Combined exposure to benzo(a)pyrene and dibutyl phthalate aggravates pro-inflammatory macrophage polarization in spleen via pyroptosis involving cathepsin B

Humans are often simultaneously exposed to benzo(a)pyrene (BaP) and dibutyl phthalate (DBP) through consumption of food and water. Yet, direct evidence of the link between BaP and DBP co-exposure and the risk of splenic injury is lacking. In the present study, we established the rats and primary splenic macrophages models to evaluate the effects of BaP or/and DBP exposure on spleen and underlying mechanisms. Compared to the single exposure or control groups, the co-exposure group showed more severe spleen damage and higher production of pro-inflammatory cytokines. Co-exposure to BaP and DBP resulted in a 1.79-fold, 2.11-fold and 1.9-fold increase in the M1 macrophage markers iNOS, NLRP3 (pyroptosis marker protein) and cathepsin B (CTSB), respectively, and a 0.8-fold decrease in the M2 macrophage marker Arg1 in vivo. The more prominent effects in perturbation of imbalance in M1/M2 polarization (iNOS, 2.25-fold; Arg1, 0.55-fold), pyroptosis (NLRP3, 1.43-fold), and excess CTSB (1.07-fold) in macrophages caused by BaP and DBP co-exposure in vitro were also found. Notably, MCC950 (the NLRP3-specific inhibitor) treatment attenuated the pro-inflammatory macrophage polarization and following pro-inflammatory cytokine production triggered by BaP and DBP co-exposure. Furthermore, CA-074Me (the CTSB-specific inhibitor) suppressed the macrophages pyroptosis, pro-inflammatory macrophage polarization, and secretion of pro-inflammatory cytokine induced by BaP and DBP co-exposure. In conclusion, this study indicates co-exposure to BaP and DBP poses a higher risk of spleen injury. Pro-inflammatory macrophage polarization regulated by pyroptosis involving CTSB underlies the spleen injury caused by BaP and DBP co-exposure.