New England Eye Center/Department of Ophthalmology, Tufts University School of Medicine, Boston, Massachusetts, United States.
Department of Medicine, Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States.
Invest Ophthalmol Vis Sci. 2023 Feb 1;64(2):11. doi: 10.1167/iovs.64.2.11.
The current study was designed to examine the role of the NLRP3 inflammasome pathway in the clearance of Pseudomonas aeruginosa (PA) infection in mouse corneas.
Corneas of wild type and NLRP3-/- mice were infected with PA. The severity of bacterial keratitis was graded on days 1 and 3 post-infection by slit lamp, and then corneas were harvested for: (i) bacterial enumeration, (ii) immune cell analysis by flow cytometry, (iii) immunoblotting analysis of cleaved caspase-1 and IL-1β, and (iv) IL-1β quantification by ELISA. In parallel experiments, severity of keratitis was examined in the wild-type mice receiving a subconjunctival injection of a highly selective NLRP3 inhibitor immediately prior to infection.
Compared to wild type mice, NLRP3-/- mice exhibited more severe infection, as indicated by an increase in opacity score and an increase in bacterial load. The hallmark of inflammasome assembly is the activation of proinflammatory caspase-1 and IL-1β by cleavage of their precursors, pro-caspase-1 and pro-IL-1β, respectively. Accordingly, increased severity of infection in the NLRP3-/- mice was associated with reduced levels of cleaved forms of caspase-1 and IL-1β and reduced IL-1β+ neutrophil infiltration in infected corneas. Likewise, corneas of mice receiving subconjunctival injections of NLRP3 inhibitor exhibited increased bacterial load, and reduced IL-1β expression.
Activation of NLRP3 pathway is required for the clearance of PA infection in mouse corneas.
本研究旨在探讨 Nlrp3 炎性小体通路在清除小鼠角膜铜绿假单胞菌（PA）感染中的作用。
用 PA 感染野生型和 Nlrp3-/- 小鼠的角膜。在感染后第 1 天和第 3 天通过裂隙灯对细菌角膜炎的严重程度进行分级，然后采集角膜用于：（i）细菌计数，（ii）通过流式细胞术分析免疫细胞，（iii）免疫印迹分析裂解的 caspase-1 和 IL-1β，以及（iv）通过 ELISA 定量 IL-1β。在平行实验中，在感染前立即向野生型小鼠的结膜下注射高选择性 Nlrp3 抑制剂，观察角膜炎的严重程度。
与野生型小鼠相比，Nlrp3-/- 小鼠的感染更为严重，表现为混浊评分增加和细菌负荷增加。炎性小体组装的标志是前体 pro-caspase-1 和 pro-IL-1β 的切割，导致炎症性 caspase-1 和 IL-1β 的激活。因此，Nlrp3-/- 小鼠感染的严重程度增加与裂解形式的 caspase-1 和 IL-1β 水平降低以及感染角膜中 IL-1β+中性粒细胞浸润减少有关。同样，接受结膜下注射 NLRP3 抑制剂的小鼠的角膜细菌负荷增加，IL-1β 表达减少。
Nlrp3 通路的激活是清除小鼠角膜 PA 感染所必需的。
