title: Reactive oxygen species activated NLRP3 inflammasomes initiate inflammation in hyperosmolarity stressed human corneal epithelial cells and environment-induced dry eye patients.
abstract: In studies on dry eye (DE) disease, an association has been identified between tear film hyperosmolarity and inflammation severity elicited through receptor-induced increases in proinflammatory cytokine and chemokine release. These immune reactions might be mediated by inflammasomes, macromolecular complexes mounted around the NLRP3 protein and can be activated by reactive oxygen species (ROS) over-generation. Hence in this study we determine whether: a) ROS activated NLRP3 inflammasomes mediate hyperosmotic stress-induced inflammation in human corneal epithelial cells (HCECs); b) the ROS-NLRP3-IL-1β axis activation is associated with environment-induced DE. Immortalized HCECs were exposed to 500 mOsm medium in the presence and absence of a ROS inhibitor, N-acetyl-l-cysteine (NAC). HCECs transfected with NLRP3 siRNA or a negative control (NC) siRNA. Intracellular ROS was measured by fluorometric analysis using the probe 2',7'-dichlorofluorescin diacetate (DCFH-DA). Real-time PCR evaluated NLRP3, ASC, pro-caspase-1 and pro-IL-1β mRNA levels. Western blot analysis assessed NLRP3 protein expression whereas caspase-1 activity was determined with a fluorometric assay. Bioactive IL-1β release was assessed by ELISA. ROS production, NLRP3 inflammasome and pro-IL-1β gene expression as well as IL-1β secretion were also evaluated in the conjunctival epithelial cells and tear fluid samples of environment-induced DE patients and normal subjects. NAC suppressed hyperosmolarity-induced rises in ROS levels, NLRP3 inflammasome formation and activation, caspase-1 activity and IL-1β release. On the other hand, NLRP3 siRNA knockdown inhibited hyperosmotic stress-induced NLRP3 activation, which led to ASC, pro-caspase-1 and pro-IL-1β mRNA down-regulation followed by suppression of associated caspase-1 activity and IL-1β secretion. In addition, in ocular surface samples of environment-induced DE patients, ROS generation, NLRP3, ASC, pro-caspase-1 and pro-IL-1β gene expression as well as IL-1β secretion were upregulated. Taken together, NLRP3 mediated innate immune responses triggered by rises in ROS generation induce inflammation in hyperosmotic stressed HCECs. ROS-NLRP3-IL-1β signaling pathway might play a priming role in environment-induced DE development.
authors.affiliation: School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Zhejiang 325027, China.
authors.forename: Qinxiang
authors.initials: Q
authors.lastname: Zheng
authors.affiliation: School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Zhejiang 325027, China.
authors.forename: Yueping
authors.initials: Y
authors.lastname: Ren
authors.affiliation: School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Zhejiang 325027, China.
authors.forename: Peter S
authors.initials: PS
authors.lastname: Reinach
authors.affiliation: School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Zhejiang 325027, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Xiao
authors.affiliation: School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Zhejiang 325027, China.
authors.forename: Huihui
authors.initials: H
authors.lastname: Lu
authors.affiliation: School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Zhejiang 325027, China.
authors.forename: Yirui
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Zhejiang 325027, China. Electronic address: jqu@wzmc.net.
authors.forename: Jia
authors.initials: J
authors.lastname: Qu
authors.affiliation: School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Zhejiang 325027, China. Electronic address: chenweimd@hotmail.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000328:Adult; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D020170:Caspase 1 / Q000378:metabolism; D002460:Cell Line; D015352:Dry Eye Syndromes / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000378:metabolism*; D005260:Female; D055785:Gene Knockdown Techniques; D006801:Humans; D058847:Inflammasomes / Q000378:metabolism*; D007249:Inflammation / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D007634:Keratitis / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D009997:Osmotic Pressure / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D014162:Transfection; D055815:Young Adult
chemical_list: D002352:Carrier Proteins; C508585:IL1B protein, human; D058847:Inflammasomes; D053583:Interleukin-1beta; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D020170:Caspase 1; D000111:Acetylcysteine
keywords: Caspase-1; Dry eye; IL-1β; Inflammasomes; Inflammation; NLRP3; Reactive oxygen species; Tear film hyperosmolarity