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

Treatment of multiple-level tracheobronchial stenosis secondary to endobronchial tuberculosis using bronchoscopic balloon dilatation with topical mitomycin-C

Background: Tracheobronchial stenosis is a known complication of endobronchial tuberculosis. Despite antituberculous and steroid therapy, the development of bronchial stenosis is usually irreversible and requires airway patency to be restored by either bronchoscopic or surgical interventions. We report the use of balloon dilatation and topical mitomycin-C to successful restore airway patency. Case presentation: We present a 24-year old lady with previous pulmonary tuberculosis and laryngeal tuberculosis in 2007 and 2013 respectively who presented with worsening dyspnoea and stridor. She had total left lung collapse with stenosis of both the upper trachea and left main bronchus. She underwent successful bronchoscopic balloon and manual rigid tube dilatation with topical mitomycin-C application over the stenotic tracheal segment. A second bronchoscopic intervention was performed after 20 weeks for the left main bronchus stenosis with serial balloon dilatation and topical mitomycin-C application. These interventions led to significant clinical and radiographic improvements. Conclusion: This case highlights that balloon dilatation and topical mitomycin-C application should be considered in selected patients with tracheobronchial stenosis following endobronchial tuberculosis, avoiding airway stenting and invasive surgical intervention.Published versio

The Effect of Aspergillus fumigatus Infection on Vitamin D Receptor Expression in Cystic Fibrosis

Rationale: Aspergillus fumigatus (A. fumigatus) in cystic fibrosis (CF) is increasingly recognized. Although allergic bronchopulmonary aspergillosis (ABPA) leads to deterioration of pulmonary function, the effect of A. fumigatus colonization in the absence of ABPA remains unclear. Objectives: To address this, we examined individuals with CF with A. fumigatus who were ABPA negative to identify the effects of itraconazole therapy on Aspergillus-induced lung inflammation. Methods: The effect of A. fumigatus on nuclear vitamin D receptor (VDR) expression was investigated using qRT-PCR and Western blotting. IL-5 and IL-13 levels were quantified by ELISA. The effect of itraconazole was assessed by a combination of high-resolution computed tomography, lung function test, and microbiological analysis. Measurements and Main Results: We demonstrate that A. fumigatus down-regulates VDR in macrophages and airway epithelial cells and that the fungal metabolite gliotoxin (Gt) is the main causative agent. Gt overcame the positive effect of 1,25-OH vitamin D3 on VDR expression in vitro, resulting in increased IL-5 and IL-13 production. In vivo, A. fumigatus positivity was associated with increased Gt in CF bronchoalveolar lavage fluid and increased bronchoalveolar lavage fluid levels of IL-5 and IL-13. After airway eradication of A. fumigatus with itraconazole, we observed decreased Gt, IL-5 and IL-13, improved respiratory symptoms, and diminished high-resolution computed tomography mosaic pattern consistent with sustained pulmonary function. Conclusions: This study provides a rationale for the therapeutic effect of itraconazole and implied that the therapeutic potential of vitamin D supplementation in preventing ABPA is only feasible with concurrent elimination of A. fumigatus to permit VDR expression and its positive functional consequences