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Jiangyuan Gao, Haohang Rachel Chen, Sijia Cao, Jing Z Cui, Aikun Wang, Joanne A Matsubara; The reduction of XIAP is associated with NLRP3 inflammasome activation in RPE: implications for AMD pathogenesis. Invest. Ophthalmol. Vis. Sci. 2018;59(9):347. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Age-related macular degeneration (AMD) is a complex disease with many contributing risk factors. So far, several candidate cellular and biochemical pathways have been hypothesized to play a role in AMD pathogenesis. Our work and those of others, point towards the activation of the NLRP3 inflammasome as a pathway associated with retinal pigment epithelium (RPE) cell demise. X-linked inhibitor of apoptosis protein (XIAP) is a potent anti-apoptosis factor, which has been recently shown to regulate NLRP3 inflammasome activation in non-ocular cells. However, it is not clear whether other types of inflammasomes are involved in AMD. The purpose of this study is to characterize XIAP’s immune regulatory role in RPE and identify the relevant inflammasome subtype(s).
An in vitro inflammasome activation model was established using L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) stimulation on low-passage RPE cells. Validated Silencer® Select human XIAP 21-mer siRNA and the recommended control siRNA were purchased from Life Technologies and applied before Leu-Leu-OMe stimulation. mRNA levels of XIAP, NLRP1, NLRP6, NLRC4, caspase-4 and 18S RNA were quantified by RT-PCR. Cell culture supernatants were concentrated using a StrataClean resin method for the detection of secreted IL-18. Cell protein lysates were collected for the measurement of cleaved caspase-1 p20, XIAP and GAPDH. Rat eye tissue protein lysates were utilized for the analysis of XIAP protein levels. All data are presented as Mean ± SD. p<0.05 is considered statistically significant.
The XIAP protein level was significantly increased when inflammasome was inhibited at the activation, but not the priming step, in vivo. XIAP siRNA knockdown alone reduced the mRNA levels of NLRP1, NLRP6, NLRC4 and caspase-4 in vitro. However, when XIAP siRNA was given as a pre-treatment, Leu-Leu-OMe triggered enhanced IL-18 secretion with no change on the caspase-1 cleavage, compared to cells stimulated with Leu-Leu-OMe alone.
Together, these data suggest NLRP3 inflammasome might be the major subtype in response to XIAP mediated dual inhibition in RPE cell death. It also provides insights into the biological consequences of inflammasome activity in RPE and reveals the caspase-1/XIAP/IL-18 axis as a target for broader applications in AMD biology and treatment design.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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