Immunology Letters 162 (2014) 258–263
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
CCR5 small interfering RNA ameliorated joint inflammation in rats with adjuvant-induced arthritis Hongmei Duan, Pingting Yang, Fang Fang, Shuang Ding, Weiguo Xiao ∗ Department of Rheumatology and Immunology, The 1st Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Province, China
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Article history: Available online 7 October 2014 Keywords: Rheumatoid arthritis Adjuvant-induced arthritis CCR5 siRNA Electroporation
a b s t r a c t Rheumatoid arthritis (RA) is a systemic inflammatory disease. C-C chemokine receptor type 5 (CCR5) is found in inflamed synovium of RA patients and is necessary for formation of RA. We aimed to check whether delivery of CCR5-specific small interfering RNA (siRNA) via electroporation suppresses local inflammation in arthritis rats. Vectors encoding siRNA that target CCR5 or negative control siRNA were constructed for gene silencing and the silencing effects of suppressing CCR5 expression in synovium examined by western blot. The vector with strongest effect was delivered into the knee joint of adjuvantinduced arthritis (AIA) rats by the in vivo electroporation method 7, 10, 13, and 16 days after immunization with Complete Freund’s adjuvant. During an observation of 28 days, behavior, paw swelling, arthritis and histopathologic scoring were estimated. The expression level of CCR5 in synovium was evaluated by western blot and real-time PCR. Anti-CCR5 D1 siRNA was effectively inhibited CCR5 expression in vitro. Moreover, delivery of the siRNA into inflammatory joint also suppressed the expression of CCR5 in vivo and markedly suppressed paw swelling and inflammation. Local electroporation of anti-CCR5 siRNA into the left inflamed joints could achieve the silencing of CCR5 gene and alleviate local inflammation just in the knee joint injected with siRNA other than the opposite joint. Inhibition of CCR5 expression may provide a potential for treatment of RA. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a systemic inflammatory disease characterized by infiltration of leukocytes into the synovial membranes of joints as well as progressive destruction of the bone and cartilage [1] and that affects approximately 1% of population. These leukocytes especially T cells and B cells could give rise to excessive activation and proliferation of synoviocytes and fibroblasts which secrete cytokines through autocrine and paracrine mechanisms. The cytokines will induce the production of other proinflammatory cytokines and together participate in the pathogenesis of RA and eventually lead to joint damage [2,3]. Recently, the role of chemokines and their receptors in the regulation of RA has been the focus of a number of researchers. Accordingly, blockades for specific cytokines or immune regulators have applied in clinical therapy in RA. Although the role of cytokines in RA still
∗ Corresponding author at: Department of Rheumatology and Immunology, The 1st Affiliated Hospital of China Medical University, No. 155, North Nanjing Street, Heping District, Shenyang 110001, Liaoning Province, China. Tel.: +86 024 83282549/13804042268; fax: +86 024 83282549. E-mail address: [email protected] (W. Xiao). http://dx.doi.org/10.1016/j.imlet.2014.09.018 0165-2478/© 2014 Elsevier B.V. All rights reserved.
remains unclear, a variety of inflammatory cytokines are crucially involved in the pathogenesis. C-C chemokine receptor type 5 (CCR5) is specific receptor for the known ligands including macrophage inflammatory protein ␣ (MIP-1␣), MIP-1 and chemokine (C-C motif) ligand 5 (CCL5), which also known as RANTES (regulated upon activation, normal T cell expressed and secreted). Among the known chemokine receptor, CCR5 and its ligands play an important role in the pathological formation of RA [4–6]. In the synovial tissue of RA patients and animals as arthritis models, expression of CCR5 increased obviously, as well as its ligands [6,7]. Supportive evidences have proved that CCR5-delta 32 polymorphism might be a genetic marker related to the severity of RA [8]. To attempt to cure RA, antagonists of CCR5 are used to ameliorate symptoms of this disease. Yang et al. showed that CCR5 antagonist could inhibit the development of arthritis in collagen-induced arthritis (CIA) model in mice by interfering with their migration to joint injury [9]. Moreover, in rhesus monkeys, a CCR5 antagonist also had an obvious effect of alleviating the symptoms of CIA [10]. Importantly, Okamoto and co-workers demonstrated that TAK-779, a non-peptide compound which inhibited the development of adjuvant-induced arthritis (AIA), had anti-arthritis effect in Complete Freund’ s adjuvant (CFA) treated rats [11].
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The discovery of small interfering RNA (siRNA)-mediated RNA interference (RNAi) has made it possible to induce sequencespecific silencing of target messenger RNAs. Over recent years, siRNAs have been shown to effectively suppress gene expression in human cells. Although RNAi has a relatively short history in human applications, targeted silencing of gene expression via siRNA was one of the most widely used experimental tools. Now, the therapeutic potential of RNAi was highlighted in experiments indicating the efficacy of the approach against viral infection, such as human immunodeficiency virus [12,13], hepatitis B virus [14] and simplex virus 2 [15]. In addition, the siRNA-dependent strategy also be used in RA model [16–18], neurodegenerative diseases [19,20] and cancers [21,22]. Moreover, RNAi-based therapy for age-related macular degeneration has already reached clinical trials in 2004 [23]. Unfortunately, the exact effect of CCR5 knockdown with specific siRNA in RA has not been clearly elucidated in the present study. In this study, we aimed to assess the effects of silenced CCR5 via siRNA-mediated RNAi in AIA rats. First of all, plasmid containing anti-CCR5 siRNA was constructed and tested in human embryonic kidney 293 cells (Hek293T). Then the plasmid was delivered into AIA rats induced by Complete Freund’s adjuvant (CFA) via electroporation. The effects of anti-CCR5 siRNA on AIA rats were examined by calculating arthritis score and histopathologic score. Meanwhile, CCR5 expression also was test by means of western blot and real time PCR (RT-PCR) analysis.
2. Materials and methods 2.1. Animals Male Wistar rats, approximately 6 to 8 weeks old, were kindly provided by the experimental center of China Medical University and maintained under clean condition in animal facility. All animal experiments were conducted according to the Guidelines regarding Committee on Laboratory Animals of the First Affiliated Hospital of China Medical University. 2.2. siRNA duplexes Four siRNA duplexes directed against rat CCR5 and a negative control (NC) siRNA were synthesized by GeneChem (Shanghai, China). The sense sequences for each of the anti-CCR5 siRNAs were as follows: siRNA-D1, 5 -CAGGGAUCUAUCACAUUGGUUd(TT)-3 ; siRNA-D2, 5 -CAGGGCUGUAAGGCUCATCUUd(TT)-3 ; siRNA-D3, 5 -GACCACCUUCCAGGAAUACUUd(TT)-3 ; siRNA-D4, 5 -CAGCAAGUCAAUCCUGAUCGUd(TT)-3 ; NC siRNA, 5 -UUCUCCGAACGUGUCACGUd(TT)-3 . 2.3. Plasmid construction and siRNA transfection SD11 vector containing a green fluorescent protein (GFP) reporter gene was used to generate constructs encoding anti-CCR5 siRNAs or NC siRNA under the control of the rat U6 promoter. These oligonucleotides were annealed and ligated to the BamHI and HindIII sites upstream of the GFP of SD11 vector to get plasmids pU6-siRNA-GFP. The pEGFP-N1-3FLAG containing enhanced GFP (EGFP) reporter gene and triple FLAG tags was used for the construction of an overexpressed system for rat gene CCR5. XhoI and KpnI were the inserting sites. To assess silenced efficiency in vitro, pU6-siRNA-GFP (0.3 g) and pEGFP-N1-3FLAG-CCR5 (0.5 g) plasmids were co-transfected into human embryonic kidney 293 cells (Hek293T) using lipofectamine 2000 (Invitrogen, Carlsbad, California, USA).
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2.4. Observation of EGFP expression The 6 male Wistar rats were randomly selected to observe the expression of EGFP. Rats with EGFP signal were sacrificed at 1 (n = 2), 3 (n = 2) and 5 (n = 2) days after delivery of plasmid pU6siRNA-GFP into the left knee joint. Synovial membrane was stripped from the region surrounding the patella of the left knee joint of EGFP rats. After washed with PBS and fixed in 4% formaldehyde for 72 h, the synovium was dehydrated in sucrose gradient from 10% to 30%. The specimens were cut into 14-m-sections after embedded by OCT, and then were stained with hematoxylin and eosin (H&E) and examined under fluorescence microscope (BX51, Olympus, Tokyo, Japan). 2.5. Induction and evaluation of adjuvant-induced arthritis The remaining 48 male Wistar rats were randomly assigned to 4 groups (12 rats per group), including one control group (group 1) and 3 experimental groups (group 2, 3 and 4). To induce AIA, Complete Freund’s adjuvant (CFA) (Sigma, Santa Clara, CA, USA) was mixed with heat-killed Bacille Calmette–Guerin (BCG) freezedried powder (Institute of Biological Products Co. Ltd., Changchun, China). Briefly, on day 0, 36 rats in experimental groups were intradermally injected with 100 l CFA solution at the base of the tail. Rats in the control group were injected with an equal volume of physiology saline instead of CFA. After electro-transduction of plasmids encoding siRNA into rats in group 2 or group 3 for 4 times (once every 3 days, i.e., 7, 10, 13, and 16 days after immunization), rats were observed for pelage color, activity, appetite and body weight once a week for 4 weeks. Then we measured the foot volume using a water replacement plethysmometer once a week for 4 weeks. The severity of involvement of each paw and the degree of edema were measured once every 3 days for 21 days by arthritis scoring as described [24]. The left knee and ankle joints were removed from rats 17 or 28 days after disease induction, fixed in 4% paraformaldehyde, decalcified with formic acid, dehydrated with gradient ethanol, embedded in paraffin and sectioned to 6m-thick slices. Then sections were stained with H&E. Arthritic changes, such as synovial proliferation, infiltration of inflammatory cells, bone erosion and destruction of articular cartilage were evaluated using the histopathologic scores as described [25]. 2.6. Electroporation-assisted siRNA transduction in vivo Rats in group 2 or 3 were anesthetized by an intraperitoneal injection of 0.3 ml/100 g chloral hydrate, and 50 g pU6-siRNA-GFP encoding anti-CCR5 siRNA or NC siRNA diluted in 40 l PBS was administered into the left knee joint using a syringe equipped with a 50-gauge needle. Immediately after injection, a pair of electrode pads of 1.0 cm diameter was placed onto keratin cream coating around the knee joint and the gap between the pads was fixed at 0.7 cm [26]. Electric pulses were emerged by an electroporator ECM399 (Harvard apparatus, Holliston, MA, USA) and then three square-wave pulses with a pulse length of 100 ms were given at a frequency of 1 s−1 followed by three other pulses with the opposite polarity. 2.7. Western blot analysis Western blot was performed with anti-FLAG or anti-CCR5 antibody to detect CCR5 and anti-GAPDH antibody to detect GAPDH. At 36 h after transfection using lipofectamine 2000, Hek293T cells were harvested and lysed on ice in lysis buffer containing 1 M Tris–HCl, 2% mercaptoethanol, 4% SDS and 20% glycerol. Cellular debris was discarded and protein was collected by centrifugation. Total proteins were separated by 12% SDS-PAGE and
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electrotransferred onto PVDF membranes (Amresco, Solon, OH, USA). Then the membranes were blocked in 5% non-fat milk-TBST (1.0 M Tris–HCl, Ph 8.0, 150 mM NaCl, 0.05% Tween-20), incubated with mouse anti-Flag or mouse anti-GAPDH antibodies (Abcam, Cambridge, England) at a dilution of 1:500, and followed by probing with alkaline phosphatase-labeled goat anti-mouse IgG antibody at a dilution of 1:5000 (Asia Pioneer Pharmaceutical Co. Ltd., Shanghai, China). Protein CCR5 and GAPDH obtained from synovium were also examined with this method. Anti-CCR5 (Abcam, Cambridge, England) and anti-GAPDH (Sigma, Santa Clara, CA, USA) antibodies with the dilution of 1:200 and 1:400 were used. The second antibody was same to the aforementioned one. 2.8. RT-PCR To analyze CCR5 mRNA expression in vivo, the synovium was removed from the region around the left patella of AIA rats. Total RNA was extracted and subjected to RT-PCR using a pair primers for CCR5 (forward: 5 -AGAAGAAGAGGCACAGG-3 and reverse: 5 -AAAGGCATAGATGACAGG-3 ) or GAPDH (forward: 5 -TGACTTCAACAGCGACACCCA-3 and reverse: 5 -CACCCTGTTGCTGTAGCCAAA-3 ) genes. PCR amplification of these two genes was performed using the following conditions: predenaturing at 95 ◦ C for 15 s, denaturing 95 ◦ C for 5 s, annealing and extension at 60 ◦ C for 30 s, for a total of 45 cycles. RT-PCR was performed on real time PCR TP800 (TAKARA, Otsu, Shiga, Japan). 2.9. Statistical analysis Student’s t-test was used to examine the significance of differences. P value of less than 0.05 was considered as significance. On-way ANOVA test was performed for multiple comparisons and p value less than 0.05 was considered to be significant. 3. Results 3.1. Silencing of the CCR5 gene in vitro To test the silencing efficiency of siRNA for target gene CCR5, plasmids pU6-siRNA-GFP containing different anti-CCR5 siRNA (D1–D4) and NC siRNA were co-transfected with pEGFP-N1-3FLAGCCR5 into Hek293T cell. We accomplished different levels of CCR5 silencing after transfection with lipofectamine 2000 containing unmodified anti-CCR5-siRNA compared to NC group (Fig. 1). An obviously silencing of CCR5 with D1 siRNA was visualized in results and it was used in next treatment in this study.
3.2. GFP expression in synovium of rat knee joint in vivo To investigate the delivery rate in vivo with electroporation method and the continuance of anti-CCR5-siRNA, GFP expression was observed in synovium of knee joint. The signal of green fluorescence at the synovial tissue surrounding the patella emerged within 24 h after the transduction (Fig. 2) and increased remarkably with time. The intensity of green fluorescence reached a maximum at the third day and dropped down in the next time.
3.3. Therapeutic effects of siRNA Therapeutic effects of siRNA targeting CCR5 were investigated in rats with AIA. Arthritis began to appear in rats injected with CFA and meanwhile the symptoms of bad appetite, listlessness, back alopecia and slow growth of body weight also emerged. Interestingly, these symptoms in group 2 ameliorated after 14 days electroporation of pU6-D1-siRNA-GFP compared with group 3 (injected with pU6-NC-siRNA-GFP) or group 4 (only injected with CFA) (Fig. 3A). In addition, arthritis score was calculated to estimate the degree of paw swelling and the results indicated that anti-CCR5 siRNA transduction significantly suppressed paw swelling, as well as remarkably decrease of paw volume (Fig. 3B and C). These effects were not observed in the group 3 infected with NC siRNA. Meanwhile, histopathologic findings were evaluated on day 28 after CFA injection. The rats in group 3 or 4, which were treated with NC siRNA or CFA, showed typical symptoms of arthritis including massive inflammatory cell infiltration into the articular cavity, degeneration of articular cartilage, prominent hypertrophy of synovium and bone erosion (Fig. 4A). However, the paws of rats in group 2 were virtually normal and the bone and cartilage remained almost unaffected. Moreover, the histopathologic features of arthritis were statistically significant between the opposite knee joint of rats in group 2 and bilateral knee joints of rats in group 3 (Fig. 4B). Disappointingly, the inhibitory effects of anti-CCR5 siRNA to arthritis were only observed in the hind limb on the infected side, but not on the opposite side. As shown in Fig. 5A and B, western blot analysis for synovium tissue revealed that there was not significant expression of CCR5 in the control group. However, the expression of CCR5 was higher after CFA immunization both in bilateral knee synovium. Importantly, CCR5 D1 siRNA was capable of suppressing CCR5 expression in AIA rats by 70% compared to group 3. The silencing effect of anti-CCR5 siRNA was further confirmed by RT-PCR and the similar results were obtained (Fig. 5C).
Fig. 1. Efficiency of gene knockdown by anti-CCR5 siRNA in vitro. (A) Expression levels of CCR5 was assessed at the protein level by western blot in each group after cotransfection of vectors including four anti-CCR5 shRNAs (D 1–4), one negative control shRNA (NC shRNA) and gene CCR5. (B) Relative density values are expressed as the ratio of CCR5 to GAPDH (loading control), and the NC group is set at 1.0. Values are the mean ± SEM of Hek293T cells per group. *P < 0.01 versus NC.
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Fig. 2. Electro-transfer of siRNA into the knee joint. (A–D) Vector containing CCR5-specific siRNA was transduced into the knee joint of rats by in vivo electroporation. The synovium tissue was observed under fluorescence microscope 24 h (A), 3 days (B) and 5 days (C) after the transduction. The opposite joint synovium also shown in D. Original magnification was 100×.
4. Discussion RNAi has quickly become the choice for gene knockdown in experiments, and based on this standpoint, siRNA can be widely
used to study gene function in RA [16,27,28]. In addition, due to its crucial role in RA as an essential co-receptor, CCR5 is an ideal therapeutic target by means of RNAi strategy. In our study, with the aim to evaluate the role of CCR5 in the regulation of local joint
Fig. 3. Therapeutic effects of CCR5-specific siRNA in adjuvant-induced arthritis (AIA) rats. (A and C), rats were immunized with complete Freund’s adjuvant or saline (black diamonds) (day 0), and 7, 10, 13 and 16 days later CCR5-specific CCR5 (purple diamonds) or NC (green diamonds) siRNA duplexes were electro-transduced into the knee joint. A group of rats were untreated (aqua diamonds). Body weight and paw volume were observed every week but arthritis score was calculated (B) every three days. P < 0.01 and P < 0.05, versus the control group; P < 0.05, versus the CFA group. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)
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Fig. 4. Histopathological demonstration of therapeutic outcome of siRNA therapy. AIA rats were treated with anti-CCR5 specific siRNA as in Fig. 3. (A) Ankle joints were sectioned and stained with H&E observed under microscope. Original magnification was 100×. A-a represents the control group. A-b represents (CFA + D1) group. A-c represents (CFA + NC) group. A-d represents CFA group. (B) Histopathological scores (mean ± SD) of the CCR5-specific siRNA (left and right joint), NC siRNA (left and right joint) and CFA groups are plotted. *P < 0.05, versus the CFA group.
inflammation resulting from induced arthritis, we derived a highly potent D1 siRNA capable of suppression of CCR5 expression. First of all, a rat model with arthritis was induced with intrathecally injection of CFA into the base of tail. Then, to suppress the CCR5 expression in synovium, plasmid containing D1 siRNA or NC siRNA was injected into the left knee joint cavity of rats in group 2 or 3. We observed that both in vitro transfection and in vivo injection of antiCCR5 siRNA were found to be highly effective. Moreover, treatment with anti-CCR5 siRNA on day 14 following CFA injection induced a significant reduction in paw edema and joint destruction in AIA rats. This suggests that CCR5 has a critical role in the generation of joint inflammation and silencing of CCR5 may be benefit for curing RA. There are few studies of systemic utilization of anti-CCR5 siRNA by the intravenous route as a therapeutic tool in RA. Undoubtedly, viral vectors are highly efficient delivery systems to transduce a
gene into the joint tissue [29]. However, it may cause systemic side effects including induction of toxic immune responses, tumorigenicity, antigenicity and cytotoxicity [30,31]. Thus, we believe that inhibiting gene expression via non-viral transduction would be safer than viral mediated transfection. On the contrary, the electroporation has several advantages including delivery of siRNA into specific site of diseased tissues, easy to operate and little side effect. Therefore, several studied have suggested that electroporation is a feasible method of achieving gene silencing therapy and inhibiting inflammatory cytokines against joint diseases [16,26,28]. It is extraordinary that local siRNA delivery into the knee joint effectively suppressed paw inflammation. It is consistent with the previous studies [9,10] which demonstrated that inflammation in the local joint was inhibited by CCR5 antagonists in CIA rats. However, the efficacy was local which barely appeared in the joint injected anti-CCR5 siRNA but not in the opposite joint.
Fig. 5. Suppression of CCR5 in the synovial tissue of AIA rats. (A) AIA rats were treated with CCR5-specific siRNA. Protein and RNA were extracted from the synovial tissue and subjected to western blot and RT-PCR using indicated antibodies and primers. (B) Relative density values are expressed as the ratio of CCR5 to GAPDH (loading control). Values are the mean ± SD of synovial tissue per group. *P < 0.01 versus the NS control. # P < 0.01 versus the NC group.
H. Duan et al. / Immunology Letters 162 (2014) 258–263
Nevertheless, it is strongly suggested that CCR5 released from the knee joint may affect progression of arthritis, and the gene silencing for CCR5 might have therapeutic potential on local arthritis. Furthermore, suppressed effects of anti-CCR5 siRNA on paw inflammation were confirmed with western blot and RT-PCR. Our results were in accordance with the findings of reports by Joosten et al. [32]. As a result, we speculate that inflammatory cytokines in knee joint may inhibit the progression of paw joint, and gene silencing in knee joint perhaps has a therapeutic effect on adjacent arthritis. Although CCR5 antagonists AZD5672 and SCH351125 have been demonstrated to be failure in clinical RA treatment [33], the crucial role of CCR5 in RA development cannot be ignored. The abovedescribed failures in utilizing CCR5 inhibitors for RA target therapy may be explained by the importance of a strict and spatial control of CCR5 expression. More recently, manipulation of the drugs dosing regimen is also critical in clinical trials. Accordingly, findings regarding RA target therapy with CCR5 blockades need more evidences to be confirmed in future study. In conclusion, anti-CCR5 siRNA is capable of silencing CCR5 both in vitro and in vivo. Local electroporation of anti-CCR5 siRNA in joint tissue can suppress AIA in rats. Thus, interference with CCR5 may provide a novel treatment option for joint disorders including RA. Given the recent advances in chemistry and delivery, siRNA will be soon proved to be an important and widely used in clinical applications in human diseases.
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Acknowledgement
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This study was supported by grant from Liaoning Province Medical Peak Scientific Research Project (No. 2010006).
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
CCR5 small interfering RNA ameliorated joint inflammation in rats with adjuvant-induced arthritis Hongmei Duan, Pingting Yang, Fang Fang, Shuang Ding, Weiguo Xiao ∗ Department of Rheumatology and Immunology, The 1st Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Province, China
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Article history: Available online 7 October 2014 Keywords: Rheumatoid arthritis Adjuvant-induced arthritis CCR5 siRNA Electroporation
a b s t r a c t Rheumatoid arthritis (RA) is a systemic inflammatory disease. C-C chemokine receptor type 5 (CCR5) is found in inflamed synovium of RA patients and is necessary for formation of RA. We aimed to check whether delivery of CCR5-specific small interfering RNA (siRNA) via electroporation suppresses local inflammation in arthritis rats. Vectors encoding siRNA that target CCR5 or negative control siRNA were constructed for gene silencing and the silencing effects of suppressing CCR5 expression in synovium examined by western blot. The vector with strongest effect was delivered into the knee joint of adjuvantinduced arthritis (AIA) rats by the in vivo electroporation method 7, 10, 13, and 16 days after immunization with Complete Freund’s adjuvant. During an observation of 28 days, behavior, paw swelling, arthritis and histopathologic scoring were estimated. The expression level of CCR5 in synovium was evaluated by western blot and real-time PCR. Anti-CCR5 D1 siRNA was effectively inhibited CCR5 expression in vitro. Moreover, delivery of the siRNA into inflammatory joint also suppressed the expression of CCR5 in vivo and markedly suppressed paw swelling and inflammation. Local electroporation of anti-CCR5 siRNA into the left inflamed joints could achieve the silencing of CCR5 gene and alleviate local inflammation just in the knee joint injected with siRNA other than the opposite joint. Inhibition of CCR5 expression may provide a potential for treatment of RA. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a systemic inflammatory disease characterized by infiltration of leukocytes into the synovial membranes of joints as well as progressive destruction of the bone and cartilage [1] and that affects approximately 1% of population. These leukocytes especially T cells and B cells could give rise to excessive activation and proliferation of synoviocytes and fibroblasts which secrete cytokines through autocrine and paracrine mechanisms. The cytokines will induce the production of other proinflammatory cytokines and together participate in the pathogenesis of RA and eventually lead to joint damage [2,3]. Recently, the role of chemokines and their receptors in the regulation of RA has been the focus of a number of researchers. Accordingly, blockades for specific cytokines or immune regulators have applied in clinical therapy in RA. Although the role of cytokines in RA still
∗ Corresponding author at: Department of Rheumatology and Immunology, The 1st Affiliated Hospital of China Medical University, No. 155, North Nanjing Street, Heping District, Shenyang 110001, Liaoning Province, China. Tel.: +86 024 83282549/13804042268; fax: +86 024 83282549. E-mail address: [email protected] (W. Xiao). http://dx.doi.org/10.1016/j.imlet.2014.09.018 0165-2478/© 2014 Elsevier B.V. All rights reserved.
remains unclear, a variety of inflammatory cytokines are crucially involved in the pathogenesis. C-C chemokine receptor type 5 (CCR5) is specific receptor for the known ligands including macrophage inflammatory protein ␣ (MIP-1␣), MIP-1 and chemokine (C-C motif) ligand 5 (CCL5), which also known as RANTES (regulated upon activation, normal T cell expressed and secreted). Among the known chemokine receptor, CCR5 and its ligands play an important role in the pathological formation of RA [4–6]. In the synovial tissue of RA patients and animals as arthritis models, expression of CCR5 increased obviously, as well as its ligands [6,7]. Supportive evidences have proved that CCR5-delta 32 polymorphism might be a genetic marker related to the severity of RA [8]. To attempt to cure RA, antagonists of CCR5 are used to ameliorate symptoms of this disease. Yang et al. showed that CCR5 antagonist could inhibit the development of arthritis in collagen-induced arthritis (CIA) model in mice by interfering with their migration to joint injury [9]. Moreover, in rhesus monkeys, a CCR5 antagonist also had an obvious effect of alleviating the symptoms of CIA [10]. Importantly, Okamoto and co-workers demonstrated that TAK-779, a non-peptide compound which inhibited the development of adjuvant-induced arthritis (AIA), had anti-arthritis effect in Complete Freund’ s adjuvant (CFA) treated rats [11].
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The discovery of small interfering RNA (siRNA)-mediated RNA interference (RNAi) has made it possible to induce sequencespecific silencing of target messenger RNAs. Over recent years, siRNAs have been shown to effectively suppress gene expression in human cells. Although RNAi has a relatively short history in human applications, targeted silencing of gene expression via siRNA was one of the most widely used experimental tools. Now, the therapeutic potential of RNAi was highlighted in experiments indicating the efficacy of the approach against viral infection, such as human immunodeficiency virus [12,13], hepatitis B virus [14] and simplex virus 2 [15]. In addition, the siRNA-dependent strategy also be used in RA model [16–18], neurodegenerative diseases [19,20] and cancers [21,22]. Moreover, RNAi-based therapy for age-related macular degeneration has already reached clinical trials in 2004 [23]. Unfortunately, the exact effect of CCR5 knockdown with specific siRNA in RA has not been clearly elucidated in the present study. In this study, we aimed to assess the effects of silenced CCR5 via siRNA-mediated RNAi in AIA rats. First of all, plasmid containing anti-CCR5 siRNA was constructed and tested in human embryonic kidney 293 cells (Hek293T). Then the plasmid was delivered into AIA rats induced by Complete Freund’s adjuvant (CFA) via electroporation. The effects of anti-CCR5 siRNA on AIA rats were examined by calculating arthritis score and histopathologic score. Meanwhile, CCR5 expression also was test by means of western blot and real time PCR (RT-PCR) analysis.
2. Materials and methods 2.1. Animals Male Wistar rats, approximately 6 to 8 weeks old, were kindly provided by the experimental center of China Medical University and maintained under clean condition in animal facility. All animal experiments were conducted according to the Guidelines regarding Committee on Laboratory Animals of the First Affiliated Hospital of China Medical University. 2.2. siRNA duplexes Four siRNA duplexes directed against rat CCR5 and a negative control (NC) siRNA were synthesized by GeneChem (Shanghai, China). The sense sequences for each of the anti-CCR5 siRNAs were as follows: siRNA-D1, 5 -CAGGGAUCUAUCACAUUGGUUd(TT)-3 ; siRNA-D2, 5 -CAGGGCUGUAAGGCUCATCUUd(TT)-3 ; siRNA-D3, 5 -GACCACCUUCCAGGAAUACUUd(TT)-3 ; siRNA-D4, 5 -CAGCAAGUCAAUCCUGAUCGUd(TT)-3 ; NC siRNA, 5 -UUCUCCGAACGUGUCACGUd(TT)-3 . 2.3. Plasmid construction and siRNA transfection SD11 vector containing a green fluorescent protein (GFP) reporter gene was used to generate constructs encoding anti-CCR5 siRNAs or NC siRNA under the control of the rat U6 promoter. These oligonucleotides were annealed and ligated to the BamHI and HindIII sites upstream of the GFP of SD11 vector to get plasmids pU6-siRNA-GFP. The pEGFP-N1-3FLAG containing enhanced GFP (EGFP) reporter gene and triple FLAG tags was used for the construction of an overexpressed system for rat gene CCR5. XhoI and KpnI were the inserting sites. To assess silenced efficiency in vitro, pU6-siRNA-GFP (0.3 g) and pEGFP-N1-3FLAG-CCR5 (0.5 g) plasmids were co-transfected into human embryonic kidney 293 cells (Hek293T) using lipofectamine 2000 (Invitrogen, Carlsbad, California, USA).
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2.4. Observation of EGFP expression The 6 male Wistar rats were randomly selected to observe the expression of EGFP. Rats with EGFP signal were sacrificed at 1 (n = 2), 3 (n = 2) and 5 (n = 2) days after delivery of plasmid pU6siRNA-GFP into the left knee joint. Synovial membrane was stripped from the region surrounding the patella of the left knee joint of EGFP rats. After washed with PBS and fixed in 4% formaldehyde for 72 h, the synovium was dehydrated in sucrose gradient from 10% to 30%. The specimens were cut into 14-m-sections after embedded by OCT, and then were stained with hematoxylin and eosin (H&E) and examined under fluorescence microscope (BX51, Olympus, Tokyo, Japan). 2.5. Induction and evaluation of adjuvant-induced arthritis The remaining 48 male Wistar rats were randomly assigned to 4 groups (12 rats per group), including one control group (group 1) and 3 experimental groups (group 2, 3 and 4). To induce AIA, Complete Freund’s adjuvant (CFA) (Sigma, Santa Clara, CA, USA) was mixed with heat-killed Bacille Calmette–Guerin (BCG) freezedried powder (Institute of Biological Products Co. Ltd., Changchun, China). Briefly, on day 0, 36 rats in experimental groups were intradermally injected with 100 l CFA solution at the base of the tail. Rats in the control group were injected with an equal volume of physiology saline instead of CFA. After electro-transduction of plasmids encoding siRNA into rats in group 2 or group 3 for 4 times (once every 3 days, i.e., 7, 10, 13, and 16 days after immunization), rats were observed for pelage color, activity, appetite and body weight once a week for 4 weeks. Then we measured the foot volume using a water replacement plethysmometer once a week for 4 weeks. The severity of involvement of each paw and the degree of edema were measured once every 3 days for 21 days by arthritis scoring as described [24]. The left knee and ankle joints were removed from rats 17 or 28 days after disease induction, fixed in 4% paraformaldehyde, decalcified with formic acid, dehydrated with gradient ethanol, embedded in paraffin and sectioned to 6m-thick slices. Then sections were stained with H&E. Arthritic changes, such as synovial proliferation, infiltration of inflammatory cells, bone erosion and destruction of articular cartilage were evaluated using the histopathologic scores as described [25]. 2.6. Electroporation-assisted siRNA transduction in vivo Rats in group 2 or 3 were anesthetized by an intraperitoneal injection of 0.3 ml/100 g chloral hydrate, and 50 g pU6-siRNA-GFP encoding anti-CCR5 siRNA or NC siRNA diluted in 40 l PBS was administered into the left knee joint using a syringe equipped with a 50-gauge needle. Immediately after injection, a pair of electrode pads of 1.0 cm diameter was placed onto keratin cream coating around the knee joint and the gap between the pads was fixed at 0.7 cm [26]. Electric pulses were emerged by an electroporator ECM399 (Harvard apparatus, Holliston, MA, USA) and then three square-wave pulses with a pulse length of 100 ms were given at a frequency of 1 s−1 followed by three other pulses with the opposite polarity. 2.7. Western blot analysis Western blot was performed with anti-FLAG or anti-CCR5 antibody to detect CCR5 and anti-GAPDH antibody to detect GAPDH. At 36 h after transfection using lipofectamine 2000, Hek293T cells were harvested and lysed on ice in lysis buffer containing 1 M Tris–HCl, 2% mercaptoethanol, 4% SDS and 20% glycerol. Cellular debris was discarded and protein was collected by centrifugation. Total proteins were separated by 12% SDS-PAGE and
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electrotransferred onto PVDF membranes (Amresco, Solon, OH, USA). Then the membranes were blocked in 5% non-fat milk-TBST (1.0 M Tris–HCl, Ph 8.0, 150 mM NaCl, 0.05% Tween-20), incubated with mouse anti-Flag or mouse anti-GAPDH antibodies (Abcam, Cambridge, England) at a dilution of 1:500, and followed by probing with alkaline phosphatase-labeled goat anti-mouse IgG antibody at a dilution of 1:5000 (Asia Pioneer Pharmaceutical Co. Ltd., Shanghai, China). Protein CCR5 and GAPDH obtained from synovium were also examined with this method. Anti-CCR5 (Abcam, Cambridge, England) and anti-GAPDH (Sigma, Santa Clara, CA, USA) antibodies with the dilution of 1:200 and 1:400 were used. The second antibody was same to the aforementioned one. 2.8. RT-PCR To analyze CCR5 mRNA expression in vivo, the synovium was removed from the region around the left patella of AIA rats. Total RNA was extracted and subjected to RT-PCR using a pair primers for CCR5 (forward: 5 -AGAAGAAGAGGCACAGG-3 and reverse: 5 -AAAGGCATAGATGACAGG-3 ) or GAPDH (forward: 5 -TGACTTCAACAGCGACACCCA-3 and reverse: 5 -CACCCTGTTGCTGTAGCCAAA-3 ) genes. PCR amplification of these two genes was performed using the following conditions: predenaturing at 95 ◦ C for 15 s, denaturing 95 ◦ C for 5 s, annealing and extension at 60 ◦ C for 30 s, for a total of 45 cycles. RT-PCR was performed on real time PCR TP800 (TAKARA, Otsu, Shiga, Japan). 2.9. Statistical analysis Student’s t-test was used to examine the significance of differences. P value of less than 0.05 was considered as significance. On-way ANOVA test was performed for multiple comparisons and p value less than 0.05 was considered to be significant. 3. Results 3.1. Silencing of the CCR5 gene in vitro To test the silencing efficiency of siRNA for target gene CCR5, plasmids pU6-siRNA-GFP containing different anti-CCR5 siRNA (D1–D4) and NC siRNA were co-transfected with pEGFP-N1-3FLAGCCR5 into Hek293T cell. We accomplished different levels of CCR5 silencing after transfection with lipofectamine 2000 containing unmodified anti-CCR5-siRNA compared to NC group (Fig. 1). An obviously silencing of CCR5 with D1 siRNA was visualized in results and it was used in next treatment in this study.
3.2. GFP expression in synovium of rat knee joint in vivo To investigate the delivery rate in vivo with electroporation method and the continuance of anti-CCR5-siRNA, GFP expression was observed in synovium of knee joint. The signal of green fluorescence at the synovial tissue surrounding the patella emerged within 24 h after the transduction (Fig. 2) and increased remarkably with time. The intensity of green fluorescence reached a maximum at the third day and dropped down in the next time.
3.3. Therapeutic effects of siRNA Therapeutic effects of siRNA targeting CCR5 were investigated in rats with AIA. Arthritis began to appear in rats injected with CFA and meanwhile the symptoms of bad appetite, listlessness, back alopecia and slow growth of body weight also emerged. Interestingly, these symptoms in group 2 ameliorated after 14 days electroporation of pU6-D1-siRNA-GFP compared with group 3 (injected with pU6-NC-siRNA-GFP) or group 4 (only injected with CFA) (Fig. 3A). In addition, arthritis score was calculated to estimate the degree of paw swelling and the results indicated that anti-CCR5 siRNA transduction significantly suppressed paw swelling, as well as remarkably decrease of paw volume (Fig. 3B and C). These effects were not observed in the group 3 infected with NC siRNA. Meanwhile, histopathologic findings were evaluated on day 28 after CFA injection. The rats in group 3 or 4, which were treated with NC siRNA or CFA, showed typical symptoms of arthritis including massive inflammatory cell infiltration into the articular cavity, degeneration of articular cartilage, prominent hypertrophy of synovium and bone erosion (Fig. 4A). However, the paws of rats in group 2 were virtually normal and the bone and cartilage remained almost unaffected. Moreover, the histopathologic features of arthritis were statistically significant between the opposite knee joint of rats in group 2 and bilateral knee joints of rats in group 3 (Fig. 4B). Disappointingly, the inhibitory effects of anti-CCR5 siRNA to arthritis were only observed in the hind limb on the infected side, but not on the opposite side. As shown in Fig. 5A and B, western blot analysis for synovium tissue revealed that there was not significant expression of CCR5 in the control group. However, the expression of CCR5 was higher after CFA immunization both in bilateral knee synovium. Importantly, CCR5 D1 siRNA was capable of suppressing CCR5 expression in AIA rats by 70% compared to group 3. The silencing effect of anti-CCR5 siRNA was further confirmed by RT-PCR and the similar results were obtained (Fig. 5C).
Fig. 1. Efficiency of gene knockdown by anti-CCR5 siRNA in vitro. (A) Expression levels of CCR5 was assessed at the protein level by western blot in each group after cotransfection of vectors including four anti-CCR5 shRNAs (D 1–4), one negative control shRNA (NC shRNA) and gene CCR5. (B) Relative density values are expressed as the ratio of CCR5 to GAPDH (loading control), and the NC group is set at 1.0. Values are the mean ± SEM of Hek293T cells per group. *P < 0.01 versus NC.
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Fig. 2. Electro-transfer of siRNA into the knee joint. (A–D) Vector containing CCR5-specific siRNA was transduced into the knee joint of rats by in vivo electroporation. The synovium tissue was observed under fluorescence microscope 24 h (A), 3 days (B) and 5 days (C) after the transduction. The opposite joint synovium also shown in D. Original magnification was 100×.
4. Discussion RNAi has quickly become the choice for gene knockdown in experiments, and based on this standpoint, siRNA can be widely
used to study gene function in RA [16,27,28]. In addition, due to its crucial role in RA as an essential co-receptor, CCR5 is an ideal therapeutic target by means of RNAi strategy. In our study, with the aim to evaluate the role of CCR5 in the regulation of local joint
Fig. 3. Therapeutic effects of CCR5-specific siRNA in adjuvant-induced arthritis (AIA) rats. (A and C), rats were immunized with complete Freund’s adjuvant or saline (black diamonds) (day 0), and 7, 10, 13 and 16 days later CCR5-specific CCR5 (purple diamonds) or NC (green diamonds) siRNA duplexes were electro-transduced into the knee joint. A group of rats were untreated (aqua diamonds). Body weight and paw volume were observed every week but arthritis score was calculated (B) every three days. P < 0.01 and P < 0.05, versus the control group; P < 0.05, versus the CFA group. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)
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Fig. 4. Histopathological demonstration of therapeutic outcome of siRNA therapy. AIA rats were treated with anti-CCR5 specific siRNA as in Fig. 3. (A) Ankle joints were sectioned and stained with H&E observed under microscope. Original magnification was 100×. A-a represents the control group. A-b represents (CFA + D1) group. A-c represents (CFA + NC) group. A-d represents CFA group. (B) Histopathological scores (mean ± SD) of the CCR5-specific siRNA (left and right joint), NC siRNA (left and right joint) and CFA groups are plotted. *P < 0.05, versus the CFA group.
inflammation resulting from induced arthritis, we derived a highly potent D1 siRNA capable of suppression of CCR5 expression. First of all, a rat model with arthritis was induced with intrathecally injection of CFA into the base of tail. Then, to suppress the CCR5 expression in synovium, plasmid containing D1 siRNA or NC siRNA was injected into the left knee joint cavity of rats in group 2 or 3. We observed that both in vitro transfection and in vivo injection of antiCCR5 siRNA were found to be highly effective. Moreover, treatment with anti-CCR5 siRNA on day 14 following CFA injection induced a significant reduction in paw edema and joint destruction in AIA rats. This suggests that CCR5 has a critical role in the generation of joint inflammation and silencing of CCR5 may be benefit for curing RA. There are few studies of systemic utilization of anti-CCR5 siRNA by the intravenous route as a therapeutic tool in RA. Undoubtedly, viral vectors are highly efficient delivery systems to transduce a
gene into the joint tissue [29]. However, it may cause systemic side effects including induction of toxic immune responses, tumorigenicity, antigenicity and cytotoxicity [30,31]. Thus, we believe that inhibiting gene expression via non-viral transduction would be safer than viral mediated transfection. On the contrary, the electroporation has several advantages including delivery of siRNA into specific site of diseased tissues, easy to operate and little side effect. Therefore, several studied have suggested that electroporation is a feasible method of achieving gene silencing therapy and inhibiting inflammatory cytokines against joint diseases [16,26,28]. It is extraordinary that local siRNA delivery into the knee joint effectively suppressed paw inflammation. It is consistent with the previous studies [9,10] which demonstrated that inflammation in the local joint was inhibited by CCR5 antagonists in CIA rats. However, the efficacy was local which barely appeared in the joint injected anti-CCR5 siRNA but not in the opposite joint.
Fig. 5. Suppression of CCR5 in the synovial tissue of AIA rats. (A) AIA rats were treated with CCR5-specific siRNA. Protein and RNA were extracted from the synovial tissue and subjected to western blot and RT-PCR using indicated antibodies and primers. (B) Relative density values are expressed as the ratio of CCR5 to GAPDH (loading control). Values are the mean ± SD of synovial tissue per group. *P < 0.01 versus the NS control. # P < 0.01 versus the NC group.
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Nevertheless, it is strongly suggested that CCR5 released from the knee joint may affect progression of arthritis, and the gene silencing for CCR5 might have therapeutic potential on local arthritis. Furthermore, suppressed effects of anti-CCR5 siRNA on paw inflammation were confirmed with western blot and RT-PCR. Our results were in accordance with the findings of reports by Joosten et al. [32]. As a result, we speculate that inflammatory cytokines in knee joint may inhibit the progression of paw joint, and gene silencing in knee joint perhaps has a therapeutic effect on adjacent arthritis. Although CCR5 antagonists AZD5672 and SCH351125 have been demonstrated to be failure in clinical RA treatment [33], the crucial role of CCR5 in RA development cannot be ignored. The abovedescribed failures in utilizing CCR5 inhibitors for RA target therapy may be explained by the importance of a strict and spatial control of CCR5 expression. More recently, manipulation of the drugs dosing regimen is also critical in clinical trials. Accordingly, findings regarding RA target therapy with CCR5 blockades need more evidences to be confirmed in future study. In conclusion, anti-CCR5 siRNA is capable of silencing CCR5 both in vitro and in vivo. Local electroporation of anti-CCR5 siRNA in joint tissue can suppress AIA in rats. Thus, interference with CCR5 may provide a novel treatment option for joint disorders including RA. Given the recent advances in chemistry and delivery, siRNA will be soon proved to be an important and widely used in clinical applications in human diseases.
[10]
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[15]
[16]
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[18]
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[21]
Acknowledgement
[22]
This study was supported by grant from Liaoning Province Medical Peak Scientific Research Project (No. 2010006).
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