Philippe Joubert

Molecular Signature of Smoking in Human Lung Tissues

Cigarette smoking is the leading risk factor for lung cancer. To identify genes deregulated by smoking and to distinguish gene expression changes that are reversible and persistent following smoking cessation, we carried out genome-wide gene expression profiling on nontumor lung tissue from 853 patients with lung cancer. Gene expression levels were compared between never and current smokers, and time-dependent changes in gene expression were studied in former smokers. A total of 3,223 transcripts were differentially expressed between smoking groups in the discovery set (n = 344, P < 1.29 × 10(-6)). A substantial number of smoking-induced genes also were validated in two replication sets (n = 285 and 224), and a gene expression signature of 599 transcripts consistently segregated never from current smokers across all three sets. The expression of the majority of these genes reverted to never-smoker levels following smoking cessation, although the time course of normalization differed widely among transcripts. Moreover, some genes showed very slow or no reversibility in expression, including SERPIND1, which was found to be the most consistent gene permanently altered by smoking in the three sets. Our findings therefore indicate that smoking deregulates many genes, many of which reverse to normal following smoking cessation. However, a subset of genes remains altered even decades following smoking cessation and may account, at least in part, for the residual risk of lung cancer among former smokers. Cancer Res; 72(15); 3753-63. ©2012 AACR.

Oseltamivir-Resistant Pandemic A/H1N1 Virus Is as Virulent as Its Wild-Type Counterpart in Mice and Ferrets

The neuraminidase inhibitor oseltamivir is currently used for treatment of patients infected with the pandemic A/H1N1 (pH1N1) influenza virus, although drug-resistant mutants can emerge rapidly and possibly be transmitted. We describe the characteristics of a pair of oseltamivir-resistant and oseltamivir-susceptible pH1N1 clinical isolates that differed by a single change (H274Y) in the neuraminidase protein. Viral fitness of pH1N1 isolates was assessed in vitro by determining replication kinetics in MDCK α2,6 cells and in vivo by performing experimental infections of BALB/c mice and ferrets. Despite slightly reduced propagation of the mutant isolate in vitro during the first 24 h, the wild-type (WT) and mutant resistant viruses induced similar maximum weight loss in mice and ferrets with an identical pyrexic response in ferrets (AUC of 233.9 and 233.2, P = 0.5156). Similarly, comparable titers were obtained for the WT and the mutant strains on days 1, 3, 6 and 9 post-infection in mouse lungs and on days 1–7 in ferret nasal washes. A more important perivascular (day 6) and pleural (days 6 and 12) inflammation was noted in the lungs of mice infected with the H274Y mutant, which correlated with increased pulmonary levels of IL-6 and KC. Such increased levels of IL-6 were also observed in lymph nodes of ferrets infected with the mutant strain. Furthermore, the H274Y mutant strain was transmitted to ferrets. In conclusion, viral fitness of the H274Y pH1N1 isolate is not substantially altered and has the potential to induce severe disease and to disseminate.

Targeted Prostaglandin E2 Inhibition Enhances Antiviral Immunity through Induction of Type I Interferon and Apoptosis in Macrophages

Aspirin gained tremendous popularity during the 1918 Spanish Influenza virus pandemic, 50 years prior to the demonstration of their inhibitory action on prostaglandins. Here, we show that during influenza A virus (IAV) infection, prostaglandin E2 (PGE2) was upregulated, which led to the inhibition of type I interferon (IFN) production and apoptosis in macrophages, thereby causing an increase in virus replication. This inhibitory role of PGE2 was not limited to innate immunity, because both antigen presentation and T cell mediated immunity were also suppressed. Targeted PGE2 suppression via genetic ablation of microsomal prostaglandin E-synthase 1 (mPGES-1) or by the pharmacological inhibition of PGE2 receptors EP2 and EP4 substantially improved survival against lethal IAV infection whereas PGE2 administration reversed this phenotype. These data demonstrate that the mPGES-1-PGE2 pathway is targeted by IAV to evade host type I IFN-dependent antiviral immunity. We propose that specific inhibition of PGE2 signaling might serve as a treatment for IAV.

Downregulation of MicroRNA-126 Contributes to the Failing Right Ventricle in Pulmonary Arterial Hypertension.

Background— Right ventricular (RV) failure is the most important factor of both morbidity and mortality in pulmonary arterial hypertension (PAH). However, the underlying mechanisms resulting in the failed RV in PAH remain unknown. There is growing evidence that angiogenesis and microRNAs are involved in PAH-associated RV failure. We hypothesized that microRNA-126 (miR-126) downregulation decreases microvessel density and promotes the transition from a compensated to a decompensated RV in PAH. Methods and Results— We studied RV free wall tissues from humans with normal RV (n=17), those with compensated RV hypertrophy (n=8), and patients with PAH with decompensated RV failure (n=14). Compared with RV tissues from patients with compensated RV hypertrophy, patients with decompensated RV failure had decreased miR-126 expression (quantitative reverse transcription–polymerase chain reaction; P<0.01) and capillary density (CD31+ immunofluorescence; P<0.001), whereas left ventricular tissues were not affected. miR-126 downregulation was associated with increased Sprouty-related EVH1 domain-containing protein 1 (SPRED-1), leading to decreased activation of RAF (phosphorylated RAF/RAF) and mitogen-activated protein kinase (MAPK); (phosphorylated MAPK/MAPK), thus inhibiting the vascular endothelial growth factor pathway. In vitro, Matrigel assay showed that miR-126 upregulation increased angiogenesis of primary cultured endothelial cells from patients with decompensated RV failure. Furthermore, in vivo miR-126 upregulation (mimic intravenous injection) improved cardiac vascular density and function of monocrotaline-induced PAH animals. Conclusions— RV failure in PAH is associated with a specific molecular signature within the RV, contributing to a decrease in RV vascular density and promoting the progression to RV failure. More importantly, miR-126 upregulation in the RV improves microvessel density and RV function in experimental PAH.

Effects of Bronchial Thermoplasty on Airway Smooth Muscle and Collagen Deposition in Asthma

RATIONALE The aim of bronchial thermoplasty is to improve asthma symptoms by reducing central airway smooth muscle mass. Up to now, the reduction of smooth muscle mass has been documented for only 1 group of 10 patients who had 15% or more of their pretreatment total bronchial biopsy area occupied by smooth muscle. OBJECTIVES To evaluate the effects of bronchial thermoplasty on airway smooth muscle mass and airway collagen deposition in adult patients with asthma, regardless of pretreatment smooth muscle area. METHODS Seventeen patients with asthma underwent bronchial thermoplasty over the course of three visits. At Visit 1, bronchial biopsies were taken from the lower lobe that was not treated during this session. At Visit 2 (3-14 wk after the first visit), all 17 patients underwent biopsy of the lower lobe treated during the first procedure. At Visit 3 (7-22 wk after the first visit), nine patients agreed to undergo biopsy of the same lower lobe. Histological and immunohistochemical analyses were performed on the biopsy specimens. MEASUREMENTS AND MAIN RESULTS Bronchial thermoplasty decreased airway smooth muscle from 12.9 ± 1.2% of the total biopsy surface at Visit 1 to 4.6 ± 0.8% at Visit 2 (P < 0.0001). For the nine patients who underwent a third biopsy, mean airway smooth muscle area was 5.3 ± 1.3% at Visit 3 (P = 0.0008 compared with baseline). Bronchial thermoplasty also decreased Type I collagen deposition underneath the basement membrane from 6.8 ± 0.3 μm at Visit 1 to 4.3 ± 0.2 μm at Visit 2 (P < 0.0001) and to 4.4 ± 0.4 μm for nine patients at Visit 3 (P < 0.0001 compared with baseline). Over the course of 1 year after treatment, the doses of inhaled corticosteroid, the number of severe exacerbations, and asthma control all improved (P ≤ 0.02). CONCLUSIONS For patients with severe asthma, bronchial thermoplasty reduced the smooth muscle mass of treated airway segments, regardless of the baseline level of muscle mass. Treatment also altered the deposition of collagen. At follow-up, bronchial thermoplasty improved asthma control; however, the limited number of subjects did not allow us to evaluate possible correlations between these improvements and the studied histological parameters. Further studies are needed to confirm these results and evaluate their persistence.

Corticosteroids and Antigen Avoidance Decrease Airway Smooth Muscle Mass in an Equine Asthma Model

Recent studies suggest that airway smooth muscle remodeling is an early event in the course of asthma. Little is known of the effects of long-term antigen avoidance and inhaled corticosteroids on chronically established airway remodeling. We sought to measure the effects of inhaled corticosteroids and antigen avoidance on airway remodeling in the peripheral airways of horses with heaves, a naturally occurring asthma-like disease. Heaves-affected adult horses with ongoing airway inflammation and bronchoconstriction were treated with fluticasone propionate (with and without concurrent antigen avoidance) (n = 6) or with antigen avoidance alone (n = 5). Lung function and bronchoalveolar lavage were performed at multiple time points, and peripheral lung biopsies were collected before and after 6 and 12 months of treatment. Lung function improved more quickly with inhaled corticosteroids, but eventually normalized in both groups. Inflammation was better controlled with antigen avoidance. During the study period, corrected smooth muscle mass decreased from 12.1 ± 2.8 × 10(-3) and 11.3 ± 1.2 × 10(-3) to 8.3 ± 1.4 × 10(-3) and 7.9 ± 1.0 × 10(-3) in the antigen avoidance and fluticasone groups, respectively (P = 0.03). At 6 months, smooth muscle mass was significantly smaller compared with baseline only in the fluticasone-treated animals. The subepithelial collagen area was lower at 12 months than at baseline in both groups. During the study period, airway smooth muscle remodeling decreased by approximately 30% in both groups, although the decrease was faster in horses receiving inhaled corticosteroids. Inhaled corticosteroids may accelerate the reversal of smooth muscle remodeling, even if airway inflammation is better controlled with antigen avoidance.

The 2009 Pandemic H1N1 D222G Hemagglutinin Mutation Alters Receptor Specificity and Increases Virulence in Mice but Not in Ferrets

BACKGROUND The D222G (H1 numbering) hemagglutinin (HA) mutation within the receptor-binding site was detected with higher frequencies in severe cases of 2009 pandemic H1N1 (pH1N1) infections. We investigated the impact of this mutation in vitro and in animal models using recombinant pH1N1 viruses. METHODS The recombinant D222G HA mutant was generated from a wild-type (WT) clinical strain by using reverse genetics and site-directed mutagenesis. Replicative capacities were determined in MDCK and MDCK-α2,6 cells. Antigenicity was characterized by HA inhibition and microneutralization assays. HA titers were determined using human, chicken, and resialylated turkey red blood cells (RBCs). Virulence and contact-transmissibility were analyzed in mice and ferrets. RESULTS The recombinant D222G virus grew to significantly higher titers and generated larger viral plaques compared with the WT in MDCK but not in MDCK-α2,6 cells. The mutant also showed a significant reduction in HA titers using α2,6-expressing RBCs. The 2 recombinants were antigenically similar. The D222G mutant virus induced higher lung viral titers and alveolar inflammation in mice whereas the 2 recombinants had similar impacts in ferrets. CONCLUSIONS The D222G HA mutation alters receptor binding specificity, resulting in higher lung titers in mice. This could contribute to the higher case fatality rates reported in humans.

Correlation between CCL26 production by human bronchial epithelial cells and airway eosinophils: Involvement in patients with severe eosinophilic asthma

BACKGROUND High pulmonary eosinophil counts are associated with asthma symptoms and severity. Bronchial epithelial cells (BECs) produce CC chemokines, notably CCL26 (eotaxin-3), which recruits and activates eosinophils from asthmatic patients. This suggests that CCL26 production by BECs might be involved in persistent eosinophilia in patients with severe asthma despite treatment with high corticosteroid doses. OBJECTIVE We sought to determine whether CCL26 levels correlate with eosinophilia and asthma severity. METHODS Human CC chemokine expression was assessed by means of quantitative PCR or a quantitative PCR array in vehicle- or IL-13-treated BECs. CCL26 was quantitated by means of ELISA. Immunohistochemistry analyses of CCL26 and major basic protein were done on bronchial biopsy specimens. RESULTS IL-13 selectively induced CCL26 expression by BECs. This increase was time-dependent and more prominent in BECs from patients with severe eosinophilic asthma. CCL26 levels measured in supernatants of IL-13-stimulated BECs also increased with asthma severity as follows: patients with severe eosinophilic asthma > patients with mild asthma ≈ healthy subjects. Immunohistochemistry analyses of bronchial biopsy specimens confirmed increased levels of CCL26 in the epithelium of patients with mild and those with severe eosinophilic asthma. Tissue eosinophil counts did not correlate with CCL26 staining. However, sputum CCL26 levels significantly correlated with sputum eosinophil counts (P < .0001), suggesting that CCL26 participates in the movement of eosinophils from the tissues to the airway lumen. CONCLUSIONS These results show a relation between CCL26 production by IL-13-stimulated BECs, sputum eosinophil counts, and asthma severity. They also suggest a role for CCL26 in the sustained inflammation observed in patients with severe eosinophilic asthma and reveal CCL26 as a potential target for treating patients with eosinophilic asthma that are refractory to classic therapies.

Sex-related discordance between aortic valve calcification and hemodynamic severity of aortic stenosis : is valvular fibrosis the explanation?

Rationale: Calcific aortic stenosis (AS) is characterized by calcium deposition in valve leaflets. However, women present lower aortic valve calcification loads than men for the same AS hemodynamic severity. Objective: We, thus, aimed to assess sex differences in aortic valve fibrocalcific remodeling. Methods and Results: One hundred and twenty-five patients underwent Doppler echocardiography and multidetector computed tomography within 3 months before aortic valve replacement. Explanted stenotic tricuspid aortic valves were weighed, and fibrosis degree was determined. Sixty-four men and 39 women were frequency matched for age, body mass index, hypertension, renal disease, diabetes mellitus, and AS severity. Mean age (75±9 years), mean gradient (41±18 mm Hg), and indexed aortic valve area (0.41±0.12 cm2/m2) were similar between men and women (all P≥0.18). Median aortic valve calcification (1973 [1124–3490] Agatston units) and mean valve weight (2.36±0.99 g) were lower in women compared with men (both P<0.0001). Aortic valve calcification density correlated better with valve weight in men (r2=0.57; P<0.0001) than in women (r2=0.26; P=0.0008). After adjustment for age, body mass index, aortic valve calcification density, and aortic annulus diameter, female sex was an independent risk factor for higher fibrosis score in AS valves (P=0.003). Picrosirius red staining of explanted valves showed greater amount of collagen fibers (P=0.01), and Masson trichrome staining revealed a greater proportion of dense connective tissue (P=0.02) in women compared with men. Conclusions: In this series of patients with tricuspid aortic valve and similar AS severity, women have less valvular calcification but more fibrosis compared with men. These findings suggest that the pathophysiology of AS and thus potential targets for drug development may be different according to sex.

Functional characterization of the Ucp1 -associated oxidative phenotype of human epicardial adipose tissue

Brown fat presence and metabolic activity has been associated with lower body mass index, higher insulin sensitivity and better cardiometabolic profile in humans. We, and others, have previously reported the presence of Ucp1, a marker of brown adipocytes, in human epicardial adipose tissue (eAT). Characterization of the metabolic activity and associated physiological relevance of Ucp1 within eAT, however, is still awaited. Here, we validate the presence of Ucp1 within human eAT and its ‘beige’ nature. Using in-vitro analytical approaches, we further characterize its thermogenic potential and demonstrate that human eAT is capable of undergoing enhanced uncoupling respiration upon stimulation. Direct biopsy gene expression analysis reveals a negative association between thermogenic markers and oxidative stress-related genes in this depot. Consistently, isoproterenol (Iso) stimulation of eAT leads to a downregulation of secreted proteins included in the GO terms ‘cell redox homeostasis’ and ‘protein folding’. In addition, cardiac endothelial cells exhibit a downregulation in the expression of adhesion markers upon treatment with Iso-stimulated eAT derived conditioned media. Overall, these observations suggest that Ucp1- associated metabolic activity plays a significant role in local tissue homeostasis within eAT and can plausibly alter its communication with neighboring cells of the cardiovascular system.