June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Destructive neuroinflammation triggered by activation of the NLRP3 inflammasome in the glaucomatous optic nerve head
Author Affiliations & Notes
  • Meredith S Gregory-Ksander
    Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary/HMS, Boston, Massachusetts, United States
  • Fei Fei
    Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary/HMS, Boston, Massachusetts, United States
    Ophthalmology, Fourth Military Medical University, Xian, Shannxi Province, China
  • anitha krishnan
    Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary/HMS, Boston, Massachusetts, United States
  • Te-Chen Tzeng
    Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, United States
  • Deepa Subramanian
    Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary/HMS, Boston, Massachusetts, United States
  • Douglas Golenbock
    Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, United States
  • Ann Marshak-Rothstein
    Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States
  • Bruce Ksander
    Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary/HMS, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Meredith Gregory-Ksander, None; Fei Fei, None; anitha krishnan, None; Te-Chen Tzeng, None; Deepa Subramanian, None; Douglas Golenbock, None; Ann Marshak-Rothstein, None; Bruce Ksander, None
  • Footnotes
    Support  Bright Focus Foundation, Thomas R. Lee Award for Glaucoma
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 2021. doi:
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      Meredith S Gregory-Ksander, Fei Fei, anitha krishnan, Te-Chen Tzeng, Deepa Subramanian, Douglas Golenbock, Ann Marshak-Rothstein, Bruce Ksander; Destructive neuroinflammation triggered by activation of the NLRP3 inflammasome in the glaucomatous optic nerve head. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2021.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The importance of inflammation in glaucoma has been realized, but the mechanism by which it is triggered is incompletely understood. There is substantial evidence that the initial injury in glaucoma occurs in the optic nerve head (ONH), where activated astrocytes produce mediators that can damage axons and trigger inflammatory cell recruitment. We discovered that human and mouse ONH astrocytes constitutively express NLRP3, a critical regulator of inflammation and part of a multi-protein complex that contains the adapter protein ASC and activates caspase-1 to produce IL-1β and IL-18. We hypothesize that activation of the NLRP3 inflammasome in ONH astrocytes initiates inflammation and axonal damage in glaucoma.

Methods : NLRP3 expression in human and mouse ONH astrocytes was determined by immunofluorescence, PCR, and Western blot. Speck formation, a hallmark of inflammasome activation, was monitored in vivo using a fluorescent reporter mouse (ASC-citrine/Cre+). Intracameral injection of microbeads (controls: uninjected or saline-only) was used to elevate IOP in WT C57BL/6J mice and C57BL/6 KO mice (NLRP3 KO, ASC KO, IL-1R KO, and IL-18 KO). IOP was monitored by rebound tonometry. At designated time points, mice were euthanized and the ONH and retina were processed for: (i) qPCR (GFAP, IL-1β, IL-18, TNFα), (ii) WB (NLRP3, caspase-1, IL-1β, IL-18), (iii) immunostaining (Iba1, GFAP, NLRP3), and (iv) RGC and axon counts.

Results : NLRP3 is constitutively expressed in the astrocytes of normal human and mouse ONH. WT and KO mice displayed a similar increase in IOP for 21 days as compared with controls. The absence of NLRP3, ASC, IL-1R or IL-18 resulted in a significant reduction in RGC and axon loss, as compared to WT controls. At 7 days post microbead injection, the ASC-citrine reporter mouse revealed increased speck formation in the ONH, but not retina, that co-localized with NLRP3 staining. The formation of ASC specks in the ONH coincided with the induction of pro-inflammatory cytokines (GFAP, IL-1β, IL-18, and TNFα) and accumulation of Iba1+ immune cells in the ONH.

Conclusions : The components of the NLRP3 inflammasome are constitutively expressed in normal ONH astrocytes but remain inactive. Our data indicate that elevated IOP-induced axonal damage and death of RGCs is dependent upon destructive neuroinflammation triggered by the activation of the NLRP3 inflammasome in the ONH.

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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