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Question: Pseudomonas aeruginosa ( Pa ) is a common pathogen that contributes to progressive lung disease in Cystic Fibrosis (CF). Genetic factors other than CF-causing CFTR variations contribute approximately 85% of the variation in chronic Pa infection age in CF according to twin studies, but the susceptibility loci remain unknown. Our objective is to advance understanding of the genetic basis of host susceptibility to Pa infection., Materials and Methods: We conducted a genome-wide association study (GWAS) of chronic Pa infection age in 1037 Canadians with CF. We subsequently assessed the genetic correlation between chronic Pa infection age and lung function through polygenic risk score (PRS) analysis and inferred their causal relationship through bi-directional Mendelian Randomization analysis., Results: Two novel genome-wide significant loci with lead SNPs rs62369766 (chr5p12; p-value= 1.98 ×10 -8 ) and rs927553 (chr13q12.12; p-value= 1.91 × 10 -8 ) were associated with chronic Pa infection age. The rs62369766 locus was validated using an independent French cohort (N=501). Furthermore, PRS constructed from CF lung function-associated SNPs was significantly associated with chronic Pa infection age (p-value=0.002). Finally, our analysis presented evidence for a causal effect of lung function on the chronic Pa infection age (Beta=0.782 years, p-value= 4.24 × 10 -4 ). In the reverse direction, we observed a moderate effect (Beta=0.002, p-value=0.012)., Conclusions: We identified two novel loci that are associated with chronic Pa infection age in individuals with CF. Additionally, we provided evidence of common genetic contributors and a potential causal relationship between Pa infection susceptibility and lung function in CF. Therapeutics targeting these genetic factors may delay the onset of chronic infections which accounts for significant remaining morbidity in CF., (Copyright ©The authors 2024. For reproduction rights and permissions contact permissions@ersnet.org.)

Airway epithelial cells form a physical barrier against inhaled pathogens and coordinate innate immune responses in the lungs. Bronchial cells in people with cystic fibrosis (pwCF) are colonized by Pseudomonas aeruginosa because of the accumulation of mucus in the lower airways and an altered immune response. This leads to chronic inflammation, lung tissue damage, and accelerated decline in lung function. Thus, identifying the molecular factors involved in the host response in the airways is crucial for developing new therapeutic strategies. The septin (SEPT) cytoskeleton is involved in tissue barrier integrity and anti-infective responses. SEPT7 is critical for maintaining SEPT complexes and for sensing pathogenic microbes. In the lungs, SEPT7 may be involved in the epithelial barrier resistance to infection; however, its role in cystic fibrosis (CF) P. aeruginosa infection is unknown. This study aimed to investigate the role of SEPT7 in controlling P. aeruginosa infection in bronchial epithelial cells, particularly in CF. The study findings showed that SEPT7 encages P. aeruginosa in bronchial epithelial cells and its inhibition downregulates the expression of other SEPTs. In addition, P. aeruginosa does not regulate SEPT7 expression. Finally, we found that inhibiting SEPT7 expression in bronchial epithelial cells (BEAS-2B 16HBE14o- and primary cells) resulted in higher levels of internalized P. aeruginosa and decreased IL-6 production during infection, suggesting a crucial role of SEPT7 in the host response against this bacterium. However, these effects were not observed in the CF cells (16HBE14o-/F508del and primary cells) which may explain the persistence of infection in pwCF. The study findings suggest the modification of SEPT7 expression as a potential approach for the anti-infective control of P. aeruginosa, particularly in CF., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Pseudomonas aeruginosa (Pa) infection is detrimental to people with cystic fibrosis (pwCF). Several clinical and genetic factors predispose to early Pa infections. However, the role of earlier infections with other pathogens on the risk of Pa infection in paediatric pwCF remains unknown., Methods: Using Kaplan-Meier method, we computed the cumulative incidences of bacterial and fungal initial acquisition (IA) and chronic colonisation (CC) in 1,231 French pwCF under 18 years of age for methicillin-susceptible and resistant Staphylococcus aureus (MSSA and MRSA), Stenotrophomonas maltophilia, Haemophilus influenzae, Achromobacter xylosoxidans, and Aspergillus species. Previous infections were analysed as Pa-IA and Pa-CC risk factors using Cox regression models., Results: By 2 years of age, 65.5% pwCF had experienced at least one bacterial or fungal IA, and 27.9% had experienced at least one CC. The median age of Pa-IA was 5.1 years, and Pa-CC was present in 25% pwCF by 14.7 years. While 50% acquired MSSA at 2.1 years, 50% progressed to chronic MSSA colonisation at 8.4 years. At 7.9 and 9.7 years, 25% pwCF were infected by S. maltophilia and Aspergillus spp., respectively. The risk of Pa-IA and Pa-CC increased with IAs of all other species, with hazard ratios (HR) up to 2.19 (95% Confidence interval (CI) 1.18-4.07). The risk of Pa-IA increased with the number of previous bacterial/fungal IAs (HR=1.89, 95% CI 1.57-2.28), with a 16% increase per additional pathogen; same trend was noted for Pa-CC., Conclusions: This study establishes that the microbial community in CF airways can modulate Pa occurrence. At the dawn of targeted therapies, it paves the way for characterizing future trends and evolution of infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Bronchial epithelial cells (BEC) play a crucial role in innate immunity against inhaled fungi. Indeed, in response to microorganisms, BEC synthesize proinflammatory cytokines involved in the recruitment of neutrophils. We have recently shown that BEC exert antifungal activity against Aspergillus fumigatus by inhibiting filament growth. In the present study, we first analyzed the inflammatory and antifungal responses of BEC infected by several fungal species such as Aspergillus spp., Scedosporium apiospermum and Candida albicans , which are frequently isolated from the sputum of people with chronic pulmonary diseases. The airways of these patients, such as people with cystic fibrosis (pwCF), are mainly colonized by P. aeruginosa and secondary by fungal pathogens. We have previously demonstrated that BEC are capable of innate immune memory, allowing them to increase their inflammatory response against A. fumigatus following a previous contact with Pseudomonas aeruginosa flagellin. To identify the impact of bacteria exposure on BEC responses to other fungal infections, we extended the analysis of BEC innate immune memory to Aspergillus spp., Scedosporium apiospermum and Candida albicans infection. Our results show that BEC are able to recognize and respond to Aspergillus spp., S. apiospermum and C. albicans infection and that the modulation of BEC responses by pre-exposure to flagellin varies according to the fungal species encountered. Deepening our knowledge of the innate immune memory of BEC should open new therapeutic avenues to modulate the inflammatory response against polymicrobial infections observed in chronic pulmonary diseases such as CF.

Although cystic fibrosis (CF) is recognized as a monogenic disease, due to variants within the CFTR ( Cystic Fibrosis Transmembrane Regulator ) gene, an extreme clinical heterogeneity is described among people with CF (pwCF). Apart from the exocrine pancreatic status, most studies agree that there is little association between CFTR variants and disease phenotypes. Environmental factors have been shown to contribute to this heterogeneity, accounting for almost 50% of the variability of the lung function of pwCF. Nevertheless, pwCF with similar CFTR variants and sharing the same environment (such as in siblings) may have highly variable clinical manifestations not explained by CFTR variants, and only partly explained by environmental factors. It is recognized that genetic variants located outside the CFTR locus, named "modifier genes", influence the clinical expression of the disease. This short review discusses the latest studies that have described modifier factors associated with the various CF phenotypes as well as the response to the recent CFTR modulator therapies.

Secretory phospholipase A2 (sPLA2) regulates the first step of inflammatory cascade and is involved in several pathological processes. sPLA2 also plays a role in preterm labor and parturition, since they are triggered by inflammatory mediators such as prostaglandins. Interestingly, chorioamnionitis (i.e., the presence of intrauterine inflammation) is also often associated with preterm birth. We aimed to verify if chorioamnionitis with fetal involvement modifies sPLA2 activity and expression profile in mothers and neonates delivered prematurely. We collected maternal plasma and amniotic fluid, as well as bronchoalveolar lavage fluid from preterm neonates born to mothers with or without clinical chorioamnionitis with fetal involvement. We measured concentrations of sPLA2 subtype-IIA and -IB, total enzyme activity, and proteins. Urea ratio was used to obtain epithelial lining fluid concentrations. Enzyme activity measured in maternal plasma ( P < 0.001) and amniotic fluid ( P < 0.001) was higher in chorioamnionitis cases than in controls. This was mainly due to the increased production of sPLA2-IIA, as the subtype -IB was present in a smaller amount and was similar between the two groups; sPLA2-IIA was increased in epithelial lining fluid ( P = 0.045) or increased, although without statistical significance, in maternal plasma ( P = 0.06) and amniotic fluid ( P = 0.08) of chorioamnionitis cases. Cytokines that are known to increase sPLA2-IIA expression (TNF-α and IL-1β) or whose expression was increased by sPLA2-IIA (IL-8) were higher in histologically confirmed chorioamnionitis [TNF-α ( P = 0.028), IL-1β ( P < 0.001), and IL-8 ( P = 0.038)]. These data represent the basis for future studies on sPLA2-IIA inhibition to prevent deleterious consequences of chorioamnionitis and preterm birth.

Cystic fibrosis (CF), due to pathogenic variants in CFTR gene, is associated with chronic infection/inflammation responsible for airway epithelium alteration and lung function decline. Modifier genes induce phenotype variability between people with CF (pwCF) carrying the same CFTR variants. Among these, the gene encoding for the amino acid transporter SLC6A14 has been associated with lung disease severity and age of primary airway infection by the bacteria Pseudomonas aeruginosa . In this study, we investigated whether the single nucleotide polymorphism (SNP) rs3788766, located within SLC6A14 promoter, is associated with lung disease severity in a large French cohort of pwCF. We also studied the consequences of this SNP on SLC6A14 promoter activity using a luciferase reporter and the role of SLC6A14 in the mechanistic target of rapamycin kinase (mTOR) signaling pathway and airway epithelial repair. We confirm that SLC6A14 rs3788766 SNP is associated with lung disease severity in pwCF ( p = 0.020; n = 3,257, pancreatic insufficient, aged 6-40 years old), with the minor allele G being deleterious. In bronchial epithelial cell lines deficient for CFTR , SLC6A14 promoter activity is reduced in the presence of the rs3788766 G allele. SLC6A14 inhibition with a specific pharmacological blocker reduced 3 H-arginine transport, mTOR phosphorylation, and bronchial epithelial repair rates in wound healing assays. To conclude, our study highlights that SLC6A14 genotype might affect lung disease severity of people with cystic fibrosis via mTOR and epithelial repair mechanism modulation in the lung., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mercier, Calmel, Mésinèle, Sutanto, Merabtene, Longchampt, Sage, Kicic, Boëlle, Corvol, Ruffin and Guillot.)

Lumacaftor/ivacaftor (LUMA-IVA) therapy is prescribed to people with cystic fibrosis (pwCF) homozygous for the Phe508del- CFTR variant to restore CFTR protein function. There is, however, large inter-individual variability in treatment response. Here, we seek to identify clinical and/or genetic factors that may modulate the response to this CFTR modulator therapy. A total of 765 pwCF older than 12 years under LUMA-IVA therapy and with lung function and nutritional measurements available before and after treatment initiation were included. Response to treatment was determined by the change in lung function and nutritional status, from baseline and over the first two years after initiation, and it was assessed by weighted generalized estimating equation models. Gains in lung function and nutritional status were observed after 6 months of treatment (on average 2.11 ± 7.81% for percent predicted FEV 1 and 0.44 ± 0.77 kg/m 2 for BMI) and sustained over the 2 years. We observed that the more severe patients gained the most in lung function and nutritional status. While females started with a nutritional status more impaired than males, they had a larger response and regained BMI Z-score values similar to men after 2 years of treatment. We observed no association between variants in solute carrier ( SLC ) genes and the respiratory function response to LUMA-IVA, but the SLC6A14 rs12839137 variant was associated with the nutritional response. Further investigations, including other genomic regions, will be needed to fully explore the inter-individual variability of the response to LUMA-IVA.

Background Cystic fibrosis (CF) lung disease is characterised by recurrent Pseudomonas aeruginosa (Pa) infections, leading to structural lung damage and decreased survival. The epidemiology of Pa infection and its impact on lung function in people with CF (pwCF), especially in recent birth cohorts, remain uncertain. Methods We included 1,231 French pwCF under 18 years of age. Age at initial acquisition (Pa-IA), chronic colonisation (Pa-CC), and duration from Pa-IA to Pa-CC were estimated using the Kaplan-Meier method. Demographic, clinical, and genetic characteristics were analysed as risk factors for Pa infection using Cox regression models. Lung function decline was assessed by modelling percent-predicted forced expiratory volume in 1 s (ppFEV1) before Pa infection, after Pa-IA, and after Pa-CC. Results Among the 1,231 pwCF, 50% had Pa-IA by the age of 5.1 years [95% confidence interval (CI) 3.8-6.2] and 25% had Pa-CC by the age of 14.7 years (95% CI 12.1 to ∞). We observed that CF-related diabetes and liver disease were risk factors for Pa, while gender, CFTR variants, and CF centre size were not. Genetic variants of TNF, DCTN4, SLC9A3, and CAV2 were confirmed to be associated with Pa. The annual rate of ppFEV1 decline before Pa was -0.38% predicted/year (95% CI -0.59 to -0.18), which decreased significantly after Pa-IA to -0.93% predicted/year (95% CI -1.14 to -0.71) and after Pa-CC to -1.51% predicted/year (95% CI -1.86 to -1.16). Conclusions We identified and replicated several risk factors associated with Pa infection and showed its deleterious impact on lung function in young pwCF. This large-scale study confirmed that Pa airway infection is a major determinant of lung disease severity., (Copyright © 2021 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

In the coronavirus disease 2019 (COVID-19) health crisis, one major challenge is to identify the susceptibility factors of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in order to adapt the recommendations for populations, as well as to reduce the risk of COVID-19 development in the most vulnerable people, especially patients with chronic respiratory diseases such as cystic fibrosis (CF). Airway epithelial cells (AECs) play a critical role in the modulation of both immune responses and COVID-19 severity. SARS-CoV-2 infects the airway through the receptor angiotensin-converting enzyme 2, and a host protease, transmembrane serine protease 2 (TMPRSS2), plays a major role in SARS-CoV-2 infectivity. Here, we show that Pseudomonas aeruginosa increases TMPRSS2 expression, notably in primary AECs with deficiency of the ion channel CF transmembrane conductance regulator (CFTR). Further, we show that the main component of P. aeruginosa flagella, the protein flagellin, increases TMPRSS2 expression in primary AECs and Calu-3 cells, through activation of Toll-like receptor-5 and p38 MAPK. This increase is particularly seen in Calu-3 cells deficient for CFTR and is associated with an intracellular increased level of SARS-CoV-2 infection, however, with no effect on the amount of virus particles released. Considering the urgency of the COVID-19 health crisis, this result may be of clinical significance for CF patients, who are frequently infected with and colonized by P. aeruginosa during the course of CF and might develop COVID-19., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ruffin, Bigot, Calmel, Mercier, Givelet, Oliva, Pizzorno, Rosa-Calatrava, Corvol, Balloy, Terrier and Guillot.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

COVID-19 is caused by the Severe Acute Respiratory Syndrome (SARS) coronavirus (Cov)-2, an enveloped virus with a positive-polarity, single-stranded RNA genome. The initial outbreak of the pandemic began in December 2019, and it is affecting the human health of the global community. In common with previous pandemics (Influenza H1N1 and SARS-CoV) and the epidemics of Middle east respiratory syndrome (MERS)-CoV, CoVs target bronchial and alveolar epithelial cells. Virus protein ligands (e.g., haemagglutinin or trimeric spike glycoprotein for Influenza and CoV, respectively) interact with cellular receptors, such as (depending on the virus) either sialic acids, Dipeptidyl peptidase 4 (DPP4), or angiotensin-converting enzyme 2 (ACE2). Host proteases, e.g., cathepsins, furin, or members of the type II transmembrane serine proteases (TTSP) family, such as Transmembrane protease serine 2 (TMPRSS2), are involved in virus entry by proteolytically activating virus ligands. Also involved are Toll Like Receptor (TLR) family members, which upregulate anti-viral and pro-inflammatory mediators [interleukin (IL)-6 and IL-8 and type I and type III Interferons among others], through the activation of Nuclear Factor (NF)-kB. When these events (virus cellular entry and innate immune responses) are uncontrolled, a deleterious systemic response is sometimes encountered in infected patients, leading to the well-described "cytokine storm" and an ensuing multiple organ failure promoted by a downregulation of dendritic cell, macrophage, and T-cell function. We aim to describe how the lung and systemic host innate immune responses affect survival either positively, through downregulating initial viral load, or negatively, by triggering uncontrolled inflammation. An emphasis will be put on host cellular signaling pathways and proteases involved with a view on tackling these therapeutically., (Copyright © 2020 Sallenave and Guillot.)

Aspergillus fumigatus is an environmental filamentous fungus that can be pathogenic for humans, wherein it is responsible for a large variety of clinical forms ranging from allergic diseases to life-threatening disseminated infections. The contamination occurs by inhalation of conidia present in the air, and the first encounter of this fungus in the human host is most likely with the bronchial epithelial cells. Although alveolar macrophages have been widely studied in the Aspergillus -lung interaction, increasing evidence suggests that bronchial epithelium plays a key role in responding to the fungus. This review focuses on the innate immune response of the bronchial epithelial cells against A. fumigatus , the predominant pathogenic species. We have also detailed the molecular interactants and the effects of the different modes of interaction between these cells and the fungus., (Copyright © 2020 Bigot, Guillot, Guitard, Ruffin, Corvol, Balloy and Hennequin.)

Background: The use of electronic cigarettes (ECIGs) is increasing, but the impact of ECIG-vapor on cellular processes like inflammation or host defense are less understood. The aim of the present study was to compare the acute effects of traditional cigarettes (TCIGs) and ECIG-exposure on host defense, inflammation, and cellular activation of cell lines and primary differentiated human airway epithelial cells (pHBE)., Methods: We exposed pHBEs and several cell lines to TCIG-smoke or ECIG-vapor. Epithelial host defense and barrier integrity were determined. The transcriptome of airway epithelial cells was compared by gene expression array analysis. Gene interaction networks were constructed and differential gene expression over all groups analyzed. The expression of several candidate genes was validated by qRT-PCR., Results: Bacterial killing, barrier integrity and the expression of antimicrobial peptides were not affected by ECIG-vapor compared to control samples. In contrast, TCIGs negatively affected host defense and reduced barrier integrity in a significant way. Furthermore ECIG-exposure significantly induced IL-8 secretion from Calu-3 cells but had no effect on NCI-H292 or primary cells. The gene expression based on array analysis distinguished TCIG-exposed cells from ECIG and room air-exposed samples., Conclusion: The transcriptome patterns of host defense and inflammatory genes are significantly distinct between ECIG-exposed and TCIG-treated cells. The overall effects of ECIGs on epithelial cells are less in comparison to TCIG, and ECIG-vapor does not affect host defense. Nevertheless, although acute exposure to ECIG-vapor induces inflammation, and the expression of S100 proteins, long term in vivo data is needed to evaluate the chronic effects of ECIG use.

Aspergillus fumigatus is an environmental filamentous fungus that may act as an opportunistic pathogen causing a variety of diseases, including asthma or allergic bronchopulmonary aspergillosis, and infection, ranging from asymptomatic colonization to invasive pulmonary form, especially in immunocompromised patients. This fungus is characterized by different morphotypes including conidia which are the infective propagules able to germinate into hyphae. Due to their small size (2-3 µm), conidia released in the air can reach the lower respiratory tract. The objective of this study was to characterize the interactions between conidia and bronchial epithelial cells. To this end, we studied the role of bronchial epithelial cells, i.e., the BEAS-2B cell line and human primary cells, in conidial germination of a laboratory strain and three clinical strains of A. fumigatus. Microscopic observations and galactomannan measurements demonstrated that contact between epithelial cells and conidia leads to the inhibition of conidia germination. We demonstrated that this fungistatic process is not associated with the release of any soluble components nor internalization by the epithelial cells. We highlight that this antifungal process involves the phosphoinositide 3-kinase pathway on the host cellular side and the lectin FleA on the fungal side. Collectively, our results show that bronchial epithelial cells attenuate fungal virulence by inhibiting germination of extracellular conidia, thus preventing the morphological change from conidia to filaments, which is responsible for tissue invasion.

Ivacaftor is a drug used to treat cystic fibrosis (CF) patients carrying specific gating CFTR mutations. Interpatient variability in the lung response has been shown to be partly explained by rs7512462 in the Solute Carrier Family 26 Member 9 ( SLC26A9 ) gene. In an independent and larger cohort, we aimed to evaluate whether SLC26A9 variants contribute to the variability of the lung phenotype and if they influence the lung response to ivacaftor. We genotyped the French CF Gene Modifier Study cohort ( n = 4,840) to investigate whether SLC26A9 variants were involved in the lung phenotype heterogeneity. Their influence in the response to ivacaftor was tested in the 30 treated patients who met the inclusion criteria: older than 6 years of age, percent-predicted forced expiratory volume measured in 1 s (FEV 1pp ) in the 3 months before treatment initiation ranging between 40 and 90%. Response to treatment was determined by the change in FEV 1pp from baseline, averaged in 15-75 days, and the 1st-year post-treatment. We observed that SLC26A9 variants were not associated with lung function variability in untreated patients and that gain of lung function in patients treated with ivacaftor was similar to clinical trials. We confirmed that rs7512462 was associated with variability in ivacaftor-lung response, with a significant reduction in lung function improvement for patients with the C allele. Other SLC26A9 SNPs also contributed to the ivacaftor-response. Interindividual variability in lung response to ivacaftor is associated with SLC26A9 variants in French CF patients. Pharmacogenomics and personalized medicine will soon be part of CF patient care.

Pseudomonas aeruginosa ( Pa ) is the leading cause of chronic lung infection in Cystic Fibrosis (CF) patients. It is well recognized that CF epithelial cells fail to develop an appropriate response to infection, allowing bacterial colonization and a chronic inflammatory response. Since long non-coding RNAs (lncRNAs), are known to play a key role in regulating mammalian innate immune response, we hypothesized that CF cells exposed to Pa could express a specific lncRNA signature responsible of the maladaptative CF response. We analyzed transcriptomic datasets to compare the expression profiles of lncRNAs in primary CF and non-CF epithelial cells infected with Pa at 0, 2, 4, and 6 h of infection. Our analysis identified temporal expression signatures of 25, 73, 15, and 26 lncRNA transcripts differentially expressed at 0, 2, 4, and 6 h post-infection respectively, between CF and non-CF cells. In addition, we identified profiles specific to CF and non-CF cells. The differential expression of two candidate lncRNAs were independently validated using real-time PCR. We identified a specific CF signature of lncRNA expression in a context of Pa infection that could potentially play a role in the maladaptive immune response of CF patients.

Background: Lung disease progression is variable among cystic fibrosis (CF) patients and depends on DNA mutations in the CFTR gene, polymorphic variations in disease modifier genes, and environmental exposure. The contribution of genetic factors has been extensively investigated, whereas the mechanism whereby environmental factors modulate the lung disease is unknown. In this project, we hypothesized that (i) reiterative stress alters the epigenome in CF-affected tissues and (ii) DNA methylation variations at disease modifier genes modulate the lung function in CF patients., Results: We profiled DNA methylation at CFTR , the disease-causing gene, and at 13 lung modifier genes in nasal epithelial cells and whole blood samples from 48 CF patients and 24 healthy controls. CF patients homozygous for the p.Phe508del mutation and ≥18-year-old were stratified according to the lung disease severity. DNA methylation was measured by bisulfite and next-generation sequencing. The DNA methylation profile allowed us to correctly classify 75% of the subjects, thus providing a CF-specific molecular signature. Moreover, in CF patients, DNA methylation at specific genes was highly correlated in the same tissue sample. We suggest that gene methylation in CF cells may be co-regulated by disease-specific trans -factors. Three genes were differentially methylated in CF patients compared with controls and/or in groups of pulmonary severity: HMOX1 and GSTM3 in nasal epithelial samples; HMOX1 and EDNRA in blood samples. The association between pulmonary severity and DNA methylation at EDNRA was confirmed in blood samples from an independent set of CF patients. Also, lower DNA methylation levels at GSTM3 were associated with the GSTM3*B allele, a polymorphic 3-bp deletion that has a protective effect in cystic fibrosis., Conclusions: DNA methylation levels are altered in nasal epithelial and blood cell samples from CF patients. Analysis of CFTR and 13 lung disease modifier genes shows DNA methylation changes of small magnitude: some of them are a consequence of the disease; other changes may result in small expression variations that collectively modulate the lung disease severity.

Cystic fibrosis (CF) is the most common life-threatening recessive genetic disease in the Caucasian population. This multiorgan disease is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, a chloride channel recognized as regulating several apical ion channels. The gene mutations result either in the lack of the protein at the apical surface or in an improperly functioning protein. Morbidity and mortality because of the mutation of CFTR are mainly attributable to lung disease resulting from chronic infection and inflammation. Since its discovery as the causative gene in 1989, much progress has been achieved not only in clinical genetics but also in basic science studies. Recently, combinations of these efforts have been successfully translated into development and availability for patients of new therapies targeting specific CFTR mutations to correct the CFTR at the protein level. Current technologies such as next gene sequencing and novel genomic editing tools may offer new strategies to identify new CFTR variants and modifier genes, and to correct CFTR to pursue personalized medicine, which is already developed in some patient subsets. Personalized medicine or P4 medicine ("personalized," "predictive," "preventive," and "participatory") is currently booming for CF. The various current and future challenges of personalized medicine as they apply to the issues faced in CF are discussed in this review., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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

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., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Variants in FAM13A have been found in genome-wide association studies (GWAS) to associate with lung function in the general population as well as in several common chronic lung diseases (CLD) such as chronic obstructive pulmonary disease (COPD), asthma, as well as in idiopathic interstitial pneumonias (IIP). The gene was cloned in 2004, yet the encoded protein has not been characterised and its function is unknown. The redundancy of its genetic contribution in CLD suggests a major function of this gene both in lung physiology and CLD. This review provides a brief summary of the current knowledge of FAM13A, and demonstrates the necessity to resolve its biological function besides its well accepted genetic contribution. Further interpretations of FAM13A variants may help in the understanding of CLD mechanisms and reveal opportunity for intervention., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: 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., Methods: 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., Results: 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; p<0.05), 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., Conclusion: 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.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

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-κB and MAPK pathways through direct interaction with AP-1 and NF-κB 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-κB and MAPK pathways, after stimulation with TNF-α. We demonstrate that the synthetic glucocorticoid dexamethasone (Dex) significantly decreases IL-8 secretion, AP-1 and NF-κB 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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

ABCA3 (ATP-binding cassette subfamily A, member 3) is expressed in the lamellar bodies of alveolar type II cells and is crucial to pulmonary surfactant storage and homeostasis. ABCA3 gene mutations have been associated with neonatal respiratory distress (NRD) and pediatric interstitial lung disease (ILD). The objective of this study was to look for ABCA3 gene mutations in patients with severe NRD and/or ILD. The 30 ABCA3 coding exons were screened in 47 patients with severe NRD and/or ILD. ABCA3 mutations were identified in 10 out of 47 patients, including 2 homozygous, 5 compound heterozygous and 3 heterozygous patients. SP-B and SP-C expression patterns varied across patients. Among patients with ABCA3 mutations, five died shortly after birth and five developed ILD (including one without NRD). Functional studies of p.D253H and p.T1173R mutations revealed that p.D253H and p.T1173R induced abnormal lamellar bodies. Additionally, p.T1173R increased IL-8 secretion in vitro. In conclusion, we identified new ABCA3 mutations in patients with life-threatening NRD and/or ILD. Two mutations associated with ILD acted via different pathophysiological mechanisms despite similar clinical phenotypes.

Cystic fibrosis is a hereditary disease caused by a mutation in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene that encodes a chloride (Cl(-)) channel. Cystic fibrosis pulmonary pathophysiology is characterised by chronic inflammation and bacterial infections. Azithromycin, a macrolide antibiotic, has shown promising anti-inflammatory properties in some inflammatory pulmonary diseases. Moreover, all clinical studies have presented an improvement of the respiratory condition of cystic fibrosis patients, but the molecular and cellular mechanisms remain unknown. The aim of this study was to investigate, in bronchial epithelial cells, the effects of azithromycin on inflammatory pathways involved in cystic fibrosis. We have analysed the effects of azithromycin on cystic fibrosis and non-cystic fibrosis bronchial epithelial cell lines but also in non-immortalized non-cystic fibrosis human glandular cells. To create an inflammatory context, cells were treated with Tumor Necrosis Factor (TNF)-α or Interleukin (IL)1-β. Activation of the NF-κB pathway was investigated by luciferase assay, western blotting, and by Förster Resonance Energy Transfer imaging, allowing the detection of the interaction between the transcription factor and its inhibitor in live cells. In all conditions tested, azithromycin did not have an anti-inflammatory effect on the cystic fibrosis human bronchial epithelial cells and on CFTR-inhibited primary human bronchial glandular cells. More, our data showed no effect of azithromycin on IL-1β- or TNF-α-induced IL-8 secretion and NF-κB pathway activation. Taken together, these data show that azithromycin is unable to decrease in vitro inflammation in cystic fibrosis cells from airways., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: 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., Aims and Methods: 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., Results: 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., Conclusion: 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., (Copyright © 2012 S. Karger AG, Basel.)

There is strong clinical evidence for the effectiveness of macrolides in the treatment of a number of chronic airway diseases through their immunomodulatory effects. Recently, new information has been released supporting the view that macrolides may also be beneficial in pathologic situations associated with altered repair of the alveolar structure, such as those observed in interstitial lung diseases and fibrosis. It is proposed that macrolides may contribute to lung regeneration through their actions on several components of the remodeling process. The present review provides new insights on the effects of macrolides on the regenerative response of alveolar epithelium to injury. It also discusses novel findings which suggest that macrolides may contribute to alveolar surfactant homeostasis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

Study Objectives: To present diagnosis and treatment modalities of children with interstitial lung disease associated with frequent or rare surfactant protein C gene (SFTPC) mutation., Patients: Twenty-two children with chronic lung disease associated with SFTPC mutation in a heterozygous form., Results: Mutations located in the BRICHOS domain ('BRICHOS domain' group) were identified in six children, whereas 16 children carried mutations located outside the BRICHOS domain ('non-BRICHOS domain' group). The median age of onset was 3 (0-24) months. Four patients had neonatal respiratory distress, and symptom onset was associated with acute bronchiolitis in nine patients. Cough, tachypnoea and failure to thrive were initially noticed in all the children. Physical examination at presentation revealed tachypnoea (n=22), clubbing (n=1) and crackles (n=5). Low oxygen saturation (<95%) was observed in 18 patients. The predominant findings on initial high-resolution CT (HRCT) scans were basal-predominant ground-glass opacity (n=21) and cystic spaces (n=3). Bronchoalveolar lavage fluid (BALF) cell counts showed 379+/-56x10(3) cells/ml with increased neutrophil percentage (18+/-4%) independent of the mutation status. The median follow-up was 3.2 (1-18.3) years. Eighteen patients were treated by monthly methylprednisolone pulses associated with oral prednisolone (n=16), hydroxychloroquine (n=11) and/or azithromycin (n=4). Fifteen patients benefited from enteral nutrition., Conclusion: Initial diagnosis is based on clinical presentation, radiological features and BALF analysis, but the definitive diagnosis requires genetic analysis. Although progressive improvement was seen in most patients, the development of new therapeutic strategies with minimal side effects is needed.

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&#95;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"., ((c) 2009 Wiley-Liss, Inc.)

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.

Genetically regulated mechanisms of host defense against Cryptococcus neoformans infection are not well understood. In this study, pulmonary infection with the moderately virulent C. neoformans strain 24067 was used to compare the host resistance phenotype of C57BL/6J with that of inbred mouse strain SJL/J. At 7 days or later after infection, C57BL/6J mice exhibited a significantly greater fungal burden in the lungs than SJL/J mice. Characterization of the pulmonary innate immune response at 3 h after cryptococcal infection revealed that resistant SJL/J mice exhibited significantly higher neutrophilia, with elevated levels of inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and keratinocyte-derived chemokine (KC)/CXCL1 in the airways, as well as increased whole-lung mRNA expression of chemokines KC/CXCL1, MIP-1alpha/CCL3, MIP-1beta/CCL4, MIP-2/CXCL2, and MCP-1/CCL2 and cytokines interleukin 1beta (IL-1beta) and IL-1Ra. At 7 and 14 days after infection, SJL/J mice maintained significantly higher levels of TNF-alpha and KC/CXCL1 in the airways and exhibited a Th1 response characterized by elevated levels of lung gamma interferon (IFN-gamma) and IL-12/IL-23p40, while C57BL/6J mice exhibited Th2 immunity as defined by eosinophilia and IL-4 production. Alveolar and resident peritoneal macrophages from SJL/J mice also secreted significantly greater amounts of TNF-alpha and KC/CXCL1 following in vitro stimulation with C. neoformans. Intracellular signaling analysis demonstrated that TNF-alpha and KC/CXCL1 production was regulated by NF-kappaB and phosphatidylinositol 3 kinase in both strains; however, SJL/J macrophages exhibited heightened and prolonged activation in response to C. neoformans infection compared to that of C57BL/6J. Taken together, these data demonstrate that an enhanced innate immune response against pulmonary C. neoformans infection in SJL/J mice is associated with natural resistance to progressive infection.

Background: Cryptococcus 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., Methods: Immortalized BEAS-2B bronchial epithelial cells and primary normal human bronchial epithelium (NHBE) were stimulated in vitro with encapsulated or acapsular C. neoformans cultivated at room temperature or 37 degrees 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., Results: Viable C. neoformans is 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 acapsular C. neoformans that was grown at 37 degrees C. C. neoformans was also able to induce the expression of the chemokine CXCL1 and the transcription factor CAAT/enhancer-binding protein beta (CEBP/beta) in BEAS-2B cells. NHBE was highly responsive to stimulation with C. 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 of C. neoformans., Conclusion: This study demonstrates that human bronchial epithelial cells mediate an acute inflammatory response to C. neoformans and are susceptible to damage by this fungal pathogen. The presence of capsular polysaccharide and in vitro fungal culture conditions modulate the host inflammatory response to C. neoformans. Human bronchial epithelial cells are likely to contribute to the initial stages of pulmonary host defense in 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.

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 Toll-like 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-alpha and interleukin (IL)-1beta, 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-kappaB 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-beta 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.

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-kappaB signaling pathway and up-regulation of cytokine synthesis such as TNF-alpha 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.