Abstract
Phosphoinositide 3-kinases (PI3Ks) contribute to the pathogenesis of asthma by regulating the activation of inflammatory mediators, inflammatory cell recruitment and immune cell function. Recent findings have indicated that PI3Ks also regulate the expression of interleukin (IL)-17, which has been recognised as an important cytokine involved in airway inflammation.
In the present study, we investigated a role of PI3Kδ in the regulation of IL-17 expression in allergic airway disease using a murine model of asthma.
After ovalbumin inhalation, administration of a selective p110δ inhibitor, IC87114, significantly attenuated airway infiltration of total cells, lymphocytes, neutrophils and eosinophils, as well as airway hyperresponsiveness, and attenuated the increase in IL-17 protein and mRNA expression. Moreover, IC87114 reduced levels of IL-4, -5 and -13, expression of keratinocyte chemoattractant protein and mRNA, and nuclear factor (NF)-κB activity. In addition, a NF-κB inhibitor, BAY 11-7085 substantially reduced the increase in IL-17 protein levels. Our results also showed that inhibition of IL-17 activity with an anti-IL-17 antibody remarkably reduced airway inflammation and hyperresponsiveness.
These findings suggest that inhibition of the p110δ signalling pathway suppresses IL-17 expression through regulation of NF-κB activity and, thus, has therapeutic potential in asthma.
Asthma is a chronic inflammatory disease of the airways characterised by reversible airway obstruction and airway hyperresponsiveness 1. Airway inflammation in asthma increases expression of various components of the inflammatory cascade, which include cytokines, chemokines, adhesion molecules, growth factors and enzymes. Central to this inflammatory cascade is a complex interaction between receptor signalling, and downstream lipid and protein kinases. In particular, phosphoinositide 3-kinase (PI3K) is an important kinase involved in the inflammatory process.
The PI3K is a large family of signalling kinases that generate lipid second messengers responsible for regulating a number of cellular activities. PI3Ks mediate key signal transduction reactions during immune and inflammatory responses and, thus, represent an attractive target for therapeutic development in various inflammatory diseases 2, 3. Research conducted over the past few years has highlighted the importance of PI3Ks in bronchial asthma. Our previous study with a murine model of asthma has shown that PI3K activity increases after ovalbumin (OVA) challenge compared with the pre-challenge period 4. In addition, administration of PI3K inhibitor, wortmannin or LY-294002, reduces T-helper cell (Th) type 2 cytokine production, pulmonary eosinophilia, airway inflammation and bronchial hyperresponsiveness 4. There is increasing evidence that PI3Ks contribute to the pathogenesis of asthma by regulating the expression and activation of inflammatory mediators, inflammatory cell recruitment and immune cell function 2, 4. Recent work has begun to explore specific roles of different PI3K isoforms, and p110δ and p110γ have been suggested to be the dominant isoforms involved in PI3K-mediated immune responses 5. Two commercially available PI3K inhibitors used to investigate the biological roles of PI3Ks and to analyse PI3K-driven pathways, wortmannin and LY-294002, are poorly selective among the four members of PI3K class I. Therefore, development of PI3K isoform-specific inhibitors has been an area of considerable interest. A selective inhibitor of p110δ catalytic activity, IC87114, has recently been synthesised and used to explore the role of PI3Kδ in neutrophil migration 6, 7. In addition, previous studies using IC87114 have suggested that p110δ plays an important role in the pathogenesis of allergic asthma 8, 9.
The interleukin (IL)-17 family is a recently described group of cytokines that consists of six members, namely IL-17 (also called IL-17A), IL-17B, IL-17C, IL-17D, IL-17E and IL-17F 10. IL-17, the most extensively investigated member of this family, has been implicated in many immune and inflammatory responses, primarily as a proinflammatory regulator, inducing expression of many inflammatory mediators, such as cytokines, chemokines, adhesion molecules and growth factors 11, 12. IL-17 mRNA is upregulated in inflamed airways after allergen inhalation in a mouse model of allergic asthma 13. Consistent with the animal study, IL-17 mRNA and/or protein expression is increased in sputum, bronchoalveolar lavage (BAL) fluids and peripheral blood in asthmatics 14–18. Moreover, the levels of IL-17 expression in airways correlate with the severity of airway hypersensitivity in asthmatic patients 14. These findings have indicated the potential role of IL-17 in the pathogenesis of asthma. Recent studies, including our own, have found that IL-17 production is mediated by the activation of PI3K/Akt pathway 12, 19–21. However, the contributions of individual PI3K isoforms to the up-regulation of IL-17 expression have not been examined. In the present study, we investigated an involvement of PI3Kδ in IL-17 expression and its molecular mechanism using a murine model of asthma.
MATERIALS AND METHODS
A more detailed methodology is available in the online supplementary material.
Animals and experimental protocol
Female C57BL/6 mice, 8–10 weeks of age and free of murine-specific pathogens, were obtained from Orientbio Inc. (Seoungnam, South Korea). All animal experiments were approved by the Institutional Animal Care and Use Committee (Chonbuk National University, Jeonju, South Korea). Mice were sensitised on days 1 and 14 by intraperitoneal injection of 20 μg OVA (Sigma–Aldrich, St Louis, MO, USA) emulsified in 1 mg of aluminum hydroxide (Pierce Chemical Co., Rockford, IL, USA) in a total volume of 200 μL, as described previously 8, 9. On days 21, 22 and 23 after the initial sensitisation, the mice were challenged for 30 min with an aerosol of 3% (weight/volume) OVA in saline (or saline alone as a control) using an ultrasonic nebuliser (NE-U12; Omron, Tokyo, Japan). BAL was performed 48 h after the last challenge. At the time of lavage, mice were sacrificed with an overdose of sodium pentobarbital (100 mg per kg of body weight, administered intraperitoneally).
Administration of IC87114, LY-294002, anti-IL-17 antibody, isotype-control monoclonal antibody, Akt inhibitor or BAY 11-7085
IC87114 (0.1 or 1 mg·kg−1·day−1), LY-294002 (a selective inhibitor of PI3K; 1.5 mg·kg−1·day−1; BIOMOL Research Laboratories Inc., Plymouth Meeting, PA, USA) or vehicle control (dimethylsulfoxide (DMSO)) diluted with 0.9% NaCl, was administered in a volume of 50 μL by intratracheal instillation to each animal, once on day 21 (1 h before the first OVA challenge) and again on day 23 (3 h after the last OVA challenge) 8, 9. Anti-IL-17 antiobody (Ab) or isotype-control monoclonal (m)Ab (5 mg·kg−1·day−1; R&D Systems, Inc., Minneapolis, MN, USA) was administered intraperitoneally to each animal, once on day 21 and again on day 24 (24 h after the last OVA challenge). An Akt inhibitor, 1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate (3 mg·kg−1·day−1; Calbiochem, Darmstadt, Germany), dissolved in DMSO and diluted with 0.9% NaCl, was administered by intraperitoneal injection to each animal, once on day 21 and again on day 24. An inhibitor of nuclear factor (NF)-κB activation, BAY 11-7085 (20 mg·kg−1·day−1; BIOMOL Research Laboratories Inc.), dissolved in DMSO and diluted with 0.9% NaCl, was administered by intraperitoneal injection to each animal, once on day 21 and again on day 24.
Western blot analysis
Protein expression levels were analysed by means of Western blot analysis, as described previously 8. The primary antibodies used were: anti-IL-17 Ab (R&D Systems, Inc.), anti-IL-4 Ab (Serotec Ltd, Oxford, UK), anti-IL-5 Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-IL-13 Ab (R&D Systems, Inc.), biotinylated anti-mouse keratinocyte chemoattractant (KC) Ab (R&D Systems, Inc.), anti-Akt Ab (Cell Signaling Technology Inc., Beverly, MA, USA), anti-phosphorylated Akt (p-Akt) Ab (Cell Signaling Technology Inc.), and anti-inhibitor of κBα (I-κBα) Ab (Santa Cruz Biotechnology).
RNA isolation and RT-PCR
Levels of mRNA expression were analysed by RT-PCR assay using total RNA isolated from lung tissues by a rapid extraction method (TRI-Reagent; Sigma–Aldrich) as previously described 12.
Quantitative real-time RT-PCR
Quantitative real-time RT-PCR analysis was performed using the LightCycler® FastStart DNA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany) as described previously 12.
Measurement of Th2 cytokines
Levels of IL-4, IL-5 and IL-13 were quantified by enzyme immunoassays (R&D Systems, Inc.).
Cytosolic or nuclear protein extractions for analysis of NF-κB p65
Cytosolic or nuclear extractions were performed as described previously 9.
Measurement of PI3K enzyme activity in lung tissues
PI3K enzyme activity in lung tissues was measured as described previously 9. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) was quantified using a PIP3 competition enzyme immunoassay (Echelon, Inc., Salt Lake City, UT, USA).
Determination of airway responsiveness
Airway responsiveness was assessed as a change in airway function after challenge with aerosolised methacholine, as described previously 9.
Densitometric analyses and statistics
All immunoreactive and phosphorylation signals were analysed by densitometric scanning (Gel Doc XR; Bio-Rad Laboratories Inc., Hercules, CA, USA). Data are presented as mean±sem. Statistical comparisons were performed using one-way ANOVA followed by Scheffe’s test. Statistical significance was set at p<0.05.
RESULTS
Effect of IC87114 on IL-17 protein levels and mRNA expression in lung tissues of OVA-sensitised and OVA-challenged mice
Western blot analysis revealed that the levels of IL-17 protein in lung tissues were significantly increased 48 h after the last inhalation of OVA compared with those in the control mice (figs 1a and 1c). The increase in the IL-17 protein levels after OVA inhalation was attenuated significantly by the administration of IC87114. RT-PCR and real-time RT-PCR analysis showed that the increase in the IL-17 mRNA expression after OVA inhalation was significantly reduced by the administration of IC87114 (figs 1b and 1d).
Effect of IC87114 on interleukin (IL)-17 protein levels and mRNA expression in lung tissues of ovalbumin (OVA)-sensitised and OVA-challenged mice. Sampling was performed 48 h after the last challenge in saline-nebulised mice administered drug vehicle (dimethylsulfoxide) (SAL+VEH), OVA-nebulised mice administered drug vehicle (OVA+VEH), OVA-nebulised mice administered 0.1 mg·kg−1 IC87114 (OVA+IC87114 0.1 mg·kg−1), and OVA-nebulised mice administered 1.0 mg·kg−1 IC87114 (OVA+IC87114 1.0 mg·kg−1). a) Western blot analyses of IL-17 protein. b) Representative RT-PCR analyses of IL-17 mRNA expression. c) Densitometric analyses are presented as the relative ratio of IL-17 in OVA+VEH, OVA+IC87114 1.0 mg·kg−1 to those in SAL+VEH. The relative ratio of IL-17 in the lung tissues of SAL+VEH is arbitrarily presented as 1. d) Quantitative analyses of IL-17 mRNA expression by means of real-time RT-PCR. Data represent mean±sem from seven mice per group. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. #: p<0.05 versus SAL+VEH; ¶: p<0.05 versus OVA+VEH.
Effect of IC87114 on IL-17 protein levels in BAL fluids of OVA-sensitised and OVA-challenged mice
Western blot analysis revealed that the levels of IL-17 protein in BAL fluids were significantly increased 48 h after OVA inhalation compared with those in the control mice (fig. 2). The increase in the IL-17 protein levels after OVA inhalation was significantly inhibited by the administration of IC87114.
Effect of IC87114 on interleukin (IL)-17 protein levels in bronchoalveolar lavage fluids of ovalbumin (OVA)-sensitised and OVA-challenged mice. Sampling was performed 48 h after the last challenge in saline-nebulised mice administered drug vehicle (SAL+VEH), OVA-nebulised mice administered drug vehicle (OVA+VEH), OVA-nebulised mice administered 0.1 mg·kg−1 IC87114 (OVA+IC87114 0.1 mg·kg−1), and OVA-nebulised mice administered 1.0 mg·kg−1 IC87114 (OVA+IC87114 1.0 mg·kg−1). a) Western blot analyses of IL-17 protein. b) Densitometric analyses are presented as the relative ratio of IL-17 to actin. The relative ratio of IL-17 in the lung tissues of SAL+VEH is arbitrarily presented as 1. Data are presented as mean±sem of seven mice per group. #: p<0.05 versus SAL+VEH; ¶: p<0.05 versus OVA+VEH.
Effect of IC87114 or anti-IL-17 Ab on cellular changes in BAL fluids
Numbers of total cells, lymphocytes, neutrophils and eosinophils in BAL fluids were increased significantly 48 h after OVA inhalation compared with the numbers after saline inhalation (fig. 3a). The increases in the numbers of total cells, lymphocytes, neutrophils and eosinophils were significantly reduced by the administration of IC87114. In addition, administration of anti-IL-17 Ab to OVA-sensitised and -challenged mice significantly decreased the increases in the numbers of total cells, lymphocytes, neutrophils and eosinophils compared with the numbers in OVA-nebulised mice administered drug vehicle or isotype-control mAb.
a) Effect of IC87114 or anti-interleukin-17 antibody (anti-IL-17 Ab) on total and differential cell counts in bronchoalveolar lavage (BAL) fluids of ovalbumin (OVA)-sensitised and OVA-challenged mice. Differential cell counts in BAL fluids from saline-nebulised mice administered drug vehicle (SAL+VEH; □), OVA-inhaled mice administered drug vehicle (OVA+VEH; ░), OVA-nebulised mice administered 0.1 mg·kg−1 IC87114 (OVA+IC87114 0.1 mg·kg−1; ▓), OVA-nebulised mice administered 1.0 mg·kg−1 IC87114 (OVA+IC87114 1.0 mg·kg−1; ▒), OVA-nebulised mice administered anti-IL-17 Ab (OVA+anti-IL-17 Ab; ▪), and OVA-nebulised mice administered isotype-control monoclonal Ab (mAb) (OVA+control mAb; 
). b–e) Effect of IC87114 or anti-IL-17 Ab in lung tissues of OVA-sensitised and OVA-challenged mice. Representative haematoxylin and eosin-stained sections of the lungs. Sampling was performed 48 h after the last challenge in b) SAL+VEH, c) OVA+VEH, d) OVA+IC87114 0.1 mg·kg−1 and e) OVA+anti-IL-17 Ab mice. Scale bars = 20 μm. Results were similar in seven mice per group. f) Effect of IC87114 or anti-IL-17 Ab on airway responsiveness in OVA-sensitised and OVA-challenged mice. Airway responsiveness was measured 48 h after the last challenge in SAL+VEH (♦), OVA+VEH (▪), OVA+IC87114 0.1 mg·kg−1 (○), OVA+IC87114 1.0 mg·kg−1 (⋄), OVA+anti-IL-17 Ab (•) and OVA+control mAb (▴). Data are presented as mean±sem from seven mice per group. Rrs: resistance of respiratory system. #: p<0.05 versus SAL+VEH; ¶: p<0.05 versus OVA+VEH; +: p<0.05 versus OVA+control mAb.
Effect of IC87114 or anti-IL-17 Ab on pathological changes of OVA-induced asthma
Histological analysis revealed the typical pathological features of asthma in the OVA-exposed mice. Numerous inflammatory cells infiltrated around the bronchioles, and mucus and debris had accumulated in the lumens of bronchioles (fig. 3c) compared with the control (fig. 3b). Mice treated with IC87114 (fig. 3d) or anti-IL-17 Ab (fig. 3e) showed marked reductions in the infiltration of inflammatory cells in the peribronchiolar region and in the amount of debris in the airway lumens.
Effect of IC87114 or anti-IL-17 Ab on airway hyperresponsiveness
The airway responsiveness was assessed as a percentage increase in the resistance of the respiratory system (Rrs) in response to increasing doses of methacholine. In OVA-sensitised and -challenged mice, the dose–response curve of percentage Rrs shifted to the left compared with that of the control mice (fig. 3f). In addition, the percentage Rrs produced by methacholine administration (at a dose of 50 mg·mL−1) increased significantly in the OVA-nebulised mice compared with the control mice. OVA-sensitised and -challenged mice treated with IC87114 showed a dose–response curve of percentage Rrs was that shifted to the right and a significant reduction in the percentage Rrs produced by 50 mg·mL−1 methacholine compared with those of untreated mice. In addition, the administration of anti-IL-17 Ab to OVA-sensitised and -challenged mice also decreased significantly the percentage Rrs produced by 50 mg·mL−1 methacholine compared with that of OVA-nebulised mice administered drug vehicle or isotype-control mAb. These results indicate that treatment of IC87114 or anti-IL-17 Ab reduces OVA-induced airway hyperresponsiveness.
Effect of IC87114 on levels of IL-4, IL-5 and IL-13 in lung tissues and BAL fluids of OVA-sensitised and OVA-challenged mice
Western blot analysis revealed that the protein levels of IL-4, IL-5 and IL-13 in lung tissues were increased 48 h after OVA inhalation compared with those in the control mice (fig. 1Sa in the online supplementary material). The increases in these cytokine levels were reduced by the administration of IC87114. Consistent with the Western blot analysis results, enzyme immunoassays of IL-4, IL-5 and IL-13 showed that the levels of these cytokines in BAL fluids were significantly reduced by the administration of IC87114 (fig. 1Sb in the online supplementary material).
Effect of IC87114 on KC protein levels and mRNA expression in lung tissues of OVA-sensitised and OVA-challenged mice
Western blot analysis revealed that the levels of KC protein in lung tissues were significantly increased 48 h after OVA inhalation compared with those in the control mice (fig. 4a and c). The increase in the KC protein levels after OVA inhalation was decreased significantly by the administration of IC87114. RT-PCR and real-time RT-PCR analysis showed that the increase in the KC mRNA expression after OVA inhalation was significantly reduced by the administration of IC87114 (fig. 4e and g).
Effect of IC87114 or anti-interleukin-17 antibody (anti-IL-17 Ab) on keratinocyte chemoattractant (KC) protein levels and mRNA expression in lung tissues of ovalbumin (OVA)-sensitised and OVA-challenged mice. a, c, e, g) Sampling was performed 48 h after the last challenge in saline-nebulised mice administered drug vehicle (SAL+VEH), OVA-nebulised mice administered drug vehicle (OVA+VEH), OVA-nebulised mice administered 0.1 mg·kg−1 IC87114 (OVA+IC87114 0.1 mg·kg−1) and OVA-inhaled mice administered 1.0 mg·kg−1 IC87114 (OVA+IC87114 1.0 mg·kg−1). b, d, f, h) Sampling was performed 48 h after the last challenge in saline-inhaled mice administered saline (SAL+SAL), OVA-nebulised mice administered isotype-control monoclonal Ab (OVA+control mAb) and OVA-nebulised mice administered anti-IL-17 Ab (OVA+anti-IL-17 Ab). a and b) Western blot analyses of KC protein. c and d) Densitometric analyses are presented as the relative ratio of KC to actin. The relative ratio of KC in the lung tissues of SAL+VEH (c) or SAL+SAL (d) is arbitrarily presented as 1. e and f) Representative RT-PCR analyses of KC mRNA expression. g and h) Quantitative analyses of KC mRNA expression by means of real-time RT-PCR. Data are presented as mean±sem of seven mice per group. GAPDF: glyceraldehyde-3-phosphate dehydrogenase. #: p<0.05 versus SAL+VEH; ¶: p<0.05 versus OVA+VEH; +: p<0.05 versus SAL+SAL; §: p<0.05 versus OVA+control mAb.
Effect of anti-IL-17 Ab on KC protein levels and mRNA expression in lung tissues of OVA-sensitised and OVA-challenged mice
Western blot analysis revealed that administration of anti-IL-17 Ab to OVA-sensitised and -challenged mice significantly decreased the levels of KC protein in lung tissues compared with the levels in OVA-inhaled mice administered isotype control mAb (fig. 4b and d). In addition, RT-PCR and real-time RT-PCR analysis showed that the administration of anti-IL-17 Ab to OVA-sensitised and -challenged mice significantly decreased the KC mRNA expression compared with that in OVA-nebulised mice administered isotype-control mAb (fig. 4f and h).
Effect of LY-294002 or IC87114 on p-Akt and Akt protein levels, and PI3K enzyme activity in lung tissues of OVA-sensitised and OVA-challenged mice
The levels of p-Akt protein in the lung tissues were significantly increased at 48 h after OVA inhalation compared with levels in the control mice (fig. 5a and c). However, no significant changes in the Akt protein levels were observed in any of the groups tested. The increase of p-Akt but not Akt protein levels in the lung tissues 48 h after OVA inhalation was significantly reduced by the administration of LY-294002 or IC87114. Consistent with these results, PIP3 levels in the lung tissues were significantly increased 48 h after OVA inhalation compared with those in the control mice (fig. 5e). The increase of the PIP3 levels in the lung tissues 48 h after OVA inhalation was significantly reduced by the administration of LY-294002 or IC87114.
Effect of LY-294002 or IC87114 on phosphorylated Akt (p-Akt) and Akt protein levels, phosphoinositide 3-kinase (PI3K) enzyme activity and inhibitor of κBα (I-κBα) protein levels in lung tissues of ovalbumin (OVA)-nebulised and OVA-challenged mice. Sampling was performed 48 h after the last challenge in saline-inhaled mice administered drug vehicle (dimethylsulfoxide) (SAL+VEH), OVA-nebulised mice administered drug vehicle (OVA+VEH), OVA-inhaled mice administered LY-294002 (OVA+LY-294002), and OVA-inhaled mice administered 1.0 mg·kg−1 IC87114 (OVA+IC87114). a) Western blotting of p-Akt and Akt protein. b) Western blotting of I-κBα protein. c) Densitometric analyses are presented as the relative ratio of p-Akt to Akt. The relative ratio of p-Akt in the lung tissues of SAL+VEH is arbitrarily presented as 1. d) Densitometric analyses are presented as the relative ratio of I-κBα to actin. The relative ratio of I-κBα in the lung tissues of SAL+VEH is arbitrarily presented as 1. e) Enzyme immunoassay of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generation by PI3Ks in lung tissues. Data are presented as mean±sem from six mice per group. #: p<0.05 versus SAL+VEH; ¶: p<0.05 versus OVA+VEH.
Effect of LY-294002 or IC87114 on I-κBα protein levels in lung tissues of OVA-sensitised and OVA-challenged mice
Western blot analysis revealed that the I-κBα protein levels in the lung tissues were significantly decreased 48 h after OVA inhalation compared with those in the control mice (fig. 5b and d). The decrease in the I-κBα protein levels after OVA inhalation was significantly inhibited by the administration of LY-294002 or IC87114.
Effect of Akt inhibitor on I-κBα and NF-κB p65 protein levels in lung tissues of OVA-sensitised and OVA-challenged mice
The I-κBα protein levels decreased after OVA inhalation was significantly increased by the administration of Akt inhibitor (fig. 6a and c). Western blot analysis revealed that the levels of NF-κB p65 in nuclear protein extracts from the lung tissues were increased 48 h after OVA inhalation compared with the levels in the control mice administered saline (fig. 6b and 6d). The administration of Akt inhibitor significantly reduced the increase in the NF-κB p65 levels in nuclear protein extracts after OVA inhalation. In contrast, the levels of NF-κB p65 in cytosolic protein fractions from the lung tissues were decreased after OVA inhalation compared with the levels in the control mice administered saline. The decrease in the NF-κB p65 levels in cytosolic protein fractions was substantially inhibited by the administration of Akt inhibitor.
Effect of Akt inhibitor on inhibitor of κBα (I-κBα) and nuclear factor (NF)-κB p65 protein levels in lung tissues of ovalbumin (OVA)-sensitised and OVA-challenged mice. Sampling was performed 48 h after the last challenge in saline-nebulised mice administered drug vehicle (dimethylsulfoxide) (SAL+VEH), OVA-inhaled mice administered drug vehicle (OVA+VEH) and OVA-inhaled mice administered Akt inhibitor (OVA+Akt inhibitor). a) Western blotting of I-κBα protein. b) Western blot analyses of NF-κB p65 levels in nuclear (Nuc) and cytosolic (Cyt) protein extracts from lung tissues. c) Densitometric analyses are presented as the relative ratio of I-κBα to actin. The relative ratio of I-κBα in the lung tissues of SAL+VEH is arbitrarily presented as 1. d) Densitometric analyses are presented as the relative ratio of NF-κB p65 levels in OVA+VEH or OVA+Akt inhibitor to those in SAL+VEH. The relative ratio of NF-κB in nuclear protein extracts from the lung tissues of SAL+VEH is arbitrarily presented as 1. Data are presented as mean±sem of six mice per group. □: Nuc; ▓: Cyt. #: p<0.05 versus SAL+VEH; ¶: p<0.05 versus OVA+VEH.
Effect of IC87114 on NF-κB p65 protein levels in lung tissues of OVA-sensitised and OVA-challenged mice
The increase in the NF-κB p65 levels in nuclear protein extracts of the lung tissues after OVA inhalation was significantly reduced by the administration of IC87114 (fig. 7). In contrast, the decrease in the NF-κB p65 levels in cytosolic protein fractions of the lung tissues was substantially inhibited by the administration of IC87114.
Effect of IC87114 on nuclear factor (NF)-κB p65 protein levels in lung tissues of ovalbumin (OVA)-sensitised and OVA-challenged mice. Levels of NF-κB p65 were measured 48 h after the last challenge in saline-nebulised mice administered drug vehicle (SAL+VEH), OVA-nebulised mice administered drug vehicle (OVA+VEH), OVA-inhaled mice administered 0.1 mg·kg−1 IC87114 (OVA+IC87114 0.1 mg·kg−1) and OVA-inhaled mice administered 1.0 mg·kg−1 IC87114 (OVA+IC87114 1.0 mg·kg−1). a) Western blot analyses of NF-κB p65 levels in nuclear (Nuc) and cytosolic (Cyt) protein extracts from lung tissues. b) Densitometric analyses are presented as the relative ratio of NF-κB p65 levels in OVA+VEH, OVA+IC87114 0.1 mg·kg−1 or OVA+IC87114 1.0 mg·kg−1 to those in SAL+VEH. The relative ratio of NF-κB in nuclear protein extracts from the lung tissues of SAL+VEH is arbitrarily presented as 1. Data are presented as mean±sem of seven mice per group. □: Nuc; ▓: Cyt. #: p<0.05 versus SAL+VEH; ¶: p<0.05 versus OVA+VEH.
Effect of BAY 11-7085 on IL-17 protein levels in lung tissues of OVA-sensitised and OVA-challenged mice
Western blot analysis showed that the IL-17 protein levels increase after OVA inhalation was significantly reduced by the administration of BAY 11-7085, an inhibitor of NF-κB activation (fig. 8).
Effect of BAY11-7085 on interleukin (IL)-17 protein levels in lung tissues of ovalbumin (OVA)-sensitised and OVA-challenged mice. Sampling was performed 48 h after the last challenge in saline-nebulised mice administered drug vehicle (dimethylsulfoxide) (SAL+VEH), OVA-inhaled mice administered drug vehicle (OVA+VEH) and OVA-inhaled mice administered BAY11-7085 (OVA+BAY 11). a) Western blot analyses of IL-17. b) Densitometric analyses are presented as the relative ratio of IL-17 to actin. The relative ratio of IL-17 in the lung tissues of SAL+VEH is arbitrarily presented as 1. Data are presented as mean±sem of seven mice per group. #: p<0.05 versus SAL+VEH; ¶: p<0.05 versus OVA+VEH.
DISCUSSION
In this study, we demonstrated that inhibition of the p110δ signalling pathway suppressed IL-17 protein and mRNA expression in the lungs, and attenuated allergen-induced airway inflammation and airway hyperresponsiveness. The OVA-induced model of asthma used in this study revealed the following typical pathological features of asthma: increased numbers of inflammatory cells in airways, airway hyperresponsiveness, and increased levels of Th2 cytokines (IL-4, IL-5 and IL-13), chemokine KC and nuclear NF-κB. In addition, the expression of IL-17 protein and mRNA in the lungs was increased after OVA inhalation. The administration of a selective PI3Kδ inhibitor, IC87114, significantly attenuated airway infiltration of total cells, lymphocytes, neutrophils and eosinophils, as well as airway hyperresponsiveness, and also decreased IL-17 protein and mRNA expression. Moreover, IC87114 reduced levels of Th2 cytokines, expression of KC protein and mRNA, Akt phosphorylation, I-κBα degradation and NF-κB activity. Supporting these results, an Akt inhibitor also increased I-κBα protein levels while reducing NF-κB activity, and an NF-κB inhibitor, BAY 11-7085, substantially reduced the increase in IL-17 protein levels after OVA inhalation. In addition, the inhibition of IL-17 activity with an anti-IL-17 Ab remarkably reduced bronchial inflammation and airway hyperresponsiveness. These results suggest that the therapeutic effect of a PI3Kδ inhibitor in asthma is partly mediated by suppressing IL-17 expression via modulation of Akt-mediated NF-κB activation. To the best of our knowledge, this is the first study clarifying a role of PI3Kδ in regulation of IL-17 expression.
The PI3K family is divided into class I, II and III, and the class I PI3Ks are further subdivided into class IA (PI3Kα, PI3Kβ and PI3Kδ isoforms) and class IB (PI3Kγ isoform) on the basis of structure and substrate specificities 22. Structurally, the class IA PI3K consists of a heterodimer composed of a 110-kd (p110α, β or δ) catalytic subunit and a regulatory subunit (p85α, p85β, p55α, p55γ or p50α) 23. These differences allow PI3K isoforms to mediate distinct functions. Of these isoforms of PI3K, p110δ has been implicated in immune responses and inflammation 24. Recent studies have demonstrated a novel biological role of p110δ signalling in allergic airway inflammation and emphasised the importance of PI3Kδ isoform as a potential therapeutic target in asthma 8, 9. The genetic inactivation of p110δ has resulted in reduction of antigen receptor function, Th2 cytokine production and Th2 cell-mediated inflammatory reactions 25. In the present study, the results showed that administration of a specific inhibitor of PI3Kδ substantially reduced the pathological features of asthma, including airway inflammation, airway hyperresponsiveness and the increases in Th2 cytokine levels. These results are consistent with the findings of previous studies 8, 9, 25, confirming the therapeutic potential of the PI3Kδ inhibitor in asthma.
IL-17 is a new cytokine that has recently captured the attention of many scientists due to its involvement in immune and inflammatory responses 26. IL-17 in airways is most likely produced by a unique Th lineage called Th17 cells 27. Recent studies have indicated that eosinophils, neutrophils and monocytes are also sources of IL-17 15, 26. The biological function of IL-17 appears to be associated with neutrophil-dominated inflammation as a promoter of granulopoiesis, neutrophil accumulation and neutrophil activation in the lung 11, 26. Intratracheal administration of IL-17 has been shown to increase the absolute number of neutrophils in BAL fluids 28. In addition, IL-17 increases the expression of IL-8 mRNA in bronchial epithelial cells and the neutrophil chemotactic effect of IL-17 is blocked by an anti-IL-8 Ab 29. Therefore, it is likely that IL-17 is able to recruit neutrophils selectively into airways by inducing the release of the potent neutrophil-directed chemokine IL-8 29, 30. In this study, we evaluated an effect of an anti-IL-17 Ab on the expression of KC, which is a functional murine homologue of IL-8. Consistent with previous studies, we found that inhibiting IL-17 activity with the anti-IL-17 Ab remarkably reduced the increase in airway infiltration of neutrophils, and KC protein and mRNA expression after allergen inhalation. These results suggest that IL-17 orchestrates neutrophilic influx into airways by inducing the release of IL-8 in our model of allergic airway inflammation. It is well established that the pathognomonic features of asthma are mediated by eosinophils, mast cells and Th2 cells, as well as their cytokines 31. However, it has been reported that the numbers of neutrophils in airways are increased in severe asthma 32. In asthma, neutrophils potentially contribute to airway gland hypersecretion, airway obstruction and airway remodelling 17, 33. Data from the present study support the notion that IL-17-induced neutrophil recruitment into airways is implicated in the pathogenesis of asthma.
Interestingly, a recent study has demonstrated that IL-17 receptor gene-deficient mice show a reduced recruitment of not only neutrophils but also eosinophils into airways upon antigen challenge 34. In addition, eosinophil peroxidase activity in lung tissues and OVA-specific serum immunoglobulin E concentrations are reduced in the absence of IL-17 receptor signalling 35. Furthermore, an involvement of IL-17 in the activation of allergen-specific T-cells has also been reported 36. IL-17-deficient or IL-17 receptor-deficient mice show decreases in Th2 cytokine levels, which are associated with reduced airway hypersensitivity 34, 35. A more recent study has shown that IL-17 mRNA levels correlate positively with IL-5 mRNA levels in sputum from asthmatic patients 18. Therefore, IL-17 seems to contribute to Th2 cell-mediated and eosinophilic inflammation in asthma. Consistent with these observations, our present data showed that inhibition of IL-17 activity with an anti-IL-17 Ab remarkably reduced antigen-induced airway infiltration of eosinophils and airway hyperresponsiveness. We also observed that inhibition of IL-17 activity significantly reduced the increases in Th2 cytokine levels in BAL fluids. Taken together, these findings indicate that IL-17 plays a crucial role in the pathogenesis of asthma, contributing to neutrophilic inflammation as well as Th2 cell-mediated eosinophilic inflammation.
IL-17 production is mediated by activation of the PI3K/Akt pathway 23, 19–21. It has been reported that LY-294002 inhibits IL-17 production and mRNA expression in CD4+ T cells 20. Consistent with these in vitro data, we have previously reported that administration of wortmannin or LY-294002 reduces the increase in IL-17 levels after allergen inhalation in a murine model of asthma 12. However, these inhibitors do not differentiate the four isoforms of class I PI3K. In the present study, pharmacological blockade of p110δ activity with IC87114 substantially decreased the expression of IL-17 protein and mRNA in the OVA-inflamed lungs. In addition, IC87114 was able to reduce the levels of Th2 cytokines as well as the expression of KC protein and mRNA, which are associated with IL-17 expression. PI3Kδ has been shown to play a prominent role in the accumulation and activation of neutrophils in inflamed tissues 6, 7. However, the signalling pathways for the contribution of PI3Kδ to neutrophil chemotaxis have not been well defined. The results of the present study suggest that IL-17 is a key regulator underlying neutrophilic inflammation mediated by PI3Kδ. Taken together, these findings suggest that the therapeutic effect of the PI3Kδ inhibitor in the asthma is exerted by down-regulating IL-17 expression, thereby attenuating Th2 cell-mediated eosinophilic inflammation and neutrophilic inflammation.
A transcription factor, NF-κB, plays an essential role in immune and inflammatory responses and is, consequently, associated with the pathogenesis of asthma. As expected, nuclear NF-κB protein levels were substantially increased in our OVA-induced asthma model, indicating that NF-κB is activated. Activation of this transcription factor induces many inflammatory genes encoding cytokines (tumour necrosis factor-α, IL-4, IL-5, IL-6, IL-9 and IL-13), chemokines (RANTES (regulated on activation, normal T-cell expressed and secreted), eotaxin and IL-8), adhesion molecules (intracellular cell adhesion molecule-1 and vascular cell adhesion molecule-1), growth factors and receptors that are potentially relevant to the pathogenesis of asthma 37. Furthermore, in vitro and in vivo studies have shown that inhibition of NF-κB activation reduces IL-17 production 13, 19–21. These findings suggest that NF-κB is an important regulator of IL-17 expression. Activation of the PI3K/Akt pathway leads to a pronounced augmentation of NF-κB activity through I-κBα degradation 37, 38. However, no data are available on the role of PI3K catalytic subunit p110δ in the activation of NF-κB. The present study showed that administration of a PI3Kδ inhibitor substantially decreased allergen-induced Akt phosphorylation with a significant reduction of I-κBα degradation and NF-κB activity in lung tissues. Consistent with the published data 38, we also found that an Akt inhibitor increased I-κBα protein levels while reducing NF-κB activity in lung tissues of OVA-challenged mice. In addition, the increase in IL-17 protein and mRNA expression after OVA inhalation was decreased by administration of an inhibitor of NF-κB activation, BAY 11-7085, or an Akt inhibitor (fig. 2S in the supplementary material). These findings suggest that p110δ activates Akt and, thereby, enhances I-κBα degradation and subsequent NF-κB activation, which regulates IL-17 expression in allergic airway inflammation.
In conclusion, our results have demonstrated that a specific inhibitor of PI3Kδ, IC87114, reverses all pathological features of asthma, including airway infiltration of inflammatory cells, Th2 cytokine levels and KC expression. In addition, IC87114 decreased the expression of IL-17 protein and mRNA in lungs increased by OVA challenge. These results suggest that IC87114 down-regulates IL-17 expression, and attenuates Th2 cell-mediated eosinophilic inflammation and neutrophilic inflammation. In addition, we have also found that this PI3Kδ inhibitor inhibits NF-κB activity and that NF-κB inhibition reduces the increase in IL-17 expression. Based on these observations, we suggest that inhibition of the p110δ signalling pathway has therapeutic potential in asthma by down-regulating the NF-κB pathway and the resultant suppressing IL-17 expression. Accordingly, the present study provides an important mechanism for the use of PI3Kδ inhibitor to prevent and/or treat asthma and other allergic airway diseases.
Acknowledgments
We would like to thank M.J. Im (Chonbuk National University Medical School, Jonju, South Korea) for critical reading of the manuscript.
Footnotes
This article has supplementary material available from www.erj.ersjournals.com
Support Statement
This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, South Korea (A084144).
Statement of Interest
A statement of interest for K.D. Puri can be found at www.erj.ersjournals.com/site/misc/statements.xhtml
- Received July 8, 2009.
- Accepted March 15, 2010.
- ©ERS 2010
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