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Cellular imaging in rheumatic diseases

Robert A. Benson, Iain B. McInnes, James M. Brewer and Paul Garside
Abstract | Developments in cellular imaging now enable the real-time visualization of the choreographed
sequence of events that underlie the development of immune responses invivo. The previously unappreciated
dynamics and anatomical context of cellular interactions, revealed in these studies, can have profound
consequences for the decision by the immune system to induce immunological priming versus immunological
tolerance. Importantly, dysregulation of this balance can result in autoimmune diseases such as rheumatoid
arthritis (RA). By further developing our understanding of how, where and when cells interact during immune
responses, we can further dissect these events to assess how cell interactions might be aberrant in
autoimmunity. A better knowledge of the mechanisms involved in cellular interactions by means of cellular
imaging can help the development and targeting of therapies to particular disease stages and tissues in
patients with RA in efforts to restore immune homeostasis.
Benson, R. A. etal. Nat. Rev. Rheumatol. advance online publication 24 March 2015; doi:10.1038/nrrheum.2015.34

Introduction
The pathogenesis of rheumatoid arthritis (RA) can be use-
fully parsed into several phases, namely an initial breach
of tolerance to self-antigens, transition to autoreactivity
and tissue localization, and failed resolution presaging
chronicity (Figure1).1,2 Pathogenesis maps have been
proposed to guide the use of the most appropriate animal
models to dissect the mechanistic basis of each of these
phases.3,4 Despite the various molecular pathways and cel-
lular effector functions already described in the context
of RA pathology, arising from the use of these models
together with appropriate exvivo human studies, fun-
damental questions concerning the basic organization
of disease-driving immune responses remain. Where
doesimmune activation originate in patients with RA?
Does immune triggering happen in joints, mucosal sur-
faces or in the bone marrow? How and where are critical
fate determination decisions for effector and memory cells
made? Why do such pathways bypass normal immune
homeostatic mechanisms to perpetuate damage, and how
does such damage (putatively) drive chronicity? In this
Review, we describe how intact tissue imaging invivo can
answer some of these questions. We summarize develop-
ments in cellular imaging over the past 15years that have
facilitated these studies, outlining new insights into the
dynamic nature of immune responses and their applica-
tion in the treatment of RA. Additionally, we highlight
Centre for some of the remaining challenges in our understanding of
Immunobiology,
Institute of Immunology, immune responses which might be addressed by cellular
Infection and imaging studies.
Inflammation, Glasgow
Biomedical Research
Centre, University of Developments in cellular imaging
Glasgow, Glasgow Our understanding of the cellular dynamics in immune
G128TA, UK (R.A.B.,
I.B.M., J.M.B., P.G.). responses has been improved by the advancement of
Correspondence to: P.G.
paul.garside@ Competing interests
glasgow.ac.uk The authors declare no competing interests.
intravital imaging techniques, and imaging approaches
have benefited the investigation of cellular dynamics
(Table1). For example, multiphoton laser-scanning micro
scopy (MPLSM) and widespread accessibility to fluores-
cent reporter transgenic animals have been particularly
useful for the visualization of cellular interactions.
The biophysics of invivo light microscopy have been
reviewed elsewhere.5 Multiphoton excitation has several
advantages over conventional single-photon excita-
tion systems such as wide-field fluorescence or confocal
microscopy. The use of near-infrared light rather than
visible light enables excitation of fluorophores located
deeper in tissues, reduces light scattering and absorp-
tion by tissue pigments and, owing to its lower photon
energy, reduces photodamage.6 In multiphoton micro
scopy, excitation occurs only in areas of high photon
densitythe focal point of the lensreducing out-of-
focus excitation of fluorophores and negating the need
for a pinhole. As a result, the efficiency of light recovery
from tissues is enhanced and enables the use of sensitive
photodiode detectors instead of charge-coupled device
(CCD) detectors, which enable enhanced spatiotemporal
control over fluorophore excitation, deeper tissue pen-
etration and decreased photodamage in comparison with
conventionalmicroscopy.
Even though deep imaging of tissues or organs at cel-
lular resolution involves many challenges, accessibility to
this technique has improved. The development of trans-
genic reporter animals that express fluorescent proteins
under the control of specific promoters, which label spe-
cific tissues or cell types, can be combined with imaging
techniques to yield new insights about the function,
location and interactions of the cells labelled (Table2).
Thus, the ability to visualize the dynamic behaviour of
cells within living tissues, mapping the dynamics of their
migration and cellular interactions, allows for a better

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Key points
Cellular imaging of immune cells such as Tcells, Bcells and antigen-presenting
cells has improved our understanding of immune homeostasis, immune
responses and autoimmune diseases
Cellular interactions underlie multiple stages of an immune response, including
its initiation, maintenance, regulation and termination
The details of how, where and when cells migrate and interact during the
multiple phases of immune responses remain unclear; cellular imaging
techniques can elucidate some of these aspects
The real-time visualization and assessment of cellular interactions and
functions invivo has been made possible by new developments in cellular
imaging techniques
A better understanding of the cellcell interactions underlying immune
responses will contribute to improvements in rheumatoid arthritis therapy
understanding and contextualization of molecules and
pathways of importance in health and disease. Several
studies710 support the utility of such methodologies
in investigations of the pathogenesis of inflamma-
tory arthritis, particularly in the imaging of important
immunoregulatorypathways.
Imaging immune tolerance mechanisms
Central tolerance
Several studies have found associations between RA and
genes involved in Tcell or Bcell receptor signal tuning
(for example PTPN2211,12 and CTLA412) and sensitivity
to cytokines (such as STAT413 and IL2RA12). When com-
bined with RAassociated HLA polymorphisms (HLA
molecules mediate peptide presentation to Tcell recep-
tors [TCR]), these minor associations probably contrib-
ute to the development of an autoimmune-prone immune
system. In addition to mature effector Tcell function, the
generation and maintenance of central tolerance (involv-
ing appropriate selection of Tcells in the thymus and of
Bcells in the bone marrow) and of peripheral tolerance
(dependant on appropriate regulatoryT [TREG] cell func-
tion) can contribute to an over-reactive immune system.
Notably, several mouse models with perturbations in
thymic selection (namely K/BN,14 TS1HACII,15 Il6st
(gp130) mutant16 and SKG17 mice) develop erosive arthri-
tis. The Tcell repertoire is defined during Tcell devel-
opment in the thymus, where positive selection screens
CD4+CD8+ (double positive, DP) thymocytes for their
ability to weakly recognize self-peptide-bound MHC (self-
pMHC), whereas a negative selection mechanism removes
DP, single positive (SP) CD4+ and SP CD8+ thymocytes
with high affinity for self-pMHC (Figure1). These mecha-
nisms, when working appropriately, ensure a mature Tcell
compartment that is responsive to foreign antigens yet tol-
erant to self.18,19 To date, cellular imaging studies in this
area that could be directly linked with RA pathogenesis
have been rare or nonexistent.
The anatomical position of the thymus poses a problem
for intravital studies, but this limitation can be circum-
vented by using murine models of thymic lobe explants,
slices and engraftment in the kidney capsule.2026 Of note,
MPLSM studies of thymocyte migration from the cortex
to the medulla demonstrated considerable conserva-
tion between mouse and human in thymocytestroma
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interactions and chemokine cues.21 Analysis of Ca2+ flux
by MPLSM after TCR ligation has revealed that mecha-
nisms of thymocyte motility, localization and interaction
with thymic stroma (namely with antigen-presentingcells
[APCs] such as thymic epithelial cells and dendritic
cells[DCs]) are crucial for the Tcell selection processes,
and can be a source of abnormal selection of autoreactive
Tcell clones.23 These studies aim to provide insights into
how the integration of signals from the same receptor can
give way to disparate outcomes of Tcell selection and to
potential autoimmune consequences.
Peripheral tolerance
Disruption in peripheral Tcell tolerance has been postu-
lated as a mechanism contributing to RA pathogenesis,27,28
but the origin of peripheral tolerance defects remains
unclear. Some studies cite a failure of TREGcell activity,
whereas others suggest that pathogenic Tcells can become
refractory to regulation.27 The mechanisms by which TREG
cells mediate peripheral tolerance are of great interest to
the scientific and clinical communities, as they are poten-
tial therapeutic targets. Various mechanisms have been
proposed to explain the suppressive activity of TREG cells,
including secretion of soluble factors, direct contact with
target Thelper (TH) cells and functional modification
of APCs (comprehensively reviewed elsewhere29). Most
data on how TREG cells interact with other immune cells to
achieve immune tolerance have been derived from invitro
studies; whether such mechanisms are an accurate repre-
sentation of interactions invivo is mostly unknown. To
date, imaging data relating to TREGcell function is scarce,
but lymph node MPLSM studies suggest that TREG cells
do not form stable interactions with TH cells, implicating
APCs as the probable target of TREG cells. Stable contact
between TREG cells and DCs has been observed invivo,30
with reduced contact time between subsequent auto
reactive Tcell and autoantigen-bearing DCs resulting
in abortive TH cell activation.30,31 Further insights into
TREGcell function might be obtained by comparing these
imaging data with studies of TcellAPC interactions
during priming and tolerance events.
Adaptive immunity in action
A breach in immune tolerance will result in the activation
of adaptive immunity, a process which has been clearly
implicated in the pathogenesis of RA.1113 Imaging the
cellular dynamics underlying the temporal and spatial
organization of adaptive responses has been particularly
informative. Several events in the generation of adap-
tive immune responses have been visualized, namely
the migration of DCs out of tissues, their interactions
withand activation of Tcells in secondary lymphoid
organs, and the subsequent interactions of Tcells and
Bcells in establishing germinal centre (GC) reactions
(Figure1). The current understanding of these processes
has been revolutionized by invivo imaging.3236
Visualizing lymph node dynamics
Conventional histological techniques have demonstrated
the intricate nature of lymph node structure and
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Genetic and environmental factors

Disease progression
Break of Immune cell Immune cell Joint
tolerance activation infiltration pathology
Thymus Lymph node Joint

Cell migration

T cell Stage 1 Monocyte/

08 h macrophage
cTEC DN
Transient (<5 min) interactions B cell
Developing 2
thymocyte Stage 2 TEFF
CXCR4 TREG
824 h
and CCR7

Capillary
Transient (<5 min) Long (>10 min)
interactions interactions
DP DC Neutrophil
Apoptotic Stage 3
1 DP 48+ h
1

SP
mTEC Transient (<5 min) Transient (<5 min) Long (>10 min) ?
CCR7 interactions interactions interactions
APC
Trabeculae Interaction
Tolerance Priming TFH time unknown

Figure 1 | Pathogenesis of rheumatoid arthritis. Disease progression can be considered in several phases: breach of
self-tolerance; activation of adaptive immune cells in joint-draining lymph nodes; autoreactive immune cell infiltration
ofarticular tissue; and pathology. Perturbations in cellular interactions, chiefly APCTcell communications, can contribute
to these phases. Genetic and environmental factors can influence thymocyte interactions with thymic epithelial cells and
DCs, which can affect selection events and allow the escape of autoreactive Tcells to the periphery (1) or deficient
generation ofTREG cells (2). Presentation of self or modified peptides or neoepitopes by DCs to CD4 + Tcells prolongs their
interaction, promoting Tcell activation and differentiation in joint-draining lymph nodes. The behaviour of immune cells in
the joint is mostly unknown, in particular the relevance of antigen-specific interactions between APCs and T EFF cells.
Abbreviations: APC, antigen-presenting cell; CCR7, CC chemokine receptor type7; cTEC cortical thymic epithelial cell;
CXCR4, CXC chemokine receptor type4; DC, dendritic cell; DP, double positive thymocyte; mTEC medullary thymic
epithelial cell; SP, single positive thymocyte; TEFF, Teffector; TFH, follicular helper T; TREG, regulatoryT.

associated this complexity to function. However, dynamic
imaging of cells within lymph nodes enables a better
understanding of the complexity of immune reactions.
The combination of MPLSM with exogenous adminis-
tration of fluorescent molecules has shown that antigens
can arrive in lymph nodes within seconds after admin
istration, as these compounds can be tracked through the
subcapsular sinus and lymphatic circulation over time.37
The physical characteristics of the antigen (such as its
size) define the access to these anatomical structures and
subsequent contact with various lymph node-resident
populations of APCs (for example, paracortical DCs, fol-
licular DCs [FDC] and Bcells).37 Specialized anatomical
locations have been identified within the lymph node,
such as peripherally-located Bcell-containing follicles
organized around a network of FDCs, or the deeper
lying paracortical Tcell zone structured on a fibroblastic
reticular cell network.
Blood enters lymph nodes through the hilar side of the
lymph node, disseminating through capillaries before
exiting through the same side. Lymph node blood vessels
include specialized high endothelial venules (HEVs)
through which Bcells (as observed by intravital fluo-
rescent microscopy 38) and Tcells enter the lymph node
and interact with the fibroblastic reticular cell stromal
network,39 a process that can be visualized by MPLSM.40
Bcells and Tcells express different chemokine receptors
(CXCR5 and CCR7, respectively) which mediate their
migration along chemokine cues (CXCL13 in the follicle
and CCL19/21 in the paracortex). These receptors allow
lymphocytes to find the appropriate anatomical location
where antigen is displayed in a relevant form (on FDCs in

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Table 1 | Imaging modalities

Technique Resolution Detection Advantages Disadvantages
threshold
Bioluminescence ~3mm ~100 cells Image whole organism, low running Low resolution
costs
Confocal laser 0.1m 1 cell Can image intracellular compartments; Phototoxicity, images thin
scanning microscopy good axial resolution sections of tissue
MRI Up to 25m 1 cell Good resolution; excellent tissue Too slow to track dynamic cellular
contextualization; whole-body imaging interactions; labelling difficulties
MPLSM 0.2m 1 cell High-resolution and fast; allows Small viewing area; requires
single-cell tracking with greater surgery
depththan other single-cell
resolutionsystems
Radioactive tracer ~2mm ~100 cells Can image metabolic processes Use of radioactivity;
imaging lowresolution; high space and
financial investment owing to
the need for a cyclotron
Spinning disk confocal 0.1m 1 cell Fast, high resolution; better cell Poor depth of imaging
viability than confocal
Whole-body ~1mm ~100 cells Image whole organism Resolution does not allow
fluorescence (with ICG) tracking of cellular interactions
X-ray CT <50m ~50 cells Whole animal imaging, good resolution Poor cell labelling methods
Abbreviations: ICG, indocyanine green; MPLSM, multiphoton laser scanning microscopy.

the follicles and DCs in the paracortex). Thus, both struc-
tural and chemical factors control the segregation and
migration of different cell populations. Upon encounter-
ing antigen, both Bcells and Tcells undergo transforma-
tions that facilitate their subsequent interactions with each
other. Activated Tcells downregulate CCR7 and upregu-
late CXCR5,41 whereas activated Bcells do the opposite;42
subsequently, antigen-specific cells of both types can be
seen migrating towards each other by MPLSM to interact
at the follicular border before establishing a GC.42
T-cellAPC interactions
The interaction of an antigen-specific Tcell with an
appropriate ligand-bearing DC in the paracortex of
lymph nodes, an event that precedes follicular migra-
tion, is necessary to determine the future differentiation
of Tcells. Using intravital MPLSM imaging techniques,
researchers have demonstrated that Tcell priming occurs
in three distinct phases (Figure1). Interactions within
the first 8h of exposure to antigen are generally transient
(<5min). In the second phase, 820h after exposure to
antigen, Tcells and DCs form long-term interactions
(>10min) in which Tcells will be primed and an immune
response will ensue.43,44 After ~48h, Tcells recover
motility and subsequent interactions with DCs are short.
In contrast to immune activation, TcellAPC inter
actions in the second phase of tolerance induction remain
transient,45 with cells forming small, short-lived clusters.46
In this case, whereas Tcells have clearly seen an antigen
and exhibit NFAT (nuclear factor of activated Tcells)
translocation to the nucleus, they do not fully differen-
tiate into effector cells, adopting a tolerant phenotype45
that inhibits its migration into Bcell follicles and prevents
the support of Bcell responses.47 These processes are
suggestive of different stages in the interaction between
Tcells and APCs, and are key checkpoints in determining
whether a TcellDC interaction will result in tolerance
or priming. Dysregulation of this step could underlie the
induction of autoimmunity and provide opportunities for
therapeutic targeting.
Germinal centre reactions
As the interactions between Tcells, Bcells and APCs
become better understood, models describing their
dynamics are being constantly refined. New informa-
tion on how cellcell interactions influence, for example,
the induction of tolerance or the development of follicu-
lar helperT (TFH) cells has contributed to these models
(Figure1). Once a Tcell has become activated and has
migrated towards a Bcell follicle, sustained antigen
presentation, typically (though not necessarily) from an
antigen-specific Bcell48 (R. A. Benson, P. Garside and
J.M. Brewer, unpublished observations), and other long-
term interactions might be required for full differentia-
tion into the TFHcell lineage.49 Once differentiated, TFH
cells support GC responses in the production of high-
affinity-matured, isotype-switched antibodies. The inher-
ently dangerous process that allows Bcells to undergo
random somatic hypermutation, which can generate
autoantibodies, is further regulated by the requirement
for migration and interaction between antigen-specific
Tcells and Bcells in the dark and light zones of the GC.
MPLSM has been pivotal in addressing outstanding
questions regarding GC development. By defining the
patterns and kinetics of cellular migration and interac-
tions in the GC, this technique has revealed prolonged
BcellFDC interactions within GCs, bidirectional migra-
tion of Bcells between light and dark zones, selection of
higher-affinity Bcells through competition for Tcell
help, and newly arrived cells entering established GCs
outcompeting lower affinity Bcells.5053 These obser-
vations have led to a refined model of GC activity.54,55

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Table 2 | Fluorescent reporter animals and labels

Imaging Example Reporter expression Findings
application
Specific cell LysMEGFP103 Transgenic mouse with Effective in tracking neutrophil motility during various
populations enhancedGFP insertion into inflammatory processes (extravasation, bacterial
lysozymeM locus infection, sterile inflammation and arthropathy)
CD11cYFP104 CD11c-promoter-driven Definition of DC networks in lymphoid tissue and
YFPmouse interactions with Tcells
a3GFP105 Tcirg1 (encoding Vtype H+ Visualizing osteoclasts;9,105 real-time imaging of
ATPase a3 subunit)-driven bone-destructive processes
GFPmouse
Cell-tracker dyes Exogenous labels (for instance Labelling purified cells prior to transfer into animal
(various colours) CFDA, CMTPX) recipient; imaging of cellular behaviour
Cell Nur77GFP106 Nr4a1 (Nur77) promoter-driven Antigen receptor stimulation in lymphocytes
activation GFPBAC transgenic mouse drivesstrong GFP expression; used to visualize
thymocyteselection
NFATGFP Retroviral-encoded construct; NFAT nucleocytoplasmic shuttling in Tcells as a
expression in infected cells readout of TCR signalling45
Calcium indicator Exogenous labels (such as In-cell measurement of calcium flux triggered upon
(various) Fluo4, Rhod3-AM) T-cell stimulation
Exogenously-expressed Fab molecules (various Insitu labelling of cells (such as DISC)92
cell surface markers of fluorophores)
activation
Cytokine 4GET107 IL4-reporter mouse Identifying TEFFcell responses and lineage commitment
production in several autoimmune and infectious disease models
YETI108 IFN-reporter mouse Identifying TEFFcell responses and lineage commitment
in several autoimmune and infectious disease models
Il17aCreR26ReYFP109 IL17A-reporter mouse Identifying TEFFcell responses and lineage commitment
in several autoimmune and infectious disease models
VertX110 IL10-reporter mouse Identifying TEFFcell responses and lineage commitment
in several autoimmune and infectious disease models
Tissue Col2GFP GFP expression controlled by Mature chondrocytes express GFP, allowing cartilage
visualization the murine collagen typeII imaging, particularly its growth and repair
promoter/enhancer
Prox1mOrange2111 Lymphatic endothelial-cell- Visualisation of lymphatic vessels, allowing the
specific Prox1 promoter-driven monitoring of cellular, particulate and fluid egress
fluorescent reporter from tissues
Fate-mapping Kaede112 Ubiquitous expression of the Tracking lymphocyte migration within and between
photoconvertible kaede protein tissues93,112114
Brainbow115 Stochastic expression of Distinguishing an individual cell from others of the
multiple fluorescent proteins, same lineage; most successfully used in mapping
generating cells of specific hues neuronal and glial networks
Abbreviations: CFDA, carboxyfluorescein diacetate; CMTPX, CellTrackerRed CMTPX (Life Technologies, CA, USA); DISC, dynamic insitu cytometry; Fab, fragment
antigen-binding; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; LysM, lysozymeM; NFAT, nuclear factor of activated Tcells; TCR, Tcell
receptor; YFP, yellow fluorescent protein.

Therecent combination of MPLSM and photoactivatable-
fluorescence reporter mice has demonstrated a dynamic
exchange of TFH cells between multiple GCs, increas-
ing the effectiveness of the GC reaction and enhancing
antigenic variation in the antibody response.56
Lymph node egress
In a productive adaptive immune response, lymphocytes
must leave the lymph node to exert their effector func-
tion in somatic tissues. During this process, differentiated
cells must disengage from the stroma within the lymph
node and respond to chemoattractants within exit routes
in a coordinated fashion. Chemokines and their recep-
tors (such as the CCL21CCR7 pair) are implicated in
this process, which also involves sphingosine-containing
lipids via the sphingosine 1phosphate (S1P) receptor1
(S1PR1). In a set of experiments using MPLSM, Tcells
were shown to continuously probe cortical sinuses as
they migrated through GC Tcell zones.57 Signalling
through the surface activation marker CD69 down-
regulates S1PR1 expression and prevents detection of
local S1P, thus retaining cells within the lymph node. As
CD69 expression decreases, S1PR1 is able to respond to
its ligand within cortical sinuses, and at this stage Tcells
can be seen entering the sinusesthe first step to exiting
the lymph node via efferent lymphatics. The details of this
process have been dissected elegantly and reviewed by the
J. Cyster group.34
Real-time imaging the dynamics of DC, Tcell and
Bcell interactions in response to model antigens in

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murine systems has provided unprecedented insights into
the development of adaptive immune responses invivo.
All steps in these processes are points at which cell migra-
tion and interaction might fail or be enhanced to initiate
disease. Thus, therapeutic intervention during the initia-
tion of immune responses might be useful in a time and
location-specific manner. The detailed information pro-
vided by imaging studies is also improving the mechanistic
understanding of previous findings and existing therapeu-
tics: for example, the effects of costimulation blockade on
the migration of Tcells to Bcell follicles, with subsequent
reduction in TFH cell and antibody responses, suggest the
most appropriate indications for such approaches.58
Imaging immune responses in RA
Imaging at cellular resolution is now being applied in the
context of autoimmunity in general and RA in particu-
lar. Progress has focused on immune responses in lymph
nodes and joints in experimental disease models.
Secondary lymphoid tissues
Cellular imaging studies in the context of arthritic
dise ase are in their infancy. Studies focusing on the
immune responses in the lymph node in preclinical and
early RA1,5960 have begun to show the importance of
events occurring in these organs. In lymph nodes from
proteoglycan-induced arthritis animal models, the basic
modes of Tcell behaviour previously described in model
antigen systems seem to be recapitulated by antigen-
experienced, proteoglycan-specific Tcells.7 However,
these model systems rely on priming of naive Tcells
with an exogenous antigen. Autoreactive TCRpMHC
complexes have reduced stability compared with those
associated with exogenous antigen recognition.61 As
such, this difference might affect DCTcell interactions.
Supporting this speculation, data from imaging studies
have shown that autoreactive Tcells do not form conven-
tional immune synapses:62 unlike pathogen-restricted
Tcell clones, autoreactive clones have no TCR-induced
stop signals nor consequent arrest of motility upon invitro
recognition of self-pMHC, despite successful TCR signal-
ling.62 How this difference might influence the motility
of autoreactive Tcells invivo remains to be understood.
The articular microenvironment
Somatic tissues have an important role in orchestrating
immune responses, and in many instances become targets
of autoimmune diseases such as RA; the exact mechanisms
involved are unclear, but the visualization of cellular inter-
actions as they occur in an intact inflammatory articular
environment could offer valuable insights. Studies in both
humans and rodent models have identified populations
of immune cells within diseased synovia, including DCs,
neutrophils, macrophages, mast cells, innate lymphoid
cells, Tcells and Bcells. However, the temporal dynam-
ics and cellular interactions that lead to destruction of
the articular tissues remain poorly defined (Figure1).
After the success of lymph node imaging studies of cel-
lular spatial distribution, entry and exit routes and reten-
tion mechanisms, these critical questions can now be
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addressed within the articular environment. Despite being
technically demanding, imaging studies are beginning to
reveal the intricacies of early joint-infiltration by immune
cells and the ensuing pathology.
Innate immune cells
The recruitment of neutrophils to synovial tissues is a
hallmark of inflammatory arthritis. Imaging studies in
models of pathogen-driven and sterile inflammation
demonstrate three phases of neutrophil trafficking:63
rolling and arrest within the vasculature, with some
extravasation; extravascular motility, with initial chemo-
taxis to sites of damage and subsequent amplification of
chemotactic cues; and neutrophil clustering or swarming.
Imaging data suggests that this model also holds true in
experimental arthritis, as neutrophils can be seen rolling
on the luminal face of synovial vasculature after induction
of inflammatory arthritis.6466
The interaction of neutrophils with vascular endothe-
lium is pivotal in the development of inflammation (the
mechanistic details of which are reviewed elsewhere67).
Imaging the synovial vasculature has revealed the adhe-
sion of neutrophils to, and their transmigration across,
the vascular endothelium to be granulocyte colony-
stimulating factor (GCSF)-dependent,64 with further
requirement for chemokine (CC motif ) receptor2
(CCR2)-expressing monocytes to facilitate transmigra-
tion.66 Interestingly, CCR2+ monocytes are dispensable
in the immune response to microbial stimuli, suggest-
ing a unique role for these cells in mediating neutrophil
extravasation in sterile inflammation.66
After transmigration into the articular compartment,
the initial neutrophil influx has weak chemotaxis,64,66 sim-
ilarly to the scouting-like behaviour reported in a sterile
skin-injury model.68 Binding of complementC5 (also
known as C5a), released as a consequence of immune
complex deposition and activation of the alternative
complement cascade,69 induces neutrophil release of
leukotrieneB4. This early scouting behaviour results in
neutrophil accumulation at sites of C5a deposition, where
leukotriene B4 provides a chemotactic cue that drives
formation of neutrophil clusters. Imaging studies have
delineated the temporal dynamics of neutrophil infiltra-
tion implied by molecular studies of C5a anaphylatoxin
chemotactic receptors and Fc receptors in arthritic
inflammation.7073 The recruitment of macrophages or
monocytes to the edges of these neutrophil swarms has
been observed in K/BN serum transfer 66 and collagen-
induced arthritis models8. The functional consequence
of the clustering of neutrophils and monocytes is
unknown, but a distinct anatomical cytokine profile can
be detected histologically, with neutrophils in the centre
of the cluster producing IL1, whereas the surrounding
monocytes and macrophages produce TNF and IL6.8
Once established, these structures condition the syno-
vial microenvironment, acting directly as a source of
proinflammatory mediators, promoting further leukocyte
recruitment that precedes periarticular boneerosion.
Neutrophil activity can also contribute to autoreactive
responses through the release of chromatin in the form
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of neutrophil extracellular traps (NETs).7477 Antibodies
that bind to citrullinated histone4 (present in NETs) have
been identified in patients with RA,76 but why NETosis
occurs in this disease is unclear. Intravital imaging studies
have allowed the visualization of the process during
infection, providing insight as to the function of NETs in
immunity.78Advances in deep-tissue imaging, labelling
and intravital surgical techniques will allow visualiza-
tion of the articular environment, potentially revealing
additional information about NET activity during
inflammatory arthritis.
Adaptive immune cells
Despite the known importance of CD4+ Tcells for the
pathology of RA, the timing and mechanisms of their
pathogenicity are unclear. The contribution of CD4+
Tcells to RA pathology is likely to be multifaceted, ranging
from cellular interactions in lymph nodes and support-
ing autoreactive Bcell responses, to actively coordinating
destructive processes in the joint, both through cell
contact and secretion of soluble mediators.
Imaging these processes within articular tissues has
been technically demanding. Constraints imposed by the
imaging procedure enable the visualization of only a small
area of a single joint. This limitation, combined with rela-
tively low numbers of infiltrating Tcells, unknown tempo-
ral regulation of inflammation and poorly defined relevant
Tcell specificities, increases the complexity of imaging
studies in this tissue. Initial approaches have not been
able to visualize Tcell infiltrates in synovial tissues, despite
their presence in the synovial fluid, probably also reflect-
ing the complexity of the model.10 However, that CD4+
Tcells are present in synovial tissues is widely accepted,
with current open questions involving the nature of cel-
lular interactions that mediate articular Tcell survival,
retention, and the relevance of their antigen specificity in
coordinating destruction of the joint.7984
Despite these limitations, cellular imaging studies are
beginning to provide insights into the mechanisms by
which CD4+ Tcells might contribute to articular pathol-
ogy. Infiltrating CD4+ Tcells are a major source of RANKL
(receptor activator of nuclear factor B ligand, also known
as TNF ligand superfamily member11), an important
cytokine that promotes osteoclastogenesis-driven peri
articular bone erosion. Using a green fluorescent protein
(GFP) reporter driven by Tcirg1 (VH+-ATPasea3),85
Kikuta and colleagues successfully imaged mature osteo
clasts in live bone.9 Mature osteoclasts express many
VH+-ATPases (also known as vacuolar H+-ATPases)
along their ruffled border (the membrane area adher-
ing to the bone surface), which mediate acid resorption
through high secretion of protons. Exposure and imaging
of calvarial bone showed the presence of two osteoclast
populations, motile and nonresorptive, and nonmotile and
bone-resorptive, with RANKL expression regulating the
transition between the two states.9 Critically, type17 TH
(TH17) cells, but not type1 TH (TH1) cells, were observed
in contact with osteoclasts in a RANKL-dependent
process to promote nonmotile, bone-resorptive osteoclast
phenotypes.9 Interestingly, this study 84 demonstrated that
NATURE REVIEWS | RHEUMATOLOGY ADVANCE ONLINE PUBLICATION | 7
2015 Macmillan Publishers Limited. All rights reserved
REVIEWS
a polyclonal population of activated CD4+ Tcells could
migrate to bone tissues and interact with osteoclasts in an
immunologically naive setting. Why Tcells are recruited
to noninflammed tissues and whether this happens
independently of an antigen remains to be understood.
Comparative studies using animal models and transla-
tion of these findings into inflammatory arthritis will
proveinformative.
Future challenges
A detailed understanding of the fundamental processes
underlying the induction, maintenance and resolution of
RA should provide useful insights for the development
ofnew therapeutic approaches. Furthermore, defining
where and how existing therapeutic research is of impor-
tance, so that treatments can be better targeted to the most
appropriate anatomical location (for example, lymph
nodes versus joints) and phase of disease (for example,
whether blockade of costimulation is most appropriate in
the earliest indication of disease and anti-cytokine thera-
pies in established disease). Observing tolerance and
resolution at the molecular and functional level is vital to
understand when and where tolerance is breached, and
whether or not it can be re-established.
The poor applicability of current technological and
experimental practices in the clinic is a central hurdle to
the imaging of cellular processes in RA. The use of animal
models enables these challenges to be readily addressed,
but findings from these studies will have to be translated to
treatments for patients. The main imaging challenges have
included labelling and tracking cells, and analysing the
key interactions between cells of the immune system and
stromal components. Accessing tissues with minimally
invasive techniques has also been an important considera-
tion. Developments in transgenic reporter animal models,
alongside advances in microscopy, have allowed particular
cell types and their physiological functions to be visualized
invivo (Figure2).
Developments in animal models
Resolution and tissue access
The resolution of any type of invivo imaging is limited
by the inherent light-scattering properties of tissues.86
Consequently, imaging at cellular resolutions requires
access to the tissue being imaged and usually involves
invasive surgery. Surgical approaches to improve direct
visualization of the tissue include orthotopic window
implantation87 and tissue transplantation,88 but these
approaches require surgery prior to imaging (Figure2).
Alternatively, microendoscopy using bundled fibre lenses89
or gradient refractive index (GRIN) lenses enable implan-
tation of lenses (within tissues) that can be used as a light
conduit, allowing light collection at cellular resolution,
potentially at any depth. The GRIN lens approach can also
be optimized for multiphoton imaging, enabling imaging
of undisturbed tissue beyond the tip of the GRIN lens.90
Labelling
Owing to the improved transmission of long wavelength
light in tissues, near-infrared-shifted fluorescent proteins
REVIEWS
Tissue access Labelling
Tissue transplantation Infra-red flourescence
Orthotopic window In vivo cytometry
GRIN microendoscopy Photoswitching/photoactivation
Fibre bundle microendoscopy Barcoding
Quantitation Clinical translation
Negative dispersion Sentinel lymph node (e.g. ICG)
Adaptive optics enzymatically-activated FRET probes
Post-acquisition compensation Inherent tissues signals (e.g. SHG)
Figure 2 | Challenges in tissue imaging. The main obstacles to visualizing dynamic
immune processes include accessing tissues in a minimally invasive manner and
labelling, tracking and analysing the key interactions between immune system cells
and stromal components. Approaches to address tissue accessibility include
transplantation of tissues to more accessible imaging sites, insertion of an
imagingwindow or use of endoscopy techniques. Labelling with near-infrared-
shifted fluorescent proteins or dyes provides improved transmission of emitted
light, beneficial for deep imaging. Insitu labelling with Fab molecules enables the
detection of specific surface molecules using DISC; fate-mapping is possible using
photoconvertible proteins such as kaede or barcoding (use of randomly-expressed
combinations and ratios of fluorescent proteins, as in brainbow mice for example).
Strategies to overcome limitations in quantitation can involve modification of the
excitation light path to anticipate and accommodate inhomogeneity of light
behaviour in tissue (for example, negative dispersion and adaptive optics);
alternatively, modelling light behaviour in tissue can be used after acquisition to
compensate for inhomogeneity. Mechanistic discoveries from studies of animal
models are a major source of translatable knowledge in imaging. Some
technologies such as ICG are directly translatable, with exciting prospects on the
horizon such as enzymatically-activated FRET probes and use of intrinsic tissue
signals such as SHG. Abbreviations: DISC, dynamic insitu cytometry; Fab, fragment
antigen-binding; FRET, fluorescence resonance energy transfer; GRIN, gradient
refractive index; ICG, indocyanine green; SHG, second harmonic generation.
are desirable for imaging of deep tissues (Figure2). In
whole-body fluorescence imaging, sensitivity has been
improved by several orders of magnitude with near-
infrared and infrared fluorescent proteins.91 Even though
exogenous labelling with antibodies is problematic (owing
to issues such as antibody clearance and bioavailability),
approaches such as dynamic insitu cytometry 92 have been
applied to show Tcell signalling associated with immune
synapses invivo. However, as the recovered signal inten-
sity is a function of the imaging depth, these approaches
are only applicable where cells operate within the same
8 | ADVANCE ONLINE PUBLICATION
2015 Macmillan Publishers Limited. All rights reserved
zplane. Owing to the limited distribution of antibodies
invivo, most studies rely on tracking fluorescent proteins
expressed endogenously in transgenic cells or under cell-
specific promoters in animal models. In the past decade,
fate mapping of cells has been achieved invivo with
advanced approaches such as photoactivation53 and photo
switching.93 In the future, barcoding approaches such as
brainbow94 and confetti mice95 might also be compatible
with invivo imaging techniques. Such approaches should
improve our understanding of how migration of cells
between lymphoid organs and target tissues is regulated
in the context of RA.
Quantitation
Signal intensity invivo varies due to the strength of the
originating fluorescence signal. However, other less con-
trollable factors, such as tissue depth and heterogeneity
(for example, the presence of pigments or changing light-
scattering properties), also affect the delivery and recov-
ery of light from tissues (Figure2). Consequently, true
quantitative imaging invivo is problematic. Controlling
laser power with depth can help normalize signal intensity,
and precompensation and prechirping of laser pulses can
abrogate pulse elongation, increasing depth and reducing
zaxis signal distortion. Advanced approaches to address
these limitations include post-acquisition compensation
(for example, using phantoms) to model tissue inhomo-
geneity, and implementation of adaptive optical devices
in the beam path to compensate for tissue heterogeneity
during image acquisition.96
Cellular imagingclinical translation
The current limitations of tissue imaging at cellular reso-
lution are considerable obstacles to invivo translational
imaging of cellular behaviour in humans. Consequently,
clinical imaging strategies have traditionally involved
low-resolution ultrasound, magnetic resonance and
radiological imaging techniques. However, noninvasive
invivo imaging with near-infrared fluorescence using
indocyanine green (ICG) has been applied, for example,
in sentinel lymph node mapping or to assess lymphatic
function.97 Currently, ICG remains one of the few near-
infrared fluorescence imaging agents applied for medical
diagnostics in off-label investigational studies (Figure2).
However, ICG functionalization by attachment to anti-
bodies or therapeutic proteins has proven difficult, and
new, exogenously administered functional probes such as
enzyme-activated fluorescence resonance energy trans-
fer (FRET) probes are needed.98 Acquisition of intrinsic
signals in tissues is already being used experimentally
with multiple techniques: second or third harmonic gen-
eration to reveal collagen; coherent anti-stokes Raman
spectroscopy to detect lipids; and fluorescent lifetime
microscopy analysis of autofluorescence signals, such as
those derived from NADH, to reveal metabolic activity.99
Notably, these types of intrinsic signals from an optical
biopsy could bear useful diagnostic information in the
context of RA.100102
Although not all imaging techniques developed in
animal models will be directly applicable to the clinic, the
www.nature.com/nrrheum
 REVIEWS

mechanistic discoveries they enable are undoubtedly rele
vant. A good example of a major development in clinical
translational research in RA has been the study of disease
establishment prior to disease manifestation.13
Conclusions
Preventative strategies and therapeutics for patients with
RA will benefit from the interpretation of directly visual
ized cellular and molecular events associated with the
disease. These events might be applicable to interventions
with existing therapeutic strategies (such as costimula-
tion and cytokine inhibitor therapy and cell depletion
protocols). Additionally, new molecular mechanisms
might help understand and rectify the complexities of
failed lymphoid homeostasis that mediate progression to
disease. Clinically, the identification of the pivotal events
by invivo imaging methodologies will contribute to an
informed, optimal intervention. Preventative studies in
humans will be challenging, (for example, biomarker
outcomes do not yet exist for tolerance induction or
maintenance) and predictive modelling will be required
to build confidence in the preclinical development phase
for new compounds.
In the field of inflammatory arthritis, the study of
effector immune responses in specific tissues has been
the focus of intense interest, namely the study of skin
and joints in psoriatic arthritis, and the gut and eye in
spondyloarthropathies. With the constant development
of invivo imaging modalities, a detailed evaluation of the
cellular origins, fate and migration patterns of leukocyte
subsets should become possible, contributing to a better
understanding of the processes that lead to initiation and
perpetuation of the inflammatory lesions in a spectra of
complex tissue diseases. For this reason, we have invested
considerably in exploring such responses in the context
of inflammatory arthritis, and consider this endeav-
our a vital part of the future evaluation of mechanisms
ofpathogenesis.

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Acknowledgements
The authors acknowledge Arthritis Research UK
forfunding their research, and the Rheumatoid
Arthritis pathogenesis Centre of Excellence (RACE)
forsupport.
Author contributions
All authors contributed equally to researching data
forthe article and made substantial contributions to
discussion of content, writing and reviewing/editing
ofthe manuscript before submission.