Abstract
Purpose :
Nucleotide binding oligomerization domain 2 (NOD2) is an intracellular sensor of bacterial peptidoglycan and belong to the Nod-like receptor (NLR) family of innate immune proteins that play pleiotropic roles in inflammatory responses. Docosahexaenoic acid (DHA)-derived lipid mediators, elovanoids (ELVs), are downregulated in the retinas and retinal pigment epithelium (RPE) of mutant Adipor1 and Mfrp mice, leading to a selective reduction of ELV and NPD1 precursors. This leads to impaired retinal function and loss of photoreceptor cells. Now we aim to determine whether ELVs reduce chronic inflammation by modulating NOD2 activation in the NLRP3 inflammasome response.
Methods :
In vitro analysis on RPE cells were pre-treated for 30 min with 300 nM ELV. Cellular stress was induced using IL-1α (24 h) followed by H2O2 (1200 μM for 6-8 h) or oxidized LDL (ox-LDL, 120-150 µg/ml) for 24 h. In vivo analyses were conducted with eyes from Adipor1-/- and Mfrprd6 mice. Detection of NOD2 and NRLP3 inflammasome markers was done using qPCR, Western Blot, and immunocytochemistry/histochemistry.
Results :
We showed that IL-1α or ox-LDL increased the expression of NLRP3 pro-inflammatory signaling markers in RPE cells. ELVs suppress expression of IL-1β, IL-6, IL-8, and NOD2 by 80%, 90%, 65%, and 60%, respectively (IL-1α activation), and 80% and 65% (ox-LDL) for IL-8 and NOD2, respectively. Adipor1-/- and Mfrprd6 had a 47% and 189% increase in caspase 1 activity, respectively, reported by FAM-FLICA assay.
Conclusions :
ELVs downregulate the expression of innate immunity markers, e.g., NOD2 activation signaling cascade: IL-1β, IL-6, as well as NLRP3 inflammasome and its activation response genes (IL-23α and IL-1β). Adipor1-/- and Mfrprd6 are susceptible to uncontrolled inflammatory events leading to microglial/macrophage invasion and caspase 1-dependent tissue damage, suggesting pyroptosis action. Thus, ELVs open potential therapeutics avenues targeting NLR family proteins.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.