Apolipoprotein A-I modulates regulatory T cells in autoimmune LDLr-/-, ApoA-I-/- mice

Abstract

The immune system is complex, with multiple layers of regulation that serve to prevent the production of self-antigens. One layer of regulation involves regulatory T cells (Tregs) that play an essential role in maintaining peripheral self-tolerance. Patients with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis have decreased levels of HDL, suggesting that apoA-I concentrations may be important in preventing autoimmunity and the loss of self-tolerance. In published studies, hypercholesterolemic mice lacking HDL apoA-I or LDLr(-/-), apoA-I(-/-) (DKO), exhibit characteristics of autoimmunity in response to an atherogenic diet. This phenotype is characterized by enlarged cholesterol-enriched lymph nodes (LNs), as well as increased T cell activation, proliferation, and the production of autoantibodies in plasma. In this study, we investigated whether treatment of mice with lipid-free apoA-I could attenuate the autoimmune phenotype. To do this, DKO mice were first fed an atherogenic diet containing 0.1% cholesterol, 10% fat for 6 weeks, after which treatment with apoA-I was begun. Subcutaneous injections of 500 μg of lipid-free apoA-I was administered every 48 h during the treatment phase. These and control mice were maintained for an additional 6 weeks on the diet. At the end of the 12-week study, DKO mice showed decreased numbers of LN immune cells, whereas Tregs were proportionately increased. Accompanying this increase in Tregs was a decrease in the percentage of effector/effector memory T cells. Furthermore, lipid accumulation in LN and skin was reduced. These results suggest that treatment with apoA-I reduces inflammation in DKO mice by augmenting the effectiveness of the LN Treg response.

FIGURE 1.

Study design for apoA-I treatment. Panel A, study design shows 6–7-week-old mice were fed an atherogenic diet for 6 weeks before beginning 6 weeks of subcutaneous injections of 500 μg of lipid-free human apoA-I or BSA every 48 h while maintained on the atherogenic diet for a total of 12 weeks; panel B, timed plasma samples were assayed for human apoA-I following injection of lipid-free apoA-I; panel C, lipoprotein cholesterol distribution following FPLC of mouse plasma; panel D, Western blot of 4–30% nondenaturing gradient gel of timed plasma samples following injection of lipid-free human apoA-I. LDLr^−/−, apoA-I^−/− = DKO mice and LDLr^−/− = SKO mice. All data represent a minimum of 5–8 male mice per group.

FIGURE 2.

Effects of apoA-I treatment on lymph node cell expansion and cholesterol content. Panel A shows the total number of cells isolated from four sets of skin draining LNs that include the brachial, inguinal, axillary, and superficial cervical subsets per mouse. Panels B–D show the total cholesterol (TC), free cholesterol (FC), and esterified cholesterol (EC), respectively. LDLr^−/−, apoA-I^−/− = DKO mice and LDLr^−/− = SKO mice. Mice were fed an atherogenic diet for 6 weeks before beginning 6 weeks of subcutaneous injections of 500 μg of lipid-free human apoA-I (shown as DKO + A-I) or BSA (shown as DKO and SKO) every 48 h while being maintained on the atherogenic diet for a total of 12 weeks. Data represent mean ± S.D. for n = 5–15 mice per group. Different lowercase letters indicate significant differences at p < 0.05.

FIGURE 3.

Alterations in lymph node macrophage and dendritic cell populations following apoA-I treatment. Panels A and B show the number and percent of DCs in total LN cells, respectively. Panels C and D show the total number and percent of macrophages in total LN cells, respectively. LN cells were stained with CD3, CD11c CD11b, and F4/80 surface markers, and the CD3⁻CD11c⁺ population was measured for DCs and CD11c⁻CD11b⁺F4/80⁺ population was measured for macrophages. All cell populations were determined by FACS. Total LN cells were isolated from four sets of skin draining LNs that include the brachial, inguinal, axillary, and superficial cervical subsets per mouse. Data represent mean ± S.D. for a minimum of n = 5–14 mice per group. Different lowercase letters indicate significant differences at p < 0.05.

FIGURE 4.

ApoA-I treatment attenuates LN regulatory T cell and effector/effector-memory populations. Panels A and B show the total number of LN cells staining for CD4⁺CD25⁺FoxP3 or the percent of the total CD4⁺ cells, respectively. Panel C, left, shows representative dot plots for CD4⁺ CD25⁺ gate drawn off viable cells; panel C, right, for CD25⁺FoxP3⁺ cells, showing DKO + A-I (top), DKO + BSA (middle), an, SKO + BSA (bottom) mice. Panel D shows total number of cells staining positive for CD4⁺CD62L^low effector/memory T cells. Panel E show the ratio of Treg to Teff (CD4⁺CD25⁻FoxP3⁻) cells. Data represent mean ± S.D. for a minimum of n = 6–8 mice per group. Different lowercase letters indicate significant differences at p < 0.05.

FIGURE 5.

ApoA-I treatment modulates T cell response to stimulation. Panels A–C show the percent [³H]thymidine incorporation within immune cells isolated from lymph nodes and panels D–F from spleen. Panels A and D show incorporation of [³H]thymidine in unstimulated cells; panels B and E show incorporation of [³H]thymidine in cells stimulated with anti-CD3 and CD28 for 24 h. Panels C and F shows the relative fold increase in response to stimulation (Stim). Data represent means ± S.D. for cells obtained from n = 3–6 mice per treatment group. Different lowercase letters indicate significant differences at p < 0.05.

FIGURE 6.

ApoA-I treatment restores skin neutral lipid content. Panel A shows total skin cholesterol content expressed as mg/g wet weight. Panel B shows the total skin triglyceride content expressed as mg/g wet weight. Data represent mean ± S.D. for a minimum of n = 5–8 mice per group. Different lowercase letters indicate significant differences at p < 0.05. Panel C shows Oil Red O staining of 10-μm-thick skin sections from DKO 12-week chow-fed mice; DKO 6-week diet-fed mice; DKO 12-week diet-fed mice; DKO 12-week diet-fed mice + 6-week apoA-I treatment. Sections shown are representative of at least four sections per animal with at least 4–6 different animals per genotype and treatment group. There were no regional skin staining differences observed among any of the genotypes or treatment groups.

FIGURE 7.

ApoA-I treatment reduces immune cell infiltrate into the skin. Panels A–D show H&E staining of skin sections from each of the four study groups; panels E–H show immunofluorescence staining for CD68⁺ (macrophages); panels I–L show immunofluorescence staining for CD11c⁺ (dendritic cells). Panels A, E, and I are skin sections from 12-week diet-fed SKO mice; panels B, F, and J are skin sections from 6-week diet-fed DKO mice; panels C, G, and K are skin sections from 12-week diet-fed DKO mice; panels D, H, and L are skin sections from 12-week diet-fed DKO mice treated for 6 weeks with apoA-I. Sections shown are representative of at least four sections per animal with at least 4–6 different animals per genotype and treatment group.