Lucie Bartoníčková

ZIB seminar – 12th January 2009

1 Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.2 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.

transmitted by sand fly Phlebotomus (Old World) or Lutzomia (New World)

Symptoms: fever, weight loss, damage to the spleen and liver, anaemia

Phlebotomus dubosci; WHO, TDR, Stammers

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

cutaneous form visceral form (kala-azar)skin ulcers (→scars)on exposed parts of body

Treatment: drugs based on antimony, amphotericin B

mucocutaneous form (espundia)lesions on skin+mucosa of the nose, mouth

fatal if untreated

parasites present in the internal organs

more than 90 percent of the world's cases occur in Brazil, Bangladesh, India, Nepal and Sudan

LEISHMANIASIS

more detailed maps available at: http://www.who.int/leishmaniasis/leishmaniasis_maps/en/index.html

LEISHMANIA

www.sanger.ac.uk/Info/Press/gfx/070617_leishmania_300.jpg

Domain: Eukaryota

Phylum: Euglenozoa/ Sarcomastigophorea

Class: Zoomastigophorea

Family: Trypanosomatidae

Order: Kinetoplastida

obligate intracellular protozoan parasite

invade macrophages of skin, liver, spleen and bone marrow

Leishmania promastigote (green and inset) invading a macrophage (yellow)

Research Unit for Tropical Diseases TROP

 species form host  region

L. major cutaneous zoonosis (human, rodents)

N Africa, Sahel, Middle East, W Asia

L. tropica cutaneous only humanMediterranean region, SW Asia (India)

L.(Viannia) brasiliensis

muco-cutaneus

zoonosis(human, dogs)

South America

L. donovani visceral human  E India, N + E Africa

L. infantum/ L.chagasi

visceral zoonosis(human, dogs,cats)

Meditarranean region/ Latin America

Kingdom: Protista

LIFE CYCLE OF LEISHMANIA

(© DPDx: CDC's web site for laboratory identification of parasites)

AIM

Elucidation of the role of neu in sand-fly transmitted Leishmania infection

Initial target cells for Leishmania: macroФ or neutrophils ?

(previous studies: infected neutrophils after needle injection of L. major)

In vivo imaging...

MATERIALS & METHODS

dynamic two-photon intravital microscopy (2P-IVM)

flow cytometry

Mice: C57BL/6 C57BL/6 LYS-eGFP (eGFP expression from lysosyme M promoter)C57BL/6 MHCII-eGFP

Sand flies: Phlebotomus duboscqi

Leishmania major – RFP = stable transfected promastigotes

organisms:

techniques:

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

Advantages:

- Lower energy → less damage to specimen (in particular live specimens)- longer wavelength → photons can enter deeper into specimen

Same fluorochromes as with normal fluorescence microcopy can be used.

METHODS

1) sand fly transmission

2) needle inoculation

2-4-day oldsand flies

artificial feeding:

mouse blood with L. major

13-19 days

feeding 30 min–3 h in the dark on anesthesized mice

control: uninfected sand flies of the same age

L. major infective-stage promastigotesisolated from stationary cultures (4-5-day old)

intradermal injection

specified number of parasites

rel. low & variable number of parasites

1a) Host response at the site of sand fly bite

Numbers of phagocytes recruited into the ear

infected x uninfected sand flies feeding on C57BL/6 mice

neutrophilic infiltration & recruitment of macroΦ regardless of the presence of parasite

Flo

w c

yto

met

ric

anal

ysis

1b) Visualisation of the bite site: recruitment of neu

neutrophils - CD11b+Gr-1+7/4+F4/80- MHCII- Ly6G+

macroФ – CD11b+F4/80+ MHCII+Gr-1-

monocytes - CD11b+F4/80+ MHCII- Gr-1-

Ear sections from LYS-eGFP micebitten with

eGFPhi neutrophils accumulate at bite sites ( )

eGFPhi = neutrophils

eGFPlo = macroΦ/monocytes

ear

cells

infected x uninfected sand flies, LYS-eGFP mice (= lysosyme M promoter)

neutrophils started to accumulate around the

bite sites 40 min after exposure to

sand flies

1b) Visualisation of the bite site: recruitment of neu

2P-IVM, infected x uninfected sand flies, LYS-eGFP mice 2P

-IV

M im

ages

ST

AR

TIN

G 4

0 m

in A

FT

ER

EX

PO

SU

RE

circles = sites of bite

1c) Visualisation of the bite site: parasite phagocytosis by neutrophils

See also: MOVIE s5 or s6

2P-IVM time-lapse image of the ear

3h after exposure to L.m.-RFP infected sand flies

Neutrophiles with phagocytosed Leishmania ( )

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

5.105 L. major promastigotes in 10 μl

exposure to 4 infected sand flies for 3h

comparible early recruitment of neutrophils

only sand fly bite maintains neu at site

2b) Recruitment of neutrophils following needle inoculation:independent of parasite

i.d. injection: PBS x L. major

Flo

w a

nal

ysis

106 L.major7.105 L.m.

fluorescent beads x L. major-RFP

2P-I

VM

imag

es f

rom

ear

sta

rtin

g

30s

po

st in

ocu

lati

on

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

Neutrophil extravasation

Cell migration paths(from 3 indep.exper.)

normalized for their origin & position relative to the

parasite deposition

LYS-eGFP mice, 2P-IVM 30 min p.i. with 104 L.m.-RFP

Neutrophil migration + phagocytosis of parasites

Phagocytosis concurrent with migration arrest

4) L. major transitions from neu to macroФafter intradermal inoculation of L.major-RFP

RFP+ gated cells from ears of MHCII-eGFP mice

6 d

ays

(B)

o

r

18h

(A

)

p.i.

wit

h 5

.106

L. m

.-R

FP

Mean of the ratio of

RFP+ infected neu

to RFP+ infected macroΦ/mono

neutrophils - CD11b+Gr-1+ MHCII- → ↓macroФ – CD11b+ MHCII+ Gr-1- → ↑

over time

6 days

18 h

5) Neutrophils harbour viable parasites and promote productive infections

WT mice injected in the ear with 103:

Number of viable parasites per 2500 neu (RFP- or RFP+)

(triplicate samples) infected neutrophils

Parasite load in individual ears/ pooled draining lymph nodes

21 days after inj.

Mean ear lesion diameter(n = 8)

6a) Neutrophil depletion with monoclonal Ab

GL113 = control AbRB6-8C5 = neutrophil depleting Ab

Treatment with monoAb 16h before exposure

MacroΦ/mono remained unaffected

neutrophils - CD11b+F4/80- Ly6G+

macroФ/mono – CD11b+ F4/80+ Ly6G-

CD11b+gated cells on day 1 p.i.

Total number of phagocytes per ear

6b) Neutrophil depletion reduced number of parasites

GL113 = control AbRB6-8C5 = neutrophil depleting Ab

Treatment with monoAb 16h before exposure to infected sand fly

Viable L. m. detected per ear

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

- 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

Hypotheses explaining reduced disease after neu depletion:

Neutrophil Soldiers or Trojan Horses?

Beena J & Christopher A. H. (15 August 2008) Science 321 (5891), 917

neu (containing parasites) undergo apoptosis

and are phagocytosed

silent entry of parasites into macroФ

anti-inflammatory environment created by the

uptake of apoptotic neu

parasites released from dying neu are engulfed, but not killed by macroФ

Thank you for your attention!

7) Response to needle inoculation after neu depletion

Number of infected (RFP+) macroФ/mono Total number of macroФ/mono

h post infection

i.d. inoculation of 105 L.m. in Ab-treated C57BL/6 mice

MacroФs may be compromised in microbicidal function by engagement in clearing apoptic

neutrophils

GL113 = control AbRB6-8C5 = neutrophil depleting Ab

Increase in the spontaneus release of IL-1 and 1β by ear cells from neutrophil depleted animals

?