Immunomodulation in atherosclerosis : Impact of Th balance and CD1d-restricted NKT cells

Abstract: Atherosclerosis is a slow and progressive disease consisting of lipid deposition in arterial walls. Studies in both human and experimental animal models have shown the involvement of the immune system in the pathogenesis. The colocalization of macrophages and activated T cells suggests that the atherosclerotic lesions could be the sites of an antigen specific activation. Given their abundance in the lesion, lipids might also be some of these antigens. The Natural killer T (NKT) cells can recognize lipid antigens presented by the CD I molecules. After activation, the NKT cell secretes large amount of both IFNgamma and IL-4, Upon activation, naïve CD4+ T cell, can follow two different pathways of differentiation and become either Th1 or Th2 cells. Th1 cells produce pro-inflammatory cytokines such as IFNgamma, TNFalpha and IL-2 while Th2 cells produce IL-4, IL-5 and IL-13. Therefore, the presence of the cytokines IL-12, IL-18 (two pro-Th1 cytokines), IFNgamma and TNFalpha in atherosclerotic plaques, while very little of IL-4 and IL-5 can be found suggests that the net effect of the activation of the immune system in atherosclerosis may depend on the Th1/Th2 balance. The aim of this thesis was to 1) evaluate the effect on atherosclerosis of an in vivo blocking of the Th1 differentiation pathway by a drug administration, 2) develop an in vivo T cell gene transfer method, 3) determine the role of NKT cells in atherosclerosis and 4) analyze the mechanisms by which NKT cells influence atherosclerosis. In the first study, we modulated the immune response of the atherosclerosis-prone apoE-/- mice by injection of pentoxifylline (PTX), an inhibitor of the Th1 differentiation pathway. 12-week PTX treatment reduced atherosclerotic lesion size by 60%. In parallel, the proportion of IFNgamma-producing Th1 spleen lymphocytes was significantly reduced by PTX, and lesion size was correlated to the proportion of IFNgamma T cells. This study demonstrates that the Th1 immune response associated with atherosclerosis is deleterious. We developed an in vivo intrasplenic electroporation (ISE) technique, a non viral gene transfer method. We found ISE to be an efficient gene transfer method which can be used to express secreted or intracellular proteins transiently. We also found that after ISE, spleen T lymphocytes could be transfected and recirculate into extrasplenic locations. This strategy constitutes a new method to manipulate the immune response that can be used in various experimental setups. The role of the NKT cells in atherosclerosis was investigated in two different ways: either by stimulating specifically the NKT cells by injections of alphaGalCer in apoE-/- mice or by crossing this mouse strain with CD1-/mice. We demonstrated that CD1d-restricted NKT cells exacerbate atherosclerosis. Indeed, the apoE-/-CD1d-/- mice had a 25% decrease in lesion size compared to control apoE-/- mice while alphaGalCer treatment induced a 50% increase compared to PBS-injected mice. In the follow-up study, we showed that the NKT cells play a pro-atherogenic role during several month of lesion development in apoE-/- mice. However, the effect was most pronounced during lesion initiation and/or early progression and became less prominent during later phases. We also propose that the activation of NKT cells influence atherosclerosis development in mice by inducing an increase in chemokines (RANTE, MCP-1, eotaxin) and adhesion molecules (ICAM-1 and VCAM-I) expression, therefore favoriting the leukocyte recruitment to the vessel wall. In conclusion, we found that a controlled immunomodulation of Th balance in atherosclerosis could lead to a beneficial effect. We developed a method that will allow us to perform such modulation in situ. Finally, we have discovered a new key player in atherosclerosis, the NKT cell. Future studies aiming at controlling NKT cell activation will be of great potential for future therapy.