Differential composition of lymphocyte subpopulations and activation between the hypertensive Bph/2 and normotensive Bpn/3 mouse strains

Figures

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  Figure 1. Bph/2 mice have elevated arterial blood pressure and lower body weights. Blood pressure was determined by tail cuff plethysmography. Shown in the graphs are the (A) systolic, diastolic, and mean arterial pressures, (B) heart rates and (C) body weights, (D) spleen weights, and (E) spleen-to-body weight ratio for Bpn/3 and Bph/2 mice. Data are presented as scatter plots or scatter plots plus bar graphs and include the mean ± SEM (n = 5). p values were determined by unpaired Student's t test.

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  Figure 2. Bpn/2 mice have elevated percentages of splenic neutrophils and decreased percentages of macrophages and dendritic cells in the mesenteric lymph nodes. Splenocytes and lymph nodes (brachial, inguinal, and mesenteric) were isolated from twelve-week-old Bph/2 and Bpn/3 mice, labeled with fluorochrome-conjugated antibodies (Table 1) and analyzed by flow cytometry. The graphs depict (A) eosinophils, (B) neutrophils (PMNs, splenic only), (C) dendritic cells (y-axis: CD3-AF488; x-axis: CD11c-AF647), (D) macrophages, and (E) splenic macrophages as percentages of the live cell population. The graphs represent the mean ± SEM values for each respective group, n = 5.* p<0.05 as determined by unpaired Student’s t-test.

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  Figure 3. Bph/2 mice have altered percentages of CD4+ and CD8+ T cells in brachial and inguinal lymph nodes compared to Bpn/3 mice. Single-cell suspensions were extracted from spleens and lymph nodes of twelve-week-old Bpn/3 and Bph/2 mice, labeled with fluorochrome-conjugated antibodies (Table 1) and analyzed by flow cytometry. (A) CD4+ and CD8+ T cell percentages relative to CD3+ T cell population (y-axis: CD8-PE/Cy5.5; x-axis: CD4-APC/Cy7). Representative flow density plots are shown above, and graphs shown below illustrate the mean ± SEM values for each respective group, n = 5. (B) Pie graphs showing the relative proportions of CD4+ and CD8+ T cells in relation to the whole T cell population. Other refers to T cell populations that were CD4+ CD8+ double-positive or CD4- CD8- double-negative *p<0.05, as determined by unpaired Student’s t-test.

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  Figure 4. Lower percentage of IgM+ IgD-Lo and IgM+ IgD- B cells in spleens of Bph/2 mice. Single-cell suspensions were extracted from spleens and lymph nodes of twelve-week-old Bpn/3 and Bph/2 mice, labeled with fluorochrome-conjugated antibodies (Table 2) and analyzed by flow cytometry. The following B cell populations were quantified: (A) IgM+ IgDHi B cells (red gates), IgM+ IgDLo B cells (blue gates), IgM+ IgD- B cells (green gates), and IgM- IgD- B cells (purple gates) B cells (y-axis: IgD-BV605; x-axis: IgM-AF488), (B) IgM+ IgDLo CD23- B cells, which includes marginal zone B cells (y-axis: CD23-AF700; x-axis: CD19-PEdazzle594), and recently activated (C) CD86+ (y-axis: CD19-PEdazzle594; x-axis: CD86-APC/Cy7) and (D) CD69+ (y-axis: CD19-PEdazzle594; x-axis: CD69-PE/Cy7) B cells. (E) The pan B cell population (CD19+) was also quantified (histogram: CD19-PEdazzle594). Representative flow density plots are shown above, and graphs shown below illustrate the mean ± SEM values for each respective group, n = 5. *p<0.05 **p<0.01 and ****p<0.0001 as determined by unpaired Student’s t-test.

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  Figure 5. Lower percentages of CD4 effector memory T cells and CD8 central memory T cells in Bph/2 mice. Single-cell suspensions were extracted from spleens and lymph nodes of twelve-week-old Bpn/3 and Bph/2 mice, labeled with fluorochrome-conjugated antibodies (Table 3) and analyzed by flow cytometry (y-axis: CD44-PEdazzle; x-axis: CD62L-Pacific Blue; pre-gated on either (A) CD4 T cells or (B) CD8 T cells). Naïve (CD62LHi CD44Lo), effector memory (CD62LLo CD44Hi) and central memory (CD62LHi CD44Hi) T cell subsets were quantified in the A) CD4 and B) CD8 T cell populations. No differences were observed in the effector memory (CD44Hi CD62LLo) or naïve (CD44Lo CD62LHi) CD8+ T cells. Bars are presented as the mean ± SEM. n = 5. ***p<0.001 and ****p<0.0001 as determined by unpaired Student’s t-test.

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  Figure 6. Decreased proliferation by activated splenic T cells from Bph/2 mice. Freshly isolated splenocytes were (2.5x104 cells/well), stained with IncuCyte Nuclight Rapid Red reagent, and cultured for 48 (h) Fluorescence was quantified every 8 h using the Incucyte S3 Live-Cell Analysis System. (A) Representative images of cells from Bph/2 and Bpn/2 mice. (B) Line graph depicting cell counts at 0–48 h post activation. The data are presented as the mean ± SEM, n=5. **p<0.01, ***p<0.001 and ****p<0.0001 as determined by 2-way ANOVA followed by Dunnett's post hoc analysis.

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  Figure 7. Diminished induction of T cell activation markers in CD8 T cells, and to a lesser extent, CD4 T cells from Bph/2 mice. Freshly isolated splenocytes were cultured in the presence or absence anti-CD3/anti-CD28 activating antibodies for 24h. The cells were then labeled with fluorochrome-conjugated antibodies (Table 3) and analyzed by flow cytometry. A) Decreased percentages of CD4+ T cells from BPH/2J mice were CD44Hi(y-axis: CD44-PEdazzle; x-axis: CD62L-Pacific Blue), CD137+,(y-axis: CD137-PE/Cy7; x-axis: CD4-APC/Cy7) and OX-40+ (y-axis: OX40-BV605; x-axis: CD4-APC/Cy7) 24-hours post-activation, whereas no differences were observed in percentages of CD25+(y-axis: CD25-BV711; x-axis: CD4-APC/Cy7), CD69+(y-axis: CD69-AF700; X-axis: CD4-APC/Cy7), and CD62LHi(y-axis: CD44-PEdazzle; x-axis: CD62L-Pacific Blue). B) BPH/2J mice had decreased percentages of CD8+ T cells expressing the inducible cell surface markers: CD69 (y-axis: CD69-AF700; X-axis: CD8-PE/Cy5.5), CD25 (y-axis: CD25-BV711; x-axis: CD8-PE/Cy5.5), CD44 (y-axis: CD44-PEdazzle; x-axis: CD62L-Pacific Blue), CD137 (y-axis: CD137-PE/Cy7; x-axis: CD8-PE/Cy5.5) and OX-40 (y-axis: OX40-BV605; x-axis: CD8-PE/Cy5.5). CD62L (y-axis:…

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  Figure 8. Reduced induction of T cell cytokines by splenic T cells from Bph/2 mice 24 h after activation. Freshly isolated splenocytes were activated with anti-CD3/anti-CD28 antibodies for 24 h prior to collection of cell supernatants for cytokine analysis by EVE Technologies (Calgary, Alberta, Canada). Data are presented as the mean ± SEM, n = 5. *p<0.05 and **p<0.01 as determined by two-tailed Student’s t test.

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  Figure 9. Decreased induction of IL-1β and VEGF by myeloid cells from Bph/2 mice 24 h after activation. Freshly isolated splenocytes were activated with anti-CD3/anti-CD28 antibodies for 24 h prior to collection of cell supernatants for cytokine analysis by EVE Technologies (Calgary, Alberta, Canada). Induction of cytokines was likely the result of indirect stimulation of myeloid cells by cytokines released by T cells. Data are presented as the mean ± SEM, n = 5. * p<0.05 as determined by two-tailed Student’s t test.

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  Figure 10. Reduced induction of the chemokines, RANTES and MIP3α, by splenic T cells from Bph/2 mice 24 h after activation. Freshly isolated splenocytes were activated with anti-CD3/anti-CD28 antibodies for 24 h prior to collection of cell supernatants for cytokine analysis by EVE Technologies (Calgary, Alberta, Canada). Data are presented as the mean ± SEM, n = 5. *p<0.05 and **p<0.01 as determined by two-tailed Student’s t test.

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  Figure 11. Decreased induction of T cell cytokines by splenic T cells from Bph/2 mice 120 h after activation. Freshly isolated splenocytes were activated with anti-CD3/anti-CD28 antibodies for 120 h prior to collection of cell supernatants for cytokine analysis by EVE Technologies (Calgary, Alberta, Canada). Data are presented as the mean ± SEM, n = 5. *p<0.05 and **p<0.01 as determined by two-tailed Student’s t test.