Use of molecular imaging to quantify response to IKK-2 inhibitor treatment in murine arthritis.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 56, No. 1, January 2007, pp 117–128 DOI 10.1002/art.22303 © 2007, American College of Rheumatology Use of Molecular Imaging to Quantify Response to IKK-2 Inhibitor Treatment in Murine Arthritis Elena S. Izmailova,1 Nancy Paz,1 Herlen Alencar,2 Miyoung Chun,1 Lisa Schopf,1 Michael Hepperle,1 Joan H. Lane,1 Geraldine Harriman,1 Yajun Xu,1 Timothy Ocain,1 Ralph Weissleder,2 Umar Mahmood,2 Aileen M. Healy,1 and Bruce Jaffee1 Objective. The NF-B signaling pathway promotes the immune response in rheumatoid arthritis (RA) and in rodent models of RA. NF-B activity is regulated by the IKK-2 kinase during inflammatory responses. To elucidate how IKK-2 inhibition suppresses disease development, we used a combination of in vivo imaging, transcription profiling, and histopathology technologies to study mice with antibodyinduced arthritis. Methods. ML120B, a potent, small molecule inhibitor of IKK-2, was administered to arthritic animals, and disease activity was monitored. NF-B activity in diseased joints was quantified by in vivo imaging. Quantitative reverse transcriptase–polymerase chain reaction was used to evaluate gene expression in joints. Protease-activated near-infrared fluorescence (NIRF) in vivo imaging was applied to assess the amounts of active proteases in the joints. Results. Oral administration of ML120B suppressed both clinical and histopathologic manifestations of disease. In vivo imaging demonstrated that NF-B activity in inflamed arthritic paws was inhibited by ML120B, resulting in significant suppression of multiple genes in the NF-B pathway, i.e., KC, epithelial neutrophil–activating peptide 78, JE, intercellular adhesion molecule 1, CD3, CD68, tumor necrosis factor ␣, interleukin-1␤, interleukin-6, inducible nitric oxide synthase, cyclooxygenase 2, matrix metalloproteinase 3, cathepsin B, and cathepsin K. NIRF in vivo imaging demonstrated that ML120B treatment dramatically reduced the amount of active proteases in the joints. Conclusion. Our data demonstrate that IKK-2 inhibition in the murine model of antibody-induced arthritis suppresses both inflammation and joint destruction. In addition, this study highlights how gene expression profiling can facilitate the identification of surrogate biomarkers of disease activity and treatment response in an experimental model of arthritis. NF-B signaling is thought to play an important role in the inflammatory response underlying the pathogenesis of autoimmune diseases, including rheumatoid arthritis (RA). NF-B activity is regulated by the IKK complex. This complex consists of 2 kinases, IKK-1 and IKK-2, and a regulatory subunit, IKK␥/NEMO. Recent data indicate that IKK-2, rather than IKK-1, participates in the pathway by which proinflammatory stimuli induce NF-B function (1). NF-B activity is implicated in promoting both inflammation and tissue remodeling, by activating the transcription of many key genes (2). Recent data from both clinical RA studies and studies in rodent models of arthritis suggest that inflammation and destruction of articular structures occur independent of one another (3,4). It is well established that NF-B is involved in the regulation of multiple proinflammatory mechanisms. Activation of NF-B is necessary and sufficient for transcriptional activation of intercellular adhesion mol- 1 Elena S. Izmailova, PhD, Nancy Paz, MS (current address: Merrimack Pharmaceuticals, Cambridge, Massachusetts), Miyoung Chun, PhD (current address: University of California, Santa Barbara), Lisa Schopf, PhD, Michael Hepperle, PhD (current address: Phenomix Corporation, San Diego, California), Joan H. Lane, DVM, DACVP, Geraldine Harriman, PhD, Yajun Xu, PhD, Timothy Ocain, PhD, Aileen M. Healy, PhD, Bruce Jaffee, PhD: Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts; 2Herlen Alencar, MD, Ralph Weissleder, MD, PhD, Umar Mahmood, MD, PhD: Massachusetts General Hospital, Charlestown. Drs. Izmailova, Schopf, Lane, Harriman, Ocain, and Jaffee have or have had stock or stock options in Millennium Pharmaceuticals, Inc. Drs. Weissleder and Mahmood have stock or stock options in Visen Medical. Dr. Mahmood holds patents on activatable optical agents. Address correspondence and reprint requests to Elena S. Izmailova, PhD, Millennium Pharmaceuticals, Inc., 35 Lansdowne Street, Cambridge, MA 02139. E-mail: Elena.Izmailova@mpi.com. Submitted for publication March 10, 2006; accepted in revised form September 22, 2006. 117 118 ecule 1 (ICAM-1) and the chemokines monocyte chemotactic protein 1 and interleukin-8 (IL-8). These molecules facilitate infiltration of inflammatory cells into the diseased joint. NF-B also regulates transcription of tumor necrosis factor ␣ (TNF␣), IL-1␤, IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX-2) (5), all of which are required for the initiation, amplification, and maintenance of chronic inflammation. The role of NF-B in the mechanisms regulating tissue remodeling is less well defined. Several lines of evidence indicate that heightened protease activity is a contributing factor in the tissue remodeling associated with RA (6,7). Proteases play a role in pannus growth and degradation of the articular structure. Both matrix metalloproteinases (MMPs) and cysteine proteases are implicated in the development of RA (8–11). NF-B regulates the production of some MMPs, such as MMP-3 and MMP-13 (12), but its effect on cysteine proteases remains unclear. One of the major challenges in assessing the efficacy of antirheumatic drugs is the inability to estimate protease activity in the joints directly and noninvasively in vivo. Radiographic assessment of the joint does not generally take place until late in the course of the disease, which limits its usefulness. The recent development of near-infrared fluorescence (NIRF) imaging probes allows for the localization, visualization, and quantification of signal in areas with increased proteolytic activity in vivo (13). Such molecular reporters may serve as sensitive indicators for use in objective assessment of treatment response. ML120B is a potent and selective small molecule inhibitor of IKK-2 (14) that suppresses the clinical and histologic disease manifestations of antibody-induced arthritis in mice. IKK-2 inhibition also results in significant suppression of multiple genes in the NF-B pathway that are involved in the development of murine antibody-induced arthritis. In the present study, we combined molecular imaging with gene expression profiling and histopathologic examination to examine the effects of IKK-2 inhibition in murine antibody-induced arthritis. In vivo imaging demonstrated direct inhibition of NF-B activity by ML120B in inflamed paws. We found that the IKK-2 inhibitor suppressed expression, in the joints, of several proinflammatory genes, including the adhesion molecule ICAM-1, the chemokines JE, KC, and epithelial neutrophil–activating peptide 78 (ENA-78), the inflammation mediators TNF␣, IL-1␤, IL-6, COX-2, and iNOS, as well as 2 markers of mononuclear cell infiltra- IZMAILOVA ET AL tion, CD3 and CD68. Of note, ML120B treatment also inhibited the expression of the tissue remodeling genes MMP-3 and the cysteine proteases cathepsin B and cathepsin K. Furthermore, IKK-2 suppression dramatically reduced the level of destructive enzymes in diseased joints as demonstrated by noninvasive NIRF imaging, and these findings were concordant with histologic evidence of cartilage damage and bone resorption. Thus, our data demonstrate that IKK-2 inhibition of antibody-induced arthritis in mice is likely the result of suppression of both inflammation and degradation of bone and cartilage. MATERIALS AND METHODS Induction of arthritis in mice. Female BALB/c mice were purchased from Charles River (Wilmington, MA). All mice were housed at Millennium Pharmaceuticals with free access to standard rodent chow diet and water and were studied at 8–9 weeks of age. Animal studies were performed according to Institutional Animal Care and Use Committee standards. For induction of arthritis, mice were injected intravenously with 2 mg anti–type II collagen (anti-CII) antibody, according to the protocol recommended by the manufacturer (Chemicon, Temecula, CA). Three days later, animals received an intraperitoneal injection of 12.5 g lipopolysaccharide (LPS) (Escherichia coli O111:B4). Administration of the IKK-2 inhibitor ML120B. The IKK-2 inhibitor ML120B was administered orally at 10 mg/kg, 30 mg/kg, or 60 mg/kg in 0.5% hydroxypropyl-methylcellulose/ 0.2% Tween twice daily starting on day 0 after anti-CII antibody injection. Untreated animals and animals injected with LPS only were used as nonarthritic controls. Clinical disease scoring. The severity of arthritis was graded visually by assessing the level of swelling in each paw, including the tarsus (ankle) and carpus (wrist) joints. Scores were assessed consistently 1 hour after morning dosing, at the following time points: day 0 (before administration of anti-CII antibody), day 3 (before disease boost with the LPS injection), day 5 or 6 (when clinical symptoms became clearly visible), and days 7 and 9 (peak of disease activity). The following scoring system was used: 0.5 ⫽ slight redness, 1.0 ⫽ 1 or more digits swollen, 1.5 ⫽ 1 or more digits swollen and red/swollen tarsus, 2.0 ⫽ moderate swelling, 2.5 ⫽ moderate swelling and swollen digits, 3.0 ⫽ severe swelling, 3.5 ⫽ severe swelling and moderate ankylosis, and 4.0 ⫽ severe swelling and complete ankylosis (maximum possible total score per animal 16). Histopathology. Hind paws from one side of each mouse were fixed for 4 days in 10% buffered formalin, decalcified for 2 weeks in 5% formic acid, and processed for paraffin embedding. Eight-micrometer sections were stained with toluidine blue and scored, under blinded conditions, by a veterinary pathologist (at BoulderPath, Boulder, CO) for inflammation, pannus formation/cartilage loss, and bone erosion. Severity was scored on a 0–5 scale. In vivo NF-B imaging. For NF-B imaging studies (15), antibody-induced arthritis was introduced into transgenic RESPONSE TO IKK-2 INHIBITION IN ARTHRITIC MICE female BALB/c mice as described above. ML120B was administered orally at 60 mg/kg twice daily. Imaging experiments were performed 1 hour after compound administration (the time of maximum concentration in peripheral blood) on days 0, 3, 7, and 9. For the imaging procedure, mice were anesthetized with 2% isoflurane in oxygen, and luciferin (150 mg/kg) was injected intraperitoneally. Ten minutes after luciferin injection, the animals were imaged in an IVIS200 system (Xenogen, Alameda, CA), with a 1-minute bioluminescence exposure. Signal quantification was based on region of interest analysis. RNA extraction and polymerase chain reaction (PCR) analysis. Total RNA was prepared from each animal separately using left rear paw whole-joint homogenate. RNA was extracted by the single-step method using RNA Stat-60 (TelTest, Friendswood, TX). After treatment with DNase I (Qiagen, Valencia, CA), complementary DNA (cDNA) was synthesized using the MultiScribe Reverse Transcription kit (Applied Biosystems, Foster City, CA). Gene expression was measured by TaqMan real-time PCR according to the protocol recommended by the manufacturer (Applied Biosystems). Target gene probes were labeled with 6-FAM, and the internal reference probe, rodent GAPDH, was labeled with VIC. PCRs were performed with the forward and reverse primers (200 nM) and the probe (100 nM) for GAPDH and the forward and reverse primers (600 nM) and probe (200 nM) for the gene of interest. The experiments were performed on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) under the following conditions: 2 minutes at 50°C and 10 minutes at 95°C, followed by 2-step PCR for 40 cycles of 95°C for 15 seconds followed by 60°C for 1 minute. The number of PCR cycles needed for FAM and VIC fluorescence to cross a threshold of a statistically significant increase in fluorescence (threshold cycle [Ct]) was measured using Applied Biosystems software. Relative target gene expression was determined using the following formula: relative expression ⫽ 2-⌬⌬Ct, where ⌬⌬Ct ⫽ (Ct target gene – Ct reference gene in experimental cDNA sample) ⫺ (Ct target gene – Ct reference gene in mock reverse-transcribed RNA sample). In vivo protease optical imaging and image analysis. Fluorescence reflectance imaging was performed using an epifluorescence system (bonSAI; Siemens, Erlangen, Germany) that is capable of near simultaneous data acquisition in multiple channels, including a broad-spectrum visible white light channel providing anatomic detail and an NIR channel providing molecular imaging information. The system consists of a 150W halogen excitation light source connected to the acquisition box through an optical waveguide. The built-in filter wheel is set to deliver light at 400–745 nm for white light images and at 645–675 nm for NIR images. On the detection side, a second filter wheel uses a 4-step neutral optical density filter for white light and a 720–750–nm filter for activated Prosense (Visen Medical, Woburn, MA) NIR fluorescence detection. A charge coupled device camera with a matrix size of 1,360 ⫻ 1,024 pixels and a resolution of 0.116 mm/pixel was used for image acquisition. Anesthesia was maintained by mask inhalation of isoflurane vaporized at concentrations of up to 4% in the induction phase, and 1.5% during imaging. The isoflurane was delivered along with 100% oxygen at a flow 119 rate of 2 liters/minute. During imaging, body temperature was kept constant at 37°C. Previous experiments aimed at optimizing imaging time indicated that statistically significant differences in signal intensity between control and diseased animals can be detected as early as day 6. Fluorescence signal intensity reaches its maximum at the peak of disease activity, between days 7 and 10. We performed imaging on days 6 and 9 after anti-CII antibody injection, to detect early disease response to treatment and to assess the effect of the added compound when disease activity is maximal. The imaging procedure was carried out 24 hours after intravenous injection of the proteaseactivated probe Prosense, which contained a total of 2 nmoles of quenched NIRF dye. This dose and timing were based on doses that had previously been used in tumor model systems. Signal intensity of this class of activated probe is linear with respect to enzyme concentration. Exposure time was 0.3 seconds for all fluorescence images, and the data were stored in DICOM (Digital Imaging and Communications in Medicine) format for subsequent analysis. System control and data storage were performed with a PC using Syngo software (Siemens). Using custom software (CMIRImage; Center for Molecular Imaging Research, Massachusetts General Hospital), signal intensities from all fluorescence images were determined using a circular region of interest placed over ankle joints, skin (snout), and a reference standard. The fluorescence signal from the mouse paws, expressed as relative fluorescence intensity, was calibrated to the standard, subtracting the skin value as background. Immediately after the last imaging session, animals were killed and paws were obtained for RNA expression analysis and histologic study. Statistical analysis. Summary statistics are reported as the mean ⫾ SEM for each treatment group. Data were tested for normal distribution. One-way analysis of variance with Dunnett’s multiple comparison test was used to identify significant differences between experimental and vehicle-treated control groups. Pearson’s correlation coefficient was used to express the correlation between fluorescence intensity and the means of total clinical scores. All statistics were generated using GraphPad Prism software (GraphPad, San Diego, CA). P values less than 0.05 were considered significant. RESULTS Increase in NF-B activity in vivo in murine arthritis. To assess NF-B activity during arthritis, we induced antibody-induced arthritis in transgenic mice expressing the luciferase gene, under control of the NF-B–inducible promoter (15). We visualized NF-B activity in diseased joints by in vivo bioluminescence imaging, captured images, and quantified the luminescence signal intensity in nonarthritic controls and arthritic paws during disease progression. On day 7 after injection of anti-CII antibodies, signal intensity was 6.5-fold higher in arthritic animals than in the control 120 IZMAILOVA ET AL Figure 1. Up-regulation of NF-B activity in vivo in the murine arthritis model. The model was generated in transgenic mice expressing the luciferase gene under the control of the NF-B–inducible promoter, as described in Materials and Methods. A, In vivo bioluminescence imaging. Shown are overlays of photographic and color-coded bioluminescence images of the front and hind paws of nonarthritic control mice (day 0) and arthritic mice on days 3, 7, and 9 after the injection of anti–type II collagen antibodies. B, Quantitative analysis of total bioluminescence signal intensity in front and hind paws. Values are the mean ⫾ SEM signal intensity in nonarthritic controls (day 0) and in arthritic mice on days 3, 7, and 9, and are representative of 3 independent experiments. Each group of mice consisted of 6–8 animals. ⴱ ⫽ P ⬍ 0.05 versus controls. group. On day 9, signal intensity was 9.4-fold higher in the arthritic animals compared with controls (Figure 1). Inhibition of the development of antibodyinduced arthritis by ML120B treatment. Clinical disease symptoms. To determine if inhibition of the NF-B pathway affects antibody-induced arthritis development, we used ML120B, a potent and selective inhibitor of IKK-2 kinase (14). The antibody-induced arthritis model was initiated by injection of anti-CII antibodies, and animals were treated orally twice daily with various concentrations of ML120B or vehicle alone, as described above. Vehicle-treated animals developed severe arthritis, with clinical disease symptoms (redness, swelling, ankylosis) appearing on day 5 and peaking between days 7 and 9 (Figure 2A). Clinical disease symptoms were almost completely inhibited with 60 mg/kg of ML120B. A small number of animals (2 of 6) developed scores of 0.5 in 1 paw on days 7 and 10 with ML120B at this dose level. Mice treated with ML120B exhibited a dose-dependent decrease in clinical symptoms compared with vehicle-treated animals (Figure 2A). Based on these data, further imaging studies characterizing the effects of IKK-2 inhibition were performed using the maximally efficacious dose evaluated, i.e., 60 mg/kg. Histopathologic assessment. We compared nonarthritic, vehicle-treated, and ML120B-treated animals on days 6 and 9 after disease initiation. Histologic staining RESPONSE TO IKK-2 INHIBITION IN ARTHRITIC MICE 121 Figure 2. Suppression of disease development in the murine antibody-induced arthritis model by administration of the IKK-2 inhibitor ML120B. A, Mean ⫾ SEM clinical scores on days 0, 3, 5, 7, and 10 in mice administered anti–type II collagen (anti-CII) antibodies and subsequently treated with vehicle or with ML120B twice daily at the doses shown. Each group of mice consisted of 6–8 animals. Values shown are representative of 5 independent experiments. ⴱⴱ ⫽ P ⬍ 0.01 versus vehicle-treated animals. B, Histopathologic assessment, on days 6 and 9 after administration of anti-CII, of joint sections from representative mice treated with vehicle or with ML120B at 60 mg/kg (toluidine blue–stained; original magnification ⫻ 10). C, Total pathology scores in the paws of nonarthritic control, vehicle-treated, and ML120B-treated mice on days 6 and 9 after administration of anti-CII. Values are the mean and SEM. 122 IZMAILOVA ET AL Figure 3. Suppression of NF-B activity in vivo in arthritic mice treated with the IKK-2 inhibitor ML120B. A, Overlays of photographic and color-coded bioluminescence images of the front and hind paws of nonarthritic control mice, vehicle-treated arthritic mice, and 60 mg/kg ML120B–treated mice on days 7 and 9. B, Quantitative analysis of total bioluminescence signal intensity. ⴱⴱⴱ ⫽ P ⬍ 0.001 versus vehicle-treated animals. Embedded graph shows the total clinical scores of the animals used in the imaging experiments. Values are the mean ⫾ SEM. of paw sections (Figure 2B) indicated that as early as day 6, joints from the vehicle-treated group had moderate cellular infiltration, edema, and minimal growth of pannus into the cartilage and subchondral zone. Cellular infiltrates were composed predominantly of neutrophils, accompanied by fibroblast-like cells and smaller numbers of lymphocytes and macrophages. The joints displayed minimal-to-mild chondrocyte loss or collagen disruption, while a few animals (2 of 8) had small areas of bone resorption and rare osteoclasts in affected joints at this time point. On day 9, animals in the vehicletreated group showed marked cellular infiltrates and edema, pronounced pannus formation, mild-to-marked chondrocyte and collagen loss, larger areas of bone resorption, and more frequent osteoclasts compared with day 6. In contrast, joints from ML120B-treated animals were mostly disease free on both day 6 and day 9. A small number of animals (2 of 8 on day 6) showed some cell infiltration, mild edema, minimal pannus formation, and cartilage loss (Figure 2C). Thus, animals treated with 60 mg/kg of ML120B twice daily exhibited suppressed disease development as judged by histopathologic as well as clinical features. Inhibition of NF-B activity in arthritic paws by ML120B treatment. To determine whether ML120B treatment directly inhibits NF-B activity in vivo, we measured NF-B activity by quantifying the luminescence signal intensity in paws of compound-treated animals and compared the findings with those in the vehicle-treated group. Luminescence signal intensity in ML120B-treated animals was significantly lower than that in the vehicle-treated animals and similar to the levels detected in nonarthritic controls (Figure 3A). Moreover, luminescence signal intensity correlated RESPONSE TO IKK-2 INHIBITION IN ARTHRITIC MICE 123 Figure 4. Suppressed expression of disease-mediating genes in arthritic mice treated with the IKK-2 inhibitor ML120B. Gene expression in the paws of nonarthritic control mice, vehicle-treated arthritic mice, and 60 mg/kg ML120B–treated mice on days 6 and 9 was measured by quantitative reverse transcriptase–polymerase chain reaction. Values are the mean ⫾ SEM relative expression (RE) of each gene. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001, versus vehicle-treated animals. ICAM-1 ⫽ intercellular adhesion molecule 1; ENA-78 ⫽ epithelial neutrophil–activating peptide 78; TNFa ⫽ tumor necrosis factor ␣; IL-1b ⫽ interleukin-1␤; iNOS ⫽ inducible nitric oxide synthase; COX-2 ⫽ cyclooxygenase 2; MMP-3 ⫽ matrix metalloproteinase 3. 124 with clinical scores during disease progression (r ⫽ 0.6, P ⬍ 0.001). Thus, these results demonstrate a correlation between the inhibition of NF-B activity in arthritic paws in vivo and clinical disease activity (Figure 3B). Suppression of the expression of diseasemediating genes by ML120B treatment. The development of RA is associated with chronic active inflammation in the synovial tissue and damage to articular surfaces. Antibody-induced arthritis exhibits both periarticular inflammation and cartilage destruction in a subacute to chronic-active setting. To gain insight into the pathogenic processes blocked by ML120B, we examined gene expression in whole-joint homogenates from nonarthritic control, vehicle-treated, and 60 mg/kg ML120B–treated mice, by quantitative reverse transcriptase–PCR. First, we examined molecules involved in the development of inflammation. We found an 11–13-fold increased induction of ICAM-1 on days 6 and 9 in the paws of vehicle-treated animals compared with the control group. A similar expression profile was observed for ENA-78, KC, and JE, neutrophil and monocyte chemoattractants induced by NF-B (Figure 4). Compared with vehicle-treated animals, ML120Btreated mice exhibited significantly suppressed ICAM-1 levels on days 6 (61%) and 9 (86%). Moreover, on both day 6 and day 9, ML120B-treated animals displayed significantly inhibited gene expression for chemokines KC (88% and 110%), ENA-78 (98% and 99%), and JE (77% and 58%) compared with vehicle-treated animals. We estimated modulation of the composition of cellular infiltrate in joints via examination of CD3 and CD68 expression, for T cells and macrophages, respectively. The expression of CD3 was increased 3.3-fold and CD68 expression was increased 7.2-fold on day 9 in the vehicle-treated animals compared with the control group, indicating increased infiltration of both T cells and macrophages. Levels of CD3 and CD68 expression in the joints of ML120B-treated animals were comparable with levels in the joints of the nonarthritic controls; this was corroborated histologically by observation of decreased cellular infiltrate. With regard to expression of the inflammation mediators TNF␣, IL-1␤, IL-6, iNOS, and COX-2, we detected a modest but significant increase in expression of TNF␣ (3-fold; P ⫽ 0.008) and COX-2 (2.5-fold; P ⫽ 0.0004) in the vehicle-treated mice, and these animals exhibited a dramatic increase in levels of messenger IZMAILOVA ET AL RNA (mRNA) for IL-1␤ (11-fold), IL-6 (76-fold), and iNOS (10-fold). ML120B treatment caused a profound inhibition of all proinflammatory markers on both day 6 and day 9 (TNF␣ 48% and 96%, respectively, IL-1␤ 91% and 98%, respectively, IL-6 94% and 100%, respectively, iNOS 80% and 66%, respectively, and COX-2 128% and 116%, respectively) (Figure 4). We next investigated whether IKK-2 inhibition ameliorates joint damage via matrix remodeling, by quantifying the expression of matrix-degrading enzymes. Expression of MMP-3 (stromelysin) was elevated up to 7.3-fold in the vehicle-treated group. ML120B administration suppressed MMP-3 expression to levels comparable with those in control mice. We also measured expression of genes for cysteine proteases and found that cathepsin B expression was elevated 3.2-fold on day 6 and 6-fold on day 9 in vehicle-treated mice. Expression of cathepsin K was significantly increased only on day 9 (12-fold). ML120B administration significantly inhibited the expression of cathepsin B on both day 6 and day 9 (73% and 66%, respectively). Cathepsin K expression was inhibited by 70% on day 9 (Figure 4). The gene expression profiling results are summarized in Table 1. Reduction of levels of destructive proteases in the joint by ML120B treatment. Since we detected increased mRNA levels for cathepsins B and K in arthritic joints, which were inhibited in the ML120B-treated group, we selected an optical probe activated by cathepsin proteases to monitor protease activity in vivo. The Prosense “smart” probe is an enzyme-cleavable fluorescent probe specific for a defined subset of proteases, including cathepsins B and K. Probe fluorescence remains quenched when the probe is administered intravenously, until activation by enzyme cleavage in the target tissue (16). The Prosense probe was injected into control, vehicle-treated, and 60 mg/kg ML120B–treated animals, and an in vivo image was captured and fluorescence intensity in paws quantified (Figure 5A). The fluorescence signal was increased 4.2-fold in the paws of vehicle-treated mice on day 6 after the injection of anti-CII antibodies and 8-fold on day 9 (Figure 5B), which indicates augmented probe enzymatic cleavage in arthritic paws, resulting in fluorescence activation. The increased fluorescence signal during disease progression correlated with clinical scores (r ⫽ 0.95, P ⬍ 0.001) (Figure 5C). The signal intensity was significantly lower in the animals receiving ML120B and was similar to the levels found in the control group (Figures 5A and B). RESPONSE TO IKK-2 INHIBITION IN ARTHRITIC MICE 125 Table 1. Regulation of gene expression in the antibody-induced arthritis model and suppression by the IKK-2 inhibitor ML120B* Day 6 Day 9 Gene Mean fold induction over nonarthritic controls Mean % inhibition with ML120B CD3 CD68 ICAM-1 JE KC ENA-78 TNF␣ IL-6 IL-1␤ iNOS COX-2 MMP-3 Cathepsin B Cathepsin K 2.2 3 13 40 8.8 22 3 76 11 10 2.5 7.3 3.2 1.7 75 40 61 77 88 98 48 94 91 80 128 97 73 ⫺14 P† Mean fold induction over nonarthritic controls Mean % inhibition with ML120B P† ⬍0.05 ⬍0.01 ⬍0.01 ⬍0.01 ⬍0.001 ⬍0.001 ⬍0.05 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 NS 3.3 7.2 11 29 5.2 37 3.4 72 10 8.4 3.2 3.5 6 12 89 87 86 58 110 99 96 100 98 66 116 119 66 70 NS ⬍0.001 ⬍0.05 NS ⬍0.001 ⬍0.001 ⬍0.05 ⬍0.001 ⬍0.001 ⬍0.05 ⬍0.001 ⬍0.05 ⬍0.01 ⬍0.01 * NS ⫽ not significant; ICAM-1 ⫽ intercellular adhesion molecule 1; ENA-78 ⫽ epithelial neutrophil–activating peptide 78; TNF␣ ⫽ tumor necrosis factor ␣; IL-6 ⫽ interleukin-6; iNOS ⫽ inducible nitric oxide synthase; COX-2 ⫽ cyclooxygenase 2; MMP-3 ⫽ matrix metalloproteinase 3. † Significance of the percent inhibition with ML120B. DISCUSSION Our results demonstrate that the combination of molecular imaging methods, gene expression profiling, and histopathologic analysis is a powerful approach to understanding the mechanism of action of small molecule inhibitors in complex models of disease. We assessed the effects of the IKK-2 inhibitor ML120B in a murine arthritis model and demonstrated that, at the efficacious dose, this compound suppressed NF-B activity and inhibited mediators of inflammation and joint destruction. There are some differences between the pathogenesis of murine antibody-induced arthritis and that of RA in humans, i.e., the antibody-induced arthritis is subacute while RA is generally chronic with periodic flares. Nevertheless, the known shared features of the characteristic inflammatory response to injury render the model informative with respect to the pathways of interest for potential therapeutic intervention. Several studies have assessed in vitro NF-B– DNA binding activity in synovial tissue both from humans and from animals with experimental disease. NFB–DNA binding was significantly greater in human RA tissue compared with that from patients with osteoarthritis, and moreover, was localized to the sites of maximum tissue destruction (17). Increased NF-B– DNA binding activity has been demonstrated in synovial extracts from rats and mice following disease development (18,19). The inhibition of NF-B binding after IKK-2 inhibitor treatment has also been demonstrated in the mouse collagen-induced arthritis model (19) and the rat adjuvant-induced arthritis model (18). In vivo imaging enabled us to visualize and quantify target activity in the inflamed joints of mice. Our data extend previous observations by providing in vivo evidence of NF-B activity during disease development and its suppression in arthritic joints via the administration of the IKK-2 inhibitor ML120B. NF-B regulates the expression of multiple genes involved in arthritis pathogenesis. This regulation may be direct, via activation of gene transcription, or indirect, via the secondary downstream effects of NF-B–regulated gene products. For example, TNF␣ production is regulated by NF-B directly via the TNF␣ promoter, which contains an NF-B binding site (20,21). NF-B also regulates the expression of cell adhesion molecules and inflammatory cell chemoattractants (5), and therefore, indirectly regulates cell migration. Our mRNA expression analyses provide evidence of decreased cell infiltration into the joints of ML120B-treated animals, which was confirmed by histopathologic analyses. One potential mechanism ex- 126 IZMAILOVA ET AL Figure 5. Reduced levels of destructive proteases in the joints of arthritic mice treated with the IKK-2 inhibitor ML120B. Protease-activated probe was injected intravenously 24 hours prior to the imaging experiment. A, Superimposition of white light and near-infrared fluorescence images of the paws of nonarthritic control, vehicle-treated, and 60 mg/kg ML120B–treated mice on days 0, 6, and 9. B, Time course of fluorescence intensity. Embedded graph represents the means of total clinical scores of the animals used in the imaging experiments. Values are the mean ⫾ SEM. ⴱⴱⴱ ⫽ P ⬍ 0.001 versus vehicle-treated animals. C, Correlation between mean total clinical scores and relative fluorescence intensity in arthritic hind paws 24 hours after injection of Prosense probe injection. Fluorescence intensities in arthritic animals correlated strongly with clinical scores (r ⫽ 0.95, P ⬍ 0.001). RESPONSE TO IKK-2 INHIBITION IN ARTHRITIC MICE plaining this observation may be inhibition of chemotactic chemokines and/or leukocyte adhesion molecules in the joint. Previous reports on the effects of IKK-2 inhibitors in animal models were limited to genes directly regulated by NF-B (19,22). We investigated the impact of IKK-2 inhibition on genes that are both directly and indirectly regulated by NF-B in the development of experimental arthritis. As expected, we observed the inhibition of several genes that are directly regulated by NF-B and involved in cell migration, inflammation, and matrix degradation, including ICAM-1, TNF␣, IL-1␤, IL-6, KC, ENA-78, JE, iNOS, COX-2, and MMP-3, in the diseased paws of animals treated with the IKK-2 inhibitor. Reports concerning the role of NF-B in cathepsin expression are controversial, and it is not clear whether IKK-2 inhibition has a direct or indirect effect (23,24). However, in vivo IKK-2 inhibition by ML120B suppressed the transcription of the cathepsin B and cathepsin K genes. Overall, our data indicate that IKK-2 inhibition has effects on multiple genes involved in the inflammatory and destructive components of disease. We observed increased expression of mRNA for several proteases, including cathepsins B and K, in arthritic animals. Since reduced expression of these genes was found in the ML120B-treated group, we hypothesized that ML120B therapy decreases the levels of destructive enzymes in joints. To test this, we used NIRF imaging with a protease-specific probe. We selected a probe that is activated by the cysteine protease cathepsin, because the results of previous studies demonstrated that cysteine proteases are up-regulated in RA synovial tissue and fluid (25). Increased cysteine protease expression is predominantly restricted to the synovium at sites of joint damage and can be detected as early as 2 weeks after the onset of disease symptoms (26). These enzymes directly degrade cartilage and bone matrices. In addition to their destructive activities, they can activate MMPs (27). Consistent with these findings, we observed a dramatic increase in fluorescence signal intensity in the joints of vehicle-treated animals, which indicated increased amounts of active proteases. In contrast, signal intensity in the ML120B-treated group was similar to that in the nonarthritic control animals. The reduction of protease-activated fluorescence signal in response to methotrexate treatment has been observed in the murine collagen-induced arthritis model (16). However, that study did not include analysis of the correlation between the decrease in protease-activated 127 fluorescence signal and clinical disease indices such as redness, swelling, and ankylosis. In the present study we demonstrated that protease-activated NIRF imaging probes can be used as sensitive biomarkers of subacute to chronic active joint disease activity and treatment efficacy in a murine antibody-induced arthritis model. We observed complete disease suppression with the IKK-2 inhibitor at the efficacious concentration used in this study. Current efforts are aimed at testing the prognostic utility of the Prosense probe for predicting clinical and histopathologic disease amelioration. In conclusion, we have demonstrated, using in vivo imaging and gene expression profiling, that the ML120B compound offers protection from inflammation and joint destruction in subacute to chronic active murine antibody-induced arthritis. Moreover, we showed a direct correlation between the drug’s diseasemodifying effects and biochemical target activity. These studies highlight how gene expression profiling can be implemented to identify surrogate biomarkers of disease activity and treatment response in experimental models of arthritis. ACKNOWLEDGMENTS The authors would like to thank K. Anderson and E. Grant for technical assistance, and A. Parker and C. Fraser for critical review of the manuscript. AUTHOR CONTRIBUTIONS Dr. Izmailova had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study design. Drs. Izmailova, Alencar, Chun, Schopf, Xu, Ocain, Weissleder, Mahmood, Healy, and Jaffee. Acquisition of data. Dr. Izmailova, Ms Paz, and Drs. Alencar, Mahmood, and Jaffee. Analysis and interpretation of data. Drs. Izmailova, Alencar, Schopf, Lane, Xu, Ocain, Mahmood, Healy, and Jaffee. Manuscript preparation. Dr. Izmailova, Ms Paz, and Drs. Alencar, Schopf, Lane, Ocain, Mahmood, Healy, and Jaffee. Statistical analysis. Dr. Izmailova, Ms Paz, and Dr. Jaffee. Medicinal chemistry. Dr. Hepperle. Invention of chemical matter, progression of key compound. Dr. Harriman. Project leadership. Dr. Xu. Manuscript editing. Dr. Weissleder. REFERENCES 1. Ghosh S, Karin M. Missing pieces in the NF-B puzzle. Cell 2002;109 Suppl:S81–96. 2. Tak PP, Firestein GS. NF-B: a key role in inflammatory diseases. J Clin Invest 2001;107:7–11. 3. Van den Berg WB, van Riel PL. Uncoupling of inflammation and 128 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. IZMAILOVA ET AL destruction in rheumatoid arthritis: myth or reality? [editorial]. Arthritis Rheum 2005;52:995–9. Smolen JS, Han C, Bala M, Maini RN, Kalden JR, van der Heijde D, et al, for the ATTRACT Study Group. Evidence of radiographic benefit of treatment with infliximab plus methotrexate in rheumatoid arthritis patients who had no clinical improvement: a detailed subanalysis of data from the Anti–Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy study. Arthritis Rheum 2005;52:1020–30. Pahl HL. Activators and target genes of Rel/NF-B transcription factors. Oncogene 1999;18:6853–66. Bresnihan B. Pathogenesis of joint damage in rheumatoid arthritis. J Rheumatol 1999;26:717–9. Tak PP, Bresnihan B. The pathogenesis and prevention of joint damage in rheumatoid arthritis: advances from synovial biopsy and tissue analysis [review]. Arthritis Rheum 2000;43:2619–33. Trabandt A, Gay RE, Fassbender HG, Gay S. Cathepsin B in synovial cells at the site of joint destruction in rheumatoid arthritis. Arthritis Rheum 1991;34:1444–51. Murphy G, Knauper V, Atkinson S, Butler G, English W, Hutton M, et al. Matrix metalloproteinases in arthritic disease. Arthritis Res 2002;4 Suppl 3:S39–49. Mengshol JA, Mix KS, Brinckerhoff CE. Matrix metalloproteinases as therapeutic targets in arthritic diseases: bull’s-eye or missing the mark? [review]. Arthritis Rheum 2002;46:13–20. Hansen T, Petrow PK, Gaumann A, Keyszer GM, Eysel P, Eckardt A, et al. Cathepsin B and its endogenous inhibitor cystatin C in rheumatoid arthritis synovium. J Rheumatol 2000;27:859–65. Han Z, Boyle DL, Manning AM, Firestein GS. AP-1 and NF-B regulation in rheumatoid arthritis and murine collagen-induced arthritis. Autoimmunity 1998;28:197–208. Rudin M, Weissleder R. Molecular imaging in drug discovery and development. Nat Rev Drug Discov 2003;2:123–31. Nagashima K, Sasseville VG, Wen D, Bielecki A, Yang H, Simpson C, et al. Rapid TNFR1 dependent lymphocyte depletion in vivo with a selective chemical inhibitor of IKK␤. Blood 2006; 107:4266–73. Carlsen H, Moskaug JO, Fromm SH, Blomhoff R. In vivo imaging of NF-B activity. J Immunol 2002;168:1441–6. Wunder A, Tung CH, Muller-Ladner U, Weissleder R, Mahmood U. In vivo imaging of protease activity in arthritis: a novel approach for monitoring treatment response. Arthritis Rheum 2004;50:2459–65. 17. Benito MJ, Murphy E, Murphy EP, van den Berg WB, FitzGerald O, Bresnihan B. Increased synovial tissue NF-B1 expression at sites adjacent to the cartilage–pannus junction in rheumatoid arthritis. Arthritis Rheum 2004;50:1781–87. 18. Schopf L, Savinainen S, Anderson K, Kujawa J, DuPont M, Silva M, et al. IKK␤ inhibition protects against bone and cartilage destruction in a rat model of rheumatoid arthritis. Arthritis Rheum 2006;54:3163–73. 19. Podolin PL, Callahan JF, Bolognese BJ, Li YH, Carlson K, Davis TG, et al. Attenuation of murine collagen-induced arthritis by a novel, potent, selective small molecule inhibitor of IB kinase 2, TPCA-1 (2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide), occurs via reduction of proinflammatory cytokines and antigen-induced T cell proliferation. J Pharmacol Exp Ther 2005;312:373–81. 20. Ghosh S, May MJ, Kopp EB. NF- B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998;16:225–60. 21. Baldwin AS Jr. The NF- B and I B proteins: new discoveries and insights. Annu Rev Immunol 1996;14:649–83. 22. McIntyre KW, Shuster DJ, Gillooly KM, Warrier RR, Connaughton SE, Hall LB, et al. Reduced incidence and severity of collagen-induced arthritis in interleukin-12-deficient mice. Eur J Immunol 1996;26:2933–8. 23. Li Q, Bever CT Jr. Effect of protein kinase modulators on the regulation of cathepsin B activity in THP-1 human monocytic leukemia cells. Oncol Rep 1998;5:227–33. 24. Liuzzo JP, Petanceska SS, Moscatelli D, Devi LA. Inflammatory mediators regulate cathepsin S in macrophages and microglia: a role in attenuating heparan sulfate interactions. Mol Med 1999;5: 320–33. 25. Maciewicz RA, Wotton SF. Degradation of cartilage matrix components by the cysteine proteinases, cathepsins B and L. Biomed Biochim Acta 1991;50:561–4. 26. Cunnane G, FitzGerald O, Hummel KM, Youssef PP, Gay RE, Gay S, et al. Synovial tissue protease gene expression and joint erosions in early rheumatoid arthritis. Arthritis Rheum 2001;44: 1744–53. 27. Eeckhout Y, Vaes G. Further studies on the activation of procollagenase, the latent precursor of bone collagenase: effects of lysosomal cathepsin B, plasmin and kallikrein, and spontaneous activation. Biochem J 1977;166:21–31.