Data from: Upregulation of NLRP3 inflammasome in the tears and ocular surface of dry eye patients

Purpose: To evaluate the mRNA and protein expressions of NLRP3 inflammasome and its downstream inflammatory factors in human dry eye. Methods: We recruited 54 patients with Sjögren’s syndrome dry eye (SSDE), 50 patients with non-Sjögren’s syndrome dry eye (NSSDE), and 46 healthy controls. Tear film breakup time (TBUT), Schirmer I test, and fluorescein staining (FL) were performed on all subjects. Tear samples were obtained to analyze the inflammatory cytokine levels of IL-1β and IL-18 via enzyme-linked immunosorbent (ELISA). Conjunctival impression cytology (CIC) specimens were collected to detect the mRNA expression of NLRP3, caspase-1, IL-1β, and IL-18 using quantitative RT-PCR, and the protein expression of NLRP3 and caspase-1 by Western blotting. Results: NLRP3 mRNA expression showed higher levels in both dry eye groups compared with controls, with a comparably significant elevation in the SSDE group (relative 2.47-fold upregulation, p<0.05). NLRP3 protein expression was also increased in SSDE group (relative1.94-fold upregulation) compared with the controls. mRNA expression of caspase-1 was significantly upregulated in both SSDE (relative 1.44-fold upregulation, p<0.05) and NSSDE (relative 1.32-fold upregulation, p<0.05). Procaspase-1 protein level was increased in SSDE (relative 1.84-fold upregulation) and NSSDE (relative 1.12-fold upregulation) versus controls; and caspase-1 protein expression was also increased in SSDE (relative 1.49-fold upregulation) and NSSDE (relative 1.17-fold upregulation) compared with the controls. The patients with SSDE and NSSDE had higher IL-1β and IL-18 mRNA values and protein expressions than the controls did. The relative mRNA expression of IL-1β upregulated 3.59-fold (p<0.001) in SSDE and 2.13-fold (p<0.01) in NSSDE compared with the controls. IL-1β protein level also showed significant upregulation in SSDE (p=0.01; vs. controls groups). IL-18 mRNA expression levels were significantly upregulated in the SSDE (relative 2.97-fold upregulation, p=0.001) and NSSDE (relative 2.05-fold upregulation, p=0.001) groups compared with the controls; tear IL-18 concentrations were also significantly increased in the SSDE (p<0.001) and NSSDE (p<0.05) groups. Conclusions: In the current study, we found that mRNA and protein expressions of NLRP3 inflammasome were upregulated in human dry eyes, especially in SSDE; the downstream inflammatory factors caspase-1, IL-1β, and IL-18 were also elevated in dry eye patients. These observations suggest the involvement of NLRP3 inflammasome in the onset and development of the inflammation in dry eye.

研究目的：评估NLRP3炎性小体（NLRP3 inflammasome）及其下游炎症因子在人干眼症中的mRNA与蛋白表达水平。研究方法：本研究共纳入54例干燥综合征相关性干眼症（Sjögren’s syndrome dry eye, SSDE）患者、50例非干燥综合征相关性干眼症（non-Sjögren’s syndrome dry eye, NSSDE）患者及46例健康对照受试者。所有受试者均接受泪膜破裂时间（tear film breakup time, TBUT）、泪液分泌试验I（Schirmer I test）及荧光素染色（fluorescein staining, FL）检查。采集泪液样本，通过酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）检测IL-1β与IL-18的炎症细胞因子水平；采集结膜印迹细胞学（conjunctival impression cytology, CIC）标本，采用定量RT-PCR检测NLRP3、caspase-1、IL-1β及IL-18的mRNA表达水平，并通过蛋白质印迹法（Western blotting）检测NLRP3与caspase-1的蛋白表达水平。研究结果：相较于健康对照组，两个干眼症组的NLRP3 mRNA表达水平均显著升高，其中干燥综合征相关性干眼症组的上调幅度更为显著（相对表达上调2.47倍，p<0.05）。干燥综合征相关性干眼症组的NLRP3蛋白表达水平同样高于对照组（相对表达上调1.94倍）。caspase-1的mRNA表达水平在干燥综合征相关性干眼症组（相对表达上调1.44倍，p<0.05）与非干燥综合征相关性干眼症组（相对表达上调1.32倍，p<0.05）中均显著上调。与对照组相比，干燥综合征相关性干眼症组的procaspase-1蛋白水平（相对表达上调1.84倍）与非干燥综合征相关性干眼症组的procaspase-1蛋白水平（相对表达上调1.12倍）均有所升高；同时，干燥综合征相关性干眼症组（相对表达上调1.49倍）与非干燥综合征相关性干眼症组（相对表达上调1.17倍）的caspase-1蛋白表达水平也均高于对照组。干燥综合征相关性干眼症与非干燥综合征相关性干眼症患者的IL-1β、IL-18的mRNA表达水平及蛋白表达水平均高于对照组。与对照组相比，干燥综合征相关性干眼症组的IL-1β mRNA相对表达上调3.59倍（p<0.001），非干燥综合征相关性干眼症组上调2.13倍（p<0.01）；干燥综合征相关性干眼症组的IL-1β蛋白水平同样显著高于对照组（p=0.01）。与对照组相比，干燥综合征相关性干眼症组（相对表达上调2.97倍，p=0.001）与非干燥综合征相关性干眼症组（相对表达上调2.05倍，p=0.001）的IL-18 mRNA表达水平均显著升高；泪液中IL-18的浓度在干燥综合征相关性干眼症组（p<0.001）与非干燥综合征相关性干眼症组（p<0.05）中也同样显著升高。研究结论：本研究发现，人干眼症患者的NLRP3炎性小体mRNA与蛋白表达水平均出现上调，尤以干燥综合征相关性干眼症患者为著；其下游炎症因子caspase-1、IL-1β及IL-18的表达水平在干眼症患者中也均有所升高。上述结果提示，NLRP3炎性小体参与了干眼症炎症反应的发生与发展过程。