TLR9-mediated innate immune signaling contributes to acetaminophen-induced injury in the developing lung

Acetaminophen (APAP) exposures during human development are common. Emerging epidemiological and experimental evidence links these exposures to subsequent pulmonary morbidity; however, the underlying mechanisms remain incompletely defined. Cell-type-specific expression of the xenobiotic enzyme CYP2E1 is a critical determinant of susceptibility to APAP toxicity. CYP2E1-mediated formation of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) induces mitochondrial injury, which can trigger sterile inflammation through Toll-like receptor 9 (TLR9)-dependent innate immune signaling. The developing mouse lung is particularly vulnerable to APAP exposure at postnatal day 14 (P14), a time point within peak alveologenesis that coincides with maximal pulmonary CYP2E1 expression; however, the role of TLR9 signaling in this developmental window has remained unclear. In a preclinical model, wild-type (WT) and TLR9 knockout (TLR9 KO) male and female mice received a single, nonhepatotoxic dose of APAP (140 mg/kg, ip) at P14. Acute lung injury occurred in both genotypes, but TLR9 deficiency reduced proinflammatory target gene (Il1b, Il6) expression and associated STAT3 activation and target gene (Mmp9, Ptgs2, Cxcl2, Bclxl, Pim1) expression. Although APAP-exposed WT mice demonstrated persistent structural and functional abnormalities at P28, attenuation of the early inflammatory response in TLR9 KO mice preserved lung architecture and pulmonary function. These findings identify TLR9-dependent innate immune activation as a mechanistic link between early-life APAP exposure and impaired lung development. The data further suggest that limiting inflammation during the critical window of alveologenesis may preserve normal lung maturation and reduce later pulmonary morbidity. Further studies are warranted to define the clinical relevance of these findings.NEW & NOTEWORTHY Acute lung injury following acetaminophen (APAP) exposures during the key developmental window of alveologenesis has been previously described. Here, we highlight that eliminating TLR9-mediated immune signaling and associated induction of key proinflammatory mediators attenuated impaired lung development and associated dysfunction. We propose that an underlying mechanism linked with sterile inflammation contributes to abnormal pulmonary development following APAP exposure, and that further investigation is necessary for application in a clinical environment.