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Abstract
Objective Tofacitinib, which is a Janus kinase (JAK) inhibitor, has shown clinical effects in the treatment of rheumatoid arthritis. JAKs are important kinases in lymphocyte differentiation; however, their function in dendritic cells (DCs) is unknown. In this study, the function of JAKs in DCs was investigated with tofacitinib.
Methods The effects of tofacitinib on the maturation of human monocyte-derived DCs induced by lipopolysaccharide (LPS) stimulation were investigated. In addition, its effects on T cell stimulatory capability was investigated by coculturing with naïve CD45RA-positive T cells.
Results Tofacitinib decreased expression of CD80/CD86 in a concentration-dependent manner in LPS-stimulated DCs; however, it did not affect HLA-DR expression. Tofacitinib suppressed tumour necrosis factor, interleukin (IL)-6 and IL-1β production without affecting transforming growth factor (TGF)-β and IL-10 production. Meanwhile, CD80/CD86 expression in DCs was enhanced by type I interferon (IFN) stimulation, and the LPS-induced CD80/CD86 expression was inhibited by an antibody to type I IFN receptor. Furthermore, tofacitinib suppressed production of type I IFN and activation of interferon regulatory factor (IRF)-7, which is a transcription factor involved in CD80/CD86 and type I IFN expression. Tofacitinib also decreased the T cell stimulatory capability of DCs and increased expression of indoleamine 2,3-dioxygenase (IDO)-1 and IDO-2.
Conclusions Tofacitinib, a JAK1/JAK3 inhibitor, affected the activities of human DCs. It decreased CD80/CD86 expression and T cell stimulatory capability through suppression of type I IFN signalling. These results suggest a novel mode of action for tofacitinib and a pivotal role for JAKs in the differentiation of DCs.
- Rheumatoid Arthritis
- DMARDs (synthetic)
- Cytokines
- Pharmacokinetics
- Systemic Lupus Erythematosus
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Introduction
Janus kinase (JAK) family members, constitutively bound to cytokine receptors, play an important role in the biological activation of cytokines through activation of the signal transducer and activator of transcription (STAT), which is a transcription factor. The JAK family consists of JAK1, JAK2, JAK3 and tyrosine kinase (TYK)2. Different JAK family members are activated by different cytokine receptors. JAK1 is activated by the class 1, class 2 and γc cytokines, while JAK3 is activated by γc cytokines. Therefore, JAK family members are not only essential for immune function, but they also play an important role in inflammation response.1–4 Tofacitinib, which is selective for JAK1 and JAK3,5 ,6 is effective for patients with rheumatoid arthritis (RA).7–10 This finding supports the notion that JAK1 and JAK3 play an important role in autoimmune diseases. Furthermore, it is thought that elucidation of the mode of action of JAK family members in vivo will lead to a better understanding and treatment of autoimmune diseases.
Dendritic cells (DCs) play a key role in bridging natural immunity and acquired immunity. Immature DCs are potent phagocytes, and they mature through toll-like receptor (TLR) signalling. They also show antigen-specific T-cell activation abilities, which are accompanied by induction of expression of major histocompatibility complex (MHC) and costimulus molecules. Moreover, DCs play an important role in autoimmune diseases. They suppress antigen-specific responses and cause induction of immunotolerance relative to the degree of their differentiation and functional modification,11–15 while suppression of DC apoptosis destroys immunotolerance, resulting in induction of autoimmune diseases.16 Hence, DCs are potential targets not only for immune responses but also for autoimmune diseases.
We have previously shown that tofacitinib selectively suppresses production of cytokines and proliferation of lymphocytes.17 These functions can be predicted to some degree by the important role that JAK family members play in the differentiation and proliferation of lymphocytes. We have reported that DCs express JAK1, JAK2 and JAK3; however, DCs derived from a JAK3-deficient mouse have been shown to overproduce interleukin (IL)-10 and exhibit anti-inflammatory activity.18 However, how the inhibition of JAK signalling affects the phenotype, differentiation and antigen presentation of human DCs, which initiate immune responses, has not been investigated. Elucidation of the effects of tofacitinib on DC function may increase basic scientific knowledge of the clinical efficacy of tofacitinib
An increase in the number of invasive DCs has been observed in the synovitis tissues in RA, and monocyte-derived DCs (MoDCs) from patients with RA produce increased IL-6.19 In addition, an increase in the number of DCs that express high levels of TLR4 ligands in RA synovial fluid has been reported,20 ,21 suggesting activation of DCs and disruption of immunotolerance. Furthermore, JAK expression increases in synovial DCs in active RA,22–24 indicating its involvement in the regulation of DCs during the pathological processes. This study was conducted in order to investigate the effects of inhibition of JAK1 and JAK3 by tofacitinib and signalling mechanisms in human MoDCs.
Methods
Inhibitors
Tofacitinib and PF95698025 were kindly provided by Pfizer (New York, New York, USA). The following inhibitors were purchased; JAK2 kinase inhibitor, G6 (Sigma-Aldrich, St Louis, Missouri, USA), Syk inhibitor I, Syk inhibitor II, PP1, PP2 (Merck, Darmstadt, Germany), anti-IL-6 receptor α antibody, tocilizumab (Chugai Pharmaceutical Co, Tokyo, Japan).
Generation of MoDCs and cell cultures
Peripheral blood mononuclear cells were isolated with lymphocyte separation medium (ICN/Cappel Pharmaceuticals, Aurora, Ohio, USA). Monocytes were obtained by positive magnetic selection using anti-CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). To generate immature MoDCs, we cultured monocytes at 0.5×106 cells/mL in the presence of IL-4 (50 ng/mL; R&D systems, Minneapolis, Minnesota, USA) and granulocyte macrophage colony-stimulating factor (50 ng/mL; Peprotech, Rocky Hill, New Jersey, USA) for 6 days. The medium was replaced with one supplemented with cytokines on day 3.
Immature MoDCs were washed and replated in fresh medium at 2.5×105 cells/mL and pretreated with tofacitinib (10, 100, 300, 1000 nM), PF956980 (300 nM), G6 (300 nM), PP1 (300 nM), PP2 (300 nM), cycloheximide (5 μg/mL), tocilizumab (5 μg/mL) or interferon (IFN)α/β receptor antibody (10 μg/mL) (Abcam, Cambridge, UK) for 6 h and matured with lipopolysaccharide (LPS) (Escherichia coli; Sigma, 100 ng/mL) for 48 h. The concentration of each drug was chosen on the basis of previous studies.6 ,26–30 MoDCs were washed twice and used for coculture with T cells. Production of tumour necrosis factor (TNF)α, IL-6, IL-1β and IFNα was determined with the BD Cytometric Bead Array (CBA) human Flex Set (BD Pharmingen, Franklin Lakes, New Jersey, USA), and that of TGFβ and IFNβ was analysed by ELISA (eBioscience, San Diego, California, USA). Apoptosis was analysed with the Apoptosis Detection kit II (BD Pharmingen).
DC–T cell cocultures
CD4 T cells were negatively selected from peripheral blood mononuclear cells with the CD4 T Cell Isolation Kit II (Miltenyi Biotec), and CD45RA+ naive T cells were positively isolated with anti-CD45RA microbeads (Miltenyi Biotec). MoDCs were cocultured with allogeneic human CD45RA+ naive T cells at a 1:10 ratio for 6 days in Roswell Park Memorial Institute medium. IL-10 was analysed by CBA, and T cell proliferation was assessed by [3H]thymidine incorporation in the last 16 h. IFNγ production was analysed after restimulation of T cells with CD3 (1 μg/mL) and CD28 (0.5 μg/mL) monoclonal antibodies (R&D Systems) for 72 h after coculture.
Flow cytometric analysis
MoDCs were incubated in blocking buffer (0.25% human globulin in phosphate-buffered saline) for 15 min and then suspended in 100 μL FACS solution (0.5% human albumin and 0.1% NaN3 in phosphate-buffered saline) with fluorochrome-conjugated monoclonal antibodies at 4°C for 30 min and then washed with FACS solution and analysed with a FACSVerse (Becton–Dickinson, San Jose, California, USA). The following fluorochrome-conjugated mouse monoclonal antibodies were purchased from BD Pharmingen: fluorescein thiocyanate (FITC)-conjugated anti-CD80, PerCP-conjugated anti-HLA-DR, and antigen presenting cell (APC)-conjugated anti-CD86.
Quantitative real-time PCR
Total RNA was prepared using the RNeasy Mini Kit (Qiagen, Chatsworth, California, USA). First-strand cDNA was synthesised, and quantitative real-time PCR was performed in the Step One Plus instrument (Applied Biosystems, Foster City, California, USA). TaqMan target mixes for tryptophan indoleamine-pyrrole 2,3-dioxygenase (IDO1), IDO paralogue IDO2 (IDO2), CD80 and CD86 were purchased from Applied Biosystems. Expression levels were expressed relative to that of glyceraldehyde-3-phosphate dehydrogenase. The relative quantity was calculated using the quantification-comparative cycle threshold formula–referenced sample of immature DCs.
Western blot analysis
MoDCs were lysed in Nonidet P-40 buffer containing NaCl, Tris/HCl (pH 8.0), distilled water and protease inhibitor. Lysates were mixed with an equal volume of sample buffer solution (2-mercaptoethanol; Wako Pure Chemical Industries) and boiled for 5 min. Proteins were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, transferred on to nitrocellulose membranes (Whatman, Tokyo, Japan), blocked with 5% skimmed milk, and immunoblotted with antibodies to human phospho-NF-κB p65, human PU.1, human IFN regulatory factor 7 (IRF7), human phospho-STAT1, STAT1, phospho-STAT2 and STAT2 (Cell Signaling Technology, Tokyo, Japan) and horseradish peroxidase-labelled anti-secondary antibodies (NA931V and NA934V; GE Healthcare, Osaka, Japan), using immunoreaction enhancer solution (Can Get Signal, Toyobo, Osaka). Blots were developed with ECL Western Blotting Detection Reagents (GE Healthcare) and visualised with a light-capture instrument (ATTO, Tokyo, Japan).
Statistical analysis
Differences were examined using the Mann–Whitney test. p<0.05 denoted the presence of a significant difference.
Results
Tofacitinib inhibited expression of CD80/CD86 without cytotoxicity in human MoDCs stimulated with LPS
The effects of tofacitinib on the expression of costimulators of human MoDCs were investigated. CD80/CD86 and HLA-DR were induced 48 h after LPS stimulation. However, CD80/CD86 expression in MoDCs was suppressed in a concentration-dependent manner by the addition of tofacitinib, whereas expression of HLA-DR was not affected (figure 1A,B). Moreover, induction of CD80/CD86 expression by LPS stimulation was suppressed by PF956980, which is a different JAK1/3 inhibitor, while CD80/CD86 expression was not suppressed by PP1, PP2 or JAK2 inhibitors. These findings suggest that suppression of CD80/CD86 expression was dependent on inhibition of JAK1/3 (figure 1C). Furthermore, cluster formation was observed 24 h after LPS stimulation, which was inhibited in a concentration-dependent manner by the addition of tofacitinib (figure 2A).
Tofacitinib suppresses CD80/CD86 expression. Immature monocyte-derived dendritic cells (DCs) were washed and cultured with or without tofacitinib or other tyrosine kinases during lipopolysaccharide (LPS; 100 ng/mL) stimulation for 48 h. The DC phenotype was evaluated using flow cytometry. (A) Expression of HLA-DR, CD80 and CD86. (B) Representative histogram data of HLA-DR, CD80 and CD86 expression. (C) Expression of HLA-DR, CD80 and CD86 in the presence of tofacitinib and other tyrosine kinase inhibitors. Representative results of three independent experiments with similar findings. Data are mean±SD. *p<0.05 and **p<0.01 (Mann–Whitney test).
Tofacitinib suppresses cluster formation of dendritic cells (DCs) and inflammatory cytokine production in DCs. Immature monocyte-derived DCs were washed and cultured with or without tofacitinib during lipopolysaccharide (LPS; 100 ng/mL) stimulation for 48 h. (A) Morphology of DC populations as shown by phase-contrast microscopy. Representative results of three independent experiments with similar findings are shown. (B) Tumour necrosis factor (TNF)α, interleukin (IL)-1β and IL-6 concentrations in the supernatants were measured. (C) Transforming growth factor (TGF)β concentration was determined by ELISA. Data of three independent experiments are shown. Data are mean±SD. *p<0.05 (Mann–Whitney test).
In the next set of experiments, cytokine production in DCs was investigated. The production of TNFα, IL-1β and IL-6 was induced by stimulation of MoDCs with LPS for 48 h, while the production of these cytokines was suppressed by tofacitinib in a concentration-dependent manner (figure 2B). However, tofacitinib affected neither the expression of TGFβ mRNA (see online supplementary figure S1A) nor the production of TGFβ (figure 2C), IL-10 and IL-12p70 (see online supplementary figure S1B).
To examine whether these suppressive effects were the result of cytotoxicity of tofacitinib on DCs, the cells were stained with annexin V and propidium iodide. DCs died at a high frequency without stimulation for 48 h, while apoptosis was inhibited by LPS stimulation (figure 3A,B). Tofacitinib did not induce apoptosis, even at concentrations as high as 1000 nM, and it did not cause cytotoxicity. When DCs were pulsed with FITC-labelled albumin, tofacitinib did not affect their capability for micropinocytosis (figure 3C). These results suggest that tofacitinib suppressed cluster formation and changed the phenotype of DCs without causing their cell death.
Tofacitinib does not induce cell death. Immature monocyte-derived dendritic cells (MoDCs) were washed and cultured with or without tofacitinib during lipopolysaccharide (LPS; 100 ng/mL) stimulation for 48 h. Early and late apoptosis of DCs were evaluated with flow cytometry. (A) Representative histogram data of annexin V/propidium iodide (PI) staining. (B) Rate of viable cells (annexin Vneg/PIneg). (C) MoDCs were pulsed with 5 μg/mL fluorescein isothiocyanate (FITC)-conjugated albumin for 60 min. Cells were incubated at 0°C for background uptake and at 37°C for albumin uptake. Data indicate the ratio compared with MoDCs stimulated with LPS. Data of three independent experiments are shown. Data are mean±SD. **p<0.01 (Mann–Whitney test). MFI, mean fluorescence intensity.
Expression of CD80 and CD86 was inhibited by antibody to type I IFN receptor
Expression of CD80/CD86 mRNA after LPS stimulation in DCs was suppressed by cycloheximide treatment (figure 4A). After LPS activation, no JAK–STAT pathway involvement was indicated in the signalling pathway downstream of TLR4; therefore, an indirect mechanism was considered in which the suppression of CD80/CD86 expression by tofacitinib occurred through a proteinogenic mechanism.
Lipopolysaccharide (LPS) induces the expression of CD80 and CD86 through type I interferon (IFN). Immature monocyte-derived dendritic cells (DCs) were washed and cultured with or without cycloheximide, an antibody to interleukin (IL)-6 receptor (tocilizumab) or to type I IFN receptor (IFNAR1) during LPS (100 ng/mL) stimulation for 48 h. (A) CD80 and CD86 mRNA expression were measured by quantitative real-time PCR. (B and C) Expression of HLA-DR, CD80 and CD86 determined by flow cytometry. Data are mean±SD. *p<0.05 and **p<0.01 (Mann–Whitney test). RQ, relative quantity.
We next assessed if CD80/CD86 is induced by cytokines in MoDCs. CD80/CD86 expression was not induced by IL-6, whereas it was induced by LPS stimulation. Furthermore, the CD80/CD86 expression that was induced by LPS was not affected by tocilizumab, which is an IL-6 receptor antibody (figure 4B). In contrast with IL-6, expression of CD80/CD86 was induced by type I IFN stimulation and was completely inhibited by tofacitinib. Expression of CD80/CD86, which was induced by LPS, was suppressed by a type I IFN receptor antibody (figure 4C). These results suggest that the inhibition of JAK1/3 in MoDCs partially suppressed the expression of CD80/CD86 by suppressing type I IFN signalling.
Tofacitinib inhibited expression of CD80 and CD86 through reduction of IRF7 signalling
The involvement of NF-κB,31 PU.132 and IRF7,33 which are transcription factors that regulate CD80/CD86 expression, was investigated. NF-κB and PU.1 were activated within 5 min, while IRF7 was activated within 3 h in DCs after LPS stimulation. The activation of NF-κB and PU.1 by LPS stimulation was not suppressed by tofacitinib, while the activation of IRF7 was suppressed by tofacitinib (figure 5A). Consistent results were achieved on analysis of nuclear and cytoplasmic fractions. LPS stimulation for 5 min induced phospho-IκBα and concurrently induced phospho-NF-κB and the subsequent translocation of phospho-NF-κB and PU.1 into the nucleus. However, tofacitinib affected the translocation of neither phospho-NF-κB nor PU.1 induced by LPS (see online supplementary figure S2). On the other hand, expression and nuclear translocation of IRF7 was induced after 3 h stimulation with LPS, and the induced translocation of IRF7 was suppressed by tofacitinib (see online supplementary figure S2). In addition, tofacitinib decreased IFNβ production (figure 5B), while IFNα production was undetectable (data not shown). Tofacitinib also suppressed phospho-STAT1/STAT2 induced by exogenous type I IFN (figure 5C). Furthermore, IFNα/β receptor was constitutively expressed and not affected by tofacitinib (data not shown). These results indicate that tofacitinib suppressed the phosphorylation of STAT1/STAT2 induced by autocrine stimulation with type I IFN, continuously suppressing IRF7 expression and the production of type I IFN, which decreased CD80/CD86 expression in MoDCs.
Tofacitinib suppresses interferon regulatory factor (IRF) activation. Immature monocyte-derived dendritic cells (MoDCs) were washed and cultured with or without tofacitinib during lipopolysaccharide (LPS; 100 ng/mL) stimulation for 48 h. The time course of suppression was evaluated as shown. (A) Phospho-NF-κB, PU.1, IRF7 and β-actin were detected by western blotting. (B) Interferon (IFN)β concentration in the supernatant from MoDCs cultured for 48 h was measured. (C) Phospho-signal transducer and activator of transcription 1 (pSTAT1), STAT1, phospho-STAT2 (pSTAT2), STAT2 and β-actin were detected by western blotting after stimulation by type-1 IFN for 15 min.
Tofacitinib reduced T cell stimulatory ability and induced expression of IDO in MoDCs
Finally, the T cell stimulation capability of MoDCs treated with tofacitinib was examined. MoDCs were cultured for 48 h in the presence of tofacitinib and LPS, washed, and then cocultured with allogeneic CD4+CD45RA+-naïve T cells for 6 days. MoDCs that were treated with LPS exhibited increased T cell growth capability, and IFNγ production capability was induced. However, MoDCs that were pretreated with tofacitinib exhibited decreased T cell stimulatory capability and demonstrated a concentration-dependent decrease in IFNγ production (figure 6A–C), while IL-10 production was increased (figure 6C) without any effects on regulatory T cell population (see online supplementary figure S3).
Tofacitinib decreased T cell stimulatory capability of dendritic cells (DCs) and increased indoleamine 2,3-dioxygenase (IDO) expression. Immature monocyte-derived DCs were cultured with or without tofacitinib during lipopolysaccharide (LPS; 100 ng/mL) stimulation for 48 h and washed. They were then cocultured with allogeneic CD4+CD45RA+-naïve T cells for 6 days. (A) Clustering of T cells as shown by phase-contrast microscopy. (B) [3H]Thymidine incorporation was measured during the last 18 h of the 72 h culture. (C) Interferon (IFN)γ and interleukin (IL)-10 concentrations in the supernatant. (D) Indoleamine 2,3-dioxygenase (IDO)1 and IDO2 mRNA expression in DCs measured by quantitative real-time PCR. Data of three independent experiments are shown. Data are mean±SD. *p<0.05 (Mann–Whitney test). RQ, relative quantity.
It has been reported that DCs that express IDO, which is an enzyme with catalytic activity on tryptophan, show decreased T cell stimulatory capability.34 Therefore, the IDO mRNA in DCs was measured. Both IDO1 and IDO2 were significantly induced by tofacitinib (figure 6D). These results suggest that inhibition of JAK1/3 in DCs with tofacitinib suppressed cell maturation and induced DCs with decreased T cell stimulatory capability.
Discussion
It is shown here that tofacitinib, a JAK inhibitor, promoted a tolerogenic phenotype in human DCs. The data indicate that inhibition of JAK1/JAK3 by tofacitinib regulated transcription of IRF7 by suppressing type I IFN signalling and CD80/CD86 expression. Tofacitinib was approved for treatment of RA in the USA and Japan in 2012 and 2013, respectively. The therapeutic efficacy of tofacitinib has been shown to be equivalent to TNF inhibitors,35 and it was also found to be effective in patients who did not respond to TNF inhibitors.36 These clinical study results indicate that JAK1/JAK3 plays an important role in inflammatory immune diseases such as RA. However, the direct suppressive effect of tofacitinib on T cells alone does not completely explain the mechanism. The results in this report suggest a novel mechanism of tofacitinib involving the induction of immunotolerance in DCs.
Tofacitinib did not affect the expression of MHC class II molecules, whereas it did suppress CD80/CD86 expression. Tofacitinib has been shown to exhibit a suppressive effect on JAK1/JAK3, while its suppressive effects on JAK2/Tyk2 are limited.5 Furthermore, we found that the JAK2 inhibitor (G6) did not show any effects on CD80/86 expression (figure 1), indicating that JAK1 is involved in CD80/86 induction. Recent clinical trials proved that a JAK1/JAK2 inhibitor possesses similar clinical efficacy to tofacitinib,37 and there could be a similar inhibitory action of JAK1-mediated signalling on DCs by this JAK1/JAK2 inhibitor. Furthermore, therapies targeting suppression of CD80/CD86-mediated T cell stimulation, such as abatacept, have been successful in the treatment of autoimmune diseases, and TNF inhibitors are also able to suppress CD80/86 expression.38 Therefore, suppression of costimulators is considered an important mechanism of action of tofacitinib.
CD80/86 expression is regulated by three transcription factors, NF-κB,31 PU.132 and IRF7.33 NF-κB and PU.1 are directly induced by TLR4 stimulation,39 ,40 while IRF7 is induced through JAK1/Tyk2, which are activated by type I IFN and its downstream signals, STAT1/STAT2.41 Furthermore, IRF7 promotes type I IFN production, which results in the formation of a positive feedback pathway.42 The results of our study indicate that tofacitinib did not affect activation of NF-κB and PU.1, whereas it did suppress IRF7 expression. Moreover, CD80/CD86 expression was suppressed in the presence of an antibody to type I IFN receptor. According to a report by Lim et al,33 IRF7 bound to the promoter lesion of CD80 and regulated its expression. Although the regulation of CD80/86 remains unclear, there may be coordinated regulatory mechanisms among NF-κB, PU.1 and IRF7, and tofacitinib may inhibit CD80/86 expression partly through IRF7.
The most significant finding of this study is that JAK1/JAK3 inhibition by tofacitinib in human DCs suppressed induction of their T cell stimulatory capability. A decrease in costimulator expression, as well as an increase in IDO expression, was observed after tofacitinib treatment. IDO is a rate-limiting enzyme in tryptophan metabolism; however, it has a strong immunomodulation effect and plays an important role in the expression of tolerogenic DC function.34 ,43 ,44 Expression of costimulators and cytokine production capability were suppressed, and expression of IDO was increased, in MoDCs in the presence of tofacitinib. Although the mechanisms of IDO induction remain unclear, we assume that the inhibition of IL-4 played a role in IDO induction by tofacitinib for the following reasons: IL-4 is produced by DCs45; IL-4 activates JAK1/JAK3; tofacitinib inhibits IL-4-mediated signalling; IL-4 is known to inhibit IDO expression.46
To clarify the functions of IDO in DCs, MoDCs were pretreated with tofacitinib and cocultured with allogeneic CD4 T cells in the presence of 1-methyltryptophan (1-MT), an IDO inhibitor. However, the treatment of MoDCs with 1-MT did not cancel the tofacitinib-mediated suppressive effects on T cell stimulation (data not shown). Furthermore, other molecules involved in immune tolerance such as programmed death ligand (PDL)-1 and PDL-2 were not induced by tofacitinib (see online supplementary figure S4). Thus, the functional significance of IDO expression in DCs remains unclear in our studies, and we suppose that the suppressive effects of tofacitinib-treated DCs on T cell stimulation mainly depend on the inhibition of CD80/86 expression in DCs.
On oral administration of tofacitinib 5 or 10 mg twice a day, serum levels of approximately 100–300 nM are achieved, and such therapeutic levels are known to last for 4–6 h. The in vitro levels of tofacitinib used in our studies were almost comparable to the therapeutic levels achieved. Although the in vivo half-life of tofacitinib is 2–3 h, an effective concentration could be obtained in vitro by administration twice a day.
These findings suggest that the inhibition of JAK1 and JAK3 responses after LPS stimulation in human DCs was involved in the regulation of disease states through a novel mechanism. In addition to the known effects of tofacitinib on lymphocytes, we discovered novel effects on human MoDCs: tofacitinib suppressed a production and stimulation loop of type I IFN through JAK1/JAK3, decreased CD80/CD86 expression, induced IDO expression, and suppressed T cell stimulatory capabilities. Thus, tofacitinib not only suppressed cytokine production, but also suppressed expression of costimulators by inhibiting the positive loop of type I IFN–IRF7 in DCs, which leads to immunomodulatory effects.
References
Supplementary materials
Supplementary Data
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Footnotes
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Handling editor Tore K Kvien
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Contributors 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. YT 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.
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Competing interests Y Tanaka received consulting fees, lecture fees, and/or honoraria from Mitsubishi-Tanabe Pharma, Eisai, Chugai Pharma, Abbott Japan, Astellas Pharma, Daiichi-Sankyo, Abbvie, Janssen Pharma, Pfizer, Takeda Pharma, Astra-Zeneca, Eli Lilly Japan, GlaxoSmithKline, Quintiles, MSD, Asahi-Kasei Pharma, and received research grants from Bristol-Myers, Mitsubishi-Tanabe Pharma, Abbvie, MSD, Chugai Pharma, Astellas Pharma, Daiichi-Sankyo. M Kondo is an employee of the Mitsubishi Tanabe Pharma Corporation. K Yamaoka received consulting fees from Pfizer.
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Ethics approval The institutional review board of the University of Occupational and Environmental Health Japan.
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Provenance and peer review Not commissioned; externally peer reviewed.