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 vivoLabelling 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 cellsTransfer 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 tracersInjection 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

1. Sporozoites enter liver through liver sinusoides

2. Gliding along the sinusoidal epithelium

3. Enter and pass through Kupffer cells into the liver parenchyma

Plasmodium gliding along liver sinusoid &Encounter of Kupffer cell

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

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

Neutrophils accumulate near bites Initially most of the parasites remained viable inneutrophils

Release of parasites by apoptotic neutrophils

Later recruited macrophages take up apoptotic neutrophils and parasites

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

Only definitive host for T. gondii Felidae

Intermediate host bird, rodents, pigs

Uptake by contaminated food, soil, water

Localization in neuronal and muscle tissue Development of cysts

T. gondii in the brainEstablishment 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 infectionin 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

Neutrophils exhibit biphasic swarming formation in the SCS following T. gondii infection driven by neutrophil derived chemoattractants

Stage 1:Clustering of some neutrophils (GFP expressing)Stage 2: Migration of large numbers of neutrophils

Recruitment of neutrophils to SCS

Red = T. gondii, green = CD8 T-cells, orange = CD169+ macrophages

- 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

-These T-Cells occasionally infected themselves after lysis of the target cells

Parasite takes advantage of close APC-T-Cell contact

Adaptive immune response after T. gondii infectionin lymph nodes

Vesicular stomatitis virus (VSV)

Visualization of fluorescently labelled virusesin vivoVSV 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 = BCGgreen = Kupffer cells

Davis, Immunity 28; 146-148, 2008

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

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)