Langerhans Cells – The Macrophage in Dendritic Cell Clothing

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Review

Langerhans Cells – The Macrophage in Dendritic Cell Clothing Thomas Doebel,1,2 Benjamin Voisin,1,2 and Keisuke Nagao1,* Our assumptions on the identity and functions of Langerhans cells (LCs) of the epidermis have undergone considerable changes. Once thought to be prototypic representatives of the dendritic cell (DC) lineage, they are now considered to be a specialized subset of tissue-resident macrophages. Despite this, LCs display a remarkable mixture of properties. Like many tissue macrophages, they self-maintain locally. However, unlike tissue macrophages and similar to DCs, they homeostatically migrate to lymph nodes and present antigen to antigen-specific T cells. Current evidence indicates that the immune responses initiated by LCs are complex and dependent on antigenic properties and localization of the stimulus. This complexity is reflected in the recently demonstrated roles of LCs in type 17, regulatory, and humoral immune responses.

Trends Based on their developmental origin, LCs are a specialized epidermal tissue macrophage subset that is seeded into skin prenatally. Under inflammatory conditions, LCs can arise from bone-marrow-derived precursors. The epidermal microenvironment seems to determine the LC identity of the different precursors. In contrast to other tissue macrophages, LCs display a striking mixture of macrophage (self-maintaining) and DC properties (migration to lymph nodes, and T cell stimulation).

Historical Perspective LCs are myeloid cells unique in the epidermis [1]. Not only are they found in mammalian skin, but they also exist in the skin of other vertebrates including birds and reptiles [2,3] and are thus widely conserved. The beginning of LC research dates back to the publication “Ueber die Nerven, der menschlichen Haut” (translated: On the nerves of the human skin) by Paul Langerhans in 1868 [4]. Langerhans weighed up arguments for LCs to be either cells of connective tissue or neural origin due to their striking dendritic morphology. He made suggestions for the latter based on the limited evidence accessible to him. However, he remained cautious and considered the question of their nature as not definitely settled. Interestingly, this question has only begun to be answered in recent years, after 140 years of research. Important questions regarding certain properties (macrophage origin vs migratory potential) and functions (immunogenicity vs tolerance induction) have yet to be fully elucidated. In this review, we summarize recent literature on ontogeny (see Glossary) and functions of LCs in health and disease focusing on results in the murine system.

The immune responses initiated by LCs are context dependent, ranging from tolerance to inflammatory immune responses.

Ontogeny and Maintenance Current Assumptions about [23_TD$IF]the Identity of LCs At the present stage of research, the evidence points towards LCs being a tissue-specific (epidermal) macrophage subset. However, LCs share typical features with DCs; especially in terms of migratory potential and ability to stimulate T cells. Indeed, the apparent DC-like properties of LCs led for many years to the assumption that LCs are a prototypic example of DCs. This past identification of LCs as DCs was additionally standing to reason because tissue-resident macrophages were considered nonmigratory, which is in striking contrast to the homeostatic migration of LCs to lymph nodes. Because the classification of cell types in immunology has strongly been aiming towards a unifying ontogenetic perspective on relatedness, the developmental origin of LCs is now considered to be the cell-type defining feature (Box 1).

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Dermatology Branch, National Cancer Institute (NCI), Bethesda, MD, USA 2 These authors contributed equally to the work

[21_TD$IF]*Correspondence: [email protected] (K. Nagao).

http://dx.doi.org/10.1016/j.it.2017.06.008 © 2017 Published by Elsevier Ltd.

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[214_TD$IF]Box 1. Macrophages versus DCs and [215_TD$IF]the Identity of LCs The conclusion that LCs are a specialized macrophage subset rests on the premise that cell types should be classified based on their developmental relationship. Under steady-state conditions LCs are derived from embryonic precursors, are long-lived, and self-maintained locally. In contrast, DCs (sensu strictu) are derived from hematopoietic stem cells of the bone marrow, are short-lived, and need to be continuously replenished. Lymphoid and nonlymphoid tissue DCs arise from common DC precursors that, at least under homeostatic conditions, do not give rise to epidermal LCs. DC subsets depend on Flt3L and its receptor Flt3 and both fate mapping and barcoding experiments have established that they share developmental trajectories. However, if the developmental relationship is disregarded and cells are classified based on their function, then it remains valid to argue that LCs are a subset of (functionally defined) DCs. Additional complexity is added if we consider monocyte-derived DCs (moDCs). During hematopoiesis these cells split earlier from the developmental path that leads to DCs at a stage called monocyte/macrophage DC progenitor. moDCs are independent of Flt3 but depend on CSF-1R. They display DC-like properties in vivo and it has recently been shown that moDCs have a different monocytic precursor than monocyte-derived macrophages [85]. Classical DCs derive from a progenitor that expresses the transcription factor zDC (Zbtb46) but moDCs do not, indicating the more distal relationship of moDCs to DCs (sensu strictu). In contrast, LCs clearly express Zbtb46, demonstrating their DC-like properties even on the level of transcription factors [86].

Ontogenetic Origin of LCs Macrophages precursors emerge early during embryonic development (Figure 1). The first macrophage progenitors (mesodermal erythromyeloid progenitors, EMPs) emerge in the extraembryonic blood islands of the yolk sac around embryonic day 8.5 [5–7]. These progenitors are part of the primitive hematopoiesis that, in contrast to the hematopoietic stem cell (HSC)-dependent definitive hematopoiesis, has restricted erythromyeloid potential. EMPs of the yolk sac that are independent of the transcription factor Myb (a transcription factor that is essential for definitive hematopoiesis) enter the embryo and seed the fetal liver from where they colonize the developing organs at the onset of organogenesis around embryonic day 9.5. This process involves a transient differentiation state called premacrophages (pMacs), in which the developing macrophages acquire a core macrophage signature comprising transcription factors, cytokine/chemokine receptors, and pattern recognition receptors. The acquisition of tissue-specific properties starts shortly after entering the developing organs in a process that is guided by organ- or location-specific microenvironmental factors. The development of organs and differentiation of tissue-specific macrophages are, therefore, tightly coupled processes. For this reason, the differentiation of tissue-specific macrophages with a unique epigenome, transcriptome, and proteome can be regarded as an aspect of organogenesis. [24_TD$IF]The work described above applied single-cell transcriptomic analyses to establish the developmental trajectories of macrophage subsets. However, the developmental processes that [25_TD$IF]were described are not undisputed. Thorough work of other authors argues for a two-wave model of organ colonization by macrophages [8,9]. In this model, the first wave of EMPs directly enters the developing organs. A second wave of late yolk-sac-derived EMPs enters the fetal liver and differentiates into Myb-dependent fetal monocytes. These fetal monocytes then again seed the tissues and, except for microglia, replace the first wave of macrophages, including most of the LC precursors of the first wave. Another recent publication surprisingly argues that fetal monocytes of the second wave are derived from HSCs, so that the origin of most tissue macrophages, including LCs, would not be the primitive hematopoiesis but the early definitive hematopoiesis [10]. This is a substantial difference to the work of other authors whose data leave the possibility for only a small contribution of definitive hematopoiesis. More work and more models are needed to settle these discrepancies but irrespective of the assumed origin of LCs, the fate of their precursors after seeding fetal skin would be similar. Upon entering the skin, pMacs or fetal monocytes likely receive tissue-specific cues that guide their differentiation into LCs and under steady-state conditions, the developing LCs are maintained without further contribution of HSC-derived cells.

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Glossary Blood islands: first site of hematopoiesis during embryonic development. Located in the yolk sac outside of the embryo proper. Vessels sprouting from the blood islands connect to the developing embryonic circulation. Definitive hematopoiesis: hematopoiesis that leads to the full spectrum of hematopoietic cells. Occurs in the fetal liver and postnatally in bone marrow. Dermoepidermal junction: interface of epidermis and dermis where basal cells of the epidermis and cells of the dermis express specific anchoring filaments or anchoring fibrils to establish a connection to the basement membrane. Attaches the epidermis to the rest of the body. Filaggrin: important structural protein of the stratum corneum and a major source for natural moisturizing factors. Loss-of-function mutation of this gene is a major predisposing factor for atopic dermatitis. Hair follicle: besides its long-known function as hair-producing organ, it is an immunologically active organ that shapes composition and function of immune cells within skin. Hapten: small molecules that lead to an immunogenic structure when bound to larger molecules such as proteins. Elicit immune responses only in the bound form. Hapten-induced contact hypersensitivity (CHS): animal models of allergic contact dermatitis in which haptens are sensitized through the skin. A type IV delayed hypersensitivity reaction. Leishmaniasis: protozoan infection that is transmitted by sandflies and, in its cutaneous form, manifests as open sores or chronic non-healing ulcers. Considered to be a neglected tropical disease by various organizations. Microenvironment: not well defined small-scale environment of a cell, usually encompassing the surrounding tissue and cells, in which a specific sphere of influence of other cells and molecules shapes the properties of this cell. Neutralizing antibodies: antibodies that upon binding neutralize the biological function of their antigen. Noncanonical NF-kB pathway: refers to pathways of NF-kB

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Fate of LC Precursors after Seeding Fetal Skin After entering fetal skin, developing LCs remain sparsely distributed and lack the typical dendritic LC morphology. They also maintain a myeloid marker profile (CX3C chemokine receptor 1; CX3CR1) without the expression of typical LC markers [MHC class II, Langerin (Box 2), and CD11c], which are only upregulated after birth [11]. In agreement with this, the transcriptome of developing LCs changes only moderately during the fetal period, and the major transcriptomic changes that give them their ultimate LC identity only occur after birth [5]. This postnatal upregulation of LC markers coincides with a proliferative burst and morphological maturation during the first 1 or 2 weeks, after which [26_TD$IF]the LC network is fully established [11]. Considering recent data on the establishment of macrophage identity in different tissues by modification of the enhancer landscape through integration of microenvironmental cues, it is likely that tissue-derived factors play a major role during differentiation of precursors towards LCs [12,13]. Because LCs gain their full identity in a short timeframe after birth, it needs to be investigated whether additional factors like the establishment of the microbiome either directly or indirectly contribute to the differentiation of LCs. Homeostasis of the LC Network LCs continuously migrate to the lymph nodes under steady-state conditions. To maintain their network, LCs need to be replenished, which in principle could be mediated by immigration of precursor cells. It appears, however, that under homeostatic conditions, replenishment of LCs is mediated by a constant low-level proliferation ( <5% of all LCs), which is similar to the maintenance of other tissue-resident macrophages [11,14]. Tissue-resident macrophages, including LCs, proliferate in a differentiated state, which is in striking contrast to the replenishment of other differentiated tissue cells from undifferentiated stem cells. Despite this, the homeostatic maintenance is controlled by the same self-renewal gene network in both tissue stem cells and tissue-resident macrophages [15]. Interestingly, tissue-resident macrophages access this self-renewal gene network by a different set of enhancers from that of other stem cells. The activity of the enhancers in macrophages is controlled by macrophage-specific Maf transcription factors. From this perspective, an LC stem cell may be dispensable for the maintenance of the LC network [15]. Of note, the proliferation of fully differentiated LCs does not preclude that there may be phenotypic differences among LCs. It has been reported that proliferating LCs are enriched in a population with lower expression of CD11b, MHC class II, and CD86. This could represent a precursor-like LCs subset or terminally differentiated LCs that have recently undergone proliferation, but this distinction has yet to be thoroughly addressed [14].

activation that bypass the inducible degradation of inhibitors of kB (IkB). Usually leads to the activation of more specific forms of the NF-kB family of transcription factors. Ontogeny of cell types: developmental history of a cell type within an organism, either during the whole lifetime of an organism or at a certain period of its development. Paracortex: one of the three major regions of the lymph node. Localized between cortex and medulla. The paracortex mainly contains T cells while the cortex mainly contains B cells. Pathogen-associated molecular patterns (PAMPs): highly conserved molecules of different substance classes that are associated with pathogens or groups of pathogens and that can be recognized by germline-encoded receptors of the innate immune system. Primitive hematopoiesis: hematopoiesis with restricted myeloid and erythroid potential that occurs in the blood islands. Yolk sac: membranous cavity connected to the embryo. Important for early blood supply of the embryo. Usually disappears before birth.

Several essential factors for the establishment and maintenance of the LC network have been identified. Among them, transforming growth factor (TGF)- [27_TD$IF]b1 and transcription factors and other members of the TGF-b1-signaling network like runt-related transcription factor (Runx)3, ID2, PU.1, and P14 are the most thoroughly studied. TGF-b1 is constitutively expressed by both suprabasal keratinocytes and LCs [16,17] and active TGF-b1 signaling is required to maintain epidermal residence of LCs [18–20]. Furthermore, the conditional ablation of Tgfb1 or its receptors (Tgfbr1 or Tgfbr2) from LCs have demonstrated that autocrine/paracrine signaling is required to maintain the LC network [18]. TGF-b is secreted as an inactive complex with the latency-associated peptide (LAP) [21]. Recent data have shown that epidermal keratinocytes activate latent TGF-b on LCs via avb6 and avb8 integrins to provide continuous TGF-b signaling. UV-induced perturbation leads to the [28_TD$IF]downregulation of avb6 and avb8 integrins on keratinocytes, thereby disrupting TGF-b signaling in LCs and leading to LC emigration from the epidermis [22]. Thus, TGF-b signaling acts as a retention signal for LCs to stay in the epidermis.

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Figure 1. Ontogeny of LCs during Steady State and Inflammation. The first LC precursors are macrophage progenitors of the primitive hematopoiesis (red) that emerge in the blood islands of the yolk sac. From there they either directly seed the skin or migrate to the fetal liver where they differentiate into fetal monocytes and then seed the skin. One recent study also argues for a major contribution of the definitive hematopoiesis to these fetal liver monocytes (blue). During embryonic development, LCs remain immature and sparsely distributed but proliferate to set up the LC network and adopt the full LC phenotype shortly after birth. Under homeostatic conditions, LCs self-maintain locally by low-level proliferation without a major contribution of the bone marrow. Under inflammatory conditions or during depletion bone marrowderived monocytes are recruited to the epidermis via hair follicles and give rise to LCs. AGM, Aorta–gonad–mesonephros; E, embryonic day; LC, Langerhans cell.

The lack of establishment of the LC network in Runx3 and Id2 knockout mice can be attributed to impaired TGF-b signaling because the knockout of both genes targets the downstream signaling cascade of TGF-b [23,24]. Similarly, knockout of Spi1 (PU.1) targets Runx3 regulatory elements and PU.1 binding to the Runx3 promoter depends on functional TGF-b receptor (TGF-bR) signaling [25]. P14 regulates the expression of TGF-bR and Lamtor2 (P14)-deficient LCs appear to be unresponsive to TGF-b. P14-depleted LCs also fail to undergo mitosis and are prone to apoptosis leading to a lack of persistence of mature LCs in the epidermis [26,27]. It is reported that TGF-b signaling via the conventional Smad-dependent pathways (Smad2/3/4 in the case of TGF-b1) is not required for the homeostasis of LCs [28,29]. This could point towards a role of noncanonical TGF-b signaling pathways [30], but simultaneous ablation of multiple Smads are required to better understand the signaling events downstream of TGF-b1. Bone morphogenic protein (BMP)7, a member of the TGF-b superfamily, and signaling via the BMPR1A–Smad1/5/8 axis was recently suggested to play a role in the early steps of LC

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Box 2. Langerin, Birbeck Granules and LCs [216_TD$IF]Langerin is a type II transmembrane protein that belongs to the calcium-dependent C-type lectin family. Extracellularly, Langerin displays a carbohydrate recognition domain and a neck region to from trimers. The carbohydrate recognition domain binds mannosylated residues, allowing for instance the recognition of viral antigens such as the HIV surface protein gp120. Moreover, a recent report unveiled the importance of Langerin to restrict HIV infection by routing its autophagic degradation in a E3-ubiquitin ligase tri-partite-[217_TD$IF]motif-containing 5a (TRIM5a)-mediated pathway [87]. Langerin is present on the surface of LCs but is continuously internalized and accumulates in endosomal compartments called Birbeck granules. These granules display a characteristic tennis racket-shaped morphology. The immunological function of Birbeck granules remains poorly understood although a role in pathogen retention and degradation has been demonstrated. Originally, Langerin was thought to be restricted to LCs. This so far holds true for humans, but in mice it is also expressed in subsets of dermal DCs as well as CD8+ DCs of lymphoid organs. Birbeck granules have never been unequivocally found in other Langerin+ DC subsets and are therefore still considered to be a hallmark of LCs.

differentiation. BMP7 was expressed in the epidermis and the epidermis of newborn Bmp7deficient mice had reduced numbers of LCs that appeared morphologically immature [31]. It was also shown that BMP7 can induce LC-like cells in human CD34+[20_TD$IF] hematopoietic progenitor cell cultures. The requirement [29_TD$IF]of BMP7 for the homeostasis of LCs in adult skin is unclear because Bmp7-deficient mice die shortly after birth. Colony-stimulating factor-1 receptor (CSF-1R) is expressed on all myeloid cells in the steady state and its ligand CSF-1 is an essential myeloid growth factor. Csf1R-deficient mice lack LCs but Csf1-deficient mice only have a mild reduction of LCs. This is due to the tissue-specific expression of the alternative CSF-1R ligand interleukin (IL)-34 in keratinocytes, which allows for the maintenance of the LC network under homeostatic conditions [32,33]. In summary, the LC network is maintained by low-level proliferation of differentiated LCs under homeostatic conditions. Cytokines and growth factors like TGF-b1, IL-34, CSF-1, and BMP7 are important for the establishment and maintenance of the LC network. TGF-b1 directly regulates the DC-like migratory properties in LCs. The LC Network under Inflammatory Conditions Skin inflammation affects proliferation, maturation, and migration of LCs. Increased migration during inflammation can lead to a partial depletion of epidermal LCs, which in various models, is compensated by a recruitment of bone-marrow-derived cells into the epidermis. Not all precursors of these inflammatory LCs have been identified, but a major population is a subset of monocytes that have been reported to either partially [34] or fully [35,36] acquire the phenotype of steady-state LCs. These monocyte-derived cells possess a distinct morphology from that of steady-state LCs after they enter the epidermis. They are guided into the skin through production of the chemokine [230_TD$IF](C-C motif) ligand (CCL)2 and CCL20 by isthmus and infundibulum of the hair follicle, respectively, which engage chemokine CC receptor (CCR)2 and CCR6, respectively [36]. It has been reported that recruited monocytes give rise to relatively short-lived inflammatory LCs, however, other work has shown that recruited monocytes can become rather long-lived cells in the epidermis [7,25]. This is in line with data from other organs that demonstrated that yolk sac macrophages, and fetal and adult monocytes can generate identical tissue-resident macrophages that then serve their tissue-specific function [37]. There may be bone-marrow-dependent cells other than monocytes that can replenish LCs of the epidermis. The mucosal counterparts of epidermal LCs are developmentally derived from the DC lineage as well as from monocytes. Mucosal LCs are neither radioresistant nor self-maintaining and are, therefore, not bona fide tissue-resident macrophages [38]. Human CD1c+ DCs have the potential to become LC-like cells in vitro [39,40]. Whether there are conditions in which epidermal LCs can be replenished by the DC lineage in vivo has to be investigated. The fact that epidermal and mucosal LCs have similar transcriptomes, phenotypes, and functions highlights how tissue-derived microenvironmental cues can lead to a convergent differentiation of distinct precursor cells.

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LC Function Unique Migratory Capacity of LCs Owing to their localization at the outermost interface between the body and the environment, LCs represent the first line of immunological defense. Despite their recent demonstration as belonging to the macrophage lineage, LCs remarkably share with DCs their ability to migrate to lymph nodes, both during steady state and inflammation. This migration is a multistep process that likely requires LCs to disengage from their liaisons with surrounding keratinocytes; in part through downregulation of E-cadherin; a homophilic cell adhesion molecule that is expressed by keratinocytes and LCs [41]. Upregulation of MHC II reflects LC activation [42], but whether this is a prerequisite for LCs to emigrate from the epidermis remains undetermined. LCs reach the dermoepidermal junction and cross the basement membrane after confined degradation of the extracellular matrix via directed release of metalloproteinases 2 and 9 [43]. This extravasation step is spatially oriented by the chemokine CXC ligand (CXCL)12 that is produced by dermal fibroblasts and is sensed by LCs via chemokine CXC receptor (CXCR)4 [44]. Once in the dermis, CCL19 and CCL21 expressed by lymphatic endothelial cells engage CCR7 on LCs and guide their migration into lymphatic vessels [45,46]. Upon arrival to skin draining lymph nodes, LCs preferentially position themselves to the inner paracortex (closer to the medulla), a migratory pattern distinct from that of Langerin dermal DCs, which position themselves to the outer paracortex (closer to the cortex) [47]. Utilization of a transgenic mouse model expressing an improved photoconvertible fluorescent protein has enabled the monitoring of the temporal turnover and fate of skin DCs within the lymph nodes. Under steady-state conditions, LCs were found to persist for 4 days in lymph nodes without any sign of further recirculation into lymphatics [48]. This is in marked contrast to CD103 dermal DCs (equivalent to Langerin dermal DCs), which turned over within 1–2 days. It is established that the transcription factor nuclear factor (NF)-kB plays an important role in DC activation and potentially for migration under inflammatory conditions [49,50]. However, in contrast to dermal DCs, neither activation nor nuclear translocation of RelB occurs in LCs [51]. Therefore, it is unlikely that LC activation or migration relies on the RelB-mediated canonical NFkB pathway during inflammation. A recent report underscores the importance of an IKKBdependent non-canonical NF-kB pathway for homeostatic migration of skin DCs that is critical for maintaining peripheral tolerance [50]. While evidence was provided for the contribution of this pathway to dermal DC activation and migration, as well as LC activation, its involvement in LC migration remains unclear. Multiple cytokines have been reported to play a role in LC migration. Intradermal injection of neutralizing antibodies against IL-1a, IL-1b, tumor necrosis factor-a, and IL-18 each impair LC migration [52–54]. However, the specific ablation of MyD88 (myeloid differentiation primary response protein 88) in LCs, the adaptor protein involved in IL-1 receptor (IL-1R), IL-18R, as well as Toll-like receptor intracellular signaling, does not impair LC migration either in steadystate or during hapten-induced inflammation [55]. These data suggest that these cytokines do not directly target LCs, but rather indirectly, perhaps through their effects on surrounding keratinocytes. Thus, while it is well established that LCs are endowed with migratory capacity similar to that of conventional DCs, their ultimate localization within lymph nodes and the signals driving their motility appear to be distinct. Initial Reports on LC Function in vivo The longstanding assumption held that LCs were the major antigen-presenting cell (APC) subset responsible for initiating immune responses in the skin (described as the LC paradigm [56]). It therefore came as a surprise when it was demonstrated that LCs did not participate in protective immunity against infection by herpes simplex virus [57]. Furthermore, a series of

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independent studies utilizing different lines of genetically modified mice, in which LCs can be depleted either constitutively (Langerin-DTA; diphtheria toxin A) [58] or inducibly (LangerinDTR; diphtheria toxin receptor) [47,59] failed to demonstrate essential roles for LCs during hapten-induced contact hypersensitivity (CHS), an animal model for allergic contact dermatitis. The lack of contribution of LCs in these models does not exclude the possibility that variation of experimental conditions like type and dose of hapten or timing of LC depletion could provide positive evidence for a unique role of LCs [60]. Antigen Uptake by LCs While CHS or infection models against intradermally injected pathogens have provided insight into mechanisms that are involved in skin immune responses, they may not have been optimal systems to study nonredundant roles of LCs. This is mainly because the route of antigen delivery in these models does not reflect the way LCs naturally take up antigens. When haptens are applied epicutaneously, they penetrate the epidermal barriers, readily reaching the dermis [61]. This likely results in the uptake of haptenized proteins not only by LCs but also by dermal DCs. Considering that CD103+[84_TD$IF] dermal DCs [62] (which are also depleted in Langerin-DTR mouse models) and CD11b+ dermal DCs play important roles in initiating CHS responses [63], their contributions may mask LC functions. Similarly, intradermally delivered pathogens are likely to be primarily taken up by dermal DCs. As such, antigen-specific responses that are attributed to LCs in such settings need to be carefully interpreted. The lack of LC contribution during herpes simplex infection, can now be explained by the fact that CD103+ DCs, but not LCs, are the primary DC subset that crosspresent antigens to induce CD8+ T cell responses [57]. To understand physiological functions of LCs, it is important to consider the processes by which LCs acquire skin-associated antigens (Figure 2). The epidermis is equipped with two sets of barriers; the stratum corneum and epidermal tight junctions [64,65]. The stratum corneum is a three-layered barrier, of which each layer has distinct hydrophobic and metal ion barrier properties [66]. Tight junctions are water-tight barriers between cells that prevent the passage of macromolecules. Thus, foreign macromolecules that breach the barrier of the stratum corneum can still be excluded by tight junctions. It has become clear in recent years that LCs constantly probe for antigens that have breached the stratum corneum. In response to minor trauma, such as tape-stripping, LCs become activated and extend their dendrites vertically through epidermal tight junctions, where they engage in endocytosis via dendrite tips to acquire foreign antigens [67,68]. During this process, LCs form de novo tight junctions with keratinocytes to maintain barrier integrity. Activated LCs can be identified in epidermal sheets by upregulated MHC II. Notably, all activated MHC IIhigh[231_TD$IF] LCs, but none of the nonactivated LCs, extend their dendrites vertically through tight junctions, suggesting that these activated LCs are specialized to acquire foreign antigens [67]. Thus, upon sensing perturbations of the skin, LCs actively engage in surveillance of the microenvironment that exists beyond tight junctions. Surveillance of Microbes by LCs By surveying the microenvironment beyond tight junctions, LCs acquire protein antigens and bacterial toxins that have breached the stratum corneum and participate in the induction of antigen-specific IgG responses, primarily of the subclass IgG1. For example, patch-immunization with Staphylococcus aureus-derived exfoliative toxin A (ETA), a toxin that impairs keratinocyte cell-cell adhesion by cleaving the desmosomal protein desmoglein 1, induces the generation of ETA-specific neutralizing antibodies in an LC-dependent manner [68]. When immunized mice are systemically challenged with ETA to induce experimental staphylococcal scalded skin syndrome, a potentially fatal blistering disease in humans, they are spared from developing skin blistering. Thus, LCs confer systemic protection after local acquisition of ETA.

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[213_TD$IF]Figure 2. LC Functions. Under homeostatic conditions and with intact barriers, epidermal LCs are believed to induce tolerance, which is suggested by targeting experiments with antigen-coupled antibodies. A minor barrier disruption, that leads to a breach of the stratum corneum induces LCs to extend their dendrites through tight junctions and to sample the space beyond the tight junctions. This can lead to the induction of antigen-specific IgG1 antibodies. After barrier disruption and invasion by Candida albicans, LCs mediate the induction of type 17 immune responses. LC, Langerhans cell; Th17, T helper 17.

Because LCs mediate the immune responses before ETA breaches the epidermal barriers, this process is referred to as pre-emptive immunity and may represent an important host defense mechanism against potentially pathogenic microbial agents that exist on the skin surface. Previous studies have demonstrated the presence of circulating anti-ETA antibodies in humans without any history of staphylococcal scalded skin syndrome, suggesting that pre-emptive immunity had taken place in these individuals [69]. The selective induction of humoral immunity is reasonable, because it would come into effect only when pathogen/toxin breach occurs. In contrast, T cell-mediated inflammation would obscure the epidermal integrity; an undesirable outcome against a pathogen/toxin that does not pose an immediate threat. LC-mediated IgG1 responses are reported to be dependent on follicular T helper cells [70]. Repetitive epicutaneous application of ovalbumin leads to the generation of LC-mediated IgE responses in a thymic stromal lymphopoietin (TSLP)-dependent manner [61], suggesting that mechanisms involved in pre-emptive immunity may also be relevant during percutaneous sensitization in allergic diseases.

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LCs initiate T cell-mediated immunity under barrier-deficient conditions. Mutations in the gene encoding filament aggregating protein (filaggrin) are a major predisposing factor in atopic dermatitis, leading to enhanced antigen penetration in filaggrin-deficient mice [71]. Filaggrindeficient mice exhibit enhanced type 17 responses (both Th17 and gd T cells) against topically inoculated S. aureus, which are abrogated when filaggrin-deficient mice are crossed to Langerin-DTA mice (constitutive lack of LCs) [72], reiterating the role of LCs in surveying skin-associated bacteria. Candida albicans is a commensal yeast of the oral cavity, genitourinary tract, and gut and commonly causes local skin infections in immunocompetent as well as systemic infections in immunocompromised hosts [73]. During epicutaneous C. albicans infection, LCs recognize the yeast and pseudohyphal forms of C. albicans via the C-type lectin Dectin-1 and induce Th17 immune responses in an IL-6-dependent manner [74,75]. While CD11b+ dermal DCs also recognize pseudohyphal forms via Dectin-1 and induce Th17 responses, CD103+ dermal DCs induce Th1 responses in a Dectin-1-independent manner. It is interesting to compare these host-defense immune responses to those in inflammatory skin diseases. Perhaps the immune responses elicited against C. albicans are analogous to the type 17 immune responses triggered by LCs and skin DCs in mouse models of psoriasis [76,77]. Thus, whereas LCs induce humoral immunity against antigens that they acquire through intact tight junctions, they are capable of inducing T cell-mediated immunity when epidermal barriers are breached, orchestrating the immune responses in conjunction with dermal DCs. These in vivo roles for LCs in mediating antibacterial or antifungal responses could only be demonstrated by carefully targeting the antigens so that LCs were the major APC subset to take up the antigens. Immunosuppressive Function of Langerhans Cells While the immunogenicity of LCs has been extensively studied over the past few decades, recent evidence also supports an immunosuppressive role under specific inflammatory conditions. Ablation of LCs during leishmaniasis results in attenuated skin lesions and decreased parasite load, together with a reduction of CD4+ T regulatory (Treg) cells [78]. Skin sensitization to the hapten dinitrothiocyanobenzene, as well as antibody-based LC targeting during imiquimod-induced inflammation, unveiled the capacity for LCs to induce CD8+ T cell anergy/ deletion [79,80]. Furthermore, in a bone marrow chimeric model, in which antigen presentation was solely restricted to LCs, peripheral CD4+ T cell tolerance was observed after skin immunization [51]. More recently, LCs were reported to generate Treg cells upon exposure of skin to ionizing radiation [81]. Thus, LCs display a marked functional plasticity, and the response they induce seems to depend on the immunological context. The selective expression of pattern recognition receptors by LCs might partially explain these different responses. While direct recognition of certain pathogen-associated molecular patterns (PAMPs) would trigger inflammatory immune responses (e.g., during C. albicans infection), absence of direct recognition of other PAMPs could skew LC function towards tolerogenic responses. With regard to autoantigens, the capacity of LCs to induce tolerance remains unclear. Results on tolerance induction obtained during induced inflammation may not reflect the route of selfantigen acquisition by LCs under homeostatic conditions. While targeting experiments, using an anti-Langerin antibody coupled with myelin oligodendrocyte glycoprotein peptide, provided clues for a potential tolerogenic role of LCs in the steady state [82], the epidermal source of selfantigens as well as their physiological acquisition by LCs remain elusive. The visualization of an LC loaded with melanin and crossing the basement membrane supported the ability of LCs to take up antigens derived from the hair follicle during the steady state [83].

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Concluding Remarks

Outstanding Questions

From many aspects, LCs are a unique and fascinating constituent of the macrophage family. Like other tissue-resident macrophages, they arise from embryonic precursors and are maintained within the epidermis by local self-renewal under steady-state conditions. However, in contrast to other tissue macrophages, LCs display DC functions and constitutively migrate to peripheral lymph nodes to interact with naïve T cells. Endowed with functional plasticity, LCs can adapt their responses to the immunological context within the skin to either activate or suppress proinflammatory adaptive immune responses. Understanding how LCs integrate different microenvironmental signals to adapt their immunological responses represents a necessary challenge in the context of vaccine research, inflammatory skin diseases, and tumor immunology. Naturally, the classification of LCs as DCs over the past few decades has driven the scientific community to extensively study their DC-like functions. However, as macrophages, it is probable that LCs exhibit homeostatic functions in situ. The observation that the loss of LCs in human and murine zincdeficiency-associated dermatitis correlates with increased levels of ATP within the epidermis may reflect this macrophage side of LC functions [84]. Future investigations in this direction should give exciting new insights into the functional repertoire of LCs [23_TD$IF](see Outstanding Questions).

If LCs are a highly specialized macrophage subset of the epidermis, what are the functions of LCs beyond acting as APCs?

Acknowledgments This work was supported by the National Institutes of Health (NIH) NCI Intramural Research Programs.

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