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Dipyrithione inhibits IFN-γ-induced JAK/STAT1 signaling pathway activation and IP-10/CXCL10 expression in RAW264.7 cells

Objective This study investigates the effects of dipyrithione (PTS2) on the expression of IP-10/CXCL10, which has been observed in a wide variety of chronic inflammatory disorders and autoimmune conditions. Methods RAW264.7 cells (a murine macrophage-like cell line) were cultured in the absence or in the presence of PTS2 (3–10 μM) together with or without IFN-γ (10 ng/ml). IP-10/CXCL10 expression was measured by specific enzyme-amplified immunoassays and reverse transcriptase-PCR (RT-PCR). Phosphorylation of JAK1, JAK2 and STAT1 were detected by Western blot analysis. Results We found that PTS2 inhibited IFN-γ-induced up-regulation of IP-10/CXCL10 protein level in a dose- and time-dependent manner in RAW264.7 cells. RT-PCR experiments showed that PTS2 suppressed IFN-γ-induced IP-10/CXCL10 expression at mRNA levels. Mechanistically, PTS2 prevented phosphorylation of JAK1, JAK2 and STAT1, but did not interfere with the p38 pathway. Furthermore, the inhibitory effect of PTS2 on IP-10/CXCL10 up-regulation was slightly stronger than JAK2 inhibitor AG490. Conclusion PTS2 inhibits IFN-γ-induced IP-10/CXCL10 expression in RAW264.7 cells by targeting the JAK/STAT1 signaling pathway, suggesting that PTS2 could exert anti-inflammatory effects through attenuating the formation of chemokine IP-10/CXCL10.


Introduction
Mononuclear phagocytes play an essential role in the regulation of host defense and homeostasis, and in the development of acute and chronic inflammatory diseases. Interferon-c secreted by activated T cells and NK cells is intimately involved in the innate and acquired immune responses [1][2][3]. As a major macrophage activation factor, IFN-c binds to its receptor on the macrophage cell surface and reprograms gene expression necessary for the execution of host defense functions by modulating signal transduction pathways. IFN-induced proteins include enzymes, transcription factors, cell surface glycoproteins, cytokines, chemokines and a large number of factors with unknown functions [4].
IP-10/CXCL10 is a non-ELR (Glu-Leu-Arg) CXC chemokine first cloned in 1985 as a protein secreted by peripheral blood mononuclear cells (PBMC), fibroblasts and endothelial cells [5]. IP-10/CXCL10 binds its receptor CXCR3 to play a biological role in stimulation of monocytes, natural killer (NK) and T cell migration, regulation of T cell and bone marrow progenitor maturation, modulation of adhesion molecule expansion and inhibition of angiogenesis [6][7][8][9][10][11][12][13]. The expression of IP-10/CXCL10 has also been observed in a wide variety of chronic inflammatory disorders and auto immune conditions [14][15][16][17][18]. Recently, it is reported that SARS-CoV may instigate an excessive immune response through overproduction of IP-10/CXCL10, MCP-1 and IL-8, resulting in transendothelial infiltration and accumulation of neutrophils, alveolar macrophages and Th1 lymphocytes into lung tissue causing pulmonary inflammation and destruction [19]. Previous studies have shown that IP-10/CXCL10 could be an excellent prognostic marker for SARS disease progression [20,21]. IP-10/CXCL10 is also induced by hepatitis C virus (HCV) in hepatocytes for recruiting inflammatory cells into the lobules and acts as an important predictor of chronic HCV infection progression [22]. Thus, IP-10/CXCL10 is considered as a predictive marker of successful treatment response in HCV/HIV-coinfected patients [23]. In addition, Kong et al. [24] have reported that IP-10/CXCL10 levels are increased in SLE, and serum IP-10/CXCL10 may represent a more sensitive marker for monitoring disease activity than standard serological tests.
In various cells, including macrophages, the intracellular signaling pathway triggered by IFN-c receptors involves the rapid and direct activation of Janus tyrosine protein kinases JAKs-STAT signaling pathways [2,[25][26][27][28]. Numerous biological actions of IFN-c have been demonstrated to be dependent on STAT1 signal transduction pathway-associated gene products [29]. Another transcription factor involved in IFN-c-induced IP-10/CXCL10 expression is nuclear factor jB (NF-jB) [30]. In addition, IFN-c has also been shown to activate the p38 signaling pathway to enhance IP-10/CXCL10 gene expression in macrophages [31].
Here we show evidence that PTS2 inhibits IFN-c-induced up-regulation of IP-10/CXCL10 in RAW264.7 cells. We also found that IFN-c-induced increase of IP-10/CXCL10 mRNA level was suppressed significantly by PTS2 treatment. The mechanism utilized by PTS2 to prevent IP-10/ CXCL10 expression is related to its inhibitory effects on JAK/STAT1 signal transduction induced by IFN-c. These results suggest that PTS2 may affect host immune responses by down-regulating IFN-c-induced gene expression.

Materials and methods
Cell culture RAW264.7, a murine macrophage-like cell line purchased from the CBCAS (Cell Bank of the Chinese Academic of Sciences, Shanghai, China) was maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (Hyclone) and antibiotics (100 U/ml penicillin and 100 lg/ml streptomycin) (HyClone) at 37°C in an atmosphere of 5% CO 2 .

Antibodies and reagents
Polyclonal antibodies against JAK1, phospho-JAK1 (Tyr1022/1023), JAK2, phospho-JAK2 (Tyr1007/1008), phospho-STAT1 (Tyr701), p38 MAPK and phospho-p38 MAPK (Thr180/Tyr182) were obtained from Cell Signaling Technology. These antibodies were diluted at the ratio of 1:1,000 according to protocol. Antibody to STAT1 was from Santa Cruz Biotechnology, diluted at the ratio of 1:1,500 according to protocol. All secondary antibodies used for Western blot were purchased from Rockland Immunochemical. Recombinant Murine Interferon-c (IFN-c) was purchased from Peprotech. AG490 (a JAK2 inhibitor) was purchased from Calbiochem. PTS2 was purchased from J&K Chemical Ltd. IP-10/CXCL10 ELISA RAW264.7 cells were seeded in 24-well plates at 5 9 10 5 cells/well the day before the experiment. After IFN-c and PTS2 treatment, media were collected and centrifuged at 10,000 rpm for 5 min. IP-10/CXCL10 level of the media was then determined by a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using a mouse-specific IP-10/CXCL10 ELISA (R&D Systems) according to the manufacturer's instructions.

Statistics
Analysis of variance (ANOVA) was used to compare the results between the two groups. Individual points were compared using a Student's t test (STATISTICA, Statsoft Inc., Tulsa, USA) and differences were considered significant for P \ 0.01. Data are presented as mean ± SD.
Western blotting analysis experiments were repeated three times with similar trends.

Results
PTS2 inhibits IFN-c-induced up-regulation of IP-10/ CXCL10 in RAW 264.7 cells In order to determine whether PTS2 could affect IFN-c-induced IP-10/CXCL10 protein up-regulation, we measured IP-10/CXCL10 level in the cultural medium of RAW264.7 cells by ELISA. We pretreated RAW264.7 cells with 0, 3.0, 5.0 or 10.0 lM PTS2 for 2 h, and then stimulated cells with IFN-c (10 ng/ml) for 24 h followed by medium collection. The result from ELISA assay showed that IFN-c stimulated IP-10/CXCL10 secretion dramatically and PTS2 dose-dependently inhibited the elevation of IP-10/CXCL10 level with significant reductions at doses of 5.0 and 10.0 lM. PTS2 alone, even at concentration of 10.0 lM, did not influence IP/CXCL10 protein level in normal RAW264.7 cells (Fig. 2a).
Further experiments were performed by treating RAW264.7 cells with 5.0 lM PTS2 for 2 h or not, followed by incubating these cells with IFN-c (10 ng/ml) for 0-24 h. The cell culture supernates were subjected to ELISA analysis to measure IP-10/CXCL10 protein level. As shown in Fig. 2b, IP-10/CXCL10 protein increased apparently 8, 12 and 24 h after IFN-c stimulation, whereas PTS2 treatment led to a decrease in IFN-c-induced IP-10/ CXCL10 up-regulation. We also detected the inhibitory ability of PTO, the monomer of PTS2, on IFN-c-induced IP-10/CXCL10 up-regulation. RAW264.7 cells were treated with PTO (5.0 lM) or PTS2 (5.0 lM) for 2 h and then were stimulated with IFN-c (10 ng/ml) for 24 h. Comparing PTS2 with PTO, we found that PTS2 was a little more effective in inhibiting IFN-c-induced IP-10/CXCL10 expression. In addition, aspirin was used as control, which is an acetylated salicylate utilized to treat inflammation and arthritis pain [36]. The data suggested that aspirin did not influence IFN-c-induced IP-10/CXCL10 up-regulation (Fig. 2c).
PTS2 inhibits IFN-c-induced increase of IP-10/ CXCL10 mRNA level in RAW 264.7 cells Since the above results indicated that PTS2 inhibited the increase of IP-10/CXCL10 protein level induced by IFN-c, we then performed RT-PCR to analyze the effect of PTS2 on IFN-c-induced IP-10/CXCL10 expression at transcription levels. RAW264.7 cells were pretreated with PTS2 (5.0 lM) for 2 h or not, and then were stimulated with IFN-c (10 ng/ml). Twelve hours after IFN-c stimulation, total RNA was isolated and mRNA level of IP-10/CXCL10 was determined by RT-PCR. As shown in Fig. 3, IFN-c stimulation elevated endogenous mRNA levels of IP-10/ CXCL10, whereas PTS2 suppressed IFN-c-induced increase of IP-10/CXCL10 mRNA levels. The housekeeping gene GAPDH did not change with treatment. This result, together with data shown above in Fig. 2, demonstrated that PTS2 inhibited IFN-c-induced IP-10/CXCL10 expression.

PTS2 prevents IFN-c-induced JAK/STAT1 signaling in RAW 264.7 cells
It has been demonstrated that the transcriptional activation of the IP-10/CXCL10 gene is dependent upon IFN-c-induced JAK/STAT1 signaling activation [29]. We thus evaluated the effects of PTS2 on IFN-c-induced JAK1, JAK2 and STAT1 activation in RAW264.7 cells. The cells were pretreated with 3.0, 5.0, 10.0 lM of PTS2 for 2 h or not, and then incubated with IFN-c (10 ng/ml) for 30 min. Western blot analysis showed that JAK1, JAK2 and STAT1 were markedly activated by IFN-c stimulation, whereas PTS2 treatment led to a reduction in IFN-cinduced JAK1, JAK2 and STAT1 phosphorylation in a concentration-dependent manner (Fig. 4a). Next, RAW264.7 cells were treated with PTS2 (5.0 lM) for 2 h or not, and then incubated with IFN-c (10 ng/ml) for 0-60 min. As assessed by Western blotting, IFN-c rapidly (within 3 min) increased the phosphorylation of tyrosine residues 1022/1023 of JAK1 and 1007/1008 of JAK2, and tyrosine 701 of STAT1 (Fig. 4b). PTS2 decreased the phosphorylation of JAK1, JAK2 and STAT1 in a timedependent manner (Fig. 4b). When RAW264.7 cells were treated with different concentration of IFN-c (1-10 ng/ml), a dramatic and concentration-dependent JAK1, JAK2 and STAT1 phosphorylation was observed (Fig. 4c). PTS2 significantly attenuated IFN-c-induced JAK1, JAK2 and  b Cells were pretreated with 5.0 lM PTS2 for 2 h or not, and then stimulated with IFN-c (10 ng/ml) for 0-24 h, respectively. c Cells were pretreated with 5.0 lM PTS2, PTO or Aspirin for 2 h followed by incubating with IFN-c (10 ng/ml) for 24 h. Cell culture supernates from a, b and c were subjected to ELISA for measuring IP-10/CXCL10 protein level. Data are presented as mean ± SD. **P \ 0.01 compared with the IFN-cstimulated cells Fig. 4 Effect of PTS2 on IFN-c-induced JAK/STAT1 signaling in RAW 264.7 cells. a RAW264.7 cells were pretreated with 3.0, 5.0 or 10.0 lM PTS2 or not, respectively, for 2 h, and then incubated with IFN-c (10 ng/ ml) for 30 min. Western blot analysis was performed by using antibodies to JAK1, p-JAK1, JAK2, p-JAK2, STAT1, or p-STAT1, respectively. GAPDH was used as control. b Cells were incubated with PTS2 (5.0 lM) for 2 h or not, followed by stimulating with IFN-c (10 ng/ml) for 0-60 min, respectively. Cell lysates were subjected to Western blot analysis. c Cells were pretreated with 5.0 lM PTS2 for 2 h or not, and then stimulated with different concentration of IFN-c (1-10 ng/ml). d Cells were pretreated with PTS2 (5.0 lM), AG490 (30.0 lM) or both of them, respectively, for 2 h, and then incubated with IFN-c (10 ng/ml) for 24 h. The cell culture supernates were subjected to ELISA analysis to measure IP-10/CXCL10 protein level. Data are presented as mean ± SD. **P \ 0.01 compared with the IFN-cstimulated control cells STAT1 phosphorylation (Fig. 4c), whereas the amount of total JAK1, JAK2 and STAT1 proteins in RAW264.7 cells were not affected by both IFN-c and PTS2 treatment (Fig. 4a-c). Previous reports showed that the selective pharmacological JAK2 inhibitor AG490 could significantly inhibit upregulated chemokine expression induced by IFN-c in mouse mesangial cells [29]. Therefore, we compared the effects of PTS2 and AG490 on IFN-c-induced IP-10/CXCL10 expression. RAW264.7 cells were pretreated with PTS2 (5.0 lM) or AG490 (30.0 lM) or both of them, respectively, for 2 h, and then incubated with IFN-c (10 ng/ml) for 24 h. The cell culture supernates were subjected to ELISA analysis to measure IP-10/CXCL10 protein levels. As shown in Fig. 4d, PTS2, at concentration of 5.0 lM, proved more effective in suppressing IFN-c-induced IP-10/CXCL10 expression than 30.0 lM of AG490. Furthermore, IFN-c-induced IP-10/CXCL10 expression in the cells co-incubated with PTS2 and AG490 together was lower than in those treated with either PTS2 or AG490. Taken together, these results suggested that PTS2 inhibited IFN-c-induced IP-10/CXCL10 expression in RAW 264.7 cells through suppressing activation of JAK/STAT1 signaling pathway.
PTS2 dose not influence IFN-c-induced p38 activation P38 has been reported to play a critical role in regulation of the expression of a number of cytokines and chemokines including IP-10/CXCL10 under IFN-c stimulation in macrophages [31]. We thus evaluated the effect of PTS2 on IFN-c-induced p38 activation in RAW264.7 cells. The cells were pretreated with 5.0 lM PTS2 for 2 h or not, and then were incubated with IFN-c (10 ng/ml) for 0-1.5 h respectively. Figure 5 shows that IFN-c-induced phosphorylation of p38 reached maximum at 0.5 h after IFN-c treatment and PTS2 pretreatment did not affect this p38 activation.

Discussion
A number of steroidal agents, such as glucocorticoids, have been used as anti-inflammatory drugs for a long time, but frequent association of serious side effects, such as liver damage, cancers, stroke and growth stoppage, have been a long-standing dilemma in clinical steroid anti-inflammatory therapy. Non-steroidal anti-inflammatory drugs (NSAIDs) represent one of the most widely used drug classes, including aspirin, indomethacin, phenylbutazone, etc. Moreover, new anti-inflammatory drugs are being discovered and developed based on their effects on signal transduction and as anti-cytokine agents [37]. Some pyridine derivates, such as pyroxin and sulfasalazine, have been known for their anti-inflammatory effects. As a member of the pyridinethione compound family, PTS2 has been used as bactericide, pesticide and fungicide for a long time [38]. However, relatively fewer studies have focused on the anti-inflammatory activity and the mechanisms of PTS2.
IFN-c is a key cytokine involved in the synergistic generation of many inflammatory responses. Activated T lymphocytes and NK cells produce IFN-c which is the major macrophage activation factor. Activation of JAK1, JAK2 and STAT1, plays a central role in the primary transcriptional response to IFN-c [2,28]. It has been well documented that excess IFN-c release could result in IP-10/ CXCL10 overexpression, which regulates almost all stages in the development of inflammation, in particular, the early stage of T cells and NK cells transmigration to the sites of inflammation [2]. Some medicines, such as glucocorticoids, which reduce the incidence of the inflammatory disease process, show inhibitory effects on IP-10/CXCL10 expression, and some chemicals with efficiency of inhibiting IP-10/CXCL10 overexpression are also considered to have the immense potential for treating a broad range of and inflammatory diseases including SARS, chronic hepatitis C, SLE, etc. [20,39,40].
In this study we focused on the influence of PTS2 on the expression of IP-10/CXCL10, an IFN-c inducible chemokine whose transcriptional regulation is mainly dependent on JAK/STAT1 signaling pathway activation. Sakaeda et al. [41] showed that IFN-c-stimulated IP-10/CXCL10 expression in RAW264.7 cells. In the current study, we demonstrated that IFN-c-induced up-regulation of IP-10/ CXCL10 in RAW264.7 cells was dose and Fig. 5 Effect of PTS2 on IFN-c-induced p38 activation. RAW264.7 cells were pretreated with 5.0 lM PTS2 for 2 h or not, followed by incubating with 10 ng/ml IFN-c for 0-1.5 h. Western blot was performed by using antibodies to p38 or p-p38. GAPDH was used as control. The results were representative of three independent experiments time-dependently inhibited by PTS2. Although IFN-c induces a rapid tyrosine phosphorylation of JAK1 and JAK2 and activation of STAT1 in RAW264.7 cells, when treating cells with IFN-c in the presence of PTS2, JAK/ STAT1 activation was depressed. Moreover, we found that the inhibitory effect of PTS2 on IP-10/CXCL10 up-regulation was slightly stronger than JAK2 inhibitor AG490 while RAW264.7 cells co-incubated with PTS2 and AG490 showed marked reduction of IP-10/CXCL10, suggesting the synergistic effect of PTS2 and AG490. These results indicate that PTS2 inhibits IFN-c-induced IP-10/ CXCL10 expression through preventing activation of the JAK/STAT1 signaling pathway. A similar result revealed in our recent report indicates that PTS2 inhibits the LPSinduced iNOS expression by depressing phosphorylation of STAT1 in RAW264.7 cells [34]. It has been demonstrated that activation of p38 appears to play a critical role in regulation of the expression of a number of chemokines and cytokines induced by IFN-c in macrophages [31], but in the present study, PTS2 did not alter IFN-c-induced p38 phosphorylation. Majumder et al. [42] showed that the induction of IP-10/CXCL10 was dependent on the ISRE (interferon-stimulated response element) and one of the two NF-jB recognition sites. We also tried to study the effect of PTS2 on NF-jB signaling, however, we did not find NF-jB activation by IFN-c stimulation through detecting ubiquitination of IjBa and IjBb in RAW264.7 cells (data not shown). The result is consistent with the results that NF-jB can regulate IP-10/CXCL10 expression but IFN-c did not detectably activate NF-jB in both Myd88 -/and wild-type macrophages [43]. In addition, our work also indicates that PTS2 has no significant effect on cell viability (data not shown).
In conclusion, our study demonstrates that PTS2 suppresses IFN-c-induced IP-10/CXCL10 expression via attenuating the activation of JAK/STAT1 signaling pathways. This finding provides a new insight for understanding the anti-inflammatory activities and clinical therapeutic potential of PTS2 and other pyridine derivates.