signal-regulated kinase (ERK) activation.⁴⁹ Both cross-talk of the pathway leads to the secretion of the diverse amount of cytokines which induce the NF-kβ pathway and mediate allergic inflammation in asthma by the activation and recruitment of various immune cells. Further study shows the possible role of IL-17F in the induction of chemokine (C-C motif) ligand 20 (CCL20) synthesis, which attracts the Th17 cells into the airway, resulting in the recruitment of additional Th17 cells and induces allergic airway inflammation.⁵¹ Intriguingly, CD4⁺ mediated IL-16 dependent activation of eosinophils was observed to be associated with the release of leukotriene C4 (LTC4) as well as chemokines such as RANTES, IL-6, eotaxin, and IL-4.⁵² IL-4 along with IL-5, IL-6, IL-13 produced can contribute to Th2 differentiation and IgE class switching stimulating allergic inflammatory response.⁵³

Functional interaction between CD32 and αMß2 has been observed to play an important role in eosinophil degranulation.^54,55 A potential role of αMß2 inte-grin on eosinophil degranulation in an IgG-mediated manner has been described previously.⁵⁶ An elevated level of IL-3 in serum has been positively associated with asthma <page> cells are IL-3 positive. IL-3 is also secreted by activated Th2 lymphocytes, mast cells, and neutrophils. An increase in IL-3 receptor expression on eosinophils suggests a possible role of IL-3 in the stimulation of eosinophil activation.⁵⁹ In response to allergen challenges, the production of IL-3 induces increased translation of semaphorin A by stimulating the p90S6K/RPS6 signaling pathway.⁶⁰ Earlier evidence indicates cytokines such as IL-3, GM-CSF, and IL-5 activate semaphorin A production by eosinophils. Existing evidence indicates that semaphorin A activates the TGF-β1 signaling pathway, which induces lung fibrosis/airway remodeling in asthma.⁶¹ Earlier findings suggest activation of eosinophils by IL-3 stimulating prolonged expression of CD32 and αMß2 integrin, as well as maintaining their activation state (Figure 2).⁶²

Figure 2 Signaling network modulating eosinophil activation and asthma phenotype displaying the potential role of cellular, molecular, and environmental factors inducing the complex signaling cross-talks. This ultimately leads to eosinophil activation which is characterized by overexpression of membrane-bound or secreted molecules. These molecular intermediates activate specific signaling, culminating directly or indirectly into complex abnormal phenotypes such as allergic response, airway remodeling, epithelial damage, or bronchodilation, modulating clinical severity in asthma.

The role of thymic stromal lymphopoietin (TSLP), a cytokine in the induction of allergic response, in diseases such as asthma has been well studied.^63,64 The protein plays an important role in atopic dermatitis, allergic rhinitis, nasal polyposis, and chronic allergic keratoconjunctivitis.⁶⁵ TSLP, a member of the hematopoietic cytokine family, has been found to interact with its receptor TSLPR and inter-leukin 7 receptor-alpha chain (IL-7α) expressed on eosinophils and also in other cell types.⁶⁶ Although, the role of TSLP in promoting the Th2 differentiation was initially attributed in a dendritic cell-mediated manner, however, subsequent results indicate its expression by various cell types which indicate that it is like eosinophils, mast cells, B cells, and T cells.⁶⁷ Further analysis on real-time quantitative PCR study indicates mRNA expression of TSLPR and IL-7α in eosinophils upon treatment with TNFα and IL-5 family cytokines. Stimulation of eosinophils with TSLP induces STAT3 signaling and promotes eosinophil-dependent neurotoxin (EDN) production leading ultimately to eosinophil degranulation, which can be subsequently blocked by the functional blocking antibody to TSLPR. EDNs are stored in eosinophil granules in response to a range of immobilized ligands like IgA and IgG.⁶⁸ Further study indicates a potential role of leukocyte Ig-like receptor (LER7) activation through an immunoglobulin mediated manner that can lead to EDN release from eosinophils.⁶⁹ A possible role of platelet-activating factor (PAF) in aggravating the chemokinesis and chemotaxis of eosinophils has been previously reported. PAF is secreted by macrophages/monocytes, platelets, neutrophils, endothelial cells as well as eosinophils.^70,71

An increased level of PAF can induce G-protein coupled receptor (GPCR) expressed by eosinophils and stimulates eosinophilotactic activity by activating the MAPK signaling pathway. In support of this notion, further studies also revealed the possible role of PAF inducing MAPK signaling in asthma.^72–74 Further evidence from a mice model indicates that the administration of prostaglandin D2 (PGD2) is significantly associated with tissue eosinophilia.⁷⁵Prostaglandin D2 (PGD2) mediated function is accomplished by its interaction with two receptors; DP1 and DP2 (D prostanoid receptor 1 and 2) expressed by T helper type 2 cells (Th2 cells) and eosinophils.⁷⁶ Furthermore, it was established that DP2 receptor activation is highly crucial in mediating activation and eosinophilotactic activity. In vivo study revealed that prior exposure of allergens or IL-5 can significantly increase airway eosinophilia in mice induced by DP2 agonist or PGD2 by coupling with inhibitory Gα subunit (Gαi), which leads to the diminished synthesis of cyclic adenosine monophosphate (cAMP) and induced accumulation of intracellular calcium.⁷⁷

The proteolytic activity of matrix metalloproteinase (MMP) can lead to extracellular matrix protein degradation, which is significantly associated with tissue injury and remodeling. MMP9 causes tissue remodeling by epithelial mesenchyme transition by the release of TGF-β, profibrotic factors that can contribute to angiogenesis. Accumulating evidence indicates the role of MMP9 secretion by eosinophils when activated by IL-3, IL-5, IL-8, TNF-α, GM-CSF, complement 5a, and PAF.^78–83 Activation of eosinophil GPCR by PAF, endothelin-1, RANTES, eotaxin, cyteinyl leukotrienes, prostaglandins, complement component 3a (C3a), C5a and other stimuli can activate phospholipase Cβ {Phospholipase C-β (PLC-β)}, which in turn causes hydrolysis of phosphatidylinositol bisphosphate into inositol triphosphate and diacylglycerol inducing calcium accumulation and protein kinase C (PKC) activation.^84–86 Furthermore, it was also reported that the combined action of IL-3 and TNF-α can activate eosinophils inducing activation of NF-kB and MAP kinase pathway involving ERK1/2 and p38 but not (c-Jun N-terminal kinase) JNK pathway leading to the synthesis and release of MMP9.⁸⁷ Recent studies have also shown the secretion of MMP-9 by eosinophils on activation by IL-3 and TNF-α.⁸⁸ The release of MMP-9, NF-kB activation has also been associated with the regulation of IL-8 and expression of intracellular cell adhesion molecule 1 (ICAM-1).⁸⁹ It was also reported that TNF-α can induce synthesis of eotaxin by activation of eosinophils and P38 MAPK signaling pathway.^90,91 Earlier evidence indicates an accumulation of a large number of CXC and CC chemokines in human and pig models of allergic inflammation.^92–94 Both eosinophils and basophils possess eotaxin receptor CCR3 which plays a crucial role in inflammation and allergic response.^95,96 A study on biopsy specimens in asthmatic patients also indicates the expression of eotaxin protein by eosinophils in inflammatory sites. Further study significantly revealed the potential ability of C5a, TNF-α, and IL-4 in the activation and release of eotaxin by eosinophils.⁹⁷ Stimulation of eosinophil CCR3 by eotaxin induces tyrosine phosphorylation of numerous proteins such as Lyn, hematopoietic cell kinase (Hck), and Fgr leading to eosinophil chemotaxis and recruitment (Figure 2).⁹⁸

A possible role of IL-5 and nitric oxide metabolites was reported and intriguingly it was observed that patients with a higher concentration of IL-5 and nitric oxide (NO) possess a higher concentration of ECP.⁹⁹ NO also plays a crucial role in the pathophysiology of asthma. A higher concentration of NO released by inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) exerts its effect by promoting mucus secretion, vasodilation, plasma extravasation, edema, and T lymphocyte and eosino-phil recruitment, whereas NO released by neuronal nitric oxide synthase (nNOS) induces bronchodilation by relaxing smooth muscles, possibly by non-noradrenergic, non- cholinergic (NANC) nerve activation. It has been previously described that activation of a variety of cell populations such as eosinophils, epithelial cells, endothelium, muscle cells, neurons, fibroblasts, T and B lymphocytes, mast cells, macrophages, and dendritic cells releases substance P (SP).¹⁰⁰ The role of SP in the induction of eosinophil activation and NO synthesis has been observed in several models.^100–102 In addition to SP potential role of other peptides like formyl-methionyl-leucyl-phenylalanine (FMLP) and melittin in contributing to eosinophil activation in guinea pig models has been described, where SP and melittin act presumably through non-specific peptide membrane phospholipid interactions and FMLP acts through receptor-mediated manner.¹⁰³ Accumulation of cytokines such as TNF-α, IL-1, IFN-γ, and lipopolysacharides (LPS) can induce L-arginine transport and thus activate arginase and iNOS activity leading to NO production.^104,105 A high concentration of NO can stimulate metalloproteinase activity, activation of oxidative stress pathway, airway remodeling, cellular activation and recruitment, inflammatory response, airway hyperresponsiveness, and allergic stimulation.¹⁰⁶

The expression and activation state of several integrins by eosinophils is significantly associated with the migration and adhesion of different extracellular matrix (ECM) proteins. Eosinophils express α4β1, α6β1, αLβ2, αMβ2, αXβ2, αDβ2, and α4β7 integrins, where each integrin interacts with its ligand, which are the counter receptors on different cell or ECM components.^55,107,108 Eosinophils via these integrins can adhere to ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), laminin, periostin, fibrin/fibrinogen, vitronectin upon stimulation with different signals such as IL-5, IL-3, GM-CSF, and P-selectin.^55,109,110 Results from both in vivo and in vitro models indicate eosinophil integrin expressions are highly crucial in the modulation of the inflammatory response in asthma.^{31,109,111,112} Different signals such as epinephrine and thrombin, express vascular endothelial growth factor (VEGF), histamine and adenosine phosphate (ADP) induce the generation of P-selectin from platelets and endothelial cells which act on P-selectin glycoprotein ligand-1 (PSGL-1) expressed by eosinophils and other leukocytes, which could lead to the activation of β1 integrin and induce its adhesion to VCAM-1.^113–116 Although P-selectin has the potential to induce eosinophil β1 inte-grin activation, there is, however, considerable discrepancy about the exact mechanism of the signaling pathway involved. The influence of different leukotrienes such as C4, D4, E4 in activation of eosinophils was also reported in asthma.^37,117,118

Oxidative stress has proven to be an intrinsic modulator in triggering the pathological complexity of asthma characterized by oxidant-antioxidant imbalance.¹¹⁹ Elevation of endogenous and exogenous reactive oxygen species (ROS) and nitrogen species (RNS), decreased level of cellular antioxidants, inflammatory cell infiltration, metabolic disorders, increased level of allergen-induced environmental peroxidants, genetic and epigenetic alterations, and poor lung functions are significantly associated with oxidative stress. It was also previously described that asthma severity and airway inflammations are significantly affected by endogenous and exogenous ROS and RNS. Oxidative stress plays a crucial role in aggravating the inflammation and pathogenesis of both mild and symptomatic cases. Exogenous sources of ROS include cigarette smoke, hyper-oxia, heavy metal poisoning, UV light, ozone, ionizing radiation, pollutants, organic solvents, and chemotherapeutic agents, whereas the source of endogenous substances includes enzyme activities and the activation of immune and non-immune cells. Augmented production of ROS was also previously reported in both children and adults, with distinct severity.¹²⁰ Tremendous generation of ROS such as superoxide radicals, hydrogen peroxide, hydroxyl radicals, singlet oxygen, hypochlorous acid, peroxyl, alkoxyl, and hydroperoxyl free radicals, like RNS such as nitric oxide (NO), peroxynitrite, and nitrite, modulate airway inflammation and asthma severity.¹²¹ Activation of eosinophils, macrophages, neutrophils, and monocytes is associated with the generation of superoxides. Heterocellular activation, allergen exposure, and challenge lead to a diverse amount of production of chemokines, lipid mediators, cigarette smoke and potent air pollutants such as ozone and diesel exhaust particles that can promote ROS production.^122,123 High blood eosinophil counts are associated with moderate to severe asthma in comparison to the high blood neutro-phil count, which is associated with only moderate but not severe asthma phenotype.¹²⁴ Activation of cell organelles such as mitochondria, endoplasmic reticulum, peroxisome, allergen exposure, activates of xanthine oxidase, P450 monooxygenase, cyclooxygenase, lipoxygenase, indoleamine dioxygenase, monoamine oxidases, dehydrogenases, dioxygenases activity leads to the generation of free radicals. Earlier evidence also indicates an elevated level of ROS and RNS in patients with asthma.^125,150 Several markers of oxidative stress have been reported in the exhaled airway of patients with asthma. An excess concentration of ROS has been observed to be associated with airway hyper-responsiveness, vascular permeability, production of chemoattractants, and lipid peroxidation.¹²⁵ The interaction of ROS with biomolecules such as proteins to form carbonyl; this subsequently reacts with nitrogen species and tyrosine to form nitrotyrosine. This was also previous evidence that patients with asthma are characterized by increased activity of eosinophil peroxidase (EPO) and myeloperoxidase (MPO) in eosinophils as well as neutrophils.¹²⁶ The product of these enzymes such as 3-bromotyrosine and chlorotyro-sine is reported to increase in asthmatic subjects compared to the controls.^127,128 Increased levels of NO and superoxide can induce epithelial cell shedding leading to neuropeptide release and contribute to bronchoconstriction.

Multiple lines of evidence also indicate the potential ability of eosinophils/neutrophils contributing ROS production in the lung airways in comparison to healthy controls. It was previously described that eosinophils isolated from asthmatic patients are characterized by the production of more hydrogen peroxide as well as ROS in comparison to healthy controls. The increased concentration of ROS can overwhelm the endogenous antioxidant status and leads to a loss in beta-adrenergic function, mucin secretion, acetylcholine-mediated muscle sensitization and contraction, activation of mitogen-activated protein kinase in tracheal myocytes. Activated neutrophils, macrophages, and monocytes have the ability to generate superoxide and hydrogen peroxides. Eosinophils have the tremendous potential to release these free radicals in comparison to the neutrophils, due to high EPO activity than MPO activity in neutrophils.¹²⁹

The effect of CC chemokine eotaxin has been observed to be associated with eosinophil activation and the production of ROS by eosinophils.¹³⁰ The possible role of ROS in stimulating expression of soluble intracellular adhesion molecule 1 (ICAM-1) by eosinophils was also previously reported. A previous study has also demonstrated the possible role of eotaxin and RANTES in activation of CCR3 in a GPCR-mediated manner involving the activation of phosphatidylinositol-3-kinase and tyrosine kinase.⁹⁵ The influence of cytokines on the eosinophil NADPH activity was studied in vitro.¹³¹ It was evident that IFN-γ and TNF-α can induce NADPH activity and gp91-phox gene in human myeloid leukemia 60 (HL-60) clone 15 cells which induce eosinophil differentiation and release of reactive oxygen intermediates in the chronic inflammatory condition in asthmatic patients.¹³² The treatment of chemokines such as CCL11, CCL24, CCL26 can stimulate isolated eosinophils to produce superoxides. The concentration-dependent degranulation of eosinophil peroxidase (EPO) and calcium ion influx was also observed. Potentiation of the response was observed maximally in the case of CCL11 and CCL24 even in Phorbol 12-myristate 13-acetate (PMA) stimulated conditions. Pre-treatment of cells with anti-CCR3 antibody significantly inhibits superoxide generation, indicating the possible role of CCR3 as a target for eotaxins.¹³³ Upregulation of CCR3 by butyric acid (BA) and IL-5 as observed in HL-60 clone 15 cells indicates a cooperative action of chemokines for the effective inflammatory response in asthmatic patients.^134,135 Further study also revealed the potential effect of IL-5 and PMA in eosinophil priming and the subsequent generation of oxygen-free radicals possibly by activation of the MEK-ERK1/2 pathway.¹³⁶ A subsequent study reported that peripheral blood eosinophils are characterized by enhanced production of ROS, chemotaxis, and a diminished level of apoptosis during allergen-induced late-phase airway inflammation.¹³⁷

Increased activity of NADPH oxidase in eosinophils was observed during infection stages which release a massive quantity of superoxides.¹³⁸ Several potent signaling molecules are associated with eosinophil NADPH oxidase activation, as epidemiological evidence in both human and guinea pig model indicates phospholipase C (PLC) and phospholipase D (PLD), activation MAP kinase/tyrosine kinase plays a crucial role in inducing eosinophil NADPH activity.^139,140 Activation of PLD is associated with phosphatidylcholine (PC) and phosphatidic acid (PA). PA observed to phosphorylate NADPH oxidase by activation of protein kinases, in addition to this C5a also plays a crucial role in PLD activation. It was earlier evidence that the potential ability of arachidonic acid (AA) stimulates H₂O₂ generation in a concentration- dependent manner in guinea pig eosinophils. Activation of PLA2 cleaves membrane lipids gives rise to AA production which induces rac p21 translocation stimulating NADPH activation. In addition to this, AA also activates PKC, MAP kinase and increases intracellular Ca²⁺ concentration. Activation of MAP kinases such as ERK1/2, JNK46/54, and p38 kinase are implicated in NADPH oxidase activation.^141,142 Further study revealed the possible role of CLC3, a chloride channel and H⁺/Cl^- antiporter in activation of eosinophil NADPH oxidase and induction of oxidative burst leading to eosinophil activation and migration.¹⁴³ Since sustained activation of NADPH oxidase is solely dependent on the costimulation of the ion transporter which maintains the charge balance generated by O₂^- production from NADPH oxidase. It was also proposed that PKC plays an important role in the phosphorylation of CLC3 inducing eosinophil degranulation, superoxide generation by stimulating NADPH oxidase activity.¹⁴⁴ A potent role of IL-33 has been observed to activate the CCR3 expressed by eosinophils and induces NADPH oxidase 2 (NOX2) expression in eosinophils.¹⁴⁵

The action of NADPH oxidase generates superoxide in activated eosinophils which is subsequently converted into H₂O₂ which is a potent substrate candidate for EPO and converted into hypobromous acid, which in turn reacts with amine and tyrosine amino acid to give rise to 3-bro-motyrosine. It was also previously evident that activated eosinophils have 3–10 times superoxide producing ability as compared to the neutrophils.¹⁴⁶ Plenty of evidence indicates that EPO activity is significantly associated with asthma severity and tissue inflammation and is also a predominant factor for the production of hypobromous acid in the airways.^147,148 In response to the allergen challenge, there is a 10-fold increase in the level of 3-bromotyrosine in the proteins isolated from BAL and sputum asthmatic patients in comparison to non-asthmatic subjects.^128,147 In addition to the oxide generation ability, eosinophils have a prodigious ability to produce RNS on reaction with NO derived intermediates.^150,151 RNS generation by eosinophils in asthmatic patients mostly contributed by alterations in NO metabolism. Activation of NOs by different cytokines such as IL-13, IL-14, IFN-γ, and TNF-α leads to the production of NO from L-arginine enzymatically. The NO generated can combine with superoxide to form peroxynitrite, which in turn combines with CO₂ to form peroxycarboxynitrite. EPO activation can use halides and pseudohalides to form a more potent form of oxidants.¹⁵² Parallel gas chromatography and mass spectrometry results significantly revealed that there is a 10-fold increase in 3-nitrotyrosine in proteins recovered from BAL from asthmatic subjects in comparison to non-asthmatic subjects.¹²⁷ Nitrosothiol and nitrotyrosine production can lead to protein inactivation. It has also been observed that an elevated level of nitrotyrosine is found in the airways of patients during an asthma attack.^153,154 Eosinophils use EPO, which plays a crucial role in the oxidative modification of proteins.

Eosinophilia asthma phenotypes are the most prognostic biomarkers and indicators to assess the disease severity, although the distinct phenotypes are associated with airway microbiology. High blood eosinophilia and its association with disease exacerbation were explained by a previous study.¹⁹ Eosinophils have an intrinsic ability to rapidly release a vast number of tissue mediators such as growth factors, cytokines, chemokines, and eosinophil- derived granules. Activation of eosinophils, expression and activation state of cell adhesion molecule (CAM), interaction of eosinophils with other cells such as platelets and neutrophils instigated by several confounding factors, is significantly associated with airway hyperresponsiveness, airway remodeling, epithelial cell damage, vascular cell adhesion molecule expression culminating in an inflammatory response. Eosinophil recruitment and priming in a chemoattractant-mediated mechanism involves transmigration, extravasations, possibly by tethering and rolling over the vascular endothelium play an important role in inducing an inflammatory response. PGD2 derived from the mast cell activation induces chemotaxis in eosinophils in a CR2H2-mediated manner and stimulates oxidative burst in eosinophils. CR2H2 antagonists have been observed to be associated with the suppression of eosinophil chemotaxis and oxidative burst.^155,156 Activated eosinophils also express vascular endothelial growth factor (VEGF) in response to the GM-CSF and IL-5. VEGF is reported to play a crucial role in mediating endothelial cell-specific response and vascular leakage contributing to tissue edema formation at the site of allergic inflammation.¹⁵⁷

Eosinophils in modulating epithelial injury

Impairment in epithelial function was observed in asthma, presumably by disruption of epithelial tight junctions, accompanied by tissue injury and aberrant repair orchestrated inflammatory response in asthma. Although epithelial cells are reported to recognize and engulf apoptotic eosinophils, activated eosinophils release potent cytotoxic granules, EPO, Mannan-binding protein (MBP), EDN, ROS, RNS, TGF-β, IL-13, IL-4, IL-8, IL-11, ECP, and other chemokines, which leads to epithelium damage.¹⁵⁸ IL-13 has been observed to activate STAT-6 signaling and stimulates airway hyperresponsiveness and mucus secretion in epithelial cells.^159–161 Epithelial injury during this stress condition is associated with the activation of NF-kB, AP-1, growth factors, and chemokines which aggravates the inflammation process. Production of TGF-β, TGF-α, PAF, IL-13, IL-5, IL-17, IL-10, leukotrienes, thromboxanes, prostaglandins, and other cytokines by activated epithelial cells and eosinophils coupled with stimulation of myofibroblasts due to accumulation of type-III collagen. In response to allergen and LPS, it induces TL4 signaling epithelial cells ultimately leading to the production of TSLP, IL-33, and IL-25 to regulate the adaptive immune response. CCL2 and CCL20 produced by activated epithelial cells also activate dendritic cells, which in turn regulate allergen processing and the presentation to T lymphocytes. Eosinophil MBP and EPO with H₂O₂ and halide reported activating basophils and mast cells which produce leukotrienes and histamine causing epithelial injury. The toxic cationic granules, viral and bacterial infection also cause desquamation of bronchial epithelial cells inducing bronchial hyperreactivity and loss of epithelium-derived relaxing factors. Intratracheal instillation of eosinophil in lungs increases the concentration of chemokines such as MCP-1/CCL2, which are the potent chemoattractant for macrophages and lymphocytes producing an inflammatory response. The prospective role of eosinophils in inducing the epithelial-mesenchymal transition (EMT), and release of collagen, fibroblast growth factors (FGF), TGF-B1 was elucidated previously.^162,163 EMT, a molecular reprogramming process, involves increased migration of mesenchymal cells to subepithelial connective tissues and is characterized by increased secretion of extracellular matrix proteins (ECM) leading to bronchial wall fibrosis in injured sites.^164,165 In vitro evidence also indicates secretion of fibroproliferative and fibrogenic growth factors including FGF-2, insulin-like growth factors 1 (IGF-1), PDGF, ET-1, and TGF-β, which can induce collagen gene expression.^166–169 It was observed that eosinophil and epithelial cell interaction causes the release of TGF-β1 by bronchial epithelial cells, which activates JNK/Smad3 pathway causing EMT of airway epithelial cells.¹⁷⁰ EMT plays a significant role in airway remodeling. The secretion of IL-37 by epithelial cells as well as macrophages and neutrophils induced by allergen exposure, bacterial and viral infections, which mediates eosinophil recruitment and activation upon interaction with N-formyl peptide receptor 2 (FPR2) and P2X7R and induces cysteinyl leukotrienes (CysLT) by activating p38-MAPK and nf-kB signaling pathway.^171,172 Epidemiological evidence revealed the expression of macrophage inflammatory protein 1-α (MIP-1α), IL-8, epidermal growth factor receptor (EGFR), and CD44 expression by epithelial cells; this indicates a potential marker of epithelial injury. In vitro results from airway epithelial cell indicate epithelium dysfunction is associated with increased secretion of cytokines, chemokines, eicosanoids such as prostaglandin E₂, 15-hydroxyeicosatetraenoic acid, IL-6, which induces an inflammatory response.^173,174

Drug approach to eosinophilic asthma

The complex clinicopathological features of asthma enable it to behave as a heterogeneous disease. Until recently, the current scenario indicated that the basic pathophysiological picture of the phenotype is characterized with exacerbated severity. Understanding the complexity of the phenotype is highly crucial in order to improve the patient outcome and better therapeutic efficacy. Although tissue-specific eosinophil activation plays an important role, associated organ dysfunction is less frequent in patients with asthma. Inhibition of eosinophil activation and priming could diminish the tissue eosinophilia and protect from end-organ damage. Corticosteroid therapy has been observed to be effective in controlling the symptoms of many patients when taken with or without long-acting beta-agonists. It was also observed that the intake of oral steroids with high dose intramuscular injection triamcino-lone is associated with a decrease in sputum eosinophilia. A previous study has also demonstrated the dose-response relationship between inhaled corticosteroid therapy (ICS) and blood eosinophils, which indicates that ICS at a dose of 800 µg/day could significantly inhibit the tissue eosinophilia and eosinophil cationic protein (ECP).¹⁷⁵ The study also showed that inverse agonist propanolol produced no significant effect when given with low dose ICS, whereas with higher dose ICS there is a significant improvement in airway responsiveness and inflammation was observed.¹⁷⁵ Although long-term corticosteroid therapy could lead to corticosteroid resistance, previous investigations have elucidated that it is highly desirable to use corticosteroid therapy along with bronchodilators to boost a better response in patients with asthma.^176–178 The role of leukotrienes in modulating the immune function and bronchial smooth muscle constriction was elucidated previously.^38,39,86,117 The compounds have 5000 times more potent bronchoconstrictors such as histamine, prostanoids, and platelet-activating factors. Potent CysLT₁ receptor antagonists such as montelukast, zafirlukast, and pranlukast are clinically approved and conventionally used in asthma. Although the exact incidence of eosinophilic asthma is yet to be understood, severe asthma groups that account for 5–10% of the asthmatic population exhibit sputum eosinophilia (≥2%), whereas 32–40% of the population exhibit a blood count (≥300 cells/µL).¹⁷⁸ A double- blind randomized placebo-controlled trial revealed that treatment with montelukast significantly decreased the serum concentration of IL-4, soluble intercellular adhesion molecule-1 (sICAM-1), ECP and peripheral blood eosinophil count indicating potential clinical benefits with asthmatic patients from bronchial hyperresponsiveness.¹⁷⁹ Another double-blind placebo-controlled trial also indicated the inhibitory action of zafirlukast on inhaled leukotriene D4 in both asthmatic patients and normal subjects. Significant attenuation was observed in terms of early and late phase response to inhaled antigen and a decrease in bronchial hyperresponsiveness to histamine on treatment with zafirlukast.¹⁸⁰ The potential inhibitory activity of salmeterol and zileuton on LTB4, LTA4, 5-lipoxygenase activity and its associated products makes it an ideal agent to act as a potent leukotriene inhibitor.¹⁸¹ Existing evidence indicates an inhibitory effect of Levalbuterol on airway smooth muscle cell proliferation and cell growth, possibly by activating the cAMP/PKA pathway and inhibiting phosphoinositide 3-kinase (PI-3 kinase), NF-kB and retinoblastoma (Rb) protein expression.¹⁸² Despite increasing the dose of inhaled corticosteroids, the addition of long-acting β-agonist salmeterol offers a better therapeutic response in asthmatic control.²¹² Bronchodilators such as β-agonist and anticholinergic agents have been a good choice in patients with asthma and chronic obstructive pulmonary disease (COPD); however, the administration of β-agonist has been associated with side effects including the risk of cardiovascular complicacy, arrhythmias, hypoxemia, tachycardia, palpitations, and inflammatory response. Conversely, regarding this context, anticholinergic agents have shown limited toxicity and longer duration of bronchodilator.²¹³ Owing to the specificity and potency, human-based monoclonal antibodies have become the intriguing target, showing maximal clinical efficacy with limited toxicity. Existing evidence indicates that the administration of mepolizumab neutralizes IL-5 and significantly reduces the eosinophil count, activation, extra-cellular matrix protein deposition, and eosinophilic-induced inflammation (Table 1).²¹⁴ A reduction in eosinophil activation markers such as ECP and EDN, with a concomitant decrease in peripheral blood eosinophilia by benralizumab, an anti-IL-5αR mediates its action by antibody-dependent cell-mediated cytotoxicity.²¹⁵ Phase III clinical trials have also demonstrated the role of benralizumab in significant ameliorating effect and improvement in lung function.²¹⁶ Furthermore, a study on reslizumab significantly revealed potential clinical benefits in asthmatic patients from nasal polyps. A phase II clinical study revealed that 3 mg/kg body weight of reslizumab is biologically active and significantly reduced the sputum eosinophil count.²²³ However, until recently, there has been a limited study in the context of the possible role of reslizumab in controlling eosinophil function. IL-4 plays a crucial role in the initiation of allergic airway response and humoral response, whereas IL-13 was observed to play a potent role in the induction of allergic manifestations such as AHR, allergic inflammation, mucus production, sub-epithelial fibrosis, and airway smooth muscle alterations.²²⁴ It is interesting to note that administration with anti-IL-13 and anti-IL-4α therapy (tralokinumab, lebrikizumab, and dupilumab) although it has shown a considerable protective effect and improvement in lung function, there is a lack of significant protection in patients with asthma in terms of clinical response, indicating the need for a further massive investigation.²¹⁷ In addition to antibody therapy, the possible ameliorating effect of cromolyn and its derivative in asthma has been elucidated previously.^218,219 The compound has potent anti-inflammatory properties, and cytokine inhibitory activity, antihistamine activity, and a mast cell stabilizer.^220–222

Conclusions

This review has described the role and behavior of eosinophils in the modulation of clinical complicacy and pathogenesis in patients with asthma. Cellular, molecular, and environmental agents could instigate the eosinophils, culminating in a more complex, comprehensive, hyperreactive phenotype in terms of morphology and biochemical complexity. Coordinated action and complex cross-talk of signaling molecules, tissue factors, cytokines, interleukins, NO, ROS, and signaling intermediates lead to altered physiology. Blood eosinophil levels represent prominent indicators of severity and act as specific biomarkers for assessing asthma severity. Eosinophil activation is associated with a myriad of signaling networks inducing a more complex heterogeneous phenotype. Tissue and blood eosinophilia could contribute to inflammation and associated infections. Assessing the exact status of eosinophils in an individual with the asthma phenotype is highly crucial for the identification of symptomatic to asymptomatic and moderate to severe phenotype. Eosinophilic inflammation represents the common patho-physiological complication, providing the rationale for the use of the potential therapeutic molecules beyond asthma. Understanding the mechanistic underpinnings at both intra-cellular and extracellular levels governing the eosinophil function is highly crucial. More recently, drug development has focused on blocking eosinophil recruitment into organs and impairing their survival and activation. Identification of accurate sub-phenotype with other associated eosinophilic and non-eosinophilic diseases could be an ideal strategy for a better diagnostic and therapeutic intervention. More recently, drug development has focused on blocking eosino-phil recruitment into organs and impairing their survival and activation. Clinical trials with first-generation eosinophil-targeted therapeutic agents are now underway and appear to be particularly promising. Corticosteroid therapy is limited due to its toxicity and variable degree.