Högskolan i Skövde

N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone, commonly called 6PPD-quinone (6PPD-Q), is an environmental transformation product formed when the antiozonant and antioxidant 6PPD, widely used in tires, reacts with ozone. 6PPD-Q enters the environment through tire wear particles and roadway runoff and has been linked to acute toxicity in aquatic and mammalian models, raising concerns about its potential effects on human health. However, its impact on human immune responses remains unclear. This study aimed to investigate the effects of 6PPD-Q on inflammasome formation and activation, as well as on inflammatory signaling, in differentiated THP-1-ASC-GFP macrophage-like cells. Specifically, this study focused on NLRP3 inflammasome-associated ASC speck formation and interleukin-1 beta (IL-1β) secretion after inflammasome activation. ASC speck formation was assessed by live-cell imaging every 30 minutes for 12 hours after exposure to 0, 1, 10, 50, 100, or 200 ng/mL 6PPD-Q, while IL-1β secretion was measured by ELISA at 1, 3, and 12 hours in cells exposed to 0, 10, 50, or 100 ng/mL 6PPD-Q. No statistically significant differences in IL-1β release were observed between 6PPD-Q-treated cells and untreated controls at any time point. Similarly, live-cell imaging did not reveal a significant dose-dependent effect of 6PPD-Q on ASC speck formation. High variability and outliers were noted in both assays, potentially limiting the detection of subtle effects. Overall, these findings suggest that, under the conditions tested, 6PPD-Q does not significantly alter IL-1β secretion or ASC speck formation in human macrophage-like cells. The observed variability underscores the need for further studies with improved experimental consistency, increased biological replication, and assessment of additional functional and mechanistic endpoints, such as caspase-1 activation, cell viability, or mitochondrial function, to clarify the immunomodulatory potential of 6PPD-Q in human cells.