Particulate matter-induced NETosis promotes peroxisomal degradation via pexophagy in neutrophils

Neutrophil extracellular traps (NETs) are chomatin-based structures that contribute to the defense mechanisms of a host; however, NETs can also drive inflammation when dysregulated. Particulate matter (PM), a major air pollutant, has been shown to induce the formation of NETs, yet the impact of PM on peroxisomal homeostasis in neutrophils remains poorly understood. Thus, this study aimed to examine the effects of PM exposure on NETosis and peroxisomal dynamics in bone marrow-derived neutrophils (BMDNs) and differentiated HL-60 (dHL-60) cells. RNA sequencing was performed on PM-treated BMDNs, identifying 2436 differentially expressed genes (DEGs). These included the upregulation of NETosis-related genes (Padi4, Mpo, H3f3b) and genes involved in peroxisomal quality control and degradation, such as Pex5, Ubb, Rps27a, Nbr1, Map1lc3b, and Sqstm1. Functionally, PM exposure (50-100 μg/mL) increased reactive oxygen species (ROS) levels and induced PAD4 and citrullinated histone H3 (citH3) expression, indicating that NETosis is dependent on ROS. In parallel, peroxisomal markers, including ACOX1, PEX5, PMP70, and catalase, were dynamically regulated. Despite transient upregulation of peroxisomal markers, pexophagy was activated, as shown by increased levels of p62, beclin-1, NBR1, and LC3B, and colocalization of LC3B with peroxisomal protein PEX13. Both ROS inhibition by DPI or NAC and NET inhibition by Cl-amidine significantly attenuated pexophagy activation, indicating that PM-induced peroxisomal degradation is driven by ROS-dependent NET formation. These results reveal a ROS-NET-peroxisome axis in PM-induced neutrophil activation, offering novel insights into immune dysregulation under environmental stress and identifying potential targets for PM-induced inflammation.