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, 9 (1), 1095

Apolipoprotein AI Prevents Regulatory to Follicular Helper T Cell Switching During Atherosclerosis

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Apolipoprotein AI Prevents Regulatory to Follicular Helper T Cell Switching During Atherosclerosis

Dalia E Gaddis et al. Nat Commun.

Abstract

Regulatory T (Treg) cells contribute to the anti-inflammatory response during atherogenesis. Here we show that during atherogenesis Treg cells lose Foxp3 expression and their immunosuppressive function, leading to the conversion of a fraction of these cells into T follicular helper (Tfh) cells. We show that Tfh cells are pro-atherogenic and that their depletion reduces atherosclerosis. Mechanistically, the conversion of Treg cells to Tfh cells correlates with reduced expression of IL-2Rα and pSTAT5 levels and increased expression of IL-6Rα. In vitro, incubation of naive T cells with oxLDL prevents their differentiation into Treg cells. Furthermore, injection of lipid-free Apolipoprotein AI (ApoAI) into ApoE-/- mice reduces intracellular cholesterol levels in Treg cells and prevents their conversion into Tfh cells. Together our results suggest that ApoAI, the main protein in high-density lipoprotein particles, modulates the cellular fate of Treg cells and thus influences the immune response during atherosclerosis.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
ExTreg cells are increased during atherogenesis. a Schematic diagram with a representative flow cytometry plot of the Treg lineage tracker-ApoE−/− (LT-ApoE−/−) mouse model used to track Treg development during atherosclerosis. The diagram shows two population of Treg; current Treg (RFP+YFP+) cells, which express Foxp3, and exTreg (RFP+YFP) cells, which previously expressed Foxp3. bd LT-ApoE−/− mice were fed a western diet for 15 weeks. Bar graphs compare the numbers of total CD4 T cells and effector CD62Llo cells (b), the percentages and numbers of exTreg and current Treg (c) in the aorta, and the ratio of current Treg to exTreg in the aorta and PaLN (d) of western fed-diet to chow controls. c Representative flow cytometry plots and graphs showing the percentages of exTreg and current Treg in the aorta of the above mice. (e) Current and exTreg cells were sorted from the PaLN of LT-ApoE−/− mice and mRNA levels for Foxp3 were examined in the extracted RNA and normalized to β-actin. f Foxp3 protein expression levels in current Treg, exTreg cells and TCRβ negative cells. Representative flow cytometry histograms and bar graphs show the MFI of Foxp3 in the two cell populations. g Treg suppression assay using sorted current and exTreg from spleens and peripheral LNs from LT-ApoE−/− mice. Sorted current and exTreg cells were cultured with CD4 depleted feeder cells from spleens of B6 mice and increasing numbers of naive effector CD45.1 CD4 T cells (CD25CD44loCD62Lhi) that were labeled with Cell Trace Violet (CTV). Cells were stimulated with soluble αCD3 for 4 days, harvested and effector cells (CD4+CD45.1+) were examined for the dilution of CTV by flow cytometry. h Flow cytometry histograms showing expression pattern of CD25 (IL-2Rα), CD62L, Nrp1, CD44 and PD1 on current Treg and exTreg cells. Results are expressed as mean ± s.e.m. from two independent experiments (n = 14) (bd), a representative of two independent experiments (n = 3) (e, g), and a representative of two independent experiments (n = 11) (f). Statistical significant differences were at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (unpaired Student’s t-test)
Fig. 2
Fig. 2
A portion of exTreg population is converting to T follicular helper cells (Tfh) and Th-1 cells. a The expression of Prdm1, Bcl6, Il21 and IFNγ genes by RT-qPCR from RNA extracted from current and exTreg cells sorted from PaLN from chow and western diet-fed LT-ApoE−/− mice. The data were analyzed using the 2−ΔΔCt method and it was normalized to β-actin. b Distribution of different T cell subsets by cell numbers within the exTreg population. Graphs show the number of Tfh, Th1 and Th17 cells within the exTreg population in the PaLN. c Representative flow cytometry plots showing the percentages of Tfh (CXCR5+PD1+) exTreg cells in PaLN of above mice. Graphs show numbers of Tfh exTreg cells in the aorta, PaLN and spleens of above mice. d Representative flow cytometry plots showing the percentages of Tfh (CXCR5+Bcl6+) exTreg cells in PaLN of above mice. Graphs show numbers of Tfh exTreg cells in the PaLN and spleens of above mice. Results are expressed as mean ± s.e.m. from two independent experiments (n = 3) (a), and mean ± s.e.m from one experiment (n = 7 (chow) and n = 8 (WD)) (b), and mean ± s.e.m from one experiment (n = 7 for aorta and spleen, and n = 4 for PaLN) (c), and n = 4 (d). Statistical significant differences were at *P < 0.05, **P < 0.01, and ****P < 0.0001 (two-way ANOVA followed by Tukey’s multiple comparisons test) (a) and (unpaired Student’s t-test) (bd)
Fig. 3
Fig. 3
Blocking ICOSL reduces exTreg Tfh cells and atherosclerosis. ae LT-ApoE−/− mice were fed a western diet for 9 weeks and then were injected with antibody against ICOSL or isotype i.p. (100 μg per mouse, twice per week) for 6 weeks. Graphs show the numbers of exTreg (a), exTreg Tfh cells (b) in aorta, PaLN and spleen, germinal center B cells in PaLN (c) and current Treg cells in aorta and spleen (d). e Oil Red O stain of aortic roots from the above mice. Images are representative section from each group of mice. Each individual dot in the graph is the average of at least 4 sections per aorta per mouse. Scale bars = 100 μm. Results are expressed as mean ± s.e.m. from two independent experiments (For aorta, n = 6 (isotype) and n = 10 (αICOSL). For PaLN and spleen, n = 8 (isotype) and n = 10 (αICOSL). For aortic roots, n = 8 (isotype) and n = 11 (αICOSL). Statistical significant differences were at *P < 0.05, **P < 0.01, and ***P < 0.001 (unpaired Student’s t-test)
Fig. 4
Fig. 4
Tfh cells are pro-atherogenic. ad B6 or Bcl6fl/flCD4-Cre+ BM chimera mice on Ldlr−/− background were fed high cholesterol diet for 15 weeks. ab Flow cytometry plots and graphs showing the percentages and numbers of Tfh cells in PaLN, aorta and spleen (a) and percentages of Treg cells (b) in the PaLN. c Oil Red O stain of the aortic roots from the above mice. Images are representative sections from each group of mice. The amount of red pixels in each section was determined and the corresponding area was calculated. Each individual dot in the graph is the average of at least 4 sections per aorta per mouse. Scale Bars = 100 µm. d En face Oil Red O staining in the aorta of the above mice. Graph shows quantification of the lesion area as a percentage of the total aortic surface area. Results are expressed as mean ± s.e.m. from one experiment (n = 12 (B6) and n = 14 (Bcl6fl/flCD4Cre+) (ab), n = 8 (B6) and n = 13 (Bcl6fl/flCD4Cre+) for PaLN and spleen, n = 7 (B6) and n = 6 (Bcl6fl/fl/CD4Cre+) for aorta (c), and n = 10 (B6) and n = 13 (Bcl6fl/flCD4Cre+) (d)). Statistical significant differences were at *P < 0.05, **P < 0.01, and ****P < 0.0001 (unpaired Student’s t-test). NS none significant
Fig. 5
Fig. 5
IL-2 and IL-6 receptors expression is altered between current Treg and exTreg Tfh cells. a Flow cytometry histograms and graphs of mean fluorescence intensity of IL-2Rα (CD25) expression on current Treg, exTreg and exTreg Tfh cells from LT-ApoE−/− mice on western diet. b Histogram plots and differential expression of phosphorylated STAT5 by phosphoflow in current Treg cells in the western diet-fed and chow controls LT-ApoE−/− following IL-2 stimulation. Graph shows the fold change between unstimulated and IL-2 stimulated samples. c Flow cytometry histograms and graph of mean fluorescence intensity of IL-6Rα (CD130) expression on current Treg, exTreg and exTreg Tfh from LT-ApoE−/− mice on western diet. d Plasma levels of IL-6 in the mice described above. ei Naive CD4 T cells from ApoE−/− mice were isolated and stimulated with αCD3/CD28 and TGFβ to induce Treg in vitro with or without the addition of oxLDL or IL-2. e Flow cytometry plots and graphs showing the percentages of induced Treg following 4 days in culture. f Expression of IL-2Rα (CD25) and IL-6Rα (CD130) on total CD4 T cells in culture following 4 days of stimulation. hg Supernatant (h) and RNA (g) were harvested from the above cultures for the assessment of IL-2 by ELISA (h) and Foxp3 mRNA by qRT-PCR (g). i Flow cytometry plots and graphs showing the percentage of induced Treg with the addition of exogenous IL-2. Results are expressed as the mean ± s.e.m. from one of two independent experiments (n = 7) (a, c), from one of two independent experiments (n = 4 (chow) and 6 (WD)) (b), from three independent experiments (n = 14 (chow) and 18 (WD)) (d), from one of two independent experiments (n = 3) (eg) and (n = 6) (h), and one of two experiments (n = 4) (i). Statistical significant differences were at * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 (one-way ANOVA) (a, c, i) and (Unpaired Student’s t-test) (b, dh)
Fig. 6
Fig. 6
ApoAI administration reduces exTreg during atherogenesis. (a, b) LT-ApoE−/− mice were fed western diet and after 6 weeks, human lipid free ApoAI (500 μg per mouse)(or BSA as control) were given subcutaneously 3 times per week for 9 weeks. Mice were sacrificed after 15 weeks and aorta and PaLN were harvested for analysis. Flow cytometry plots and graphs showing the percentages and/or numbers of RFP+YFP (exTreg) and RFP+YFP+ (current Treg) in the aorta (a) and PaLN (b). Results are expressed as the mean ± s.e.m. from four independent experiments (n = 29 (Chow), n = 27 (WD) and n = 26 (WD/ApoAI)). c Aortic roots from the above mice groups were sectioned and stained with Oil Red O. Images are representative sections from each group of mice. The amount of red pixels in each section was determined and the corresponding area was calculated. Each individual dot in the graph is the average of 8 sections per aorta per mouse. Scale bars = 100 μm. Results are expressed as the mean ± s.e.m. from three independent experiments (n = 10 (Chow and WD) and n = 9 (WD/ApoAI)). Statistical significant differences were at *P < 0.05, **P < 0.01, and ****P < 0.0001 (one-way ANOVA)
Fig. 7
Fig. 7
ApoAI selectively prevents the conversion of exTreg into Tfh cells and reduces B cells in the aorta during atherogenesis. a Flow cytometry plots and graphs showing percentages and numbers of Tfh cells (CD44hiCD62LloCXCR5+PD1+) from exTreg (CD4+RFP+YFP) in the aorta, PaLN and spleen in LT-ApoE−/− western diet-fed mice with and without ApoAI treatment or chow controls. b Graphs showing numbers of B cells (CD19+), germinal center B cells (CD19+Fas+GL7+) and plasma cells (CD19+CD138+IgD) in the aorta of the mice above. c Graphs showing numbers of CD11b+ cells in the aorta by flow cytometry and area of CD68+ in aortic roots by immunofluorescence of the mice above. d, e Graphs showing numbers of IFNγ+ from exTreg, and IFNγ+ from total CD4 T cells (d, e, respectively) in the PaLN of the above mice, following stimulation with PMA/ionomycin for 5 h. Results are expressed as mean ± s.e.m from three independent experiments (n = 19 (Chow), n = 16 (WD and WD/ApoAI)) (a), mean ± s.e.m from two independent experiments (n = 10) (b), mean ± s.e.m from one experiment (n = 6 (Chow and WD), n = 7 (WD/ApoAI)) (c) and n = 7 (de). Statistical significant differences were at *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA)
Fig. 8
Fig. 8
ApoAI administration increases IL-2Rα expression and reduces IL-6 production and Bcl6 expression during atherogenesis. a Expression of IL-2Rα (CD25) and IL-6Rα (CD130) on current Treg in the aorta of LT-ApoE−/− western diet-fed mice with and without ApoAI treatment or chow controls. b Plasma levels of IL-6 in the mice described above. c Flow cytometry plots and graphs showing the percentages and numbers of CXCR5+Bcl6+ Tfh in exTreg cells of above mice. Plots are gated on CD44+RFP+YFP CD4 T cells. d Naive CD4 T cells from ApoE−/− mice were isolated and stimulated with αCD3/CD28 and TGFβ to induce Treg in vitro with or without the addition of oxLDL or ApoAI. Flow cytometry plots and graphs showing the percentages of induced Treg following 4 days in culture. Results are expressed as the mean ± s.e.m. from one of two independent experiments (n = 6 (Chow and WD/ApoAI), n = 7 (WD)) (a), mean ± s.e.m. from 3 different experiments (n = 18 (Chow), n = 17 (WD) and n = 13 (WD/ApoAI)) (b), the mean ± s.e.m. from two independent experiments (n = 11 (Chow and WD) and n = 10 (WD/ApoAI) (c), and the mean ± s.e.m. from one of two experiments (n = 4) (d). Statistical significant differences were at *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA)
Fig. 9
Fig. 9
ApoAI administration reduces the cholesterol content in current Treg cells through increasing ABCA1 expression. a Plasma from LT-ApoE−/− western diet-fed mice with and without ApoAI treatment or chow controls were tested for total cholesterol and HDL. b Sorted current Treg (RFP+YFP+) cells from PaLN of the above mice were analyzed for the amounts of intracellular free and total cholesterol using mass spectroscopy. The amount of esterified cholesterol was calculated followed by the ratio of esterified to total cholesterol. c The mRNA expression of ABCA1 by RT-qPCR from RNA extracted from current Treg sorted from the PaLN of chow and western diet-fed LT-ApoE−/− mice, with and without ApoAI treatment. The data were analyzed using the 2−ΔΔCt method and it was normalized to β-actin. Results are expressed as the mean ± s.e.m from one experiment (n = 7) (a, b), and mean ± s.e.m from one of two independent experiments (n = 3) (c). Statistical significant differences were at *P < 0.05, **P < 0.01 and ****P < 0.0001 (one-way ANOVA)

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