Introduction

Infection with parasites of the genus Leishmania causes a spectrum of clinical manifestations involving the skin, viscera and mucous membranes. Each form of disease is initiated by introduction of parasites into the skin during the blood meal of an infected sandfly. In the host, the parasites are obligatory intracellular and invade macrophages and dendritic cells. The outcome of infection depends on the species of parasite and on the genetic background of the host that determines the type of developing CD4+ T helper cells (Th1 or Th2). Macrophages and dendritic cells are important accessory cells for regulation of the Leishmania-specific T-cell immune response. The two types of cells have specialized tasks. Macrophages are professional scavenger cells and the most important effector cells for elimination of the parasites. Their infiltration and activation is regulated by chemokines and lymphokines. Dendritic cells, on the other hand, have the distinctive ability to stimulate resting T cells after transport of Leishmania parasites from the infected skin to the draining lymph node. After cure of cutaneous lesions, dendritic cells carrying persistent parasites may be involved in the sustained stimulation of protective memory T cells. This is based on an increased stability of their MHC class II-antigen complexes.

As compared to bacterial and viral infections, the means to combat parasites are incomplete, and no vaccines for use in humans are available yet. Experimental leishmaniasis provides a valuable model for the development of novel vaccination strategies. We are presently exploring the use of dendritic cells as an antigen-delivery system for vaccination against infectious diseases and immunotherapy.

Our studies focus on different aspects of the immune response to Leishmania:

Immunoregulatory functions of dendritic cells during infection with Leishmania parasites. Leishmania parasites are transmitted by sandflies and the infection commences at the site of the vector’s bite in the skin. In the mammalian  host, the parasites are obligatory intracellular and reside predominantly in macrophages. Macrophages serve not only as host cells for Leishmania but also as antigen-presenting cells modulating the specific cellular immune response and, after appropriate activation, as effector cells for killing of the parasites via the production of nitric oxide (NO). In addition to macrophages, dendritic cells (DC) play a critical role in cutaneous leishmaniasis. DC in the skin can phagocytose Leishmania parasites and have the unique capacity to transport the ingested organisms from the site of infection in the skin to the T cell areas of the draining lymph node. During this migration, DC mature and acquire the ability to activate resting T cells with specificity for Leishmania antigen. Macrophages are unable to induce a primary response. Thus, the distinctive function of DC in the early phase of cutaneous leishmaniasis seems to be the uptake of parasites in the dermis and their transport to the regional lymph node, where they provide the principal sensitizing signal and initiate the specific cellular immune response. Moreover, they are the cellular source of interleukin 12 (IL-12). Whereas macrophage IL-12 production is inhibited by Leishmania, the parasites directly stimulate DC to release this cytokine which supports Th1 development and protection against leishmaniasis. Furthermore, Leishmania-infected DC express unusually stable complexes of antigen and major histocompatibility complex (MHC) class II molecules and may therefore also be important for the maintenance of long-term immunity to leishmaniasis.

Dendritic cells as a tool to combat infectious diseases

Dendritic cells (DC) are heterogeneous populations of immune cells with exquisite antigen presentation functions. They play a critical role in the initiation, amplification and regulation of the specific immune response to microbial pathogens such as Leishmania parasites. Therefore, they are attractive candidates for use as an antigen delivery system and the design of novel vaccination and immune intervention strategies. We previously demonstrated that Langerhans cells (LC), a population of epidermal DC, pulsed with crude L. major antigen induce long-lasting protection in otherwise susceptible mice against subsequent challenges with the parasites. This protection correlated with a shift of the cytokine expression pattern towards a Th1 response with high levels of IFN-gamma and low or no expression of IL-10 and IL-4 (Flohé et al. 1998). In addition, we showed that Langerhans cells that had been loaded with the recombinant Leishmania antigens LeIF, LACK, gp63, PSA and KMP-11 can confer protection (Berberich et al. 2003). In a subsequent study, we demonstrated that L. major antigen-pulsed bone marrow-derived DC (BMDC) generated in vitro required additional stimulation in order to induce a protective immune response against Leishmania infection. BMDC solely conditioned with Leishmania lysate failed to induce protection against leishmaniasis. In contrast, mice vaccinated with antigen-loaded BMDC that had been stimulated by overnight exposure to the TLR9 ligand CpG were completely protected and developed an antigen-specific Th1 response (Ramírez-Pineda et al. 2004). Surprisingly, IL-12 expression by the immunizing BMDC was not required for induction of resistance to L. major, since the protective efficacy of antigen-loaded BMDC from IL-12-deficient mice was virtually identical with that of BMDC obtained from wild-type mice. This finding is in sharp contrast to the previous study with epidermal LC as the source of DC. Antigen-pulsed LC from IL-12-deficient mice, as opposed to those from wild-type mice, completely failed to mediate protection against leishmaniasis (Berberich et al. 2003).

We further demonstrated that L. major lysate-loaded plasmacytoid DC (pDC) are also able to induce protective immunity against a challenge infection (Remer et al. 2007). Vaccination with pDC did not require additional CpG activation and was not associated with substantial IL-12 secretion. Although pDC have a very low T cell-stimulatory capacity in vitro, vaccination with L. major antigen-loaded pDC induced a vigorous T cell-mediated immune response with a mixed Th1/Th2 profile. A local Th1 bias in the cytokine profile was sufficient for a highly protective immune response. In summary, we provided evidence that different populations of DC, once properly conditioned ex vivo, mediate robust and long-lasting protection against infection with L. major as a model microbial pathogen. Critical parameters determining the efficiency of DC-based vaccination include the origin of DC, the choice of antigen used for DC loading, the route of immunization and the state of DC maturation and activation. Moreover, we showed that natural killer (NK) cells support the induction of protective immunity during DC-mediated vaccination against Leishmania major (Remer et al. 2010).     

Recently, we investigated whether viable DC are required for inducing protection. We showed that L. major antigen-loaded DC that had been fixed with paraformaldehyde or exposed to UV irradiation, and even disrupted cells, are able to serve as an effective vaccine (Schnitzer et al. 2010). Furthermore, we demonstrate the potential of DC-derived exosomes to mediate protective immunity against cutaneous leishmaniasis. The route of antigen presentation to recipient T cells involves uptake of intravenously injected DC fragments into late endosomal compartments of splenic DC in the recipient. This finding suggests that the development of a cell-free vaccine for immunoprophylaxis against leishmaniasis and other infectious diseases is feasible.

Identification and functional analyses of new leishmanicidal compounds  

At present, only few drugs are available for the treatment of human leishmaniasis. The pentavalent antimony agents (sodium stibogluconate and meglumine antimoniate), amphotericin B, pentamidine, and paramomycine have limitations including parenteral administration, long courses of treatment, toxic side effects and high costs. Miltefosine - the first drug for effective oral treatment of leishmaniasis - is highly teratogenic. Moreover, parasite resistance against classical drugs is increasing and the development of vaccination strategies against leishmaniasis has not yet been successful. New leishmanicidal drugs are urgently needed. To this end, our laboratory collaborates with different groups of the Faculty of Chemistry and Pharmacy of the University of Würzburg within the collaborative research center 630 (www.sfb630.uni-wuerzburg.de). Using a fast and cheap colorimetric alamarBlue® assay, natural as well as chemically synthesized compounds are tested in a first screening step against L. major promastigotes. The most interesting compounds selected with this initial screening assay are further tested using a luciferase-transgenic Leishmania amastigote assay recently developed in our laboratory. Finally, the leishmanicidal potential of drugs is evaluated in preclinical trials with L. major-infected mice. Several molecular biological approaches are currently used to characterize the mode of action of leishmanicidal compounds. Beside structure-activity relationship studies, the results of these functional analyses of drug candidates will provide a scientific basis for further improvement of compound selectivity and delivery. Among other methods, plasmon surface resonance, active site labeling of proteases and transmission electron microscopy are applied to confirm the postulated drug-target interactions. Based on protein structural information, docking studies are in progress to explore the binding mode of leishmanicidal compounds to the identified binding partners. This computational technique will enable us to improve the positive effects against L. major and to minimize unwanted side effects induced by these compounds in macrophages and dendritic cells.

The papain-like cysteine cathepsins expressed by Leishmania play a key role in the life cycle of these parasites, turning them into attractive targets for the development of new drugs. We previously demonstrated in cooperation with the laboratory of Prof. T. Schirmeister (www.sfb630.uni-wuerzburg.de/projects/a4_schirmeister/) that two compounds of a series of peptidomimetic aziridine-2,3-dicarboxylate [Azi(OBn)2]-based inhibitors, Boc-(S)-Leu-(R)-Pro-(S,S)-Azi(OBn)2 (compound 13b) and Boc-(R)-Leu-(S)-Pro-(S,S)-Azi(OBn)2 (compound 13e), reduced the growth and viability of L. major and the infection rate of macrophages while not showing cytotoxicity against host cells (Ponte-Sucre et al. 2006). We showed that leishmanicidal activities against intracellular amastigotes were caused by direct effects of aziridine-2,3-dicarboxylate-based inhibitors on the parasite as well as by modulation of the cytokine secretion and nitric oxide release by macrophages (Ponte-Sucre et al. 2006). Both inhibitors targeted leishmanial cathepsin B-like cysteine cathepsin CPC, as shown by fluorescence proteinase activity assays and active-site labeling with biotin-tagged inhibitors (Schurigt et al. 2010). Furthermore, compounds 13b and 13e were potent inducers of cell death in promastigotes, characterized by cell shrinkage, reduction of mitochondrial transmembrane potential and increased DNA fragmentation (Schurigt et al. 2010). Transmission electron microscopic studies revealed the enrichment of undigested debris in lysosome-like organelles participating in micro- and macroautophagy-like processes (Schurigt et al. 2010). The release of digestive enzymes into the cytoplasm after rupture of membranes of lysosome-like vacuoles resulted in the significant digestion of intracellular compartments. However, the plasma membrane integrity of compound-treated promastigotes was maintained for several hours. Taken together, our results suggest that the induction of cell death in L. major by cysteine cathepsin inhibitors 13b and 13e is different from mammalian apoptosis and is caused by incomplete digestion in autophagy-related lysosome-like vacuoles. Furthermore we demonstrated in collaboration with the laboratory of Prof. G. Bringmann (www.sfb630.uni-wuerzburg.de/projects/a2_bringmann/) that Ancistrocladinium A and Ancistrocladinium B, two alkaloids isolated from tropical lianas, and their simplified chemically synthesized analogs have excellent leishmanicidal activities (Ponte-Sucre et al. 2007, Ponte-Sucre et al. 2009). N,C-coupled naphthylisochinolines (NIQ) were active against promastigotes and reduced the infection rate of amastigote-infected macrophages significantly. The IC50 values of these agents were comparable to amphotericin B. Our studies documented that the antiparasitic activities of NIQ were caused primarily by direct effects on the parasite and that the modulation of macrophage-specific effector functions played no or only a minor role (Ponte-Sucre et al. 2009). Structure-activity relationship studies demonstrated that the cationic nitrogen and the lipophilicity are important features of N,C-coupled NIQ with leishmanicidal  activity. Additionally, we demonstrated that N,C-coupled NIQ accumulated in acidic compartments and caused the formation of large cytoplasmatic vacuoles in promastigotes (Ponte-Sucre et al. 2010). The formation of large vacuoles followed by a total destruction of the cell strongly suggests an autophagic- or necrotic-like cell death. The results of our functional analyses with N,C-coupled NIQ and aziridine-2,3,-dicarboxylate-based inhibitors provide a basis for further improvement of the compound selectivity and for development of novel strategies for a more direct delivery of drugs to parasitic organelles.

The biogenesis of Leishmania major-harboring vacuoles in murine dendritic cells

Protozoa of the genus Leishmania are obligatory intracellular parasites in mammals. They invade macrophages and dendritic cells (DC) where they reside in membrane-bound compartments, called parasitophorous vacuoles (PV). Most of the present knowledge about the characteristics of PV harboring Leishmania is derived from studies with infected macrophages. Since it has been demonstrated that DC play a key role in host resistance against leishmaniasis, there is a need to understand the properties and biogenesis of the PV in Leishmania-infected DC. Therefore, we determined the appearance and distribution of endosomal and lysosomal markers at several time points after infection by fluorescence labeling and confocal laser microscopical analysis of L.major-containing bone marrow-derived DC (BMDC) at different stages of maturation. Newly formed phagosomes in DC mature rapidly into late endosomal compartments and persist in this stage. The small GTPase Rab7, which regulates late fusion processes, was found only in PV of mature BMDC and was absent in immature BMDC, suggesting an arrest of their PV biogenesis at the stage of late endosomes. Indeed, fusion assays with endocytic tracers showed that the fusion activity of L.major-harboring PV towards lysosomes is higher in mature BMDC than in immature BMDC. Importantly, the inhibition of endosome-lysosome fusion in DC is dependent upon the viability and life cycle stage of the parasite; living promastigotes blocked the fusion almost completely, whereas a considerable number of heat-killed organisms- and amastigotes-containing PV fused with lysosomes. Differences in the fusion competence of immature and mature DC may explain their distinct functional activities in the uptake, processing and presentation of microbial antigens.

Facts to leishmaniasis:

• In humans, leishmaniasis presents itself in four different forms with a broad range of clinical manifestations. All forms can have devastating consequences.
• Visceral leishmaniasis (VL), also known as kala azar, is the most severe form of the disease, which, if untreated, has a high mortality rate. It is characterized by irregular bouts of fever, substantial weight loss, swelling of the spleen and liver, and anaemia.
• Mucocutaneous leishmaniasis (MCL), or espundia, produces lesions which can lead to extensive and disfiguring destruction of mucous membranes of the nose, mouth and throat cavities.
• Cutaneous leishmaniasis (CL) can produce large numbers of skin ulcers”as many as 200 in some cases ”on the exposed parts of the body, such as the face, arms and legs, causing serious disability and leaving the patient permanently scarred.
• Diffuse cutaneous leishmaniasis (DCL) never heals spontaneously and tends to relapse after treatment. The cutaneous forms of leishmaniasis are the most common and represent 50-75% of all new cases.

Increased Prevalence

• In the last decade, regions that are endemic for leishmaniasis have expanded significantly.
• The geographic spread is due to factors related mostly to development. These include massive rural-urban migration and agro-industrial projects that bring non-immune urban dwellers into endemic rural areas. Man-made projects with environmental impact, like dams, irrigation systems and wells, as well as deforestation, also contribute to the spread of leishmaniasis.
• AIDS and other immunosuppressive conditions increase the risk of Leishmania-infected individuals to develop visceral illness. In certain areas of the world the risk of co-infection with HIV is rising due to epidemiological changes.

Geographic Distribution

• The leishmaniases are now endemic in 88 countries on five continents ”Africa, Asia, Europe, North America and South America ”with a total of 350 million people at risk. 72 are developing countries and 13 are among the least developed.
• It is estimated that worldwide 12 million people are affected by leishmaniasis; this figure includes cases with overt disease and those with no apparent symptoms. Of the 1.5-2 million new cases of leishmaniasis estimated to occur annually, only 600 000 are officially declared.
• Of the 500 000 new cases of VL which occur annually, 90% are in five countries: Bangladesh, Brazil, India, Nepal and Sudan.
• The number of annual deaths is about 80.000 worldwide.
• 90% of all cases of MCL occur in Bolivia, Brazil and Peru.
• 90% of all cases of CL occur in Afghanistan, Brazil, Iran, Peru, Saudi Arabia and Syria, with 1-1.5 million new cases reported annually worldwide.
• The geographical distribution of leishmaniasis is limited by the distribution of the sandfly, its susceptibility to cold climates and its and its tendency to take blood from humans or animals.