Download Dynamic imaging of host–pathogen interactions in vivo

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Dynamic imaging of host–pathogen
interactions in vivo
Janine L. Coombes and Ellen A. Robey
Abstract | In the past decade, advances in microscopic imaging methods, together with
the development of genetically encoded fluorescent reporters, have made it possible to
directly visualize the behaviour of cells in living tissues. At the same time, immunologists
have been turning their attention from the traditional focus on responses to model
antigens to a new focus on in vivo infection models. Recently, these two trends have
intersected with exciting results. Here we discuss how dynamic imaging of in vivo
infection has revealed fascinating and unexpected details of host–pathogen interactions
at a new level of spatial and temporal resolution.
Confocal microscopy
A focused beam of light is
scanned across the sample.
Emitted light not from the focal
point of the lens on the tissue
is prevented from reaching the
detector by a pinhole, reducing
out-of-focus signal. The
microscope obtains a series
of in focus images at varying
tissue depths, termed optical
sectioning.
Two-photon laser-scanning
microscopy
(TPLSM). An imaging method
based on the excitation of
fluorophores by absorption of
energy from two photons, with
each photon contributing half
of the energy required for
excitation. Consequently, each
photon is twice the wavelength
that would be required for
single photon excitation
leading to deeper tissue
penetration compared to
other imaging methods.
Department of Molecular and
Cell Biology, Life Sciences
Addition, University of
California, Berkeley,
California 94720, USA.
Correspondence to E.A.R.
e‑mail: [email protected]
doi:10.1038/nri2746
Published online 16 April 2010
The interaction between pathogens and the host immune
system is multilayered, with each player having to recognize, respond and adapt to the other. Pathogens have
evolved strategies to manipulate and evade host immune
responses to optimize their survival or transmission.
For example, Streptococcus pyogenes produces virulence
factors that can modulate the recruitment of immune
cells to the site of infection, whereas Toxoplasma gondii
is thought to use the migratory pathways of immune
cells to spread throughout the body 1–4. Meanwhile, the
host immune system must balance the requirement
to control the pathogen with the potential for damaging its own tissues. Further complexity occurs when
the influence of the local tissue environment on both
immune cells and the invading pathogen is taken into
account. For example, bacterial pathogens might use
two component regulatory systems to sense their environment and alter the expression of virulence factors,
whereas the function of dendritic cells (DCs) can be
altered in response to epithelial cell-derived factors5,6.
An understanding of how the tissue environment influences the interaction between the host and
pathogens is crucial for the development of effective
vaccines and therapies. To this end, there are many
useful animal infection models that allow the study of
immune responses occurring in physiologically relevant tissues. Using such models, ex vivo assays and
static imaging studies have taught us much about the
interaction between the host and pathogens. However,
as we discuss in this Review, the dynamic behaviour
of a cell or pathogen in an intact living tissue has the
potential to reveal much more. Dynamic in situ imaging can be achieved using widefield epifluorescence
microscopy or confocal microscopy , but both techniques are limited with respect to the depth of tissue
that can be penetrated, which limits analysis to surface
events. Recently, two-photon laser-scanning microscopy
(TPLSM) has been used to image immune responses
in intact tissues, providing increased tissue penetration
and decreased photodamage and improving the ability to carry out time-lapse imaging of living tissues7.
An additional advantage of TPLSM over other types
of fluorescence microscopy is the generation of second
harmonic signals that allow the identification of characteristic tissue structures, such as lymph node capsules
and reticular fibres (BOX 1). Combined with methods
for the identification and tracking of specified host cells
and structures, TPLSM studies have provided surprising new insights into how immune cells interact with
their environment, and one another, and what the functional outcomes of such interactions are (BOX 1). For
example, in the lymph nodes, T cells were shown to
migrate along a network of fibroblastic reticular cells
with which DCs are also associated8–10. This behaviour
is likely to have important implications for the ability
of T cells to survey the lymph node for antigen.
Methods for objectively quantifying cell motility
and cell–cell interactions further increase the value of
dynamic in situ imaging. Image analysis software can
be used to track individual cells in the three dimensional area being imaged over time. Parameters such
as the average speed of the cell and how long it spends
in contact with defined cells or structures can then be
measured. Furthermore, time-lapse imaging is often
most informative when it is combined with other
approaches that allow investigation of the functional
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Box 1 | Approaches used to identify host cells and structures for time-lapse imaging in situ
Expression of fluorescent reporters using cell-type-specific promoters
• CD11c promoter driving expression of enhanced yellow fluorescent protein (CD11c–EYFP), which marks dendritic
cells4,15,40,51,57,60. However, not all reporter-expressing cells are surface CD11c positive.
• Lysozyme M promoter driving expression of enhanced green fluorescent protein (LysM–EGFP), which marks
neutrophils, monocytes and macrophages. Neutrophils express higher levels of the reporter and have different
morphology and motility to macrophages18,22–24,29,32,61.
• CX3C-chemokine receptor 1 (CX3CR1) promoter driving expression of EGFP (CX3CR1–EGFP), which marks monocytes
and dendritic cells62,13.
• MHC class II-expressing cells, such as B cells and CD11c+ cells, can be visualized by replacing the gene encoding the
I–Ab β-chain with a construct encoding an EGFP-tagged version (MHC class II–EGFP)14,16.
Adoptive transfer of labelled host cells in vivo
• Cell populations can be labelled using dyes, such as 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) or by
isolating them from mice expressing fluorescent proteins ubiquitously; for example, under the control of the ubiquitin
or actin promoters. Genetic labels are preferable owing to the potential for dyes to be diluted with cell division. Either
technique requires the transferred host cell population to migrate to the appropriate location after in vivo transfer. It is
typically used to introduce labelled antigen-specific T and B cells4,7,23,32,36–41,49,51,53,56–58.
Illumination of stromal cells
• Mice expressing fluorescent proteins ubiquitously can be irradiated and transferred with non-fluorescent bone marrow,
illuminating the stromal cell network8,39.
Endogenous signals
• Tissue structures such as the lymph node capsule and reticular fibres can be illuminated in two-photon laser-scanning
microscopy by the generation of second harmonic signals. This occurs when two incident photons pass through a noncentrosymmetric structure (such as collagen-rich structures) and emerge as one photon with half the incident
wavelength39,58.
• The natural autofluorescence of the tissue can also be used to define cells and structures of interest. However, these
signals lack specificity for a particular cell type and can be difficult to visualize in the presence of a brighter fluorescent
protein28,29.
Injection of vascular tracers
• Intravenous injection of quantum dot tracers can be used to identify blood vessels and follow changes in vascular
permeability22,32. Subcapsular sinus macrophages can be labelled by subcutaneous injection of antibodies specific for
CD169 taking advantage of lymphatic drainage to the lymph node capsule4,23,36.
Second harmonic signals
Signals that occur when two
incident photons pass through
a structure with no centre of
symmetry and emerge as one
photon with half the incident
wavelength. In biological
tissues this is mostly a
property of collagen.
consequences of the observations made during imaging. For example, flow cytometry of dissociated cell
populations can be used to determine how changes
in motility might correlate with changes in cell activation or cytokine production. Static imaging of tissue sections can be used to assess global changes in
the distribution of a cell population and, finally, new
observations made during the imaging process may
suggest target pathways for genetic manipulation,
allowing assessment of their role in the generation of
an optimal immune response.
However, until recently, the dynamics of the immune
response to pathogens remained under-explored.
Fortunately, improved availability of tools for labelling
and tracking of pathogens in vivo has allowed researchers to address questions such as how pathogens alter
the behaviour of infected cells, how infected cells are
recognized by the immune system and how the pathogen spreads through the host in intact tissues in real
time (FIG. 1; see Supplementary information S1, S2, S3,
S4 (movies)).
Here, we discuss how recent studies have revealed
unique features of the dynamics of the host immune
response to pathogens. Although we focus on timelapse imaging of intact tissues, key supporting data
from static imaging, analysis of dissociated ex vivo
samples and in vitro studies are also mentioned. Host–
pathogen interactions are extremely diverse and we
therefore do not attempt to provide a comprehensive
discussion of all of the interesting imaging studies that
have been published (TABLE 1). Rather, we focus on a
handful of studies that provide new insights into the
dynamics of the interaction between the host immune
system and pathogens across various locations and
infection models.
The initial encounter
The site where a pathogen breaches the hosts’ epithelial
cell defences is often the first opportunity for contact
between the host immune system and the pathogen, and
therefore it may be the first opportunity for clinical intervention. Tissue-resident populations of DCs and macrophages are among the first to encounter the pathogen
and may be involved in transport of the pathogen across
epithelial cell barriers11,12. In addition, monocytes constitutively patrol tissue blood vessels, ready to respond
rapidly to infection or tissue damage13. Subsequently,
large infiltrates of neutrophils arrive in the tissue. our
understanding of these processes — how they contribute
to the control of infection and how they are subverted by
pathogens — has been substantially advanced by recent
dynamic imaging studies.
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Aa
T. gondii
Ab
1 min
0 min
1.5 min
2 min
Release of
parasite
Cyst
Release
of parasite
Ac
20 min
19 min
BCG
0 min
B
3.5 min
7 min
Saphenous
vein
Capsule
SCS
Parenchyma
VSV
SCS floor
0 min
10 min
29 min
Figure 1 | Examples of visualizing pathogens. A | Protozoan parasites, such as
Plasmodium spp., Leishmania major and Toxoplasma gondii areNature
sufficiently
large
that
Reviews
| Immunology
individual fluorescently labelled parasites can be readily tracked within tissues using
widefield epifluorescence, confocal or two-photon laser-scanning microscopy. In these
examples, red fluorescent protein-labelled T. gondii parasites (red) were observed
invading a T cell (green) during an antigen-dependent contact with another invaded cell
in the lymph node (a) and emerging from a rupturing cyst in the brain (b). Bacteria
engineered to express fluorescent proteins have also been detected by two-photon
laser-scanning microscopy in tissues14,32. This example (c) shows uptake of
Mycobacterium bovis bacille Calmette–Guérin (BCG) (red) by Kupffer cells (green) in the
liver32. B | Bulk movement of fluorescently labelled virus particles has been visualized
in vivo39. In this example, fluorescently labelled, inactivated vesicular stomatitis virus
(VSV) particles (green) were seen to accumulate in discrete patches in the subcapsular
sinus (SCS) of draining lymph nodes within minutes of subcutaneous injection.
Virus-infected host cells have also been visualized using viruses that are engineered to
express green fluorescent protein derivatives40,68. The image in part Aa is reproduced,
with permission, from REF. 4 © (2009) Elsevier Science. The image in part Ab is
reproduced, with permission, from REF. 57 © (2009) The American Association of
Immunologists, Inc. The image in part Ac is reproduced, with permission, from REF. 32 ©
(2008) Elsevier Science. The image in part B is reproduced, with permission, from Nature
REF. 39 © (2007) Macmillan Publishers Ltd. All rights reserved.
Response of tissue-resident cells. Populations of peripheral tissue-resident innate immune cells function as
sentinels for infection and tissue damage. DCs in both
the skin and small intestine have been observed rapidly extending processes towards microorganisms
following infection, confirming earlier static imaging
studies11,12,14,15. In the intestine, the DCs (identified
using MHC class II–enhanced green fluorescent protein
(eGFP)) (BOX 1) extended processes across the epithelial
cell layer and into the lumen where they were occasionally seen to interact with non-invasive Salmonella enterica
subsp. enterica serovar Typhimurium organisms14,16. This
is consistent with the hypothesis that DC processes may
be involved in the transport of microorganisms across
the intestinal epithelium11,12,14.
Use of TPLSM of DCs in the skin (using CD11c
promoter-driven expression of yellow fluorescent protein (yFP)) (BOX 1) led to the surprising finding that,
unlike sessile Langerhans cells in the epidermis, dermal
DCs could crawl actively through the tissue. However,
following intradermal injection of Leishmania major,
dermal DCs slowed substantially, gained a more pronounced dendritic morphology and took up L. major
through the extension of dendrites15. Loss of migratory behaviour in vivo also occurred in the presence of
lipopolysaccharide15. By contrast, DCs did not acquire
a dendritic morphology in response to inert beads or
Mycobacterium bovis bacille Calmette–Guérin (BCG).
Moreover, the uptake of beads and BCG was inefficient compared with uptake of L. major. The reasons
for this difference in DC behaviour warrant further
investigation. For example, comparing the responses
that dermal DCs have towards BCG, virulent mycobacterial strains and other intradermal vaccines might
help to determine whether engineering vaccines to
provide signals that alter DC behaviour would make
them more effective.
Real-time imaging studies have revealed that DCs are
supported in their sentinel function by a population of
monocytes expressing high levels of CX3C-chemokine
receptor 1 (CX 3 CR1) (visualized using CX 3 CR1
promoter-driven GFP expression) (BOX 1). CX3CR1hi
monocytes have an unusual patrolling behaviour for
surveying blood vessels and tissues for signs of infection or damage. In the steady state, the monocytes were
observed crawling along the luminal surface of the
blood vessel endothelium, independently of the direction of blood flow13. This behaviour resulted in extensive monitoring of the surface of the vessel, but not in
extravasation of the monocytes. However, following
intraperitoneal infection with Listeria monocytogenes,
CX3CR1hi monocytes extravasated into the peritoneal
cavity with faster kinetics than neutrophils or GR1+
inflammatory monocytes and were an important early
source of tumour necrosis factor 13. Importantly, disruption of steady-state patrolling behaviour in CX3CR1deficient mice led to delayed monocyte extravasation
following infection, showing the importance of constitutive surveillance of the vessels. It will be of interest to
determine whether some pathogens target the behaviour
of CX3CR1hi monocytes to avoid detection.
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Table 1 | Time-lapse imaging in mammalian in vivo infection models
Pathogen
Imaged
tissue
Imaged host cell population
Refs
Protozoan parasites
Plasmodium spp.
Skin
Myelomonocytic cells, dermal cells
and blood vessel endothelial cells
Lymph nodes
T cells
Liver
Kupffer cells and hepatocytes
Brain
T cells
Skin
Neutrophils, dendritic cells and T cells
Lymph nodes
NK cells, T cells and dendritic cells
Lymph nodes
T cells, dendritic cells, macrophages
and neutrophils
Brain
T cells, dendritic cells and astrocytes
Vaccinia virus
Lymph nodes
T cells and dendritic cells
40
Vesicular
stomatitis virus
Lymph nodes
B cells
39
LCMV
CNS
Neutrophils, monocytes and T cells
22
Vaccinia virus
Ankara
BALT
T cells and dendritic cells
68
Mycobacterium
bovis BCG
Liver
Kupffer cells, macrophages and T cells
32
Listeria
monocytogenes
Spleen
Dendritic cells and T cells
70
Skin
Neutrophils
71
Streptococcus
pyogenes
Skin
Neutrophils
18
Escherichia coli
Kidney
Blood vessels
72
Salmonella
typhimurium
Intestine
Dendritic cells
14
Staphylococcus
aureus
Blood
Blood vessels
73
Borrelia
burgdorferi
Blood
Blood vessels
74,75
Leishmania major
Toxoplasma gondii
26–28
53
29,33–35
69
24,25,56
41
4,23,51
57,58
Viruses
Bacteria
BALT, bronchial-associated lymphoid tissue; BCG, bacille Calmette–Guérin; CNS, central
nervous system; LCMV, lymphocytic choriomeningitis virus; NK, natural killer.
Bacille Calmette–Guérin
(BCG). A strain of live
attenuated Mycobacterium
bovis used for vaccination
against Mycobacterium
tuberculosis in humans.
Subcapsular sinus
The outer region of the lymph
node where afferent lymph
first enters the lymph node.
It consists of a sponge-like
network of reticular fibroblast
cells encasing collagen fibres
and is separated from the
lymph node cortex by a layer
of sinus-lining cells and a
discontinuous basement
membrane.
Neutrophil interactions with pathogens. The initial
encounter with sentinel cells is followed quickly by the
influx of leukocytes, of which neutrophils are often
the most prominent. once present in the tissue, neutrophils have many functions, including the destruction
of microorganisms, tissue remodelling, the production of chemokines and the eventual resolution of the
inflammatory response17. Dynamic in situ imaging of
neutrophils during infection has provided new insights
into these various functions.
Imaging of neutrophil recruitment to the site of infection following bacterial infection suggests that the rate
of neutrophil extravasation is important for protection
against infection and may be subject to modification by
bacterial virulence factors18. Streptolysin S is a broadspectrum cytolysin, the expression of which might be
regulated by conditions in the tissue environment 19.
It is thought to decrease the neutrophilic infiltrate at
the site of infection through a direct cytocidal effect but
recent imaging studies have revealed another potential
mechanism18,20. TPLSM studies of LysM–eGFP mice (in
which eGFP expression is driven by the lysozyme M
promoter (BOX 1)) showed that infection with both wildtype and streptolysin S-mutant S. pyogenes led to similar levels of neutrophil rolling and firm adherence in
blood vessels, as well as similar neutrophil migration
in the infected tissue18. However, the amount of time
that the neutrophils remained in the vessels before
extravasating was substantially shorter in the presence
of the streptolysin S mutants. It is possible that streptolysin S may increase the virulence of S. pyogenes by
impairing neutrophil diapedesis and that the delay in
the arrival of neutrophils provides a short window of
opportunity for the bacteria to begin replicating and
establish a niche. This provides an excellent example
of how dynamic imaging has helped to define a precise
mechanism by which a pathogen virulence factor can
modulate the host immune response. It also suggests
that targeting signals in the local tissue environment
that modulate expression of pathogen virulence factors
could be beneficial in fine tuning the immune response
to control infection while limiting pathology.
neutrophil extravasation is known to be associated
with vascular leakage and tissue injury 21. This process has been captured in real time during lymphocytic
choriomeningitis virus (LCMv) infection of the central nervous system, in which vascular leakage results
in fatal seizures. neutrophils were observed accumulating in the vasculature before a coordinated and rapid
extravasation that was accompanied by leakage from the
vasculature of a quantum dot tracer 22 (Supplementary
information S5 (movie)). Seizure-induced death was
prevented in mice depleted of both monocytes and
neutrophils, suggesting that blocking their recruitment could be useful in preventing immune-mediated
pathology in the central nervous system. Dynamic
imaging has also provided evidence of neutrophils
altering local tissue architecture. Following T. gondii
infection, swarming of neutrophils in the lymph node
corresponded in space and time with disruption of the
layer of CD169+ macrophages in the subcapsular sinus23.
Although it remains to be determined, it is possible
that neutrophil-mediated remodelling of the extracellular matrix contributed to the migration of these cells
to other regions of the lymph node23.
Imaging the neutrophil response to L. major
infection provided insight into how a pathogen may
use neutrophils in the dermis for its own benefit 24.
A rapid and sustained recruitment of LysM–eGFPhi
neutrophils from the blood to the site of infection
was observed. neutrophils accumulated near both
infected and uninfected sand fly bites, seeming to
form plugs within them. Initially, most of the parasites were observed in neutrophils, but they were later
also found in macrophages. Interestingly, parasites in
neutrophils remained viable and infectious and were
observed being released from apoptotic neutrophils
in the vicinity of macrophages24. This real-time observation, combined with the finding that depletion of
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neutrophils decreased infection levels, suggested that
the parasite could exploit the neutrophil response to
tissue injury to enhance its survival and replication. It
has been suggested that uptake of infected neutrophils
by macrophages might allow the parasite to reach its
preferred host cell without initiating an inflammatory response24. Alternatively, the microbicidal function of the macrophages may already be compromised
by uptake of apoptotic neutrophils, enabling released
parasites to infect them more easily.
The interplay between vector, parasite and host
immune cells revealed by these studies has important
implications for vaccine design. Certain experimental, non-living vaccine strategies for leishmaniasis
are effective against needle challenge with L. major
but not against natural challenge with a sand fly bite
harbouring live parasites25. TPLSM studies revealed
that infected sand fly exposure led to a more sustained
neutrophil response than needle inoculation, suggesting that neutro phils may dampen the antiparasite
response and promote infection25. Consistent with
this explanation 25, neutrophil depletion following
sand fly challenge enhanced the protective effect
of a killed vaccine. These findings emphasize the
importance of natural challenge to assess vaccine
efficacy and the power of imaging to reveal aspects
of the host–pathogen interaction that are relevant to
vaccine design.
Liver sinusoids
Specialized blood vessels lined
by a fenestrated endothelium
and interspersed Kupffer cells.
Kupffer cells
A specialized population of
macrophages that reside in
the liver.
Granuloma
An organized structure
containing macrophages
and other immune cells.
Migration and invasion of Plasmodium spp. in the
skin. The malaria-causing parasites Plasmodium spp.
have provided some of the most rewarding infection
models for imaging studies, both because of the high
relevance for human health and because of the varied
and dynamic events that take place during the parasite
life cycle in the mammalian host (FIG. 2a,b). This cycle
begins when parasites are injected into the dermis by
the bite of an infected mosquito (FIG. 2a). Time-lapse
imaging of Plasmodium berghei sporozoites (which
infect mammals other than humans and are therefore
used in animal model studies) in the dermis of mice has
added to our appreciation of how the sporozoites disseminate from the site of infection and how this process
can be locally modulated by the host immune response
(Supplementary information S6 (movie)). Sporozoites
remained motile for some time after infection and were
seen to invade both blood and lymphatic vessels26–28.
Their motility depended on the ability to traverse host
cells, and this allowed them to avoid becoming trapped
in stromal cells or cleared by phagocytes26. non-motile
parasites rarely penetrated as far as the vasculature,
limiting dissemination.
Blood borne, P. berghei sporozoites invade the liver
rapidly, limiting the time frame during which they are
susceptible to neutralization with antibodies in the
blood76. However, these studies26–28 revealed that there
was a substantial period of time during which motile
parasites and the host immune system could interact in
the dermis, suggesting a larger window of opportunity
for vaccines that target this stage of the infection than
had previously been appreciated. In fact, when mice,
previously immunized with radiation-attenuated parasites, were challenged by mosquito bite, sporozoites lost
motility within one minute of infection28. Consistent
with this, the sporozoites did not gain access to the
vasculature in immunized mice. A similar effect was
achieved with passive transfer of antibodies28.
The liver stage of Plasmodium spp. infection
Sporozoite transit through Kupffer cells. Like many
pathogens, Plasmodium spp. disseminate from the
initial site of infection through the blood and travel
to the liver. There, the parasites enter liver sinusoids,
where they encounter specialized liver macrophages
known as Kupffer cells. when P. berghei sporozoites
entered the liver, they bound to the sinusoidal cell
layer and arrested. They were then observed gliding
along the sinusoidal endothelium until they encountered a LysM–eGFPlow Kupffer cell29 (BOX 1; FIG. 2c,d;
see Supplementary information S7 (movie)). The
sporozoites then entered and passed through the
Kupffer cells into the liver parenchyma where they
traversed through multiple hepatocytes. This realtime observation confirmed the long-held hypothesis
that Kupffer cells could act as a gateway to the liver
for Plasmodium spp. sporozoites30. The importance of
Kupffer cells for entry of the parasite into the liver is
further supported by a recent study in which a reduction in Kupffer cell numbers led to a decrease in parasite load 31. Real-time imaging has also shown that
Kupffer cells can participate in the uptake of BCG from
the blood32 (FIG. 1Ac; see Supplementary information S3
(movie)). These Kupffer cells subsequently nucleated
granuloma formation32.
How merozoites avoid phagocytosis by Kupffer cells.
After traversing several hepatocytes, Plasmodium spp.
sporozoites invade a final hepatocyte and develop into
exo-erythrocytic forms that enter exo-erythrocytic
schizogony, potentially yielding thousands of merozoites in a single infected cell (FIG. 2c,e). Merozoites are
then released into the blood where they infect erythrocytes. Kupffer cells are thought to have an important
role in this stage of infection, as merozoites are highly
susceptible to phagocytosis. However, recent intravital imaging studies have provided important details
concerning how the mouse parasites P. berghei and
Plasmodium yoelii are released from the liver and how
they can evade phagocytosis33–35. Groups of merozoites
were observed budding off from infected hepatocytes
in the form of vesicles with host cell-derived membranes termed merosomes (FIG. 2e; see Supplementary
information S8 (movie)). Merosomes reached the
blood, possibly through endothelial cell fenestrations,
and their formation was eventually followed by disintegration of the host cell. Importantly however, the merosomes did not expose phosphatidylserine on their outer
surface, protecting them from phagocytosis33,34. By contrast, disintegration or rupture of infected hepatocytes
left merozoites susceptible to phagocytosis and led to
inflammatory cell recruitment and the formation of
small granulomatous structures33.
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a
c
Mosquito
Epidermis
Dermis
Sporozoite
Blood
vessel
Sinusoidal endothelial cell
Hepatocyte
Sinusoidal lumen
Lymphatics
Lymph
node
Merozoite
Merosome
Kupffer cell
Sporozoite
Liver
Infected hepatocyte
Parasitophorous
vacuole
b
48
0
270
180
120
108
72
d
90
0–270 sec
e
Merosome
bud
282 sec
Merosome
288 sec
300 sec
Figure 2 | Dynamic imaging of Plasmodium spp. infection in mammals. a | Plasmodium spp. sporozoites are injected into
the dermis by a mosquito bite, and they migrate through the dermis before actively invading across
the endothelium
of blood
Nature
Reviews | Immunology
or lymphatic vessels26–28. b | These images show the path of a Plasmodium sporozoite migrating in the dermis, represented by
maximum intensity projection of fluorescent signal. The sporozoite glides in the dermis (gliding sporozoite shown in red)
before gliding along a blood vessel wall (green, with blood vessel marked in blue) and finally crossing the blood vessel wall
(yellow). c | After dissemination through the blood to the liver, sporozoites glide along sinusoids and enter the liver
parenchyma through Kupffer cells. After converting into merozoites in the hepatocytes, the parasites avoid being
phagocytosed by Kupffer cells by budding as membrane covered merosomes29,33–35. d | The path of a Plasmodium spp.
sporozoite (green) gliding along a liver sinusoid and then encountering a Kupffer cell (outlined with dotted line).
The sporozoite is then seen traversing the Kupffer cell and continuing its migration in the liver tissue (not depicted).
e | These images show a merosome (green) that is budding off from a green fluorescent exo-erythrocytic form into a
sinusoid (red). The image in part b is reproduced, with permission, from Nature Medicine REF. 27 © (2006) Macmillan
Publishers Ltd. All rights reserved. The image in part d is reproduced from REF. 29. The image in part e is reproduced,
with permission, from REF. 34 © (2006) American Association for the Advancement of Science.
Real-time imaging of Plasmodium infection has
undoubtedly been successful in confirming the elegant
way in which the parasite uses Kupffer cells to access
the liver but later successfully avoids a potentially lethal
interaction with this same cell type.
First encounters in lymph nodes
Encounters between pathogens and subcapsular sinus
macrophages. From the site of infection, microorganisms
and microbial particles drain to the lymph nodes and are
deposited in the subcapsular sinus. There, they interact
with a specialized subset of macrophages that protrude
through the layer of sinus-lining cells36–39. These macrophages express CD169 (sialoadhesin) and differ from
conventional macrophage populations in that they are
poorly endocytic and poorly degradative36. However, they
do have an important role in selectively trapping large
and particulate antigen that enters via the lymph with
important consequences for the generation of immune
responses and limiting pathogen dissemination36–39.
For example, fluorescently labelled inactivated vesicular stomatitis virus (vSv), injected into the footpad, rapidly accumulated on the surface of CD169+ macrophages
protruding across the subcapsular sinus floor 39 (FIG. 1B).
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Depletion of subcapsular sinus macrophages showed
their importance in retaining the virus in the lymph
node, thereby limiting its spread throughout the body.
Similarly, P. berghei travelled from the dermis to the
draining lymph node, where it was initially concentrated in the subcapsular sinus and was found associated with CD11c + cells 27. Parasites gradually lost
motility and did not penetrate further than the lymph
node, suggesting that the pathogen-trapping function
of cells in the subcapsular sinus was also important in
this model.
Although subcapsular sinus macrophages efficiently
limit pathogen dissemination, trapping of intracellular
pathogens at the cell surface may expose these cells to
invasion. within an hour of subcutaneous infection,
T. gondii arrives in the draining lymph node and is
found in CD169+ macrophages23. Many of the parasites
were surrounded by dense granule protein 6 (GRA6),
indicating that they resided in parasitophorous vacuoles and therefore had actively invaded the macrophages4. Furthermore, expression of virus-encoded GFP
by infected host cells was used to show that vaccinia
virus could also infect CD169+ macrophages following
subcutaneous infection40.
Activation of innate immunity: neutrophils and natural
killer cells. The arrival of intact viable pathogens in the
lymph node necessitates the local generation of an innate
immune response. Indeed, it seems that an innate immune
response analogous to that seen in non-lymphoid tissues
can also be initiated in the lymph nodes following the
arrival of lymph-borne pathogens. Both neutrophils and
natural killer (nK) cells circulate in the blood and can
be recruited to lymph nodes following infection. Recent
studies have revealed new information concerning the
dynamics and spatial organization of the response of
these two cell types to infection with protozoan parasites
in the lymph nodes23,41 (TABLE 1). For example, following
T. gondii infection, neutrophils formed dynamic swarms
around foci of infection in the subcapsular sinus region.
Interestingly, many swarms were initiated by clustering of
a few ‘pioneer’ neutrophils followed by migration of large
numbers of neutrophils several minutes later 23 (FIG. 3a;
see Supplementary information S9 (movie)). This suggested that the formation of neutrophil swarms was
cooperative: swarming neutrophils produced chemoattractants that brought new neutrophils to the swarm.
neutrophil swarms also formed rapidly in response to
egress of T. gondii from an infected cell23 (Supplementary
information S10 (movie)).
It is likely that a diverse range of pathogens and vaccine strains will accumulate in the subcapsular sinus and
initiate an inflammatory response. Given the profound
effect that the neutrophil response to L. major in the skin
has on vaccine efficacy, it may be informative to investigate the effect of neutrophil swarming in the lymph
node on the ability of pathogens or vaccines to elicit
adequate immune responses. It will also be important
to determine the nature of the signals that trigger this
swarming behaviour, so that they may be targeted for
the generation of an optimal immune response42,43.
nK cells have an important role in early defence against
various infectious agents. Their effector functions can be
initiated following engagement of activating cell surface
receptors or in response to cytokines produced by macrophages and DCs. In fact, nK cell activation by DCs is
thought to be cell-contact dependent, and nK cells have
been observed forming long-lasting contacts with DCs
in vitro44–46. Similarly, optimal production of interferon-γ
by nK cells following L. monocytogenes infection depends
on their ability to cluster around foci of myeloid cells in the
spleen47. Recently, the dynamics of the nK cell response
in lymph nodes and the nature of their contacts with DCs
have begun to be explored using time-lapse imaging 41,48,49.
For example, in L. major infection, nK cells interacted
with DCs in the lymph node paracortex, suggesting a
means by which they may receive activating signals41.
Priming of the adaptive immune response
The observation that lymph-borne pathogens became
associated with CD169 + subcapsular sinus macrophages suggested that the region directly beneath the
subcapsular sinus may be a previously under-appreciated
location where lymphocytes could recognize their cognate
antigen and be primed, and it indicates that the CD169+
macrophages may be involved in the optimal generation
of adaptive immune responses.
Recognition of pathogens by B cells at the subcapsular
sinus. As described in the previous section, subcapsular sinus macrophages can extend across the floor of the
subcapsular sinus into the lumen, and viruses arriving
through lymph accumulate on their surface. This allows
the macrophages to transport and present viruses to B cells
in the superficial follicle39 (FIG. 3b; see Supplementary
information S11 (movie)). TPLSM studies showed that
these macrophages were largely immobile, suggesting that
virus was transported along the surface of the cell. Specific
B cells congregated beneath the floor of the subcapsular sinus, probably as a result of receiving a stop signal
through the B cell receptor during random surveying of
macrophages in the region. Although other pathways exist,
trapping of virus by subcapsular sinus macrophages was
necessary for efficient antigen presentation to B cells soon
after infection. This might prove to be important, as even
minor alterations to the kinetics of an immune response
can affect the ability of the host to control infection.
Immune complexes and other particulate antigens
have also been shown to accumulate on the surface of
subcapsular sinus macrophages and be transported
along the cell surface into the B cell follicle36–38. Immune
complexes can then be captured by non-cognate B cells
and transported to the germinal centre. Disruption of
this process leads to impaired affinity maturation36.
Together, these imaging studies suggest a pathway for
antigen capture and transport involved in the optimal
generation of B cell responses.
Recognition of pathogens by T cells at the subcapsular sinus. The prevailing spatial and temporal model
of T cell priming in the lymph nodes is based largely
on dynamic imaging of responses to model antigens7,50.
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a
Capsule
Stage 1
Stage 2
Subcapsular
sinus
Neutrophil
Toxoplasma gondii
7 min
24 min
b
Virus
Subcapsular
sinus macrophage
Sinus-lining
cell
Virus-specific
B cell
Non-specific
B cell
c
Toxoplasma
gondii
T cell
Infected
macrophage
Dendritic
cell
0 min
13 min
25 min
Infected
T cell
Figure 3 | Dynamic imaging of immune responses in the lymph node subcapsular sinus. a | Neutrophils (expressing
enhanced green fluorescent protein driven by the lysozyme M promoter) exhibit biphasic swarm
formation
Nature
Reviewsin| the
Immunology
subcapsular sinus following Toxoplasma gondii (red) infection. The tracks of the neutrophils in stage 1 and stage 2
of swarm formation are depicted as white lines. b | Viral particles (green) accumulate on CD169+ macrophages in the
subcapsular sinus following subcutaneous injection. This leads to accumulation of virus-specific B cells (red) within and
below the subcapsular sinus floor. c | During T. gondii (red) infection, memory CD8+ T cells (green) form clusters around
uninfected dendritic cells and infected CD169+ macrophages (orange), exposing themselves to invasion by parasites. The
image in part a is reproduced, with permission, from REF. 23 © (2008) Elsevier Science. The image in part b is reproduced,
with permission, from Nature REF. 39 © (2007) Macmillan Publishers Ltd. All rights reserved. The image in part c is
reproduced, with permission, from REF. 4 © (2009) Elsevier Science.
Briefly, soluble antigens arrive first in the lymph node
and filter through a network of conduits extending into
the cortex. DCs that reside in the cortical ridge can
sample this antigen and present it to T cells arriving
through high endothelial venules. Later, antigen-loaded
tissue DCs arrive in the lymph node and also localize to
the cortical ridge, where they present antigen to T cells.
However, it remains unclear where priming of the T cell
response occurs during infection and if the location of
priming affects the outcome of the response.
Three recent studies have carried out imaging of
specific CD8+ T cell responses in the subcapsular sinus
region following infection with pathogens engineered
to express the model antigen ovalbumin4,40,51. In each
case, the pathogen studied was shown to accumulate in
the subcapsular sinus region and was found in CD169+
macrophages. This suggested that T cells might initially
encounter parasite-derived antigens in this region of
the lymph node. Indeed, both naive and memory CD8+
T cells accumulated in the region of the subcapsular sinus
following infection. In the case of T. gondii infection,
T cell relocalization was antigen independent, suggesting
that the T cells responded to chemoattractants produced
at the site of infection4,51. By contrast, in vaccinia virus
infection, redistribution of the CD8+ T cells was shown
to be more dependent on the presence of cognate antigen,
implying that retention of T cells by antigen recognition
has a role in relocalization40.
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The nature of the antigen-presenting cells (APCs)
for T cells at the subcapsular sinus is of considerable
interest. In the case of naive T cell priming by viruses,
T cells did not cluster around virus-infected CD169+
macrophages but, instead, around CD11c–yFP+ cells
present beneath the macrophage layer. These interactions seemed to result in T cell priming, as T cells
upregulated activation markers in the first 12 hours
following infection and rapidly migrated back to the
site of infection40.
For memory CD8+ T cells responding to T. gondii,
long-lived low-motility clusters formed around infected
CD169+ macrophages in an antigen-dependent manner,
suggesting that subcapsular sinus macrophages do serve
as APCs in this setting 4 (FIG. 3c; see Supplementary
information S12 (movie)). In this 4 and the related
studies40,51, T cell clusters were also observed around
CD11c–yFP+ cells that did not harbour visible fluorescent pathogens. Thus both direct presentation by
macrophages and bystander presentation by DCs
seem to contribute to T cell antigen recognition in the
subcapsular sinus.
Collectively, these studies suggest that the region of
the lymph node beneath the subcapsular sinus is a site
for early priming or reactivation of the immune response
to pathogens. It is likely that tissue microenvironments
have an important role in determining the course of an
immune response. It is therefore of substantial interest
that early CD8+ T cell responses can be activated in this
region of the lymph node. It will be important to determine whether these cells differ from those primed later
in the response or deeper within the lymph node, and
how priming in this region shapes the overall character
of the immune response.
Pathogens fight back. The presence of live pathogens
in the lymph node during priming of the adaptive
response might allow the pathogen to manipulate this
phase of the response for its own benefit. For example,
CD8+ T cells clustered around T. gondii-infected cells
were occasionally invaded themselves following lysis of
the infected target cell4 (FIGS 1A,3c; see Supplementary
information S1 (movie)). This finding implies that the
parasite may take advantage of close APC–T cell contacts to invade T cells and disseminate systemically
within them. Accordingly, T cells accounted for 50%
of infected cells in the mesenteric lymph node following oral infection4. Furthermore, blocking egress
of lymphocytes from the lymph node was effective in
reducing parasite spread4.
Pathogen products may also influence priming of
the response to subsequent challenges. Malaria infection is known to result in impaired responsiveness to
secondary infections, presenting a significant barrier
to vaccination. Uptake of the malaria pigment haemozoin by DCs is thought to contribute to suppression of
the immune response in this setting 52. TPLSM was
used to better define how plasmodium infection might
modulate DC function and, consequently, the interaction of DCs with T cells following secondary challenge53. Mice received an intraperitoneal injection with
Plasmodium chabaudi-infected erythrocytes and 12 days
later were administered 5,6-carboxyfluorescein diacetate
succinimidyl ester (CFSe)-labelled Do11.10 CD4+ T cells
and immunized with ovalbumin and lipopolysaccharide.
Immunization of uninfected animals resulted in a marked
decrease in T cell average speed and displacement. This
decrease was less pronounced in malaria-infected mice,
although T cells still upregulated early activation markers, indicating that they recognized antigen but failed
to form stable contacts with DCs. Mechanistically, this
was thought to be due to uptake of haemozoin by DCs.
Antigen-specific DC–T cell interactions were inhibited
in vivo when DCs were pulsed with haemozoin before
adoptive transfer. This is similar to decreased stable contacts formed when regulatory T cells are present 54,55. It
might therefore be informative to visualize regulatory
T cell populations in this setting.
T cell responses in peripheral tissues
During activation in lymphoid tissues, T cells receive
signals from DCs that lead to upregulation of tissuehoming receptors, allowing them to migrate to sites of
infection. Few studies have addressed the dynamics
of the interaction between T cells, infected cells and
other APCs at this stage of the immune response. How
do effector T cells migrate through inflamed tissues?
Do all infected cells have the potential to be recognized by T cells and what are the dynamics of T cell
interactions with uninfected APCs at sites of infection? These issues have recently been examined in the
settings of L. major infection in the skin56, T. gondii
infection in the brain57,58 and mycobacterial infection
in the liver 32.
In the skin. visualization of CD4 + T cell effector
responses to infection with L. major in the skin revealed
several unexpected features56. effector T cells entered
infected regions of the skin regardless of antigen specificity, while T cells specific for an immunodominant
L. major antigen slowed down and accumulated near
parasites. A subset of antigen-specific T cells arrested
and formed stable contacts with infected cells, whereas
others made transient contacts while continuing to
crawl over infected cells. Interestingly, the distribution of T cells was not uniform, with some infected
areas being extensively patrolled by T cells and others
seeming to be poorly accessible. Furthermore, many
infected cells failed to engage T cells, even when antigen-specific T cells were in close proximity. Although
this study largely focused on T helper 2 (TH2)-polarized
cells and a genetically susceptible mouse strain, similar
behaviour was also seen with TH1-polarized cells and in
mice genetically resistant to the parasite, implying that
these are general features of the CD4+ effector T cell
response to L. major. These observations raise several
interesting questions for future studies: what limits
the ability of T cells to access certain infected regions?
what determines whether T cells respond to particular
APCs? And what are the consequences of the heterogeneous response of T cells to infected cells on their
ability to control infection and induce pathology?
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In the brain. The establishment of chronic infection by
the parasite T. gondii is accompanied by stage conversion, in which parasites convert to a slowly dividing
bradyzoite that forms cysts in the brain. Interestingly,
effector CD8+ T cells in the brain seemed to ignore
the intact cysts, but they accumulated and migrated
more slowly in the vicinity of isolated parasites, which
resembled those seen to emerge from rupturing cysts57
(FIG. 1Ab; Supplementary information S13 (movie)).
Activated CD8+ T cells could enter the brains of infected
mice independent of their antigen specificity, although
antigen-specific T cells preferentially slowed and accumulated near parasites and were retained in the infected
tissue for longer periods57. Rather than forming only
one-to-one contacts with individual APCs, antigenspecific CD8+ T cells were seen to interact with granuloma-like aggregates of CD11b+ cells, some of which
contained visible parasites. Moreover, T cells slowed
when in contact with an aggregate but did not undergo
further slowing when approaching a parasite within an
aggregate, suggesting that the entire granuloma-like
structure, rather than an individual infected cell, may
be the antigen-presenting unit in this setting. Unlike
in L. major-infected skin, effector CD8+ T cells in the
brains of T. gondii-infected mice seemed to patrol large
areas of the brain, including those that did not contain visible parasites57. Another study revealed T cells
migrating along a network of reticular fibres, similar to
that found in lymph nodes, that was induced in the brain
in response to infection58. This intriguing observation
suggests a mechanism to explain how effector T cells
could access large regions of the brain parenchyma. It
Box 2 | Imaging host–pathogen interactions in zebrafish models
Interactions between bacterial pathogens and macrophages can also be visualized in
real time in zebrafish embryo infection models63. The zebrafish embryo is transparent,
and the entire intact organism can be readily imaged by widefield or confocal
microscopy at single cell resolution. This makes it considerably easier to locate regions
of interest and to follow the spread of the pathogen over extended periods of time.
Adult zebrafish have both an innate and adaptive immune system, with many
similarities to the mammalian immune system. The optically accessible embryos have
only an innate immune system, but macrophage-like cells can already be observed at
the start of blood circulation and have been shown to take up various bacterial
pathogens and have an important host protective role64,65,67.
So far studies have not only shown that the responses of mammalian and zebrafish
immune cell populations to infection are similar but also that factors important for
pathogen virulence in mammalian models are similar in zebrafish. For example,
infection with streptolysin S-mutant Streptococcus pyogenes led to enhanced
neutrophil infiltration in both zebrafish and mouse models18.
Perhaps the most important contribution of intravital imaging in these models has
been in revealing how bacterial pathogens use macrophages for their dissemination
and growth. Zebrafish embryos clear infection with Listeria innocua but not its more
pathogenic relative Listeria monocytogenes. A comparison of macrophage behaviour
in infected zebrafish revealed that macrophages that had internalized L. innocua lost
motility, whereas those that internalized L. monocytogenes remained motile. This
suggests a possible role for motile macrophages in spreading infection66. Furthermore,
dynamic imaging of nascent granulomas formed in response to Mycobacterium
marinum infection revealed greater recruitment of macrophages to granulomas and
increased motility of macrophages within granulomas when compared to the response
to a strain lacking the RD1 virulence determinant59. This allows arriving macrophages to
find and phagocytose infected macrophages, contributing both to early increases in
bacterial number and to dissemination59,67.
also raises the possibility that the formation of a structure for T cell migration could be a limiting factor for
T cell migration within infected tissues in other settings,
such as L. major infection in the skin56.
The dynamics of the CD8+ T cell response in the
brain has also been investigated following intracerebral
inoculation with LCMv22. As discussed previously,
extravasation of neutrophils and monocytes is associated
with vascular leakage and fatal seizures in this model. It
has also been known for some time that CD8+ T cells
are required for the development of seizures, but their
extravasation or positioning was not associated with vascular leakage. Similar to what was observed in chronic
T. gondii infection, CD8+ T cells in the brains of LCMvinfected mice made short antigen-dependent contacts,
but not the prolonged interactions associated with killing of a target cell22,57. notably, however, marked recruitment of myelomonocytic cells did not occur until after
the arrival of T cells in the brain, and depletion of CD8+
T cells resulted in reduced infiltration of monocytes and
neutrophils and prevented the development of seizures22.
Therefore, chemokines produced by CD8+ T cells or in
response to CD8+ T cells might direct the recruitment of
neutrophils and monocytes involved in the development
of pathology, revealing a new therapeutic target.
In the liver. Interactions between effector T cells and
aggregates of macrophages are an important feature of
mycobacterial infection, and imaging of the liver after
infection of mice with BCG revealed key aspects of the
dynamic nature of these structures32. Macrophages within
BCG-induced granulomas were largely non-motile, but
actively extended processes, suggestive of sampling of
the local environment. By contrast, polyclonal T cells
were highly motile but their migration was restricted
within the granuloma by a macrophage-defined border.
Although few T cells were observed entering or leaving
mature granulomas, this restricted migration was unlikely
to be due to a physical barrier, as in vitro-activated T cells
could be recruited rapidly and efficiently to mature granulomas. Instead, the authors suggest that macrophages
provide a scaffold over which T cells migrate, analogous
to reticular fibres in lymph nodes32.
Granulomas are diverse and dynamic structures, and
there is a great deal more to be learnt from time-lapse
imaging, including from studies in non-mammalian species. For example, imaging studies in a zebrafish embryo
model of mycobacterial infection showed that nascent
granulomas forming in the absence of T cells were highly
dynamic structures and actually contributed to the growth
and spread of the bacteria59. This difference could reflect
either the lack of T cells or the use of a more virulent
mycobacterial strain that can use the early granulomatous
response to its own benefit. Dynamic imaging in zebrafish
infection models is discussed further in BOX 2.
Summary and future challenges
The complexity of the interaction between pathogens
and the host immune system can only begin to be
properly understood when studied in the appropriate
tissue environment. The studies discussed here have
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REVIEWS
used various imaging techniques to study this interaction in tissues in real time, revealing some previously
unknown or under-appreciated facets of the host–pathogen interaction. Combining these initial observations
with genetic manipulation of host or pathogen, as well
as with other complimentary experimental approaches,
should further our understanding of the delicate
balance between host and pathogen.
There are several exciting possibilities for future
investigation. First, it is hoped that technological
advances will allow intravital imaging of previously inaccessible tissue environments, enabling the use of more
physiological infection models. Second, tools are being
developed that will allow functional information to be
derived directly from imaging studies. These include
methods for delivering and regulating agents that interfere with biological processes while intravital imaging is
taking place, pathogen strains that express fluorescent
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Acknowledgements
We thank J. Halkias for helpful comments. This work was supported by US National Institutes of Health grants AI065537
and AI065831 (E.A.R.). J.L.C. is a Sir Henry Wellcome
Postdoctoral Fellow (Wellcome Trust grant: WT085494MA).
Competing interests statement
The authors declare no competing financial interests.
DATABASES
UniProtKB: http://www.uniprot.org
CD11c | CD169 | CX3CR1
FURTHER INFORMATION
Ellen A. Robey’s homepage:
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