Platelet-regulated CD4+ T effector cell responses in atherosclerosis

Atherosclerosis (AS) is a thrombotic and inflammatory disease of the medium- and largesized arteries. Platelets participate in all stages of its formation, forming thrombus at the ruptured plaque; meanwhile, CD4+ T cells are a key driving force of inflammation, plaque formation and instability. Therefore, the interaction between CD4+ T cells and platelets are important for AS. It is vital and urgent to explore effective prevention and new treatment methods. Our earlier studies demonstrated that platelets had profound regulatory effects on CD4+ T cells. However, the mechanisms of interaction between platelets and T cell subsets, such as CD4+ effector memory T cells (Tem), CD4+ central memory T cells (Tcm), and CD4+ naïve T cells (Tn), is still elusive. The present doctoral thesis research is to elucidate the inflammatory mechanism of platelet regulation in atherosclerosis and to identify potential intervention sites for the development of a new generation of antiplatelet/antiatherosclerotic drugs. The specific goals of this thesis are: (1) To dissect the mechanisms of platelets regulating in individual CD4+ T cell subsets of Tem, Tcm, and Tn; and (2) To elucidate the effects of individual platelet-derived mediators such as transforming growth factor β (TGFβ); to investigate the impact of platelet-derived TGFβ1 deficiency in AS and CD4+ T effector responses with a murine model.

Firstly, we conducted a study on the regulation of platelets on CD4+ Tem. Specifically, when platelets were co-cultured with CD4+ Tem, they enhanced the type 1 T helper (Th1) response transiently, while continuously enhanced the activation of regulatory T (Treg) cells. Platelet factor 4 (PF4) was the key to regulate Tem response. PF4 acted through the receptor CXCR3 to attenuate the activity of protein kinase B (Akt) and reduce the peroxisome proliferatoractivated receptor γ coactivator 1 (PGC1α) phosphorylation, leading to elevated mitochondrial transcription factor A (TFAM) expression and mitochondrial biogenesis. The latter increased adenosine triphosphate (ATP) and reactive oxygen species (ROS) production and subsequently enhanced Treg and Th1 responses. Similar to that for Tem, PF4 exerted regulatory effects on Tcm via CXCR3-initiated intracellular signalling and mitochondrial biogenesis and enhanced Th1 and Treg responses. Unlike Tem, we found that platelets and PF4 enhanced Tcm responses mainly via cell proliferation, which was related to mitochondrial biogenesis and metabolism. Hence, platelets and PF4 enhanced Tcm responses through cell proliferation, while potentiated Tem responses via cell activation.

After exploring the regulation mechanisms of platelets on the memory T cell (TM), we continued to explore the interaction between Tn and platelets. Platelets regulated the effector responses of Th1, Th2, Th17, and Treg cells in a complex manner that involved close cooperation of TGFβ and PF4. Briefly, PF4 with low concentrations enhanced TGFβ signalling through heteromerizing with type III TGFβ receptor (TGFBRIII) and subsequently enhanced type II TGFβ receptor (TGFBRII) expression and TGFβ signalling. However, high concentrations of PF4 directly bound to TGFBRII, blocked TGFβ-TGFBRII ligation, thereby inhibited TGFβ signalling and produced an opposite effect. In addition, the blockade of platelet-Tn cell contact weakened platelet effects, and the injection of PF4-immobilized particles into the spleen of PF4-deficient mice mimicked the platelet effect, indicating the importance of direct platelet-Tn contact and platelet-bound PF4 for optimal regulation of platelets. Thus, platelets exerted a context-dependent regulation on the effector response of Tn cells through a PF4-TGFβ duet.

As interruption of TGFβ signalling exacerbates atherosclerosis, the effect of platelet-derived TGFβ on atherosclerosis is, however, still unclear. So the fourth research aim was to clarify the effect of platelet-specific TGFβ1 deficiency on CD4+ T effector cell responses and atherosclerosis with a murine atherosclerosis model. We created a murine strain with plateletspecific TGFβ-deficiency (plt-TGFβ-/-), and established the mouse model of atherosclerosis through low-density lipoprotein receptor (LDLR) functional knockout and a high-fat diet during 10-15 weeks, in plt-TGFβ-/- mice and their control littermates. We found that plt- TGFβ-/- increased the total cholesterol (Cho), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG), and that plt-TGFβ-/- was related to decreases of Treg cells and Treg cell activities, as well as altered Th1 and Th17 activation. The aortic root cryosections and en face oil red O (ORO) staining of the aorta showed significantly enhanced atherosclerotic lesion formation in plt-TGFβ-/- mice. RNA sequencing and proteomic analyses also showed signs of CD4+ T effector cell and macrophage activation in plt-TGFβ-/- mice. Therefore, plateletderived TGFβ has a protective effect against atherosclerosis.

Together, the thesis work shows how platelets regulate the different CD4+ T cell subsets Tem (Paper I), Tcm (Paper II), and Tn (Paper III); and that platelet-selective TGFβ deficiency aggravates atherogenesis in vivo (Paper IV).

List of scientific papers

I. Shuai Tan, Shuijie Li, Yanan Min, Anton Gisterå, Noah Moruzzi, Junhao Zhang, Yang Sun, John Andersson, Rickard E. Malmström, Miao Wang, Per-Olof Berggren, Susanne Schlisio, Wangjun Liao, Daniel F. J. Ketelhuth, Chunhong Ma, Nailin Li. Platelet factor 4 enhances CD4+ T effector memory cell responses via Akt-PGC1α-TFAM signaling-mediated mitochondrial biogenesis. J Thromb Haemost. 2020;18(10):2685-2700.
https://doi.org/10.11111/jth.15005

II. Shuai Tan, Junhao Zhang, Yang Sun, Anton Gisterå, Zi Sheng, Rickard E. Malmström, Ming Hou, Jun Peng, Chunhong Ma, Wangjun Liao, Nailin Li. Platelets enhance CD4+ central memory T cell responses via platelet factor 4-dependent mitochondrial biogenesis and cell proliferation [published online ahead of print, 2021 Jun 17]. Platelets. 2021;1-11.
https://doi.org/10.1080/09537104.2021.1936479

III. Yanan Min, Long Hao, Xinguang Liu, Shuai Tan, Hui Song, Hao Ni, Zi Sheng, Natalie Jooss, Xuena Liu, Rickard E. Malmström, Yang Sun, Jianguo Liu, Hua Tang, Chunhong Ma, Jun Peng, Ming Hou, Nailin Li. Platelets finetune effector responses of naïve CD4+ T cells via platelet factor 4-regulated transforming growth factor β signaling. [Manuscript]

IV. Shuai Tan†, Yang Sun†, Yanan Min, Anton Gisterå, Junhao Zhang, Zi Sheng, Yanmei Lu, Daniel F.J. Ketelhuth, Wangjun Liao, John Andersson, Hu Hu, Miao Wang, Ming Hou, Mingxian Zhang, Jun Peng, Chunhong Ma, Nailin Li. Platelet-specific TGFβ1 deficiency aggravates atherosclerosis, vascular inflammation, and hypercholesterolemia in mice. †These authors contributed equally. [Manuscript]