Effect of Th17 and Treg axis disorder on outcomes of pulmonary arterial hypertension in connective tissue diseases

Abstract

This prospective cohort study is to verify the hypothesis that the balance of Th17 and Treg cells frequencies in the peripheral circulation is disturbed in patients with varying degrees of connective tissue diseases-associated pulmonary arterial hypertension (CTD-aPAH) and to prove the influence of Th17/Treg imbalance on prognosis. We detected the frequencies and absolute counts of Th17 and Treg cells and related serum cytokines secretion and expressions of key transcription factors in 117 patients with connective tissue diseases (CTD), 53 patients with CTD-aPAH, and 48 healthy volunteers. Moreover, the median value according to levels of Th17/Treg ratios in patients with CTD-aPAH was chosen as basis of group division for survival analysis. CTD-aPAH patients revealed significant increase in peripheral Th17 cells, Th17-related cytokines, and ROR γt mRNA levels. They also presented a significant decrease in Treg cells, Treg-related cytokines, and Foxp3 mRNA levels as compared with CTD patients and healthy controls. More importantly, the Th17/Treg ratio was significantly related to the severity and prognosis of CTD-aPAH. This study indicated that the Th17/Treg axis disorder plays a critical role in CTD-aPAH. Furthermore, the dynamic balance between Th17 and Treg cells was likely to influence prognosis of patients with CTD-aPAH.

Figure 1

Frequencies and absolute counts of circulating Treg and Th17 cells as well as the ratio of Th17/Treg in CTD and CTD-aPAH patients and healthy controls. PBMCs from studied subjects were stained with labeled anti-human antibodies as described in Section 2. Flow cytometry dot-plots showed that the gating strategies were used in identification of Treg (gated on CD4⁺ cells for detection of CD4⁺ CD25⁺ Foxp3⁺ and CD4⁺CD25⁺ CD127⁻ cells) and Th17 cells (gated on CD4⁺ cells for detection of CD4⁺IL-17⁺ cells). (a) The dot-plots showed the gating strategies of circulating CD4⁺CD25⁺ Foxp3⁺ Treg, CD4⁺CD25⁺ CD127⁻ Treg and Th17 cells in control, CTD and CTD-aPAH groups. (b) Statistical analysis revealed the frequencies of CD4⁺ CD25⁺ Foxp3⁺ Treg and CD4⁺ CD25⁺ CD127⁻ Treg populations among the CD4⁺ T cells in healthy controls (n = 20) and in patients with CTD (n = 18) or CTD-aPAH (n = 23). (c) Frequencies (left) and absolute counts (right) of circulating Treg cells (CD4⁺CD25⁺ Foxp3⁺) in control, CTD and CTD-aPAH groups. (d) Frequencies (left) and absolute counts (right) of circulating Th17 cells in control, CTD and CTD-aPAH groups. (e) The ratios of Th17/Treg in control, CTD and CTD-aPAH groups. (f) Th17 and Treg cells frequencies and the ratios of Th17/Treg in subgroups of mild to moderate CTD-aPAH and severe CTD-aPAH. *Compared with control group, P < 0.05; ^#Compared with CTD group, P < 0.05.

Figure 2

Expression of ROR γt and Foxp3 mRNA in CTD and CTD-aPAH patients and healthy controls. mRNA expression in PBMCs from studied subjects was measured by real-time quantitative polymerase chain reaction. The result was normalized relative to GAPDH. (a) The expression of ROR γt mRNA in CTD, CTD-aPAH, and control group. (b) The expression of Foxp3 mRNA in CTD, CTD-aPAH, and control group. (c) The expression of RORγt and Foxp3 mRNA in subgroups of mild to moderate CTD-aPAH and severe CTD-aPAH. *Compared with control group, P < 0.01; ^#Compared with CTD group, P < 0.01.

Figure 3

Correlation between Th17/Treg ratios and related cytokines; expression of ROR γt and Foxp3 mRNA and markers of disease severity in patients with CTD-aPAH. (a) Ratios of Treg to CD4⁺ cells negatively correlate with ratios of Th17 to CD4⁺ cells. (b) Ratios of Th17/Treg positively correlate with IL-17 concentrations. (c) Ratios of Th17/Treg positively correlate with expression of ROR γt mRNA. (d) Ratios of Th17/Treg negatively correlate with TGF-β concentrations. (e) Ratios of Th17/Treg negatively correlate with expression of Foxp3 mRNA. (f) Ratios of Th17/Treg positively correlate with levels of sPAP. (g) Ratios of Th17/Treg positively correlate with NT-pro BNP concentrations.

Figure 4

Kaplan-Meier survival analysis of patients with CTD-aPAH. Curves showed overall survival time for patients with CTD-aPAH who had lower or higher Th17/Treg ratios.