Naseem Mushtaq

Cigarette smoke and platelet-activating factor receptor dependent adhesion of Streptococcus pneumoniae to lower airway cells

Background Exposure to cigarette smoke (CS) is associated with increased risk of pneumococcal infection. The mechanism for this association is unknown. We recently reported that the particulate matter from urban air simulates platelet-activating factor receptor (PAFR)-dependent adhesion of pneumococci to airway cells. We therefore sought to determine whether CS stimulates pneumococcal adhesion to airway cells. Methods Human alveolar (A549), bronchial (BEAS2-B), and primary bronchial epithelial cells (HBEpC) were exposed to CS extract (CSE), and adhesion of Streptococcus pneumoniae determined. The role of PAFR in mediating adhesion was determined using a blocker (CV-3988). PAFR transcript level was assessed by quantitative real-time PCR, and PAFR expression by flow cytometry. Lung PAFR transcript level was assessed in mice exposed to CS, and bronchial epithelial PAFR expression assessed in active-smokers by immunostaining. Results In A549 cells, CSE 1% increased pneumococcal adhesion (p<0.05 vs control), PAFR transcript level (p<0.01), and PAFR expression (p<0.01). Pneumococcal adhesion to A549 cells was attenuated by CV-3988 (p<0.001). CSE 1% stimulated pneumococcal adhesion to BEAS2-B cells and HBEpC (p<0.01 vs control). CSE 1% increased PAFR expression in BEAS2-B (p<0.01), and in HBEpC (p<0.05). Lung PAFR transcript level was increased in mice exposed to CS in vivo (p<0.05 vs room air). Active smokers (n=16) had an increased percentage of bronchial epithelium with PAFR-positive cells (p<0.05 vs never smokers, n=11). Conclusion CSE stimulates PAFR-dependent pneumococcal adhesion to lower airway epithelial cells. We found evidence that CS increases bronchial PAFR in vivo.

Adhesion of Streptococcus pneumoniae to human airway epithelial cells exposed to urban particulate matter

BACKGROUND Epidemiologic studies report an association between pneumonia and urban particulate matter (PM) less than 10 microns (μm) in aerodynamic diameter (PM(10)). Streptococcus pneumoniae is a common cause of bacterial pneumonia worldwide. To date, the mechanism whereby urban PM enhances vulnerability to S pneumoniae infection is unclear. Adhesion of S pneumoniae to host cells is a prerequisite for infection. Host-expressed proteins, including the receptor for platelet-activating factor (PAFR), are co-opted by S pneumoniae to adhere to lower airway epithelial cells. OBJECTIVES To define whether inhalable urban PM enhances the adhesion of S pneumoniae to airway epithelial cells. METHODS A549 cells were cultured with PM(10) from Leicester (United Kingdom [UK]) and PM(10) and PM less than 2.5 μm in aerodynamic diameter (PM(2.5)) from Accra (Ghana), then infected with S pneumoniae strain D39. Pneumococcal adhesion to human primary bronchial epithelial cells was also assessed. Bacterial adhesion was determined by quantitative culture and confocal microscopy. The role of oxidative stress was assessed by N-acetyl cysteine, and the role of PAFR was assessed by mRNA transcript level, receptor expression, and receptor blocking. RESULTS PM(10) (UK) increased S pneumoniae adhesion to both A549 airway epithelial cells and human primary bronchial epithelial cells. PM(10) (Ghana) and PM(2.5) (Ghana) also increased adhesion. Culture of A549 cells by PM(10) (UK) increased PAFR mRNA transcript level and PAFR expression. PM(10) (UK)-stimulated adhesion to A549 cells was attenuated by a PAFR blocker and N-acetyl cysteine. CONCLUSION Urban PM increases adhesion of S pneumoniae to human airway epithelial cells. PM-stimulated adhesion is mediated by oxidative stress and PAFR.

Cycling to work in London and inhaled dose of black carbon

Modelling studies suggest that urban cycling is associated with an increased inhaled dose of fossil fuel-derived black carbon (BC). Using the amount of black material in airway macrophages as a marker of long-term inhaled BC, we sought to compare inhaled BC dose in London (UK) cyclists and non-cyclists. Airway macrophage carbon was assessed in 28 (58%) out of 48 healthy adults (14 cyclists and 14 non-cyclists) who attended for induced sputum. Short-term (24 h) exposure to BC was assessed on a representative working day in 27 out of 28 subjects. Serum interleukin (IL)-1&bgr;, IL-2, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor and tumour necrosis factor (TNF)-&agr; were assessed in 26 out of the 28 subjects. Cyclists were found to have increased airway macrophage carbon when compared with non-cyclists (mean±se 1.81±0.21 versus 1.11±0.07 μm2; p<0.01). Short-term monitoring showed no difference in 24 h BC exposure between the two groups. However, cyclists were exposed to higher concentrations of BC during commuting (p<0.01). Airway macrophage carbon was associated with monitored commute BC (n=28; r=0.47, p<0.05). TNF-&agr; was found to be increased in cyclists (p<0.05), but no other cytokines were increased. Commuting to work by bicycle in London is associated with increased long-term inhaled dose of BC. Whether cycling per se increases inhaled BC dose remains unclear.

Differential potentiation of the virulence of the Pseudomonas aeruginosa cystic fibrosis liverpool epidemic strain by oral commensal Streptococci.

BACKGROUND The Pseudomonas aeruginosa Liverpool epidemic strain (LES) is an important cystic fibrosis (CF) pathogen and is associated with increased morbidity and a worsened prognosis, compared with other CF-associated strains. However, interactions of common LES phenotypic variants with other members of the polymicrobial biofilms associated with chronic CF respiratory disease, such as oral commensal streptococci, have not been investigated. METHODS Biofilm population dynamics, virulence factor production, and pathogenicity in Galleria mellonella larvae of common LES phenotypes (ie, low production, intermediate production, and overproduction of pyocyanin) in the presence or absence of anginosus group streptococci (AGS) were compared. RESULTS AGS populations isolated from biofilm cocultures were P. aeruginosa phenotypic variant dependent, with higher AGS cell densities than those in monoculture frequently observed. Coexistence of AGS with a producer of low or intermediate levels of pyocyanin was found to result in enhancement of virulence factor production. In addition, the LES formed pathogenic partnerships with AGS in the G. mellonella infection model, with killing dependent on LES phenotype and AGS species. CONCLUSIONS The pathogenic potential of LES phenotypic variants can be enhanced by the presence of oral commensal streptococci. As adaptive mutations leading to reduced virulence factor production are commonplace, the observations made are relevant in the general context of the biology of P. aeruginosa infection during CF.

Carbon in airway macrophages from children with asthma

Background Airway macrophage (AM) phagocytosis is impaired in severe asthma. Prostaglandin (PG) E2 and D2 are increased in severe asthma and suppress AM phagocytic function in vitro. In this study, we sought evidence for PG-mediated impairment of phagocytosis of inhalable carbonaceous particulate matter (PM) by AM in children with severe asthma compared with mild asthmatics and healthy controls. Methods AM were obtained from children with asthma and healthy controls using induced sputum. AM carbon area (μm2) was assessed by image analysis. In a subgroup of asthmatics, urinary PGE2 and PGD2 metabolites were measured by high-performance liquid chromatography, and PM exposure at the home address was modelled. Phagocytosis of PM by human monocyte-derived macrophages and rat AM was assessed in vitro by image analysis. Results AM carbon was 51% lower in children with moderate-to-severe asthma (n=36) compared with mild asthmatics (n=12, p<0.01) and healthy controls (n=47, p<0.01). There was no association between modelled PM exposure and AM carbon in 33 asthmatics who had a urine sample, but there was an inverse association between AM carbon and urinary metabolites of PGE2 and D2 (n=33, rs=−0.40, p<0.05, and rs=−0.44, p<0.01). PGE2 10−6 M, but not PGD2 10−6 M, suppressed phagocytosis of PM10 by human macrophages in vitro (p<0.05 vs control). PGE2 10−6 M also suppressed phagocytosis of PM10 by rat AM in vitro (p<0.01 vs control). Conclusions Phagocytosis of inhaled carbonaceous PM by AMs is impaired in severe asthma. PGE2 may contribute to impaired AM phagocytic function in severe asthma.

Respiratory tract dendritic cells in paediatric asthma.

Airway dendritic cells (DC) are critical mediators of lung inflammation in asthma, but the characteristics of DC in the airways of healthy children, and children with asthma, are currently unknown.

Phagocytosis of fossil fuel particulates by macrophages in children with asthma

Abstract Background Airway macrophages clear inhaled soot (black carbon) from traffic fumes. Macrophages from children with asthma have impaired phagocytosis of bacteria, and prostaglandin E2 (PGE2) is associated with impaired phagocytosis by macrophages. We sought to assess whether macrophage phagocytosis of soot was impaired, and to measure prostanoid metabolites, in children with asthma. Methods In this cross-sectional study, airway macrophages were obtained from 72 children aged 7–14 years by sputum induction with nebulised 4·5% saline. Children were classified as: healthy controls (n=39); mild asthma, defined as British Thoracic Society (BTS) step 1–2 (n=13); or moderate-to-severe asthma (BTS step 3–5) (n=20), and recruited from a UK paediatric outpatient clinic. Healthy controls were recruited as part of the London Low Emission Zone study. Macrophage black carbon was assessed with image analysis by an assessor masked to asthma severity. Exposure to air pollution was calculated by means of the London Air Quality Toolkit. Urinary PGE2 metabolites were measured with high performance liquid chromatography-tandem mass spectrometry. The effect of PGE2 on phagocytosis of black carbon by rat airway macrophages was assessed with an in-vitro phagocytosis assay using urban particulate matter collected from air filters placed at city centre sites in Leicester, UK. Written informed consent was obtained from children and parents (Research Ethics Committee reference 11-LO-1732 and 08/H0704/139). Findings Children with moderate-to-severe asthma had lower airway macrophage black carbon than did controls (median 0·19 mm 2 [IQR 0·11–0·26] vs 0·35 [0·16–0·49], p U test). Airway macrophage black carbon in children with mild asthma was much the same as in controls. Air pollution exposure at the home address did not differ between groups. Moderate to severe asthma was associated with an increase in the urinary PGE2 metabolite, 13,14-dihydro-15-keto-tetranor-PGE2 (median 668 pg/mg creatinine [IQR 293–937] vs 471 [315–623], p U test). In the 33 children with asthma, this urinary PGE2 metabolite was inversely associated with macrophage carbon (Spearman r =−0·47, p Interpretation Macrophage phagocytosis of black carbon derived from fossil fuel is impaired in moderate-to-severe childhood asthma. This process is associated with raised urinary concentrations of 13,14-dihydro-15-keto-tetranor-PGE2. PGE2 also impairs phagocytosis of black carbon and urban particulate matter by rat airway macrophages in vitro, and is therefore a plausible mediator underlying the impaired phagocytosis seen in macrophages from children with asthma. We now aim to replicate these findings in differentiated human macrophages and to establish airway concentrations of PGE2 from exhaled breath condensate samples taken from these children. Our results suggest a possible mechanism underlying the observation that traffic-derived air pollution adversely affects children with asthma, because they may be less able to clear inhaled particles effectively. Funding Barts and The London Charity.

G160 Association Between External and Internal Dose of Diesel Soot (Black Carbon) in Healthy Schoolchildren: A Pilot Study

Aims Exposure to diesel soot (black carbon, BC) is linked to adverse health in children. A cross-sectional study reported that BC in airway macrophages (AM BC), a marker of inhaled dose of diesel soot, is associated with decreased lung function in healthy children [1]. These data are compatible with the reduction in growth of lung function associated with long-term exposure to elemental carbon reported in an 8 year epidemiological study of schoolchildren [2].To date, the determinants of AM BC are unknown. This is an important evidence gap since it is unclear whether policy-makers should target background BC, or peaks of freshly generated BC from roads. Using a newly developed portable monitor for BC, we sought to determine whether peaks in BC exposure are associated with airway macrophage black carbon (AM BC) in healthy schoolchildren. Methods Sputum inductions were carried out at schools as previously described [1]. Following processing, mean AM BC (µm2) for 50 randomly selected AM was calculated using Image J software. Personal exposure to BC was measured by a portable aethalometer (MicroAeth AE51, Magee Scientific). This monitor continuously samples BC in the air and data is downloaded after 24 h using the microAethCOM PC-based software (Fig 1). The number of peaks of BC above 10000 ng/300 sec was determined for each child by inspection of the 24 h plot. Results Twenty three children underwent sputum inductions. In the 15 children (65%) who produced sufficient AM for analysis, the median AM BC was 0.26278 µm2 (interquartile range (IQR) of 0.16164 to 0.42842 µm2). Personal 24 hour BC data was obtained in 13/15 children. The median BC exposure was 783758 ng (IQR: 336583.5 to 1321364.5 ng). Exposure peaks were caused by the school journey and cooking. No significant correlation was found between the number of peaks of carbon exposure above 10000 ng/300 sec and average AM BC (µm2) (Fig 2). However the positive association (r = 0.40, Pearson coefficient) suggests that this pilot study may be underpowered. Conclusion Linking external and inhaled dose of BC is feasible in schoolchildren, and may provide important insights into the determinants of inhaled dose of BC. Abstract G160 Figure 1 Example 24 hour aethalometer trace. Peaks are associated with daily commute (1 and 3) and cooking (2). Abstract G160 Figure 2 Scatter graph showing AM BC vs. number of BC exposure peaks above 10000 ng/300 sec The correlation coeffi cient for the number of peaks above 10000 ng/300 sec against AM BC (r=0.4028, n=13, p=0.1724) shows a non-signifi cant positive correlation. References Kulkarni N, Pierse N, Rushton L, Grigg J. “Carbon in airway macrophages and lung function in children.” The New England Journal of Medicine 6, 355 (2006): 21–30. Gauderman, WJ, E Avol, F Gilliland, H Vora, D Thomas, K Berhane, R McConnell, N Kuenzli, F Lurmann, E Rappaport, H Margolis, D Bates and J Peters. “The effect of air pollution on lung development from 10 to 18 years of age.” The New England Journal of Medicine 351, 11 (2004): 1057–1067.