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Arch Toxicol. 2009 Nov 11. [Epub ahead of print]

Dectin-1 and inflammation-associated gene transcription and
expression in mouse lungs by a toxic (1,3)-beta-D: glucan.

Rand TG, Sun M, Gilyan A, Downey J, Miller JD.

Department of Biology, Saint Mary's University, 923 Robie
St., Halifax, NS, B3H 3C3, Canada, thomas.rand@smu.ca.

The form of (1-3)-beta-D: glucan found in the cell walls of
the anamorphic Trichocomaceae that grow on damp building
materials is considered to have potent toxic and
inflammatory effects on cells of the respiratory system. It
is also considered to have a potential role in the
development of non-allergenic respiratory health effects.
While human studies involving experimental exposures all
point to the inflammatory potential of pure curdlan, a
linear (1-3)-beta-D: glucan in a triple helix
configuration, animal experiments result in conflicting
conclusions concerning the inflammatory potency of this
glucan. However, because mice appear to be a better model
than guinea pigs for exploring the respiratory effects of
curdlan and because molecular mechanisms associated with
this glucan remain largely unknown, we conducted further
work to clarify the role of curdlan on the inflammatory
response using our mouse model of lung disease. This study
used in situ hybridization (ISH) to probe dectin-1 mRNA
transcription with a digoxigenin-labeled cDNA probe, with
reverse transcription (RT)-PCR based arrays used to measure
inflammation gene and receptor transcriptional responses.
Also, immunohistochemistry (IHC) was used to probe dectin-1
as well as anti-mouse Ccl3, Il1-alpha, and TNF-alpha
expression to evaluate dose and time-course (4 and 12 h)
postexposure (PE) response patterns in the lungs of
intratracheally instilled mice exposed to a single 50 mul
dose of curdlan at 10(-7), 10(-8), 10(-9), and 10(-10)
M/animal (=4 mug to 4 ng curdlan/kg lung wt). Dectin-1 mRNA
transcription and expression was observed in bronchiolar
epithelium, alveolar macrophages (AMs), and alveolar type
II cells (ATIIs) of lungs exposed to 4 mug to 40 ng
curdlan/kg lung wt, at both time points. Compared to
controls, array analysis revealed that 54 of 83 genes
assayed were significantly modulated by curdlan. mRNA
transcription patterns showed both dose and time
dependency, with highest transcription levels in 10(-7) and
10(-8) M treatment animals, especially at 4-h PE. Nine gene
mRNA transcripts (Ccl3, Ccl11, Ccl17, Ifng, Il1alpha, Il-
20, TNF-alpha, Tnfrsf1b, and CD40lg) were significantly
expressed at all doses suggesting they may have a central
role in immunomodulating curdlan exposures. IHC revealed
Ccl3, Il1-alpha, and TNF-alpha expression in bronchiolar
epithelium, AMs and ATIIs illustrate the important
immunomodulatory role that these cells have in the
recognition of, and response to glucan. Collectively, these
results confirm the inflammatory nature of curdlan and
demonstrate the complex of inflammation-associated gene
responses induced by (1-3)-beta-D: glucan in triple helical
forms. These observations also provide a biological basis
for the irritant and inflammatory response to curdlan
observed in humans and animals in experimental studies.

Chem Biol Interact. 2010 Jan 5;183(1):113-24.

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

Miller JD, Sun M, Gilyan A, Roy J, Rand TG.

Department of Chemistry, Carleton University, Ottawa,
Ontario, Canada K1S 5B6.

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

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