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Moisture-damaged buildings are associated with respiratory symptoms and underlying diseases among building occupants, but the causative agent(s) remain a mystery. We first identified specific fungal and bacterial taxa in classrooms with moisture damage in Finnish and Dutch primary schools. We then investigated associations of the identified moisture damage indicators with respiratory symptoms in more than 2700 students. Finally, we explored whether exposure to specific taxa within the indoor microbiota may explain the association between moisture damage and respiratory health. Schools were assessed for moisture damage through detailed inspections, and the microbial composition of settled dust in electrostatic dustfall collectors was determined using marker-gene analysis. In Finland, there were several positive associations between particular microbial indicators (diversity, richness, individual taxa) and a respiratory symptom score, while in the Netherlands, the associations tended to be mostly inverse and statistically non-significant. In Finland, abundance of the Sphingomonas bacterial genus and endotoxin levels partially explained the associations between moisture damage and symptom score. A few microbial taxa explained part of the associations with health, but overall, the observed associations between damage-associated individual taxa and respiratory health were limited.

In vitro models mimicking the human respiratory system are essential when investigating the toxicological effects of inhaled indoor air particulate matter (PM). We present a pulmonary cell culture model for studying indoor air PM toxicity. We exposed normal human bronchial epithelial cells, grown on semi‐permeable cell culture membranes, to four doses of indoor air PM in the air‐liquid interface. We analyzed the chemokine interleukin‐8 concentration from the cell culture medium, protein concentration from the apical wash, measured tissue electrical resistance, and imaged airway constructs using light and transmission electron microscopy. We sequenced RNA using a targeted RNA toxicology panel for 386 genes associated with toxicological responses. PM was collected from a non‐complaint residential environment over 1 week. Sample collection was concomitant with monitoring size‐segregated PM counts and determination of microbial levels and diversity. PM exposure was not acutely toxic for the cells, and we observed up‐regulation of 34 genes and down‐regulation of 17 genes when compared to blank sampler control exposure. The five most up‐regulated genes were related to immunotoxicity. Despite indications of incomplete cell differentiation, this model enabled the comparison of a toxicological transcriptome associated with indoor air PM exposure.

Additional file 8 Figure S4. Viability (A), Comet assay (B) and IL-8 secretion (C) of A549 control cells after exposed one hour at incubator with CO2, incubator without CO2 or Tox-ALI to clean air. Each bar shows mean ± SEM, n = 3.

Additional file 4 Figure S2. Size of the particles measured by SMPS per day to day. The geometric mean diameters are shown with the solid lines, while GSDs are represented with dashed lines. (average of 3 experiments)

Additional file 7 Table S3. Inorganic elements from spruce and pine combustion emissions. Organic compounds were sampled over 4-h exposure. Concentrations are calculated as averages from three test cycles ± standard deviation.

Additional file 9 Table S4. Top 5 significant ingenuity canonical pathways of spruce aerosol exposure.

Additional file 5 Figure S3. Size of the particles measured by SMPS. The geometric mean diameters (a) and mass median diameters (b) are shown with the solid lines, while GSDs are represented with dashed lines. Average of 3 experiments.

Additional file 11 Figure S5. Area under curve (AUC) calculated from exposed A549 cells from three different timepoints of H2DCFDA measurements. Each bar shows mean ± SEM, n = 3.

Exposure to moisture-damaged indoor environments is associated with adverse respiratory health effects, but responsible factors remain unidentified. In order to explore possible mechanisms behind these effects, the oxidative capacity and hemolytic activity of settled dust samples (n = 25) collected from moisture-damaged and non-damaged schools in Spain, the Netherlands, and Finland were evaluated and matched against the microbial content of the sample. Oxidative capacity was determined with plasmid scission assay and hemolytic activity by assessing the damage to isolated human red blood cells. The microbial content of the samples was measured with quantitative PCR assays for selected microbial groups and by analyzing the cell wall markers ergosterol, muramic acid, endotoxins, and glucans. The moisture observations in the schools were associated with some of the microbial components in the dust, and microbial determinants grouped together increased the oxidative capacity. Oxidative capacity was also affected by particle concentration and country of origin. Two out of 14 studied dust samples from moisture-damaged schools demonstrated some hemolytic activity. The results indicate that the microbial component connected with moisture damage is associated with increased oxidative stress and that hemolysis should be studied further as one possible mechanism contributing to the adverse health effects of moisture-damaged buildings.

Aiming to identify factors causing the adverse health effects associated with moisture damaged indoor environments, we analyzed immunotoxicological potential of settled dust from moisture damaged and reference schools in relation to their microbiological composition. Mouse RAW264.7 macrophages were exposed to settled dust samples (n= 25) collected from moisture damaged and reference schools in Spain, The Netherlands and Finland. After exposure, we analyzed production of inflammatory markers [nitric oxide (NO), tumor necrosis factor (TNF)α, interleukin (IL)-6 and macrophage inflammatory protein (MIP)2] as well as mitochondrial activity, viability, apoptosis and cell cycle arrest. Furthermore, particle counts, concentration of selected microbial groups as well as chemical markers ergosterol, 3-hydroxy fatty acids, muramic acid, endotoxins and glucans were measured as markers of exposure. Dust from moisture damaged schools in Spain and The Netherlands induced stronger immunotoxicological responses compared to samples from reference schools; the responses to Finnish samples were generally lower with no difference between the schools. In multivariate analysis, IL-6 and apoptosis responses were most strongly associated with moisture status of the school. The measured responses correlated with several microbial markers and numbers of particles, but the most important predictor of the immunotoxicological potential of settled dust was muramic acid concentration, a marker of Gram positive bacteria. This article is protected by copyright. All rights reserved.

Aiming to identify factors causing the adverse health effects associated with moisture damaged indoor environments, we analyzed immunotoxicological potential of settled dust from moisture damaged and reference schools in relation to their microbiological composition. Mouse RAW264.7 macrophages were exposed to settled dust samples (n= 25) collected from moisture damaged and reference schools in Spain, The Netherlands and Finland. After exposure, we analyzed production of inflammatory markers [nitric oxide (NO), tumor necrosis factor (TNF)α, interleukin (IL)-6 and macrophage inflammatory protein (MIP)2] as well as mitochondrial activity, viability, apoptosis and cell cycle arrest. Furthermore, particle counts, concentration of selected microbial groups as well as chemical markers ergosterol, 3-hydroxy fatty acids, muramic acid, endotoxins and glucans were measured as markers of exposure. Dust from moisture damaged schools in Spain and The Netherlands induced stronger immunotoxicological responses compared to samples from reference schools; the responses to Finnish samples were generally lower with no difference between the schools. In multivariate analysis, IL-6 and apoptosis responses were most strongly associated with moisture status of the school. The measured responses correlated with several microbial markers and numbers of particles, but the most important predictor of the immunotoxicological potential of settled dust was muramic acid concentration, a marker of Gram positive bacteria. This article is protected by copyright. All rights reserved.