European Respiratory Society statement for defining respiratory exacerbations in children and adolescents with bronchiectasis for clinical trials.

Bronchiectasis is being diagnosed increasingly in children and adolescents. Recurrent respiratory exacerbations are common in children and adolescents with this chronic pulmonary disorder. Respiratory exacerbations are associated with an impaired quality of life, poorer long-term clinical outcomes, and substantial costs to the family and health systems. The 2021 European Respiratory Society (ERS) clinical practice guideline for the management of children and adolescents with bronchiectasis provided a definition of acute respiratory exacerbations for clinical use but to date there is no comparable universal definition for clinical research. Given the importance of exacerbations in the field, this ERS Task Force sought to obtain robust definitions of respiratory exacerbations for clinical research. The panel was a multidisciplinary team of specialists in paediatric and adult respiratory medicine, infectious disease, physiotherapy, primary care, nursing, radiology, methodology, patient advocacy, and parents of children and adolescents with bronchiectasis. We used a standardised process that included a systematic literature review, parent survey, and a Delphi approach involving 299 physicians (54 countries) caring for children and adolescents with bronchiectasis. Consensus was obtained for all four statements drafted by the panel as the disagreement rate was very low (range 3.6-7.2%). The panel unanimously endorsed the four consensus definitions for 1a) non-severe exacerbation and 1b) severe exacerbation as an outcome measure, 2) non-severe exacerbation for studies initiating treatment, and 3) resolution of a non-severe exacerbation for clinical trials involving children and adolescents with bronchiectasis. This ERS Task Force proposes using these internationally derived, consensus-based definitions of respiratory exacerbations for future clinical paediatric bronchiectasis research.

New severity assessment in cystic fibrosis: signal intensity and lung volume compared to LCI and FEV1: preliminary results.

OBJECTIVES: Magnetic resonance imaging (MRI) aids diagnosis in cystic fibrosis (CF) but its use in quantitative severity assessment is under research. This study aims to assess changes in signal intensity (SI) and lung volumes (Vol) during functional MRI and their use as a severity assessment tool in CF patients. METHODS: The CF intra-hospital standard chest 1.5 T MRI protocol comprises of very short echo-time sequences in submaximal in- and expiration for functional information. Quantitative measurements (Vol/SI at in- and expiration, relative differences (Vol_delta/SI_delta), and cumulative histograms for normalized SI values across the expiratory lung volume) were assessed for correlation to pulmonary function: lung clearance index (LCI) and forced expiratory volume in 1 s (FEV1). RESULTS: In 49 patients (26 male, mean age 17 ± 7 years) significant correlation of Vol_delta and SI_delta (R = 0.86; p < 0.0001) during respiration was observed. Individual cumulated histograms enabled severity disease differentiation (mild, severe) to be visualized (defined by functional parameter: LCI > 10). The expiratory volume at a relative SI of 100% correlated significantly to LCI (R = 0.676 and 0.627; p < 0.0001) and FEV1 (R = - 0.847 and - 0.807; p < 0.0001). Clustering patients according to Vol_SI_100 showed that an amount of ≤ 4% was related to normal, while an amount of > 4% was associated with pathological pulmonary function values. CONCLUSION: Functional pulmonary MRI provides a radiation-free severity assessment tool and can contribute to early detection of lung impairment in CF. Lung volume with SI below 100% of the inspiratory volume represents overinflated tissue; an amount of 4% of the expiratory lung volume was a relevant turning point. KEY POINTS: • Signal intensity and lung volumes are used as potential metric parameters for lung impairment. • Quantification of trapped air impacts on therapy management. • Functional pulmonary MRI can contribute to early detection of lung impairment.

A semiquantitative MRI-Score can predict loss of lung function in patients with cystic fibrosis: Preliminary results.

OBJECTIVES: To evaluate the applicability of a semiquantitative MRI scoring system (MR-CF-S) as a prognostic marker for clinical course of cystic fibrosis (CF) lung disease. METHODS: This observational study of a single-centre CF cohort included a group of 61 patients (mean age 12.9 ± 4.7 years) receiving morphological and functional pulmonary MRI, pulmonary function testing (PFT) and follow-up of 2 years. MRI was analysed by three raters using MR-CF-S. The inter-rater agreement, correlation of score categories with forced expiratory volume in 1 s (FEV1) at baseline, and the predictive value of clinical parameters, and score categories was assessed for the whole cohort and a subgroup of 40 patients with moderately impaired lung function. RESULTS: The inter-rater agreement of MR-CF-S was sufficient (mean intraclass correlation coefficient 0.92). MR-CF-S (-0.62; p < 0.05) and most of the categories significantly correlated with FEV1. Differences between patients with relevant loss of FEV1 (>3%/year) and normal course were only significant for MR-CF-S (p < 0.05) but not for clinical parameters. Centrilobular opacity (CO) was the most promising score category for prediction of a decline of FEV1 (area under curve: whole cohort 0.69; subgroup 0.86). CONCLUSIONS: MR-CF-S is promising to predict a loss of lung function. CO seems to be a particular finding in CF patients with an abnormal course. KEY POINTS: • Lung imaging is essential in the diagnostic work-up of CF patients • MRI serves as a powerful, radiation-free modality in paediatric CF patients • Observational single-centre study showed significant correlation of MR-CF score and FEV 1 • MR-CF score is promising in predicting a loss of lung function.

Progress in Definition, Prevention and Treatment of Fungal Infections in Cystic Fibrosis.

Cystic fibrosis (CF) is a chronic lethal multi-system condition; however, most of the morbidity and mortality is dependent on the status of the respiratory system. Progressive respiratory decline is mediated by chronic infection and inflammation, punctuated by important acute events known as pulmonary exacerbations which can lead to accelerated decline. The main bacterial species causing infections include Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae and Achromobacter xylosoxidans. In addition to bacteria, fungi are detected in a significant number of patients. The impact of fungal colonization of the airways is still not completely elucidated, but an increasing body of evidence suggests an important role for moulds and yeasts. Although fungal infections are rare, fungi can cause severe pneumonia requiring appropriate targeted treatment. The most common fungi in respiratory samples of patients with CF are Aspergillus fumigatus, Aspergillus terreus and Scedosporium species for filamentous fungi, and yeasts such as Candida albicans and Candida glabrata. Therapeutic strategies depend on the detected fungus and the underlying clinical status of the patient. The antifungal therapy can range from a simple monotherapy up to a combination of three different drugs. Treatment course may be indicated in some patients for two weeks and in others for up to six months, and in rare cases even longer. New antifungal drugs have been developed and are being tested in clinical studies offering the hope of therapeutic alternatives to existing drugs. Identifying relevant risk factors and diagnostic criteria for fungal colonization and infection is crucial to enabling an adequate prevention, diagnosis and treatment.

Chitinase activation in patients with fungus-associated cystic fibrosis lung disease.

BACKGROUND: Chitinases have recently gained attention in the field of pulmonary diseases, particularly in asthma and chronic obstructive pulmonary disease, but their potential role in patients with cystic fibrosis (CF)-associated lung disease remains unclear. OBJECTIVE: The aim of this study was to assess chitinase activity systemically and in the airways of patients with CF and asthma compared with healthy subjects. Additionally, we assessed factors that regulate chitinase activity within the lungs of patients with CF. METHODS: Chitinase activities were quantified in serum and bronchoalveolar lavage fluid from patients with CF, asthmatic patients, and healthy control subjects. Mechanistically, the role of CF airway proteases and genetic chitinase deficiency was assessed. RESULTS: Chitinase activity was systemically increased in patients with CF compared with that in healthy control subjects and asthmatic patients. Further stratification showed that chitinase activity was enhanced in patients with CF colonized with Candida albicans compared with that in noncolonized patients. CF proteases degraded chitinases in the airway microenvironment of patients with CF. Genetic chitinase deficiency was associated with C albicans colonization in patients with CF. CONCLUSION: Patients with CF have enhanced chitinase activation associated with C albicans colonization. Therefore chitinases might represent a novel biomarker and therapeutic target for CF-associated fungal disease.

SLy2 controls the antibody response to pneumococcal vaccine through an IL-5Rα-dependent mechanism in B-1 cells.

The adaptor protein SLy2 (Src homology domain 3 lymphocyte protein 2) is located on human chromosome 21 and was reported to be among a group of genes amplified in Down's syndrome (DS) patients. DS patients characteristically show an impaired immunity to pneumococcal infections. However, molecular mechanisms linking gene amplifications with specific DS phenotypes remain elusive. To investigate the effect of SLy2 gene amplification on the mammalian immune system, we studied SLy2 overexpressing transgenic-SLy2 (TG) mice. We found that baseline immunoglobulin M (IgM) levels as well as IgM responses following Pneumovax immunizations were reduced in TG mice. Moreover, B-1 cells, the major natural IgM-producing population in mice, were reduced in the peritoneal cavity of TG mice, while other immune cell compartments were unaltered. Mechanistically, SLy2 overexpression attenuated the expression of the IL-5 receptor α chain on B-1 cells, resulting in decreased B-1 cell numbers and decreased differentiation into Ab-secreting cells. Since B-1 cells essentially contribute to immunity against Streptococcus pneumoniae, the present study provides a novel molecular link between SLy2 expression and pneumococcal-specific IgM responses in vivo. These studies suggest that the adaptor protein SLy2 is a potential future target for immunomodulatory strategies for pneumococcal infections.

RNA and imidazoquinolines are sensed by distinct TLR7/8 ectodomain sites resulting in functionally disparate signaling events.

TLRs 7 and 8 are pattern recognition receptors controlling antiviral host defense or autoimmune diseases. Apart from foreign and host RNA, synthetic RNA oligoribonucleotides (ORN) or small molecules of the imidazoquinoline family activate TLR7 and 8 and are being developed as therapeutic agonists. The structure-function relationships for RNA ORN and imidazoquinoline sensing and consequent downstream signaling by human TLR7 and TLR8 are unknown. Proteome- and genome-wide analyses in primary human monocyte-derived dendritic cells here showed that TLR8 sensing of RNA ORN versus imidazoquinoline translates to ligand-specific differential phosphorylation and transcriptional events. In addition, TLR7 and 8 ectodomains were found to discriminate between RNA ORN and imidazoquinolines by overlapping and nonoverlapping recognition sites to which murine loss-of-function mutations and human naturally occurring hyporesponsive polymorphisms map. Our data suggest TLR7 and TLR8 can signal in two different "modes" depending on the class of ligand. Considering RNA ORN and imidazoquinolines have been regarded as functionally interchangeable, our study highlights important functional incongruities whose understanding will be important for developing TLR7 or 8 therapeutics with desirable effector and safety profiles for in vivo application.

Regulatory T-cell impairment in cystic fibrosis patients with chronic pseudomonas infection.

RATIONALE: Patients with cystic fibrosis (CF) lung disease have chronic airway inflammation driven by disrupted balance of T-cell (Th17 and Th2) responses. Regulatory T cells (Tregs) dampen T-cell activation, but their role in CF is incompletely understood. OBJECTIVES: To characterize numbers, function, and clinical impact of Tregs in CF lung disease. METHODS: Tregs were quantified in peripheral blood and airway samples from patients with CF and from lung disease control patients without CF and healthy control subjects. The role of Pseudomonas aeruginosa and CF transmembrane conductance regulator (CFTR) in Treg regulation was analyzed by using in vitro and murine in vivo models. MEASUREMENTS AND MAIN RESULTS: Tregs were decreased in peripheral blood and airways of patients with CF compared with healthy controls or lung disease patients without CF and correlated positively with lung function parameters. Patients with CF with chronic P. aeruginosa infection had lower Tregs compared with patients with CF without P. aeruginosa infection. Genetic knockout, pharmacological inhibition, and P. aeruginosa infection studies showed that both P. aeruginosa and CFTR contributed to Treg dysregulation in CF. Functionally, Tregs from patients with CF or from Cftr(-/-) mice were impaired in suppressing conventional T cells, an effect that was enhanced by P. aeruginosa infection. The loss of Tregs in CF affected memory, but not naive Tregs, and manifested gradually with disease progression. CONCLUSIONS: Patients with CF who have chronic P. aeruginosa infection show an age-dependent, quantitative, and qualitative impairment of Tregs. Modulation of Tregs represents a novel strategy to rebalance T-cell responses, dampen inflammation, and ultimately improve outcomes for patients with infective CF lung disease.

Localization and functionality of the inflammasome in neutrophils.

Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein containing a CARD (ASC), AIM2, and caspase-1. Subcellular fractionation and microscopic analyses further showed that inflammasome components were localized in the cytoplasm and also noncanonically in secretory vesicle and tertiary granule compartments. Whereas IL-1ß and IL-18 were expressed at the mRNA level and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1ß protein compared with partially purified neutrophils. Serine proteases and caspases were differentially involved in IL-1ß release, depending on the stimulus. Spontaneous activation of the NLRP3 inflammasome in neutrophils in vivo affected IL-1ß, but not IL-18 release. In summary, these studies show that human neutrophils express key components of the inflammasome machinery in distinct intracellular compartments and release IL-1ß and IL-18, but not IL-1α or IL-33 protein. Targeting the neutrophil inflammasome may represent a future therapeutic strategy to modulate neutrophilic inflammatory diseases, such as cystic fibrosis, rheumatoid arthritis, or sepsis.

Increased CCL17 serum levels are associated with improved survival in advanced melanoma.

BACKGROUND: Prognostic factors of melanoma patients with distant metastases remain poorly established. This study aimed to compare the prognostic impact of putative serum biomarkers, namely S100B, YKL-40 or CCL17, in stage IV melanoma patients. PATIENTS AND METHODS: Serum concentrations were analyzed by ELISA. Disease-specific survival of 80 patients according to S100B, YKL-40 or CCL17 and clinical factors were calculated by univariate Kaplan-Meier survival and multivariate analysis. RESULTS: Low serum levels of S100B, high concentrations of CCL17 and female gender correlated with improved survival. A trend for favorable prognosis was observed for the M categories M1a/b versus M1c according to the AJCC classification. No correlation with survival was evident for YKL-40 serum levels and age. In multivariate analysis, S100B (HR 2.1; p = 0.005) and CCL17 (HR 1.8; p = 0.029) had independent prognostic impact. Patients with a combination of normal S100B and high CCL17 had a high chance for long-term survival, which was 43 % after 3 years. CONCLUSION: Serum levels of CCL17 and S100B represent independent prognostic markers for melanoma patients with distant metastases. These biomarkers were more powerful than the M category according to the AJCC classification to indicate overall survival. CCL17 represents a promising biomarker upon immune checkpoint blockade in melanoma.

CXCR4+ granulocytes reflect fungal cystic fibrosis lung disease.

Cystic fibrosis airways are frequently colonised with fungi. However, the interaction of these fungi with immune cells and the clinical relevance in cystic fibrosis lung disease are incompletely understood.We characterised granulocytes in airway fluids and peripheral blood from cystic fibrosis patients with and without fungal colonisation, non-cystic fibrosis disease controls and healthy control subjects cross-sectionally and longitudinally and correlated these findings with lung function parameters.Cystic fibrosis patients with chronic fungal colonisation by Aspergillus fumigatus were characterised by an accumulation of a distinct granulocyte subset, expressing the HIV coreceptor CXCR4. Percentages of airway CXCR4(+) granulocytes correlated with lung disease severity in patients with cystic fibrosis.These studies demonstrate that chronic fungal colonisation with A. fumigatus in cystic fibrosis patients is associated with CXCR4(+) airway granulocytes, which may serve as a potential biomarker and therapeutic target in fungal cystic fibrosis lung disease.

Flagellin induces myeloid-derived suppressor cells: implications for Pseudomonas aeruginosa infection in cystic fibrosis lung disease.

Pseudomonas aeruginosa persists in patients with cystic fibrosis (CF) and drives CF lung disease progression. P. aeruginosa potently activates the innate immune system, mainly mediated through pathogen-associated molecular patterns, such as flagellin. However, the host is unable to eradicate this flagellated bacterium efficiently. The underlying immunological mechanisms are incompletely understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells generated in cancer and proinflammatory microenvironments and are capable of suppressing T cell responses. We hypothesized that P. aeruginosa induces MDSCs to escape T cell immunity. In this article, we demonstrate that granulocytic MDSCs accumulate in CF patients chronically infected with P. aeruginosa and correlate with CF lung disease activity. Flagellated P. aeruginosa culture supernatants induced the generation of MDSCs, an effect that was 1) dose-dependently mimicked by purified flagellin protein, 2) significantly reduced using flagellin-deficient P. aeruginosa bacteria, and 3) corresponded to TLR5 expression on MDSCs in vitro and in vivo. Both purified flagellin and flagellated P. aeruginosa induced an MDSC phenotype distinct from that of the previously described MDSC-inducing cytokine GM-CSF, characterized by an upregulation of the chemokine receptor CXCR4 on the surface of MDSCs. Functionally, P. aeruginosa-infected CF patient ex vivo-isolated as well as flagellin or P. aeruginosa in vitro-generated MDSCs efficiently suppressed polyclonal T cell proliferation in a dose-dependent manner and modulated Th17 responses. These studies demonstrate that flagellin induces the generation of MDSCs and suggest that P. aeruginosa uses this mechanism to undermine T cell-mediated host defense in CF and other P. aeruginosa-associated chronic lung diseases.

Free DNA in cystic fibrosis airway fluids correlates with airflow obstruction.

Chronic obstructive lung disease determines morbidity and mortality of patients with cystic fibrosis (CF). CF airways are characterized by a nonresolving neutrophilic inflammation. After pathogen contact or prolonged activation, neutrophils release DNA fibres decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). NETs have been described to act in a beneficial way for innate host defense by bactericidal, fungicidal, and virucidal actions. On the other hand, excessive NET formation has been linked to the pathogenesis of autoinflammatory and autoimmune disease conditions. We quantified free DNA structures characteristic of NETs in airway fluids of CF patients and a mouse model with CF-like lung disease. Free DNA levels correlated with airflow obstruction, fungal colonization, and CXC chemokine levels in CF patients and CF-like mice. When viewed in combination, our results demonstrate that neutrophilic inflammation in CF airways is associated with abundant free DNA characteristic for NETosis, and suggest that free DNA may be implicated in lung function decline in patients with CF.

The chemokine CCL18 characterises Pseudomonas infections in cystic fibrosis lung disease.

Cystic fibrosis (CF) lung disease is characterised by chronic Pseudomonas aeruginosa infection and leukocyte infiltration. Chemokines recruit leukocytes to sites of infection. Gene expression analysis identified the chemokine CCL18 as upregulated in CF leukocytes. We hypothesised that CCL18 characterises infection and inflammation in patients with CF lung disease. Therefore, we quantified CCL18 protein levels in the serum and airway fluids of CF patients and healthy controls, and studied CCL18 protein production by airway cells ex vivo. These studies demonstrated that CCL18 levels were increased in the serum and airway fluids from CF patients compared with healthy controls. Within CF patients, CCL18 levels were increased in P. aeruginosa-infected CF patients. CCL18 levels in the airways, but not in serum, correlated with severity of pulmonary obstruction in CF. Airway cells isolated from P. aeruginosa-infected CF patients produced significantly higher amounts of CCL18 protein compared with airway cells from CF patients without P. aeruginosa infection or healthy controls. Collectively, these studies show that CCL18 levels characterise chronic P. aeruginosa infection and pulmonary obstruction in patients with CF. CCL18 may, thus, serve as a potential biomarker and therapeutic target in CF lung disease.

Inhalation treatment with glutathione in patients with cystic fibrosis. A randomized clinical trial.

RATIONALE: Glutathione is the major antioxidant in the extracellular lining fluid of the lungs and depleted in patients with cystic fibrosis (CF). OBJECTIVES: We aimed to assess glutathione delivered by inhalation as a potential treatment for CF lung disease. METHODS: This randomized, double-blind, placebo-controlled trial evaluated inhaled glutathione in subjects with CF 8 years of age and older and FEV1 of 40-90% of predicted. Subjects were randomized to receive 646 mg glutathione in 4 ml (n = 73) or placebo (n = 80) via an investigational eFlow nebulizer every 12 hours for 6 months. MEASUREMENTS AND MAIN RESULTS: FEV1 (absolute values), both as pre-post differences (P = 0.180) and as area under the curves (P = 0.205), were the primary efficacy endpoints, and were not different between the glutathione group and the placebo group over the 6-month treatment period. Exploratory analysis showed an increase of FEV1 from baseline over placebo of 100 ml or 2.2% predicted; this was significant at 3 months, but not later. Subjects receiving glutathione had neither fewer pulmonary exacerbations, nor better scores for quality of life. Whereas increased glutathione and metabolites in sputum demonstrated significant delivery to the lungs, there was no indication of diminished oxidative stress to proteins or lipids, and no evidence for anti-inflammatory or antiproteolytic actions of glutathione supplemented to the airways. The adverse event incidence was similar between glutathione and placebo. CONCLUSIONS: Inhaled glutathione in the dose administered did not demonstrate clinically relevant improvements in lung function, pulmonary exacerbation frequency, or patient-reported outcomes. Glutathione delivery to the airways was not associated with changes in markers of oxidation, proteolysis, or inflammation. Clinical trial registered with www.clinicaltrials.gov (NCT00506688) and https://eudract.ema.europa.eu/index.html (EudraCT 2005-003870-88).

A core outcome set for bronchiectasis in children and adolescents for use in clinical research: an international consensus study.

Improving the treatment of non-cystic fibrosis bronchiectasis in children and adolescents requires high-quality research with outcomes that meet study objectives and are meaningful for patients and their parents and caregivers. In the absence of systematic reviews or agreement on the health outcomes that should be measured in paediatric bronchiectasis, we established an international, multidisciplinary panel of experts to develop a core outcome set (COS) that incorporates patient and parent perspectives. We undertook a systematic review from which a list of 21 outcomes was constructed; these outcomes were used to inform the development of separate surveys for ranking by parents and patients and by health-care professionals. 562 participants (201 parents and patients from 17 countries, 361 health-care professionals from 58 countries) completed the surveys. Following two consensus meetings, agreement was reached on a ten-item COS with five outcomes that were deemed to be essential: quality of life, symptoms, exacerbation frequency, non-scheduled health-care visits, and hospitalisations. Use of this international consensus-based COS will ensure that studies have consistent, patient-focused outcomes, facilitating research worldwide and, in turn, the development of evidence-based guidelines for improved clinical care and outcomes. Further research is needed to develop validated, accessible measurement instruments for several of the outcomes in this COS.

Expression and regulation of interferon-related development regulator-1 in cystic fibrosis neutrophils.

A genome-wide association study identified interferon-related development regulator-1 (IFRD1), a protein expressed by neutrophils, as a key modifier gene in cystic fibrosis (CF) lung disease. Here, we investigated the expression and regulation of IFRD1 in CF neutrophils. IFRD1 expression was quantified in peripheral blood and airway neutrophils from patients with CF, patients with non-CF lung disease, and healthy control subjects. The regulation of IFRD1 expression was analyzed using isolated neutrophils and ex vivo stimulation assays with CF airway fluids. IFRD1 single-nucleotide polymorphisms (SNPs) were analyzed in a CF cohort (n = 572) and correlated with longitudinal lung function and IFRD1 expression. Patients with CF expressed higher protein levels of IFRD1 in peripheral blood neutrophils compared with healthy or non-CF disease control subjects. Within patients with CF, IFRD1 protein expression levels in neutrophils were lower in airway fluids compared with peripheral blood. High IFRD1 expression was positively associated with the production of reactive oxygen species (ROS) in CF neutrophils. In vitro regulation studies showed that CF airway fluid and the CF-characteristic chemokines CXCL8 and CXCL2 down-regulated IFRD1 expression in neutrophils, an effect that was mediated through CXCR2. Genetic analyses showed that three IFRD1 SNPs were associated with longitudinal declines in lung function, and modulated IFRD1 expression. These studies demonstrate that IFRD1 expression is systemically up-regulated in human CF neutrophils, is linked to the production of ROS, and is modulated by chemokines in CF airway fluids, depending on the IFRD1 genotype. Understanding the regulation of IFRD1 may pave the way for novel therapeutic approaches to target neutrophilic inflammation in CF.

Neutrophils express distinct RNA receptors in a non-canonical way.

RNAs are capable of modulating immune responses by binding to specific receptors. Neutrophils represent the major fraction of circulating immune cells, but receptors and mechanisms by which neutrophils sense RNA are poorly defined. Here, we analyzed the mRNA and protein expression patterns and the subcellular localization of the RNA receptors RIG-I, MDA-5, TLR3, TLR7, and TLR8 in primary neutrophils and immortalized neutrophil-like differentiated HL-60 cells. Our results demonstrate that both neutrophils and differentiated HL-60 cells express RIG-I, MDA-5, and TLR8 at the mRNA and protein levels, whereas TLR3 and TLR7 are not expressed at the protein level. Subcellular fractionation, flow cytometry, confocal laser scanning microscopy, and immuno-transmission electron microscopy provided evidence that, besides the cytoplasm, RIG-I and MDA-5 are stored in secretory vesicles of neutrophils and showed that RIG-I and its ligand, 3p-RNA, co-localize at the cell surface without triggering neutrophil activation. In summary, this study demonstrates that neutrophils express a distinct pattern of RNA recognition receptors in a non-canonical way, which could have essential implications for future RNA-based therapeutics.