Immunology of cutaneous leishmaniasis: the role of mast cells, phagocytes and dendritic cells for protective immunity

ARTICLE

Auteur(s) :, Esther Von Stebut^*

Department of Dermatology, Johannes Gutenberg-University, Langenbeckstrasse 1, 55131 Mainz

accepté le 7 Decembre 2006

Immunity against Leishmania major

Most of the immunological facts described above were the result of extensive research using murine experimental leishmaniasis. Some findings have now been confirmed in humans. Murine leishmaniasis is generally induced by inoculation of “supra” physiological doses (≥ 10⁶ parasites subcutaneously into foot pads) of L. major. Recently, a more physiological infection model was established that closely mimics natural transmission of the parasite by the bite of the sand fly. The differences between the former “high dose” model and this model are (i) inoculation of only 10-1,000 parasites, and (ii) intradermal inoculation imitating the bite of the sand fly. The resulting ‘physiological’ anti-Leishmania immune response can be separated into distinct phases with regard to the cell types activated in each respective phase [5]: In the first phase, silent invasion of skin resident MΦ occurs. In the second phase, immigration and activation of cells of the innate immune system (e.g. resident mast cells, neutrophils, monocytes) is most prominent and skin lesions develop. The third phase is characterized by the immigration of dendritic cells (DC) and T cells and coincides with lesion involution. Finally, in the chronic phase, persisting parasites may contribute to the maintenance of a life-long resulting immunity. Using this “low-dose” model, critical observations from the standard high dose model were confirmed and supplemented by identification of interesting additional factors (e.g. the requirement for antigen-specific CD8 responses) [5, 6].

In this review the induction of protective immunity will be described in more detail. The complex interplay of cells of the innate immune system (e.g. phagocytes), which are the first to be in contact with invading Leishmania parasites, with cells of the adaptive immune response regulates if and how protective immunity evolves. Modulation of life-long immunity, which is the goal of vaccination strategies, can only be achieved when we understand how the induction of protection takes place. Here, the focus will lie on a description of the events taking place in mouse strains which are genetically resistant to Leishmania infection (e.g. C57BL/6 mice), since they best recapitulate major hallmarks of human infections. C57BL/6 mice, as well as humans, develop self-healing skin lesions that become prominent several weeks post infection and resolve within months post infection (figure 3).

The silent phase

Invasion of resident MΦ

From an immunological point of view, the CR3-mediated early infection of resident skin MΦ with L. major is a silent process and, furthermore, results in selective inhibition of the IL-12 synthesis pathway in infected MΦ [2, 7, 8]. Once within MΦ, the infectious stage promastigotes transform into the amastigote life form of the parasite capable of surviving inside of phagosomes. During this early silent phase, lasting 4-5 weeks, post infection without visible clinical skin affection, the parasites replicate (up to 1,000-fold) until finally more infectious amastigotes are released into the tissue from lysed MΦ [5].

The second phase

Recruitment of inflammatory MΦ and neutrophils

How the inflammatory ‘wave’ in this second phase is initiated is not fully understood yet, but it appears that complement [10] and cutaneous mast cells (MC) [11] critically contribute to this response. C3 cleavage is required for the attraction of neutrophils [10]. In addition, using different disease models (foreign body granuloma induction, L. major infection), we have recently shown that MC mediate the recruitment of neutrophils and MΦ to the inflamed tissue [9, 11]. Release of TNFα from MC promotes influx of neutrophils, which release chemokines (such as MIP-1α/β, MIP-2), which in turn results in the recruitment of MΦ. Interestingly and surprisingly, if one factor in this chain of events is missing, inefficient immigration of inflammatory MΦ into skin is the result. This finding was confirmed by the facts that, in leishmaniasis, extensive dermal MC degranulation is found at sites of early infection [11, 12] and that MC co-incubated with L. major in vitro reportedly release preformed TNFα within minutes [13]. In addition, van Zandbergen et al. have recently demonstrated that infected human neutrophils secrete high levels of MIP-1β, thus attracting MΦ [14].

Function of neutrophils in cutaneous leishmaniasis?

The third phase

Recruitment and activation of DC

Interestingly, and in contrast to MΦ, DC have been shown to phagocytose only the amastigote life form, but not infectious stage promastigotes [20-25]. We have shown that DC take up L. major via a different receptor [18]. Unlike MΦ, which initially utilize CR3 to bind and phagocytose Leishmania promastigotes, DC mainly acquire the parasite through Fcγ receptor (FcγR) I and FcγRIII-mediated uptake of amastigotes. In mice without B cells or functional FcγR, decreased numbers of infected lesional CD11c⁺ DC were found [18].

Infection of DC with L. major results in activation of the cells associated with upregulation of MHC class I and II expression as well as costimulatory molecules [22, 26]. In parallel, infected cells release a variety of proinflammatory cytokines including IL-12 and various types of infected DC efficiently promoted protection against infections with L. major via release of IL-12 [23, 26-29]. In addition, pre-treatment of susceptible BALB/c mice with recombinant Flt3 ligand induced an expansion of the CD11c⁺ DC compartment, resulting in increased IL-12 production and partial protection of infected mice [30].

Induction of adaptive T cell responses by DC

It was recently shown that activated DC are the only cells capable of processing Leishmania antigen in both the MHC class I and II pathways [5]. After infection, LC downregulate E-cadherin expression [22], which mediates LC adherence to surrounding keratinocytes, and thus migrate towards the skin draining lymph nodes. Infected DC were able to prime both CD4⁺ as well as CD8⁺ T cells [32, 33]. MΦ, in contrast, express only low levels of MHC class II and costimulatory molecules and are unable to prime naïve T cells against Leishmania antigen [34]. In addition, MΦ were unable to present parasite antigen in a MHC class I context and did not re-stimulate Leishmania-specific CD8 T cells [5]. The reason for the different behaviours of DC and MΦ after infection may be the utilization of different receptors to recognize and ingest L. major [18]. Cross-presentation of Leishmania antigen in an MHC class I context to CD8 cells is the result of FcγR-mediate phagocytosis, whereas CR3-mediated phagocytosis by MΦ leads exclusively to MHC class II-restricted antigen presentation. These results bear some similarity to experiments evaluating the role of FcγR in anti-tumor immunity: effective cross-presentation of tumor antigens by DC and the generation of cytotoxic CD8⁺ T cells was also dependent on FcγR-dependent activation [35, 36]. Later on, in established infections, MΦ may also develop into more potent APC by exposure to mediators such as IFNγ and GM-CSF [37].

Mature DC cannot only trigger T cells, but also shape adaptive immunity by regulating Th development. Most of the time, activated DC induce Th1 immunity, but under certain conditions, DC are also capable of inducing Th2 development [38]. The ability of DC to induce Th1 or Th2 immunity may depend on the milieu that is present in the periphery when DC encounter antigen. However, in cutaneous infections DC preferentially induce Th1/Tc1 immunity. Although IL-12 was initially detected in MΦ, prior data indicates that DC are the primary source of IL-12 in lymphoid tissues [22, 39]. IL-12 is the key cytokine for induction of Th1 immunity. Recently, other members of the IL-12 family, e.g. IL-27 and IL-23, or proinflammatory cytokines (e.g. IL-1) have also been shown to contribute to the induction and maintenance of Th1 responses [40-42]. In Leishmania infections, IL-12 and related cytokines (IL-27, IL-1) are primarily released by infected DC very early on post infection thus efficiently inducing Th1-mediated protection [22, 23, 27, 28, 42-44]. Interestingly, sustained release of IL-12 is also important for the perpetuation of Th1 immunity [45]. The cellular origin of the continuous IL-12 release at later stages post infection has not been identified so far.

DC, parasite persistence and regulatory T cells

What is the source of IL-10 that promotes parasite persistence? Several cells may be responsible for IL-10 production at these later stages post infection. Among these, MΦ and regulatory T cells (Treg) are critically involved. Mosser and co-workers have demonstrated that at later time points post infection, FcγR-mediated Leishmania uptake of amastigotes by MΦ may also become important. FcγR-ligation on infected MΦ induced IL-10 release, which in turn prevented parasite elimination and promoted disease progression [50]. In addition, blocking of the IL-10 receptor prevented antibody-mediated disease exacerbation [51].

Endogenous CD4⁺ CD25⁺ FoxP3⁺ Treg have recently been described for their capacity to control excessive or misdirected immune responses. There is growing evidence that Treg play a fundamental role in various infectious diseases, e.g. L. major infections [52]. During leishmaniasis, many features characteristic of Treg function, such as high levels of IL-10, TGF-β or immunosuppression, have been described. Treg appear to control L. major infections by modulating the effector immune response. They control protective Th1 responses allowing for parasite survival and maintenance of memory responses [52]. Even though a precise role of DC for the generation of memory T cells as well as Treg against Leishmania is not known yet, it can be assumed that infected or antigen-loaded DC modulate these resulting T cell-dependent immune functions as well.

Which DC subtype is responsible for the induction of effective anti-parasite immunity?

Initial studies have critically involved epidermal LC [21, 54] as the primary cells to transport L. major antigen to the lymph nodes and initiate efficient T cell priming in vivo. In mice with MHC class II expression restricted to LC, these cells were the key initiators of primary immune responses after infection [55]. In addition, in L. major-infected CCR2^–/– mice, impaired migration of LC induced a non-healing phenotype in these otherwise genetically resistant mice [19]. But it is unclear how epidermal LC would gain access to a parasite that is inoculated into the upper dermis and later on primarily resides in dermal MΦ. Some studies suggested that proinflammatory cytokines and chemokines released by e.g. infected MΦ (such as IL-1, IL-6) may mobilize LC to migrate from the epidermis to the site of inflammation allowing for the uptake of viable L. major amastigotes [34, 56].

Recent studies have shown that cells other than LC, e.g. dermal DC [57, 58] or lymph node resident DC [59], transport antigen to the draining lymph nodes and initiate protective immunity. Most of these studies, however, used high doses of L. major inocula for infection (5 × 10⁵ – 2 × 10⁷ stationary phase promastigotes) [19, 55, 57-60]. In addition, some authors determined infection of DC subtypes by detecting live or whole parasites inside of DC [57, 60, 61], whereas others concentrated on the DC’s capacity to present L. major antigen to T cells [19, 55, 58, 59] independent of how and where they acquired the antigen. In the infection models using un-physiologically high doses of parasites, the preparations consist of live, but also dead parasites as well as soluble antigen that may gain access to DC at various sites distant from the inoculation site (skin) via draining through the conduit network [59]. Myeloid DC, skin immigrants residing in T cell areas of lymph nodes, take up this antigen and induce efficient priming of naïve T cells within a few hours after antigen inoculation [62, 63]. In addition, accumulation of antigen-bearing skin-derived DC in lymph nodes was found to peak ~24 hours post inoculation [62]. The question remains as to which of the described pathways is more relevant (and beneficial?) for immunity against infectious organisms. Effector memory T cells preferentially home to the skin only if antigen-presentation occurred via the skin route [64]. Using infections with physiologically low doses of parasites (1,000 metacyclic promastigotes of L. major mimicking natural transmission), Baldwin and co-workers found the highest parasite burden 3 weeks post infection in dermal DC and in LC that have migrated to lymph nodes, but additionally reported that all other CD11c⁺ DC subsets in lymphoid tissue also contained viable parasites in vivo [61].

Despite the interesting controversy about the nature of the DC subset, all authors independently found that CD11c⁺ DC are the primary cells responsible for the initiation of protective immunity in vivo. Whereas Gr-1⁺ neutrophils and CD11b⁺ MΦ were the predominant cell population infected in the skin, CD11c⁺ DC represented the most frequently infected cells in draining lymph nodes [60].

Role of B cells for protection

Reasons for impaired immunity against L. major

In murine experimental cutaneous leishmaniasis, genetically determined disease outcome is an important aspect and has been studied extensively [2]. Several factors contribute to genetic disposition. Susceptibility is a multigenetic phenomenon and T cell dependent mechanisms are very important (for reviews compare [2, 4]). These include a relative lack of IL-12 release, IL-4-mediated aberrant downregulation of the IL-12Rβ2 chain on Th2 cells and more. But, in addition, genetically determined factors on the level of neutrophils, MΦ, B cells and DC are also present, which are relevant for disease outcome (compare table 1( Table 1 )).

These cells of the innate immune system have been the focus of the present review. But how do they contribute to disease outcome? Increased numbers of neutrophils were found in lesions of susceptible as compared to genetically resistant animals [72, 73] and persisting tissue infiltration with neutrophils plays an important role in disease susceptibility [16, 74]. What regulates both recruitment and delayed/absent neutrophil clearance is unclear to date, but depletion of neutrophils from BALB/c mice improved disease outcome, reduced parasite loads and suppressed Th2 development [16, 74]. In contrast to C57BL/6 MΦ, in which neutrophils induce parasite killing, interaction of infected BALB/c MΦ with dying neutrophils led to uncontrolled parasite replication via TGF-β and PGE₂[16].

Several weeks post infection, a significantly higher percentage of MRP14-positive MΦ was detected in BALB/c lesions as compared to lesional infiltrates in resistant C57BL/6 mice [75] and these less mature MΦ were impaired in their ability to kill L. major parasites [76]. In contrast to DC, MΦ do not release IL-12 after infection with L. major. However, otherwise activated MΦ from BALB/c mice showed significantly reduced production of IL-12 [8, 77]. Thus, during later stages post infection when IL-12 may be produced by IFNγ-activated MΦ as well as DC, impaired IL-12 production from BALB/c MΦ may contribute to disease outcome.

Since infected DC are the critical cells for the induction of protective immunity, we and others have studied the biological behaviour of DC from BALB/c mice in great detail [32, 33]. The phagocytic capacity of BALB/c and C57BL/6 DC was similar, as was their activation status, IL-12 production and immigration into skin of L. major-infected mice [23, 33]. Interestingly, however, C57BL/6 DC produced more IL-1α/β than BALB/c DC [42, 43]. IL-1α/β facilitates Th1 priming and protective immunity [41-43, 78-80]. Additional DC-derived factors, not fully characterized yet, might also contribute to genetic determination of disease outcome.

Polyclonal activation of human B cells leads to production of parasite-specific and nonspecific IgM and IgG [81]. IgG-mediated effects differ significantly, depending on the genetic background of the mice [18]. As described above, in mice on a genetically resistant background, B cells and B cell-derived IgG are beneficial for the host response. BALB/c mice, in contrast, are characterized by excessive production of IgG during later phases of infection, probably due to their strong Th2 cytokine profile. In addition, the presence of functional B cells or administration of IgG worsened disease outcome in BALB/c mice [82-84] via induction of IL-10 release from infected MΦ [50].

In summary, mast cells, skin phagocytes (MΦ and neutrophils) as well as DC all critically contribute to the development of a protective immunity against intracellular pathogens such as Leishmania major. Vaccines that utilized e.g. infected DC or DC loaded with antigen have already proven to be effective against leishmaniasis [85]. Thus, modulation of the function of cells of the innate immune system may represent new therapeutic approaches to achieve long-lasting protection.
Table 1 Genetic differences in phagocyte behaviour in cutaneous leishmaniasis

Acknowledgements

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