Painful Pathways Induced by TLR Stimulation of Dorsal Root Jia Qi, Krisztina Buzas, Huiting Fan, Jeffrey I. Cohen, Kening Wang, Erik Mont, Dennis Klinman, Joost J. This information is current as Oppenheim and O. M. Zack Howard of September 24, 2021. J Immunol 2011; 186:6417-6426; Prepublished online 22 April 2011; doi: 10.4049/jimmunol.1001241

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Supplementary http://www.jimmunol.org/content/suppl/2011/04/22/jimmunol.100124 Material 1.DC1 http://www.jimmunol.org/ References This article cites 49 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/186/11/6417.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Painful Pathways Induced by TLR Stimulation of Neurons

Jia Qi,* Krisztina Buzas,*,1 Huiting Fan,* Jeffrey I. Cohen,† Kening Wang,† Erik Mont,‡ Dennis Klinman,x Joost J. Oppenheim,* and O. M. Zack Howard*

We hypothesize that innate immune signals from infectious and/or injured tissues may activate peripheral neuronal signals. In this study, we demonstrated that TLRs 3, 7, and 9 are expressed by human dorsal root ganglion neurons (DRGNs) and in cultures of primary mouse DRGNs. Stimulation of murine DRGNs with TLR ligands induced expression and production of proin-

flammatory chemokines and cytokines CCL5 (RANTES), CXCL10 (IP-10), IL-1a, IL-1b, and PGE2, which have previously been shown to augment pain. Further, TLR ligands upregulated the expression of a nociceptive receptor, transient receptor potential vanilloid type 1 (TRPV1), and enhanced calcium flux by TRPV1-expressing DRGNs. Using a tumor-induced temperature sensi- tivity model, we showed that in vivo administration of a TLR9 antagonist, known as a suppressive oligodeoxynucleotide, blocked Downloaded from tumor-induced temperature sensitivity. Taken together, these data indicate that stimulation of peripheral neurons by TLR ligands can induce pain. The Journal of Immunology, 2011, 186: 6417–6426.

oll-like receptors play a fundamental and essential role in Pain is generated by a combination of sensory and affective host defense during pathogen infection by regulating and components and classified as physiological, normal, or chronic T linking innate and adaptive immune responses (1, 2). The pain. Chronic pain, including injury-associated inflammatory http://www.jimmunol.org/ 12 mammalian TLRs belong to a family of receptors that recog- pain and -associated neuropathic pain, is often more nize pathogen-associated molecular patterns and can be divided intense than the underlying tissue damage would predict. The into those that are expressed in the membrane and those lo- vanilloid receptor 1 (VR1), which is also known as transient re- cated in endosomes. The ones located in endosomes, TLR3, ceptor potential vanilloid type 1 (TRPV1), is an re- TLR7/8, and TLR9, are activated by double-stranded and single- ceptor that has been validated as a pain target by chemical stranded nucleotides of viral or cellular origin. Innate immune stimulation, using capsaicin (CAP) or by endogenous anandamide cells sense viral infection by detecting viral proteins and/or (Ana), and by genetic deletion (4). Our earlier studies have shown nucleic acids. TLR3 is known to be a major mediator of the cel- that signals initiated by chemokine receptors (5, 6), which are lular response to viral infection because it responds to dsRNA, expressed by both immune and , enhance expression by guest on September 24, 2021 a common by-product of viral replication (3), whereas TLR7 and and function of TRPV1 (7). This led us to question if pain sen- TLR9 are activated by ssRNA and cytosine-guanosine (CpG) sation in peripheral neurons could also be en- DNA, respectively. hanced by cross-talk between classic innate immune receptors like TLRs and TRPV1. There is considerable evidence showing that TLRs participate in nerve injury in the peripheral nervous system and CNS (8–10) but *Laboratory of Molecular Immunoregulation, Cancer and Inflammatory Program, little evidence showing that neurons respond to innate immune Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD stimuli. TLR3 has a role in the activation of spinal glial cells and 21702; †Laboratory of Infectious Diseases, National Institute of Allergy and Infec- tious Diseases, National Institutes of Health, Bethesda, MD 20892; ‡Nova Scotia the development of tactile , which is pain in response x Medical Examiner Service, Halifax, Nova Scotia B3J1H6, Canada; and Laboratory to inoffensive stimulation after nerve injury (11). Intrathecal of Experimental Immunology, Cancer and Inflammatory Program, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702 administration of TLR3 agonist polyinosinic-polycytidylic acid (poly:IC) induced behavioral, morphological, and biochemical 1Current address: Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary. changes similar to those observed after nerve injury (11). Con- Received for publication April 16, 2010. Accepted for publication March 22, 2011. versely, downregulation of TLR3 inhibited injury b This work was supported by the intramural research programs of the National Cancer induced by proinflammatory cytokines, such as IL-1 , IL-6, and Institute and the National Institute of Allergy and Infectious Diseases. TNF-a (11). Furthermore, TLR3 antisense oligodeoxynucleotide The sequence presented in this article has been submitted to the Gene Expression (ODN) suppressed nerve injury-induced tactile allodynia and de- Omnibus database under accession number GSE27579. creased the phosphorylation of p38 MAPK in spinal glial cells The content of this publication does not necessarily reflect the views or policies of the (11). Lafon et al. reported that human neurons, in the absence of Department of Health and Human Services, nor does mention of trade names, com- , expressed TLR3 and sensed viral dsRNA, thus neurons have mercial products, or organization imply endorsement by the U.S. Government. the intrinsic machinery to trigger robust inflammatory, chemo- Address correspondence and reprint requests to Dr. O.M. Zack Howard, Laboratory of Molecular Immunoregulation, Cancer and Inflammatory Program, Center for Can- attractive, and antiviral responses (12). However, whether TLR3 cer Research, National Cancer Institute-Frederick, P.O. Box B, 1050 Boyles Street, contributes to pain signals remains unknown. By examining the Frederick, MD 21702. E-mail address: [email protected] role of glial cells in neuropathic pain and opioid The online version of this article contains supplemental material. actions, Hutchinson et al. demonstrated that TLR4-dependent glial Abbreviations used in this article: Ana, anandamide; CAP, capsaicin; DRG, dorsal activation is pivotal to the maintenance of neuropathic pain, and root ganglion; DRGN, dorsal root ganglion ; NGS, normal goat serum; ODN, oligodeoxynucleotide; poly:IC, polyinosinic-polycytidylic acid; Sup ODN, suppres- TLR4-dependent opioid-induced glial activation is fundamental sive oligodeoxynucleotide; TRPV1, transient receptor potential vanilloid type 1. to reducing morphine analgesia and producing dependence (13). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001241 6418 DRG NEURONS EXPRESS FUNCTIONAL TLRs

Thus, some TLRs provide a key link between the innate immune Subjects Research at the National Institutes of Health deemed this research system and the nervous system (14–16). This led us to hypothesize exempt. Cultured DRGNs and human DRG cryostat sections were fixed that TLR ligands generated by viral infections or cell death may with 4% paraformaldehyde for 10 min, washed with PBS three times each for 5 min, permeabilized with 0.1% Triton X-100 in PBS containing induce painful signals in the peripheral nervous system by stim- 1% normal goat serum (NGS) (Sigma-Aldrich) for 10 min, and washed ulating peripheral sensory neurons exemplified by dorsal root with PBS three times each for 5 min. Samples were then blocked in 5% ganglion neurons (DRGNs). We therefore investigated whether NGS at room temperature for 1 h. After blocking, the samples were then DRGNs express TLRs and whether the TLRs participate in the incubated with primary Abs, which included rabbit polyclonal Ab against TLR3 (cat. no. 3643), TLR7 (cat. no. 3269), TLR9 (cat. no. 3739) (1:200, pain signals when stimulated by TLR 3, 7, or 9 ligands. respectively; Prosci, Poway, CA; these polyclonal Abs are mouse and In the current study, we demonstrate that both human and mouse human reactive), TRPV1 (cat. no. NB100-98886, 1:1000, human and DRGNs express TLR3/7/9 and that stimulating mouse DRGNs mouse reactive; Novus Biologicals, Littleton, CO), and mouse monoclonal with TLR3/7/9 ligands increased TLR3/7/9 expression. Murine anti-neuron–specific b-III tubulin (1:200; R&D Systems) in 2% NGS DRGNs stimulated with TLR ligands increase mRNA expression overnight at 4˚C. Rabbit IgG (1:200; R&D Systems) was used as the negative control for TLRs and TRPV1. The samples were then washed and protein production of many inflammatory cytokines and with PBS three times each for 5 min and incubated with the appropriate chemokines, which have previously been identified as mediators of secondary Abs including Alexa Fluor 546 goat anti-mouse (Invitrogen, pain hypersensitivity. Further, TLR ligands upregulated the ex- Eugene, OR) or Alexa Fluor 488 goat anti-rabbit (Invitrogen) at a dilution pression of TRPV1, a nociceptive receptor, and also enhanced of 1:1000 in 2% NGS in PBS for 1 h at room temperature. Samples were 2+ then washed with PBS three times each for 5 min and mounted using calcium (Ca ) flux mediated by TRPV1. These results provide prolong gold antifade reagent with DAPI (P36935; Invitrogen) and ex- new insights into the role of TLRs in pain signaling by peripheral amined with a Zeiss LSM 510 laser-scanning microscope (Carl Zeiss neurons. MicroImaging, Thornwood, NY) equipped with argon (excitation 488 nm), Downloaded from DPSS (excitation 561 nm), and diode (excitation 405 nm) lasers. We used the following procedure to calculate the average intensity of TRPV1 in the Materials and Methods neuron surface and before and after stimulation with TLR Primary DRGN culture ligands: Otsu’s global thresholding algorithm (24) was used to identify the b-III tubulin in the red channel. The resulting binary mask of the red These studies were performed in compliance with the principles and pro- channel was then used to calculate the average intensity of TRPV1 in the cedures outlined in the National Research Council’s Guide for the Care and neuron surface and dendrites (green channel). All image-processing steps Use of Laboratory and were approved by the National Cancer were performed using a custom ImageJ (National Institutes of Health; http://www.jimmunol.org/ Institute-Frederick Care and Use Committee under Animal Study http://rsb.info.nih.gov/ij/) script. Protocols 08-005 and 10-218. DRGN cultures were prepared as described previously (17, 18) with modifications (19). A detailed description of the Western blot analysis procedure for the removal of rat dorsal root (DRGs), used in this article for both mice and rats, can be found elsewhere (20) as can concerns DRGN cultures were lysed in ice-cold lysis buffer (62.5 mM Tris-HCl, pH about plating surface coatings (21). Briefly, DRGs were dissected from 13- 6.8, 2% w/v SDS, 10% glycerol, 50 mM DTT), briefly sonicated, and 3 d-old mouse embryos of NIH Swiss mice [NIH Swiss is N:NIH(S), which centrifuged at 17,000 g at 4˚C for 10 min. Total protein in the super- was derived from the N:GP(S) colony in 1936], C57BL/6N mice (NCI natant was determined using the BCA Protein assay kit (Pierce, Thermo Animal Production Program 1999 restart), or MyD88 knockout mice (from Scientific, Logan, UT). As a positive control, spleen extract was treated as described above. Equal amounts of protein were subjected to electropho- Dr. S. Akira, Research Institute for Microbial Diseases, Osaka University, by guest on September 24, 2021 Osaka, Japan; further back-crossed to C57BL/6N by Dr. D. Klinman, Na- resis on NuPAGE Novex Bis-Tris (Invitrogen, Carlsbad, CA) in 4–12% tional Cancer Institute), respectively. Trypsin–EDTA 0.05% (Life Tech- gradient gels in MOPS buffer and then transferred to polyvinylidene nologies, Carlsbad, CA) was added to the ganglions and incubated at 37˚C fluoride membranes (Millipore, Billerica, MA). The membranes were for 10 min. After centrifugation at 350 3 g for 5 min, the cells were blocked in blocking buffer (TBST: 0.25 M Tris base, 1.37 M NaCl, 0.03 M resuspended in DMEM (Mediatech, Herndon, VA) containing 57˚C heat- KCl, and 0.1% Tween 20, pH 7.4, with 5% nonfat milk) at room tem- inactivated 10% FBS (nuclease containing; HyClone, Thermo Scientific, perature for 1 h. The membranes were then incubated with rabbit poly- clonal Ab against TLR3, TLR7, and TLR9 (1:1000; Prosci), rabbit anti- Logan, UT), 4 mM L-glutamine (Quality Biological, Gaithersburg, MD), and penicillin–streptomycin (100 U/ml and 100 mg/ml, respectively; TRPV1 (1:1000; Novus Biologicals), and rabbit anti-GAPDH (1:1000, cat. Quality Biological). The cells (7.5 3 105) were then plated onto 4-well or no. 2118; Cell Signaling Technology, Danvers, MA) overnight at 4˚C. The 8-well chambered coverglass (Nunc, Rochester, NY) precoated with poly- blots were washed three times in TBST and incubated with HRP- conjugated anti-rabbit secondary Ab (1:2000; Cell Signaling Technol- L-lysine (Sigma-Aldrich, St. Louis, MO) and collagen type 1 (Inamed Biomaterials, Fremont, CA) for 1 h. Subsequently, cells were grown in ogy) for 1 h at room temperature. Blots were then visualized with ECL neurobasal medium (Life Technologies) supplemented with 10% heat- reagent (GE Healthcare, Pittsburgh, PA). inactivated horse serum (nuclease containing; Sigma-Aldrich), 2% B-27 To quantify Western blot data, developed films were scanned, the im- supplement (Life Technologies), 3 mg/ml glucose, 0.5 mg/ml mitotic in- munoreactive bands were digitized, and the densitometry was performed hibitor (Sigma-Aldrich) to inhibit cell division, and 100 ng/ml nerve growth using a custom ImageJ (National Institutes of Health; http://rsb.info.nih. factor (R&D Systems, Minneapolis, MN) to promote neuronal survival and gov/ij/) script. The signal for each lane was calculated by summing the area 3 intensity of immunoreactivity (gray level of immunoreactive band- differentiation. DRGN cultures were maintained in the medium in a 5% CO2 incubator at 37˚C for 4 d, at which point well-differentiated dendrites were background level) of TLR3/7/9 and TRPV1 and normalized with internal 6 observed. Primary DRGN cultures were used on the fifth day of culture. control bands (GAPDH). The results, which were presented as mean SEM, were expressed as percentages of levels in control group (100%) and TLR ligand treatment statistically analyzed with Student t test for two-group comparisons. The level of significance was taken as p , 0.05. All experiments were per- TLR ligands, poly:IC (25 mg/ml, TLR3), gardiquimod (3 mg/ml, TLR7 and formed at least three times. TLR8), ODN 1826 (32 mg/ml, TLR9 agonist), and ODN 2088 (50 mg/ml, TLR9 antagonist) (InvivoGen, San Diego, CA) were dissolved in media PGE2 detection and added to the cultures on day 5 for 16 h. Suppressive oligodeox- ynucleotides (Sup ODNs) for in vivo studies were provided by Dr. Klin- Supernatants from DRGN cultures, which were pretreated with TLR man (22, 23). Sup ODN A151 had a phosphorothiate backbone of sequence ligands, poly:IC, gardiquimod, ODN 1826, or ODN 2088 (InvivoGen, San 59-TTAGGGTTAGGGTTAGGGTTAGGG-39 and was synthesized by the Diego, CA) were collected, respectively, and PGE2 was detected with PGE2 Center for Biologics Evaluation and Research Core Facility. enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) as indicated by the manufacturer. Immunochemistry RNA isolation, cDNA synthesis, and quantitative real-time Immunochemical analysis was performed on primary cultured embryonic PCR array murine DRGNs and adult human DRG sections. Human ganglions were collected at autopsy less than 24 h after death, frozen on dry ice, and storedat DRGN cultures were treated with TLR ligands for 16 h. Total RNA –80˚C before cryostat sections were obtained. The Office of Human was isolated from samples by RT2 qPCR-Grade RNA isolation kit The Journal of Immunology 6419

(SABiosciences, Frederick, MD). After checking the purification and (SABiosciences) and standard deviations calculated as recommended (31). quality by nanodrop, RNA was reverse-transcribed into cDNA (PTC-200; Ca2+ responses in activated cells were individually identified and their MJ Research, Watertown, MA) using the RT2 first strand kit (SABio- correspondence with 488-nm emission measured using the Fluoview ver- sciences). Each PCR array for quantitative PCR reaction (mouse In- sion 1.7b software. Data from activated cells on 8-well coverglass flammatory Response and Autoimmunity PCR Array, cat. no. PAMM3803; SABiosciences) was carried out in a 10-ml final volume per well con- taining cDNA (50 ng) and RT Real-Time SYBR Green/Rox PCR Master Mix (SABiosciences). Thermal cycling was performed in 384-well format plates using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Beverly, MA) according to the manufacturer’s instructions. All data were captured using the ABI Prism 7900HT Sequence Detector Software version 2.1 (Applied Biosystems) (25) and analyzed by RT2 Profiler PCR Array Data Analysis software (SABiosciences). Cytokine quantification The expression of selected cytokines, chemokines, and their receptors in the supernatant of DRGN cultures was determined by multiplex array (Aushon Biosystems, Woburn, MA). Ca2+ flux DRGNs were maintained in 8-well chambered coverglass for 5 d. Time- lapse images (phase contrast) were captured with an Olympus Confocal Downloaded from Laser Scanning Microscope, Fluoview FV1000 (Olympus, Center Valley, PA; equipped with a heated stage maintained at 25˚C and with a constant 2+ CO2 source [5%]). For Ca imaging, the culture medium was removed, and DRGNs were washed with Krebs–Ringer solution (124 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 1.3 mM NaH2PO4,26mM NaHCO3, 10 mM dextrose, pH 7.4) (26). DRGNs in the coverglass chambers were loaded with fluo-3-AM (final concentration 5 mMin http://www.jimmunol.org/ Krebs–Ringer solution) dissolved in 0.1% DMSO (Sigma-Aldrich) at 37˚C for 30 min. DRGNs were then gently washed using Krebs–Ringer solution to remove free dye. Cultures were illuminated with 488-nm light from a multi Ar+HeNe (argon and HeNe) laser through an epifluorescence Olympus FV1000 IX81 inverted microscope with a 103, 0.4 numerical aperture Olympus UPLAPO objective. Light passing through the aperture was filtered by a 505–600 nm band-pass filter (BA505-600). The basal fluorescence level was recorded for 90 s just before the application of each reagent. Subsequently, 50 ml CAP (Sigma-Aldrich, Steinheim, Switzer- land; final concentration 0.03, 0.1, 1, and 10 mM dissolved in 0.1%

DMSO) or Ana (Sigma-Aldrich; final concentration 1.86, 5.57, 16.7, and by guest on September 24, 2021 50 mM dissolved in 0.1% DMSO) were added for 10 s. To confirm the Ca2+ flux was mediated by TRPV1, we investigated the effects of SB- 366791 (Biomol International, Plymouth Meeting, PA), the selective TRPV1 antagonist (27). After being washed following dye fluo-3-AM loading, the neurons were incubated for 30 min at 25˚C with SB-366791 (1 mM) or without the compound (control). The chambers were then placed onto the microscope plate before the addition of various activators of TRPV1. Images were analyzed for changes in intensity of Ca2+-medi- ated fluorescence using Fluoview version 1.7b software (Olympus, Center Valley, PA) and converted into Rainbow color (26). Tumor-induced temperature sensitivity A tumor-induced temperature sensitivity model was adapted to our labo- ratory from earlier studies (28–30) to investigate whether TLR9 antagonist (Sup ODN) could decrease temperature sensitivity, which is mediated by TRPV1. This model is considered a neuropathic pain model because the tumor-bearing mice experience temperature hypersensitivity. The model was developed in conjunction with and approved by the National Cancer Institute-Frederick Animal Care and Use Committee (Animal Study Pro- tocol 10-218). Two million S-180 cancer cells were inoculated into the muscular tissue of female Swiss Webster mice in the immediate vicinity of the sciatic nerve near the trochanter, immediately distal to where the posterior biceps semitendinosus branches off the common sciatic nerve. A negative control group was injected with PBS instead of tumor cells. Paw withdrawal latencies to radiant heat stimulation at 55˚C were measured before any procedure and on days 2, 5, 7, 9, 11,13, and 15 after tumor in- oculation. Treatment groups consisted of $8 animals. Synthetic Sup ODNs were delivered i.p. in a dose range from 5 mg/mouse to 320 mg/mouse (22); the tumor control groups received PBS i.p.

Statistics FIGURE 1. DRGNs express TLR3, TLR7, and TLR9. Confocal images of primary cultured mouse DRGNs isolated from E13 embryos are shown. Statistical determination of PGE2 and protein levels in supernatants of DRGN cultures were performed using Student t test comparison with DRGNs were stained with TLR3 (A), TLR7 (B), and TLR9 (C) (green), control (GraphPad Prism, version 4.0c; GraphPad, San Diego, CA). The p neuron-specific b-III tubulin (red), and DAPI (blue). Shown are separate values ,0.05 were considered statistically significant. Data for RNA array monochrome images of the green, red, and blue fluorescence channel and were analyzed by RT2 Profiler PCR Array Data Analysis software merged color images from all channels. Scale bars, 50 mm. 6420 DRG NEURONS EXPRESS FUNCTIONAL TLRs chambers in three dependent experiments (a total of 30 cells) were ana- lyzed in each condition (CAP, Ana only, or pretreated with TLR ligands), and Ca2+ responses (mean of the peak values) were plotted by using Sigmaplot 8.0 software (Fig. 8). The comparison of ligands-induced ac- tivation of TRPV1 between nontreated and TLR ligands-pretreated cells Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 3. Human DRG sections express TLR3, TLR7, and TLR9. Confocal images of DRG cryostat sections. Samples were stained with TLR3 (A), TLR7 (B), and TLR9 (C) (green), neuron-specific b-III tubulin (red), and DAPI (blue). Shown are separate monochrome images of the green, red, and blue fluorescence channel and merged color images from all channels. Scale bars, 50 mm.

FIGURE 2. Negative control staining for TLRs and TRPV1 in DRGNs (A), TRPV1 in human DRGN sections (B), and magnified images from was carried out by two-way ANOVA with SPSS version 13.0. Linear re- human sections (C). Scale bars, 50 mm(A), 100 mm(B), and 10 mm(C), gression and least squares comparison (GraphPad Prism, version 4.0c; respectively. GraphPad) were used to determine the slope and correlation within The Journal of Immunology 6421

treatment groups and one-way ANOVA with post tests used to determine the probability and dosing trend.

Results TLR3/7/9 expressed by DRGNs To show that DRGNs express TLR3/7/9, we established primary cultures from day 13 mouse embryos. In these cultures, .95% of the cells showed phenotypic properties of neurons (32, 33). Im- munofluorescence staining on day 5 showed that TLR3, TLR7, or TLR9, which are pseudo-colored green in the micrographs, are expressed on neurons isolated from DRGs (Fig. 1A,1B,1C, re- spectively). Immunofluorescent controls are shown in Fig. 2A.We also found that TLR 3, 7, and 9 were expressed by neurons in adult human DRG tissue sections (Fig. 3A,3B,3C, respectively; negative controls shown in Fig. 2B,2C). b-III tubulin, which is a neuron-specific marker, is pseudo-colored red in the micro- graphs, verifying that the cells expressing TLRs were neurons.

TLR ligands enhanced TLR3/7/9 expression by DRGNs Downloaded from Based on our pilot studies (K. Buzas, J. Cohen, O.M.Z. Howard, and J.J. Oppeneheim, reported at the 2007 National Institutes of Health-Immunology Interest Group, Warrenton, VA), we deter- http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. TLR ligands induce corresponding receptor expression by DRGNs. Western blot showing TLR3, TLR7, and TLR9 expression by DRGNs after ligand stimulation are shown. A, Poly-IC (25 mg/ml) in- duced TLR3 protein expression in DRGNs. B, Gardiquimod (3 mg/ml) induced TLR7 protein expression in DRGNs. C, ODN 1826 (32 mg/ml) induced TLR9 expression in DRGNs. D, Densitometry from the blots. Cells were stimulated for 16 h then harvested, lysed, and the protein extracts were probed with anti-TLR3, anti-TLR7, and anti-TLR9 Ab. Control (Con) samples were cultured for 16 h without additional stimu- lation. Protein loading is reported using an anti-GPDH Ab. Spleen lysate was used as a positive control. One representative of more than three in- dependent experiments is shown. Image densitometry was performed using FIGURE 5. TLR ligands induce PGE2 production by DRGNs. Super- ImageJ software. The densitometry results were determined for five sites natants of DRGN cultures were collected and PGE2 was detected using m within each band, presented as mean 6 SEM, and are expressed as per- enzyme immunoassay after stimulation by poly-IC (25 g/ml), gardiqui- m m centages of levels in control group (100%). GAPDH was used as internal mod (3 g/ml), or ODN 1826 (32 g/ml) for 16 h. Each bar represents the 6 control. Statistics analysis was performed using Student t test for two- mean SEM (n = 10). A, PGE2 production by DRGNs from Swiss em- group comparisons. **p , 0.01, ***p , 0.001 (compared with control). bryonic mice. B, PGE2 production by DRGNs from C57 and MyD88 knockout embryonic mice. Statistical analysis was performed using Stu- dent t test for two-group comparisons. **p , 0.01, ***p , 0.001 (com- pared with corresponding control), ##p , 0.01 (compared with corresponding C57 mice treated with gardiquimod and ODN 1826). 6422 DRG NEURONS EXPRESS FUNCTIONAL TLRs

Table I. Fold changes in cytokine and chemokine mRNA levels and protein concentrations after stimulation of DRGNs with TLRs ligands

Cytokines and Chemokines

Ligand CCL5 CXCL10 IL-1a IL-1b mRNA Fold Increase Compared with Control Poly:IC (25 mg/ml) 289.9 6 7.4* 73.8 6 5.4* 9.7 6 1.9* 10.9 6 1.9* Gardiquimod (3 mg/ml) 16.9 6 1.56* 7.4 6 0.35* 8.9 6 0.69* 10.4 6 1.7* ODN 1826 (32 mg/ml) 9.1 6 0.69* 7.6 6 0.17* 6.4 6 1.7* 6.6 6 1.4*

Protein Concentration (pg/ml) Control 16.6 6 2.9 193.9 6 58.3 0.6 6 0.17 1.2 6 0.34 Poly:IC (25 mg/ml) 1,899.6 6 762.6* 26,455.8 6 9,714.8* 2.3 6 1.2* 7.8 6 2.9* Gardiquimod (3 mg/ml) 76.8 6 25.8 505.7 6 225.1 3.2 6 0.17* 2.8 6 0.52* ODN 1826 (32 mg/ml) 25.0 6 5.2 410.0 6 206.0 1.6 6 0.34* 3.3 6 0.69* DRG cultures were treated with TLRs ligands for 16 h. Total RNA was isolated from neurons by RNA isolation kit and reverse-transcribed into cDNA. Each PCR array for quantitative PCR reaction was carried out. The protein levels in super- natants of DRG cultures were determined by multiplex array. The results represent the mean 6 SD. Data for RNA array were analyzed by RT2 Profiler PCR Array Data Analysis software (SABiosciences). Statistical analysis for the protein levels was performed by Student t test.

*p , 0.05 compared with control (n = 3). Downloaded from mined the optimal nontoxic dose of stimulants to be 25 mg/ml duction, markedly. These data indicate that DRGNs respond to poly:IC, 3 mg/ml gardiquimod, or 32 mg/ml ODN 1826 over a 16- ligands of endosomal TLRs by producing high levels of proin- h incubation period. We applied these individual treatments to flammatory mediators. determine if DRGNs could respond to TLR ligand stimulation.

TLR ligands increased TRPV1 expression and translocation by http://www.jimmunol.org/ Western blotting analysis indicated that incubation with the DRGNs ligands promoted TLR3/7/9 expression (Fig. 4A–C). Spleen lysate was used as the positive control to confirm the expression of TLR3 The effect of TLR stimulation on TRPV1, the pain detector and (∼105 kDa), TLR7 (∼140 kDa), and TLR9 (∼95 kDa). Densi- integrator, was evaluated. As shown in Fig. 6, TLR ligands mark- tometry results are shown in Fig. 4D. edly increased TRPV1 (∼100 kDa) expression by DRGNs. More- over, TLR ligands enhanced translocation of TRPV1 proteins TLR3/7/9 ligands stimulated PGE2 release by mouse DRGN from cell bodies in DRGNs to endings (Fig. 7). The cultures elevated TRPV1 expression and translocation suggested that the PGE2 is a central mediator of febrile response triggered by the nociceptive neurons stimulated by TLR ligands are better able to by guest on September 24, 2021 inflammatory process, and intradermal PGE2 is hyperalgesic in the peripheral nervous system (34, 35). We determined the PGE2 levels in the supernatants of 5-d DRGN cultures after stimulation for 16 h. As shown in Fig. 5A, PGE2 concentration increased after stimulation by each of the TLR3/7/9 ligands compared with that of the control group (p , 0.005), which suggests that ligands for endosomal TLRs could induce DRGNs to produce this mediator of hyperalgesia and inflammation, namely PGE2. Moreover, we found that ODN 2088 (50 mg/ml), which is an antagonist of TLR9, reduced PGE2 levels induced by ODN 1826 (32 mg/ml) by 31.8% compared with ODN 1826 alone of maximum stimulation (p = 0.009). We also determined the PGE2 concentration in the super- natants of DRGN cultures of MyD88 knockout mice. As shown in Fig. 5B, gardiquimod and ODN 1826 failed to induce PGE2 production significantly in MyD88 knockout mice (p , 0.01), but as anticipated poly:IC did induce PGE2 in the knockout DRGN. TLR ligands upregulated mRNA levels and protein expression of proinflammatory cytokines and chemokines by DRGNs To confirm that ligands of endosomal TLRs induce a proin- flammatory response by DRGNs, we performed real-time quan- titative PCR (mouse Inflammatory Response and Autoimmunity PCR Array) to analyze mRNA levels in DRGNs after stimulation for 16 h with poly:IC, gardiquimod, or ODN 1826, respectively. TLR ligands dramatically increased CCL5, CXCL10, IL-1a, and FIGURE 6. TRPV1 expression is increased on DRGNs stimulated with ligands for endosomal TLRs. A, TRPV1 expression by DRGNs was de- IL-1b mRNA levels (Table I) (GSE27579, http://www.ncbi.nlm. tected by Western blot after stimulation by poly-IC (25 mg/ml), gardi- nih.gov/geo/query/acc.cgi?acc=GSE27579). These factors were quimod (3 mg/ml), or ODN 1826 (32 mg/ml) for 16 h. B, Densitometry previously identified as important mediators of pain hypersensi- from the blots (percentage of control). Image densitometry was performed tivity (5–7, 36, 37). Furthermore, as shown in Table I, TLR3/7/9 using ImageJ software. GAPDH was applied as internal control. One ligands also increased IL-1a and IL-1b protein production. representative of more than three independent experiments is shown. Moreover, poly:IC increased CXCL10 and CCL5 protein pro- ***p , 0.001 (compared with control). The Journal of Immunology 6423 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 7. TRPV1 is expressed by DRGNs and relocates after TLR ligands stimulation. Confocal images of TRPV1 expressed by primary cultured mouse DRGNs are shown. DRGNs were stained with TRPV1 (green), neuron-specific b-III tubulin (red), and DAPI (blue). Shown are monochrome images of the green fluorescence channel and merged color images from all channels. A–D, Control (A), poly-IC (25 mg/ml) (B), gardiquimod (3 mg/ml) (C), and ODN 1826 (32 mg/ml) (D) stimulation for 16 h induced TRPV1 expression in DRGNs. Scale bars, 50 mm. E, The average intensity of TRPV1 in the neuron surface and dendrites. Image intensity analysis was performed using ImageJ software. Statistical analysis was performed using Student t test for two-group comparisons. **p , 0.01 (compared with control). respond to pain stimuli. We next evaluated an in vitro correlate for (poly-IC p = 0.009, gardiquimod p = 0.029, or ODN 1826 p = pain sensation, calcium flux. 0.048). The Ca2+ influx induced by CAP in DRGNs was totally blocked by SB-366791 (Supplemental Fig. 1), a selective TRPV1 TLR ligands enhanced Ca2+ influx induced by CAP and Ana antagonist. Conversely, no Ca2+ influx in DRGNs was observed 2+ For studies of Ca influx, neurons were maintained in culture for when TLR ligands alone were applied (Supplemental Fig. 2). 5 d and only small to medium-sized neurons were studied. Murine These studies show that in addition to inducing the production of DRGNs responded with a persistent and concentration-dependent inflammatory pain-inducing mediators, TLR ligands also upreg- 2+ increase in Ca influx after stimulation with CAP as indicated by ulated expression of functional TRPV1 pain signaling receptors. an increase in the fluorescence intensity of fluo-3 emission at 488- nm excitation (Fig. 8). This demonstrated that TRPV1 in primary Sup ODN treatment blocks tumor-induced pain sensitivity cultured DRGNs was functional. As shown in Fig. 8A, DRGNs Sup ODNs, which mimic the suppressive effect of self-DNA by responded to 0.1 mM CAP with a much greater change in fluo- decreasing the immune activating signals of TLR9, were tested in rescence intensity when pretreated with poly:IC, gardiquimod, or a mouse model of tumor-induced neuropathic pain for their ability ODN 1826 for 16 h at 37˚C. Also, activation of TLR3, TLR7, or to decrease temperature sensitivity. As can be seen in Fig. 9, fi- TLR9 resulted in marked increases in the sensitivity of TRPV1 to brosarcoma tumor cells implanted near the sciatic nerve result in various concentrations of CAP (0.03, 0.1, and 1 mM) (Fig. 8B) decreased time of paw withdrawal, indicating hypersensitivity to (p = 0.008, p = 0.039, and p = 0.041, respectively). We further heat in the tumor-bearing mice. When compared with negative confirmed the enhanced sensitization of TRPV1 induced by TLR control or naive mice, the tumor-bearing mice show a significantly ligands by using an endogenous ligand of TRPV1, Ana. DRGNs faster withdrawal from the hot plate that increases over time (p , consistently responded to Ana with an enhanced Ca2+ influx when 0.001). We were unable to evaluate longer time points because pretreated with poly:IC, gardiquimod, or ODN 1826 (Fig. 8C). tumor-bearing mice begin to show stress by day 15 and must be The Ca2+ influx in neurons pretreated with TLR ligands was euthanized. Treating the tumor-bearing mice with Sup ODNs, higher in the presence of 1.86, 5.57, or 16.7 mM Ana (Fig. 8C) which block TLR9-mediated immune stimulation, resulted in an 6424 DRG NEURONS EXPRESS FUNCTIONAL TLRs

FIGURE 9. Sup ODN treatment blocks tumor-induced temperature Downloaded from sensitivity. The latency required for paw withdrawal from a 55˚C hot plate was measured. Each point represents the mean time 6 SEM (n = 8). Comparisons are made between the naive (Neg. Control) mice without S- 180 tumor (shown with closed circles) and tumor-bearing mice (shown with closed squares) or tumor-bearing mice treated with 80 mg/mouse Sup ODN (shown with closed triangles). The probability of difference between http://www.jimmunol.org/ Neg. Control and tumor-bearing mice (Tumor Control group, p = 0.001) or Sup ODN (80 mg/mouse Sup ODN group, p = 0.06) was determined using one-way ANOVA and Dunnett’s test. The trends were graphed by linear regression and the one-way ANOVA post tested for linear trend (p = 0.02) supporting a dose response.

increase in time of paw withdrawal from the heat challenge, in- dicating a reduction in heat sensitivity. Fig. 9 shows a dose re- sponse from no treatment to treatment with 320 mg/mouse Sup by guest on September 24, 2021 ODN. Beginning at the 20 mg/mouse dose, the difference between the negative control and the tumor-bearing and treated groups becomes suppressed, indicating that treatment with Sup ODN reduced tumor-induced pain. The reduction in heat sensitivity becomes significant at the 80 mg/mouse dose. This was not due to inhibition of tumor growth because a reduction in tumor growth was not observed until the highest tolerated dose (320 mg/mouse) of Sup ODN (data not shown). The paw withdrawal latencies for naive mice receiving 320 mg/mouse Sup ODN were not signifi- cantly different from those of the control group (data not shown). Thus, our data indicate that suppression of TLR9 leads to reduced sensitivity to heat in a neuropathic pain model despite the pres- ence of a growing tumor when the Sup ODNs are delivered out- side of the CNS. Our observations suggest a connection between suppression of a heat-activated pain receptor in the peripheral nervous system, when a mouse is treated with a suppressive TLR9 oligonucleotide.

Discussion One of the cardinal signals of inflammation is pain. Pathogens, FIGURE 8. TLRs stimulation enhances the Ca2+ ion channel activity of injury, and stressors stimulate TLRs on innate immune cells TRPV1 in primary cultured DRGNs. A, In DRGNs, CAP (0.1 mM) induced thereby initiating the proinflammatory signal-transduction path- a greater Ca2+ flux in cells pretreated with poly-IC (25 mg/ml, green line), ways that ultimately trigger cytokine production. In the current gardiquimod (3 mg/ml, light blue line), and ODN 1826 (32 mg/ml, dark study, we found that TLRs, which are also expressed by peripheral blue line) for 16 h than that without TLR ligands pretreatment (pink line). neuronal cells in response to synthetic ligands, produce cytokine CAP was added at 90 s for 10 s. The curves were obtained from single cells and chemokine protein products, such as IL-1a, IL-1b, RANTES, of different treatment groups. Time in seconds is shown on the x-axis, and relative fluorescent units are shown on the y-axis. B, Dose-response curve of CAP-induced Ca2+ flux in the presence or absence of TLR ligands pretreatment. C, Dose-response curve of Ana-induced Ca2+ influx in the mean 6 SEM (n = 30). The data were analyzed by two-way ANOVA. *p , presence or absence of TLR ligands pretreatment. Each bar represents the 0.05, **p , 0.01 (compared with control). The Journal of Immunology 6425 and IP-10, which have previously been shown to act as pain tide. Substance P and calcitonin gene-related peptide synergize to mediators (5, 6, 36–38). induce a phenomenon called neurogenic inflammation that results The current results provide the first evidence, to our knowledge, in increased blood– barrier permeability (49). Investigating that primary cultured mouse DRGNs constitutively express TLR3/ the effect of delivering TLR agonists to the peripheral nervous 7/9 and respond to their ligands. We focused on embryonic system on blood–brain barrier permeability will require future in DRGNs because the resulting cultures generate highly pure vivo studies. (.95%) single neurons, thereby allowing us to investigate selec- Inflammation induces neuropathic pain, which is experienced as tively neuronal TLR expression and responsiveness to TLR hypersensitivity to thermal, mechanical, or chemical stimuli. The ligands. Our results indicate that DRGNs have the potential to model of neuropathic pain used in this study, implantation of fi- recognize viral and cellular products and initiate an inflammatory brosarcoma S-180 cells near the sciatic nerve, is thought to induce response in the peripheral nervous system without prior activation pain through two pathways, mechanical restriction of the nerve (28) of immune cells to produce proinflammatory cytokines. Indeed, in as the tumor grows and tumor-produced soluble factors like PGE2 our study, stimulation by the TLR ligands (poly-IC, gardiquimod, (50) and chemokines (51). In this study, we have shown that and ODN 1826) induced cytokine (IL-1a and IL-1b) and che- suppressive oligonucleotides that block TLR9-mediated immune mokine (CCL5 and CXCL10) gene expression and protein pro- activation (22) also block sensitivity to heat, which is thought to duction by DRGNs. Moreover, the activation of TLRs produced be mediated by TRPV1 (52). Taken with our in vitro data, these PGE2, which acts as a pain inducer. TLR ligand stimulation also in vivo data indicate that there is functional cross-talk between enhanced expression and translocation of TRPV1. Furthermore, innate immune receptors (TLR) and pain sensory receptors

TRPV1 relocated from the DRGN cell bodies into dendrites after (TRPV) in peripheral neurons. Downloaded from TLR ligand stimulation. TPRV1 relocalization has been reported In summary, our findings provide an intriguing example of a set to be involved in the development of hyperalgesia in vivo (39, 40). of receptors shared between cells of the immune and the nervous Notably, pretreatment with TLR ligands also enhanced the systems that are likely to enhance the responses of both systems. TRPV1-mediated Ca2+ flux induced by CAP in DRGNs. Thus, These studies show that TLRs have the potential to have both direct peripheral neurons function to bridge the innate immune and and indirect effects on pain initiation and regulation. Therefore,

sensory systems. DRGNs and TLRs are likely to be important contributors to chronic http://www.jimmunol.org/ Previous studies demonstrated that TLRs are widely expressed pain and a rewarding target in the development of novel therapeutic in the CNS, particularly by (41). Activation of TLRs on strategies in the prevention of development and in reduction of microglia and leads to production of cytokines, cellular chronic pain. adhesion molecules, chemokines, and the expression of surface Ags that results in a nervous system immune cascade. Therefore, Acknowledgments TLRs also invoke inflammation in the nervous system (42, 43). We thank Drs. Emilia Madarasz and R. Douglas Fields for sharing neuron TLRs expressed on microglia appear to trigger microglial acti- culture conditions and providing hands-on training. We thank animal tech- vation, which might be a driving force of chronic pain. Acosta nician Steve Stull for invaluable support. by guest on September 24, 2021 et al. have shown that LPS, by stimulating TLR4, regulates the Disclosures expression of the peptide nociceptin/orphanin FQ, which con- The authors have no financial conflicts of interest. tributes to feeding behavior. These observations show that TLR4 potentially acts as a key initiator of behavioral responses medi- ated by DRGNs (44). In addition, immunohistochemical analysis References of human and rat trigeminal neurons demonstrated that CAP- 1. Marshak-Rothstein, A. 2006. Toll-like receptors in systemic autoimmune dis- sensitive express TLR4, thus enabling sensory neu- ease. Nat. Rev. Immunol. 6: 823–835. 2. Busconi, L., C. M. Lau, A. S. Tabor, M. B. Uccellini, Z. Ruhe, S. Akira, rons to respond to tissue levels of bacterial substances such as LPS G. A. Viglianti, I. R. Rifkin, and A. Marshak-Rothstein. 2006. DNA and RNA (45). autoantigens as autoadjuvants. J. Endotoxin Res. 12: 379–384. 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