To analyze the effects of a novel compound, NK-007, on the prevention and treatment of collagen-induced arthritis (CIA) and the underlying mechanisms.
To analyze the effects of a novel compound, NK-007, on the prevention and treatment of collagen-induced arthritis (CIA) and the underlying mechanisms.
We determined the effect of NK-007 on lipopolysaccharide (LPS)–triggered tumor necrosis factor α (TNFα) production by murine splenocytes and a macrophage cell line (RAW 264.7) by enzyme-linked immunosorbent assay, intracellular cytokine staining, and Western blotting. The LPS-boosted CIA model was adopted, and NK-007 or vehicle was administered at different time points after immunization. Mice were monitored for clinical severity of arthritis, and joint tissues were used for histologic examination, cytokine detection, and immunohistochemical staining. Finally, stability of TNFα production and Th17 cell differentiation were studied using quantitative polymerase chain reaction and flow cytometry.
NK-007 significantly suppressed LPS-induced TNFα production in vitro. Administration of NK-007 completely blocked CIA development and delayed its progression. Furthermore, treatment with NK-007 at the onset of arthritis significantly inhibited the progress of joint inflammation. Administration of NK-007 also suppressed production of TNFα, interleukin-6 (IL-6), and IL-17A in the joint and reduced percentages of IL-17+ cells among CD4+ and γ/δ T cells in draining lymph nodes. We further demonstrated that NK-007 acted on the stability of TNFα messenger RNA and reduced Th17 cell differentiation. In addition, it significantly inhibited levels of IL-6 and IL-17A in human coculture assay.
For its effects on the development and progression of CIA and for its therapeutic effect on CIA, NK-007 has great potential to be a therapeutic agent for human rheumatoid arthritis.
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint inflammation, progressive cartilage destruction, and bone erosion. Inflammatory cytokines produced by innate immune cells, such as interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and IL-1, play a critical role in the pathogenesis of inflammatory arthritis (1–3). Indeed, several approved biologic agents, including anti-TNFα antibodies (infliximab, adalimumab) (4, 5), a recombinant soluble TNF receptor–Fc fusion protein (etanercept) (6, 7), a recombinant human IL-1 receptor (IL-1R) antagonist (anakinra) (8, 9), and a recombinant human anti–IL-6R monoclonal antibody of the IgG1 subclass (tocilizumab) (10, 11), showed promising therapeutic effects in RA patients. However, given the cost of such biologic agents and their limited efficacy, there is still a great need for the development of novel therapeutic agents, especially small molecules.
IL-17 is an important cytokine produced by a newly defined CD4+ T cell subset, Th17 cells. Both IL-17A and IL-17F have been implicated in the pathogenesis of RA (12–16). Induction of inflammatory arthritis in SKG mice (a strain that develops an autoimmune arthritis that clinically and immunologically resembles RA) depends on IL-6 produced by antigen-presenting cells (17), which is consistent with IL-6 as being one of the key factors in Th17 cell differentiation (18, 19). IL-17A induces IL-1β and TNFα secretion by several joint cells including synovial fibroblasts (20, 21), synergizes with their proinflammatory function (22, 23), and can directly influence cartilage destruction and bone erosion (24, 25). One of the key effects of IL-17 is to attract monocytes into the synovium, and this is mediated by p38 MAPK (26). Therefore, small molecules that block Th17 cell differentiation may serve as potential therapeutic agents.
We previously reported that a novel tylophorine analog, DCB 3503, showed significant effects on development and progression of collagen-induced arthritis (CIA) through inhibiting innate immune responses (27). However, several characteristics of this compound, including the complicated synthesis process, water insolubility, and light sensitivity, have driven us to further synthesize more derivatives of this compound. One of these compounds, NK-007, exhibited a strong inhibition of lipopolysaccharide (LPS)–triggered TNFα production by splenocytes and by a macrophage cell line (28). Therefore, we chose to use NK-007 in the current study.
In this study, we demonstrated that NK-007 significantly blocked the development and progression of LPS-boosted CIA and suppressed the onset of arthritis. Moreover, NK-007 significantly inhibited LPS-triggered TNFα production through a posttranscriptional mechanism and suppressed differentiation of Th17 cells. Given its low toxicity and high bioabsorbency, NK-007 has the potential to become a novel therapeutic agent for human RA.
Male DBA/1 mice were maintained under specific pathogen–free conditions at the College of Life Sciences of Nankai University with the approval of the Institutional Animal Care and Use Committee of the College of Life Sciences of Nankai University. Male mouse strain BALB/c-Tg(Tnf-α-luc)-Xen (Inf-α-luc LPTA) were purchased from Xenogen.
NK-007 was synthesized in the laboratory of Dr. Qingmin Wang at the State Key Laboratory of Elemento-Organic Chemistry, Nankai University. RAW 264.7 cells were obtained from ATCC. All antibodies for flow cytometry and Western blotting were purchased from commercial sources as indicated.
To test the antiinflammatory effects of NK-007, CIA was induced as described in our previous studies (27). Briefly, male DBA/1 mice ages 8–10 weeks were immunized intradermally at the base of the tail with bovine type II collagen (CII; Chondrex) emulsified in Freund's complete adjuvant (no. F5881; Sigma). On day 21, the mice were given a second injection of bovine CII dissolved in Freund's incomplete adjuvant (no. F5506; Sigma). Thereafter, mice were closely monitored and scored daily in a blinded manner for signs of arthritis severity. Arthritis symptoms were graded using a scoring system as previously described, with a maximum clinical score of 16 per mouse (29).
In certain cases, LPS (50 μg/mouse, Escherichia coli serotype O111:B4) was used to boost the arthritis incidence and severity on day 28 after the initial immunization, as previously reported (27). NK-007 was injected intraperitoneally (IP) at 6 mg/kg body weight twice daily every 3 days. For the prevention of arthritis, NK-007 administration was started 1 day prior to LPS challenge, whereas for progression of arthritis, NK-007 was administered 2 days after LPS treatment. To determine the effect of NK-007 treatment at onset of CIA, mice with a score of 2–4 after the second immunization were randomly divided into 2 equal cohorts and were administered either vehicle or NK-007 twice daily every 3 days until day 47.
For histologic analysis, mice were killed and paws were skinned and fixed in 4% buffered formaldehyde and then decalcified in 12% disodium EDTA for 15 days. Tissues were then paraffin embedded, sectioned, and stained with hematoxylin and eosin. Histopathologic changes were scored in a blinded manner based on cell infiltration, cartilage destruction, and bone erosion parameters, as previously described (30).
For local TNFα staining in vehicle- and NK-007–treated joint tissues, deparaffinized sections were subjected to antigen retrieval in 0.01M citrate buffer solution, pH 6.0, at 100°C for 15 minutes. Immunohistochemistry was performed according to a standard protocol (31). Rabbit anti-mouse TNFα polyclonal antibody (ab6671; Abcam) was used as primary antibody, and horseradish peroxidase–conjugated goat anti-rabbit antibody was used as secondary antibody.
A suspension of BALB/c mouse splenocytes was cultured in complete RPMI 1640 medium (HyClone) containing 10% fetal bovine serum (FBS; Gibco). RAW 264.7 cells were grown in Dulbecco's modified Eagle's medium (DMEM; HyClone) supplemented with 10% FBS. Bone marrow–derived dendritic cells (BMDCs) were cultured for 7 days using granulocyte–macrophage colony-stimulating factor (R&D Systems) as described previously (32, 33). Cultured cells were treated in the presence or absence of NK-007 for 2 hours followed by stimulation with LPS, and the supernatants were collected for cytokine analysis at different time points after LPS addition.
Fibroblast-like synoviocytes (FLS) obtained from RA patients undergoing total joint replacement surgery were prepared as described previously (34) and seeded in 24-well plates at 50,000/well in 1 ml serum-free DMEM/insulin–transferrin–selenium A (Life Technologies). Subsequently, peripheral blood mononuclear cells (PBMCs; 5 × 105/well) in complete medium were added to FLS monolayers, and different concentrations of NK-007 were added to the coculture system for 24 hours. The culture supernatants were then collected and subjected to enzyme-linked immunosorbent assays (ELISAs) for detection of IL-6 (R&D Systems) and IL-17 (QuantoBio). All cultures were set up in triplicate.
For intracellular cytokine staining, RAW 264.7 cells were stimulated with LPS (100 ng/ml) in the presence of GolgiPlug (BD Biosciences). For interferon-γ (IFNγ) or IL-17 staining ex vivo, lymphocytes were isolated from spleens or draining lymph nodes on day 35 after immunization and stimulated with phorbol myristate acetate (PMA) (50 ng/ml; Sigma) and ionomycin (1 μg/ml; Sigma) in the presence of GolgiPlug for 4 hours. Cells were then stained with allophycocyanin-conjugated anti-mouse CD4 (Sungene Biotech) followed by fixation and permeabilization, and IFNγ (BioLegend) or IL-17 (BD Biosciences) intracellular staining was performed as described previously (35). For TNFα staining in peritoneal macrophages from immunized mice, 5 ml of 3% (weight/volume) Brewer thioglycollate medium was injected into the peritoneal cavity of each mouse. After 3 days, cells were collected and were stained first with PerCP-conjugated anti-mouse CD11b (Sungene Biotech) against surface molecules, and then fixed and permeabilized for intracellular TNFα staining.
Gene expression was quantified by real-time quantitative polymerase chain reaction (PCR) using SYBR Premix Ex Taq (Takara) and a Mastercycler Realplex (Eppendorf). Data were normalized using a β-actin reference. The following primer sequences were used: for TNFα, 5′-ATGAGCACAGAAAGCATGAT-3′ (forward) and 5′-TACAGGCTTGTCACTCGAAT-3′ (reverse); for IL-17, 5′-TTTAACTCCCTTGGCGCAAA-3′ (forward) and 5′-CTTTCCCTCCGCATTGACA-3′ (reverse); for IL-6, 5′-GTCGGAGGCTTAATT-3′ (forward) and 5′-AAGTGCATCATCGTTGTTC-3′ (reverse); for IL-1β, 5′-TGTAATGAAAGACGGCACA-3′ (forward) and 5′-CTCCACTTTGCTCTTGACTTC-3′ (reverse); for IL-23, 5′-TTCCTTCATGCGCATTCTC-3′ (forward) and 5′-CAAATCTGGCTGGCTCTG-3′ (reverse); and for β-actin, 5′-ATGCTCCCCGGGCTGTA-3′ (forward) and 5′-CATAGGAGTCCTTCTGACCCATTC-3′ (reverse). Cycling conditions were 30 seconds at 95°C, followed by 55 repeats of 95°C for 10 seconds and 60°C for 30 seconds. Splenocytes from the TNFα-luciferase reporter mouse model were stimulated by LPS (100 ng/ml) in the presence or absence of NK-007 (100 nM) for 4 hours. Luciferase assay was performed using the luciferase assay system according to the manufacturer's instructions. Briefly, cells were lysed and analyzed for luciferase activity using the Luciferase Reporter Assay kit (catalog no. E4550; Promega) on a GloMax 96 Microplate Luminometer (Promega).
For TNFα mRNA stability assays, actinomycin D (5 μg/ml) was used to inhibit transcription. TNFα gene expression was quantified by real-time PCR.
RAW 264.7 cells were pretreated with or without NK-007 (100 nM) for 2 hours before the addition of LPS (10 ng/ml). Total protein was extracted at different time points, and immunoblotting was performed as previously described (31). Briefly, the concentration of proteins was quantified by the BCA protein assay kit (Beyotime), and the total protein was separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a PVDF membrane. The membranes were blocked with 5% skim milk in phosphate buffered saline–Tween at room temperature for 2 hours, probed with appropriate antibodies, and visualized with enhanced chemiluminescence reagent (Millipore). The membranes were scanned using a VersaDoc MP5000 (Bio-Rad), and images were quantified using Quantity One software (Bio-Rad). Fold changes in target protein expression were normalized using control proteins.
Naive CD4+ T cells (CD4+CD44lowCD25−) were differentiated on plates coated with anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) with a cytokine cocktail of transforming growth factor β (1 ng/ml), IL-6 (20 ng/ml), anti-IFNγ (5 μg/ml), anti–IL-12 (5 μg/ml), and anti–IL-4 (5 μg/ml). The culture media used were RPMI 1640 for supernatants of BMDCs and optimized Iscove's modified Dulbecco's medium (IMDM) for skewing Th17 cell differentiation (36). Cells were restimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of GolgiPlug for 4 hours on day 5 after initiation of cultures for measurements of intracellular cytokines.
Splenocytes and draining lymph nodes from mice with CIA were used for analysis of T cell activation by flow cytometry. Serum anti-CII antibodies and their isotypes were measured by ELISA as described previously (37).
Data are shown as the mean ± SD. The SPSS 13.0 software package was used for statistical analysis. One-way analysis of variance with Bonferroni post hoc correction was used for comparison of arthritis scores in each group. An independent 2-sample Student's t-test was used to compare all other experimental measurements.
In our recent studies, NK-007 stood out from a series of DCB 3503 derivatives and showed a strong inhibitory effect on LPS-triggered TNFα production from both splenocytes and macrophages (28). NK-007 significantly suppressed LPS-triggered TNFα production at all time points measured (Figure 1A), and the inhibitory effect was dose dependent (Figure 1B). Similarly, NK-007 inhibited LPS-induced TNFα production from RAW 264.7 cells in a dose-dependent manner (Figure 1C). This effect was further confirmed by intracellular cytokine staining in RAW 264.7 cells and indicated by the mean fluorescence intensity of TNFα-positive cells (Figure 1D). Finally, the total amounts of TNFα from the activated macrophages were also significantly reduced in the presence of NK-007, as shown by Western blotting (Figure 1E). These results collectively demonstrated a strong dose-dependent inhibitory effect of NK-007 on LPS-triggered TNFα production.
To test the effect of NK-007 on the development of LPS-enhanced CIA, NK-007 was given 1 day prior to LPS treatment. Earlier treatment with NK-007 completely blocked LPS-enhanced arthritis development (Figure 2A). This was further confirmed with joint histology (Figure 2B). Upon LPS treatment, a destroyed joint space was observed with large amounts of inflammatory cell infiltration and pannus formation. In contrast, the joint space was almost completely preserved in those mice treated with NK-007. A representative example of joint histology is shown (Figure 2B).
We next tested the effect of NK-007 on the progression of LPS-enhanced arthritis development. NK-007 was administered by IP injection 2 days after LPS treatment (day 30 of the initial immunization) every 3 days until day 42. Administration of NK-007 significantly blocked the progression of arthritis development (Figure 2C). Histology of joints from mice treated with vehicle showed severe infiltration of inflammatory cells and bone erosion, whereas treatment with NK-007 suppressed these changes (Figure 2D, top). Interestingly, NK-007 also significantly suppressed TNFα production by joint tissues, as demonstrated by immunohistochemical staining (Figure 2D, bottom). Taken together, NK-007 effectively blocked LPS-enhanced CIA development and progression.
To further analyze the effect of NK-007 on developed arthritis, on day 29 after immunization mice with arthritis scores of 2–4 were randomly divided into 2 equal cohorts and treated either with NK-007 (n = 15) or with vehicle (n = 12) twice daily every 3 days. Treatment with NK-007 significantly blocked the progression of arthritis (Figure 3A), and this finding was supported by histologic staining (Figure 3B).
To define the underlying mechanisms that mediated the antiinflammatory properties of NK-007, mice with CIA were treated with either NK-007 or vehicle control using the protocol described in Figure 2A, and on day 35 after immunization, joint tissues were collected and the relative mRNA levels of TNFα, IL-6, IL-17, IL-1β, and IL-23 were measured by real-time PCR. Treatment with NK-007 significantly reduced the expression level of these proinflammatory cytokines in the joint tissues (Figure 4A). To explore whether NK-007 also had a systemic effect, serum samples collected from mice with CIA as described above on day 28 (4 hours after LPS injection) and day 35 after immunization were measured by ELISA. Treatment with NK-007 showed a significant effect on the systemic level of TNFα (Figure 4B). To determine the effect of NK-007 treatment on the ability of macrophages to produce TNFα ex vivo, mice with CIA treated with NK-007 on day 35 after immunization as described in Figure 2A were injected IP with Brewer thioglycollate medium, and peritoneal macrophages were collected for intracellular TNFα detection. Indeed, treatment with NK-007 significantly decreased the ability of macrophages to produce TNFα ex vivo (Figure 4C).
To further determine whether treatment with NK-007 also altered IL-17–producing cells systemically, cells from draining lymph nodes obtained from mice with CIA on day 35 after immunization as described above were activated with PMA and ionomycin and analyzed for their intracellular IL-17A production. Consistent with results of joint expression level as described above, treatment with NK-007 significantly reduced the percentage of IL-17A–producing CD4+ and γ/δ T cells (Figure 4D). One representative result from these 2 populations is shown (Figures 4E and F). Our results collectively demonstrated that treatment with NK-007 significantly reduced the expression level of TNFα and IL-17 both locally at the joints and systemically.
To determine the molecular mechanisms by which NK-007 inhibited TNFα production, we first tested whether NK-007 affected TNFα mRNA transcription. RAW 264.7 cells were treated with NK-007 or vehicle followed by stimulation with LPS, and at different time points, cells were collected for detection of TNFα mRNA by real-time PCR analysis. Interestingly, NK-007 treatment did not decrease the transcription of TNFα mRNA, and to some extent, it even enhanced the mRNA expression level (Figure 5A). This was further confirmed using TNFα-Luc–transgenic mice, and NK-007 treatment had no effect on luciferase activity (Figure 5B). These results demonstrated that the effect of NK-007 did not take place at the level of transcription, but possibly at the posttranscriptional level instead.
To further determine whether treatment with NK-007 affected the stability of TNFα mRNA, actinomycin D was used to inhibit new transcription in cell culture medium. At different time points after treatment with actinomycin D, cells were collected and used for determining the TNFα mRNA level by real-time PCR. Upon addition of actinomycin D, the TNFα mRNA level was significantly decreased in NK-007–treated cells, with no effect on vehicle-treated cells (Figure 5C), indicating the effect of NK-007 on the stability of TNFα mRNA. Based on the previous finding that the p38MAPK/MAPKAPK-2/tristetraprolin (TTP) pathway plays a critical role in the posttranscriptional regulation of TNFα mRNA (38), we analyzed the level of these signaling proteins. RAW 264.7 cells were stimulated with LPS in the presence of either NK-007 (100 nM) or vehicle, and cells were collected at the indicated time points for protein extraction and Western blotting analysis. Treatment with NK-007 resulted in an ∼5-fold decrease in the level of phospho-p38 30 minutes after stimulation with LPS, and in a nearly 70% reduction of MAPKAPK-2 protein levels 60 and 90 minutes after stimulation with LPS (Figure 5D). TTP has been identified as a negative regulator of mRNA stability; NK-007–treated cells expressed significantly higher levels of TTP at 4 and 6 hours after stimulation with LPS (Figure 5D). These results collectively determined the possible mechanisms of NK-007 in reducing the production of TNFα through a posttranscriptional mechanism.
To explain why the expression level of IL-17 in joints was significantly lower and why Th17 cells in draining lymph nodes were significantly fewer in NK-007–treated mice with CIA, we hypothesized that NK-007 treatment might affect Th17 cell differentiation. One possible mechanism was through affecting IL-6 production by DCs. Indeed, treatment with NK-007 significantly inhibited proinflammatory cytokine production, especially IL-6 (Figure 6A). Supernatant from NK-007–treated DCs induced significantly fewer Th17 cells (Figure 6B). To test whether NK-007 could directly affect Th17 cell differentiation, naive CD4+ T cells were primed using optimized IMDM for skewing Th17 cell differentiation. NK-007 treatment significantly inhibited IL-17A production without affecting IFNγ production in CD4+ T cells under conditions of Th17 cell differentiation (Figure 6C). To further explore the effect of NK-007 in human RA, a coculture of FLS and PBMCs from RA patients was set up. NK-007 treatment significantly decreased the production of IL-17A and IL-6 in a dose-dependent manner, possibly by T cells and FLS, respectively (Figure 6D). Therefore, these results demonstrated that NK-007 suppressed IL-17 production in inflammatory arthritis, both in mice and in humans, suggesting that NK-007 may serve as a novel therapeutic agent for RA patients.
To determine the effect of NK-007 treatment on adaptive immune responses, spleens and draining lymph nodes were obtained from immunized mice treated with vehicle or NK-007 as described in Figure 2, and single-cell suspensions were used for analysis of CD44 and CD62L expression by gated CD4+ and CD8+ T cells. No significant differences between the NK-007– and vehicle-treated groups were observed (further information is available at www.sky.nankai.edu.cn). To further determine the effect of NK-007 treatment on CII-specific antibody responses, serum samples from immunized mice treated with vehicle or NK-007 as described in Figure 2 were collected on days 14, 28, and 35 after immunization and used for detection of collagen-specific antibodies by ELISA. NK-007 treatment had no significant effect on antigen-specific antibody production (further information is available at www.sky.nankai.edu.cn). These results indicated that NK-007 suppressed CIA development and progression without affecting adaptive immune responses.
In our previous study, we determined the effect of a novel tylophorine analog, DCB 3503, on CIA through inhibition of the innate immune response (27). Among DCB 3503 derivatives, we selected one of the compounds, NK-007, based on its inhibitory effect on LPS-triggered TNFα production, in order to elucidate such an effect on CIA and to understand the underlying mechanisms. In the present study, we demonstrated that NK-007 significantly blocked the development and progression of LPS-enhanced CIA, and had a therapeutic effect on developed arthritis, through suppression of TNFα production and inhibition of Th17 cell differentiation.
With progressive joint inflammation and destruction, RA is a common autoimmune disease that causes a huge financial burden for medical care and has a significant effect on the quality of life. Although significant progress has been made in the treatment of RA with anti-TNFα antibodies or IL-1R antagonists, there is still a great need for novel therapeutic agents, especially small molecules. We demonstrated that NK-007 completely blocked LPS-enhanced CIA development when given prior to LPS challenge (Figures 2A and B) and significantly suppressed the progression of arthritis when it was given after LPS treatment (Figures 2C and D). Moreover, NK-007 showed a promising therapeutic effect on developed arthritis (Figures 3A and B). These conclusions were well supported by both the arthritis scores and the histopathology results. Although NK-007 had effects on CIA very similar to those of DBC 3503, NK-007 showed several advantages. First, NK-007 is a water-soluble compound and its synthesis is much less complicated than that of DBC 3503. Second, NK-007 is a stable, light-resistant compound. Third, in our preliminary studies, 60% of NK-007 was absorbed orally without obvious toxicity to the animals, with a tolerable oral dose reaching 200 mg/kg body weight (data not shown). These advantages made NK-007 a better candidate therapeutic agent for RA.
A striking finding from our study was that NK-007 significantly blocked LPS-triggered TNFα production through posttranscriptional mechanisms. Pretreatment with NK-007 significantly suppressed LPS-triggered TNFα production from splenocytes as well as from a macrophage cell line by different experimental approaches (Figure 1). Consistently, treatment of mice with CIA with NK-007 significantly reduced joint and serum TNFα levels (Figures 4A and B), reduced TNFα-positive cells in arthritic joints (Figure 2D, bottom), and also inhibited the ability of macrophages to produce TNFα (Figure 4C). Given the critical role of TNFα in the pathogenesis of CIA and human RA, we believe that the effect of NK-007 on CIA might be due to blockage of TNFα production, especially in the LPS-enhanced CIA model. In our future studies, it would be interesting to know whether NK-007 has any synergistic effect with anti-TNFα monoclonal antibodies or TNFα receptor blocking agents. It has to be emphasized that NK-007 also significantly ameliorated LPS-induced shock (further information is available at www.sky.nankai.edu.cn) and reduced Dextran sulfate sodium–induced colitis through suppressing TNFα production (28). Taken together, these results indicated that NK-007 had broad antiinflammatory effects besides those on LPS-accelerated CIA.
How did NK-007 affect TNFα secretion? Was it at transcription or at the posttranscriptional level? Interestingly, treatment with NK-007 had no significant effect on the transcription of TNFα mRNA (Figures 5A and B); instead, the TNFα mRNA from NK-007–treated cells was unstable under treatment with actinomycin D (Figure 5C), indicating a posttranscriptional mechanism for the effect of NK-007. TNFα mRNA stability is precisely controlled by several signaling pathways, especially p38 MAPK, its downstream MAPKAPK-2, and TTP, a well-characterized RNA binding protein (38–40). NK-007 significantly reduced the level of phospho-p38 and MAPKAPK-2, which in turn enhanced the expression of TTP, and this may explain the instability of TNFα mRNA in NK-007–treated cells (Figure 5). The discrepancy between this posttranscriptional mechanism and the reduced TNFα mRNA level found in joints of mice with CIA treated with NK-007 was possibly due to the effects of NK-007 in vivo. The inflammation in joints was ameliorated by NK-007, and consequently the proinflammatory cytokine levels were reduced.
It has been well documented that IL-17 plays a critical role in the pathogenesis of CIA as well as RA (14, 41). Blocking either Th17 cell differentiation or recruitment of IL-17+ cells into the joints could improve the outcomes of inflammatory arthritis (42, 43). Human IL-17 was found to be highly produced by RA synovium (12, 44). IL-17 stimulated IL-6 secretion in fibroblastic cells, inducing the expression of RANKL, which is involved in joint destruction (45, 46).
Interestingly, treatment with NK-007 significantly reduced the level of IL-17A in joints of mice with CIA, and reduced the percentage of IL-17A+ cells in both CD4+ and γ/δ T cells isolated from draining lymph nodes of mice with CIA (Figures 4D–F). Furthermore, the supernatant from NK-007–treated BMDCs showed significantly less ability to prime naive CD4+ T cells toward differentiation of Th17 cells (Figure 6B). One possible mechanism is through affecting the production of the proinflammatory cytokine IL-6 from antigen-presenting cells. Indeed, treatment of BMDCs with NK-007 significantly reduced the level of IL-6 (Figure 6A). In addition, NK-007 also had a direct effect on Th17 cell differentiation under IMDM culture conditions, but the details of the mechanism were unclear at this stage. Further studies are required to fully address the details of the molecular mechanism of NK-007 in Th17 cell differentiation. Furthermore, NK-007 treatment inhibited the levels of IL-6 and IL-17 in human PBMC/FLS coculture systems (Figure 6D), indicating a possible effect of NK-007 in human RA. Further studies are needed to see whether pretreatment of arthritic mice with NK-007 would enhance the therapeutic effect of anti–IL-17 antibodies, and whether this combination would have a synergistic effect on inflammatory arthritis.
Similar to DCB 3503, treatment with NK-007 showed no significant effect on activation of T cells and antibody production by B cells (further information is available at www.sky.nankai.edu.cn). The exact mechanisms for this are unclear. This may give this compound an advantage for potential clinical use to dampen joint inflammation without affecting adaptive immune responses. Further studies will be required to see whether NK-007 can affect other types of antigen-specific immune responses.
In summary, we determined the significant effect of a novel tylophorine compound, NK-007, on development and progression of LPS-enhanced CIA, as well as its therapeutic effect on developed arthritis, through inhibiting production of TNFα and Th17 cell differentiation. For its advantage over DCB 3503, NK-007 has the potential to be developed as a novel therapeutic agent for human RA.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Drs. Q. Wang and Yin had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Wen, Q. Wang, Yin.
Acquisition of data. Wen, Y. Li, M. Wu, Sun, Bao, Lin, Han, Cao, Z. Wang, Liu.
Analysis and interpretation of data. Wen, Hao, Z. Wu, Hong, P. Wang, Zhao, Z. Li, Q. Wang, Yin.