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Fang Bian, Eugene Volpe, Stephen C Pflugfelder, De-Quan Li, Cintia S. De Paiva, Ocular Surface Center, Department of Ophthalmology, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas; Activation of NLRP3 Inflammasome Pathway in Murine Corneal Alkali Burn Model. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4612.
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Alkali burns of the ocular surface are among the most devastating injuries to the eye. The potential role of innate immunity in the inflammatory process of ocular alkali burns remains unclear. This study tested the hypothesis that innate immunity activates the Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome pathway after ocular injury.
A unilateral alkali burn was produced with 1 N NaOH in the cornea of C57BL/6 mice under general anesthesia. Whole corneal tissues from normal and alkali-burned mice were collected at days 2 and 5 post-injury for histology. The mRNA expression of NLRP3 was measured by reverse transcription and RT-PCR and protein expression of NLRP3 and its adaptor molecule, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), were investigated by immunofluorescent staining in frozen sections. The activity of caspase-1 in whole cornea lysates was measured using a fluorometric assay. Expression of the downstream inflammasome cytokine, IL-1β, was evaluated by RT-PCR.
On day 2 post-injury, a significant increase of NLRP3 (up to 31-fold) and IL-1β (up to 54-fold) mRNA transcripts was observed in the alkali-burned corneas compared with corneal controls (P<0.0001 for both). Although levels of NLRP3 (13-fold) and IL-1β (5-fold) transcripts in the alkali-burned cornea decreased at 5 days, they remained elevated compared to normal corneas (P<0.0001 and P<0.05). Immunostaining confirmed the RT-PCR results with increased immunoreactivity of NLRP3 and ASC in all layers of the corneal epithelium at days 2 and 5 post-injury. Caspase-1 activity significantly increased in the alkali-burned cornea at days 2 and 5 post-injury compared to control corneas (P<0.05).
Corneal alkali burns activate the NLRP3-ASC-caspase-1 immune signaling pathway in cornea, leading to the proteolytic processing and secretion of the proinflammatory cytokine, IL-1β. Together, these results identified a novel innate immune signaling pathway (NLRP3-ASC-caspase-1-IL-1β) activated by ocular alkali burn and provided a mechanistic basis for optimizing therapeutic intervention in patients.
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