Synergy in Cystic Fibrosis Therapies: Targeting SLC26A9

SLC26A9, a constitutively active Cl− transporter, has gained interest over the past years as a relevant disease modifier in several respiratory disorders including Cystic Fibrosis (CF), asthma, and non-CF bronchiectasis. SLC26A9 contributes to epithelial Cl− secretion, thus preventing mucus obstruction under inflammatory conditions. Additionally, SLC26A9 was identified as a CF gene modifier, and its polymorphisms were shown to correlate with the response to drugs modulating CFTR, the defective protein in CF. Here, we aimed to investigate the relationship between SLC26A9 and CFTR, and its role in CF pathogenesis. Our data show that SLC26A9 expression contributes to enhanced CFTR expression and function. While knocking-down SLC26A9 in human bronchial cells leads to lower wt- and F508del-CFTR expression, function, and response to CFTR correctors, the opposite occurs upon its overexpression, highlighting SLC26A9 relevance for CF. Accordingly, F508del-CFTR rescue by the most efficient correctors available is further enhanced by increasing SLC26A9 expression. Interestingly, SLC26A9 overexpression does not increase the PM expression of non-F508del CFTR traffic mutants, namely those unresponsive to corrector drugs. Altogether, our data indicate that SLC26A9 stabilizes CFTR at the ER level and that the efficacy of CFTR modulator drugs may be further enhanced by increasing its expression.

Copper-Associated Oxidative Stress Contributes to Cellular Inflammatory Responses in Cystic Fibrosis

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF Transmembrane Conductance Regulator (CFTR), an apical chloride channel. An early inflammation (EI) in the lung of CF patients occurring in the absence of any bacterial infection has been reported. This EI has been proposed to be associated with oxidative stress (OX-S), generated by deregulations of the oxidant/antioxidant status. Recently, we demonstrated that copper (Cu), an essential trace element, mediates OX-S in bronchial cells. However, the role of this element in the development of CF-EI, in association with OX-S, has never been investigated. Using healthy (16HBE14o-; HBE), CF (CFBE14o-; CFBE), and corrected-wild type CFTR CF (CFBE-wt) bronchial cells, we characterized the inflammation and OX-S profiles in relation to the copper status and CFTR expression and function. We demonstrated that CFBE cells exhibited a CFTR-independent intrinsic inflammation. These cells also exhibited an alteration in mitochondria, UPR (Unfolded Protein Response), catalase, Cu/Zn- and Mn-SOD activities, and an increase in the intracellular content of iron, zinc, and Cu. The increase in Cu concentration was associated with OX-S and inflammatory responses. These data identify cellular Cu as a key factor in the generation of CF-associated OX-S and opens new areas of investigation to better understand CF-associated EI.

Benzo[ghi]perylene Induces Morphological and Genotoxic Effects in Human Bronchial Cells Independently of The AhR Pathway

Polycyclic aromatic hydrocarbons (PAH) are organic compounds found in the contaminated atmosphere of Mexico City, where the PAH present with highest concentration is benzo[ghi]perylene [B[ghi]p]. We recently demonstrated that double-stranded breaks in DNA appear after 3h of exposure, whereas cellular changes and activation of aryl hydrocarbon receptor (AHR) pathway occur after 48h in bronchial cells under exposure to B[ghi]p, these findings have led us to explore if the AHR pathway participates in morphological changes and genotoxic effects due to B[ghi]p in human NL-20 bronchial cells. Cells of the NL-20 human bronchial line were exposed to B[ghi]p in the presence, or absence, of the AHR receptor antagonist, CH-223191. Cell viability was quantified by the MTT assay, which revealed 76 and 66% at 6h and 24h, respectively (p<0.001), regardless of the presence of CH-223191. RT-qPCR showed an increase in the expression of the AHR and CYP1A1 cytochrome genes only after 24h of exposure, and the expression was inhibited by CH-223191. Genotoxicity assays revealed the presence of comets, nuclear buds (NB) and DNA fragmentation in cells exposed to B[ghi]p after 6h and in cells exposed to B[ghi]p plus CH-223191 at 24h. These results verify that B[ghi]p induces morphological and genotoxic effects, and these effects are independent of the AHR pathway.

Iron-Rich Magnetic Coal Fly Ash Particles Induce Apoptosis in Human Bronchial Cells

Svalbard is an arctic archipelago where coal mining generates all electricity via the local coal-fired power station. Coal combustion produces a waste product in the form of particulate matter (PM) coal fly ash (CFA), derived from incombustible minerals present in the feed coal. PM ≤10 µm (diameter) may be “inhaled” into the human respiratory system, and particles ≤2.5 µm may enter the distal alveoli to disrupt normal pulmonary functions and trigger disease pathways. This study discovered that Svalbard CFA contained unusually high levels of iron-rich magnetic minerals that induced adverse effects upon human lungs cells. Iron is a well-characterised driver of reactive oxygen species (ROS) generation, a driving force for cell death and disease. CFA physicochemical characterisation showed non-uniform particle morphologies indicative of coal burnt at inefficient combustion temperatures. The bioreactivity (ROS generation) of PM2.5/10 fractions was measured using plasmid scission assay (PSA, DNA damage) and haemolysis assays (erythrocyte lysis), with PM2.5 CFA showing significant bioreactivity. CFA leached in mild acid caused a significant increase in toxicity, which could occur in CFA waste-stores. The CFA and leachates were exposed to a surrogate model of human bronchial epithelia that confirmed that CFA induced apoptosis in bronchial cells. This study shows that CFA containing magnetic iron-rich minerals mediated adverse reactions in the human lung, and thus CFA should be considered to be an environmental inhalation hazard.

The solvent and treatment regimen of sodium selenite cause its effects to vary on the radiation response of human bronchial cells from tumour and normal tissues

Sodium selenite is often given to moderate the side effects of cancer therapy to enhance the cellular defence of non-cancerous cells. To determine whether sodium selenite during radiotherapy protects not only normal cells but also cancer cells, which would imply a reduction of the desired effect of irradiation on tumour during radiotherapy, the effect of the combined treatment of irradiation and sodium selenite was investigated. Human bronchial cells from carcinoma (A549) and normal tissue (BEAS-2B) were treated with sodium selenite and effects on growth and in combination with radiation on metabolic activity and cell cycle distribution were studied. The influence on radiosensitivity was determined via colony forming assays using different solvents of sodium selenite and treatment schedules. It was shown that sodium selenite inhibits growth and influences cell cycle distribution of both normal and tumour cells. Metabolic activity of normal cells decreased more rapidly compared to that of cancer cells. The influence of sodium selenite on radiation response depended on the different treatment schedules and was strongly affected by the solvent of the agent. It could be shown that the effect of sodium selenite on radiation response is strongly dependent on the respective experimental in vitro conditions and ranges from lead to an initially suspected but ultimately no real radioprotection to radiosensitizing up to no effect in one and the same cell line. This might be a reason for controversially described cell responses to radiation under the influence of sodium selenite in studies so far.

Regulation of the ACE2 locus in human airways cells

The angiotensin-converting enzyme 2 (ACE2) receptor is the gateway for SARS-CoV-2 to airway epithelium1,2 and the strong inflammatory response after viral infection is a hallmark in COVID-19 patients. Deciphering the regulation of the ACE2 gene is paramount for understanding the cell tropism of SARS-CoV-2 infection. Here we identify candidate regulatory elements in the ACE2 locus in human primary airway cells and lung tissue. Activating histone and promoter marks and Pol II loading characterize the intronic dACE2 and define novel candidate enhancers distal to the genuine ACE2 promoter and within additional introns. dACE2, and to a lesser extent ACE2, RNA levels increased in primary bronchial cells treated with interferons and this induction was mitigated by Janus kinase (JAK) inhibitors that are used therapeutically in COVID-19 patients. Our analyses provide insight into regulatory elements governing the ACE2 locus and highlight that JAK inhibitors are suitable tools to suppress interferon-activated genetic programs in bronchial cells.