The Role of Micrornas in Alpha-1 Antitrypsin Deficiency Disease.

The hereditary disorder alpha-1 antirypsin deficiency (AATD) results from mutations in the SERPINA1 gene. The most common form of AATD occurs because of the Z mutation causing the protein to fold aberrantly and accumulate in the endoplasmic reticulum (ER) as opposed to being secreted into the circulation. The loss-of-function of AAT in neutralising neutrophil elastase and other proinflammatory enzymes contribute to the pathogenesis of emphysema. Meanwhile, the gain-of-function effects are thought to derive from the toxicity of the ZAAT forming insoluble polymers within hepatocytes leading to liver disease. The unfolded protein response (EIPR), a component of the ER stress has been shown to occur in monocytes due to ZAAT accumulation intracellularly. In view of both loss-and gain-of-function effects, numerous strategies have been examined for the development of gene therapies to address both aspects of the disease. MicroRNAs (miRNAs) are small non-coding RNAs that regulate expression by translational repression or messenger RNA (mRNA) degradation. The regulatory role of miRNAs in regulating both the SERPINA1 gene and the pathophysiology of the UPR and inflammation in AATD has not been investigated. Very few experimental methods can comprehensively identify multiple miRNAs that target a single mRNA. Elere, an experimental approach to search for miRNAs targeting a specific mRNA using a capture affinity assay involving a biotinylated DNA anti-sense oligonucleotide (miR-CATCH) was developed for capture of AAT, interleukin- 8 and secretory leucoprotease inhibitor mRNAs. AAT mRNA-specific and total miRNAs from monocytic THP-1, bronchial epithelial 16HBE14o- and liver HepG2 cell lines were profiled and validated revealing cell-specific miRNA regulation. Overexpression of miR- 455-3p that was captured as an AAT-targeting miRNA identified in THP-1 monocytic cell lines in primary human monocytes with and without AATD (ie MM and ZZ monocytes respectively) led to significant decreases in both M- and ZAAT mRNA and protein. To investigate miRNA expression and function in ZZ and MM monocytes, miRNA expression profiling was performed. Forty-three miRNAs were shown to be differentially expressed, with miR-199a-5p most highly upregulated in asymptomatic ZZ (individuals with no evidence of chronic obstructive pulmonary disease or COPD) compared to MM monocytes. MiR-199a-2 promoter hypermethylation inhibits miR-199a- 5p expression and was increased in symptomatic (individuals with COPD) MM and ZZ monocytes compared to their asymptomatic counterparts. The expression of components of the UPR arms, namely GRP 78, ATF6, p50 and p65 was increased in symptomatic versus asymptomatic ZZ monocytes. Reciprocal down- or upregulation of these markers was observed after miRNA modulation using precursors and inhibitors of miR-199a-5p. Direct miR-199a-5p targeting of these markers was demonstrated using luciferase reporter systems. To examine the effects of weekly AAT augmentation therapy (AATAT) on miRNA expression, miRNA profiling was performed. This revealed sets of differentially expressed miRNAs between (i) MM and ZZ monocytes in asymptomatic individuals, and (ii) between symptomatic ZZ monocytes receiving AATAT or not. The canonical dimer of NFkB was downregulated in ZZ monocytes receiving AATAT in vivo and in vitro. NFkB modulation using an NFkB inhibitor or activator altered the expression of miR-199a-5p, - 320a and -528 with potential NFkB binding sites in the regulatory regions of these miRNA genes. Using bioinformatic tools, target genes of these NFicB-regulated miRNAs were identified in a selection of pathways that include inflammation, hypoxia and antioxidant responses. This study provides the first evidence of innate miRNAs selectively targeting and modulating AAT mRNA expression in a cell-specific manner in which modulation of AAT using miRNAs in human primary cells was successfully performed. MiR-199a-5p was identified as a key regulator of the UPR in AATD monocytes and epigenetic silencing of its expression regulates this process in emphysema. Another anti-inflammatory property of AATAT in which NFkB was downregulated in ZZ monocytes receiving AATAT altered the expression of miRNAs with potential NFkB binding sites in their regulatory region. These findings in tandem to current knowledge of AATD, could provide potential avenues for future research and new generation gene therapies involving miR-based therapeutics that could tailor organ-specific treatment in AATD

The role of proteases, endoplasmic reticulum stress and SERPINA1 heterozygosity in lung disease and α-1 anti-trypsin deficiency.

The serine proteinase inhibitor α-1 anti-trypsin (AAT) provides an antiprotease protective screen throughout the body. Mutations in the AAT gene (SERPINA1) that lead to deficiency in AAT are associated with chronic obstructive pulmonary diseases. The Z mutation encodes a misfolded variant of AAT that is not secreted effectively and accumulates intracellularly in the endoplasmic reticulum of hepatocytes and other AAT-producing cells. Until recently, it was thought that loss of antiprotease function was the major cause of ZAAT-related lung disease. However, the contribution of gain-of-function effects is now being recognized. Here we describe how both loss- and gain-of-function effects can contribute to ZAAT-related lung disease. In addition, we explore how SERPINA1 heterozygosity could contribute to smoking-induced chronic obstructive pulmonary diseases and consider the consequences

Lung involvement at presentation predicts disease activity and permanent organ damage at 6, 12 and 24 months follow - up in ANCA - associated vasculitis.

BACKGROUND: Antineutrophilic cytoplasmic antibody (ANCA)-associated vasculitis (AAV) may present with pulmonary involvement ranging from mild to life-threatening disease such as diffuse alveolar hemorrhage. There is a paucity of information regarding morbidity outcomes for AAV subjects presenting with lung involvement. This study determines the relationship between disease activity and damage in these subjects using the Birmingham Vasculitis Activity Score v 3 (BVAS 3) and Vasculitis Damage Index (VDI) respectively. RESULTS: 151 patients with AAV were included with 59 presenting initially with pulmonary involvement. The initial BVAS scores recorded at time of diagnosis were positively correlated with the final VDI scores at 24 months (p \u3c 0.0001, rs = 0.5871). No differences between BVAS and VDI scores were seen for both groups, however in the lung-involvement group only, BVAS scores were significantly higher at 6, 12 and 24 months whilst the VDI scores were significantly higher at 12 and 24 months. Subjects presenting with pulmonary involvement had an increased likelihood for cardiovascular (OR 1.31, 95% CI 0.89, 1.54; p = 0.032) and renal (OR 1.32, 95% CI 1.22, 1.39; p = 0.005) involvement. Subjects presenting with lung involvement with granulomatosis with polyangiitis and microscopic polyangiitis had 24-month VDI scores that were significantly higher (p = 0.027, p = 0.045), and more likely to develop pulmonary fibrosis (OR 1.79, 95% CI 1.48, 2.12; p \u3c 0.001). CONCLUSION: AAV subjects with lung involvement at presentation had a higher disease activity and damage scores at 6, 12 and 24 months follow-up representing a considerable burden of disease despite improvement in overall survival due to the introduction of immunosuppressive therapy

Isolation and identification of cell-specific microRNAs targeting a messenger RNA using a biotinylated anti-sense oligonucleotide capture affinity technique.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate expression by translational repression or messenger RNA (mRNA) degradation. Although numerous bioinformatic prediction models exist to identify miRNA-mRNA interactions, experimental validation of bona fide interactions can be difficult and laborious. Few methods can comprehensively identify miRNAs that target a single mRNA. We have developed an experimental approach to search for miRNAs targeting any mRNA using a capture affinity assay involving a biotinylated DNA anti-sense oligonucleotide. This method identifies miRNAs targeting the full length of the mRNA. The method was tested using three separate mRNA targets: alpha-1 antitrypsin (AAT) mRNA, interleukin-8 mRNA and secretory leucoprotease inhibitor mRNA. AAT mRNA-specific and total miRNAs from three different cell lines (monocytic THP-1, bronchial epithelial 16HBE14o- and liver HepG2 cells) were profiled, and validation studies revealed that AAT mRNA-specific miRNAs functionally target the AAT mRNA in a cell-specific manner, providing the first evidence of innate miRNAs selectively targeting and modulating AAT mRNA expression. Interleukin-8 and secretory leucoprotease inhibitor mRNAs and their cognate miRNAs were also successfully captured using this approach. This is a simple and an efficient method to potentially identify miRNAs targeting sequences within the full length of a given mRNA transcript

miR-199a-5p silencing regulates the unfolded protein response in chronic obstructive pulmonary disease and α1-antitrypsin deficiency.

RATIONALE: Retention of abnormal α1-antitrypsin (AAT) activates the unfolded protein response in AAT-deficient monocytes. The regulatory role of microRNAs (miRNAs) in unfolded protein responses and chronic obstructive pulmonary disease pathogenesis has not been investigated. OBJECTIVES: To investigate miRNA expression and function in MM and ZZ monocytes and identify miRNA(s) regulating the unfolded protein response. METHODS: Peripheral blood monocytes were isolated from asymptomatic and symptomatic MM and ZZ individuals for miRNA expression profiling and pyrosequencing analysis. miRNA/gene and protein expression was measured with quantitative polymerase chain reaction and Western blotting. Overexpression and inhibition studies were performed with pre-miR or anti-miR, respectively. Luciferase reporter genes were used to elucidate direct miRNA-target interactions. Inflammatory cytokines were detected using the Meso Scale Discovery Plex assays. MEASUREMENTS AND MAIN RESULTS: Forty-three miRNAs were differentially expressed, with miR-199a-5p most highly up-regulated in asymptomatic ZZ versus MM monocytes. miR-199a-2 promoter hypermethylation inhibits miR-199a-5p expression and was increased in symptomatic MM and ZZ monocytes compared with asymptomatic counterparts. GRP78, activating transcription factor 6, p50, and p65 were increased in symptomatic versus asymptomatic ZZ monocytes. Reciprocal down- or up-regulation of these markers was observed after miRNA modulation. Direct miR-199a-5p targeting of activating transcription factor 6, p50, and p65 by miR-199a-5p was demonstrated using luciferase reporter systems. Overexpression of miR-199a-5p also decreased other arms of the UPR and expression of cytokines that are not putative targets. CONCLUSIONS: miR-199a-5p is a key regulator of the unfolded protein response in AAT-deficient monocytes, and epigenetic silencing of its expression regulates this process in chronic obstructive pulmonary disease

Immunological corollary of the pulmonary mycobiome in bronchiectasis:The Cameb study

Understanding the composition and clinical importance of the fungal mycobiome was recently identified as a key topic in a “research priorities” consensus statement for bronchiectasis. Patients were recruited as part of the CAMEB study: an international multicentre cross-sectional Cohort of Asian and Matched European Bronchiectasis patients. The mycobiome was determined in 238 patients by targeted amplicon shotgun sequencing of the 18S–28S rRNA internally transcribed spacer regions ITS1 and ITS2. Specific quantitative PCR for detection of and conidial quantification for a range of airway Aspergillus species was performed. Sputum galactomannan, Aspergillus specific IgE, IgG and TARC (thymus and activation regulated chemokine) levels were measured systemically and associated to clinical outcomes. The bronchiectasis mycobiome is distinct and characterised by specific fungal genera, including Aspergillus, Cryptococcus and Clavispora. Aspergillus fumigatus (in Singapore/Kuala Lumpur) and Aspergillus terreus (in Dundee) dominated profiles, the latter associating with exacerbations. High frequencies of Aspergillus-associated disease including sensitisation and allergic bronchopulmonary aspergillosis were detected. Each revealed distinct mycobiome profiles, and associated with more severe disease, poorer pulmonary function and increased exacerbations. The pulmonary mycobiome is of clinical relevance in bronchiectasis. Screening for Aspergillus-associated disease should be considered even in apparently stable patients.MOE (Min. of Education, S’pore)NMRC (Natl Medical Research Council, S’pore)Published versio

<i>Neisseria</i> species as pathobionts in bronchiectasis

Neisseria species are frequently identified in the bronchiectasis microbiome, but they are regarded as respiratory commensals. Using a combination of human cohorts, next-generation sequencing, systems biology, and animal models, we show that bronchiectasis bacteriomes defined by the presence of Neisseria spp. associate with poor clinical outcomes, including exacerbations. Neisseria subflava cultivated from bronchiectasis patients promotes the loss of epithelial integrity and inflammation in primary epithelial cells. In vivo animal models of Neisseria subflava infection and metabolipidome analysis highlight immunoinflammatory functional gene clusters and provide evidence for pulmonary inflammation. The murine metabolipidomic data were validated with human Neisseria-dominant bronchiectasis samples and compared with disease in which Pseudomonas-, an established bronchiectasis pathogen, is dominant. Metagenomic surveillance of Neisseria across various respiratory disorders reveals broader importance, and the assessment of the home environment in bronchiectasis implies potential environmental sources of exposure. Thus, we identify Neisseria species as pathobionts in bronchiectasis, allowing for improved risk stratification in this high-risk group.Published versio

Distinct 'Immuno-Allertypes' of Disease and High Frequencies of Sensitisation in Non-Cystic-Fibrosis Bronchiectasis

Rationale: Allergic sensitization is associated with poor clinical outcomes in asthma, chronic obstructive pulmonary disease, and cystic fibrosis; however, its presence, frequency, and clinical significance in non–cystic fibrosis bronchiectasis remain unclear. Objectives: To determine the frequency and geographic variability that exists in a sensitization pattern to common and specific allergens, including house dust mite and fungi, and to correlate such patterns to airway immune-inflammatory status and clinical outcomes in bronchiectasis. Methods: Patients with bronchiectasis were recruited in Asia (Singapore and Malaysia) and the United Kingdom (Scotland) (n = 238), forming the Cohort of Asian and Matched European Bronchiectasis, which matched recruited patients on age, sex, and bronchiectasis severity. Specific IgE response against a range of common allergens was determined, combined with airway immune-inflammatory status and correlated to clinical outcomes. Clinically relevant patient clusters, based on sensitization pattern and airway immune profiles (“immunoallertypes”), were determined. Measurements and Main Results: A high frequency of sensitization to multiple allergens was detected in bronchiectasis, exceeding that in a comparator cohort with allergic rhinitis (n = 149). Sensitization was associated with poor clinical outcomes, including decreased pulmonary function and more severe disease. “Sensitized bronchiectasis” was classified into two immunoallertypes: one fungal driven and proinflammatory, the other house dust mite driven and chemokine dominant, with the former demonstrating poorer clinical outcome. Conclusions: Allergic sensitization occurs at high frequency in patients with bronchiectasis recruited from different global centers. Improving endophenotyping of sensitized bronchiectasis, a clinically significant state, and a “treatable trait” permits therapeutic intervention in appropriate patients, and may allow improved stratification in future bronchiectasis research and clinical trials.Ministry of Education (MOE)Ministry of Health (MOH)National Medical Research Council (NMRC)Published versionSupported by the Singapore Ministry of Health’s National Medical Research Council under its Transition Award NMRC/TA/0048/2016 (S.H.C.) and Changi General Hospital Research grant CHF2016.03-P (T.B.L.). The work performed at NUS was supported by the Singapore Ministry of Education Academic Research Fund, SIgN, and National Medical Research Council grants N-154-000-038-001, R-154-000-404-112, R-154-000-553-112, R-154-000-565-112, R-154-000-630-112, R-154-000-A08-592, R-154-000-A27-597, SIgN-06-006, SIgN-08-020, and NMRC/1150/2008 (F.T.C.); J.D.C. is supported by the GSK/British Lung Foundation Chair of Respiratory Research

Alpha-1 antitrypsin augmentation therapy decreases miR-199a-5p, miR-598 and miR-320a expression in monocytes via inhibition of NFκB

Alpha-1 antitrypsin (AAT) augmentation therapy involves infusion of plasma-purified AAT to AAT deficient individuals. Whether treatment affects microRNA expression has not been investigated. This study's objectives were to evaluate the effect of AAT augmentation therapy on altered miRNA expression in monocytes and investigate the mechanism. Monocytes were isolated from non-AAT deficient (MM) and AAT deficient (ZZ) individuals, and ZZs receiving AAT. mRNA (qRT-PCR, microarray), miRNA (miRNA profiling, qRT-PCR), and protein (western blotting) analyses were performed. Twenty one miRNAs were differentially expressed 3-fold between ZZs and MMs. miRNA validation studies demonstrated that in ZZ monocytes receiving AAT levels of miR-199a-5p, miR-598 and miR-320a, which are predicted to be regulated by NFκB, were restored to levels similar to MMs. Validated targets co-regulated by these miRNAs were reciprocally increased in ZZs receiving AAT in vivo and in vitro. Expression of these miRNAs could be increased in ZZ monocytes treated ex vivo with an NFκB agonist and decreased by NFκB inhibition. p50 and p65 mRNA and protein were significantly lower in ZZs receiving AAT than untreated ZZs. AAT augmentation therapy inhibits NFκB and decreases miR-199a-5p, miR-598 and miR-320a in ZZ monocytes. These NFκB-inhibitory properties may contribute to the anti-inflammatory effects of AAT augmentation therapy.Alpha One Foundatio