Leishmaniasis is a vector-borne neglected disease ranked among the six most important tropical infectious diseases (http://www.who.int/leishmaniasis/en/). This disease is associated with a spectrum of clinical manifestations ranging from self-healing cutaneous lesions to fatal visceral infections, principally depending on the parasite species and the immune status of the infected mammalian host. It is the second-largest parasitic killer in the world (after malaria) threatening about 350 million people in 88 countries, particularly in Africa, Latin America, South and Central Asia, the Middle East and the Mediterranean Basin.
Leishmaniasis is caused by several species of parasitic protozoa belonging to the genus Leishmania that are transmitted to mammalian host by various species of phlebotomine sand flies during a blood meal. The parasite is inoculated into the host dermis as extracellular flagellated motile metacyclics promastigote, the highly infectious parasite form. There, promastigotes infect mainly phagocytic cells in which they differentiate into oval shaped amastigotes, the obligatory intracellular form of the parasite.
Visceral Leishmaniasis (VL) is the most severe form of Leishmaniasis in humans, characterized by high fever, substantial weight loss, swelling of the spleen and liver, and anemia. The disease has a fatality rate as high as 100% within two years in developing countries if left untreated (http://www.who.int/leishmaniasis/epidemic/en/). This disease is caused by Leishmania donovani, a parasite species that has the astonishing ability to migrate and establish into visceral organs such as liver, spleen and bone marrow. In these organs parasites proliferate and/or achieve a state of long-lived persistence giving rise either to the acute and symtomatic form of VL or to the chronic and asymptomatic form of the disease. L. donovani is responsible for 500.000 cases of new infections per year. During the past 10 years, the endemic regions have been spreading with a massive increase in the number of recorded cases of VL. Significantly, the number of infected people is underestimated since VL often develops as a chronic/asymptomatic infection. Therefore such patients are not tested for VL, considered as negative and consequently they constitute dangerous reservoir of parasites.
VL recently is becoming an emerging opportunistic disease (a category I emerging disease according to the WHO) throughout the world mainly due to its association with HIV infection. For instance today, up to 70% of cases of VL in adults are associated with HIV infection in southern Europe. HIV-infected people are particularly vulnerable to VL, increasing dramatically the human reservoir of Leishmania parasites. In turn, VL infection accelerates HIV replication and progression to AIDS. In areas endemic for VL, many people have asymptomatic infection and concomitant HIV infection, which dramatically increase the risk of developing active VL. The impact of co-infection will be devastating in the near future, with important clinical, diagnostic and epidemiological implications, especially in the Indian subcontinent and Sudan, and possibly in Brazil and the Mediterranean, where it may change the epidemiology of visceral leishmaniasis.
The aim of our team is to study the complex relationships that develop between Leishmania donovani parasites and its mammalian host during the infectious process and elucidate the key aspects of host-parasite interactions that drive pathogenesis. For this purpose we use a highly interdisciplinary approach combining molecular parasitology, cellular biology and immunology together with state of the art microscopy imaging technologies and address three central questions:
The liver constitutes a primary target for bloodborne pathogens. It represents an important site of visceral/chronic infection in part due to its location at the intersection of the conventional arterial system and two venous systems such that 30% of the total blood passes through this organ every minute, 80% of which coming from the gastrointestinal tract. Little information is available about the mechanisms of liver immuno-surveillance for hepatotropic pathogens. From an immunological perspective, the liver shows many original and interesting aspects, which markedly distinguish this organ from the conventional primary and secondary lymphoid organs. The profile of hepatic immune cells is unique. It contains several types of resident antigen presenting cells including Kupffer cells, hepatic dendritic cells, sinusoidal endothelial cells and ITO cells and an unusual intrahepatic lymphocyte composition, which differs from the lymphoid organs. The liver is selectively enriched in “innate-like” lymphocytes such as invariant NKT cells (iNKT) representing up to 30% of hepatic lymphocytes while present at trace levels in primary lymphoid organs. iNKT cells share structural and functional features with innate NK cells and express a conserved TCR which recognizes glycolipid antigens in the context of the non polymorphic MHC class I-like molecule, CD1. iNKT cells display “innate-like” characteristics such that they produce very rapidly upon activation large amounts of various types of cytokines, including those within the Th1-, Th2- and Th17-type groups as well as numerous chemokines which dramatically influence the ensueing adaptive immune response.
Based on the characteristics and the previously identified role of iNKT cells in several types of immune regulation including infectious diseases, the goal of our team is to decipher iNKT cell behavior during infection by the hepatotropic pathogen, Leishmania donovani.
Infection of vertebrate host cells by flagellated Leishmania promastigote parasites remains poorly characterized. Axis 1 focuses on the analysis of the encounter between Leishmania donovani promastigote and its specific host phagocytic cells from the initial moment of parasite attachment to the establishment of intracellular infection using real time, static and high resolutive microscopy.
The goal of Axis 1 is to elucidate the role of parasite motility and host cell lysosomes at the early stages of the host cell infection process.
VL is a disease that may be “acute/symptomatic” or “chronic/asymptomatic”, depending on both the infectious parasites and the immune status of the host. Using different Leishmania donovani parasite isolates we have established two distinct and novel experimental mouse models of VL in initially susceptible but self-curing wild type C57BL/6 mice. These two models reproduce important clinical symptoms featuring independently the two forms of VL expressed in human, mimicking either the acute or the chronic form of Leishmaniasis.
The “acute/symptomatic” VL mouse model, is characterized by hepato-splenomegaly, massive cell infiltration and intra-tissular parasite multiplication. The acute phase is eventually followed by an asymptomatic period associated with the presence of persistent intra-tissular parasites. In contrast, “chronic/asymptomatic” mouse VL, is characterized by a lack of clinical symptoms but is associated with significant number of long-term persistent parasites in visceral organs.
The goal of Axis 2 is to study the hepatic immune response and more specifically determine the function of liver iNKT cells in “acute/symptomatic” versus “chronic/asymptomatic” mouse model of VL.
The liver is a favorable organ for intra-vital microscopy. After anesthesia and small incision of the abdomen, the liver can be gently pulled out and placed in the view of a microscope lens. One major advantage of this organ as compared to spleen and lymph node resides in its homogenous 3D architecture/anatomy, which allows the use of a conventional scanning or spinning disk microscope to perform intra vital imaging. The IHU institute, has acquired a spinning disk confocal microscope specifically dedicated to this present project. This spinning disk is equipped with ultra-rapid camera and a CO2 heated chamber and has been placed in a Biosafety level 3 laboratory to insure optimal hygiene and security conditions while performing intravital imaging of L. donovani- infected mice.
The goal of Axis 3 is to reveal NKT cell dynamics and functions during the infectious process.
Movie Legend: Intravital imaging was performed in the liver from CXCR6 gfp/gfp C57BL/6 mice using a scanning confocal microscope (SP5, leica). Objective 63x. Red: blood flow labeled by injection of BSA-Alexa 647; green: NKT cells.
1. Claire-Lise Forestier * et al. Exosome-like vesicles export the Leishmania virulence factor LPG and control the production of IFNg by iNKT cells. Under Review. * Corresponding author
2. Claire-Lise Forestier. Imaging host-Leishmania interactions: significance in visceral Leishmaniasis. Parasite Immunology. 2013.
3. C.Loussert, Claire-Lise Forestier and Bruno M. Humbel. Correlative Light and Electron Microscopy in Parasite Research. Methods Cell Biology. 2012. 111:59-73.
4. Claire-Lise Forestier * et al. Dynamic and correlative imaging reveals intracellular parasite movement, lysosome recruitment and host cell wounding during infection by Leishmania donovani. Cell Host Microbe. 2011 Apr 21;9(4):319-30. * Corresponding author