Dynamic imaging of host-pathogen interactions in vivo

1. Dynamic imaging of host-pathogen interactionsin vivo Janine L. Coombes & Ellen A Robey Nature Reviews, 2010

2. Methods for imaging 2. Two-photon laser-scanning microscopy • Confocal imaging • + 3D images • + sharp images of thick samples at various depths • - Limited depth of tissue which can be penetrated + increased tissue penetration + decreased photodamage + identification of characteristic tissue structures + time-lapse imaging of living tissues

3. 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

4. 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

5. Plasmodium • Obligate eukaryotic parasites • etiological agent of malaria • 300-500 million cases of debilitating or fatal disease worldwide • Invasion and migration

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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.

13. 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 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

14. Adaptive immune response after T. gondii infectionin 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 • These T-Cells occasionally infected themselves after lysis of the target cells •  Parasite takes advantage of close APC-T-Cell contact Red = T. gondii, green = CD8 T-cells, orange = CD169+ macrophages

15. 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

16. 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  Macrophages provide a scaffold over which T-cells migrate Davis, Immunity 28; 146-148, 2008

17. 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)