Impact of Cigarette Smoke Exposure on Innate Immunity: A Caenorhabditis elegans Model

BACKGROUND: Cigarette smoking is the major cause of chronic obstructive pulmonary disease (COPD) and lung cancer. Respiratory bacterial infections have been shown to be involved in the development of COPD along with impaired airway innate immunity. METHODOLOGY/PRINCIPAL FINDINGS: To address the in vivo impact of cigarette smoke (CS) exclusively on host innate defense mechanisms, we took advantage of Caenorhabditis elegans (C. elegans), which has an innate immune system but lacks adaptive immune function. Pseudomonas aeruginosa (PA) clearance from intestines of C. elegans was dampened by CS. Microarray analysis identified 6 candidate genes with a 2-fold or greater reduction after CS exposure, that have a human orthologue, and that may participate in innate immunity. To confirm a role of CS-down-regulated genes in the innate immune response to PA, RNA interference (RNAi) by feeding was carried out in C. elegans to inhibit the gene of interest, followed by PA infection to determine if the gene affected innate immunity. Inhibition of lbp-7, which encodes a lipid binding protein, resulted in increased levels of intestinal PA. Primary human bronchial epithelial cells were shown to express mRNA of human Fatty Acid Binding Protein 5 (FABP-5), the human orthologue of lpb-7. Interestingly, FABP-5 mRNA levels from human smokers with COPD were significantly lower (p = 0.036) than those from smokers without COPD. Furthermore, FABP-5 mRNA levels were up-regulated (7-fold) after bacterial (i.e., Mycoplasma pneumoniae) infection in primary human bronchial epithelial cell culture (air-liquid interface culture). CONCLUSIONS: Our results suggest that the C. elegans model offers a novel in vivo approach to specifically study innate immune deficiencies resulting from exposure to cigarette smoke, and that results from the nematode may provide insight into human airway epithelial cell biology and cigarette smoke exposure

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Etude structure/fonction d'une proteine ABC : SUR, le récepteur des sulfonylurées

ATP-sensitive K+ channels (KATP channels) are metabolic sensors formed by the association of an ATP-inhibited inwardlyrectifying K+ channel (Kir6.2) and a regulatory subunit, SUR, of the ABC protein family. SUR adjusts channel gating as afunction of internal ATP and ADP. It is also the target of pharmaceutical KATP channel openers (KCO) or blockers.We have worked on several structure/function relationships of SUR as a model of eucaryotic ABC protein. The naturalassociation of SUR with an ion channel greatly facilitates its study as it allows the use of the patch clamp technique.Pursuing work on the site of action of KCO, we have shown that the molecular selectivity of the SUR isoforms for KCOsresults from the compactness of SUR2A Thr 1253 as opposed to the bulkiness of SUR1 Met1290. This size parameterappears as the most important criteria for the pharmacological activation of KATP channels.Our work then focused on a glutamic acid reach domain of SUR (succession of 15 glutamic or aspartic residues).Extensive mutagenesis of this domain suggested that it is not involved in channel function in our expression system.We tested the intracellular effects of Zn2+ and Cd2+ on KATP channels and showed that these ions can activate the channelsby binding to SUR. This binding site remains to be determined.To examine the respective role of each nucleotide binding domain, we have created hybrid SUR2A proteins withduplicated or switched NBDs (SUR2A NBD1-NBD1, SUR2A NBD2-NBD2 and SUR2A NBD2-NBD1). Besidedemonstrating that these proteins remain functional when co-expressed with Kir6.2, our results suggest that (1) the NBDsare interchangeable (2) ADP activation requires both NBDs (3) KCOs are effective in the absence of NBD2.Le canal KATP résulte de l'assemblage d'un canal potassique inhibé par l'ATP intracellulaire (Kir6.2) et d'un transporteurABC, le récepteur des sulfonylurées (SUR) de la famille MRP/ABCC. SUR a un rôle régulateur essentiel : il confère aucanal une sensibilité accrue à l'inhibition par l'ATP, provoque son activation lorsque l'ADP augmente, et est la cible desactivateurs et bloqueurs pharmacologiques du canal.Nous nous sommes intéressés à divers aspects structure/fonction de SUR en tant que modèle de transporteur ABCeucaryote. Son couplage naturel à un canal ionique en facilite grandement l'étude grâce à la techniqueélectrophysiologique du patch-clamp.La poursuite des travaux pour déterminer la nature moléculaire de la sélectivité des isoformes de SUR aux ouvreurspharmacologiques nous a permis de conclure que seul le faible encombrement de la Thr1253 de SUR2A, contre la Met1290 de SUR1, serait le critère important pour l'activation pharmacologique des canaux KATP.Nos travaux ont ensuite porté sur un domaine de la sous-unité SUR riche en acides aminés chargés négativement(succession de 15 résidus glutamates ou aspartates) qui s'est avéré ne pas être impliquée dans la fonction du canal dansnotre système d'expression.Nous avons étudié l'effet des ions Zn2+ et Cd2+ intracellulaires sur les canaux KATP et montré que ces ions peuvent activerles canaux via leur liaison à SUR. Ce site de liaison reste encore à déterminer.Nous avons enfin essayé de comprendre le rôle de chacun des domaines de liaison des nucléotides et nous avons pour celaconçu des protéines SUR2A possédant des NBD identiques (NBD1-NBD1 et NBD2-NBD2) ou inversés (NBD2-NBD1).Nos résultats suggèrent que (1) les NBD sont interchangeables (2) l'activation pas le Mg-ADP requiert les deux NBD (3)l'action des ouvreurs est indépendante du NBD2

Etude structure/fonction d'une protéine ABC (SUR, le récepteur des sulfonylurées)

Le canal KATP résulte de l'assemblage d'un canal potassique inhibé par l'ATP intracellula ire (Kir6.2) et d'un transporteur ABC, le récepteur des sulfonylurées (SUR) de la famille MRP/ABCC. SUR a un rôle régulateur essentiel : il confère au canal une sensibilité accrue à l'inhibition par l'ATP, provoque son activation lorsque l'ADP augmente, et est la cible des activateurs et bloqueurs pharmacologiques du canal. Nous nous sommes intéressés à divers aspects structure/fonction de SUR en tant que modèle de transporteur ABC eucaryote. Son couplage naturel à un canal ionique en facilité gradement l'étude gra ce à la technique électrophysiologique du patch-clamp. La poursuite des travaux pour déterminer la nature moléculaire de la sélectivité des isoformes de SUR aux ouvreurs pharmacologiques nous a permis de conclure que seul le faible encombrement de la Thr1253 de SUR2A, contre la Met 1290 de SURI, serait le critère important pour l'activation pharmacologique des canaux KATP. Nos travaux ont ensuite porté sur un domaine de la sous-unité SUR riche en acides aminés chargés négativement (succession de 15 résidus glutamates ou aspartates) qui s'est avéré ne pas être impliquée dans la fonction du canal dans notre système d'expression. Nous avons étudié l'effet des ions Zn2+ et Cd2+ intracellulaires sur les canaux KATP et montré que ces ions peuvent activer les canaux via leur liaison à SUR. Ce site de liaison reste encore à déterminer. Nous avons enfin essayé de comprendre le rôle de chacun des domaines de liaison des nucléotides et nous avons pour cela conçu des protéines SUR2A possédant des NBD identiques (NBDl-NBDI et NBD2-NBD2) ou inversés (NBD2-NBDl). Nos résultats suggèrent que (1) les NBD sont interchangeables (2) l'activation pas le Mg-ADP requiert les deux NBD (3) l'action des ouvreurs est indépendante du NBD2.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Cigarette smoke inhibits LPS-induced FABP5 expression by preventing c-Jun binding to the FABP5 promoter.

Cigarette smoking is the primary cause of chronic obstructive pulmonary disease (COPD) with repeated and sustained infections linked to disease pathogenesis and exacerbations. The airway epithelium constitutes the first line of host defense against infection and is known to be impaired in COPD. We have previously identified Fatty Acid Binding Protein 5 (FABP5) as an important anti-inflammatory player during respiratory infections and showed that overexpression of FABP5 in primary airway epithelial cells protects against bacterial infection and inflammation. While cigarette smoke down regulates FABP5 expression, its mechanism remains unknown. In this report, we have identified three putative c-Jun binding sites on the FABP5 promoter and show that cigarette smoke inhibits the binding of c-Jun to its consensus sequence and prevents LPS-induced FABP5 expression. Using chromatin immunoprecipitation, we have determined that c-Jun binds the FABP5 promoter when stimulated with LPS but the presence of cigarette smoke greatly reduces this binding. Furthermore, cigarette smoke or a mutation in the c-Jun binding site inhibits LPS-induced FABP5 promoter activity. These data demonstrate that cigarette smoke interferes with FABP5 expression in response to bacterial infection. Thus, functional activation of FABP5 may be a new therapeutic strategy when treating COPD patients suffering from exacerbations

Immunomodulatory functions of FABP5 in primary NHBE cells.

<p>FABP5 exerts host defense and anti-inflammatory functions against <i>P. aeruginosa</i> bacterial infection indirectly by stimulating PPAR-γ activity. PPAR-γ activity increases β defensin-2 expression thus preventing bacterial growth and inhibits inflammatory cytokine (e.g., IL-8) production.</p

CS exposure prevents bacteria-induced FABP5 expression.

<p><b>A.</b> FABP5 mRNA expression is increased post <i>P. aeruginosa</i> infection, however CS exposure decreases FABP5 mRNA expression and prevents bacteria-induced FABP5 mRNA expression. <b>B.</b> Representative Western blot detection of FABP5 and β-actin proteins shows that FABP5 protein levels increased post Pa infection but CS exposure decreases Pa-induced FABP5 protein levels. Data are representative of 3 independent experiments and are expressed as mean ± SEM.</p

FABP5 down regulation increases inflammation but decreases innate immunity in primary NHBE cells.

<p><b>A.</b> CS exposure increases <i>P. aeruginosa</i> colony forming units (CFU) on FABP5 knocked down primary NHBE cells. <b>B.</b> CS exposure significantly enhances IL-8 secretion in the basolateral supernatant of primary NHBE cell cultures knocked down for FABP5. <b>C.</b> CS exposure decreases β-defensin 2 mRNA expression on FABP5 knocked down primary NHBE cells. FABP5 shRNA − indicates cells that were knocked down for Firefly luciferase and FABP5 shRNA + indicates cells that were knocked down for FABP5. Data are representative of 3 independent experiments and are expressed as mean ± SEM.</p