Human Lung Mast Cells: Therapeutic Implications in Asthma

Mast cells are strategically located in different compartments of the lung in asthmatic patients. These cells are widely recognized as central effectors and immunomodulators in different asthma phenotypes. Mast cell mediators activate a wide spectrum of cells of the innate and adaptive immune system during airway inflammation. Moreover, these cells modulate the activities of several structural cells (i.e., fibroblasts, airway smooth muscle cells, bronchial epithelial and goblet cells, and endothelial cells) in the human lung. These findings indicate that lung mast cells and their mediators significantly contribute to the immune induction of airway remodeling in severe asthma. Therapies targeting mast cell mediators and/or their receptors, including monoclonal antibodies targeting IgE, IL-4/IL-13, IL-5/IL-5Rα, IL-4Rα, TSLP, and IL-33, have been found safe and effective in the treatment of different phenotypes of asthma. Moreover, agonists of inhibitory receptors expressed by human mast cells (Siglec-8, Siglec-6) are under investigation for asthma treatment. Increasing evidence suggests that different approaches to depleting mast cells show promising results in severe asthma treatment. Novel treatments targeting mast cells can presumably change the course of the disease and induce drug-free remission in bronchial asthma. Here, we provide an overview of current and promising treatments for asthma that directly or indirectly target lung mast cells.


Introduction
Mast cells, first identified in humans by Paul Ehrlich in 1878 [1], play a role in allergic [2,3] and autoimmune disorders [4], microbial infections [5], cardiovascular diseases [6,7], immunodeficiencies [8], and cancer [9][10][11]. Mast cells are derived from CD34 + haemopoietic progenitors that migrate from the bone marrow to the blood and mature in almost all tissues [12]. These cells release a plethora of mediators and display several surface receptors [13,14]. Mast cells uniquely express the cell surface receptor of stem cell factor (SCF) [15], also known as KIT or CD117. SCF plays a critical role in the differentiation, proliferation, and modulation of human and rodent mast cells [16].
In the 1990s, human mast cells that contain only tryptase were termed MC T , whereas those that express both tryptase and chymase were classified as MC TC [17,18]. There are also definitions of mast cells being inflammatory, pro-, or anti-tumorigenic [19][20][21]. The transcriptional profiles of mast cells clearly demonstrate the heterogeneity of mast cells and their different gene expression [14,[22][23][24]. Moreover, human mast cells analyzed ex vivo or differentiated in vitro showed significant differences [24]. Human and mouse mast cells have distinct proteomes and unique gene expressions compared to other immune cells [22,25]. Single-cell transcriptomics of human lungs provide evidence of mast cells [26,27]. Different triggers (e.g., IgE-mediated or IL-33) can induce distinct genomic and transcriptional changes in human mast cells [28]. Individual mast cells are exposed to their local environment (e.g., cytokines, different pH, growth factors, etc.) and, over time, are tuned by many different activating and inhibitory signals. Mast cells in different organs differ in their receptor and mediator expression, but there is also considerable heterogeneity among human lung mast cells [29]. It is possible to speculate that individual mast cells could all be unique to some extent.
In this review, we provide an overview of current and promising treatments for asthma that directly or indirectly target lung mast cells.

Activating and Inhibitory Receptors on Human Mast Cells
Human mast cells display a wide spectrum of cell surface receptors that can be activated by several immunologic and non-immunologic stimuli that modulate their development and effector functions [11,30]. Figure 1 schematically illustrates the main activating and inhibitory receptors on human lung mast cells relevant to bronchial asthma. vivo or differentiated in vitro showed significant differences [24]. Human and mouse mast cells have distinct proteomes and unique gene expressions compared to other immune cells [22,25]. Single-cell transcriptomics of human lungs provide evidence of mast cells [26,27]. Different triggers (e.g., IgE-mediated or IL-33) can induce distinct genomic and transcriptional changes in human mast cells [28]. Individual mast cells are exposed to their local environment (e.g., cytokines, different pH, growth factors, etc.) and, over time, are tuned by many different activating and inhibitory signals. Mast cells in different organs differ in their receptor and mediator expression, but there is also considerable heterogeneity among human lung mast cells [29]. It is possible to speculate that individual mast cells could all be unique to some extent.
In this review, we provide an overview of current and promising treatments for asthma that directly or indirectly target lung mast cells.

VEGF-A
The main angiogenic factor released by HLMCs. VEGFs released by macrophages, basophils, and neutrophils contribute to mast cell infiltration in bronchial asthma. [56,117,125-128]

Role of Mast Cells in Asthma
FcεRI cross-linking by allergens, anti-IgE, or super allergens results in the release of histamine, cytokines/chemokines, enzymes such as tryptase and chymase, and the generation of eicosanoids (i.e., LTC 4 and PGD 2 ) from human mast cells [32,134]. Mast cell-derived mediators are responsible for bronchoconstriction, airway inflammation, and remodeling in different asthma endotypes [2]. The density of mast cells within airway smooth muscle (ASM) bundles is increased in asthmatic patients compared to controls [90]. There is an inverse correlation between the number of mast cells in the ASM and airway hyperresponsiveness (AHR) in asthmatics [90]. HLMCs adhere avidly to ASM cells [135], which favor mast cell survival and activation [136]. Elevated circulating mast cell progenitors are correlated with reduced lung function in allergic asthma [137]. The rapid IgE-dependent release of histamine and eicosanoids (e.g., LTC 4 and PGD 2 ) from isolated HLMCs [138] correlates with these mediators in bronchoalveolar lavage fluids following bronchial allergen challenge [139][140][141]. Histamine can promote mucus secretion and bronchoconstriction. Asthma is accompanied by airway remodeling [142] and angiogenesis [143,144], and lung mast cells may contribute to this by the release of several cytokines, chemokines [88], and VEGFs [58, 126,145,146]. Submucosal mast cells, which are abundant in healthy controls, are shifted from the submucosal compartment to the epithelium in asthma [147]. IL-33activated mast cells increase the expression of epithelial IL33, which in turn upregulates the production of type-2 cytokines (i.e., IL-5, IL-13, IL-4) in mast cells. These findings demonstrate a shift in the location of mast cells to the epithelium in asthma and identify intraepithelial mast cells as critical modulators of inflammation in asthma. Psychological stress is thought to induce mast cell activation via the stimulation of peripheral nerves and the release of substance P and corticotropin-release hormone (CRH) [148]. Human mast cells express CRH receptors and their activation induces the selective release of VEGF-A without degranulation [149]. These findings provide a hypothetical link between stress, mast cell activation, and asthma exacerbations [150]. The role of HLMCs in inducing the symptoms of human airway inflammation is also supported by the efficacy of drugs, which block either their function or target mediators primarily released by these cells. Figure 2 schematically illustrates the multiple interactions between HLMCs and several cells of the innate and adaptive immune system through the release of mediators. HLMCs can also interact with non-immune cells involved in bronchial asthma ( Figure 3).

Tryptase
Circulating β-tryptase levels were increased in asthmatics independently of type 2 inflammation and associated with lesser omalizumab response [92]. MTPS9579A, a mAb that inhibits the activity of tryptase, is in a phase II trial in patients with moderate-to-severe asthma (NCT04092582). E104 and 31A.v11 are anti-tryptase mAbs showing promising effects in preclinical models of allergic reactions [92,158].

Cysteinyl Leukotrienes
Leukotriene inhibitors (i.e., montelukast, zafirlukast, pranlukast, and zileuton) have been used with mixed results in allergic diseases. Montelukast, approved by the Food and Drug Administration (FDA) for the treatment of asthma and allergic rhinitis [162], is less effective compared to inhaled or intranasal glucocorticoids [163]. AZD5718, a reversible 5-lipoxygenase activating protein (FLAP) that suppresses leukotriene synthesis, is currently in Phase II trial for moderate-to-severe asthma treatment compared to montelukast (NCT05251259).

Mast Cell Cytokines and Their Receptors
IL-13 and IL-5 are produced by HLMCs [107]. Phase III studies demonstrated that two anti-IL-13 mAbs, lebrikizumab [164] and tralokinumab [165,166], did not reduce asthma exacerbation rates but did improve lung function in patients with severe asthma [166][167][168]. In contrast, dupilumab, a mAb which is a dual inhibitor of IL-4 and IL-13 through blockade of their shared IL-4Rα subunit, is approved for the treatment of severe uncontrolled asthma and chronic rhinosinusitis with nasal polyps [169][170][171]. The anti-IL-5 mAbs, mepolizumab [172,173] and reslizumab [174], and the anti-IL-5Rα mAb benralizumab [175] are approved as add-on therapy for the treatment of severe eosinophilic asthma [176]. These drugs markedly deplete blood eosinophils and decrease the frequency of asthma exacerbations and improve lung function in patients with severe uncontrolled asthma [174,177,178]. TNF-α is released by mouse mast cells [5], but its production by HLMCs is still controversial. Golimumab, a human mAb anti-TNF-α, showed an increase in adverse events and inconsistent efficacy in severe asthma patients [179].
A mAb anti-IL-17R, brodalumab, did not demonstrate efficacy in asthma [180]. Although mast cells are a major source of several cytokines, their production of other cytokines (i.e., IL-17A) may be selectively restricted to mast cell subtypes (e.g., synovial and skin mast cells [181]). Mast cells are also a source of IL-23 and express IL-23R [181]. Risankizumab, a mAb anti-IL-23, showed increased asthma worsening in a phase 2a trial [182].

Alarmins and Their Receptors
There is increasing evidence that bronchial epithelial cells represent not only a physical barrier but also an immune organ, which plays a central role in asthma pathobiology [183,184]. TSLP, IL-33, and IL-25 are upstream epithelial-derived cytokines, collectively known as alarmins [40,43,185]. These cytokines also activate downstream a broad range of cellular targets, including mast cells, to propagate the release of several cytokines involved in asthma [40].

Tezepelumab
Tezepelumab is a human IgG2λ mAb, which binds to TSLP, which is involved in different asthma phenotypes [40]. TSLP is overexpressed by the airway epithelium of asthmatics [115]. TSLP levels are increased in the BAL fluid of asthmatics [186] and serum during asthma exacerbations [187]. Tezepelumab was recently approved by the FDA and European Medicines Agency (EMA) for severe asthma treatment with no phenotype or biomarker limitations. In RCTs, tezepelumab reduced annual exacerbation rates regardless of blood eosinophil count, with an increase in prebronchodilator FEV 1 compared to the placebo group [188,189]. In two different RCTs, tezepelumab reduced AHR, suggesting an effect on lung mast cell activation [190,191]. TSLP can promote airway remodeling in asthma through different mechanisms: it activates human lung fibroblasts [192] and causes angiogenic and lymphangiogenic factor release from HLMs [193]. In the phase II CASCADE study, the effects of tezepelumab on airway remodeling were examined in moderate-to-severe asthmatics [190]. Tezepelumab reduced airway submucosal eosinophils compared to placebo. A human mAb anti-TSLP (HBM9378) [194] and an inhaled antibody fragment against TSLP (CSJ117) [195] (NCT03138811; NCT04410523; NCT04946318) are under development for asthma treatment. Figure 4 schematically illustrates the inhibition of mast cell activation by different biologics and drugs.

Itepekimab
Itepekimab is a human IgG4 mAb that binds to IL-33. In a phase 2 trial, the safety and efficacy of itepekimab, dupilumab, itepekimab plus dupilumab, or placebo were compared in moderate-to-severe asthmatics [200]. Loss of asthma control was similar in the three groups but better than in the placebo. Itepekimab and dupilumab monotherapies increased pre-bronchodilator FEV 1 , reduced peripheral blood eosinophils, and improved asthma control and quality of life compared to placebo.

Astegolimab and Etokimab
Astegolimab is a human IgG2 mAb that targets ST2, the IL-33 receptor, and blocks IL-33 signaling [46,202]. In the phase 2b ZENYATTA study, astegolimab was well-tolerated and reduced the number of exacerbation rates in severe asthma patients [202]. Astegolimab did not significantly modify FEV 1 compared to placebo in the entire population of asthmatics. FEV1 improvement appeared to be higher in patients with low blood eosinophils.
Etokimab (ANB020) is a humanized mAb that binds to IL-33. A preliminary study found that etokimab has the potential to desensitize subjects allergic to peanuts [201].

Tozorakimab
Tozorakimab (formerly MEDI3506) is a mAb that binds to IL-33 [216]. RCTs are evaluating the safety and efficacy of tozorakimab compared to placebo in adults with moderate-to-severe asthma (NCT04570657) and chronic obstructive pulmonary disease (COPD) with a history of exacerbations (NCT05166889). mAbs blocking IL-25 have shown beneficial effects in a mouse model of allergic asthma [217].

FcεRI and IgE
Omalizumab, a humanized IgG1-k mAb that binds to Fcε, was the first mAb approved by the FDA for the treatment of patients with moderate and severe asthma [218]. It binds to free IgE and inhibits the IgE-FcεRI interaction by preventing the binding of IgE to FcεRI on human mast cells and basophils. Omalizumab also downregulates FcεRI expression [219]. Omalizumab did not improve FEV 1 in RCTs [220][221][222], but there is some evidence that it can improve FEV 1 in real-life settings [212,222]. It reduces asthma symptoms and exacerbations [223][224][225].
Ligelizumab is a second-generation humanized anti-IgE mAb, which has a higher affinity for the Cε3 domain of IgE compared to omalizumab and may affect IgE production by B cells [213]. Ligelizumab failed to meet the primary endpoints in phase II clinical trials of asthma, and it was discontinued (NCT02075008, NCT02336425). The safety and efficacy of ligelizumab are presently investigated in chronic urticaria (NCT05024058, NCT04513548, NCT03580356, NCT03580369, NCT02477332, NCT04903613). There are several promising compounds targeting FcεRI and/or IgE under investigation. GI-301, an IgE trap-Fc fusion protein, and the anti-IgE mAb UB-221 showed higher affinity to IgE compared to omalizumab (NCT05298215). Combined treatment with omalizumab and omalizumab-resistant IgE-Fc fragment (IgE-R419N-Fc3-4 mutant) caused more inhibition of basophil activation than either agent alone [226]. It has been proposed that exon skipping of the β-subunit of FcεRI in mast cells eliminated FcεRI expression and function in these cells [227].
Several BTK inhibitors are used for the management of hematological tumors [237] and are in development for the treatment of mast cell-driven diseases, including acalabrutinib for anaphylaxis [238] (NCT05038904), remibrutinib for CSU and food allergy (NCT05432388, NCT05032157, NCT05170724, NCT05513001), fenebrutinib for CSU (NCT036933625), and ibrutinib for food allergy [239] and anaphylaxis (NCT03149315). Concern has risen regarding the risk of cardiovascular adverse events associated with BTK inhibitors [237].
A phase I study assessed the safety and efficacy of GDC-0214, an inhaled JAK inhibitor, in adults with mild asthma [214]. This compound caused a dose-dependent reduction in fractional exhaled nitric oxide (FeNo) in patients with mild asthma. Additional studies on the effects of JAK inhibitors are expected for asthma treatment [240].
AK006, a humanized IgG1 agonistic Siglec-6 mAb, inhibits mast cell activation in vitro. Interestingly, co-culturing human mast cells with macrophages in the presence of AK006 induces antibody-dependent phagocytosis of mast cells [215]. These findings represent a novel strategy to selectively reduce mast cells via Siglec-6 targeting.

Depleting Mast Cells
Human mast cells express high levels of KIT throughout their development [16]. Activation of KIT by SCF influences several aspects of mast cell responses. Dysregulation of the SCF/KIT pathway markedly alters mast cell homeostasis. For instance, loss-of-function mutations in SCF or KIT result in mast cell deficiency; in contrast, gain-of-function mutations in KIT lead to mast cell hyperplasia and activation, as found in mastocytosis [250][251][252]. The blockage of the SCF/KIT pathway has been investigated in several models of allergic disorders [253][254][255][256][257]. A bispecific antibody cross-linking KIT and CD300a [247] inhibit SCF-induced human mast cell differentiation and survival and skin reactions induced by SCF in mice [247].
Mast cell apoptosis can be achieved via neutralization of the effects of SCF and/or blockage of its receptor KIT (CD117) ( Figure 5). CDX-0159 (Celldex Therapeutics, NJ, USA) is a humanized mAb that binds to the extracellular dimerization domain of KIT [258,259]. This mAb is under investigation in CSU (NCT04538794) and chronic inducible urticaria (NCT04548869). In a phase Ia trial, CDX-0159 administration showed a favorable safety profile and caused a marked reduction of peripheral blood tryptase, suggestive of systemic mast cell depletion (NCT04146129). It remains to be evaluated whether this mAb may work in experimental models of asthma [256].  260,261]. Preliminary evidence indicates that prolonged administration of imatinib [262] and masitinib [263,264] can influence airway hyperresponsiveness or reduce asthma exacerbations in asthma patients. These beneficial effects have been tentatively attributed to the inhibition of mast cell activation and/or depletion of mast cells. Another approach to induce mast cell apoptosis is through blockage of SCF-KIT interaction. CDX-0159 is a mAb that targets the extracellular dimerization domain of KIT [258,259] and causes a marked reduction of peripheral blood tryptase, suggesting systemic mast cell depletion (NCT04146129).
Another approach to block KIT signaling in mast cells is to use specific tyrosine kinase inhibitors (TKIs) ( Figure 5). There are several classes of KIT-targeting TKIs, which display distinct pharmacologic characteristics on human mast cells in vitro [260]. KITtargeting drugs can inhibit mast cell activation and mediator-induced symptoms in allergic disorders [265][266][267][268]. There are very preliminary data on the in vivo efficacy of KITspecific or multitargeted TKIs in the treatment of patients with severe allergic disorders (e.g., severe asthma). The administration of masitinib to patients with severe glucocorticoid-dependent asthma was associated with steroid-sparing effects [264]. Imatinib did not influence lung function. In another study, imatinib reduced airway hyperresponsiveness in patients with severe asthma compared to controls [262].  260,261]. Preliminary evidence indicates that prolonged administration of imatinib [262] and masitinib [263,264] can influence airway hyperresponsiveness or reduce asthma exacerbations in asthma patients. These beneficial effects have been tentatively attributed to the inhibition of mast cell activation and/or depletion of mast cells. Another approach to induce mast cell apoptosis is through blockage of SCF-KIT interaction. CDX-0159 is a mAb that targets the extracellular dimerization domain of KIT [258,259] and causes a marked reduction of peripheral blood tryptase, suggesting systemic mast cell depletion (NCT04146129).
Another approach to block KIT signaling in mast cells is to use specific tyrosine kinase inhibitors (TKIs) ( Figure 5). There are several classes of KIT-targeting TKIs, which display distinct pharmacologic characteristics on human mast cells in vitro [260]. KITtargeting drugs can inhibit mast cell activation and mediator-induced symptoms in allergic disorders [265][266][267][268]. There are very preliminary data on the in vivo efficacy of KIT-specific or multitargeted TKIs in the treatment of patients with severe allergic disorders (e.g., severe asthma). The administration of masitinib to patients with severe glucocorticoid-dependent asthma was associated with steroid-sparing effects [264]. Imatinib did not influence lung function. In another study, imatinib reduced airway hyperresponsiveness in patients with severe asthma compared to controls [262].
In a phase III trial, masitinib reduced asthma exacerbations compared to placebo in severe asthma patients [263]. Avapritinib (BLU-285), a potent inhibitor of mutant KIT and PDGFRA with activation loop mutations, induces mast cell cytoreduction and remission in the majority of advanced systemic mastocytosis patients [269,270]. It should be emphasized that some of these TKIs also inhibit IgE-dependent basophil activation [271][272][273]. This is relevant because basophils play a role in allergic disorders [88,274]. Future studies should evaluate the safety and efficacy of imatinib, masitinib, and possibly newer TKIs in patients with different phenotypes of severe asthma.

Discussion and Conclusions
Human mast cells were identified and named over 140 years ago by Paul Ehrlich [1]. IgE was discovered by Kimishige and Teruko Ishizaka [275] and Gunnar Johansson [276]. The approval of omalizumab, the first mAb anti-IgE for the treatment of asthma in 2003, was a breakthrough in the treatment of patients with mast cell-driven diseases, such as asthma and CSU. Since then, several biologics targeting mast cells directly or indirectly have been approved for the treatment of severe asthma. In particular, mAbs targeting IL-5 (mepolizumab [204][205][206][207] and reslizumab [208,209]), IL-5Rα (benralizumab) [175,210,211], IL-4Rα (dupilumab) [169][170][171]212], and TSLP (tezepelumab) [188,189] have been demonstrated to reduce annual exacerbation rates and also certain features (e.g., FEV 1 ) of airway remodeling in severe asthmatic patients. The efficacy and safety of the above mAbs have been recently discussed in detail [142]. Collectively, these clinical findings support the involvement of lung mast cells in central features of severe asthma.
We are going through an exciting and promising era for understanding human mast cell biology. However, we must consider that many aspects of mast cell biology and their complex phenotypic and functional heterogeneity remain largely unknown. Mast cells are exposed to their local environment that, over time, can modify their phenotype and biochemical machinery [277]. More studies using novel techniques (e.g., single-cell mRNA seq, CyTOF) will more accurately reveal mast cell heterogeneity [278,279]. These techniques will contribute to identifying the role of mast cell subtypes in different asthma phenotypes. Another level of complexity derives from the species differences in extrapolating findings from mouse mast cell models to human settings [19,43].
Human mast cells and basophils have some similarities (e.g., FcεRI) but also striking differences [43]. Basophils have been recently identified in the human lung [26,27,280], where they play a prominent role in macrophage differentiation [281]. Macrophages represent the most prominent immune cells in human lung tissue [282,283]. There is also evidence that basophils and their mediators (i.e., IL-4, IL-13) play a role in Th2 and M2 polarization in allergic asthma [43]. Likely, some biologics that primarily target mast cells (e.g., omalizumab, mepolizumab, benralizumab) may also target human basophils [284,285].
Mast cells and their mediators play homeostatic and protective roles in several pathophysiological conditions [19,286]. Moreover, several normal cell types, such as germ cells, hematopoietic stem cells, and melanoblasts, express KIT, and the chronic administration of TKIs and mAbs targeting KIT may be associated with long-term adverse effects. Caution will be necessary in the future when drugs able to markedly reduce tissue mast cells in humans will be available for the treatment of mast cell-driven diseases.

Acknowledgments:
The authors apologize to the many researchers who have contributed importantly to this field and whose work was not cited due to space and citation limitations. The authors thank the administrative staff (Roberto Bifulco, Anna Ferraro, and Maria Cristina Fucci) and the medical graphic artist Fabrizio Fiorbianco for the elaboration of figures.