Effect of erythromycin on cigarette-induced histone deacetylase protein expression and nuclear factor-κB activity in human macrophages in vitro
Highlights
► EM increases cigarette-induced declines in HDAC1, 2, 3 protein expression. ► EM inhibits cigarette-increased NF-κB activity and protein expression. ► EM suppresses cigarette-mediated IL-8 and TNF-a protein release. ► EM increases HDAC expression which may be associated with inhibition of NF-κB and cytokine.
Introduction
In addition to their well-known antimicrobial activity, macrolide antibiotics possess anti-inflammatory and immunomodulatory properties that may confer beneficial effects to patients with respiratory diseases associated with chronic inflammation, including diffuse panbronchiolitis (DPB), cystic fibrosis (CF), asthma and chronic obstructive pulmonary disease (COPD) [1], [2]. Recently, macrolides have been proposed for the treatment of patients with moderate to severe stable COPD and acute exacerbations of COPD [3], [4]. The anti-inflammatory and immunomodulatory properties of macrolides are well documented; they have been postulated to reduce airway inflammation via several mechanisms [5], [6]. These effects include regulation of leukocyte function and the production of inflammatory mediators, control of mucus hypersecretion, resolution of inflammation, and modulation of host defense mechanisms. However, the molecular mechanism of the anti-inflammatory action of macrolides remains unclear.
COPD is a common and debilitating chronic inflammatory disease characterized by progressive airflow limitation that is poorly reversible [7]. In COPD, oxidative stress due to cigarette smoke exposure is considered to be the main etiologic factor in disease pathogenesis [8]. Cigarette smoking is the major factor for the ongoing inflammation in the airways and lung parenchyma, and the severity of airflow limitation is correlated with the degree of pulmonary inflammation [9]. Cigarette smoke causes airway inflammation by activating macrophages, neutrophils, and T lymphocytes, which release proteases and reactive oxygen species (ROS), leading to cellular injury [10], [11]. Alveolar macrophages are considered to be an important component in perpetuating the inflammatory responses to cigarette smoke; following exposure, they secrete many inflammatory mediators, oxidants, proteins and proteinases [12]. Thus, macrophages are thought to be the main orchestrators of the chronic inflammatory response and tissue destruction observed in patients with COPD [13]. IL-8 is a multifunctional cytokine that has significant neutrophil chemoattractant and activating properties. It is produced by a variety of inflammatory and pulmonary cell types in response to oxidative stress. IL-8 and proinflammatory cytokines such as TNF-a are important inflammatory mediators in COPD; both mediators are increased in the sputum of patients with COPD [14]. It has also been shown that alveolar macrophages from patients with COPD release increased levels of IL-8 and TNF-a [15].
Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are families of enzymes that regulate chromatin structure and thus affect inflammatory gene expression [16], [17]. The acetylation of core histones by coactivator proteins that possess intrinsic HAT activity leads to the unwinding of chromatin, which subsequently allows transcription factors and RNA polymerase II to switch on gene transcription. Conversely, the deacetylation of core histones is generally associated with transcriptional repression [18]. HDACs are key molecules for repressing the production of proinflammatory cytokines [19]. The HDAC enzyme family consists of 18 isoforms grouped into three classes. Various members of the class I HDAC family (HDAC1, -2, -3, -8, and -11) have been shown to play roles in regulating cell proliferation and inflammatory responses [20]. Recently, HDAC2 has been reported to be required for corticosteroid-mediated anti-inflammatory activity [21]. Corticosteroid resistance is known to occur in COPD; inflammation becomes unresponsive to treatment [22]. Even high doses of inhaled and oral glucocorticoids have no effect on the inflammatory cell and cytokine profile and fail to reverse the protease–antiprotease imbalance [23]. Bronchoalveolar lavage (BAL) macrophages isolated from patients with COPD also display resistance to corticosteroid-mediated suppression of inflammation [24]. It has been shown that there are decreases in HDAC activity and HDAC2 expression in peripheral lung and bronchial biopsy specimens and in alveolar macrophages from COPD patients; this is correlated with disease severity and with increased gene expression levels of IL-8. This may account for the amplified inflammation and resistance to corticosteroids that occur as COPD progresses [25].
COPD is characterized by the increased expression of multiple inflammatory genes that are regulated by proinflammatory transcription factors, such as NF-κB, that bind to and activate coactivator molecules, which then acetylate core histones to switch on gene transcription. Conversely, gene repression is mediated via HDACs and other corepressors [26]. Previous studies have shown that cigarette smoke causes a decrease in HDAC activity and in HDAC1, -2, -3 protein levels and upregulates NF-κB-dependent proinflammatory cytokine release in MonoMac6 cells [27]. Therefore, in this report, we evaluated the effects of erythromycin on CSE-induced HDAC activity and HDAC1, -2, -3 protein expression, the correlation with NF-κB activity, and proinflammatory cytokine synthesis in human macrophages.
Section snippets
U937 cell culture
The human monocytic cell line (U937) [28], [29], obtained from the ATCC (CRL-1593.2; Manassas, VA, USA), was grown in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 2 mM l-glutamine, 100 μg/ml penicillin, 100 U/ml streptomycin, 1% nonessential amino acids, 1% sodium pyruvate, 1 μg/ml human holotransferrin, and 1 mM oxaloacetic acid. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The
Effects of CSE and EM on the growth of human macrophages
Because CSE can be toxic to cells, we confirmed cell survival rates under the various conditions using MTT assays. The MTT assay demonstrated that neither CSE (0.1% and 1%) nor EM (0.1, 1 and 10 μg/ml) affected the proliferation of human macrophage cells (p > 0.05). Microscopically, no morphological changes were visible in either control cells or cells treated with CSE (0.1% and 1%) or EM (0.1, 1 and 10 μg/ml). However, 2.5% CSE affected the proliferation of human macrophage cells (p < 0.05).
Discussion
Macrolides have anti-inflammatory and immunomodulatory effects that appear to be the reason for the clinical benefits of DPB. A literature search was conducted for studies on the clinical effectiveness of macrolides in other chronic lung conditions [37]. Preliminary data from studies of patients with COPD have shown improvements in symptom scores and the forced expiratory volume in 1 s (FEV1) after macrolide treatment. It was recently reported that low-dose, long-term treatment with the
Acknowledgment
This study was supported by grants from the National Nature Science Foundation of China (30760085).
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