Multicentric investigations of the role in the disease severity of accelerated phospholipid changes in COVID-19 patient airway.

CONTEXT: The changes in host membrane phospholipids are crucial in airway infection pathogenesis. Phospholipase A2 hydrolyzes host cell membranes, producing lyso-phospholipids and free fatty acids, including arachidonic acid (AA), which contributes significantly to lung inflammation. AIM: Follow these changes and their evolution from day 1, day 3 to day 7 in airway aspirates of 89 patients with COVID-19-associated acute respiratory distress syndrome and examine whether they correlate with the severity of the disease. The patients were recruited in three French intensive care units. The analysis was conducted from admission to the intensive care unit until the end of the first week of mechanical ventilation. RESULTS: In the airway aspirates, we found significant increases in the levels of host cell phospholipids, including phosphatidyl-serine and phosphatidyl-ethanolamine, and their corresponding lyso-phospholipids. This was accompanied by increased levels of AA and its inflammatory metabolite prostaglandin E2 (PGE2). Additionally, enhanced levels of ceramides, sphingomyelin, and free cholesterol were observed in these aspirates. These lipids are known to be involved in cell death and/or apoptosis, whereas free cholesterol plays a role in virus entry and replication in host cells. However, there were no significant changes in the levels of dipalmitoyl-phosphatidylcholine, the major surfactant phospholipid. A correlation analysis revealed an association between mortality risk and levels of AA and PGE2, as well as host cell phospholipids. CONCLUSION: Our findings indicate a correlation between heightened cellular phospholipid modifications and variations in AA and PGE2 with the severity of the disease in patients. Nevertheless, there is no indication of surfactant alteration in the initial phases of the illness.

Should We Interfere with the Interleukin-6 Receptor During COVID-19: What Do We Know So Far?

Severe manifestations of COVID-19 consist of acute respiratory distress syndrome due to an initially local reaction leading to a systemic inflammatory response that results in hypoxia. Many therapeutic approaches have been attempted to reduce the clinical consequences of an excessive immune response to viral infection. To date, systemic corticosteroid therapy is still the most effective intervention. More recently, new hope has emerged with the use of interleukin (IL)-6 receptor inhibitors (tocilizumab and sarilumab). However, the great heterogeneity of the methodology and results of published studies obfuscate the true value of this treatment, leading to a confusing synthesis in recent meta-analyses, and the persistence of doubts in terms of patient groups and the appropriate time to treat. Moreover, their effects on the anti-infectious or pro-healing response are still poorly studied. This review aims to clarify the potential role of IL-6 receptor inhibitors in the treatment of severe forms of COVID-19.

TMEM16A/ANO1: Current Strategies and Novel Drug Approaches for Cystic Fibrosis.

Cystic fibrosis (CF) is the most common of rare hereditary diseases in Caucasians, and it is estimated to affect 75,000 patients globally. CF is a complex disease due to the multiplicity of mutations found in the CF transmembrane conductance regulator (CFTR) gene causing the CFTR protein to become dysfunctional. Correctors and potentiators have demonstrated good clinical outcomes for patients with specific gene mutations; however, there are still patients for whom those treatments are not suitable and require alternative CFTR-independent strategies. Although CFTR is the main chloride channel in the lungs, others could, e.g., anoctamin-1 (ANO1 or TMEM16A), compensate for the deficiency of CFTR. This review summarizes the current knowledge on calcium-activated chloride channel (CaCC) ANO1 and presents ANO1 as an exciting target in CF.

Novel Anti-Inflammatory Approaches for Cystic Fibrosis Lung Disease: Identification of Molecular Targets and Design of Innovative Therapies.

Cystic fibrosis (CF) is the most common genetic disorder among Caucasians, estimated to affect more than 70,000 people in the world. Severe and persistent bronchial inflammation and chronic bacterial infection, along with airway mucus obstruction, are hallmarks of CF lung disease and participate in its progression. Anti-inflammatory therapies are, therefore, of particular interest for CF lung disease. Furthermore, a better understanding of the molecular mechanisms involved in airway infection and inflammation in CF has led to the development of new therapeutic approaches that are currently under evaluation by clinical trials. These new strategies dedicated to CF inflammation are designed to treat different dysregulated aspects such as oxidative stress, cytokine secretion, and the targeting of dysregulated pathways. In this review, we summarize the current understanding of the cellular and molecular mechanisms that contribute to abnormal lung inflammation in CF, as well as the new anti-inflammatory strategies proposed to CF patients by exploring novel molecular targets and novel drug approaches.

miR-636: A Newly-Identified Actor for the Regulation of Pulmonary Inflammation in Cystic Fibrosis.

Cystic fibrosis (CF) results from deficient CF transmembrane conductance regulator (CFTR) protein activity leading to defective epithelial ion transport. Pulmonary degradation due to excessive inflammation is the main cause of morbidity and mortality in CF patients. By analysing miRNAs (small RNAseq) in human primary air-liquid interface cell cultures, we measured the overexpression of miR-636 in CF patients compared to non-CF controls. We validated these results in explant biopsies and determined that the mechanism underlying miR-636 overexpression is linked to inflammation. To identify specific targets, we used bioinformatics analysis to predict whether miR-636 targets the 3'-UTR mRNA regions of IL1R1 and RANK (two pro-inflammatory cytokine receptors), IKBKB (a major protein in the NF-κB pathway), and FAM13A (a modifier gene of CF lung phenotype implicated in epithelial remodelling). Using bronchial epithelial cells from CF patients to conduct a functional analysis, we showed a direct interaction between miR-636 and IL1R1, RANK, and IKBKB, but not with FAM13A. These interactions led to a decrease in IL1R1 and IKKß protein expression levels, while we observed an increase in RANK protein expression levels following the overexpression of miR-636. Moreover, NF-κB activity and IL-8 and IL-6 secretions decreased following the transfection of miR-636 mimics in CF cells. Similar but opposite effects were found after transfection with an antagomiR-636 in the same cells. Furthermore, we demonstrated that miR-636 was not regulated by Pseudomonas aeruginosa in our model. We went on to show that miR-636 is raised in the blood neutrophils, but not in the plasma, of CF patients and may have potential as a novel biomarker. Collectively, our findings reveal a novel actor for the regulation of inflammation in CF, miR-636, which is able to reduce constitutive NF-κB pathway activation when it is overexpressed.

Emerging microRNA Therapeutic Approaches for Cystic Fibrosis.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and remains the most common life-shortening diseases affecting the exocrine organs. The absence of this channel results in an imbalance of ion concentrations across the cell membrane and results in more abnormal secretion and mucus plugging in the gastrointestinal tract and in the lungs of CF patients. The direct introduction of fully functional CFTR by gene therapy has long been pursued as a therapeutical option to restore CFTR function independent of the specific CFTR mutation, but the different clinical trials failed to propose persuasive evidence of this strategy. The last ten years has led to the development of new pharmacotherapies which can activate CFTR function in a mutation-specific manner. Although approximately 2,000 different disease-associated mutations have been identified, a single codon deletion, F508del, is by far the most common and is present on at least one allele in approximately 70% of the patients in CF populations. This strategy is limited by chemistry, the knowledge on CFTR and the heterogenicity of the patients. New research efforts in CF aim to develop other therapeutical approaches to combine different strategies. Targeting RNA appears as a new and an important opportunity to modulate dysregulated biological processes. Abnormal miRNA activity has been linked to numerous diseases, and over the last decade, the critical role of miRNA in regulating biological processes has fostered interest in how miRNA binds to and interacts explicitly with the target protein. Herein, this review describes the different strategies to identify dysregulated miRNA opens up a new concept and new opportunities to correct CFTR deficiency. This review describes therapeutic applications of antisense techniques currently under investigation in CF.

[miRNA as a bullet against cystic fibrosis]. / Mucoviscidose : dans la ligne des miR.

Cystic fibrosis is the most common lethal genetic disease in the Caucasian population, characterized by CFTR gene mutations, which is a chloride channel. Whereas this gene has been known since 1989, the curative therapeutic solutions proposed to patients remain limited. New therapeutic strategies are therefore being explored, such as those targeting miRNA participating in the regulation of target mRNA expression. This review focuses on the involvement of miRNA in cystic fibrosis including ion channel control, inflammation, infection and bronchial obstruction and their therapeutic potential.

Opposite Expression of Hepatic and Pulmonary Corticosteroid-Binding Globulin in Cystic Fibrosis Patients.

Cystic fibrosis (CF) is characterized by a chronic pulmonary inflammation. In CF, glucocorticoids (GC) are widely used, but their efficacy and benefit/risk ratio are still debated. In plasma, corticosteroid-binding globulin (CBG) binds 90% of GC and delivers them to the inflammatory site. The main goal of this work was to study CBG expression in CF patients in order to determine whether CBG could be used to optimize GC treatment. The expression of CBG was measured in liver samples from CF cirrhotic and non-CF cirrhotic patients by qPCR and Western blot and in lung samples from non-CF and CF patients by qPCR. CBG binding assays with 3H-cortisol and the measurement of the elastase/α1-antitrypsin complex were performed using the plasmas. CBG expression increased in the liver at the transcript and protein level but not in the plasma of CF patients. This is possibly due to an increase of plasmatic elastase. We demonstrated that pulmonary CBG was expressed in the bronchi and bronchioles and its expression decreased in the CF lungs, at both levels studied. Despite the opposite expression of hepatic and pulmonary CBG in CF patients, the concentration of CBG in the plasma was normal. Thus, CBG might be useful to deliver an optimized synthetic GC displaying high affinity for CBG to the main inflammatory site in the context of CF, e.g., the lung.

Small RNA and transcriptome sequencing reveal the role of miR-199a-3p in inflammatory processes in cystic fibrosis airways.

Cystic fibrosis (CF) is the most common lethal genetic disease, caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations. CF is characterized by an ionic imbalance and thickened mucus, which impair mucociliary clearance and promote bacterial colonization and the establishment of infection/inflammation cycles. However, the origin of this inflammation remains unclear, although microRNAs (miRNAs) are suspected to be involved. MiRNAs are small non-coding RNAs that bind to the 3'-untranslated regions (UTRs) of target gene mRNA, thereby repressing their translation and/or inducing their degradation. The goal of this study was to investigate the role of microRNAs associated with pulmonary inflammation in CF patients. Through the analysis of all miRNAs (miRNome) in human primary air-liquid interface cultures, we demonstrated that miR-199a-3p is the only miRNA downregulated in CF patients compared to controls. Moreover, through RNA sequencing (transcriptome) analysis, we showed that 50% of all deregulated mRNAs are linked directly or indirectly to the NF-κB pathway. To identify a specific target, we used bioinformatics analysis to predict whether miR-199a-3p targets the 3'-UTR of IKBKB, which encodes IKKß, a major protein in the NF-κB pathway. Subsequently, we used bronchial explants from CF patients to show that miR-199a-3p expression is downregulated compared to controls and inversely correlated with increases in expression of IKKß and IL-8. Through functional studies, we showed that miR-199a-3p modulates the expression of IKBKB through a direct interaction at its 3'-UTR in bronchial epithelial cells from CF patients. In miR-199a-3p overexpression experiments, we demonstrated that for CF cells, miR-199a-3p reduced IKKß protein expression, NF-κB activity, and IL-8 secretion. Taken together, our findings show that miR-199a-3p plays a negative regulatory role in the NF-κB signalling pathway and that its low expression in CF patients contributes to chronic pulmonary inflammation. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MicroRNA-9 downregulates the ANO1 chloride channel and contributes to cystic fibrosis lung pathology.

Cystic fibrosis results from reduced cystic fibrosis transmembrane conductance regulator protein activity leading to defective epithelial ion transport. Ca2+-activated Cl- channels mediate physiological functions independently of cystic fibrosis transmembrane conductance regulator. Anoctamin 1 (ANO1/TMEM16A) was identified as the major Ca2+-activated Cl- channel in airway epithelial cells, and we recently demonstrated that downregulation of the anoctamin 1 channel in cystic fibrosis patients contributes to disease severity via an unknown mechanism. Here we show that microRNA-9 (miR-9) contributes to cystic fibrosis and downregulates anoctamin 1 by directly targeting its 3'UTR. We present a potential therapy based on blockage of miR-9 binding to the 3'UTR by using a microRNA target site blocker to increase anoctamin 1 activity and thus compensate for the cystic fibrosis transmembrane conductance regulator deficiency. The target site blocker is tested in in vitro and in mouse models of cystic fibrosis, and could be considered as an alternative strategy to treat cystic fibrosis.Downregulation of the anoctamin 1 calcium channel in airway epithelial cells contributes to pathology in cystic fibrosis. Here the authors show that microRNA-9 targets anoctamin 1 and that inhibiting this interaction improves mucus dynamics in mouse models.

Translating the genetics of cystic fibrosis to personalized medicine.

Cystic fibrosis (CF) is the most common life-threatening recessive genetic disease in the Caucasian population. This multiorgan disease is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, a chloride channel recognized as regulating several apical ion channels. The gene mutations result either in the lack of the protein at the apical surface or in an improperly functioning protein. Morbidity and mortality because of the mutation of CFTR are mainly attributable to lung disease resulting from chronic infection and inflammation. Since its discovery as the causative gene in 1989, much progress has been achieved not only in clinical genetics but also in basic science studies. Recently, combinations of these efforts have been successfully translated into development and availability for patients of new therapies targeting specific CFTR mutations to correct the CFTR at the protein level. Current technologies such as next gene sequencing and novel genomic editing tools may offer new strategies to identify new CFTR variants and modifier genes, and to correct CFTR to pursue personalized medicine, which is already developed in some patient subsets. Personalized medicine or P4 medicine ("personalized," "predictive," "preventive," and "participatory") is currently booming for CF. The various current and future challenges of personalized medicine as they apply to the issues faced in CF are discussed in this review.

Normal and Cystic Fibrosis Human Bronchial Epithelial Cells Infected with Pseudomonas aeruginosa Exhibit Distinct Gene Activation Patterns.

BACKGROUND AND AIMS: In cystic fibrosis (CF), Pseudomonas aeruginosa is not eradicated from the lower respiratory tract and is associated with epithelial inflammation that eventually causes tissue damage. To identify the molecular determinants of an effective response to P. aeruginosa infection, we performed a transcriptomic analysis of primary human bronchial epithelial cells from healthy donors (CTRL) 2, 4, and 6 h after induced P. aeruginosa infection. Compared to noninfected cells, infected cells showed changes in gene activity, which were most marked 6 h postinfection and usually consisted in upregulation. RESULTS: By comparing for each time point of infection, the transcriptomic response of epithelial cells from CF patients and healthy donors, we identified 851, 638, 667, and 980 differentially expressed genes 0, 2, 4, and 6 h postinfection, respectively. Gene selection followed by bioinformatic analysis showed that most of the differentially expressed genes, either up- or downregulated, were in the protein-binding and catalytic gene-ontology categories. Finally, we established that the protein products of the genes exhibiting the greatest differential upregulation (CSF2, CCL2, TNF, CSF3, MMP1, and MMP10) between CF patients and CTRL were produced in higher amounts by infected cells from CF patients versus CTRL. CONCLUSIONS: The differentially expressed genes in CF patients may constitute a signature for a detrimental inflammatory response and for an inefficient P. aeruginosa host-cell response.

New insights about miRNAs in cystic fibrosis.

The molecular basis of cystic fibrosis (CF) is a mutation-related defect in the epithelial-cell chloride channel called CF transmembrane conductance regulator (CFTR). This defect alters chloride ion transport and impairs water transport across the cell membrane. Marked clinical heterogeneity occurs even among patients carrying the same mutation in the CFTR gene. Recent studies suggest that such heterogeneity could be related to epigenetic factors and/or miRNAs, which are small noncoding RNAs that modulate the expression of various proteins via post-transcriptional inhibition of gene expression. In the respiratory system, it has been shown that the dysregulation of miRNAs could participate in and lead to pathogenicity in several diseases. In CF airways, recent studies have proposed that miRNAs may modulate disease progression by affecting the production of either CFTR or various proteins that are dysregulated in the CF lung. Herein, we provide an overview of studies showing how miRNAs may modulate CF pathology and the efforts to develop miRNA-based treatments and/or to consider miRNAs as biomarkers. The identification of miRNAs involved in CF disease progression opens up new avenues toward treatments targeting selected clinical components of CF, independently from the CFTR mutation.

Transcription factors and miRNAs that regulate fetal to adult CFTR expression change are new targets for cystic fibrosis.

The CFTR gene displays a tightly regulated tissue-specific and temporal expression. Mutations in this gene cause cystic fibrosis (CF). In this study we wanted to identify trans-regulatory elements responsible for CFTR differential expression in fetal and adult lung, and to determine the importance of inhibitory motifs in the CFTR-3'UTR with the aim of developing new tools for the correction of disease-causing mutations within CFTR. We show that lung development-specific transcription factors (FOXA, C/EBP) and microRNAs (miR-101, miR-145, miR-384) regulate the switch from strong fetal to very low CFTR expression after birth. By using miRNome profiling and gene reporter assays, we found that miR-101 and miR-145 are specifically upregulated in adult lung and that miR-101 directly acts on its cognate site in the CFTR-3'UTR in combination with an overlapping AU-rich element. We then designed miRNA-binding blocker oligonucleotides (MBBOs) to prevent binding of several miRNAs to the CFTR-3'UTR and tested them in primary human nasal epithelial cells from healthy individuals and CF patients carrying the p.Phe508del CFTR mutation. These MBBOs rescued CFTR channel activity by increasing CFTR mRNA and protein levels. Our data offer new understanding of the control of the CFTR gene regulation and new putative correctors for cystic fibrosis.

Lung disease modifier genes in cystic fibrosis.

Cystic fibrosis (CF) is recognized as a single gene disorder. However, a considerable diversity in its clinical phenotype has been documented since the description of the disease. Identification of additional gene alleles, so called "modifier genes" that directly influence the phenotype of CF disease became a challenge in the late '90ies, not only for the insight it provides into the CF pathophysiology, but also for the development of new potential therapeutic targets. One of the most studied phenotype has been the lung disease severity as lung dysfunction is the major cause of morbidity and mortality in CF. This review details the results of two main genetic approaches that have mainly been explored so far: (1) an "a priori" approach, i.e. the candidate gene approach; (2) a "without a priori" approach, analyzing the whole genome by linkage and genome-wide association studies (GWAS), or the whole exome by exome sequencing.

Enhanced F508del-CFTR channel activity ameliorates bone pathology in murine cystic fibrosis.

In patients with cystic fibrosis (CF), rib and thoracic vertebral fractures can have adverse effects on lung health because the resulting pain and debilitation can impair airway clearance. The F508del mutation in the CF transmembrane conductance regulator (Cftr) gene induces an osteopenic phenotype in humans and mice. N-butyldeoxynojyrimicin (miglustat), an approved drug for treating type 1 Gaucher disease, was found to normalize CFTR-dependent chloride transport in human F508del CFTR lung cells and in nasal mucosa of F508del CF mice. Herein, we investigated whether targeting F508del-CFTR may rescue the skeletal osteopenic phenotype in murine CF. We found that oral administration of low-dose miglustat (120 mg/kg once a day for 28 days) improved bone mass and microarchitecture in the lumbar spine and femur in F508del mice. The increased bone density was associated with an increased bone formation rate and reduced bone resorption. This effect was associated with increased 17ß-estradiol but not with insulin-like growth factor 1 serum levels in miglustat-treated F508del mice. Exposure of primary F508del osteoblasts to miglustat partially restored the deficient CFTR-dependent chloride transport in these bone-forming cells. This study provides evidence that reversal of CFTR-dependent chloride transport in osteoblasts normalizes bone mass and microarchitecture in murine CF. These findings may provide a potential therapeutic strategy to prevent or correct the bone disease in patients with CF.

Anoctamin 1 dysregulation alters bronchial epithelial repair in cystic fibrosis.

Cystic fibrosis (CF) airway epithelium is constantly subjected to injury events due to chronic infection and inflammation. Moreover, abnormalities in CF airway epithelium repair have been described and contribute to the lung function decline seen in CF patients. In the last past years, it has been proposed that anoctamin 1 (ANO1), a Ca(2+)-activated Cl(-) channel, might offset the CFTR deficiency but this protein has not been characterized in CF airways. Interestingly, recent evidence indicates a role for ANO1 in cell proliferation and tumor growth. Our aims were to study non-CF and CF bronchial epithelial repair and to determine whether ANO1 is involved in airway epithelial repair. Here, we showed, with human bronchial epithelial cell lines and primary cells, that both cell proliferation and migration during epithelial repair are delayed in CF compared to non-CF cells. We then demonstrated that ANO1 Cl(-) channel activity was significantly decreased in CF versus non-CF cells. To explain this decreased Cl(-) channel activity in CF context, we compared ANO1 expression in non-CF vs. CF bronchial epithelial cell lines and primary cells, in lung explants from wild-type vs. F508del mice and non-CF vs. CF patients. In all these models, ANO1 expression was markedly lower in CF compared to non-CF. Finally, we established that ANO1 inhibition or overexpression was associated respectively with decreases and increases in cell proliferation and migration. In summary, our study demonstrates involvement of ANO1 decreased activity and expression in abnormal CF airway epithelial repair and suggests that ANO1 correction may improve this process.

Alveolar epithelial cells: master regulators of lung homeostasis.

The lung interfaces with the environment across a continuous epithelium composed of various cell types along the proximal and distal airways. At the alveolar structure level, the epithelium, which is composed of type I and type II alveolar epithelial cells, represents a critical component of lung homeostasis. Indeed, its fundamental role is to provide an extensive surface for gas exchange. Additional functions that act to preserve the capacity for such unique gas transfer have been progressively identified. The alveolar epithelium represents a physical barrier that protects from environmental insults by segregating inhaled foreign agents and regulating water and ions transport, thereby contributing to the maintenance of alveolar surface fluid balance. The homeostatic role of alveolar epithelium relies on the regulated/controlled production of the pulmonary surfactant, which is not only a key determinant of alveolar mechanical stability but also a complex structure that participates in the cross-talk between local cells and the lung immune and inflammatory response. In regard to these critical functions, a major point is the maintenance of alveolar surface integrity, which relies on the renewal capacity of type II alveolar epithelial cells, and the contribution of progenitor populations within the lung.