The role of dendritic cells in asthma

Transcription

1 Mechanisms of allergic diseases Series editors: Joshua A. Boyce, MD, Fred Finkelman, MD, William T. Shearer, MD, and Donata Vercelli, MD The role of dendritic cells in asthma Michelle Ann Gill, MD, PhD Dallas, Tex INFORMATION FOR CATEGORY 1 CME CREDIT Credit can now be obtained, free for a limited time, by reading the review articles in this issue. Please note the following instructions. Method of Physician Participation in Learning Process: The core material for these activities can be read in this issue of the Journal or online at the JACI Web site: The accompanying tests may only be submitted online at Fax or other copies will not be accepted. Date of Original Release: April Credit may be obtained for these courses until March 31, Copyright Statement: Copyright Ó All rights reserved. Overall Purpose/Goal: To provide excellent reviews on key aspects of allergic disease to those who research, treat, or manage allergic disease. Target Audience: Physicians and researchers within the field of allergic disease. Accreditation/Provider Statements and Credit Designation: The American Academy of Allergy, Asthma & Immunology (AAAAI) is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians. The AAAAI designates these educational activities for a maximum of 1 AMA PRA Category 1 Creditä. Physicians should only claim credit commensurate with the extent of their participation in the activity. List of Design Committee Members: Michelle Ann Gill, MD, PhD Activity Objectives 1. To understand the role of dendritic cells (DCs) in the mucosal surfaces of the lung and during allergic inflammation, serving as innate sensors of foreign antigens/pathogens, and to determine whether the response to an inhaled antigen will entail the induction of tolerance or allergic inflammation. 2. To present the complexities of molecular targets and chemokine interactions responsible for allergen-mediated chemotaxis of DCs to the airway and of the tuning of the magnitude of the DC response amenable to therapeutic interventions that can reduce or abolish the asthmatic allergic lung inflammation. Recognition of Commercial Support: This CME activity has not received external commercial support. Disclosure of Significant Relationships with Relevant Commercial Companies/Organizations: M. A. Gill declares that she has no relevant conflicts of interest. Dendritic cells (DCs) are known to play a central role in sensing the presence of foreign antigens and infectious agents and in initiating appropriate immune responses. More recently, an additional role has been discovered for DCs in determining whether the response to potential environmental allergens will be one of tolerance or whether a vigorous response along allergic pathways will be initiated. This review discusses ways in which DCs participate specifically in initiating allergic responses, particularly those associated with allergic asthma, and how interventions focused on DCs might lead to new therapeutic approaches to asthma. (J Allergy Clin Immunol 2012;129: ) Key words: Dendritic cells, asthma, allergen, T H 2 inflammation From the Department of Pediatrics, Division of Infectious Diseases, University of Texas Southwestern Medical Center at Dallas. Received for publication November 23, 2011; revised February 21, 2012; accepted for publication February 23, Corresponding author: Michelle Ann Gill, MD, PhD, Department of Pediatrics, Division of Infectious Diseases, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Mail Code 9063, Dallas, TX Michelle.Gill@UTSouthwestern.edu /$36.00 Ó 2012 American Academy of Allergy, Asthma & Immunology doi: /j.jaci Abbreviations used BALF: Bronchoalveolar lavage fluid BDCA: Blood dendritic cell antigen cdc: Conventional dendritic cell DC: Dendritic cell HDM: House dust mite IDO: Indoleamine 2,3-dioxygenase ILT7: Immunoglobulin-like transcript 7 mdc: Myeloid dendritic cell MMP-9: Matrix metalloproteinase 9 OVA: Ovalbumin OX40L: OX40 ligand PAR: Protease-activated receptor pdc: Plasmacytoid dendritic cell SIRP-a: Signal regulatory protein a TARC: Thymus and activation-regulated chemokine TLR: Toll-like receptor TRAIL: TNF-related apoptosis-inducing ligand TSLP: Thymic stromal lymphopoietin WT: Wild-type Dendritic cells (DCs) play critical roles in initiating and directing immune responses, serving as sentinels at the mucosal surfaces, where they constantly sample the antigens at the interface between the external and internal environment. DCs 889

2 890 GILL J ALLERGY CLIN IMMUNOL APRIL 2012 are unique in their capacity to induce primary lymphocyte responses, representing the principal cells involved in directing T H 1 responses to infectious agents. DCs also play primary roles in determining the nature of T-lymphocyte differentiation in the face of allergen exposure. 1 Instructive cytokines, including IL-4, IL-12, IL-10, IL-6, and TGF-b, are known to participate in T-lymphocyte differentiation. Most of these cytokines, with the exception of IL-4, are produced by DCs themselves and are reviewed elsewhere. 2,3 Through mechanisms that are not yet clearly understood in the lung in vivo, DCs can drive the differentiation of uncommitted T H cells into T H 2 cells, which are key mediators of allergic airway inflammation. DCs are therefore of primary importance in determining the type of T H response that is elicited on aeroallergen inhalation, and in light of this strategic role, they are poised to both direct immune responses to allergen and affect the development and perpetuation of allergic inflammation associated with asthma. The purpose of this review is to acquaint the clinician with the role of DCs in initiating and sustaining allergic inflammation associated with asthma and to point out how this knowledge could lead to new therapeutic approaches. In general, discussions begin with studies in mice and extend to available data in human subjects. KEY STUDIES: ROLE OF DCs IN ANIMAL MODELS OF ASTHMA Evidence supporting the participation of DCs in asthma pathogenesis is derived mainly from animal models. The role of DCs in promoting allergic inflammation and the clinical features of asthma has been primarily established in mice using an ovalbumin (OVA) sensitization model. 4 After sensitization, which is usually induced by intraperitoneal injection of OVA in a T H 2-inducing adjuvant such as alum, repeated aerosol challenges result in lung eosinophilic infiltrates and enhanced secretion of mucus by airway epithelial cells. These changes are accompanied by airway obstruction and airway hyperresponsiveness after methacholine challenge, both of which are key features of asthma. Data from animal models are robust; summary of these data into 3 key observations clearly establishes a role for DCs in the development of experimental allergic asthma. 5 First, significant increases in the numbers of airway DCs after exposure to allergen have been observed in both murine and rat models of asthma. 6-8 Potential mechanisms for this allergen-mediated recruitment of DCs include activation of Toll-like receptor (TLR) 4 and synthesis of b-d-glucan and matrix metalloproteinase 9 (MMP-9; see DC recruitment section). Second, it has been demonstrated that placement of OVApulsed DCs directly into the airways of naive animals results in not only OVA sensitization but also an ensuing T H 2 response, eosinophilic airway inflammation, goblet cell hyperplasia, and bronchial hyperreactivity after rechallenge with OVA aerosol. 9,10 Antigen-pulsed DCs also have been shown to promote the development of allergic inflammation when administered intratracheally in primed mice, even in the absence of antigen aerosol Finally, it has been shown that depletion of DCs from OVAsensitized mice abrogates aeroallergen-induced airway hyperreactivity and that repletion of these cells restores the asthma phenotype in these animals. 11,13 All of these observations together support critical roles for DCs in both the development and maintenance of allergen-induced airway inflammation and hyperreactivity in murine models. In evaluating the evidence for a DC-asthma association in human subjects, parallels can be made only for the first observation that numbers of airway DCs are increased in human subjects after allergen challenge. These studies are reviewed below in the DC recruitment to the airway section. DCs IN THE LUNG ENVIRONMENT DCs have been identified in dense networks throughout the epithelium of the respiratory tract, including the nose, nasopharynx, large conducting airways, bronchi, bronchioles, and alveolar interstitium Populations of DCs exist both above and beneath the basement membrane of the respiratory epithelium, 20 positioning these cells as first responders to incoming antigens. Defining the division of labor between lung DC subsets in murine models has contributed to the current understanding of the role of DCs in the airway, especially how, on allergen inhalation, they contribute to the development of tolerance versus allergic inflammation. 14,21,22 Several lung DC subsets with distinct functions have been identified in specific anatomic locations. 23 Although there is significant overlap in the expression of DC surface proteins among these subsets, certain subsets, defined by unique surface marker profiles, have been shown to be particularly associated with allergic inflammatory responses. DC subsets in the murine lung can be broadly separated into 2 categories: conventional dendritic cells (cdcs), which express high levels of the integrin CD11c, and plasmacytoid dendritic cells (pdcs), which express Siglec-H, Ly6C, and B220 but low levels of CD11c. Importantly, the 3 DC subsets described in Table I have distinct anatomic locations and division of labor. The cdc subset can be further subdivided into the CD103 1 cdc group and the CD11b 1 cdc group. CD103 is an ae integrin that is highly expressed at mucosal sites. The first subset, CD103 1 cdcs, is intimately associated with the respiratory epithelium. Here they project their dendritic extensions between epithelial cells, allowing them to directly sample airway luminal contents. This subset of lung DCs has been termed the intraepithelial subset. These cells express tight junction proteins, which allow them to anchor themselves within the epithelial cell layer. Enzyme activity of allergens, such as the cysteine protease of Der p 1 (Dermatophagoides pteronyssinus allergen), causes cleavage of epithelial tight junctions, 24 providing a potential mechanism by which CD103 1 DCs begin migration to lymph nodes, where they can transfer antigen to resident lymph node DCs. A second lung DC subset is the CD11b 1 cdc subset that resides beneath the basement membrane in conducting airways and lung parenchyma. This subset displays an efficient capacity for priming and restimulating effector CD4 1 T cells in the lung. 11,25 CD11b 1 DCs play a major role in influencing allergic inflammation by providing a rich source of proinflammatory chemokines, such as TNF-a and thymus and activation-regulated chemokine (TARC)/CCL ,26 Secretion of these chemokines results in attraction of T H 2 CD4 1 and CD8 1 effector T cells to the lung, a critical step in the development of allergic inflammation. Although cdcs contribute to the development of tolerance to inhaled allergens, 27 tolerance is particularly dependent on the presence of a third lung DC subset, pdcs. After taking up inhaled

3 J ALLERGY CLIN IMMUNOL VOLUME 129, NUMBER 4 GILL 891 TABLE I. Major murine lung DC subsets DC subset Surface markers Specialized function Anatomic location CD103 1 cdcs intraepithelial CD11b 2 CD103 1 CD11c 1 Langerin 1 Tight junction proteins CD11b 1 cdcs CD11b 1 CD103 2 CD11c 1 pdcs Siglec-H, Ly6C, B220 CD11c int, Gr-1 int Periscope surveillance of airway luminal surface, antigen uptake Efficient priming and restimulating effector CD4 T cells in lung; rich source of proinflammatory chemokines, attracting effector CD4 and CD8 cells Role in inducing tolerance to inhaled antigens, induce regulatory T-cell development; secretion of type I IFN Associated with epithelium of large conducting airways above basement membrane Submucosa/lamina propria of conducting airways beneath basement membrane and lung parenchyma Large conducting airways beneath basement membrane; lining alveolar septum TABLE II. Human lung DC subsets DC subset Surface markers Examples of studies identifying DCs in human lung mdcs (type 1) CD11c 1 BDCA1 1 (CD1c) MHC class II (HLA-DR) 1 mdcs (type 2) CD11c 1 BDCA3 1 (CD141) MHC class II (HLA-DR) 1 pdcs CD123 1, CD11c 2 BDCA2 1, BDCA4 1 MHC class II (HLA-DR) 1 ILT7 1 d Demedts et al 34 : 3 subsets in digests of normal lung specimens: 1. HLA-DR 1 CD11c 1 BDCA1 1 mdc subset 2. HLA-DR 1 CD11c 1 BDCA3 1 mdc subset 3. CD123 1 BDCA2 1 pdc subset d Masten et al 35 : CD1c 1 CD11c 1 CD14 2 HLA-DR 1 mdcs and CD123 1 CD11c 2 CD14 2 HLA-DR 1 pdcs (BDCA2 1 pdcs identified) in normal lung specimens d Bratke et al 36 : CD11c 1 HLA-DR 1 mdcs and CD123 1 HLA-DR 1 pdcs in BALF after allergen challenge d Lommatzsch et al 37 : CD1a 1 CD11c 1 HLA-DR 1 mdcs, CD1a 2 CD11c 1 HLA-DR 1 mdcs, and CD123 1 HLA-DR 1 pdcs in BALF from healthy and disease states; evidence of influence of pulmonary inflammatory diseases on DC airway recruitment and phenotype antigens, including allergens, pdcs have the capacity to drive the development of regulatory T cells, a cell type critical to the development of tolerance to foreign antigens. 28 Evidence that pdcs provide intrinsic protection against inflammatory responses to harmless antigen has been established in murine models of OVA-induced asthma. 28,29 de Heer et al 28 demonstrated that depletion of pdcs with either anti Gr-1 antibody or the pdcspecific antibody 120G8 30 during OVA inhalation resulted in the development of cardinal features of asthma, including airway eosinophilia and T H 2 cytokine production. Moreover, adoptive transfer of pdcs into mice before OVA sensitization prevented disease in this model. A possible mechanism by which pdcs inhibit allergic airway inflammation lies in the capacity of pdcs to release massive concentrations of IFN-a on stimulation with viruses or certain TLR agonists. 31 It should be emphasized that this mechanism has been demonstrated in human studies. Recent in vitro studies of human circulating naive CD4 1 T cells revealed that IFN-a blocks T H 2 development through suppression of GATA-3, the primary T H 2 transcription factor. In addition, IFN-a inhibited secretion of IL-4, IL-5, and IL-14 from committed T H 2 cells. 32 Thus pdcs might exert inhibitory effects on the development of T H 2 inflammatory responses in vivo through their secretion of IFN-a. This aspect of pdc biology as it relates to asthma is discussed in more detail in the subsequent section entitled Relationships among viruses, IFN-a, IgE,DCs,andasthma. The above scheme represents a simplified view of lung DC subset functions; division of labor and specialized functions can be further subdivided based on location in the airways versus lung parenchyma. 33 A greater delineation of the precise functions of these distinct lung DC subsets will provide opportunities for the development of potential new therapies targeting DC-driven development of allergic inflammation. In human subjects knowledge regarding lung DC subsets is not as complete. As in mice, DCs can be broadly divided into 2 major groups: myeloid dendritic cells (mdcs; also referred to as conventional DCs) and pdcs. 14 Expression of the integrin CD11c combined with expression of a set of blood dendritic cell antigen (BDCA) antibodies has recently been used to define/differentiate mdc and pdc subsets (Table II) mdcs can be further subdivided based on the expression of surface BDCA1 and BDCA3: type 1 mdcs express BDCA1 (CD1c), and type 2 mdcs express BDCA3 (CD141). 34 CD1a expression also delineates another subset of mdcs and has been demonstrated on human lung DCs as well. CD11c 1 CD1a 1 DCs can differentiate to Langerhans cells at epithelial surfaces, whereas CD11c 1 CD1a 2 mdcs replenish mdcs within interstitial compartments. 38 Demedts et al 34 identified CD1a 1 mdcs in human lung tissues; interestingly, these DCs were most abundant in the epithelium, whereas the CD1c 1 mdcs were found more frequently in the submucosa. pdcs express BDCA2 (CD303), CD123 (the IL-3 receptor), and immunoglobulin-like transcript 7 (ILT7). Both mdcs and pdcs have been identified in human lung tissue; examples of studies demonstrating this are shown in Table II. 34,35 Compared with the knowledge of murine lung DC subsets, little is known

4 892 GILL J ALLERGY CLIN IMMUNOL APRIL 2012 FIG 1. DCs regulate allergic inflammation in the lung. A simplified scheme demonstrating the multiple locations and roles DCs play in the development of allergic inflammatory responses associated with the pathogenesis of asthma is shown. regarding DC subset anatomic localization and specific functions in the human lung during health and disease. MODULATION OF DC FUNCTION BY ALLERGEN For more information on modulation of DC function by inhaled allergens, see Fig 1. DC recruitment to the airway Studies in mice. Numbers of DCs in the airway increase substantially after allergen exposure. The mechanisms underlying this allergen-driven chemotaxis of DCs have been the focus of several studies. In one such study challenge with house dust mite (HDM) allergen resulted in rapid recruitment of monocytederived CD11b 1 DCs to the lungs in mice. 6 Interestingly, activation of TLR4 was found to be an important step in triggering this DC migration. Experiments were performed in mice chimeric for TLR4 expression to determine the relative contribution of hematopoietic cell (including DCs) versus structural airway cell TLR4 expression on lung DC recruitment. Briefly, TLR4-deficient and wild-type (WT) mice were sublethally irradiated to deplete hematopoietic cells and subsequently reconstituted with hematopoietic cells from either TLR4-deficient or WT mice. Results revealed that TLR4 expression on airway structural cells but not on DCs or other hematopoietic cells was required in the development of HDM-driven allergic airway inflammation. The absence of TLR4 on airway cells inhibited the development of such inflammation. Additionally, TLR4 blockade, through inhalation of a TLR4 antagonist, suppressed HDM-driven inflammation, secretion of T H 2 cytokines, and bronchial hyperreactivity. 6 Taken together, these data highlight a critical role for TLR4 expression on airway cells in mediating DC responses to allergic stimuli. In another study, trafficking of DCs to the lung on inhalational OVA challenge was found to be dependent on MMP This protease, which is secreted by immune cells, including eosinophils, neutrophils, and alveolar macrophages, facilitates the migration of inflammatory cells between tissue compartments through its ability to degrade collagen IV, a major constituent of basement membranes. In this study deletion of the gene encoding MMP-9 resulted in specific inhibition of DC transmigration into the airways after OVA challenge. 39 Additionally, MMP-9 deficient mice had both decreased local airway concentrations of the DC-derived T H 2-attracting chemokines CCL17 and TARC and significant attenuation of OVA-induced peribronchial airway inflammation. These data indicate an important role for MMP-9 in DC recruitment to the airways during allergic inflammatory responses. Increased airway epithelial cell secretion of CCL-2 (monocyte chemoattractant protein 1) has also been linked to the influx of DCs to the lung after OVA sensitization and challenge. 43 CCL-2 is the ligand for CCR2, a chemokine receptor expressed on DCs. Investigators used mixed bone marrow chimeric mice containing both WT and knockout cells for several chemokine receptors in an OVA sensitization model to explore the role of CCR2 expression in DC recruitment to the airways. After induction of an allergic airway inflammatory response with OVA, receptor knockout versus WT mice DC populations were tracked through various lung compartments. Using this approach, the authors demonstrated that the allergen-mediated increase in pulmonary DC numbers was dependent on CCR2. 43

5 J ALLERGY CLIN IMMUNOL VOLUME 129, NUMBER 4 GILL 893 FIG 2. DC airway epithelium interactions relevant to the pathogenesis of allergic asthma. A simplified scheme depicting the mediators involved in epithelial cell DC cross-talk that contributes to allergic inflammation is shown. NF-kB, Nuclear factor kb. Another crucial chemokine receptor in DC recruitment to the lungs is CCR6, the receptor for the chemokine CCL20 (macrophage inflammatory protein 3a). CCR6 is expressed on circulating immature DCs, 44 and its interaction with CCL20 leads to the recruitment of DCs to the lungs after allergen inhalation. 45,46 CCL20 is secreted by airway epithelial cells on allergic stimulation. Studies in human subjects. Studies in human subjects have also demonstrated allergen-induced DC migration into the airways. In one such study bronchial biopsy specimens were obtained from patients with allergic asthma (patients with both asthma and allergic sensitization to >_1 environmental allergens) before and 4 to 5 hours after local challenge with HDM allergen. Immunofluorescent staining of bronchial mucosal sections revealed a significant increase in CD1c 1 HLA-DR 1 DC numbers (a subset of mdcs) induced by allergen challenge. A concomitant decrease in numbers of circulating blood CD11c 1 DCs (mdcs) in the postallergen specimens suggested that these mdcs were recruited from the blood into the airways by means of allergen stimulation. 47 In a similar study, increased numbers of mdcs and pdcs were observed in bronchoalveolar lavage fluid (BALF) samples after segmental allergen challenge in patients with allergic asthma. 36 In this study increased BALF DC numbers were observed 24 hours after segmental allergen challenge (allergens included dust mite, rye grass, and birch). A concomitant decrease in blood DC subset numbers was observed also after allergen challenge, suggesting that the increased BALF DC numbers were recruited from the blood. 36 Another study in asthmatic patients revealed a reduction in blood CD11c 1 DC numbers as early as 3 hours after allergen challenge with ragweed or dust mite allergen, suggesting recruitment of these cells from the blood to the lung and indicating that recruitment occurs quickly in response to allergen challenge. 48 DC interactions with airway epithelial cells The close physical association of DCs with airway epithelial cells suggests a potential role for DC epithelial cell interactions in modulating inflammatory responses to allergens. DCs and epithelial cells express specific cell-surface proteins and cytokines that can modulate each other s functions. 49,50 The conversation between DCs and epithelial cells through expression, secretion, or both of such proteins as thymic stromal lymphopoietin (TSLP), OX40 ligand (OX40L), GM-CSF, CCL20, and others (Fig 2), promotes inflammatory cytokine production and the recruitment of inflammatory cells. Thus in light of this extensive cross-talk, it is not surprising that these 2 cell types have been shown to play key roles in the pathogenesis of asthma. 2 Much of these data are obtained from animal models, thus presenting some limitations of the extension of these findings to human disease. TSLP. TSLP is a 140-amino-acid IL-7 like epitheliumderived cytokine that contributes to lymphopoiesis. TSLP is produced by epithelial cells, fibroblasts, keratinocytes, and stromal cells, and its receptor has been demonstrated on human mdcs and pdcs. 51 Expression of TSLP is induced by a variety of

6 894 GILL J ALLERGY CLIN IMMUNOL APRIL 2012 stimuli, including TLR ligands, certain proinflammatory cytokines (TNF, IL-1a, and IL-1b), and T H 2 cytokines, such as IL-4 and IL Importantly, increased epithelial TSLP expression has been demonstrated in asthmatic patients by means of in situ hybridization of bronchial biopsy specimens, and this expression correlates with asthma disease severity and T H 2-attracting chemokine expression. 53 Moreover, TSLP also promotes allergic inflammation by inducing DCs to drive the differentiation of naive CD4 1 T cells into inflammatory T H 2 cells that secrete IL-4, IL-5, and IL-13, as well as large concentrations of TNF. 51 This inflammatory T H 2 cell differentiation is mediated through TSLPinduced DC expression of the TNF superfamily protein OX40L. 54 DC-expressed OX40L interacts with OX40 on naive T cells, resulting in T H 2 lineage commitment by initiating signaling events that lead to the production of IL-4 and GATA-3 transcription. 55 T H 2 T-cell polarization is further enhanced by IL-25, another cytokine secreted by epithelial cells. 56 Increased epithelial cell expression of IL-25 in the airways of allergic asthmatic patients represents another mechanism by which epithelial cell DC interactions promote development of T H 2 inflammation. 56 TSLP stimulates DCs to synthesize high concentrations of the T H 2 cell attractants TARC (CCL17) and macrophage-derived chemokine (CCL22). 54 TSLP also activates DCs to secrete IL-8 and eotaxin 2, resulting in the recruitment of granulocytes and eosinophils. 57 Thus, although TSLP does not induce direct DC secretion of T H 1orT H 2 cytokines, it induces both the differentiation of naive T cells into T H 2 lymphocytes and chemotaxis of already differentiated lymphocytes. GM-CSF. GM-CSF (granulocyte-macrophage colony stimulating factor) represents another airway epithelial cell product that influences DCs to propagate T H 2 lymphocyte responses. Exposure to allergen-derived proteases associated with HDM and German cockroach allergens induces airway epithelial cell GM- CSF secretion. 58,59 GM-CSF induces DC maturation, resulting in increased expression of DC costimulatory molecules and increased priming of T-lymphocyte responses. In mice overexpression of GM-CSF causes animals to become sensitized to OVA after aerosol exposure. Mice infected with an adenovirus construct expressing GM-CSF (Ad/GM-CSF) had OVA-induced eosinophilic airway inflammation, whereas mice exposed to OVA alone did not, indicating that localized airway GM-CSF can promote a T H 2 inflammatory response to an otherwise innocuous antigen. 60 Interestingly, other elicitors of T H 2-mediated inflammation, including diesel exhaust particles and cigarette smoke, can induce DC maturation. This maturation occurs indirectly as a result of DC stimulation by GM-CSF released from human bronchial epithelial cells after exposure to such airway irritants. 61 GM-CSF therefore constitutes another epithelial cell product that, on release (stimulated by allergens and irritants), leads to DC activation with subsequent skewing of lymphocyte responses toward T H 2 pathways. CCL20. Multiple studies in both human subjects and mice have demonstrated the role of epithelial cell secreted CCL20 in DC recruitment to the respiratory tract. Human bronchial epithelial cells exposed to HDM secrete CCL20, a known chemoattractant for DCs. 46,62 Using a human airway epithelial cell line, Nathan et al 62 demonstrated that HDM exposure induced specific secretion of CCL20. Interestingly, this HDM-induced CCL20 secretion was mediated through epithelial cell recognition of b-d-glucan moieties in HDM allergen, a distinct molecular pattern specific to this allergen. This novel pathway of stimulation by HDM allergen led to airway epithelial cell secretion of CCL20, 62 a chemokine crucial for the initial recruitment of DCs to the lung during allergic airway responses. 59 Similar results were demonstrated in asthmatic patients in a study conducted by Pichavant et al. 46 Stimulation with the dust mite allergen Der p 1, the major component of Dermatophagoides pteronyssinus, induced increased CCL20 production from bronchial epithelial cells of asthmatic patients sensitized to HDM, leading to increased migration of Langerhans cell precursors, a subpopulation of mdcs that reside at mucosal surfaces. 46 Another mechanism underlying allergen-induced production of CCL20 involves interactions between protease-activated receptors (PARs) expressed on airway epithelial cells and allergen-associated proteases. PARs, a family of G protein coupled receptors, can be cleaved and activated by proteases, such as those found in dust mite and cockroach allergens. This signaling by allergen-associated proteases through PARs has been demonstrated to play a role in modulating airway hyperresponsiveness through the activation of PAR-2 on airway epithelial cells. Through the activation of PAR-2, allergen-derived proteases induce both CCL20 and GM-CSF production in the airways, leading to increased recruitment, differentiation, or both of mdc populations in the lungs. 59 IL-1b and TNF. IL-1b and TNF are pleiotropic cytokines that have major roles in initiating inflammatory and innate immune responses. IL-1b, an important participant in airway epithelial cell DC interactions, is secreted by airway epithelial cells after exposure to Der p 1 allergen. IL-1b can induce epithelial cell production of CCL20, a DC chemoattractant, and boost epithelial TLSP and GM-CSF secretion (Fig 2). 63,64 TNF, another inflammatory cytokine, also plays a key role in DC epithelial cell interactions. Studies in mice have shown that TNF can break tolerance to inhaled allergens through activation of lung DCs. 65 The airway epithelium secretes increased amounts of TNF on stimulation with known asthma exacerbators, such as allergens, cigarette smoke, and diesel exhaust particles, thus potentially skewing DCs toward the promotion of T H 2 inflammatory responses. TNF-related apoptosis-inducing ligand. TNF-related apoptosis-inducing ligand (TRAIL), which is abundantly expressed in the airway epithelium of allergic mice, has been demonstrated to play a role in DCs and T H 2 recruitment to the respiratory tract. Epithelial cells produce enhanced TRAIL in response to allergen challenge (and adoptive transfer of T H 2 cells). This in turn leads to increased production of CCL20 with resulting chemoattraction of CCR6 1 DCs and T H 2 effector cells, thus perpetuating allergic inflammation. 45 Blockade of TRAIL signaling impairs CCL20-induced homing of mdcs to the airway in this murine model. Whether TRAIL is important in the pathogenesis of human disease is not known. Direct effects of allergens on DCs In addition to epithelium-associated allergen effects on DCs, several direct effects of allergens on DCs have been described. HDM, through its interaction with the receptor dectin-2 on bone marrow derived murine DCs, results in the synthesis and release of cysteinyl leukotrienes by DCs. 66 Also, allergens containing proteases have been shown to activate PAR-2 on pulmonary mdcs. This activation has been shown to induce both allergic airway inflammation and airway

7 J ALLERGY CLIN IMMUNOL VOLUME 129, NUMBER 4 GILL 895 hyperresponsiveness in a murine model of cockroach allergy. 67 DC lymph node trafficking and influence of allergens DCs migrate from mucosal surfaces of the lung to T cell dependent areas of regional lung lymph nodes, where they present antigens to T lymphocytes and initiate cognate T-cell responses. This migration occurs in response to a gradient of chemokines CCL19 and CCL21 interacting with their receptor (CCR7), which is expressed by DCs after antigen uptake. 68 Several lines of evidence indicate that CCR7-mediated DC lymph node migration plays an important role in the balance between allergic versus tolerant responses to antigens. In a murine model (Runx3 knockout mice) enhanced CCR7 expression on bone marrow derived DCs resulted in increased migration of CD11c 1 DCs to regional lymph nodes. Strikingly, this was accompanied by the development of asthma-associated features, including increased serum IgE levels and airway hyperreactivity. 69 Overexpression of DC CCR7 might therefore promote the development of allergic lung responses. However, in another model CCR7-mediated DC transport of innocuous antigens to bronchial lymph nodes was demonstrated to be critical in developing tolerance to inhaled antigens. 70 Other chemokine receptors also contribute to DC lymph node trafficking. Mice with the plt (paucity of lymph node) mutation, a mutation resulting in disruption of CCR7 signaling, still display appreciable T H 2 responses to OVA challenge. 71 In addition to CCR7, another protein, the integrin-associated CD47, and its binding partner ligand, signal regulatory protein a (SIRP-a), have been shown to induce DC migration from lung tissues to pulmonary lymph nodes in an allergen-induced model of asthma. 72 Blockade of this SIRP-a 1 CD47 pathway by administration of a SIRP-a Fc molecule protected mice from OVA-induced allergic airway inflammation, whereas adoptive transfer of SIRPa 1 CD47 1 DCs into CD47 2/2 mice resulted in restoration of strong T H 2 responses. 72 Other murine models of allergic asthma provide evidence that, like OVA, HDM allergen exposure induces migration of lung DCs to lung lymph nodes, 6,73 a phenomenon that occurs as early as 1 day after allergen inhalational exposure and peaks at day 3. The importance of DC migration to the lymph node in the development of airway inflammation also has been demonstrated in a humanized severe combined immunodeficiency mouse model. In this model inhibition of HDM-induced airway inflammation was abolished by antibodies specific for CCL In summary, a variety of chemokines and ligands not only direct the migration of DCs from the respiratory tract to lung lymph nodes but can also alter the function of DCs by modulating the immune response to the antigens they transport. Additional molecules, including CCR8, cysteinyl leukotrienes, and prostaglandins, also contribute to the complex pathways mediating DC migration to pulmonary lymph nodes and are reviewed elsewhere. 75 RELATIONSHIP AMONG VIRUSES, IFN-a, IgE, DCs AND ASTHMA Type I interferon (IFN-a/b) is a family of cytokines with potent antiviral activity. pdcs represent the major source of IFN-a on stimulation with viruses 31 and are recruited to the airway during FIG 3. IFN-a blocks allergic inflammation. IFN-a negatively regulates the development of T H 2 and T H 17 lymphocyte responses. In the face of allergic sensitization and exposure, activation of IgE-mediated pathways on pdcs results in suppression of virus-induced IFN-a release by pdcs. This diminished pdc IFN-a secretion in patients with allergic asthma could result in loss of this suppression of allergic inflammation and contribute to virusinduced asthma exacerbations. This figure was adapted with permission from J. David Farrar, PhD. respiratory tract viral infections Respiratory tract viral infections are common precipitants of asthma exacerbations. 79,80 Although the specific mechanisms by which viral infections precipitate wheezing episodes remain unclear, research from our laboratory and from others suggests that asthmatic subjects might have deficient viral-induced IFN-a responses In studies of pdcs purified from blood, patients with allergic asthma had diminished influenza-induced IFN-a secretion from pdcs compared with healthy control subjects. 81 Tversky et al 84 also found a similar impairment of IFN-a responses in pdcs from allergic subjects after stimulation with oligodeoxynucleotide-containing unmethylated CpG motifs (TLR9 agonist). Another effect of IFN-a/b that is particularly relevant to asthma pathogenesis is its influence on T H development. 32,85,86 Huber et al 32 recently demonstrated that IFN-a/b blocks human T H 2 development and inhibits IL-4, IL-5, and IL-13 secretion from committed T H 2 cells by suppressing GATA-3, a major T H 2 transcription factor. 32 IFN-a also has been shown to negatively regulate T H 17 development in mice 86,87 and human subjects. 86 Thus these data taken together suggest that the impaired capacity of pdcs to secrete IFN-a/b in patients with allergic asthma might contribute to the development of T H 2 and T H 17 inflammatory responses and that these responses might be at least partially responsible for virus-induced asthma exacerbations (Fig 3). Recent data support the notion that mechanisms involving IgE and the expression of its high-affinity receptor, FcεRI, might underlie the defect in pdc IFN-a secretion present in patients with allergic asthma. We recently reported that the magnitude of pdc IFN-a responses to in vitro viral challenge is reduced in subjects with increased serum IgE concentrations. 81 Similarly, Tversky et al 84 reported an inverse correlation

8 896 GILL J ALLERGY CLIN IMMUNOL APRIL 2012 FIG 4. IgE-mediated suppression of pdc antiviral IFN-a responses. A schematic depicting the role of IgE in pdc antiviral responses is shown. A, IgE is constitutively bound to its high-affinity receptor, FcεRI, on pdcs. In the absence of allergic stimulation, exposure to respiratory tract viruses, such as influenza and rhinovirus, results in upregulation of pdc TLR7 and interferon regulatory factor 7 (IRF-7), resulting in robust IFN-a release. B, In the presence of allergen (or cross-linking anti-ige), suppression of virus-induced TLR7 and IRF-7 responses occurs, with resulting impairment in IFN-a release. We propose that this could occur in vivo through cross-linking of allergen-specific IgE on pdcs in the face of allergic sensitization and exposure. between TLR9-mediated pdc IFN-a production and pdc FcεRI expression in allergic subjects. In addition to these findings, we found that IgE cross-linking significantly inhibited virus-induced pdc IFN-a secretion (Fig 4), 81 thus creating a T H 2-promoting environment (Fig 3). Schroeder et al 88 also demonstrated similar counterregulation between IgE-mediated signaling and pdc IFN-a responses. Interestingly, treatment of subjects with cat allergy with anti-ige (omalizumab; Genentech, Inc, South San Francisco, Calif, and Novartis, Basel, Switzerland) resulted in diminished capacity for allergen-exposed DCs to elicit T H 2 cytokine responses, illustrating the potential relevance of this IgE-mediated pathway in vivo. 89 Taken together, these data suggest a link between IgE and pdc antiviral responses that could, in part, explain the increased risk of asthma exacerbations associated with atopy and respiratory tract viral infections mdcs also express the FcεRI receptor. 93 In a murine model of virus-induced airway hyperreactivity, cross-linking of FcεRI on lung mdcs led to secretion of CCL28, recruitment of CD4 1 T cells, and mucous cell metaplasia. 94 CCL28 can also chemoattract eosinophils, effector T H 2 cells, and resting CD4 1 and CD8 1 T lymphocytes. 95 FcεRI 2/2 mice exhibited decreased virus-induced CCL28 and diminished IL-13 producing CD4 1 T-cell recruitment to the lung. 94 Additionally, CCL28 blockade resulted in abrogation of virus-induced mucus cell metaplasia in this model. Taken together, these results suggest that FcεRI expression on mdcs, as well as pdcs, might be important in eliciting key features of asthma after viral infection. Omalizumab, a recombinant monoclonal antibody that selectively binds to human immunoglobulin E, reduces IgE levels in vivo, 96 and has been shown to reduce the degree of airway inflammation in patients with allergic asthma. 97 The effect of omalizumab in clinical studies has been promising. The recent National Institute of Allergy and Infectious Diseases sponsored Inner City Anti-IgE Therapy of Asthma trial evaluated the addition of omalizumab to guidelines-based therapy in children with asthma. 98 Participants who received omalizumab experienced a significant overall decrease in asthma morbidity compared with the guidelines-based group. Of particular interest was the near-complete elimination of the fall increase in asthma exacerbations in the omalizumab group, a seasonal increase known to be associated with the fall peak in rhinovirus respiratory tract viral infections. One potential mechanism for omalizumab s effect on allergic inflammation might involve its effect on DCs. In vivo, omalizumab has been shown to decrease FcεRI expression on blood DCs in human subjects. 99 Moreover, a recent study of subjects with intermittent persistent asthma revealed that anti-ige therapy significantly decreased the number of airway mdcs identifiable in bronchial biopsy specimens, a finding that correlated with improved allergen-induced airway hyperreactivity. 93 In contrast, the numbers of airway pdcs were slightly increased in the same study, suggesting that pathways involving IgE-mediated events might affect DC subsets differentially. DCs IN THE DEVELOPMENT OF T H 2 INFLAMMATION Sensitization and effector responses Evidence demonstrating the role of DCs in the induction of T H 2 responses to inhaled allergens and their relevance to the pathogenesis of allergic asthma is extensive. 2,21 Recently, the comparative

9 J ALLERGY CLIN IMMUNOL VOLUME 129, NUMBER 4 GILL 897 role of basophils and DCs in the induction of T H 2 immunity to inhaled HDM allergen was investigated in a murine model of allergic asthma. Although basophils were found to contribute to the T H 2 responses elicited on HDM inhalation, inflammatory DCs, specifically FcεRI-expressing inflammatory DCs, were both necessary and sufficient for the induction of HDM-induced T H 2 immune responses. 73 Moreover, results from another study showed that a specific CD103-expressing subset of lung DCs plays a significant role in priming T H 2 responses to inhaled antigens, including OVA, HDM allergen, and cockroach allergen. 100 In this model mice deficient in CD103 1 DCs were actually refractory to the development of allergic sensitization through the airway, although skin-associated allergic sensitization was intact, findings that underscore the complexity of DC subsets and their roles in inducing allergic inflammation at different sites. 100 Allergic subjects are rarely hypersensitive to only 1 allergen, suggesting that sensitization to 1 antigen facilitates sensitization to multiple other allergens/antigens. DCs also have been implicated in mechanisms leading to multiple sensitization. Using a murine model, van Rijt et al 101 demonstrated that chronic inflammation induced by 1 antigen (FBS proteins) causes lung DCs to promote T H 2-like inflammatory responses when other potential allergens (OVA) are encountered subsequently, despite the absence of prior sensitization to these allergens. 101 Although DCs do not produce the T H 2-instructive cytokine IL-4, 21 multiple potential mechanisms leading to their induction of T H 2 responses have been demonstrated. Although beyond the scope of this review, lung DC IL-6 secretion, 102 OX40L 54 expression, and instructive signals mediated by such cell-surface molecules as c-kit 102 and Notch ligand jagged also have been shown to contribute to DC-driven T H 2 cell development. In addition to their essential role in initiation of primary T-cell responses to allergens, DCs also are very efficient in restimulating memory T cells. Lung DCs represent the predominant source of the chemokines CCL17 and CCL22, and these cells have been shown to recruit T H 2 cells to the lungs in mice challenged with allergen. 103 In a humanized severe combined immunodeficiency model of asthma, inhalational HDM challenge after reconstitution with blood mononuclear cells from donors with HDM allergy led to CCL17 and CCL22 production by the DCs and subsequent attraction of human CCR4 1 T H 2 cells to the lungs. 104 Indoleamine 2,3-dioxygenase Expression of indoleamine 2,3-dioxygenase (IDO), an enzyme that regulates tryptophan metabolism, has been linked to DC induction of T H 2 inflammatory responses. Tryptophan, an amino acid, is essential for protein synthesis during cellular processes, including activation and proliferation of T cells. The expression of IDO by a subset of DCs inhibits T-cell proliferation, suggesting a role for IDO-expressing DCs in the development of immune tolerance. 105 Another study demonstrated that allergens might subvert this suppressive effect of DC-expressed IDO on T-cell responses. Der p 1 exposure suppressed IDO activity in DCs from HDM-sensitive asthmatic patients, resulting in T H 2 skewing of DC-induced T-cell responses. 106 Although these results point to IDO as a tolerance-generating signal in DCs, other studies provide conflicting data. In contrast to the studies described above, IDO in lung DCs was shown to promote T H 2 responses and allergic inflammation in a murine model of OVA sensitization. 107 Furthermore, in an IDO 2/2 murine model, challenge with OVA resulted in marked attenuation of allergic airway disease, suggesting that IDO expressed in lung DCs might contribute to asthma pathogenesis. ATP and purinergic signaling: Linking DCs to T H 2 airway inflammation Purines, in particular ATP, have been implicated as important mediators in the pathogenesis of allergic inflammation associated with asthma. 108 Accumulation of extracellular ATP in BALF after airway allergen challenge has been demonstrated in both human subjects with allergic asthma and OVA-sensitized mice. 109 Additionally, neutralization of airway ATP before OVA challenge abrogated the cardinal features of asthma in this murine model. Inhibition of P2-purigenic receptors administered before OVA challenge blocked the capacity of mdcs to drive ATPdependent T H 2 responses in the lung. 109 P2X 7 R, a purinergic receptor that is highly expressed on DCs, has been shown to be upregulated during acute and chronic asthmatic airway inflammation in both mice and human subjects. 110 P2X 7 R-deficient DCs displayed a reduced capacity to induce T H 2 inflammation in the lung. Taken together, these data highlight purinergic signaling as another pathway contributing to DCmediated allergic inflammation. POTENTIAL THERAPEUTIC TARGETS BASED ON MODULATING DC FUNCTION Although significant accomplishments in reducing asthma mortality and morbidity have been made over the past 20 years, new therapeutic strategies are needed to further alleviate disease burden. 111 Airway DCs represent novel targets for directed therapy for asthma. Several general principles in targeting DCs for potential therapy have been suggested. 2 With respect to current therapies, it is notable that inhaled corticosteroids, one of the cornerstones of asthma treatment, result in a significant reduction in the number of DCs in the bronchial mucosa of subjects with allergic asthma. 112 With respect to future therapies, there is considerable promise in continued dissection of the mechanisms governing epithelial cell modulation of DC responses in the face of allergen challenge. The suggestion that viewing asthma as an epithelial disease will lead to much-needed therapeutic strategies aimed at improving airway resistance to the inhaled environment 113 places DCs in the spotlight as potential therapeutic targets at the epithelial interface. For example, specific blocking of TSLP or IL-25 at the respiratory tract interface might alter DC-driven T H 2 inflammatory airway responses to allergen. Although generalized blocking of the DC chemoattractant CCL20 seems potentially hazardous because of potential inhibition of DC responses to respiratory tract pathogens, targeting specific receptors used by allergens as they upregulate epithelial cell secreted CCL20 might be a more preferable option. Examples of this approach include specific inhibition of PAR-2 or b-d-glucan receptors, both targets of HDM allergen, on epithelial cells, to prevent HDM-induced secretion of CCL20. However, such inhibition also has the potential to interrupt the induction of tolerance to inhaled allergens. Thus a more attractive strategy would be to inhibit DC signals leading to T H 2 inflammation. For example, selective inhibition of DC secretion of CCL22 and CCL17, major

10 898 GILL J ALLERGY CLIN IMMUNOL APRIL 2012 attractants of T H 2 effector cells to the lung, could more precisely inhibit T H 2-driven allergic responses. Further molecular characterization of the mechanisms by which DCs induce and maintain T H 2 responses in the lung, such as those involving OX40L expression or IDO, might reveal potential therapeutic targets. The finding that allergen challenge results in enhanced extracellular ATP, subsequently promoting DC-driven T H 2 responses in the airway, suggests that inhaled purinergic receptor antagonists might have therapeutic potential in allergic asthmatic patients through their capacity to interrupt ATP binding with purinergic receptors on DCs. 109 The discovery of the role of TLR4 in epithelial cell recognition of HDM allergen and that this interaction results in DC activation led to evaluation of an intrapulmonary TLR4 antagonist in a murine model of HDM-induced experimental asthma. TLR4 blockade in this model was encouraging, resulting in reduced airway eosinophilia and suppression of bronchial hyperresponsiveness in HDM-sensitized mice. 6 TLR agonists that specifically activate and potentially improve the function of pdcs in patients with allergic asthma also have potential therapeutic value. It is now recognized that pdcs play a central role in balancing the response to allergen (ie, the development of tolerance vs allergic inflammation). Consideration should therefore be given to identifying methods that shift this balance to favor antigen tolerance. Because omalizumab has shown considerable promise in reducing the sensitivity of DCs and other cells to allergens by decreasing IgE levels, additional methods of reducing IgE levels or inhibiting the interaction of IgE with its cellular receptors on DCs might be expected to have similar beneficial effects. This type of therapy might be particularly helpful in atopic subjects with virus-induced asthma, in whom the interaction of allergenspecific IgE on DCs with inhaled allergen might interrupt the release of antiviral type I interferons. Decreasing IgE levels in these persons might improve their antiviral responses, decrease associated T H 2 inflammation, and make them less susceptible to virus-induced wheezing. SUMMARY AND FUTURE PERSPECTIVES Knowledge of the role of DCs in generating allergic responses continues to expand. Among the most interesting recent observations are that the number of DCs arriving at lymph nodes, their state of activation, or both seem to correlate directly with breaking of tolerance and the induction of immune responses. Thus the development of methods of regulating the number and activation status of DCs migrating to lymph nodes might have therapeutic potential, and future efforts might be directed to this area of research. Further identification of DC subtypes, with clarification of their role in directing inflammatory responses toward or away from the allergic phenotype, will be important steps. Additional animal models of asthma are needed because murine models do not reflect chronic asthma but instead become progressively refractory to continued allergic sensitization. Meanwhile, learning how DCs contribute to this refractory state in the murine model would be of great therapeutic interest. Methods of altering the allergic milieu, either by changing the predominant cytokines present or by influencing the way epithelial cells and DCs interact should be explored. Finally, decreasing serum IgE levels or finding other methods of blocking the IgE receptor might improve antiviral immune responses in atopic subjects. I would like to acknowledge Boyd Jacobson for his assistance with the illustrations included in this review. Key concepts and therapeutic implications d DCs are present at the pulmonary mucosal interface, where they serve as innate sensors of foreign antigens/ pathogens and transmit this information to the immune system. d DCs influence whether the response to an inhaled antigen will entail the induction of tolerance to the antigen or allergic inflammation. d Subsets of DCs have differential roles in the development of allergic airway responses. Agents designed to selectively inhibit specific functions of these subsets could provide potential therapeutic targets for regulation of allergic responses. d The interaction of DCs with airway epithelial cells is critical in inducing allergic responses. Disruption of this cross-signaling might reduce allergic inflammation in the lung. d Certain chemokines are important in the process of allergen-mediated chemotaxis of DCs to the airway. Blockade of these specific chemokines might provide targeted inhibition of inflammatory responses to allergens. d A reduction in DC numbers or the degree of DC activation after allergen challenge reduces the likelihood that an allergic response will develop. Therapeutic agents that fine tune the magnitude of DC responses could be especially beneficial in patients with allergic asthma. d Increased IgE levels are linked to impaired DC antiviral responses. This might explain the enhanced susceptibility of atopic subjects to virus-induced wheezing, as well as indicate new therapeutic approaches. REFERENCES 1. Bates EE, Dieu MC, Ravel O, Zurawski SM, Patel S, Bridon JM, et al. CD40L activation of dendritic cells down-regulates DORA, a novel member of the immunoglobulin superfamily. Mol Immunol 1998;35: Lambrecht BN, Hammad H. The role of dendritic and epithelial cells as master regulators of allergic airway inflammation. 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