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De-Quan Li, Wei Chi, XIN CHEN, Xia Hua, Fang Bian, Cintia S De Paiva, Stephen C Pflugfelder; A Novel Innate Immunity Pathway in Dry Eye: ROS Dysregulates Activity of NLRP3/NLRP6 Inflammasomes via BRCC36 and Caspase-8 Signaling. Invest. Ophthalmol. Vis. Sci. 2017;58(8):798.
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© ARVO (1962-2015); The Authors (2016-present)
The concept of innate immunity has been expanded to recognize environmental pathogens other than microbes. However, whether and how the innate immunity is initiated by epithelium in response to environmental physical challenges is largely unknown. This study was to explore potential mechanism by which ocular epithelium responds to environmental stress via NLRs-mediated innate immunity pathway in dry eye.
Two models were used: in vitro primary human corneal epithelial cells (HCECs) exposed to hyperosmolarity, and in vivo mouse ocular surface facing desiccating stress, with untreated as controls. Confluent HCEC cultures were treated for 4-24 hours in medium at iso- and hyper- osmolarity (312-450 mOsM), without or with inhibitors for NLRP3, caspase-1, caspase-8 or ROS. The cells and medium supernatant from culture, as well as eyeballs, corneal epithelium and conjunctival tissues from mouse model were collected for different assays. Gene expression was determined by RT-qPCR, protein levels were evaluated by immunostaining, Western blotting, ELISA and caspase activity assays. Intracellular ROS was measured by DCFDA assay.
In HCECs exposed to hyperosmolarity, the activated NLRP3 with suppressed NLRP6 stimulated caspase-1 activation leading to IL-1β and IL-18 maturation and secretion. NLRP3-independent caspase-8 activation was revealed to noncanonically activate caspase-1 via reciprocal regulation of NLRP3/NLRP6-mediated inflammasomes. ROS overproduction was observed to induce mitochondrial DNA oxidative damage and BRCC36 deubiquitinating activity. Furthermore, exogenous 8-OHdG significantly suppressed 450mOsM-stimulated maturation and secretion of IL-1β and IL-18 while dG had no effect. In vivo model of mouse facing desiccating environment displayed an imbalanced activation of NLRP3/NLRP6 inflammasomes and IL-1β and IL-18 mediated inflammation with stimulated caspase-8 and BRCC3 activity in corneal and conjunctival epithelia, the responses similar to in vitro HCECs under environmental stress.
Our findings uncover a novel innate immunity pathway by ocular epithelium where ROS-induced mitochondrial DNA oxidation dysregulates the activity of NLRP3/NLRP6 inflammasomes via BRCC36 and caspase-8 signaling in response to environmental stress, which provide a missing link between inflammation and environmental stress in dry eye.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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