Divergent TLR2 and TLR4 Activation by Fungal Spores and Species Diversity in Dust from Waste Sorting Plants

Abstract

This manuscript presents the results of an exploratory study on the relationships between NF-κB response through Toll-like receptor (TLR) activation by dust characterized by fungal spore concentrations and species diversity. Personal total dust samples were collected from Norwegian waste sorting plants and then characterized for fungal spores and fungal species diversity, as well as for other bioaerosol components, including endotoxins and actinobacteria. The ability of the dust to induce an NF-κB response by activating TLR2 and TLR4 in vitro was evaluated, as well as the relationship between such responses and quantifiable bioaerosol components. The average concentrations of bioaerosols were 7.23 mg total dust m^-3, 4.49 × 10⁵ fungal spores m^-3, 814 endotoxin units m^-3, and 0.6 × 10⁵ actinobacteria m^-3. The mean diversity measurements were 326, 0.59, and 3.39 for fungal richness, evenness, and Shannon index, respectively. Overall, fungal operational taxonomic units (OTUs) belonging to the Ascomycota phylum were most abundant (55%), followed by Basidiomycota (33%) and Mucoromycota (3%). All samples induced significant NF-κB responses through TLR2 and TLR4 activation. While fungal spore levels were positively associated with TLR2 and TLR4 activation, there was a trend that fungal species richness was negatively associated with the activation of these receptors. This observation supports the existence of divergent immunological response relationships between TLR activation and fungal spore levels on one hand and between TLR activation and fungal species diversity on the other. Such relationships seem to be described for the first time for dust from waste facilities. IMPORTANCE This manuscript presents results on multifactorial characterization of bioaerosol exposure in Norwegian waste sorting plants and the potential of such airborne dust to induce NF-κB reactions through TLR2 and TLR4 activations in an in vitro reporter cell model system. Our data revealed that increasing fungal spore levels in the dust is associated with increased activation of TLR2 and TLR4, whereas increasing fungal OTU richness is associated with decreasing activation of these receptors. The NF-κB-induced responses by the collected dust represent, therefore, effective measures of potential key immunological effects induced by a complex mixture of hazardous components, including characterized factors such as endotoxins, fungal spores, bacteria, and many other uncharacterized components. The key immunological events reported here are suggested as holistic alternatives to today's bioaerosol exposure characterization approaches for epidemiological studies in the future.

Keywords: TLR2 and TLR4; bioaerosols; mycobiome; waste workers.

FIG 1

Micrographs of spores from fungi (A) and actinobacteria (B).

FIG 2

Taxonomic distribution of the fungal communities detected from four recycling plants (companies 1 to 4). (a and b) Proportional relative abundances of airborne fungal compositions at phyla (a) and order (b) levels. The data represent average reads per sample. (c) Hierarchical clustering-based heat plots for proportional abundances of different genera that significantly vary in their abundance among the four companies.

FIG 3

Nonmetric multidimensional scaling (NMDS) ordination analysis for fungal communities detected in personal airborne samples from workers in the four recycling plants. (a) Plot represents samples color coded by companies 1 to 4. Vectors (TLR2 and TLR4 activation variables) shown with the arrow had significant effects (P < 0.05) on the ordination configuration. (b) Plot showing the fungal OTU composition of four recycling plants. The ordination plot is based on all fungal OTUs present, but only the most common 25 OTUs are shown here, which accounted for 66% of the total reads. The number codes correspond to Rhizopus (19), Melamspora (20), Alternaria (21), Penicillium (22), Cyllindrobasidium (23), Hypholoma (24), and Pycnopezia (25).

FIG 4

Picture gallery from the waste treatment plants. (1) Volunteer worker at the reception and inspection with bag pack containing sampling devices. (2) Pressed waste materials for stockage. (3) Worker with personal dust-collecting devices in the breathing zone. (4) Operating machine. (5) Bulk of waste materials for energy production.