Proinflammatory macrophage polarization is driven by NOX4/PPARγ axis-mediated oxidative-inflammatory crosstalk in CEES-induced lung injury

Sulfur mustard (SM), a well-known vesicant chemical warfare agent, induces acute lung injury through mechanisms that remain incompletely understood. This study aimed to delineate the specific contributions of oxidative stress and inflammatory pathways in SM-mediated pulmonary damage. A C57BL/6 J mouse model exposed to aerosolized 2-chloroethyl ethyl sulfide (CEES, a sulfur mustard analogue) and an in vitro macrophage model were established. Evaluations were conducted using pulmonary function tests, transcriptome sequencing, RT-qPCR, Western blot, and ELISA. Functional validation was performed through pharmacological activation of peroxisome proliferator-activated receptor γ (PPARγ) and application of a NADPH oxidase 4 (NOX4) inhibitor. CEES exposure was found to induce airway obstruction in mice, accompanied by alveolar structural damage and significant infiltration of pro-inflammatory macrophages. Levels of ROS and MDA were elevated in lung tissues, and antioxidant enzyme activity showed an initial compensatory increase followed by exhaustion. Increased levels of TNF-α and IL-6, together with decreased levels of Arg-1 and IL-10, were observed in serum and lung homogenates. Transcriptomic analysis and subsequent validation experiments indicated that CEES up-regulated the expression of NOX4 and suppressed PPARγ. In vitro, overexpression of PPARγ promoted the expression of IL-10, Arg-1, and CD206, while suppressing TNF-α, IL-6, and iNOS. Inhibition of NOX4 expression reduced ROS and MDA levels, restored PPARγ expression, and promoted a shift from M1 to M2 polarization. Collectively, these findings elucidate a central regulatory axis wherein CEES triggers macrophage polarization imbalance via the NOX4/PPARγ-mediated integration of oxidative stress and inflammatory signaling, thereby amplifying lung injury. This study lays the foundation for developing targeted dual-action therapeutic strategies addressing both oxidative and inflammatory injury, with significant relevance to the field of chemical warfare agent protection.