Dendritic cells: Bridging innate and adaptive immunity in atopic dermatitis

Dendritic cells: Bridging innate and adaptive immunity in atopic dermatitis Natalija Novak, MD, Susanne Koch, PhD, Jean-Pierre Allam, MD, and Thomas Bieber, MD, PhD Much knowledge has been gained about the multifaceted functions of dendritic cells (DCs). The central role of various DC subtypes as bridges between innate and adaptive immunity has become more and more evident. However, a high number of differences exist in the expression of pattern-recognition receptors, the first sensors of the innate immune system, in particular Toll-like receptors (TLRs) by distinct DC subtypes (including myeloid and plasmacytoid DCs), their maturation stage, and tissue distribution, as well as state of health or disease. Furthermore, a plethora of variations in human and murine model systems have to be considered. This review sheds some light on this complex and rapidly growing field. It summarizes the most recent findings and deals with the role of TLR-expressing DCs as promoters of chronic inflammatory immune responses in patients with atopic dermatitis, as well as tolerogenic pathways. Therefore TLR-bearing DCs represent promising targets, which might help to improve tolerance induction during immunotherapeutic approaches in the future. (J Allergy Clin Immunol 2010;125:50-9.) Key words: Dendritic cells, innate immunity, adaptive immunity, Toll-like receptors

Rapid recognition of invading pathogens that have circumvented physical barriers, such as the epithelium or the upper skin, is accomplished by pattern-recognition receptors (PRRs). PRRs are expressed by a plethora of immune cells, including monocytes, macrophages, granulocytes, and dendritic cells (DCs), as well as keratinocytes or epithelial cells.1 DCs are subdivided into myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) and are present in the peripheral organs and lymphatic and blood systems. Because of their frequent location at the border zones of the organism and close proximity to the environment, they are regarded as important sentinels of the

From the Department of Dermatology and Allergy, University of Bonn. Supported by grants from the Deutsche Forschungsgemeinschaft (SFB704 TPA4 and TPA15 and FOR208 TPA1) and a BONFOR grant of the University of Bonn. N.N. is supported by a Heisenberg-Professorship of the DFG NO454/5-2. Disclosure of potential conflict of interest: N. Novak has received research support from the German Research Council, is an advisory board member for Novartis, and has received speaker’s fees from ALK-Abello´, Stallergenes, Astellas, Allergy Therapeutics, and Novartis. J.-P. Allam has received research support from ALK-Abello´ and the Deustche Forschungsgemeinschaft; is a speaker for ALK-Abello´, Allergy Therapeutics, and Stallergenes; and has provided legal consultation or expert witness testimony on the topic of sublingual immunotherapy. The rest of the authors have declared that they have no conflict of interest. Received for publication September 8, 2009; revised November 4, 2009; accepted for publication November 16, 2009. Reprint requests: Thomas Bieber, MD, PhD, Department of Dermatology and Allergy, University of Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany. E-mail: [email protected]. 0091-6749/$36.00 Ó 2010 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2009.11.019

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Abbreviations used AD: Atopic dermatitis AIT: Allergen-specific immunotherapy DC: Dendritic cell dDC: Dermal dendritic cell dsRNA: Double-stranded RNA LC: Langerhans cell mDC: Myeloid dendritic cell MoDC: Monocyte-derived dendritic cell MPL: Monophosphoryl lipid A NOD: Nucleotide-binding oligomerization domain protein oLC: Oral mucosal Langerhans cell pDC: Plasmacytoid dendritic cell PRR: Pattern-recognition receptor SNP: Single nucleotide polymorphism ssRNA: Single-stranded RNA TLR: Toll-like receptor TSLP: Thymic stromal lymphopoietin

immune system.2 Hence DCs are equipped with a plethora of extracellular and intracellular PRRs, including Toll-like receptors (TLRs), the mannose receptor (CD206), dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DCSIGN) (CD209), dectin-1, and nucleotide-binding oligomerization domain proteins (NODs) to sense the environment for danger signals and pathogen-associated molecular patterns.3 The nature of the signal transduction pathways initiated by PRRs determines the behavior of DCs and is modulated by the dose and type of the PRR ligand, other PRRs involved, the duration and time point of exposition, and the microenvironment in which the DCs are located.4 As an example, stimulation of TLR4 on DCs in the presence of TGF-b and IL-10 or other tolerogenic factors favors the outcome of regulatory T cells after priming of naive T cells by DCs, whereas inflammation, together with TLR4 activation on DCs, promotes TH1 immune responses (Fig 1). In contrast, TLR4 ligation combined with thymic stromal lymphopoetin (TSLP) and histamine stimulation promotes TH2 immune responses, whereas TGF-b and IL-6 in the microenvironment of TLR4-activated DCs favor the outcome of TH17 T cells (Fig 1). Recognition of pathogen-derived lipids, proteins, and nucleic acids is mediated by different TLR molecules and complexes. To date, 10 (TLR1-TLR10) and 12 (TLR1-TLR9 and TLR11TLR13) members of the TLR family have been identified in human subjects and mice, respectively. In general, pathogenderived lipids and lipopeptides are sensed through TLR1, TLR2, TLR4, and TLR6; proteins are recognized by TLR5 and TLR11; and TLR3 and TLR7 through TLR10 are engaged in binding bacterial and viral nucleic acids. TLR structures, their adaptor molecules used to accomplish signal transduction, and their ligand specificity important for pathogen sensing have been summarized in several reviews; a comprehensive and recent review was published by Kawai and Akira.5 Important

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FIG 1. Influence of TLR4 stimulated by LPS and factors in the micromilieu of DCs on naive T-cell priming by TLR-bearing DCs. Stimulation of TLR4 on DCs by LPS in an inflammatory microenvironment (A) induces the release of IL-12p70 by DCs, which promotes T-cell immune responses of the TH1 type, whereas stimulation of TLR4 on DCs in the presence of histamine or TSLP (B) reduces the capacity of DCs to release IL-12p70 and favors immune responses of the Th2 type. TLR4 ligation together with IL-6 and TGF-b in the microenvironment (C) enforces DCs to produce IL-23, which channels the induction of TH17 cells, whereas activation of DCs through TLR4 in the presence of tolerogenic soluble factors, such as TGF-b, IL-10, vitamin D3 (Vit-D), glucocorticoids (GCs), or vasoactive intestinal peptide (VIP; D) enhances their release of IL-10, TGF-b, and activation of indoleamine 2,3-dioxygenase (IDO), strengthening their tolerogenic properties and the outcome of T cells with regulatory functions (Treg).

components of the cellular immune system, such as DC subtypes and T cells, are recruited in the initial phase of atopic dermatitis (AD) to the epidermal and dermal skin compartment. This is mainly mediated by an accurately modulated release of different chemotactic mediators by keratinocytes and local DCs.6,7 AD is a chronic inflammatory skin disease with a complex pathophysiology, including genetic, environmental, and immunologic cofactors promoting the manifestation of the disease.8 Epidermal Langerhans cells (LCs) and different DC subtypes recruited from the blood to the skin play a pivotal role in the initiation and amplification of the allergic immune response. As an important disease-promoting factor, a genetically predetermined impaired skin barrier enables allergens and microbial antigens to come into contact with skin DCs and thereby affects diseasespecific immunologic changes. As a consequence, cross-linking of allergen-specific IgE molecules bound to the high-affinity receptor for IgE, FceRI, on skin DCs, as well as stimulation of other surface receptors by microbial antigens, induces a cascade of events, including the release of a plethora of soluble mediators. Increased numbers of activated circulating CD41 and CD81 T cells and high numbers of CD41 T cells infiltrating the dermis are characteristic features of patients with AD.9 In particular, T cells

bearing the cutaneous lymphocyte antigen with features of allergen-specific T cells of the TH2 type are recruited. The initial phase of AD is dominated by T cells producing IL-4, IL-5, and IL-13, whereas the number of TH1 cells producing IFN-g increases later.10 Not only T cells but also keratinocytes are capable of aggravating the inflammatory reaction in the skin through upregulated release of cytokines and chemokines.9 It is assumed that acute and chronic inflammation in the skin affects the adaptive immune system and might be responsible for the initiation and continuation of a TH2 response. Thereby TSLP, an IL-7–like cytokine produced in high amounts by keratinocytes in response to microbes, trauma, and inflammation, plays an important role.11 DCs primed by TSLP promote T-cell responses of the TH2 type.12 Together, enhanced TSLP release triggered by frequent allergen challenge, microbial infections, and inflammation might initiate and perpetuate TH2 immune responses in AD. Among T cells infiltrating the inflamed skin, it has been suggested that the so-called TH17 cells might be operative not only in patients with psoriasis but also in those with AD. Indeed, some reports from animal models and studies using atopy patch tests in human subjects13-15 have suggested that TH17 cells might

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FIG 2. Expression of TLRs by human and murine DC subsets. Blood/spleen DCs, DCs isolated from peripheral blood or spleen; sLCs, LCs from skin. Green, Positive; red, negative; white, no data available; yellow, not clear.

be induced in the skin by the topical application of allergens and could also in part contribute to skin infection in patients with AD. Later, it was shown that subpopulations of CD41 and CD81 T cells producing IL-22 might be present in higher amounts in patients with AD, whereas the number of IL-17 cells is relatively low.16 However the role of TH17 cells remains controversial because it has been shown that the contribution of TH17 cells to AD is rather weak compared with their contribution to psoriasis.

MOLECULAR MECHANISMS OF LIGAND BINDING, SIGNAL TRANSDUCTION, AND GENE ACTIVATION THROUGH TLRS ON DCS Structural studies of TLR/ligand complexes have provided insights into recognition of a plethora of ligands by TLRs expressed by DCs, as well as subsequent TLR receptor dimerization.17 Hydrophobic ligands of TLR1, TLR2, and TLR4 interact with internal protein pockets. In contrast, double-stranded RNA (dsRNA), as a hydrophilic ligand, interferes with the solvent-exposed surface of TLR3. Binding of agonistic ligands, lipopeptides, or dsRNA induces dimerization of the ectodomains of the various TLRs, forming dimers that are strikingly similar in shape. All TLRs contain an intracellular Toll/IL-1R18 domain that initiates by recruiting 1 or more of 5 identified intracellular Toll/IL-1R–containing adapter proteins.5 Recently, an additional control mechanism for TLR activity was discovered19: UNC93B1, a multiple membrane–spanning endoplasmic reticulum protein, biased the TLR response to nucleic acids in DCs toward DNA sensing through TLR9 in a stronger way than RNA recognition by TLR7. Thus by ensuring that only 1 of these TLRs leaves the endoplasmic reticulum at the same time, the production of type I IFN by pDCs is kept under control. TLRs regulate gene expression in DCs through a conserved signaling pathway that leads to the activation of several transcription factors, including nuclear factor kB, mitogen-activated protein kinase, and IFN regulatory factors.20,21 Signal transduction induced on ligand binding to TLRs favors a complex interplay of a growing number of adaptor proteins, of which MyD88 was the first identified.22

TLR-MEDIATED DC ACTIVATION DCs are the mediators of pathogen pattern recognition and are responsible for the transfer of the information that such a

recognition has occurred to the adaptive part of the immune systems. Therefore TLR expression by DCs is of central importance for the successful initiation of an immune response. Several DC subtypes with immense phenotypic and functional plasticity have been described thus far. At least 5 phenotypically defined DC populations have been identified in the mouse.23 A significant heterogeneity in TLR expression has been observed among DC subpopulations, as well as differences in expression between murine and human DCs, as already known for other types of innate and adaptive immune mechanisms.24 It is important to consider these differences when using mice as preclinical models for human diseases. Another aspect that has to be taken into account when interpreting TLR data is that the expression of TLRs is not static but rather modulated as a complex cascade of events in response to pathogens, a variety of cytokines, and environmental stressors together. For example, it has been shown that TLR signaling through TLR4 can be deactivated by engagement of other TLR molecules by their specific ligands.25 Moreover, because TLRs are specific for products of certain types of microbes, the DC subset activated by a pathogen or a pathogen product depends on the specific TLR expression pattern of this DC subset (Fig 2).

RECOGNITION OF LIPID COMPONENTS BY TLR1-, TLR2-, TLR4-, AND TLR6-EXPRESSING DCS Most TLR molecules function as monomers, with exception of TLR2, the major task of which is, in addition to recognition of viral components,26,27 binding of lipoproteins from gram-negative bacteria and gram-positive bacterial peptidoglycans.5 TLR2 forms a heterodimeric complex with either TLR1, which recognizes 2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-R-cysteine, or with TLR6, which binds lipoteichoic acid. TLR2/ TLR6 specifically recognizes diacylated lipopeptides, whereas TLR1/TLR2 is involved in the recognition of triacylated bacterial lipoproteins. Thus TLR1 and TLR6 differentially recognize TLR2 ligands, distinguishing the degree of acylation of the lipopeptides.28 TLR1 RNA has been identified in epidermal LCs and dermal dendritic cells (dDCs),29 blood-derived DCs,30 and monocytederived dendritic cells (MoDCs).29-32 In contrast to human DCs, TLR1 is expressed by murine blood-derived DCs, as well as pDCs.33 Murine epidermal LCs can be activated through TLR2 ligands,34,35 whereas controversial reports about TLR2 expression

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of human LCs exist. Flacher et al,30 in contrast to van der Aar et al,29 demonstrated TLR2 RNA expression and function in human LCs. Both human30 and murine33 mDCs from peripheral blood express TLR2, whereas human,36 but not murine,33 pDCs are negative for this receptor. TLR2 is also expressed by human oral mucosal Langerhans cells (oLCs)37 and MoDCs.29,31,32 For TLR2 expression by dDCs, contradictory reports exist: TLR2 RNA expression29 but no TLR2 protein expression38 could be found. Angel et al38 observed that only the immature CD141CD1a2 dDC subset expressed TLR2, whereas mature dDCs were TLR22. TLR6 is, besides TLR1, the second TLR receptor that forms a heterodimeric complex with TLR2. Interestingly, TLR6 is expressed by the same DC subpopulations as TLR1, namely LCs,29,30 MoDCs,29,31 dDCs,29 and blood-derived DCs,30,33 in both human and murine DCs. This means that all mDCs of human or murine origin are capable of binding ligands of TLR1/TLR2 and TLR1/TLR6 derived from gram-positive bacteria, namely diacyl and triacyl lipopeptides, lipoteichoic acid, and peptidoglycans. In contrast, only murine,33 and not human,36 pDCs express TLR6. In addition, various ligands for a single TLR or TLR complex exist, such as human b-defensin, Staphylococcus aureus, and its products yeasts and herpes simplex virus, which have all been demonstrated to bind to TLR2 and TLR2-associated structures.39-42 The second TLR-sensing component of gram-negative bacteria is TLR4, which together with CD14 is the major cellular receptor for LPS.43 As their most striking difference to human LCs, murine LCs respond to TLR4 ligands,34,35 whereas human LCs are devoid of TLR4 RNA.29,30 Interestingly, in contrast to human epidermal LCs, oLCs express TLR4 and are thereby capable of responding to LPS.37 In addition, TLR4 was detected on human MoDCs29,31,32 and their murine equivalent, bone marrow–derived DCs,44,45 as well as on subsets of murine splenic DCs.33 As another difference to the murine system, TLR4 mRNA expression was not detectable on human DCs isolated from the blood,30 and human pDCs do not display TLR4 expression.33,36

RECOGNITION OF PROTEINS BY TLR5- AND TLR11EXPRESSING DCS TLR5 serves, in addition to TLR1, TLR2, TLR4, and TLR6, as a recognition structure for material derived from bacteria, namely for flagellin, the major protein of flagellated bacteria.46 TLR5 is absent from human LCs29 but was found on MoDCs,29,31,32,47 dDCs,29 and blood DCs.48 In addition, subsets of murine splenic mDCs express TLR5,33 in contrast to pDCs, which are devoid of this receptor both in human subjects36 and mice.33 In mice TLR11 is expressed by splenic DCs, which are activated by its ligand, the protozoan-derived profilin-like molecule.49 The human TLR11 gene contains several stop codons and does not code for a fulllength protein. RECOGNITION OF NUCLEIC ACID COMPONENTS BY TLR3-, TLR7-, TLR8-, AND TLR9-EXPRESSING DCS Bacterial and viral DNA contains a high amount of unmethylated CpG motifs, which are sensed through TLR9 and through the closely related TLR7 and TLR8 molecules. TLR7 and TLR8 are also engaged by viral single-stranded RNA (ssRNA) and by

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small synthetic molecules mimicking features of nucleic acids, such as imiquimod.50,51 Additionally, these immune response modifiers are agonists for human, but not murine, TLR8.52 Thus murine TLR7 and human TLR8 mediate species-specific recognition of ssRNA.5,53 In addition, viral dsRNA is recognized by TLR3. In human subjects TLR3 was detected on LCs,29,30 MoDCs,29,31,32 dDCs,29 and mDCs from the blood.30 Human pDCs and murine pDCs were negative for TLR3.33,48 In contrast, murine bone marrow–derived DCs, as well as LCs, expressed TLR3 and responded to its ligands.35,54 For TLR7 and TLR8, contradictory results concerning its expression by human epidermal LCs exist.29,30 Burns et al55 reported on functional activation of epidermal LCs by TLR7/TLR8 ligands. However, Gorden et al56 have provided functional evidence for the dependence of TLR7- and TLR8-mediated signaling in human PBMCs in the presence of pDCs. In mice the CD11cbright/CD8a2 subpopulation of splenic DCs has been typed positive for TLR7 RNA expression and has shown ssRNA-mediated activation.57 Murine LCs were devoid of TLR7,34 and ligands to TLR7 did not activate murine bone marrow2derived DCs.58 Unmethylated bacterial DNA serves as a sensor for immune cells through TLR9. Although TLR9 is expressed on all DC types in mice,33,35,45,57 TLR9 expression is restricted to pDCs in human subjects.59 Most importantly, the sequences of motifs recognized by TLR9 differ between human and murine cells and are much less defined in human compared with murine cells.60 Expression of TLR10 mRNA has been shown in human CD341 stem cell2derived DCs and in pDCs isolated from tonsils.61 In contrast to these results, other groups36,48 could not detect TLR10 mRNA or protein expression in pDCs. Additionally, blood-derived pDCs are devoid of TLR10 RNA.48 In mice the TLR10 gene is truncated and nonfunctional. For TLR12 and TLR13, no data concerning its expression or function in DCs exist thus far. In view of the data presented here, it has to be taken into account that conflicting results might be based on the use of different techniques (PCR, FACS, and function) and reagents for detection of TLRs in different studies.62 Nevertheless, taken together, the data summarized here challenge the often cited dogma63 that mDC and pDC subsets are equipped with mutually exclusive groups of TLRs. According to this dogma, it was assumed for a long time that mDCs, such as LCs, dDCs, MoDCs, and blood-derived DCs, express TLR1 through TLR6 but not TLR7 through TLR9 and that TLR7 through TLR9 was confined to pDCs. However, the summary of the most recent data shows that there are several exceptions to this rule. Moreover, TLR expression by murine DCs is much more widespread compared with that seen for their human counterparts. Major differences between human and murine immunology have been noted before24 and could be attributed to the different environments in mice and human subjects, especially the more restricted pathogen load in the human environment. As a conclusion, TLRs expressed by murine mDCs and pDCs should be carefully discriminated.

MODIFIED TLR EXPRESSION AND SIGNALING ON HUMAN ANTIGEN-PRESENTING CELLS AND MACROPHAGES IN PATIENTS WITH AD Impaired TLR2-mediated production of proinflammatory cytokines by monocytes isolated from the peripheral blood of

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FIG 3. Proposed model for dysfunctions of TLR2 signaling on the levels of monocytes, macrophages, and mDCs and TLR9 signaling on pDCs in patients with AD, which might increase the susceptibility of patients with AD to microbial infections. HSV, herpes simplex virus; IDEC, inflammatory dendritic epidermal cell; Mac, macrophage; Mo, monocyte; SEB, Staphylococcus aureus enterotoxin B.

patients with AD has been linked to a higher expression of FceRI of these cells (Fig 3).64 In contrast to TLR2, cytokine response after TLR4 ligation on monocytes of patients with AD did not differ significantly from cytokine responses observed in healthy control subjects.64 Furthermore, selective impairment of TLR2-mediated signaling has been linked to higher susceptibility of patients with AD to cutaneous infections with S aureus and herpes simplex virus, which both bind to TLR2. Interestingly, higher expression of TLR2 and TLR4 has been observed on monocytes isolated from the peripheral blood of patients with intrinsic AD in one study.65 This upregulated expression has been interpreted as a secondary effect of repetitive inflammatory immune reactions in AD.65 Monocytes from patients with AD carrying the TLR2 R753Q polymorphism produced significantly more IL-6 and IL-12 on TLR2 stimulation than monocytes of patients with the wild-type TLR2 variant.66 In contrast, monocyte-derived macrophages of patients with AD expressed lower TLR2 protein levels than macrophages derived from healthy control donors.66 Consequently, the TLR2mediated cytokine response, including IL-6, IL-8, and IL1b production, after stimulation with respective TLR2 ligands of macrophages derived from monocytes of patients with AD was lower (Fig 3).66 These functional differences in TLR2 on macrophages might in vivo contribute to the higher susceptibility to bacterial and viral infections seen in patients with AD.67 In contrast, MoDCs from highly atopic individuals with profoundly high serum IgE levels did not show any significant differences in their qualitative and quantitative TLR expression repertoires.68 As a consequence, no modifications in TLR-related inflammatory cytokine responses have been observed in in vitro assays.68

The TLR2 ligand S aureus enterotoxin B plays an important role as a trigger factor in the skin of patients with AD. In vitro studies with monocytes and MoDCs of patients with AD revealed induction of TH2-prone immune responses by S aureus enterotoxin B, and this overemphasized TH2 response was mainly related to the inhibition of the IL-12p70 production of DCs.69 These data have been further verified in monocytes from patients with AD,70 leading to very similar results. In contrast, data from murine model systems provided evidence for a role of TLR2 in the induction of TH1 immune responses to cutaneous sensitization71 because TLR22/2 mice showed decreased local ear swelling, cellular infiltration, and IFN-g expression.71 This was mainly based on a higher induction of IL-10– and IFN-g–producing regulatory T-cell subtypes by those DCs.72 Another TLR ligand, the TLR7/TLR8 agonist imidazoquinoline has been demonstrated to inhibit the TSLP-mediated TH2 immune responses inducing properties of DCs in vitro.72 Consequently, the protective effect of this TLR agonist on allergic immune responses might be useful for the treatment of atopic disorders in vivo. Considering TLR expression and TLR-mediated responses as predictive markers for the development of atopic disorders, it has been demonstrated with the help of peripheral blood cells isolated from cord blood that maternal allergy was associated with higher TLR2-, TLR3-, and TLR4-mediated IL-12 and IFN-g responses.73 In addition, higher IL-6 and TNF-a production induced by TLR2, TLR4, and TLR5 ligation was observed in newborns with allergic diseases. These data indicate that higher risk for the development of allergic disorders might go along with increased release of soluble mediators after TLR activation.

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TABLE I. Overview of genetic studies on associations of SNPs in gene regions encoding TLRs with AD Gene

Association with AD

Phenotypic characteristics

TLR2

(1)

recurrent bacterial infections AD AD AD Pure AD AD AD AD

TLR2 TLR3 TLR4 TLR9 IRAKM TOLLIP NOD1

(2) (2) (2) (1) (2) (1/2) (1)

Reference

associated with AD in general. This might explain the lack of associations observed in many of the studies, most likely because of relatively low study power or variations of the results related to the phenotypic criteria used to select the patients.

Mrabet-Dhabi et al75 Weidinger et al74 Terhorst et al68 Weidinger et al74 Novak et al76 Beygo et al77 Schimming et al78 Weidinger et al79

IRAKM, IL-1 receptor–associated kinase M; TOLLIP, Toll-interacting protein.

GENETIC STUDIES ON ASSOCIATIONS OF TLR POLYMORPHISMS WITH AD While investigating and interpreting the expression and function of TLRs on DCs in patients with AD, genetic modifications in gene regions encoding TLRs and TLR-related structures have to be carefully kept in mind (Table I).68,74-79 Genetically modified expression and function of PRRs leading to impaired recognition of microbial components and initiation of innate immune responses in patients with atopic disorders, such as AD, allergic rhinitis, or asthma, represents a plausible hypothesis. Possible variations in both expression pattern and intensity of expression of TLRs by immunocompetent cells combined with defective TLR-mediated signaling mirrored by increased or decreased activation of the immune system might not only influence the maturation of the immune system but also modify profoundly the nature of defensive immune responses. Therefore several studies have investigated the putative association of TLR polymorphisms with AD. Lack of association of TLR2/TLR4 polymorphisms with AD has been reported in one study,74 whereas another study, which focused on a lower number of selected adult patients with AD and concomitant bacterial infections with S aureus and severe forms of the disease, reported an association of a single nucleotide polymorphism (SNP) in the TLR2 gene with AD.75 This SNP has been further linked to functional impairment, including lower TLR2 protein expression and receptor-mediated cytokine production.75 No association of SNPs in the TLR3 gene with AD has been detected in one study.68 In contrast, association of an SNP in the TLR9 promoter region, which affected the promoter activity in functional assays, has been observed to be associated with pure AD but not with AD and high serum IgE levels.76 Furthermore, no evidence for an association of SNPs in the IL1 receptor–associated kinase M gene (IRAKM), which encodes an IL-2 receptor–associated kinase M negatively regulating TLR signaling with AD, was detected.77 Additionally, only borderline association of an SNP in a gene region encoding the Toll-interacting protein, an inhibitory adaptor protein of the TLR pathway, with AD has been observed.78 Another important pathogen-recognition receptor is NOD1, a cytosolic receptor that recognizes muropeptide, a component of gram-negative bacteria. Significant association of an NOD1 haplotype with AD and asthma has been described for a populationbased cohort, as well as a case-control population.79 Based on these data, it can be concluded that genetic modifications in gene regions encoding TLRs might occur in subgroups of patients with severe courses but seem not to be strongly

COREGULATION OF FCeRI AND TLR9 ON PDCS pDCs link innate and adaptive immune responses and are important sensors for nucleic acids in the environment to initiate effective defense of bacterial or viral infections. This sensing is achieved with the help of PRRs, mainly TLR7 and TLR9.80 Human pDCs are characterized by the expression of the IL-3 receptor a-chain (CD123) and blood dendritic cell antigen 2 on their cell surface and are regarded as the only professional IFN-producing cells.81 Most interestingly, human pDCs bear a trimeric variant of FceRI on their cell surfaces.82,83 Similar to mDCs, the surface expression of FceRI on pDCs in the peripheral blood correlates directly with IgE serum levels and the atopic state of the subjects.82 The capacity to release IFN-a and IFN-b in response to TLR9 stimulation with CpG motifs is profoundly downregulated in pDCs after FceRI aggregation and allergen challenge in vitro, which implies a direct cross-talk of FceRI with TLR9.82 Furthermore, increased IL-10 production of pDCs might enhance, together with the TH2-dominated micromilieu in the skin, pDC apoptosis and reduction of the number of pDCs recruited from the blood and detectable in epidermal AD lesions in patients with AD (Fig 3).82,84 A close cross-talk of FceRI with TLR9 might be of particular importance in atopic subjects, in whom IgE-mediated allergen challenge might result in frequent FceRI activation followed by the reduction of their properties to release IFN-a/b. Lower production of IFN-a by human blood DCs from allergic subjects after TLR9 stimulation,85 as well as downregulation of FceRI expression on pDCs after TLR9 activation and reduced TLR9 expression after FceRI cross-linking, support the concept of a close interaction of these structures on pDCs.86 This implies that TLR-mediated mechanisms of the innate immune system directly interact on different levels with IgE-dependent immune mechanisms. Taken together, the reduced capability of pDCs to release IFN in response to TLR stimulation by viral antigens after FceRI activation/allergen challenge might explain in part the increased susceptibility of allergic patients to viral infections observable in vivo (Fig 3).87 TLR ACTIVATION ON DCS ENFORCES TOLERANCE: THE OTHER SIDE OF THE COIN In the context of immune responses to invading pathogens, the induction of the proinflammatory arm of the immune systems on TLR activation represents a key mechanism. Without doubt, TLR activation on DCs preferentially leads to their maturation, followed by the enforcement of effector T-cell immune responses, mainly of the TH1 subtype. However, in patients with AD, regulatory mechanisms inducing regulatory T cells and counteracting inflammation seem to take part only in an attenuated way or might be completely missing. Absence of forkhead box protein 3–expressing, functionally active regulatory T cells from the skin has been described in patients with AD.88,89 In addition, 2 subtypes, namely CD41CD251CCR61 and CD41CD251CCR62, with the phenotype of regulatory T cells have been observed to be present in the blood of patients with AD. Regulatory functions

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FIG 4. Ligation of CD14/TLR4 on oLCs by natural ligands, such as bacterial components in the micromilieu or adjuvants applied during vaccines combined with allergens binding to IgE molecules on the surface of oLCs, leads to the release of tolerogenic mediators, such as IL-10 by oLCs, and enforces their tolerogenic properties, partially also through upregulated expression of coinhibitory molecules, such as B7-H1. Therefore oLCs prime naive T cells primarily into regulatory T-cell subtypes with high forkhead box protein 3 (Foxp3) expression and IL-10/TGF-b release. Furthermore, oLCs promote the outcome of TH1 cells in part through the production of IL-12 and IL-18. These TLR-mediated properties of oLCs might be useful in the context of sublingual allergen-specific immunotherapy.

have been designated to the CD41CD251CCR62 T cells only, whereas CD41CD251CCR61 T cells display characteristics of TH2 cells.90 More insights into physiologic mechanisms of tolerance induction would help for a better understanding of pathways deregulated in patients with AD on the one hand and for development of therapeutic strategies to restore immunologic homeostasis on the other hand. Recent evidence emerges that TLRs on DCs at specific anatomic sites, such as the oral mucosa, might also silence T-cell immune responses and induce regulatory T cells to control inflammation. Thereby they are assumed to prevent excessive damage of surrounding tissue at inflammatory regions on the one hand and perpetuate immunostasis of mucosal surface regions highly exposed to bacterial products, such as gastrointestinal or oral mucosal tissue on the other hand.91-93 Especially in regard to the latter, ‘‘endotoxin tolerance’’ represents a key phenomenon described by an abrogated or reduced response to repetitive exposure to LPS that is mainly sensed by TLR4.94 It is most likely that TLRs on DCs play a pivotal role in this context. In this regard it has been reported recently that constitutive IL-10

production might maintain DCs’ unresponsiveness to TLR ligation.95 On the other hand, it could be demonstrated that regulatory properties of DCs are directly enhanced by activation of TLR2 and TLR4 on DCs on restimulation in an IL-6–, p38 mitogen-activated protein kinase–, extracellular signal–regulated kinase 1/2– , and TNF receptor-associated factor 3–dependent manner, as reflected by increased IL-10 production.96,97 Thus constant exposure to TLR ligands, such as LPS, might enforce the regulatory functions of DCs and thereby silence the T-cell immune response in favor of the immunologic immunostasis predominating in the gut. However, it has been reported that TLR ligation might induce regulatory DCs through IFN-g–induced protein 10.98 In this study stimulation of DCs with TLR2, TLR4, TLR3, and TLR9 ligands upregulated IFN-g–induced protein 10 production, leading to an inhibition of TH1 proliferation.98 Furthermore, recent data suggest a synergistic effect of dual stimulation of TLRs on DCs, which induced both regulatory and proinflammatory cytokine production of naive T cells. In this regard it has been shown that concomitant activation of TLR4 and TLR7 on DCs induced

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IL-10 production through the Janus kinase pathway.99 This is in line with another study reporting that LPS stimulation of TLR4 and TLR7/TLR8 together on DCs promoted T cells to secrete the regulatory cytokine IL-10.100 Moreover, it has been demonstrated that combinatory stimulation of TLR6 and TLR2 enforced induction of regulatory T cells producing IL-10, whereas in the same model the simultaneous stimulation of TLR1 and TLR2 resulted in a proinflammatory immune response.101 Altogether, it appears that the interaction between certain TLRs on DCs is a critical factor directing DCs into a regulatory or proinflammatory state. Another critical parameter in this regard is the strength of TLR signaling on DCs. It has been reported recently that only strong TLR4 signaling prompted DCs to induce regulatory T cells in vitro.102 In conclusion, the picture emerges that tolerogenic DC properties through TLR signaling are critically influenced by constant exposure to TLR ligands, the coactivation of certain TLRs, and the strength of TLR activation.

EFFECT OF TLR-ACTIVATED DCS ON ALLERGENSPECIFIC IMMUNOTHERAPY TLR ligands have been used as adjuvants not only in the context of vaccination strategies but also of allergen-specific immunotherapy (AIT). Recently, it has been shown that TLR9 agonists could improve clinical efficiency of ragweed subcutaneous AIT (specific immunotherapy).103 It has been reported that activation of TLR9 on DCs through CpG motifs not only induces a TH1 cytokine pattern but also leads to the generation of regulatory T cells.104 Interestingly, in preclinical asthma models the therapeutic response is rather related to the regulatory immune response than to the induction of TH1 cells.104 In this context the induction of IL-10–producing T cells and the expression of indoleamine 2,3-dioxygenase in DCs appears to be critical. This is in line with a recent investigation demonstrating that high doses of CpG-activating TLR9 in pDCs results in the induction of regulatory T cells, depending on indoleamine 2,3-dioxygenase expression.105 However, it is still unclear which DC subpopulations process allergens during specific immunotherapy and which TLRs these cells express. Nevertheless, recent data from mice suggest that the critical DC population in allergen-specific sublingual immunotherapy comprises local oral mucosal DCs.106 It has been shown that human LCs are the predominant DC population within the oral epithelium.107 A recently published study demonstrated further that human oLCs express the LPS receptor/CD14 and TLR4. Ligation of TLR4 on oLCs resulted in the upregulation of the expression of the coinhibitory molecules B7-H1 and B7-H3 on oLCs. Furthermore, increased IL-10 production could be detected on TLR4 ligation on oLCs along with the induction of regulatory forkhead box protein 3-expressing T cells (Fig 4).108 Together, these in vitro data strongly argue for a positive effect of TLR4 ligands, such as monophosphoryl lipid A (MPL), on the intended immune deviation and induction of tolerogenic mechanisms during AIT in vivo. Moreover, the combinatory effect of allergens and the TLR4 ligand MPL might form the basis for the positive results observable after treatment of patients with allergic rhinitis with commercially available allergen vaccines, which contain MPL as an adjuvant.109 Similar results were obtained from a murine model, in which TLR2 activation on mucosal DCs increased their IL-10 production and induced regulatory T-cell responses.100 In view of these data, it is tempting to speculate that even physiologic TLR ligands

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from the surrounding microenvironment of oral mucosal tissue, such as LPS, might act as natural adjuvants at this site. Altogether, TLR-expressing DCs appear to be central targets for adjuvants in sublingual immunotherapy, which, in addition to mucoadhesive formulations, facilitate allergen uptake.110

CONCLUSION Based on the data from the current literature presented here, we conclude that DCs express a high variety of PRRs and that the specific expression of these PRRs is dependent on the maturation stage of DCs, the DC subtype, and the surrounding micromilieu. Furthermore, thus far, complex interactions of pathogen-associated molecular patterns with PRRs on various DC subtypes in the human and murine system have been observed. Stimulation of DCs is orchestrated by the composition of PRR ligands, as well as the intensity and frequency of stimulation, which determine the nature of the outgoing T-cell immune response. Both enhanced and reduced TLR expression and signaling, as modified by genetic variations or secondarily by disease-specific factors, play a role in the pathophysiology of chronic inflammatory diseases, such as AD. Furthermore, the protolerogenic character of several immune responses initiated and promoted by TLRbearing DCs in the oral mucosa and putatively also other tissue sites predetermines TLR-expressing DCs as promising targets for immunotherapeutic approaches, in which adjuvants binding to TLRs represent an option to enhance the efficacy of the treatment. REFERENCES 1. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol 2004;5:987-95. 2. Novak N, Haberstok J, Geiger E, Bieber T. Dendritic cells in allergy. Allergy 1999;54:792-803. 3. Diebold SS. Activation of dendritic cells by toll-like receptors and C-type lectins. Handb Exp Pharmacol 2009;(188):3-30. 4. Schroder NW, Maurer M. The role of innate immunity in asthma: leads and lessons from mouse models. Allergy 2007;62:579-90. 5. Kawai T, Akira S. The roles of TLRs, RLRs and NLRs in pathogen recognition. Int Immunol 2009;21:317-37. 6. Homey B, Steinhoff M, Ruzicka T, Leung DY. Cytokines and chemokines orchestrate atopic skin inflammation. J Allergy Clin Immunol 2006;118:178-89. 7. Gros E, Bussmann C, Bieber T, Forster I, Novak N. Expression of chemokines and chemokine receptors in lesional and nonlesional upper skin of patients with atopic dermatitis. J Allergy Clin Immunol 2009;124:753-60. 8. Novak N. New insights into the mechanism and management of allergic diseases: atopic dermatitis. Allergy 2009;64:265-75. 9. Werfel T. The role of leukocytes, keratinocytes, and allergen-specific IgE in the development of atopic dermatitis. J Invest Dermatol 2009;129:1878-91. 10. Grewe M, Walther S, Gyufko K, Czech W, Schopf E, Krutmann J. Analysis of the cytokine pattern expressed in situ in inhalant allergen patch test reactions of atopic dermatitis patients. J Invest Dermatol 1995;105:407-10. 11. Esnault S, Rosenthal LA, Wang DS, Malter JS. Thymic stromal lymphopoietin (TSLP) as a bridge between infection and atopy. Int J Clin Exp Pathol 2008;1: 325-30. 12. Ebner S, Nguyen VA, Forstner M, Wang YH, Wolfram D, Liu YJ, et al. Thymic stromal lymphopoietin converts human epidermal Langerhans cells into antigen-presenting cells that induce proallergic T cells. J Allergy Clin Immunol 2007;119:982-90. 13. Koga C, Kabashima K, Shiraishi N, Kobayashi M, Tokura Y. Possible pathogenic role of Th17 cells for atopic dermatitis. J Invest Dermatol 2008;128: 2625-30. 14. Oyoshi MK, Murphy GF, Geha RS. Filaggrin-deficient mice exhibit TH17-dominated skin inflammation and permissiveness to epicutaneous sensitization with protein antigen. J Allergy Clin Immunol 2009;124:485-93. 15. Eyerich K, Pennino D, Scarponi C, Foerster S, Nasorri F, Behrendt H, et al. IL-17 in atopic eczema: Linking allergen-specific adaptive and microbial-triggered innate immune response. J Allergy Clin Immunol 2009;123:59-66.

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