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1 Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA. Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA. 3 Laboratory of Bacterial, Parasitic, and Unconventional Agents, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD 20892, USA. 4 Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA. 2 Lucie Bartoníčková ZIB seminar – 12th January 2009 LEISHMANIASIS Synonyms: Orient Boils, Baghdad Boil, kala azar (Hindi for “black fever” ), sandfly disease, Dum-Dum fever, in Latin America also espundia and Chiclero's disease transmitted by sand fly Phlebotomus (Old World) or Lutzomia (New World) Phlebotomus dubosci; WHO, TDR, Stammers cutaneous form skin ulcers (→scars)on exposed parts of body mucocutaneous form (espundia) lesions on skin+mucosa of the nose, mouth visceral form (kala-azar) parasites present in the internal organs Symptoms: fever, weight loss, damage to the spleen and liver, anaemia fatal if untreated Treatment: drugs based on antimony, amphotericin B LEISHMANIASIS more than 90 percent of the world's cases occur in Brazil, Bangladesh, India, Nepal and Sudan more detailed maps available at: http://www.who.int/leishmaniasis/leishmaniasis_maps/en/index.html LEISHMANIA obligate intracellular protozoan parasite Domain: Eukaryota Kingdom: Protista Phylum: Euglenozoa/ Sarcomastigophorea Class: Zoomastigophorea Order: Kinetoplastida Family: Trypanosomatidae www.sanger.ac.uk/Info/Press/gfx/070617_leishmania_300.jpg Leishmania promastigote (green and inset) invading a macrophage (yellow) invade macrophages of skin, liver, spleen and bone marrow species Research Unit for Tropical Diseases TROP form host zoonosis L. major cutaneous L. tropica cutaneous only human L.(Viannia) brasiliensis mucocutaneus (human, dogs) L. donovani visceral human L. infantum/ L.chagasi visceral (human, rodents) zoonosis zoonosis (human, dogs,cats) region N Africa, Sahel, Middle East, W Asia Mediterranean region, SW Asia (India) South America E India, N + E Africa Meditarranean region/ Latin America LIFE CYCLE OF LEISHMANIA (© DPDx: CDC's web site for laboratory identification of parasites) Initial target cells for Leishmania: macroФ or neutrophils ? (previous studies: infected neutrophils after needle injection of L. major) AIM Elucidation of the role of neu in sand-fly transmitted Leishmania infection In vivo imaging... MATERIALS & METHODS techniques: dynamic two-photon intravital microscopy (2P-IVM) flow cytometry organisms: Leishmania major – RFP = stable transfected promastigotes Sand flies: Phlebotomus duboscqi Mice: C57BL/6 C57BL/6 LYS-eGFP (eGFP expression from lysosyme M promoter) C57BL/6 MHCII-eGFP Two Photon Confocal Microscopy One Photon CM Two Photon CM Excitation only at one focal point One Photon CM Two Photon CM used for live (or fixed) specimens Two identical laser beams of separate lasers have to hit the same point of the specimen at the exact same time sapphire lasers - low energy, long wavelength IR light Same fluorochromes as with normal fluorescence microcopy can be used. Advantages: - Lower energy → less damage to specimen (in particular live specimens) - longer wavelength → photons can enter deeper into specimen METHODS 1) sand fly transmission feeding 30 min–3 h in the dark on anesthesized mice 2-4-day old sand flies rel. low & variable number of parasites 13-19 days artificial feeding: mouse blood with L. major control: uninfected sand flies of the same age 2) needle inoculation intradermal injection specified number of parasites L. major infective-stage promastigotes isolated from stationary cultures (4-5-day old) 1a) Host response at the site of sand fly bite infected x uninfected sand flies feeding on C57BL/6 mice Flow cytometric analysis Numbers of phagocytes recruited into the ear neutrophilic infiltration & recruitment of macroΦ regardless of the presence of parasite 1b) Visualisation of the bite site: recruitment of neu infected x uninfected sand flies, LYS-eGFP mice (= lysosyme M promoter) Ear sections from LYS-eGFP mice bitten with eGFPhi = neutrophils eGFPlo = macroΦ/monocytes - - ear cells neutrophils - CD11b+Gr-1+7/4+F4/80 MHCII Ly6G+ macroФ – CD11b+F4/80+ MHCII+Gr-1 monocytes - CD11b+F4/80+ MHCII Gr-1 eGFPhi neutrophils accumulate at bite sites ( ) 1b) Visualisation of the bite site: recruitment of neu 2P-IVM images STARTING 40 min AFTER EXPOSURE 2P-IVM, infected x uninfected sand flies, LYS-eGFP mice circles = sites of bite neutrophils started to accumulate around the bite sites 40 min after exposure to sand flies 1c) Visualisation of the bite site: parasite phagocytosis by neutrophils 2P-IVM time-lapse image of the ear 3h after exposure to L.m.-RFP infected sand flies Neutrophiles with phagocytosed Leishmania ( ) See also: MOVIE s5 or s6 http://www.sciencemag.org/cgi/content/full/321/5891/970/DC1 2a) Response to needle inoculation vs infected sand fly bite Total number of neutrophils recruited per ear exposure to 4 infected sand flies for 3h 5.105 L. major promastigotes in 10 μl comparible early recruitment of neutrophils only sand fly bite maintains neu at site 2b) Recruitment of neutrophils following needle inoculation: independent of parasite fluorescent beads x L. major-RFP Flow analysis 2P-IVM images from ear starting 30s post inoculation i.d. injection: PBS x L. major 7.105 106 L.major L.m. LYS-eGFP mice 3a) Analysis of neu recruitment + parasite uptake after intradermal inoculation of L.major-RFP promastigotes RFP+ gated cells from ears of LYS-eGFP mice 2 h p.i. with 5.105 L. m.-RFP neutrophils - CD11bhiGr-1hi eGFPhi 3b) Neutrophil recruitment + parasite uptake after intradermal inoculation of L.major-RFP LYS-eGFP mice, 2P-IVM 30 min p.i. with 104 L.m.-RFP Neutrophil extravasation Neutrophil migration + phagocytosis of parasites Cell migration paths (from 3 indep.exper.) normalized for their origin & position relative to the parasite deposition Phagocytosis concurrent with migration arrest 4) L. major transitions from neu to macroФ after intradermal inoculation of L.major-RFP Mean of the ratio of RFP+ infected neu to RFP+ infected macroΦ/mono 18 h 6 days 6 days (B) or 18h (A) p.i. with 5.106 L. m.-RFP RFP+ gated cells from ears of MHCII-eGFP mice neutrophils - CD11b+Gr-1+ MHCII- → ↓ over time macroФ – CD11b+ MHCII+ Gr-1→↑ 5) Neutrophils harbour viable parasites and promote productive infections Number of viable parasites per 2500 neu (RFP- or RFP+) Parasite load in individual ears/ pooled draining lymph nodes 21 days after inj. WT mice injected in the ear with 103: (triplicate samples) infected neutrophils Mean ear lesion diameter (n = 8) 6a) Neutrophil depletion with monoclonal Ab CD11b+gated cells on day 1 p.i. Treatment with monoAb 16h before exposure GL113 = control Ab RB6-8C5 = neutrophil depleting Ab Total number of phagocytes per ear neutrophils - CD11b+F4/80- Ly6G+ macroФ/mono – CD11b+ F4/80+ Ly6G- MacroΦ/mono remained unaffected 6b) Neutrophil depletion reduced number of parasites Viable L. m. detected per ear Treatment with monoAb 16h before exposure to infected sand fly GL113 = control Ab RB6-8C5 = neutrophil depleting Ab Incidence of ears with detectable parasites SUMMARY • rapid infiltration of neu at sand fly bite sites • neu efficiently captured L.major early after inoculation • phagocytosed L. major remained viable • infected neu efficiently inniciated infection • neu depletion reduced establishment of productive infections Hypotheses explaining reduced disease after neu depletion: - neu may rescue parasites from death in extracel spaces - neu may release parasites better adapted for macroФ uptake & survival - macroФ may be compromised in microbicidal function by engagement in clearing apoptic neu Neutrophil Soldiers or Trojan Horses? neu (containing parasites) undergo apoptosis and are phagocytosed anti-inflammatory environment created by the uptake of apoptotic neu silent entry of parasites into macroФ parasites released from dying neu are engulfed, but not killed by macroФ Beena J & Christopher A. H. (15 August 2008) Science 321 (5891), 917 Thank you for your attention! 7) Response to needle inoculation after neu depletion i.d. inoculation of 105 L.m. in Ab-treated C57BL/6 mice Number of infected (RFP+) macroФ/mono h post infection Total number of macroФ/mono ? MacroФs may be compromised in microbicidal function by engagement in clearing apoptic neutrophils Increase in the spontaneus release of IL-1 and 1β by ear cells from neutrophil depleted animals GL113 = control Ab RB6-8C5 = neutrophil depleting Ab