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Nan Gao, Fushin X Yu; ISG15 as a Key player in corneal innate defense against Pseudomonas aeruginosa keratitis. . Invest. Ophthalmol. Vis. Sci. 2017;58(8):5766.
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© ARVO (1962-2015); The Authors (2016-present)
To elucidate the role of ISG15 in controlling corneal inflammation and innate immunity in B6 mouse model of Pseudomonas aeruginosa keratitis.
Cultured primary HCECs were challenged with 100:1 HK PA. Cells or conditioned media were collected at 4-16 h for Western blotting analysis. To determine the role of ISG15 signaling in P. aeruginosa keratitis, the corneas of WT or ISG15 KO mouse corneas were inoculated with 104 P. aeruginosa. Disease progress was monitored by digital photograph, clinical scoring, and MPO measurement for PMN infiltration. The expressions of various pro-inflammatory and innate defense genes were determined by real-time PCR. Cryostat sections of mouse corneas were immunostained with antibodies against F4/80, iNOS, ARG1 and PMN. Macrophages were collected from WT or ISG15 KO mice peritoneal cavity and treated with heat-killed PA for 1 hour and subjected to real-time PCR analysis.
Heat-killed ATCC infection induced the expressions of ISG15 in PHECEs and conditioned media. Knockdown of ISG15 greatly increased severity of keratitis, including markedly increased bacterial burden and PMN infiltration. Knockdown ISG15 induce downregulation AMPs while and upregulate the expression of IL1β. ISG15 deficiency increased infiltration of PMN and macrophages, both type 1 and 2, in infected corneas. In vitro analysis of cultured macrophages revealed that challenging of isolated macrophages with heat-killed PA markedly induced the expression of IL-1β, IL-23, Arg1, CXCL2, iNOS in an ISG15-dependent manner as well as CD206 and CXCL10 in an ISG15-independent manner.
ISG15 is an innate immune response gene and its deficiency increases corneal susceptibility to P. aeruginosa and altered the expressions of many cytokines and defense molecules in vivo and in cultured macrophages.
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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