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

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Key points interactions and chemokine cues.21 Analysis of Ca2+ flux


by MPLSM after TCR ligation has revealed that mecha-
Cellular imaging of immune cells such as Tcells, Bcells and antigen-presenting
cells has improved our understanding of immune homeostasis, immune
nisms of thymocyte motility, localization and interaction
responses and autoimmune diseases with thymic stroma (namely with antigen-presentingcells
Cellular interactions underlie multiple stages of an immune response, including [APCs] such as thymic epithelial cells and dendritic
its initiation, maintenance, regulation and termination cells[DCs]) are crucial for the Tcell selection processes,
The details of how, where and when cells migrate and interact during the and can be a source of abnormal selection of autoreactive
multiple phases of immune responses remain unclear; cellular imaging Tcell clones.23 These studies aim to provide insights into
techniques can elucidate some of these aspects how the integration of signals from the same receptor can
The real-time visualization and assessment of cellular interactions and
give way to disparate outcomes of Tcell selection and to
functions invivo has been made possible by new developments in cellular
imaging techniques potential autoimmune consequences.
A better understanding of the cellcell interactions underlying immune
responses will contribute to improvements in rheumatoid arthritis therapy Peripheral tolerance
Disruption in peripheral Tcell tolerance has been postu-
lated as a mechanism contributing to RA pathogenesis,27,28
understanding and contextualization of molecules and but the origin of peripheral tolerance defects remains
pathways of importance in health and disease. Several unclear. Some studies cite a failure of TREGcell activity,
studies710 support the utility of such methodologies whereas others suggest that pathogenic Tcells can become
in investigations of the pathogenesis of inflamma- refractory to regulation.27 The mechanisms by which TREG
tory arthritis, particularly in the imaging of important cells mediate peripheral tolerance are of great interest to
immunoregulatorypathways. the scientific and clinical communities, as they are poten-
tial therapeutic targets. Various mechanisms have been
Imaging immune tolerance mechanisms proposed to explain the suppressive activity of TREG cells,
Central tolerance including secretion of soluble factors, direct contact with
Several studies have found associations between RA and target Thelper (TH) cells and functional modification
genes involved in Tcell or Bcell receptor signal tuning of APCs (comprehensively reviewed elsewhere29). Most
(for example PTPN2211,12 and CTLA412) and sensitivity data on how TREG cells interact with other immune cells to
to cytokines (such as STAT413 and IL2RA12). When com- achieve immune tolerance have been derived from invitro
bined with RAassociated HLA polymorphisms (HLA studies; whether such mechanisms are an accurate repre-
molecules mediate peptide presentation to Tcell recep- sentation of interactions invivo is mostly unknown. To
tors [TCR]), these minor associations probably contrib- date, imaging data relating to TREGcell function is scarce,
ute to the development of an autoimmune-prone immune but lymph node MPLSM studies suggest that TREG cells
system. In addition to mature effector Tcell function, the do not form stable interactions with TH cells, implicating
generation and maintenance of central tolerance (involv- APCs as the probable target of TREG cells. Stable contact
ing appropriate selection of Tcells in the thymus and of between TREG cells and DCs has been observed invivo,30
Bcells in the bone marrow) and of peripheral tolerance with reduced contact time between subsequent auto
(dependant on appropriate regulatoryT [TREG] cell func- reactive Tcell and autoantigen-bearing DCs resulting
tion) can contribute to an over-reactive immune system. in abortive TH cell activation.30,31 Further insights into
Notably, several mouse models with perturbations in TREGcell function might be obtained by comparing these
thymic selection (namely K/BN,14 TS1HACII,15 Il6st imaging data with studies of TcellAPC interactions
(gp130) mutant16 and SKG17 mice) develop erosive arthri- during priming and tolerance events.
tis. The Tcell repertoire is defined during Tcell devel-
opment in the thymus, where positive selection screens Adaptive immunity in action
CD4+CD8+ (double positive, DP) thymocytes for their A breach in immune tolerance will result in the activation
ability to weakly recognize self-peptide-bound MHC (self- of adaptive immunity, a process which has been clearly
pMHC), whereas a negative selection mechanism removes implicated in the pathogenesis of RA.1113 Imaging the
DP, single positive (SP) CD4+ and SP CD8+ thymocytes cellular dynamics underlying the temporal and spatial
with high affinity for self-pMHC (Figure1). These mecha- organization of adaptive responses has been particularly
nisms, when working appropriately, ensure a mature Tcell informative. Several events in the generation of adap-
compartment that is responsive to foreign antigens yet tol- tive immune responses have been visualized, namely
erant to self.18,19 To date, cellular imaging studies in this the migration of DCs out of tissues, their interactions
area that could be directly linked with RA pathogenesis withand activation of Tcells in secondary lymphoid
have been rare or nonexistent. organs, and the subsequent interactions of Tcells and
The anatomical position of the thymus poses a problem Bcells in establishing germinal centre (GC) reactions
for intravital studies, but this limitation can be circum- (Figure1). The current understanding of these processes
vented by using murine models of thymic lobe explants, has been revolutionized by invivo imaging.3236
slices and engraftment in the kidney capsule.2026 Of note,
MPLSM studies of thymocyte migration from the cortex Visualizing lymph node dynamics
to the medulla demonstrated considerable conserva- Conventional histological techniques have demonstrated
tion between mouse and human in thymocytestroma 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 lying paracortical Tcell zone structured on a fibroblastic
imaging of cells within lymph nodes enables a better reticular cell network.
understanding of the complexity of immune reactions. Blood enters lymph nodes through the hilar side of the
The combination of MPLSM with exogenous adminis- lymph node, disseminating through capillaries before
tration of fluorescent molecules has shown that antigens exiting through the same side. Lymph node blood vessels
can arrive in lymph nodes within seconds after admin include specialized high endothelial venules (HEVs)
istration, as these compounds can be tracked through the through which Bcells (as observed by intravital fluo-
subcapsular sinus and lymphatic circulation over time.37 rescent microscopy 38) and Tcells enter the lymph node
The physical characteristics of the antigen (such as its and interact with the fibroblastic reticular cell stromal
size) define the access to these anatomical structures and network,39 a process that can be visualized by MPLSM.40
subsequent contact with various lymph node-resident Bcells and Tcells express different chemokine receptors
populations of APCs (for example, paracortical DCs, fol- (CXCR5 and CCR7, respectively) which mediate their
licular DCs [FDC] and Bcells).37 Specialized anatomical migration along chemokine cues (CXCL13 in the follicle
locations have been identified within the lymph node, and CCL19/21 in the paracortex). These receptors allow
such as peripherally-located Bcell-containing follicles lymphocytes to find the appropriate anatomical location
organized around a network of FDCs, or the deeper 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- whether a TcellDC interaction will result in tolerance
tural and chemical factors control the segregation and or priming. Dysregulation of this step could underlie the
migration of different cell populations. Upon encounter- induction of autoimmunity and provide opportunities for
ing antigen, both Bcells and Tcells undergo transforma- therapeutic targeting.
tions that facilitate their subsequent interactions with each
other. Activated Tcells downregulate CCR7 and upregu- Germinal centre reactions
late CXCR5,41 whereas activated Bcells do the opposite;42 As the interactions between Tcells, Bcells and APCs
subsequently, antigen-specific cells of both types can be become better understood, models describing their
seen migrating towards each other by MPLSM to interact dynamics are being constantly refined. New informa-
at the follicular border before establishing a GC.42 tion on how cellcell interactions influence, for example,
the induction of tolerance or the development of follicu-
T-cellAPC interactions lar helperT (TFH) cells has contributed to these models
The interaction of an antigen-specific Tcell with an (Figure1). Once a Tcell has become activated and has
appropriate ligand-bearing DC in the paracortex of migrated towards a Bcell follicle, sustained antigen
lymph nodes, an event that precedes follicular migra- presentation, typically (though not necessarily) from an
tion, is necessary to determine the future differentiation antigen-specific Bcell48 (R. A. Benson, P. Garside and
of Tcells. Using intravital MPLSM imaging techniques, J.M. Brewer, unpublished observations), and other long-
researchers have demonstrated that Tcell priming occurs term interactions might be required for full differentia-
in three distinct phases (Figure1). Interactions within tion into the TFHcell lineage.49 Once differentiated, TFH
the first 8h of exposure to antigen are generally transient cells support GC responses in the production of high-
(<5min). In the second phase, 820h after exposure to affinity-matured, isotype-switched antibodies. The inher-
antigen, Tcells and DCs form long-term interactions ently dangerous process that allows Bcells to undergo
(>10min) in which Tcells will be primed and an immune random somatic hypermutation, which can generate
response will ensue.43,44 After ~48h, Tcells recover autoantibodies, is further regulated by the requirement
motility and subsequent interactions with DCs are short. for migration and interaction between antigen-specific
In contrast to immune activation, TcellAPC inter Tcells and Bcells in the dark and light zones of the GC.
actions in the second phase of tolerance induction remain MPLSM has been pivotal in addressing outstanding
transient,45 with cells forming small, short-lived clusters.46 questions regarding GC development. By defining the
In this case, whereas Tcells have clearly seen an antigen patterns and kinetics of cellular migration and interac-
and exhibit NFAT (nuclear factor of activated Tcells) tions in the GC, this technique has revealed prolonged
translocation to the nucleus, they do not fully differen- BcellFDC interactions within GCs, bidirectional migra-
tiate into effector cells, adopting a tolerant phenotype45 tion of Bcells between light and dark zones, selection of
that inhibits its migration into Bcell follicles and prevents higher-affinity Bcells through competition for Tcell
the support of Bcell responses.47 These processes are help, and newly arrived cells entering established GCs
suggestive of different stages in the interaction between outcompeting lower affinity Bcells.5053 These obser-
Tcells and APCs, and are key checkpoints in determining 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- lipids via the sphingosine 1phosphate (S1P) receptor1
fluorescence reporter mice has demonstrated a dynamic (S1PR1). In a set of experiments using MPLSM, Tcells
exchange of TFH cells between multiple GCs, increas- were shown to continuously probe cortical sinuses as
ing the effectiveness of the GC reaction and enhancing they migrated through GC Tcell zones.57 Signalling
antigenic variation in the antibody response.56 through the surface activation marker CD69 down-
regulates S1PR1 expression and prevents detection of
Lymph node egress local S1P, thus retaining cells within the lymph node. As
In a productive adaptive immune response, lymphocytes CD69 expression decreases, S1PR1 is able to respond to
must leave the lymph node to exert their effector func- its ligand within cortical sinuses, and at this stage Tcells
tion in somatic tissues. During this process, differentiated can be seen entering the sinusesthe first step to exiting
cells must disengage from the stroma within the lymph the lymph node via efferent lymphatics. The details of this
node and respond to chemoattractants within exit routes process have been dissected elegantly and reviewed by the
in a coordinated fashion. Chemokines and their recep- J. Cyster group.34
tors (such as the CCL21CCR7 pair) are implicated in Real-time imaging the dynamics of DC, Tcell and
this process, which also involves sphingosine-containing Bcell interactions in response to model antigens in

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murine systems has provided unprecedented insights into addressed within the articular environment. Despite being
the development of adaptive immune responses invivo. technically demanding, imaging studies are beginning to
All steps in these processes are points at which cell migra- reveal the intricacies of early joint-infiltration by immune
tion and interaction might fail or be enhanced to initiate cells and the ensuing pathology.
disease. Thus, therapeutic intervention during the initia-
tion of immune responses might be useful in a time and Innate immune cells
location-specific manner. The detailed information pro- The recruitment of neutrophils to synovial tissues is a
vided by imaging studies is also improving the mechanistic hallmark of inflammatory arthritis. Imaging studies in
understanding of previous findings and existing therapeu- models of pathogen-driven and sterile inflammation
tics: for example, the effects of costimulation blockade on demonstrate three phases of neutrophil trafficking:63
the migration of Tcells to Bcell follicles, with subsequent rolling and arrest within the vasculature, with some
reduction in TFH cell and antibody responses, suggest the extravasation; extravascular motility, with initial chemo-
most appropriate indications for such approaches.58 taxis to sites of damage and subsequent amplification of
chemotactic cues; and neutrophil clustering or swarming.
Imaging immune responses in RA Imaging data suggests that this model also holds true in
Imaging at cellular resolution is now being applied in the experimental arthritis, as neutrophils can be seen rolling
context of autoimmunity in general and RA in particu- on the luminal face of synovial vasculature after induction
lar. Progress has focused on immune responses in lymph of inflammatory arthritis.6466
nodes and joints in experimental disease models. The interaction of neutrophils with vascular endothe-
lium is pivotal in the development of inflammation (the
Secondary lymphoid tissues mechanistic details of which are reviewed elsewhere67).
Cellular imaging studies in the context of arthritic Imaging the synovial vasculature has revealed the adhe-
dise ase are in their infancy. Studies focusing on the sion of neutrophils to, and their transmigration across,
immune responses in the lymph node in preclinical and the vascular endothelium to be granulocyte colony-
early RA1,5960 have begun to show the importance of stimulating factor (GCSF)-dependent,64 with further
events occurring in these organs. In lymph nodes from requirement for chemokine (CC motif ) receptor2
proteoglycan-induced arthritis animal models, the basic (CCR2)-expressing monocytes to facilitate transmigra-
modes of Tcell behaviour previously described in model tion.66 Interestingly, CCR2+ monocytes are dispensable
antigen systems seem to be recapitulated by antigen- in the immune response to microbial stimuli, suggest-
experienced, proteoglycan-specific Tcells.7 However, ing a unique role for these cells in mediating neutrophil
these model systems rely on priming of naive Tcells extravasation in sterile inflammation.66
with an exogenous antigen. Autoreactive TCRpMHC After transmigration into the articular compartment,
complexes have reduced stability compared with those the initial neutrophil influx has weak chemotaxis,64,66 sim-
associated with exogenous antigen recognition.61 As ilarly to the scouting-like behaviour reported in a sterile
such, this difference might affect DCTcell interactions. skin-injury model.68 Binding of complementC5 (also
Supporting this speculation, data from imaging studies known as C5a), released as a consequence of immune
have shown that autoreactive Tcells do not form conven- complex deposition and activation of the alternative
tional immune synapses:62 unlike pathogen-restricted complement cascade,69 induces neutrophil release of
Tcell clones, autoreactive clones have no TCR-induced leukotrieneB4. This early scouting behaviour results in
stop signals nor consequent arrest of motility upon invitro neutrophil accumulation at sites of C5a deposition, where
recognition of self-pMHC, despite successful TCR signal- leukotriene B4 provides a chemotactic cue that drives
ling.62 How this difference might influence the motility formation of neutrophil clusters. Imaging studies have
of autoreactive Tcells invivo remains to be understood. delineated the temporal dynamics of neutrophil infiltra-
tion implied by molecular studies of C5a anaphylatoxin
The articular microenvironment chemotactic receptors and Fc receptors in arthritic
Somatic tissues have an important role in orchestrating inflammation.7073 The recruitment of macrophages or
immune responses, and in many instances become targets monocytes to the edges of these neutrophil swarms has
of autoimmune diseases such as RA; the exact mechanisms been observed in K/BN serum transfer 66 and collagen-
involved are unclear, but the visualization of cellular inter- induced arthritis models8. The functional consequence
actions as they occur in an intact inflammatory articular of the clustering of neutrophils and monocytes is
environment could offer valuable insights. Studies in both unknown, but a distinct anatomical cytokine profile can
humans and rodent models have identified populations be detected histologically, with neutrophils in the centre
of immune cells within diseased synovia, including DCs, of the cluster producing IL1, whereas the surrounding
neutrophils, macrophages, mast cells, innate lymphoid monocytes and macrophages produce TNF and IL6.8
cells, Tcells and Bcells. However, the temporal dynam- Once established, these structures condition the syno-
ics and cellular interactions that lead to destruction of vial microenvironment, acting directly as a source of
the articular tissues remain poorly defined (Figure1). proinflammatory mediators, promoting further leukocyte
After the success of lymph node imaging studies of cel- recruitment that precedes periarticular boneerosion.
lular spatial distribution, entry and exit routes and reten- Neutrophil activity can also contribute to autoreactive
tion mechanisms, these critical questions can now be responses through the release of chromatin in the form

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of neutrophil extracellular traps (NETs).7477 Antibodies a polyclonal population of activated CD4+ Tcells could
that bind to citrullinated histone4 (present in NETs) have migrate to bone tissues and interact with osteoclasts in an
been identified in patients with RA,76 but why NETosis immunologically naive setting. Why Tcells are recruited
occurs in this disease is unclear. Intravital imaging studies to noninflammed tissues and whether this happens
have allowed the visualization of the process during independently of an antigen remains to be understood.
infection, providing insight as to the function of NETs in Comparative studies using animal models and transla-
immunity.78Advances in deep-tissue imaging, labelling tion of these findings into inflammatory arthritis will
and intravital surgical techniques will allow visualiza- proveinformative.
tion of the articular environment, potentially revealing
additional information about NET activity during Future challenges
inflammatory arthritis. A detailed understanding of the fundamental processes
underlying the induction, maintenance and resolution of
Adaptive immune cells RA should provide useful insights for the development
Despite the known importance of CD4+ Tcells for the ofnew therapeutic approaches. Furthermore, defining
pathology of RA, the timing and mechanisms of their where and how existing therapeutic research is of impor-
pathogenicity are unclear. The contribution of CD4+ tance, so that treatments can be better targeted to the most
Tcells to RA pathology is likely to be multifaceted, ranging appropriate anatomical location (for example, lymph
from cellular interactions in lymph nodes and support- nodes versus joints) and phase of disease (for example,
ing autoreactive Bcell responses, to actively coordinating whether blockade of costimulation is most appropriate in
destructive processes in the joint, both through cell the earliest indication of disease and anti-cytokine thera-
contact and secretion of soluble mediators. pies in established disease). Observing tolerance and
Imaging these processes within articular tissues has resolution at the molecular and functional level is vital to
been technically demanding. Constraints imposed by the understand when and where tolerance is breached, and
imaging procedure enable the visualization of only a small whether or not it can be re-established.
area of a single joint. This limitation, combined with rela- The poor applicability of current technological and
tively low numbers of infiltrating Tcells, unknown tempo- experimental practices in the clinic is a central hurdle to
ral regulation of inflammation and poorly defined relevant the imaging of cellular processes in RA. The use of animal
Tcell specificities, increases the complexity of imaging models enables these challenges to be readily addressed,
studies in this tissue. Initial approaches have not been but findings from these studies will have to be translated to
able to visualize Tcell infiltrates in synovial tissues, despite treatments for patients. The main imaging challenges have
their presence in the synovial fluid, probably also reflect- included labelling and tracking cells, and analysing the
ing the complexity of the model.10 However, that CD4+ key interactions between cells of the immune system and
Tcells are present in synovial tissues is widely accepted, stromal components. Accessing tissues with minimally
with current open questions involving the nature of cel- invasive techniques has also been an important considera-
lular interactions that mediate articular Tcell survival, tion. Developments in transgenic reporter animal models,
retention, and the relevance of their antigen specificity in alongside advances in microscopy, have allowed particular
coordinating destruction of the joint.7984 cell types and their physiological functions to be visualized
Despite these limitations, cellular imaging studies are invivo (Figure2).
beginning to provide insights into the mechanisms by
which CD4+ Tcells might contribute to articular pathol- Developments in animal models
ogy. Infiltrating CD4+ Tcells are a major source of RANKL Resolution and tissue access
(receptor activator of nuclear factor B ligand, also known The resolution of any type of invivo imaging is limited
as TNF ligand superfamily member11), an important by the inherent light-scattering properties of tissues.86
cytokine that promotes osteoclastogenesis-driven peri Consequently, imaging at cellular resolutions requires
articular bone erosion. Using a green fluorescent protein access to the tissue being imaged and usually involves
(GFP) reporter driven by Tcirg1 (VH+-ATPasea3),85 invasive surgery. Surgical approaches to improve direct
Kikuta and colleagues successfully imaged mature osteo visualization of the tissue include orthotopic window
clasts in live bone.9 Mature osteoclasts express many implantation87 and tissue transplantation,88 but these
VH+-ATPases (also known as vacuolar H+-ATPases) approaches require surgery prior to imaging (Figure2).
along their ruffled border (the membrane area adher- Alternatively, microendoscopy using bundled fibre lenses89
ing to the bone surface), which mediate acid resorption or gradient refractive index (GRIN) lenses enable implan-
through high secretion of protons. Exposure and imaging tation of lenses (within tissues) that can be used as a light
of calvarial bone showed the presence of two osteoclast conduit, allowing light collection at cellular resolution,
populations, motile and nonresorptive, and nonmotile and potentially at any depth. The GRIN lens approach can also
bone-resorptive, with RANKL expression regulating the be optimized for multiphoton imaging, enabling imaging
transition between the two states.9 Critically, type17 TH of undisturbed tissue beyond the tip of the GRIN lens.90
(TH17) cells, but not type1 TH (TH1) cells, were observed
in contact with osteoclasts in a RANKL-dependent Labelling
process to promote nonmotile, bone-resorptive osteoclast Owing to the improved transmission of long wavelength
phenotypes.9 Interestingly, this study 84 demonstrated that light in tissues, near-infrared-shifted fluorescent proteins

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Tissue access Labelling zplane. Owing to the limited distribution of antibodies


Tissue transplantation Infra-red flourescence invivo, most studies rely on tracking fluorescent proteins
Orthotopic window In vivo cytometry
GRIN microendoscopy Photoswitching/photoactivation
expressed endogenously in transgenic cells or under cell-
Fibre bundle microendoscopy Barcoding 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-
Quantitation Clinical translation ery of light from tissues (Figure2). Consequently, true
Negative dispersion Sentinel lymph node (e.g. ICG) quantitative imaging invivo is problematic. Controlling
Adaptive optics enzymatically-activated FRET probes
Post-acquisition compensation Inherent tissues signals (e.g. SHG) 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
Figure 2 | Challenges in tissue imaging. The main obstacles to visualizing dynamic in the beam path to compensate for tissue heterogeneity
immune processes include accessing tissues in a minimally invasive manner and during image acquisition.96
labelling, tracking and analysing the key interactions between immune system cells
and stromal components. Approaches to address tissue accessibility include Cellular imagingclinical translation
transplantation of tissues to more accessible imaging sites, insertion of an The current limitations of tissue imaging at cellular reso-
imagingwindow or use of endoscopy techniques. Labelling with near-infrared-
lution are considerable obstacles to invivo translational
shifted fluorescent proteins or dyes provides improved transmission of emitted
light, beneficial for deep imaging. Insitu labelling with Fab molecules enables the
imaging of cellular behaviour in humans. Consequently,
detection of specific surface molecules using DISC; fate-mapping is possible using clinical imaging strategies have traditionally involved
photoconvertible proteins such as kaede or barcoding (use of randomly-expressed low-resolution ultrasound, magnetic resonance and
combinations and ratios of fluorescent proteins, as in brainbow mice for example). radiological imaging techniques. However, noninvasive
Strategies to overcome limitations in quantitation can involve modification of the invivo imaging with near-infrared fluorescence using
excitation light path to anticipate and accommodate inhomogeneity of light indocyanine green (ICG) has been applied, for example,
behaviour in tissue (for example, negative dispersion and adaptive optics); in sentinel lymph node mapping or to assess lymphatic
alternatively, modelling light behaviour in tissue can be used after acquisition to
function.97 Currently, ICG remains one of the few near-
compensate for inhomogeneity. Mechanistic discoveries from studies of animal
models are a major source of translatable knowledge in imaging. Some infrared fluorescence imaging agents applied for medical
technologies such as ICG are directly translatable, with exciting prospects on the diagnostics in off-label investigational studies (Figure2).
horizon such as enzymatically-activated FRET probes and use of intrinsic tissue However, ICG functionalization by attachment to anti-
signals such as SHG. Abbreviations: DISC, dynamic insitu cytometry; Fab, fragment bodies or therapeutic proteins has proven difficult, and
antigen-binding; FRET, fluorescence resonance energy transfer; GRIN, gradient new, exogenously administered functional probes such as
refractive index; ICG, indocyanine green; SHG, second harmonic generation. enzyme-activated fluorescence resonance energy trans-
fer (FRET) probes are needed.98 Acquisition of intrinsic
are desirable for imaging of deep tissues (Figure2). In signals in tissues is already being used experimentally
whole-body fluorescence imaging, sensitivity has been with multiple techniques: second or third harmonic gen-
improved by several orders of magnitude with near- eration to reveal collagen; coherent anti-stokes Raman
infrared and infrared fluorescent proteins.91 Even though spectroscopy to detect lipids; and fluorescent lifetime
exogenous labelling with antibodies is problematic (owing microscopy analysis of autofluorescence signals, such as
to issues such as antibody clearance and bioavailability), those derived from NADH, to reveal metabolic activity.99
approaches such as dynamic insitu cytometry 92 have been Notably, these types of intrinsic signals from an optical
applied to show Tcell signalling associated with immune biopsy could bear useful diagnostic information in the
synapses invivo. However, as the recovered signal inten- context of RA.100102
sity is a function of the imaging depth, these approaches Although not all imaging techniques developed in
are only applicable where cells operate within the same animal models will be directly applicable to the clinic, the

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

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The authors acknowledge Arthritis Research UK
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forfunding their research, and the Rheumatoid
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