Distinct pro-inflammatory responses to pristine and microbially contaminated PET nanoplastics in a human alveolar cell co-culture

Background: The small size of nanoplastics (NPs; <1 & micro;m in diameter) facilitates airborne transport, inhalation, and deposition in the lungs, raising significant concerns about potential effects on human health. In occupational settings, such as waste management and recycling facilities, exposure to NPs carrying microbial contaminants may pose an additional health risk to workers. In the present study, we investigated pulmonary cytotoxicity and pro-inflammatory responses after exposure to polyethylene terephthalate nanoplastics (PET-NPs) with or without microbial contaminants. Methods: PET-NPs were synthesized from a post-consumer juice bottle (PET b001) and commodity PET pellets (PET c000). The presence of microbial contaminants was assessed via receptor activation in HEK293 Toll-like receptor (TLR) reporter cells expressing TLR2 or TLR4. Co-cultures of human alveolar epithelial cells (A549) and monocyte-derived macrophages (dTHP-1) were exposed to PET-NPs (0, 10 or 100 mu g/mL) that tested either negative or positive for TLR2 and TLR4 activation. After 24 h, cell viability was measured, and cytokine responses were quantified at both mRNA and protein levels. Results: PET b001 activated TLR2 and TLR4, indicating the presence of biologically active microbial components, whereas PET c000 showed no activation. In A549/dTHP-1 co-cultures, PET b001 (10 and 100 mu g/mL) significantly increased IL-1B, IL-6, IL-8, and TNF mRNA levels and IL-6 and IL-8 protein secretion. In comparison, PET c000 selectively increased IL-8 mRNA levels and protein secretion, and only at the highest tested concentration (100 mu g/mL). No changes in cell viability were observed for either particle type. Conclusion: We found that the pro-inflammatory responses to PET-NPs are largely mediated by associated microbial components rather than the polymer itself, highlighting the importance of accounting for environmental context when evaluating their health risks. No evidence of cytotoxicity was observed, as cell viability remained unchanged. Our results further emphasize the need to assess microbial contamination prior to toxicity testing and point to potential occupational health risks in plastic waste and recycling environments.