Loss of ABCG1 influences regulatory T cell differentiation and atherosclerosis

Abstract

ATP-binding cassette transporter G1 (ABCG1) promotes cholesterol accumulation and alters T cell homeostasis, which may contribute to progression of atherosclerosis. Here, we investigated how the selective loss of ABCG1 in T cells impacts atherosclerosis in LDL receptor-deficient (LDLR-deficient) mice, a model of the disease. In LDLR-deficient mice fed a high-cholesterol diet, T cell-specific ABCG1 deficiency protected against atherosclerotic lesions. Furthermore, T cell-specific ABCG1 deficiency led to a 30% increase in Treg percentages in aorta and aorta-draining lymph nodes (LNs) of these mice compared with animals with only LDLR deficiency. When Abcg1 was selectively deleted in Tregs of LDLR-deficient mice, we observed a 30% increase in Treg percentages in aorta and aorta-draining LNs and reduced atherosclerosis. In the absence of ABCG1, intracellular cholesterol accumulation led to downregulation of the mTOR pathway, which increased the differentiation of naive CD4 T cells into Tregs. The increase in Tregs resulted in reduced T cell activation and increased IL-10 production by T cells. Last, we found that higher ABCG1 expression in Tregs was associated with a higher frequency of these cells in human blood samples. Our study indicates that ABCG1 regulates T cell differentiation into Tregs, highlighting a pathway by which cholesterol accumulation can influence T cell homeostasis in atherosclerosis.

Figure 1. Tregs are increased in mice with T cell–specific deficiency of ABCG1.

(A) ABCG1 protein expression was completely absent in T cells isolated from spleen and thymus of Lck-Cre⁺ Abcg1^fl/fl mice (Cre⁺). T cells from Lck-Cre^– Abcg1^fl/fl (Cre^–) littermate controls expressed normal levels of ABCG1. T cells from mice with a systemic deficiency of ABCG1 (Abcg1^–/–) and WT C57BL/6 mice served as negative and positive controls, respectively. Each lane represents a pool of 3 mice. (B) Treg levels were elevated in the peripheral LNs of Cre⁺ mice. Top panel: Representative FACS plots of CD4⁺CD25⁺FOXP3⁺ Tregs in Cre^– and Cre⁺ mice. Bottom panel: Percentages of Tregs and IFN-γ– and IL-17–producing Teffs in the peripheral LNs of Cre^– and Cre⁺ mice. Data were averaged from 4 to 5 mice per group and are presented as individual mice. **P < 0.01, by unpaired Student’s t test to determine differences between 2 groups.

Figure 2. The advantage of Lck-Cre⁺ Abcg1^fl/fl Treg development is intrinsic.

(A) CD45.1 (WT) and CD45.2 (Lck-Cre⁺ Abcg1^fl/fl) bone marrow cells were injected at a 1:1 ratio into irradiated CD45.1/2 heterozygous recipient mice, which were analyzed 10 weeks later. Gating strategy shows the splenic CD45.1/CD45.2 Treg composition. Representative FACS plot shows CD45.1 (blue box) versus CD45.2 (red box) Tregs. Graph shows the percentages of CD45.1⁺ (WT) and CD45.2⁺ (Lck-Cre⁺ Abcg1^fl/fl) Tregs in recipient mice. N = 5 mice per group. FSC, forward scatter; SSC, side scatter; W, width. (B and C) Increased Treg differentiation from naive Lck-Cre⁺ Abcg1^fl/fl CD4⁺ cells. (B) Naive CD4⁺ T cells from Lck-Cre⁺ Abcg1^fl/fl mice and Lck-Cre^– Abcg1^fl/fl littermates were cultured with CD3/CD28 Abs plus TGF-β for 72 hours. N = 5–6 mice per group. (C) Naive CD4⁺ T cells isolated from WT (CD45.1) and Lck-Cre⁺ Abcg1^fl/fl (CD45.2) donor mice were transferred into Rag1^–/– recipient mice at a 1:1 ratio, and 10 days later, the recipients’ peripheral LNs were analyzed. Representative FACS plot shows CD45.1 (WT; blue box) versus CD45.2 (Lck-Cre⁺ Abcg1^fl/fl; red box) Tregs. Dot plot shows the percentages of WT- and Lck-Cre⁺ Abcg1^fl/fl–expressing Tregs in the recipient mice. N = 5–6 mice per group. (D) An in vitro suppression assay was performed. CellTrace Violet–labeled WT naive CD4⁺CD25⁻CD62L⁺CD44^– T cells (Teff) were cocultured with or without CD4⁺CD25⁺ Tregs from Lck-Cre^– Abcg1^fl/fl and Lck-Cre⁺ Abcg1^fl/fl mice at a Teff/Treg ratio of 2:1. Representative plots of Teff cell division are shown. Graph shows the percentages of CellTrace Violet–diluted Teff cells (divided cell percentage) at an increasing Teff/Treg ratio. Results were averaged from 4 to 6 mice per group. **P < 0.01 and ****P < 0.0001, by unpaired Student’s t test to determine differences between 2 groups.

Figure 3. Mice with T cell–specific deletion of ABCG1 are protected against atherosclerosis.

(A) Lck-Cre⁺ Abcg1^fl/fl Ldlr^–/– and Lck-Cre^– Abcg1^fl/fl Ldlr^–/– littermates were fed a high-cholesterol diet for either 4 or 15 weeks, and the percentages of Tregs were determined in the aorta and aortic LNs. Each dot represents an individual mouse. N = 4–5 mice per group. (B) In vitro suppression assay performed with Tregs from mice on an Ldlr^–/– background and fed a high-cholesterol diet for 15 weeks. Results showed no change in Treg-suppressive activity in the absence of ABCG1. Data were averaged from 4 mice per group. (C) Atherosclerotic lesion area in aorta after 15 weeks of high-cholesterol diet feeding was analyzed in Lck-Cre⁺ Abcg1^fl/fl Ldlr^–/– mice and Lck-Cre^– Abcg1^fl/fl Ldlr^–/– littermates. Representative oil red O staining (red) in the aortic arches of Lck-Cre⁺ Abcg1^fl/fl Ldlr^–/– (Cre⁺) mice and Lck-Cre^– Abcg1^fl/fl Ldlr^–/– (Cre^–) littermates. Graph shows the quantification of lesion area as a percentage of aortic surface area in matched Cre⁺ and Cre^– mice after 15 weeks of high-cholesterol diet feeding. Results are shown for individual mice. N = 14–16 mice per group. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by unpaired Student’s t test to determine differences between 2 groups.

Figure 4. T cells from mice with T cell–specific deletion of ABCG1 show increased PD-1 expression and decreased activation during atherosclerosis development.

Lck-Cre⁺Abcg1^fl/fl (Cre⁺) mice and Lck-Cre^– Abcg1^fl/fl (Cre^–) littermates on an Ldlr^–/– background were fed a high-cholesterol diet (1.25% cholesterol) for 15 weeks, and the percentages of activated (CD44⁺CD62L^–) CD4 T cells (A), the percentages and MFI of PD-1 expression on CD4⁺ T cells (B), and the MFI of TCRβ (C) were determined in cells obtained from either the aorta or aortic LNs. Results are shown for individual mice. N = 4–10 mice per group. *P < 0.05, by unpaired Student’s t test to determine differences between 2 groups.

Figure 5. Mice with FOXP3-specific deficiency of ABCG1 show decreased atherosclerosis and an increase in Tregs and IL-10.

(A) Expression of Abcg1 mRNA was undetectable in Tregs sorted from the blood of Foxp3-Cre⁺ Abcg1^fl/fl Ldlr^–/– mice. Results are shown for individual mice. N = 4–5 mice per group. (B–C) Foxp3-Cre⁺ Abcg1^fl/fl Ldlr^–/– and Ldlr^–/– control mice were fed a high-cholesterol diet for 15 weeks, and the atherosclerotic lesion area in aortae (B) and Treg percentages in the aortic LNs (C) were analyzed. (B) Representative oil red O staining (red) in the aortic arches of Ldlr^–/– and Foxp3-Cre⁺ Abcg1^fl/fl Ldlr^–/– mice. Graph shows the quantification of the lesion area as a percentage of aortic surface area in matched Ldlr^–/– control and Foxp3-Cre⁺ Abcg1^fl/fl Ldlr^–/– mice after 15 weeks of high-cholesterol diet feeding. Results are shown for individual mice. N = 13–15 (B) or 6 (C) mice per group. (D–E) Foxp3-Cre⁺ Abcg1^fl/fl Ldlr^–/– mice and Foxp3-Cre⁺ Ldlr^–/– controls were fed a high-cholesterol diet for 15 weeks, and the capacity of CD4 T cells to produce IL-10 (D) and IL-17 (E) was determined in aortic LNs following 4 hours of stimulation with PMA and ionomycin. Data shown represent individual mice. N = 5–7 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by unpaired Student’s t test to determine differences between 2 groups.

Figure 6. Absence of ABCG1 results in increased cellular cholesterol and lipid rafts, inhibition of mTOR signaling, and increased p-STAT5 signaling.

(A–C) Cholesterol content using filipin staining (A) p-S6 (B) and p-STAT5 (C) in Lck-Cre⁺ Abcg1^fl/fl Ldlr^–/– Tregs compared with Tregs from Lck-Cre^– Abcg1^fl/fl Ldlr^–/– littermates. Cells were isolated from aortic LNs in A–C and were stimulated with 100 U/ml IL-2 for 15 minutes in B and C. Red line indicates Lck-Cre⁺ Abcg1^fl/fl Ldlr^–/– Tregs; black line indicates Lck-Cre^– Abcg1^fl/fl Ldlr^–/– Tregs. An unstimulated sample is shown in gray in B and C. Results are shown for individual mice. N = 5–6 mice per group. (D) Filipin staining showed an increase in intracellular cholesterol in Tregs in aortic LNs from Foxp3-Cre⁺ Abcg1^fl/fl Ldlr^–/– mice (red) compared with Ldlr^–/– control mice (black) following 15 weeks of high-cholesterol diet feeding. Results are shown for individual mice. N = 4–5 mice per group. (E–F) Foxp3-Cre⁺ Abcg1^fl/fl Ldlr^–/– and Foxp3-Cre⁺ Ldlr^–/– control mice were fed a high-cholesterol diet for 15 weeks, and lipid rafts and p-S6 were measured in Tregs in the aortic LNs. Plots represent 2 separate experiments (Exp1 and Exp2). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by unpaired Student’s t test to determine differences between 2 groups.

Figure 7. A negative correlation exists between ABCG1 expression and Treg levels in human blood.

ABCG1 mRNA levels from sorted Tregs (CD4⁺CD25⁺) was plotted against the percentage of Tregs (CD4⁺CD25⁺CD127^–FOXP3⁺) in the same PBMC sample. PBMCs were prepared from blood drawn from patients in the cardiovascular clinic at the University of Virginia. PBMC samples were divided into 2 portions. One was used for Treg sorting (CD4⁺CD25⁺) and qRT-PCR quantification of ABCG1 levels from sorted Tregs. The other was used for flow cytometric determination of Treg (CD4⁺CD25⁺CD127^–FOXP3⁺) percentages. A moderate negative correlation between ABCG1 levels and Treg percentages was observed (R = –0.34). Linear regression was used to determine the association between ABCG1 expression levels and Treg percentages.