Blood pressure regulation by CD4+ lymphocytes expressing choline acetyltransferase

Abstract

Blood pressure regulation is known to be maintained by a neuro-endocrine circuit, but whether immune cells contribute to blood pressure homeostasis has not been determined. We previously showed that CD4⁺ T lymphocytes that express choline acetyltransferase (ChAT), which catalyzes the synthesis of the vasorelaxant acetylcholine, relay neural signals. Here we show that these CD4⁺CD44^hiCD62L^lo T helper cells by gene expression are a distinct T-cell population defined by ChAT (CD4 T_ChAT). Mice lacking ChAT expression in CD4⁺ cells have elevated arterial blood pressure, compared to littermate controls. Jurkat T cells overexpressing ChAT (JT_ChAT) decreased blood pressure when infused into mice. Co-incubation of JT_ChAT and endothelial cells increased endothelial cell levels of phosphorylated endothelial nitric oxide synthase, and of nitrates and nitrites in conditioned media, indicating increased release of the potent vasorelaxant nitric oxide. The isolation and characterization of CD4 T_ChAT cells will enable analysis of the role of these cells in hypotension and hypertension, and may suggest novel therapeutic strategies by targeting cell-mediated vasorelaxation.

Figure 1. Gene expression analysis reveals differences between CD4⁺ CD44^hi CD62L^low ChAT-eGFP⁺ and ChAT-eGFP⁻ T cells

a) RNA from the ChAT-eGFP⁺ and ChAT-eGFP⁻ subsets of CD4⁺ CD44^hi CD62L^low T cells was analyzed by Affymetrix Gene ST 1.0 arrays. Unsupervised hierarchical clustering of the whole transcriptome of six samples, each from ≥3 pooled murine spleens, is shown. b) The 1288 transcripts with significantly different expression between the subsets (P<0.05 with Benjamini Hochberg correction) were studied using unsupervised hierarchical clustering and plotted in a heat map. 1281 out of 1288 transcripts had a fold change of >1.5 (n=3). c) Bottom: Unsupervised global hierarchical clustering of gene expression in ChAT-eGFP⁺ T lymphocytes and 198 other immune cell subsets from the ImmGen dataset. Top: Expression level of ChAT across the 199 different immune cell subsets. Bar color indicates cell type as shown in the figure insert. | Gene expression in CD4⁺ CD44^hi CD62L^low ChAT-eGFP⁺ T cells was analyzed in the context of 13 other subsets isolated from spleen in the ImmGen dataset. d) Unsupervised hierarchical clustering of gene expression in the T cell subsets derived from spleen. e) Heat map of pair-wise Euclidian distances and f) principal component analysis of gene expression in the splenic T cell subsets.

Figure 1. Gene expression analysis reveals differences between CD4⁺ CD44^hi CD62L^low ChAT-eGFP⁺ and ChAT-eGFP⁻ T cells

a) RNA from the ChAT-eGFP⁺ and ChAT-eGFP⁻ subsets of CD4⁺ CD44^hi CD62L^low T cells was analyzed by Affymetrix Gene ST 1.0 arrays. Unsupervised hierarchical clustering of the whole transcriptome of six samples, each from ≥3 pooled murine spleens, is shown. b) The 1288 transcripts with significantly different expression between the subsets (P<0.05 with Benjamini Hochberg correction) were studied using unsupervised hierarchical clustering and plotted in a heat map. 1281 out of 1288 transcripts had a fold change of >1.5 (n=3). c) Bottom: Unsupervised global hierarchical clustering of gene expression in ChAT-eGFP⁺ T lymphocytes and 198 other immune cell subsets from the ImmGen dataset. Top: Expression level of ChAT across the 199 different immune cell subsets. Bar color indicates cell type as shown in the figure insert. | Gene expression in CD4⁺ CD44^hi CD62L^low ChAT-eGFP⁺ T cells was analyzed in the context of 13 other subsets isolated from spleen in the ImmGen dataset. d) Unsupervised hierarchical clustering of gene expression in the T cell subsets derived from spleen. e) Heat map of pair-wise Euclidian distances and f) principal component analysis of gene expression in the splenic T cell subsets.

Figure 1. Gene expression analysis reveals differences between CD4⁺ CD44^hi CD62L^low ChAT-eGFP⁺ and ChAT-eGFP⁻ T cells

a) RNA from the ChAT-eGFP⁺ and ChAT-eGFP⁻ subsets of CD4⁺ CD44^hi CD62L^low T cells was analyzed by Affymetrix Gene ST 1.0 arrays. Unsupervised hierarchical clustering of the whole transcriptome of six samples, each from ≥3 pooled murine spleens, is shown. b) The 1288 transcripts with significantly different expression between the subsets (P<0.05 with Benjamini Hochberg correction) were studied using unsupervised hierarchical clustering and plotted in a heat map. 1281 out of 1288 transcripts had a fold change of >1.5 (n=3). c) Bottom: Unsupervised global hierarchical clustering of gene expression in ChAT-eGFP⁺ T lymphocytes and 198 other immune cell subsets from the ImmGen dataset. Top: Expression level of ChAT across the 199 different immune cell subsets. Bar color indicates cell type as shown in the figure insert. | Gene expression in CD4⁺ CD44^hi CD62L^low ChAT-eGFP⁺ T cells was analyzed in the context of 13 other subsets isolated from spleen in the ImmGen dataset. d) Unsupervised hierarchical clustering of gene expression in the T cell subsets derived from spleen. e) Heat map of pair-wise Euclidian distances and f) principal component analysis of gene expression in the splenic T cell subsets.

Figure 1. Gene expression analysis reveals differences between CD4⁺ CD44^hi CD62L^low ChAT-eGFP⁺ and ChAT-eGFP⁻ T cells

a) RNA from the ChAT-eGFP⁺ and ChAT-eGFP⁻ subsets of CD4⁺ CD44^hi CD62L^low T cells was analyzed by Affymetrix Gene ST 1.0 arrays. Unsupervised hierarchical clustering of the whole transcriptome of six samples, each from ≥3 pooled murine spleens, is shown. b) The 1288 transcripts with significantly different expression between the subsets (P<0.05 with Benjamini Hochberg correction) were studied using unsupervised hierarchical clustering and plotted in a heat map. 1281 out of 1288 transcripts had a fold change of >1.5 (n=3). c) Bottom: Unsupervised global hierarchical clustering of gene expression in ChAT-eGFP⁺ T lymphocytes and 198 other immune cell subsets from the ImmGen dataset. Top: Expression level of ChAT across the 199 different immune cell subsets. Bar color indicates cell type as shown in the figure insert. | Gene expression in CD4⁺ CD44^hi CD62L^low ChAT-eGFP⁺ T cells was analyzed in the context of 13 other subsets isolated from spleen in the ImmGen dataset. d) Unsupervised hierarchical clustering of gene expression in the T cell subsets derived from spleen. e) Heat map of pair-wise Euclidian distances and f) principal component analysis of gene expression in the splenic T cell subsets.

Figure 1. Gene expression analysis reveals differences between CD4⁺ CD44^hi CD62L^low ChAT-eGFP⁺ and ChAT-eGFP⁻ T cells

a) RNA from the ChAT-eGFP⁺ and ChAT-eGFP⁻ subsets of CD4⁺ CD44^hi CD62L^low T cells was analyzed by Affymetrix Gene ST 1.0 arrays. Unsupervised hierarchical clustering of the whole transcriptome of six samples, each from ≥3 pooled murine spleens, is shown. b) The 1288 transcripts with significantly different expression between the subsets (P<0.05 with Benjamini Hochberg correction) were studied using unsupervised hierarchical clustering and plotted in a heat map. 1281 out of 1288 transcripts had a fold change of >1.5 (n=3). c) Bottom: Unsupervised global hierarchical clustering of gene expression in ChAT-eGFP⁺ T lymphocytes and 198 other immune cell subsets from the ImmGen dataset. Top: Expression level of ChAT across the 199 different immune cell subsets. Bar color indicates cell type as shown in the figure insert. | Gene expression in CD4⁺ CD44^hi CD62L^low ChAT-eGFP⁺ T cells was analyzed in the context of 13 other subsets isolated from spleen in the ImmGen dataset. d) Unsupervised hierarchical clustering of gene expression in the T cell subsets derived from spleen. e) Heat map of pair-wise Euclidian distances and f) principal component analysis of gene expression in the splenic T cell subsets.

Figure 1. Gene expression analysis reveals differences between CD4⁺ CD44^hi CD62L^low ChAT-eGFP⁺ and ChAT-eGFP⁻ T cells

a) RNA from the ChAT-eGFP⁺ and ChAT-eGFP⁻ subsets of CD4⁺ CD44^hi CD62L^low T cells was analyzed by Affymetrix Gene ST 1.0 arrays. Unsupervised hierarchical clustering of the whole transcriptome of six samples, each from ≥3 pooled murine spleens, is shown. b) The 1288 transcripts with significantly different expression between the subsets (P<0.05 with Benjamini Hochberg correction) were studied using unsupervised hierarchical clustering and plotted in a heat map. 1281 out of 1288 transcripts had a fold change of >1.5 (n=3). c) Bottom: Unsupervised global hierarchical clustering of gene expression in ChAT-eGFP⁺ T lymphocytes and 198 other immune cell subsets from the ImmGen dataset. Top: Expression level of ChAT across the 199 different immune cell subsets. Bar color indicates cell type as shown in the figure insert. | Gene expression in CD4⁺ CD44^hi CD62L^low ChAT-eGFP⁺ T cells was analyzed in the context of 13 other subsets isolated from spleen in the ImmGen dataset. d) Unsupervised hierarchical clustering of gene expression in the T cell subsets derived from spleen. e) Heat map of pair-wise Euclidian distances and f) principal component analysis of gene expression in the splenic T cell subsets.

Figure 2. Increased blood pressure in mice with genetic ablation of choline acetyltransferase⁺ CD4⁺ cells

a) Blood from ChAT-eGFP reporter mice 6–12 weeks of age obtained by tail bleed was analyzed by flow cytometry to determine the fraction of ChAT-eGFP⁺ cells of CD3⁺ cells in each mouse. A histogram of the compiled data is plotted. b) Blood pressure measured by telemetry over 48 h in awake 12 weeks old ChAT-deficient CD4-Cre^0/+ x ChAT^loxP/loxP (ChAT^−/−, n=6) and their CD4-Cre^0/0 x ChAT^loxP/loxP (ChAT^+/+, n=4) littermate controls. Data were analyzed using repeated measures ANOVA. c) Blood pressure (BP) as measured by an indwelling catheter in the left carotid artery in anesthetized ChAT^+/+ (+/+, n=3) and ChAT^−/− (−/−, n=4) mice. D – diastolic, M – mean arterial, S – systolic. * - P < 0.05 (two-tailed Student’s t test). d) Echocardiographic measurements in 10–11 weeks old ChAT^+/+ (n=6) and ChAT^−/− (n=5) mice. CO – cardiac output, SV – stroke volume, FS – fractional shortening, EF – ejection fraction. * - P < 0.05 (two-tailed Student’s t test).

Figure 2. Increased blood pressure in mice with genetic ablation of choline acetyltransferase⁺ CD4⁺ cells

a) Blood from ChAT-eGFP reporter mice 6–12 weeks of age obtained by tail bleed was analyzed by flow cytometry to determine the fraction of ChAT-eGFP⁺ cells of CD3⁺ cells in each mouse. A histogram of the compiled data is plotted. b) Blood pressure measured by telemetry over 48 h in awake 12 weeks old ChAT-deficient CD4-Cre^0/+ x ChAT^loxP/loxP (ChAT^−/−, n=6) and their CD4-Cre^0/0 x ChAT^loxP/loxP (ChAT^+/+, n=4) littermate controls. Data were analyzed using repeated measures ANOVA. c) Blood pressure (BP) as measured by an indwelling catheter in the left carotid artery in anesthetized ChAT^+/+ (+/+, n=3) and ChAT^−/− (−/−, n=4) mice. D – diastolic, M – mean arterial, S – systolic. * - P < 0.05 (two-tailed Student’s t test). d) Echocardiographic measurements in 10–11 weeks old ChAT^+/+ (n=6) and ChAT^−/− (n=5) mice. CO – cardiac output, SV – stroke volume, FS – fractional shortening, EF – ejection fraction. * - P < 0.05 (two-tailed Student’s t test).

Figure 2. Increased blood pressure in mice with genetic ablation of choline acetyltransferase⁺ CD4⁺ cells

a) Blood from ChAT-eGFP reporter mice 6–12 weeks of age obtained by tail bleed was analyzed by flow cytometry to determine the fraction of ChAT-eGFP⁺ cells of CD3⁺ cells in each mouse. A histogram of the compiled data is plotted. b) Blood pressure measured by telemetry over 48 h in awake 12 weeks old ChAT-deficient CD4-Cre^0/+ x ChAT^loxP/loxP (ChAT^−/−, n=6) and their CD4-Cre^0/0 x ChAT^loxP/loxP (ChAT^+/+, n=4) littermate controls. Data were analyzed using repeated measures ANOVA. c) Blood pressure (BP) as measured by an indwelling catheter in the left carotid artery in anesthetized ChAT^+/+ (+/+, n=3) and ChAT^−/− (−/−, n=4) mice. D – diastolic, M – mean arterial, S – systolic. * - P < 0.05 (two-tailed Student’s t test). d) Echocardiographic measurements in 10–11 weeks old ChAT^+/+ (n=6) and ChAT^−/− (n=5) mice. CO – cardiac output, SV – stroke volume, FS – fractional shortening, EF – ejection fraction. * - P < 0.05 (two-tailed Student’s t test).

Figure 2. Increased blood pressure in mice with genetic ablation of choline acetyltransferase⁺ CD4⁺ cells

a) Blood from ChAT-eGFP reporter mice 6–12 weeks of age obtained by tail bleed was analyzed by flow cytometry to determine the fraction of ChAT-eGFP⁺ cells of CD3⁺ cells in each mouse. A histogram of the compiled data is plotted. b) Blood pressure measured by telemetry over 48 h in awake 12 weeks old ChAT-deficient CD4-Cre^0/+ x ChAT^loxP/loxP (ChAT^−/−, n=6) and their CD4-Cre^0/0 x ChAT^loxP/loxP (ChAT^+/+, n=4) littermate controls. Data were analyzed using repeated measures ANOVA. c) Blood pressure (BP) as measured by an indwelling catheter in the left carotid artery in anesthetized ChAT^+/+ (+/+, n=3) and ChAT^−/− (−/−, n=4) mice. D – diastolic, M – mean arterial, S – systolic. * - P < 0.05 (two-tailed Student’s t test). d) Echocardiographic measurements in 10–11 weeks old ChAT^+/+ (n=6) and ChAT^−/− (n=5) mice. CO – cardiac output, SV – stroke volume, FS – fractional shortening, EF – ejection fraction. * - P < 0.05 (two-tailed Student’s t test).

Figure 3. Infusion of JT_ChAT lymphocytes lowers blood pressure

a–c) Mean arterial blood pressure was measured by a catheter inserted in the left carotid artery in anesthetized wild-type C57Bl/6 mice. a) Mean MAP over ~9 min. after infusion of JT_ChAT lymphocytes or saline. Open circle – saline (n=14). Closed squares - JT_ChAT (n=7–13). * - p<0.05, *** - p<0.001 JT_ChAT vs Saline by ANOVA followed by Bonferroni post-hoc test. b) Tracings of mean arterial pressure (MAP) over time in mice infused with (1) saline, (2) JT lymphocytes, or (3) JT_ChAT lymphocytes, or infusion of JT_ChAT lymphocytes after with (4) L-NAME or (5) atropine. Arrows indicate the time for infusion start. The cut in the tracings indicate the period of infusion-related measurement artifacts. c) Mean MAP change from baseline x time expressed as area under the curve ± SEM is shown for mice injected with saline (n=19), JT lymphocytes (n=13) or JT_ChAT lymphocytes (n=9). Mice were pre-treated with L-NAME (n=7) or atropine (n=8) as indicated. * - p<0.05, *** - p<0.001 (ANOVA with Bonferroni post-hoc analysis).

Figure 3. Infusion of JT_ChAT lymphocytes lowers blood pressure

a–c) Mean arterial blood pressure was measured by a catheter inserted in the left carotid artery in anesthetized wild-type C57Bl/6 mice. a) Mean MAP over ~9 min. after infusion of JT_ChAT lymphocytes or saline. Open circle – saline (n=14). Closed squares - JT_ChAT (n=7–13). * - p<0.05, *** - p<0.001 JT_ChAT vs Saline by ANOVA followed by Bonferroni post-hoc test. b) Tracings of mean arterial pressure (MAP) over time in mice infused with (1) saline, (2) JT lymphocytes, or (3) JT_ChAT lymphocytes, or infusion of JT_ChAT lymphocytes after with (4) L-NAME or (5) atropine. Arrows indicate the time for infusion start. The cut in the tracings indicate the period of infusion-related measurement artifacts. c) Mean MAP change from baseline x time expressed as area under the curve ± SEM is shown for mice injected with saline (n=19), JT lymphocytes (n=13) or JT_ChAT lymphocytes (n=9). Mice were pre-treated with L-NAME (n=7) or atropine (n=8) as indicated. * - p<0.05, *** - p<0.001 (ANOVA with Bonferroni post-hoc analysis).

Figure 3. Infusion of JT_ChAT lymphocytes lowers blood pressure

a–c) Mean arterial blood pressure was measured by a catheter inserted in the left carotid artery in anesthetized wild-type C57Bl/6 mice. a) Mean MAP over ~9 min. after infusion of JT_ChAT lymphocytes or saline. Open circle – saline (n=14). Closed squares - JT_ChAT (n=7–13). * - p<0.05, *** - p<0.001 JT_ChAT vs Saline by ANOVA followed by Bonferroni post-hoc test. b) Tracings of mean arterial pressure (MAP) over time in mice infused with (1) saline, (2) JT lymphocytes, or (3) JT_ChAT lymphocytes, or infusion of JT_ChAT lymphocytes after with (4) L-NAME or (5) atropine. Arrows indicate the time for infusion start. The cut in the tracings indicate the period of infusion-related measurement artifacts. c) Mean MAP change from baseline x time expressed as area under the curve ± SEM is shown for mice injected with saline (n=19), JT lymphocytes (n=13) or JT_ChAT lymphocytes (n=9). Mice were pre-treated with L-NAME (n=7) or atropine (n=8) as indicated. * - p<0.05, *** - p<0.001 (ANOVA with Bonferroni post-hoc analysis).