July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Human Retinal Pigment Epithelial (hRPE) single cell genomics of NLRP3 inflammasome is modulated by Elovanoid (ELV32-6) and NeuroprotectinD1 (NPD1) in response to Uncompensated Oxidative Stress (UOS).
Author Affiliations & Notes
  • Aram Asatryan
    Neuroscience, LSUHSC, New Orleans, Louisiana, United States
  • Marie-Audrey Ines Kautzmann
    Neuroscience, LSUHSC, New Orleans, Louisiana, United States
  • Jessica Heap
    Neuroscience, LSUHSC, New Orleans, Louisiana, United States
  • Nicolas G Bazan
    Neuroscience, LSUHSC, New Orleans, Louisiana, United States
  • Footnotes
    Commercial Relationships   Aram Asatryan, None; Marie-Audrey Kautzmann, None; Jessica Heap, None; Nicolas Bazan, Elovanoid PCT# PCT/US16/21429 (P)
  • Footnotes
    Support   NEI EY005121 and the Eye, Ear, Nose and Throat Foundation
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 5561. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Aram Asatryan, Marie-Audrey Ines Kautzmann, Jessica Heap, Nicolas G Bazan; Human Retinal Pigment Epithelial (hRPE) single cell genomics of NLRP3 inflammasome is modulated by Elovanoid (ELV32-6) and NeuroprotectinD1 (NPD1) in response to Uncompensated Oxidative Stress (UOS).. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5561.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : Activation of innate immunity and UOS unresolution are associated with RPE cell damage and progression of Age-related Macular Degeneration (AMD). Drusen accumulation beneath the RPE can be “sensed” by the NLRP3 inflammasome, climaxing in the release of pro-inflammatory cytokines. The goal of this study was to delineate the significance of the newly discovered lipid mediators termed "elovanoids" in regulating inflammasome transcriptional activation on a single RPE cell using microfluidic technology.

Methods : hRPE cells were either subjected to UOS (600μM H2O2/10ng/ml TNFα) for six hours or treated with ELV32-6 and NPD1 (400nM each) with UOS then dissociated by trypsin and loaded on a medium size C1 chip (Fluidigm). Briefly: RPE cells were separated in automated C1 system which utilizes microfluidics technologies. Cells were lysed in micro-chambers and pre-amplified with human apoptosis and inflammation panel of genes. Pre-amplified samples were transferred into Biomark HD 96x96 chip for a RT-PCR amplification. Results were analyzed using Singular and Partek statistical packages.

Results : PCA and tSNE analysis show distinct clusters of gene expression for control, UOS, NPD1 and ELV32-6 treated samples. Consistent with our previous results BIRC3 (11-fold), RelB (13-fold) and cREL (7.6-fold) were upregulated with NPD1. ELV32-6 up-regulated both cREL and RelB moderately but not the BIRC3. However, both ELV32 and NPD1 suppress NLRP3 (5.5 and 15-fold) expression as well as downregulate the expression of IL1β (7 and 15-fold respectively), IL23A (2 and 4-fold) when compared with oxidative stress. NLRP3 expressing hRPE controls and UOS cells were represented by a single distribution density. In contrast, the distribution was bimodal in ELV32-6 and NPD1 treated samples.

Conclusions : Our results indicate that both ELV32-6 and NPD1 downregulate the expression of NLRP3 inflammasome and its activation response genes (IL23A and IL1β). Moreover, ELV32-6 and NPD1 seem to have distinct mechanisms of inflammasome suppression. While NPD1 acts through cREL-BIRC3 axis, ELV32 appear to have a distinct path of regulation. Despite a robust decrease in NLRP3 expression a small portion of cells did not responds to the treatment. This suggests that unveiling different-sub populations in cell culture is imperative.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×