The development and activation of T cells
The development and activation of T citric acid sale play an important role in the progression of atherosclerosis. As for activation and differentiation of T cells, TCR signalling pathway is pivotal. ZAP70 plays a crucial role in formulation of TCR–microclusters, initiation of TCR signalling pathway, moreover activation of T cells and secretion of cytokines (Hashimoto-Tane and Saito, 2016). Thus, inhibition of ZAP70 by interacting with the TCR–CD3 may be effective for treating patients with autoimmune diseases, organ transplants or chronic inflammatory diseases. A previous study from our group found that lck, which regulates ZAP70 phosphorylation in TCR signalling, is associated with HSP65 inducing atherosclerosis. Down-regulating lck could enhance cholesterol efflux capacity of T cells (Luo et al., 2016). However, studies investigating the connection between ZAP70 and RCT or atherosclerosis are scarce. Previous study from our group showed that ZAP70 also regulated HSP65-induced RCT dysfunction (Hu et al., 2018). Using a ZAP70 mutant cell line, P116 cell line, we also demonstrated that ZAP70-deficiency had influence on RCT after activation of T cell by CD3/CD28. ZAP70 deletion significantly benefited RCT of T cells. Cholesterol efflux capacity, representative of RCT, was found remarkably improved in ZAP70-defient cells (Fig. 3A). Additionally, mRNA and proteins expression of ABCA1, ABCG1, SR-BI, which regulate RCT, were found increased in the ZAP70-mutant cell line (Fig. 3B–D). Together, these data demonstrated that ZAP70-deficiency could enhance RCT of T cells. In addition to immune cells, cytokines could also regulate the development of atherosclerosis. Several novel strategies for inhibiting pro-atherosclerotic cytokines have been found to be effective for anti-atherosclerosis (Back and Hansson, 2015). Canakinumab, a monoclonal antibody binding to IL-1b and blocking IL-1b interaction with its receptor, was demonstrated be effective on reducing atherosclerosis. Recently, the CANTOS trial has reported that canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering (Ridker et al., 2017). In our results, both mRNA and proteins secretion of pro-atherosclerotic cytokines, TNF-α, IFN-γ, IL-2 and IL-6, were found reduced in P116 cells compared with Jurkat cells (Fig. 2A and B). ZAP70 deficiency also reduced phosphorylation of ERK, JNK and p38 MAPK (Fig. 2C and D), which contributed to secretion of cytokines and activation of T cells. Besides, either lack of ZAP70 or inhibiting the phosphorylation of ERK, JNK and p38 MAPK with their specific inhibitors could reduce TNF-α, IFN-γ and IL6. Inhibiting the phosphorylation of ERK had additional effect of reducing IL-2 like lack of ZAP70. These findings demonstrated that ZAP70 deficiency reduces inflammatory regulatory pathway and secretion of inflammatory factors probably through MAPK pathway. MAPK cascades are part of the downstream pathways of ZAP70 and have been shown to stimulate inflammatory response and promote progression of atherosclerosis (Muslin, 2008). Mouse peritoneal macrophages treated with ox-LDL were found that ERK1/2, p38 MAPK and JNK1/2 were all activated within 15 min, with ERK1/2 activation occurring at the earliest time point. Treating macrophages with JNK pathway inhibitor SP600125 blocked ox-LDL-induced foam cell formation (Rahaman et al., 2006). Another study found p38 MAPK inhibitor SB203580 had a similarly effect on foam cell formation in J774 macrophage cell line. Moreover, ApoE-/-mice treated with the p38 inhibitor SB203580 had reduced atheromatous lesions and decreased atherosclerotic disease progression (Seeger et al., 2010). Treating rat peritoneal macrophages with ERK inhibitor U0126 resulted in a marked reduction of lipid deposition, upregulation of ABCA1/G1 expression and suppression of CD36 expression in Ox-LDL-stimulated macrophages (Xue et al., 2016). Our study suggested that, ERK instead of JNK or p38 MAPK contributed to the increase of T-cell RCT. Treatment of U0126 enhanced cholesterol efflux capacity of T cells (Fig. 5A). Additionally, protein and mRNA expression of RCT regulatory proteins, ABCA1, ABCG1 and SR-BI, were increased after inhibiting ERK phosphorylation (Fig. 5, Fig. 6). However, inhibiting JNK or p38 MAPK did not impact on cholesterol efflux capacity or the expression of RCT regulatory proteins. Together, these data demonstrated that ZAP70 make influence on RCT of T cells probably through ERK pathway.