American Journal of Respiratory and Critical Care Medicine

Rationale: There is an increasing prevalence of reduced responsiveness to glucocorticoid therapy in severe asthma and chronic obstructive pulmonary disease (COPD). The molecular mechanism of this remains unknown. Recent studies have shown that histone deacetylase activity, which is critical to glucocorticoid function, is altered by oxidant stress and may be involved in the development of glucocorticoid insensitivity.

Objectives: To determine the role of phosphoinositol-3-kinase (PI3K) in the development of cigarette smoke–induced glucocorticoid insensitivity.

Methods: Wild-type, PI3Kγ knock-out and PI3Kδ kinase dead knock-in transgenic mice were used in a model of cigarette smoke–induced glucocorticoid insensitivity. Peripheral lung tissue was obtained from six healthy nonsmokers, nine smokers with normal lung function, and eight patients with COPD.

Measurements and Main Results: In vitro oxidative stress activates PI3K and induced a relative glucocorticoid resistance, which is restored by PI3K inhibition. In vivo, cigarette smoke exposure in mice increased tyrosine nitration of histone deacetylase 2 in the lung, correlating with reduced histone deacetylase 2 activity and reduced glucocorticoid function. Histone deacetylase 2 activity and the antiinflammatory effects of glucocorticoids were restored in PI3Kδ kinase dead knock-in but not PI3Kγ knock-out smoke-exposed mice compared with wild type mice, correlating with reduced histone deacetylase 2 tyrosine nitration. Glucocorticoid receptor expression was significantly reduced in smoke-exposed mice, in smokers with normal lung function, and in patients with COPD.

Conclusions: These data show that therapeutic inhibition of PI3Kδ may restore glucocorticoid function in oxidative stress–induced glucocorticoid insensitivity.

Scientific Knowledge on the Subject

Glucocorticoid unresponsiveness in severe asthma chronic obstructive pulmonary disease may involve an oxidant-mediated impairment of glucocorticoid receptor alpha function through reduction of histone deacetylase activity and co-repressor expression.

What This Study Adds to the Field

Histone deacetylase 2 activity is reduced in smoke-exposed mouse lungs, correlating with reduced glucocorticoid function, which is restored by PI3Kγ but not PI3Kδ inhibition. GRα expression is reduced in smoke-exposed mouse lungs and in the lungs of patients with chronic obstructive pulmonary disease.

Glucocorticoid-mediated immunosupression is impaired in severe asthma and chronic obstructive pulmonary disease (COPD) compared with their effectiveness in mild/moderate asthma (1). Both these conditions have a strong component of oxidative stress that may contribute to the development of this reduction in glucocorticoid responsiveness (2); however, the precise molecular mechanism(s) of this impairment remain unclear.

Glucocorticoid antiinflammatory action is predominantly mediated through the glucocorticoid receptor–α (GR-α) transrepression of proinflammatory genes (3). GR-α transrepression is dependent on the recruitment of histone deacetylase 2 (HDAC-2), which facilitates the termination of gene expression through deacetylation of the core histone proteins and recondensation of the DNA (2, 4). Reduced HDAC-2 expression/activity is proposed to play a role in the chronic inflammation, relative glucocorticoid insensitivity, and disease severity of severe asthma and COPD (1, 5, 6). Indeed, knock-down of HDAC-2 expression in bronchoalveolar macrophages induces a relative glucocorticoid insensitivity, whereas HDAC-2 overexpression restores glucocorticoid function (7).

Oxidative stress not only reduces HDAC-2 activity and thereby glucocorticoid immunosupression (2) but also activates the phosphoinositol-3-kinases (PI3K) PI3K/Akt pathway (8). Furthermore, in vitro pharmacological restoration of glucocorticoid function has been shown in alveolar macrophages from patients with COPD using theophylline (9, 10), which can inhibit PI3K (11). PI3K generates secondary lipid signaling molecules, which regulate a plethora of cellular functions including cell growth, proliferation, motility, and survival (12). The class I PI3K isoforms γ and δ are predominantly expressed in leukocytes where PI3Kδ is activated by receptor tyrosine kinases and PI3Kγ by G protein–coupled receptors (12). Specifically, signaling pathways downstream of antigen receptors such as FcεRI are sensitive to PI3Kδ but not PI3Kγ inhibition, whereas chemokine-receptor signaling in leukocytes is sensitive to PI3Kγ inhibition (13). However, PI3Kδ and PI3Kγ can also function in the same pathway, such as in formylmethionylleucylphenylalanine-stimulated respiratory burst in neutrophils primed with tumor necrosis factor (TNF)-α (14). A number of studies have shown that both isoforms play key roles in innate and adaptive inflammatory responses, including lymphocyte signaling, mast cell–mediated allergic response, and neutrophil activation, making them attractive potential antiinflammatory targets (1320).

This study explores the impact of PI3K on glucocorticoid immunosuppression in the presence of oxidative stress in vitro and the roles of the γ and δ isoforms in vivo using transgenic mice in an animal model of cigarette smoke–induced relative glucocorticoid insensitivity. Furthermore, we investigate the relative expression of GR in cigarette smoke–exposed mice and peripheral lung tissue from smokers with normal lung function and patients with COPD. Some of the results of these studies have been previously reported in the form of an abstract (21, 22).

Cell Culture and Treatments

U937 cells were cultured in RPMI 1640 GlutaMAX media with 10% fetal calf serum and 5% l-glutamate. All cell culture reagents were purchased from Invitrogen (Paisley, UK) unless otherwise stated. The following reagents were used: H2O2 (Sigma Dorset, UK), LY294002 (Merck Biosciences, Nottingham, UK), budesonide (Sigma), and TNF-α (R&D Systems, Abingdon, UK). The following antibodies were used: Akt (New England BioLabs, Herts, UK); HDAC-2, GR-α, lamin A/C, and actin (Santa Cruz Biotechnology, Santa Cruz, CA); GAPDH, nitrotyrosine, and phosphoserine (Abcam, Cambridge, UK); and GR-β (Affinity Bioreagents, Rockford, IL).

Cigarette Smoke–induced Glucocorticoid Insensitive Mouse Model

The studies described herein were performed under a Project License issued by the UK Home Office, and protocols were approved by the Local Ethical Review Process. PI3Kδ kinase dead knock-in (PI3KδD910A/D910A) and PI-3Kγ knockout (PI3Kγ−/−) mice have been described previously (19, 20). Wild-type (BALB/c; WT) and PI3Kγ−/− and PI3KδD910A/D910A mice were exposed to cigarette smoke (5 × 1 R3F cigarettes/d) or room air on three consecutive days as previously described (23) and dosed with budesonide (1 mg/kg) or vehicle (saline with 2% N-methyl-2-pyrrolidione [NMP]) by intranasal administration 1 hour before exposure. Air-exposed animals were subjected to the exact treatment conditions and regime as the smoke–exposed animals. The budesonide dose that was selected inhibits ovalbumin-induced lung inflammation (24). Only high levels of glucocorticoid may be effective against cigarette smoke exposure; such levels are not therapeutically relevant (25). Animals were killed after 24 hours, and bronchoalveolar lavage and differential cell counts were performed as previously described (23).

Protein Extraction, Immunoblotting, and Immunoprecipitation

Cytosolic proteins were extracted using a hypotonic lysis buffer (10 mM Tris HCl [pH 6.5], 0.5 mM Na bisulfite, 10 mM MgCl2, 8.6% sucrose, 0.5% NP-40 phosphatase inhibitors, and protease inhibitors). Nuclear proteins were extracted using a high-salt extraction buffer (15 mM Tris HCL [pH 7.9], 450 mM NaCl, 10% glycerol, phosphatase inhibitors, and protease inhibitors) and nuclear extract salt concentrations normalized with 2 volumes of a Tris-glycerol buffer (15 mM Tris HCL [pH 7.9], 10% glycerol, phosphatase inhibitors, and protease inhibitors). Protein quantification was assessed by BCA assay (Perbio, Northumberland, UK). Immunoblotting and immunoprecipitation was performed as previously described (26). All blots were stripped and reprobed for loading controls as previously described (26).

ELISA and HDAC Activity Assay

Keratinocyte-derived chemokine (KC) and IL-6 and IL-8 levels were measured using Quantikine ELISA kits (R&D Systems), and HDAC activity was measured by HDAC activity assay kit (Biomol International, PA) according to the manufacturer's instructions.

Human Study Subjects

All subjects were recruited from the Section of Respiratory Medicine of the University Hospital of Ferrara, Italy, with approval by the local ethics committee of the University Hospital of Ferrara (Table 1). All the subjects were undergoing elective surgery for lung cancer, and COPD was diagnosed retrospectively; therefore, subjects were free from bronchodilator, theophylline, antibiotic, antioxidant, and/or glucocorticoid therapy in the last month before surgery. Pulmonary function tests were performed as previously described (27). COPD and chronic bronchitis were defined, according to international guidelines, as the presence of postbronchodilator FEV1/FVC ratio <70% or the presence of cough and sputum production for at least 3 months in each of two consecutive years, respectively (28). Lung tissue processing and immunohistochemistry was performed as previously described (27).

TABLE 1. CHARACTERISTICS OF SUBJECTS FOR THE STUDY*


Subjects

n

Age

Sex (M/F)

Smoking History

Pack-years

Chronic Bronchitis

FEV1 % Predicted

FEV1/ FVC (%)
Nonsmoker670.0 ± 0.3.04/2None0None102.2 ± 5.677.7 ± 3.1
Smoker967.3 ± 2.68/15 Ex, 4 Cur30.1 ± 5.56 yes, 3 no95.9 ± 6.176.0 ± 1.3
COPD
8
70.0 ± 1.6
8
4 Ex, 4 Cur
39 ± 5.2
6 yes, 2 no
85.3 ± 3.7
66.5 ± 1.0

Definition of abbreviations: COPD = chronic obstructive pulmonary disease; Cur = current smoker; Ex = exsmoker.

*For COPD and smokers with normal lung function, FEV1 % predicted and FEV1/FVC% are postbronchodilator values. Data are expressed as mean ± SEM.

Statistical Analysis

PI3Kγ−/− and PI3KδD910A/D910A mice were compared separately with their own WT control groups. The data are presented graphically with both WT groups combined; however, statistical analysis was performed using the appropriate WT group for both studies. For all experiments, the statistical analysis was first performed for all groups using a one-way ANOVA to determine statistical significant variance between the groups for each endpoint assessed. Statistical significance between groups was then calculated using t test (Mann-Whitney test). All statistical analysis was performed using GraphPad Prism software, and data are expressed as mean ± SEM. Differences were considered significant at P < 0.05.

Oxidative Stress Induces PI3K-Dependent Akt Phosphorylation and Reduced Glucocorticoid Function In Vitro

To confirm that oxidative stress activates PI3K, the monocytic cell line U937 was treated with a noncytotoxic concentration of hydrogen peroxide (H2O2), and Akt phosphorylation was assessed as a measure of PI3K activation. H2O2 (200 μM) induced Akt phosphorylation in a time-dependent manner, which was abolished by pretreatment with the pan PI3K inhibitor LY294002 (Figure 1A). U937 cells were stimulated with H2O2 to evaluate the impact of oxidative stress on inflammatory mediator production and thereafter repression by glucocorticoid. H2O2 elevated TNF-α stimulated IL-8 release and impaired dexamethasone function (Figure 1B). H2O2 exposure alone induced a small 0.5- to 1-fold increase in IL-8 release but augmented the levels of IL-8 release induced by TNF-α (Figure 1C). Dexamethasone significantly reduced IL-8 release in TNF-α–stimulated cells (49.4 ± 5.7%) but failed to reduce IL-8 levels in cells treated with H2O2 below that seen in TNF-α treatment alone (Figure 1C). Inhibition of PI3K using a low concentration of LY294002 had little impact on TNF-α–stimulated IL-8 release in H2O2–treated cells but restored dexamethasone suppression of IL-8 release (Figure 1C).

Inhibition of PI3Kδ Enables Glucocorticoid Suppression of Cigarette Smoke–induced Lung Inflammation

BAL neutrophil counts (Figure 2A) and whole lung tissue levels of the proinflammatory cytokines KC (the functional murine homolog of IL-8) and IL-6 (Figures 2B and 2C) were measured as markers of cigarette smoke–induced lung inflammation. Cigarette smoke exposure induced a marked inflammatory response with no significant differences in neutrophil number (Figure 2A) or cytokine lung tissue levels (Figures 2B and 2C) between the WT and PI3KδD910A/D910A or PI3Kγ−/− sham-exposed mice. Cigarette smoke exposure resulted in an influx of neutrophils into the lung and increased KC and IL-6 lung tissue levels. Budesonide treatment at 1 mg/kg failed to reduce the neutrophil influx or tissue cytokine levels in WT mice, confirming glucocorticoid insensitivity in this model (Figures 2B and 2C). However, budesonide treatment in PI3KδD910A/D910A mice, but not PI3Kγ−/− mice, reduced the neutrophil influx and lung tissue cytokine levels, indicating that the PI3Kδ signaling may play a role in the development of cigarette smoke–induced glucocorticoid insensitivity.

Inhibition of PI3Kδ Prevents Cigarette Smoke–mediated Reduction in HDAC Activity In Vivo

Oxidative stress, including cigarette smoke, can impair HDAC-2 activity, which is implicated in the development of glucocorticoid insensitivity (2, 26). Consistent with this, total nuclear HDAC and nuclear HDAC-2 activity was reduced in WT and PI3Kγ−/− mouse lungs (Figures 3A and 3B). However, in smoke-exposed PI3KδD910A/D910A mice, total nuclear and nuclear HDAC-2 activity was unaffected (Figures 3A and 3B). Budesonide treatment had no significant effect on total nuclear HDAC or HDAC-2 activity. There was no difference in nuclear HDAC-2 expression levels between any of the groups (data not shown), indicating that the observed reduction in HDAC-2 activity was due to reduced activity alone rather than to a reduction in expression. Oxidative stress, including cigarette smoke, can induce an elevation in posttranslational modification that can affect protein function (26, 29). Assessment of immunoprecipitated HDAC-2 revealed that tyrosine nitration and serine phosphorylation were elevated in the smoke-exposed WT mice but had no effect on PI3Kγ−/− (Figures 4A and 4B). However, tyrosine nitration and serine phosphorylation of HDAC-2 were reduced in smoke-exposed PI3KδD910A/D910A mice compared with WT control mice (Figures 4A and 4B), correlating with restored HDAC-2 activity and thereby suggesting that these cigarette smoke–induced modifications were, at least in part, responsible for the reduction in HDAC-2 activity. Budesonide treatment had no additional impact on tyrosine nitration of serine phosphorylation in any group.

GR Expression Is Reduced by Cigarette Smoke and Is Further Reduced in COPD

The reduction in glucocorticoid function in the smoke-exposed mice may also be due to an alteration in the expression and/or translocation of GRα. Neither PI3Kγ−/− nor PI3Kδ D910A/D910A affects GR expression or localization. Cigarette smoke exposure significantly reduced GRα protein expression but had no effect on PI3Kδ or PI3Kγ (Figures 5A and 5B). However, there was also no difference in the GRα cytosolic/nuclear ratios in the smoke-exposed mice with and without budesonide treatment (data not shown), indicating that budesonide-mediated GRα translocation is ineffective in smoke-exposed animals. To assess if this reduction in GRα expression was translated in humans in an oxidant-driven glucocorticoid insensitive disease, peripheral lung from patients with COPD, age-matched normal subjects, and smokers with normal lung function was assessed (Table 1). Immunohistochemical analysis demonstrated GRα staining of the bronchiolar and alveolar epithelial cells, bronchiolar smooth muscle cells, endothelial cells, and infiltrating cells and revealed no significant difference in localization between patients with COPD (Figures 6A–6F). Similar to the cigarette smoke–exposed mice, there was a significant reduction in the expression of GRα and GRβ protein in the peripheral lung smokers with normal lung function compared with nonsmokers (Figures 6G and 6H). The expression of GR-α and GR-β was further reduced in the peripheral lung of patients with COPD (Figures 6G and 6H).

PI3Kγ and PI3Kδ are proposed to be central in innate and allergic immune responses (12, 13). Oxidative stress from exogenous (cigarette smoke/pollution) and endogenous (inflammatory cell respiratory burst) sources is believed to have a major contribution to the progression of chronic inflammation (1, 30, 31). Oxidative stress may also contribute to the development of a chronic inflammation with relatively reduced glucocorticoid insensitivity (1, 30, 31). However, the role of PI3K in oxidant-mediated inflammation remains unknown. Our preliminary studies showed that oxidative stress activated PI3K in vitro and impaired glucocorticoid suppression of reduced proinflammatory mediator release. The impairment, rather than the total inhibition of glucocorticoid function, represent at least one glucocorticoid-sensitive and one relatively glucocorticoid-insensitive component in the assay and may reflect different mechanisms of GRα inflammatory gene expression. Inhibition of PI3K restored glucocorticoid function in oxidant stressed cells, implicating a potential role for PI3K in oxidant-induced relative glucocorticoid insensitivity. Transgenic mice, deficient in PI3Kγ or PI3Kδ activity, were then used in a model of cigarette smoke–induced relative glucocorticoid insensitivity to investigate the relative roles of these isoforms. Cigarette smoke exposure induced a similar inflammatory response in the lungs of PI3Kγ−/− and PI3KδD910A/D910A mice, indicating that, acutely, neither of these isoforms affects oxidant-induced inflammation. Pharmacological and transgenic studies show that PI3Kγ and PI3Kδ inhibition is in itself antiinflammatory (1420). However, cigarette smoke exposure represents a complex cocktail of oxidants and chemicals that is likely to mediate a complex inflammatory response (32). Indeed, recent evidence suggests that it may take 3 weeks before a reduction in cigarette smoke–induced inflammation is seen in vivo with PI3K inhibition (33). Therefore, 3 days of smoke exposure may not have been sufficient for any antiinflammatory effect of PI3Kγ−/− or PI3KδD910A/D910A to be seen. Budesonide pretreatment had no impact on the inflammatory response in PI3Kγ−/− mice but reduced the inflammatory response in PI3KδD910A/D910A mice, indicating that cigarette smoke–induced glucocorticoid insensitivity may be linked to activation of PI3Kδ-specific signaling. The specific signaling pathways of the PI3Kδ isoforms remain unclear.

Cigarette smoke exposure reduced GR expression in the mouse lungs but had no apparent effect on PI3K inhibition. Similarly, smokers with normal lung function also displayed reduced GRα expression, indicating that cigarette smoke reduces GRα expression. There was no significant elevation of GRα translocation in the mouse lungs with budesonide, indicating that cigarette smoke exposure may affect GRα translocation. However, glucocorticoid function was restored in PI3KδD910A/D910A mice, and therefore the reduction of GRα observed in this model, as with the reduction seen in the smokers, may not be sufficient to significantly affect glucocorticoid (GC) function. The reduction in GRα is therefore unlikely to be a primary mechanism of reduced glucocorticoid sensitivity seen in this model. GRα expression in the lungs of patients with COPD was further reduced compared with smokers with normal lung function, which may be due to the enhanced oxidative stress seen in COPD (1, 30, 31). This further reduction may take GRα levels below the threshold needed for effective glucocorticoid-mediated inflammatory repression and therefore, unlike the reduction seen in the smokers in this model, may play a role in the mechanism(s) of reduced glucocorticoid responsiveness in COPD. GRβ expression was also reduced in smokers and patients with COPD, indicating that negative regulation of GRα by GRβ is unlikely to play a significant role in the development of glucocorticoid insensitivity in patients with COPD (34).

HDACs play a critical role in the regulation of gene silencing but require recruitment to the site of gene expression by co-repressors, including GR-α (4). HDAC-2 activity is critical to GRα transrepression, and impaired HDAC-2 activity may be central in glucocorticoid insensitivity in severe asthma and COPD (1, 5, 6). Consistent with this, total nuclear HDAC and nuclear HDAC-2 activity in the lung were reduced in smoke-exposed mice. However, removal of PI3Kδ but not PI3Kγ activity protected nuclear total HDAC and HDAC-2 activity, correlating with the glucocorticoid insensitivity in smoke-exposed WT and PI3Kγ−/− but not PI3KδD910A/D910A mice. HDAC-2 expression remained unchanged in all groups, indicating that reduction of activity must be posttranslational rather than an effect on expression per se. We have previously shown that HDAC-2 is subject to oxidative modifications that may alter its activity and that hyperphosphorylation may disrupt HDAC-2 interactions with other co-repressors (26, 29, 35). Indeed, HDAC-2 tyrosine nitration and serine phosphorylation were elevated in smoke-exposed animals, which is consistent with the HDAC-2 activity and glucocorticoid function. Only the PI3KδD910A/D910A mice had reductions in both tyrosine nitration and phosphorylation. This correlation between HDAC-2 activity, modifications, and glucocorticoid function with PI3Kδ inhibition may therefore provide a potential mechanism and therapeutic target for the restoration of glucocorticoid function. However, the specific PI3Kδ signaling pathways or downstream targets that are distinct from PI3Kγ have not been elucidated and remain unclear. A direct/indirect pathway link between P13Kδ and HDAC-2 remains unclear and requires substantial future investigation that would include extensive immunoprecipitation and mass spectrometry with multiple inhibitor/siRNA studies. Inhibition of p38, ERK, cAMP, and calcium signaling using specific inhibitors had no impact on HDAC activity or glucocorticoid function in the in vitro oxidant-induced relative glucocorticoid insensitive assay (data not shown). These pathways are therefore unlikely to be involved in the PI3Kδ effects.

Combined, these data show that cigarette smoke confers a relative glucocorticoid insensitivity in the airways during cigarette smoke–induced inflammation that is protected by specific inhibition of the PI3Kδ by a mechanism that involves the restoration of HDAC-2 activity. Furthermore, cigarette smoke reduced GR expression, which is further reduced in COPD and may therefore represent an additional mechanism by which the chronic elevated oxidative stress in COPD impairs glucocorticoid function. Clinically, the development of PI3Kδ-specific inhibitors may provide a means of overcoming the relative glucocorticoid insensitivity induced by oxidative stress in conditions such as COPD and severe asthma that affect millions of patients worldwide and whose current therapy is suboptimal.

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Correspondence and requests for reprints should be addressed to Dr. John Marwick, Ph.D., National Heart & Lung Institute, Airways Disease Section, Imperial College London, Dovehouse Street, London SW3 6LY, UK. E-mail:

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