High-dose steroid therapy has been proven effective in AIDS-related Pneumocystis pneumonia (PCP) but not in non-AIDS-related cases. We evaluated the effects on survival of steroids in HIV-negative patients with PCP.
Retrospective study patients admitted to the ICU with hypoxemic PCP. We compared patients receiving HDS (≥1 mg/Kg/day prednisone equivalent), low-dose steroids (LDS group, <1 mg/Kg/day prednisone equivalent), and no steroids (NS group). Variables independently associated with ICU mortality were identified.
139 HIV-negative patients with PCP were included. Median age was 48 [40–60] years. The main underlying conditions were hematological malignancies (n=55, 39.6%), cancer (n=11, 7.9%), and solid organ transplantation (n=73, 52.2%). ICU mortality was 26% (36 deaths). The HDS group had 72 (51.8%) patients, the LDS group 35 (25%) patients, and the NS group 32 (23%) patients. Independent predictors of ICU mortality were SAPS II at ICU admission (odds ratio [OR], 1.04/point; [95%CI], 1.01-1.08, P=0.01), non-hematological disease (OR, 4.06; [95%CI], 1.19-13.09, P=0.03), vasopressor use (OR, 20.31; 95%CI, 6.45-63.9, P<0.001), and HDS (OR, 9.33; 95%CI, 1.97-44.3, P=0.02). HDS was not associated with the rate of ICU-acquired infections.
HDS were associated with increased mortality in HIV-negative patients with PCP via a mechanism independent from an increased risk of infection.
Pneumocystis jiroveci infection; Immunocompromised host; Mortality
Idiopathic pulmonary fibrosis (IPF) is a chronic, fibrosing interstitial lung disease that primarily affects older adults. Median survival after diagnosis is 2–3 years. The clinical course of IPF may include periods of acute deterioration in respiratory function, which are termed acute exacerbations of IPF (AEx-IPF) when a cause cannot be identified. AEx-IPF may represent a sudden acceleration of the underlying disease process of IPF, or a biologically distinct pathological process that is clinically undiagnosed. An AEx-IPF can occur at any time during the course of IPF and may be the presenting manifestation. The incidence of AEx-IPF is hard to establish due to variation in the methodology used to assess AEx-IPF in different studies, but AEx-IPF are believed to occur in between 5 and 10% of patients with IPF every year. Risk factors for AEx-IPF are unclear, but there is evidence that poorer lung function increases the risk of an AEx-IPF and reduces the chances of a patient surviving an AEx-IPF. The presence of comorbidities such as gastroesophageal reflux disease (GERD) and pulmonary hypertension may also increase the risk of an AEx-IPF. AEx-IPF are associated with high morbidity and mortality. Patients who experience an AEx-IPF show a worsened prognosis and AEx-IPF are believed to reflect disease progression in IPF. Current treatments for AEx-IPF have only limited data to support their effectiveness. The latest international treatment guidelines state that supportive care remains the mainstay of treatment for AEx-IPF, but also give a weak recommendation for the treatment of the majority of patients with AEx-IPF with corticosteroids. There is emerging evidence from clinical trials of investigational therapies that chronic treatment of IPF may reduce the incidence of AEx-IPF. Additional clinical trials investigating this are underway.
Acute exacerbation; Idiopathic pulmonary fibrosis (IPF); Impact; Management; Prevention; Treatment
The carcinoembryonic antigen (CEA)-related cell adhesion molecules CEACAM1 (BGP, CD66a), CEACAM5 (CEA, CD66e) and CEACAM6 (NCA, CD66c) are expressed in human lung. They play a role in innate and adaptive immunity and are targets for various bacterial and viral adhesins. Two pathogens that colonize the normally sterile lower respiratory tract in patients with chronic obstructive pulmonary disease (COPD) are non-typable Haemophilus influenzae (NTHI) and Moraxella catarrhalis. Both pathogens bind to CEACAMs and elicit a variety of cellular reactions, including bacterial internalization, cell adhesion and apoptosis.
To analyze the (co-) expression of CEACAM1, CEACAM5 and CEACAM6 in different lung tissues with respect to COPD, smoking status and granulocyte infiltration, immunohistochemically stained paraffin sections of 19 donors were studied. To address short-term effects of cigarette smoke and acute inflammation, transcriptional regulation of CEACAM5, CEACAM6 and different CEACAM1 isoforms by cigarette smoke extract, interferons, Toll-like receptor agonists, and bacteria was tested in normal human bronchial epithelial (NHBE) cells by quantitative PCR. Corresponding CEACAM protein levels were determined by flow cytometry.
Immunohistochemical analysis of lung sections showed the most frequent and intense staining for CEACAM1, CEACAM5 and CEACAM6 in bronchial and alveolar epithelium, but revealed no significant differences in connection with COPD, smoking status and granulocyte infiltration. In NHBE cells, mRNA expression of CEACAM1 isoforms CEACAM1-4L, CEACAM1-4S, CEACAM1-3L and CEACAM1-3S were up-regulated by interferons alpha, beta and gamma, as well as the TLR3 agonist polyinosinic:polycytidylic acid (poly I:C). Interferon-gamma also increased CEACAM5 expression. These results were confirmed on protein level by FACS analysis. Importantly, also NTHI and M. catarrhalis increased CEACAM1 mRNA levels. This effect was independent of the ability to bind to CEACAM1. The expression of CEACAM6 was not affected by any treatment or bacterial infection.
While we did not find a direct correlation between CEACAM1 expression and COPD, the COPD-associated bacteria NTHi and M. catarrhalis were able to increase the expression of their own receptor on host cells. Further, the data suggest a role for CEACAM1 and CEACAM5 in the phenomenon of increased host susceptibility to bacterial infection upon viral challenge in the human respiratory tract.
CEACAM1; CEACAM5 (CEA); CEACAM6; Lung; Normal human bronchial epithelial (NHBE) cells; Interferon; Polyinosinic:polycytidylic acid (poly I:C); Non-typable Haemophilus influenzae (NTHI); Moraxella catarrhalis
Chronic obstructive pulmonary disease (COPD) is characterized by structural changes in alveoli and airways. Our aim was to analyse the numbers of alpha-smooth muscle actin (α-SMA) positive cells, as a marker of myofibroblasts, in different lung compartments in non-smokers and smokers with normal lung function or COPD.
α-SMA, tenascin-C (Tn-C) and EDA-fibronectin in alveolar level and airways were assayed by immunohistochemistry and quantified by image analysis. Immunohistochemical findings were correlated with clinical data. α-SMA protein was also analysed by Western blotting from fibroblastic cells cultured from peripheral lung of non-smokers, smokers without COPD and smokers with COPD.
In many cases, the endings of the detached alveolar walls were widened, the structures of which were named as widened alveolar tips. Widened alveolar tips contained α-SMA positive cells, which were obviously myofibroblasts. There were less alveolar tips containing positive cells for α-SMA in alveoli and α-SMA positive cells in bronchioles in smokers and in COPD compared to non-smokers. The quantity of α-SMA positive cells was increased in bronchi in COPD. Tn-C was elevated in bronchi in COPD and smokers’ lung. The α-SMA protein level was 1.43-fold higher in stromal cells cultured from non-smokers than in those of smokers.
Myofibroblasts are localized variably in normal and diseased lung. This indicates that they have roles in both regeneration of lung and pathogenesis of COPD. The widened alveolar tips, these newly characterized histological structures, seemed to be the source of myofibroblasts at the alveolar level.
Bronchiolus; Bronchus; Emphysema; Electron microscopy; Fibroblast; Western blotting
Lung growth in utero and lung function loss during adulthood can be affected by exposure to environmental tobacco smoke (ETS). The underlying mechanisms have not been fully elucidated. Both ETS exposure and single nucleotide polymorphisms (SNPs) in Glutathione S-Transferase (GST) Omega genes have been associated with the level of lung function. This study aimed to assess if GSTO SNPs interact with ETS exposure in utero and during adulthood on the level of lung function during adulthood.
We used cross-sectional data of 8,128 genotyped participants from the LifeLines cohort study. Linear regression models (adjusted for age, sex, height, weight, current smoking, ex-smoking and packyears smoked) were used to analyze the associations between in utero, daily and workplace ETS exposure, GSTO SNPs, the interaction between ETS and GSTOs, and level of lung function (FEV1, FEV1/FVC). Since the interactions between ETS and GSTOs may be modified by active tobacco smoking we additionally assessed associations in never and ever smokers separately. A second sample of 5,308 genotyped LifeLines participants was used to verify our initial findings.
Daily and workplace ETS exposure was associated with significantly lower FEV1 levels. GSTO SNPs (recessive model) interacted with in utero ETS and were associated with higher levels of FEV1, whereas the interactions with daily and workplace ETS exposure were associated with lower levels of FEV1, effects being more pronounced in never smokers. The interaction of GSTO2 SNP rs156697 with in utero ETS associated with a higher level of FEV1 was significantly replicated in the second sample. Overall, the directions of the interactions of in utero and workplace ETS exposure with the SNPs found in the second (verification) sample were in line with the first sample.
GSTO genotypes interact with in utero and adulthood ETS exposure on adult lung function level, but in opposite directions.
Genes; Environmental tobacco smoke; Lung function
The redox-active pyocyanin (PCN) is a toxic, secondary metabolite secreted by the respiratory pathogen Pseudomonas aeruginosa (PA). Previously, we have shown that mouse lungs chronically exposed to PCN develop goblet cell hyperplasia and metaplasia (GCHM) and mucus hypersecretion, fibrosis and emphysema. These pathological features are commonly found in the airways of several chronic lung diseases, including cystic fibrosis (CF), as well as in mouse airways deficient in the forkhead box A2 (FOXA2), a transcriptional repressor of goblet GCHM and mucus biosynthesis. Furthermore, PCN inhibits FOXA2 by activating the pro-GCHM signaling pathways Stat6 and EGFR. However, it is not known whether PCN-generated reactive oxygen (ROS) and nitrogen (RNS) species posttranslationally modify and inactivate FOXA2.
We examined the posttranslational modifications of FOXA2 by PCN using specific antibodies against oxidation, nitrosylation, acetylation and ubiquitination. Electrophoretic mobility shift assay (EMSA) was used to examine the ability of modified FOXA2 to bind the promoter of MUC5B mucin gene. In addition, we used quantitative real time PCR, ELISA, immunofluorescence and mouse lung infection to assess whether the loss of FOXA2 function caused GCHM and mucin overexpression. Finally, we examined the restoration of FOXA2 function by the antioxidant glutathione (GSH).
We found that PCN-generated ROS/RNS caused nitrosylation, acetylation, ubiquitination and degradation of FOXA2. Modified FOXA2 had reduced ability to bind the promoter of the MUC5B gene. The antioxidant GSH alleviated the modification of FOXA2 by PCN, and inhibited the overexpression of MUC5AC and MUC5B mucins.
These results suggest that PCN-mediated posttranslational modifications of FOXA2 are positively correlated with GCHM and overexpression of airway mucins. Furthermore, antioxidant treatment restores the function of FOXA2 to attenuate GCHM and mucus hypersecretion.
Pyocyanin; Reactive oxygen and nitrogen species; Posttranslational modifications; FOXA2; Mucus hypersecretion
Interstitial lung disease is a major cause of morbidity and mortality in systemic sclerosis (SSc), with insufficiently effective treatment options. Progression of pulmonary fibrosis involves expanding populations of fibroblasts, and the accumulation of extracellular matrix proteins. Characterisation of SSc lung fibroblast gene expression profiles underlying the fibrotic cell phenotype could enable a better understanding of the processes leading to the progressive build-up of scar tissue in the lungs. In this study we evaluate the transcriptomes of fibroblasts isolated from SSc lung biopsies at the time of diagnosis, compared with those from control lungs.
We used Affymetrix oligonucleotide microarrays to compare the gene expression profile of pulmonary fibroblasts cultured from 8 patients with pulmonary fibrosis associated with SSc (SSc-ILD), with those from control lung tissue peripheral to resected cancer (n=10). Fibroblast cultures from 3 patients with idiopathic pulmonary fibrosis (IPF) were included as a further comparison. Genes differentially expressed were identified using two separate analysis programs following a set of pre-determined criteria: only genes significant in both analyses were considered. Microarray expression data was verified by qRT-PCR and/or western blot analysis.
A total of 843 genes were identified as differentially expressed in pulmonary fibroblasts from SSc-ILD and/or IPF compared to control lung, with a large overlap in the expression profiles of both diseases. We observed increased expression of a TGF-β response signature including fibrosis associated genes and myofibroblast markers, with marked heterogeneity across samples. Strongly suppressed expression of interferon stimulated genes, including antiviral, chemokine, and MHC class 1 genes, was uniformly observed in fibrotic fibroblasts. This expression profile includes key regulators and mediators of the interferon response, such as STAT1, and CXCL10, and was also independent of disease group.
This study identified a strongly suppressed interferon-stimulated gene program in fibroblasts from fibrotic lung. The data suggests that the repressed expression of interferon-stimulated genes may underpin critical aspects of the profibrotic fibroblast phenotype, identifying an area in pulmonary fibrosis that requires further investigation.
SSc-ILD; IPF; Pulmonary fibroblasts; Interferon regulated genes
Patients with chronic obstructive pulmonary disease (COPD) can be categorized as having frequent (FE) or infrequent (IE) exacerbations depending on whether they respectively experience two or more, or one or zero exacerbations per year. Although most patients do not change category from year to year, some will, and the factors associated with this behaviour have not been examined.
1832 patients completing two year follow-up in the Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points (ECLIPSE) study were examined at baseline and then yearly. Exacerbations were defined by health care utilisation. Patient characteristics compared between those patients who did or did not change exacerbation category from year 1 to year 2.
Between years 1 and 2, 221 patients (17%) changed from IE to FE and 210 patients (39%) from FE to IE. More severe disease was associated with changing from IE to FE and less severe disease from FE to IE. Over the preceding year, small falls in FEV1 and 6-minute walking distance were associated with changing from IE to FE, and small falls in platelet count associated with changing from FE to IE.
No parameter clearly predicts an imminent change in exacerbation frequency category.
SCO104960, clinicaltrials.gov identifier NCT00292552
COPD; Exacerbations; Exacerbation frequency; Exacerbation phenotype
Aspergillus fumigatus conidia can exacerbate asthma symptoms. Phagocytosis of conidia is a principal component of the host antifungal defense. We investigated whether allergic airway inflammation (AAI) affects the ability of phagocytic cells in the airways to internalize the resting fungal spores.
Using BALB/c mice with experimentally induced AAI, we tested the ability of neutrophils, macrophages, and dendritic cells to internalize A. fumigatus conidia at various anatomical locations. We used light microscopy and differential cell and conidium counts to determine the ingestion potential of neutrophils and macrophages present in bronchoalveolar lavage (BAL). To identify phagocyte-conidia interactions in conducting airways, conidia labeled with tetramethylrhodamine-(5-(and-6))-isothiocyanate were administered to the oropharyngeal cavity of mice. Confocal microscopy was used to quantify the ingestion potential of Ly-6G+ neutrophils and MHC II+ antigen-presenting cells located in the intraepithelial and subepithelial areas of conducting airways.
Allergen challenge induced transient neutrophil recruitment to the airways. Application of A. fumigatus conidia at the acute phase of AAI provoked recurrent neutrophil infiltration, and consequently increased the number and the ingestion potential of the airway neutrophils. In the absence of recurrent allergen or conidia provocation, both the ingestion potential and the number of BAL neutrophils decreased. As a result, conidia were primarily internalized by alveolar macrophages in both AAI and control mice at 24 hours post-inhalation. Transient influx of neutrophils to conducting airways shortly after conidial application was observed in mice with AAI. In addition, the ingestion potential of conducting airway neutrophils in mice with induced asthma exceeded that of control mice. Although the number of neutrophils subsequently decreased, the ingestion capacity remained elevated in AAI mice, even at 24 hours post-conidia application.
Aspiration of allergen to sensitized mice enhanced the ingestion potential of conducting airway neutrophils. Such activation primes neutrophils so that they are sufficient to control dissemination of non-germinating A. fumigatus conidia. At the same time, it can be a reason for the development of sensitivity to fungi and subsequent asthma exacerbation.
Allergic airway inflammation; Aspergillus fumigatus; Neutrophils; Mucosal dendritic cells; Conducting airway; Confocal laser scanning microscopy
Meta-analyses of genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) spanning the 5-hydroxytryptamine receptor 4 (5-HT4R) gene (HTR4) associated with lung function. The aims of this study were to i) investigate the expression profile of HTR4 in adult and fetal lung tissue and cultured airway cells, ii) further define HTR4 gene structure and iii) explore the potential functional implications of key SNPs using a bioinformatic approach.
Following reverse transcription (RT)-PCR in human brain, 5′ rapid amplification of cDNA ends (5′ RACE) was used to examine the exonic structure of HTR4 at the 5′ end. Quantitative (Q)-PCR was used to quantify HTR4 mRNA expression in total RNA from cultured airway cells and whole lung tissue. Publically available gene microarray data on fetal samples of estimated gestational age 7–22 weeks were mined for HTR4 expression. Immunohistochemistry (IHC; in adult and fetal lung tissue) and a radioligand binding assay (in cultured airway cells) were used to analyze 5HT4R protein expression.
IHC in adult lung, irrespective of the presence of chronic obstructive pulmonary disease (COPD), suggested low level expression of 5-HT4R protein, which was most prominent in alveolar pneumocytes. There was evidence of differential 5-HT4R protein levels during gestation in fetal lung, which was also evident in gene expression microarray data. HTR4 mRNA expression, assessed by Q-PCR, was <0.5% relative to brain in total adult lung tissue and in human airway smooth muscle (HASM) and bronchial epithelial cells (HBEC) derived from adult donors. Radioligand binding experiments also indicated that HBEC and HASM cells did not express a significant 5-HT4R population. 5′ RACE in brain identified a novel N-terminal variant, containing an extended N-terminal sequence. The functional significance of key HTR4 SNPs was investigated using the encyclopedia of DNA elements consortium (ENCODE) dataset. These analyses identified multiple alterations in regulatory motifs for transcription factors implicated in lung development, including Foxp1.
Taken together, these data suggest a role for HTR4 in lung development, which may at least in part explain the genetic association with lung function.
5-hydroxytryptamine; HTR4; 5-HT4R; Splice variant; Lung development; COPD; GPCR
The International Study on Asthma and Allergies in Childhood (ISAAC) reported a prevalence of asthma symptoms in 17 centers in nine Latin American countries that was similar to prevalence rates reported in non-tropical countries. It has been proposed that the continuous exposure to infectious diseases in rural populations residing in tropical areas leads to a relatively low prevalence of asthma symptoms. As almost a quarter of Latin American people live in rural tropical areas, the encountered high prevalence of asthma symptoms is remarkable. Wood smoke exposure and environmental tobacco smoke have been identified as possible risk factors for having asthma symptoms.
We performed a cross-sectional observational study from June 1, 2012 to September 30, 2012 in which we interviewed parents and guardians of Warao Amerindian children from Venezuela. Asthma symptoms were defined according to the ISAAC definition as self-reported wheezing in the last 12 months. The associations between wood smoke exposure and environmental tobacco smoke and the prevalence of asthma symptoms were calculated by means of univariate and multivariable logistic regression analyses.
We included 630 children between two and ten years of age. Asthma symptoms were recorded in 164 of these children (26%). The prevalence of asthma symptoms was associated with the cooking method. Children exposed to the smoke produced by cooking on open wood fires were at higher risk of having asthma symptoms compared to children exposed to cooking with gas (AOR 2.12, 95% CI 1.18 - 3.84). Four percent of the children lived in a household where more than ten cigarettes were smoked per day and they had a higher risk of having asthma symptoms compared to children who were not exposed to cigarette smoke (AOR 2.69, 95% CI 1.11 - 6.48).
Our findings suggest that children living in rural settings in a household where wood is used for cooking or where more than ten cigarettes are smoked daily have a higher risk of having asthma symptoms.
Asthma[Mesh]; Wheeze; Woodsmoke; Tobacco smoke pollution[Mesh]; Smoke[Mesh]; Indigenous children
Patients with chronic obstructive pulmonary disease (COPD) have a modified clinical presentation of venous thromboembolism (VTE) but also a worse prognosis than non-COPD patients with VTE. As it may induce therapeutic modifications, we evaluated the influence of the initial VTE presentation on the 3-month outcomes in COPD patients.
COPD patients included in the on-going world-wide RIETE Registry were studied. The rate of pulmonary embolism (PE), major bleeding and death during the first 3 months in COPD patients were compared according to their initial clinical presentation (acute PE or deep vein thrombosis (DVT)).
Of the 4036 COPD patients included, 2452 (61%; 95% CI: 59.2-62.3) initially presented with PE. PE as the first VTE recurrence occurred in 116 patients, major bleeding in 101 patients and mortality in 443 patients (Fatal PE: first cause of death). Multivariate analysis confirmed that presenting with PE was associated with higher risk of VTE recurrence as PE (OR, 2.04; 95% CI: 1.11-3.72) and higher risk of fatal PE (OR, 7.77; 95% CI: 2.92-15.7).
COPD patients presenting with PE have an increased risk for PE recurrences and fatal PE compared with those presenting with DVT alone. More efficient therapy is needed in this subtype of patients.
Chronic obstructive pulmonary diseases; Deep venous thrombosis; Prognosis; Pulmonary embolism; Venous thromboembolism
Acute exacerbation of idiopathic pulmonary fibrosis has become an important outcome measure in clinical trials. This study aimed to explore the concept of suspected acute exacerbation as an outcome measure.
Three investigators retrospectively reviewed subjects enrolled in the Sildenafil Trial of Exercise Performance in IPF who experienced a respiratory serious adverse event during the course of the study. Events were classified as definite acute exacerbation, suspected acute exacerbation, or other, according to established criteria.
Thirty-five events were identified. Four were classified as definite acute exacerbation, fourteen as suspected acute exacerbation, and seventeen as other. Definite and suspected acute exacerbations were clinically indistinguishable. Both were most common in the winter and spring months and were associated with a high risk of disease progression and short-term mortality.
In this study one half of respiratory serious adverse events were attributed to definite or suspected acute exacerbations. Suspected acute exacerbations are clinically indistinguishable from definite acute exacerbations and represent clinically meaningful events. Clinical trialists should consider capturing both definite and suspected acute exacerbations as outcome measures.
Clinical trials; Endpoints; Acute exacerbation; Pulmonary fibrosis
COPD exacerbations are associated with neutrophilic airway inflammation. Adhesion molecules on the surface of neutrophils may play a key role in their movement from blood to the airways. We analysed adhesion molecule expression on blood and sputum neutrophils from COPD subjects and non-obstructed smokers during experimental rhinovirus infections.
Blood and sputum were collected from 9 COPD subjects and 10 smoking and age-matched control subjects at baseline, and neutrophil expression of the adhesion molecules and activation markers measured using flow cytometry. The markers examined were CD62L and CD162 (mediating initial steps of neutrophil rolling and capture), CD11a and CD11b (required for firm neutrophil adhesion), CD31 and CD54 (involved in neutrophil transmigration through the endothelial monolayer) and CD63 and CD66b (neutrophil activation markers). Subjects were then experimentally infected with rhinovirus-16 and repeat samples collected for neutrophil analysis at post-infection time points.
At baseline there were no differences in adhesion molecule expression between the COPD and non-COPD subjects. Expression of CD11a, CD31, CD62L and CD162 was reduced on sputum neutrophils compared to blood neutrophils. Following rhinovirus infection expression of CD11a expression on blood neutrophils was significantly reduced in both subject groups. CD11b, CD62L and CD162 expression was significantly reduced only in the COPD subjects. Blood neutrophil CD11b expression correlated inversely with inflammatory markers and symptom scores in COPD subjects.
Following rhinovirus infection neutrophils with higher surface expression of adhesion molecules are likely preferentially recruited to the lungs. CD11b may be a key molecule involved in neutrophil trafficking in COPD exacerbations.
Chronic obstructive pulmonary disease; Exacerbations; Respiratory viruses; Neutrophils
Respiratory syncytial virus (RSV) is the leading cause of respiratory infections in children, elderly, and immunocompromised individuals. Severe infection is associated with short- and long-term morbidity including pneumonia, recurrent wheezing, and abnormal pulmonary function, and several lines of evidence indicate that impaired adaptive immune responses during infection are critical in the pathophysiology of RSV-mediated disease. Myeloid Dendritic cells (mDCs) play a pivotal role in shaping antiviral immune responses in the respiratory tract; however, few studies have examined the interactions between RSV and individual mDC subsets. In this study, we examined the effect of RSV on the functional response of primary mDC subsets (BDCA-1+ and BDCA-3+) isolated from peripheral blood.
BDCA-1+ and BDCA-3+ mDCs were isolated from the peripheral blood of healthy adults using FACS sorting. Donor-matched BDCA-1+ and BDCA-3+ mDCs were infected with RSV at a multiplicity of infection (MOI) of 5 for 40 hours. After infection, cells were analyzed for the expression of costimulatory molecules (CD86, CD80, and PD-L1), cytokine production, and the ability to stimulate allogenic CD4+ T cell proliferation.
Both BDCA-1+ and BDCA-3+ mDCs were susceptible to infection with RSV and demonstrated enhanced expression of CD86, and the inhibitory costimulatory molecules CD80 and PD-L1. Compared to BDCA-3+ mDCs, RSV-infected BDCA-1+ mDC produced a profile of cytokines and chemokines predominantly associated with pro-inflammatory responses (IL-1β, IL-6, IL-12, MIP-1α, and TNF-α), and both BDCA-1+ and BDCA-3+ mDCs were found to produce IL-10. Compared to uninfected mDCs, RSV-infected BDCA-1+ and BDCA-3+ mDCs demonstrated a reduced capacity to stimulate T cell proliferation.
RSV infection induces a distinct pattern of costimulatory molecule expression and cytokine production by BDCA-1+ and BDCA-3+ mDCs, and impairs their ability to stimulate T cell proliferation.
The differential expression of CD86 and pro-inflammatory cytokines by highly purified mDC subsets in response to RSV provides further evidence that BDCA-1+ and BDCA-3+ mDCs have distinct roles in coordinating the host immune response during RSV infection. Findings of differential expression of PD-L1 and IL-10 by infected mDCs, suggests possible mechanisms by which RSV is able to impair adaptive immune responses.
RSV; Myeloid dendritic cells; BDCA-1+ mDCs; BDCA-3+ mDCs; Primary DCs; Immune response; Costimulatory molecules; CD80; CD86; PD-L1; Cytokine profiles
Although use of the mechanical ventilator is a life-saving intervention, excessive tidal volumes will activate NF-κB in the lung with subsequent induction of lung edema formation, neutrophil infiltration and proinflammatory cytokine/chemokine release. The roles of NF-κB and IL-6 in ventilator-induced lung injury (VILI) remain widely debated.
To study the molecular mechanisms of the pathogenesis of VILI, mice with a deletion of IкB kinase in the myeloid cells (IKKβ△mye), IL-6-/- to WT chimeric mice, and C57BL/6 mice (WT) were placed on a ventilator for 6 hr.
WT mice were also given an IL-6-blocking antibody to examine the role of IL-6 in VILI.
Our results revealed that high tidal volume ventilation induced pulmonary capillary permeability, neutrophil sequestration, macrophage drifting as well as increased protein in bronchoalveolar lavage fluid (BALF). IL-6 production and IL-1β, CXCR2, and MIP2 expression were also increased in WT lungs but not in those pretreated with IL-6-blocking antibodies. Further, ventilator-induced protein concentrations and total cells in BALF, as well as lung permeability, were all significantly decreased in IKKβ△mye mice as well as in IL6-/- to WT chimeric mice.
Given that IKKβ△mye mice demonstrated a significant decrease in ventilator-induced IL-6 production, we conclude that NF-κB–IL-6 signaling pathways induce inflammation, contributing to VILI, and IкB kinase in the myeloid cells mediates ventilator-induced IL-6 production, inflammation, and lung injury.
Mechanical ventilator; Inflammation; IL-6; NF-κB; Alveolar macrophage; Chimeric mice
Ciliated cells play a central role in cleansing the airways of inhaled contaminants. They are derived from basal cells that include the airway stem/progenitor cells. In animal models, the transcription factor FOXJ1 has been shown to induce differentiation to the ciliated cell lineage, and the RFX transcription factor-family has been shown to be necessary for, but not sufficient to induce, correct cilia development.
To test the hypothesis that FOXJ1 and RFX3 cooperatively induce expression of ciliated genes in the differentiation process of basal progenitor cells toward a ciliated cell linage in the human airway epithelium, primary human airway basal cells were assessed under conditions of in vitro differentiation induced by plasmid-mediated gene transfer of FOXJ1 and/or RFX3. TaqMan PCR was used to quantify mRNA levels of basal, secretory, and cilia-associated genes.
Basal cells, when cultured in air-liquid interface, differentiated into a ciliated epithelium, expressing FOXJ1 and RFX3. Transfection of FOXJ1 into resting basal cells activated promoters and induced expression of ciliated cell genes as well as both FOXJ1 and RFX3, but not basal cell genes. Transfection of RFX3 induced expression of RFX3 but not FOXJ1, nor the expression of cilia-related genes. The combination of FOXJ1 + RFX3 enhanced ciliated gene promoter activity and mRNA expression beyond that due to FOXJ1 alone. Corroborating immunoprecipitation studies demonstrated an interaction between FOXJ1 and RFX3.
FOXJ1 is an important regulator of cilia gene expression during ciliated cell differentiation, with RFX3 as a transcriptional co-activator to FOXJ1, helping to induce the expression of cilia genes in the process of ciliated cell differentiation of basal/progenitor cells.
Lung epithelium; Ciliated cell differentiation; Human; FOXJ1; RFX3; Basal cell
Secondary pulmonary alveolar septal formation requires platelet derived growth factor (PDGF-A) and platelet derived growth factor receptor-alpha (PDGFRα), and their regulation influences alveolar septal areal density and thickness. Insufficient PDGFRα expression in lung fibroblasts (LF) results in failed septation.
Mice in which the endogenous PDGFRα-gene regulates expression of the green fluorescent protein were used to temporally and spatially track PDGFRα-signaling. Transition from the G1/Go to the S-phase of the cell cycle was compared in PDGFRα-expressing and non-expressing LF using flow cytometry. Laser scanning confocal microscopy was used to quantify p27kip1 and forkhead box "other" 3a (FoxO3a) in the nuclei of alveolar cells from mice bearing the PDGFRα-GFP knock-in, and p27kip1 in mice with a conditional deletion of PDGFRα-gene function. The effects of PDGF-A on the phosphorylation and the intracellular location of FoxO3a were examined using Western immuoblotting and immunocytochemistry.
In neonatal mouse lungs, entry of the PDGFRα-expressing LF subpopulation into the S-phase of the cell cycle diminished sooner than in their non-expressing LF counterparts. This preferential diminution was influenced by PDGFRα-mediated signaling, which phosphorylates and promotes cytoplasmic localization of FoxO3a. Comparative observations of LF at different ages during secondary septation and in mice that lack PDGFRα in alveolar LF demonstrated that nuclear localization of the G1 cyclin-dependent kinase inhibitor p27kip1 correlated with reduced LF entry into S-phase.
Nuclear localization of FoxO3a, an important regulator of p27kip1 gene-expression, correlates with diminished proliferation of the PDGFRα-expressing LF subpopulation. These mechanisms for diminishing the effects of PDGFRα-mediated signaling likely regulate secondary septal formation and their derangement may contribute to imbalanced fibroblast cell kinetics in parenchymal lung diseases.
Alveolar development; Emphysema; Pulmonary fibrosis; Cell cycle-regulation; Secondary septation; Platelet-derived Growth factor Receptor-alpha
Epithelial-mesenchymal transition (EMT) plays a crucial role in small airway fibrosis of patients with chronic obstructive pulmonary disease (COPD). Increasing evidence suggests that the urokinase plasminogen activator receptor (uPAR) is involved in the pathogenesis of COPD. Increased uPAR expression has been implicated in the promotion of EMT in numerous cancers; however the role of uPAR in EMT in small airway epithelial cells of patients with COPD remains unclear. In this study, we investigated the degree of EMT and uPAR expression in lung epithelium of COPD patients, and verified the effect of uPAR on cigarette smoke extract (CSE)-induced EMT in vitro.
The expression of EMT biomarkers and uPAR was assessed in lung epithelium specimens from non-smokers (n = 25), smokers (n = 25) and non-smokers with COPD (n = 10) and smokers with COPD (n = 18). The role of uPAR on CSE-induced EMT in human small airway epithelial cells (HSAEpiCs) was assessed by silencing uPAR expression in vitro.
Markers of active EMT and uPAR expression were significantly increased in the small airway epithelium of patients with COPD compared with controls. We also observed a significant correlation between uPAR and vimentin expression in the small airway epithelium. In vitro, CSE-induced EMT in HSAEpiCs was associated with high expression of uPAR, and targeted silencing of uPAR using shRNA inhibited CSE-induced EMT. Finally, we demonstrate that the PI3K/Akt signaling pathway is required for uPAR-mediated EMT in HSAEpiCs.
A uPAR-dependent signaling pathway is required for CSE-induced EMT, which contributes to small airway fibrosis in COPD. We propose that increased uPAR expression in the small airway epithelium of patients with COPD participates in an active EMT process.
Urokinase plasminogen activator receptor; Epithelial-mesenchymal transition; Small airway epithelial cells; Chronic obstructive pulmonary disease and cigarette smoke
Exercise limitation is an important issue in patients with chronic obstructive pulmonary disease (COPD), and it often co-exists with obstructive sleep apnoea (overlap syndrome). This study examined the effects of nocturnal continuous positive airway pressure (CPAP) treatment on walking capacity in COPD patients with or without obstructive sleep apnoea.
Forty-four stable moderate-to-severe COPD patients were recruited and completed this study. They all underwent polysomnography, CPAP titration, accommodation, and treatment with adequate pressure. The incremental shuttle walking test was used to measure walking capacity at baseline and after two nights of CPAP treatment. Urinary catecholamine and heart rate variability were measured before and after CPAP treatment.
After two nights of CPAP treatment, the apnoea-hypopnoea index and oxygen desaturation index significantly improved in both overlap syndrome and COPD patients, however these changes were significantly greater in the overlap syndrome than in the COPD group. Sleep architecture and autonomic dysfunction significantly improved in the overlap syndrome group but not in the COPD group. CPAP treatment was associated with an increased walking capacity from baseline from 226.4 ± 95.3 m to 288.6 ± 94.6 m (P < 0.05), and decreased urinary catecholamine levels, pre-exercise heart rate, oxygenation, and Borg scale in the overlap syndrome group. An improvement in the apnoea-hypopnoea index was an independent factor associated with the increase in walking distance (r = 0.564).
Nocturnal CPAP may improve walking capacity in COPD patients with overlap syndrome.
Chronic obstructive pulmonary disease; Obstructive sleep apnoea; Walking capacity; Autonomic dysfunction; Continuous positive airway pressure
De novo lymphatic vessel formation has recently been observed in lungs of patients with moderate chronic obstructive pulmonary disease (COPD). However, the distribution of lymphatic vessel changes among the anatomical compartments of diseased lungs is unknown. Furthermore, information regarding the nature of lymphatic vessel alterations across different stages of COPD is missing. This study performs a detailed morphometric characterization of lymphatic vessels in major peripheral lung compartments of patients with different severities of COPD and investigates the lymphatic expression of molecules involved in immune cell trafficking.
Peripheral lung resection samples obtained from patients with mild (GOLD stage I), moderate-severe (GOLD stage II-III), and very severe (GOLD stage IV) COPD were investigated for podoplanin-immunopositive lymphatic vessels in distinct peripheral lung compartments: bronchioles, pulmonary blood vessels and alveolar walls. Control subjects with normal lung function were divided into never smokers and smokers. Lymphatics were analysed by multiple morphological parameters, as well as for their expression of CCL21 and the chemokine scavenger receptor D6.
The number of lymphatics increased by 133% in the alveolar parenchyma in patients with advanced COPD compared with never-smoking controls (p < 0.05). In patchy fibrotic lesions the number of alveolar lymphatics increased 20-fold from non-fibrotic parenchyma in the same COPD patients. The absolute number of lymphatics per bronchiole and artery was increased in advanced COPD, but numbers were not different after normalization to tissue area. Increased numbers of CCL21- and D6-positive lymphatics were observed in the alveolar parenchyma in advanced COPD compared with controls (p < 0.01). Lymphatic vessels also displayed increased mean levels of immunoreactivity for CCL21 in the wall of bronchioles (p < 0.01) and bronchiole-associated arteries (p < 0.05), as well as the alveolar parenchyma (p < 0.001) in patients with advanced COPD compared with never-smoking controls. A similar increase in lymphatic D6 immunoreactivity was observed in bronchioles (p < 0.05) and alveolar parenchyma (p < 0.01).
This study shows that severe stages of COPD is associated with increased numbers of alveolar lymphatic vessels and a change in lymphatic vessel phenotype in major peripheral lung compartments. This novel histopathological feature is suggested to have important implications for distal lung immune cell traffic in advanced COPD.
Chronic obstructive pulmonary disease; Alveolar; Lymphatic vessel; CCL21; D6; Immunohistochemistry; Inflammation
Long-acting beta-agonists were one of the first-choice bronchodilator agents for stable chronic obstructive pulmonary disease. But the impact of long-acting beta-agonists on mortality was not well investigated.
National Emphysema Treatment Trial provided the data. Severe and very severe stable chronic obstructive pulmonary disease patients who were eligible for volume reduction surgery were recruited at 17 clinical centers in United States during 1988–2002. We used the 6–10 year follow-up data of patients randomized to non-surgery treatment. Hazard ratios for death by long-acting beta-agonists were estimated by three models using Cox proportional hazard analysis and propensity score matching were measured.
The pre-matching cohort was comprised of 591 patients (50.6% were administered long-acting beta-agonists. Age: 66.6 ± 5.3 year old. Female: 35.4%. Forced expiratory volume in one second (%predicted): 26.7 ± 7.1%. Mortality during follow-up: 70.2%). Hazard ratio using a multivariate Cox model in the pre-matching cohort was 0.77 (P = 0.010). Propensity score matching was conducted (C-statics: 0.62. No parameter differed between cohorts). The propensity-matched cohort was comprised of 492 patients (50.0% were administered long-acting beta-agonists. Age: 66.8 ± 5.1 year old. Female: 34.8%. Forced expiratory volume in one second (%predicted) 26.5 ± 6.8%. Mortality during follow-up: 69.1%). Hazard ratio using a univariate Cox model in the propensity-matched cohort was 0.77 (P = 0.017). Hazard ratio using a multivariate Cox model in the propensity-matched cohort was 0.76 (P = 0.011).
Long-acting beta-agonists reduce mortality of severe and very severe chronic obstructive pulmonary disease patients.
Bronchodilator agents; Treatment outcome; Case-control study; Propensity score
Pro-inflammatory/cytotoxic T cells (IFNγ, TNFα, granzyme B+) are increased in the peripheral circulation in COPD. NKT-like and NK cells are effector lymphocytes that we have also shown to be major sources of pro-inflammatory cytokines and granzymes. P-glycoprotein 1 (Pgp1) is a transmembrane efflux pump well characterised in drug resistant cancer cells. We hypothesized that Pgp1 would be increased in peripheral blood T, NKT-like and NK cells in patients with COPD, and that this would be accompanied by increased expression of IFNγ, TNFα and granzyme B. We further hypothesized that treatment with cyclosporine A, a Pgp1 inhibitor, would render cells more sensitive to treatment with corticosteroids.
Pgp1, granzyme B, IFNγ and TNFα expression were measured in peripheral blood T, NK and NKT-like cells from COPD patients and control subjects (± cyclosporine A and prednisolone) following in vitro stimulation and results correlated with uptake of efflux dye Calcein-AM using flow cytometry.
There was increased Pgp1 expression by peripheral blood T, NKT-like and NK cells co-expressing IFNγ, TNFα and granzyme B in COPD patients compared with controls (e.g. %IFNγ/Pgp1 T, NKT-like, NK for COPD (Control): 25(6), 54(27), 39(23)). There was an inverse correlation between Pgp1 expression and Calcein-AM uptake. Treatment with 2.5 ng/ml cylosporin A and10-6 M prednisolone resulted in synergistic inhibition of pro-inflammatory cytokines in Pgp1 + cells (p < 0.05 for all).
Treatment strategies that target Pgp1 in T, NKT-like and NK cells may reduce systemic inflammatory mediators in COPD and improve patient morbidity.
COPD; Pgp1; Pro-inflammatory lymphocytes; Granzyme B