Biology subjects

Pathinayake, P. S.

Publications and source records attributed to Pathinayake, P. S..

4 recordsLinked to original sources

bioRxiv2023

Comparison of commercially available differentiation media on morphology, function, and virus-host interaction in conditionally reprogrammed human bronchial epithelial cells

IntroductionPrimary air liquid interface (ALI) cultures of bronchial epithelial cells are used extensively to model airway responses. A recent advance is the development of conditional reprogramming that enhances proliferative capability. Several different media and protocols are utilized, yet even subtle differences may influence cellular responses. We compared the morphology and functional responses, including innate immune responses to rhinovirus infection in conditionally reprogrammed primary bronchial epithelial cells (pBECs) differentiated using two commonly used culture media. MethodspBECs from healthy participants (n = 5) were CR using {gamma}-irradiated 3T3 fibroblasts and Rho Kinase inhibitor. CRpBECs were differentiated at ALI in either PneumaCult (PN-ALI) or Bronchial Epithelial Growth Medium (BEGM)-based differentiation media (BEBM:DMEM, 50:50, Lonza) - (AB-ALI) for 28 days. Transepithelial electrical resistance (TEER), immunofluorescence, histology, cilia activity, ion channel function, and expression of cell markers were analyzed. Viral load was assessed by RT-qPCR and anti-viral factors quantified by Legendplex following Rhinovirus-A1b (RVA1b) infection. ResultsCRpBECs differentiated in PneumaCult were smaller and had a lower TEER and cilia beat frequency (CBF) compared to BEGM media. PneumaCult media cultures exhibited significantly increased FOXJ1 expression, more ciliated cells with a larger active area, increased intracellular mucins, and increased calcium-activated chloride channel current. However, there were no significant changes in viral RNA or host antiviral responses. ConclusionThere are distinct structural and functional differences in CRpBECs cultured in the two commonly used ALI differentiation media. Such factors need to be taken into consideration when designing and comparing CRpBECs ALI experiments.

cell biology↗

bioRxiv2022

Single cell RNA-seq identifies inflammation-induced loss of CFTR-expressing airway ionocytes in non-eosinophilic asthma

Asthma is the most common chronic airways disease worldwide and the severe treatment resistant subtype of asthma is responsible for the majority of disease burden. Asthma is heterogeneous in nature and can be classified according to airway infiltrates as eosinophilic or non-eosinophilic (sometimes referred to as Type 2 low), which is further divided into paucigranulocytic (low levels of granulocytes), or neutrophilic asthma characterized by elevated neutrophils, and mixed Type 1 and Type 17 cytokines in airway tissue, sputum, and bronchoalveolar lavage. Severe non-eosinophilic asthma currently has fewer effective treatment options and many of these patients fail to qualify for newer biologic monoclonal therapies. The cystic fibrosis transmembrane conductance regulator (CFTR) is a key protein whose function is dysregulated in multiple respiratory diseases including cystic fibrosis and chronic obstructive pulmonary disease (COPD) and has proven a valuable therapeutic target. Using human bronchial epithelial cells (hBECs) isolated differentiated at air-liquid interface we demonstrated a reduced function of the CFTR in non-eosinophilic asthma. Characterization of the cell and molecular differences in airway epithelial cells between severe asthma subtypes using single cell RNA-sequencing (scRNAseq) revealed that airway epithelial cells from non-eosinophilic asthma, and in particular neutrophilic asthma patients, fail to differentiate into CFTR-expressing ionocytes compared with eosinophilic asthma or healthy donors. We identified a novel ionocyte transcriptional signature, which was present in both bronchial and tracheal airway epithelial samples indicating conserved anatomical gene regulation. Using protein markers and immunofluorescent quantification loss of ionocytes was confirmed in non-eosinophilic asthma hBECs. Similarly, ioncytes were also diminished in the airways of a murine model of neutrophilic-dominant but not eosinophilic allergen asthma models. Furthermore, treatment of hBECs from healthy donors with a neutrophilic asthma-like inflammatory cytokine mixture, but not IL-13, led to loss of ionocytes primarily due to IFN-{gamma}. Inflammation-induced loss of CFTR-expressing ionocytes in airway cells from non-eosinophilic asthma may represent a key feature of disease pathogenesis and a novel drug target for this difficult-to-treat disease.

cell biology↗

bioRxiv2020

SARS-CoV-2 Spike protein promotes hyper-inflammatory response that can be ameliorated by Spike-antagonistic peptide and FDA-approved ER stress and MAP kinase inhibitors in vitro

SARS-CoV-2 infection causes an inflammatory cytokine storm and acute lung injury. Currently there are no effective antiviral and/or anti-inflammatory therapies. Here we demonstrate that 2019 SARS-CoV-2 spike protein subunit 1 (CoV2-S1) induces high levels of NF-{kappa}B activations, production of pro-inflammatory cytokines and mild epithelial damage, in human bronchial epithelial cells. CoV2-S1-induced NF-{kappa}B activation requires S1 interaction with human ACE2 receptor and early activation of endoplasmic reticulum (ER) stress, and associated unfolded protein response (UPR), and MAP kinase signalling pathways. We developed an antagonistic peptide that inhibits S1-ACE2 interaction and CoV2-S1-induced productions of pro-inflammatory cytokines. The existing FDA-approved ER stress inhibitor, 4-phenylburic acid (4-PBA), and MAP kinase inhibitors, trametinib and ulixertinib, ameliorated CoV2-S1-induced inflammation and epithelial damage. These novel data highlight the potentials of peptide-based antivirals for novel ACE2-utilising CoVs, while repurposing existing drugs may be used as treatments to dampen elevated inflammation and lung injury mediated by SARS-CoV-2.

immunology↗

bioRxiv2019

Anti-microbial immunity is impaired in COPD patients with frequent exacerbations

BackgroundPatients with frequent exacerbations represent a chronic obstructive pulmonary disease (COPD) sub-group requiring better treatment options. The aim of this study was to determine the innate immune mechanisms that underlie susceptibility to frequent exacerbations in COPD.\n\nMethodsWe measured sputum expression of immune mediators and bacterial loads in samples from patients with COPD at stable state and during virus-associated exacerbations. Ex vivo immune responses to rhinovirus infection in differentiated bronchial epithelial cells (BECs) sampled from patients with COPD were additionally evaluated. Patients were stratified as frequent exacerbators ([≥]2 exacerbations in the preceding year) or infrequent exacerbators (<2 exacerbations in the preceding year) with comparisons made between these groups.\n\nResultsFrequent exacerbators had reduced sputum cell mRNA expression of the anti-viral immune mediators type I and III interferons and reduced interferon-stimulated gene (ISG) expression when clinically stable and during virus-associated exacerbation. RV-induction of interferon and ISGs ex vivo was also impaired in differentiated BECs from frequent exacerbators. Frequent exacerbators also had reduced sputum levels of the anti-microbial peptide mannose-binding lectin (MBL)-2 with an associated increase in sputum bacterial loads at 2 weeks following virus-associated exacerbation onset. MBL-2 levels correlated negatively with bacterial loads during exacerbation.\n\nConclusionThese data implicate deficient airway innate immunity in the increased propensity to exacerbations observed in some patients with COPD. Therapeutic approaches to boost innate antimicrobial immunity in the lung could be a viable strategy for prevention/treatment of frequent exacerbations.

immunology↗