FINNISH RESEARCH PROGRAMME
ON ENVIRONMENTAL HEALTH
MECHANISMS OF ADVERSE HEALTH EFFECTS OF MOULDY HOUSE MICROBES: IN VITRO AND IN VIVO STUDIES ON TOXIC EFFECTS AND INFLAMMATORY RESPONSES
Maija-Riitta Hirvonen, National Public Health Institute (KTL), Laboratory of
Environmental Microbiology, P.O.Box 95, FIN-70701 Kuopio, Finland, tel.
Consortium: Moisture, mould
KEY WORDS: Toxicity,
inflammatory responses, in vivo, in vitro, microbes
There is an increasing concern about the adverse health effects including increased frequency of respiratory symptoms among the inhabitants or occupants in association with moisture and mould problems in buildings. At present, the causal relationship between detected exposures and health end points are not known because the most important causative microbes among the mixed population of microbes and interactions between them have not been satisfactorily identified. Moreover, next to nothing is known about the cellular mechanisms by which relatively low exposure levels may cause the reported adverse health effects. Also the components or metabolites of the microbes responsible for the induced effects as well as the interactions between the microbes need to be clarified. Altogether, there is a serious lack of data based on biochemical evidence of a link between objective biomarkers, qualitative characteristics of the microbial emissions and subjective symptoms. There is also need for methods that can be used for direct evaluation of biological potential of collected airborne samples to identify the health-relevant exposures associated with moisture in buildings. Thus, experimental data are needed for the risk assessment of the health effects of the mouldy house microbes to fill the current gap between the exposure and the symptoms observed. To prioritize the actions taken, the most harmful microbes should be identified and their effects have to be known.
The overall aim of the study was to identify which microbes among the mixed population of the microbes present in the mouldy houses are able to cause adverse respiratory health effects and what are their mechanisms. The studies were focused on inflammatory responses and cytotoxic effects 1) in human and mouse cells, 2) in a mouse model and 3) in upper and lower airways of people living or working in mouldy buildings.
Microbes: The microbes used in in vitro and in vivo studies were Streptomyces anulatus, Streptomyces californicus, Aspergillus versicolor, Stachybotrys chartarum, Penicillium spinulosum, Mycobacterium avium complex, and Mycobacterium terrae. All these microbes were isolated from indoor air or building material sample of moisture damaged buildings. In addition, Bacillus cereus and Pseudomonas fluorescens were used as examples of common gram positive and gram negative bacterium. For the experiments, the mycobacteria isolates were cultured on 7H11-agar with OADC-enrichment, and identified according to their chemical composition and characteristics. S. californicus was identified in Deutsche Sammlung von Microorganismen und Zellkulturen GmbH and the strains of B. cereus and P. fluorescens in National Veterinary and Food Research Institute, Kuopio Regional Laboratory, Finland. Bacterial strains were cultured on trypton yeast glucose agar. A. versicolor, P. spinulosum and S. chartarum were identified by the CBS identification service (Centraal Bureau of Schimmelcultures, Utrecht, the Netherlands) and cultured on 2% malt extract agar.
In vitro studies: Three different cell lines were used: 1. Human macrophage cell line (SC) 2. Mouse macrophage cell line (RAW264.7) and 3. Human alveolar type II epithelial-like cell line (A-549). All the cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in cell line specific culture medium. Both human cell lines were primed with IFN-gamma (10 ng/ml) and anti-microbial agents (nystatin and penicillin-streptomycin) were added after the dispension of the cells. The cells were incubated with different doses of microbes, and the exposures were terminated in selected timepoints. The culture medium and cells were stored at -80oC for the analyses.
In vivo studies: Male NIH/S
mice were used in in vivo studies. The animals were exposed intratracheally
to a single- or repeated doses of the microbes or their spores. In single
dose studies, both dose-responses and time-courses were studied. Responses
after a single dose of the exposure have been studied with four microbial
species: S. californicus, A. versicolor, P. spinulosum and M. terrae. In the
repeated dose study, dose-responses were determined after the exposure to
the spores of S. californicus. Bronchoalveolar lavage (BAL) was made, and
other samples were collected at the selected time points after the
Analyses: The methods listed in the Table 1 were used in the studies.
Table 1: Analyses in cell culture studies, in animal studies (bronchoalveolar lavage fluid) and in human clinical studies (nasal lavage fluid, induced sputum)
3 Results and discussion
In vitro studies. We have demonstrated, that: 1) The bacterial species, isolated from moldy buildings i.e. streptomycetes and mycobacteria as well as gram the negative bacterium Ps. fluorescens which was used as positive control, were more potent inducers of inflammatory mediators than than the fungal species and the gram positive bacterium B. cereus Ref. 3, 8, 9, 11, 15, 23. Based on our results the studied microbes can be ranked by their inflammatory potential to the following order: Pseudomonas fluorescens > Streptomyces californicus > Mycobacterium avium complex > Bacillus cereus > Stachybotrys chartarum > Aspergillus versicolor > Penicillium spinulosum. 2) Mouse macrophage cell line is more sensitive and produces a different cytokine profile than human cell lines after the exposure. However, both mouse and human cell lines react essentially to the same microbes. Out of the measured variables, interleukin-6 is the most coherent inflammatory mediator between the different cell lines 23. 3) The responses induced by different strains of streptomycetes in macrophages are not dependent on the viability of the spores of the microbes. 4) Spores of satratoxin-producing strains of Stachybotrys sp. cause a significant production of ROS and are highly cytotoxic to mouse macrophages, whereas atranone producers trigger production of proinflammatory cytokines in association with low cytotoxicity. However, atranones as such are not responsible for the inflammatory response of the macrophages 19. 5) Nutrient content and pH level of building materials or in growth media play an important role in the ability of the microbes to induce production of inflammatory mediators and to cause cytotoxicity in macrophages. Thus, the growth condition needs to be carefully considered when evaluating the inflammatory potential and/or toxicity of these organisms 6, 8, 9, 15. 6) Co-exposure to two microbes at the same time can lead to enhanced reaction; a synergistic effect on the exposed cells. Out of the studied microbes, only S. chartarum caused a synergistic reaction together with S. californicus. At least one metabolite of Stachybotrys, was observed to be able to cause such a synergistic reaction with S. californicus.
In vivo studies. We used an animal model, where mice are exposed to the spores or cells of moldy house microbes by using intratracheal instillation. In the single dose studies done with the spores of S. californicus, A. versicolor, P. spinulosum and with the M. terrae cells we observed that 1) acute inflammation in mouse lungs was induced by S. californicus, M. terrae and A. versicolor whereas P. spinulosum induce only a slight and transient response. 2) These microbes, however, differed significantly from each other in potency, time-course, and induced spectrum of inflammatory mediators 7, 20, 21, 22. 3) M. terrae caused also a delayed inflammatory response up to one month after a single dose exposure whereas the other microbes did not cause such effect 7, 20, 21, 22. 4) There was a good correlation between the in vitro and in vivo data. Repeated dosing of the spores of S. californicus demonstrated that 1) both adaptive and non-adaptive mechanisms of host defense were activated during the exposure. 2) The spore exposure was able to cause both strong immunostimulation (i.e. inflammation) and immunotoxicity (e.g. depletion of spleen cellularity). 3) Effects were also detected at relatively low exposure levels25 4) Some of the responses were stronger but some were weaker than the responses observed in the single dose study 7, 25.
Biomonitoring and Human Clinical Studies. Based on our results in human clinical studies following insights can be gained: 1) Both IS and NAL methods and measurement of inflammatory mediators were highly reproduciple 4, 6. Within-subject variation in the basal levels of measured inflammatory mediators in NAL fluid is low16. However, due to the considerable differences between subjects the comparison should be made using test subjects as their own controls, e.g. measuring the markers in NAL and IS before and after the environmental exposure 2) There is an association between inflammatory markers in NAL, the high prevalence of respiratory symptoms, and chronic exposure to microbes in the indoor environment 10, 13, 14, 17, 26. 3) Intense occupational fungal spore exposure (e.g. in sawmill) as such do not always cause inflammatory changes in upper airways. In line with in vitro this finding points to significant role of the growth conditions of the microbes affecting to the inflammatory potential and/or toxicity of these organisms13. 4) Both NAL method and exposure of the mouse cells to the environmental particle samples discriminate between subjects with high and low microbial exposure 26.
1. There are significant
differences between mouldy house indicator microbes in their ability to
cause cytotoxicity and to induce production of inflammatory mediators (NO,
ROS, cytokines) in human and mouse cells.