Peripheral Th17/Treg imbalance in patients with atherosclerotic cerebral infarction

Abstract

Objective: CD4(+)CD25(+) regulatory T (Treg) cells and Th17 cells play important roles in peripheral immunity. Oxidized low-density lipoprotein (ox-LDL) is an instrumental factor in atherogenesis. However, the changes of Th17/Treg cells in patients with acute cerebral Infarction (ACI) and impact on Th17/Treg by ox-LDL are not clear. Here, we examined the balance of Th17/Treg in ACI patients and the effect of ox-LDL on this balance.

Materials and methods: The frequencies of Th17 and Treg cells, key transcription factors and relevant cytokines were examined in patients with ACI, Transient ischemic attack (TIA) and controls. The correlations of cytokines, inflammatory biomarkers and ox-LDL in serum to Th17/Treg frequency, and the effects of ox-LDL on Th17/Treg cells in vitro were analyzed.

Results: ACI patients have shown a significant increase of Th17 frequency, RORγt expression and Th17 related cytokines (IL-17 and IL-6 ) levels, and a clear decline of Treg frequency, Foxp3 expression, suppressive function and regulatory cytokines (IL-10 and TGF-β1) levels. Furthermore, TIA patients also have notable variation as compared to control group. Serum ox-LDL and inflammatory biomarkers were positively correlated with the frequency of Th17 cells and negatively correlated with the frequency of Treg cells. Treg and Th17 cells from ACI patients were significantly susceptible to ox-LDL-mediated alterations in vitro.

Conclusions: Th17/Treg cells were imbalanced in ACI patients, and ox-LDL may contribute to this imbalance and lead to the occurrence of ACI suggesting their pathogenetic role in ACI.

Keywords: Cerebral infarction; T helper 17; atherosclerosis; oxidized low-density lipoprotein; regulatory T cells; transient ischemic attack.

Figure 1

Flow sorting of Tregs and responder T cells. A. The dot plots show the CD4⁺CD25⁺CD127^low (right box) and CD4⁺CD25^- cells (left box) T cell-gating strategy pre-flow sorting and the subsequent purified subsets, and the purity of CD4⁺CD25⁺CD127^low and CD4⁺CD25^- T cells isolated were > 90% for each fraction. B. Over 90% of the freshly purified CD4⁺CD25⁺CD127^low T cells (marked with green)express Foxp3, and over 90% of the freshly purified CD4⁺CD25^- T cells (marked with red) did not express Foxp3.

Figure 2

Treg frequencies decreased and Th17 Frequencies increased in patients with ACI. A. Comparison of Treg expression among the 3 groups. B. Comparison of Th17 expression among the 3 groups. ◆P < 0.05 vs. Control; *P < 0.01 vs. Control; #P < 0.05 vs. TIA; ★P < 0.01 vs. TIA. C, D. Representative FACS figures of Treg (C) and Th17 cells (D) from a single patient in each group. The percentage of positive cells is shown in each panel. ACI: acute myocardial infarction; TIA: unstable angina; Control: control subjects.

Figure 3

Expression of RORγt and Foxp3 in PBMCs from controls, TIA and ACI patients was determined by real time-polymerase chain reaction (PCR). A. The ratios of RORγt/β-actin mRNA were compared in the 3 groups. B. The ratios of Foxp3/β-actin and RORγt/β-actin for the gray-scale value were compared in 3 groups. ◆P < vs. Control; *P < 0.01 vs. Control; #P < 0.05 vs. TIA.

Figure 4

Comparison of the suppressive rate of Treg cells among the controls, TIA and ACI groups (n = 5 in each group). *P < 0.01 vs. Control; #P < 0.05 vs. TIA.

Figure 5

Effects of Ox-LDL on Treg and Th17 cells from the control, TIA and ACI groups in vitro. A. Co-culture assay was performed as described in the Methods section, PBMCs from the Control, TIA and ACI groups were incubated with 1.0 μg/ml ox-LDL for 48 h. ◆P < 0.05 vs. ox-LDL-untreated group; *P < 0.01 vs. ox-LDL-untreated group. B. Average change of Treg and Th17 numbers in the 3 groups after a 48 h co-culture with 1.0 μg/ml ox-LDL. *P < 0.01 vs. control; ◆P < 0.05 vs. control; #P < 0.05 vs. TIA. C. Treg cells from the 3 groups are compromised in their suppressive properties after a 48 h co-culture with 1.0 μg/ml ox-LDL. *P < 0.05 vs no-ox-LDL group; #P < 0.01 vs. no-ox-LDL group. D. Average attenuation of the suppressive function of Treg cells in the 3 groups when exposed to ox-LDL. *P < 0.01 vs control; #P < 0.01 vs. TIA.