6:2 Cl-PFESA induces sex-dependent lung inflammation through NLRP12 inflammasome activation and pulmonary microbiota dysbiosis

Widespread detection of 6:2 chlorinated polyfluoroalkyl ether sulfonic acid (6:2 Cl-PFESA), a major substitute for perfluorooctane sulfonate (PFOS), has been reported in various environmental and human biological samples. Yet, its lung inflammation and sex-dependent effects have not been studied. In this study, mice were orally gavaged a single dose of 10 mg/kg/BW 6:2 Cl-PFESA or vehicle control for 28 days. Males exhibited greater susceptibility than females, as evidenced by earlier body weight loss, more severe lung histopathology, elevated inflammatory cytokine expression, and increased pulmonary lipopolysaccharide accumulation. The transcriptomic analysis also showed extensive sexual dimorphism, with a significantly larger number of differentially expressed genes in the female lungs compared with males, with minimal overlap between sexes. The NOD-like receptor signaling pathway was identified as a major responsive pathway, with upregulation of NLRP12 in both sexes and sex-specific down-regulation of Interferon regulatory factor 7 ( Irf7 ) in males and Bcl-2-like protein 1 ( Bcl2l1 ( Bcl-xL ) in females. In vitro siRNA-mediated knockdown of NLRP12 in A549 and THP-1 cells confirmed its role as a positive mediator of the 6:2 Cl-PFESA-induced inflammatory response. Furthermore, 6:2 Cl-PFESA promoted NLRP12 inflammasome-associated activation, characterized by enhanced NLRP12 expression, caspase-1 cleavage, mature IL-1β processing, increased IL-18 secretion, and elevated caspase-1 enzymatic activity. Notably, 6:2 Cl-PFESA significantly altered the lung microbial community, enriched by Proteobacteria and depleted by Firmicutes and Bacteroidetes . Pro-inflammatory genera, including Stenotrophomonas (e.g., S. maltophilia ) and Chryseobacterium (e.g., C. indologenes ), were enriched predominantly in males. Collectively, these findings identified NLRP12 inflammasome-associated signaling and sex-dependent microbial dysbiosis as key mechanisms underlying male-biased lung inflammation, informing sex-specific risk assessment of PFAS alternatives.