Neutrophils and TNF: two key players in the immune response induced by "Leishmania major" infection

Summary Resolution of the inflammation is as important as its induction. In this thesis, we investigated the contributions of two prominent factors involved in inflammation, Tumour Necrosis Factor (TNF) and neutrophils. We studied their role in the resolution óf the inflammatory lesion induced by the infection with the protozoan parasite Leishmania major. In mice susceptible to infection with L. major, unhealing lesions are characterized by an elevated number and sustained presence of inflammatory neutrophils in the infected tissue, illustrating an acute inflammatory process. In contrast, mice from resistant strains, which resolve their lesions, can control the presence of neutrophils at the site of infection. Neutrophil persistence in the infected tissue may result from several events including an increased survival of neutrophils mediated by factors produced by the pathogen or the microenvironment. Following infection with L. major, the cellular composition of the inflammatory lesion differs significantly between susceptible and resistant mice and a higher proportion of macrophages is present in the lesions of resistant strains. In an attempt to clarify the factors involved in neutrophil persistence, we investigated the mechanisms modulating neutrophil cell death. We demonstrated that macrophages could induce neutrophil apoptosis in a process involving TNF. TNF is an essential cytokine with pro- and anti-inflammatory properties, which is expressed as a transmembrane protein that can be cleaved releasing the secreted form. Our data show the essential role of the transmembrane form of TNF (mTNF) in the induction of neutrophil apoptosis by macrophages, revealing macrophages and mTNF as important regulators of neutrophil apoptosis. TNF is critical in the resolution of the inflammatory lesion induced by L. major infection, and in L. major resistant strains its absence results in increased swelling of the lesions. We investigated the contribution of mTNF in the outcome of L. major infection. Our data demonstrate that following infection with L. major, mTNF is sufficient to support the resolution of the inflammatory lesion and optimal parasite killing. In addition, we show that the presence of mTNF is essential to induce neutrophil clearance in the infected tissue. While the persistence of neutrophils is deleterious for the host, we could demonstrate an early anti-inflammatory role of neutrophils. Altogether, this study demonstrates the importance of mTNF in the induction of neutrophil apoptosis, a process involved in the resolution of the inflammatory lesion induced by L. major infection. Résumé La résolution de l'inflammation est toute aussi importante que son initiation. Durant ce travail de thèse, nous avons étudié les contributions de deux facteurs importants impliqués dans l'inflammation, le TNF (Facteur Nécrosant des Tumeurs) et les neutrophiles, dans la résolution de la lésion inflammatoire induite par l'infection avec le parasite protozoaire Leishmania major. Chez les souris sensibles à l'infection avec L. major, des lésions importantes qui ne guérissent pas se développent ; celles-ci sont caractérisées par un nombre élevé et une présence soutenue de neutrophiles dans les tissus infectés, ce qui illustre un processus inflammatoire aigu. Au contraire, les souris résistantes à l'infection qui guérissent leurs lésions, sont capables de contrôler la présence des neutrophiles au site d'infection. La persistance des neutrophiles dans la lésion inflammatoire peut être la conséquence de plusieurs événements, dont une augmentation de la survie des neutrophiles induite par des facteurs produits par le pathogène ou le micro-environnement. Suite à l'infection avec L. major, la composition cellulaire de la lésion inflammatoire est significativement différente entre les souris sensibles et résistantes à l'infection, et une plus grande proportion de macrophages est présente dans les lésions des souris résistantes. Dans l'objectif de clarifier les facteurs impliqués dans la persistance des neutrophiles dans les tissus infectés par L. major, nous avons étudié les mécanismes de régulation de la mort des neutrophiles. Nous avons démontré que les macrophages pouvaient induire l'apoptose des neutrophiles dans un procédé impliquant le TNF. Le TNF est une cytokine aux propriétés pro- et anti-inflammatoires, exprimée sous une forme transmembranaire qui peut être clivée pour relâcher la forme sécrétée. Nos expériences illustrent le rôle essentiel de la forme transmembranaire du TNF (mTNF) dans l'induction de l'apoptose des neutrophiles par les macrophages. Lé TNF est une cytokine importante dans la résolution de la réaction inflammatoire induite par L. major, et chez les souris résistantes l'absence de TNF provoque des lésions inflammatoires plus importantes. Nous avons étudié la contribution du mTNF dans la résolution de l'infection avec L. major. Nos résultats démontrent que suite à une infection avec le parasite, la présence du mTNF est suffisante pour guérir la lésion inflammatoire et contrôler efficacement la réplication du parasite. De plus, le mTNF joue un rôle essentiel dans l'élimination des neutrophiles du tissu infecté. Alors que la persistance des neutrophiles est nocive pour l'hôte, nous avons montré que les neutrophiles avaient un rôle précoce anti-inflammatoire. En résumé, cette étude révèle l'importance du mTNF dans l'induction de l'apoptose des neutrophiles par les macrophages, un procédé impliqué dans la résolution de la lésion inflammatoire induite par l'infection avec L. major.

Implication of TNF family members in the immune response to the infection with the intracellular protozoan parasite "Leishmania major"

Suite à une infection avec le protozoaire Leishmania major (L. major), les souris sensibles de souche BALB/c développent des lésions progressives associées à une maturation des cellules CD4+ TH2 sécrétant de l'IL-4. A l'inverse, les souris résistantes de souche C57BL/6 guérissent à terme, sous l'influence de l'expansion des cellules CD4+ TH1 produisant de l'IFNy qui a un effet synergique avec le TNF ("tumor necrosis factor") sur l'activation des macrophages et leur fonction leishmanicide. Lors de notre étude nous avons montré que des souris C57BL/6 doublement déficientes en TNF et FasL ("Fas ligand") infectées par L. major ne guérissaient ni leur lésions ni ne contrôlaient la réplication de parasites malgré une réponse de type TH1. Bien que l'activité de synthétase inductible de l'oxyde nitrique ("iNOs") soit comparable chez les souris doublement ou simplement déficientes, seules celles déficientes en FasL ont démontré une incapacité à contrôler la réplication parasitaire. De surcroît il est apparu que le FasL a un effet synergique avec l'IFNy. L'adjonction de FasL à une culture cellulaire de macrophages stimulés par l'IFNy conduit à une activation de ces cellules. Celle-ci est démontrée par l'augmentation de la production de TNF et de NO par les macrophages ainsi que par l'élimination des parasites intracellulaires par ces mêmes cellules. Alors que le FasL et l'IFNy semblent essentiels au contrôle de la réplication des pathogènes intracellulaires, la contribution de TNF s'oriente davantage vers le contrôle de l'inflammation. L'activation macrophagique via Fas précède la mort cellulaire qui survient quelques jours plus tard. Cette mort cellulaire programmée était indépendante de la cascade enzymatique des caspases, au vu de l'absence d'effet de l'inhibiteur non-spécifique ZVAD-fmk des caspases. Ces résultats suggèrent que l'interaction Fas-FasL agit comme une costimulation nécessaire à une activation efficace des macrophages, la mort cellulaire survenant consécutivement à l'activation des macrophages.¦-¦Upon infection with the protozoan parasite Leishmania major (L. major), susceptible BALB/c mice develop non healing lesions associated with the maturation of CD4+ TH2 cells secreting IL-4. In contrast, resistant C57BL/6 mice are able to heal their lesions, because of CD4+ TH1 cell expansion and production of high levels of IFNy, which synergizes with tumour necrosis factor (TNF) in activating macrophages to their microbicidal state. In our study we showed that C57BL/6 mice lacking both TNF and Fas ligand (FasL) infected with L. major neither resolved their lesions nor controlled L. major replication despite a strong TH1 response. Although comparable inducible nitric oxide synthase (iNOs) was measured in single or double deficient mice, only mice deficient in FasL failed to control the parasite replication. Moreover FasL synergized with IFNy for the induction of leishmanicidal activity within macrophages infected with L. major in vitro. Addition of FasL to IFNy stimulated macrophages led to their activation, as reflected by the secretion of tumour necrosis factor and nitrite oxide, as well as the induction of their microbicidal activity, resulting in the killing of intracellular L. major. While FasL along with IFNy and iNOs appeared to be essential for the complete control of intracellular pathogen replication, the contribution of TNF appeared more important in controlling the inflammation on the site of infection. Macrophage activation via Fas pathway preceded cell death, which occurred a few days after Fas mediated activation. This program cell death was independent of caspase enzymatic activities as revealed by the lack of effect of ZVAD-fmk, a pan-caspase inhibitor. These results suggested that the Fas-FasL pathway, as part of the classical activation pathway of the macrophages, is essential in the stimulation of macrophage leading to a microbicidal state and to AICD, and may thus contribute to the pathogenesis of L. major infection.

Type I signal peptidase from Leishmania is a target of the immune response in human cutaneous and visceral leishmaniasis.

The gene encoding type I signal peptidase (Lmjsp) has been cloned from Leishmania major. Lmjsp encodes a protein of 180 amino residues with a predicted molecular mass of 20.5 kDa. Comparison of the protein sequence with those of known type I signal peptidases indicates homology in five conserved domains A-E which are known to be important, or essential, for catalytic activity. Southern blot hybridisation analysis indicates that there is a single copy of the Lmjsp gene. A recombinant SPase protein and a synthetic peptide of the L. major signal peptidase were used to examine the presence of specific antibodies in sera from either recovered or active individuals of both cutaneous and visceral leishmaniasis. This evaluation demonstrated that sera from cutaneous and visceral forms of leishmaniasis are highly reactive to both the recombinant and synthetic signal peptidase antigens. Therefore, the Leishmania signal peptidase, albeit localised intracellularly, is a significant target of the Leishmania specific immune response and highlights its potential use for serodiagnosis of cutaneous and visceral leishmaniasis.

Neutrophils contribute to development of a protective immune response during onset of infection with Leishmania donovani.

Neutrophils are key components of the inflammatory response and as such contribute to the killing of microorganisms. In addition, recent evidence suggests their involvement in the development of the immune response. The role of neutrophils during the first weeks post-infection with Leishmania donovani was investigated in this study. When L. donovani-infected mice were selectively depleted of neutrophils with the NIMP-R14 monoclonal antibody, a significant increase in parasite numbers was observed in the spleen and bone marrow and to a lesser extent in the liver. Increased susceptibility was associated with enhanced splenomegally, a delay in the maturation of hepatic granulomas, and a decrease in inducible nitric oxide synthase expression within granulomas. In the spleen, neutrophil depletion was associated with a significant increase in interleukin 4 (IL-4) and IL-10 levels and reduced gamma interferon secretion by CD4(+) and CD8(+) T cells. Increased production of serum IL-4 and IL-10 and higher levels of Leishmania-specific immunoglobulin G1 (IgG1) versus IgG2a revealed the preferential induction of Th2 responses in neutrophil-depleted mice. Altogether, these data suggest a critical role for neutrophils in the early protective response against L. donovani, both as effector cells involved in the killing of the parasites and as significant players influencing the development of a protective Th1 immune response.

Immunization with H1, HASPB1 and MML Leishmania proteins in a vaccine trial against experimental canine leishmaniasis.

The protective capabilities of three Leishmania recombinant proteins - histone 1 (H1) and hydrophilic acylated surface protein B1 (HASPB1) immunized singly, or together as a protein cocktail vaccine with Montanide, and the polyprotein MML immunized with MPL-SE adjuvant - were assessed in beagle dogs. Clinical examination of the dogs was carried out periodically under blinded conditions and the condition of the dogs defined as asymptomatic or symptomatic. At the end of the trial, we were able to confirm that following infection with L. infantum promastigotes, five out of eight dogs immunized with H1 Montanide, and four out of eight dogs immunized with either the combination of HASPB1 with Montanide or the combination of H1+HASPB1 with Montanidetrade mark, remained free of clinical signs, compared with two out of seven dogs immunized with the polyprotein MML and adjuvant MPL-SE, and two out of eight dogs in the control group. The results demonstrate that HASPB1 and H1 antigens in combination with Montanide were able to induce partial protection against canine leishmaniasis, even under extreme experimental challenge conditions.

Histone H1 regulates chromatin condensation in Leishmania parasites.

We investigated the functional role of the Leishmania histone H1 and demonstrate for the first time that addition of histone H1 has a strong effect on microccocal digestion, chromatin condensation of parasite nuclei and that its overexpression can modulate parasite infectivity in vivo.

Role and regulation of polyphosphate in leishmania parasites

Le protozoaire unicellulaire Leishmania est l'agent responsable de la leishmaniose, une maladie parasitaire humaine qui se manifeste par des lésions de la peau, se résolvant le plus souvent spontanément, jusqu'à des lésions viscérales fatales. Le parasite est transmis de l'insecte à l'hôte mammifère lors d'un repas sanguin de la mouche des sables et y réside respectivement sous formes extra- et intracellulaires. On estime que cette maladie touche environ 12 millions de personnes dans 98 pays. Etant donné que les médicaments disponibles à ce jour sont faiblement efficaces et/ou hautement toxiques, il est indispensable de consolider les connaissances sur le fonctionnement et la survie du parasite pour pouvoir développer de nouvelles stratégies de traitements et de préventions. Tous les organismes vivants, dont Leishmania, contiennent du polyphopshate (polyP). Cette molécule chargée négativement est constituée de trois jusqu'à plusieurs centaines de résidus de phosphates reliées par des liaisons à haute énergie. Le polyP sert donc de source d'énergie et de réservoir de phosphate; dans certaines espèces, il joue aussi un rôle dans l'adaptation au stress et la virulence de pathogènes. Ceci nous a amené à étudier le rôle du polyP dans le parasite Leishmania. L'enzyme responsable de la synthèse de polyP a été identifié récemment dans la levure : il s'agit de la chaperone de transport vacuolaire 4 (Vtc4). Nous avons identifié un homologue de Vtc4 chez les Trypanosomatidae, et avons donc décidé d'examiner sa fonction dans le métabolisme du polyP chez Leishmania. En éliminant l'expression de Vtc4 chez L. major et L. guyanensis, nous avons pu démontrer qu'il est indispensable pour la production de polyP chez Leishmania. De plus, nous avons constaté que ces parasites possèdent des chaînes de polyP allant de trois jusqu'à environ 300 résidus de phosphate. Le taux de polyP dans la cellule est précisément régulé et varie entre un très haut niveau durant la phase proliférative des promastigotes à un niveau bas en phase stationnaire tardive, alors que l'expression de Vtc4p reste stable. Dans les amastigotes intracellulaires, seulement des petites quantités de polyP et de Vtc4p sont détectées. En outre, l'absence de Vc4p et de polyP n'a pas d'effet significatif sur les infections in vivo de souris, ce qui indique que le polyP n'est pas nécessaire au développement de la leishmaniose. Ceci suggère que Vtc4p n 'est pas une bonne cible pour le développement de nouveaux traitements contre Leishmania. "Néanmoins, la présence du polyP favorise fortement la survie du parasite suite à un choc de température (37°C) et aide ainsi à sa persistance intracellulaire pendant les premiers jours d'infection de macrophages. En résumé, nos résultats indiquent que si le polyP a peu d'importance pendant l'infection et le développement de la leishmaniose chez la souris, il est par contre crucial pour l'adaptation à des situations de stress comme l'augmentation de la température. Le fait que le polyP a été conservé dans tous les organismes durant l'évolution suggère toutefois que cette molécule joue un rôle fondamental. Etant donné que l'absence de polyP n'a pas d'effet sur la survie des amastigotes, il pourrait être plus important dans la forme promastigote infectant la mouche des sables. - The unicellular protozoan parasite Leishmania is the causative agent of the human disease leishmaniasis, which can range from self-healing skin lesions to fatal visceral lesions. The parasite is transmitted from the insect vector to the mammalian host when the sand fly takes its blood meal and exists in an extra- and an intracellular form, respectively. The disease is estimated to affect 12 million people in 98 countries and currently available drug treatments are of relatively low potency and/or high toxicity. Thus, investigating parasite survival mechanisms and parasite adaptation to the two host environments contributes to the general understanding of Leishmania propagation and might therefore help to develop future treatments or preventions. All living cells, including Leishmania, contain a negatively charged polymer of a few up to several hundred phosphate residues. These so-called polyphosphates (polyPs) serve as an energy source and phosphate reservoir. In some organisms, polyP is also involved in adaptation to stresses and virulence of pathogens. Therefore we were interested in investigating the importance of polyP in Leishmania parasites. Recently, an eukaryotic enzyme responsible for polyP synthesis has been identified as the vacuolar transporter chaperone 4 (Vtc4) in yeast. We, and others, found a Vtc4 homologue in trypanosomatids and decided to examine its potential function in polyP metabolism. By generating VTC4 knock-out cell lines in L. major and Vtc4 knock-down cell lines in L. guyanensis, we were able to demonstrate that Vtc4p is responsible for the total amount of cellular polyP. We also observed that Leishmania polyP chain length ranges from a few up to around 300 residues and that its level is tightly regulated. PolyP abundance is highest during the logarithmic proliferating phase of promastigotes and decreases in the stationary phase, while Vtc4 protein expression remains stable during both phases. In the intracellular amastigote form, only low amounts of polyP and Vtc4p were detectable. Furthermore, absence of Vtc4p and polyP did not have a significant effect on in vivo mouse infections, indicating that polyP is not necessary for Leishmania disease progression. This suggests that Vtc4p would be a poor drug target against Leishmania infection. However, presence of the polymer strongly supported parasite survival during heat shock (37°C) and thereby promoted intracellular persistence during the first days of macrophage infections. Taken together, we found that polyP has little importance in Leishmania {in vivo) infection but that it plays a crucial role during adaptation to stress, such as heat shock. Given that polyP has been preciously conserved in all organisms during evolution it seems to play a fundamental role. Since absence of polyP does not affect amastigote survival, it might be significant for promastigote existence in the sand fly vector.

IL-10 producing CD8+ T cells in human infection with Leishmania guyanensis.

Cytokines are increasingly recognized as important components of the cellular immune responses to intracellular pathogens. In this study, we analyzed the production of TGF-beta, IL-10 and IFN-gamma by PBMC of unexposed naïve subjects and LCL patients after stimulation with live Leishmania guyanensis (L.g.). We demonstrated that IFN-gamma is produced in controls and LCL patients, IL-10 only in LCL patients and TGF-beta only in naïve subjects. Furthermore, in naive subjects, neutralization of TGF-beta induced IL-10 production. IL-10 produced in naïve subjects when TGF-beta is neutralized or in LCL patients did not modify the IFN-gamma production but inhibit reactive nitrogen species production. Analysis of the phenotype of IL-10 producing cells in naive subjects when TGF-beta is neutralized clearly showed that they are memory CD45RA- CD8+ T cells. In LCL patients, IL-10 producing cells are both CD45RA- CD4 and CD8+ T cells. The role of these IL-10 producing CD8+ T cells in the development of the diseases should be carefully evaluated.

Regulatory B cells shape the development of Th2 immune responses in BALB/c mice infected with Leishmania major through IL-10 production.

Recent evidence indicates that B cells are required for susceptibility to infection with Leishmania major in BALB/c mice. In this study, we analyzed the role of the IL-10 produced by B cells in this process. We showed that B cells purified from the spleen of BALB/c mice produced IL-10 in response to stimulation with L. major in vitro. In vivo, early IL-10 mRNA expression is detected after L. major infection in B cells from draining lymph nodes of susceptible BALB/c, but not of resistant C57BL/6 mice. Although adoptive transfer of naive wild-type B cells prior to infection in B cell-deficient BALB/c mice restored Th2 cell development and susceptibility to infection with L. major of these otherwise resistant mice, adoptive transfer of IL-10(-/-) B cells mice did not. B cells stimulated by L. major, following in vitro or in vivo encounter, express the CD1d and CD5 molecules and the IL-10 produced by these cells downregulate IL-12 production by L. major-stimulated dendritic cells. These observations indicate that IL-10 secreting B cells are phenotypically and functionally regulatory B cells. Altogether these results demonstrate that the IL-10 produced by regulatory CD1d+ CD5+ B cells in response to L. major is critical for Th2 cell development in BALB/c mice.

Sense and antisense transcripts in the histone H1 (HIS-1) locus of Leishmania major.

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by two genes, HIS-1.1 and HIS-1.2. These genes are separated by approximately 20 kb of sequence and are located on the same DNA strand of chromosome 27. When Northern blots of parasite RNA were probed with HIS-1 strand-specific riboprobes, we detected sense and antisense transcripts that were polyadenylated and developmentally regulated. When the HIS-1.2 coding region was replaced with the coding region of the neomycin phosphotransferase gene, antisense transcription of this gene was unaffected, indicating that the regulatory elements controlling antisense transcription were located outside of the HIS-1.2 gene, and that transcription in Leishmania can occur from both DNA strands even in the presence of transcription of a selectable marker in the complementary strand. A search for other antisense transcripts within the HIS-1 locus identified an additional transcript (SC-1) within the intervening HIS-1 sequence, downstream of adenine and thymine-rich sequences. These results show that gene expression in Leishmania is not only regulated polycistronically from the sense strand of genomic DNA, but that the complementary strand of DNA also contains sequences that could drive expression of open reading frames from the antisense strand of DNA. These findings suggest that the parasite has evolved in such a way as to maximise the transcription of its genome, a mechanism that might be important for it to maintain virulence.

In susceptible mice, Leishmania major induce very rapid interleukin-4 production by CD4+ T cells which are NK1.1-.

Susceptibility of BALB/c mice to infection with Leishmania major is associated with a T helper type 2 (Th2) response. Since interleukin-4 (IL-4) is critically required early for Th2 cell development, the kinetics of IL-4 mRNA expression was compared in susceptible and resistant mice during the first days of infection. In contrast to resistant mice, susceptible mice exhibited a peak of IL-4 mRNA in their spleens 90 min after i.v. injection of parasites and in lymph nodes 16 h after s.c. injection. IL-12 and interferon-gamma (IFN-gamma) down-regulated this early peak of IL-4 mRNA; the effect of IL-12 was IFN-gamma dependent. Treatment of resistant C57BL/6 mice with anti-IFN-gamma allowed the expression of this early IL-4 response to L. major. The increased IL-4 mRNA expression occurred in V beta 8, 7, 2- CD4+ cells in BALB/c mice and NK1.1- CD4+ cells in anti-IFN-gamma treated C57BL/6 mice. These results show that the NK1.1+ CD4+ cells, responsible for the rapid burst of IL-4 production after i.v. injection of anti-CD3, do not contribute to the early IL-4 response to L. major.

Setting in motion the immune mechanisms underlying genetically determined resistance and susceptibility to infection with Leishmania major.

The murine model of infection with Leishmania major has allowed the demonstration of a causal relationship between, on the one hand, genetically determined resistance to infection and the development of a Th1 CD4+ cell response, and on the other hand, genetically determined susceptibility and Th2 cell maturation. Using this murine model of infection, the role of cytokines in directing the functional differentiation pathway of CD4+ T cell precursors, has been demonstrated in vivo. Thus, IL-12 and IFN-gamma have been shown to favour Th1 cell development and IL-4 is crucial for the differentiation of Th2 responses. Maturation of a Th2 response in susceptible BALB/c mice following infection with L. major is triggered by the IL-4 produced during the first two days after parasite inoculation. This IL-4 rapidly renders parasite specific CD4+ T cells precursors unresponsive to IL-12. A restricted population of CD4+ T cells expressing the V beta 4V alpha 8 TCR heterodimer and recognizing a single epitope on the LACK (Leishmania Activated C-Kinase) antigen of L. major is responsible for this rapid production of IL-4, instructing subsequent differentiation towards the Th2 phenotype of CD4+ T cells specific for several parasite antigens.

Characterization of a surface metalloprotease from Herpetomonas samuelpessoai and comparison with Leishmania major promastigote surface protease.

The monogenetic kinetoplastid protozoan parasite Herpetomonas samuelpessoai expresses a surface-exposed metalloprotease. Comparable to the Leishmania promastigote surface protease, or PSP, the protease of Herpetomonas is active at the surface of fixed and live organisms, and both enzymes display an identical cleavage specificity toward a nonapeptide substrate. The protease was enriched 440 times by partition into Triton X-114 followed by 2 steps of anion exchange chromatography. The 56-kDa enzyme is inhibited by the metal chelator 1,10-phenanthroline and is susceptible to cleavage by glycosyl-phosphatidylinositol phospholipase C (GPI-PLC). The conservation of an identical surface protease activity in these monogenetic and digenetic trypanosomatids suggests that the enzyme has a physiological function in the promastigote (insect) stage of these parasites.

Structure and function of a "yellow" protein from saliva of the sand fly Lutzomyia longipalpis that confers protective immunity against Leishmania major infection.

LJM11, an abundant salivary protein from the sand fly Lutzomyia longipalpis, belongs to the insect "yellow" family of proteins. In this study, we immunized mice with 17 plasmids encoding L. longiplapis salivary proteins and demonstrated that LJM11 confers protective immunity against Leishmania major infection. This protection correlates with a strong induction of a delayed type hypersensitivity (DTH) response following exposure to L. longipalpis saliva. Additionally, splenocytes of exposed mice produce IFN-γ upon stimulation with LJM11, demonstrating the systemic induction of Th1 immunity by this protein. In contrast to LJM11, LJM111, another yellow protein from L. longipalpis saliva, does not produce a DTH response in these mice, suggesting that structural or functional features specific to LJM11 are important for the induction of a robust DTH response. To examine these features, we used calorimetric analysis to probe a possible ligand binding function for the salivary yellow proteins. LJM11, LJM111, and LJM17 all acted as high affinity binders of prohemostatic and proinflammatory biogenic amines, particularly serotonin, catecholamines, and histamine. We also determined the crystal structure of LJM11, revealing a six-bladed β-propeller fold with a single ligand binding pocket located in the central part of the propeller structure on one face of the molecule. A hypothetical model of LJM11 suggests a positive electrostatic potential on the face containing entry to the ligand binding pocket, whereas LJM111 is negative to neutral over its entire surface. This may be the reason for differences in antigenicity between the two proteins.

Leishmania RNA virus: when the host pays the toll.

The presence of an RNA virus in a South American subgenus of the Leishmania parasite, L. (Viannia), was detected several decades ago but its role in leishmanial virulence and metastasis was only recently described. In Leishmania guyanensis, the nucleic acid of Leishmania RNA virus (LRV1) acts as a potent innate immunogen, eliciting a hyper-inflammatory immune response through toll-like receptor 3 (TLR3). The resultant inflammatory cascade has been shown to increase disease severity, parasite persistence, and perhaps even resistance to anti-leishmanial drugs. Curiously, LRVs were found mostly in clinical isolates prone to infectious metastasis in both their human source and experimental animal model, suggesting an association between the viral hyperpathogen and metastatic complications such as mucocutaneous leishmaniasis (MCL). MCL presents as chronic secondary lesions in the mucosa of the mouth and nose, debilitatingly inflamed and notoriously refractory to treatment. Immunologically, this outcome has many of the same hallmarks associated with the reaction to LRV: production of type 1 interferons, bias toward a chronic Th1 inflammatory state and an impaired ability of host cells to eliminate parasites through oxidative stress. More intriguing, is that the risk of developing MCL is found almost exclusively in infections of the L. (Viannia) subtype, further indication that leishmanial metastasis is caused, at least in part, by a parasitic component. LRV present in this subgenus may contribute to the destructive inflammation of metastatic disease either by acting in concert with other intrinsic "metastatic factors" or by independently preying on host TLR3 hypersensitivity. Because LRV amplifies parasite virulence, its presence may provide a unique target for diagnostic and clinical intervention of metastatic leishmaniasis. Taking examples from other members of the Totiviridae virus family, this paper reviews the benefits and costs of endosymbiosis, specifically for the maintenance of LRV infection in Leishmania parasites, which is often at the expense of its human host.