Accepted Manuscript Update on Molecular Mechanisms of Corticosteroid Resistance in Chronic Obstructive Pulmonary Disease Zhilong Jiang, Lei Zhu PII:

S1094-5539(16)30002-5

DOI:

10.1016/j.pupt.2016.01.002

Reference:

YPUPT 1514

To appear in:

Pulmonary Pharmacology & Therapeutics

Received Date: 1 September 2015 Revised Date:

14 January 2016

Accepted Date: 20 January 2016

Please cite this article as: Jiang Z, Zhu L, Update on Molecular Mechanisms of Corticosteroid Resistance in Chronic Obstructive Pulmonary Disease, Pulmonary Pharmacology & Therapeutics (2016), doi: 10.1016/j.pupt.2016.01.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Obstructive Pulmonary Disease

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Update on Molecular Mechanisms of Corticosteroid Resistance in Chronic

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Zhilong Jiang*, Lei Zhu* Department of Pulmonary Medicine Zhongshan Hospital, Fudan University No. 180, Feng Lin Road Shanghai 200032 China

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* Corresponding authors： Department of Pulmonary Medicine Zhongshan Hospital, Fudan University No. 180, Feng Lin Road Shanghai, China 200032 Email: [email protected] and [email protected] Tel.: +86 21 64041990 Fax: +86 21 64035399

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Abstract Chronic obstructive pulmonary disease (COPD) is an inflammatory and irreversible pulmonary disorder that is characterized by inflammation and airway destruction. In recent years, COPD has become a global epidemic due to increased air pollution and exposure to cigarette

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smoke. Current therapeutics using bronchiodialator and anti-inflammatory corticosteroids are most widely used for all patients with persistent COPD, but these approaches are disappointing due to

limited improvement in symptom control and survival rate. More importantly, a certain number of COPD patients are resistant to the corticosteroid treatment and thus their symptoms worsen.

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Therefore, more effective anti-inflammatory drugs and combinational treatment are required. Understanding of the underlying molecular and immunological mechanisms is critical to

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developing new therapeutics. Lung inflammation and the released pro-inflammatory cytokines affect glucocorticoid receptor (GR), histone deacetylase 2 (HDAC2) and surfactant protein D (SP-D) activities in many cell types. Macrophages, neutrophils, airway epithelial cells and lymphocytes are involved in the induction of corticosteroid resistance. This review updated the recent advances in molecular and immunological mechanisms of steroid resistance among patients and animal models with COPD. Meanwhile we discussed novel therapeutic approaches

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in controlling lung inflammation and improving corticosteroid sensitivity among the steroid resistant patients with COPD.

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Keywords: Chronic obstructive pulmonary disease (COPD), corticosteroid resistance,

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glucocorticoid receptor (GR), histone deacetylase 2 (HDAC2), surfactant protein D (SP-D)

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Introduction Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease, characterized by progressive lung tissue destruction, shortness of breath, chronic coughing and mucus production. Recent clinical surveys revealed that COPD accounted for 1.6% of all

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hospital admission in China and ranked fourth as a leading cause of mortality in urban areas and third in rural areas of China [1]. Due to the increased prevalence and incidence, COPD places a burden on employers and individuals in terms of lost productivity and lost income related to absenteeism, activity limitation, and disability [2]. Cigarette smoke (CS) and biomass fuel use

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are major contributors to the high incidence of COPD in China [1]. Other factors include occupational hazards and pathogen infections [3]. Mice with experimental COPD are

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predisposed to influenza infection. Rhinovirus (RV)-infected mice have high risk of COPD exacerbation [4, 5]. CS-exposed animals develop emphysema, characterized by irreversible alveolar destruction and airspace enlargement [6]. In CS-exposed A549 cells, a large amount of hydrogen peroxide (H2O2) is produced and responsible for cell apoptosis and autophagy [7, 8]. In additional to the debilitating effect on lung epithelial cells, CS acts on alveolar macrophages

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via oxidative stress, under which macrophage phagocytosis activity is largely suppressed [9, 10].

Under oxidative stress, multiple pro-inflammatory cytokines and chemokines such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta), interleukin-6

(IL-6),

CXCL8,

monocyte

chemoattractant

protein-1

(MCP-1),

matrix

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metalloproteinase-12 (MMP-12), macrophage inflammatory protein-2 (MIP-2), MIP-1alpha are increasingly produced from activated macrophages, airway epithelial cells, neutrophils,

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lymphocytes and other cells types. They contribute to the pathogenesis of COPD among patients and animal models [11, 12]. Anti-inflammatory drugs are major therapeutic agents for relieving COPD symptoms. Dexamethasone (Dex) is a synthetic corticosteroid and has been widely used for the treatment of asthma and COPD. However, its effectiveness was recently challenged by developing corticosteroid resistance among 30% of treated patients [13, 14]. A high dose of inhaled or systemic corticosteroid is required for symptom control, but in rare cases, high inhaled doses of steroids fail to control symptoms. Recent research suggests that pro-inflammatory cytokines and other mediators contribute to the development of corticosteroid resistance. In association with increased lung inflammation, the expression and activity of corticosteroid 3

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receptor (GR), histone deacetylase-2 (HDAC2) and other important molecules are reduced. This review updated the recent advances in molecular and immunological mechanisms of COPD and discussed the novel therapeutic approaches in overcoming corticosteroid resistance during the

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COPD treatment.

The role of inflammation in COPD

CS exposure creates great damage to lung epithelial cells and activates alveolar macrophages. The CS-exposed lung epithelial cells produce a greater amount of H2O2 and superoxide radicals,

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contributing to cell apoptosis [7, 15]. The CS-exposed lung develops emphysema and the lung parenchyma destruction is irreversible even after smoking cessation [7, 14, 16-18]. CS-exposed

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mice and human subjects have increased bronchoalveolar lavage (BAL) protein levels, hyperplasia of airway epithelial cells and alveolar-capillary barrier permeability [19, 20]. Current smokers have more pro-inflammatory cytokines than ex-smokers [21]. Multiple lung resident cell types such as airway epithelial cells, alveolar macrophages, smooth muscle cells and fibroblasts, are activated and release greater amount of cytokines and chemokines [22-24]. Serum IL-27 and IL-33 levels are elevated in serum, sputum and BAL of COPD patients and

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their expression levels are correlated to disease exacerbation [23, 25, 26]. In addition, IL-33 can up-regulate IL-6 and IL-8 expression in alveolar macrophages in ex vivo [27].

Studies among human COPD samples and mouse models indicate that neutrophils are major

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cell infiltrates in the inflamed lung of COPD. Other infiltrates include peripheral monocytes, CD4+ and CD8+ T cell lymphocytes are recruited into the lung and contribute to lung

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inflammation, tissues destruction and remodeling [28]. The activated neutrophils and alveolar macrophages releases multiple pro-inflammatory cytokines, chemokines and other mediators such as myeloperoxidase (MPO), TNF-alpha, IL-1beta, IL-6, IL-17, MCP-1, CCL2, CXCL2, CXCL5, CXCL8 [29, 30]. The increased production of MMP-9 can break down the extracellular matrix of lung tissues into small peptides that are subsequently recognized by T cells for T cell activation and infiltration [31, 32]. Macrophages constitute a heterogeneous cell population, composed of classically activated macrophages (M1 cells) and alternatively activated macrophages (M2 cells). There are altered macrophage phenotypes in the inflamed lung of COPD. Most of them are presented as intermediate phenotypes [33]. A study by Kunz et al 4

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indicated that the percentage of CD163+ M2 macrophages was increased, but the total number decreased among COPD ex-smokers compared to that of current smokers, indicating that smoking cessation affects macrophage phenotypes and related function [34]. In general, COPD smokers have dominant M2 type macrophages, characterized by high expression of MMP-2,

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MMP-7 and adenosine A3 receptor (ADORA3) [35]. However information about the dynamic changes and involvement of alveolar macrophage phenotypes is limited in the pathogenesis and inflammation of COPD. More investigation on patients and animal models will address these

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issues in the future.

Recent studies also indicated that CD4+ and CD8+ T cells play an important role in the

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pathogenesis of COPD via releasing IFN-gamma, TNF-alpha, serine protease and granzyme B. COPD exacerbation was inversely associated with the proportion of circulating CD4+ and CD8+ T lymphocytes, but positively associated with the lung T lymphocyte infiltrates. The differences are possibly caused by T cell extravasation from peripheral blood circulation into inflammatory sites and the circulating T cell apoptosis may result in decreases in circulating T cell numbers [36-38]. Hodge et al reported that bronchial brushing-derived CD8+ T cells were increased

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among COPD patients and the expression of TNF-alpha was elevated [39]. In vitro stimulation of the lung derived CD8+ T cells with IL-18 and IL-12 leads to greater production of IFNgamma and TNF-alpha from the stimulated CD8+ T cells [40]. The role of CD8+ T cells in the pathogenesis of COPD is also recently demonstrated in CD8 knock-out mice, in which long-term

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exposure to CS did not induce emphysema. Lung inflammation and cytokine expression, such as IFN-gamma-inducible protein-10 (IP-10) and MMP-12 were greatly reduced [41]. A low-dose of

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azithromycin can suppress CD8+ infiltration and airway inflammation in COPD patients [39]. Additional study in ex vivo showed that targeting CD8+ T cells, natural killer (NK) cells and , natural killer T (NKT)-like cells through anti-CD137 antibody can efficiently reduce IFNgamma, TNF-alpha and granzyme B from the isolated peripheral mononuclear cells (PBMC) [42]. Thus T lymphocytes are critically involved in the pathogenesis of COPD. Suppressing T lymphocyte activation and cytokine expression via molecular intervention may have therapeutic potential in COPD control.

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Additional study also indicated that there was increased expression of IL-17A in the infiltrating macrophages, neutrophils, NK cells, NKT cells and gamma delta T-cells after CS exposure [43]. As a major source of IL-17A, Th17 cells also play an important role in lung inflammation. A high number of Th17 cells was observed in the peripheral blood and BAL fluids

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of COPD patients. The Th17 cell number and IL-17 levels are related to disease severity [44-46]. In contrast, anti-inflammatory T lymphocytes, cytokines and mediators such as regulatory T cells, IL-10 and indoleamine 2,3-dioxygenase (IDO) are reduced among COPD patients [44, 47, 48]. Therefore, COPD progression is associated with increased pro-inflammatory responses, but

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reduced anti-inflammatory responses.

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The role of inflammation in corticosteroid resistance

Inhaled corticosteroids (ICSs) are anti-inflammatory agents and have become major therapeutic agents in symptom control, particularly those with bronchial reversibility. The antiinflammatory effects are thought to be a result of targeting myeloid macrophages among patients with COPD and other chronic inflammatory diseases [49].

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However ICSs have not been shown to prevent disease progression or reduce mortality in clinical trials. Around 30% of the treated patients are low and unresponsive to the steroid treatment. A body of evidence in clinics and animal models has confirmed the involvement of neutrophils, alveolar macrophages, lymphocytes and mast cells in the induction of corticosteroid

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resistance [13, 31, 50, 51]. Sputum neutrophils and bronchial CD8+ T cells cannot be reduced in persistent and ex-smokers in patients after short-term steroid treatment [52]. A high dose of ICSs

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has to be administered to partially achieve symptom control [50], but high doses of ICSs result in patient susceptibility to pneumonia, diabetes, dysphonia and other complications [53].

Extensive evidence showed that there are increased amounts of IL-8, MMP-9, phosphoinositide 3-kinase delta, macrophage migration inhibitory factor (MIF) and GR-beta in corticosteroid resistant patients than in steroid sensitive patients. In contrast, the activities of histone deacetylase 2 (HDAC2) [31] and mitogen-activated protein kinase phosphatase 1 [31, 54] are attenuated in steroid resistant patients. In ex vivo experiments showed that Dex failed to suppress lipopolysaccharide (LPS)-induced up-regulation of IL-6, IL-8, GM-CSF, MCP-1 and 6

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MMP-9 in alveolar macrophages isolated from the steroid resistant patients with COPD [13, 55]. Therefore, not only does inflammation greatly contribute to the pathogenesis of COPD, but it also induces steroid resistance. Neutrophils, lymphocytes and macrophages contribute to the development of steroid resistance. Thus these cells are potential cell targets for molecular

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intervention in overcoming steroid resistance.

Recent studies indicate that combinational treatment with other agents are more effective in improving steroid sensitivity. CXCR2 antagonists, phosphodiesterase 4 inhibitors (Roflumilast

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N-oxide, RNO), p38 mitogen-activating protein kinase inhibitors, and antibodies against IL-1 and IL-17 can increase steroid sensitivity via targeting neutrophils [31, 56]. Other agents such as rosiglitazone,

PPARgamma

agonist

(10-nitro-oleic

acid),

theophylline

and

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synthetic

phosphoinositide 3-kinase delta inhibitors are also effective in suppressing release of multiple pro-inflammatory cytokines from the cigarette smoke extract (CSE)-exposed macrophages [57, 58]. Resveratrol is an activator of sirtuins, has class III HDAC activity. Pre-incubation of the cultivated airway smooth muscle cells (HASMCs) of COPD with resveratrol can suppress CCL2, IL-6 and IL-8 expression after lipoteichoic acid (LTA)-exposure than pre-incubation with

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dexamethasone. Thus the results provided scientific rationale for the combinational use of antioxidant reagents with corticosteroids for increasing steroid sensitivity in the clinic [22].

The role of glucocorticoid receptor isoforms in corticosteroid resistance

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Inflammatory cytokines attenuate steroid sensitivity through modulating multiple protein expression and activity involved in corticosteroid signal pathways. The expression and activity

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of glucocorticoid receptor (GR) are crucial for efficient steroid-mediated anti-inflammatory function. GR is an intracellular receptor for corticosteroid ligand and is coupled with chaperone protein heat shock protein 90 (HSP90) to avoid protein degradation. After GR is bound to the corticosteroid, HSP90 is released to promote GR maturation and binding to glucocorticoid response element (GRE) [59]. Two GR isoforms including GR-alpha and GR-beta exist. GRalpha is abundantly localized in the cytoplasm and suppresses activation of the pro-inflammatory transcription factor NF-kappaB through up-regulation of IKB-alpha and direct binding to NFkappaB. NF-kappaB exerts pro-inflammatory effects through up-regulation of histone acetyltransferase (HAT) and formation of NF-kappaB/HAT complex. After GR-alpha is 7

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activated via engagement with ligand steroid, GR-alpha can suppress formation of NFkappaB/HAT complex via increasing HDAC2 recruitment to the protein complex, subsequently leading to deacetylation of histones on the promoter region of key pro-inflammatory cytokines (Figure 1) [60, 61]. In contrast, GR-beta is less abundant and can directly bind to GRE with no

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requirement of ligand [62]. Thus GR-beta is considered an antagonist of GR-alpha and counteracts the anti-inflammatory activity of GR-alpha. In vitro studies have indicated that TNFalpha, IL-1beta, IL-17 and IL-23 can up-regulate GR-beta in PBMCs and other cell types [63, 64]. There is elevated GR-beta expression in the patients infected with respiratory syncytial virus

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(RSV) and patients with severe allergic rhinitis [65, 66]. However information about the GR-beta expression in COPD patients is limited, but a few studies showed that expression and activity of

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GR-alpha was reduced in alveolar epithelial cells and macrophages after exposure to CSE [57, 58]. In addition, GR activity was reduced or lost in CD8+CD28-/- T cells of COPD patients. Meanwhile IFN-gamma and TNF-alpha release were increased from T and NKT-like cells. Thus GR isoform affects steroid sensitivity that can be improved by up-regulation of GR-alpha and down-regulation of GR-beta in alveolar macrophages and T lymphocytes [67].

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The role of GR post- translational modification in corticosteroid resistance The GR activity is critically influenced by post-translational modification. Recent studies have revealed that differential GR post-translational modifications such as phosphorylation, acetylation and small ubiquitin-like modifier 1 (SUMO1) affect GR activities [58, 60, 68, 69].

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Thus a balanced GR post-translational modification is critical for effective GR-mediated antiinflammatory effects. Multiple stimuli such as pro-inflammatory cytokines and oxidative stress

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modulate GR activity through post-translational modification.

HSP90 is an important protein for GR stability and maturation. Under physiological conditions, HSP90 and cochaperone p23 are coupled with GR to prevent GR from degradation. However oxidative stress increases HSP90 acetylation and attenuates the protective activity of HSP90. HDAC6 activity is an important deacetylase. Under oxidative stress, HDAC6 activity is reduced and HSP90 acetylation is increased [59, 70, 71]. In addition, GR activity is critically influenced by post-translational phosphorylation. GR phosphorylation at distinct residues affect GR activity differentially [58]. GR phosphorylation at Ser211 residue promotes GR nuclear 8

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transportation and transcriptional activity; whereas GR phosphorylation at Ser226 and Ser203 residues increase GR nuclear export and suppress nuclear transport [72, 73]. IL-2 and IL-4 are potent inducers of GR phosphorylation. In steroid resistant patients with asthma and Crohn's disease, GR phosphorylation is increased in association with reduced GR binding and nuclear

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transportation activities in PBMC and epithelial cells. The p38 mitogen-activated kinase (MAPK) and transcription factor 2 are activated [68, 74]. However there is limited information about the effects of GR phosphorylation on steroid resistance in COPD.

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In addition to the critical role of GR phosphorylation in GR activity, aberrant GR acetylation affects GR activity and steroid sensitivity. Under physiological conditions, GR

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activity inversely oscillates in response to diurnal changes of serum glucocorticoid levels. A balanced GR activity is important for antagonizing the biological action of diurnally fluctuating circulating glucocorticoids [75, 76]. Circadian genes Clock and brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1(Bmal1) form protein complex and control the circadian rhythm activity of GR through affecting GR acetylation [77, 78]. In CS-exposed mice, Bmal1 expression and activity were reduced and subsequently decrease GR activity. CXCL5

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expression was elevated from alveolar macrophages and neutrophil infiltrates were increased in the CS-exposed mice. The decreased steroid sensitivity was also observed in Bmal1 knock-out mice after exposure to aerosolized LPS [79]. The silent mating type information regulation 2 homologue 1 (SIRT1), a class III histone deacetylase is potent activator of Bmal1 and other

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intracellular mediators. Under oxidative stress, SIRT1 activity is suppressed and correlated with the reduced Bmal1 activity [80]. However, so far there is limited information of altered GR

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acetylation in COPD patients and effect of SIRT1 on GR acetylation and activity.

The role of histone deacetylases (HDACs) in corticosteroid resistance Similar to SIRT1, histone deacetylases (HDACs) are potent deacetylases and critically involved in the biological activities of many transcription factors, receptors and histones. The expression levels and activities of HDACs are critically associated with their pro-and antiinflammatory effects. HDACs are currently divided into class I (1, 2, 3, 8), class II (4, 5, 6, 7, 9, 10) and class IV (11). Different classes of HDACs have distinct activities via epigenetic modification of histone H2A, H2B, H3 and H4 [81]. HDAC7 is pro-inflammatory and promotes 9

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toll-like receptor 4-dependent IL-12p40 and IL-6 expression [82], whereas HDAC6 and HDAC11 exert pro-inflammatory effects through suppressing anti-inflammatory gene IL-10 expression in antigen presenting cells (APC) [83]. In contrast, HDAC2 and HDAC3 have antiinflammatory properties through suppressing pro-inflammatory gene expression. During pro-

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inflammatory responses, NF-kappaB is activated and forms protein complex with the CREB binding protein (CBP). Recruitment of HAT promotes histone acetylation and pro-inflammatory gene transcription, such as TNF-alpha, IL-8 and IL-1beta, etc. HDAC2 and HDAC3 counteract with HAT activity through deacetylation of histone on target gene promoter region, and decrease

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transcriptional activity via tightening chromatin [13, 60, 81, 84-90]. In addition, HDAC2 promotes phagocytosis activity of alveolar macrophages and facilitates the clearance of invading

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pathogens [10]. Thus a balanced activity between HAT and HDAC2 is important for effective suppression of inflammation and inflammation resolution [8, 91-94].

A body of evidence has indicated the important role of HDAC2 in the induction of steroid resistance. HDAC2 is greatly reduced in alveolar macrophages of patients with COPD and smokers, the activity is correlated to disease severity and exacerbation [13, 54, 60, 85]. Li M et al

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studies in vitro showed that CSE exposure to human macrophages significantly reduced HDAC2 expression, but increased NF-kappaB-dependent pro-inflammatory cytokine expression such as IL-8 and TNF-alpha [95]. HDAC2 expression was also reduced in PBMCs of patients with COPD and smokers [96]. Oxidative stress is a major factor in HDAC2 reduction. Nitric oxide

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and superoxide in macrophages reduce HDAC2 activity through inducing S-nitrotyrosine and aldehyde-adduct modification of HDAC2. Exposure to CSE greatly reduced HDAC2 activity via

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S-nitrotyrosine modification at Cys262 and Cys274 residues in human macrophage-like cell lines [13, 88].

The reduced HADC2 is considered a major factor in the induction of steroid resistance in patients with COPD. A low acetylated GR is required for effective suppression of protein complex formation with NF-kappaB and AP-1. Oxidative stress reduces HDAC2 and GR activities through increasing protein post-translational modification, but it is still elusive whether oxidative stress modulates GR activity through other deacetylase such as SIRT1. Modulation of HDAC2 activity has recently emerged as a major therapeutic strategy in the treatment of 10

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inflammatory diseases as well as an improvement in steroid sensitivity. Theophylline is a potent HDAC activator and can enhance steroid sensitivity in patients with COPD. Cosio et al reported that a low dose of theophylline treatment significantly suppressed NF-kappaB activity, sputum levels of TNF-alpha, IL-6 and IL-8 in the COPD patients with exacerbation [97]. Alveolar

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macrophages are major targets for the HDAC2 activator treatment. Sulforaphane is a smallmolecule activator of transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and can restore steroid sensitivity through increasing HDAC2 activity. Alveolar macrophages isolated from steroid resistant patients with COPD demonstrated greater responses to the steroid

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after the treatment with sulforaphane in vitro [13]. The beneficial effects are considered in association with elevated Nrf2 binding to anti-oxidant responsive element (ARE) of anti-oxidant

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gene and increased expression of anti-oxidant gene proteins such as heme-oxygenase-1 (HO-1) [87, 98]. The anti-oxidant gene protein can increase HDAC2 activity through induction of HDAC2 denitrosylation. A variety of anti-oxidants, such as Chinese herbs, fruits, dietary polyphenols including curcumin, resveratrol, and green tea catechins and quercetin, have been used in the treatment of autoimmune and inflammatory diseases. They are also confirmed effective in increasing steroid sensitivity through restoring HDAC2 activity. For example,

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monocytes treated with curcumin treatment have restored HDAC2 activity after CSE exposure [86]. Thus anti-oxidants have effective therapeutic potential in overcoming steroid resistance among patients with COPD and other inflammatory diseases.

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The role of surfactant D in COPD

The role of surfactant protein D (SP-D) in the pathogenesis of COPD and its possible

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involvement in the induction of steroid resistance are not well defined so far. SP-D is a family member of proteins termed collagen-like lectins. It is mainly produced from type II alveolar epithelial cells. Recent studies have revealed that SP-D plays an important role in modulating innate and adaptive immune responses under physiological and pathological conditions. A body of evidence indicated that SP-D can facilitate uptake/binding by alveolar macrophages via interaction with pathogens and cell debris, promote macrophage phagocytosis and facilitate inflammation resolution [99-102]. In addition, SP-D suppresses adaptive immune responses in the inflamed lung tissues to avoid excess inflammation-induced lung tissue injury [103, 104]. Recent observation has revealed that SP-D exerts the immune modulatory effects via interaction 11

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with dendritic cell (DC)-specific ICAM-grabbing non-integrin (DC-SIGN) and transmembrane receptor signal inhibitory regulatory protein alpha (SIRP-alpha) on antigen presenting cells [99, 105]. The role of SP-D in immune responses is well studied in the asthma animal model, but there is limited information on COPD. SP-D deficient mice developed more severe asthma than

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wild type mice after the allergen challenge. Th2 cytokines IL-4 and IL-13 were highly produced in the SP-D deficient asthmatic mice [106], indicating the potent role of SP-D in protecting mice from developing Th2 type allergic responses.

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Recent studies in clinic and animal models have revealed an elevation of serum SP-D level in COPD and smokers. In contrast, SP-D was reduced in the lung tissues, but it was reversible after

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inhaled corticosteroid treatment [107-109]. The serum SP-D level is correlated with symptom severity, thus serum SP-D level has been currently considered a risk biomarker for COPD [108]. Multiple factors affect SP-D level, including corticosteroids, TNF-alpha, IL-4, IL-6 and IL-13 [104]. Up-regulation of SP-D by corticosteroid has been demonstrated in vitro and in vivo studies. However SP-D promoter lacks GRE, our previous study has observed that SP-D upregulation by corticosteroids was mediated by GR-tethered c/EBP-beta binding to c/EBP binding

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site at proximal region of SP-D promoter (Jiang Z, et al. Am J Respir Crit Care Med 2014, 198: A2355). In addition, the elevated serum SP-D may be caused by increased alveolar-capillary barrier permeability and leakage of pulmonary SP-D into blood system, due to the increased lung epithelial cells and endothelial cell apoptosis under oxidative stress [19, 110, 111]. It is not

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excluded that SP-D may be down regulated in type II alveolar epithelial cells under oxidative stress that was demonstrated in LPS-induced rat lung injury [109], but the reduction of SP-D

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expression cannot be explained merely by altered gene expression and transportation, other mechanisms maybe involved in the process. It is possible that oxidative stress in COPD affects SP-D biological function. A multimeric structure is required for efficient binding of SP-D to cell receptors, however studies in vitro showed that oxidative stress can dissemble multimeric SP-D into a monomeric structure that is not functional and even has pro-inflammatory and chemoattractive property [112]. Thus oxidative stress promotes lung inflammation that may be in association with altered SP-D expression and biological function. Given the role of SP-D in modulating lung inflammation, the reduced pulmonary SP-D may contribute to the induction of steroid resistance not only in COPD, but also in the inflammation-related other lung diseases, 12

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such as acute lung injury and acute respiratory distress syndrome (ARDS). Modulation of SP-D expression and activity may be a potential therapeutic approach in controlling lung inflammation and improving steroid sensitivity among smokers and patients with COPD.

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Conclusion and Perspective

In patients with COPD and smokers, oxidative stress activates lung epithelial cells and alveolar macrophages, from which a variety of pro-inflammatory cytokines and other mediators are released. These inflammatory cytokines are potent mediators for the induction of steroid

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resistance during steroid therapy by suppressing GR-alpha and HDACs activity. Pulmonary SPD has both an immune protection function against pathogen infection and an immune regulatory

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property to prevent tissue damage during lung inflammation. However, there are unbalanced levels of SP-D in the inflamed lung and blood stream of patients with COPD and smokers. The altered SP-D expression level potentially causes the development of steroid resistance. Oxidative stress may play an important role in the alteration of SP-D levels via affecting alveolar-capillary barrier permeability, SP-D gene expression and biological function. Anti-oxidants and some transcription factor activators of anti-oxidant genes can suppress variety of pro-inflammatory

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cytokine gene expression, greatly improve steroid sensitivity. The beneficial effects are related to the increased expression and activities of HDACs and GR-alpha in the target immune cells. Thus targeting HDACs and GR-alpha activities and modulation of SP-D expression through molecular intervention have therapeutic potential in overcoming steroid resistance among patients with

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COPD.

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Acknowledgements

The work was supported by the National Natural Science Foundation of China to L.Z. (No. 81270137) and Research Grant from Zhongshan Hospital, Fudan University of China to Z.J. We thank Kelly Yiting Jiang for her scientific editing assistance in the preparation of this manuscript.

Disclosure The authors declare no competing financial interests. None of the authors affiliated with this manuscript have any commercial or associations that might pose a conflict interest.

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Declaration of Interest The authors report no conflicts of interest. The author alone are responsible for the content and writing of the paper.

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Legends

Figure 1. Schematic diagram of steroid resistance in the patients with COPD and smokers. Cigarette smoke exposure and pro-inflammatory stimuli activate p38MAPK, but attenuate HDAC2 expression and activity in alveolar macrophages and epithelial cells through oxidative stress. The GR-alpha expression and activity are reduced, but GR-beta expression is enhanced, 19

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lead to enhanced activation of NF-kappaBp65 and unresponsiveness to corticosteroid. The attenuated GR-alpha activity negatively affects transactivation and transrepression of steroid through different signaling pathways: complex protein tethered GR-alpha indirect binding and

Abbreviations Acute respiratory distress syndrome (ARDS) Adenosine A3 receptor (ADORA3) Airway smooth muscle cells (HASMCs)

Antigen presenting cells (APC) Anti-oxidant responsive element (ARE)

M AN U

Alternatively activated macrophages (M2 cells)

SC

RI PT

direct GR-alpha binding to GRE of target gene promoters.

Brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1(Bmal1) Bronchoalveolar lavage (BAL)

Chronic obstructive pulmonary disease (COPD)

TE D

Cigarette smoke (CS) Cigarette smoke extract (CSE)

Classically activated macrophages (M1 cells) Corticosteroid receptor (GR)

EP

CREB binding protein (CBP)

Dendritic cell (DC)-specific ICAM-grabbing non-integrin (DC-SIGN)

AC C

Dexamethasone (Dex)

Glucocorticoid response element (GRE) Heme-oxygenase-1 (HO-1)

Histone acetyltransferase (HAT) Histone deacetylase-2 (HDAC2) Hydrogen peroxide (H2O2) IFN-gamma-inducible protein-10 (IP-10) Indoleamine 2,3-dioxygenase (IDO) 20

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Inhaled corticosteroids (ICSs) Interferon-gamma (IFN-gamma) Interleukin-1beta (IL-1beta)

RI PT

Interleukin-6 (IL-6) Lipopolysaccharide (LPS) Lipoteichoic acid (LTA) Macrophage inflammatory protein-2 (MIP-2)

SC

Matrix metalloproteinase-12 (MMP-12) Monocyte chemoattractant protein-1 (MCP-1)

Natural killer (NK) cells Natural killer T (NKT) cells

M AN U

Myeloperoxidase (MPO)

Nuclear factor erythroid 2-related factor 2 (Nrf2) p38 mitogen-activated kinase (p38MAPK) Peripheral mononuclear cells (PBMC)

Rhinovirus (RV)

TE D

Respiratory syncytial virus (RSV)

Roflumilast N-oxide (RNO)

EP

Shock protein 90 (HSP90)

Signal inhibitory regulatory protein alpha (SIRP-alpha)

AC C

Silent mating type information regulation 2 homologue 1 (SIRT1) Small ubiquitin-like modifier 1 (SUMO1) Surfactant protein D (SP-D)

Tumor necrosis factor-alpha (TNF-alpha)

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Cigarette smoking (CS) Pro-inflammatory stimuli Corticosteroid (GC)

p38 MAPK

SC

HDAC2 Acetyl

M AN U

GRα

p65

IkBα

IL-10 IL-1Ra

TE D

+

HDAC2

-

Co-activator HAT

+

TSLP

AC C

SP-D

c/EBP

CXCL5 GM-CSF

p65

EP

Co-activator HAT

GRβ

p65

STATs

Co-activator HAT

Co-activator HAT

Figure 1