CD4+ Regulatory T Lymphocytes Prevent Impaired Cerebral Blood Flow in Angiotensin II-Induced Hypertension

Abstract

Background Immune cells are key regulators of the vascular inflammatory response characteristic of hypertension. In hypertensive rodents, regulatory T lymphocytes (Treg, CD 4⁺ CD 25⁺) prevented vascular injury, cardiac damage, and endothelial dysfunction of mesenteric arteries. Whether Treg modulate the cerebrovascular damage induced by hypertension is unknown. Methods and Results C57 BL /6 mice were perfused with angiotensin II (Ang II ; 1000 ng/kg per minute) for 14 days and adoptive transfer of 3×10⁵ CD 4⁺ CD 25⁺ T cells was performed via 2 intravenous injections. Control mice received a sham surgery and PBS . Treg prevented Ang II -induced neurovascular uncoupling ( P<0.05) and endothelial impairment ( P<0.05), evaluated by laser Doppler flowmetry in the somatosensory cortex. The neuroprotective effect of Treg was abolished when they were isolated from mice deficient in interleukin-10. Administration of interleukin-10 (60 ng/d) to hypertensive mice prevented Ang II -induced neurovascular uncoupling ( P<0.05). Treg adoptive transfer also diminished systemic inflammation induced by Ang II ( P<0.05), examined with a peripheral blood cytokine array. Mice receiving Ang II + Treg exhibited reduced numbers of Iba-1+ cells in the brain cortex ( P<0.05) and hippocampus ( P<0.001) compared with mice infused only with Ang II. Treg prevented the increase in cerebral superoxide radicals. Overall, these effects did not appear to be directly modulated by Treg accumulating in the brain parenchyma, because only a nonsignificant number of Treg were detected in brain. Instead, Treg penetrated peripheral tissues such as the kidney, inguinal lymph nodes, and the spleen. Conclusions Treg prevent impaired cerebrovascular responses in Ang II -induced hypertension. The neuroprotective effects of Treg involve the modulation of inflammation in the brain and periphery.

Keywords: angiotensin; cerebral blood flow; inflammation; interleukin‐10; lymphocyte.

Figure 1

Schematic representation of the T lymphocyte isolation procedure and experimental timeline. A, CD4⁺ CD25⁺ T lymphocytes were isolated via magnetic bead purification from the spleens of two 8–10‐wk‐old pathogen‐free male C57BL/6 or Il10 ^−/− mice. B, Mice received 2 intravenous injections of 3×10⁵ CD4⁺ CD25⁺ cells or CD4⁺ CD25⁺ Il10 ^−/− cells (isolated from Il10 ^−/− mice) or PBS (for the control group). The adoptive transfer injections were done 7 d before and the day of Ang II (1000 ng/kg per min) or IL‐10 (60 ng/d for 14 d) minipump implantation. Systolic blood pressure was monitored the day before cerebral blood flow (CBF) analysis and tissue collection. Plasma cytokine analysis, microglia counts, and assessment of superoxide anion production were performed afterwards.

Figure 2

Effect of CD4⁺ CD25⁺ regulatory T lymphocytes on CBF. CBF responses to whisker stimulation (A) and to the endothelium‐dependent vasodilator, acetylcholine (B) following adoptive transfer of CD4⁺ CD25⁺ cells (3×10⁵) or PBS in mice infused s.c. with Ang II (1000 ng/kg per min, 14 d) or in control mice (CTL). Graphs depict the percentage increase in CBF following the stimulation with respect to its initial value, measured by laser Doppler flowmetry. Results represent mean±SEM; n=7 CTL/PBS, n=8 Ang II/PBS, n=6 CTL/CD4⁺ CD25⁺ and n=9 Ang II/CD4⁺ CD25⁺ mice in (A), n=5 CTL/PBS and Ang II/CD4⁺ CD25⁺, n=4 Ang II/PBS and CTL/CD4⁺ CD25⁺ mice in (B). Data were analyzed with 2‐way ANOVA followed by Bonferroni correction (^§ or *P<0.05, **P<0.01). Ang II indicates angiotensin II; CBF, cerebral blood flow; CTL, control.

Figure 3

IL‐10 is a key mediator of the cerebrovascular protective effect of CD4⁺ CD25⁺ regulatory T cells. CBF responses to whisker stimulation (A) and to acetylcholine (B) following adoptive transfer of CD4⁺ CD25⁺ cells (3×10⁵) isolated from mice lacking the gene for IL‐10. CD4⁺ CD25⁺ Il10 ^−/− or PBS injections were given to mice infused s.c. with Ang II (1000 ng/kg per min, 14 d) or to control mice (CTL). Graphs depict the percentage increase in CBF following the stimulation with respect to its initial value, measured by laser Doppler flowmetry. Results represent mean±SEM; n=7 CTL/PBS, n=9 Ang II/PBS and CTL/CD4⁺ CD25⁺ ll10 ^−/− and n=5 Ang II/CD4⁺ CD25⁺ ll10 ^−/− mice in (A), n=5 CTL/PBS, Ang II/PBS and Ang II/CD4⁺ CD25⁺ Il10 ^−/− and n=6 CTL/CD4⁺ CD25⁺ Il10 ^−/− mice in (B). Data were analyzed with 2‐way ANOVA followed by Bonferroni correction (**P<0.01, **** or ^#### P<0.0001). Ang II indicates angiotensin II; CBF, cerebral blood flow; CTL, control mice; IL‐10, interleukin‐10.

Figure 4

IL‐10 rescues the neurovascular coupling deficit induced by Ang II. CBF responses to whisker stimulation (A) and to acetylcholine (B) following simultaneous administration of Ang II (1000 ng/kg per min) and IL‐10 (60 ng/d) or Ang II or IL‐10 alone for 14 d. Graphs depict the percentage increase in CBF following the stimulation with respect to its initial value, measured by laser Doppler flowmetry. Results represent mean±SEM; n=8 CTL/PBS, n=12 Ang II/PBS, n=6 CTL/IL‐10, and n=11 Ang II/IL‐10 mice in (A), n=6 CTL/PBS and CTL/IL‐10, n=8 Ang II/PBS, and n=11 Ang II/IL‐10 mice in (B). Data were analyzed by 2‐way ANOVA followed by Bonferroni correction (*P<0.05 and ****P<0.0001). Ang II indicates angiotensin II; CBF, cerebral blood flow; CTL, control; IL‐10, interleukin‐10.

Figure 5

CD4⁺ CD25⁺ regulatory T cells prevent systemic immune responses induced by Ang II. Analysis of plasma cytokines and chemokines following adoptive transfer of CD4⁺ CD25⁺ or CD4⁺ CD25⁺ Il10 ^−/− cells (3×10⁵) in mice infused s.c. with Ang II (1000 ng/kg per min, 14 d) and in control mice (CTL). Cytokines were grouped into 4 categories: pro‐inflammatory cytokines (IL‐1α, IL‐6, IL‐17, TNF‐α, and LIF) (A), neutrophil chemoattractants (KC, LIX, MIP‐2) and stimulators of their development (G‐CSF) (B), stimulators of Th1‐driven responses (IL‐12p40, IL12p70, MIP‐1β, RANTES, MIG, and IP‐10) (C), and stimulators of Th2 responses (IL‐4, IL‐5, IL‐9, IL‐10, IL‐13, and MCP‐1) (D). A composite score was calculated by converting each marker to a standardized Z score and then added. Results represent mean±SEM; n=11 CTL, n=7 Ang II and Ang II+ CD4⁺ CD25⁺, n=4 Ang II+ CD4⁺ CD25⁺ Il10 ^−/− mice. *P<0.05, by Kruskal‐Wallis and Dunn's post hoc correction (A, C, D) or 1‐way ANOVA followed by Bonferroni correction (B). Ang II indicates angiotensin II; CTL, control mice; G‐CSF, granulocyte colony‐stimulating factor; IL, interleukin; IP‐10, Interferon gamma‐induced protein 10; KC, Keratinocyte chemoattractant; LIF, Leukemia inhibitory factor; LIX, lipopolysaccharide (LPS)‐induced CXC chemokine; MCP, Monocyte chemoattractant protein; MIG, Monokine induced by gamma interferon; MIP, Macrophage Inflammatory Protein; RANTES, Regulated on activation, normal T cell expressed and secreted; TNF, tumor necrosis factor.

Figure 6

CD4⁺ CD25⁺ Il10 ^−/− cells affect systemic immune responses in naïve mice. Analysis of plasma cytokines and chemokines following adoptive transfer of CD4⁺ CD25⁺ or CD4⁺ CD25⁺ Il10 ^−/− cells (3×10⁵) or PBS to naïve mice. Cytokines were grouped into 4 categories: pro‐inflammatory cytokines (IL‐1α, IL‐6, IL‐17, TNF‐α, and LIF) (A), neutrophil chemoattractants (KC, LIX, MIP‐2) and stimulators of their development (G‐CSF (B)), stimulators of Th1 responses (IL‐12p40, IL12p70, MIP‐1β, RANTES, MIG, and IP‐10) (C), and stimulators of Th2 responses (IL‐4, IL‐5, IL‐9, IL‐10, IL‐13 and MCP‐1) (D). A composite score was calculated by converting each marker to a standardized Z score and then added. Results represent mean±SEM; n=11 PBS, n=4 CD4⁺ CD25⁺ and n=7 CD4⁺ CD25⁺ Il10 ^−/− mice. *P<0.05 by 1‐way ANOVA followed by Bonferroni correction (A, D) or Kruskal‐Wallis followed by Dunn's post hoc correction (B, C). G‐CSF, granulocyte colony‐stimulating factor; IL, interleukin; IP‐10, Interferon gamma‐induced protein 10; KC, Keratinocyte chemoattractant; LIF, Leukemia inhibitory factor; LIX, lipopolysaccharide (LPS)‐induced CXC chemokine; MCP, Monocyte chemoattractant protein; MIG, Monokine induced by gamma interferon; MIP, Macrophage Inflammatory Protein; RANTES, Regulated on activation, normal T cell expressed and secreted; TNF, tumor necrosis factor.

Figure 7

Effect of CD4⁺ CD25⁺ regulatory T cells on cerebral gliosis and oxidative stress. A, Microglia/monocytes were stained and visualized with Iba‐1. A semiquantitative assessment of the number of Iba‐1+ cells per micrograph was performed in the somatosensory cortex and hippocampus. Representative micrographs are shown. Scale bar=50 μm. Graphs represent mean±SEM; n=3 CTL and Ang II, n=5 Ang II+CD4⁺ CD25⁺ mice, *P<0.05, by 1‐way ANOVA followed by Bonferroni correction (cortex) or **P<0.01, ***P<0.001 by Kruskal‐Wallis test followed by Dunn's post hoc correction (hippocampus). B, Superoxide anion production was measured by lucigenin‐enhanced chemiluminescence of cortical tissue incubated with NADPH and extrapolated from the area under the curve (AUC) counts/time. Graphs represent mean±SEM; n=6 CTL n=7 Ang II, n=9 Ang II+CD4⁺ CD25⁺ and n=4 Ang II+CD4⁺ CD25⁺ Il10 ^−/− mice, *P<0.05, by 1‐way ANOVA followed by Bonferroni correction. C, Cerebral NOX‐2‐derived radicals were determined based on the extent of the inhibition of superoxide anion production following the addition of its specific inhibitor, gp91ds‐tat. Graphs represent mean± SEM (n=5 CTL, n=7 Ang II, n=8 Ang II+ CD4⁺ CD25⁺ and n=3 Ang II+CD4⁺ CD25⁺ Il10 ^−/− mice, *P<0.05, by 1‐way ANOVA followed by Bonferroni. Ang II indicates angiotensin II; CTL, control; NOX, NADPH oxidase.