The Role of IL-1R8 in Modulating Macrophage Inflammation: Implications for Atherosclerosis.

Abstract

Atherosclerosis is a chronic inflammatory disease of the cardiovascular system characterized by macrophage-driven arterial plaque accumulation. Interleukin (IL)-37 is an antiinflammatory cytokine within the IL-1 family which suppresses inflammatory immune response by complexing with the receptors IL-1R8 and IL-18Rα. Because IL-37 and its receptor are robustly expressed in the macrophage, I sought to determine if IL-1R8 plays a key role in macrophage inflammation that may in turn influence the development of atherosclerosis. Initially the proposed anti-inflammatory effects of IL-37 was investigated by co-treating mice bone marrow-derived macrophages (BMDM) with recombinant IL-37 protein and various inflammatory stimuli. Although inflammation was not quelled by IL-37, interestingly, IL-1R8 was consistently downregulated by LPS and IFNg treatment. To test whether IL-1R8 inhibits inflammation, I overexpressed IL-1R8 in the mouse macrophage cell line, RAW 264.7 cells, and BMDM by transfection with IL-1R8 or GFP expression plasmids. Transfected cells were treated with pro-inflammatory (LPS, IFNg, TNFa) or anti-inflammatory (IL-4) mediators, as well as IL- 37. Transfection of RAW 264.7 cells with IL-1R8 resulted in 35-to-60-fold increases of IL-1R8 transcript expression compared to controls as measured by RT-qPCR, whereas IL-1R8 protein level was increased 6 to9 folds compared to GFP-transfected control cells, as measured by Western blot. Interestingly, transfection with IL-1R8 resulted in robust overexpression of IL-1R8 with a molecular weight of 70-95 kDa, indicating significant glycosylation, which was confirmed by glycosidase treatment. In contrast to my initial hypothesis, overexpression of IL-1R8 in RAW cells and BMDM had a pro-inflammatory effect overall. However, when treated with recombinant IL-37 protein as the ligand for IL-1R8, the IL-1R8-transfected RAW cells showed reduced inflammatory gene expression compared to GFP controls treated with the same stimulus. iv Additionally, siRNA knockdown of IL-1R8 resulted in significantly higher inflammatory gene expression, such as IL-6, IL-1b, iNOS, MCP1/CCL2, and TNFa in both RAW 264.7 cells and BMDM. The experiments described show that IL-1R8, along with its ligand IL-37, plays a role in regulating macrophage inflammation, although future studies are needed to further elucidate its exact molecular mechanism.