Download L. major

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