Abstract 217: PCSK6 Is A Key Regulator Of Immune Status In Mice And Its Ablation Increases Atherosclerotic Plaque Burden In A Bone Marrow Transplant Model

Abstract

Introduction: Proprotein convertase subtilisins/kexins (PCSKs) activate cytokines and growth factors and have been implicated in various cancers. We have previously shown that PCSK6 is a key protease modulating smooth muscle cell response in atherosclerosis, but its expression also correlated positively with typical markers of T lymphocytes and macrophages in plaques and it was localised in the proximity of these cells. Here, we hypothesized that PCSK6 may be involved in modulating inflammatory responses and aimed to elucidate its role in a hyperlipidemic mice model. Methods: Detailed immunophenotyping using histology, FACS-, OLINK- and ELISA-based analyses and primary cell cultures was used to compare Pcsk6 -/- and littermate controls. Atherosclerosis was evaluated in Ldlr -/- mice upon bone marrow transplant with Pcsk6 -/- or wild-type bone-marrow. Results: At baseline Pcks6 -/- mice showed an enrichment of pro-inflammatory cytokines Ccl2, Ccl3, Ccl20, Cxcl1 and in particular Il17a and Il17f in plasma compared controls. Spleens of Pcsk6 -/- mice had an increased number of germinal centres and FACS analysis showed that they contained significantly more CD8+ T cells. Microarray analysis of spleens from Pcsk6 -/- vs. controls confirmed that T cell markers CD4, CD3E and CD3G were upregulated. In vitro , splenocytes isolated from Pcsk6 -/- mice secreted more IFN-γ, IL-2 and IL-10 than controls upon stimulation with α-CD3 and α-CD28 antibodies. Moreover, peritoneal macrophages from Pcsk6 -/- mice secreted more TNF-α, MCP-1, IL-6 and IL-10 compared to control mice upon LPS stimulation. Interestingly, bone marrow derived macrophages from Pcsk6 -/- mice were also more prone to lipid uptake. Finally, in vivo transplantation of Pcsk6 -/- bone marrow to Ldlr -/- mice led to increased atherosclerotic plaque burden compared to controls, as quantified in the aortic root. Conclusions: PCSK6 ablation led to increased number of CD8+ T cells, as well as macrophage and cytokine activation. Transplantation of Pcsk6 -/- bone marrow resulted in an increase in atherosclerotic plaque burden compared to controls. Taken together, these results indicate that PCSK6 is a key regulator of the immune system, though the exact mechanisms involved require further investigation.