L-selectin deficiency decreases aortic B1a and Breg subsets and promotes atherosclerosis

Abstract

There is a significant recruitment of leucocytes into aortas during atherogenesis. L-selectin regulates leucocyte migration into secondary lymphoid and peripheral tissues and was proposed to play a role in leucocyte homing into aortas. Here, we determine the role of L-selectin in atherosclerosis. L-selectin-deficient Apoe-/- (Sell-/-Apoe-/-) mice had a 74% increase in plaque burden compared to Apoe-/- mice fed a chow diet for 50 weeks. Elevated atherosclerosis was accompanied by increased aortic leucocyte content, but a 50% reduction in aortic B cells despite elevated B cell counts in the blood. Follicular B cells represented 65%, whereas B1a and regulatory B cells (Breg) comprised 5% of aortic B cells. B1a and Breg cell subsets were reduced in Sell-/-Apoe-/- aortas with accompanied two-fold decrease in aortic T15 antibody and 1.2-fold decrease of interleukin-10 (IL-10) levels. L-selectin was required for B1 cell homing to the atherosclerotic aorta, as demonstrated by a 1.5-fold decrease in the migration of Sell-/-Apoe-/- vs Apoe-/- cells. Notably, we found a 1.6-fold increase in CD68hi macrophages in Sell-/-Apoe-/- compared to Apoe-/- aortas, despite comparable blood monocyte numbers and L-selectin-dependent aortic homing. L-selectin had no effect on neutrophil migration into aorta, but led to elevated blood neutrophil numbers, suggesting a potential involvement of neutrophils in atherogenesis of Sell-/-Apoe-/- mice. Thus, L-selectin deficiency increases peripheral blood neutrophil and lymphocyte numbers, decreases aortic B1a and Breg populations, T15 antibody and IL-10 levels, and increases aortic macrophage content of Sell-/-Apoe-/- mice. Altogether, these data provide evidence for an overall atheroprotective role of L-selectin.

Keywords: Atherosclerosis; B cell subsets; L-selectin.

Figure 1. L-selectin deficiency increases plaque burden within the aortas of Sell^−/−Apoe^−/− mice

(A) Quantification of positive Oil Red O staining in aortas of 50–60 week old Sell^−/−Apoe^−/− (black circles) and Apoe^−/− mice (black squares) (n=16 and 17, respectively) (B) Representative images of Sell^−/−Apoe^−/− and Apoe^−/− aortas stained by Oil Red O. Unpaired student’s t-tests were used for statistical analysis; ***P≤0.0005.

Figure 2. L-selectin deficient Apoe^−/− mice have an increased number of total aortic leucocytes but a decrease in aortic B cells

Total leucocyte and CD19⁺ cell numbers in the (A) Aorta (C) Peripheral lymph nodes, and (D) Blood of 40–50 week old Sell^−/−Apoe^−/− (black bars) and Apoe^−/− (gray bars) mice (n=19 and 24, respectively). B cells were quantified as CD19⁺ percentages of leucocytes by flow cytometry. B cell numbers were calculated from their percentage determined by flow cytometry and total leucocyte counts. (B) Representative flow cytometric plots of CD19⁺ B cells within CD45⁺ cells of Sell^−/−Apoe^−/− and Apoe^−/− aortas.* P≤0.05, *** P≤0.0005.

Figure 3. Absence of L-selectin results in an altered distribution of B cell subsets within Apoe^−/− aorta

(A) Distribution of aortic B cell subsets within CD19⁺ B cell population of 40–50 week old Sell^−/−Apoe^−/− and Apoe^−/− mice. B cell subsets were characterized based on CD19, CD43, CD5, and IgD expression (Table 1) using flow cytometry. Percentages were calculated from the gated CD19⁺ population for Sell^−/−Apoe^−/− (n=20) and Apoe^−/− (n=27) mice. Pie size reflects total aortic B cell number. (B) Total number of B cell subsets in the aorta of Sell^−/−Apoe^−/− (black bars, n=17) and Apoe^−/− mice (gray bars, n=24). (C) Representative plots of flow cytometric analysis of aortic B cell subsets from Sell^−/−Apoe^−/− and Apoe^−/− mice. (D) Representative immunofluorescence images of B cells or B1a and B1b subsets (total B1 cells) in Apoe^−/− mice (Images at 20× magnification, Inset images at 63×, Scale bar represents 10 µm). * P≤0.05, *** P≤0.0005.

Figure 4. Altered splenic B cell subset distribution and diminished T15 and IL-10 levels in the aortas of Sell^−/−Apoe^−/− mice

(A) Total numbers of B cell subsets in the spleens of 40–50 week old Sell^−/−Apoe^−/− (n=7) and Apoe^−/− (n=14) mice. (B) L-selectin expression on splenic B cell subsets of 50 week old Apoe^−/− mice (n=6). One-way ANOVA was used for statistical analysis. (C) Fold difference of T15 antibody in Sell^−/−Apoe^−/− and Apoe^−/− in serum and aortic supernatants. Values were normalized to the average T15 in Apoe^−/− serum in each independent experiment (n=6–9 per genotype). (D) IL-10 production was measured in serum and aortic supernatants of Sell^−/−Apoe^−/− and Apoe^−/− mice using cytomix beads (n=5–8 per genotype) Black bars represent Sell^−/−Apoe^−/− mice and gray bars represent Apoe^−/− mice.* P≤0.05, *** P≤0.0005.

Figure 5. B1 cells require L-selectin for homing to the aorta

(A) Fold difference of CD68^hi leucocytes from Sell^−/−Apoe^−/− (black bar) versus Apoe^−/− (gray bar) aortas (n=5 per genotype). (B) Number of neutrophils and monocytes in the peripheral blood of Sell^−/−Apoe^−/− (black bar) and Apoe^−/− (gray bar) mice. Neutrophils were defined as CD11b⁺Gr1⁺Ly6G⁺ cells and monocytes as CD11b⁺Gr1⁺Ly6G⁻ cells by flow cytometry. Cell numbers were calculated from their percentage by flow cytometry and total leucocyte counts. (C) L-selectin-dependent monocyte homing into atherosclerotic aortas. Sell^−/−Apoe^−/− and Apoe^−/− peripheral blood leukocytes were labeled with CFSE or CellTrace^TM Violet dye, mixed in the ratio 1:1, and adoptively transferred to aged Apoe^−/− recipients. After 16 hours, aortas of Apoe^−/− recipients were stained for CD45, CD11b, Gr-1 and Ly6G and evaluated by flow cytometry. Values were normalized based on neutrophil and monocyte populations within the start population as described in Materials and Methods. Migration of Apoe^−/− monocytes or neutrophils was determined as 1 (gray bars). Ratios were created from the migrated Sell^−/−Apoe^−/− versus Apoe^−/− cells (n=4). Results show mean±SE from at least 4 recipients from 3 independent experiments. (D) Aortic homing of FO and B1 cells into the aorta of Apoe^−/− recipients. Splenic CD19⁺ cells were purified from Sell^−/−Apoe^−/− and Apoe^−/− mice, labeled with CFSE or CellTrace^TM Violet dye, mixed in the ratio 1: 1, and injected into aged Apoe^−/− mice. After 16 hours, aortas were stained for CD45, CD19, CD43, and IgD, and then evaluated by flow cytometry. Values were normalized based on B1 and FO B cell populations within the start population as described in Materials and Methods. Migration of Apoe^−/− B1 and FO B cells was determined as 1 (gray bars), and the homing of Sell^−/−Apoe^−/− B cell subsets was expressed relative to appropriate Apoe^−/− B cell subsets (black bars). Ratios were created from the migrated Sell^−/−Apoe^−/− versus Apoe^−/− cells (n=4). * P≤0.05, ** P≤0.005, *** P≤0.0005.