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Keywords:

  • Dasatinib;
  • Interferon;
  • Plasmacytoid dendritic cells (pDCs);
  • TLR9

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of Interest
  9. References
  10. Supporting Information

Plasmacytoid dendritic cells (pDCs) produce a vast amount of interferon (IFN)-α in response to nucleic acids from viruses and damaged self-cells through Toll-like receptor (TLR)7 and TLR9. Pharmaceutical agents that suppress IFN-α production by pDCs are instrumental in elucidating the mechanisms behind IFN-α production, and in developing novel therapies for inflammatory disorders that involve pDCs. Here, we show that a tyrosine kinase inhibitor for chronic myeloid leukemia with multiple targets, dasatinib, strongly suppresses production of IFN-α and proinflammatory cytokines by human pDCs stimulated with multimeric CpG oligodeoxynucleotides (CpG-A) without reducing viability. In contrast, other tyrosine kinase inhibitors, imatinib, and nilotinib, did not suppress the cytokine production at clinically relevant concentrations. Inhibitors of SRC family kinases (SFKs), which are prominent targets of dasatinib, also suppressed the cytokine production. Notably, however, dasatinib, but not SFK inhibitors, abrogated prolonged localization of CpG-A in early endosomes, which is a critical step for pDCs to produce a large amount of IFN-α. This study suggests that dasatinib suppresses IFN-α production by pDCs by inhibiting SFK-dependent pathways and SFK-independent endosomal retention of CpG DNA. Kinases controlling the distinctive endosomal trafficking in pDCs may be exploited as targets to develop novel therapies for pDC-related inflammatory disorders.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of Interest
  9. References
  10. Supporting Information

Plasmacytoid DCs (pDCs) are a distinctive immune cell type that produces a vast amount of IFN-α in response to virus-derived CpG DNA or ssRNA through TLR9 or TLR7, respectively [1], thus playing an important role in antiviral immunity. pDCs also produces IFN-α in response to nucleic acids derived from damaged self-tissues, and are thereby implicated in provoking inflammatory disorders such as lupus and psoriasis [2, 3]. Thus, pharmaceutical agents that suppress IFN-α production by pDCs are instrumental in elucidating mechanisms of the production of a large amount of IFN-α and in developing novel therapies for inflammatory disorders that involve pDCs.

A variety of protein kinases is involved in signaling pathways in immune responses. Antitumor kinase inhibitors that have different targets may be useful to dissect such pathways. Three tyrosine kinase inhibitors (TKIs), imatinib, nilotinib, and dasatinib, have been approved for the treatment of chronic myeloid leukemia (CML) and Philadelphia (Ph)+ acute lymphoblastic leukemia (ALL), which are caused by constitutive activation of an ABL tyrosine kinase [4]. Notably, dasatinib is capable of inhibiting a broad array of tyrosine kinases in addition to ABL, among which SRC family kinases (SFKs) are prominent targets [5]. As a consequence, it has been shown that dasatinib inhibits activation of T cells [6, 7] and NK cells [8, 9] in vitro. However, it has not been reported whether dasatinib affects immunostimulatory activity of DCs, which play a pivotal role in the induction of innate and adaptive immune responses.

Here, we investigated the effect of dasatinib on human pDCs in comparison with the effects of the other TKIs, imatinib, and nilotinib [10]. We show that clinically relevant concentrations of dasatinib, but not imatinib or nilotinib, strongly suppressed the production of IFN-α and proinflammatory cytokines by pDCs without impairing viability. Mechanistic analysis suggests that dasatinib suppresses IFN-α production by pDCs through inhibiting both SFK-dependent pathways and SFK-independent endosomal retention of CpG DNA, which is a critical step for pDCs to produce a large amount of IFN-α [11, 12]. These results have significant implications for dissecting the mechanisms of IFN-α production by pDCs and for developing novel therapies for pDC-related inflammatory disorders.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of Interest
  9. References
  10. Supporting Information

Dasatinib suppresses cytokine production by pDCs stimulated with TLR9 and TLR7 ligands

There are two major types of CpG oligodeoxynucleotides (ODNs), CpG-A, and CpG-B [13]. CpG-A forms large multimeric aggregates, whereas CpG-B are monomeric and do not form such high order structure [14]. CpG-A shares its particle-like physical features with viral particles and an aggregated self-DNA-antimicrobial peptide complex observed in psoriatic lesions [15]. These particle-like nucleic acids induce pDCs to produce a large amount of IFN-α due to their prolonged retention in early endosomes [11, 12]. Thus, we first examined whether dasatinib suppresses IFN-α production by pDCs stimulated with two TLR9 ligands, ODN2216 (CpG-A) or HSV-1. We also stimulated pDCs with a TLR7 ligand, influenza virus, which similarly induces a large amount of IFN-α production by pDCs. When we stimulated PBMCs depleted of pDCs with ODN2216, IFN-α was scarcely detected in the supernatant (Supporting Information Fig. 1), indicating that pDCs are virtually the only cell type among PBMCs that secretes a detectable level of IFN-α in response to CpG-A. Thus, we pretreated PBMCs for 1 h with one of the three ABL kinase inhibitors dasatinib, imatinib, and nilotinib at clinically relevant concentrations observed in blood after administration. We then added the TLR ligands to each condition and cultured PBMCs for 24 h, and concentrations of IFN-α in the supernatants were measured by ELISA (Fig. 1A). Dasatinib strongly suppressed IFN-α production by pDCs stimulated with ODN2216 as well as natural ligands, HSV-1 or influenza virus, in a dose-dependent manner, and a low concentration (10 nM) was sufficient to induce significant suppression. Imatinib and nilotinib suppressed the IFN-α production to a lesser extent, and high concentrations (5000 and 1000 nM) were necessary to exhibit substantial suppression. When we calculated absolute amounts of IFN-α secreted from a single pDC, we obtained similar results (Supporting Information Fig. 2).

image

Figure 1. Dasatinib suppresses production of IFN-α and proinflammatory cytokines by pDCs stimulated with CpG-A or viruses. (A) PBMCs were stimulated with ODN2216, HSV-1, or influenza virus in the absence or presence of the indicated concentrations of TKIs for 24 h. The concentration of IFN-α in the supernatants was measured in duplicate by ELISA. Because the absolute concentrations were variable depending on the donors, the cytokine concentrations were normalized to the maximum value obtained without the TKIs. The data are shown as means + SE of 7 (ODN2216), 3 (HSV-1), or 6 (influenza virus) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 between the data obtained without TKIs and those obtained with each concentration of TKIs, paired two-tailed t-test. The means and ranges of absolute concentrations of IFN-α obtained without TKIs are as follows: ODN2216 3874 pg/mL (703–9619 pg/mL); HSV-1 3014 pg/mL (632–5262 pg/mL); influenza virus 5334 pg/mL (2117–10 764 pg/mL). (B) Purified pDCs were stimulated with ODN2216 in the absence or presence of the indicated concentrations of TKIs for 24 h. The concentration of IFN-α in the supernatants was measured in duplicate by ELISA, and normalized to the maximum value obtained without TKIs. The data are shown as means + SE of four experiments. **p < 0.01, ***p < 0.001, paired two-tailed t-test. The mean and range of absolute concentrations of IFN-α obtained without TKIs are 62 960 pg/mL (34 078–85 289 pg/mL).(C) Purified pDCs were stimulated with ODN2216 (CpG-A) or ODN2006 (CpG-B) in the absence or presence of the indicated concentrations of dasatinib for 24 h. The concentrations of IFN-α, TNF-α, and IL-6 in the supernatants were measured in duplicate by ELISA, and normalized to the maximum value obtained without dasatinib. The data are shown as means + SE of 4 (IFN-α) and 3 (TNF-α, IL-6) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, paired two-tailed t-test. The means and ranges of absolute concentrations of IFN-α obtained without dasatinib are as follows: ODN2216 62 960 pg/mL (34 078–85 289 pg/mL); ODN2006 398 pg/mL (66–822 pg/mL). TNF-α: ODN2216 1443 pg/mL (1026–1935 pg/mL); ODN2006 849 pg/mL (750–915 pg/mL). IL-6: ODN2216 3883 pg/mL (2173–5160 pg/mL); ODN2006 4156 pg/mL (1483–7571 pg/mL).

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We also examined the effect of dasatinib on IFN-α production by purified pDCs to exclude indirect effects from other cell types. Dasatinib suppressed the production of IFN-α by purified pDCs, whereas imatinib did so to a lesser extent (Fig. 1B), as observed with PBMCs.

Whereas CpG-A induces pDCs to produce both IFN-α and proinflammatory cytokines (TNF-α and IL-6), CpG-B induces production of TNF-α and IL-6 but only a low level of IFN-α [16]. Thus, we next examined whether dasatinib suppresses cytokine production by pDCs stimulated with ODN2216 (CpG-A) or ODN2006 (CpG-B) to compare the suppressive effect of dasatinib on the two types of CpG DNA. Dasatinib strongly suppressed production of IFN-α, TNF-α, and IL-6 induced by ODN2216 (Fig. 1C). In contrast, dasatinib significantly suppressed IFN-α and TNF-α production induced by ODN2006 only at a high concentration (100 nM), and did not suppress IL-6 production (Fig. 1C). Dasatinib reduced the frequency of pDCs bearing intracellular IFN-α and TNF-α (Supporting Information Fig. 3), excluding the possibility that dasatinib simply blocks secretion of the cytokines. In addition, dasatinib suppressed upregulation of CD86 induced by ODN2216 but not by ODN2006 (Supporting Information Fig. 4). Dasatinib also suppressed IL-6 production induced by HSV-1 and influenza virus (Supporting Information Fig. 5).

Neither dasatinib nor imatinib significantly reduced the viability of pDCs stimulated with ODN2216 or ODN2006 (Supporting Information Fig. 6).

Collectively, these data indicate that dasatinib strongly suppresses the production of IFN-α, TNF-α, and IL-6 and the expression of CD86 by pDCs stimulated with CpG-A or natural viral ligands but not with CpG-B without reducing viability.

Dasatinib reduces the IFN-α-producing capacity of pDCs in vivo

To examine whether administration of dasatinib also reduce the IFN-α-producing capacity of pDCs in vivo, we stimulated PBMCs from patients of CML or Ph+ ALL with ODN2216 before starting dasatinib (100 mg once a day) or nilotinib (400 mg twice a day) and two time points after starting them, and measured concentrations of IFN-α in the supernatants by ELISA. Because the proportion of pDCs among PBMCs may vary during the clinical course in each patient, we calculated absolute amounts of IFN-α secreted from a single pDC to exclude the influence of fluctuations of the pDC frequency. Information of the patients is described in Supporting Information Table 1. Dasatinib but not nilotinib significantly reduced the IFN-α-producing capacity of pDCs (Fig. 2). This indicates that dasatinib suppresses the IFN-α-producing capacity of pDCs in vivo as well.

image

Figure 2. Dasatinib suppresses IFN-α production by pDCs in patients with CML and Ph+ ALL. PBMCs were obtained from 17 CML patients and 3 Ph+ ALL patients (ten patients for dasatinib and nilotinib each) at three time points (before starting dasatinib or nilotinib (“before”), 2–3 weeks after starting the drugs (“after #1”), 4–8 weeks after starting the drugs (“after #2”)). PBMCs were stimulated with ODN2216 for 24 h. The concentration of IFN-α in the supernatants was measured by ELISA, and the absolute amounts of IFN-α secreted from a single pDC were calculated. The amounts of IFN-α after starting dasatinib or nilotinib were normalized to the control value obtained before starting them. Statistical significance was determined by paired two-tailed t-test. n.s.: not significant. The means and ranges of the amounts of IFN-α before starting dasatinib or nilotinib are as follows: dasatinib 0.39 pg (0.02–0.92 pg); nilotinib 0.25 pg (0.04–0.63 pg).

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SFK inhibitors suppress IFN-α production by pDCs stimulated with CpG-A

Next, we investigated mechanisms by which dasatinib suppresses cytokine production by pDCs. Because dasatinib strongly suppresses SFKs [5], we examined whether SFK inhibitors PP2 [17] and SU6656 [18], suppress the production of IFN-α, TNF-α, and IL-6 by pDCs stimulated with ODN2216. Whereas PP2 and SU6656 did not significantly reduce the viability of pDCs (data not shown), these reagents suppressed the cytokine production in a dose-dependent manner (Fig. 3A). In contrast, PP2 did not suppress TNF-α and IL-6 production induced by ODN2006, and SU6656 significantly did so only at the highest concentration (Fig. 3B). Thus, it is likely that inhibition of SFKs is responsible for the suppressive effect of dasatinib on pDCs stimulated with CpG-A at least in part.

image

Figure 3. SFK inhibitors suppress production of IFN-α and proinflammatory cytokines by pDCs stimulated with CpG-A. (A) Purified pDCs were stimulated with ODN2216 in the absence or presence of the indicated concentrations of inhibitors or 10 μM PP3 for 24 h. The concentrations of IFN-α, TNF-α, and IL-6 in the supernatants were measured in duplicate by ELISA, and normalized to the maximum value obtained without the inhibitors. The data are shown as means + SE of four (IFN-α) and three (TNF-α, IL-6) experiments. *p < 0.05, **p < 0.01, ***p < 0.001 between data obtained without kinase inhibitors and those obtained with each concentration of inhibitors, paired two-tailed t-test. The mean and range of absolute concentrations of IFN-α, TNF-α, and IL-6 obtained without the inhibitors are 55 157 pg/mL (26 553–12 1608 pg/mL), 421 pg/mL (194–590 pg/mL), and 2059 pg/mL (783–4389 pg/mL), respectively. (B) Purified pDCs were stimulated with ODN2006 in the absence or presence of the inhibitors for 24 h. The concentrations of TNF-α and IL-6 in the supernatants from three experiments were analyzed as in (A). The mean and range of absolute concentrations of TNF-α and IL-6 obtained without the inhibitors are 263 pg/mL (70–598 pg/mL) and 1027 pg/mL (427–1902 pg/mL), respectively.

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Dasatinib inhibits nuclear translocation of IRF7 and NF-κB in pDCs stimulated with CpG-A

The earliest event leading to CpG ODN-induced IFN-α production is endocytosis of CpG ODN by pDCs. Thus, we examined whether dasatinib inhibits uptake of CpG ODN by pDCs. We observed the intracellular localization of FITC-conjugated ODN2216 in the absence or presence of dasatinib by confocal microscopy (Supporting Information Fig. 7). Whereas pDCs kept on ice did not endocytose ODN2216, pDC cultured at 37°C did. Dasatinib did not inhibit the endocytosis of ODN2216. Thus, dasatinib targets a signaling pathway(s) further downstream.

Upon stimulation with CpG ODN, TLR9 rapidly moves from the ER to endosomes [19]. We have recently shown that a proteasome inhibitor bortezomib inhibits the intracellular trafficking of TLR9 in pDCs [20]. Thus, we next examined whether dasatinib also inhibits this step. We stimulated purified pDCs with ODN2216 in the absence or presence of dasatinib, and examined whether TLR9 colocalizes with an ER marker (ER-Tracker) or early endosomal markers (EEA1 and Rab5) by confocal microscopy. To quantitatively compare the degrees of colocalization between culture conditions, we analyzed the data using Manders’ Colocalization Coefficients [21, 22] with the Costes’ method of automatic thresholding [22, 23]. Supporting Information Fig. 8A and B shows representative confocal images and statistical analysis of pooled microscopy data, respectively. TLR9 colocalized with ER-Tracker but not with EEA1 or Rab5 without stimulation. In contrast, TLR9 did not colocalize with ER-Tracker and instead colocalized with EEA1 and Rab5 after stimulation with ODN2216, indicating trafficking of TLR9 from the ER to early endosomes. TLR9 also colocalized with EEA1 and Rab5 but not with ER-Tracker even in the presence of dasatinib. Thus, dasatinib does not inhibit the traffic-king of TLR9 from the ER to endosomes induced by CpG ODN.

Stimulation of pDCs with CpG ODN induces the nuclear translocation of two major transcription factors, IFN regulatory factor (IRF)7 and NF-κB [24]. IRF7 and NF-κB induce the production of IFN-α and proinflammatory cytokines at the final step of TLR signaling, respectively. Thus, we examined whether dasatinib inhibits the nuclear translocation of these transcription factors induced by ODN2216 in pDCs. Whereas IRF7 and NF-κB are located in the cytoplasm in untreated pDCs, both of the transcription factors moved to the nucleus after stimulation with ODN2216 (Fig. 4). Dasatinib inhibited the nuclear translocation of IRF7 and NF-κB, consistently with the suppression of production of IFN-α and proinflammatory cytokines induced by ODN2216.

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Figure 4. Dasatinib inhibits the nuclear translocation of IRF7 and NF-κB in pDCs.Purified pDCs were stimulated with ODN2216 in the absence or presence of dasatinib for 24 h. The cells were stained with rabbit anti-IRF7 (top) or anti-NF-κBp65 (bottom, both green). Nuclei were identified using TOTO-3 dye (blue). The data shown are representative of three experiments performed. Scale bars, 10 μm.

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Dasatinib and a SFK inhibitor inhibit intracellular events upstream of TLR9 engagement

Intracellular events after endocytosis of CpG DNA resulting in the nuclear translocation of transcription factors are composed of (i) trafficking of endosomes carrying CpG DNA (upstream of TLR9) and (ii) signaling triggered by TLR9 engagement (downstream of TLR9). Chloroquine suppresses CpG-induced cellular activation by inhibiting endosomal acidification and thus cleavage of the ectodomain of TLR9 necessary for downstream signaling [25], resulting in suppression of IFN-α production by pDCs (data not shown). Thus, we compared the effects of the three TKIs, PP2, and chloroquine on CpG-induced IFN-α production and tyrosine phosphorylation in pDCs. Because we could not extract sufficient amounts of proteins from primary pDCs to obtain unambiguous results in Western blotting, we used a cell line derived from blastic pDC neoplasm, CAL-1 [26]. As mixing ODN2216 with cationic liposome 1,2-dioleoyloxy-3-trimethylammonium-propane (DOTAP) was necessary to induce CAL-1 to produce IFN-α, we stimulated CAL-1 with the mixture of ODN2216 and DOTAP. Dasatinib, PP2, and chloroquine suppressed IFN-α production by CAL-1, as they did for primary pDCs (Supporting Information Fig. 9A). The stimulation induced tyrosine phosphorylation of several proteins (Supporting Information Fig. 9B). Chloroquine did not inhibit the phosphorylation, indicating that the tyrosine phosphorylation induced by CpG DNA occurs upstream of TLR9 engagement. In contrast, dasatinib, but not imatinib or nilotinib, inhibited the phosphorylation in a dose-dependent manner. PP2 also did so. These data suggest that dasatinib and PP2 inhibit intracellular events upstream of TLR9.

Dasatinib, but not SFK inhibitors, abrogates retention of CpG-A in early endosomes

It has been shown that CpG-A is retained in early endosomes together with the MyD88-IRF7 complex for a long period in pDCs but not in conventional DCs [11, 12]. This spatiotemporal regulation of CpG trafficking is likely to enable prolonged activation of the signaling complex, leading to prodigious production of IFN-α by pDCs. Thus, we examined whether the TKIs (dasatinib, imatinib) and the SFK inhibitors (PP2, SU6656) affect trafficking of endosomes carrying CpG-A in pDCs by confocal microscopy. We used EEA1 and Rab5 as early endosome markers, Rab7 as a late endosome marker, and LAMP-1 as a lysosome marker. Figure 5A and B shows representative confocal images and statistical analysis of pooled microscopy data, respectively. ODN2216 colocalized with EEA1 and Rab5 rather than with Rab7 and LAMP-1 without any of the inhibitors, indicating retention of ODN2216 in early endosomes. Notably, ODN2216 colocalized with Rab7 and LAMP-1 rather than with EEA1 and Rab5 in the presence of dasatinib, indicating that dasatinib abrogates the retention of ODN2216 in early endosomes. In contrast, ODN2216 remained colocalized with EEA1 and Rab5 in the presence of imatinib. Unexpectedly, ODN2216 also remained colocalized with EEA1 and Rab5 in the presence of PP2 or SU6656, indicating that the SFK inhibitors as well as imatinib do not abrogate the retention of ODN2216 in early endosomes. Collectively, these data suggest that dasatinib, but not imatinib, abrogates retention of CpG-A in early endosomes and facilitates trafficking to late endosomes and lysosomes in pDCs by inhibiting protein kinases other than SFKs.

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Figure 5. Dasatinib, but not SFK inhibitors, inhibits the retention of CpG-A in early endosomes. Purified pDCs were cultured with FITC-conjugated ODN2216 in the absence or presence of the TKIs or the SFK inhibitors for 90 min. The cells were stained with anti-EEA1 or anti-Rab5 mAb for early endosomes, or anti-Rab7 mAb for late endosomes, followed by Alexa Fluor 555-conjugated goat anti-rabbit IgG. Alternatively, the cells were stained with PE-conjugated mouse anti-LAMP-1 mAb for late endosomes and lysosomes. (A) Data shown are representative of three to five experiments. Positive signals for both probes are shown in white. Scale bars, 5 μm. (B) Statistical analysis of microscopy data pooled from three to –five experiments. Each symbol represents a cell, and 15 cells were analyzed for each condition. The coefficient value represents the fraction of green in compartments containing red. Statistical significance was determined by Mann–Whitney U-test with Bonferroni correction following Kruskal–Wallis H-test.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of Interest
  9. References
  10. Supporting Information

pDCs represent a unique immune cell type in that they rapidly produce a vast amount of IFN-α in response to nucleic acids derived not only from pathogens but also from damaged self-cells. Here, we showed that a TKI dasatinib potently suppresses IFN-α production by pDCs without reducing cell viability. Importantly, dasatinib abrogated retention of CpG-A in early endosomes and facilitated the movement of CpG-A to late endosomes/lysosomes. It is of note that this effect is independent of SFKs. This is the first study showing the pharmacological interruption of the critical step for pDCs to produce IFN-α: prolonged localization of CpG DNA in early endosomes [11, 12]. It sheds new light on the molecular mechanisms by which pDCs perform their immune functions and on the development of novel therapies for pDC-related inflammatory disorders.

Among the three TKIs (dasatinib, imatinib, nilotinib), dasatinib most strongly suppressed IFN-α production by pDCs stimulated with representative IFN-α inducers, ODN2216 (CpG-A), HSV-1 (TLR9 ligand), or influenza virus (TLR7 ligand) at clinically relevant, low concentrations. Although imatinib and nilotinib also diminished IFN-α production, the effects were much weaker than that of dasatinib and needed high concentrations. Dasatinib also potently suppressed production of TNF-α and IL-6 as well as expression of CD86 by pDCs stimulated with ODN2216 (CpG-A). However, the suppression of such responses induced by ODN2006 (CpG-B) was less marked. The main difference between CpG-A and CpG-B is in the mode of their endosomal transport; CpG-A is retained in early endosomes for a longer time than CpG-B [11, 12]. Thus, the differential effects of dasatinib on the stimulation with CpG-A and CpG-B imply that dasatinib may target certain functions of early endosomes.

Importantly, dasatinib, but not nilotinib, significantly diminished the IFN-α-producing capacity of pDCs in patients treated with these drugs. These data are consistent with the in vitro data, and suggest that dasatinib suppresses the IFN-α-producing capacity of pDCs in vivo as well.

Next, we examined the mechanisms by which dasatinib suppresses cytokine production by pDCs stimulated with CpG-A. Two inhibitors of SFKs (PP2, SU6656) significantly suppressed production of IFN-α, TNF-α, and IL-6 by pDCs stimulated with CpG-A but not with CpG-B, consistent with the previous report [27]. Correspondingly, dasatinib and PP2 suppressed CpG-triggered global tyrosine phosphorylation in a pDC cell line CAL-1, but chloroquine did not, suggesting that dasatinib and PP2 inhibits CpG-induced intracellular events upstream of TLR9. Notably, however, dasatinib but not PP2 or SU6656 abrogated prolonged localization of CpG-A in early endosomes, and facilitated transport of CpG-A to late endosomes/lysosomes. These data suggest that dasatinib suppresses IFN-α production by pDCs through inhibiting both SFK-dependent and SFK-independent signaling pathways. Although both of the pathways appears to be involved in the functions of early endosomes necessary for responses to CpG-A, the data of confocal microscopy indicates that only the SFK-independent pathway is responsible for the prolonged localization of CpG-A in early endosomes.

The mechanisms by which an aggregated form of CpG DNA is retained in early endosomes in pDCs for a long period remain to be elucidated. It has been shown that an aggregated form of CpG DNA rapidly goes to late endosomes/lysosomes in conventional DCs in mice [11], suggesting that pDCs have distinctive mechanisms for retention of CpG DNA in early endosomes. A wide variety of protein kinases is involved in endocytic pathways [28]. Dasatinib targets a broad array of tyrosine kinases and several serine/threonine kinases [29-32]. Thus, dasatinib may affect endosomal trafficking in pDCs by inhibiting certain protein kinases other than SFKs involved in early-to-late endosome transition.

In this context, two recent studies have shown that the machinery of lysosome-related organelle biogenesis is essential for TLR7 and TLR9 signaling in pDCs but not in conventional DCs. Sasai et al [33] have reported that the adaptor protein (AP)-3 complex is responsible for trafficking of TLR9 to IRF7+ endosomes but not to NF-κB+ endosomes using mouse macrophages, and that AP-3 is necessary for the production of IFN-α but not IL-12 by mouse pDCs. Blasius et al. [34] have reported that an oligo-peptide transporter Slc15a4 and three protein complexes involved in Hermansky–Pudlak syndrome (AP-3, biogenesis of lysosome-related organelles complex (BLOC)-1, BLOC-2) are required for production of both IFN-α and proinflammatory cytokines by pDCs stimulated with TLR7 or TLR9 ligands. Slc15a4 [35] and the three complexes [36] are located in early endosomes, and BLOC-1 is necessary for sorting certain cargoes from early endosomes to lysosome-related organelles [37]. Together with these findings, the present study suggests that distinctive properties of early endosomes in pDCs, which is affected by dasatinib, are crucial for the large amount of IFN-α production. Although vesicle traffic pathways for TLR7 ligands to induce IFN-α has not been reported, the involvement of Slc15a4 [34] and AP-3 [33] in type I IFN production by pDCs stimulated with TLR7 ligands suggests that dasatinib targets similar machineries for IFN-α induction by TLR7 and TLR9 ligands.

In conclusion, dasatinib strongly suppresses the production of IFN-α and proinflammatory cytokines by pDCs stimulated with CpG-A, most likely by inhibiting both SFK-dependent and independent pathways, the latter of which is responsible for the prolonged localization of an aggregated form of CpG DNA in early endosomes. Protein phosphorylation is at the heart of controlling the physical properties of endocytosis and of integrating them with signal transduction networks of the cell [28]. Thus, the present study provides a clue to dissect molecular mechanisms for the distinctive behavior of endosomes in pDCs as well as possibilities to develop novel therapies for inflammatory disorders by targeting the endosomal trafficking in pDCs.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of Interest
  9. References
  10. Supporting Information

Culture media and reagents

RPMI 1640 (Wako, Osaka, Japan) supplemented with 10% heat-inactivated FBS (Equitec-Bio, Inc., Kerrville, TX, USA), 2 mM L-glutamine, penicillin G, streptomycin (Gibco BRL, Carlsbad, CA, USA), and 10 mM HEPES (Nacalai Tesque, Kyoto, Japan) were used for cell culture. Dasatinib (provided by Bristol-Myers Squibb Company), imatinib, and nilotinib (provided by Novartis Pharma) were dissolved in DMSO (Nacalai Tesque) at 100 mM as a stock solution and were stored at −20°C. A SFK inhibitor PP2 and its negative control PP3 were purchased from Calbiochem (Darmstadt, Germany), and a SFK inhibitor SU6656 was purchased from Cayman Chemical (Ann Arbor, MI, USA). HSV-1 (KOS strain, a gift from Dr. Masaki Yasukawa (Ehime University, Ehime, Japan)) was attenuated with UV irradiation. Influenza virus (105.3 median tissue culture infective dose/0.2 mL of A/Niigata/05F254/2006, a kind gift from Dr. Reiko Saito (Niigata University, Niigata, Japan)) was inactivated at 56°C for 30 min. A cell line derived from blastic plasmacytoid dendritic cell neoplasm CAL-1 was described previously by Maeda et al. [26].

Isolation of pDCs

This study was approved by the Institutional Review Board of the Graduate School of Medicine at Kyoto University and abides by the tenets of the Declaration of Helsinki. PBMCs were obtained from healthy donors with written informed consent. CD4+CD11clin cells were isolated as pDCs as described [38], using FACSAria cell sorter (BD Biosciences, San Jose, CA, USA). Reanalysis of the sorted cells confirmed a purity of more than 98%.

Cell culture

PBMCs were plated in flat-bottom, 96-well plates at 4 × 105 cells/200 μL. Purified pDCs were plated in round-bottom, 96-well plates at 4 × 104 cells/200 μL, except culture for stimulation with ODN2006 where the cells were plated at 1 × 105 cells/200 μL. The cells were pretreated with indicated concentrations of TKIs or SFK inhibitors for 1 h, and were stimulated with 0.5 μM ODN2216 (CpG-A) [16], ODN2006 (CpG-B) [39] (Operon Biotechnologies, Huntsville, AL, USA), 106 PFU/mL HSV-1, or 0.5% vol/vol influenza virus without removing the inhibitors for 24 h. For control culture, DMSO was added instead of the inhibitors. The cells were used for flow cytometry, and the supernatants were used for ELISA. For confocal analyses, pDCs were cultured for indicated time periods.

Analysis of cell viability

The cultured pDCs were stained with FITC-conjugated CELL LAB ApoScreen annexin V (Beckman Coulter, Orange County, CA, USA) and propidium iodide, and were analyzed for viability by flow cytometry with the FACSCalibur, and data were analyzed with CellQuest software (BD Biosciences).

Analysis of cytokine production by ELISA

Concentrations of cytokines in the supernatants were measured by ELISA. The following reagents were used: the human IFN-α module set (Bender MedSystems, Vienna, Austria) and the human TNF-α and IL-6 ELISA MAX Standard sets (BioLegend, San Diego, CA, USA).

Analysis of patient samples

Peripheral blood samples were obtained from 17 CML (chronic phase) patients and three Ph+ ALL patients who newly started dasatinib or nilotinib, at three time points (before, 2–3 weeks after, and 4–8 weeks after starting dasatinib or nilotinib) under the approval by the Institutional Review Board of the Graduate School of Medicine at Kyoto University and with written informed consent. PBMCs isolated with Lympholyte-H (CEDARLANE, Burlington, Ontario, Canada) were stained with FITC-conjugated anti-BDCA-2 (CD303) mAb (Miltenyi Biotec, Bergisch Gladbach, Germany), and pDCs were identified as BDCA-2+ cells with the FACSCalibur. Dead cells were excluded by staining with propidium iodide. Absolute numbers of pDCs in the wells were calculated from numbers of PBMCs and percentages of BDCA-2+ cells among PBMCs. The PBMCs were cultured in flat-bottom, 96-well culture plates at 4 × 105 cells/200 μL in the presence of 0.5 μM ODN2216 for 24 h. Culture supernatants were analyzed for concentrations of IFN-α by ELISA. The amounts of IFN-α secreted from a single pDC were calculated by dividing the amounts of IFN-α in the supernatants by absolute numbers of pDCs.

Western blotting

CAL-1 cells were pretreated for 1 h with 10 μM PP3, 10 μM PP2, 10 μM chloroquine (WAKO), 5 μM imatinib, 1 μM nilotinib, or indicated concentrations of dasatinib. The cells were stimulated with DOTAP Liposomal Transfection Reagent (Roche Applied Science, Penzberg, Germany) alone or 1 μM ODN2216 plus DOTAP for 1 h without removing the inhibitors. The cells were lysed directly in sample buffer containing 1%SDS, boiled, and diluted tenfold with 1% Triton X-100-containing lysis buffer. The cell extracts were fractionated by SDS-PAGE and transferred to Immobilon-P transfer membranes (Millipore, Billerica, MA, USA), using a wet transfer apparatus (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were incubated with HRP-conjugated anti-phosphotyrosine mAb (PY20) (Transduction Laboratories, Lexington, KY, USA) or anti-β-actin mAb (Sigma, St. Louis, MO, USA) for 1 h. After a wash with TBS-T (25 mM Tris-HCl (pH 7.4), 137 mM NaCl, 2.7 mM KCl, and 0.1% Tween 20), peroxidase activity was detected with Pierce Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA).

Confocal microscopy

To observe uptake of CpG ODN, purified pDCs were cultured in the absence or presence of 100 nM dasatinib for 1 h, and were incubated with 6 μM FITC-conjugated ODN2216 (InvivoGen, San Diego, CA, USA) for 3 h without removing dasatinib. The cells were harvested, washed, stained with PE-conjugated anti-HLA-DR mAb (BD Biosciences), and fixed with 2% paraformaldehyde. pDCs cultured without dasatinib and with FITC-conjugated ODN2216 on ice were used as a negative control for endocytosis.

To observe translocation of TLR9 from ER to endosomes, pDCs were cultured in the absence or presence of 30 nM dasatinib for 1 h, and were stimulated with 0.5 μM ODN2216 without removing dasatinib for 2 h. ER-Tracker Red (Invitrogen, Carlsbad, CA) was added for 30 min before harvest. After fixation with 4% paraformaldehyde for 5 min at room temperature, permeabilization with 0.1% Triton X-100 for 5 min at −20°C, and blocking with 10% goat serum, the cells were stained with rabbit anti-EEA1 mAb (C45B10) or rabbit anti-Rab5 mAb (C8B1) (Cell Signaling Technology, Danvers, MA, USA) followed by Alexa Fluor 555-conjugated goat anti-rabbit IgG (Cell Signaling Technology), and were stained with biotinylated mouse anti-TLR9 mAb (26C593.2) (IMGENEX, San Diego, CA, USA) followed by Alexa Fluor 488-conjugated streptavidin (Invitrogen).

To observe nuclear translocation of IRF7 and NF-κB, pDCs were cultured in the absence or presence of 30 nM dasatinib for 1 h, and were stimulated with 0.5 μM ODN2216 without removing dasatinib for 3 h. After fixation with 2% paraformaldehyde for 15 min at 37°C, permeabilization with 100% methanol for 10 min at −20°C, and blocking with 10% goat serum, the cells were stained with rabbit anti-IRF7 (sc-9083) or NF-κBp65 (sc-109) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by Alexa Fluor 488-conjugated goat-anti rabbit IgG (Invitrogen). Nuclei were identified using TOTO-3 dye (Invitrogen).

To observe intracellular trafficking of CpG ODN, pDCs were cultured in the absence or presence of 30 nM dasatinib, 5 μM imatinib, 10 μM PP2, or 5 μM SU6656 for 1 h, and were incubated with 6 μM FITC-conjugated ODN2216 without removing the inhibitors for 90 min. After fixation with 4% paraformaldehyde for 5 min at room temperature, permeabilization with 0.1% Triton X-100 for 5 min at −20°C, and blocking with 10% goat serum, the cells were stained with anti-EEA1, anti-Rab5 mAb, or rabbit anti-Rab7 XP mAb (D95F2), followed by Alexa Fluor 555-conjugated goat anti-rabbit IgG (Cell Signaling Technology). Alternatively, the cells were stained with PE-conjugated mouse anti-LAMP-1 mAb (BioLegend) after blocking for 30 min using Image-iT FX signal enhancer (Invitrogen).

The cells were attached to slide glass by cytospin centrifuge and examined with an oil immersion objective (×60 Plan Apo, numerical aperture 1.4) by LSM510 META confocal microscope (Carl Zeiss, Oberkochen, Germany). Data were acquired with LMS5 software Version 3.2 (Carl Zeiss).

Colocalization analysis

Images were analyzed with Fiji (http://fiji.sc/wiki/index.php/Fiji), an image-processing package based on ImageJ. Colocalization amounts were analyzed using Manders’ Colocalization Coefficients [21, 22] with the Costes’ method of automatic thresholding [22, 23] using the Coloc_2 plug-in. The coefficient value represents the fraction of green in compartments containing red.

Statistical analyses

Data are presented as means ± SE. The significance of differences was determined by paired two-tailed t-test for cytokine and viability data and by Mann–Whitney U-test with Bonferroni correction following Kruskal–Wallis H-test for microscopy data. Difference with p < 0.05 was considered significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of Interest
  9. References
  10. Supporting Information

We thank Bristol-Myers Squibb Company for providing dasatinib, Novartis Phama for providing imatinib and nilotinib, Masaki Yasukawa for providing HSV-1, Reiko Saito for providing influenza virus, Keiko Fukunaga (Kyoto University) for her excellent technical assistance, Takashi Uchiyama (Kyoto University) for his supervision, the physicians who provided blood samples from patients treated with the TKIs, and Shin-ichiro Fujii (RIKEN, Research Center for Allergy and Immunology, Japan) for reviewing the manuscript. This study was supported by research funding from Ministry of Education, Culture, Sports, Science, and Technology of Japan (17016034) and from Japan Science and Technology Agency, Core Research for Evolutional Science and Technology (CREST) (to N.K.).

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of Interest
  9. References
  10. Supporting Information
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Abbreviations
ALL

acute lymphoblastic leukemia

AP

adaptor protein

BLOC

biogenesis of lysosome-related organelles complex

CML

chronic myeloid leukemia

DOTAP

1,2-dioleoyloxy-3-trimethylammonium-propane

IRF

IFN regulatory factor

ODN

oligodeoxynucleotide

pDC

plasmacytoid DC

Ph

Philadelphia

SFK

SRC family kinase

TKI

tyrosine kinase inhibitor

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of Interest
  9. References
  10. Supporting Information

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Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset.

Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FilenameFormatSizeDescription
eji2489-sup-0001-FigureS1.pdf762K

Figure S1. Depletion of pDCs from PBMCs abrogates IFN-α production induced by CpG-A (A) PBMCs were stained with PE-labeled anti-BDCA2 mAb (Miltenyi Biotec) and propidium iodide, and PE- and propidium iodide-negative viable cells were obtained by FACSAria cell sorter. All the propidium iodide-negative cells were obtained as total PBMCs. The percentages of BDCA-2+ cells are indicated on the plot. (B) Total PBMCs or PBMCs depleted of pDCs were stimulated with ODN2216 for 24 h. The Concentration of IFN-α in the supernatants was measured in duplicate by ELISA. The data shown are representative of 2 experiments.

Figure S2. Dasatinib suppresses IFN-α production by pDCs stimulated with CpG-A when calculated at a single cell level PBMCs were stimulated with ODN2216 in the absence or presence of the indicated concentrations of TKIs for 24 h. The concentration of IFN-α in the supernatants was measured in duplicate by ELISA, and the absolute amounts of IFN-α secreted from a single pDC were calculated. The cytokine concentrations were normalized to the maximum value obtained without the TKIs. The data are shown as means +/- SE of 3 independent experiments. *P < 0.05; ***P < 0.001. P values refer to the comparison between the data obtained without TKIs and those obtained with each concentration of TKIs. The means and ranges of absolute concentrations of IFN-α obtained without TKIs are 1.21 pg (0.92-1.47 pg).

Figure S3. Dasatinib reduces the frequency of pDCs bearing intracellular IFN-α and TNF-α pDCs were enriched by depleting lin+ cells from PBMCs using anti-CD3, anti-CD14, and anti-CD16 mAbs, and were stimulated with ODN2216 in the absence or presence of the indicated concentrations of dasatinib for 6 h. Brefeldin A was added during the last 2 h. After stimulation, the cells were stained with FITC-labeled anti-CD3, CD14, CD16, CD20, PE-Cy7 labeled anti-CD4, and APC-labeled anti-CD11c mAbs. Then the cells were fixed, permeabilized, and stained with PE-labeled anti-IFN-α and anti-TNF-α mAbs (BD Biosciences). Lin-CD4+CD11c- cells were gated and intracellular IFN-α and TNF-α were detected by FACSCalibur. The percentages in each quadrant are indicated on the plot. The data shown are representative of 2 experiments.

Figure S4. Dasatinib suppresses upregulation of CD86 induced by CpG-A but not by CpG-B Purified pDCs were stimulated with ODN2216 or ODN2006 in the absence or presence of the indicated concentrations of TKIs for 24 h, stained with FITC-conjugated anti-CD86 mAb (BD Biosciences), and were analyzed by FACSCalibur. Open histograms represent cells stained with isotype-matched control mAbs. The numbers shown with each histogram represent ratios of mean fluorescence intensity of CD86 to that of isotype-matched control. Data are representative of 3 experiments.

Figure S5. Dasatinib suppresses IL-6 production by pDCs stimulated with virus Purified pDCs were stimulated with HSV-1 or influenza virus in the absence or presence of the indicated concentrations of dasatinib for 24 h, The concentration of IL-6 in the supernatants was measured in duplicate by ELISA, and were normalized to the maximum value obtained without dasatinib. The data are shown as means +/- SE of 3 experiments. *P < 0.05; **P < 0.01. The significance of differences was determined by paired two-tailed t-test. The means and ranges of absolute concentrations of IL-6 obtained without dasatinib are as follows: HSV-1 219 pg/mL (182-260 pg/mL); influenza virus 253 pg/mL (157-421 pg/mL).

Figure S6. Dasatinib and imatinib do not reduce the viability of pDCs Purified pDCs were stimulated with ODN2216 (A) or ODN2006 (B) in the presence of the indicated concentrations of dasatinib or imatinib for 24 h. Percentages of Annexin V- and propidium iodide-double negative viable cells were measured by flow cytometry, and were normalized to the value obtained without TKIs. The data are shown as means +/- SE of 3 independent experiments. The significance of differences was determined by paired two-tailed t-test, and was not detected between the viability obtained without and with TKIs.

Figure S7. Dasatinib does not inhibit endocytosis of CpG ODN by pDCs Purified pDCs were cultured with FITC-conjugated ODN2216 in the absence or presence of dasatinib for 3 h. The cells were stained with PE-conjugated anti-HLA-DR mAb. pDCs cultured with FITC-conjugated ODN2216 in the absence of dasatinib on ice were used as a negative control for endocytosis. The cells were observed by confocal microscopy. The data are representative of 3 experiments. Scale bars, 10 μm.

Figure S8. Dasatinib does not inhibit the trafficking of TLR9 from the ER to early endosomes Purified pDCs were stimulated with ODN2216 in the absence or presence of dasatinib for 2 h. ER-Tracker Red was added for the last 30 min. Alternatively, the cells were stained with anti-EEA1 or anti-Rab5 mAb followed by Alexa Fluor 555-conjugated goat anti-rabbit IgG. The cells were stained with biotinylated anti-TLR9 mAb followed by Alexa Fluor 488-conjugated streptavidin, and were observed by confocal microscopy. The micrographs shown in (A) are representative of 3 experiments. Pixels with positive signals for both probes are shown in white. Scale bars, 5 μm. (B) Statistical analysis of microscopy data pooled from 3 experiments. Each symbol represents a cell, and 15 cells were analyzed for each condition. The coefficient value represents the fraction of green in compartments containing red. n.s. : not significant. The significance of differences was determined by Mann-Whitney U-test with Bonferroni correction following Kruskal-Wallis H-test.

Figure S9. Dasatinib and PP2, but not chloroquine, suppress CpG-triggered global tyrosine phosphorylation CAL-1 cells were stimulated with ODN2216 plus DOTAP in the absence or presence of PP3, PP2, chloroquine (CQ), dasatinib, imatinib (Ima) or nilotinib (Nilo) for 24 h. (A) The concentration of IFN-α in the supernatants was measured in duplicate by ELISA, and were normalized to the maximum value obtained without the inhibitors. The data are shown as means +/- SE of 3 experiments. *P < 0.05; **P < 0.01; ***P < 0.001. The significance of differences was determined by paired two-tailed t-test. The mean and range of absolute concentrations of IFN-α obtained without the inhibitors are 1411 pg/mL (857-2426 pg/mL). (B) Cell lysates were fractionated by SDS-PAGE followed by immunoblotting with anti-phosphotyrosine mAb or anti-β-actin mAb. The data are representative of 3 experiments.

Table S1. Patient information

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