Low-dose multi-walled carbon nanotubes are non-inflammatory alone but amplify lipopolysaccharide-induced cytokine responses in A549 lung epithelial spheroids.

Background: Airborne pollutants comprise biological agents such as lipopolysaccharide (LPS) and engineered nanomaterials, including carbon nanotubes (CNTs), which are increasingly prevalent in industrial and consumer applications. Although the pulmonary toxicity of high-dose CNT exposure is established, the inflammatory consequences of low-dose, non-cytotoxic CNT exposure, particularly in combination with other aerosols, remain poorly understood. Methods: We investigated cytotoxicity and proinflammatory responses to multi-walled CNTs in three-dimensional A549 human epithelial spheroids, compared with A549 monolayers and MM6 monocytes. Cells were exposed to two CNTs differing in diameter (CNT1: 7-15 nm; CNT2: 110-170 nm), alone or combined with LPS. Cell viability was assessed by WST-1 assay and F-actin junction staining, while IL-8 and IL-6 production was quantified by ELISA and qPCR. Results: A549 spheroids were more susceptible to CNT-induced cytotoxicity than monolayers, with CNT1 inducing greater cytotoxicity than CNT2. At non-cytotoxic concentrations, CNTs alone did not elicit cytokine release; however, co-exposure with LPS significantly enhanced cytokine secretion in spheroids, particularly in the presence of CNT2. This synergistic inflammatory response was not observed in MM6 monocytes. Conclusion: Our study demonstrates that non-cytotoxic CNTs can potentiate LPS-driven inflammation in A549 epithelial spheroids, suggesting potential respiratory risks from combined exposure to environmental endotoxins and nanomaterials, even at low doses.