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 (µm(2)) 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.

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.

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)-1ß, IL-2, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor and tumour necrosis factor (TNF)-α 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 µm(2); 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-α 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.

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.

Prevention of arthritis by interleukin 10-producing B cells.

In this study we have shown that activation of arthritogenic splenocytes with antigen and agonistic anti-CD40 gives raise to a B cell population that produce high levels of interleukin (IL)-10 and low levels of interferon (IFN)-gamma. Transfer of these B cells into DBA/1-TcR-beta-Tg mice, immunized with bovine collagen (CII) emulsified in complete Freund's adjuvant inhibited T helper type 1 differentiation, prevented arthritis development, and was also effective in ameliorating established disease. IL-10 is essential for the regulatory function of this subset of B cells, as the B cells population isolated from IL-10 knockout mice failed to mediate this protective function. Furthermore, B cells isolated from arthritogenic splenocytes treated in vitro with anti-IL-10/anti-IL-10R were unable to protect recipient mice from developing arthritis. Our results suggest a new role of a subset of B cells in controlling T cell differentiation and autoimmune disorders.

Airway dendritic cell maturation in children exposed to air pollution.

Urban particulate matter (PM) enhances airway dendritic cell (DC) maturation in vitro. However, to date, there are no data on the association between exposure to urban PM and DC maturation in vivo. We sought to determine whether exposure of school-age children (8 to 14 y) to PM was associated with expression of CD86, a marker of maturation of airway conventional DCs (cDC). Healthy London school children underwent spirometry and sputum induction. Flow cytometry was used to identify CD86 and CCR7 expression on cDC subsets (CD1c+ cDC2 and CD141+ cDC1). Tertiles of mean annual exposure to PM ≤ 10 microns (PM10) at the school address were determined using the London Air Quality Toolkit model. Tertiles of exposure from the 409 children from 19 schools recruited were; lower (23.1 to 25.6 µg/m3, n = 138), middle (25.6 to 26.8 µg/m3, n = 126), and upper (26.8 to 31.0 µg/m3, n = 145). DC expression was assessed in 164/370 (44%) children who completed sputum induction. The proportion (%) of cDC expressing CD86 in the lower exposure tertile (n = 47) was lower compared with the upper exposure tertile (n = 49); (52% (44 to 70%) vs 66% (51 to 82%), p<0.05). There was a higher percentage of cDC1 cells in the lower tertile of exposure (6.63% (2.48 to 11.64) vs. 2.63% (0.72 to 7.18), p<0.05). Additionally; children in the lower exposure tertile had increased FEV1 compared with children in the upper tertile; (median z-score 0.15 (-0.59 to 0.75) vs. -0.21 (-0.86 to 0.48), p<0.05. Our data reveal that children attending schools in the highest areas of PM exposure in London exhibit increased numbers of "mature" airway cDCs, as evidenced by their expression of the surface marker CD86. This data is supportive of previous in vitro data demonstrating an alteration in the maturation of airway cDCs in response to exposure to pollutants.

Treatment of experimental Escherichia coli infection with recombinant bacteriophage-derived capsule depolymerase.

OBJECTIVES: The aim of this study was to investigate the effect of single doses of the capsule depolymerizing enzyme endosialidase E (endoE) on the course of systemic infection due to Escherichia coli K1 strains in neonatal rats. We also determined the capacity of the enzyme to increase the sensitivity of K1 strains to rat peritoneal macrophages. METHODS: Bacteraemia was established in Wistar rats by induction of gastrointestinal colonization with the virulent K1 strain A192PP; colonization preceded a lethal bacteraemia. Decreasing single doses of endoE were administered intraperitoneally. Macrophage engulfment of K1 strain A192PP was evaluated by staining and microscopy in the presence and absence of endoE. RESULTS: A192PP colonized the gastrointestinal tract of all 2-day-old animals and produced bacteraemia in over 90%. A single endoE dose of 0.25 microg curtailed bacteraemia and prevented death in at least 80% of infected animals. Older animals (up to 5 days of age) were less susceptible to systemic infection following intestinal colonization. EndoE-mediated removal of K1 capsular polysaccharide led to increased ingestion by macrophages. CONCLUSIONS: A small single dose of capsule-depolymerizing enzyme has therapeutic utility in lethal systemic infection in a non-invasive model that has characteristics of the infectious process in humans. We propose that the enzyme reduces the virulence of E. coli K1 by rapid removal of the protective capsular polysaccharide, sensitizing the pathogen to host defences such as phagocytosis by macrophages. Thus, whilst endoE-mediated therapy may not be a viable approach to the treatment of systemic infection in humans, it does support the concept that alteration of the cell wall phenotype is a valid therapeutic strategy.

Prevention and cure of systemic Escherichia coli K1 infection by modification of the bacterial phenotype.

Escherichia coli is a common cause of meningitis and sepsis in the newborn infant, and the large majority of isolates from these infections produce a polysialic acid (PSA) capsular polysaccharide, the K1 antigen, that protects the bacterial cell from immune attack. We determined whether a capsule-depolymerizing enzyme, by removing this protective barrier, could alter the outcome of systemic infection in an animal model. Bacteriophage-derived endosialidase E (endoE) selectively degrades the PSA capsule on the surface of E. coli K1 strains. Intraperitoneal administration of small quantities of recombinant endoE (20 micro g) to 3-day-old rats, colonized with a virulent strain of K1, prevented bacteremia and death from systemic infection. The enzyme had no effect on the viability of E. coli strains but sensitized strains expressing PSA to killing by the complement system. This study demonstrates the potential therapeutic efficacy of agents that cure infections by modification of the bacterial phenotype rather than by killing or inhibition of growth of the pathogen.