ARTHRITIS & RHEUMATOLOGY

Vol. 67, No. 11, November 2015, pp 2933–2943

DOI 10.1002/art.39291
C 2015, American College of Rheumatology
V

Damage-Associated Molecular Patterns Generated in

Osteoarthritis Directly Excite Murine Nociceptive Neurons
Through Toll-like Receptor 4

Rachel E. Miller,1 Abdelhak Belmadani,2 Shingo Ishihara,1 Phuong B. Tran,1 Dongjun Ren,2
Richard J. Miller,2 and Anne-Marie Malfait1

Objective. To determine whether selected damage- in DRG cultures, and the neurons rapidly responded to
associated molecular patterns (DAMPs) present in the S100A8 and a2-macroglobulin with increased [Ca21]i.
osteoarthritic (OA) joints of mice excite nociceptors Blocking TLR-4 inhibited these effects. Neurons within
through Toll-like receptor 4 (TLR-4). intact DRGs responded to the TLR-4 agonist lipopoly-
Methods. The ability of S100A8 and a2-macro- saccharide. In both of the calcium-imaging assays, it was
globulin to excite nociceptors was determined by mea- primarily the nociceptor population of neurons that
suring the release of monocyte chemoattractant protein responded to TLR-4 ligands. TLR-4–/– mice were not pro-
1 (MCP-1) by cultured dorsal root ganglion (DRG) cells tected from mechanical allodynia or from joint damage
as well as by measuring the intracellular calcium concen- associated with DMM.
tration ([Ca21]i) in cultured DRG neurons from naive Conclusion. Our experiments suggest a role of
mice or from mice that had undergone surgical destabili- TLR-4 signaling in the excitation of nociceptors by
zation of the medial meniscus (DMM) 8 weeks previous- selected DAMPs. Further research is needed to delineate
ly. The role of TLR-4 was assessed using TLR-4–/– cells or the importance of this pathway in relation to OA pain.
a TLR-4 inhibitor. The [Ca21]i in neurons within ex vivo
intact DRGs was measured in samples from Pirt- Pain represents a protective response to poten-
GCaMP3 mice. Neuronal expression of the Tlr4 gene was tially damaging stimuli and is therefore essential to
determined by in situ hybridization. DMM surgery was health. Under normal circumstances, both the stimuli
performed in wild-type and TLR-4–/– mice; mechanical that cause pain and the ensuing response are relatively
allodynia was monitored, and joint damage was assessed transient in nature. However, pain may also be experi-
histologically after 16 weeks. enced in the context of chronic pathology. In these
Results. DRG neurons from both naive and DMM chronic pain syndromes, nerves that normally transmit
mice expressed Tlr4. Both S100A8 and a2-macroglobulin information about pain to the brain undergo numerous
stimulated release of the proalgesic chemokine MCP-1 changes in their properties, resulting in hypersensitivity
to painful stimulation or in allodynia (defined as pain in
Dr. R. E. Miller’s work was supported by an Arthritis Founda- response to a normally innocuous stimulus) or even
tion Postdoctoral Fellowship and by NIH/National Institute of Arthritis resulting in pain in the absence of a stimulus (1).
and Musculoskeletal and Skin Diseases (NIAMS) grant F32-AR-
062927. Dr. R. J. Miller’s work was supported by NIH/NIAMS grant Chronic joint disease is frequently accompanied
R01-AR-064251. Dr. Malfait’s work was supported by NIH/NIAMS by pain. Notably, osteoarthritis (OA), the most common
grants R01-AR-064251 and R01-AR-060364. form of arthritis, is a major source of chronic pain (2).
1
Rachel E. Miller, PhD, Shingo Ishihara, MS, Phuong B.
Tran, PhD, Anne-Marie Malfait, MD, PhD: Rush University Medical The lifetime risk of developing symptomatic OA of the
Center, Chicago, Illinois; 2Abdelhak Belmadani, PhD, Dongjun Ren, knee is ;45%, based on Johnston County Osteoarthritis
BS, Richard J. Miller, PhD: Northwestern University, Chicago, Illinois.
Address correspondence to Anne-Marie Malfait, MD, PhD,
Project data (3), and OA is a leading cause of chronic
Department of Internal Medicine, Division of Rheumatology, Rush pain worldwide (4). Treatment options for OA pain
University Medical Center, 1611 West Harrison Street, Suite 510, include acetaminophen, nonsteroidal antiinflammatory
Chicago, IL 60612. E-mail: [email protected].
Submitted for publication January 25, 2015; accepted in drugs, opioids, and intraarticular therapy with glucocor-
revised form July 16, 2015. ticoid or hyaluronan preparations, but their efficacy is
2933
2934 MILLER ET AL

limited, and long-term use can be associated with S100A8 synovial expression (24). The plasma protein,
adverse effects (5). a2-macroglobulin, is detectable in synovial fluid in both
In order to develop improved analgesics, a better early and late-stage knee OA (23,25–27). S100A8 and
understanding of the mechanisms that generate and a2-macroglobulin have been shown to promote catabol-
maintain OA pain is necessary. Current evidence sug- ic pathways in macrophages through TLR-4 (22,23).
gests that ongoing nociceptive input from the joint con- We therefore investigated the ability of these
tributes to the maintenance of OA knee pain (6). The DAMPs to excite sensory neurons through TLR-4 and
pathology of knee OA affects all joint tissues, including to promote a proalgesic state. We have previously
the cartilage, subchondral bone, menisci, ligaments, and shown that wild-type mice develop sustained mechanical
synovium (7). With the exception of articular cartilage, allodynia for 16 weeks after destabilization of the medial
joint tissues are innervated by sensory neurons. The cell meniscus (DMM), but not after sham surgery, indicating
bodies of these afferents are contained in the dorsal that maintenance of mechanical allodynia is associated
root ganglia (DRGs). These neurons are pseudounipo- with joint damage in this model (28,29). Therefore, we
lar, extending one axon toward the periphery and the tested the role of TLR-4 in the development of mechan-
other toward the dorsal horn of the spinal cord, where ical allodynia, a behavioral measure of sensitization, in
synapses are formed with second-order neurons. Noci- the DMM model.
ceptive neurons, either small nonmyelinated C-fibers or
medium-sized lightly myelinated Ad fibers, are a subtype MATERIALS AND METHODS
of sensory afferents that detect mechanical, chemical, or
thermal noxious stimuli in the innervated tissues (1). Animals and surgery. A total of 125 mice were used.
All animal experiments were approved by the Institutional
Therefore, it is of great interest to define mediators in Animal Care and Use Committees at Rush University Medical
the OA joint that can sensitize nociceptors and lead to Center and Northwestern University. Animals were housed
changes that contribute to pain. Numerous mediators of with food and water ad libitum and kept on 12-hour light
inflammation, including a number of cytokines and che- cycles. Wild-type C57BL/6 mice (The Jackson Laboratory),
mokines present in the arthritic joint, are capable of TLR-4–/– mice (C57BL/6 background; courtesy of Dr. David
activating nociceptors and may thus contribute to joint Hackam, University of Pittsburgh, Pittsburgh, PA) (30), and
Pirt-GCaMP3 mice (C57BL/6 background; courtesy of Dr.
pain (8–10). Xinzhong Dong, Johns Hopkins University, Baltimore, MD)
In addition to traditional inflammatory cytokines (31) were used. DMM surgery was performed as previously
such as interleukin-1b (IL-1b), IL-6, and tumor necrosis described (29,32) in the right knee of 10-week-old male mice.
factor (TNF), damage-associated molecular patterns Briefly, after medial parapatellar arthrotomy, the anterior fat
(DAMPs) (11,12) have been implicated as drivers of the pad was dissected to expose the anterior medial meniscotibial
ligament, which was severed. The knee was flushed with saline,
chronic, low-level inflammation associated with OA
and the incision was closed.
(11,13). DAMPs can activate pattern-recognition recep- DRG cell culture and stimulations. Cells were isolat-
tors (PRRs) on chondrocytes and synovial macrophages, ed from knee-innervating DRGs (L3–L5) obtained from 3–4
thus promoting cartilage degradation and synovitis in OA mice (male or female naive C57BL/6 or TLR-4–/– mice at least
(14). These PRRs include Toll-like receptors (TLRs) and 10 weeks of age or male C57BL/6 mice 8 weeks after DMM
the receptor for advanced glycation end products surgery), plated onto glass coverslips, and cultured in adult
neurogenic medium, as previously described (29). Overnight
(RAGE). In particular, TLR-4 has been highlighted as a stimulations with S100A8 (0–1 mg/ml; ProSpec) or a2-macro-
potential target for disease-modifying OA drugs (15). globulin (0–100 mg/ml; EMD Millipore), with or without the
Since it has recently been reported that DRG selective small-molecule TLR-4 inhibitor TAK-242 (1 mM;
neurons also express PRRs (16–19), including TLR-4 EMD Millipore), were carried out from day 3 to day 4. Stimu-
(16,20,21), we hypothesized that DAMPs generated in lation with 10 ng/ml of IL-1a (PeproTech) was used as a posi-
the OA joint can directly excite nociceptors and, thus, tive control. Similar results were obtained with or without
polymyxin B (10 mg/ml; Sigma-Aldrich), an inhibitor of lipo-
provide a putative link between tissue damage and pain polysaccharide (LPS), which was used to control for endotoxin
development. To test this hypothesis, we chose to contamination. In addition, endotoxin levels in S100A8 and
investigate 2 endogenous activators of TLR-4 present a2-macroglobulin were confirmed to be less than 0.01 endotox-
in OA joints, namely, S100A8 (22) and a2-macroglobu- in units/mg by Limulus amebocyte cell lysate assay (Fisher
lin (23). Synovial biopsy samples taken from patients Scientific). As a positive control for TLR-4 activation, stimula-
tions were performed using the TLR-4 ligand LPS (1 mg/ml)
with early symptomatic knee OA had significantly ele-
(ultrapure LPS from Escherichia coli O111:B4, purified so that
vated expression of the alarmin S100A8, which corre- only TLR-4 is activated [33], catalog no. tlrl-3pelps; Invivo-
lated with the presence of synovitis (24). Patients Gen). Supernatants were collected for protein analysis. Three
undergoing total joint replacement also had abundant independent experiments were performed per stimulus.
OA DAMAGE PRODUCTS EXCITE NEURONS 2935

Figure 1. Candidate osteoarthritis damage-associated molecular patterns (DAMPs)–induced production of monocyte chemoattractant protein 1
(MCP-1) by dorsal root ganglion (DRG) cells primarily through Toll-like receptor 4 (TLR-4). A, Wild-type mouse DRG cells left unstimulated or stimu-
lated with 0.5 or 1 mg/ml of S100A8, with or without 1 mM TAK-242 (Tak; a small-molecule TLR-4 inhibitor), or with 10 ng/ml of interleukin-1a (IL-1a;
positive control). B, Wild-type mouse DRG cells left unstimulated or stimulated with 50 or 100 mg/ml of a2-macroglobulin (a2m), with or without 1 mM
TAK-242. C, Fold change in wild-type naive mouse DRG cells over no stimulation, using combined data from the 3 independent experiments for S100A8
and a2-macroglobulin. ** 5 P , 0.01 versus no stimulation. D, Wild-type mouse DRG cells stimulated with 1 mg/ml of lipopolysaccharide (LPS), with or
without 1 mM TAK-242. E, TLR-4–/– mouse DRG cells without stimulation or after stimulation with 1 mg/ml of S100A8, 100 mg/ml of a2-macroglobulin,
or 10 ng/ml of IL-1a. F, DRG cells obtained 8 weeks after destabilization of the medical meniscus (DMM18), incubated in the presence or absence of 1
mM TAK-242. Results in A, B, E (for S100A8), and F are representative of 3 independent experiments; those in D and E (for a2-macroglobulin) are repre-
sentative of 2 independent experiments. Horizontal lines with bars show the mean 6 SEM. * 5 P , 0.05; ** 5 P , 0.01; *** 5 P , 0.001.

Protein analysis of supernatant. Total protein levels intracellular Ca21 imaging, following standard protocols
were determined by bicinchoninic acid assay (ThermoFisher). using Fura 2 AM (2 mM; Life Technologies) (29,35). S100A8 (1
MCP-1 protein levels were determined via enzyme-linked mg/ml) or a2-macroglobulin (100 mg/ml) was applied for 3
immunosorbent assay (R&D Systems). minutes by adding 0.5–1 ml of solution to the bath chamber.
In situ hybridization. Ipsilateral L3–L5 DRGs were Cells were washed with balanced salt solution before apply-
harvested, embedded, and sectioned as previously described ing controls (potassium [50 mM] and capsaicin [10 mM]). LPS
(29). For the generation of TLR-4 probes, a 405-bp Tlr4 comple- (1 mg/ml) was used as a positive control for TLR-4 activation.
mentary DNA (cDNA) fragment (GenBank no. NM_021297) Three independent experiments, each using DRGs pooled
was cloned by polymerase chain reaction (PCR) by using from 4 naive wild-type C57BL/6 mice, 3 independent experi-
mouse brain cDNA. The resulting PCR product was subcloned ments each using DRGs pooled from 4 wild-type C57BL/6
into a pGEM-T Easy Vector (Promega) and verified by restric- mice 8 weeks after DMM surgery, and 2 independent experi-
tion analysis and automated DNA sequencing (PerkinElmer). ments using DRGs pooled from 4 TLR-4–/– naive mice, were
The TLR-4 template was linearized with Xba I to generate an performed.
antisense probe by using SP6 polymerase. The sense probe was Ex vivo calcium imaging of intact DRGs. Intact
linearized with Hind III by using T7 polymerase. In situ hybrid- DRGs (L4 or L5) were isolated from naive male or female Pirt-
ization histochemistry for TLR-4 was performed by using GCaMP3 mice and equilibrated in artificial cerebrospinal fluid
digoxigenin-labeled riboprobes (Roche Applied Science) as (CSF) (31) bubbled with 95% O2/5% CO2 on ice. After 30
previously described (34). minutes, explants were placed in a perfusion chamber in artifi-
In vitro calcium imaging. The response of cultured cial CSF and imaged at a 488-nm wavelength using a CSU-X1
DRG neurons to selected DAMPs was recorded though spinning-disk confocal microscope (Intelligent Imaging Innova-
2936 MILLER ET AL

tions) at 203 magnification. Explants were stimulated by inject- in B to test for endotoxin contamination. Similar results
ing 10 ml of LPS solution into a continuously running perfusion were found in the presence and absence of polymyxin B
chamber at a total volume of 1 ml (50 mg/ml of LPS). Positive
(see Supplementary Figure 1, available on the Arthritis &
controls (potassium and capsaicin) were applied as for in vitro
calcium imaging. Image analysis was performed using an Rheumatology web site at http://onlinelibrary.wiley.com/
ImageJ (36) macro to determine the change in fluorescence doi/10.1002/art.39291/abstract). In 3 independent experi-
intensity over time. Neurons with spontaneous responses to per- ments, both S100A8 and a2-macroglobulin induced signif-
fusion buffer were excluded. icantly greater amounts of MCP-1 than did unstimulated
Testing with von Frey fibers. Wild-type mice or cells (Figure 1C).
TLR-4–/– mice were tested for mechanical allodynia using von
Frey fibers as previously described (29,37,38). Baseline thresh- Role of TLR-4 in MCP-1 production. Since the
olds were assessed prior to surgery and at weeks 2, 4, 8, 12, TLR-4 pathway has been reported to mediate cytokine pro-
and 16 after DMM. Results are representative of 2 indepen- duction in response to these DAMPs in other cell types,
dent experiments. including macrophages and phagocytes (22,23,40), we
Histopathology of the knee. Sixteen weeks after investigated whether the observed MCP-1 up-regulation
DMM, histopathologic changes in the knee joint were evaluated
according to the recommendations of the Osteoarthritis
was mediated through TLR-4. A small-molecule TLR-4
Research Society International (39) (Alison Bendele, Bolder inhibitor, TAK-242 (1 mM), significantly inhibited the
BioPATH, Inc., Boulder, CO). Joints were fixed in 10% forma- MCP-1 production induced by S100A8 (mean 6 SEM
lin, decalcified, embedded in the frontal plane, sectioned, and inhibition 83 6 2%; n 5 3 independent experiments) and
stained with toluidine blue, as described (29). Medial and later- by a2-macroglobulin (88 6 2%; n 5 3 independent experi-
al femoral condyles and tibial plateaus (3 zones per component)
ments) (Figures 1A and B). The pathogen-associated
were scored for severity of cartilage degeneration using a scale
of 0–5, where 5 represents the most damage (maximum molecular pattern (PAMP) lipopolysaccharide (TLR-4–
summed score 60). Scoring of the osteophytes on the medial selective LPS from E coli O111:B4 [33]) was tested as a
and lateral sides (the largest on the tibial or femoral surface positive control for the ability of the TLR-4 pathway to
under evaluation) and categorization as small, medium, and induce MCP-1 release. LPS stimulated the production of
large were done with the use of an ocular micrometer and were MCP-1 a mean 6 SEM of 19.0 6 6.6–fold over that of
scored on a scale of 0–3, where 3 represents large.
Statistical analysis. For MCP-1 stimulation experi- unstimulated cells, and TAK-242 completely blocked
ments, one-way analysis of variance (ANOVA) with Bonfer- LPS-stimulated MCP-1 release (Figure 1D).
roni post test or unpaired t-tests assuming equal variances In TLR-4–/– mouse cells, stimulation of MCP-1
were used to compare the groups of interest. For calcium production by S100A8 and by a2-macroglobulin was inhib-
imaging experiments, Fisher’s exact test was used to compare ited to a similar extent as was seen with TAK-242, while
the number of responses in capsaicin-sensitive neurons versus
non–capsaicin-sensitive neurons. For von Frey testing, one-
IL-1a still provoked robust stimulation of MCP-1 (Figure
way ANOVA with Bonferroni post test was used to compare 1E). Specifically, S100A8 stimulated only a 3.0 6 0.3–fold
each time point versus time 0. For joint histopathology, an production of MCP-1 as compared to unstimulated TLR-
unpaired t-test assuming equal variance was used to compare 4–/– mouse cells, whereas a2-macroglobulin stimulation
TLR-4–/– mice and wild-type mice. P values less than 0.05 were was completely inhibited (1.4 6 0.03–fold versus unstimu-
considered significant for all tests. All analyses were carried
lated TLR-4–/– mouse cells).
out using GraphPad Prism software version 5.00 for Windows.
Results are presented as the mean 6 SEM. Together, these results suggest that S100A8 and
a2-macroglobulin primarily signal through TLR-4 on
RESULTS DRG neurons.
TLR-4 blocking and reduction of MCP-1 pro-
MCP-1 production by cultured DRG cells. We duced by DRG cells harvested 8 weeks after DMM
previously found that MCP-1 is up-regulated by DRG surgery. We have previously shown that 8 weeks after
neurons in the DMM model and acts as a key mediator DMM surgery, cultured DRG cells produce increased
of pain (29). In order to determine whether selected amounts of MCP-1 as compared to sham-operated neu-
DAMPs can promote a proalgesic state in DRG cells, rons and to age-matched naive neurons (29). Here, we
we stimulated primary cultures of DRG cells and mea- confirmed our previous results, finding that DRG cells
sured the release of the chemokine MCP-1 (CCL2) into from DMM mice 8 weeks after surgery produced more
the culture medium. Overnight stimulation of DRG MCP-1 than did naive mouse cells (Figures 1A and F).
cells with S100A8 or a2-macroglobulin induced the Incubating cultures of DRGs from DMM mice with
release of MCP-1 in a concentration-dependent manner TAK-242 significantly reduced MCP-1 production, sug-
(Figures 1A and B). IL-1a was included as a positive gesting that part of the up-regulated expression of
control for MCP-1 stimulation (Figure 1A). A subset of MCP-1 by these cells in OA is driven by activation of
experiments was performed in the presence of polymyx- TLR-4 (Figure 1F).
OA DAMAGE PRODUCTS EXCITE NEURONS 2937

studies using an immunohistochemical approach (20,21).

A sense probe was used as a negative control (Figure 2B).
Similar numbers and sizes of neurons expressed Tlr4 at
8 weeks after DMM as compared to that in DRG sections
from wild-type naive mice (Figure 2C).
Calcium imaging in cultured naive mouse DRG
neurons. We next examined the ability of S100A8 and
a2-macroglobulin to elicit calcium signals by determining
whether they could directly excite DRG neurons. Cultured
DRG neurons rapidly responded to S100A8 (1 mg/ml)
and to a2-macroglobulin (100 mg/ml), as indicated by
increased intracellular calcium levels, suggesting that DRG
neurons express receptors for these proteins (Figures 3A
and B). Response to potassium depolarization for the
activation of voltage-dependent calcium channels was
used to confirm that each traced cell was a viable senso-
ry neuron. Both S100A8 and a2-macroglobulin induced
responses in small-to-medium–diameter neurons,
which is consistent with the size of nociceptors (Figures
3C and D).
We divided the responses into 2 categories:
responses by capsaicin-sensitive neurons (marker for
transient receptor potential vanilloid channel 1 [TRPV1]–
expressing nociceptive neurons) and responses by
non–capsaicin-sensitive neurons. Both S100A8 and a2-
macroglobulin induced more responses in capsaicin-
sensitive neurons than in non–capsaicin-sensitive neurons
(Table 1), although only a2-macroglobulin induced signif-
icantly greater responses in capsaicin-sensitive neurons.
Increases in LPS-induced [Ca21]i were also primarily
observed in capsaicin-sensitive neurons (Table 1).
In DRG neurons obtained from TLR-4–/– mice,
responses to S100A8 and a2-macroglobulin were seen in a
reduced number of neurons as compared to those from
wild-type mice. Combined data from 2 independent experi-
ments in TLR-42/2 mouse neurons showed that 3 of 113
neurons (2.7%) responded to S100A8 and 1 of 99 neurons
(1.0%) responded to a2-macroglobulin. This again con-
firms that S100A8 and a2-macroglobulin primarily signal
Figure 2. A, Representative image of in situ hybridization using through TLR-4 on DRG neurons.
an antisense probe for Tlr4 in dorsal root ganglion (DRG) sec- Calcium imaging in cultured DRG neurons
tions obtained from wild-type naive mice (n 5 2). B, Sense probe from mice that had undergone DMM. We also tested
control. C, Representative image of in situ hybridization using an
antisense probe for Tlr4 in DRG sections obtained 8 weeks after
whether DRG neurons obtained from DMM mice 8 weeks
surgical destabilization of the medial meniscus in wild-type mice after surgery could be directly excited by S100A8 or by
(n 5 2). Bars 5 100 mm. a2-macroglobulin. Increased numbers of neurons from
DMM mice responded to S100A8 as compared to neu-
Expression of TLR-4 by DRG neurons. In order rons from naive mice (P 5 0.0004) (Table 1). In contrast,
to determine which cells in the DRG express Tlr4, in situ similar numbers of neurons from DMM mice responded
hybridization was performed. Staining DRG sections to a2-macroglobulin as compared to those from naive
from naive mice with the antisense probe revealed that mice, perhaps reflecting differences in the way these 2
Tlr4 was widely expressed by small-to-medium–diameter stimuli engage and signal through the TLR-4 receptor. In
neurons (Figure 2A), which is consistent with previous both cases, responses were primarily in small-to-medium–
2938 MILLER ET AL

Figure 3. Candidate osteoarthritis DAMP induction of an increased intracellular calcium concentration ([Ca21]i) in cultured primary DRG neu-
rons. A and B, Representative tracings showing [Ca21]i increases in a wild-type mouse DRG neuron in response to S100A8 (A) and a2-macro-
globulin (a2m) (B) at the left. Responses to 50 mM potassium (to verify neuronal identity and viability) and 10 mM capsaicin (cap) (to identify
transient receptor potential vanilloid channel 1–expressing nociceptors) are shown at the right. C and D, Histograms showing the areas of naive
mouse neurons responding to S100A8 (C) and a2-macroglobulin (D) relative to all imaged neurons, both responsive and nonresponsive. E and
F, Histograms showing the areas of neurons obtained from mice 8 weeks after DMM surgery responding to S100A8 (E) and a2-macroglobulin
(F) relative to all imaged neurons, both responsive and nonresponsive. See Figure 1 for other definitions.

diameter capsaicin-sensitive neurons, similar to the find- DRG neurons, providing additional evidence that TLR-4
ings in naive mice (Figures 3E and F and Table 1). expression and activation by DRG neurons is not a cell cul-
Ex vivo calcium imaging in intact DRG ex- ture artifact. As visualized using this ex vivo technique, LPS
plants. In order to produce a more complete picture of induced rapid [Ca21]i increases in DRG neurons (Figures
the effects of TLR-4 activation on sensory ganglia, ex 4A and B and see Supplementary Videos 1–4, available on
vivo calcium imaging was performed using intact DRG the Arthritis & Rheumatology web site at http://onlinelibrary.
explants from naive Pirt-GCaMP3 mice. Pirt-GCaMP3 wiley.com/doi/10.1002/art.39291/abstract). Responses to LPS
mice express the genetically encoded fluorescent calci- were seen in neurons of small-to-medium diameter (Figure
um indicator GCaMP3 in almost all primary sensory 4C). All neurons responding to LPS also responded to cap-
neurons via the phosphoinositide-interacting regulator saicin (Table 1 and Figure 4D). Together, these findings
of transient potential channels (PIRT) promoter, which are consistent with the notion that LPS induces responses
is selective for DRG neurons and absent from other specifically in TRPV1-expressing nociceptors.
peripheral tissues, glia, and the central nervous system Role of TLR-4 in mechanical allodynia associ-
(31). In contrast to in vitro calcium imaging of cultured ated with experimental OA. In order to assess whether
cells, this technique visualizes in situ [Ca21]i changes in TLR-4 is necessary for the development of mechanical
OA DAMAGE PRODUCTS EXCITE NEURONS 2939

Table 1. Calcium imaging of dorsal root ganglion neurons*

No. (%) of neuronal responses
Per total no. of Per total no. of No. of
Protein tested, capsaicin-sensitive non–capsaicin- Per total no. of independent
imaging type neurons sensitive neurons neurons P experiments
S100A8 (1 mg/ml)
Naive in vitro 10/127 (8) 4/134 (3) 14/261 (5) 0.1011 3
DMM in vitro 18/82 (22) 5/52 (10) 23/134 (17) 0.0984 3
a2-macroglobulin (100 mg/ml)
Naive in vitro 23/98 (23) 10/120 (8) 33/218 (15) 0.0023 3
DMM in vitro 15/76 (20) 7/81 (9) 22/157 (14) 0.0646† 3
LPS (1 mg/ml)
Naive in vitro 5/52 (10) 1/54 (2) 6/106 (6) 0.11 1
Naive ex vivo 30/104 (29) 0/82 (0) 30/186 (16) ,0.0001† 2

* Neurons were obtained from naive mice or from mice that had undergone destabilization of the medial meniscus (DMM)
8 weeks previously. P values are for the comparison of capsaicin-sensitive versus non–capsaicin-sensitive neuron groups.
LPS 5 lipopolysaccharide.
† Statistically significant.

allodynia, DMM surgery was performed in TLR-4–/– DISCUSSION

mice, and mechanical allodynia was monitored for 16
These experiments are the first to demonstrate
weeks. Mechanical allodynia developed by week 2 after
that DRG neurons can respond to representative mole-
surgery and was maintained through week 16, similar to
cules from 2 classes of DAMPs present in OA joints:
what was seen in wild-type mice (29) (Figures 5A and B). alarmins, represented by S100A8, and plasma proteins,
In addition, 16 weeks after DMM, TLR-4–/– mice (mean 6 represented by a2-macroglobulin. TLR-4 is the primary
SEM cartilage degeneration score 19.1 6 3.6 and osteo- receptor through which S100A8 and a2-macroglobulin
phyte score 1.9 6 0.3; n 5 9) showed similar joint damage excite DRG neurons, as shown by blockade using a
as wild-type mice (cartilage degeneration score 19.1 6 2.6 small-molecule TLR-4 inhibitor and as observed in
and osteophyte score 1.9 6 0.3; n 5 11). TLR-4–/– mice. Our previous results demonstrated that

Figure 4. LPS induction of increases in intracellular calcium concentration ([Ca21]i) in neurons contained within intact DRGs. A and B, Repre-
sentative images (A) and tracing (B) of Pirt-GCaMP3 mouse DRG neuron responses to the TLR-4 agonist LPS (50 mg/ml). Bars 5 10 mm. The
tracing plots the relative change in fluorescence (DF/F), indicative of increases in [Ca21]i. Potassium (50 mM) was used to verify neuronal identi-
ty and viability, capsaicin (cap; 10 mM) was used to identify transient receptor potential vanilloid channel 1–expressing nociceptors, and phos-
phate buffered saline (PBS) was used as control. Results are representative of 2 independent experiments. C, Histogram showing areas of
neurons responding to LPS relative to all imaged neurons, both responsive and nonresponsive. D, Venn diagram showing the relationship among
neurons responding to potassium, capsaicin, and LPS. See Figure 1 for other definitions.
2940 MILLER ET AL

up-regulate MCP-1 expression, and S100A9 stimulation

was mediated through TLR-4 signaling (S100A8 stimu-
lation of MCP-1 was not tested for TLR-4 dependency
in that study) (42). S100A8 has also been associated
with RAGE signaling under certain circumstances (43).
In the present study, we demonstrated that
S100A8-stimulated MCP-1 release by DRG neurons is
primarily mediated by TLR-4. Our findings suggest that
in addition to acting on chondrocytes, extracellular
S100A8 may be able to act directly on joint nociceptors
in early OA.
Synovial fluid levels of the endoproteinase inhibi-
tor a2-macroglobulin are elevated in both early and
late-stage OA (23,25–27). A wide range of proteases
that are present in synovial fluid, including matrix met-
alloproteinases and ADAMTS, are inhibited by a2-
macroglobulin (27,44,45), and intraarticular administration
of a2-macroglobulin has been found to inhibit cartilage
degradation in the rat anterior cruciate ligament transec-
tion model of OA, consistent with its protease-inhibiting
ability (46). Conversely, this protein may also promote
Figure 5. Development of ipsilateral mechanical allodynia in wild-type
inflammation by stimulating macrophages to produce cyto-
(WT) mice (A) and TLR-4–/– mice (B), as measured through 16 weeks
post–destabilization of the medical meniscus. Results are representative kines (47) through TLR-4 signaling (23). In the present
of 2 independent experiments. Values are the mean 6 SEM of 6–12 study, a2-macroglobulin stimulated DRG cells to produce
mice per time point. *** 5 P , 0.001 versus time 0. MCP-1 through TLR-4.
Using calcium imaging assays, we demonstrated
that in both naive and DMM mouse cells, increases in
MCP-1 and its receptor, CCR2, are up-regulated by [Ca21]i in response to S100A8 and to a2-macroglobulin
DRG neurons 8 weeks after DMM surgery (29). In addi- occurred in small-to-medium–diameter neurons and
tion, CCR2–/– mice are protected from persistent pain in that the majority of responses occurred in neurons that
the DMM model. The ability of DAMPs such as S100A8 express TRPV1 (capsaicin-sensitive neurons). Consis-
and a2-macroglobulin to up-regulate MCP-1 production tent with this finding, in situ hybridization showed that
in DRG neurons and the ability of a TLR-4 inhibitor to
neurons of mainly small-to-medium diameter expressed
reduce MCP-1 released by DRG cells 8 weeks after
DMM surgery suggest that the TLR-4 pathway may be Tlr4, both in naive mice and in mice 8 weeks after
an important upstream pathway in the promotion of DMM. Together, these findings suggest that it is the
pain. However, TLR-4–/– mice were not protected from nociceptor population that is able to directly respond to
developing mechanical allodynia associated with joint these DAMPs. This is consistent with recent studies
damage after DMM surgery, indicating that pathways showing that the majority of calcium responses by cul-
other than TLR-4 contribute to the development of OA- tured neurons to another TLR-4 agonist, high mobility
associated mechanical allodynia in this model. group box chromosomal protein 1 (HMGB-1), were in
Chondrocytes were found to have increased capsaicin-sensitive neurons (16,48). In addition, 6% of
S100A8 mRNA expression during the first 2 weeks after neurons responded to HMGB-1, which again, is consis-
DMM, while chondrocyte immunostaining in load- tent with the percentage of naive mouse neurons shown
bearing areas of cartilage was lost (41). This led the in the current studies to respond to S100A8 (5%) and to
authors of that study to speculate that S100A8 is secret- a2-macroglobulin (15%). Although the numbers of neu-
ed into the extracellular space, where it may act as a rons responding to S100A8 and to a2-macroglobulin
cytokine-like molecule during early OA (41). Extracellu- were decreased in TLR-4–/– mice, a few neurons still
lar homodimeric S100A8 and S100A9, but not the het- responded, indicating that other pathways, including
erodimer S100A8/A9, have been shown to induce RAGE, may also facilitate responses to these DAMPs.
catabolic responses in human (42) and ovine (41) chon- Through the use of genetically modified Pirt-
drocytes. S100A8 and S100A9 stimulation of chondro- GCaMP3 mice, it was possible to visualize increases in
cyte monolayers derived from OA patients was shown to [Ca21]i in DRG neurons within seconds of application
OA DAMAGE PRODUCTS EXCITE NEURONS 2941

of LPS, a TLR-4 ligand, while neurons were still con- intrathecal injection of a TLR-4 antagonist reduced
tained in intact DRGs. Intact DRGs are more relevant mechanical allodynia associated with several models of
to the in vivo situation, providing evidence that these acute inflammation (56). Here, we did not observe a
functional responses to TLR-4 ligands are not an arti- reduction in mechanical allodynia in TLR-4–/– mice in
fact of cell isolation and culture and that neurons are the DMM model, which may be due to differences in
able to directly respond to these stimuli. These results both peripheral and central levels of inflammation in the
support previous work in cell cultures showing TLR-4– DMM model as compared to the models above.
mediated LPS-induced [Ca21]i increases in dissociated It is interesting to note that TLR-4–/– mice devel-
DRG neurons (16,20,49,50). Again, the fact that oped similar joint damage as wild-type mice by 16 weeks
responses were seen primarily in capsaicin-sensitive after surgery. This is consistent with previous reports that
neurons suggests that LPS-responsive neurons are a S100A9–/– mice (which are also functional S100A8–/– mice
subpopulation of TRPV1-expressing nociceptors, which [57]) were not protected from joint damage after DMM
is consistent with recent reports studying calcium fluxes (24,58). In a murine partial meniscectomy model of
induced by LPS in cell culture (16,20). Although we OA, TLR-4–/– mice also developed similar joint destruc-
have not identified the source of the [Ca21]i signal in tion as compared to wild-type mice (59). In contrast, in
cell culture or whole ganglion experiments, published collagenase-induced arthritis, which has more synovitis
observations that activation of TLR-4 can depolarize than the DMM model, synovial expression of S100A8 and
populations of DRG neurons indicate that a rise in S100A9 has been shown to be up-regulated for prolonged
[Ca21]i represents direct excitation of neurons by this periods (24,58), and S100A9–/– mice were protected from
mechanism (20). synovitis, cartilage degradation, and osteophyte formation
Collectively, our findings suggest a role of TLR-4 in this model (24,58). Taken together, these findings sug-
in mediating nociceptor sensitization by tissue damage gest that in a joint environment with low-level inflamma-
products such as S100A8 and a2-macroglobulin. Although tion, other pathways besides TLR-4 are likely to be
our experiments and other reports clearly establish that driving the joint damage. TLR-4–/– mice have yet to be
the cell bodies of DRG neurons express TLR-4 tested in the collagenase-induced model of OA.
(16,20,21), their presence and location on joint termini In summary, our experiments suggest a role of
need to be investigated. TLR-4 signaling in mediating the excitation of DRG
Based on our in vitro findings, we tested whether neurons by DAMPs, S100A8 and a2-macroglobulin,
TLR-4–/– mice developed mechanical allodynia, an evoked which may contribute to nociceptor sensitization in OA.
measure of sensitization, after DMM. We found that
Further research is needed to understand the relative
TLR-4–/– mice developed secondary mechanical allody-
importance of this pathway in comparison to other PRR
nia in the presence of experimental OA after DMM and
pathways in the development of mechanical allodynia
that this was maintained for 16 weeks, just like in wild-
and other OA-associated pain behaviors. In addition,
type mice (29). This may reflect the great complexity of
the complex and diverse actions of DAMPs on a variety
the OA joint milieu, where many tissue damage prod-
of cell types indicate that more research is needed to
ucts are generated in the course of progressive disease.
understand how intervening in these PRR pathways at
In addition to the TLR-4 pathway, OA DAMPs can sig-
different stages of disease and in varying inflammatory
nal through TLR-2 and RAGE (11), and other proalge-
environments augments both pain and joint damage.
sic cytokines and chemokines signal through additional
pathways, which often involve the transcription factor
NF-kB (9,51,52). Our results are consistent with the AUTHOR CONTRIBUTIONS
idea that it is unlikely that OA-associated pain would All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
arise from damage products signaling through a single the final version to be published. Dr. Malfait had full access to all of
pathway. Further, secondary mechanical allodynia in the data in the study and takes responsibility for the integrity of the
the hindpaw also reflects central sensitization (53). data and the accuracy of the data analysis.
Study conception and design. R. E. Miller, R. J. Miller, Malfait.
Although we focused on the role of TLR-4 in the Acquisition of data. R. E. Miller, Belmadani, Ishihara, Tran, Ren.
peripheral nervous system, there is evidence that this Analysis and interpretation of data. R. E. Miller, Ishihara, R. J.
receptor can play a role in the regulation of neuroim- Miller, Malfait.
mune processes in the central nervous system. In a nerve
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