Age-related arterial immune cell infiltration in mice is attenuated by caloric restriction or voluntary exercise

Abstract

Age-related arterial inflammation is associated with dysfunction of the arteries and increased risk for cardiovascular disease. To determine if aging increases arterial immune cell infiltration as well as the populations of immune cells principally involved, we tested the hypothesis that large elastic and resistance arteries in old mice would exhibit increased immune cell infiltration compared to young controls. Additionally, we hypothesized that vasoprotective lifestyle interventions such as lifelong caloric restriction or 8weeks of voluntary wheel running would attenuate age-related arterial immune cell infiltration. The aorta and mesenteric vasculature with surrounding perivascular adipose was excised from young normal chow (YNC, 4-6months, n=10), old normal chow (ONC, 28-29months, n=11), old caloric restricted (OCR, 28-29months, n=9), and old voluntary running (OVR, 28-29months, n=5) mice and digested to a single cell suspension. The cells were then labeled with antibodies against CD45 (total leukocytes), CD3 (pan T cells), CD4 (T helper cells), CD8 (cytotoxic T cells), CD19 (B cells), CD11b, and F4/80 (macrophages) and analyzed by flow cytometry. Total leukocytes, T cells (both CD4⁺ and CD8⁺ subsets), B cells, and macrophages in both aorta and mesentery were all 5- to 6-fold greater in ONC compared to YNC. Age-related increases in T cell (both CD4⁺ and CD8⁺), B cell, and macrophage infiltration in aorta were abolished in OCR mice. OVR mice exhibited 50% lower aortic T cell and normalized macrophage infiltration. B cell infiltration was not affected by VR. Age-related mesenteric CD8⁺ T cell and macrophage infiltration was normalized in OCR and OVR mice compared to young mice, whereas B cell infiltration was normalized by CR but not VR. Splenic CD4⁺ T cells from ONC mice exhibited a 3-fold increase in gene expression for the T helper (Th) 1 transcription factor, Tbet, and a 4-fold increase in FoxP3, a T regulatory cell transcription factor, compared to YNC. Splenic B cells and mesenteric macrophages from old mice exhibited decreased proinflammatory cytokine gene expression regardless of treatment group. These results demonstrate that aging is associated with infiltration of immune cells around both the large-elastic and resistance arteries and that the vasoprotective lifestyle interventions, CR and VR, can ameliorate age-related arterial immune cell infiltration.

Keywords: Aorta; B cells; Inflammation; Macrophages; Mesentery; T cells.

Figure 1. Leukocyte infiltration of aorta and mesenteric vascular arcade

Aortas (A) and mesenteric vascular arcade (B) from young normal chow (YNC), old- (O) normal chow (NC), voluntary running (VR) and calorie restricted (CR) mice were digested to a single cell suspension and stained with antibodies against CD45 to assess total leukocytes. Representative flow cytometry plots are shown on the left of each panel, summary data is shown on the right. n = 5–11/group. Differences were assessed with one-way ANOVA with LSD post hoc tests. * different from YNC, † different from ONC, p≤0.05. Data are means ± SEM.

Figure 2. T cell infiltration of aorta and mesenteric vascular arcade

Aortas (A) and mesenteric vascular arcade (B) from young normal chow (YNC), old- (O) normal chow (NC), voluntary running (VR) and calorie restricted (CR) mice were digested to a single cell suspension and incubated with antibodies against CD45 and CD3 to assess total leukocytes CD4⁺ and CD8⁺ T cell populations were assessed in the same groups by addition of antibodies against CD4 and CD8 in aorta (C) and mesentery (D). Representative flow cytometry plots are shown on the left of each panel, summary data is shown on the right. n = 5–11/group. Differences were assessed with one-way ANOVA with LSD post hoc tests. * different from YNC, † different from ONC, p≤0.05. Data are means ± SEM.

Figure 3. B cell infiltration of aorta and mesenteric vascular arcade

Aortas (A) and mesenteric vascular arcade (B) from young normal chow (YNC), old- (O) normal chow (NC), voluntary running (VR) and calorie restricted (CR) mice were digested to a single cell suspension and stained with antibodies against CD45 and CD19 to assess B cell infiltration. Representative flow cytometry plots are shown on the left of each panel, summary data is shown on the right. n = 5–11/group. Differences were assessed with one-way ANOVA with LSD post hoc tests. * different from YNC, † different from ONC, p≤0.05. Data are means ± SEM.

Figure 4. Macrophage infiltration of aorta and mesenteric vascular arcade

Aortas (A) and mesenteric vascular arcade (B) from young normal chow (YNC), old- (O) normal chow (NC), voluntary running (VR) and calorie restricted (CR) mice were digested to a single cell suspension and stained with antibodies against CD45, CD11b and F4/80 to assess macrophage infiltration. Representative flow cytometry plots are shown on the left of each panel, summary data is shown on the right., n = 5–11/group. Differences were assessed with one-way ANOVA with LSD post hoc tests. * different from YNC, † different from ONC, p≤0.05. Data are means ± SEM.

Figure 5. Immune cell pro- and anti-inflammatory gene expression. Splenic

CD4⁺ T cells (A) and B cells (B) and mesenteric macrophages (C) were isolated using Flow Activated Cell Sorting (FACS). Following RNA isolation and cDNA synthesis qPCR was employed to assess gene expression for inflammatory cytokines TNF-α (tnfa), IFN-γ (ifng), anti-inflammatory cytokine Interleukin-10 (il10) and T cell transcription factors Tbet (tbet), GATA3 (gata3) and FoxP3 (foxp3). 18s RNA was used as a reference gene and mRNA fold change was calculated using the ΔΔCt method. n = 4–11/group. Differences were assessed with one-way ANOVA with LSD post hoc tests. * different from YNC, † different from ONC, p 0.05. Data are means ± SEM.