Transfer of Th17 from Adult Spontaneous Hypertensive Rats Accelerates Development of Hypertension in Juvenile Spontaneous Hypertensive Rats

Abstract

Hypertension develops in the recipient rats that are transferred with the activated T helper (Th) 17 cells of the donor rats exposed to high-fructose or high-salt intake. This result suggests that a pathologic Th17 cell plays a role in the development and maintenance of hypertension. Here, we tested the hypothesis that the transfer of Th17 cells from adult spontaneous hypertensive rats (SHR) accelerates the development of hypertension in juvenile SHR. The tail-cuff method was used to measure systolic blood pressure. T cell (Th17 and regulatory T (Treg)) profiling was analyzed by flow cytometry. The expressions of Th17-related interleukin- (IL-) 17A and Treg-related IL-10 were measured by ELISA. Th17 cells isolated from adult SHR were intraperitoneally injected into juvenile recipient SHR and Wistar-Kyoto rats (WKY). SHR exhibited prominent development of hypertension at 15 weeks. The proportion of CD4⁺IL-17A⁺ (Th17) cells among Th cells increased whereas the proportion of CD4⁺FoxP3⁺ (Treg) cells decreased in SHR, as compared to WKY. The serum levels of IL-17A increased gradually with aging in SHR, but the serum levels of IL-10 did not. The serum levels of IL-17A and IL-10 seemed to be well related to the proportion of Th17 cells and Treg cells, respectively. Injection of Th17 cells isolated from adult SHR accelerates the development of hypertension in juvenile SHR but not in juvenile WKY though it increased the proportion of Th17 cells in juvenile recipient WKY and SHR. The transfer of Th17 cells from adult SHR accelerates the development of hypertension in juvenile SHR. These results implicate that the hypertension in SHR is ascribed to activation of Th17 cells.

Figure 1

The systolic blood pressure (SBP), body weight, and serum levels of cytokines in Wistar-Kyoto rats (WKY) and spontaneous hypertensive rats (SHR). (a) SBP was measured using the tail-cuff method. SBP of SHR significantly higher at adult (15 weeks) compared with juvenile (5 weeks). (b) The body weights of the WKY and SHR revealed comparable in 5 weeks and 15 weeks. The serum levels of (c) a proinflammatory cytokine (IL-17A) and (d) an anti-inflammatory cytokine (IL-10) were measured using ELISA in juvenile and adult rats. The serum levels of IL-17A were higher in SHR than in WKY, whereas the levels of IL-10 were lower in SHR than in WKY. Data are the mean ± SEM of 6 independent experiments. Most SEMs were too small to be seen out of symbols (^∗P < 0.05, ^∗∗P < 0.01 vs. WKY; ^##P < 0.01 vs. corresponding juvenile rats by two-way ANOVA followed by the post hoc Duncan test).

Figure 2

T cell profiles of the peripheral blood mononuclear cells (PBMCs) and spleen in WKY and SHR. Quantification of flow cytometric analysis revealed the proportion of CD3⁺CD4⁺ (Th) cells of the (a) PBMCs and (b) spleen in juvenile and adult rats. The proportion of CD3⁺CD4⁺ (Th) cells of PBMCs was higher in WKY than in SHR. The proportion of CD3⁺CD4⁺ (Th) cells of the spleen revealed comparable in 5 weeks and 15 weeks, but increased at 15 weeks compared with 5 weeks (^∗P < 0.05, ^∗∗P < 0.01 vs. WKY; ^##P < 0.01 vs. corresponding juvenile rats by two-way ANOVA followed by the post hoc Duncan test). Quantification of flow cytometric analysis revealed the proportion of CD4⁺IL-17A⁺ (Th17) and CD4⁺FoxP3⁺ (Treg) cells among CD3⁺CD4⁺ (Th) cells of the (c) PBMCs and (d) spleen in adult WKY and SHR by the gating strategy of flow cytometry. There was a higher proportion of CD4⁺IL-17A⁺ (Th17) cells of PBMCs in SHR than in WKY, and there was a lower proportion of CD4⁺FoxP3⁺ (Treg) cells in SHR than in WKY. Data are the mean ± SEM of 6 independent experiments (^∗P < 0.05 vs. WKY by Student's t-test).

Figure 3

Accelerated development of hypertension in juvenile SHR by transfer of adult Th17 cells. (a) Schematic illustration shows that Th17 cells from adult donor SHR or the vehicle was transferred into juvenile recipient WKY or SHR. Intraperitoneal injections with Th17 cells from adult SHR or the vehicle were administrated into juvenile recipient WKY or SHR. SBP was measured in juvenile recipient (b) WKY and (c) SHR for 4 days using the tail-cuff method. The transfer of Th17 cells from adult SHR into juvenile recipient SHR significantly increased SBP than the vehicle group, which was not seen in juvenile recipient WKY. Data are the mean ± SEM of 6 independent experiments (^∗∗P < 0.01 vs. vehicle group by repeated measures ANOVA).

Figure 4

T cell profiles of PBMCs in juvenile recipient WKY and SHR for 4 days after transferring Th17 cells from adult donor SHR. Representative flow cytometric analysis revealed the proportion of (a) CD4⁺IL-17A⁺ (Th17) cells and (b) CD4⁺FoxP3⁺ (Treg) cells of PBMCs in juvenile recipient WKY or SHR. (c) Quantification revealed that CD4⁺IL-17A⁺ (Th17) cells after the transfer of Th17 cells significantly increased compared with the vehicle in juvenile recipient WKY and SHR. (d) Quantification revealed that the proportion of CD4⁺FoxP3⁺ (Treg) cells after transfer of Th17 cells significantly increased compared with the vehicle in juvenile recipient SHR. Data are the mean ± SEM of 6 independent experiments (^∗P < 0.05, ^∗∗P < 0.01 vs. vehicle group by two-way ANOVA followed by the post hoc Duncan test).

Figure 5

T cell profiles of the spleen in juvenile recipient WKY and SHR for 4 days after transferring Th17 cells from adult donor SHR. Representative flow cytometric analysis revealed the proportion of (a) CD4⁺IL-17A⁺ (Th17) cells and (b) CD4⁺FoxP3⁺ (Treg) cells of the spleen in juvenile recipient WKY or SHR. (c) Quantification revealed that the proportion of CD4⁺IL-17A⁺ (Th17) cells after transfer of Th17 cells significantly increased compared with the vehicle in juvenile recipient WKY and SHR. (d) Quantification revealed that the proportion of CD4⁺FoxP3⁺ (Treg) cells after transfer of Th17 cells significantly increased compared with the vehicle in juvenile recipient SHR. Data are the mean ± SEM of 6 independent experiments (^∗P < 0.05, ^∗∗P < 0.01 vs. vehicle group by two-way ANOVA followed by the post hoc Duncan test).

Figure 6

T cell profiles of the kidney in juvenile recipient WKY and SHR for 4 days after transferring Th17 cells from adult donor SHR. Representative flow cytometric analysis revealed the proportion of (a) CD4⁺IL-17A⁺ (Th17) cells and (b) CD4⁺FoxP3⁺ (Treg) cells of kidney lymphocytes in juvenile recipient WKY or SHR. Quantification revealed that the proportion of (c) CD4⁺IL-17A⁺ (Th17) cells and (d) CD4⁺FoxP3⁺ (Treg) cells after transfer of Th17 cells was comparable with transferring the vehicle in juvenile recipient WKY and SHR. Data are the mean ± SEM of 4 independent experiments.