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.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
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.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
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.