Pathogenesis, imaging and clinical characteristics of CF and non-CF bronchiectasis

Bronchiectasis is a common feature of severe inherited and acquired pulmonary disease conditions. Among inherited diseases, cystic fibrosis (CF) is the major disorder associated with bronchiectasis, while acquired conditions frequently featuring bronchiectasis include post-infective bronchiectasis and chronic obstructive pulmonary disease (COPD). Mechanistically, bronchiectasis is driven by a complex interplay of inflammation and infection with neutrophilic inflammation playing a predominant role. The clinical characterization and management of bronchiectasis should involve a precise diagnostic workup, tailored therapeutic strategies and pulmonary imaging that has become an essential tool for the diagnosis and follow-up of bronchiectasis. Prospective future studies are required to optimize the diagnostic and therapeutic management of bronchiectasis, particularly in heterogeneous non-CF bronchiectasis populations

Obesity exacerbates influenza-induced respiratory disease via the arachidonic acid-p38 MAPK pathway

Obesity is a risk factor for severe influenza, and asthma exacerbations caused by respiratory viral infections. We investigated mechanisms that increase the severity of airway disease related to influenza in obesity using cells derived from obese and lean individuals, and in vitro and in vivo models. Primary human nasal epithelial cells (pHNECs) derived from obese compared with lean individuals developed increased inflammation and injury in response to influenza A virus (IAV). Obese mice infected with influenza developed increased airway inflammation, lung injury and elastance, but had a decreased interferon response, compared with lean mice. Lung arachidonic acid (AA) levels increased in obese mice infected with IAV; arachidonic acid increased inflammatory cytokines and injury markers in response to IAV in human bronchial epithelial (HBE) cells. Obesity in mice, and AA in HBE cells, increased activation of p38 MAPK signaling following IAV infection; inhibiting this pathway attenuated inflammation, injury and tissue elastance responses, and improved survival. In summary, obesity increases disease severity in response to influenza infection through activation of the p38 MAPK pathway in response to altered arachidonic acid signaling

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

Geographic variation in the aetiology, epidemiology and microbiology of bronchiectasis

Bronchiectasis is a disease associated with chronic progressive and irreversible dilatation of the bronchi and is characterised by chronic infection and associated inflammation. The prevalence of bronchiectasis is age-related and there is some geographical variation in incidence, prevalence and clinical features. Most bronchiectasis is reported to be idiopathic however post-infectious aetiologies dominate across Asia especially secondary to tuberculosis. Most focus to date has been on the study of airway bacteria, both as colonisers and causes of exacerbations. Modern molecular technologies including next generation sequencing (NGS) have become invaluable tools to identify microorganisms directly from sputum and which are difficult to culture using traditional agar based methods. These have provided important insight into our understanding of emerging pathogens in the airways of people with bronchiectasis and the geographical differences that occur. The contribution of the lung microbiome, its ethnic variation, and subsequent roles in disease progression and response to therapy across geographic regions warrant further investigation. This review summarises the known geographical differences in the aetiology, epidemiology and microbiology of bronchiectasis. Further, we highlight the opportunities offered by emerging molecular technologies such as -omics to further dissect out important ethnic differences in the prognosis and management of bronchiectasis.NMRC (Natl Medical Research Council, S’pore)MOH (Min. of Health, S’pore)Published versio

Identification of the molecular partners that regulate MEIS1A function

Meis1 encodes a homeodomain-containing transcription factor which performs a vital role during embryonic development as well as in adult physiological processes, particularly hematopoiesis. It is an oncogene in many kinds of leukemia, most notably acute myeloid leukemia (AML) and mixed lineage leukemia (MLL) as well as solid tumors like neuroblastoma. In addition, Meis1 has been implicated in genetic disorders like restless leg syndrome. MEIS1 functions upstream of many target genes to regulate their expression. HOX and PBX proteins are the most important and well-studied interaction partners of MEIS1. Recently, however, MEIS1 has been shown to interact with CRTC transcription factors and function in a PKA-dependent signaling pathway. Considering the significance and the diversity of the processes regulated by MEIS1, we hypothesized that it could interact with a large repertoire of proteins in order to carry out its functions. Hence my work involved the purification and identification of novel interaction partners of MEIS1A, the widely expressed isoform of MEIS1. To accomplish this goal, I used a recently described technique for the purification of interaction partners. It involved the in vivo biotinylation of MEIS1A followed by a single-step purification of the MEIS1A-interacting proteins using streptavidin-affinity purification. Using mass spectrometry, I identified approximately 40 different proteins that specifically co-purified with MEIS1A. Through co-immunoprecipitation assays, I validated the interaction of MEIS1A with some of these proteins including NONO, NOP56, NOP58, CBX3 and MLL1. I was also able to show that the interaction with NONO, NOP56 and NOP58 is conserved among other MEIS1-related proteins like PKNOX1 and PKNOX2. NONO is of interest because of its reported interaction with CRTC1. I was able to show that NONO does not mediate the interaction between CRTC1 and MEIS1A. In addition, NONO does not contribute to the transactivation potential of MEIS1A under our experimental conditions. The physiological relevance of the MEIS1A-NONO interaction is therefore yet to be understood. The MEIS1A-MLL1 interaction that I identified was very interesting because both proteins play physiologically important roles during hematopoiesis and leukemia and function in a common pathway. We were able to show that MLL1 and MEIS1 interact endogenously in RS4;11 cells. The MEIS1A interaction is retained by MLL-AF4 which is a leukemogenic fusion protein of MLL1. I was also able to map the interaction of MLL1 to the C-terminal region of MEIS1A. Together, my data provide insight into the interactome and mechanisms of MEIS1A function, and open multiple avenues for further investigation.DOCTOR OF PHILOSOPHY (SBS

Protein Disulfide Isomerase A3 Regulates Influenza Neuraminidase Activity and Influenza Burden in the Lung

Influenza (IAV) neuraminidase (NA) is a glycoprotein required for the viral exit from the cell. NA requires disulfide bonds for proper function. We have recently demonstrated that protein disulfide isomerase (PDI)A3 is required for oxidative folding of IAV hemagglutinin (HA), and viral propagation. However, it not known whether PDIs are required for NA maturation or if these interactions represent a putative target for the treatment of influenza infection. We sought to determine whether PDIA3 is required for disulfide bonds of NA, its activity, and propagation of the virus. Requirement of disulfides for NA oligomerization and activity were determined using biotin switch and redox assays in WT and PDIA3−/− in A549 cells. A PDI specific inhibitor (LOC14) was utilized to determine the requirement of PDIs in NA activity, IAV burden, and inflammatory response in A549 and primary mouse tracheal epithelial cells. Mice were treated with the inhibitor LOC14 and subsequently examined for IAV burden, NA activity, cytokine, and immune response. IAV-NA interacts with PDIA3 and this interaction is required for NA activity. PDIA3 ablation or inhibition decreased NA activity, viral burden, and inflammatory response in lung epithelial cells. LOC14 treatment significantly attenuated the influenza-induced inflammatory response in mice including the overall viral burden. These results provide evidence for PDIA3 inhibition suppressing NA activity, potentially providing a novel platform for host-targeted antiviral therapies

A new therapeutic avenue for bronchiectasis : dry powder inhaler of ciprofloxacin nanoplex exhibits superior ex vivo mucus permeability and antibacterial efficacy to its native ciprofloxacin counterpart

Non-cystic fibrosis bronchiectasis (NCFB) characterized by permanent bronchial dilatation and recurrent infections has been clinically managed by long-term intermittent inhaled antibiotic therapy among other treatments. Herein we investigated dry powder inhaler (DPI) formulation of ciprofloxacin (CIP) nanoplex with mannitol/lactose as the excipient for NCFB therapy. The DPI of CIP nanoplex was evaluated against DPI of native CIP in terms of their (1) dissolution characteristics in artificial sputum medium, (2) ex vivo mucus permeability in sputum from NCFB and healthy individuals, (3) antibacterial efficacy in the presence of sputum against clinical Pseudomonas aeruginosa strains (planktonic and biofilm), and (4) cytotoxicity towards human lung epithelial cells. Despite their similarly fast dissolution rates in sputum, the DPI of CIP nanoplex exhibited superior mucus permeability to the native CIP (5-7 times higher) attributed to its built-in ability to generate highly supersaturated CIP concentration in the sputum. The superior mucus permeability led to the CIP nanoplex's higher antibacterial efficacy (>3 log10 CFU/mL). The DPI of CIP nanoplex exhibited similar cytotoxicity towards the lung epithelial cells as the native CIP indicating its low risk of toxicity. These results established the promising potential of DPI of CIP nanoplex as a new therapeutic avenue for NCFB.MOH (Min. of Health, S’pore)Accepted versio