Ozone (O₃) is a prevalent air pollutant causing lung inflammation. Previous studies demonstrate that O₃ oxidizes lipids, such as cholesterol, in the airway to produce oxysterols, such as secosterol A (SecoA), which are electrophiles that are capable of forming covalent linkages preferentially with lysine residues and that consequently modify protein function. The breadth of proteins modified by this oxysterol as well as the biological consequences in the lung are unknown. By using an alkynyl-tagged form of SecoA and shotgun proteomics, we identified 135 proteins as being modified in bronchial epithelial cells. Among them was NLRP2 (NLR family pyrin domain–containing protein 2), which forms an alkynyl-tagged SecoA–protein adduct at lysine residue 1019 (K1019) in the terminal leucine-rich repeat region, a known regulatory region for NLR proteins. NLRP2 expression in airway epithelial cells was characterized, and CRISPR-Cas9 knockout (KO) and shRNA knockdown of NLRP2 were used to determine its function in O₃-induced inflammation. No evidence for NLPR2 inflammasome formation or an NLRP2-dependent increase in caspase-1 activity in response to O₃ was observed. O₃-induced proinflammatory gene expression for CXCL2 and CXCL8/IL8 was further enhanced in NLRP2-KO cells, suggesting a negative regulatory role. Reconstitution of NLRP2-KO cells with the NLRP2 K1019 mutated to arginine partially blocked SecoA adduction and enhanced O₃-induced IL-8 release as compared with wild-type NLRP2. Together, our findings uncover NLRP2 as a highly abundant, key component of proinflammatory signaling pathways in airway epithelial cells and as a novel mediator of O₃-induced inflammation.

臭氧 (O ₃ ) 是一种常见的空气污染物，可引起肺部炎症。以前的研究表明，O ₃氧化气道中的脂质，如胆固醇，产生氧甾醇，如 secosterol A (SecoA)，它们是亲电子试剂，能够优先与赖氨酸残基形成共价键，从而改变蛋白质功能。这种氧甾醇修饰的蛋白质的广度以及在肺中的生物学后果是未知的。通过使用 SecoA 的炔基标记形式和鸟枪法蛋白质组学，我们确定了 135 种蛋白质在支气管上皮细胞中被修饰。其中包括 NLRP2（含有 NLR 家族 pyrin 结构域的蛋白质 2），它在富含亮氨酸的末端重复区（NLR 蛋白的已知调节区）的赖氨酸残基 1019 (K1019) 处形成炔基标记的 SecoA 蛋白加合物。NLRP2 在气道上皮细胞中的表达被表征，_3-诱发炎症。没有观察到 NLPR2 炎性体形成或响应于 O ₃的 NLRP2 依赖性 caspase-1 活性增加的证据。O ₃诱导的CXCL2和CXCL8 / IL8促炎基因表达在 NLRP2-KO 细胞中进一步增强，表明其具有负调节作用。与野生型 NLRP2 相比，用突变为精氨酸的 NLRP2 K1019 重建 NLRP2-KO 细胞部分阻断了 SecoA 加合并增强了 O _{3诱导的 IL-8 释放。}总之，我们的研究结果揭示了 NLRP2 是气道上皮细胞中促炎信号通路的高度丰富的关键成分，也是 O _{3的新介质}-引起的炎症。