Complement Components As Potential Therapeutic Targets for Asthma Treatment Mohammad Afzal Khan A,*, Mark R

Respiratory Medicine (2014) 108, 543e549

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REVIEW Complement components as potential therapeutic targets for asthma treatment Mohammad Afzal Khan a,*, Mark R. Nicolls b, Besiki Surguladze c, Ismail Saadoun a a Department of Applied Biology, College of Sciences, University of Sharjah, Sharjah, United Arab Emirates b Division of Pulmonary and Critical Care Medicine, VA Palo Health Care System, Stanford University, School of Medicine, Palo Alto, CA, USA c Innovative Bio-Medical Technologies Ltd, Toronto, Canada

Received 6 February 2013; accepted 7 January 2014 Available online 15 January 2014

KEYWORDS Summary Complement Asthma is the most common respiratory disorder, and is characterized by distal airway inflam- mediated injury; mation and hyperresponsiveness. This disease challenges human health because of its Asthma; increasing prevalence, severity, morbidity, and the lack of a proper and complete cure. e Anaphylatoxins Asthma is characterized by TH2 skewed inflammation with elevated pulmonary levels of IL- 4, IL-5, and IL-13 levels. Although there are early forays into targeting TH2 immunity, less- specific corticosteroid therapy remains the immunomodulator of choice. Innate immune injury mediated by complement components also act as potent mediators of the allergic inflammatory responses and offer a new and exciting possibility for asthma immunotherapy. The complement cascade consists of a number of plasma- and membrane-bound proteins, and the cleavage products of these proteins (C3 and C5) regulate the magnitude of adaptive immune responses. Complement protein are responsible for many pathophysiological features of asthma, including inflammatory cell infiltration, mucus secretion, increases in vascular permeability, and smooth muscle cell contraction. This review highlights the complement-mediated injury during asthma inflammation, and how blockade of active complement mediators may have therapeutic application. ª 2014 Elsevier Ltd. All rights reserved.

Abbreviations: AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; ASM, airway smooth muscle; MAC, membrane attack complex; Treg, regulatory T cells. * Corresponding author. Applied Biology and Biotechnology, College of Sciences, University of Sharjah, Sharjah, United Arab Emirates. Tel.: þ971 6 505 3829; fax: þ971 6 5053814. E-mail addresses: [email protected], [email protected] (M.A. Khan).

Contents Introduction ...... 544 Generation of c3a and c5a in asthma ...... 545 Complement mediators-immune cell interaction in asthma pathogenesis ...... 546 Summary ...... 546 Conflict of interest ...... 547 References ...... 547

Introduction classical, alternative, lectin pathways, and by the direct action of certain proteolytic enzymes on C3 or C5 [7] Asthma is a chronic inﬂammatory disease of the bronchi (Fig. 1), participate in AHR induction. Infections, and al- arising because of inappropriate immunological responses lergens of respiratory tract activate local complement e to common environmental antigens in genetically suscep- activation participate in AHR [8 10] because of their ability tible individuals [1]. It is thought to be mediated by CD4þ T to recruit, activate leukocytes, increase vascular perme- lymphocytes that produce T 2 cytokines linked with ability, stimulate contraction of smooth muscle, and trigger H e elevated speciﬁc IgE, eosinophilia, and airway hyper- degranulation of mast cells [9,11 13]. In addition to al- responsiveness (AHR) [2e4]. This perspective will explore lergens, other triggers of asthma have been shown to how an important component of the innate immunity, the activate complement cascade in human, and in animal complement system, normally a key defense against models [13]. It has been demonstrated that bronchoalveo- mucosal bacteria, viruses, fungi, helminthes, and other lar lavage (BAL) of asthma individuals contain quantita- pathogens, may also play an important role in the patho- tively higher levels of C3a and C5a as compared to healthy genesis of asthma. Although complement factors have been control subjects at baseline [14]. associated with development of pathophysiology of asthma In asthma, overproduction of activated complement [5,6], the role of individual complement components in the fragments may promote asthma susceptibility [13]. This pathogenesis of allergic asthma is not clear. Biologically imbalance results in up regulation of biologically active active fragments (C3a, C5a), generated through the fragments, C3a and C5a, which may act on cells of the

Classical Lectin Alternative

C4b2a C3bBb

C3b

C4b2a3b C3bBb3b C3aR C5

C5b

C3a bind to C3a bind MAC

C5a

Release of histamine

Mast cell CD4+

Release of IL-17 and Airway narrowing and AHR IL-4, Il-13, IL-5 (neutrophil inflammation)

Figure 1 Model explains the generation of C3a and C5a through classical, lectin and alternative pathway during airway inﬂammation. Further, C3a binds to C4aR on CD4þ T cells and promotes recruitment of IL-17þCD4þ cells, neutrophil inﬂammation and activation of Mast cells that leads to histamine mediated AHR. Complement components for asthma treatment 545

IL-2 involvement of T-lymphocytes, eosinophils, altered immu- nosuppressive macrophages, excessive nitric oxide through C3a C5a Th1 response inducible nitric oxide synthase, overproduction of proinflammatory cytokines and immunoglobulins [22] during the C5a blockade asthma development. Asthmatic inflammation may be initiated or exacerbated Th2 response by amplification of the complement cascade [11e13]. Complement components, especially C5 and C3 with their associated cleavage products C5a and C3a, regulate the magnitude of adaptive immune responses via ligation of their respective receptors expressed on antigen-presenting cells, and T lymphocytes, as well as on pulmonary struc- tures, and stromal cells [5,22]. These immune responses involve many pathophysiological features of asthma that Figure 2 Model explains Th1 to Th2 shift during the devel- include inflammatory cell infiltration, mucus secretion, in- opment of asthma pathogenesis, and, C3a and C5a as a potential crease vascular permeability, and smooth muscle contrac- targets to rescue asthma by blocking local T cell recruitment. tion [23]. This review summarizes the crucial role of complement mediators in airway inflammation, and how it affects the pathogenesis of asthma disease. innate immune system to favor asthma development [9,11]. The anaphylatoxins C3a and C5a have been characterized as potent mediators of the effector phase of the allergic Generation of c3a and c5a in asthma response [8e10,15] with C3a regulating TH2 cytokine production possibly through the recruitment, and activation of Asthma is associated with activation of complement TH2 cells [13]. C5a plays a dual immunoregulatory role by cascade and allergen induced complement generates C3a protecting against the TH2-polarized adaptive immune and C5a [3]. It has been demonstrated that C3a plays a response and mediates type 2 inflammatory responses once crucial role in asthma primarily by regulating mast cell-ASM inflammation proceeds [13] (see Fig. 2). Complement may (Airway Smooth Muscle) cell interaction [14]. C3a and C5a participate in the development of susceptibility to asthma, are released as key active factors in complement cascade despite a normal level of complement fragments generated that modulate innate immunity [3,4]. C5a is, however, during complement activation. involved in a number of inflammatory diseases [24] such as Different models of experimental allergic asthma sug- immune-complex-mediated lung injury, microvascular gest that the C3a and C5a not only promote pro-allergic injury in rejecting allografts [20] and in sepsis [14]. Levels effector functions during the allergic effector phase, but of C3a are found elevated in bronchoalveolar lavage fluid also regulate the development of TH2 immunity during after allergen challenge in asthmatic but not among allergen sensitization [16]. Generation of C3a on airway healthy controls [3]. The C3a and C5a peptides regulate surfaces induce TH2-mediated inflammatory responses to a inflammatory functions by interacting with their receptors variety of environmental triggers of asthma (i.e., allergens, C3aR and C5aR [25,26]. These receptors were mostly pre- pollutants, viral infections, cigarette smoke) [9,11]. C5a is sent only on myeloid cells such as macrophages, neutro- dominant during allergen sensitization, and protects phils, eosinophils, basophils, and mast cells, however, the against the development of maladaptive TH2 immunity immune cells that express these receptors in the lung have [13,16]. By contrast, C3a and C5a appear to act synergis- been investigated, and their expression been examined tically and drive allergic inflammation during the effector during phase of asthma inflammation [27e30]. These find- phase [10]. In addition to its proinflammatory effector ings suggests the participation of bronchial epithelial and functions, complement regulates adaptive immunity at smooth muscle cells in the pathology of diseases such as many levels [12], and play critical role as well in causing sepsis and asthma, the data suggest a role for complement vascular injury in allografts [17,18]. receptors during lung inflammation [27]. It has been observed that allergen challenged C3aR- It has been observed that C3aR activation is associated deficient mice and guinea pigs are protected against with the development of AHR, and inflammation in bronchoconstriction and AHR [16]. Interestingly, there was different animal models of asthma [27]. However, C3aR- no difference in eosinophilic airway inflammation, TH2 deficient mice are protected from AHR in response to cytokine production, IgE production between C3a receptor- aerosolized ovalbumin challenge following intraperitoneal deficient, and in wild type animals which demonstrate that sensitization with ovalbumin [31]. Single nucleotide poly- airway inflammation, and AHR are two independent fea- morphisms in C3 and C3aR genes have been linked with tures of asthma [2,3]. Several studies have demonstrated increased susceptibility to asthma [31]. This speculates the that blocking of IL-4 reduces AHR in the lung, and that crucial role of C3a and C3aR in the development of AHR and RAG / mice, which lack Th2 cells, fail to develop AHR, inflammation [31]. BAL fluids of C3aR deficient mice also mucus hyper-secretion, and eosinophilia during the course had low levels of TH2 cytokines (IL-4, IL-5, and IL-13), IgE of asthma [19]. However, airway inflammation, and the titers, and mucous production that further support a role of immune responses at cellular and molecular levels have led C3a receptors in the development of AHR, and generalized to the proposition of a number of mechanisms such as mast inflammation [15,31,32]. It is observed that deficiency of cell degranulation [18,20,21], neurogenic dysfunction, C3aR leads to decrease airway hyperresponsiveness in a 546 M.A. Khan et al. mouse model pulmonary allergy [32]. In addition, increased Direct administration of IL-12 has shown to reduce AHR, and C3a levels have been reported in bronchial lavage samples macrophages from the C5-deficient mice were here shown from allergen-challenged asthma patients [3]. There is a to produce lower levels than the control mice [39e42]. C3b significant association has been reported between AHR and on the other hand, has been shown as capable of blocking C5 level [3], however, compared to C5 sufficient mice, the IL-12 production by its interaction with the aMb2 integrin, C5-deficient mice are more responsive to methacholine an action perfectly in keeping with a positive role for C3 challenges after allergen exposure [33]. The presence of C5 cleavage in AHR e either via C3a and its receptor or through and C5aR is necessary for a variety of immunological re- C3b and reduced levels of IL-12 [43]. sponses including inflammation and host defense [14]. C5 has been associated with dendritic cells mediated Elevated levels of complement anaphylatoxin peptides induction of Tregs (CD4þCD25þ T cells) and Tregs blockade have been observed in the lungs of asthmatic patients [27] in allergen exposed C5 sufficient mice eliminated their which further supports the significance of complement protection from the development of AHR associated with a factors in asthma pathogenesis. The C5 gene and the C5aR drop in the numbers of pulmonary dendritic cells [13].In receptor genetic regions have been identified as putative addition, depletion of dendritic cells and Tregs in mice asthma susceptible loci [27]. Finally, C3aR and C5aR results in an increased capacity to stimulate T cell prolif- expression has demonstrated on lung bronchial smooth eration and Th2 cytokine production. The balance between muscle cells implicating these receptors as mediators of C3a and C5a during early life exposures to allergens may be bronchoconstriction [34]. a crucial determinant factor in the development of tolerance to inhaled antigens [9,13]. In lungs, C3 would most probably create Th2 shift, which is consistent with data Complement mediators-immune cell suggesting that the lungs have Th2 type cell at birth in interaction in asthma pathogenesis newborn [44]. Clinical studies has shown the relatively higher levels of C3a and C5a in BAL fluid of allergen induced Cells of the innate immune system in asthmatics are asthmatic airways as compared with control subjects abnormally responsive to the regulatory effects of com- [5,10,16]. C5a contribute to the development of the pro- plement followed by the development of susceptibility to allergic environment in allergic asthma [10], and targeting asthma [10,35]. Recent research efforts have also demon- C5 in allergen-induced asthma model have demonstrated þ strated that CD4 T cells, which produce a TH2 pattern of that C5 may serve as a suitable target in treatment of cytokines, play a pivotal role in the pathogenesis of this asthma [10,45]. disease [52] and cytokines such as IL-4, IL-13, and IL-5 to C5a can bind to both C5aR and C5L2 receptors [5], and, contribute in bronchial hyper-reactivity, and mucus hyper- more specifically C5L2 acts at the dendritic cell and T cells secretion as well as orchestrate the recruitment, activation interface, and control the development of TH1 and TH17 of mast cells, and eosinophils [19,53,54]. The complement cells in response to airway antigen exposure, and drives TH2 cascade is a central player of innate immunity that co- immune responses independent of specific dendritic cells ordinates a number of inflammatory responses [35]. C3a [46]. As reported earlier, C5a, and perhaps C3a may cause activates mast cells, basophils, eosinophils, and contrac- immediate airflow obstruction, and subsequent airway hy- tion of airway smooth muscle cell [18,24]. Both C3a and C5a peractivity [27]. It has been demonstrated in murine model can induce ASM cell contraction, increase the microvascular of AHR that C5a may act directly or indirectly to stimulate permeability, and regulate vasodilation [5]. C5a has been C5aR on local mast cells and/or platelets, resulting in the widely used as standard stimulant to eosinophil/or basophil release of broncho constrictive mediators, and results in responsivity, and active C5 fragments alone can induce sensitization of the airways without cellular inflammation airway hyperresponsiveness when administered [24].In [47]. In a number of other asthma models, the role of IL-17 addition, C3a and C5a can: 1) stimulate respiratory burst in has been highlighted in inducing asthmatic response, and macrophages, neutrophils, and eosinophils; 2) stimulate AHR [48]. There has been increasing evidence suggest the the release of histamine from basophils and mast cells; and involvement of C3a in the asthma pathogenesis, and the 3) regulate the synthesis of eosinophil cationic proteins and relationship between C3a driven IL-17 and IgE-mediated adhesion to endothelial cells by eosinophils [14,20]. C3a asthmatic responses that have shown the contribution of can also stimulate serotonin release from platelets, and IL-17 to an IgE-mediated late-phase asthmatic response, modulate synthesis of IL-6 and TNF-a by B-lymphocytes and and AHR [48]. They reported that during repeated antigen monocytes [36,37]. C5a is a potent chemotactic molecule exposure, C3a mediated antibody production (IgE) results in for macrophages, neutrophils, T lymphocytes, and baso- production of IL-17þCD4þ cells in the lungs [18,24,49] phils [27]. Both C3a and C5a can induce chemotaxis of eo- (Fig. 1). sinophils and mast cells [27]. Generation of C3a at the airway surface triggers induction of AHR [13], while C5/ C5a, plays a dual immunoregulatory role by protecting Summary against the initiation of Th2-mediated immune responses during initial allergen exposure by its ability to affect Asthma, a complex airway inflammatory disease, is char- dendritic cell-T cell interactions, and a more traditional acterized by bronchoconstriction, AHR and airway remod- pro-inflammatory role once immune responses are estab- elling [50]. Current consensus suggests that TH2 cytokine lished [13,16]. producing T cells, mast cells, and ASM cells play central The interactions between C5a and the IL-12 are impor- roles in the pathogenesis of asthma [51]. This classification tant for generating AHR-associated inflammation [38]. of asthma has led to the concept that the immediate Complement components for asthma treatment 547 response after allergen challenge is mediated by mast cells, recognized that some of the endogenous complement reg- whereas eosinophils are the predominant effector cells in ulatory proteins has been proven to serve as potential the late asthmatic reaction [27]. C3 and C5 play unique therapeutic agents in blocking inappropriate activation of roles in airway inflammation associated with asthma and complement in human diseases specially asthma the release of C3a at the airway surface mediates the in- [34,45,71,72]. In this review, our aim is to focus on more duction of AHR in different asthma models, while C5/C5a translational approach in the field of asthma cure with the plays a dual immunoregulatory role by protecting against possible use of novel complement inhibition approach to Th2-mediated immune responses during initiation of re- control complement mediated airway injury, hyper- sponses, and a proinflammatory role once immune re- responsiveness and ultimately to rescue asthma. sponses are established [50]. Serine proteases generated in response to classical and alternative pathways has potential to generate C3a and C5a from C3 and C5 respectively Conflict of interest [55,56]. It is observed that different components of the complement cascade have implicated in mediating allergic The authors have no conflict of interest. inflammation [57]. 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