Chin Med J 2006; 119(8):640-648
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Original article Treatment of allergic airway inflammation and hyperresponsiveness by imiquimod modulating transcription factors T-bet and GATA-3 BIAN Tao, YIN Kai-sheng, JIN Shu-xian, ZHANG Xi-long，ZHOU Jin-yong, MA Xiu-qin, HU Jing-jing and DE Wei Keywords: asthma; T-bet; GATA-3; imiquimod Background Imiquimod is an imidazoquinoline, which class of compounds are known to have antiviral and antitumoural properties. In recent studies, it was shown that imiquimod modulates the T helper cell type Th1/Th2 response by inducing the production of Th1 cytokines like IFN-(, and by inhibiting the Th2 cytokines like interleukin (IL)-4. Several investigators have shown that T-bet and GATA-3 are master Th1 and Th2 regulatory transcription factors. This study investigated whether imiquimod treatment inhibited airway inflammation by modulating transcription factors T-bet and GATA-3. Methods Thirty-six male SD rats were randomly divided into a control group, an asthmatic group, and an imiquimod group, which was exposed to an aerosol of 0.15% imiquimod. Twenty-four hours after the last ovalbumin (OVA) challenge, airway responsiveness was measured and changes in airway histology were observed. The concentrations of IL-4, IL-5 and IFN-( in bronchoalveolar lavage fluid (BALF) and serum were measured by enzyme linked immunosorbent assay (ELISA). The mRNA expressions of IL-4, IL-5, IFN-(, T-bet and GATA-3 in lung and in CD4+ T cells were determined by reverse transcription polymerase chain reaction (RT-PCR). The protein expressions of T-bet and GATA-3 were measured by Western blot. Results It was demonstrated that imiquimod 1) attenuated OVA induced airway inflammation; 2) diminished the degree of airway hyperresponsiveness (AHR); 3) decreased the Th2 type cytokines and increased Th1 type cytokines mRNA and protein levels; 4) modulated the Th1/Th2 reaction by inhibiting GATA-3 production and increasing T-bet production. Conclusion Imiquimod treatment inhibits OVA induced airway inflammation by modulating key master switches GATA-3 and T-bet that result in committing T helper cells to a Th1 phenotype. Chin Med J 2006; 119(8):640-648
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miquimod is an immune response modifier, which belongs to the chemical class of imidazoquinolinamines. Imiquimod has been shown to modulate the Th1/Th2 response by inducing the production of Th1 cytokines such as interferon-( (IFN-(), and by inhibiting the Th2 cytokines IL-4 and IL-5.1 Several investigators have shown that T-bet and GATA-3 are master Th1 and Th2 regulatory transcription factor. However, allergic asthma is a chronic pulmonary disease associated with bronchoconstriction and chronic inflammation of the airways.2 In particular, it has been shown, in patients with asthma and in animal models, that allergic airway inflammation is associated with increased Th2 cytokine production and decreased Th1 cytokine production.3 In a recent study, Finotto et al4
suggest that T-bet might protect from asthma and Nakamura et al5 showed an increased expression of GATA-3 mRNA in asthmatic airways. Whether imiquimod inhibits airway inflammation by modulating T-bet and GATA-3 remained unclear. This study investigated the role of imiquimod on T-bet and GATA-3 in asthmatic rats. METHODS Reagents The following reagents were used: aluminium potassium Department of Respiratory Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China (Bian T, Yin KS, Jin SX, Zhang XL, Zhou JY, Ma XQ, Hu JJ and De W) Correspondence to: Dr. YIN Kai-sheng, Department of Respiratory Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China (Email:
[email protected])
Chinese Medical Journal 2006; 119(8):640-648
sulfate (Sigma-Aldrich, USA), crystalline OVA (Sigma Chemical Co., USA), imiquimod (a gift from Dr. LIU Guang-yu from Clinical Pharmacological Department of Nanjing Medical University, China), mouse monoclonal antibody anti-GATA-3 and anti-T-bet (Santa Cruz Biotechnology, Inc., USA), and mouse monoclonal antibody anti-β-actin (Sigma), horseradish peroxidase conjugated antirabbit Ig, anti-goat Ig and anti-mouse Ig (Santa Cruz Biotechnology, Inc., USA), mouse mAb OX7, OXB, OX33, OX42 and 10/78 (BD Pharmingen, USA), anti-CD4 FITC (IgG2a) (BD Pharmingen), goat antimouse IgG MicroBeads (Miltenyi Biotec, USA), TRIzol reagent and Maloney murine leukaemia virus (M-MLV), reverse transcriptase(Gibco BRL, USA), Taq DNA polymerase (TaKaRa, Japan), enzyme linked immunosorbent assay (ELISA) kits for rat recombined IFN-(, IL-4, and IL-5 ( R&D, USA). Allergen sensitization/challenge protocol Thirty-six male Sprague-Dawley rats (6 to 8 weeks) were obtained from Shanghai Laboratory Animal Inc., China and were randomly divided equally into a control group, an asthmatic group and an imiquimod group. Twenty-four rats received an intraperitoneal injection of 1 ml 10% OVA (50 mg OVA in 500 ml normal saline) complexed with 10% alum on days 0 and 7. Two weeks after the sensitization, the rats inhaled 1% OVA for 30 minutes in an exposure chamber (50 cm × 30 cm × 25 cm) to sensitize their airways. For inhalation challenge, OVA was dissolved in saline, nebulized (NE-U11B, Omuron Co., Tokyo, Japan), and delivered at 1 ml/min. The imiquimod group inhaled 0.15% imiquimod 30 minutes before 30 minutes of OVA challenge. The control group inhaled saline aerosol and received an intraperitoneal injection of 1 ml saline. Measurement of airway responsiveness to acetylcholine chloride For the measurement of airway responsiveness to acetylcholine chloride (Ach) the rats were anaesthetised by intraperitoneal injection of pentobarbital sodium (50 mg/kg) 24 hours after the final challenge. A plastic tube of 2 mm internal diameter was inserted into the trachea. The rats were then mechanically ventilated with an animal ventilator (AniRes2003, Beijing SYNOL High-Tech Co. Ltd, China) with a tidal volume of 6 ml/kg and frequency of 75 breaths per minute. Airway
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expiration resistance (Re) was continuously measured and recorded for later analysis. After measurement of baseline Re one minute following the animals were injected with acetylcholine chloride in progressively doubling doses from 10 µg/ml for one minute at each dose until the Re was twice the baseline level. Analysis of IFN-(, IL-4 and IL-5 in bronchoalveolar lavage fluid and serum Twenty-four hours after the last aerosolization with either OVA or saline at day 28, bronchoalveolar lavage (BAL) of the right lung was performed after tying off the left lung at the mainstem bronchus. Total BAL fluid (BALF; 3 x 4 ml of saline) and serum were collected. Samples were centrifuged at 1200 rpm for 5 minutes at 4˚C. Supernatants were analysed by ELISA for content of IFN-(, IL-4 and IL-5 using commercially available kit systems. Lung histology Twenty-four hours after the last aerosolization with either OVA or saline, lungs were analysed by histology (day 28). The right lung was frozen immediately and stored in liquid nitrogen until tested. The left lung was fixed in 10% formalin, dehydrated, mounted in paraffin, sectioned and stained with haematoxylin/eosin. Eosinophils in the lungs, the thicknesses of airway wall, ratio of wall area (acreage is an older, non SI, measure of area of land.) (WA)/bronchial internal perimeter (Pi) and ratio of airway smooth muscle (ASM)/Pi were quantified blindly by the same pathologist using an Olympus BX40F4 microscope, the program Photoshop, and an Image Processing FR-988 (Smart Scape 2002, China). Five high power fields in the area of peripheral bronchi were randomly selected for quantification. Counts are given as eosinophils per square millimetre. CD4+ T cells isolation，culture and flow cytometry Two weeks after immunization, spleens were excised aseptically from cervical dislocated rats and placed in RPMI. Enrichment of CD4+ T cells was performed according to the technique described by Stephens.6 Peripheral blood mononuclear cells were obtained from single cell suspension of spleen (after lysis of red blood cells). Enrichment for CD4+ T cells involved incubation for 40 minutes on ice with mouse
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mAb OX7, OXB, OX33, OX42 and 10/78 (BD Pharmingen), followed by washing and two consecutive rounds of depletion with goat antimouse IgG MicroBeads. The final composition of purified CD4+ T cells was determined by flow cytometric analysis with CD4+ T cell enrichment of 50% to 75%. Purified CD4+ T cells were cultured in RPMI 1640 (Life Technologies, USA) supplemented with 10% foetal calf serum, 2 mmol/L L-glutamine (Life Technologies), 100 U/ml penicillin (Life Technologies), and 100 µg/ml streptomycin (Life Technologies) at a density of 1 × 106/ml (6 ml/well) in 6-well culture plates. CD4+ T cells were cultured with 25 ng/ml phorbol myristate acetate (PMA) (Sigma Chemical Co, USA) and with or without 100 µg/ml OVA and 20 µg/ml imiquiod dissolved in DMSO. The mixtures were incubated in a humidified atmosphere of 5% CO2 at 37˚C. Supernatants and cell pellets harvested after culture for 24 hours were analysed for IL-4, IL-5, IFN-(, T-bet and GATA-3 protein and mRNA expression as described below. RT-PCR for cytokine and transcription factor gene expression The total RNA was extracted from three groups of lungs and cultured CD4+ T cells, and the RNA was extracted with the TRIzol reagent according to the recommendation of the manufacturer. First strain cDNA was synthesized from equal amounts of total RNA with M-MLV reverse transcriptase. Genes were amplified by PCR using sense and antisense primers of IL-4, IL-5, IFN-(, T-bet and GATA-3 as described before7 with some modifications. T-bet primers were designed corresponding to the mouse T-bet gene sequence outside of the T box domain to maintain its specificity for T cells. GATA-3, IFN-(, IL-5 and IL-4 PCR primers were designed according to the corresponding structures of rat genes. Primers were as follows: T-bet: sense: 5'-AACCAGTATCC TGTTCCCAGC-3', antisense: 5'-TGTCGCCACTG GAAGGATAG-3'; GATA-3: sense: 5'-CAGTCCGCAT CTCTTCAC-3', antisense: 5'-TAGTGCCCAGTAC CATCTC-3'; IFN-(: sense: 5'-CGTCTTGGTTTT GCAGCTC-3', antisense: 5'-ACTCCTTTTCCTCTT CCTTA-3'; IL-4: sense: 5'-TTGCTGTCACCCTGTT CTGC-3', antisense: 5'-GTTGTGAGCGTGGACTC ATTCACG-3'; IL-5, sense: 5'-ACGATGAGGC TTCC TGTTCC-3', antisense: 5'-TTCCATTGCCCACTCT GTAC-3'; β-actin: sense: 5'-TCCTG TGGCATC
Chin Med J 2006; 119(8):640-648
CATGAAACT-3', antisense 5'- GAAGCATTTGCGGT GCACGAT -3'. PCR annealing temperature: T-bet: 58˚C; GATA-3: 59˚C; (-IFN: 59˚C; IL-4: 58˚C; IL-5: 58˚C; β-actin: 58˚C. Semiquantitative RT-PCR was performed using β-actin as an internal control to normalize gene expression for the PCR templates. The PCR products were studied on a 1% agarose gel and the amplified bands were visualized after staining with ethidium bromide. The size of the amplified fragments was determined by comparison with a standard DNA marker. Measurement of T-bet and GATA-3 protein by Western blot analysis Fifty µg of total proteins isolated from the whole lung and CD4+ T cells were separated by 10% SDS-PAGE and blotted onto a nitrocellulose membrane for 2 hours.8 The membrane was then incubated in blocking solution (5% dry milk in PBS/0.05% Tween 20) for 1 hour at room temperature and subsequently exposed to 0.8 µg/ml of monoclonal antibodies anti-GATA-3, anti-T-bet or anti-β-actin overnight at 4˚C. The next day the membrane was incubated with peroxidase conjugated goat antimouse IgG (1:1000) for 2 hours at room temperature and enhanced chemiluminescence according to the manufacturer's instructions. Statistical analysis Data are given as mean values±standard error (SE). Statistical analysis was carried out using one way analysis of variance (ANOVA), followed by multiple comparisons by Dunnett's test using SPSS 11.5. P< 0.05 was considered statistically significant. RESULTS Inhaling imiquimod suppresses airway hyperreactivity in OVA sensitized rats Asthmatic rats showed an increase in airway hyperreactivity compared with saline treated control rats after challenge (P < 0.01, Fig. 1). Furthermore, it was found that administration of imiquimod resulted in a significant downregulation of ACh responsiveness compared with asthmatic rats after challenge (P < 0.01, Fig. 1). However, the levels of Re showed no difference in control rats and imiquimod
Chinese Medical Journal 2006; 119(8):640-648
Fig. 1. Airway responsiveness to Ach in three different groups. Airway responsiveness was monitored by Re as described in Methods. *Values are significantly different from the imiquimod treated group and control group (P0.05). BL: baseline; SAL: saline.
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Imiquimod inhibits infiltration of eosinophils in the lung and reduces thickness of bronchial wall and area of airway smooth muscle We also assessed infiltration of eosinophils and thicknesses of bronchial wall and area of smooth muscle in the lung. (“morphology” is to do with appearance and shape in 3D. In this experiment the numbers of eosinophils in a known area were counted.) The number of eosinophils into the lung interstitium was significantly reduced after administration of imiquimod compared with the asthmatic rats, but increased compared with the control rats (P
