Pulmonary Dendritic Cells

Chapter 33

Pulmonary Dendritic Cells Donald N. Cook and Hideki Nakano

Chapter Outline 1. 2. 3. 4. 5.

Overview of Dendritic Cells DC Subsets in the Lung DC Development DC Activation DC Trafficking

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1. OVERVIEW OF DENDRITIC CELLS The lung is particularly vulnerable to infection, and it is essential that vertebrates are able to rapidly recognize and clear inhaled pathogens before they establish overwhelming infections. The ability to generate robust immune and inflammatory responses to infection is therefore critical to health, but these responses can also damage delicate lung tissue and thereby prevent efficient gas exchange. Accordingly, vertebrates must generate effective immune responses to pathogens while also remaining tolerant to self-antigens and exogenous antigens that do not pose a threat to health. The cellular and molecular mechanisms that control immunologic responses to inhaled antigens are therefore of upmost importance in maintaining homeostasis. A wealth of evidence over the last several decades has shown that a bone marrowderived cell known as the dendritic cell (DC) is largely responsible for the initiation of immune responses to both exogenous and self-antigens. DCs were identified by Ralph Steinman as cells whose morphology and ability to stimulate naïve T cells distinguished them from monocytes and macrophages (Steinman and Cohn, 1973, 1974; Steinman et al., 1974; Steinman and Witmer, 1978). In subsequent years, the importance of DCs for initiating adaptive immune responses gained increasing appreciation, culminating in the 2011 Noble Prize in Physiology or Medicine for Steinman (shared with Bruce Beutler and Jules Hoffmann). We now know that DCs are present in virtually all tissues but are particularly abundant in organs that are in intimate contact

Comparative Biology of the Normal Lung. http://dx.doi.org/10.1016/B978-0-12-404577-4.00033-3 Copyright © 2015 Elsevier Inc. All rights reserved.

6. DC-Mediated Induction of T Helper Cell Differentiation 7. Activation of Memory T Cells during Antigen Challenge 8. Interaction of DCs with Other Cell Types 9. Summary and Future Directions References

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with the environment, such as the skin, gut, and lung (Sertl et al., 1986). In keeping with their role of sensing the antigenic content of inhaled air, DCs reside close to or within the respiratory epithelium of the entire airway, including the nose and nasopharynx, trachea, bronchi, bronchioles, and alveoli. Like DCs in other tissues, pulmonary DCs take up antigens and process them into peptides that are displayed on the cell surface in association with molecules of the major histocompatibility (MHC) locus. There are two types of MHC, class I and class II, which stimulate CD8þ cytotoxic T cells and CD4þ T helper cells, respectively. Class I is displayed on most cells in the body, whereas display of class II is restricted to antigen presenting cells, including DCs, macrophages, and B cells. In addition to their potent antigen presenting ability, DCs also possess the ability to migrate from peripheral tissue to draining lymph nodes (LNs), which contain large numbers of naïve T cells. Thus, pulmonary DCs function as sentinels of the pulmonary adaptive immune system by taking up inhaled antigens and transporting them to thoracic LNs. In this way, pulmonary DCs present to naïve T cells a dynamic array of antigens that are present within the lung (Cook and Bottomly, 2007; Lambrecht and Hammad, 2009; Randolph et al., 2005). It is likely that the migratory ability of DCs evolved to increase the frequency of their encounters with naïve T cells. The number of naïve T cells specific for any given antigen is very small (w1/100,000), and chance encounters between such a cell and a DC displaying that antigen are likely to be very rare. The ability of DCs to migrate from peripheral 651

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tissue to naïve T cell-rich draining LNs undoubtedly increases the frequency of encounters between those two cell types. Efficient T cell activation by DCs requires more than antigen presentation. Otherwise, self-antigens and harmless exogenous antigens would continually trigger immune responses that would ultimately lead to organ failure and death. The ability of DCs to initiate productive immune responses depends on the innate immune system that includes cells such as macrophages, neutrophils, epithelial cells, and DCs themselves (Iwasaki and Medzhitov, 2010). In the absence of signals from these cells, DCs do not become sufficiently activated to provide strong costimulation to T cells. Under these circumstances, antigen presentation by DCs leads to either T cell anergy (the inability to respond to subsequent stimuli) or to the proliferation of regulatory T cells that negatively regulate effector immune responses. Thus, the activation state of DCs determines whether or not antigen inhalation leads to the induction of effector T cell responses.

2. DC SUBSETS IN THE LUNG 2.1 Anatomic Distribution of DCs DCs are sometimes classified according to whether they are located in the large airways or in the lung parenchyma, which is comprised of small airways and alveoli. Confocal microscopic analysis of rat trachea has revealed that approximately 20% of resident are in close contact with the resting epithelium, whereas 80% of DCs reside within the subepithelial region (Jahnsen et al., 2006). DCs in the airways of the rat and mouse turn over rapidly with an estimated half-life of 12 h, whereas parenchymal DCs have a longer half-life, about several days (Holt et al., 1988; von Garnier et al., 2005). The replenishment of DCs during

both steady-state and inflammatory conditions is mediated in part by the chemokine receptors, CCR1 and CCR5 (McWilliam et al., 1994; Stumbles et al., 2001). DCs prepared from the airway are more endocytic and present peptides more efficiently to CD4þ T cells than DCs prepared from the lung parenchyma (von Garnier et al., 2005). These findings suggest that DCs residing in the airway and parenchyma of the lung might fulfill different functions. In addition to the mucosa and parenchyma, the vasculature of the lung also contains DCs that are able to take up antigens in the blood and present them to T cells in mediastinal LNs (Willart et al., 2009). Interestingly, T cells that have been activated in this way have a propensity to return to the lungs. Thus, DCs in the vasculature of the lung can also be considered as bona fide pulmonary DCs, although relatively little is known about these cells compared to mucosal or parenchymal DCs.

2.2 Classification by Cell Surface Markers Although classification of DCs according to their anatomical position is reasonable and has been instructive, most investigators classify lung DCs based on their display of cell surface proteins. In the mouse, all DCs express the integrin CD11c, and for most tissues, expression of this marker is sufficient to identify DCs by immunohistochemistry or flow cytometry. However, use of this marker alone is insufficient to identify DCs in the lung because alveolar macrophages also display CD11c (Figure 1). However, macrophages are highly autofluorescent, whereas DCs are not (Vermaelen and Pauwels, 2004), and at least for flow cytometry-based experiments, autofluorescence is a useful parameter for distinguishing between these two cell types. There are two major conventional (c)DC subsets in the lung, which are defined according to their display of the CD11b and aE(CD103)b7 integrins: CD11bhi CD103 (CD11bhi

nonautofluorescent CD11c+ cells

conventional DCs

CD11c

CD11b

P1

MHC class II

P2

(no staining)

autofluorescence

viable cells

CD11c

CD103

FIGURE 1 Resolution of the two major pulmonary DC populations by flow cytometry. (left panel) Nonautofluorescent CD11cþ cells are shown within the black rectangle (P1). (middle panel) Staining and gating for MHC class IIþ cells. (right panel) Resolution of nonautofluorescent, CD11cþMHCþ cells into the two major pulmonary DC subsets, expressing CD11b and CD103, respectively.

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DCs) and CD11blow (lo) CD103þ DCs (CD103þ DCs) (Sung et al., 2006). The lung also contains smaller populations of CD11cintermediate (int) CD11blo Ly-6Cþ B220þ plasmacytoid DCs (pDC), and CD11cint CD11bhi Ly-6Cþ monocyte-derived inflammatory DCs. The assignment of specialized functions to these various DC subsets is a rapidly evolving field of pulmonary DC biology, as discussed below.

2.3 CD103D DCs CD103þ DCs reside in the lung mucosa, in close proximity to the basal surface of bronchial epithelial cells (Sung et al., 2006). Because CD103 binds E-cadherin, which is displayed on the basolateral side of epithelial cells, the positioning of CD103þ DCs in close contact with the airway epithelia is likely mediated by interactions between these two molecules (Cepek et al., 1994; Taraszka et al., 2000). In addition, CD103þ DCs also express several tight junction proteins, including Claudin-1, Claudin-2, and ZO-2. These proteins are thought to allow CD103þ DCs to extend processes between epithelial cells and gain access to the lumen of the airway (Jahnsen et al., 2006; Sung et al., 2006). In this way, CD103þ DCs can sample luminal antigens in airways with an intact epithelial barrier. CD103þ DCs also express high levels of the chemokine receptor XCR-1, although the functional significance of this observation is not currently known. CD103þ DCs are not present in large numbers in alveolar septa, although pDCs can be found there. Recent reports have revealed that in addition to their differences in display of cell surface markers, CD103þ DCs and CD11bhi DCs also have different functions. For example, CD103þ DCs can efficiently cross-prime CD8þ T cells, a process whereby exogenous antigens are displayed on MHC class I. By contrast, CD11bhi DCs are more proficient at priming CD4þ T cells (del Rio et al., 2007). In agreement with this report, CD103þ DCs can also acquire and transport apoptotic cells to the draining LNs and cross present apoptotic cell-associated antigens to CD8þ T cells (Desch et al., 2011). CD103þ DCs also express the protein langerin, and mice that express the diphtheria toxin receptor (DTR) under control of the langerin promoter have been generated. This allows CD103þ DCs to be depleted by the administration of diphtheria toxin (DT) (Bennett et al., 2005). Using this system, it has been shown that the development of influenza virus-specific T cells is delayed, with consequent increased clinical severity and delayed viral clearance following influenza infection (GeurtsvanKessel et al., 2008). Depleting CD103þ DCs in langerin-DTR mice also reduces eosinophil recruitment to the lung following LPS-treatment of animals that had been previously sensitized and challenged with the LACK (Leishmania analog of

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the receptors of activated C kinase) antigen (Ortiz-Stern et al., 2010). Although selective depletion of DC subsets in this way has great potential for improving our understanding of their function in vivo, we and others (Zammit et al., 2005) have found that DT can cause massive cell death and fatalities in mice. Thus, it remains to be determined whether some of the effects of DT attributed to depletion of specific DC subsets might actually be due to nonspecific effects of DT that cause cell death and inflammation. Although CD103þ DCs do not generally produce large amounts of chemokines, they do produce CCL17 and CCL22, which bind the chemokine receptor CCR4 (Beaty et al., 2007). Interestingly, this receptor is on T helper (Th) 2 cells, which produce the cytokines IL-4, IL-5, and IL-13 and are associated with allergic responses. We have also found that eosinophil chemoattractant, CCL11 (eotaxin), is also more highly expressed by CD103þ DCs than by CD11bhi DCs (Nakano et al., 2012). Thus, one of the functions of CD103þ DCs might be to recruit Th2 cells to the lung during allergen challenge. CD103þ DCs are also present in the intestinal mucosa, but the extent to which these DCs differ from their counterparts in the lung remains to be determined. Gutassociated CD103þ DCs promote T regulatory (Treg) differentiation by producing aldehyde dehydrogenase 1a2 (ALDH1a2), which converts retinol to retinoic acid (RA) (Annacker et al., 2005; Coombes et al., 2007). However, to date, a similar role for lung CD103þ DCs in the induction of tolerance to inhaled antigens has not been shown.

2.4 CD11bhi DCs Unlike CD103þ DCs, CD11bhi DCs in the lung do not directly contact the airway epithelium but lie just beneath it. Consequently, the latter cells probably have limited access to antigens in the airway. However, CD11bhi DCs produce very high levels of chemokines ex vivo and can sustain allergic pulmonary inflammation during the challenge phase of allergic responses (Beaty et al., 2007; Raymond et al., 2009). Together, these findings suggest that CD11bhi DCs can respond to antigen rechallenge and produce many chemokines that serve to amplify inflammatory responses. The chemokines produced by CD11bhi DCs include the C-X-C and C-C categories, which can collectively recruit leukocytes of all types to the lung. In addition to their differences in chemokine production, CD103þ DCs and CD11bhi DCs also have distinct radiosensitivities and turnover rates. Repopulation of the CD11bhi DC subsets is complete after 10 days, whereas CD103þ DCs require 28 days (Hahn et al., 2011). It is unclear if this difference relates to differences in anatomic location, intrinsic resistance to radiation, or the ability of monocytes to replenish CD11bhi DCs but not CD103þ DCs.

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Although the vast majority of DCs in the lung are either CD11bhi or CD103þ, a small percentage of DCs are positive for both markers (Figure 1). In part because of their relatively small numbers, it is not currently known whether these double positive DCs are similar in function to one of the major DC subsets or if they possess unique functions.

2.5 Inflammatory DCs Blood-borne monocytes can crawl along the vascular endothelium of healthy tissue in a manner dependent on the integrin LFA-1 and the chemokine receptor CX3CR1. During infection, these cells can rapidly invade peripheral tissue and LNs and develop into inflammatory DCs. These DCs are particularly adept at priming Th1 responses to proinflammatory stimuli (Cheong et al., 2010; Leon et al., 2007; Nakano et al., 2009), although a subset of inflammatory DCs that display the high-affinity IgE receptor (FcεR1) is reported to have Th2 stimulating activity (Hammad et al., 2010). Several lines of evidence suggest that inflammatory DCs might contribute to the exacerbation of asthma symptoms that are often seen following viral infection. First, large numbers of monocyte-derived inflammatory DCs are recruited to the lungs of virus-infected mice (Lin et al., 2008). Second, FcεR1 is displayed at high levels on circulating monocytes during asthma exacerbations. Third, expression of this receptor is increased by type I interferons (Grayson et al., 2007), which are produced at high levels during virus infection. Thus, viruses might trigger asthma exacerbations by increasing FcεR1 on circulating monocytes and by promoting the recruitment of FcεR1-expressing monocytes and inflammatory DCs to the lung. Under this scenario, allergen/IgE complexes cause DCs to release cytokines and chemokines that can amplify existing pulmonary inflammation. Upon their maturity, monocyte-derived DCs display increased levels of DC SIGN and CD14, which distinguishes them from CD11bhi cDCs (Cheong et al., 2010). This differential display of CD14 might provide a basis to separate monocyte-derived CD11bhi cDCs from CD11bhi cDCs that arise from pre-DCs. Using this strategy, it should be possible to determine if these two types of CD11bhi DCs are functionally different.

2.6 pDCs The lung contains a minor population of pDCs, which display either moderate levels of CD11c (murine pDCs) or are negative for CD11c (human pDCs). In addition to their relatively low levels of CD11c, murine pDCs can also be distinguished phenotypically from other DC subsets by their display of Siglec H, Ly6C, bone marrow stromal cell antigen 2 (BST-2, PDCA-1, 120G8), and B220 (Asselin-

Paturel et al., 2001; Bjorck, 2001; Nakano et al., 2001). Human pDCs can be identified by their expression of CD4, IL-3Ra (CD123), BDCA-2, and BDCA-4. One of the most important features of pDCs is their ability to produce very high levels of type I interferons in response to viruses or their products (Siegal et al., 1999). Recent work has shown that this response results from the activation of TLR7 or TLR9, which are highly expressed in pDCs but not in cDCs. TLR7 and TLR9 are intracellular receptors for single-stranded RNA and unmethylated CpG DNA, respectively, nucleic acids that are not normally seen in the cytoplasm of healthy cells. The transcription factor IRF7 is critically important for the induction of type I interferon production following stimulation of either of these TLRs (Honda et al., 2005). Despite their ability to produce large amounts of type I interferons, pDCs are relatively poor inducers of effector T cell responses. In fact, pDCs have been shown to have suppressive effects on immune responses to inhaled antigens (de Heer et al., 2004). Depletion of pDCs with an antiLy-6C/G (Gr-1) antibody resulted in allergic sensitization to inhaled ovalbumin under conditions that normally induce tolerization. Because Gr-1 antibodies also bind other cell types, including monocytes and inflammatory DCs recognized by Ly-6C, and neutrophils recognized by Ly-6G, this experiment alone does not prove that pDCs suppress allergic responses. However, adoptive transfer of pDCs before sensitization resulted in reduced levels of allergic inflammation (de Heer et al., 2004). Moreover, recent findings with mice in which the DTR gene was inserted into the 30 untranslated region of the Siglech gene have supported the concept that pDCs suppress the proliferation of CD4þ T cells (Takagi et al., 2011). Interestingly, however, the latter study also showed that pDCs could support the proliferation of CD8þ T cells. This finding suggests that pDCs can promote viral clearance in two ways: by producing type I interferons and by inducing virus-specific, cytotoxic CD8þ T cells.

3. DC DEVELOPMENT All DCs are derived from the bone marrow-derived, monocyte and DC precursor (MDP), which can also give rise to monocytes (Figure 2). Phenotypically, MDPs are identified as lineage (lin)CD117hiCX3CR1þCD115þ CD135þ (Fogg et al., 2006). MDPs can differentiate into the common DC precursor (CDP), whose progeny is restricted to pDCs and the immediate precursor of cDCs, known as the pre-DC (Naik et al., 2007; Onai et al., 2007). Recent studies have suggested that CD103þ DCs and the highly related CD8aþ lymphoid DCs are derived exclusively from pre-DCs via a developmental pathway dependent on interactions between fms-like tyrosine kinase 3 (Flt3) and its ligand (Flt3L) (Bogunovic et al.,

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FIGURE 2 DC development and induction of adaptive immune responses. Pre-DCs and monocytes from the bone marrow are circulating precursors of mature DCs. Pre-DCs give rise to both CD11bhi DCs and CD103þ DCs, whereas monocytes can differentiate into only CD11bhi DCs. Inhaled antigens are taken up by lung DCs that migrate to draining LNs in a CCR7-dependent manner. Depending on the signals they receive from DCs, antigen-specific naïve T cells can differentiate into CD8þ cytotoxic T cells or CD4þ helper T cells.

2009; Ginhoux et al., 2009). Transcription factors that are required for the development of these DC subsets include IRF8, which is mutated in BXH2 recombinant inbred mice (Tailor et al., 2008), BATF3 (Hildner et al., 2008), and ID2 (Hacker et al., 2003). The development of CD11bhi DCs in the lung is also at least partly dependent on FLT3L, probably because it also contributes to the development of MDPs, which give rise to all DCs. Intraperitoneal delivery of FLT3L to mice blunts allergic responses and inhibits their ability to take up inhaled antigens and migrate to draining LNs (Shao et al., 2009a,b), suggesting that this cytokine might be of therapeutic benefit. However, because FLT3L is a growth factor, its effects might result from the expansion of immature DCs, which might not yet be capable of eliciting effector immune responses. It is conceivable, however, that these DCs could eventually acquire immunostimulatory activity, and that these cells might exacerbate inflammation in the longer term. Although CD11bhi DCs have been reported to be absent in Flt3L-deficient mice (Ginhoux et al., 2009), others have reported that CD11bhi DCs can develop from monocytes (moDCs), which do not require FLT3 or FLT3L for their differentiation into DCs (Bogunovic et al., 2009; Jakubzick et al., 2008a,b; Varol et al., 2007). This might explain the finding that although Flt3L-deficient mice have drastic reductions in lymphoid tissue DCs and parenchymal DCs, they display no defect in the total number of MHC class IIþ airway DCs or in their ability to

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reactivate CD4þ T cells that had been previously primed with antigen (Walzer et al., 2005). It is possible that the relative abundance of monocytederived DCs in the lung, and therefore the total number of DCs in lungs of Flt3L-deficient mice, depends on the conditions used to house the mice. Very clean conditions might not promote the recruitment and development of monocyte-derived DCs, and Flt3L-deficient mice might therefore have a larger deficit in total DCs than when mice are housed under dirtier conditions that might promote monocyte recruitment to the lung. Peripheral blood monocytes are comprised of two phenotypically distinct subsets. One subset is CCR2hi Gr1hiCX3CR1int inflammatory monocytes and the other CCR2loGr1loCX3CR1hi noninflammatory monocytes, respectively. Landsman and colleagues showed that when adoptively transferred into recipient mice, both subsets of monocytes can give rise to pulmonary DCs, even under steady-state conditions (Landsman et al., 2007). Under the conditions studied, only Gr1loCX3CR1hi monocytes had the potential to differentiate into lung macrophages. However, Gr1hiCX3CR1int monocytes could acquire this potential upon conversion into Gr1loCX3CR1hi cells. Thus, the lung can contain CDP-derived, Flt3L-dependent classical CD103þ DCs and CD11bhi DCs, as well as monocytederived CD11bhi DCs. Whether CD11bhi DCs that arise from these alternative developmental pathways possess different capacities and functions is currently unknown.

4. DC ACTIVATION Efficient pathogen recognition and elimination requires the coordinated interactions of many cells of the innate and adaptive immune systems. It has been more than 20 years since Charles Janeway first suggested that the innate immune system might recognize infectious organisms through germline-encoded genes and that this recognition would activate the adaptive immune system. We now know that this hypothesis was correct and that there are several families of such pattern recognition receptors (PRRs), including the TLR family, C-type lectins, nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and retinoic acid-inducible gene (RIG)I-like receptors that can fulfill this function (Joffre et al., 2009). These receptors have evolved to directly recognize pathogen-associated molecular patterns (PAMPs) and to trigger innate immune responses. Many of these receptors are expressed in DCs, which also initiate adaptive immune responses. Thus, DCs are strategically positioned at the crossroads of the innate and adaptive immune systems. Normally residing in a relatively quiescent state, DCs are able to sense danger signals from pathogen products and respond by stimulating adaptive immune responses.

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4.1 DC Activation by TLRs The TLR family is probably the best-studied family of PRRs (Iwasaki and Medzhitov, 2010). Following binding of PAMPs, individual receptors form homo- or heterodimers that transduce signals to their downstream partners. Except for TLR3, all TLRs signal through the adaptor molecule MyD88. Many TLRs are present on the cell surface, including TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11. One of the best studied of these receptors is TLR4, which recognizes lipopolysaccharide (LPS), a membranebound lipid found in both Gramve and Gramþve bacteria. Other cell-surface TLRs recognize different conserved molecular patterns, including bacterial peptidoglycan (TLR2) and flagellin (TLR5). Some TLRs, including TLR3, TLR7, TLR8, and TLR9, are found primarily within intracellular vesicles. As noted in the discussion on pDCs, these receptors recognize nucleic acids, such as doublestranded RNA (TLR3) and unmethylated CpG motifs (TLR9). The importance of TLR7 in antiviral responses is underscored by the finding that mice lacking this receptor fail to generate protective immunity following vaccination with inactivated virus (Koyama et al., 2007). Using a microarray-based comparison of gene expression in CD103þ and CD11bhi DC subsets prepared from lungs of untreated mice, we have found that that Tlr2 is highly expressed in CD11bhi DCs, whereas Tlr3 is highly expressed in CD103þ DCs. The latter finding is consistent with the observation that CD103þ DCs are important for clearance of influenza virus (GeurtsvanKessel et al., 2008). However, Tlr7 and Tlr8 are both higher in CD11bhi DCs, although a requirement for this cell type in clearing viral infections has not yet been reported. Signaling responses of individual TLRs can be sufficient to activate both innate and adaptive immune response, but activation of more than one TLR can result in complementary, synergistic, or antagonistic effects (Trinchieri and Sher, 2007). It is likely, therefore, that multiple TLRs can also cooperate in the lung, but the consequences of that cooperation remain to be determined. In addition to interactions within the TLR family, these signaling pathways can also combine with non-TLR pathways to elicit stronger signals than from either one alone. For example, TLR signaling combined with CD40 signaling leads to particularly strong DC activation (Ahonen et al., 2004). It is likely that the signals that pulmonary DCs receive are from multiple sources and the consequences are still poorly understood.

4.2 DC Activation by C-type Lectins Dectin-1 Dectin-1 is a member of the C-type lectin family, which recognizes b-glucans from fungal pathogens such as

Candida albicans or Aspergillus fumigatus (Taylor et al., 2007). C. albicans preferentially induces Th17 responses (Acosta-Rodriguez et al., 2007), and mice lacking Dectin-1 have reduced Th17 responses to A. fumigatus. These mice also have increased Th1 responses, probably because Dectin-1 on wild-type DCs reduces the production of IL-12 and IFN-g (Rivera et al., 2011). Transient depletion of monocyte-derived DCs reduced Th1 responses and increased Th17 responses, which is in agreement with previous findings that inflammatory DCs are potent inducers of Th1 responses upon LPS stimulation or influenza infection (Cheong et al., 2010; Nakano et al., 2009). However, Ly-6CþCD11bhi DCs are also reported to promote IL-17A production by CD4þ T cells through a mechanism that requires TNF-a production, and this pathway is abolished in Dectin-1-deficient mice (Fei et al., 2011). Together, these findings suggest that inflammatory DCs can promote either Th1 or Th17 responses, depending on experimental conditions. The ability of Dectin-1 to promote Th17 responses to fungi exemplifies the ability of PPRs to induce immune responses specialized at clearing specific types of pathogens.

DC-SIGN The DC-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) is another C-type lectin that is expressed by DCs in the lung. DC-SIGN was originally described as a molecule on DCs that binds ICAM-3 on resting T cells, thereby promoting T cell: DC interactions (Geijtenbeek et al., 2000). Subsequently, DC-SIGN was shown to bind also both endogenous and exogenous antigens. For example, DC-SIGN mediates the binding and internalization of Mycobacterium tuberculosis (Tailleux et al., 2003) and A. fumigatus (Serrano-Gomez et al., 2004). Interestingly, binding to DC-SIGN by a cell membrane component, ManLAM, of M. bovis suppressed DC function by interfering with TLR signaling (Geijtenbeek et al., 2003). This suppression is mediated through Raf-1 and NF-kB, and leads to increased IL-10 production and antiinflammatory responses (Gringhuis et al., 2007). Other IL-10-inducing pathways, such as those including ERK and PI3K, can also be triggered by DC-SIGN (Caparros et al., 2006). Whether this molecule is relevant to the induction of diseases such as asthma remains to be determined.

4.3 DC Activation by RIG-I-Like Receptors In addition to TLR3 and TLR7, another family of proteins known as retinoic acid-inducible gene-I (RIG-I)-like receptors are capable of recognizing viral RNA by a TLR-independent mechanism (Yoneyama et al., 2005). These proteins are RNA helicases that are expressed ubiquitously at low levels but are highly induced by type I

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interferons or viral infection. In addition to RIG-I itself, this family contains the melanoma differentiation-associated 5 (MDA5) and the laboratory of genetics and physiology 2 (LGP2) receptors that can respond to viral infection. Interestingly, RIG-I is important for the induction of type I interferons in cDCs, but not in pDCs, even though the latter produce very large quantities of these proteins (Kato et al., 2005).

5. DC TRAFFICKING The ability of pulmonary DCs to take up antigen and migrate to draining LNs provides naïve T cells with a dynamic representation of antigens in the lung. DC migration proceeds at low levels during steady-state conditions, but the rate and volume of that migration is markedly increased following exposure to proinflammatory stimuli (Vermaelen et al., 2001). This migration of DCs from the lung to draining LNs is thought to be an important event in the induction of effector responses to inhaled antigens. However, DC migration might also be important to maintain stimulation of memory T cells because after influenza infection, some DCs continue to transport viral antigen to LNs long after virus has been cleared from the lung (Kim et al., 2010). The transition from resident to migratory DCs is a multistep process that involves the MHC-II invariant chain CD74 (Faure-Andre et al., 2008). When DCs are stationary, CD74 sequesters the actin-based motor protein myosin II in the endosomal compartment and promotes loading of peptide onto the MHC. Upon DC activation, antigen uptake increases transiently and is then markedly reduced, in concert with the acquisition of mobility. Once DCs are mobile, their migration to draining LNs is guided by interactions between the chemokine receptor CCR7 and its cognate chemokine ligands CCL19 and CCL21, which are produced in the lymphatic vessels and in T cell areas of LNs (Forster et al., 1999; Gunn et al., 1998). Many CD103þ DCs display CCR7 and are dependent on this receptor for their migration to lung-draining mediastinal LNs (Hintzen et al., 2006). Consequently, CD103þ DCs are virtually absent in LNs of Ccr7/ mice. However, CD11bhi DCs can nonetheless be found in LNs of these mice, albeit at reduced levels compared to those of wildtype mice (Hintzen et al., 2006; Nakano et al., 2012). The latter observation suggests that CD11bhi DCs can gain access to mediastinal LNs by both CCR7-dependent and -independent pathways. Other studies have confirmed that under some circumstances, CD11bhi DCs can migrate to LNs (Jakubzick et al., 2006; Raymond et al., 2009), but it is unclear whether all CD11bhi DCs in the lung have this potential. The factors that induce migration of DCs to draining LNs are poorly characterized, and it is not known if all DCs have the potential for migration. Some DCs

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might reside in an anatomic position that effectively shields them from stimuli that would otherwise promote their mobilization. Alternatively, DCs might gain the ability to migrate on a stochastic basis that ensures an appropriate distribution of DCs in the lung and draining LNs. A third possibility is that some DCs are programmed to remain in the lung, even when exposed to stimuli that promote migration of other DC subsets. Future studies will be required to resolve these questions. It is well documented that the aging process includes a decline in immune function, and the elderly are more susceptible to infectious pathogens, including respiratory viruses. This segment of the population also displays diminished responses to vaccines, suggesting that the initiation of immune responses declines with age. Some insight into the underlying causes of this phenomenon was provided by a recent report showing that migration of lung DCs to draining LNs in response to viral infection diminishes with increasing age (Zhao et al., 2011). The impaired migration of lung DCs in aged mice is due at least in part to increased levels of the arachidonic acid metabolite, prostaglandin D2 (PDG2), because blockade of PDG2 synthesis with small molecule antagonists increased levels of Ccr7 expression and consequent DC migration to draining LNs. The constitutively expressed cyclooxygenase (COX)-1 and the inducible COX-2 are responsible for the synthesis of PDG2, but it remains to be seen whether nonsteroidal anti-inflammatory drugs such as aspirin can increase DC migration in the elderly and thereby confer more robust responses to pathogens.

6. DC-MEDIATED INDUCTION OF T HELPER CELL DIFFERENTIATION DC stimulation of naïve CD4þ T cells directs their differentiation into various Th cell lineages having specialized functions for pathogen clearance (Pulendran et al., 2010). Thus, Th1 cells provide protective immunity against viruses and intracellular bacteria, whereas Th17 cells promote clearance of extracellular bacteria (Zhou et al., 2009). Th2 cells help to clear helminths by promoting IgE production, eosinophil proliferation, and mucus production, but these same responses to Th2 cytokines are pathogenic in the setting on allergic diseases such as asthma. An improved understanding of the APCs and molecules that drive Th2 cell differentiation might provide new opportunities for therapeutic intervention in multiple disease settings. Naïve CD4þ T cell differentiation into specific T cell lineages is determined largely by local cytokine levels. Th1 differentiation is driven by IL-12, which is produced in very high amounts by monocyte-derived, Ly6CþCD11bhi inflammatory DCs (Leon et al., 2007; Nakano et al., 2009). Th17 cell differentiation is directed by TGF-b together with

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either IL-6 or IL-1 (Dong, 2008), whereas TGF-b on its own, or together with retinoic acid, promotes the differentiation of regulatory T cells (Coombes et al., 2007). Unlike these relatively well-characterized mechanisms, the cells and molecules that promote Th2 differentiation remain poorly understood. Although IL-4 can promote Th2 differentiation in vitro by activating STAT6 and promoting GATA3 expression, DCs do not produce IL-4 (Lambrecht and Hammad, 2009; Paul and Zhu, 2010). Accordingly, identifying the APC and cellular source of IL-4 that directs Th2 differentiation in vivo has been the focus of considerable effort and controversy during the past several years. Basophils have been shown to act as APCs and as a source of IL-4, and injection of anti-FcεRIa-specific antibodies suppress Th2 responses in mice (Perrigoue et al., 2009; Sokol et al., 2007, 2009; Yoshimoto et al., 2009). Based largely on these findings, it has been suggested that basophils are both necessary and sufficient for inducing Th2 responses in vivo. However, genetically engineered mice lacking basophils are still able to initiate Th2 cell differentiation, including allergic pulmonary inflammation (Ohnmacht et al., 2010). Moreover, several recent studies have shown a requirement for DCs in Th2 induction (Hammad et al., 2010; Lambrecht and Hammad, 2009; Ohnmacht et al., 2010; Phythian-Adams et al., 2010), and lung DCs are still widely considered to be required for the induction of allergic responses to inhaled allergens. CD103þ DCs and CD11bhi DCs were originally thought to possess similar T helper inducing activities (Beaty et al., 2007). However, this conclusion was based on experiments in which exogenous peptides were added to DC subsets ex vivo, a procedure that bypasses events related to in vivo uptake of inhaled antigens. Our own experiments in which allergen was instilled directly into the airways to allow lung resident DCs to take up the allergen in vivo revealed that CD103þ DCs from the lung or lungdraining LNs efficiently primed Th2 differentiation ex vivo, whereas CD11bhi DCs primed Th1 differentiation (Nakano et al., 2012). Moreover, mice lacking CD103þ DCs failed to undergo Th2 priming to various inhaled allergens and did not develop asthma-like responses following subsequent allergen challenge. Paradoxically, however, when stimulated with TLR ligands or PMA and ionomycin, CD103þ DCs produce the Th1-inducing cytokine IL-12, suggesting that these DCs have the potential to prime Th1 responses. Indeed, when cockroach antigen or house dust mite preparations (that include both PAMPs and allergens) are instilled into the airway, CD103þ DCs are able to prime both Th1 and Th2 responses, whereas CD11bhi DCs exclusively prime Th1 responses (Nakano et al., 2012). Thus, depending on the experimental setting, CD103þ DCs can prime either Th1 or Th2 responses. These findings might be related to the observation that low levels of TCR stimulation lead to Th2 responses by

upregulating GATA3 expression, whereas strong TCR stimulation suppresses GATA3 expression through activation of extracellular signal-regulated kinase (ERK) and thereby primes Th1 responses (Iezzi et al., 2009; Yamane et al., 2005). In agreement with these observations, we found that when the two major DC subsets were purified from the lung and cultured with low levels of peptide, CD103þ DCs primed Th2 responses, but at high levels of peptide, they primed Th1 responses. Together, these findings show that CD103þ DCs have a significant role in priming both Th1 and Th2 responses to inhaled allergens. Differential expression of Notch ligands has been proposed to contribute to T-helper cell differentiation, with Jagged-1 and Jagged-2 promoting Th2 differentiation and Delta-like 4 promoting Th1 differentiation (Amsen et al., 2004). However, a requirement of Jagged-2 for Th2 responses in vivo has been questioned (Worsley et al., 2008), and it remains to be determined whether Jagged is required for allergic sensitization through the airway. Nonetheless, Jagged-1 is upregulated on bone marrow-derived DCs (BMDCs) pulsed with allergen, and inhibition of Notch signaling results in decreased cytokine production when antigen-pulsed DCs and naïve CD4þ T cells are transferred into IL-4-deficient mice. Thus, interactions between Notch on CD4þ T cells and Jagged-1 on APCs might contribute to IL-4 production by T cells and promote Th2 differentiation for the development of airway hyperresponsiveness and allergic airway inflammation. In agreement with this concept, it has been reported that Jagged-2 is blunted in DCs from mice lacking c-Kit and that they are unable to induce robust Th2 and Th17 responses when adoptively transferred into mice (Krishnamoorthy et al., 2008). We have observed that c-Kit is much more highly expressed in CD103þ DCs than in CD11bhi DCs, but we have been unable to confirm that c-Kit-deficient CD103þ DCs are impaired in their ability to prime Th2 responses (Nakano et al., 2012).

7. ACTIVATION OF MEMORY T CELLS DURING ANTIGEN CHALLENGE In addition to their primary role in driving the differentiation of naïve T cells to CD8þ cytotoxic cells or CD4þ T helper cells, DCs are also important in the effector phase of immune responses. Following allergen challenge, DCs in the lung increase rapidly (McWilliam et al., 1994), with most of this increase being due to CD11bhi DCs (Beaty et al., 2007). The recruitment of these cells is mediated largely by the chemokine receptor CCR2 (Robays et al., 2007), which is highly expressed on inflammatory monocytes. DCs are necessary during the time of allergen challenge because their depletion by delivery of DTX to CD11c-DTR mice prevents development of asthma-like

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features, including eosinophilic inflammation, goblet cell hyperplasia, and airway hyperresponsiveness. Moreover, intratracheal delivery of bone marrow-derived CD11bhi DCs is sufficient to restore these asthma-like features (Lambrecht et al., 1998; van Rijt et al., 2005). In a similar experiment, selective depletion of CD11bþ myeloid cells in the lung by administration of DTX to CD11b-DTR mice prevented production of the chemokines CCL17 and CCL22, which likely recruit Th2 cells by binding to their receptor, CCR4 (Medoff et al., 2009). Taken together, these data suggest that CD11bhi DCs might be important to activate memory or effector T cells arriving in the lung in response to allergen challenge. Interestingly, production of these chemokines was also found to be dependent on the presence of STAT6, which also is required for the development of Th2 cells. The requirement of DCs for activating effector cells is not unique to challenges with allergen because lung resident DCs are also required to trigger release of inflammatory mediators by influenza-specific CD8þ T cells during the late phase of influenza infection, after most of the virus has been cleared (Hufford et al., 2011). It remains to be shown which specific subset of lung DCs in most important in this regard, although they might be monocyte-derived because CCR2-deficient mice have less pathology and increased survival following influenza infection (Lin et al., 2008).

8. INTERACTION OF DCs WITH OTHER CELL TYPES

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In addition to its barrier function, the airway epithelium might also sense the environment and respond by providing signals to DCs. For example, human epithelial cells can activate DCs through the production of cytokines, such as thymic stromal lymphopoietin (TSLP) (Soumelis et al., 2002). In response to TSLP, DCs upregulate MHC class II, CD40, CD80, CD86, and OX40-L (Wang et al., 2006). However, TSLP does not upregulate IL-12, the Th1promoting cytokine, which likely favors the induction of Th2 responses by DCs. Airway epithelial cell lines also increase levels of TLR3 and TLR4 on human monocytederived DCs, which consequently develop increased sensitivity to LPS (Rate et al., 2009). This axis might be highly relevant to the effects of air pollution on allergic responses. Although isolated monocyte-derived DCs fail to undergo phenotypic (CD80, CD83, CD86) or functional (T cell activation) maturation in response to exposure to diesel exhaust particles (DEP), these DCs can be activated if they are cultured with primary human bronchial epithelial cells treated with DEP (Bleck et al., 2006). It is likely that soluble molecules convey some of these signals because conditioned medium from DEP-treated epithelial cells can also promote DC maturation, and this activity is inhibited by blockade of GM-CSF. Together, these findings suggest that airway epithelial cells might promote DC maturation through production of multiple soluble cytokines, including TSLP and GM-CSF. In this way, epithelial cells can exert some control over the initial events that ultimately give rise to immune responses to inhaled antigens.

8.1 Interaction of DCs with Epithelial Cells

8.2 Interaction of DCs with T Cells

The epithelium is widely considered to provide an effective barrier to most inhaled molecules, but it has long been known that whole proteins can penetrate the epithelium because they can be found in the blood within 24 h of instillation (Bensch et al., 1967). It is unclear whether this occurs by active transport of proteins through epithelial cells, or if some gaps between epithelial cells allow molecules to gain access to the subepithelial space. Thus, although the ability of CD103þ DCs to extend processes into the lumen of the airway might suggest that they have greater access to it, relatively small molecules, including ovalbumin, can be readily taken up by DCs that do not extend processes into the lumen. In fact, CD11bhi DCs take up higher levels of ovalbumin than do CD103þ DCs (Nakano et al., 2012). Nonetheless, recent findings have revealed that the TLR3 ligand, polyinosinic:polycytidylic acid (poly(I:C)), can markedly increase the paracellular permeability of the airway epithelium in a manner dependent on protein kinase D (Rezaee et al., 2011). This finding suggests that poly(I:C) might act as an adjuvant by exposing increased numbers of subepithelial DCs to antigens that might otherwise be shielded from them.

Another cell type that can promote the ability of DCs to stimulate T cells is the T cell itself. In the rat, “immature” DCs can acquire antigen, but they lack the ability to effectively present antigen to T cells, probably because levels of costimulatory molecules such as CD86 are too low. However, levels of CD86 on mucosal DCs (not parenchymal DCs) are increased by their interaction with antigen-specific T cells (Huh et al., 2003). Interaction of allergen-specific, CD4þ T cells with DCs is also linked to enhanced allergen uptake and CD40 expression by CD11blo (CD103þ) DCs (von Garnier et al., 2007). Thus, peripheral memory T cells might indirectly promote the activation of naïve T cell or central memory T cell activation by activating airway DCS.

8.3 Interaction of DCs with Neutrophils Like T cells, neutrophils can also promote DC activation. In the setting of Aspergillus infection, neutropenia results in the increased accumulation of DCs in the lung, but these DCs have a reduced ability to migrate to draining LNs (Park et al., 2012). Neutrophils are also reported to increase

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FcεRI expression on lung DCs (Cheung et al., 2010) and to stimulate IL-12 production by DCs (Kreisel et al., 2011). These findings reveal yet another link between innate and adaptive immune responses and show that the rapid neutrophilic response to infection contributes to DC activation, and by extension, the development of adaptive immune responses.

8.4 Interaction of DCs with Macrophages The lung contains two major types of macrophages, alveolar macrophages that express high levels of CD11c but relatively low levels of F4/80 and CD11b, and interstitial macrophages that have low levels of CD11c but high levels of F4/80 and CD11b. It has long been known that macrophages can regulate the function of DCs (Holt et al., 1993, 1988), suggesting that these cells might be responsible for preventing the induction of allergic responses in healthy individuals. Recently, the inhibitory function of lung macrophages was shown to be conferred interstitial macrophages, not alveolar macrophages. The former cell type can suppress the function of bone marrow-derived DCs by an IL-10-dependent mechanism (Bedoret et al., 2009). Although this report did not directly study interactions between macrophages and lung DCs, interstitial macrophages were preferentially depleted with an anti-F4/80 antibody, and these mice developed increased allergic responses following sensitization with allergen.

9. SUMMARY AND FUTURE DIRECTIONS The past 20 years have witnessed a dramatic increase in our understanding of pulmonary DCs. This improved understanding includes knowledge of how DCs develop, are activated, traffic to draining LNs, and stimulate antigenspecific T cells to become effector and memory T cells. Specialized functions have begun to be assigned to specific DC populations in the lung, which is important for the eventual design of therapies designed to alter DC function. Because DCs play such a pivotal role in the development and control of immune responses to inhaled antigens and pathogens, it is likely that they might eventually be used in therapeutic applications to either increase responses to pathogens or decrease immune responses to harmless antigens. Currently, the relatively low number of pulmonary DCs limits their use in this regard. Consequently, bone marrow DCs are often used in experiments because they can be obtained in much higher numbers. However, it is not clear that bone marrow-derived DCs have the capacity to interact with other cell types in the lung and in the way that bone fide lung DCs are able to do. The development of procedures to expand specific populations of lung DCs in a way that maintains their functions would be an important advance. Similarly, the development of small molecules

that specifically target select subsets of DCs in the lung would also provide a major advance in modulating immune responses to inhaled antigens by targeting the actions of pulmonary DCs.

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