Inflammation-associated gene transcription and expression in mouse lungs induced by low molecular weight compounds from fungi from the built environment

Abstract

Few metabolites from fungi found indoors have been tested for inflammatory mediators endpoints in primary cultures of alveolar macrophages or in vivo. In this study, mice were intratracheally instilled with a single dose comprising 4x10(-5)moletoxin/kg lung wt dose of either atranone C, brevianamide, cladosporin, mycophenolic acid, neoechinulin A & B, sterigmatocystin or TMC-120A. These toxins are from fungi common on damp building materials. The dose used was comparable to the estimated doses of possible human exposure. Hematoxylin and eosin (H&E) histology and Alcian Blue/Periodic Acid Schiff (AB/PAS) histochemistry were used to evaluate lungs for time course (4h and 12h post-exposure (PE)) inflammatory and toxic changes. Reverse-transcription (RT)-PCR based arrays were also employed to evaluate time course inflammation-associated gene transcription in lung tissues of the different toxins. Immunohistochemistry (IHC) was used to probe MIP-2 and Tnf-alpha protein expression in treatment lungs to determine whether responses correspond with gene transcription data. Both histology and histochemistry revealed that toxin exposed lungs at 12h PE showed evidence of inflammation. H&E revealed that bronchioli were lined with irregularly thickened and sometimes sloughing epithelium and bronchiolar spaces supported infiltration of leukocytes, cellular and mucus-like debris while alveolar spaces supported swollen macrophages and modest amorphous debris accumulations. All toxin-instilled lungs exhibited copious mucus production and alveolar macrophages with red stained cytoplasm on bronchiolar surfaces, especially at 12h PE. Array analysis of 83 inflammation-associated genes extracted from lung tissue demonstrated a number of patterns, compared to controls. 82 genes assayed at 4h PE and 75 genes at 12h PE were significantly altered (p< or =0.05; >or =1.5-fold or < or =-1.5-fold change) in the different treatment animal groups. Expression of transcriptionally regulated genes was confirmed using immunohistochemistry that demonstrated MIP-2 and Tnf-alpha staining in respiratory bronchiolar epithelia, alveolar macrophages and alveolar type II cells. The transcriptional regulation in these genes in the treatment groups suggests that they may serve central roles in the immunomodulation of toxin-induced pro-inflammatory lung responses. Hierarchical cluster analysis revealed significant patterns of gene transcription linking the response of the toxins at equimolar doses in three groups: (1) brevianamide, mycophenolic acid and neoechinulin B, (2) neoechinulin A and sterigmatocystin, and (3) cladosporin, atranone C and TMC-120. The results further confirm the inflammatory nature of metabolites/toxins from such fungi can contribute to the development of non-allergenic respiratory health effects.