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.

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.

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.

Neutrophil elastase degrades cystic fibrosis transmembrane conductance regulator via calpains and disables channel function in vitro and in vivo.

RATIONALE: Cystic fibrosis transmembrane conductance regulator (CFTR) protein is a chloride channel regulating fluid homeostasis at epithelial surfaces. Its loss of function induces hypohydration, mucus accumulation, and bacterial infections in CF and potentially other lung chronic diseases. OBJECTIVES: To test whether neutrophil elastase (NE) and neutrophil-mediated inflammation negatively impact CFTR structure and function, in vitro and in vivo. METHODS: Using an adenovirus-CFTR overexpression approach, we showed that NE degrades wild-type (WT)- and ΔF508-CFTR in vitro and WT-CFTR in mice through a new pathway involving the activation of intracellular calpains. MEASUREMENTS AND MAIN RESULTS: CFTR degradation triggered a loss of function, as measured in vitro by channel patch-clamp and in vivo by nasal potential recording in mice. Importantly, this mechanism was also shown to be operative in a Pseudomonas aeruginosa lung infection murine model, and was NE-dependent, because CFTR integrity was significantly protected in NE(-/-) mice compared with WT mice. CONCLUSIONS: These data provide a new mechanism and show for the first time a link between NE-calpains activation and CFTR loss of function in bacterial lung infections relevant to CF and to other chronic inflammatory lung conditions.

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.

Restoration of chloride efflux by azithromycin in airway epithelial cells of cystic fibrosis patients.

Azithromycin (AZM) has shown promising anti-inflammatory properties in chronic obstructive pulmonary diseases, and clinical studies have presented an improvement in the respiratory condition of cystic fibrosis (CF) patients. The aim of this study was to investigate, in human airway cells, the mechanism by which AZM has beneficial effects in CF. We demonstrated that AZM did not have any anti-inflammatory effect on CF airway cells but restored Cl(-) efflux.

Reduced expression of Tis7/IFRD1 protein in murine and human cystic fibrosis airway epithelial cell models homozygous for the F508del-CFTR mutation.

12-O-tetradecanoyl phorbol-13-acetate-induced sequence 7/interferon related development regulator 1 (Tis7/IFRD1) has been recently identified as a modifier gene in lung inflammatory disease severity in patients with cystic fibrosis (CF), based upon its capacity to regulate inflammatory activities in neutrophils. In CF patients, the F508del mutation in the Cftr gene encoding a chloride channel, the CF transmembrane conductance regulator (CFTR) in airway epithelial cells results in an exaggerated inflammatory response of these cells. At present, it is unknown whether the Tis7/IFRD1 gene product is expressed in airway epithelial cells. We therefore investigated the possibility there is an intrinsic alteration in Tis7/IFRD1 protein level in cells lacking CFTR function in tracheal homogenates of F508del-CFTR mice and in a F508del-CFTR human bronchial epithelial cell line (CFBE41o(-) cells). When Tis7/IFRD1 protein was detectable, trachea from F508del-CFTR mice showed a reduction in the level of Tis7/IFRD1 protein compared to wild-type control littermates. A significant reduction of IFRD1 protein level was found in CFBE41o(-) cells compared to normal bronchial epithelial cells 16HBE14o(-). Surprisingly, messenger RNA level of IFRD1 in CFBE41o(-) cells was found elevated. Treating CFBE41o(-) cells with the antioxidant glutathione rescued the IFRD1 protein level closer to control level and also reduced the pro-inflammatory cytokine IL-8 release. This work provides evidence for the first time of reduced level of IFRD1 protein in murine and human F508del-CFTR airway epithelial cell models, possibly mediated in response to oxidative stress which might contribute to the exaggerated inflammatory airway response observed in CF patients homozygous for the F508del mutation.

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.

Cystic fibrosis transmembrane conductance regulator controls lung proteasomal degradation and nuclear factor-kappaB activity in conditions of oxidative stress.

Cystic fibrosis is a lethal inherited disorder caused by mutations in a single gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, resulting in progressive oxidative lung damage. In this study, we evaluated the role of CFTR in the control of ubiquitin-proteasome activity and nuclear factor (NF)-kappaB/IkappaB-alpha signaling after lung oxidative stress. After a 64-hour exposure to hyperoxia-mediated oxidative stress, CFTR-deficient (cftr(-/-)) mice exhibited significantly elevated lung proteasomal activity compared with wild-type (cftr(+/+)) animals. This was accompanied by reduced lung caspase-3 activity and defective degradation of NF-kappaB inhibitor IkappaB-alpha. In vitro, human CFTR-deficient lung cells exposed to oxidative stress exhibited increased proteasomal activity and decreased NF-kappaB-dependent transcriptional activity compared with CFTR-sufficient lung cells. Inhibition of the CFTR Cl(-) channel by CFTR(inh-172) in the normal bronchial immortalized cell line 16HBE14o- increased proteasomal degradation after exposure to oxidative stress. Caspase-3 inhibition by Z-DQMD in CFTR-sufficient lung cells mimicked the response profile of increased proteasomal degradation and reduced NF-kappaB activity observed in CFTR-deficient lung cells exposed to oxidative stress. Taken together, these results suggest that functional CFTR Cl(-) channel activity is crucial for regulation of lung proteasomal degradation and NF-kappaB activity in conditions of oxidative stress.

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.

Airway epithelial cell inflammatory signalling in cystic fibrosis.

Cystic fibrosis (CF) is the most common lethal monogenic disorder in Caucasians, estimated to affect one out of 2500-4000 new-borns. In patients with CF, lack of CF transmembrane conductance regulator (CFTR) Cl(-) channel function leads to progressive pulmonary damage and ultimately to death. Severe and persistent polymorphonuclear neutrophil-dominated endobronchial inflammation and chronic bacterial infection are characteristic hallmarks of CF lung disease. Whether CFTR dysfunction results directly in an increased predisposition to infection and whether inflammation arises independent of infection remains to be established. The loss of functional CFTR in airway epithelial cells promotes depletion and increased oxidation of the airway surface liquid. Activated neutrophils present in airways produce large amounts of proteases and reactive oxygen species (ROS). Together these changes are associated with diminished mucociliary clearance of bacteria, activation of epithelial cell signalling through multiple pathways, and subsequent hyperinflammatory responses in CF airways. The NF-kappaB pathway and Ca(2+) mobilization in airway epithelial cells are believed to be of key importance for control of lung inflammation through regulated production of mediators such as interleukin-8 that participate in recruitment and activation of neutrophils, modulation of apoptosis, and control of epithelial barrier integrity. In this review, the current understanding of the molecular mechanisms by which airway epithelial cells contribute to abnormal lung inflammation in CF, as well as the anti-inflammatory strategies that can be proposed are discussed.

Oxidative stress induces extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase in cystic fibrosis lung epithelial cells: Potential mechanism for excessive IL-8 expression.

Cystic fibrosis (CF) is a lethal disease caused by defective function of the cftr gene product, the CF transmembrane conductance regulator (CFTR) that leads to oxidative damage and excessive inflammatory response in lungs of CF patients. We here report the effects of oxidative stress (hyperoxia, 95% O(2)) on the expression of pro-inflammatory interleukin (IL)-8 and CXCR1/2 receptors in two human CF lung epithelial cell lines (IB3-1, with the heterozygous F508del/W1282X mutation and CFBE41o- with the homozygous F508del/F508del mutation) and two control non-CF lung epithelial cell lines (S9 cell line derived from IB3-1 after correction with wtCFTR and the normal bronchial cell line 16HBE14o-). Under oxidative stress, the expression of IL-8 and CXCR1/2 receptors was increased in CF, corrected and normal lung cell lines. The effects of oxidative stress were also investigated by measuring the transcription nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1) activities. Under oxidative stress, no increase of NF-kappaB activation was observed in CF lung cells in contrast to that observed in normal and corrected CF lung cells. The signalling of mitogen-activated protein (MAP) kinases was further studied. We demonstrated that extracellular signal-regulated kinase (ERK1/2) and AP-1 activity was markedly enhanced in CF but not non-CF lung cells under oxidative stress. Consistently, inhibition of ERK1/2 in oxidative stress-exposed CF lung cells strongly decreased both the IL-8 production and CXCR1/2 expression. Therefore, targeting of ERK1/2 MAP kinase may be critical to reduce oxidative stress-mediated inflammation in lungs of CF patients.

Proinflammatory effect of sodium 4-phenylbutyrate in deltaF508-cystic fibrosis transmembrane conductance regulator lung epithelial cells: involvement of extracellular signal-regulated protein kinase 1/2 and c-Jun-NH2-terminal kinase signaling.

Sodium 4-phenylbutyrate (4-PBA) has attracted a great deal of attention in cystic fibrosis (CF) pathology due to its capacity to traffic DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) to the cell membrane and restore CFTR chloride function at the plasma membrane of CF lung cells in vitro and in vivo. Using two different DeltaF508-CFTR lung epithelial cell lines (CFBE41o- and IB3-1 cells, characterized with DeltaF508-homozygous and heterozygous genotype, respectively) in vitro, 4-PBA induced an increase of proinflammatory cytokine interleukin (IL)-8 production in a concentration-dependent manner. This 4-PBA-induced IL-8 production was associated with a strong reduction of proteasome and nuclear factor-kappaB transcriptional activities in the two DeltaF508-CFTR lung cells either in a resting state or after tumor necrosis factor-alpha stimulation. In contrast, a strong increase of activator protein-1 transcriptional activity was observed. The inhibition of extracellular signal-regulated protein kinase 1/2 (ERK1/2) by 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126) and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) and c-Jun-NH(2)-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) by anthra[1,9-cd] pyrazol-6 (2H)-one (SP600125), respectively, was associated with a reduction (2-3.5-fold) of IL-8 production in both DeltaF508-CFTR lung cell lines treated with 4-PBA. No significant change of IL-8 production was observed after an inhibition of p38 MAPK with 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl] phenol (SB202190). Therefore, we suggest that inhibition of both ERK1/2 and JNK signaling may be a means to strongly reduce 4-PBA-induced IL-8 production in combination with 4-PBA treatment to restore CFTR Cl(-) channel function in lung epithelial cells of patients with CF.

Circulating and airway neutrophils in cystic fibrosis display different TLR expression and responsiveness to interleukin-10.

We compared blood neutrophils (PMNs) collected from healthy subjects with PMNs derived from either blood or airways collected from the same cystic fibrosis (CF) patients. When compared to healthy blood PMNs, CF blood PMNs expressed enhanced level of CD64, a marker of neutrophil activation, and lower level of Toll-like receptor-2 (TLR2). CF airway PMNs expressed enhanced level of TLR4. Interleukin-8 (IL-8) production by CF blood PMNs could be enhanced upon addition of lipopolysaccharide or peptidoglycan, and this production was inhibited by recombinant human IL-10. In contrast, CF airway PMNs released spontaneously high level of IL-8 that was neither further enhanced by microbial activators nor inhibited by recombinant human IL-10. The levels of IL-10 receptors were similar in all types of neutrophils. These data further demonstrate that circulating PMNs from CF patients display a distinct pattern of surface markers, including TLRs, as compared to PMNs from healthy donors, and that airways PMNs from CF patients are primed and resistant to anti-inflammatory signals delivered by IL-10.