Oxidants induce a corticosteroid-insensitive phosphorylation of histone 3 at serine 10 in monocytes

Oxidative stress enhances inflammation and reduces the effectiveness of corticosteroids, but the inflammatory signalling pathways induced by oxidants remain ill-defined. Phosphorylation of histone 3 at serine 10 (H3-Pser10) marks out a subset of inflammatory genes for transcription, several of which are induced in oxidant-associated inflammation. However, the influence of oxidants or of corticosteroids on this modification remains unknown. We assessed the regulation of H3-Pser10 by oxidants and lipopolysaccharide (LPS) in human blood monocytes and lung macrophages and the effectiveness of its abolition in controlling inflammatory gene expression in cells from asthmatic subjects compared to corticosteroids alone. Both oxidants and LPS promoted the induction of H3-Pser10 which was unaffected by corticosteroids. The induction of H3-Pser10 was mediated through p38α mitogen-activated protein kinase (MAPK) and IκB kinase 2 (IKK-2) signalling. Consequently, inhibitors of p38α MAPK or IKK-2 used in combination with dexamethasone were more effective at controlling inflammatory gene expression from monocytes and lung macrophages from asthmatic patients than the corticosteroid alone. Therefore, reduction of H3-Pser10 by inhibition of p38α MAPK or of IKK-2 may provide greater anti-inflammatory control than corticosteroids alone in oxidant-associated inflammation such as severe asthma

Regulation of oxidant and antioxidant enzyme expression by TGF-[beta] in airway smooth muscle cells

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

TLR3/TAK1 signalling regulates rhinovirus-induced interleukin-33 in bronchial smooth muscle cells

Background: Asthma exacerbations are commonly associated with rhinovirus (RV) infection. Interleukin-33 (IL-33) plays an important role during exacerbation by enhancing Type 2 inflammation. Recently we showed that RV infects bronchial smooth muscle cells (BSMCs) triggering production of interferons and IL-33. Here we compared levels of RV-induced IL-33 in BSMCs from healthy and asthmatic subjects, and explored the involvement of pattern-recognition receptors (PRRs) and downstream signalling pathways in IL-33 expression.Method: BSMCs from healthy and severe and non-severe asthmatic patients were infected with RV1B or stimulated with the PRR agonists poly(I:C) (Toll-like receptor 3 (TLR3)), imiquimod (TLR7) and poly(I:C)/LyoVec (retinoic acid-inducible gene 1 (RIG-I)/melanoma differentiation-associated protein 5 (MDA5)). Knockdown of TLR3, RIG-I and MDA5 was performed, and inhibitors targeting TBK1, nuclear factor-κB (NF-κB) and transforming growth factor (TGF)-β-activated kinase 1 (TAK1) were used. Gene and protein expression were assessed.Results: RV triggered IL-33 gene and protein expression in BSMCs. BSMCs from patients with non-severe asthma showed higher baseline and RV-induced IL-33 gene expression compared to cells from patients with severe asthma and healthy controls. Furthermore, RV-induced IL-33 expression in BSMCs from healthy and asthmatic individuals was attenuated by knockdown of TLR3. Inhibition of TAK1, but not NF-κB or TBK1, limited RV-induced IL-33. The cytokine secretion profile showed higher production of IL-33 in BSMCs from patients with non-severe asthma compared to healthy controls upon RV infection. In addition, BSMCs from patients with non-severe asthma had higher levels of RV-induced IL-8, TNF-α, IL-1β, IL-17A, IL-5 and IL-13.Conclusion: RV infection caused higher levels of IL-33 and increased pro-inflammatory and Type 2 cytokine release in BSMCs from patients with non-severe asthma. RV-induced IL-33 expression was mainly regulated by TLR3 and downstream via TAK1. These signalling molecules represent potential therapeutic targets for treating asthma exacerbations

Metabolic re-patterning in chronic obstructive pulmonary disease airway smooth muscle cells

COPD airways are characterised by airway smooth muscle (ASM) thickening, partly due to ASM cell (ASMC) hyperplasia. Metabolic reprogramming involving increased glycolysis and glutamine catabolism supports the biosynthetic and redox balance required for cellular growth. We examined whether COPD ASMCs show a distinct metabolic phenotype that may contribute to increased growth. We performed an exploratory intracellular metabolic profile analysis of ASMCs from healthy non-smokers, healthy smokers and COPD patients, under unstimulated or growth conditions of transforming growth factor (TGF)-β and fetal bovine serum (FBS). COPD ASMCs showed impaired energy balance and accumulation of the glycolytic product lactate, glutamine, fatty acids and amino acids compared to controls in unstimulated and growth conditions. Fatty acid oxidation capacity was reduced under unstimulated conditions. TGF-β/FBS-stimulated COPD ASMCs showed restoration of fatty acid oxidation capacity, up-regulation of the pentose phosphate pathway product ribose-5- phosphate and of nucleotide biosynthesis intermediates, and increased levels of the glutamine catabolite glutamate. TGF-β/FBS-stimulated COPD ASMCs also showed a higher reduced to oxidised glutathione ratio and lower mitochondrial oxidant levels. Inhibition of glycolysis, and glutamine depletion attenuated TGF-β/FBS-stimulated growth of COPD ASMCs. Changes in glycolysis, glutamine and fatty acid metabolism may lead to increased biosynthesis and redox balance, supporting COPD ASMC growth.MRC-ABPI COPDMAP consortium G1001367/1This study was supported by the MRC-ABPI COPD-MAP consortium (G1001367/1) and a Dunhill Medical Trust grant (R368/0714). It was also supported by the NIHR Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London. The Canadian Respiratory Research Network (CRRN) is supported by grants from the Canadian Institutes of Health Research (CIHR), Institute of Circulatory and Respiratory Health; Canadian Lung Association (CLA); British Columbia Lung Association; and industry partners Boehringer-Ingelheim Canada, AstraZeneca Canada, Novartis Canada and GlaxoSmithKlin

Characteristics of non-severe and severe asthmatic subjects.

<p>^a6 severe asthmatic subjects were on oral prednisolone at the time of the study. Data shown as mean ± SEM.</p><p>Characteristics of non-severe and severe asthmatic subjects.</p

H3-Pser10 induction is associated with inflammatory gene induction and is sensitive to p38α MAPK and IKK2 inhibition but not by corticosteroid.

<p>Panels A to C show western blot analysis of H3-Pser10 expression of blood monocytes from a normal subject exposed to H₂O₂ (A), LPS (B) or to both H₂O₂ and LPs (C) in the presence of dexamethasone (DEX), budesonide (BUD) and prednisolone (PRED) Panels D-G show the mean data of the effect of SB239063, TPCA-1 and dexamethasone on the association of at the gene promoter of IL-6 (D), CXCL-8 (E), TNFα (F) and CCL-2 (G) with H3-Pser10 as assessed by ChIP of H3-Pser10 followed by qRT-PCR for the respective gene promoter in alveolar macrophages from non-severe and severe asthmatic subjects. Data is presented as the mean ± SEM. Severe asthmatics n = 5, non-severe asthmatics n = 11. * p<0.05, **p<0.01,*** p<0.001 as compared to LPS treated non-severe asthmatics; #p<0.05, ##p<0.01 as compared to LPS-treated severe asthmatics.</p

TGF-β regulates Nox4, MnSOD and catalase expression, and IL-6 release in airway smooth muscle cells

Reactive oxygen species (ROS) are generated as a result of normal cellular metabolism, mainly through the mitochondria and peroxisomes, but their release is enhanced by the activation of oxidant enzymes such as NADPH oxidases or downregulation of endogenous antioxidant enzymes such as manganese-superoxide dismutase (MnSOD) and catalase. Transforming growth factor-β (TGF-β), found to be overexpressed in airway smooth muscle (ASM) from asthmatic and chronic obstructive pulmonary disease patients, may be a pivotal regulator of abnormal ASM cell (ASMC) function in these diseases. An important effect of TGF-β on ASMC inflammatory responses is the induction of IL-6 release. TGF-β also triggers intracellular ROS release in ASMCs by upregulation of NADPH oxidase 4 (Nox4). However, the effect of TGF-β on the expression of key antioxidant enzymes and subsequently on oxidant/antioxidant balance is unknown. Moreover, the role of redox-dependent pathways in the mediation of the proinflammatory effects of TGF-β in ASMCs is unclear. In this study, we show that TGF-β induced the expression of Nox4 while at the same time inhibiting the expression of MnSOD and catalase. This change in oxidant/antioxidant enzymes was accompanied by elevated ROS levels and IL-6 release. Further studies revealed a role for Smad3 and phosphatidyl-inositol kinase-mediated pathways in the induction of oxidant/antioxidant imbalance and IL-6 release. The changes in oxidant/antioxidant enzymes and IL-6 release were reversed by the antioxidants N-acetyl-cysteine (NAC) and ebselen through inhibition of Smad3 phosphorylation, indicating redox-dependent activation of Smad3 by TGF-β. Moreover, these findings suggest a potential role for NAC in preventing TGF-β-mediated pro-oxidant and proinflammatory responses in ASMCs. Knockdown of Nox4 using small interfering RNA partially prevented the inhibition of MnSOD but had no effect on catalase and IL-6 expression. These findings provide novel insights into redox regulation of ASM function by TGF-β

Induction of H3-Pser10 by LPS and oxidants.

<p>Panels A-F show the induction of H3-Pser10 measured by western blot analysis in blood monocytes from a normal healthy subject, with the dose-dependent effect of LPS (A), time-course (B), the comparative effect of LPS and oxidants H₂O₂, GEA and pyocyanin (C), dose-dependency and time-course effect of H₂O₂ (D, E) and the interaction of H₂O₂ and LPS (F).</p