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, 45 (6), 1311-1326

Cholesterol Accumulation in CD11c + Immune Cells Is a Causal and Targetable Factor in Autoimmune Disease

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Cholesterol Accumulation in CD11c + Immune Cells Is a Causal and Targetable Factor in Autoimmune Disease

Ayaka Ito et al. Immunity.

Abstract

Liver X receptors (LXRs) are regulators of cholesterol metabolism that also modulate immune responses. Inactivation of LXR α and β in mice leads to autoimmunity; however, how the regulation of cholesterol metabolism contributes to autoimmunity is unclear. Here we found that cholesterol loading of CD11c+ cells triggered the development of autoimmunity, whereas preventing excess lipid accumulation by promoting cholesterol efflux was therapeutic. LXRβ-deficient mice crossed to the hyperlipidemic ApoE-deficient background or challenged with a high-cholesterol diet developed autoantibodies. Cholesterol accumulation in lymphoid organs promoted T cell priming and stimulated the production of the B cell growth factors Baff and April. Conversely, B cell expansion and the development of autoantibodies in ApoE/LXR-β-deficient mice was reversed by ApoA-I expression. These findings implicate cholesterol imbalance as a contributor to immune dysfunction and suggest that stimulating HDL-dependent reverse cholesterol transport could be beneficial in the setting of autoimmune disease.

Keywords: LXR; autoantibodies; autoimmune disease; reverse cholesterol transport.

Figures

Figure 1
Figure 1. Deletion of Lxrβ on an ApoE-null background is sufficient to provoke autoimmune disease
(A) Kidney sections from indicated ages of Apoe−/− and Apoe−/−Lxrβ−/− mice immunostained for IgG, B220, CD3 and CD68. (B) Plasma samples from 8-week-old and 24-week-old Apoe−/− and Apoe−/−Lxrβ−/− mice were analyzed for anti-nuclear antibody (ANA) titers by ELISA. (C) Flow cytometric analysis of CD19+B220+ B cells from lymph node of 8-week-old Apoe−/− and Apoe−/− Lxrβ−/− mice. (D) Percentages of indicated cell populations in lymph node of Apoe−/− and Apoe−/−Lxrβ−/− mice analyzed by flow cytometry. (E) Cell counts of the indicated cell populations in lymph node of ApoE−/− and Apoe−/−Lxrβ−/− mice analyzed by flow cytometry. N=4–6 per group. Statistical analysis was performed with Student’s t test. *p < 0.05, **p < 0.01, NS, not significant. Error bars represent means +/− SEM. See also Figure S1.
Figure 2
Figure 2. Feeding a high cholesterol diet promotes autoimmunity
(A) Flow cytometric analysis of CD19+B220+ B cells from lymph node of wild-type and Lxrβ−/− mice fed Western diet for 16 weeks. (B) Percentages and cell counts of the indicated cell populations in lymph node of wild-type and Lxrβ−/− mice fed chow or Western diet for 8 weeks or 16 weeks analyzed by flow cytometry. (C) Flow cytometric analysis of CD19+B220+ B cells from lymph node of wild-type and Lxrαβ−/− mice fed Western diet for 12 weeks. (D) Percentages and cell counts of the indicated cell populations in lymph node of wild-type and Lxrαβ−/− mice fed chow or Western diet for 12 weeks analyzed by flow cytometry. (E) Plasma samples from wild-type and Lxrαβ−/− mice fed chow and Western diet for 12 weeks analyzed for the titer of ANA by ELISA. (F) Plasma samples from wild-type and Lxrαβ−/− mice fed chow or Western diet for 12 weeks analyzed for the titers of total IgM, IgM against Cu-OxLDL, MDA-LDL and EO6 by ELISA. N=4–6 per group. Statistical analysis was performed with two-way ANOVA. *p < 0.05, **p < 0.01, NS, not significant. Error bars represent means +/− SEM. See also Figure S2.
Figure 3
Figure 3. Cholesterol accumulation in lymphoid organs promotes the production of Baff and April
(A) Lipid was extracted from lymph node, spleen and liver of 8-week-old Apoe−/− and Apoe−/−Lxrβ−/− mice. Total masses of cholesterol and triglyceride were determined by colorimetric methods. N=3. (B) Gene expression in lymph node of 8-week-old Apoe−/− and Apoe−/−Lxrβ−/− mice analyzed by real-time PCR. (C) Plasma Baff concentration in 8-week-old ApoE−/− and Apoe−/−Lxrβ−/− mice determined by ELISA. (D) Gene expression in lymph node (upper) and spleen (bottom) of wild-type and Lxrβ−/− mice fed chow or Western diet for 16 weeks analyzed by real-time PCR. (E) Gene expression in lymph node (upper), spleen (middle) and CD11c+ antigen-presenting cells (APC) (bottom) of wild-type and Lxrαβ−/− mice fed chow or Western diet for 12 weeks analyzed by real-time PCR. (F) Plasma Baff concentration in wild-type and Lxrαβ−/− mice fed chow or Western diet for 12 weeks determined by ELISA. (G) Pan B cells, pan T cells and APC were isolated from spleen of wild-type mice. Gene expression was analyzed by real-time PCR. N=4–6 per group. Statistical analysis was performed with Student’s t test (A–C) and two-way ANOVA (D–F). *p < 0.05, **p < 0.01, NS, not significant. Error bars represent means +/− SEM.
Figure 4
Figure 4. Loss of LXRβ expression in hematopoietic cells promotes the development of autoimmune disease
Bone marrow cells from Apoe−/− and Apoe−/−Lxrβ−/− mice were transplanted into Apoe−/− mice. Mice were analyzed 12 weeks after the transplantation. (A) Gene expression in from lymph node and spleen of Apoe−/− mice transplanted with bone marrow cells from Apoe−/− or Apoe−/− Lxrβ−/− mice analyzed by real-time PCR. (B) Percentages of indicated cell populations in lymph node and spleen of Apoe−/− mice transplanted with bone marrow cells from Apoe−/− or Apoe−/−Lxrβ−/− mice analyzed by flow cytometry. (C) Plasma Baff concentration in Apoe−/− mice transplanted with bone marrow cells from Apoe−/− or Apoe−/−Lxrβ−/− mice determined by ELISA. N=6 per group. Statistical analysis was performed with Student’s t test. *p < 0.05, **p < 0.01, NS, not significant. Error bars represent means +/− SEM.
Figure 5
Figure 5. Loss of LXRβ in lymphocyte does not affect the development of autoimmune disease
(A) Pan-B cells and non-B cells were isolated from spleen of LxrβF/F (F/F) and LxrβF/F; CD19-Cre (B-KO) mice. Cells were treated with GW3965 (1μM) overnight. Gene expression was analyzed by real-time PCR. (B) Pan-T cells and non-T cells were isolated from spleen of LxrβF/F (F/F) and LxrβF/F; LCK-Cre (T-KO) mice. Cells were treated with GW 3965 (1μM) overnight. Gene expression was analyzed by real-time PCR. (C) Percentages and cell counts of the indicated cell populations in lymph node of Apoe−/−LxrβF/F and ApoE−/−LxrβF/F; CD19-Cre mice analyzed by flow cytometry. (D) Gene expression in from lymph node of Apoe−/−LxrβF/Fand Apoe−/−LxrβF/F; CD19-Cre mice analyzed by real-time PCR. (E) Percentages and cell counts of the indicated cell populations in lymph node of Apoe−/−LxrβF/Fand Apoe−/−LxrβF/F; Lck-Cre mice analyzed by flow cytometry. (F) Gene expression in lymph node from Apoe−/−LxrβF/Fand Apoe−/−LxrβF/F; Lck-Cre mice analyzed by real-time PCR. N=4–5 per group. Statistical analysis was performed with Student’s t test. *p < 0.05, NS, not significant. Error bars represent means +/− SEM. See also Figures S3 and S4.
Figure 6
Figure 6. Altered antigen-presenting cell function in LXR-deficient mice
(A) Lipid content in CD11c+ APCs was analyzed by staining cells from lymph node and spleen of wild-type and Lxrαβ−/− mice fed Western diet for 12 weeks with BODIPY. MFI in CD11c+ APC population was determined by flow cytometry. (B) CFSE dilution of adoptively-transferred OT-1 T cells from lymph node and spleen of wild-type and Lxrαβ−/− mice fed Western diet for 16 weeks. On day 3 after challenge with ovalbumin (200 μg/mouse), CFSE-labeled OT-1 T cells were assessed by flow cytometry gating on CD8+TCR Va2+. (C) Bone marrow-derived dendritic cells were incubated with hydroxypropyl-β-cyclodextrin (HβCD, 10 mM) for 1 hr. Gene expression was analyzed by real-time PCR. (D) Bone marrow–derived dendritic cells were pretreated with GW3965 (1 μM) overnight, followed by stimulation with Pam3CSK4 (100 ng/ml) or CpG (1 μM) for 24 hr. Gene expression was analyzed by real-time PCR. (E) CD11c+ and CD11c cells were isolated from spleen of Apoe−/−LxrβF/F and Apoe−/−LxrβF/F; Cd11c-Cre mice. Cells were treated with GW3965 (1μM) overnight. Gene expression was analyzed by real-time PCR. (F) Cell counts of the indicated cell populations in lymph node of ApoE−/−LxrβF/F and −/−LxrβF/F; Cd11c-Cre mice at 8 weeks of age analyzed by flow cytometry. (G) Gene expression in lymph node and spleen of Apoe−/−LxrβF/F and Apoe−/−LxrβF/F; CD11c-Cre mice analyzed by real-time PCR. N=4–6 per group. Statistical analysis was performed with Student’s t test (A, C, F, G) and two-way ANOVA (D). *p < 0.05, **p < 0.01. Error bars represent means +/− SEM. See also Figures S5 and S6.
Figure 7
Figure 7. ApoA-I expression ameliorates autoimmune disease in mice deficient in ApoE and LXRβ
6 week-old Apoe−/− and Apoe−/−Lxrβ−/− mice were injected with Hd-Ad-control or Hd-Ad-ApoA-I. The mice were analyzed on 12 weeks after the injection. (A) Gene expression in liver of Apoe−/− and Apoe−/−Lxrβ−/− mice injected Hd-Ad-control or Hd-Ad-ApoA-I. (B) Percentages of indicated cell populations in lymph node and spleen of Apoe−/− and Apoe−/− Lxrβ−/− mice injected Hd-Ad-control or Ad-ApoA-I were analyzed by flow cytometry. (C) Gene expression in lymph node and spleen of Apoe−/− and Apoe−/−Lxrβ−/− mice injected with Hd-Ad-control or Ad-ApoA-I was analyzed by real-time PCR. (D) Plasma samples from Apoe−/− and Apoe−/−Lxrβ−/− mice injected with Hd-Ad-control or Ad-ApoA-I were analyzed for the presence of Baff (upper) and ANA (bottom) by ELISA. N=4–6 per group. Statistical analysis was performed with two-way ANOVA. *p < 0.05, **p < 0.01. Error bars represent means +/− SEM. See also Figure S7.

Comment in

  • Immune Cell Intolerance for Excess Cholesterol
    SB Widenmaier et al. Immunity 45 (6), 1186-1188. PMID 28002726.
    Chronic metabolic challenges have severe consequences on physiological systems. In this issue of Immunity, Ito et al. (2016) show that defects in cholesterol metabolism i …
  • Grease Fires Turn Up the Heat in Autoimmune Disease
    NR Bhakta. Sci Transl Med 9 (374). PMID 28123070.
    Restoring reverse cholesterol transport ameliorates the B cell hyperexpansion and autoimmune disease found to be associated with accumulation of cholesterol in CD11c …

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