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Pulmonary epithelial cells are continuously exposed to microbial challenges as a result of breathing. It is recognized that immune myeloid cells express Toll-like receptors (TLRs), which play a major role in detecting microbes and initiating innate immune responses. In contrast, little is known concerning the expression of TLR in pulmonary epithelial cells per se, their distribution within the cell, their function, and the signaling pathways involved. In this work, we demonstrated by reverse transcription-PCR and/or immunoblot that TLR4 and the accessory molecule MD-2 are constitutively expressed in distinct human alveolar and bronchial epithelial cells. We further characterized by flow cytometry, biotinylation/precipitation, and confocal microscopy the intracellular localization of TLR4 in these cells. Despite this intracellular compartmentalization of TLR4, pulmonary epithelial cells were responsive to the TLR4 activator lipopolysaccharide (LPS), a potent Gram-negative bacteria-associated molecular pattern. Using respiratory epithelial cells isolated from TLR4 knock-out and wild type mice, we demonstrated that TLR4 is the actual activating receptor for LPS in these cells. Furthermore we showed that this cell response to LPS involves a signaling complex including the kinases interleukin-1 receptor-associated kinase (IRAK), p38, Jnk, and ERK1/2. Moreover, using vectors expressing dominant-negative forms of MyD88 and TRAF6, we established that LPS-induced activation of respiratory epithelial cells is largely dependent on TLR4 signaling intermediates. Altogether these data demonstrate that TLR4 is a key element in the response of pulmonary epithelial cells to molecules derived from Gram-negative bacteria. The intracellular localization of TLR4 in lung epithelia is expected to play an important role in the prevention of the development of chronic inflammatory disease.

The collectin surfactant protein-A (SP-A) is involved in the innate host defense and the regulation of inflammatory processes in the lung. In this work we investigated the molecular mechanisms related to the immunostimulatory activity of SP-A using macrophages from C3H/HeJ mice, which carry an inactivating mutation in the Toll-like receptor (TLR)4 gene, and TLR4-transfected Chinese hamster ovary cells. We demonstrate that SP-A-induced activation of the NF-κB signaling pathway and up-regulation of cytokine synthesis such as TNF-α and IL-10 are critically dependent on the TLR4 functional complex. These findings support the concept that TLR4 is a pattern recognition receptor that signals in response to both foreign pathogens and endogenous host mediators.

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.

Introduction Chez les patients atteints de mucoviscidose (CF), la deficience du canal chlorure CFTR est responsable de la degradation de la fonction respiratoire. En 2008, un nouveau canal chlorure est identifie : le canal ANO1, propose comme cible therapeutique pour restaurer les efflux chlorure chez le patient CF. Des travaux precedents du laboratoire ont montre que l’activite et l’expression d’ANO1 etaient diminuees en contexte CF (Ruffin et al., 2013). A ce jour, les origines de ces diminutions sont inconnues. Une hypothese serait qu’ANO1 soit regule par les microARNs (miRNAs) qui sont de petits ARNS non-codants regulant negativement l’expression des genes. L’objectif de ce travail est donc de comprendre l’origine de la deregulation d’ANO1 en contexte CF en etudiant les miRNAs. Methodes Nous avons utilise des bases de donnees bioinformatiques afin d’etudier les miRNAs qui pourraient cibler et reguler ANO1. Nous avons effectue des experiences de fonctionnalite en etudiant la liaison d’un miRNA candidat au 3′UTR d’ANO1 a l’aide d’un plasmide luciferase couple au 3′UTR d’ANO1 en condition de surexpression du miRNA, et en quantifiant l’expression d’ANO1 dans ces memes conditions dans des lignees de cellules epitheliales bronchiques. Resultats L’etude bioinformatique nous a permis d’identifier differents miRNAs dont miR-9 comme regulateurs potentiels d’ANO1. Nous avons observe la surexpression de miR-9 dans des cellules CF par rapport a des cellules non-CF. En condition de surexpression de miR-9, nous avons observe une diminution significative de l’expression d’ANO1 en ARNm et de l’activite luciferase par rapport a la condition controle. Dans les memes conditions, nous avons observe une diminution significative de la vitesse de migration des cellules et de l’activite chlorure d’ANO1 lorsque miR-9 est surexprime. Conclusions Nos resultats suggerent que miR-9 regule le canal chlorure ANO1 de facon directe en se liant au 3′UTR d’ANO1 et qu’il joue un role dans la migration ANO1-dependante des cellules. Chez les patients CF, des strategies therapeutiques alternatives a la deficience de CFTR sont d’un interet majeur et sont complementaires aux strategies proteiques proposees pour CFTR. Des tests sur l’effet d’un « target site blocker » qui empeche la fixation de miR-9 au 3′UTR d’ANO1 sont actuellement en cours pour voir s’il est possible de retablir dans les cellules CF une activite chlorure semblable aux cellules non-CF et voir si une application therapeutique est envisageable.

Introduction La colonisation par Pseudomonas aeruginosa des voies aeriennes de patients atteints de mucoviscidose et la reponse inflammatoire chronique qui en resulte conduisent a une deterioration du tissu pulmonaire et par consequence a une perte de la fonction respiratoire responsable de l’evolution pejorative de la maladie. L’epithelium respiratoire qui constitue une barriere physique de defense contre les micro-organismes a aussi la capacite d’etablir une reponse immunitaire innee. Notre etude consiste a comparer le transcriptome de cellules epitheliales bronchiques primaires (CEBP) saines (non-CF) a celui de CEBP de patients atteints de mucoviscidose (CF) au cours d’une infection a P. aeruginosa L’objectif est d’identifier les modifications d’expression de genes de l’immunite innee pouvant expliquer la persistance de P. aeruginosa et de la reponse inflammatoire dans la mucoviscidose. Methodes Des CEBP primaires de 4 sujets non-CF et de 4 patients CF (F508del homozygote) (Epithelix) ont ete infectees par P. aeruginosa (souche PAK) et une cinetique a ete realisee (0, 2, 4 et 6 heures) a une MOI de 0,25. Les ARNm ont ete purifies, analyses pour leur purete et sequences par la technique Illumina RNA-seq. Resultats L’analyse complete du transcriptome montre une expression significativement differente de nombreux genes au cours de l’infection entre les cellules CF et non-CF. Nous avons pu ainsi identifier des differences de modulations d’expression de genes impliques dans la production de cytokines (TNF, IL-17C, IL-32), de chimiokines (CXCL5, CCL5), de peptides anti-microbiens (SLPI, pentraxin 3), de peptidases (granzyme B, metalloproteases, serpines, kallikreines) et de facteurs de transcription (KLF2). Conclusions Cette etude a permis de mettre en evidence des differences d’expression de genes de l’immunite innee au cours de l’infection par P. aeruginosa de cellules CF, pouvant etre a l’origine de la forte reponse inflammatoire ou de la persistance de P. aeruginosa dans la mucoviscidose. Notre objectif est maintenant de confirmer l’expression proteique de ces differentes cibles d’interet et d’identifier les facteurs de virulence bacteriens responsables de ces modulations d’expression.

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. This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.

Homozygous loss-of-function mutations in the FOXE1 gene have been reported in several patients with partial or complete Bamforth-Lazarus syndrome: congenital hypothyroidism (CH) with thyroid dysgenesis (usually athyreosis), cleft palate, spiky hair, with or without choanal atresia, and bifid epiglottis. Here, our objective was to evaluate potential functional consequences of a FOXE1 mutation in a patient with a similar clinical phenotype.FOXE1 was sequenced in eight patients with thyroid dysgenesis and cleft palate. Transient transfection was performed in HEK293 cells using the thyroglobulin (TG) and thyroid peroxidase (TPO) promoters in luciferase reporter plasmids to assess the functional impact of the FOXE1 mutations. Primary human thyrocytes transfected with wild type and mutant FOXE1 served to assess the impact of the mutation on endogenous TG and TPO expression.We identified and characterized the function of a new homozygous FOXE1 missense mutation (p.R73S) in a boy with a typical phenotype (athyreosis, cleft palate, and partial choanal atresia). This new mutation located within the forkhead domain was inherited from the heterozygous healthy consanguineous parents. In vitro functional studies in HEK293 cells showed that this mutant gene enhanced the activity of the TG and TPO gene promoters (1.5-fold and 1.7-fold respectively vs. wild type FOXE1; p0.05), unlike the five mutations previously reported in Bamforth-Lazarus syndrome. The gain-of-function effect of the FOXE1-p.R73S mutant gene was confirmed by an increase in endogenous TG production in primary human thyrocytes.We identified a new homozygous FOXE1 mutation responsible for enhanced expression of the TG and TPO genes in a boy whose phenotype is similar to that reported previously in patients with loss-of-function FOXE1 mutations. This finding further delineates the role for FOXE1 in both thyroid and palate development, and shows that enhanced gene activity should be considered among the mechanisms underlying Bamforth-Lazarus syndrome.

International audience; 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.

Comment in :The yin and yang of cystic fibrosis transmembrane conductance regulator function: implications for chronic lung disease. [Am J Respir Crit Care Med. 2013]; International audience; 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.

NKX2.1 mutations have been identified in patients displaying complete or partial brain-lung-thyroid syndrome, which can include benign hereditary chorea (BHC), hypothyroidism and/or lung disease.We evaluated the recently developed Multiplex Ligation-dependent Probe Amplification (MLPA) method to assess the relative copy number of genes. The goal was to determine if MLPA could improve, in addition to direct sequencing, the detection rate of NKX2.1 mutations in a phenotype-selected cohort of 24 patients affected by neurological, thyroid and/or pulmonary disorders.Direct sequencing revealed two heterozygous mutations. Using MLPA, we identified two further heterozygous NKX2.1 gene deletions. MLPA increased the detection rate by 50%. All patients with gene deletions identified were affected by BHC and congenital hypothyroidism.MLPA should be considered as a complementary tool in patients with partial or total brain-lung-thyroid syndrome when direct sequencing failed to identify NKX2.1 mutations. All patients with an NKX2.1 mutation had BHC and congenital hypothyroidism, emphasizing the high prevalence of these signs associated with defective NKX2.1 alleles.

Reduction of lung inflammation is one of the goals of cystic fibrosis (CF) therapy. Among anti-inflammatory molecules, glucocorticoids (GC) are one of the most prescribed. However. CF patients seem to be resistant to glucocorticoid treatment. Several molecular mechanisms that contribute to decrease anti-inflammatory effects of glucocorticoids have been identified in pulmonary diseases, but the molecular actions of glucocorticoids have never been studied in CF. In the cytoplasm, glucocorticoids bind to glucocorticoid receptor (GR) and then, control NF-kappa B and MAPK pathways through direct interaction with AP-1 and NF-kappa B in the nucleus. Conversely. MAPK can regulate glucocorticoid activation by targeting GR phosphorylation. Together these pathways regulate IL-8 release in the lung. Using bronchial epithelial cell lines derived from non CF and CF patients, we analyzed GR-based effects of glucocorticoids on NF-kappa B and MAPK pathways, after stimulation with TNF-alpha. We demonstrate that the synthetic glucocorticoid dexamethasone (Dex) significantly decreases IL-8 secretion, AP-1 and NF-kappa B activity in CF cells in a pro-inflammatory context. Moreover, we show that p38 MAPK controls IL-8 release by determining GR activation through specific phosphorylation on serine 211. Finally, we demonstrate a synergistic effect of dexamethasone treatment and inhibition of p38 MAPK inducing more than 90% inhibition of IL-8 production in CF cells. All together, these results demonstrate the good responsiveness to glucocorticoids of CF bronchial epithelial cells and the reciprocal link between glucocorticoids and p38 MAPK in the control of CF lung inflammation.

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.

NKX2-1 (NK2 homeobox 1) is a critical regulator of transcription for the surfactant protein (SP)-B and -C genes (SFTPB and SFTPC, respectively). We identified and functionally characterized two new de novo NKX2-1 mutations c.493C>T (p.R165W) and c.786_787del2 (p.L263fs) in infants with closely similar severe interstitial lung disease (ILD), hypotonia, and congenital hypothyroidism. Functional analyses using A549 and HeLa cells revealed that NKX2-1-p.L263fs induced neither SFTPB nor SFTPC promoter activation and had a dominant negative effect on wild-type (WT) NKX2-1. In contrast,NKX2-1-p.R165W activated SFTPC, to a significantly greater extent than did WTNKX2-1, while SFTPB activation was only significantly reduced in HeLa cells. In accordance with our in vitro data, we found decreased amounts of SP-B and SP-C by western blot in bronchoalveolar lavage fluid (patient with p.L263fs) and features of altered surfactant protein metabolism on lung histology (patient with NKX2-1-p.R165W). In conclusion, ILD in patients with NKX2-1 mutations was associated with altered surfactant protein metabolism, and both gain and loss of function of the mutated NKX2-1 genes on surfactant protein promoters were associated with ILD in "Brain-Lung-Thyroid syndrome".

Background: Mutations in the surfactant protein C gene ( SFTPC ) have been recently associated with the development of diffuse lung disease, particularly sporadic and familial interstitial lung disease (ILD). Objective: We have investigated the prevalence and the spectrum of SFTPC mutations in a large cohort of infants and children with diffuse lung disease and suspected with surfactant dysfunction. Method and results: 121 children were first screened for the common SFTPC mutation, p.Ile73Thr (I73T). Ten unrelated patients were shown to carry this mutation. The I73T mutation was inherited in six cases, and appeared de novo in four. The 111 patients without the I73T mutation were screened for the entire coding sequence of SFTPC . Of these, eight (seven unrelated) subjects were shown to carry a novel mutant allele of SFTPC . All these seven new mutations are located in the BRICHOS domain except the p.Val39Ala (V39A) mutation, which is in the surfactant protein C (SP-C) mature peptide. Conclusions: Our results confirm that SFTPC mutations are a frequent cause of diffuse lung disease, and that I73T is the most frequent SFTPC mutation associated with diffuse lung disease.

Interstitial lung diseases (ILDs) comprise a group of lung disorders characterized by various levels of inflammation and fibrosis. The current understanding of the mechanisms underlying the development and progression of ILD strongly suggests a central role of the alveolar epithelium. Following injury, alveolar epithelial cells (AECs) may actively participate in the restoration of a normal alveolar architecture through a coordinated process of re-epithelialization, or in the development of fibrosis through a process known as epithelial-mesenchymal transition (EMT). Complex networks orchestrate EMT leading to changes in cell architecture and behaviour, loss of epithelial characteristics and gain of mesenchymal properties. In the lung, AECs themselves may serve as a source of fibroblasts and myofibroblasts by acquiring a mesenchymal phenotype. This review covers recent knowledge on the role of alveolar epithelium in the pathogenesis of ILD. The mechanisms underlying disease progression are discussed, with a main focus on the apoptotic pathway, the endoplasmic reticulum stress response and the developmental pathway.

An intranasal vaccine composed of Toll-like receptor 2 (TLR2) ligand Neisseria meningitidis outer membrane proteins and Toll-like receptor 4 (TLR4) ligand Shigella flexneri lipopolysaccharide (LPS) (Protollin) and enriched respiratory syncytial virus (RSV) proteins (eRSV) has been demonstrated to promote balanced Th1/Th2 responses without eosinophil recruitment and to protect against challenge in mouse models. We used TLR2, TLR4 and myeloid differentiation factor 88 (MyD88) knock-out (-/-) mice to investigate the roles of these signalling pathways on immunogenicity, protection and pulmonary infiltrates following RSV immunization and challenge. Antigen-specific systemic and mucosal antibody production was significantly impaired only in TLR4-/- mice following Protollin-eRSV immunization. In contrast, an intact MyD88 pathway was crucial to elicit a balanced type 1:type 2 immune response, characterized by increased splenocyte production of antigen-induced IFNgamma and IL-10 with concomitant reduction of IL5, IgG2a isotype switching and abrogation of pulmonary eosinophil recruitment following challenge. MyD88-dependent signalling also contributed to neutrophil recruitment to the lungs following immunization with eRSV antigen, in the presence or absence of Protollin, compared to a mock antigen or vaccine. Both TLR4 and MyD88-signalling were required for optimal protection against challenge. The upregulation of early signalling molecules IFN-beta, TNFalpha, CD40 and CD86 were studied in splenocytes isolated from naïve TLR2, TLR4 and MyD88-/- mice following stimulation with vaccine components. Splenocytes from TLR4-/- mice displayed reduced IFN-beta while those of MyD88-/- mice elicited less TNFalpha and lower expression of CD40 and CD86 on CD11c+ cells. Together, our results suggest that optimal immunogenicity and protection against RSV without risk of enhanced pulmonary inflammation requires intact TLR4/MyD88-dependent signalling.

Cryptococcus neoformans is a major cause of fungal pneumonia, meningitis and disseminated disease in the immune compromised host. Here we have used a clinically relevant model to investigate the genetic determinants of susceptibility to progressive cryptococcal pneumonia in C57BL/6J and CBA/J inbred mice. At 5 weeks after infection, the lung fungal burden was over 1000-fold higher in C57BL/6J compared to CBA/J mice. A genome-wide scan performed on 210 male and 203 female (CBA/J x C57BL/6J) F2 progeny using lung colony-forming units as a quantitative trait revealed a sex difference in genetic architecture with three loci (designated Cnes1-Cnes3) associated with susceptibility to cryptococcal pneumonia. Single locus analysis identified significant loci on chromosomes 3 (Cnes1) and 17 (Cnes2) with logarithm of the odds (LOD) scores of 4.09 (P=0.0110) and 7.30 (P

BackgroundCryptococcus neoformans(C. neoformans) is a globally distributed fungal pathogen with the potential to cause serious disease, particularly among immune compromised hosts. Exposure to this organism is believed to occur by inhalation and may result in pneumonia and/or disseminated infection of the brain as well as other organs. Little is known about the role of airway epithelial cells in cryptococcal recognition or their ability to induce an inflammatory response.MethodsImmortalized BEAS-2B bronchial epithelial cells and primary normal human bronchial epithelium (NHBE) were stimulatedin vitrowith encapsulated or acapsularC. neoformanscultivated at room temperature or 37°C. Activation of bronchial epithelial cells was characterized by analysis of inflammatory cytokine and chemokine expression, transcription factor activation, fungal-host cell association, and host cell damage.ResultsViableC. neoformansis a strong activator of BEAS-2B cells, resulting in the production of the neutrophil chemokine Interleukin (IL)-8 in a time- and dose-dependent manner. IL-8 production was observed only in response to acapsularC. neoformansthat was grown at 37°C.C. neoformanswas also able to induce the expression of the chemokine CXCL1 and the transcription factor CAAT/enhancer-binding protein beta (CEBP/β) in BEAS-2B cells. NHBE was highly responsive to stimulation withC. neoformans; in addition to transcriptional up regulation of CXCL1, these primary cells exhibited the greatest IL-8 secretion and cell damage in response to stimulation with an acapsular strain ofC. neoformans.ConclusionThis study demonstrates that human bronchial epithelial cells mediate an acute inflammatory response toC. neoformansand are susceptible to damage by this fungal pathogen. The presence of capsular polysaccharide andin vitrofungal culture conditions modulate the host inflammatory response toC. neoformans. Human bronchial epithelial cells are likely to contribute to the initial stages of pulmonary host defensein vivo.

The rising incidence of serious fungal diseases represents a growing threat to human health. Cryptococcus neoformans, an encapsulated yeast saprophyte with global distribution, has been recognized as an important emerging pathogen. Humans frequently develop asymptomatic or mild infection with C. neoformans, but individuals with impaired host defense systems may develop severe pneumonia and potentially fatal meningoencephalitis. Insight into the biology and virulence of C. neoformans is advancing rapidly and will be propelled even further by the recently completed and published genome sequences for two related strains of C. neoformans serotype D. Several mammalian model hosts including the guinea pig, rabbit, rat, and mouse have been developed for the study of cryptococcosis. The combination of microbial genomics with well-characterized model hosts that are amenable to immunologic and genetic manipulation represents a powerful resource for comprehensive study of cryptococcal disease pathogenesis as well as vaccine and antifungal drug therapy. This review provides an introduction to each mammalian model host and briefly highlights the advantages, limitations, and potential of each system for future research involving cryptococci.

Objectives Cystic fibrosis (CF) is characterized by airway colonization by Pseudomonas aeruginosa ( P.a ) resulting in a prolonged inflammatory response. Here, we performed transcriptomic analysis of P.a -infected primary bronchial epithelial cells from non-CF and CF patients to look for new leads explaining P.a persistence in lungs in spite of a strong inflammatory response. Methods Non-CF and CF cells were infected by P.a at 0.25 MOI for 0, 2, 4 and 6 hours. Biological samples (4 CF and 4 non-CF) were processed for mRNA profiling using Illumina mRNA sequencing. Statistical analysis provided lists of genes having a significant differential expression (p Results We found 310, 295, 252 and 448 genes differentially down-regulated and 541, 343, 415, 532 differentially up-regulated in CF vs non-CF cells at 0, 2, 4 and 6 hours of infection, respectively. We identified genes that could explain the intense inflammatory response, the development of chronic infection or the absence of antiinflammatory response. Several genes such as IL-6, IL-17C, KLF2, TLR4, SLPI or GSTT1 were already known to play a role in the epithelium- P.a interaction but interestingly some genes were never or sparsely described in the lung CF disease. Conclusion Different clinical approaches are used to eliminate P.a especially antibiotic prescriptions, but none of them leading to complete eradication. Here, we identified mRNA pattern associated with P.a infection that could explain the lack of P.a eradication in CF. Those results may be the first step to study the development of new diagnostic tools and therapeutic strategies.

Background Impairment of the activity of the CFTR chloride channel observed in cystic fibrosis (CF) patients is the main cause of the deterioration of lung function. In 2008, the channel ANO1 was identified and proposed as a potential therapeutic target in CF. Previously, we have shown that ANO1 activity and expression were reduced in a CF context (Ruffin et al., 2013). The aim of this work is to understand the origin of the reduced expression of ANO1 in CF by studying the role of miRNAs. Methods We performed bio-informatics approaches to study miRNAs that could target ANO1 and evaluated the expression levels of miRNAs candidates by RT-qPCR in bronchial epithelial cells lines. We performed functionality experiments studying the miRNAs candidates binding to the 3'UTR of ANO1 over-expressing miRNAs, and quantifying the expression of ANO1 in these conditions. We also evaluated cells migration rate and ANO1 chloride activity. Results We identified different miRNAs including miR-9 as potential regulator of ANO1. We observed that miR-9 is overexpressed in CF cells and a decrease of ANO1 expression and luciferase activity when we overexpress miR-9 in CF and non-CF cells. Decrease of cells migration rate and ANO1 chloride activity were also observed when miR-9 is overexpressed. Conclusions Our results showed that miR-9 directly regulates ANO1 and that modulate miR-9 allows to change cells migration rate and ANO1 chloride activity. In the context of CF, it would be interesting to increase ANO1 expression and activity, so we are doing tests using antisense oligonucleotides designed to specifically link ANO1 3'UTR by masking miR-9 fixation site.

Loïc Guillot: Supported by the Delegation General pour l’Armement Ronan Le Goffic: Supported by “Vaincre la Mucoviscidose” (Paris, France); International audience; Influenza A is a highly contagious single-stranded RNA virus that infects both the upper and lower respiratory tracts of humans. The host innate immune Tolllike receptor (TLR) 3 was shown previously in cells of myeloid origin to recognize the viral replicative, intermediate double-stranded RNA (dsRNA). Thus, dsRNA may be critical for the outcome of the infection. Here we first compared the activation triggered by either influenza A virus or dsRNA in pulmonary epithelial cells. We established that TLR3 is constitutively expressed in human alveolar and bronchial epithelial cells, and we describe its intracellular localization. Expression of TLR3 was positively regulated by the influenza A virus and by dsRNA but not by other inflammatory mediators, including bacterial lipopolysaccharide, the cytokines tumor necrosis factor-␣ and interleukin (IL)-1␤, and the protein kinase C activator phorbol 12-myristate 13-acetate. We also demonstrated that TLR3 contributes directly to the immune response of respiratory epithelial cells to influenza A virus and dsRNA, and we propose a molecular mechanism by which these stimuli induce epithelial cell activation. This model involves mitogen-activated protein kinases, phosphatidylinositol 3-kinase/ Akt signaling, and the TLR3-associated adaptor molecule TRIF but not MyD88-dependent activation of the transcription factors NF-B or interferon regulatory factor/interferon-sensitive response-element pathways. Ultimately, this signal transduction elicits an epithelial response that includes the secretion of the cytokines IL-8, IL-6, RANTES (regulated on activation normal T cell expressed and secreted), and interferon-␤ and the up-regulation of the major adhesion molecule ICAM-1.

Pulmonary epithelial cells are continuously exposed to microbial challenges as a result of breathing. It is recognized that immune myeloid cells express Toll-like receptors (TLRs), which play a major role in detecting microbes and initiating innate immune responses. In contrast, little is known concerning the expression of TLR in pulmonary epithelial cells per se, their distribution within the cell, their function, and the signaling pathways involved. In this work, we demonstrated by reverse transcription-PCR and/or immunoblot that TLR4 and the accessory molecule MD-2 are constitutively expressed in distinct human alveolar and bronchial epithelial cells. We further characterized by flow cytometry, biotinylation/precipitation, and confocal microscopy the intracellular localization of TLR4 in these cells. Despite this intracellular compartmentalization of TLR4, pulmonary epithelial cells were responsive to the TLR4 activator lipopolysaccharide (LPS), a potent Gram-negative bacteria-associated molecular pattern. Using respiratory epithelial cells isolated from TLR4 knock-out and wild type mice, we demonstrated that TLR4 is the actual activating receptor for LPS in these cells. Furthermore we showed that this cell response to LPS involves a signaling complex including the kinases interleukin-1 receptor-associated kinase (IRAK), p38, Jnk, and ERK1/2. Moreover, using vectors expressing dominant-negative forms of MyD88 and TRAF6, we established that LPS-induced activation of respiratory epithelial cells is largely dependent on TLR4 signaling intermediates. Altogether these data demonstrate that TLR4 is a key element in the response of pulmonary epithelial cells to molecules derived from Gram-negative bacteria. The intracellular localization of TLR4 in lung epithelia is expected to play an important role in the prevention of the development of chronic inflammatory disease.

Introduction Pediatric interstitial lung diseases (pILD) represent a heterogeneous group of rare and poorly defined disorders. Genetic abnormalities of surfactant proteins B (SP-B) and C (SP-C) have been shown to be related to these pathologies. However, variability in the lung disease phenotype suggests the involvement of other surfactant-associated genes. The aim of this study was to examined the contribution of TTF-1 (Thyroid Transcription Factor 1), a transcription factor especially involved in lung development in pILD. Methods TTF-1 gene sequencing was performed in two children presenting with hypotonia, pILD and hyptothyroidism without a known etiology. Surfactant proteins were assessed in bronchoalveolar lavage (BAL) fluid by western blot analysis using proSP-B, SP-B and proSP-C antibodies. TTF-1 mutated plasmids were realized by mutagenesis. SP-B/SP-C promoter analysis was realized in A549 cells using co-transfection experiment with SP-C and SP-B luciferase plasmids with wild type or mutated TTF-1 expression plasmids. Results Two new spontaneous TTF-1 mutations (M1 and M2) have been identified by sequencing in two patients (P1 and P2). The measurement of SP-B and SP-C promoter activation by plasmids encoding mutated TTF-1 (TTF-1M1 and TTF-1M2) revealed two different mecanisms. TTF-1M1 do not induces either SP- B or SP-C promoter activation and has a dominant negative effect on the wild type TTF-1. In contrast, TTF-1M2 activates SP-B and SP-C promoters Also, SP-C promoter activation was higher with TTF1M2 that the wild type TTF-1. BAL from P1 is characterized by a normal pattern of pro-SP-B and SP-B contents but exhibits an accumulation of pro-SP-C. Conclusion In this study, we identified and characterized two new TTF-1 mutations responsible for pILD with two different mecanisms.

Introduction Cystic Fibrosis (CF) is the most common lethal autosomal recessive disease in Caucasian population. The gene mutated in CF encodes a chloride channel named CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Lung dysfunction is the main cause of mortality in CF patients. Infection combined with inflammation lead to progressive destruction of the respiratory epithelium. Glucocorticoids (GC) are anti-inflammatory molecules commonly used to treat inflammation but with controversial efficiency among patients. The lack of efficiency could be due to resistance to GC already observed in others respiratory diseases. To test this hypothesis, we study the key steps in GC activation pathway in the lung to identify the molecular basis of such dysregulation. Our present studies focus on the regulation of the expression of the GR isoforms. Methods CF bronchial epithelial cell lines (IB3-1) and CF corrected (S9) were incubated with IL-1β (10ng/ml) with or without dexamethasone (dex, 1μM). Rates of secreted IL-8 were dosed by ELISA NF-κB activation was measured using NF-κB promotor coupled to luciferase. Expression of GR isoforms was measured by quantitative RT-PCR. Results In presence of IL-1β, dex can restore basal secretion of IL-8 at 16h in S9, whereas IB3-1 barely respond. Moreover, no effect of dex on NF-κB activation is observed in CF cells. There is a differential regulation of the GR isoforms expression at 16h in the two cell lines. The study of the GR activation window of activation in inflammatory conditions is currently in progress. Conclusions In this study we have shown a different regulation of inflammation by GC Our preliminary study on GR expression also suggests differences between CF and non-CF cell lines. The present results require to be assessed in other cellular models and from CF lung biopsies. We will subsequently investigate the possible dysregulation of other key steps in GC activation pathway such as GR phosphorylation and nuclear translocation.

Introduction Pediatric interstitial lung diseases (pILD) represent a heterogeneous group of rare and poorly defined disorders. Genetic abnormalities of surfactant proteins B (SP-B) and C (SP-C) have been shown to be related to these pathologies. However, variability in the lung disease phenotype suggests the involvement of other surfactant-associated genes. The aim of this study was to examined the contribution of TTF-1 (Thyroid Transcription Factor 1), a transcription factor especially involved in lung development in pILD. Methods TTF-1 gene sequencing was performed in two children presenting with hypotonia, pILD and hyptothyroidism without a known etiology. Surfactant proteins were assessed in bronchoalveolar lavage (BAL) fluid by western blot analysis using proSP-B, SP-B and proSP-C antibodies. TTF-1 mutated plasmids were realized by mutagenesis. SP-B/SP-C promoter analysis was realized in A549 cells using co-transfection experiment with SP-C and SP-B luciferase plasmids with wild type or mutated TTF-1 expression plasmids. Results Two new spontaneous TTF-1 mutations (M1 and M2) have been identified by sequencing in two patients (P1 and P2). The measurement of SP-B and SP-C promoter activation by plasmids encoding mutated TTF-1 (TTF-1M1 and TTF-1M2) revealed two different mecanisms. TTF-1M1 do not induces either SP- B or SP-C promoter activation and has a dominant negative effect on the wild type TTF-1. In contrast, TTF-1M2 activates SP-B and SP-C promoters Also, SP-C promoter activation was higher with TTF1M2 that the wild type TTF-1. BAL from P1 is characterized by a normal pattern of pro-SP-B and SP-B contents but exhibits an accumulation of pro-SP-C. Conclusion In this study, we identified and characterized two new TTF-1 mutations responsible for pILD with two different mecanisms.

NOD2/CARD15 mediates innate immune responses to mycobacterial infection. However, its role in the regulation of adaptive immunity has remained unknown. In this study, we examined host defense, T cell responses, and tissue pathology in two models of pulmonary mycobacterial infection, using wild-type and Nod2-deficient mice. During the early phase of aerosol infection with Mycobacterium tuberculosis, Nod2−/− mice had similar bacterial counts but reduced inflammatory response on histopathology at 4 and 8 wk postchallenge compared with wild-type animals. These findings were confirmed upon intratracheal infection of mice with attenuated Mycobacterium bovis bacillus Calmette-Guérin. Analysis of the lungs 4 wk after bacillus Calmette-Guérin infection demonstrated that Nod2−/− mice had decreased production of type 1 cytokines and reduced recruitment of CD8+ and CD4+ T cells. Ag-specific T cell responses in both the spleens and thoracic lymph nodes were diminished in Nod2−/− mice, indicating impaired adaptive antimycobacterial immunity. The immune regulatory role of NOD2 was not restricted to the lung since Nod2 disruption also led to reduced type 1 T cell activation following i.m. bacillus Calmette-Guérin infection. To determine the importance of diminished innate and adaptive immunity, we measured bacterial burden 6 mo after aerosol infection with M. tuberculosis and followed a second infected group for assessment of survival. Nod2−/− mice had a higher bacterial burden in the lungs 6 mo after infection and succumbed sooner than did wild-type controls. Taken together, these data indicate that NOD2 mediates resistance to mycobacterial infection via both innate and adaptive immunity.

Cystic fibrosis (CF) is due to CFTR gene pathogenic variants and has no curative treatment to date. The main cause of morbidity and mortality in CF patients is respiratory failure, due to cycles of infection and inflammation responsible for progressive lung tissue degradation.Although CF is a monogenic disease, lung disease severity varies among patients carrying the same CFTR variants. This is partly explained by other genetic factors called "cystic fibrosis modifier genes". In this study, we focused on a particular modifier gene, SLC6A14, which have been shown to be associated with CF digestive and lung disease severities. We were able to demonstrate that the G allele of the rs3788766 variant, located in the promoter sequence of SLC6A14, is associated with lung function decline as well as a decrease of SLC6A14 promoter activity. We then focused on its role in lung pathophysiology. We showed that SLC6A14 is involved in mTOR signaling and that it favors bronchial epithelial repair. We also studied SLC6A14 expression and function regulation in CF inflammatory and infectious contexts. Our results indicate an induction of SLC6A14 expression following IL-1β and Pseudomonas aeruginosa exposure. Interestingly, we identified LasB, a protease secreted by P. aeruginosa, as capable of cleaving SLC6A14. Thus, this work contributes to new knowledge acquisition on SLC6A14 as a CF modifier gene, which is essential for a better understanding of the disease and for the development of new therapeutic approaches. In this sense, our results highlight the rs3788766 impact and the host-pathogen interactions existing within the CF patients’ bronchial epithelium., La mucoviscidose est due à des variants pathogènes du gène CFTR et ne dispose d’aucun traitement curatif à ce jour. La principale cause de mortalité des patients atteints de mucoviscidose est l’atteinte pulmonaire, caractérisée par des cycles d’infections et d’inflammation menant, à terme, à une insuffisance respiratoire. La sévérité de la maladie est variable selon les patients porteurs des mêmes variants de CFTR. Cela s’explique en partie par d’autres facteurs génétiques appelés « gènes modificateurs ». Dans le cadre de cette étude, nous nous sommes intéressés au gène SLC6A14, dont certains variants sont associés à une aggravation des atteintes respiratoires de la mucoviscidose. Nous avons pu démontrer que l’allèle G du variant rs3788766, localisé dans la séquence promotrice de SLC6A14, est associé à un déclin de la fonction respiratoire ainsi qu’à une diminution de l’activité du promoteur de SLC6A14. Nous avons montré que SLC6A14 est impliqué dans la voie de signalisation mTOR et favorise la réparation épithéliale bronchique. L’expression et la fonction de SLC6A14 sont modulées en réponse à la cytokine IL-1β et à l’infection par Pseudomonas aeruginosa, contextes inflammatoire et infectieux caractéristiques de la mucoviscidose. L’apport de nouvelles connaissances sur le gène modificateur SLC6A14, qui fait l’objet de recherches depuis peu, est essentiel à une meilleure compréhension de la maladie ainsi qu’au développement de nouvelles pistes thérapeutiques. En ce sens, ce travail apporte un nouvel éclairage sur l’impact du variant rs3788766 et sur les interactions hôte-pathogène existant au sein de l’épithélium bronchique des patients atteints de mucoviscidose.