Abstract
Purpose :
Dry eye disease (DED) stresses the cornea and the conjunctiva that triggers an innate immune activation that initiates a self-perpetuating cycle of inflammation of the ocular surface. This innate immune activation and cytokine release can lead to immune cell recruitment and infiltration and inflammatory cell death, both of which beget more inflammation that can further damage the ocular surface. Inflammasomes are activated during this process, behaving as an initial signal to trigger subsequent inflammatory cascades and cell death (pyroptosis). Inflammasomes are protein complexes composed of inflammatory cytokines that bind with the cell surface through Gasdermin D pore formation and inflammasome-mediate pyroptosis. Inhibition of Gasdermin D pore formation is critical as a downstream blocking of pro-inflammatory cytokines release and inflammatory signal propagation and therefore is a very promising target.
Methods :
In vitro results indicate that hyperosmotic stress induces NLRP3/Caspase 1/Gasdermin-D-mediated pyroptosis in human corneal epithelial cells. Cryopreserved primary monocytes isolated from 3 healthy human donors were thawed, pooled, plated, and incubated at 37 celsius for 30 minutes. Trigger media followed by SPR-397 was added followed by 20-minute incubation, followed by LPS with a 4 hour incubation, and a final addition of nigericin followed by 2 hours of incubation. Following nigericin incubation, supernatants were harvested for downstream cytokine analysis. FirePlex®-HT panel 1 kit (Abcam) was used to detect relative levels of a panel of human cytokines (IL-1β, IL-6, TNF-a, IL-8, MCP-1, IL-17A, IL-10, IL-2, IFNg, IL-4).
Results :
In primary human monocytes, SPR-397 inhibits the inflammasome-mediated release of the pro-inflammatory cytokines IL-1b, IL-6, TNFa, and IL-8. Additionally, SPR-397 potently blocks pyroptotic cell death in a dose-dependent manner following stimulation with either ATP, flagellin, or LPS+nigericin.
Conclusions :
Inhibition of the Inflammasome pore formation in vitro may prove beneficial in vivo in DED. Topical ocular delivery of SPR-397 0.25% Ophthalmic Solution targeting inhibition of Gasdermin D in tissues where dysregulated inflammasome signaling drives pathological inflammation may prove as an alternative treatment modality to current immune or cytokine suppressing DED therapies such as cyclosporine or lifitigrast eye drops.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.