International Immunopharmacology 21 (2014) 128–136
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FLLL31, a derivative of curcumin, attenuates airway inflammation in a multi-allergen challenged mouse model Shaopeng Yuan, Shuhua Cao, Rentao Jiang, Renping Liu, Jinye Bai, Qi Hou ⁎ Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, People's Republic of China
a r t i c l e
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Article history: Received 15 January 2014 Received in revised form 22 April 2014 Accepted 23 April 2014 Available online 9 May 2014 Keywords: Signal transducer and activator of transcription protein 3 FLLL31 Asthma Airway inflammation Interleukin 17
a b s t r a c t Signal transducer and activator of transcription protein 3 (STAT3), one of the major regulators of inflammation, plays multiple roles in cellular transcription, differentiation, proliferation, and survival in human diseases. Dysregulation of STAT3 is related to the severe airway inflammation associated with asthma. FLLL31 is a newly developed compound based on the herbal medicine curcumin, which specifically suppresses the activation of STAT3. However, the function of FLLL31 on inflammatory diseases, especially on the regulation of airway inflammation, has not been fully studied. In our prior investigations, we developed a mouse model that was challenged with a mixture of DRA allergens (including house dust mite, ragweed, and Aspergillums species) to mimic the severe airway inflammation observed in human patients. In this study, we performed a series of experiments on the inflammatory regulation activities of FLLL31 in both in vitro cultured cells and our in vivo DRA-challenged mouse model. Our results show that FLLL31 exhibits anti-inflammatory effects on macrophage activation, lymphocyte differentiation, and pro-inflammatory factor production. Importantly, FLLL31 significantly inhibited airway inflammation and recruitment of inflammatory cells in the DRA-challenged mouse model. Based on these results, we conclude that FLLL31 is a potential therapeutic agent that can be used against severe airway inflammation diseases. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In recent years, ovalbumin (OVA) challenge models have been used to simulate asthmatic airway inflammation in mice [1,2]. The characteristics of this frequently used model include elevated cytokines in T helper type 2 (Th2) cells, eosinophilic inflammation, and airway hyperresponsiveness (AHR). As such, these mediators are considered to be the major players of airway inflammation in asthma [3,4]. However, recent data has revealed that only nearly 50% of human asthmatic cases are associated with Th1/Th2 imbalance and eosinophilic inflammation. These occurrences are even less common in individuals who have developed severe asthma [5,6]. Moreover, several drugs targeting Th2related inflammatory mediators failed in clinical trials in severe asthmatic patients [7]. This suggests that new candidate therapeutic agents
Abbreviations: STAT, signal transducer and activator of transcription; JAK, Janus kinase; LPS, lipopolysaccharide; Con A, concanavalin A; HDM, house dust mite; PBS, phosphate-buffered saline; NO, nitric oxide; Dex, dexamethasone; TNF-α, tumor necrosis factor α; IL, interleukin; IFN-γ, interferon γ; ELISA, enzyme-linked immunosorbent assay; IC50, the inhibitory concentration of the compound by 50%. ⁎ Corresponding author at: Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, 1 Xian Nong Tan Street, Beijing 100050, People's Republic of China. Tel.: +86 10 63165191. E-mail address:
[email protected] (Q. Hou).
http://dx.doi.org/10.1016/j.intimp.2014.04.020 1567-5769/© 2014 Elsevier B.V. All rights reserved.
should be tested in an animal model that more closely mimics the airway inflammation observed in patients. STAT3 is constitutively activated in inflamed human cells, including inflammatory and structural cells, and plays a pivotal role in cellular transcription, differentiation, proliferation, and survival. Dysregulation of STAT3 is crucial in the initiation and maintenance of several inflammatory diseases. Differentiation of T cells, eosinophils, and neutrophils is all associated with STAT3 regulation [8]. Moreover, the function of STAT3 is altered during the chronic airway inflammation associated with asthma [9,10]. Recently, Simon and colleagues reported that airway epithelial STAT3 mediation is required for allergic inflammation in a HDM-challenged mouse model of asthma [11]. In addition, STAT3 promotes the expression of CCL11 and IL-6 in smooth muscle cells, major structural cells involved in airway remodeling during asthma [12]. Thus, these findings suggest that STAT3 is not only involved in the differentiation of inflammatory cells, but that it also directly regulates the function of structural cells during the initiation of asthma. Importantly, STAT3 has been recently identified as the key regulator of Th17 cell differentiation during chronic inflammation [13], as attenuation of STAT3 activation inhibits the differentiation of Th17 cells [14]. Since Th17 cells and IL-17 are important mediators of severe asthma [15], inhibiting STAT3 activation might attenuate development of airway inflammation, and possibly serve as a new target for the treatment of severe asthma.
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FLLL31 is a newly developed compound based on curcumin, a herbal medicine, which selectively binds to Janus kinase 2 (JAK2) and the STAT3 Src homology-2 (SH2) domain, suppressing the activation of STAT3 in both in vitro and in vivo studies (Fig. 1A) [16,17]. However, the effects of FLLL31 on inflammatory pathways, especially those involved in the regulation of airway inflammation, have not been previously reported. Therefore, it is important to understand whether FLLL31 can attenuate airway inflammation, particularly in a mouse model more relevant to human severe asthma. In the present study, we investigated the anti-inflammatory and immunosuppressive functions of FLLL31 on macrophages and lymphocyte differentiation in vitro. Specifically, we used the DRA-challenged mouse model [18], which closely mimics the severe and steroid-resistant airway inflammation of asthma, to detect the in vivo effects of FLLL31 on airway inflammation. 2. Materials and methods 2.1. Materials FLLL31 was purchased from Sigma-Aldrich (St. Louis, Missouri, USA), and diluted in DMSO (final concentration of DMSO is lower than 0.1% in the medium) for the in vitro experiments [16]. All other materials were bought from Sigma-Aldrich, unless otherwise stated. Allergens including extracts of house dust mite (Dermatophagoides farinae), ragweed (Ambrosia artemisiifolia) and Aspergillums species are from Greer Laboratories (Lenoir, USA).
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2.2. The effects of FLLL31 on airway inflammation in a murine asthmatic model The experiment was performed according to the Institutional Guidelines for Animal Care and Use of the Chinese Academy of Medical Sciences and Peking Union Medical College. The female BALB/c mice (18–20 g, n = 8 per group) were subcutaneously injected with pooled allergens (DRA; 5 μg dust mite, 50 μg ragweed, and 5 μg A. species) in combination with aluminum hydroxide in 100 μl normal saline on day 1 and day 8. DRA allergens were then administered intra-nasally twice a week for another 8 weeks from week 3 to week 10. The control group of mice was given an equal volume of normal saline at the same time points. To determine the effect of FLLL31 on airway inflammation, either FLLL31 (15 mg/kg, p.o.), or dexamethasone (Dex, as positive control, 1 mg/kg, i.p.) was given daily in weeks 9 and 10. The treatments were administered 1 h prior to antigen exposure when challenge and treatment occurred on the same day. On the first day of week 11 (the end of experiment), the animals were sacrificed using sodium pentobarbital for bronchial alveolar lavage (BAL), serum collection, and lung histology. 2.3. Croton-oil-induced ear edema The experiment was performed according to the Institutional Guidelines for Animal Care and Use of the Chinese Academy of Medical Sciences and Peking Union Medical College. In ICR male mice (18–20 g, n = 10 per group), 0.4 mg of croton oil in 1 ml of acetone was applied to
Fig. 1. FLLL31 inhibited LPS-induced NO production, iNOS and STAT3 activation in macrophages. A. Chemical structure of FLLL31 (Tetramethylcurcumin, (E, E)-1, 7-bis (3, 4dimethoxyphenyl)-4, 4-dimethyl-1, 6-heptadiene-3, 5-dione). Formula: C25H28O6. Molecular weight: 424.49. B. Proliferation assay of FLLL31 on macrophages. The cells were cultured in the absence (Control) or presence of FLLL31 (0.5, 1, 5, 10, and 20 μM) for 48 h. The cell proliferation was expressed as the percentage of OD450 value of each concentration of FLLL31 with control. C. FLLL31 inhibits cellular lipopolysaccharide (LPS)-induced nitrite concentrations, an indicator of NO production. Macrophages were cultured in the absence of any treatment (Control) or with LPS (1 μg/ml) and FLLL31 (0, 1, 5, or 10 μM) for 24 h. Dexamethasone (Dex, 1 μM) was used as a positive control. Data are mean ± SD. *** represents P b 0.001 vs. Control; #P b 0.05, ###P b 0.001 vs. LPS. D. The effect of LPS-induced activation of iNOS, STAT3 and STAT1 in the absence of any treatment (Control) or with LPS (1 μg/ml) and FLLL31 (0, 1, 5, or 10 μM) in macrophages. β-Actin was used as a loading control. The phosphorylation changes of STAT3 and STAT1 were detected using rabbit monoclonal antiphospho-Tyr705 STAT3 and anti-phospho-Tyr701 STAT1 antibodies respectively.
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the left ear topically to induce ear edema. To determine the dose effect of FLLL31, 5, 15, and 45 mg/kg of FLLL31, Dex (0.5 mg/kg) or the diluents (control group) were administered p.o. 1 h prior to croton oil treatment. Four hours after the application of croton oil, the animals were euthanized using sodium pentobarbital, and 8 mm (diameter) punches of ear tissues were collected for measuring the weight of the left and right ear patches. The anti-inflammatory effects of FLLL31 were calculated by the weight difference of two ear patches compared with those of the control group. 2.4. Bronchial alveolar lavage fluid (BALF) collection and analysis BALF was collected as previously described [19]. Briefly, after last DRA challenge in mice as indicated in Materials and methods section 2.2, lungs were isolated under complete anesthesia, and lavaged with 1 ml ice-cold 1× PBS buffer for 3 times. The lavaged fluid was centrifuged at 1000 × g for 15 min at 4 °C. The pellet was re-suspended in 1 ml 1× PBS to count the number of inflammatory cells, including neutrophils, lymphocytes, and macrophages, using the Nihon Kohden MEK7222K Blood Analysis System. 2.5. Histological examination Lung tissue was fixed by inflating with and soaking in 10% buffered formalin. The fixed lung tissues were subsequently washed, dehydrated, and embedded in paraffin. Each 5 μm tissue section was stained with hematoxylin and eosin (H&E) to quantify inflammation, and PAS (Periodic Acid Schiff, Sigma-Aldrich) to quantify mucus-producing goblet cells. Next, the stained lung sections were examined by bright-field microscopy with an Olympus DP1T digital camera system (Olympus Optical, Tokyo, Japan). Additional lung sections were prepared for immunostaining using a rat anti-Ly-6G/Gr-1 antibody (1:500, R&D Systems) to quantify neutrophilic cells in the airway, and a rabbit monoclonal anti-phospho-Tyr705 STAT3 to examine the phosphorylation changes of STAT3 in the lung sections. 2.6. Nitrite determination in macrophages Griess reagent was used to measure nitrite, a stable metabolite of NO in aqueous solutions [20]. Briefly, peritoneal macrophages were harvested from male C57BL/6 mice after intraperitoneal injection of brewer thioglycollate medium (5 ml/100 g body weight) for 3 days. The collected macrophages were seeded in 96-well plates at a density of 1 × 105 cells/well in RPMI-1640 medium supplemented with 5% newborn calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. After growing/incubating at 37 °C for 2 h, the cells were replaced with fresh medium and challenged with 1 μg/ml LPS for 24 h. In addition, the cells were treated with varying concentrations of FLLL31 or its diluents as control. All incubations were performed with 5% CO2 in a humidified atmosphere at 37 ºC. After the designated times, 100 μl culture medium was mixed with an equal volume of Griess reagent for NO measurement. IC50 was calculated as the inhibitory concentration of the test compounds that reduces NO production by 50%. Each experiment was performed three times. 2.7. Splenic lymphocyte proliferation assay The proliferation and differentiation of splenic lymphocyte in response to Con A or LPS stimulation were assessed using the Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) according to the manufacturer's instructions [21]. Briefly, BALB/c mice were sacrificed and their spleens were removed aseptically. A single spleen cell suspension was prepared and cell debris and clumps were removed. Next, erythrocytes were lysed with Tris-buffered ammonium chloride (0.155 M NH4Cl and 16.5 mM Tris, pH 7.2). Mononuclear cells were
washed and resuspended in RPMI-1640 medium. The splenic lymphocyte suspensions (5 × 105 cells/well) were stimulated with or without Con A (5 μg/ml), LPS (10 μg/ml) as challenges, in the presence of FLLL31 or its diluents as controls in 96-well plates for 48 h. The OD value at 450 nm, an indicator of cell proliferation, was measured using a microplate reader after 3 h incubation with CCK-8 (5 mg/ml). Cell viability was indicated by reduction of OD450 between the groups of test compounds and the unstimulated control. The immunosuppressive rates were calculated by the reduction of OD450 between the groups of test compounds and the Con A- or LPS-challenged groups, respectively.
2.8. T cell receptor (TCR) crosslinking-induced primary T cell responses The proliferation and differentiation of primary T cells from the lymph node of mice were assessed with CCK-8 according to the methods mentioned above. Briefly, primary T cell suspensions (5 × 105 cells/well) were prepared from the lymph node of C57BL/6 mice. Then, erythrocytes were lysed with Tris-buffered ammonium chloride (0.155 M NH4Cl and 16.5 mM Tris, pH 7.2). Mononuclear cells were washed and resuspended in RPMI-1640 medium. Next, the cells were co-cultured with anti-CD28 (2 μg/ml) and plate-coated anti-CD3 (5 μg/ml) antibodies in 96-well plates in the presence of FLLL31 or its diluents as control for 48 h. The OD value at 450 nm was then assessed using a microplate reader after pulsation with CCK-8 for 3 h before measurement. The inhibition rates were calculated by the reduction of OD450 between the groups of test compounds and the anti-CD3/CD28 antibody challenged groups. Treated cell culture supernatants were harvested to determine IL-2 and IFN-γ levels by ELISA.
2.9. Western blotting analysis to detect the phosphorylation changes of STAT3 Adherent peritoneal macrophages from C57BL/6 male mice were seeded in 6-well plates and grown to confluence before being treated as indicated in Materials and methods section 2.6. Cells were rinsed twice with ice-cold PBS and lysed on ice for 30 min in a lysis buffer containing 20 mM Tris (pH 7.5), 2 mM EDTA, 135 mM NaCl, 2 mM DTT, 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 10 mM NaF, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM PMSF. Lysates were centrifuged (12,000 ×g) at 4 °C for 15 min. Equal amounts of soluble proteins were denatured in SDS, electrophoresed on 12% SDS-PAGE, and transferred onto PVDF membranes. Levels of iNOS were detected using a rabbit polyclonal iNOS (Santa Cruz Biotechnology), and β-actin was used as the loading control. The phosphorylation changes of STAT3 and STAT1 were detected using rabbit monoclonal anti-phospho-Tyr705 STAT3 and antiphospho-Tyr701 STAT1 (Cell Signaling Technology), respectively.
2.10. ELISA measurements The amounts of cytokines, including TNF-α, IL-1β, IFN-γ, IL-2, IL-6 and IL-17, in the supernatants of BALF, macrophages, or lymphocyte culture media were measured using ELISA kits (eBioscience, San Diego, USA).
2.11. Statistical analysis All data are the result of at least three independent experiments and are expressed as mean ± SD. The Student's t-test was used to compare the difference of two groups. A P value b0.05 was considered to be statistically significant.
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3. Results 3.1. FLLL31 inhibited LPS-induced NO production and STAT3 activation in macrophages NO promotes the chemotaxis of inflammatory cells in the asthmatic lung microenvironment. Since curcumin represses inducible NO production in macrophages and attenuates asthma initiation [22], we first investigated the activities of FLLL31 on macrophage proliferation and NO production to assess its anti-inflammatory effects in vitro. Peritoneal macrophages were harvested and treated as indicated in Materials and methods section 2.6. Briefly, the collected macrophages were seeded in 96-well plates and incubated at 37 °C for 2 h, and then were replaced with new fresh medium and challenged with 1 μg/ml LPS for 24 h. In addition, the cells were treated with varying concentrations of FLLL31 or its diluents as control. Compared to the non-treated control group, proliferation of peritoneal macrophages did not change across the FLLL31 dose range of 0.5 to 20 μM for 24 h (Fig. 1B). NO level, compared to the control (3.4 ± 0.7 pg/ml), increased when cells were challenged with LPS alone (81.7 ± 2.2 pg/ml, P b 0.001), while addition of FLLL31 decreased the LPS-stimulated NO production in a concentrationdependent manner (75.7 ± 0.8, 59.8 ± 0.4, 39.1 ± 0.9 pg/ml for 1, 5, and 10 μM FLLL31, respectively; P b 0.05 for all compared to LPS treatment). Furthermore, the NO level in peritoneal macrophages treated with 10 μM FLLL31 was similar to treatment with 1 μM Dex, and the IC50 of FLLL31 on NO production was 5.1 μM (Fig. 1C). Therefore, our results demonstrate that FLLL31 significantly inhibited LPS-stimulated macrophage NO release, while having little to no effect on macrophage proliferation. Inducible nitric-oxide synthase (iNOS) is expressed in several pathophysiological states in inflammatory cells and produces large amounts of NO in response to inflammatory signals, such as LPS and inflammatory cytokines [23]. In western blotting analysis, LPS challenge alone
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increased peritoneal macrophage iNOS expression, while FLLL31 reduced LPS-induced expression of iNOS in a concentration-dependent manner (Fig. 1D). Since STAT3 is an essential inflammatory signaling factor in the immune response to LPS, and phosphorylation at Y705 of STAT3 leads to its activation and promotion of inflammatory factor transcription, we sought to examine the effects of FLLL31 on LPS-induced STAT3 phosphorylation. FLLL31 treatment reduced the LPS-induced phosphorylation of STAT3 (Y705) in a concentration-dependent manner, similar to the prior report on the anti-inflammatory properties of baicalein [24]. However, the effect of FLLL31 on STAT1 phosphorylation (Y701), another major mediator of the STAT protein family, was not observed. Therefore, these results indicated that FLLL31 specifically targets STAT3 to inhibit LPS-induced NO production in macrophages. 3.2. FLLL31 inhibited LPS-induced macrophage cytokine release Pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, play important roles in the inflammatory process. In peritoneal macrophages, LPS significantly increased production of TNF-α, IL-1β, and IL-6 (P b 0.001, compared to control group), while treatment with FLLL31 reduced the production of these cytokines in a concentrationdependent manner (P b 0.001, compared to LPS challenge alone) (Fig. 2A–C). Thus, our results suggest that FLLL31 attenuates inflammatory cytokine production in macrophages. 3.3. FLLL31 inhibited splenic lymphocyte proliferation Next, we studied the effects of FLLL31 on Con A- and LPS-induced splenic lymphocyte proliferation. Similar to its effect on macrophages, FLLL31 treatment had little effect on the proliferation rate, compared with that of the unstimulated control group. However, proliferation rates were significantly inhibited upon challenge with Con A (5 μg/ml) and LPS (10 μg/ml) (Fig. 3A). The IC50 of FLLL31 on Con A- and LPS-
Fig. 2. FLLL31 inhibited LPS-induced productions of pro-inflammatory cytokines in macrophages. A–C. Macrophages were cultured in the absence of any treatment (Control) or with LPS (1 μg/ml) and FLLL31 (0, 1, 5, or 10 μM) for 24 h. The supernatants of the culture medium were collected for TNF-α (A), IL-1β (B), and IL-6 (C) measurement using ELISA kits. *** represents P b 0.001 vs. Control; ###P b 0.001 vs. LPS.
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Fig. 3. FLLL31 inhibited splenic lymphocyte proliferation. A. FLLL31 inhibits the proliferation of splenic lymphocytes. The splenic lymphocytes were cultured in the absence or presence of FLLL31 (1, 5, 10 μM), and stimulated with Con A (5 μg/ml) or LPS (10 μg/ml) for 48 h, respectively. Dex was used as a positive control. Cell viability was indicated by reduction of OD450 between the groups of test compounds and the un-stimulated control. # represents P b 0.05, ##P b 0.01, and ###P b 0.001 vs. Con A or LPS challenge, respectively. B. FLLL31 inhibited CD3and CD28-antibody-induced T cell responses. Primary T lymphocytes from the lymph node of C57BL/6 mice were cultured in the absence of any treatment or presence of plate-coated antiCD3 (5 μg/ml) and anti-CD28 (2 μg/ml) antibodies with FLLL31 (0, 1, 5, 10 μM) for 48 h. Dex was used as a positive control. #P b 0.05, ##P b 0.01 vs. CD3- and CD28-antibody challenges. C. FLLL31 inhibited CD3- and CD28-antibody-induced IL-2 productions in the absence of any treatment or presence of CD3- and CD28-antibodies and FLLL31 (0, 1, 5, 10 μM). *** represents P b 0.001 vs. Control; #P b 0.05, ##P b 0.01 vs. CD3- and CD28-antibody challenges. D. FLLL31 inhibits CD3- and CD28-antibody-induced IFN-γ productions in the absence or presence of CD3- and CD28-antibodies and FLLL31 (0, 1, 5, 10 μM). ** represents P b 0.05 vs. Control; #P b 0.05, and ###P b 0.001 vs. CD3- and CD28-antibody challenges.
induced splenic lymphocyte proliferation was 1.9 μM and 5.3 μM, respectively (P b 0.05 compared to challenge for all concentrations of FLLL31). Moreover, inhibition activities of 10 μM FLLL31 were similar to the activity of 1 μM Dex upon Con A- and LPS-challenges, confirming the significant immunosuppressive efficiency of FLLL31. Immune responses are greatly influenced by the balance between Th1- and Th2-type cytokines. We therefore examined the activities of FLLL31 on TCR crosslinking-induced primary T cell responses. Primary T lymphocytes from the lymph nodes of C57BL/6 mice were stimulated with anti-CD3 and anti-CD28 antibodies to mimic TCR crosslinkinginduced primary T cell responses in vitro [25]. FLLL31 inhibited the activation of primary CD4+ T lymphocytes induced by the anti-CD3/
CD28 antibodies (Fig. 3B). Anti-CD3/CD28 stimulation increased IL-2 and IFN-γ levels (390.3 ± 6.9 pg/ml, P b 0.001, and 689.9 ± 8.6 pg/ml, P b 0.01), compared to the unstimulated control group (2.8 ± 0.9 pg/ml and 75.2 ± 0.4 pg/ml, IL-2 and IFN-γ respectively). The production of IL-2 (Fig. 3C) and IFN-γ (Fig. 3D) was attenuated upon FLLL31 treatment in a dose-dependent manner (P b 0.05 compared to challenge). Since IL-2 and IFN-γ generally promote Th1 cell-mediated immune responses, FLLL31 therefore inhibits T lymphocyte activation and differentiation. Additionally, we also found that FLLL31 can significantly inhibit the differentiation of primary murine T lymphocytes to Th17 cells in vitro, when stimulated with anti-CD3/CD28 antibodies and treated with IL-6, TGF-β, and IL-23 (Supplemental data, S1).
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swelling at concentrations of 15 mg/kg (21.6 ± 4.7 mg, P b 0.05) and 45 mg/kg (18.5 ± 4.1 mg, P b 0.001) compared to control (27.0 ± 2.1 mg), similar to the effect of Dex (10.4 ± 4.2 mg, P b 0.001) (Fig. 4). Administration of 15 mg/kg FLLL31 for 6 days significantly suppressed 2,4-dinitrofluorobenzene (DNFB)-induced delayed type hypersensitivity (DTH) responses in BALB/c mice, providing additional evidence for the immunosuppressive function of FLLL31 in vivo (data not shown) [26]. Since DNFB-induced DTH reaction is a Th1 cellmediated pathologic response and Th1 lymphocytes play an important role in severe asthma, the in vivo results above further suggest the potential function of FLLL31 on attenuation of severe asthma. Fig. 4. FLLL31 inhibited acute inflammation in vivo. ICR male mice were initially sensitized to croton oil on the left ear before the experiment was initiated. FLLL31 at different doses (5 mg/kg, 15 mg/kg, and 45 mg/kg, p.o.) and Dex (0.5 mg/kg) were applied 1 h before croton oil treatment. Ear swelling was calculated as the difference between the weights of left (croton oil treated) and right (untreated) ear punches 4 h after challenge. n = 10 mice/group. Data are expressed as mean ± S.D. # represents P b 0.05; ##P b 0.01 vs. croton oil treatment group.
3.4. FLLL31 inhibited acute inflammation in vivo To further identify the function of FLLL31 on the inhibition of inflammation, we studied the effects of FLLL31 on the croton oil-induced mice ear edema model in vivo. FLLL31 (5, 15, 45 mg/kg) and Dex (0.5 mg/kg) were applied 1 h prior to croton oil treatment, and the extent of ear swelling was measured. FLLL31 attenuated croton oil-induced ear
3.5. FLLL31 inhibited DRA-induced severe asthmatic chronic inflammation in vivo To investigate the effect of FLLL31 on airway inflammation, we treated FLLL31 (15 mg/kg daily, p.o.) on DRA-challenged mice for 2 weeks. In parallel, mice were treated with Dex (1 mg/kg, i.p.) as a positive control. Histological analysis on lung and tracheal sections using H&E staining revealed that the combinations of DRA allergen significantly increased the number of recruited inflammatory cells compared with the untreated control group. In addition, recruitment and migration of inflammatory cells into the alveolar septum lead to its thickening in the DRA allergen challenge. However, FLLL31 reduced inflammatory cell recruitment and alveolar septum thickening in DRA-challenged lung sections (Fig. 5A). Analysis of the BALF further showed that inflammatory cells,
Fig. 5. Effect of FLLL31 on airway inflammation in the DRA-challenged mice model. A. Histological analysis of airway inflammation using H&E staining (magnification 100×). Mice were sensitized and challenged with DRA as described in Materials and methods. Lungs were removed after 24 h of last DRA challenge. Lung sections (5 μm) were stained with hematoxylin and eosin. Control: without sensitization and challenge; DRA: DRA sensitized/challenged section; DRA + Dex: DRA sensitized/challenged section with Dex (1 mg/kg, i.p.); DRA + FLLL31: DRA sensitized/challenged section with FLLL31 (15 mg/kg daily, p.o.). B. Quantification of inflammatory cells, including neutrophils, lymphocytes, and macrophages, in the bronchial alveolar lavage fluid (BALF) from the DRA challenged mice lungs. Values represent the means ± SD from n = 8 mice for each group. * represents P b 0.05 vs. Control; #P b 0.05, ##P b 0.01, and ###P b 0.001 vs. DRA challenge group, respectively. C. Histological analysis of airway mucus glycoprotein (magnification 100×). Lung sections were stained with Periodic Acid Schiff (PAS) as mentioned in Materials and methods.
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including neutrophils, lymphocytes, and macrophages, were reduced after pre-treatment with FLLL31, compared with the DRA-challenged group (Fig. 5B). Interestingly, FLLL31 was even more effective than Dex on inhibition of inflammatory cell recruitment. Analysis of lung sections stained with PAS indicated that DRA challenge caused significant mucus hypersecretion of glycoprotein compared with the control group (Fig. 5C). However, the hypersecretion was mild in the group pre-treated with FLLL31. These results, along with the in vitro investigations, further support the anti-inflammatory function of FLLL31 on severe asthmatic airway inflammation in vivo. Furthermore, the immunohistochemical analysis results revealed that the DRA challenge caused phosphorylation of STAT3 (Y705) on the inflamed sections. More importantly, treatment with FLLL31 inhibited DRA-induced phosphorylation of STAT3 (Fig. 6A). As mentioned, the attenuation of STAT3 activation inhibits the differentiation of Th17 cells. Since Th17 cells and IL-17 are important mediators of severe asthma, and in vitro results revealed that FLLL31 inhibited the production of IL-17, we therefore investigated the IL-17 level in the BALF of DRA-challenged mice. DRA challenge increased IL-17 (65.1 ± 22.7 pg/ml) compared to control (14.1 ± 4.1 pg/ml, P b 0.001), while FLLL31 treatment attenuated the increased IL-17 levels (16.9 ± 8.9 pg/ml, P b 0.001) (Fig. 6B). Furthermore, IL-17 inhibition activities of FLLL31 were more effective than Dex (25.4 ± 9.8 pg/ml), consistent with the results of our histological findings (Fig. 5A, B). Our previous investigations in DRA-challenged mouse models revealed that lower effectiveness of therapeutic Dex treatment was associated with severe neutrophilic airway inflammation. Therefore, we investigated the function of FLLL31 on neutrophil recruitment in the lung sections. Immunohistochemical analysis showed that DRA challenge promotes the
expression and accumulation of Ly-6G/Gr-1, a neutrophilic biomarker (Fig. 6C), while FLLL31 significantly reduces expression levels. Therefore, the anti-inflammatory effect of FLLL31 on a DRA mice model is related to its inhibitive effect on neutrophil recruitment. Moreover, these results suggest that STAT3 plays an important role in the observed chronic asthmatic inflammation in vivo and reveal that treatment with FLLL31 can significantly attenuate inflammatory cell recruitment and the onset of asthma. Taken together, our results demonstrate that the administration of FLLL31 significantly reduces airway inflammation, identifying FLLL31 as a potential therapeutic agent for use in asthma patients. 4. Discussion STAT3 regulates multiple pro-inflammatory genes that contribute to allergic asthma [9,27]. Additionally, several lines of evidence suggest a pivotal role for STAT3 in the pathogenesis of asthma [28,29]. Therapeutic agents targeting the STAT3 signaling pathway, therefore, have the potential to attenuate allergic airway inflammation [30,31]. However, specific STAT3 inhibitors as asthmatic therapeutics have poorly been investigated. FLLL31 is a new compound that was developed based on an herbal medicine, curcumin, which specifically suppresses the activation of STAT3. In this study, we investigated the ability of FLLL31 to modulate inflammation in vitro and in vivo, and measured its effects on severe chronic airway inflammation using a mice model. The in vitro results showed that FLLL31 had low toxicity, specifically inhibited STAT3 phosphorylation, and attenuated LPS-stimulated NO and inflammatory factor production in macrophages. In addition, FLLL31 inhibited the proliferation and lymphoblastic transformation of lymphocytes. Moreover,
Fig. 6. FLLL31 attenuated STAT3 phosphorylation and neutrophil recruitment in vivo. A. Immunohistochemical analysis of phosphorylation of STAT3 in the lung section. Lung sections were prepared for immunostaining using a rabbit monoclonal anti-phospho-Tyr705 STAT3, and examined the phosphorylation changes of STAT3 as mentioned above. Scale bar: 100 μm. B. Quantitation of the IL-17 concentrations in the BALF of mice (n = 8 mice for each group). *** represents P b 0.001 vs. Control; ###P b 0.001 vs. DRA challenge group. C. Immunohistochemical staining of lung-sections with Ly-6G/Gr-1 antibodies. Scale bar: 100 μm.
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the immunosuppressive function of FLLL31 was further characterized by in vivo experiments. FLLL31 significantly attenuated recruitment of inflammatory cells and mucus hypersecretion in DRA-challenged murine lung sections [32]. Importantly, FLLL31 reduced the expression of IL-17 and Ly-6G/Gr-1+ cells [6,33] in DRA-challenged murine lungs. These data are the first to report a therapeutic effect of FLLL31 in airway inflammation. These findings suggest that additional studies on FLLL31 should be carried out to further characterize its role as a potential immunoregulatory therapeutic agent for severe asthma. The pattern and progression of chronic airway inflammation in our mouse model closely resembles those seen in patients with asthma [18,34]. In our model, animals were exposed to allergens consisting of pooled extracts of dust mite, ragweed, and A. species. Our previous studies revealed that chronic multi-allergen challenge better simulates the severe asthma seen in patients, as it results in high levels of neutrophilic inflammation and airway remolding, elevated concentrations of inflammatory factors, and dexamethasone resistance. Moreover, chronic multi-allergen challenge avoids the development of tolerance often seen in mice with repeated single antigen challenge, as well as the transient nature of airway inflammation seen in the OVA-challenged mouse model. The mouse DRA model could therefore serve as a useful tool for evaluating novel therapies for the treatment of chronic airway inflammation [34]. NO is a short-lived, easily diffusible molecule with potent biological activity. Endogenous NO is considered to be a major marker of physiological deterioration of airway function, and is closely implicated in airway diseases such as asthma, adult respiratory distress syndrome, and lung fibrosis [3,4]. There is significant evidence to suggest that NO donors increase airway inflammation by enhancing the activation of inflammatory cells [35]. NO is also a potent vasodilator in the bronchial circulation, and plays a major role in airway circulation [19]. Our results demonstrate that FLLL31 decreases the expression of iNOS and NO release in macrophages. In addition, the results in the DRA-challenged mouse model revealed that FLLL31 attenuates airway inflammation by inhibiting infiltration of inflammatory cells into the lungs, which may in part be due to inhibited NO production. Moreover, our data revealed that high doses of FLLL31 prevented the activation and lymphoblastic transformation of lymphocyte subsets, indicating its ability to differentially regulate immunosuppressive activities during lymphocyte differentiation. FLLL31 therefore has the ability to modulate the immune function of lymphocytes. Importantly, the inactivation of STAT3 in diverse immune cells may cause a potent inhibition of chronic inflammation by decreasing the number of immature dendritic cells and helper T cells [25,36]. These results suggest that FLLL31 might serve as a potential therapeutic agent for use in the treatment of autoimmune and inflammatory diseases. Cytokines play a crucial role in inflammatory responses [37]. TNF-α, IL-1β, and IL-6 are the major inflammatory cytokines that regulate the activation of inflammatory cells [38]. Previous studies have demonstrated that incubation of tracheal tissues with inflammatory factors increases the contractile response to methacholine and other bronchoconstrictors. In this report, we demonstrate that FLLL31 can inhibit LPS-induced expression of TNF-α, IL-1β, and IL-6 in macrophages. This suggests that down-regulation of inflammatory cytokines by FLLL31 results in the attenuation of airway inflammation in vivo. Similarly, IL-2 and IFN-γ are produced and released upon the activation of Th1 cells, and play important roles in the regulation of the inflammatory response. Since severe airway inflammation is mainly neutrophilic and involves IFN-γ-dependent Th1 inflammation, the attenuation of airway inflammation in our studies may also be due to an immunosuppressive effect by FLLL31 in Th1 lymphocytes [29,39]. IL-1β, IL-6, and IL-17 are major inflammatory mediators in both Th17 lymphocyte differentiation and severe asthma [15], and inhibition of these inflammatory factors by FLLL31 reveals its potential effect on attenuating the initiation and development of Th17 lymphocytes. Furthermore, given that STAT3 plays a pivotal role in the Th17 cell differentiation and IL-17 production, the
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results of phosphorylation inhibition of STAT3 (Y705) in the FLLL31 pre-treatment group confirm that STAT3 plays an important role in the observed chronic asthmatic inflammation in vivo, and identify FLLL31 as a potential therapeutic agent for asthma patients. Overall, our data demonstrate that pre-treatment with FLLL31 can significantly attenuate inflammatory cell recruitment and the onset of asthma in vitro and in vivo. Administration of FLLL31 significantly reduced airway inflammation, the infiltration of inflammatory cells, and the expression of inflammatory cytokines in the chronic DRAchallenged mouse model. These findings suggest that FLLL31 may be useful as an adjuvant therapy for asthmatic patients in the future, and additional investigations are needed to further characterize the effect of FLLL31 in severe asthma and steroid-insensitive mouse models.
Acknowledgments We greatly thank Dr. Hou's lab members for their discussions and suggestions on this manuscript. This work is supported in part by a grant from the National Natural Science Foundation of China (no. 81001442) and the Central Public-Interest Scientific Institution Basal Research Fund (no. 2011CHX04).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2014.04.020.
References [1] Nials AT, Uddin S. Mouse models of allergic asthma: acute and chronic allergen challenge. Dis Models Mech 2008;1:213–20. [2] Kumar RK, Herbert C, Foster PS. The “classical” ovalbumin challenge model of asthma in mice. Curr Drug Targets 2008;9:485–94. [3] Holt PG, Macaubas C, Stumbles PA, Sly PD. The role of allergy in the development of asthma. Nature 1999;402:B12–7. [4] Wilson J. The bronchial microcirculation in asthma. Clin Exp Allergy 2000;30(Suppl. 1): 51–3. [5] Wenzel SE. Asthma phenotypes: the evolution from clinical to molecular approaches. Nat Med 2012;18:716–25. [6] Park SJ, Lee YC. Interleukin-17 regulation: an attractive therapeutic approach for asthma. Respir Res 2010;11:78. [7] Durrani SR, Viswanathan RK, Busse WW. What effect does asthma treatment have on airway remodeling? Current perspectives. J Allergy Clin Immunol 2011;128:439–48 [quiz 449–450]. [8] Gao H, Ward PA. STAT3 and suppressor of cytokine signaling 3: potential targets in lung inflammatory responses. Expert Opin Ther Targets 2007;11:869–80. [9] Pernis AB, Rothman PB. JAK–STAT signaling in asthma. J Clin Invest 2002;109: 1279–83. [10] Finotto S, Eigenbrod T, Karwot R, Boross I, Doganci A, Ito H, et al. Local blockade of IL-6R signaling induces lung CD4+ T cell apoptosis in a murine model of asthma via regulatory T cells. Int Immunol 2007;19:685–93. [11] Simeone-Penney MC, Severgnini M, Tu P, Homer RJ, Mariani TJ, Cohn L, et al. Airway epithelial STAT3 is required for allergic inflammation in a murine model of asthma. J Immunol 2007;178:6191–9. [12] Shan L, Redhu NS, Saleh A, Halayko AJ, Chakir J, Gounni AS. Thymic stromal lymphopoietin receptor-mediated IL-6 and CC/CXC chemokines expression in human airway smooth muscle cells: role of MAPKs (ERK1/2, p38, and JNK) and STAT3 pathways. J Immunol 2010;184:7134–43. [13] Yang XO, Panopoulos AD, Nurieva R, Chang SH, Wang D, Watowich SS, et al. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells. J Biol Chem 2007;282:9358–63. [14] Chang SH, Dong C. Signaling of interleukin-17 family cytokines in immunity and inflammation. Cell Signal 2011;23:1069–75. [15] Lajoie S, Lewkowich IP, Suzuki Y, Clark JR, Sproles AA, Dienger K, et al. Complementmediated regulation of the IL-17A axis is a central genetic determinant of the severity of experimental allergic asthma. Nat Immunol 2010;11:928–35. [16] Lin L, Hutzen B, Zuo M, Ball S, Deangelis S, Foust E, et al. Novel STAT3 phosphorylation inhibitors exhibit potent growth-suppressive activity in pancreatic and breast cancer cells. Cancer Res 2010;70:2445–54. [17] Kim HY, Park EJ, Joe EH, Jou I. Curcumin suppresses Janus kinase–STAT inflammatory signaling through activation of Src homology 2 domain-containing tyrosine phosphatase 2 in brain microglia. J Immunol 2003;171:6072–9. [18] Liu R, Bai J, Xu G, Xuan L, Zhang T, Meng A, et al. Multi-allergen challenge stimulates steroid-resistant airway inflammation via NF-kappaB-mediated IL-8 expression. Inflammation 2013;36(4):845–54.
136
S. Yuan et al. / International Immunopharmacology 21 (2014) 128–136
[19] Li YT, Yao CS, Bai JY, Lin M, Cheng GF. Anti-inflammatory effect of amurensin H on asthma-like reaction induced by allergen in sensitized mice. Acta Pharmacol Sin 2006;27:735–40. [20] Korhonen R, Lahti A, Hamalainen M, Kankaanranta H, Moilanen E. Dexamethasone inhibits inducible nitric-oxide synthase expression and nitric oxide production by destabilizing mRNA in lipopolysaccharide-treated macrophages. Mol Pharmacol 2002;62:698–704. [21] Kosuge H, Haraguchi G, Koga N, Maejima Y, Suzuki J, Isobe M. Pioglitazone prevents acute and chronic cardiac allograft rejection. Circulation 2006;113:2613–22. [22] Bright JJ. Curcumin and autoimmune disease. Adv Exp Med Biol 2007;595:425–51. [23] Guha M, Mackman N. LPS induction of gene expression in human monocytes. Cell Signal 2001;13:85–94. [24] Qi Z, Yin F, Lu L, Shen L, Qi S, Lan L, et al. Baicalein reduces lipopolysaccharideinduced inflammation via suppressing JAK/STATs activation and ROS production. Inflamm Res 2013;62:845–55. [25] Gogolak P, Rethy B, Horvath A, Toth GK, Cervenak L, Laszlo G, et al. Collaboration of TCR-, CD4- and CD28-mediated signalling in antigen-specific MHC class II-restricted T-cells. Immunol Lett 1996;54:135–44. [26] Zhou R, Zhang F, He PL, Zhou WL, Wu QL, Xu JY, et al. (5R)-5-hydroxytriptolide (LLDT-8), a novel triptolide analog mediates immunosuppressive effects in vitro and in vivo. Int Immunopharmacol 2005;5:1895–903. [27] Doganci A, Eigenbrod T, Krug N, De Sanctis GT, Hausding M, Erpenbeck VJ, et al. The IL-6R alpha chain controls lung CD4 + CD25+ Treg development and function during allergic airway inflammation in vivo. J Clin Invest 2005;115:313–25. [28] Hausding M, Tepe M, Ubel C, Lehr HA, Rohrig B, Hohn Y, et al. Induction of tolerogenic lung CD4+ T cells by local treatment with a pSTAT-3 and pSTAT-5 inhibitor ameliorated experimental allergic asthma. Int Immunol 2011;23:1–15. [29] Tundwal K, Alam R. JAK and Src tyrosine kinase signaling in asthma. Front Biosci 2012;17:2107–21.
[30] Foster PS, Webb DC, Yang M, Herbert C, Kumar RK. Dissociation of T helper type 2 cytokine-dependent airway lesions from signal transducer and activator of transcription 6 signalling in experimental chronic asthma. Clin Exp Allergy 2003;33:688–95. [31] Matsunaga Y, Inoue H, Fukuyama S, Yoshida H, Moriwaki A, Matsumoto T, et al. Effects of a Janus kinase inhibitor, pyridone 6, on airway responses in a murine model of asthma. Biochem Biophys Res Commun 2011;404:261–7. [32] Mantovani A, Cassatella MA, Costantini C, Jaillon S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev 2011;11:519–31. [33] Pelletier M, Maggi L, Micheletti A, Lazzeri E, Tamassia N, Costantini C, et al. Evidence for a cross-talk between human neutrophils and Th17 cells. Blood 2010;115:335–43. [34] Pavord ID. Non-eosinophilic asthma and the innate immune response. Thorax 2007;62:193–4. [35] Li H, Bradbury JA, Dackor RT, Edin ML, Graves JP, DeGraff LM, et al. Cyclooxygenase-2 regulates Th17 cell differentiation during allergic lung inflammation. Am J Respir Crit Care Med 2011;184:37–49. [36] Rajasingh J, Raikwar HP, Muthian G, Johnson C, Bright JJ. Curcumin induces growtharrest and apoptosis in association with the inhibition of constitutively active JAK– STAT pathway in T cell leukemia. Biochem Biophys Res Commun 2006;340:359–68. [37] Bill MA, Bakan C, Benson Jr DM, Fuchs J, Young G, Lesinski GB. Curcumin induces proapoptotic effects against human melanoma cells and modulates the cellular response to immunotherapeutic cytokines. Mol Cancer Ther 2009;8:2726–35. [38] Finkelman FD, Hogan SP, Hershey GK, Rothenberg ME, Wills-Karp M. Importance of cytokines in murine allergic airway disease and human asthma. J Immunol 2010;184:1663–74. [39] Simpson JL, Brooks C, Douwes J. Innate immunity in asthma. Paediatr Respir Rev 2008;9:263–70.