Abstract
Background: The P2X7 receptor (P2X7R) is an ATP-gated cation channel highly expressed on microglia. Prolonged activation induces release of the proinflammatory cytokine IL-1b and formation of a large, cytolytic pore. Development of CNS-penetrant P2X7R antagonists is aimed at reducing the persistent presence of proinflammatory cytokines and activated microglia observed in many neurodegenerative (Alzheimer's and Parkinson's disease) and neuropsychiatric conditions (Major depressive disorder). The endogenous human antimicrobial peptide LL-37 was also found to promote pore formation and IL-1b secretion,1 with elevated levels of the peptide detected in the Alzheimer's brain.2 It is still unclear if LL-37 effects are fully or partially P2X7R-mediated. We aimed to determine if LL-37 requires interaction with the P2X7R to exert its effects by using cell lines that do or do not express the P2X7R, and if LL-37-induced effects can be inhibited by P2X7R antagonists.
Method: Cells expressing the P2X7R (HEK293 cells transfected with human P2X7R, and THP-1-derived human macrophages) and cells without the P2X7R (wild type HEK293 cells) were treated with LL-37 alone, and with P2R or P2X7R-antagonists. Pore formation was measured using a dye uptake assay, and IL-1b release by THP-1 cells quantified by ELISA.
Results: Dye uptake in wild type HEK293 cells strongly suggested LL-37 did not require P2X7R expression to exert its effects. This was confirmed through the inability of orthosteric and allosteric P2X7R antagonists (and combination of the two) to inhibit LL-37-induced dye uptake. However a general P2 receptor antagonist, PPADS (100 uM), was found to inhibit LL-37. Similar inhibition of dye uptake and IL-1b release by a selective P2Y13R antagonist, MRS2211 (100 uM), strongly implicates the involvement of this P2Y receptor in LL-37-mediated effects.
Conclusions: LL-37-induced pore formation and IL-1b are not P2X7R-dependent, and may instead be mediated by P2Y13R.
(1) Elssner A. et al. (2004) J Immunol 172(8):4987-94
(2) Lee M. et al. (2015) Biochem Pharmacol 94(2):130-41
