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Dynamic imaging of host-pathogen interactions
in vivo
Janine L. Coombes & Ellen A Robey
Nature Reviews, 2010
Methods for imaging
1.
Confocal imaging
+ 3D images
+ sharp images of thick samples
at various depths
- Limited depth of tissue which
can be penetrated
2. Two-photon laser-scanning microscopy
+ increased tissue penetration
+ decreased photodamage
+ identification of characteristic tissue
structures
+ time-lapse imaging of living tissues
Approaches used to identify host cell structures for
time-lapse imaging in situ
1. Expression of fluorescent reporters using cell-type-specific promoters
2. Adoptive transfer of labelled host cells in vivo
Labelling of cells or isolation of cells from mice expressing fluorescent proteins
ubiquitously. Transferred host cell population has to migrate to appropriate location
in vivo
3. Illumination of stromal cells
Transfer of non-fluorescent bone marrow into mice expressing fluorescent protein
ubiquitously
4. Endogenous signals
- non-centrosymmetric structures (collagen-rich structures)
- Autofluorescence
5. Injection of vascular tracers
Injection of quantum dot tracers or antibodies
Time-lapse imaging in mammalian in vivo infection models
Studies regarding the initial encounter,first encounters in lymph nodes, priming of
adaptive immune response & T-cell responses in peripheral tissues
Plasmodium
- Obligate eukaryotic parasites
- etiological agent of malaria
- 300-500 million cases of debilitating or fatal disease worldwide
Invasion and migration
The liver stage of Plasmodium infection
Plasmodium gliding along liver sinusoid &
Encounter of Kupffer cell
1. Sporozoites enter liver through liver sinusoides
2. Gliding along the sinusoidal epithelium
3. Enter and pass through Kupffer cells into the liver parenchyma
The liver stage of Plasmodium infection
4. Invasion of hepatocytes
5. Development into exo-erythrocytic form (Merozoite)
6. Merozoites are released into the blood by budding off from infected hepatocytes in form of
vesicels with host-cell derived membranes (Merosome)
Merozoites highly susceptible to phagocytosis  Merosomes protect them from
phagocytosis
Recognition of Plasmodium by T-cells
Malaria infection results in impaired responsiveness to secondary infection
Uptake of malaria pigment haemozoin by DCs is thought to contribute to suppression of
immune system, but mechanism unknown
 TPLSM study!!
Interactions of DCs and CFSE-labelled T-cells after secondary challenge with
Plasmodium observed
 Decrease of T-cell avarage speed less pronounced in malaria infected mice, although
T-cells still upregulated early activation markers
 T-cells still recognize antigen but fail to form stable contacts
Leishmania major
Studies with
LysM-EGFP neutrophils
Neutrophils accumulate near bites
Initially most of the parasites remained viable in
neutrophils
Release of parasites by apoptotic neutrophils
Later recruited macrophages take up
apoptotic neutrophils and parasites
 Depletion of Neutrophils decreases infection level  Neutrophil response to tissue
injury enhances survival and replication by allowing L. major to reach ist preferred host,
the macrophages
T-cell responses to L. major in the skin
Visualization of CD4+ T effector cells after L. major infection
-Distribution of T-cells not uniform, some infected areas extensively patrolled while others
were poorly accessible
- Many infected cells failed to make contacts with T-cells even when they were in close proximity
Toxoplasma gondii
Localization in neuronal and muscle tissue
Development of cysts
Only definitive host for
T. gondii Felidae
Intermediate host bird, rodents, pigs
Uptake by contaminated food, soil, water
T. gondii in the brain
Establishment of chronic infections accompanied by conversion into slowly dividing bradyzoites
that form cysts in the brain
Red = T. gondii, green = T-Cell
- CD8+T-cells in the brain ignore intact cysts, but migrate more slowly in the vicinity of isolated
parasites
- Rather than forming only one-to-one contacts with individual antigen presenting cells,
antigenspecific CD8+ T cells were seen to interact with granuloma-like aggregates of CD11b+
(Macrophages, dendritic cells)
- No further slowing when approaching a parasite within an aggregate  entire granuloma-like
structure, rather than an individual infected cell, may be the antigen-presenting unit in this setting.
Innate immune response after T. gondii infection
in lymph nodes
Important role of CD169+ macrophages in SCS  Poorly endocytic and degradative but important role
in trapping antigen and limiting pathogen dissemination
T. gondii accumulates in the subcapsular sinus (SCS) region of the lymph node after infection and
encounters CD169+ macrophages there
In case of T. gondii trapping also exposes CD169+ macrophages to invasion. Within one hour they were
found in parasitophorous vacuoles within macrophages
Recruitment of neutrophils to SCS
Stage 1:
Clustering of some neutrophils (GFP expressing)
Stage 2:
Migration of large numbers of neutrophils
Neutrophils exhibit biphasic swarming formation
in the SCS following T. gondii infection driven by
neutrophil derived chemoattractants
Adaptive immune response after T. gondii infection
in lymph nodes
- T-Cells initially encounter parasite
antigens in the subcapsular region of the
lymph node
- Naive and memory CD8+ T-cells form
clusters around infected CD169+
macrophages
Red = T. gondii, green = CD8 T-cells, orange = CD169+
macrophages
-These T-Cells occasionally infected
themselves after lysis of the target cells
 Parasite takes advantage of close
APC-T-Cell contact
Vesicular stomatitis virus (VSV)
Visualization of fluorescently labelled viruses
in vivo
VSV accumulates in discrete patches in the
SCS after subcutaneous injection
SCS macrophages can also
extend into the lumen
TPSLM study suggests that
virus is transported along the
surface of the macrophages
Presentation of viruses to
B-cells in the superficial follicle
Bacille Calmette-Guérin (BCG)
Strain of live attenuated Mycobacterium bovis, used for vaccination against M. tuberculosis
red = BCG
green = Kupffer cells
Stages of BCG granuloma formation in the mouse liver
Macrophages in granulomas largely non-motile
T-cell highly motile but restricted within the grauloma
Few T-cells were entering or leaving mature granuloma
Davis, Immunity 28; 146-148, 2008
 Macrophages provide a scaffold over which T-cells
migrate
Conclusions & Summary
 New techniques allow in situ / in vivo imaging of a big range of previously
inaccessible tissues
 Time lapse imaging it is possible to study interactions in real time
 Imaging possible with a broad range of pathogen (protozoa, bacteria, viruses)
 Many unknown or under-appreciated facets of host-pathogen interactions
revealed
 Challenge is now to study host-pathogen interactions in humans (tissue
explants, humanized mouse models)