Hitasha Rupani

Multidimensional endotyping in patients with severe asthma reveals inflammatory heterogeneity in matrix metalloproteinases and chitinase 3-like protein 1.

Background Disease heterogeneity in patients with severe asthma and its relationship to inflammatory mechanisms remain poorly understood. Objective We aimed to identify and replicate clinicopathologic endotypes based on analysis of blood and sputum parameters in asthmatic patients. Methods One hundred ninety-four asthmatic patients and 21 control subjects recruited from 2 separate centers underwent detailed clinical assessment, sputum induction, and phlebotomy. One hundred three clinical, physiologic, and inflammatory parameters were analyzed by using topological data analysis and Bayesian network analysis. Results Severe asthma was associated with anxiety and depression, obesity, sinonasal symptoms, decreased quality of life, and inflammatory changes, including increased sputum chitinase 3–like protein 1 (YKL-40) and matrix metalloproteinase (MMP) 1, 3, 8, and 12 levels. Topological data analysis identified 6 clinicopathobiologic clusters replicated in both geographic cohorts: young, mild paucigranulocytic; older, sinonasal disease; obese, high MMP levels; steroid resistant TH2 mediated, eosinophilic; mixed granulocytic with severe obstruction; and neutrophilic, low periostin levels, severe obstruction. Sputum IL-5 levels were increased in patients with severe particularly eosinophilic forms, whereas IL-13 was suppressed and IL-17 levels did not differ between clusters. Bayesian network analysis separated clinical features from intricately connected inflammatory pathways. YKL-40 levels strongly correlated with neutrophilic asthma and levels of myeloperoxidase, IL-8, IL-6, and IL-6 soluble receptor. MMP1, MMP3, MMP8, and MMP12 levels were associated with severe asthma and were correlated positively with sputum IL-5 levels but negatively with IL-13 levels. Conclusion In 2 distinct cohorts we have identified and replicated 6 clinicopathobiologic clusters based on blood and induced sputum measures. Our data underline a disconnect between clinical features and underlying inflammation, suggest IL-5 production is relatively steroid insensitive, and highlight the expression of YKL-40 in patients with neutrophilic inflammation and the expression of MMPs in patients with severe asthma.

MicroRNAs and respiratory diseases

MicroRNAs (miRNAs) are a family of endogenous, small, noncoding RNA molecules that modulate physiological and pathological processes by post-transcriptional inhibition of gene expression. They were first recognised as regulators of development in worms and fruitflies. In recent years extensive research has explored their pivotal role in the pathogenesis of human diseases. Over 1,000 human miRNAs have been discovered to date; however, the biological function and protein targets for the majority remain to be uncovered. Within the respiratory system, miRNAs are important in normal pulmonary development and maintaining lung homeostasis. Recent studies have also begun to reveal that altered miRNA expression profiles may be associated with pathological processes within the lung and lead to the development of various pulmonary diseases, ranging from inflammatory diseases to lung cancers. Advancing our understanding of the role of miRNAs in the respiratory system will help provide new perspectives on disease mechanisms and reveal intriguing therapeutic targets and diagnostic markers for respiratory disorders.

A microRNA network dysregulated in asthma controls IL-6 production in bronchial epithelial cells.

MicroRNAs are short non-coding single stranded RNAs that regulate gene expression. While much is known about the effects of individual microRNAs, there is now growing evidence that they can work in co-operative networks. MicroRNAs are known to be dysregulated in many diseases and affect pathways involved in the pathology. We investigated dysregulation of microRNA networks using asthma as the disease model. Asthma is a chronic inflammatory disease of the airways characterized by bronchial hyperresponsiveness and airway remodelling. The airway epithelium is a major contributor to asthma pathology and has been shown to produce an excess of inflammatory and pro-remodelling cytokines such as TGF-β, IL-6 and IL-8 as well as deficient amounts of anti-viral interferons. After performing microRNA arrays, we found that microRNAs -18a, -27a, -128 and -155 are down-regulated in asthmatic bronchial epithelial cells, compared to cells from healthy donors. Interestingly, these microRNAs are predicted in silico to target several components of the TGF-β, IL-6, IL-8 and interferons pathways. Manipulation of the levels of individual microRNAs in bronchial epithelial cells did not have an effect on any of these pathways. Importantly, knock-down of the network of microRNAs miR-18a, -27a, -128 and -155 led to a significant increase of IL-8 and IL-6 expression. Interestingly, despite strong in silico predictions, down-regulation of the pool of microRNAs did not have an effect on the TGF-β and Interferon pathways. In conclusion, using both bioinformatics and experimental tools we found a highly relevant potential role for microRNA dysregulation in the control of IL-6 and IL-8 expression in asthma. Our results suggest that microRNAs may have different roles depending on the presence of other microRNAs. Thus, interpretation of in silico analysis of microRNA function should be confirmed experimentally in the relevant cellular context taking into account interactions with other microRNAs when studying disease.

Altered Epithelial Gene Expression in Peripheral Airways of Severe Asthma

Management of severe asthma remains a challenge despite treatment with glucocorticosteroid therapy. The majority of studies investigating disease mechanisms in treatment-resistant severe asthma have previously focused on the large central airways, with very few utilizing transcriptomic approaches. The small peripheral airways, which comprise the majority of the airway surface area, remain an unexplored area in severe asthma and were targeted for global epithelial gene expression profiling in this study. Differences between central and peripheral airways were evaluated using transcriptomic analysis (Affymetrix HG U133 plus 2.0 GeneChips) of epithelial brushings obtained from severe asthma patients (N = 17) and healthy volunteers (N = 23). Results were validated in an independent cohort (N = 10) by real-time quantitative PCR. The IL-13 disease signature that is associated with an asthmatic phenotype was upregulated in severe asthmatics compared to healthy controls but was predominantly evident within the peripheral airways, as were genes related to mast cell presence. The gene expression response associated with glucocorticosteroid therapy (i.e. FKBP5) was also upregulated in severe asthmatics compared to healthy controls but, in contrast, was more pronounced in central airways. Moreover, an altered epithelial repair response (e.g. FGFBP1) was evident across both airway sites reflecting a significant aspect of disease in severe asthma unadressed by current therapies. A transcriptomic approach to understand epithelial activation in severe asthma has thus highlighted the need for better-targeted therapy to the peripheral airways in severe asthma, where the IL-13 disease signature persists despite treatment with currently available therapy.

IL-1α mediates cellular cross-talk in the airway epithelial mesenchymal trophic unit

ABSTRACT The bronchial epithelium and underlying fibroblasts form an epithelial mesenchymal trophic unit (EMTU) which controls the airway microenvironment. We hypothesized that cell-cell communication within the EMTU propagates and amplifies the innate immune response to respiratory viral infections. EMTU co-culture models incorporating polarized (16HBE14o-) or differentiated primary human bronchial epithelial cells (HBECs) and fibroblasts were challenged with double-stranded RNA (dsRNA) or rhinovirus. In the polarized EMTU model, dsRNA affected ionic but not macromolecular permeability or cell viability. Compared with epithelial monocultures, dsRNA-stimulated pro-inflammatory mediator release was synergistically enhanced in the basolateral compartment of the EMTU model, with the exception of IL-1α which was unaffected by the presence of fibroblasts. Blockade of IL-1 signaling with IL-1 receptor antagonist (IL-1Ra) completely abrogated dsRNA-induced basolateral release of mediators except CXCL10. Fibroblasts were the main responders to epithelial-derived IL-1 since exogenous IL-1α induced pro-inflammatory mediator release from fibroblast but not epithelial monocultures. Our findings were confirmed in a differentiated EMTU model where rhinovirus infection of primary HBECs and fibroblasts resulted in synergistic induction of basolateral IL-6 that was significantly abrogated by IL-1Ra. This study provides the first direct evidence of integrated IL-1 signaling within the EMTU to propagate inflammatory responses to viral infection.

Case series reporting the effectiveness of mycophenolate mofetil in treatment-resistant asthma

To the Editor: Treatment-resistant asthma represents a significant unmet clinical need. Due to lack of therapeutic options many patients receive treatment with long-term oral steroids despite appreciation of the associated systemic adverse effects. While anti-IgE therapy with omalizumab affords benefit and there have been encouraging outcomes with monoclonal antibodies against interleukin (IL)-5 and IL-13 [1, 2], all such treatments target T-helper cell type 2 (Th2) orientated disease in a sub-population of patients. Mycophenolate mofetil (MMF) is a reversible inhibitor of inosine monophosphate dehydrogenase, an enzyme required by T- and B-cells for guanine synthesis [3]. It is most commonly used to prevent transplanted organ rejection, but is increasingly prescribed to treat inflammatory disorders such as connective tissue disorders and systemic vasculitides. In January 2009, a patient in our institution with lifelong severe treatment-resistant asthma, poorly controlled despite high-dose inhaled corticosteroids, regular oral steroids and nebulised bronchodilators, was prescribed MMF for co-existent undifferentiated vasculitis. Over the following year his disease improved dramatically with a reduction in hospital admissions, enabling a reduction in oral steroids with a concurrent decrease in weight, improvement in quality of life and exercise capacity. In view of this experience, another individual with severe asthma resistant to all standard therapies was placed on MMF. This patient also showed a dramatic clinical improvement. Subsequently we treated a further 20 patients with MMF with varying results to give a case series of 22, which we report here. All patients started on MMF had been under specialist asthma care for at least 12 months and despite step 4 or 5 of asthma guidelines management had poor control, as measured by the asthma control questionnaire (ACQ), and …

S116 MicroRNA Regulation of Toll-Like Receptor 7 Function in Severe Asthma: Relevance to Viral Responses

Background and Aims Viral-induced disease exacerbation is common in asthma and studies have identified that both bronchial epithelial cells and alveolar macrophages (AM) from asthmatics have a reduced interferon (IFN) response to rhinovirus infection. The mechanism behind this defect is unclear. As asthmatic peripheral blood mononuclear cells have been reported to have defective toll-like receptor (TLR) 7 function, we investigated the expression of microRNAs (miRNAs) in AM from healthy control (HC) and severe asthma (SA) volunteers with relevance to TLR7-viral interactions. MicroRNAs are non-coding RNAs that down-regulate gene expression by suppressing translation. We identified and focused on 3 miRNAs that could target TLR7. Additionally, we investigated if manipulating the expression of these miRNAs can ameliorate the defective IFN response in AM. Methods 26 HC and 30 patients with SA (BTS Step 4/5) were recruited for bronchoscopy. AM were isolated from bronchoalveolar lavage using the adherence to plastic technique. Expression of miRNAs and TLR7 was determined by qRT-PCR and western blotting. AM were transfected with a combination of antagomirs, specifically directed against the 3 miRNAs, and then treated with imiquimod (5ug/ml), a TLR7 agonist, or human rhinovirus-16 (HRV16) and IFN-β expression was determined after 24 hours using qRT-PCR and ELISA. Results Expression of all three miRNAs was significantly increased in SA compared to HC TLR7 mRNA was found to be significantly reduced in AM from volunteers with SA compared to HC. Western blotting confirmed reduced expression of TLR7 protein in AM from SA compared to HC. Compared to mock transfected AM, AM transfected with the 3 antagomirs showed significantly increased imiquimod-induced IFN-β mRNA and protein expression and significantly increased HRV16-induced IFN-β mRNA production. Conclusion TLR7 expression is significantly reduced in SA compared to HC. The differential expression of the miRNAs identified may lead to impaired viral sensing by asthmatic AM and contribute to the defective IFN response to rhinovirus. Importantly, TLR7 induced IFN-β production by human AM can be significantly augmented by inhibition of these miRNAs. The identification of these miRNAs and our ability to manipulate their expression in human AM offers the potential for future miRNA-based therapies in asthma.

Genome-wide posttranscriptional dysregulation by microRNAs in human asthma as revealed by Frac-seq.

MicroRNAs are small noncoding RNAs that inhibit gene expression posttranscriptionally, implicated in virtually all biological processes. Although the effect of individual microRNAs is generally studied, the genome-wide role of multiple microRNAs is less investigated. We assessed paired genome-wide expression of microRNAs with total (cytoplasmic) and translational (polyribosome-bound) mRNA levels employing subcellular fractionation and RNA sequencing (Frac-seq) in human primary bronchoepithelium from healthy controls and severe asthmatics. Severe asthma is a chronic inflammatory disease of the airways characterized by poor response to therapy. We found genes (i.e., isoforms of a gene) and mRNA isoforms differentially expressed in asthma, with novel inflammatory and structural pathophysiological mechanisms related to bronchoepithelium disclosed solely by polyribosome-bound mRNAs (e.g., IL1A and LTB genes or ITGA6 and ITGA2 alternatively spliced isoforms). Gene expression (i.e., isoforms of a gene) and mRNA expression analysis revealed different molecular candidates and biological pathways, with differentially expressed polyribosome-bound and total mRNAs also showing little overlap. We reveal a hub of six dysregulated microRNAs accounting for ∼90% of all microRNA targeting, displaying preference for polyribosome-bound mRNAs. Transfection of this hub in bronchial epithelial cells from healthy donors mimicked asthma characteristics. Our work demonstrates extensive posttranscriptional gene dysregulation in human asthma, in which microRNAs play a central role, illustrating the feasibility and importance of assessing posttranscriptional gene expression when investigating human disease.

Exploring the Waveform Characteristics of Tidal Breathing Carbon Dioxide, Measured Using the N-Tidal C Device in Different Breathing Conditions (The General Breathing Record Study): Protocol for an Observational, Longitudinal Study

Background In an increasingly comorbid population, there are significant challenges to diagnosing the cause of breathlessness, and once diagnosed, considerable difficulty in detecting deterioration early enough to provide effective intervention. The burden of the breathless patient on the health care economy is substantial, with asthma, chronic heart failure, and pneumonia affecting over 6 million people in the United Kingdom alone. Furthermore, these patients often have more than one contributory factor to their breathlessness symptoms, with conditions such as dysfunctional breathing pattern disorders—an under-recognized component. Current methods of diagnosing and monitoring breathless conditions can be extensive and difficult to perform. As a consequence, home monitoring is poorly complied with. In contrast, capnography (the measurement of tidal breath carbon dioxide) is performed during normal breathing. There is a need for a simple, easy-to-use, personal device that can aid in the diagnosis and monitoring of respiratory and cardiac causes of breathlessness. Objective The aim of this study was to explore the use of a new, handheld capnometer (called the N-Tidal C) in different conditions that cause breathlessness. We will study whether the tidal breath carbon dioxide (TBCO2) waveform, as measured by the N-Tidal C, has different characteristics in a range of respiratory and cardiac conditions. Methods We will perform a longitudinal, observational study of the TBCO2 waveform (capnogram) as measured by the N-Tidal C capnometer. Participants with a confirmed diagnosis of asthma, breathing pattern disorders, chronic heart failure, motor neurone disease, pneumonia, as well as volunteers with no history of lung disease will be asked to provide twice daily, 75-second TBCO2 collection via the N-Tidal C device for 6 months duration. The collated capnograms will be correlated with the underlying diagnosis and disease state (stable or exacerbation) to determine if there are different TBCO2 characteristics that can distinguish different respiratory and cardiac causes of breathlessness. Results This study’s recruitment is ongoing. It is anticipated that the results will be available in late 2018. Conclusions The General Breathing Record Study will provide an evaluation of the use of capnography as a diagnostic and home-monitoring tool for various diseases. Registered Report Identifier RR1-10.2196/9767

S88 Micrornas regulate genome-wide translation in severe asthma bronchial epithelial cells as revealed by frac-seq

Severe asthma represents a significant unmet clinical need and the molecular basis for disease persistence remains inadequately understood. Bronchial epithelial cells, at the interface of environment/tissue, are central to asthma pathogenesis. There is thus a need to evaluate genome-wide changes between health and asthma to better understand the molecular mechanisms underlying disease. The vast majority of genome-wide measurements have focused on determining changes at the DNA or mRNA levels, with little attention paid to how and which mRNAs are actually translated into protein. This may not disclose changes happening at the protein level, since mRNA and protein expression correlate poorly. To determine translation and its regulation in bronchial epithelial cells in severe asthma patients we analysed paired genome-wide expression of transcriptional (cytoplasmic) and translational (polyribosome-bound) mRNA levels employing Frac-seq (subcellular fractionation and RNA-sequencing) in primary bronchoepithelium in health and severe asthma patients. We also integrated those data with genome-wide profiling of microRNAs to understand their role in gene expression and impact on the pathophysiology of severe asthma bronchial epithelium. We found both genes (=all isoforms of a gene) and mRNA isoforms differentially expressed in severe asthma airways cells, with dysregulated transcriptional mRNA levels (194 genes) showing little overlap with dysregulated translational mRNA (243 genes) expression. We determined novel inflammatory and remodelling pathophysiological mechanisms disclosed solely by polyribosome-bound mRNAs, centred in epithelium remodelling and repair pathways. We also reveal six dysregulated microRNAs accounting for ∼90% of all cellular microRNA targeting, displaying preferential targeting of ∼50% of mRNAs undergoing translation in severe asthma airways cells. Thus, microRNAs in human severe asthma are major regulators of translation in airways epithelium and offer potential as future therapeutic targets.