ncRNAs as biomarkers and therapeutic targets for bronchiectasis

According to a recent statement from the Global Alliance for Chronic Diseases (Lung Diseases group) the effective management of chronic lung disease requires timely and accurate diagnosis. Whilst lack of access to appropriate standard diagnostics is a key barrier to the implementation of guideline-care for chronic obstructive lung disease (COPD), for example, especially in low and middle income countries, the problem is global. Compared to COPD, this is even more pronounced for bronchiectasis. There is a paucity of tools to test for bronchiectasis and clearly new methods to diagnose this disease are much needed; non-coding RNAs (ncRNA) hold great potential to fulfill this role.</p

ncRNAs as biomarkers and therapeutic targets for bronchiectasis

According to a recent statement from the Global Alliance for Chronic Diseases (Lung Diseases group) the effective management of chronic lung disease requires timely and accurate diagnosis. Whilst lack of access to appropriate standard diagnostics is a key barrier to the implementation of guideline-care for chronic obstructive lung disease (COPD), for example, especially in low and middle income countries, the problem is global. Compared to COPD, this is even more pronounced for bronchiectasis. There is a paucity of tools to test for bronchiectasis and clearly new methods to diagnose this disease are much needed; non-coding RNAs (ncRNA) hold great potential to fulfill this role.</p

Knockdown of interleukin-8 in airway epithelial cells

Introduction: Cystic fibrosis (CF) is a neutrophil-dominated lung disease. The neutrophil chemokine interleukin-8 (IL-8) is present at higher than normal levels in the lungs of individuals with CF and is a key factor responsible for neutrophil infiltration into the lungs. Lipopolysaccharide (LPS) expressed by Pseudomonas aeruginosa in the CF lung can stimulate IL-8 expression by airway epithelial cells. Inhibiting the expression of IL-8 using small-interfering RNA (siRNA) represents a potential gene therapy approach for CF. Here, the efficacy of an IL-8-directed siRNA at inhibiting IL-8 gene and protein expression is tested in basal and LPS-stimulated human bronchial epithelial cells. Methods: 16HBE14O- cells were transfected with either scrambled, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or IL-8-specific siRNAs. RNA was then isolated and used in quantitative real-time PCR to measure GAPDH, IL-8 or beta-actin (housekeeping) gene expression. Lastly, IL-8 protein production was quantified in cell supernatants by IL-8 ELISA. Results: Transfection of 16HBE14O- cells with 30nM IL-8 siRNA for 48 hours led to an 84% knockdown in IL-8 mRNA (p Conclusions: The data show that IL-8 siRNA can inhibit basal and LPS-induced IL-8 gene expression in 16HBE14O- cells. Further investigation is required to optimise the conditions for inhibition of IL-8 protein production, and to study its potential role in CF therapy.</p

Protein quality control in lung disease: it's all about cloud networking.

Protein quality control involves the comprehensive management of protein function in the cell and is called “proteostasis” [1]. It ranges from translation and chaperone-assisted three-dimensional folding, interaction with protein partners, signal-induced post-translational modifications to disposal by the proteasome or autophagy pathways. Dysfunctional protein quality control is emerging as a key pathogenic mechanism for chronic lung diseases. Two major hereditary conformational disorders of the lung, cystic fibrosis and α1-antitrypsin (α1-AT) deficiency, and some familial forms of idiopathic pulmonary fibrosis (IPF) are caused by the expression of mutant and misfolded proteins that disrupt protein homeostasis and drive the onset of pulmonary diseases [2, 3]. Disturbed proteostasis also causes sporadic respiratory diseases [1, 4]. Cigarette smoke-induced protein misfolding, aberrant proteasomal protein degradation and induction of autophagy have been observed in chronic obstructive pulmonary disease (COPD) patients and smoke-exposed mice [4, 5]. Dysregulation of autophagy and endoplasmic reticulum (ER) stress have also been implicated in cystic fibrosis, pulmonary arterial hypertension, IPF and other lung diseases [6, 7]. Impairment of protein quality control pathways exacerbates the detrimental effects of environmentally induced protein damage in lung pathogenesis [1]. The European Respiratory Society (ERS) research seminar Protein Quality Control in Lung Disease, held on March 1–2, 2014, at Lake Starnberg in Germany, brought together international experts to develop a comprehensive view of protein quality control in general and in the lung in particular. Understanding the complex interplay of protein misfolding, ER homeostasis and protein degradation as interrelated components of adaptive proteostasis will identify novel therapeutic targets for treatment of pulmonary diseases, as outlined here.</p

Investigation of host and pathogen responses to oestrogen in cystic fibrosis

Introduction: A ‘gender gap’ exists in cystic fibrosis (CF). Females acquire earlier microbial infections and have a worse prognosis. The sex hormone oestradiol (E2) has recently been highlighted as a key molecule responsible for the CF gender dichotomy. Pseudomonas aeruginosa (Ps. aeruginosa) colonises the CF lung dominating at end stages and undergoes mucoid conversion in response to E2. The aim of this project is to investigate other roles of E2 in host and pathogen, in particular its effects on the growth rate of Ps. aeruginosa and the expression of catalase and superoxide dismutase (SOD) in CF bronchial epithelial cells. Methods: Growth rate of Ps. aeruginosa (strain PA01) in the presence or absence of E2 was measured by optical density (OD600nm) and by calculating colony-forming units (cfu) per ml. Catalase and SOD gene expression in E2-treated CFBE41o- airway epithelial cells were measured using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Results: E2 had no effect on the growth rate of Ps. aeruginosa when compared to control. The expression of catalase mRNA was not altered in CFBE41o- cells in response to E2; however, there was a two-fold increase in SOD gene expression in response to 10nM E2, at 24 hours (p=0.0057). Conclusion: Oestradiol has no effect on the growth rate of Ps. aeruginosa in vitro. In CF bronchial epithelial cells, although catalase gene expression remains unchanged, E2 increases SOD expression, potentially increasing hydrogen peroxide levels and contributing to Ps. aeruginosa mucoid conversion.</p

microRNAs in asthma: potential therapeutic targets.

PURPOSE OF REVIEW: Asthma is a global disease affecting millions of people. Current treatments are largely symptomatic and, although often effective, can be associated with various side effects. microRNAs (miRNAs/miRs) are regulatory RNAs that affect protein synthesis. They represent new therapeutic targets, and medicines that target specific miRNAs may have potential in the treatment of asthma. RECENT FINDINGS: There have been a number of studies in the field of miRNA that implicate specific miRNAs in the pathophysiology of asthma. For example, studies using mouse models have identified miRNAs that are altered in response to allergen challenge. Certain miRNAs that are involved in the regulation of interleukin-13 and the TH2 response, key components of the asthmatic response, have been shown to be amenable to modulation by premiRs and antimiRs. Other studies have identified miRNAs that are implicated in bronchial smooth muscle hyperresponsiveness and proliferation. Single-nucleotide polymorphisms in miRNA responsive elements within asthma susceptibility genes, and also in miRNAs themselves, can also contribute to the asthma phenotype. SUMMARY: Developing miRNA-based medicines to treat the pulmonary manifestations of asthma could yield therapeutics with new properties that have the potential to treat both the inflammation and hyperresponsivesness associated with this disease.</p

Long non-coding RNA expression in alpha-1 antitrypsin deficient monocytes pre- and post-AAT augmentation therapy

Long non-coding RNAs (lncRNAs) regulate gene expression. Their expression in alpha-1 antitrypsin (AAT) deficiency has not been investigated. Treatment of AAT deficiency involves infusion of plasma-purified AAT and this augmentation therapy has previously been shown to alter microRNA expression in monocytes of AAT-deficient (ZZ) individuals. Here, we assess the effect of AAT augmentation therapy on the lncRNA expression profile in ZZ monocytes. Peripheral blood monocytes were isolated from ZZ individuals pre (Day 0)- and post (Day 2)-AAT augmentation therapy. Arraystar lncRNA microarray profiling was performed; a total of 17,761 lncRNAs were detectable across all samples. The array identified 7509 lncRNAs with differential expression post-augmentation therapy, 3084 were increased and 4425 were decreased (fold change ≥ 2). Expression of many of these lncRNAs were similarly altered in ZZ monocytes treated ex vivo with 27.5 μM AAT for 4 h. These properties may contribute to the manifold effects of AAT augmentation therapy </p

Measurement of the unfolded protein response (UPR) in monocytes.

In mammalian cells, the primary function of the endoplasmic reticulum (ER) is to synthesize and assemble membrane and secreted proteins. As the main site of protein folding and posttranslational modification in the cell, the ER operates a highly conserved quality control system to ensure only correctly assembled proteins exit the ER and misfolded and unfolded proteins are retained for disposal. Any disruption in the equilibrium of the ER engages a multifaceted intracellular signaling pathway termed the unfolded protein response (UPR) to restore normal conditions in the cell. A variety of pathological conditions can induce activation of the UPR, including neurodegenerative disorders such as Parkinson's disease, metabolic disorders such as atherosclerosis, and conformational disorders such as cystic fibrosis. Conformational disorders are characterized by mutations that modify the final structure of a protein and any cells that express abnormal protein risk functional impairment. The monocyte is an important and long-lived immune cell and acts as a key immunological orchestrator, dictating the intensity and duration of the host immune response. Monocytes expressing misfolded or unfolded protein may exhibit UPR activation and this can compromise the host immune system. Here, we describe in detail methods and protocols for the examination of UPR activation in peripheral blood monocytes. This guide should provide new investigators to the field with a broad understanding of the tools required to investigate the UPR in the monocyte.</p

TLR3 Sensing of Viral Infection

Viral infection is detected by the innate immune system which mounts a rapid semi-selective defence involving inflammation and production of type 1 interferons. Several sensors, both cell surface and intracellular, exist to detect different types of viral motifs. Double-stranded RNA viruses and dsRNA replication intermediates are detected by tolllike receptor 3 (TLR3) as well as by retinoid-inducible gene 1 (RIG-I) like receptors. Binding of dsRNA or its synthetic analogue poly I:C to TLR3 recruits the adaptor protein TRIF and stimulates distinct pathways leading to activation of interferon regulatory factor (IRF) and NF-KB. Here, we review the signalling cascades initiated by TLR3 and the modulation of these pathways.</p

Gain of function effects of Z alpha-1 antirypsin

The serine proteinase inhibitor alpha-1 antitrypsin (AAT) is produced principally by the liver from where it is secreted into the circulation and provides an antiprotease protective screen throughout the body. Mutations leading to deficiency in AAT are associated with liver and lung disease. The most notable is the Z mutation, which encodes a misfolded variant of the AAT protein in which the glutamic acid at position 342 is replaced by a lysine. ZAAT is not secreted effectively and accumulates intracellularly in the endoplasmic reticulum (ER) of hepatocytes and other AAT-producing cells. The ER has evolved a number of elegant mechanisms to manage the accumulation of incorrectly folded proteins; ZAAT interferes with this function and promotes ER stress responses and inflammation. Until recently it was thought that gain of function was the major cause of the liver disease whilst the lung disease was entirely due to loss of antiprotease protection in the lung. This belief is now being challenged with the discovery that ER stress is also activated in bronchial epithelial cells and inflammatory cells normally resident in the lung in ZAAT deficient individuals. Here we describe the gain of function effects of ZAAT. In particular we highlight the signalling pathways that are activated during ER stress in response to accumulation of ZAAT and how these events are linked to inflammation and may contribute to disease pathogenesis. © 2010 Bentham Science Publishers Ltd.</p