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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

Tofacitinib (CP-690,550) is a novel JAK inhibitor that is currently in clinical trials for the treatment of rheumatoid arthritis (RA). The aim of this study was to examine the effects of tofacitinib in vitro and in vivo in RA, in order to elucidate the role of JAK in the disease process.

Methods

CD4+ T cells, CD14+ monocytes, and synovial fibroblasts (SFs) were purified from the synovium and peripheral blood of patients with RA and were evaluated for the effect of tofacitinib on cytokine production and cell proliferation. For in vivo analysis, synovium and cartilage samples obtained from patients with RA were implanted in immunodeficient mice (SCID-HuRAg mice), and tofacitinib was administered via an osmotic minipump.

Results

Tofacitinib treatment of CD4+ T cells originating from synovium and peripheral blood inhibited the production of interleukin-17 (IL-17) and interferon-γ (IFNγ) in a dose-dependent manner, affecting both proliferation and transcription, but had no effect on IL-6 and IL-8 production. Tofacitinib did not affect IL-6 and IL-8 production by RASFs and CD14+ monocytes. However, conditioned medium from CD4+ T cells cultured with tofacitinib inhibited IL-6 production by RASFs and IL-8 production by CD14+ monocytes. Treatment of SCID-HuRAg mice with tofacitinib decreased serum levels of human IL-6 and IL-8 and markedly suppressed invasion of synovial tissue into cartilage.

Conclusion

Tofacitinib directly suppressed the production of IL-17 and IFNγ and the proliferation of CD4+ T cells, resulting in inhibition of IL-6 production by RASFs and IL-8 production by CD14+ cells and decreased cartilage destruction. In CD4+ T cells, presumably Th1 and Th17 cells, JAK plays a crucial role in RA synovitis.

The importance of inflammatory cytokines in the pathogenesis of rheumatoid arthritis (RA) has become apparent based on the clinical efficacy of biologic agents targeting tumor necrosis factor α (TNFα), interleukin-1 (IL-1) receptor, and IL-6. For such cytokines to exert their biologic activities, the appropriate intracellular signaling pathways must be activated via their specific receptors on the cell surface. Tyrosine kinases are the first intracellular signaling molecules to be activated following receptor binding in a cytokine response. Therefore, various tyrosine kinases are involved at the sites of inflammation (1, 2). Several recent studies have focused on tyrosine kinases as potential targets for the treatment of RA. Among these, the JAK family, consisting of JAK-1, JAK-2, JAK-3, and tyrosine kinase 2 (Tyk-2), has gathered particular attention, because JAKs are essential for the signaling pathways of various cytokines and growth factors that have been implicated in the pathogenesis of RA (e.g., IL-2, IL-6, IL-7, IL-12, IL-15, IL-17, IL-23, granulocyte–macrophage colony-stimulating factor, and interferon-γ [IFNγ]).

The importance of JAKs in development of the immune system has been demonstrated by gene deletion or mutation. According to the abundant expression of JAK-1 and JAK-2, deletion or mutation of either gene in mice has been shown to be lethal, whereas mutation of Tyk-2 or JAK-3 results in immunodeficiency in both humans and mice (3, 4). In contrast to other members of the JAK family that are widely expressed, JAK-3 expression is essentially limited to hematopoietic cells, and JAK-3 constitutively binds to the common γ-chain (γc-chain). The γc-chain is a common receptor subunit for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, some of which are known to be involved in RA. In fact, tofacitinib, an orally available selective inhibitor of JAK, “dose-dependently decreased endpoints of disease” in both murine collagen-induced arthritis and rat adjuvant-induced arthritis (5). Furthermore, tofacitinib is currently in clinical trials for the treatment of RA, and satisfactory effects, acceptable safety, and, surprisingly, efficacy comparable with that of known biologic agents have been observed (6–8).

Tofacitinib is a selective inhibitor of JAKs with nanomolar potency resulting in the inhibition of transmigration of STAT molecules to the nucleus. Initially, the high specificity of tofacitinib for JAK-3 attracted attention; however, recent efforts to investigate the mechanism of action have shown that tofacitinib interacts with multiple JAKs and presumably other kinases (9–11). Tofacitinib preferentially inhibits JAK-1, JAK-3, and STAT-1 activation, resulting in potent inhibition of γc-chain cytokines, IL-6, and IFNγ in naive CD4+ T cells (12).

Although the biologic roles of JAK in lymphocytes are well known, its function in monocyte-lineage cells remains elusive. Previously, we reported that dendritic cells (DCs) from Jak3−/− mice produce increased IL-10, but not IL-6 or TNFα, compared with wild-type DCs, in response to Toll-like receptor ligands (13). Ghoreschi et al also showed increased IL-10 production in mouse plasma after the mice received an intraperitoneal injection of lipopolysaccharide (LPS) following tofacitinib pretreatment (12). Accordingly, tofacitinib may affect not only lymphoid cells but also myeloid cells and other cells that do not express JAK-3, such as mesenchymal cells involved in synovitis, as an off-target effect.

The remarkable effects of tofacitinib observed in clinical studies thus far indicate that this agent will be widely used for the treatment of RA. Although the precise action of tofacitinib on the JAK/STAT pathway in mice has been investigated, the exact mechanism of action under inflammatory conditions in humans remains unclear. Improved knowledge of the underlying mechanisms of tofacitinib would contribute to a better understanding of the pathogenesis of RA and to further application of the drug in other diseases. In this study, we used synovium from patients with RA for in vitro and in vivo experiments to evaluate the effect of tofacitinib and elucidate the role of JAKs at the sites of inflammation in RA.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Tofacitinib (CP-690,550).

Tofacitinib (kindly provided by Pfizer) was dissolved in DMSO (Wako) and kept as a 20-μM stock solution at −80°C.

Cell isolation.

Human synovial tissue specimens were obtained from patients undergoing joint replacement surgery or synovectomy at our university and at the National Hospital Organization Kyushu Medical Center. All patients fulfilled the 1987 American College of Rheumatology criteria for the classification of RA (14) and provided written informed consent. All patients had active RA but had never received treatment with biologic agents. The tissue was digested with collagenase (1 mg/ml; Wako) and Dispase (1,000 proteolytic units/ml; Godo Shusei) for at least 2 hours at 37°C. After being filtrated, the cells were cultured in 10-cm culture dishes with RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Following overnight culture, CD14+ cells were isolated from adherent cells by positive selection, and CD4+ T cells were isolated from nonadherent cells by negative selection using a magnetic cell separation system (Miltenyi Biotec). Purities were >90%, as determined by flow cytometry (FACSCalibur; BD PharMingen). Adherent cells were subcultured in Dulbecco's modified Eagle's medium to purify RA synovial fibroblasts (RASFs). Cells between passages 3 and 6 were used for the experiments, as a homogeneous population of RASFs. Peripheral blood mononuclear cells were isolated by density-gradient centrifugation over Lymphocyte Separation Medium (ICN Pharmaceuticals), and CD4+ T cells and CD14+ monocytes were obtained as described above.

Cell proliferation, apoptosis, and cytokine production.

Synovial and peripheral blood CD4+ T cells were plated at 2 × 105 cells/200 μl with the indicated concentrations of tofacitinib and stimulated with plate-bound anti-CD3 antibodies (100 ng/well; R&D Systems) and soluble anti-CD28 antibodies (1 μg/ml; R&D Systems) for 72 hours; supernatants were harvested to measure cytokine levels and to use as culture medium for RASFs and CD14+ cells. Peripheral blood CD4+ T cells were prestimulated with anti-CD3 and anti-CD28 antibodies for 72 hours, collected and washed, and re-plated at 2 × 105 cells/200 μl with the indicated concentrations of tofacitinib; the cells were stimulated with recombinant IL-2 (100 ng/ml; R&D Systems) for 72 hours, and supernatants were harvested. A BD Cytometric Bead Array (BD Biosciences) and a DuoSet enzyme-linked immunosorbent assay (R&D Systems) were used to measure cytokine concentrations. To analyze cell proliferation, cells were pulsed with 3H-thymidine for the last 16 hours of culture. Cell apoptosis was evaluated by staining with fluorescein isothiocyanate–conjugated annexin V and propidium iodide (PI) (BD PharMingen). RASFs were plated at a density of 5 × 103 cells/200 μl and stimulated with LPS from Escherichia coli 055:B5 (10 ng/ml; Sigma-Aldrich), IL-1β (10 pg/ml; ReliaTech), or IL-17 (10 ng/ml; R&D Systems) for 48 hours. CD14+ cells were plated at 1 × 105 cells/200 μl and cultured for 24 hours.

TaqMan polymerase chain reaction analysis.

Total RNA was isolated using an RNeasy Mini Kit (Qiagen), and complementary DNA was synthesized. TaqMan Gene Expression Assays for human IL-17A (Hs99999082_m1), IFNγ (Hs99999041_m1), and GAPDH (Hs99999905_m1) (Applied Biosystems) were used to evaluate gene expression. The relative quantities were obtained using the comparative threshold (Ct) method and were normalized to GAPDH. Stimulation-dependent fold induction was calculated relative to the Ct value obtained in the unstimulated cells. All experiments were performed in triplicate.

SCID-HuRAg mice.

Male SCID mice (CB17/lcr; CLEA Japan), 6–8 weeks of age, were housed in specific pathogen–free conditions at our university animal center. Synovial tissue, articular cartilage, and bone obtained as a mass from 2 patients with RA at the time of joint replacement surgery were used. Synovium was cut into pieces 5–10 mm in diameter, and cartilage was cut into 2-mm3 pieces. Mice were anesthetized according to the guidelines established by our animal ethics committee, and synovium and cartilage were transplanted onto the backs of 9 SCID mice (day 0). One week after implantation, the 9 mice were randomly divided into 3 groups, and tofacitinib dissolved in polyethylene glycol 300 (Sigma-Aldrich) was administered continuously at dosages of 0 mg/kg/day (n = 3), 1.5 mg/kg/day (n = 3), or 15 mg/kg/day (n = 3) via Alzet osmotic minipumps (DURECT Corporation) (5, 15) implanted subcutaneously on the backs. Blood samples were collected, and the sera were stored at −80°C until measurement of IL-6 and IL-8.

Histologic evaluation of SCID-HuRAg mice.

Implanted tissues were removed from the SCID-HuRAg mice 5 weeks after implantation, paraffin embedded, and stained with hematoxylin and eosin. Immunostaining was performed with anti–IL-6 antibodies (R&D Systems) and anti–IL-8 antibodies (R&D Systems). Invasion of synovial tissue into the cartilage was quantified according to a semiquantitative score ranging from 0 to 4, based on the number of invading cell layers and the number of affected cartilage sites. Erosion was classified as previously described (16), as follows: 0 = no or minimal invasion, 0.5 = invasion of 1–2 cell layers, 1 = invasion of 3–5 cell layers, 1.5 = invasion of 3–5 cell layers at 3 independent sites of the cartilage, 2 = invasion of 6–10 cell layers, 2.5 = invasion of 6–10 cell layers at 3 independent sites, 3 = invasion of >10 cell layers, 3.5 = invasion of >10 cell layers at 2 independent sites, and 4 = invasion of >10 cell layers at ≥3 independent sites. The invasion scores were determined by counting cells at 400× magnification in 7 high-power fields in each specimen. Histologic assessments were made under double-blind conditions. Three animal researchers recorded the data on separate case record forms without exchanging any information.

Statistical analysis.

All data were evaluated by one-way analysis of variance with Dunnett's post hoc test. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Tofacitinib-induced inhibition of proliferation of CD4+ T cells from synovium and peripheral blood.

We first analyzed the effect of tofacitinib on the proliferation of CD4+ T cells isolated from the synovium and peripheral blood of patients with active RA. When CD4+ T cells were stimulated with anti-CD3 and anti-CD28 antibodies, marked proliferation was induced. However, the addition of tofacitinib to the culture inhibited the proliferation in a dose-dependent manner, with a statistically significant difference starting at 10 nM) (Figures 1A and B). Similar inhibitory effects were observed in CD4+ T cells from healthy subjects (data not shown). Next, in order to evaluate whether these inhibitory effects were mediated by the cytotoxicity of tofacitinib, synovial and peripheral blood CD4+ T cells were stained with annexin V and PI. The addition of tofacitinib did not significantly affect the percentage of apoptotic cells (annexin V–positive/PI-negative and annexin V–positive/PI-positive) even at the highest concentration of tofacitinib (300 nM) (Figures 1C and D), indicating that the effects of tofacitinib were not mediated by apoptosis.

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Figure 1. Tofacitinib inhibits proliferation of CD4+ T cells derived from the synovium and peripheral blood of patients with rheumatoid arthritis (RA), without cell toxicity. Synovial (A and C) and peripheral blood (B and D) CD4+ T cells were stimulated with anti-CD3/anti-CD28 antibodies in the presence of increasing doses of tofacitinib. A and B, To analyze cell proliferation, cells were pulsed with 3H-thymidine for the last 16 hours of culture. Values are the mean ± SD of triplicate cultures. The experiments were repeated in 3 RA patients, and the results were similar; representative data are shown. ∗ = P < 0.05; ∗∗ = P < 0.01 versus stimulated untreated cells. C and D, Cell apoptosis was evaluated by staining with fluorescein isothiocyanate–conjugated annexin V and propidium iodide. Values are the mean ± SD results of all experiments.

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Tofacitinib-induced inhibition of IL-17 and IFNγ production by CD4+ T cells.

We next assessed the effects of tofacitinib on cytokine production by CD4+ T cells obtained from patients with RA. Although stimulation of CD4+ T cells derived from both the synovium and peripheral blood with anti-CD3/anti-CD28 antibodies strongly induced the production of IL-17 and IFNγ, the addition of tofacitinib to the culture inhibited production of these cytokines in a dose-dependent manner, starting at the minimum concentration of 10 nM (Figures 2A and B). Furthermore, tofacitinib decreased messenger RNA (mRNA) levels of IL-17 and IFNγ in a dose-dependent manner, indicating inhibitory effects on gene transcription (Figures 2C and D). In contrast to its effect on IL-17 and IFNγ, tofacitinib did not affect IL-6 and IL-8 production by synovial and peripheral blood CD4+ T cells, at both the protein and mRNA levels (Figures 2E and F, and data not shown). Furthermore, when peripheral blood CD4+ T cells were restimulated with IL-2 (100 ng/ml) for 72 hours in the presence or absence of tofacitinib after prestimulation with anti-CD3/anti-CD28 antibodies, production of IL-17 and IFNγ by peripheral blood CD4+ T cells was inhibited by tofacitinib in a dose-dependent manner (Figure 2G). These results suggest that inhibition of IL-17 and IFNγ production could be associated with inhibition of the IL-2–mediated JAK-1/3/STAT-5 pathway by tofacitinib.

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Figure 2. Inhibitory effect of tofacitinib on interleukin-17 (IL-17) and interferon-γ (IFNγ) production, but not IL-6 and IL-8 production, by CD4+ T cells derived from the synovium and peripheral blood of patients with rheumatoid arthritis (RA). Synovial (A, C, E, and F) and peripheral blood (B and D) CD4+ T cells were stimulated with anti-CD3/anti-CD28 antibodies for 72 hours in the presence of increasing concentrations of tofacitinib. A, B, and E, Culture supernatants were collected for analysis of cytokine production. C, D, and F, Messenger RNA expression (fold induction versus unstimulated cells) was determined by TaqMan polymerase chain reaction. G, After prestimulation with anti-CD3/anti-CD28 antibodies for 72 hours, peripheral blood CD4+ T cells were restimulated with IL-2 (100 ng/ml) for 72 hours, and supernatants were harvested. Values are the mean ± SD of triplicate cultures. The experiments were repeated in 3 RA patients, and the results were similar; representative data are shown. ∗ = P < 0.05; ∗∗ = P < 0.01 versus stimulated untreated cells. RQ = relative quantity.

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Lack of effect of tofacitinib on IL-6 and IL-8 production by RASFs and CD14+ monocytes.

We next investigated the effect of tofacitinib on RASFs and CD14+ monocytes, which together are the major source of cytokines in RA synovium. Because RASFs expressed JAK-1 and JAK-2 abundantly but did not express JAK-3 (data not shown), we expected off-target effects of tofacitinib on RASFs. Both LPS and IL-1β, which are not implicated in JAK/STAT signaling, strongly induced the production of IL-6 and IL-8 by RASFs. The production of these cytokines was not, however, affected by the addition to the culture of tofacitinib at any concentration (Figure 3A). Furthermore, although CD14+ monocytes isolated from RA synovium produced large amounts of IL-6 and IL-8 without any stimulation, the addition of tofacitinib did not affect production of these cytokines (Figure 3B). Based on our previous studies of JAK-3–deficient DCs, we expected tofacitinib to increase IL-10 production by CD14+ monocytes. However, we did not observe the parallel phenotype in vitro (data not shown). These results indicate that the mode of action of tofacitinib appeared to be restricted to proliferation and particular cytokine production by CD4+ T cells rather than CD14+ monocytes and RASFs in patients with RA.

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Figure 3. Tofacitinib does not affect interleukin-6 (IL-6) and IL-8 production by rheumatoid arthritis synovial fibroblasts (RASFs) and CD14+ monocytes originating from RA synovium. A, RASFs were stimulated with 10 ng/ml of lipopolysaccharide (LPS), 10 pg/ml of IL-1β, or 10 ng/ml of IL-17 for 48 hours, alone or with tofacitinib in increasing concentrations. B, CD14+ monocytes were cultured for 24 hours with increasing concentrations of tofacitinib, and supernatant was collected for analysis of IL-6 and IL-8 production. Values are the mean ± SD of triplicate cultures. The experiments were repeated in 3 RA patients, and the results were similar; representative data are shown.

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Indirect effect of tofacitinib on IL-6 production by RASFs and IL-8 production by CD14+ monocytes.

Because direct inhibitory effects of tofacitinib on IL-6 and IL-8 production by RASFs and CD14+ monocytes were not observed, we next investigated the possibility of an indirect effect of tofacitinib through CD4+ T cells. We collected the culture supernatants from purified CD4+ T cells that were stimulated with anti-CD3/ anti-CD28 antibodies in the presence of tofacitinib, added the obtained conditioned medium to RASFs or CD14+ monocytes from RA synovium, and assessed IL-6 and IL-8 levels in the supernatants of RASFs and CD14+ monocytes after further incubation. When RASFs were cultured with supernatant from CD4+ T cells treated with tofacitinib, IL-6 production was reduced significantly at doses of 30 nM and higher, while IL-8 production was not affected (Figure 4A). When CD14+ monocytes were cultured with the supernatant, IL-8 production decreased significantly at doses of 30 nM and higher, whereas IL-6 production was not affected (Figure 4B). Thus, tofacitinib-induced inhibition of cytokine production by CD4+ T cells appeared to result in reduced production of IL-6 and IL-8 from RASFs and CD14+ monocytes in a cell-trophic manner.

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Figure 4. Tofacitinib indirectly suppresses IL-6 production by RASFs and IL-8 production by CD14+ monocytes. Peripheral blood CD4+ T cells were stimulated with anti-CD3/anti-CD28 antibodies for 72 hours in the presence of increasing concentrations of tofacitinib. Culture supernatants (sup) were harvested and cultured with RASFs or peripheral blood CD14+ cells. RASFs (A) and CD14+ monocytes (B) were cultured for 48 hours and 24 hours, respectively, and the supernatants were collected to measure cytokine concentration. Values are the mean ± SD of 3 individual experiments. The experiments were repeated in 3 RA patients, and the results were similar. ∗ = P < 0.05; ∗∗ = P < 0.01 versus stimulated untreated cells. See Figure 3 for other definitions.

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Effect of tofacitinib on production of human IL-6 and IL-8 and invasion of synovial cells into cartilage in SCID-HuRAg mice.

In order to clarify the mode and mechanism of action of tofacitinib, we assessed the in vivo effects of tofacitinib in SCID-HuRAg mice. Tofacitinib was continuously administered to these mice by osmotic minipump. We observed increased production of human IL-6 and IL-8 in serum from the SCID-HuRAg mice, which peaked 7–14 days after implantation and then gradually decreased and became undetectable within 21 to 28 days (data not shown). Human TNF and IL-10 were not detected in the serum. Because of the variable levels of cytokine production depending on the condition of synovium samples, IL-6 and IL-8 levels on day 14 were compared with those on day 7 to evaluate the effect of tofacitinib in vivo (Figure 5A). In the groups receiving tofacitinib at dosages of 1.5 mg/kg/day and 15 mg/kg/day, the serum level of human IL-8 was significantly lower compared with that in the control group. Human IL-6 was also inhibited by tofacitinib, although there was no significant difference compared with control.

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Figure 5. Tofacitinib suppresses human interleukin-6 (IL-6) and IL-8 production and cartilage destruction in SCID-HuRAg mice. Rheumatoid arthritis (RA) synovium and articular cartilage were co-implanted onto the backs of SCID mice. Treatment with vehicle or tofacitinib (1.5 or 15 mg/kg/day) was initiated on day 7, and thereafter serum was collected weekly. The co-implants were removed on day 35 and stained for histologic evaluation. A, Markedly decreased production of human IL-6 and IL-8 in the tofacitinib-treated groups compared with the vehicle-treated group. B, Cartilage erosion score in each treatment group. The experiments were repeated in 2 RA patients, and the results were similar. Bars show the mean ± SEM of 3 individual experiments. ∗ = P < 0.05 versus control. C and D, Immunohistochemical evaluation of human IL-6– and IL-8–positive cells (C), and light microscopic features of cartilage erosion in the engrafted specimens (D). Arrows show the invasive front of the synovial tissue. Original magnification × 400.

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To further investigate the effect of tofacitinib on cytokine expression and cartilage destruction, we removed the implanted specimens on day 35 and performed histologic evaluation. Immunohistochemical analysis demonstrated that IL-6 was highly expressed in the RA synovium grafts in mice treated with vehicle, but that the number of IL-6–positive cells was markedly reduced in the tofacitinib-treated group (Figure 5C). Tofacitinib also decreased the expression of IL-8 (Figure 5C) and IL-17 (data not shown) in the implanted RA synovium graft. Furthermore, the mice treated with vehicle alone showed prominent invasion of synovial tissue into the implanted cartilage. However, treatment with tofacitinib markedly inhibited this invasion (Figure 5D). Histologic evaluation according to the erosion score also showed a dose response, with significant differences between mice treated with high-dose tofacitinib (15 mg/kg/day) and placebo-treated controls (Figure 5B).

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

The JAK/STAT pathway is a common signaling pathway activated by inflammatory cytokines, which has recently received attention as a new potential molecular target for the treatment of RA. In this study, we used CD4+ T cells, RASFs, and CD14+ monocytes purified from the synovium and peripheral blood of patients with RA to clarify the mechanism of the JAK inhibitor tofacitinib, which has shown clinical benefit in trials involving patients with active RA (6–8). Although the high specificity of tofacitinib for JAK-3 was shown in earlier studies (9), recent in vitro evidence (10, 12) and the emergence of anemia and neutropenia in clinical trials (6, 8) have indicated that tofacitinib also exerts an inhibitory effect on other JAKs. The present study showed that the therapeutic potency of tofacitinib in patients with RA could occur via the inhibition of CD4+ T cells, especially proliferation and cytokine production for which JAK plays a critical role in physiologic processes.

IL-6 plays a pivotal role in the pathologic processes in RA, and anti–IL-6 receptor antibody is therapeutically useful in RA (17, 18). Although previous studies have shown that tofacitinib decreased the serum IL-6 level in a rodent model of arthritis (5, 11), this study is the first to show that tofacitinib inhibited both human IL-6 and IL-8 derived from RA synovium implanted in SCID mice. However, we observed that tofacitinib did not directly affect IL-6 and IL-8 production by RASFs, CD14+ monocytes, and CD4+ T cells in vitro, whereas IL-17 and IFNγ production by CD4+ T cells was markedly decreased by tofacitinib in vitro. In contrast, we also observed that production of IL-6 and IL-8 by RASFs and CD14+ monocytes was significantly reduced in a concentration-dependent manner when these cells were cultured with supernatant from CD4+ T cells treated with tofacitinib. This suggests that tofacitinib inhibited IL-6 production by RASFs and IL-8 production by CD14+ monocytes in an indirect manner through the inhibition of CD4+ T cells. Moreover, the numbers of IL-6– and IL-8–positive cells were significantly reduced, with decreased cartilage destruction in SCID-HuRAg mice treated with tofacitinib. Thus, it appears that tofacitinib-induced specific inhibition of IL-17 and IFNγ production by CD4+ T cells (presumably Th1 and Th17 cells) resulted in the suppression of IL-6 and IL-8 production by RASFs and CD14+ monocytes, with decreased cartilage destruction in SCID-HuRAg mice.

The mechanism of tofacitinib-induced inhibition of IL-17 and IFNγ production by CD4+ T cells remains unknown. We observed that the concentration of IL-2 in the culture supernatant of CD4+ T cells stimulated with anti-CD3/anti-CD28 antibodies was apparently increased when tofacitinib was added to the culture (data not shown), as reported by other investigators (19). This suggests that tofacitinib might inhibit the consumption of IL-2, which is produced by CD4+ T cells stimulated with anti-CD3/anti-CD28 antibodies. Alternatively, IL-2 production by CD4+ T cells is also thought to be enhanced by tofacitinib, because tofacitinib might cancel the IL-2–mediated negative feedback loop through activating STAT-5 (20). Furthermore, it has been reported that tofacitinib inhibited IL-2–enhanced IFNγ production by T cells in the peripheral blood of the cynomolgus monkey (21). Our study also showed that tofacitinib significantly decreased IL-2–induced production of IL-17 and IFNγ by peripheral blood CD4+ T cells (Figure 2G). Taken together, our observations suggest that IL-2–dependent activation of CD4+ T cells may be important in the pathologic processes of RA, and that tofacitinib could inhibit the IL-2–mediated JAK/STAT signaling pathway.

Although the present study confirmed the specificity of the action of tofacitinib on CD4+ T cells, it remains possible that other immune cells expressing JAKs could be targeted. Dose-related decreases in neutrophil counts (6, 8) have been observed that can be related to attenuation of inflammation (11); however, it is highly likely that neutropenia can occur through inhibition of JAK-1. Because active RA responds to rituximab, a monoclonal antibody selectively targeting CD20+ B cells (22–24), and because B cells also express JAKs, it is possible that tofacitinib directly affects B cell function. Treatment with tofacitinib was previously reported to decrease the absolute number of CD3+CD16+CD56+ natural killer cells (25), suggesting an antiinflammatory effect through natural killer cells. Additionally, a growing body of evidence indicates the involvement of mast cells in the pathogenesis of RA (26, 27), and these immune cells also express JAKs. Indeed, the majority of IL-17A–expressing cells in synovium have been reported to be mast cells (28), suggesting another possible underlying mechanism of the antiinflammatory effect.

Here, we demonstrated that tofacitinib functions through a mechanism different from that of biologic agents that target IL-6 or TNF. Our results indicate that tofacitinib is potentially useful in various autoimmune diseases involving autoreactive T cells. This JAK inhibitor could therefore be indicated widely for immunologic abnormalities and inflammatory conditions in the future. In addition to the effect of tofacitinib in RA, the clinical benefit of orally available tofacitinib in other immune diseases such as inflammatory bowel disease and psoriasis has been observed in ongoing clinical trials. We await with interest the results of these and future trials, to establish the usefulness of tofacitinib in clinical practice.

Finally, we conclude that JAKs in CD4+ T cells play an important role in RA synovitis. However, considering the dramatic effect of tofacitinib in RA, further basic and clinical research is needed to fully determine the mechanism of tofacitinib and the importance of immune cells expressing JAKs and mesenchymal cells expressing only JAK-1 and JAK-2 in RA pathology.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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. Dr. Y. Tanaka 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 conception and design. Maeshima, Yamaoka, Kubo, Nakano, Iwata, Saito, Ishii, Yoshimatsu, Y. Tanaka.

Acquisition of data. Maeshima, Yamaoka, Kubo, Nakano, Iwata, Ohishi, Miyahara, S. Tanaka, Y. Tanaka.

Analysis and interpretation of data. Maeshima, Yamaoka, Kubo, Nakano, Iwata, Saito, Ishii, Yoshimatsu, Y. Tanaka.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We thank Ms N. Sakaguchi, Ms K. Noda, and Ms T. Adachi for the excellent technical assistance. We also thank Dr. John O'Shea (National Institutes of Health, Bethesda, MD) for his helpful review of the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES