Recognition of Leishmania Parasites by Innate Immunity

Immun., Endoc. & Metab. Agents in Med. Chem., 2009, 9, 000-000

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Recognition of Leishmania Parasites by Innate Immunity Ricardo Silvestre1,2, Nuno Santarém1,2, Joana Tavares1,2, Ana Marta Silva1,2 and Anabela Cordeiro-da-Silva1,2,* 1

Parasite Disease Group, IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal

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Departamento de Bioquímica, Faculdade de Farmácia, Universidade do Porto, Portugal Abstract: The host innate immune system represents the first line of defense against invasive pathogens. During the crucial early stages of infection, the host innate immune system must be able to rapidly detect and respond to foreign pathogens, enabling an efficient and successful adaptative immune response. Leishmania parasites are obligate intracellular eukaryotic pathogens living inside cells of the mononuclear phagocytic system. Recent data has proven distinct roles of various phagocytic cells, such as neutrophils, macrophages and dendritic cells during Leishmania infection. There is growing evidence that Leishmania modifies antigen presentation, apoptosis and immunoregulatory functions on these cells, leading to persistent and chronic infection. At the molecular level, the Toll-like receptors (TLR) family is a major player in the early host-pathogen interaction. The TLRs expressed intracellularly or at the surface of the cells involved in the innate immune response recognize conserved structures on foreign pathogens, such as Leishmania, playing a pivotal role in triggering innate and adaptative immune responses. Nonetheless, the same TLRs can be considered as a potential strategic target used by these organisms for their own advantage. In this review, we discussed the findings on the cellular processes involved in the innate host defense against intracellular pathogens, focusing on the Leishmania infection, from the initial host-parasite interactions involved in the parasite recognition to the mechanisms employed to eliminate the pathogen, presenting new data on the role of TLR2 in visceral leishmaniasis.

Key Words: Leishmania, innate immunity, Toll-like receptors, TLR2, antigen presenting cells, host-parasite interaction. 1. INTRODUCTION Leishmaniasis is a vector-transmitted zoonotic disease distributed throughout the world’s tropical and subtropical regions. Approximately 20 out of 30 species belonging to the Leishmania genus are pathogenic to humans. The clinical manifestations of leishmaniasis range from mild cutaneous to fatal visceral pathologies. The disease outcome will depend not only on the infectious Leishmania species but also on the immunological status of the host. Leishmania spp. are obligate heteroxenous protozoan parasites with a dimorphic life cycle. Initially, the extracellular promastigote form multiplies and develops within the alimentary tract of the sand fly vector. During a blood meal, a female sand fly transmits to the host between 100 and 1000 metacyclic promastigotes that are deposited into the dermis [1]. It is generally accepted that the successful establishment of a promastigote infection requires silent entry into specific host cells, where the parasite differentiates into its obligate and highly resistant intracellular amastigote form that will thrive within the phagolysosomal vacuole of the host mononuclear phagocytes [2]. Leishmania infection is an excellent example of complex host-parasite interactions. Since amastigotes reside in an

*Address correspondence to this author at the Parasite Disease Group, Biology of Infection and Immunology, Instituto de Biologia Molecular e Celular da Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal; Tel: +351 226 074 900; Fax: +351 226 099 157; E-mail: [email protected]

1871-5222/09 $55.00+.00

extremely hostile environment, Leishmania spp. have developed a remarkable range of sophisticated adaptive mechanisms, which not only allow to evade and inhibit normal host immune cells functions, but also subvert innate and acquired (both cellular and humoral) immune responses into their own advantage. In general, it has been assumed that it is the cell mediated immunity (CMI) that participates in the defense against leishmaniasis. Initial studies in experimental murine and natural human leishmaniasis have underscored the central role of T-lymphocytes in the resistance to infection. Indeed, T cell deficient mice are highly susceptible and succumb rapidly after a Leishmania infection [3], yet they become resistant after the adoptive transfer of T cells [4]. The rapid and fatal development of visceral leishmaniasis (VL) in HIV patients, especially in those with low CD4+-T cells numbers, clearly illustrate the role of these cells and the CMI in the control of leishmaniasis [5]. Despite some major advances in the past years, reliable correlates of immunity for all forms of leishmaniasis are yet to be defined, as they seem to vary among the infection model used. Nonetheless, the general consensus for an effective response towards all forms of leishmaniasis is the preferential development of Th1-mediated immune response. Moreover, the quality of the Th1 cell cytokine response is a crucial determinant. Darrah and colleagues, using a multiparameter flow cytometry analysis of CD4+ T cells, have shown that the degree of protection against L. major infection in mice can be predicted by the frequency of CD4+ T cells that produce simultaneous IFN-, IL-2 and TNF- [6]. Nevertheless, different reports in mice [7] and

© 2009 Bentham Science Publishers Ltd.

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humans [8] conducted with Leishmania species causing human VL have shown that the outcome of the infection does not depend only on the differential production of Th1 and Th2 derived cytokines. Indeed, this clear Th1/Th2 dichotomy is lacking in VL and also fails to explain some nonhealing forms of cutaneous leishmaniasis in mice caused by L. mexicana, L. amazonensis and even some strains of L. major [9]. Albeit it seems clear that in the mice model of visceral disease, despite the presence of cytokines produced by the two Th subsets, exists an overshadowing of the Th2cytokines by a Th1 cell-associated response leading to the control of the infection [10]. Experimental data have demonstrated that the success of Leishmania to persist in the host depends on the outcome of the crucial early stages of infection. Here, the host innate immune system plays a prominent role, since it is the first line of defense against invasive pathogens. It must rapidly detect and orchestrate the development of an acquired immune response, which is strictly necessary to resistance. This review present new data and discuss the cellular and molecular processes involved in the innate host defense against Leishmania, an intracellular protozoan parasite. 2. INITIAL INTERACTIONS OF LEISHMANIA WITH HOST MOLECULES AND INNATE IMMUNE CELLS The inoculation of Leishmania parasites in a mammalian host triggers a serie of immunological events that will determine the outcome of the immune response. It will be during these initial steps that the parasite’s ability to evade the immune response will be challenged by the innate immune response. It should be stated that sand fly – mammalian host interactions can also play a relevant role in the outcome of the infection [11]. After Leishmania entry in a mammalian host, serum complement components are the first challenge that extracellular promastigotes should face until its complete engulfment by phagocytic cells [12]. Leishmania promastigotes are covered by a dense surface glycocalix, composed largely of molecules attached by a glycosylphosphatidylinositol (GPI) anchor. Among those, the 63-kDa surface metalloproteinase (gp63 or leishmanolysin) and lipophosphoglycans (LPG) constitute the dominant promastigote “defense” on their surface, as demonstrated by the high complement sensitivity of Leishmania parasites deficient for these molecules [13]. The LPG molecule, which is significantly longer in the metacyclic when compared to the non-infective promastigotes, prevent the attachment of the complement C5b-C9-complex in the parasite surface [14, 15]. While around 10-fold less abundant than LPG, gp63 is also involved in the resistance to complement mediated lysis. This protein, which is also upregulated in metacyclic promastigotes, inactivates C3b by converting it into a C3bi-like molecule. The role of these surface molecules extends well beyond the resistance against host serum since they also participate in the parasite “silent entry”, i.e. without activating and possibly deactivating specific antimicrobial effector mechanisms. In an in vivo system, the complement factors C3b and C3bi, will opsonize metacyclic promastigotes, enabling the binding to complement receptor 1 (CR1) and CR3, respectively. The binding to CR3 seems to provide the predominant mean by which the

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promastigote parasites accesses and enters host macrophage, while CR1 interaction is considered to be only transient [16]. The uptake through CR3 is considered an important mechanism contributing to the failure of infected macrophages to produce IL-12 [17]. Furthermore, the C3bi-like molecule resulting from the gp63 activity on C3b can also function as an opsonin facilitating parasite uptake [18]. Furthermore, due to its fibronectin-like properties, gp63 interacts with the fibronectin receptor present on the host cell surface maximizing parasite adhesion and internalization [19]. LPG is another parasite surface molecule that seems to have a broader role in the promastigotes attachment. The promastigotes uptake mediated by LPG involves either the binding to the mannose-fucose receptor [20, 21], interaction with the early inflammatory C-reactive protein (CRP), which triggers phagocytosis by the CRP receptor [22] or direct interaction with CR4 [23]. Recently, it was hypothesized that Leishmania takes advantage of the C5 cleavage fragment, C5a, a strong chemoattractant anaphylatoxin, as an evasion mechanism, since C5a has a negative impact on the toll-like receptor (TLR) 4-induced synthesis of IL-12 family of cytokines. Indeed, C5a receptor knockout mice were more resistant to L. major infection as compared with wild-type BALB/c mice, with lower number of parasites and increased numbers of IFN- producing CD4+ and CD8+ T cells [24]. The amastigotes internalization must be explained differently as they have a specie-dependent strong downregulation or even absence of surface glycoconjugates as LPG or gp63. Internalization via antibody is probably the preferred mechanism. Indeed, the amastigotes released from infected macrophages in vivo, are very likely to be coated with specific antibodies, which promote internalization by binding the Fc piece of IgGs to surface macrophage Fc receptors [25]. Recently, Van Zandbergen and colleagues have proposed a model of "silent entry", involving the uptake of apoptotic cells, to explain the early events of Leishmania infection [26]. As polymorphonuclear neutrophil granulocytes (PMNs) are the first leukocytes that encounter the parasites after infection, this model proposes that PMNs can phagocyte Leishmania parasites in an opsonin-independent manner hampering thus the cell activation and consequently increasing the parasite survival [27]. Moreover, it prolongs the PMN life-span, which is a general evasion mechanism used by pathogens to survive inside PMN. Further investigations revealed that apoptotic parasites were essential in the virulent inoculum of Leishmania promastigotes for the successful establishment of infection [28]. Indeed, the silencing effect induced by the uptake of apoptotic parasites allows the survival of the viable non-apoptotic population inside PMN. The infected PMN become apoptotic approximately two days after infection and are subsequently ingested by macrophages in the vicinity, ensuring the silencing of the macrophage effector functions. Consequently, the parasites not only survive but also multiply inside macrophages, which suggested that PMNs can be misused by Leishmania as a “Trojan horse” enabling the unchecked establishment of a macrophage infection [26, 29]. After the internalization of the parasite most of the interactions will occur at the parasitophorous vacuole (PV) or

Recognition of Leishmania Parasites by Innate Immunity

phagolysosome (results from the fusion of phagosomes with late endosomes/lysosomes), although it was also suggested a possible free cytoplasmic localization [30]. In contrast with other intracellular pathogens, such as Toxoplasma gondii or Mycobacterium spp. that prevent fusion of the lysosomes, or T. cruzi and Listeria spp. that escape from the PV, Leishmania amastigotes have adapted their metabolism to the acidic pH and hydrolytic active environment of PV. Nonetheless, Leishmania promastigotes are vulnerable to degradation in those conditions. Therefore, Desjardins and Descoteaux have proposed a model, using the cutaneous L. major and visceral L. donovani species, where promastigotes transiently retard the phagolysosomal fusion in a LPG-dependent manner, allowing the parasite to gain time in order to complete its differentiation into the more resistant amastigote form in milder conditions [31]. However, recent data have shown that although this process actually occurs with the majority of Leishmania species, it does not play a prominent role in the parasite survival [32]. Interestingly, despite all PV present similar features in general (acidic pH, hydrolases), significant differences were accounted between Leishmania species. Thus, while Old World species such as L. major and L. donovani are inside several but small PV containing one or few amastigotes, New World L. mexicana and L. braziliensis reside in a large PV containing many amastigotes. Moreover, these species do not inhibit the phagolysosome formation inside macrophages. To explain this behavior, it was suggested that the large vacuolar size might dilute the hydrolytic enzymes to a level below their effectiveness, which was considered as an alternative intrinsic mechanism in favor of the promastigote differentiation [33]. Despite several evidences point to the presence of parasites in other cells types [fibroblasts and dendritic cells (DC)], little is known about the mechanism and molecules involved in the parasite invasion and survival in these cells. Although in vitro experiments have shown that Leishmania parasites can be internalized by DCs through Fc receptors I and III [34], there is still a considerable debate of whether DC can phagocyte both parasite forms in vivo. It is strongly believed that Leishmania parasites use DCs for immunological evasion purposes. It seems that a parasite strategy may pass for inhibiting DCs maturation, allowing their amplification and the establishment of infection in an immune privileged site [35]. Recently, the DC-specific intracellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) was identified as a surface receptor exploited by several Leishmania species to gain entry into DC [36]. This receptor was reported as a mediator of DC-T cell interactions and as an escape mechanism of intracellular pathogens, which can target DC-SIGN to shift from a Th1 towards a Th2 cell response [37]. On the other hand, fibroblasts can harbor but do not support the growth of intracellular parasites. Instead, these cells, due to their inability to sustain effective inducible nitric oxide synthase (iNOS)-mediated killing, should provide a protective niche contributing to long-term survival of small numbers of parasites, which might reactivate the disease following a state of immunosuppression (e.g. HIV co-infection) [38, 39].

Immun., Endoc. & Metab. Agents in Med. Chem., 2009, Vol. 9, No. 2

3. EVASION PARASITES

STRATEGIES

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LEISHMANIA

3.1. Microbicidal Molecules Neutrophils and macrophages are the major effector cells of the innate immune response. The outcome of a Leishmania infection will depend tightly on the balance between the host ability to activate the killing machinery present in these cells and the parasite capacity to evade it. These cells synthesize two species of highly reactive molecules with recognized leishmanicidal capacity such as nitric oxide (NO), by the iNOS and reactive oxygen intermediates (ROI), by the phagocyte NADPH oxidase system (phox) [40]. Neutrophils are detected in the local of infection as early as 40 minutes after exposure to sand flies [29] being the first cells to encounter the parasite. Recognition of apoptotic parasites with phosphatidylserine (PS) in the outer leaflet of their plasma membrane drives the neutrophils to internalize these parasites without resulting in the release of an oxidative burst [27]. Wanderley and colleagues postulated that the interaction between PS-positive promastigote and neutrophils leads to the secretion of TGF-, which in an autocrine and paracrine manner will shift the L-arginine metabolism towards the production of L-ornithine [41]. Therefore, the preferential activation of arginase over iNOS is a favorable mechanism to the parasite since it will decrease NO secretion and induce the polyamine synthesis. In a similar manner, the uptake of PS-positive parasites was shown to increase intracellular Leishmania growth in macrophages through the inhibition of NO production [42]. NO is believed to be the most significant microbicidal molecule during the Leishmania infection, since mice lacking iNOS are unable to control the infection, and macrophages derived from those mice cannot control promastigote growth in vitro [43]. Nevertheless, the production of ROI is also detrimental to parasite survival, since phoxdeficient mice developed an early enhanced susceptibility to both L. donovani and L. major, yet are capable to control the infection [44, 45]. The surface and secreted metacyclic promastigote molecules play a crucial role in protecting the parasite against host microbicidal molecules. Hence, surface LPG and gp63 inhibit ROI generation [46], in a mechanism involving the inhibition of protein kinase C (PKC) in macrophages [47]. In addition, LPG was described to scavenge oxygen radicals generated during the oxidative burst [48]. Another Leishmania surface glycolipid, the glycosylinositolphospholipid (GIPL) and the phosphoglycan moiety of LPG can suppress macrophage iNOS expression and NO production even in response to IFN- and/or lipopolysaccharide (LPS) stimulus [49]. Other molecules have been described as part of the parasite machinery to detoxify ROI and NO species. Among those, a L. donovani superoxide dismutase detoxifies ROI produced by activated macrophages [50], two peroxidoxins identified in L. chagasi (LcPxn1 and LcPxn2) were shown to be capable of protecting the parasites against both reactive species [51], and two distinct L. infantum peroxiredoxins, one cytosolic (LicTXNPx) and one mitochondrial (LimTXNPx) perform complementary roles in parasite protection against the oxidative stress [52].


The parasite proliferation within a phagolysosome entails its ability to resist and inhibit host hydrolytic enzymes. It was argued that the highly anionic nature of LPG along with its unique galactose-mannose repetitions might protect the parasite against this proteolytic attack [47]. Moreover, the proteolytic activity of gp63 is optimal at acidic pH, and has been shown to protect the parasites from intraphagolysosomal cytolysis mediated by host lysosomal enzymes [53]. On the other hand, LPG is almost absent and gp63 is substantially downregulated in the amastigote form [54]. Although it was argued that in the absence of LPG, gp63 is no longer masked and may therefore play its protective role [55], other phosphoglycan-containing molecules were described to be involved in the amastigote resistance. Among those, it is noteworthy to mention proteophosphoglycans (surface and secreted) and acid phosphatases that seem to form a transient barrier against acid hydrolases due to their negative charge [47]. 3.2. Cytokine Secretion Successful infection by Leishmania implies the modulation of the secretion of different factors, preventing the activation of an effective and specific immune response. When internalized, Leishmania play a dual role in the regulation of host secreted cytokines. It will induce the secretion of suppressive cytokines and chemotactic proteins (IL-10 / TGF- / prostaglandin E2 (PGE2) and macrophage inflammatory protein (MIP)-1 / 1), but will also inhibit several factors involved in the inflammatory response or activation of T-lymphocytes (IL-1 / TNF- / IL-12) [56]. The induction of an effective immune response established in an early phase of infection, is believed to be critically dependent on the production of IL-12 [57]. This cytokine is essential for NK-mediated cytotoxicity, T-lymphocyte activation and subsequent IFN- secretion, leading to macrophage activation and parasite elimination. Several reports demonstrated that Leishmania impairs both macrophages IL-12 production and the efficient presentation by the latter of foreign antigens to naïve T-cells [58-60]. The parasite phagocytosis through complement and Fc receptors lead to the inhibition of IL-12 secretion [61, 62]. Remarkably, it was elegantly showed using a single-cell flow cytometry analysis that IL-12 secretion is shut down exactly in the cells infected by Leishmania parasites [60]. Nevertheless, albeit their immunomodulatory properties, it is not very likely that infected macrophages are the major candidates to prime naïve T cells to mount an effective immune response. Indeed, the decrease capacity of infected macrophages to secrete IL-12 creates an immunological privileged environment for establishment and propagation of the infection, nonetheless this infected macrophages must be understood as a transient stage of infection since infected macrophages will eventually be primed for killing. Although Leishmaniainfected neutrophils are believed to constitute one of the earliest sources of IL-12 in resistant C57BL/6 mice [63], it is currently believed that DCs are the main cell type responsible to coordinate the acquired immunity during leishmaniasis. Extensive in vitro experiments with L. major promastigotes have clearly demonstrated that myeloid DC (mDC) can phagocyte the antibody-coated parasites, and in clear

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contrast to macrophages, can transiently acquire a mature phenotype. This will ultimately leads to upregulation of major histocompatibility complex I (MHC I) and II, the expression of co-stimulatory molecules and the release of a variety of proinflammatory cytokines including IL-12, which allows the activation of naïve T cells [64, 65]. Also, plasmocytoid DC (pDC), although unable to phagocyte Leishmania promastigotes, release type I IFN after contact with Leishmania genomic DNA in a TLR 9 dependent mechanism [66]. Nevertheless, recent evidences points to a more important role of mDC and IL-12 than pDC and IFN in the coordination of an acquired immune response [67]. Promastigotes of other Leishmania species, such as L. mexicana, L. donovani, L. amazonensis and amastigotes are unable to induce similar effects and are considered to poorly prime naïve T cells [68-70]. Therefore, while a consensus exists about the key role of DC in the evolution of susceptibility or resistance to leishmaniasis, several results raised controversy about the magnitude and profile of DC activation, which could be explained by the type of DCs [(Langerhans cells (LC), bone marrow derived DCs, monocytederived DCs, spleen DCs)] the species and developmental stage of the parasite used (New vs. Old World species, cultured-derived promastigotes or amastigotes, lesionderived amastigotes) [70]. As described above, recent evidences have clearly point a critical role for the uptake of parasite apoptotic cells on the development of Leishmania disease [28]. Early studies showed compelling evidence that the uptake of apoptotic cells decrease the production of proinflammatory mediators [71, 72]. Indeed, IL-12 is transcriptional shutdown in macrophages and dendritic cells following apoptotic cell uptake or treatment with PS that led to a diminished capacity to stimulate T cells [73, 74]. However, a different profile was observed with the anti-inflammatory cytokines TGF- and IL-10, which were found increased [75]. Moreover, the apoptotic cell uptake through PS leads to Rho GTPases activation [76], which in turn markedly reduce phagocytosis [77]. In that sense, among the host immunosuppressive molecules induced after a Leishmania infection, a major role is played by the anti-inflammatory cytokines TGF- and IL10. Leishmania parasites exploit the PS at their surface to enter host cells (especially by using the PS receptor), which will contribute to their survival during the crucial initial events of infection [41, 42]. The recognition of PS at the amastigotes surface led to increased macrophage secretion of TGF- and IL-10, which will create a immunosupressive microenvironment resulting in an accelerated resolution of inflammation favourable to parasite multiplication. Indeed, the pretreatment with the high affinity PS-binding protein, annexin V, not only inhibited the secretion of these antiinflammatory cytokines but also resulted in a 50% decreased of infection [42]. Other major sources of TGF- and IL-10 include the infected neutrophil population. Just a few hours after Leishmania inoculation, the infected neutrophils were found to down-regulate TNF- production and secrete large amounts of MIP-1, TGF- and IL-10, which will act in autocrine and paracrine manners recruiting macrophages to the site of infection [78, 79]. In addition, TGF- and IL-10 were also described to be induced through the binding


of the opsonized parasite to the macrophage FcR [80]. Both cytokines were shown to be an important parasite escape mechanisms during the infection in studies that targeted their depletion in vivo. Genetic deletions and the application of specific mononuclear antibody strategies against IL-10 or TGF- [81-83], respectively, gave origin to a drastic reduction in the parasite burden. On the other side, transgenic mice that constitutively produce IL-10 [16] or mice administrated with recombinant adenovirus expressing TGF- [84] had large parasite burdens and were unable to control their proliferation. The parasite, due to the action of these immunosuppressive cytokines, can maintain a long-term infection within the mammalian host without killing him. IL-10 as a major suppressive cytokine and a key player in Leishmania susceptibility as been studied over the years and several major sources of IL-10 have been identified. Among those, regulatory T cells (Treg) were described as playing a highly relevant role for parasite maintenance in the site of infection [85]. These cells create an immune privileged area for sustained infection, not allowing the complete parasite eradication. Interestingly, it is believed that this sustained presence of the parasite will mediate a mutually beneficial relationship between the host and the parasite. Thus, this relation was formally described to allow transmission of L. major to the sand flies [86] and to ensure a powerful and long-term immunity against re-infection, since mice that accomplished complete elimination of the parasite evidenced reduced level of acquired resistance [85]. Nevertheless, recent studies in cutaneous or visceral disease demonstrated that Foxp3 CD25-CD4 + Th1 cells rather that Tregs are the main source of IL-10–mediated immune suppression during chronic leishmaniasis [87, 88]. This Th1 self-regulation restrains the collateral damage caused by exaggerated inflammation, but nevertheless limits the effectiveness of the cellular response resulting in a persistent infection. 3.3. Antigen Presentation and T Cell Stimulation Although a protective role against leishmaniasis had been shown for MHC I antigen presentation, through induction of effector CD8+ T cells [89], only the efficient MHC II presentation is essential for complete parasite clearance [90]. For several years now, it is known that Leishmania infection inhibits expression of both MHC I and II in their target cells [91]. Subsequently, several Leishmania induced mechanisms were already demonstrated or proposed to explain Leishmania evasion or interference with MHC II-restricted antigens in infected macrophages including: i) processing of parasite antigens (low release of immunogenic molecules, shedding of peptide epitopes by parasite surface GIPLs); ii) formation of MHC II-antigen complex (internalization and degradation of MHC II molecules, prevention of MHC II binding to exogenous peptide); iii) physical interference with MCH II intracellular trafficking to the cell surface; iv) alteration of MHC II-antigens distribution in the macrophage surface (disruption of surface lipid rafts where MHC II are present) [35]. Moreover, the level of inhibition is directly correlated with the parasite adaptation to the host, being the procyclic promastigotes the less effective and amastigotes the most capable to induce the highest inhibition [92].


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Several experiments have demonstrated an essential role for appropriate CD40 co-stimulation in the resistance mechanisms against Leishmania. Deficiency on CD40 or CD40L or inhibition of the CD40-CD40L interaction resulted in increased susceptibility towards Leishmania infections, with decreased IFN- and IL-12 secretion [93, 94]. Hence, the administration of anti-CD40 antibody was shown to increase IL-12 and IFN-, inducing a protective response against the cutaneous L. major infection [95]. A similar observation was done in VL, where the administration of an agonist anti-CD40 monoclonal antibody induced the elimination of the parasites within liver macrophages. This was accompanied by high levels of IFN- and tissue granuloma formation acting synergistically with antimony therapy in the treatment of L. donovani infected mice [96]. Therefore, in a similar manner to other protozoans, Leishmania interference with host CD40 presentation or signaling is one of the powerful strategies to avoid a protective Th1 immune response [97]. The activation of T cells also requires co-stimulation of B7-1 (CD80)/B7-2 (CD86) on APC with CD28 on naïve T cells. Nevertheless, the role of B7 co-stimulation is still controversial. Upon infection with Leishmania, a decreased expression of co-stimulatory molecules of the B7 family in macrophages is observed, whereas the expression of adhesion molecules (ICAM-1, VCAM-1) is only marginally affected. This down-regulatory mechanism has been associated with the characteristic immunosupression during leishmaniasis [98, 99]. Similar observations were made with DCs. Leishmania infection results in the downregulation of surface CD80, CD86 and CD1a (host molecule involved in the presentation of microorganism lipids and glycol [100, 101], leading to a dysfunctional priming of naïve CD4+ T cells. Altogether, these studies suggest that the inhibitory effects exerted on the B7-CD28 interconnection contribute to parasite persistence. In the spleen of L. donovani-chronic infected mice, DCs fail to migrate from the marginal zones to the T cell areas [102]. This was explained by a defective expression of the chemokine receptor CCR7 after parasite or antigen internalization. However, the defective CCR7 expression in the chronic stage of L. donovani infection has also been attributed to high levels of TNF- and IL-10 [102]. Nevertheless, the destruction of stromal cells in the spleen marginal zone plays also a role in the defective migration as it leads to a loss of recruitment of the CCR7-expressing naive T cells and mature DCs [103]. Indeed, the impairment of DCs motility or destruction of spleen architecture with concomitant loss of DC-recruiting chemokines are two alternative strategies to prevent DC-T cell communication. In Table 1 is summarized several parasite mechanisms to evade innate and adaptive immune responses. 3.4. Modulation of Host Cell Apoptosis It was already shown that Leishmania programmed cell death is a mechanism employed to achieve a successful host invasion. Nevertheless, Leishmania, as several others intracellular protozoan parasites, was shown to inhibit


the apoptotic response of its host cell, in a process that renders the host more susceptible to several critical steps of the intracellular infection. Indeed, Leishmania-infected host cells seem to have an increased threshold to apoptosis induced by FasL, TNF-, IFN-, and other apoptotic inducing-molecules [104]. Nevertheless, the interplay of Leishmania with host cells involved in the innate immune response often results in the secretion of apoptosis-inducing molecules, which can account for the pathogenesis [104]. In fact, increased rates of T lymphocyte apoptosis have been detected in the peripheral blood of VL patients [105] as well in the lymph nodes, spleen and liver of experimental murine VL models [106, 107]. This suggests that chronic infection with Leishmania might persistently alter lymphocyte physiology through an unknown mechanism(s) to date. Patients with VL were observed to have elevated plasma levels of soluble TNF-, Fas and FasL and an up-regulation of membrane bound Fas on Leishmania-infected spleen cells, which return to normal levels after successful treatment [108]. The cell death-inducing molecule Fas has been implicated in the early resistance to both cutaneous and visceral Leishmania strains in experimental mouse models, since mice bearing a mutation in Fas or FasL were more susceptible to infection despite elevated Th1 cytokine production [96, 99, 100]. Hence, Fas/FasL system may act in synergy with IFN- and NO appearing to be essential in the control of parasite replication, enhancing both the microbicidal function and the apoptosis of infected macrophages [109]. Nevertheless, it was demonstrated not to contribute to the T-cell death [106]. In opposition, TNF-, essentially in the transmembranar form, was suggested as a key mediator of T cell apoptosis within inflammatory lesions after parasite elimination [110, 111]. Overall, although the mechanism of T-cells depletion during leishmaniasis remains elusive, recent experimental data suggested that the pro-apoptotic protein Bim plays a deleterious role during chronic leishmaniasis, since it limits protective immunity by limiting the number of Leishmaniaspecific memory T cells [112]. There is still lack of information regarding the mechanisms employed by Leishmania to inhibit host cell apoptotic death. A few reports have shown that the internalization of Leishmania confers macrophages the capacity to resist apoptosis induced by diverse stimuli [113-116]. Moore and Matlashewski were the first to show that Leishmania inhibits the macrophage-apoptosis induced by the deprivation of macrophage colony stimulating factor (M-CSF) [113]. Later, Akarid and colleagues demonstrated that the enhancement of the macrophage survival was an intrinsic mechanism to Leishmania spp. infections through the control of the mitochondrial cytochrome C release and caspase 3 activation, independently of the genetic background of the host [115]. Interestingly, although Leishmania promastigotes induce the activation of several pathways, such as NF-B, the p38 mitogen-activated protein kinases (MAPK) and PI3K/Akt, only the latter was found to be involved in the anti-apoptotic phenotype [117]. The activation of the PI3K/Akt pathway leads to the phosphorylation of Bad, which results in its sequestration in the mitochondria and sequential blockage of cytochrome C release [117].

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Overall, these results suggest that the modulation of host cells survival may provide a mean to favor the invasion and persistence of these intracellular parasites contributing to the pathogenesis of leishmaniasis. Table 1.

Overview of Some Evasion Strategies Used by Leishmania Parasites

Macrophages Complement lysis evasion gp63 converts C3b in C3bi Protein kinases phosphorilate complement components LPG induces C5b-C9 shedding Modulation of anti-Leishmania products LPG scavenge ROI gp63 inhibits phagolysosomal enzymes Amastigotes have enhanced resistance to phagolysosomal H2O2, acidic pH, NO, lysosomal enzymes Leishmania phagocytosis inhibits ROI and NO burst Cytokine modulation Leishmania phagocytosis induces anti-inflammatory cytokines (TGF-, IL-10) Leishmania phagocytosis inhibits proinflammatory cytokines (IL-1 and , IL-12, TNF-) Inhibition of APC-T-cell interaction Leishmania phagocytosis downregulates MHC II and B7 molecules Leishmania phagocytosis inhibits MHC II-peptide loading Dendritic cells Inhibition of DC maturation At early stages of infection by non-opsonized or C3-coating Leishmania internalization Shift Th1 towards Th2 DC-SIGN-mediated Leishmania phagocytosis Inhibition of DC migration Defective chemokines expression Impaired antigen presentation Internalization of MHC-II molecules and impaired IL-12 secretion Regulatory T cells (Treg) Induction of regulatory T cell populations Down regulation of effector immune responses Treg and CD4+CD25-FoxP3-Th1-mediated IL-10


3.5. Modulation of Host Cell Signaling Some of the disturbances described above are caused by direct interference of Leishmania parasites with host cells intracellular signaling pathways. Therefore, the binding of an external signal, that can arise from Leishmania secreted or surface proteins or even cytokines, to a host cell, namely macrophage and dendritic cells, surface receptor will result in modifications in the phosphorylation or conformational status of the receptor, which result ultimately in the activation/ deactivation of transcription factors that change the cell’s behavior. For several years that Leishmania is known to affect second messengers as the intracellular Ca2+ concentration of phagocytes and the activation of PKC. Both Leishmania and LPG were described to have an active role in increasing the intracellular Ca2+ of phagocytes, altering Ca2+-dependent responses as chemotaxis and production of ROI [118, 119]. Similarly, PKC activity has been shown to be reduced in Leishmania-infected macrophages favouring the parasite survival [120]. Although LPG is not the only mediator, since amastigotes also inhibit PKC activity, its direct interaction with PKC at its diacylglycerol binding site or by blocking the PKC insertion into the membrane were described as effector mechanisms. Several other examples of host cell inhibition are found in the literature. Since IFN- is essential for innate host cells activation, the JAnus Kinases (JAK)/ Signal Transducers and Activators of Transcription (STAT) signaling cascade (the principal downstream signaling pathway of IFN- receptor) was found to be defectively phosphorylated upon infection, which depends on the activation of phosphotyrosine phosphatase SHP-1 induced by a Leishmania infection [101, 121]. Moreover, mice deficient for SHP-1 or treated with peroxovanadium, a potent SHP-1 inhibitor, were more resistant to L. major after a footpad challenge, which was correlated with an effective NO production. Although these experiments suggested a SHP-1 mediated role in the progression of leishmaniasis [122, 123], a recent publication have questioned the real in vivo role for this tyrosine phophatase, since SHP-1 deficient mice inoculated into the rump with L. major did not showed any modifications of intracellular parasite survival or pathology [124]. Although the role of SHP-1 for other Leishmania species is still uknown, these data strongly suggests that the presence or activation of this tyrosine phophatase is not essential for intracellular parasite survival. Leishmania manipulates other signaling pathways in their own benefit. Among those, the mitogen activated protein kinase (MAPK) members [(extracellular-regulated kinase (ERK1/2), p38 kinase and Jun N-terminal kinase (JNK)], and the PI3K/Akt are the most described [125, 126]. Early studies have demonstrated that all these pathways were modulated after a Leishmania infection [127]. Although currently it is believed that the parasite control of these pathways depends on both host cell used and the parasite species, this strategy results in impaired macrophage and DC functions modulating the cytokines and chemokines secretion. The activation of PI3K/Akt lead to a dominant negative effect on IL-12 production [128]. Two signaling


7

pathways downstream of PI3K/AKT, the mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3 (GSK3) were demonstrated to differentially contribute to the expression of IL-12 in TLR activated DCs [129]. In that sense, mTOR inhibition using rapamycin enhanced IL12 secretion and the inhibition of GSK3 by LiCl supressed a protective Th1 response to L. major in vivo [129]. In addition, ERK1/2 is activated after infection, specially if Leishmania uptake is mediated through the binding of IgG coated parasites to surface Fc receptors. ERK1/2 activation led to the phophorylation of histone H3 at the IL10 promoter [125]. Thus, the secreted IL-10 will, in an autocrine and paracrine manner, downregulate an inflammatory response favouring the parasite survival. A new group of negative regulators were recently described, characterized as members of the suppressors of cytokine signaling (SOCS) family. It has been shown that SOCS members are a major negative feedback regulator of STATactivating cytokines [130]. Although one study has shown an increase of SOCS3 in macrophages following Leishmania infection [131], mice lacking SOCS3 in T cells showed reduced immune responses to L. major infection associated with increased secretion of anti-inflammatory cytokines (TGF- and IL-10) [132]. Interestingly, SOCS5 knockout mice displayed normal resistance to infection [133]. Overall, a high amount of data have been gathered in the recent years explaining how Leishmania modulates host cell signaling in its own advantage. Nevertheless, some conflicting data have aroused from those studies suggesting that each result must be contextualized in terms of host targeted cell (e.g. macrophages vs. dendritic cells; primary vs. cell lines) and Leishmania species/stage. We anticipate that a comparative study between natural or genetically attenuated Leishmania parasites versus its virulent wild-type (WT) counterparts in primary innate cells will give an uncountable contribute to understanding the pathogenic mechanisms controling the innate host cell machinery. 4. TOLL-LIKE RECEPTORS In mammalian cells, the TLR family is an important group of receptors through which innate recognition of invading pathogens occurs [134]. All the members of the Toll family are trans-membrane proteins containing an extracellular domain composed of leucine-rich repeats and a cytoplasmic domain homologous to the cytoplasmic region of IL-1 receptor, known as TIR domain, required for downstream signaling [135]. In mammals, the family of TLRs is expressed essentially in the cells involved in the innate immune response (dendritic cells, B cells, NK cells and macrophages) and is responsible for recognizing conserved motifs termed pathogen-associated molecular patterns (PAMPs). These motifs are present in invasive pathogens and in normal condition are not found in the host cells [136, 137]. To date, 11 human and 13 mouse TLRs have been identified [138], with the particularity of each one responding to distinct PAMPs, leading to the activation of specific signaling pathways [139]. Hence, TLR3 and TLR5 recognize double-stranded RNA and bacterial flagellin, respectively. TLR4 recognizes


lipopolysaccharide (LPS) from Gram-negative bacteria and heat-shock proteins, TLR7 and TLR8 sense single-stranded viral RNA and TLR9 responds to CpG motifs of bacterial DNA [139]. TLR2 recognizes a myriad of unrelated molecules, including lipopeptides [140], peptidoglycans [141], outer membrane proteins [142], a T. cruzi protein belonging to the thiol-disulfide oxidoreductase family [143] and porins from a broad spectrum of pathogens [144]. This diversity is achieved due to heterodimerization with TLR1 or TLR6 [145] and/or accessory molecules, such as CD14 [146] and CD36 [147]. Recognition of PAMPs by TLRs stimulates the recruitment of a set of cytoplasmic adaptor molecules, of which the myeloid differentiation factor 88 (MyD88) is the best studied example [148]. It is now well established that TLRs are important for defense against every known category of human microbial pathogen. Thus, one can state that TLR signaling pathway is involved in the initial recognition of Leishmania parasites by the innate immune system of the host, in the early resistance to infection by inducing IFN- cell mediated immunity and in the development of acquired immunity as well as immunopathology [104]. An initial study has demonstrated the ability of L. major parasites to activate the IL-1 promoter in macrophages via a MyD88-dependent pathway [149]. However, the most convincing data indicating the importance of TLR in leishmaniasis resistance are those obtained from infections with MyD88-deficient mice. Mice lacking the TLR adaptor protein, MyD88, have increased susceptibility to infection in comparison with wild-type C57BL/6 mice, corroborated with high levels of IL-4 and low levels of IFN- and IL-12 [150, 151]. TLR9 was shown to be essential to NK-mediated cytotoxicity and IFN- production in acute leishmaniasis, and mice deprived of this receptor develop more lesions. Nevertheless, it was proved to be dispensable for an effective T cell response during late phase of infections since TLR9 knockout mice ultimately resolve the infection [66, 67]. Although at less extent, TLR4-deficient mice had higher parasite burdens and were less efficient in the resolution of cutaneous lesions caused by L. major, suggesting a partial role for TLR4 in host defense against Leishmania [152]. However, this receptor was shown not to interfere with the chemokines expression in Leishmania infected macrophages [153], TLR4 signaling was demonstrated to be essential for the macrophage microbicidal activity induced by neutrophils or neutrophil elastase (a serine protease released by neutrophil azurophilic granules during inflammation) [154]. Using a RNA interference approach to eliminate the expression of TLRs, it was demonstrated that NO and TNF- secretions by infectedmacrophages are, at least in part, TLR2 and TLR3-dependent [155]. A growing interest has been given to the identification of Leishmania structures that interfere with TLRs. Hence, some researchers have demonstrated that LPG, but not other surface glycolipids, is a TLR2 agonist capable of activating mouse macrophages and human NK cells, in a MyD88dependent manner [150, 156]. Also, it was demonstrated that the heat-shock protein 70 (HSP 70), a known potent activator of the immune system, signals via MyD88 pathway [157] and the P8 proteoglycolipid expressed on the amastigote

Silvestre et al.

surface induce macrophage inflammatory responses through TLR4 [158]. Therefore, the evidences so far, point to a multiple TLRs orchestrated defense against Leishmania spp. (Table 2) and other protozoan pathogens. On the one side, the TLR-dependent proinflammatory cascade that is triggered after recognition of foreign Leishmania must be tightly controlled to avoid immunopathology and eventually death. On the other side, the parasite must interfere with signaling cascades of TLR in order to assure its persistence in the host. Underhill et al. have classified the mechanism of TLR evasion in three categories: (i) manipulation of the structure and/or expression of TLR ligands; (ii) interference with intracellular signaling components proximal to TLR and (iii) delivering signal through receptors that modifies TLR signaling [159]. Although in Leishmania, examples for the first category have not been yet shown, it is well established that Leishmania parasites target macrophage signaling pathways, such as the MAPK, the STAT1 or the protein tyrosine phosphatase SHP1 for downregulation of IL-12 secretion even in the presence of LPS or IFN-. In addition, it was recently shown that L. donovani can shift the TLR2 responses towards a Th2 response through a contact dependent mechanism [160]. Moreover, for successful infection, Leishmania must subvert TLR engagement, by interacting with other receptors, such as DC-SIGN in DCs. Thus, it seems evident that TLRs present beneficial effects against pathogenic infections. Therefore, TLR-based therapeutic or prophylactic strategies against Leishmania bring high expectations in the battle against this infectious disease. As example, different TLR agonists, such as imiquimod (TLR7 agonist) and resiquimod (TLR7/8 agonist), also known as R848, were tested as potential vaccine adjuvants. Subcutaneous vaccination with Leishmania crude extract in the presence of these adjuvants mediated a Th1 response against a L. major infection with the development of protective immunity [161]. In a similar manner, TLR9 activation due to the presence of stimulatory unmethylated CpG motifs given exogenously or as plasmid DNA form decreased the parasite burden during cutaneous and visceral leishmaniasis [162, 163]. 4.1. The Role of TLR2 During VL The majority of experimental studies have point out an essential role for the MyD88 signaling in the resistance against several intracellular infections [150, 151, 164-168]. Nevertheless, studies performed in mice deficient in a single TLR were unable to show similar increases in susceptibility as observed in MyD88-deficient mice. The role of TLR2 during pathogenic infections has been classically studied recurring to TLR2 knockout mice. Hence, a major protective role of TLR2 was already described for several infections, such as Staphilococcus aureus [169], Streptococcus pneumonia [170, 171], Toxoplasma gondii [172], Mycobacterium bovis [173], M. avium [174, 175] and M. tuberculosis [176, 177]. However, several studies also suggested that TLR2-dependent mechanisms might also contribute to the evasion or inhibition of an effective immune response. As examples, Yersinia enterocolitica, Aspergillus fumigates and Candida albicans [178-181] induce immunosuppression


Table 2.

TLR

TLR4

TLR7/8

TLR9

9

Overview of TLRs Role During Leishmania Infection

Model

Major Conclusions

Reference

Cell culture - L. major

Purified Leishmania LPG activates innate immune signaling via TLR2

[150]


Purified Leishmania LPG activates NK cells through TLR2 leading to IFN- and TNF- production via NF-B

[156]

Cell culture - L. donovani

Role for TLR3 in the leishmanicidal activity of IFN- primed macrophages; the Leishmania TLR3 agonist (s) is (are) unknown

[155]

L. major

First study showing the contributive role to the effective control of Leishmania infection in vivo; TLR4-/- mice had increased parasite burdens than control mice

[152]

L. major

TLR4 contributes to the efficient control of Leishmania growth in the innate phase of infection

[152]

L. major

TLR4 does not interfere with the expression of chemokines after Leishmania infection

[153]

L. major

C5a downregulates TLR4-induced expression of IL-12 family cytokines: C5aR-/mice are substantially more resistance to L. major

[24]

L. major

Intramacrophagic microbicidal activity induced by neutrophils or purified NE require TLR4 signaling

[154]

L. pifanoi and L. amazonensis

P8 proteoglycolipid complex signaling through TLR4

[158]

L. major

Topical administration of imiquimod (TLR7 agonist) or subcutaneous injection of R848 (TLR7/8 agonist) enhances the Th1 response and increase protective immunity against cutaneous leishmaniasis

[161]

L. major

DNA vaccination decreases the number of lesion caused by L. major through TLR9 activation

[163]

L. major

TLR9-/- mice develop more lesions and have impaired NK cell response. TLR9 signaling is essential for NK cell activation, but dispensable for a protective T cell response since mice ultimately resolved the cutaneous infection

[66]

L. major, L. infantum and L. brasiliensis

NK cell activation requires mieloid DC, TLR9 and IL-12

[67]


L. major activates IL-1 expression in macrophages through a MyD88-dependent pathway

[149]

L. major

MyD88 is required for efficient clearance of L. major infection

[150]

L. major

MyD88-dependent pathways are essential for the development of protective IL-12 mediated Th1 response against L. major infection

[151]

Cell culture - recombinant HSP70

The heat-shock protein 70 (HSP 70) signals via MyD88 pathway

[157]

TLR2

TLR3


MyD88

through TLR2-derived signals that mediate increased IL-10 production and survival of Tregs. In these cases, the absence of TLR2 rendered the mice more resistant to infection. Although, TLR2-deficient mice were fully competent at an innate immunity level against these infections, they were shown to be incapable to mount a protective Th1 response as demonstrated by the high susceptibility to re-infection [180]. The authors explained this behavior through the TLR2-dependent increase of IL-10, which is essential to

the induction of Treg cells and consequently long-lasting immunity. It was proposed that TLR2-induced signals preferentially induce a Th2 profile with IL-10 secretion [182]. IL-10 is a major susceptibility factor during leishmaniasis. Since, we explored the role of TLR2 during VL caused by L. infantum, recurring to a TLR2-knockout mice model [183]. First, we evaluated the Leishmania phagocytic capacity of


Silvestre et al.

bone marrow derived-macrophages (BM-M) (F4/80 + and CD11b+ positive) and flow cytometry sorted BM-DC (over 97% CD11c positive) derived from TLR2 competent (C57BL/6) and deficient mice against L. infantum promastigotes carrying a luciferase encoding gene (LUC-L. infantum). As shown in Fig. (1A), the number of parasites, at low infection ratios, internalized by BM-M, and to a lower extent BM-DC from TLR2-deficient mice was significantly lower when compared with C57BL/6 mice. A similar result was found when BM-M derived from TLR2-competent C57BL/6 mice were pre-treated with an anti-TLR2 antibody (Fig. 1B). Interestingly, no such effect was observed in BMDC (Fig. 1C). However, when higher numbers of parasites were used (10 parasites per cell) no significant difference was found in both cell types. Flandin and colleagues had already proposed a role of TLR2 and TLR3 in the LPG-dependent phagocytosis of L. donovani [155]. Thus, our result confirmed the role played by TLR2 in the phagocytosis of Leishmania promastigotes. In vivo, however, this mechanism should be of minor importance and in the presence of higher numbers of parasites it would be overcome by other more classical receptors. The outcome of the in vitro L. infantum infection in TLR2-deficient BM-DC resulted in a significantly increase of IL-12p40 and decrease secretion of IL-10 when compared with C57BL/6 mice (Fig. 2A and C). Although a similar decrease of TNF- was observed in both strains of mice (Fig. 2B), these results suggests a preferential shift to a Th1 profile in the absence of TLR2. Indeed, a dose-dependent significantly increase of IL-12p40 was also observed in BM-M of TLR2-deficient mice (Fig. 2D). Interestingly, this result is in concordance with the previous findings described for C. albicans, A. fumigatus and Y. enterocolitica [178-181].

by L. infantum in the two major target organs, the spleen and the liver.

Nevertheless, further in vivo experiments did not support these in vitro findings. Indeed, the absence of TLR2 did not alter the course of VL in both the spleen (Fig. 3A) and liver (Fig. 3B). The only exception was at late chronic phase (120 days post-infection), where a significant decrease of parasite burden was observed in the resistant C57BL/6 mice strain. Yet, a contradictory result occurred in the draining lymph nodes. VL caused by the intraperitoneal injection of L. infantum promastigotes are known to be transient in this organ [10]. Anyhow, significant lower levels or even undetectable parasites were remarked in the absence of TLR2 (Fig. 3C). One of the major reasons that may be accounted for this behavior is a significant increase in L. infantum specific IL-12p40 and IFN- found in 10 days infected draining lymph nodes, but not in the spleen, after SLA ex-vivo stimulus (Fig. 4A and B). Since TLR2 is involved in the stimulation of regulatory T cells [184], one probable reason for this shift may be a decrease in the Treg numbers and a consequently down-regulation of immunosuppressive IL-10. Interestingly, at this time post-infection, an increase of CD4+-T cells were accounted only in the draining lymph nodes of TLR2-deficient mice (data not shown). Although we did not proceed to the phenotypic analysis of the T cell population in both organs, the IL-10 levels were not found to be different among the two strains of mice (Fig. 4C). Hence, it is acceptable to affirm that no major differences were found during the visceral infection caused

Fig. (1). TLR2 is involved in the phagocytosis of L. infantum promastigotes. Macrophage or dendritic cells (BM-M and BMDC, respectively) were derived from C57BL/6 or TLR2-/- bone marrow as previously described [186-188]. A cloned line of L. infantum expressing the luciferase (LUC) gene derived from virulent L. infantum (MHOM/MA/67/ITMAP-263) [189] was used in all in vitro experiments. (A) BM-M and BM-DC were cultured in 96-well plates at 1 x 106/ml and infected at different ratios for 4 hours. In some cases, C57BL/6 BM-M (B) or BM-DC (C) infections were preceded by incubation with an -TLR2 antibody (10 g/ml clone T2.5, Cell Sciences, MA) or the correspondent isotypic control (IgG1, 10 g/ml) for 30 minutes. After 4 hours of infection, the non-internalized parasites were washed with PBS-EGTA and the luciferase activity of the LUC-recombinant parasites was determined as described elsewhere [190]. Values were expressed as relative light units (RLU). Experiments were done in quintuplicate cultures and the result is representative of three independent experiments. * P < 0.05, ** P < 0.01.



11

Fig. (2). Secretion of IL-12p40 (A), TNF- (B) and IL-10 (C) by BM-DC and IL-12p40 and IL-10 by BM-M (D) derived from C57BL/6 or TLR2-/- mice exposed to live L. infantum promastigotes. BM-M and BM-DC were cultured in 24-well plates at 1 x 106/ml and infected at different ratios for 4 hours, after which the cells were washed to remove unphagocytosed parasites. After 24 hours the levels of IL-12p40, TNF- and IL-10 were measured in the culture supernatants by ELISA, following the manufacturer’s recommendations (BD Pharmingen for IL-10 and IL-12p70; BioLegend for IL12p40 and TNF-). Minimum detection levels were 7.8 pg/ml for IL-12p40 and TNF-, 31.3 pg/ml for IL-10 and 62.5 pg/ml for IL-12p70. The results shown are representative of three independent experiments that yielded similar results. * P < 0.05, ** P < 0.01.

Together these results suggest that TLR2 perform a minor role in initiating the synthesis of proinflammatory cytokines and the effector cellular responses during the in vivo infection with L. infantum. Since partial contributions were already observed for other TLR, such as TLR4 and TLR9 [66, 152], it is plausible to conclude that a complete activation of the innate immune response during Leishmania infection rely on the capacity of several TLRs to recognize parasite molecules. 5. CONCLUDING REMARKS While during several years macrophages were the focus of the efforts developed by researchers, recent evidences point to an urge in understanding the critical initial steps of infection that involve neutrophils and dendritic cells. Peters and colleagues have recently demonstrated that the sustained initial neutrophils recruitment caused by the sand fly bite was irrespective of the parasite presence [29]. Although the role of sand fly saliva was beyond the scope of this review, it has been linked to several immunomodulatory properties soon after parasite inoculation [185]. Therefore, an effort should be made to include sand fly inoculation or sand fly extracts in the inoculation of the parasite in in vivo models

of leishmaniasis. Also, TLR are one of the first sensors of invading pathogens. An optimal understanding of TLR manipulation by this and other protozoan parasites might hopefully lead to the development of original rational targets for alternative drug treatments against these infectious diseases. ANIMAL EXPERIMENTS Male wild-type C57BL/6 were obtained from Harlen Iberica (Spain) and TLR2-deficient mice with a C57BL/6 background [183] were obtained from Dr. S. Akira (Osaka University, Japan) via Dr. Salomé Gomes (IBMC, Porto University, Portugal). Under laboratory conditions, the animals were maintained in sterile cabinets and allowed sterile food and water ad libitum. All animals entered into experiments at 8 wk of age. All animal procedures were carried out in the IBMC approved facilities. A cloned line of virulent L. infantum [MHOM/MA/ 67/ITMAP-263, WT] was grown at 26ºC by weekly subpassages in complete culture medium – RPMIc medium - RPMI 1640 medium (GIBCO BRL) supplemented with 10% heat-inactivated FBS, 100 units/ml penicillin, 100 mg/ml


Silvestre et al.

streptomycin and 2 mM HEPES. WT parasites were frequently passed through susceptible BALB/c mice to maintain the virulence. For mice infection, TLR2-deficient and C57BL/6 wild-type mice (n=5 for each point in two independent experiments) were injected intraperitoneally with 108 WT stationary-phase promastigotes, which were collected, washed and suspended in sterile PBS.

Fig. (3). Kinetics of visceral leishmaniasis in TLR2-/- mice. Course of parasite burden progression in the spleen (A), liver (B) and draining lymph nodes (C) of C57BL/6 or TLR2-/- mice infected intraperitoneally with 1 x 108 stationary phase promastigotes of virulent L. infantum (MHOM/MA/67/ITMAP-263). The mice were sacrificed after 3, 10, 30, 60, 90 and 120 days of infection and the parasite load in the organs determined by the limiting dilution method [191]. Statistical analysis was performed using Student t-test. Statistically significant differences between C57BL/6 or TLR2-/- infected mice are indicated. * P< 0.05.

Fig. (4). Levels of cytokines in the supernatant of splenocyte and draining lymph nodes cultures from C57BL/6 or TLR2-/- mice at 10 days postinfection with L. infantum. Spleen and lymph nodes cells from infected C57BL/6 or TLR2-/- mice were cultured for 72 hours in the presence or absence of soluble Leishmania antigens (SLA; 50 g/ml) as previously described [10]. Supernatants were removed and the production of IL-12p40 (A), IFN- (B) and IL-10 (C) were measured by ELISA. * P< 0.05.

ACKNOWLEDGMENTS This work was supported by Fundação para a Ciência e Tecnologia (FCT), Programa Operacional Ciência e Inovação 2010 (POCI 2010) and FEDER in project number



POCI/CVT/59840/2004. The work was also supported by FCT, POCI 2010 and co-funded by FEDER in the project PTD/SAU-FCF/67351/2006. RS, NS and AMS are supported by fellowship from FCT , POCI 2010 and co-funded by FEDER in the number SFRH/BPD/41476/ 2007, POCI/SAU-FCT/59837/2004 and SFRH/BD/28316/ 2006, respectively.

[3]

CONFLICT OF INTEREST

[5]

The authors have no financial conflict of interest.

[4]

[6]

ABBREVIATIONS VL

=

Visceral leishmaniasis

CMI

=

Cell mediated immunity

TLR

=

Toll-like receptor

GPI

=

Glycosylphosphatidylinositol

LPG

=

Lipophosphoglycans

CR

=

Complement receptor

CRP

=

C-reactive protein

PMN

=

Polymorphonuclear neutrophil granulocyte

PV

=

Parasitophorous vacuole

DC

=

Dendritic cells

[11]

DC-SIGN

=

DC-specific intracellular adhesion molecule 3-grabbing non-integrin

[12]

[7]

[8]

[9] [10]

NO

=

Nitric oxide

iNOS

=

Inducible nitric oxide synthase

ROI

=

Reactive oxygen intermediates

PS

=

Phophatidylserine

MHC

=

Major histocompatibility complex

IFN

=

Interferon

GIPL

=

Glycosylinositolphospholipids

PAMP

=

Pathogen-associated molecules patterns

MyD88

=

Myeloid differentiation factor 88

PKC

=

Protein kinase C

LPS

=

Lipopolysaccharide

BM-M

=

Bone marrow derived-macrophages

[17]

BM-DC

=

Bone marrow derived-dendritic cells

[18]

PGE2

=

Prostaglandin E2

FcR

=

Fc receptor

Treg

=

Regulatory T cells

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[2]

Kimblin, N.; Peters, N.; Debrabant, A.; Secundino, N.; Egen, J.; Lawyer, P.; Fay, M. P.; Kamhawi, S.; Sacks, D. Quantification of the infectious dose of Leishmania major transmitted to the skin by single sand flies. Proc. Natl. Acad. Sci. USA, 2008, 105, 1012510130. Sacks, D.; Kamhawi, S. Molecular aspects of parasite-vector and vector-host interactions in leishmaniasis. Annu. Rev. Microbiol., 2001, 55, 453-483.

[13]

[14]

[15]

[16]

[19]

[20]

[21]

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Received: November 4, 2008

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Revised: March 3, 2009

Accepted: June 14, 2009