Aspergillusfumigatus Recognition by Dendritic Cells Negatively Regulates Allergic Lung Inflammation through a TLR2/MyD88 Pathway.

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for a spectrum of clinical manifestations. Dendritic cells recognize pathogen-associated molecular patterns of Aspergillus via two main receptor families, Toll-like receptors (TLRs) and C-type lectin receptors (CLR). Here, the importance of TLR and CLR signaling in the regulation of T-helper cell type 2 (Th2) responses was analyzed using a mouse model based on the transfer of bone marrow-derived dendritic cells (BMDCs) pulsed with A. fumigatus conidia. BMDCs were generated from mice deficient in either MyD88 or MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1). Both the MyD88 and MALT1 signaling pathway in BMDCs contributed to the production of inflammatory cytokines induced by A. fumigatus conidia. Mice sensitized with MyD88-/- BMDCs pulsed in vitro with A. fumigatus conidia showed an exacerbated allergic inflammation, with stronger eosinophil recruitment in the BAL and higher Th2 cytokine production compared with mice sensitized with wild-type or MALT1-/- BMDCs. This exacerbation was not observed when MyD88-/- BMDCs were pulsed with Cladosporium sphaerospermum, a nonpathogenic mold. A lack of TLR2 signaling recapitulated the exacerbation of the A. fumigatus Th2 response observed in the absence of MyD88 signaling, whereas TLR2 agonist dampened the response induced with A. fumigatus and C. sphaerospermum conidia. IL-10 production by BMDCs in response to A. fumigatus was dependent on the expression of TLR2 and MyD88. IL-10-/- BMDCs exacerbated, whereas MyD88-/- BMDCs supplemented with exogenous IL-10 decreased the allergic pulmonary inflammation. These results indicate that TLR2/MyD88-specific recognition of PAMPs from A. fumigatus conidia can upregulate IL-10 production and downregulate lung eosinophilia and the development of a Th2 response.

Allergic Asthma Favors Brucella Growth in the Lungs of Infected Mice.

Allergic asthma is a chronic Th2 inflammatory disease of the lower airways affecting a growing number of people worldwide. The impact of infections and microbiota composition on allergic asthma has been investigated frequently. Until now, however, there have been few attempts to investigate the impact of asthma on the control of infectious microorganisms and the underlying mechanisms. In this work, we characterize the consequences of allergic asthma on intranasal (i.n.) infection by Brucella bacteria in mice. We observed that i.n. sensitization with extracts of the house dust mite Dermatophagoides farinae or the mold Alternaria alternata (Alt) significantly increased the number of Brucella melitensis, Brucella suis, and Brucella abortus in the lungs of infected mice. Microscopic analysis showed dense aggregates of infected cells composed mainly of alveolar macrophages (CD11c+ F4/80+ MHCII+) surrounded by neutrophils (Ly-6G+). Asthma-induced Brucella susceptibility appears to be dependent on CD4+ T cells, the IL-4/STAT6 signaling pathway and IL-10, and is maintained in IL-12- and IFN-γR-deficient mice. The effects of the Alt sensitization protocol were also tested on Streptococcus pneumoniae and Mycobacterium tuberculosis pulmonary infections. Surprisingly, we observed that Alt sensitization strongly increases the survival of S. pneumoniae infected mice by a T cell and STAT6 independent signaling pathway. In contrast, the course of M. tuberculosis infection is not affected in the lungs of sensitized mice. Our work demonstrates that the impact of the same allergic sensitization protocol can be neutral, negative, or positive with regard to the resistance of mice to bacterial infection, depending on the bacterial species.

GLI2 promoter hypermethylation in saliva of children with a respiratory allergy.

Background: The prevalence of respiratory allergy in children is increasing. Epigenetic DNA methylation changes are plausible underlying molecular mechanisms. Results: Saliva samples collected in substudies of two longitudinal birth cohorts in Belgium (FLEHS1 & FLEHS2) were used to discover and confirm DNA methylation signatures that can differentiate individuals with respiratory allergy from healthy subjects. Genome-wide analysis with Illumina Methylation 450K BeadChips revealed 23 differentially methylated gene regions (DMRs) in saliva from 11y old allergic children (N=26) vs. controls (N=20) in FLEHS1. A subset of 7 DMRs was selected for confirmation by iPLEX MassArray analysis. First, iPLEX analysis was performed in the same 46 FLEHS1 samples for analytical confirmation of the findings obtained during the discovery phase. iPLEX results correlated significantly with the 450K array data (P <0.0001) and confirmed 4 out of the 7 DMRs. Aiming for additional biological confirmation, the 7 DMRs were analyzed using iPLEX in a substudy of an independent birth cohort (FLEHS2; N=19 cases vs. 20 controls, aged 5 years). One DMR in the GLI2 promoter region showed a consistent statistically significant hypermethylation in individuals with respiratory allergy across the two birth cohorts and technologies. In addition to its involvement in TGF-ß signaling and T-helper differentiation, GLI2 has a regulating role in lung development. Conclusion: GLI2 is considered an interesting candidate DNA methylation marker for respiratory allergy.

The comet assay in human biomonitoring: cryopreservation of whole blood and comparison with isolated mononuclear cells.

The comet assay is often applied in human biomonitoring. Most of the time the assay is performed with isolated peripheral blood mononuclear cells (PBMC). However, using whole blood instead of isolated cells reduces processing time, and only 20 µl is sufficient for analysis. In this study, a cryopreservation protocol for human whole blood for application in the comet assay was optimised by removing excess plasma before adding freezing medium. Cryopreservation of whole blood samples (n = 30) did not increase the detected level of strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites. Although there was no significant correlation with breaks measured in fresh whole blood, strand breaks detected in frozen whole blood were significantly correlated with breaks measured in frozen PBMC (Pearson correlation r = 0.54, P < 0.01). This correlation was however not observed for FPG-sensitive sites. Since we do not yet know the full extent to which cryopreservation might influence the blood cell population, care should be taken to ensure a similar cell type and storage conditions for all samples in one study.

Blood Pressure and Same-Day Exposure to Air Pollution at School: Associations with Nano-Sized to Coarse PM in Children.

BACKGROUND: Ultrafine particles (UFP) may contribute to the cardiovascular effects of particulate air pollution, partly because of their relatively efficient alveolar deposition. OBJECTIVE: In this study, we assessed associations between blood pressure and short-term exposure to air pollution in a population of schoolchildren. METHODS: In 130 children (6-12 years of age), blood pressure was determined during two periods (spring and fall 2011). We used mixed models to study the association between blood pressure and ambient concentrations of particulate matter and ultrafine particles measured in the schools' playground. RESULTS: Independent of sex, age, height, and weight of the child, parental education, neighborhood socioeconomic status, fish consumption, heart rate, school, day of the week, season, wind speed, relative humidity, and temperature on the morning of examination, an interquartile range (860 particles/cm3) increase in nano-sized UFP fraction (20-30 nm) was associated with a 6.35 mmHg (95% CI: 1.56, 11.14; p = 0.01) increase in systolic blood pressure. For the total UFP fraction, systolic blood pressure was 0.79 mmHg (95% CI: 0.07, 1.51; p = 0.03) higher, but no effects on systolic blood pressure were found for the nano-sized fractions with a diameter > 100 nm, nor PM2.5, PMcoarse, and PM10. Diastolic blood pressure was not associated with any of the studied particulate mass fractions. CONCLUSION: Children attending school on days with higher UFP concentrations (diameter < 100 nm) had higher systolic blood pressure. The association was dependent on UFP size, and there was no association with the PM2.5 mass concentration.

Airway oxidative stress and inflammation markers in exhaled breath from children are linked with exposure to black carbon.

BACKGROUND: The current study aimed at assessing the associations between black carbon (BC) exposure and markers for airway inflammation and oxidative stress in primary school children in a Western European urban area. METHODS: In 130 children aged 6-12 years old, the fraction of exhaled nitric oxide (FeNO), exhaled breath condensate (EBC) pH, 8-isoprostane and interleukin (IL)-1ß were measured in two seasons. BC concentrations on the sampling day (2-h average, 8:00-10:00 AM) and on the day before (24-h average) were assessed using measurements at a central monitoring site. Land use regression (LUR) models were applied to estimate weekly average BC exposure integrated for the time spent at home and at school, and seasonal average BC exposure at the home address. Associations between exposure and biomarkers were tested using linear mixed effect regression models. Next to single exposure models, models combining different BC exposure metrics were used. RESULTS: In single exposure models, an interquartile range (IQR) increase in 2-h BC (3.10 µg/m(3)) was linked with a 5.9% (95% CI: 0.1 to 12.0%) increase in 8-isoprostane. FeNO increased by 16.7% (95% CI: 2.2 to 33.2%) per IQR increase in 24-h average BC (4.50 µg/m(3)) and by 12.1% (95% CI: 2.5 to 22.8%) per IQR increase in weekly BC (1.73 µg/m(3)). IL-1ß was associated with weekly and seasonal (IQR=1.70 µg/m(3)) BC with respective changes of 38.4% (95% CI: 9.0 to 75.4%) and 61.8% (95% CI: 3.5 to 153.9%) per IQR increase in BC. An IQR increase in weekly BC was linked with a lowering in EBC pH of 0.05 (95% CI: -0.10 to -0.01). All associations were observed independent of sex, age, allergy status, parental education level and meteorological conditions on the sampling day. Most of the associations remained when different BC exposure metrics were combined in multiple exposure models, after additional correction for sampling period or after exclusion of children with airway allergies. In additional analyses, FeNO was linked with 24-h PM10 levels, but the effect size was smaller than for BC. 8-Isoprostane was not linked with either 2-h or 24-h concentrations of PM2.5 or PM10. CONCLUSION: BC exposure on the morning of sampling was associated with airway oxidative stress while 24-h and weekly exposures were linked with airway inflammation.

Exhaled nitric oxide and nasal tryptase are associated with wheeze, rhinitis and nasal allergy in primary school children.

Rhinitis and asthma are the most common respiratory diseases in children. We assessed whether airway inflammation markers were associated with nasal allergies and self-reported symptoms of wheeze and rhinitis in 130 children 6-12 year old in an epidemiological context. Independent of sex and age, the fraction of exhaled nitric oxide (FeNO) and nasal mast cell (MC) activation (tryptase ≥ 5 ng/mL) were positively associated with wheeze, rhinitis and with nasal allergy. Nasal eosinophil cationic protein (ECP) and exhaled breath condensate (EBC) markers (pH, 8-isoprostane, interleukin-1ß) were not associated with symptoms or with nasal allergy. In conclusion, FeNO and nasal tryptase reflect allergic inflammation in the respiratory system.

Land use regression models as a tool for short, medium and long term exposure to traffic related air pollution.

BACKGROUND AND AIMS: In the HEAPS (Health Effects of Air Pollution in Antwerp Schools) study the importance of traffic-related air pollution on the school and home location on children's health was assessed. 130 children (aged 6 to 12) from two schools participated in a biomonitoring study measuring oxidative stress, inflammation and cardiovascular markers. METHODS: Personal exposure of schoolchildren to black carbon (BC) and nitrogen dioxide (NO2) was assessed using both measured and modeled concentrations. Air quality measurements were done in two seasons at approximately 50 locations, including the schools. The land use regression technique was applied to model concentrations at the children's home address and at the schools. RESULTS: In this paper the results of the exposure analysis are given. Concentrations measured at school 2h before the medical examination were used for assessing health effects of short term exposure. Over two seasons, this short term BC exposure ranged from 514 ng/m(3) to 6285 ng/m(3), and for NO2 from 11 µg/m(3) to 36 µg/m(3). An integrated exposure was determined until 10 days before the child's examination, taking into account exposures at home and at school and the time spent in each of these microenvironments. Land use regression estimates were therefore recalculated into daily concentrations by using the temporal trend observed at a fixed monitor of the official air quality network. Concentrations at the children's homes were modeled to estimate long term exposure (from 1457 ng/m(3) to 3874 ng/m(3) for BC; and from 19 µg/m(3) to 51 µg/m(3) for NO2). CONCLUSIONS: The land use regression technique proved to be a fast and accurate means for estimating long term and daily BC and NO2 exposure for children living in the Antwerp area. The spatial and temporal resolution was tailored to the needs of the epidemiologists involved in this study.

Comparison of methods for quantification of global DNA methylation in human cells and tissues.

DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element) for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the "gold standard" of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.

Influence of ambient air pollution on global DNA methylation in healthy adults: a seasonal follow-up.

BACKGROUND: DNA methylation changes are potential pathways of environmentally induced health effects. We investigated whether exposure to ambient concentrations of NO2, PM10, PM2.5 and O3 and traffic parameters were associated with global DNA methylation in blood of healthy adults. METHODS: 48 non-smoking adults (25 males) with a median age of 39years were sampled in winter and summer. Global DNA methylation in whole blood (% 5-methyl-2'-deoxycytidine, %5mdC) was analyzed with HPLC. Exposure to air pollutants at the home address was assessed using interpolated NO2, PM10, PM2.5 and O3 concentrations for various exposure windows (60- to 1-day moving average exposures and yearly averages) and GIS-based traffic parameters. Associations between pollutants and %5mdC were tested with multiple mixed effects regression models. RESULTS: Average %5mdC (SD) was 4.30 (0.08) in winter and 4.29 (0.08) in summer. Men had higher %5mdC compared to women both in winter (4.32 vs. 4.26) and summer (4.31 vs. 4.27). When winter and summer data were analyzed together, various NO2, PM10 and PM2.5 moving average exposures were associated with changes in %5mdC (95% CI) ranging from -0.04 (-0.09 to 0.00) to -0.14 (-0.28 to 0.00) per IQR increase in pollutant. NO2, PM10, PM2.5 and O3 moving average exposures were associated with decreased %5mdC (95% CI) varying between -0.01 (-0.03 to 0.00) and -0.17 (-0.27 to -0.06) per IQR increase in pollutant in summer but not in winter. CONCLUSION: Decreased global DNA methylation in whole blood was associated with exposure to NO2, PM10, PM2.5 and O3 at the home addresses of non- adults. Most effects were observed for the 5- to 30-day moving average exposures.

Decreased mitochondrial DNA content in association with exposure to polycyclic aromatic hydrocarbons in house dust during wintertime: from a population enquiry to cell culture.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants that are formed in combustion processes. At the cellular level, exposure to PAHs causes oxidative stress and/or some of it congeners bind to DNA, which may interact with mitochondrial function. However, the influence of these pollutants on mitochondrial DNA (mtDNA) content remains largely unknown. We determined whether indoor exposure to PAHs is associated with mitochondrial damage as represented by blood mtDNA content. Blood mtDNA content (ratio mitochondrial/nuclear DNA copy number) was determined by real-time qPCR in 46 persons, both in winter and summer. Indoor PAH exposure was estimated by measuring PAHs in sedimented house dust, including 6 volatile PAHs and 8 non-volatile PAHs. Biomarkers of oxidative stress at the level of DNA and lipid peroxidation were measured. In addition to the epidemiologic enquiry, we exposed human TK6 cells during 24 h at various concentrations (range: 0 to 500 µM) of benzo(a)pyrene and determined mtDNA content. Mean blood mtDNA content averaged (± SD) 0.95 ± 0.185. The median PAH content amounted 554.1 ng/g dust (25(th)-75(th) percentile: 390.7-767.3) and 1385 ng/g dust (25(th)-75(th) percentile: 1000-1980) in winter for volatile and non-volatile PAHs respectively. Independent for gender, age, BMI and the consumption of grilled meat or fish, blood mtDNA content decreased by 9.85% (95% CI: -15.16 to -4.2; p = 0.002) for each doubling of non-volatile PAH content in the house dust in winter. The corresponding estimate for volatile PAHs was -7.3% (95% CI: -13.71 to -0.42; p = 0.04). Measurements of oxidative stress were not correlated with PAH exposure. During summer months no association was found between mtDNA content and PAH concentration. The ability of benzo(a)pyrene (range 0 µM to 500 µM) to lower mtDNA content was confirmed in vitro in human TK6 cells. Based on these findings, mtDNA content can be a target of PAH toxicity in humans.

Induction of the specific allergic immune response is independent of proteases from the fungus Alternaria alternata.

We have analyzed the importance of proteases for the induction of allergic responses against the mold Alternaria alternata. Responses induced in vivo with untreated or heat treated (protease inactivated) extracts were compared in BALB/c, C57BL/6, TLR4 KO, and MyD88 KO mice. In BALB/c mice, both extracts induced similar lung inflammation, upregulation of inflammatory mediators, Th2 cytokines, and Alternaria-specific antibodies. However heat inactivation abrogated polyclonal IgE production. Similar results were obtained in C57BL/6 albeit lung expression of some Th2 mediators was decreased in mice stimulated with the heat-treated extract. Treatment of the extract with protease inhibitors did not affect the induction of the allergic response either, except again for the polyclonal IgE response. Th2 responses and lung inflammation were readily induced in TLR4 knockout mice. In contrast, lung inflammation, Th2 responses, cytokine productions, and antibody synthesis were strongly suppressed in MyD88-deficient mice. Early lung IL-33 and IL-1-α expression were also suppressed. In conclusion, albeit some heat labile proteases are required for the stimulation of the polyclonal IgE secretion, fungal proteases, and TLR4 signaling are not required while MyD88 is essential for triggering the systemic immune response and for the development of lung allergic inflammation in response to Alternaria extracts.