Inflammation-associated gene expression in RAW 264.7 macrophages induced by toxins from fungi common on damp building materials.

Most fungi that grow on damp building materials produce low molecular weight compounds, some of which are known to be toxic. In this study, we tested the hypothesis that exposure to some metabolites of fungi common on damp building materials would result in time-, dose-, and compound-specific responses in the production of various chemokines by RAW 264.7 cells. Cell cultures were exposed to a 10-7M or 10-8M metabolite dose for 2, 4, 8 or 24h. Metabolite concentrations used were based on those that might be expected in alveolar macrophages due to inhalation exposure from living or working in a damp building. Compared to controls, exposure provoked significant time-, dose- and compound-specific responses manifest as differentially elevated secretion of three of nine cytokines tested in culture supernatant of treated cells. The greatest number of cytokines produced in response to the metabolites tested were in andrastin A-treated cells (GM-CSF, TGFß1, Tnf-α) followed by koninginin A (TGFß1 and Tnf-α) and phomenone (GM-CSF, TGFß1). Chaetoglobosin A, chaetomugilin D and walleminone exposures each resulted in significant time-specific production of Tnf-α only. This investigation adds to a body of evidence supporting the role of low molecular weight compounds from damp building materials as pathogen associated molecular patterns (PAMPs). Along with fungal glucan and chitin, these compounds contribute to the non-allergy based respiratory outcomes for people living and working in damp buildings.

Induction of Dectin-1 and asthma-associated signal transduction pathways in RAW 264.7 cells by a triple-helical (1, 3)-ß-D glucan, curdlan.

People living in damp buildings are typically exposed to spore and mycelial fragments of the fungi that grow on damp building materials. There is experimental evidence that this exposure to triple-helical (1, 3)-ß-D glucan and low molecular weight toxins may be associated with non-atopic asthma observed in damp and moldy buildings. However, the mechanisms underlying this response are only partially resolved. Using the pure (1, 3)-ß-D glucan, curdlan, and the murine macrophage cell line, RAW 264.7, there were two objectives of this study. The first was to determine whether signal transduction pathways activating asthma-associated cell signaling pathways were stimulated using mouse transduction Pathway Finder(®) arrays and quantitative real-time (QRT) PCR. The second objective was to evaluate the dose and temporal responses associated with transcriptional changes in asthma-associated cytokines, the signal transduction receptor gene Dectin-1, and various transcription factor genes related to the induction of asthma using customized RT-PCR-based arrays. Compared to controls, the 10(-7) M curdlan treatment induced significant changes in gene transcription predominately in the NFkB, TGF-ß, p53, JAK/STAT, P13/AKT, phospholipase C, and stress signaling pathways. The 10(-8) M curdlan treatment mainly induced NFkB and TGF-ß pathways. Compared to controls, curdlan exposures also induced significant dose- and time-dependent changes in the gene translations. We found that that curdlan as a non-allergenic potentiator modulates a network of transduction signaling pathways not only associated with TH-1, TH-2, and TH-3 cell responses including asthma potentiation, but a variety of other cell responses in RAW 264.7 cells. These results help provide mechanistic basis for some of the phenotypic changes associated with asthma that have been observed in in vitro, in vivo, and human studies and open up a hypothesis-building process that could explain the rise of non-atopic asthma associated with fungi.

Immunohistochemical and immunocytochemical detection of SchS34 antigen in Stachybotrys chartarum spores and spore impacted mouse lungs.

The purpose of this study was to evaluate the distribution of a 34 kD antigen isolated from S. chartarum sensu lato in spores and in the mouse lung 48 h after intra-tracheal instillation of spores by immuno-histochemistry. This antigen was localized in spore walls, primarily in the outer and inner wall layers and on the external wall surfaces with modest labelling observed in cytoplasm. Immuno-histochemistry revealed that in spore impacted mouse lung, antigen was again observed in spore walls, along the outside surface of the outer wall and in the intercellular space surrounding spores. In lung granulomas the labelled antigen formed a diffusate, some 2-3x the size of the long axis of spores, with highest concentrations nearest to spores. Collectively, these observations indicated that this protein not only displayed a high degree of specificity with respect to its location in spores and wall fragments, but also that it slowly diffuses into surrounding lungs.

Comparison of inflammatory responses in mouse lungs exposed to atranones A and C from Stachybotrys chartarum.

Stachybotrys chartarum isolates can be separated into two distinct chemotypes based on the toxins they produce. One chemotype produces macrocyclic trichothecenes; the other produces atranones (and sometimes simple trichothecenes, e.g., trichodermol and trichodermin). Studies using in vivo models of lung disease revealed that exposure to spores of the atranone producing S. chartarum isolates led to a variety of immunotoxic, inflammatory, and other pathological changes. However, it is unclear from these studies what role the pure atranone toxins sequestered in spores of these isolates exert on lung disease onset. This study examined dose-response (0.2, 1.0, 2.0, 5.0, or 20 microg atranone/animal) and time-course (3, 6, 24, and 48 h postinstillation [PI]) relationships associated with inflammatory cell and proinflammatory chemokine/cytokine responses in mouse lungs intratracheally instilled with two pure atranones (either A or C) isolated from S. chartarum. High doses (2.0 to 20 microg toxin/animal) of atranone A and C induced significant inflammatory responses manifested as differentially elevated macrophage, neutrophil, macrophage inflammatory protein (MIP)-2, tumor necrosis factor (TNF) and interleukin (IL)-6 concentrations in the bronchioalveolar lavage fluid (BALF) of intratracheally exposed mice. Compared to controls, BALF macrophage and neutrophil numbers were increased to significant levels from 6 to 48 h (PI). Except for macrophage numbers in atranone A treatment animals, cells exhibited significant dose dependent-like responses. The chemokine/cytokine marker responses were significantly and dose-dependently increased from 3 to 24 h PI and declined to nonsignificant levels at 48 h PI. The results suggest not only that atranones are inflammatory but also that they exhibit different inflammatory potency with different toxicokinetics. Data also suggest that exposure to these toxins in spores of S. chartarum in contaminated building environments could contribute to inflammatory lung disease onset in susceptible individuals.

Lead and cadmium uptake in the marine fungi Corollospora lacera and Monodictys pelagica.

This study provides observations on the effects of lead and cadmium ions on the growth of two species of marine fungi, Corollospora lacera and Monodictys pelagica. On solid media lead appeared to have no effect on the radial rate of growth of fungi. Exposure to increasing cadmium concentrations on solid media resulted in significant reduction (p < 0.05) in the radial mycelial growth rates of both fungi, especially in M. pelagica. These results reveal significant difference in species sensitivity toward cadmium and, essentially, insensitivity toward lead exposure. In liquid cultures, the metal content of mycelia (metal mass found in mycelium, in mg), and the concentration of metal in dry mycelium (metal mass in 1g of mycelium, in mg g(-1)) were both found to increase (p < 0.05) with the increase in the metal cation concentration, while mycelium dry mass decreased. As it was observed on solid media, cadmium cation affected more severely (p < 0.05) the growth of M. pelagica in liquid cultures. Ergosterol content of mycelia of C. lacera exposed to increasing cadmium cation concentration decreased, similarly to the trend observed for dry mycelial mass. It was found that ca. 93% of all lead sequestered by C. lacera is located extracellularly. M. pelagica was found to bioaccumulate over 60 mg of cadmium and over 6 mg of lead per 1 g of mycelium, while C. lacera bioaccumulated over 7 mg of cadmium and up to 250 mg of lead per 1 g of mycelium. Overall, the results indicate that both metal ions affect the growth of marine fungi with lead being accumulated extracellularly in the mycelia. Both metals accumulated by fungi may then enter the marine ecosystem food web, of which marine fungi are integral members.

Acute inflammatory responses to Stachybotrys chartarum in the lungs of infant rats: time course and possible mechanisms.

Stachybotrys chartarum has been linked to building-related respiratory problems including pulmonary hemorrhage in infants. The macrocyclic trichothecenes produced by S. chartarum have been the primary focus of many investigations. However, in addition to trichothecenes this fungus is capable of producing other secondary metabolites and a number of protein factors. This study examines the effects of intact, autoclaved, and ethanol-extracted spores on the lungs of infant rats as an approach to differentiate between secondary metabolites and protein factors. Seven-day-old infant rats were exposed intratracheally to 1 x 10(5) spores/g body weight (toxic strain JS58-17) and sacrificed at various times up to 72 h. The inflammatory response was measured by morphometric analysis of the lungs and determination of inflammatory cells and cytokine concentrations in bronchoalveolar lavage (BAL) fluid. Alveolar space was greatly reduced in animals exposed to fungal spores compared to phosphate buffered saline (PBS)-treated controls. The largest effects were observed in pups treated with intact spores where alveolar space 24 h after treatment was 42.1% compared to 56.8% for autoclaved spores, 51.1% for ethanol-extracted spores, and 60.6% for PBS-treated controls. The effects of different spore preparations on inflammatory cells, cytokine, and protein concentrations in the BAL fluid can be ranked as intact > autoclaved > extracted. Tumor necrosis factor alfa (TNF-alpha), interleukin 1-beta (IL-1beta), and neutrophils were the most sensitive indicators of inflammation. The difference between autoclaved (100% trichothecene toxicity, denatured/enzymatically inactive proteins) and intact (100% trichothecene activity, unaltered/released proteins) spores indicates the involvement of fungal proteins in the inflammatory response to S. chartarum and sheds new light on the clinical importance of "nontoxic" strains.

Localization of satratoxin-G in Stachybotrys chartarum spores and spore-impacted mouse lung using immunocytochemistry.

Satratoxin-G (SG) is the major macrocyclic trichothecene mycotoxin produced by Stachybotrys chartarum (atra) and has been implicated as a cause of a number of animal and human health problems including pulmonary hemorrhage in infants. However, there is little understanding where this toxin is localized in the spores and mycelial fragments of this species or in the lung impacted by SG-sequestered spores. The purpose of this study was to evaluate the distribution of SG in S. chartarum spores and mycelium in culture, and spore-impacted mouse lung in vivo, using immunocytochemistry. SG was localized predominately in S. chartarum spores with moderate labelling of the phialide-apex walls. Labelling was primarily along the outer plasmalemma surface and in the inner wall layer. Only modest labelling was observed in hyphae. Toxin localization at these sites supports the position that spores contain the highest satratoxin concentrations and that the toxin is constitutively produced. In impacted mouse lung, highest SG labelling was detected in lysosomes, along the inside of the nuclear membrane in nuclear heterochromatin and RER within alveolar macrophages. Alveolar type II cells also showed modest labelling of the nuclear heterochromatin and RER. There was no evidence that the toxin accumulated in the neutrophils, fibroblasts, or other cells associated with the granulomas surrounding spores or mycelial fragments. These observations indicate that SG displays a high degree of cellular specificity with respect to its uptake in mouse lung. They further indicate that the alveolar macrophages play an important role in the sequestration and immobilization of low concentrations of the toxin.