Timing is everything: dendritic cell subsets in murine Leishmania infection

Review

Timing is everything: dendritic cell subsets in murine Leishmania infection Devika Ashok and Hans Acha-Orbea Department of Biochemistry CIIL, University of Lausanne, 155 Chemin des Boveresses, 1066 Epalinges, Switzerland

Mouse models of Leishmania major infection have shown that a predominant CD4+ T helper type 1 cell (Th1) response leads to protection, while T helper type 2 cell (Th2) predominance confers susceptibility. Dendritic cells (DCs) are antigen-presenting cells that orchestrate the T cell response. The immune response to L. major involves direct antigen presentation by migrating DCs or transfer of antigens to resident DCs to prime T cells. In this review, we discuss the timing and consequences of antigen presentation by DC subsets and how this affects Leishmania susceptibility. We propose a model where dermal DCs and Langerhans cells play a role early in infection, followed by inflammatory monocyte-derived DC and lymph node (LN)-resident DCs at later time points of infection to establish the resistant Th1 response. Leishmania and the host immune interaction More than 12 million people are affected worldwide by Leishamania parasite infections [1]. There are over 20 known species of Leishmania, with approximately a dozen species associated with the various forms of leishmaniasis. The disease is caused by an obligate intracellular parasite inoculated into the skin by the bite of a sand fly [2]. Humans and dogs are the major natural reservoirs of the parasite. Ninety percent of Leishmania infections are restricted to the skin; however, other forms of infection include diffuse cutaneous leishmaniasis (L. mexicana), mucocutaneous leishmaniasis (L. guyanensis), and visceral leishmaniasis (L. donovani). Leishmania infections have been studied extensively in the laboratory, with mouse models proving to be an effective tool to analyze the immune responses that contribute to resistance versus susceptibility to disease. The manifestation of disease is dependent on the species as well as the immune response of the host. The general consensus is that an interleukin 12 (IL-12)-induced T helper type 1 cell (Th1) (see Glossary) response, characterized by high interferon g (IFNg) levels, is required for clearance and protection from infection [3]. The non-healer phenotype has been shown to be associated with Th2 cytokines, such as high levels of IL-4 and immunoglobulin E (IgE) production, and low IFNg [4,5]. The classical mouse strains used for Leishmania infection models are BALB/c (for susceptibility) and C57BL/6 (for resistance). Corresponding author: Acha-Orbea, H. ([email protected]). Keywords: dendritic cell (DC); Leishmania; susceptibility; resistance. 1471-4922/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2014.08.001

When the susceptible BALB/c mice are crossed with the resistant C57BL/6 mice, an interesting intermediate susceptibility phenotype is observed in the F1 generation, particularly after intra-dermal injection of L. major, where they develop a prolonged Th2 response [6]. In contrast, footpad injections in F1 mice develop a Th1 response. Interestingly, the early events of IL-4 and IL-12 production are similar between the two sites of injections in the F1 generation, suggesting that the early response to infection is independent, and a more defined Th1/Th2 response comes into play later in infection. This finding has important implications in light of more recent studies that have identified the role of different antigen presenting cells at varying time points during the establishment of a resistant Th1 response, and may propel future efforts to develop effective vaccines [6]. Leishmania major L. major is the best-characterized model of cutaneous leishmaniasis in murine models. Resistance in C57BL/6 versus susceptibility in BALB/c mice has been genetically mapped to six chromosomal loci [7]. The presence of all six loci was not necessary to confer resistance and no single locus alone was sufficient to confer susceptibility. Rather, a variety of combinations of these loci may be capable of interacting to confer resistance. This multi-gene interaction Glossary Antigen presentation: the process, by which naı¨ve T cells are primed by the presentation of antigen by an antigen presenting cells via the major histocompatibility complex (MHC)/peptide–T cell receptor interaction. Batf-3: basic leucine zipper transcription factor, ATF-Like 3 (Batf-3) is a transcription factor protein encoded by the batf3 gene that is required for the formation of CD8+ and dermal CD103+ DC subsets. Cross-presentation: the process by which an exogenous antigen is processed and presented via major histocompatibility complex I (MHC-I). dLN: draining lymph node (dLN) is the lymph node draining the site of vaccination, infection or tumor. F1: refers to the first generation of a monohybrid cross. In this review article, F1 refers to the first generation of mice from a C57BL/6 and BALB/c parental cross. Th1: T helper 1 (Th1) refers to a subset of CD4+ T cells that are responsible for the protective response to Leishmania. Naı¨ve CD4+ T cells differentiate into Th1 cells upon stimulation in the presence of interleukin 12 (IL-12). These T cells produce interferon g (IFNg) and other cytokines. Th2: T helper 2 (Th2) refers to a subset of CD4+ T cells that are dominant in the non-protective response to Leishmania. Naı¨ve CD4+ cells differentiate into Th2 cells upon stimulation in the presence of IL-4. These T cells produce IL-4 and other cytokines. Treg: regulatory T cells (Treg) are special subsets of CD4+ T cells that regulate the immune response. They can either be generated during maturation in the thymus, or can be induced in the periphery from naı¨ve T cells. Several subsets exist, producing inhibitory cytokines such as IL-10, transforming growth factor b (TGFb), and IL-35, or inducing co-inhibition via, for example, cytotoxic T-lymphocyte antigen 4 (CTLA-4) Trends in Parasitology, October 2014, Vol. 30, No. 10

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responsible for a healing versus non-healing phenotype demonstrates the complexity of the host response to parasite infection. Initially L. major infects neutrophils, and releases particles that then survive preferentially in macrophages, but also in other cells such as fibroblasts [8]. Th1 cells mediate the protective immune response to L. major, and successful healing depends on the strain of mouse being used. IL4 / BALB/c mice are able to resist infection with L. major by mounting a Th1 response [9], pointing to the fact that a healing response to L. major cutaneous leishmaniasis requires IL-12, while IL-4 may play a role in the susceptible response [10]. However, one report has also highlighted the role of early IL-4 in instructing dendritic cells (DCs) to release IL-12, thereby orchestrating a Th1 response, while in the later T cell priming phase, an IL-4-dependent Th2 response contributed to susceptibility [11]. A more recent study has confirmed the role for IL-4R on DCs early after L. major infection in susceptible BALB/c mice [12]. Additionally, it was found that, in contrast to macrophages, not only do DCs harbor parasites, but they also have the ability to present parasite antigens to T cells [13]. Amastigote forms of the parasite are also able to activate DCs and induce an increase in IL-12 production in skin derived DCs [14]. Given the wide variety of immune responses to the different strains of Leishmania, it must be highlighted that the balance between proinflammatory and anti-inflammatory responses will determine the outcome of disease. In the past few years, the clear discrimination between Th1 and Th2 responses is giving way to a much more complex interaction also involving Th17 and Treg cells [15,16]. Data generated using mouse models will form the basis for this review, as the immune response to L. major is best characterized in mice. A summary of mouse models deficient in different cytokines, cytokine receptors, toll-like receptor (TLR) signaling molecules, or cells in L. major infection is presented in Table 1. Dendritic cells DCs are capable of sensing various microbial patterns, as well as danger signals, due to the array of receptors on their surface, their endosomes and in the cytoplasm. Of these receptors, TLRs are the most investigated. In

response to sensing infection or danger, DCs produce a number of key cytokines and chemokines that determine the immune response (Table 2). There are many subtypes of DCs, which are classified based on function, surface phenotype, and location [17]. The two main functional groups are plasmacytoid DCs (pDCs) and conventional DCs (cDCs); the latter group also subdivides into lymph node resident and migratory DC. The surface expression of protein markers is highly variable in DC subsets, and importantly, depends on whether the DC is in homeostatic, inflammatory, or tolerogenic conditions [18]. With regard to the ontogeny of DCs, there are two hypotheses for their development: (i) functional plasticity; and (ii) a specialized lineage model. The former refers to a single precursor cell, giving rise to a functionally diverse set of differentiated cells, while the latter refers to multiple precursors, giving rise to a diverse array of differentiated cells. More often than not, a mixture of these two models is accepted [19,20]. However, a recent study provides evidence to support the finding that cDCs, pDCs, and macrophages do not share a common restricted progenitor population [21]. During inflammation, a subtype of DCs (known as inflammatory or monocyte-derived DCs, moDCs) can differentiate from monocytes [22]. Importantly, the origins of DC subtypes remains hotly debated; however, these form an important basis for the many DC-targeted mouse models used for the study of Leishmania. In mice, CD11c is an ubiquitous marker of both immature and mature DCs. This is followed by high to moderate surface expression of major histocompatibility complex II (MHC-II) and co-stimulatory molecules CD40, CD70, CD80, CD86, and CD54 [23–27]. In addition, CD4 or CD8, which are traditionally T cell markers, are found on the main cDC subsets in secondary lymphoid organs [28]. The CD8+ subset of DCs, together with the dermal CD103 equivalent, is the major cross presenting and IL-12producing DC subset in mice [29–31]. The function of CD4 and CD8 in DC subsets remains unknown. The other major tissue resident DC subsets express CD11b (the integrin a m chain of Mac-1) and are further segregated into CD4+ and CD4 populations [24]. In addition DEC-205, a multilectin domain molecule, is also a marker for several subsets of cDCs including CD8+ DC [32].

Table 1. Mouse models of Leishmania major infectiona Genetic model

Response to L. major

WT MyD88–/– IL1R–/– IL-12p40–/– IFNg R–/– IL17–/– IL4–/– IL4 RA–/– IL13–/– IL-10–/– IL-6–/– Mice lacking Vb4+ T cells (MMTV infected) TNFa–/–

C57BL/6 Resistant Susceptible Worse disease outcome Susceptible Susceptible Resistant Resistant Resistant Resistant Resistant (no memory) Resistant Resistant Susceptible

a

Refs BALB/c Susceptible Susceptible Susceptible Susceptible Susceptible Improved disease outcome Improved disease outcome Resistance/improved disease outcome in acute phase Improved disease outcome Resistant (more so with IL4–/–) Susceptible Improved disease outcome Susceptible

Abbreviations: IFNg, interferon g; IL, interleukin; MYD88, myeloid differentiation primary response gene 88; WT, wild type.

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Table 2. Role of cytokines and chemokines in the immune response. Adapted, with permission, from [98]a Cytokine IFNa IFNb IFNg

IL-12p70 IL-18 IL-27 TNFa Chemokine CXCL9 CXCL10 CCL21

Function Antiviral response, MHC-I expression Antiviral response, MHC-I expression macrophage activation, inducesTh1, suppresses Th2 Induces IFNg and Th1 Induces IFNg Induces IFNg promotes inflammation Function: co-stimulators More Th1, B, T, pDCs Activated T cells, NK cells, B cells T cells, B cells, NK cells, DCs

Produced by pDCs, leukocytes

Cytokine IL-4

Function B cell activation, IgE switch and induces Th2 Immunoglobulin secretion and eosinophil activation Inhibits Th1, inhibits macrophage inflammatory activation Enhances mast cells, Th2 stimulation

Produced by T, B, and mast cells, Basophils T cells, mast cells, B cells T cells, B cells

fibroblasts, DCs

IL-5

T cells and NK cells

IL-13

DCs and macrophages DCs and macrophages Phagocytes Macrophages, T and NK cells Receptor CXCR3A and B CXCR3A and B

IL-9 TSLP

Induces Th2

Epithelial cells

Chemokine CCL2 CCL7

Function More Th2, monocytes, DCs More Th2, monocytes, NK cells, DCs More Th2

Receptor CCR2 CCR1, 2, 3, 5, 10

CCR7

CCL8

More Th2, basophils, monocytes, DCs Treg, more Th2, thymocytes, DCs Function: co-inhibition

CCR1, 2,3, 5

CCL13 CCL17 Cytokine regulating IL-10 TGFb

Functions

Cells producing

Decrease Th1, inhibit macrophages, Inhibits cell growth, switch to IgA

Monocytes, nTreg, T cells and B cells T cells, monocytes, DCs, macrophages, B cells

Chemokine and cytokine regulating CTLA-4 PD1 PD1

T cells T cells, B cells, macrophages

T cells

CCR2, 3

CCR4 Interacting with cells expressing CD80, CD86 PD-L1 PD-L1

a

Abbreviations: CTLA-4, cytotoxic T-lymphocyte antigen 4; DC, dendritic cell; IFN, interferon; IgE, immunoglobulin E; IL, interleukin; MHC-I, major histocompatibility complex class I; NK, natural killer; PD-1, programmed cell death 1; PD-1L, programmed cell death 1 ligand; pDC, plasmacytoid dendritic cell; TGFb, transforming growth factor b; Th1, T helper type 1; TNFa, tumor necrosis factor a; Treg, regulatory T cells; TSLP, thymic stromal lymphopoietin.

Langerhans cells (LCs) are dendritic cells that reside in the epidermis and are identified by the presence of a transmembrane lectin with mannose binding specificity called Langerin [33]. Langerin is also found on some subsets of dermal DCs (dDCs) and draining lymph node (dLN) CD8+ DCs [34]. Finally, CD103 is a marker for some tissue resident DCs which are found in the dermis, gut, and lungs of mice [35]. After contact with microorganisms or inflammatory stimuli, tissue resident DCs undergo a process of maturation. This is followed by migration to the dLN where DCs present the antigen to T lymphocytes, initiating an adaptive immune response. Maturation consists of: (i) increased expression of MHC and co-stimulatory molecules; (ii) downregulation of phagocytic activity, after an initial increase for approximately 30 min; (iii) enhanced cytokine secretion; and (iv) changes in chemokine receptor expression and chemokine production [36]. These changes influence the migration and recruitment of other cell types and Th lineage decision [37]. In an inflammatory environment, DCs can give immunogenic signals, while in a noninflammatory environment, they are capable of providing tolerogenic signals [38]. Different subsets of DCs are described in Table 3. DC subsets in phases post L. major infection Functional data regarding the role of individual DC subsets has grown exponentially in recent years, contributing

to a better understanding of this heterogeneous population of antigen presenting cells. A general consensus is that DCs at the site of infection take up parasites, acquire a mature phenotype and, upon migration to the dLN, present L. major antigens to CD4+ and CD8+ T cells via MHC-II and MHC-I respectively [39,40]. However, there is discord on the role of the different DC subsets due to differences in the parasite strains used (LV39, MHOM/IL/80/FE/BNI), the dosage (low dose or high dose), and the route of administration (subcutaneous or intra dermal) [41]. Many groups have identified the early presence of Leishmania antigen presenting dermal DCs (dDCs) moDC, followed by Langerhans cells, and finally, the dLN resident CD11b+ cells themselves [42–44]. It is known that in the high dose subcutaneous model (with a slight delay in the low dose model), antigen presentation occurs in two to three waves in the dLN of infected mice: the first wave within 1 day, the second after 1 week, and the third commencing at days 15– 21 [42,43,45,46]. Besides the classical DC subsets, monocyte-derived DCs play crucial roles in the generation of a protective immune response (moDCs, inflammatoy DCs) [47]. However, not all subsets of DCs are involved to a similar extent in immunity towards Leishmania infection, and although individual studies point towards the role of specific DC subsets at different time phases post-infection, it is possible that they can act across multiple phases. In this review, we focus on what is known about the different subsets of DCs in mouse models of Leishmania infection. 501

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Table 3. Surface markers of dendritic cell subsets in micea,b Skin derived DC

Lymphoid tissue resident DC

Langerhans

Langerin+

LangerinS

CD11b+

CD8+

CD11c CD11b CD8a Langerin DEC-205 CD103 Ly6C B220 Clec9A Sirpa CD24 CD4 F4/80 MHC-II TLRs Half life Location

+ + –/+ +++ ++ – – –

+ low –/+ ++ + + – –

+ + – – – – – –

+++ ++ – – – – – –

+++

+ ND – + ++ TLR4 Slow Epidermis

ND ND – + ++ TLR 3 Fast Dermis

+ ND – – ++

CCR7 Radiation

– Resistant

* Sensitive

* Sensitive

+ low +/– – +++ TLR9, TLR7 Fast LN, spleen, Peyer’s patches, liver + Sensitive

Fast Dermis

++ + + – – – + –/low + – – +++ TLR3, TLR9 Fast LN, spleen, Peyer’s patches, thymus, liver + Sensitive

Inflammatory monocyte derived DC int ++ – ND ND ND ++ –

pDCs

ND ND – ND ++ TLR7, 9 Fast Monocytes in the blood

ND ND +/– – ++ TLR7, TLR9 Fast Blood, LN, thymus, spleen, Peyer’s patches + Sensitive

* Sensitive

int – – – – ++ ++

a

Adapted, with permission, from [19,33,99].

b

Abbreviations: Clec9A, C-type lectin receptor 9A; int, intermediate; LN, lymph node; MHC, major histocompatibility complex; ND, not determined; pDC, plasmacytoid dendritic cell; Sirpa, signal regulatory protein a; TLR, toll-like receptor.

We propose a model of infection based on DC roles at the time points at which each subset exerts their main influence post L. major infection (Figure 1). The scenario shown in Figure 1 represents the time points in high dose subcutaneous injections; however, similar kinetics are observed in the dermal low dose injection model [48]. First wave of antigen presentation Langerhans cells Langerhans cells are identified as being epidermal resident Langerin+ Birbeck granule+ cells. LCs are efficient at taking up pathogens in the skin, upon which they become activated, differentiate, gain mobility, and move to the cutaneous dLN [49]. In 1993, Moll et al. identified Langerhans cells, in contrast to macrophages, as taking up Leishmania antigen and migrating to the dLN. In addition, these cells were identified as initiating an antigen specific T cell response within the dLN [43]. In 2000, Von Stebut et al. used fetal skin derived DCs, which were similar to Langerhans cells, to induce robust functional responses in both C57BL/6 and BALB/c mice [50]. In contrast, upregulated expression of IL-4R, and the consequent decrease in IL-12 production, was observed in Langerhans cells from susceptible mice, thereby indicating their contribution to an enhanced Th2 response [51]. Since then, two divergent in vivo studies have highlighted the role of Langerhans cells, although with contrasting outcomes based on the model of infection used. Ritter et al. in 2009, using Lang-DTR mice (diptheria toxin receptor expressed using the Langerin promoter, allowing their depletion with the toxin), found that Langerin+ dDCs were responsible for early CD8 T cell priming, not Langerhans cells, when infecting mice with 3106 stationary phase 502

promastigotes subcutaneously [52]. Another study supported this finding and identified that Langerhans cells were dispensable for antigen presentation to T cells in vivo in L. major infection [42]. By contrast, intra dermal injections with 1000 metacyclic promastigotes in mice lacking Langerin+ cells showed that, not only did these cells play a role, but Langerin+ cells were actually detrimental to an effective anti-Leishmania response [44]. Dermal DCs in L. major infection The two major dDC subpopulations, CD11bhi and CD103+ Langerin+, have been identified as infected early in infection and are able to carry Leishmania antigens to the dLN as early as 24 h post-infection [53]. Within these 24 h, skin draining DCs are the source of antigen of Leishmania antigen for the CD11b dLN resident cells that are then responsible for priming T cells [45]. Functionally, the C11bhi subset of dDCs could potentially be involved in susceptibility, as mice lacking CD11b (complement receptor 3) fared marginally better than their wild type BALB/c counterparts. This is likely due to the fact that in the CD11b knock-out mice, the CD11bhi subset of cells are still present but do not express the integrin [54]. However, this study focused on CD11b expression on macrophages and neutrophils, while CD11b expression on the Langerin subset of dDCs could also contribute to this effect. While the CD11blow CD103+ dermal DCs (dDCs) have been implicated in transporting antigens, their role in L. major immunity is not established [46]. A prominent study that addressed the functional dichotomy between these two subsets of dDCs found that while the Langerin populations of DCs were initiating a CD4+ T cell response, the Langerin+ dDC subset was required for effective priming of the CD8+ T cell response [52]. Another study

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0

3

LCs dDCs moDCs cDCs : CD11b+ some CD8+

7

BATF3 dependent DCs

Wave of angen presentaon

LCs dDCs moDCs

Wave of angen presentaon

L.major

Wave of angen presentaon

Foot pad lesion progression

BATF3 dependent DCs?

moDCs cDCs : CD11b+ CD8+

14 21 Number of days

C57BL/6 ba3-/-

C57BL/6

28

35

42

TRENDS in Parasitology

Figure 1. Schematic representation of infection progression in a mouse model of Leishmania major infection. Infection progression and healing in C57Bl/6 wild type mice is shown over time by a thin black line. A thick black line represents infection progression without healing, as observed in batf3 / mice. Langerhans cells [43], dermal dendritic cells (dDCs) [42,46], and a monocyte-derived DC population (inflammatory DC, moDC)s [47] form the first wave of antigen presentation [45] following L. major infection. The second wave of antigen presentation involves mostly CD11b+, and CD8+ cDCs [46]. The third wave of antigen presentation involves CD11b+ and CD8+ cDCs [45]. Background shading indicates the progression of infection.

found that dDCs, upon vaccination with L. major/unmethylated DNA (CpG), produce IL-6 that then results in a decrease in Treg accumulation in resistant mice [55]. Dermal DCs were also identified in the same system of L. major/CpG vaccination as producing IL-2, which then induced IFNg production by natural killer (NK) cells [56]. In addition, a recent study showed that not only do dDCs take up parasites, they also perform apoptotic cell clearance, whereby they phagocytose infected apoptotic neutrophils [57]. Although this study used the ubiquitous DC marker CD11c for depletion of DCs, it is likely that the taking up of apoptotic cells is by the CD103+ dDC subset, because they have the recognition receptor DEC-205 [31]. Also, in herpes simplex virus (HSV) infections, CD103+ dDCs transfer antigen to lymphoid tissue-resident CD8+ DCs for presentation to CD4+ and CD8+ T cells, a phenomenon that could potentially be of significance to many other infection settings [58]. moDCs in L. major infection Inflammation induced monocyte derived DCs (moDCs) are potent inducers of a Th1 response by carrying antigen to the dLN, where they can prime naı¨ve T cells [47]. They are found in increased numbers early after infection, but even more so at approximately day 21 post-infection. We have, therefore, discussed them in further detail in the third wave of DC subsets, since it is then that their potent IL-12 production can markedly shape the anti-Leishmania response towards establishment of a Th1 healing response. Second wave of antigen presentation Langerhans cells, dermal DCs, and cDCs continue to increase in number, whereas moDC numbers drop at 1 week and show striking increases thereafter [47]. All DC subsets are involved during the later phases of infection. We now discuss the role of cDCs, starting early and continuing through the course of L. major infection.

Conventional lymph node resident DC The CD11b+ cDC subsets mainly exert their influence in this phase with a role for the CD8+ DCs as well. In the dLN, the CD11b+ cDC subset was demonstrated to have the capability to acquire soluble antigen that arrived via lymph and mediate a L. major-specific immune response [42,45]. In addition, further studies identified that a protein highly expressed on the CD11b cDCs, Sirpa (signal regulatory protein alpha) was required for protection against L. major [59]. CD11b+ dLN resident cells are better at antigen processing and presentation to CD4+ T cells, while producing significantly less IL-12 than the CD8+ subset upon activation [26]. The CD8+ cDC subset is known to be the predominant producer of IL-12 [60,61]. In addition, this subset is one of the only lymph node resident DC subsets (together with the migratory CD103+ dDC) capable of cross-presentation to CD8+ T cells [62,63]. CD8+ cDCs are also capable of taking up apoptotic cells via the death cell receptor DEC-205 [32], and necrotic cells via C-type lectin receptor 9A (Clec9A) [64]. Despite being the least permissive to infection by L. major promastigotes in vitro, CD8a+ DCs were the most potent producers of IL-12 [65]. This, along with their capability to efficiently cross present, makes this cDC subset an attractive candidate to study the role of crosspresentation and dLN IL-12 production at the various stages of infection. However, there is a degree of functional plasticity amongst the different subsets of DCs depending on the activation state, the nature of the antigen, and the concentration of antigen, as well as the type of receptor used to take up the antigen [26]. Third wave of antigen presentation During the third wave of antigen presentation a massive increase in the number of all the DC subsets is observed. The most striking increase is observed for moDC, whereas the CD8+ DC start decreasing in number approximately 3 weeks after infection [47]. 503

Review Dermal and epidermal DC These subsets continue to increase in number through the course of infection and, depending on the dosage used, can also be found in this third wave of antigen presenting cells the dLN [47]. moDC In this seminal study, a population of CD11cint Ly6Chi CD11b+ cells were identified that were generated early at the site of infection, acquired L. major antigen, and migrated to the dLN where they initiated a Th1 profile from CD4+ T cells of resistant mice. CCR2 / mice, which do not allow monocyte infiltration into inflamed tissues, become highly susceptible, highlighting the requirement of moDCs [66]. These moDCs are also capable of powerful IL-12 production, mediated by the myeloid differentiation primary response gene 88 (MyD88) adapter proteins [67]. L. major induced moDCs have been observed as being infected at the site of infection and migrating to the dLN, where they effectively present the L. major LACK antigen to T cells [47]. In addition, inducible nitric oxide synthase (iNOS)-producing inflammatory cells have also been implicated in the chronic phase of infection to L. major and are, therefore, not limited to the early phase [68]. Lymphoid tissue resident cDCs CD11b+ DC can present antigens during the third wave of infection and, similar to the first two waves, all the DC subsets participate in the immune response. Recently, however, the important role of CD8+ and CD103+ DCs in protection from L. major infection using batf3 / mice was described [69]. Three weeks after infection, CD8+ but not CD103+ DC could present antigen ex vivo to primed Leishmania-specific T cells. Due to the absence of the transcription factor basic leucine zipper transcription factor ATFlike 3 (BATF3), these mice lack both CD8+ and CD103+ DC subsets, but show a normal distribution of other DC including moDC [70]. These mice become highly susceptible to L. major infection, develop exacerbated lesions compared to resistant wild type C57BL/6 mice, and the lesion size remains higher even at a later time of infection (i.e., after 15–16 weeks) [69]. The same applies to the low dose injection model. In the absence of CD8+ DC, a predominant TH2 response is observed. Furthermore, by depleting the cross-presenting CD8+ and CD103+ DC subsets using cytochrome c injection from day 17–19 post-infection, C57BL/ 6 mice developed a similar enhanced susceptibility response. The return of this population soon after cessation of infection did not contribute to a protective response. The same effect was achieved by blocking the IL-12R from day 19 in C57BL/6 mice, indicating that IL-12 mediates the protective contribution of these DC subsets. Interestingly, batf3-independent mechanisms have been identified that describes a partial depletion or minor repopulation of these two subsets of DCs in batf3 / mice [31,71,72]. It is interesting to note that, despite the minor re-population, batf3 / mice displayed a highly susceptible response to L. major infection and it may be speculated that in a system without re-population, an even further susceptible response to infection would be observed. 504

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Recent studies have, therefore, highlighted the role for both inflammatory moDCs and the cross-presenting DC subsets towards a protective immune response to L. major [47,69]. We propose that a collaboration could exist between the inflammatory moDC and the CD8+ DCs, since moDCs efficiently transfer peptide/MHC complexes to CD8+ DCs for presentation (cross-dressing) during viral infection [73], so it is possible that such a process also occurs during Leishmania infection. Plasmacytoid DCs Little is known about the role of pDCs in L. major infection. Vaccination with L. major pulsed pDCs was able to induce protection against the parasite [74]. However, a more recent study demonstrated that pDCs harbored the parasites longer than other subsets of DCs [75]. This information, together with the potent type I IFN production capabilities of pDCs, has important implications for its role in L. major immunity. Using a constitutively depleted pDC mouse model, it was shown that pDCs are required for the development of an adequate innate immune response, particularly the NK cell response to TLR9 activation, as well as the recruitment of polymorphonuclear leukocytes (PMNs), monocytes, and macrophages to the site of Leishmania infection [76]. However, further studies are required to identify at what point post-infection these cells are exerting their major influence. Implications for vaccine strategies Despite our current understanding of the immune response to L. major, we lack a reliable vaccine against L. major infection. There have been vaccination attempts against Leishmania infection targeting TAT-antigen Tg from L. major, pDCs pulsed with L. major, and many others, but these are yet to be put into effect in the field [77–82]. A more recent study used an engineered L. major antigen conjugated to anti-DEC-205 monoclonal antibody in order to induce targeted anti-Leishmania effects, and this displayed promising results in the mouse model [83]. Another study identified that IL-4 R signaling was imperative for the efficiency of a successful DC based vaccine in susceptible BALB/c mice [84]. An effective vaccine against L. major would require the ability to generate Th1 differentiation, prevent induction of a strong Th2 response, and be able to generate and maintain high levels of IL-12. This is where the antigen presenting DCs play a vital role, as DCs are at the threshold of the innate and the adaptive immune systems (Box 1).

Box 1. Outstanding questions  Are the first phases of antigen presentation important for inducing protection?  Could targeting cross-presenting DC during the third phase of antigen presentation allow efficient post-infection vaccination?  Are cross-presenting DC subsets required late in infection to maintain protection?  How do moDCs and cross-presenting DC interact?  Are lymph node resident DCs required for memory responses to L. major?  Are CD8+ T cell responses affected in the absence of crosspresenting DC subsets?

Review Concluding remarks and future perspectives The recent work on batf3 / mice has highlighted the effects of targeting cross-presenting cells and IL-12 as potential mechanisms by which DCs contribute to resistance [69], and allows speculation that post-infection vaccines might be efficient if cross-presenting DC subsets are targeted. It will be important to clarify whether the early phases of the anti-Leishmania immune response are trials to adapt to the pathogen or if they are required to allow formation of a protective Th1 response during the third wave of antigen presentation. It is known that low-dose infected C57BL/6 WT mice exhibit an IFNg response from CD8+ T cells which inhibits the CD4+ Th1 phenotype [85]. Given the requirement for CD8+ T cells in the low-dose model, and the potential interaction with batf3-dependent cells, the low-dose model of infection in batf3 / mice may exhibit significantly exacerbated disease similar to, if not more than, the high-dose model. This review highlights the complexity involved in interpreting the current available information on the role of DCs in L. major infection. The review is focused on the timing post-infection, while dosage, route of administration, mouse strain, and even sub-strain of the parasite could all be potential factors that must be taken into consideration. Given the importance of DCs as potential targets for vaccination, we propose that further attention must be paid to the post-infection phase. This will have important implications to the degree of protection that can be achieved. There are still fundamental questions that will determine the success of a reliable DC based vaccination, such as how would the memory response be affected in the absence of certain DC subsets, what is the latest time point post-infection that a vaccination can be administered to confer protection, and many more. DC-based L. major vaccination is an exciting field that will only continue to become more intriguing as new mouse models develop along with our understanding of the immune response to the pathogen. References 1 Sacks, D. and Noben-Trauth, N. (2002) The immunology of susceptibility and resistance to Leishmania major in mice. Nat. Rev. Immunol. 2, 845–858 2 Reiner, S.L. and Locksley, R.M. (1995) The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13, 151–177 3 McDowell, M.A. et al. (2002) Leishmania priming of human dendritic cells for CD40 ligand-induced interleukin-12p70 secretion is strain and species dependent. Infect. Immun. 70, 3994–4001 4 Lipoldova, M. et al. (2000) Susceptibility to Leishmania major infection in mice: multiple loci and heterogeneity of immunopathological phenotypes. Genes Immun. 1, 200–206 5 Mattner, F. et al. (1997) The role of IL-12 and IL-4 in Leishmania major infection. Chem. Immunol. 68, 86–109 6 Nabors, G.S. et al. (1995) The influence of the site of parasite inoculation on the development of Th1 and Th2 type immune responses in (BALB/c x C57BL/6) F1 mice infected with Leishmania major. Parasite Immunol. 17, 569–579 7 Beebe, A.M. et al. (1997) Serial backcross mapping of multiple loci associated with resistance to Leishmania major in mice. Immunity 6, 551–557 8 Peters, N.C. et al. (2008) In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science 321, 970–974 9 Kopf, M. et al. (1996) IL-4-deficient Balb/c mice resist infection with Leishmania major. J. Exp. Med. 184, 1127–1136

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94 Belkaid, Y. et al. (2001) The role of interleukin (IL)-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti-IL-10 receptor antibody for sterile cure. J. Exp. Med. 194, 1497–1506 95 Moskowitz, N.H. et al. (1997) Efficient immunity against Leishmania major in the absence of interleukin-6. Infect. Immun. 65, 2448–2450 96 Launois, P. et al. (1997) IL-4 rapidly produced by V beta 4 V alpha 8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice. Immunity 6, 541–549 97 Wilhelm, P. et al. (2001) Rapidly fatal leishmaniasis in resistant C57BL/6 mice lacking TNF. J. Immunol. 166, 4012–4019 98 Kapsenberg, M.L. (2003) Dendritic-cell control of pathogen-driven Tcell polarization. Nat. Rev. Immunol. 3, 984–993 99 Lopez-Bravo, M. and Ardavin, C. (2008) In vivo induction of immune responses to pathogens by conventional dendritic cells. Immunity 29, 343–351

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