Role of inducible Nitric Oxide Synthase and Histone Deacetylase in Human Lung Epithelial Cell Model of Inflammation and Steroid Insensitivity

One of the hallmarks of chronic obstructive pulmonary disease (COPD) is chronic systemic and local inflammation of the lung. A majority of COPD patients show insensitivity to inhaled corticosteroids, which are usually very effective drugs for the treatment of chronic lung inflammation. Expression of inducible NO synthase (iNOS) has been shown in the lung epithelial, inflammatory and skeletal muscle cells of COPD patients and correlates well with increased NO and peroxynitrite (ONOO-) production, leading to increased nitrative stress and amplified inflammation in COPD. iNOS inhibitors have been shown to potently block inflammation in a tobacco smoke-induced steroid-insensitive mouse model of COPD. Furthermore, iNOS and iNOS-derived NO and ONOO- has been hypothesized to be involved in steroid insensitivity through tyrosine nitration of histone deacetylase 2 (HDAC2), as the anti-inflammatory action of corticosteroids works, at least partially, via HDAC-mediated histone deacetylation and reduction of the inflammatory gene transcription. In contrast, HDAC inhibition by HDAC inhibitors revealed potent anti-inflammatory effects in a wide variety of animal and cell models, which suggests an even more complex situation.The objectives of the present study were i) to characterize several human lung epithelial cell models including A549, BEAS-2B and MucilAir cells for their applicability to study lung inflammation in vitro; ii) to evaluate iNOS mRNA expression and NO production in the human lung epithelial cell line A549 and to study the anti-inflammatory effects of specific iNOS inhibitors in these cell models; iii) to identify an iNOS expressing lung epithelial cell model of steroid insensitivity and to test the hypothesis that iNOS is involved in steroid insensitivity by modulating iNOS activity and expression as well as NO/ONOO- level; iv) to analyze a putative connection between steroid sensitivity of cytokine release and HDAC inhibition; v) to explore the functional effects of HDAC inhibitors in lung epithelial cell models of inflammation by assessing release and gene expression of pro-inflammatory cytokines and chemokines, and elucidating histone hyperacetylation at IL-8 promoter region.Initially, stimulation of human lung epithelial cells using various cytokine stimuli such as IL-1beta and TNF-alpha induced the production and mRNA expression of amounts of pro-inflammatory cytokines and chemokines substantially, suggesting the critical role of lung epithelial cells in lung injury and also in regulating the inflammatory responses together with immune cells in the lung.Previously, potent inhibitory effects of several iNOS inhibitors on inflammatory cell influx (neutrophils, alveolar macrophages and T lymphocytes) into the lung and accumulation of various cytokines and chemokines [MIP-1alpha, MCP-1 and KC (IL-8 homologue)] in BALF (bronchoalveolar lavage fluid) were shown using a clinically relevant tobacco smoke mouse model of COPD. With the intention to elucidate these anti-inflammatory effects of selective iNOS inhibitors in lung epithelial cells, iNOS expression and NO production by A549 cells in response to different cytokine stimuli were characterized. The combination of IL-1beta, TNF-alpha and IFN-gamma as cytokine-mix (CM) upregulated iNOS mRNA expression and iNOS-derived NO production in A549 cells substantially and dsRNA further enhanced this induction. We could show that highly selective and potent iNOS inhibitor BYK191023 inhibited IL-8 release and gene expression in CM-stimulated A549 cells. This effect was verified by BYK402750, another selective iNOS inhibitor, by reducing release of various cytokines and chemokines in CM-stimulated lung epithelial cell line BEAS-2B and primary lung epithelial cells MucilAir.Subsequently, a cellular model of steroid insensitivity of IL-8 release using cytokine mix (CM) stimulated A549 cells was established and characterized. CM-stimulated A549 cells showed steroid insensitivity and concomitantly expressed iNOS. They also demonstrated reduction of HDAC activity upon exposure to exogenous nitrative and oxidative stress caused by SIN-1 and H2O2. Using multiple analyte profiling (Luminex) steroid insensitivity of cytokine release was shown for a wide variety of cytokines and chemokines, including GM-CSF and IL-2R. However, modulating iNOS or the level of NO/ONOO- did not affect steroid responsiveness of cytokine release, neither by using selective iNOS inhibitors or iNOS overexpression nor by usage of the NO/ONOO- donor SIN-1 in presence or absence of H2O2. Additionally, steroid sensitivity in BEAS-2B cells and in MucilAir cells was analyzed using identical conditions and preliminary data suggested steroid-insensitive responses of IL-6, IL-8 and GM-CSF release in primary lung epithelial cells but not in BEAS-2B cells. Thus, iNOS does not seem to play a role in steroid insensitivity, at least in the CM-stimulated A549 cell model used.Furthermore, the dexamethasone-mediated anti-inflammatory effects on cytokine release were not changed by HDAC inhibition in lung epithelial cells by addition of various HDAC inhibitors, again suggesting an HDAC independent mode of action of steroid under the conditions used.Studying functional effects of HDAC inhibitors on pro-inflammatory mediator release and mRNA expression in cytokine-stimulated human lung epithelial cells indicated that HDAC inhibitors can act both anti- and pro-inflammatory depending on the HDAC inhibitor used, on the cell type (primary cells vs. cancer cell line) and on the target gene, which may suggest different target gene/promoter-specific mode of action of HDAC inhibitors. For some of the cytokines and chemokines studied, this effect was partially regulated at the level of mRNA expression, which may involve the induction of histone H3 hyperacetylation at the IL-8 promoter.In conclusion, the present study demonstrated that iNOS inhibitors show anti-inflammatory effects on the release of specific cytokines and chemokines in human lung epithelial cells, which may help to define the rationale for the development of potent iNOS inhibitors for the treatment of lung inflammation. Nevertheless, our results suggest that iNOS does not influence steroid sensitivity, at least in the cell model used, and that steroid sensitivity of cytokine release in cell model used may be independent of HDAC activity. Moreover, HDAC inhibition revealed both pro- or anti-inflammatory effects, depending on the HDAC inhibitors studied and gene targeted. The induction of histone H3 hyperacetylation at the promoter of IL-8 gene may help to explain the mode of action of SAHA in its functional effects in inflammation