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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., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Rationale: Asthma is one of the most common chronic diseases worldwide, and individuals with severe asthma experience recurrent exacerbations. Exacerbations are predominantly viral associated and have been linked to defective airway IFN responses. Ascertaining the molecular mechanisms underlying this deficiency is a major research goal to identify new therapeutic targets., Objectives: We investigated the hypothesis that reduced Toll-like receptor 7 (TLR7)-derived signaling drove the impaired IFN responses to rhinovirus by asthmatic alveolar macrophages (AMs); the molecular mechanisms underlying this deficiency were explored., Methods: AMs were recovered from bronchoalveolar lavage from healthy subjects and patients with severe asthma. Expression of pattern-recognition receptors and microRNAs was evaluated by quantitative polymerase chain reaction and Western blotting. A TLR7-luciferase reporter construct was created to evaluate binding of microRNAs to the 3' untranslated region of TLR7. IFN production was measured by quantitative polymerase chain reaction and ELISA., Measurements and Main Results: The expression of TLR7 was significantly reduced in severe asthma AMs and was associated with reduced rhinovirus and imiquimod-induced IFN responses by these cells compared with healthy AMs. Severe asthma AMs also expressed increased levels of three microRNAs, which we showed were able to directly reduce TLR7 expression. Ex vivo knockdown of these microRNAs restored TLR7 expression with concomitant augmentation of virus-induced IFN production., Conclusions: In severe asthma, TLR7 deficiency drives impaired innate immune responses to virus by AMs. Blocking a group of microRNAs that are up-regulated in these cells can restore antiviral innate responses, providing a novel approach for therapy in asthma.

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.

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.