Apoptotic cells promote their own clearance and immune tolerance through activation of the nuclear receptor LXR

Abstract

Effective clearance of apoptotic cells by macrophages is essential for immune homeostasis. The transcriptional pathways that allow macrophages to sense and respond to apoptotic cells are poorly defined. We found that liver X receptor (LXR) signaling was important for both apoptotic cell clearance and the maintenance of immune tolerance. Apoptotic cell engulfment activated LXR and thereby induced the expression of Mer, a receptor tyrosine kinase critical for phagocytosis. LXR-deficient macrophages exhibited a selective defect in phagocytosis of apoptotic cells and an aberrant proinflammatory response to them. As a consequence of these defects, mice lacking LXRs manifested a breakdown in self-tolerance and developed autoantibodies and autoimmune glomerulonephritis. Treatment with an LXR agonist ameliorated disease progression in a mouse model of lupus-like autoimmunity. Thus, activation of LXR by apoptotic cells engages a virtuous cycle that promotes their own clearance and couples engulfment to the suppression of inflammatory pathways.

Figure 1. LXR signaling regulates phagocytosis of apoptotic cells

(A). Decreased engulfment of apoptotic thymocytes by Lxrαβ-/- macrophages in vitro as visualized by phagocytosis of CellTracker™ Green CMFDA-labeled AT. A cell dissociation buffer (Krysko et al., 2006) was added to distinguish engulfed from bound cells as indicated. Arrows indicate attached cells. CellTracker™ Green CMFDA-labeled AT were cultured with macrophages (stained with CellTracker™ Red CMTPX) for 1 h. In this and all subsequent experiments a ratio of 5 AT:1 macrophage was used. Attached and engulfed cells were distinguished by extensive washing with cold PBS and Enzyme Free Cell Dissociation Buffer (Invitrogen) to remove free AT. Macrophage phagocytosis was evaluated by fluorescence microscopy. B) Quantification of phagocytosis by confocal microscopy after incubation of apoptotic thymocytes (AT) for 0.5, 1 or 2 h with WT or LXRαβ-/- macrophages. C,D) LXR agonist promotes phagocytosis of labeled AT as shown by confocal microscopy. Thioglycolate-elicited peritoneal macrophages from WT or LXRαβ-/- mice were pretreated with vehicle or 1 μM GW3965 for 48 h and then incubated with apoptotic cells for 1 h. E) In vivo association of csfe-labeled apoptotic cells with WT or Lxrαβ-/- macrophages was determined by flow cytometry. Results from 3 separate mice are shown for each genotype. All experiments were repeated a minimum of three times with comparable results. Data were expressed as mean ± SD. Statistical analysis was performed using Student’s t test. *P < 0.05, **P < 0.01

Figure 2. Defective in vivo clearance of apoptotic cells in LXRαβ-/- lymphoid tissues

A) Spleen sections from 40 week-old WT and Lxrαβ-/- mice were stained with fluorescein-labeled peanut agglutinin (PNA), IgG, Mucosal addressin cell adhesion molecule (MAdCAM-1), and B220 as indicated. Bar represents 50 μm. B) Representative spleen sections (4 μm) from 40 week-old WT and LXRαβ-/- mice were analyzed by TUNEL staining (green) and staining for CD68+ macrophages (red). Free apoptotic cells are indicated by arrows (right panel). Bar represents 25 μm. C) 4-week old male WT and LXRαβ-/- mice (4 animals/group) were injected with 0.2 mg dexamethasone (dex) and thymi were isolated 24 h later. D) Thymic mass was measured in each mouse after 24 h of dex. The percentage of Annexin V+/Tunel+ cells and recruitment of macrophages was evaluated in cell suspensions by flow cytometry. E) Thymic sections were analyzed by transmission electron microscopy. **P < 0.001.

Figure 3. Regulation of Mer expression by LXR/RXR heterodimers. Apoptotic cells promote Mer expression and phagocytosis through LXR-dependent pathways

A) Thioglycolate-elicited peritoneal macrophages from WT or LXRαβ-/- mice were treated with vehicle, 1 μM GW3965 (GW), or AT (ratio of 5 AT:1 macrophage) for 24 h. Gene expression was measured by real-time PCR. *P < 0.05 versus vehicle control; §P < 0.05 versus WT control. Experiment was repeated three times with comparable results. B) LXR-dependent regulation of Mer expression in peritoneal macrophages. mRNA expression was assayed in WT, Lxrα-/- α-/-, Lxrβ-/- or Lxrαβ-/- macrophages treated for 24 h with 1 μM GW3965 and/or 100 nM LG268 (LG). C) Induction of Mer protein expression by LXR/RXR agonists in primary WT and Lxrαβ-/- macrophages. D) Induction of Mer expression in vivo. mRNA expression was evaluated in spleen and lung tissues from WT and LXRαβ-/- mice treated with GW agonist for 3 days. N=5 per group. *P < 0.05. E) Apoptotic cells promote their own uptake in an LXR-dependent manner. WT or LXRαβ-/- macrophages were challenged with CMFDA-labeled AT for 90 min. 3 or 24 h later, the same macrophages were challenged with a second round of CMTPX-labeled AT for 90 min and only CMTPX-labeled engulfed cells were scored as in Fig 1. *P < 0.05. F). Depletion of cellular sterols from AT inhibits their ability to activate LXR. Sterol content from AT was decreased by treating cells with 5 mM of methyl-beta-cyclodextran (MßCD) prior to addition to macrophages. Macrophages were cultured with untreated or sterol-depleted AT for 90 min. Unengulfed cells were removed and gene expression was measured by real-time PCR after 24 h.

Figure 4. Apoptotic cells promote tolerogenic gene expression and repression of proinflammatory mediators through LXR-dependent pathways

A) Thioglycolate-elicited peritoneal macrophages from WT or Lxrαβ-/- mice were treated with vehicle or AT (ratio of 5 AT:1 macrophage) for 24 h. Gene expression was measured by real-time PCR. *P < 0.05 versus vehicle control; §P < 0.05 versus WT control. Experiment was repeated three times with comparable results. B) Macrophages from WT and Lxrαβ-/- mice were challenged with AT for 24 h and mRNA expression was evaluated by Northern blotting. C) Exposure to apoptotic cells inhibits macrophage responses to LPS in an LXR-dependent manner. WT or Lxrαβ-/- macrophages were preincubated with AT for 1h and then challenged with LPS for 3 and 6h. Gene expression was evaluated by real-time PCR. *P<0.05.

Figure 5. Mer expression is required for LXR-dependent phagocytosis of apoptotic cells

A) Protein expression in RAW cells stably expressing LXRα (RAW-LXRα) or Mer (RAW-Mer). B) Phagocytosis of apoptotic cells was evaluated in RAW-LXRα or RAW-Mer macrophages. Cells were treated for 36 h with GW3965 and then challenged with AT for 15, 45 and 90 min. C) A blocking antibody to Mer inhibits AT uptake in WT but not LXRαβ-/- macrophages. Thioglycolate-elicited peritoneal macrophages from WT or LXRαβ-/- mice were incubated with either IgG control antibody or Mer-specific blocking antibody for 3h. Phagocytosis of apoptotic cells was evaluated by confocal microscopy after 90 min incubation of AT. *P < 0.05. D) Effect of control and Mer-specific siRNAs on protein expression in RAW cells. E) Mer knockdown blocks the effect of LXR ligand on AT phagocytosis in RAW-LXRα macrophages. F) Efficacy of control, Mer-, ApoE- and ABCA1-specific siRNAs on protein expression in mouse primary peritoneal macrophages. G) Effect of control, Mer-, ApoE- and ABCA1-specific siRNAs on the ability of LXR agonists to promote AT uptake by primary macrophages. *P < 0.05; **P < 0.001.

Figure 6. Lxrαβ-/- mice develop age-dependent autoimmune disease

A) Spleen and lymph nodes isolated from 40 week-old WT and Lxrαβ-/- mice. B) Analysis of spleen mass in WT and LXRαβ-/- mice. Spleens were isolated from WT and Lxrαβ-/- mice at the indicated age and spleen mass relative to body weight was measured. C) Total cell counts from spleen and lymph node of 40 week-old mice. D) 1:100 dilutions of serum samples obtained from 40 week-old WT (12 female mice) and Lxrαβ-/- mice (8 female mice) were analyzed for the presence of autoantibodies by ELISA. **P < 0.01, ***P < 0.001. E) Kidney sections (4 μm) from 40 week-old WT and Lxrαβ-/- female mice stained with H&E. F) Kidney sections from 40 week-old female mice stained with FITC-labeled anti-mouse IgG. Bar represents 50 μm. G) Consecutive kidney sections from 40 week-old female mice were stained with anti-mouse CD4, B220, CD68 and H&E. Bar represents 50 μm. H) Kidney sections from 40 week-old WT and LXRαβ-/- female mice stained with anti-CD8 and anti-CD4 antibodies. Bar represents 25 μm.

Figure 7. Treatment with an LXR agonist ameliorates autoimmune disease in mice

A) Peritoneal macrophages obtained from WT and B6^lpr/lpr mice were treated with GW3965 for 24 h and phagocytosis was analyzed by confocal microscopy. B) Gene expression in macrophages from WT and B6^lpr/lpr mice treated as in A. C) L-FMAC and PET/CT imaging of B6^lpr/lpr mice. Images were analyzed using AMIDE software. N = 5/group; representative images are shown. D) Lymph node cellularity in 8-week old B6^lpr/lpr female mice treated for 4 months with GW3965. N = 5/group. E). Representative kidney sections from female B6^lpr/lpr mice treated with GW3965 for 4 months. Sections were analyzed by anti-immunoglobulin staining. Bar represents 50 μm. N = 6/group. Representative images are shown.