Abstract
Apolipoprotein (Apo)A-I, the major lipid-binding protein of high-density lipoprotein, can prevent autoimmunity and suppress inflammation in hypercholesterolemic mice by attenuating lymphocyte cholesterol accumulation and removing tissue-oxidized lipids. However, whether ApoA-I mediates immune-suppressive or anti-inflammatory effects under normocholesterolemic conditions and the mechanisms involved remain unresolved. We transferred bone marrow from systemic lupus erythematosus (SLE)-prone Sle123 mice into normal, ApoA-I–knockout (ApoA-I−/−) and ApoA-I–transgenic (ApoA-Itg) mice. Increased ApoA-I in ApoA-Itg mice suppressed CD4+ T and B cell activation without changing lymphocyte cholesterol levels or reducing major ApoA-I–binding oxidized fatty acids. Unexpectedly, oxidized fatty acid peroxisome proliferator–activated receptor γ ligands 13- and 9-hydroxyoctadecadienoic acid were increased in lymphocytes of autoimmune ApoA-Itg mice. ApoA-I reduced Th1 cells independently of changes in CD4+Foxp3+ regulatory T cells or CD11c+ dendritic cell activation and migration. Follicular helper T cells, germinal center B cells, and autoantibodies were also lower in ApoA-Itg mice. Transgenic ApoA-I also improved SLE-mediated glomerulonephritis. However, ApoA-I deficiency did not have the opposite effects on autoimmunity or glomerulonephritis, possibly as the result of compensatory increases in ApoE on high-density lipoprotein. We conclude that, although compensatory mechanisms prevent the proinflammatory effects of ApoA-I deficiency in normocholesterolemic mice, increasing ApoA-I can attenuate lymphocyte activation and autoimmunity in SLE independently of cholesterol transport, possibly through oxidized fatty acid peroxisome proliferator-activated receptor γ ligands, and it can reduce renal inflammation in glomerulonephritis.
Introduction
Apolipoprotein (Apo)A-I is the major cholesterol- and lipid-binding protein component of high-density lipoprotein (HDL) and, as such, confers many of its protective properties in atherosclerosis (1). Although the ability of ApoA-I to counteract excessive cellular cholesterol accumulation and promote reverse cholesterol transport was linked to the anti-inflammatory action of HDL in rodent atherosclerosis models (2, 3), other mechanisms are also thought to be involved (4). These include the binding and hydrolysis of oxidized lipids by HDL-associated ApoA-I and paraoxonase-1 (PON-1) enzymatic activity, respectively, which contribute to the anti-inflammatory effects of HDL in hypercholesterolemic mice (5). For example, oxidized metabolites of linoleic and arachidonic acids (hydroxyoctadecadienoic acid [HODE] and hydroxyeicosatetraenoic acid [HETE], respectively) that have proinflammatory effects on vascular cells are produced abundantly in atherosclerosis by the action of lipoxygenase (LO) enzymes and reactive oxygen species (6), and they are reduced by ApoA-I in concert with its vasoprotective and antiatherogenic action in hypercholesterolemic atherosclerosis models (5, 7).
There is considerable evidence from studies in hypercholesterolemic animal models to support the notion that modulating ApoA-I could alter the levels of cholesterol in lymphoid tissue and other organs and affect immune activation and inflammation in autoimmune settings. For example, ApoA-I deficiency in hypercholesterolemic low-density lipoprotein (LDL) receptor–knockout mice causes excessive lymphocyte cholesterol accumulation in lymph nodes, resulting in hyperproliferation of T lymphocytes and the development of systemic autoimmunity resembling systemic lupus erythematosus (SLE) (8). Impaired immune cell cholesterol homeostasis caused by deficiency of the liver X receptor pathway or scavenger receptor BI (a receptor involved in hepatic cholesterol ester uptake from HDL) similarly caused lymphocyte hyperproliferation and the development of SLE-like disease (9–11). The common mechanism mediating the autoimmune phenotypes in these hypercholesterolemic settings is the abnormally high immune cell cholesterol accumulation that causes immune hyperactivation, at least in part, through modulation of membrane raft–dependent receptor signaling (2). Therefore, it was suggested that ApoA-I is essential to prevent systemic autoimmunity resulting from excessive immune cell cholesterol accumulation under conditions of hypercholesterolemia or interrupted cholesterol transport in mice. Indeed, the notion that ApoA-I suppresses autoimmunity in hypercholesterolemia by counterbalancing excessive cellular cholesterol accumulation to dampen lymphocyte activation and proliferation is also supported by data in mice showing suppressive effects of genetic disruptions in cholesterol transport pathways on cellular activation and proliferation in other systems, including the hematopoietic stem cell compartment (12).
Although data in hypercholesterolemic models provided important insights into the interactions between HDL cholesterol (HDL-C) metabolism and autoimmunity (2), their interpretation with respect to the immunomodulatory properties of ApoA-I and HDL in SLE is confounded by the extremely high levels of hypercholesterolemia and disruption of homeostatic mechanisms controlling cellular cholesterol levels in the animal models used. As a result, questions exist with regard to their physiological relevance, particularly considering the disappointing outcome of clinical trials in coronary heart disease patients of an ApoA-I mimetic peptide, 4F, which can provide robust anti-inflammatory and anti-atherogenic effects in hypercholesterolemic rodent models by recapitulating cholesterol- and oxidized lipid–binding properties of ApoA-I (13, 14). Indeed, the high expectations for 4F as a therapeutic were based largely on its effects in hypercholesterolemic animal models, with comparatively little data from normocholesterolemic animal models that are not compromised by confounding effects of hypercholesterolemia on inflammation and the immune system. Furthermore, the role of oxylipids, such as HODEs and HETEs, which are typically involved in atherosclerosis and bound by ApoA-I in concert with its anti-inflammatory action in hypercholesterolemic mouse models, have not been investigated in autoimmune settings like SLE. Therefore, there is no direct evidence that modulating ApoA-I can provide immune suppression in autoimmune settings without hypercholesterolemia, and it is not known whether ApoA-I can modify cellular and molecular pathways to suppress relevant immune processes, such as lymphocyte activation, unless they are first pushed in the opposite direction (hyperactivated) by hypercholesterolemia (15).
Further underscoring the need for studies in normocholesterolemic models to test the immune-suppressive and anti-inflammatory properties of ApoA-I, SLE is associated with lower ApoA-I (HDL) levels and the development of dysfunctional proinflammatory HDL, which may contribute to premature atherosclerosis in SLE patients (16–18). It was also suggested that these reductions in ApoA-I (HDL) levels and function in SLE might feedback onto autoimmunity itself, amplifying immune activation, autoantibody production, and organ inflammation. However, this has not been addressed in the context of normocholesterolemic SLE models with modified ApoA-I (HDL) levels. Therefore, we used a normocholesterolemic bone marrow (BM) transfer model of SLE to test whether modulating ApoA-I levels can provide immune suppression in SLE, despite an underlying predisposition for HDL to become reduced or dysfunctional, to characterize the cellular and molecular mechanisms involved and identify lipid-based immune-suppressive pathways mediated by ApoA-I that can be exploited therapeutically in SLE.
Materials and Methods
Mice
C57BL/6J (wild-type [WT]), C57BL/6J ApoA-I−/−, C57BL/6J ApoA-I–transgenic (ApoA-Itg) [containing approximately twice the normal levels of plasma ApoA-I (19)], and Sle123 mice were obtained from The Jackson Laboratory (Bar Harbor, ME), and colonies were maintained at the University of Alabama at Birmingham. Female mice were weaned at 4 wk of age and maintained on a standard rodent chow diet for the duration of the study. All studies were conducted in conformity with Public Health Service Policy on Humane Care and Use of Laboratory Animals and with approval from the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.
BM transplantation
BM transplantation (BMT) was conducted as previously described (20). To distinguish donor from recipient BM-derived cells, colonies of WT, ApoA-I−/−, and ApoA-Itg mice expressing the differential Ly5.1 (CD45.1) alloantigen were derived by crossing with the B6.SJL-Ptprca Pep3b/BoyJ strain of C57BL/6J mice (The Jackson Laboratory). Ly5.1 C57BL/6J, Ly5.1 ApoA-I−/−, and Ly5.1 ApoA-Itg recipients were lethally irradiated (two doses of 450 rad ionizing radiation separated by 3 h) and transplanted at 6 wk of age with 5 × 106 BM mononuclear cells from Ly5.2 WT or Ly5.2 Sle123 donor mice. Peripheral blood samples were collected by retro-orbital puncture and processed for flow cytometric analysis of reconstitution of major hematopoietic lineages using the following conjugated Abs: anti-CD4 (RM4-5) PerCP, anti-CD8 (53-6.7) FITC, anti-CD11b (M1/70) PE, anti-CD19 PerCP (1D3) (BD Pharmingen, San Jose, CA), anti-Ly5.1 (A20) FITC or PE, and anti-Ly5.2 (104) allophycocyanin (Southern Biotech, Birmingham, AL). No aberrant ApoA-I–specific Ab responses were generated as a result of BM transfer into WT or ApoA-I gene–targeted mice [by anti-human ApoA-I and anti-mouse ApoA-I ELISA; as in (18); data not shown].
Measurement of plasma autoantibodies
Anti-dsDNA autoantibody levels were measured by ELISA, as previously described (18). Ninety-six–well MaxiSorp plates (Thermo Scientific, Waltham, MA) were coated with dsDNA (0.24 μg/well; Sigma-Aldrich, St. Louis, MO) in bicarbonate buffer (150 mM Na2CO3, 350 mM NaHCO3 [pH 9.7]) overnight at 4°C. Plates were washed with PBS containing 0.5% Tween 20 (PBST) using an automated plate washer (BioTek, Winooski, VT) and blocked with 3% nonfat milk/PBS. Plates were subsequently incubated with diluted plasma (1:100 in PBS) for 2 h, washed, and incubated with HRP-conjugated goat anti-mouse IgG at 1:5000 (Southern Biotech) in blocking buffer. Plates were washed, developed with tetramethylbenzidine substrate (Pierce, Rockford, IL), and measured on a Bio-Rad Model 680 microplate reader (Bio-Rad, Hercules, CA) at 450 nm.
Flow cytometry
Single-cell suspensions were prepared from collagenase D (Roche, Indianapolis, IN)-digested lymph nodes (axillary, superficial cervical, brachial, inguinal, and iliac), as described previously (18+ Th1 and IL-17–secreting CD4+
FITC skin painting assay
Dendritic cell (DC) migration assays were conducted as described in Robbiani et al. (21). WT, ApoA-I−/−, and ApoA-Itg mice were anesthetized, and their sides were shaved. FITC (8 mg/ml) was dissolved in sensitizer consisting of equal volumes of acetone and dibutyl phthalate (Sigma-Aldrich). A total of 25 μl FITC-containing sensitizer was applied to the right dorsal side of the mouse, whereas 25 μl sensitizer alone was applied to the left dorsal side. Eighteen hours later, draining lymph nodes (axillary, brachial, inguinal) from each side (control lymph nodes from left and FITC lymph nodes from right) were removed, and single-cell suspensions were prepared by collagenase D digestion, as described above, for counting and flow cytometric analysis with anti-CD11c (HL3) PE Ab (BD Pharmingen).
Kidney morphometric analysis and histopathology
Kidneys were fixed in neutral buffered 10% formalin and processed routinely for paraffin sectioning. Five-micrometer-thick sections were stained with periodic acid–Schiff (PAS) stain and hematoxylin. Slides were examined with experimental classifications concealed from the observer. For morphometric analysis, nine glomeruli were evaluated per section, as previously described (22). To minimize selection bias, glomeruli were selected beginning at the capsule at the 12 o’clock position of the section and proceeding clockwise, selecting only centrally sectioned glomeruli having open capillary lumina and avoiding collapsed or tangentially sectioned glomeruli. Images of selected glomeruli were made with a SPOT Insight 4 megapixel digital camera (Diagnostic Instruments, Sterling Heights, MI) with a 40× objective. After adjustment for sharpness, contrast, and brightness to maximize visibility of PAS staining, images were analyzed by histomorphometry for total glomerular area, PAS-stained area, and nuclear area with Image Pro Plus v6.2 image analysis software (Media Cybernetics, Silver Spring, MD) by thresholding gray-scaled images, color segmentation of color images, or both. Glomeruli were also evaluated by subjective scoring, without the pathologist’s knowledge of the genotype of the mice, as previously described (22, 23). Glomerular cellularity, neutrophil infiltration, necrosis, hyaline deposits, mesangial expansion, cellular crescent formation, and interstitial inflammatory cell accumulation were evaluated. Each was scored 0 (normal), 1, 2 (mild to moderate), or 3 (severe), respectively. Equal numbers of glomeruli from superficial, middle, and deep cortex were examined. Only glomeruli sectioned through the approximate center of the tuft and including the base of the tuft were studied. Overall glomerular activity scores for each mouse were calculated as the mean of summed individual scores for each glomerulus, with scores for necrosis and crescent formation each weighted by a factor of 2: (proliferation [cellularity] + neutrophil infiltration + necrosis2 + hyaline deposits + mesangial expansion + cellular crescent formation2 + interstitial inflammatory cell infiltration).
Kidney immunofluorescence analysis
Eight-micrometer cryosections prepared from kidneys in optimum cutting temperature compound (Tissue-Tek) were fixed in acetone and stained with goat anti-mouse IgG–Alexa Fluor 594 (Life Technologies), rat anti-mouse CD4 (GK1.5), rat anti-mouse CD11b (M1/70) (BD Pharmingen), or rat anti-murine neutrophil (NIMP-R14) (Thermo Fisher) Abs (24). Goat anti-rat IgG–Alexa Fluor 555–conjugated secondary Ab (Life Technologies) was used for sections stained with anti-CD4, anti-CD11b, and anti-murine neutrophil primary Abs. Color images were captured on an Olympus BX60 microscope using a 40× objective. For quantification of IgG and CD11b, average fluorescence intensity was quantified from six representative 400× microscopic fields/kidney cryosection using the histogram function of Adobe Photoshop CS5.1 (Adobe Systems, San Jose, CA), as previously described (25). For CD4 and neutrophil quantification, the numbers of CD4+ T cells and neutrophils were enumerated from six representative 400× microscopic fields/kidney cryosection by counting Hoechst-stained nuclei that were positive for cytoplasmic CD4 or NIMPR14 staining.
Urine albumin and creatinine measurement
Urine albumin was quantified using a Mouse Albumin ELISA Quantitation Set (Bethyl Laboratories), according to the manufacturer’s instructions, at a sample dilution of 1:1000. Urine albumin and creatinine was measured in spot urine samples taken at euthanization for the control and Sle123 BMT animal groups. Urine creatinine was determined by underivatized, stable isotope dilution liquid chromatography-tandem mass spectrometry LC-MS/MS, as described (26). Briefly, samples were separated using a Waters 2795 separations module (liquid chromatography [LC]) (Waters, Milford, MA) with a TSK-Gel Amide 80 column (Tosoh Bioscience, Tokyo, Japan) with an isocratic flow of 10 mM ammonium acetate in 65% acetonitrile; creatinine was subsequently detected by a Waters Quattro micro API (tandem mass spectrometry) in the positive ion mode.
Lipoprotein and lipid analyses
Plasma lipids were processed for measurement of total cholesterol, esterified cholesterol, HDL-C, LDL cholesterol (LDL-C), triglycerides, and free fatty acids by enzymatic procedures, as previously described (27). To obtain fast-performance LC (FPLC) plasma cholesterol profiles, plasma (200 μl pooled from two mice) was fractionated using a Superose 6 column (Amersham Biosciences, GE Life Sciences, Pittsburgh, PA) on a BioLogic DuoFlow FPLC system (Bio-Rad), as previously described (20). Fractions of 0.5 ml were collected and analyzed for total cholesterol content using enzymatic cholesterol reagents (Infinity Cholesterol Reagent TR13421; Thermo Scientific). Total plasma and fractionated plasma PON-1 paraoxonase activity were measured using 1 mM Paraoxon (Supelco Analytical; Sigma-Aldrich) in 50 mM Tris/HCl (pH 8), 1 mM CaCl2, as previously described (18).
Cholesterol analysis by gas chromatography mass spectrometry
Gas chromatography–mass spectrometry (GC-MS) cholesterol analyses were performed in the Mass Spectrometer Facility of the Comprehensive Cancer Center of Wake Forest School of Medicine (Winston-Salem, NC). Lipids were extracted from 10 million lymph node lymphocyte cells that were prepared by forcing lymph nodes through 100-μm strainers, as described by Wilhelm et al. (8). Lipids were resuspended in 200 μl hexane, and 100 μl was analyzed on a Finnigan TSQ Quantum XLS mass spectrometer interfaced to a trace gas chromatograph, as described by Wilhelm et al. (8). To quantify esterified cholesterol levels, a 50-μl hexane lipid aliquot was also saponified at room temperature for 2 h with methanolic KOH, as described by Nordskog et al. (28), and then extracted with hexane, as described by Wilhelm et al. (8), prior to GC-MS.
SDS-PAGE and immunoblotting
Plasma FPLC fractions (5 μl) were separated on 4–15% mini-Protean TGX gels (Bio-Rad) and transferred to Immobilon P membranes (Millipore, Billerica, MA), as previously described (20). Membranes were blocked with PBST containing 5% nonfat milk by weight and incubated with primary Abs diluted 1:2000 in blocking buffer (rabbit anti-mouse ApoA-I, rabbit anti-mouse ApoE, and/or goat anti-human ApoA-I; Meridian Life Sciences, Memphis, TN), followed by HRP-conjugated goat anti-rabbit IgG (Bio-Rad) or donkey anti-goat IgG (Southern Biotech) secondary Abs diluted 1:4000 in blocking buffer. Immunoreactive proteins were visualized using ECL (Immun-Star; Bio-Rad). Neat plasma (1 μl) samples were separated on 4–15% mini-Protean TGX gels (Bio-Rad) and transferred to Immobilon FL membranes (Millipore). Membranes were blocked with PBST containing 5% nonfat milk by weight and incubated with primary Abs diluted 1:2,000 in blocking buffer [rabbit anti-mouse ApoA-I, rabbit anti-mouse ApoE, goat anti-human ApoA-I (Meridian Life Sciences), or rabbit anti-mouse ApoA-II (29)], followed by IRDye 800CW–conjugated donkey anti-rabbit IgG or IRDye 680RD–conjugated donkey anti-goat IgG secondary Abs (Li-Cor, Lincoln, NE) diluted 1:40,000 in blocking buffer. Immunoreactive proteins were visualized using Li-Cor’s Odyssey CLx Infrared Imaging System. Acquired images were adjusted for optimal brightness and contrast, and band intensities were quantified using Image Studio Analysis Software v3.1 (Li-Cor).
HODE/HETE measurement
For plasma HODE/HETE measurement, butylated hydroxytoluene was added to freshly isolated 100-μl plasma aliquots to a final concentration of 100 μM and overlaid with argon gas prior to storage at −80°C. Lipids were extracted from 100-μl plasma aliquots and analyzed by LC-MS/MS, as described by Imaizumi et al. (7), using a Shimadzu prominence HPLC system and an AB/Sciex API-4000 QTRAP mass spectrometer. Aliquots (100 μl) of plasma were acidified by the addition of 1.7 ml (pH 2.3) H2O, vortexed briefly, and incubated on ice for 15 min. Deuterated standards for each measured HODE and HETE (20 ng each) (Cayman Chemical, Ann Arbor, MI) were spiked into samples prior to extraction. Samples were subsequently added to solid-phase extraction columns (Oasis HLB 30 mg 1 ml; Waters, cat. no. WAT094225) while pulling a slow vacuum so that each sample took ∼2 min to pass through. After washing solid-phase extraction columns with 1 ml 5% methanol, lipids were eluted with 1 ml 100% methanol, dried under argon, and resuspended in 100 μl 80% methanol. For HODEs and HETEs, lipids (50 μl) were injected onto a Luna C18 2-HST reverse-phase LC column (2 × 100 mm, 2.5 μ; Phenomenex, Torrance, CA) using a Shimadzu autosampler with gradient elution at 0.3 ml/min (mobile phase A: 0.1% formic acid in H2O; mobile phase B: 0.1% formic acid in acetonitrile). Gradient elution was as follows: from 0 to 2 min hold at 50% B, from 2 to 3 min increase to 60% B, from 3 to 15 min increase to 65% B, from 15 to 17 min hold at 65% B, from 17 to 19 min increase to 100% B, from 19 to 21 min hold at 100% B, from 21 to 23 min decrease back to 50% B and a hold for the system to equilibrate to initial conditions until 27 min. For linoleic acid and arachidonic acid, lipids (50 μl) were injected onto a Luna C12 Proteo reverse-phase LC column (2 × 50 mm, 4 μ; Phenomenex) using a Shimadzu autosampler with gradient elution at 0.25 ml/min (mobile phase A: 10 mM ammonium acetate in H2O; mobile phase B: 10 mM ammonium acetate in isopropyl alcohol). Gradient elution was as follows: from 0 to 10 min increase from 35% B to 100% B, from 10 to 11 min decrease from 100% B to 35% B, from 11 to 17 min hold at 35% B for the system to equilibrate to initial conditions. A standard curve (0.1, 1, 2.5, 5, 7.5, 10, 25, 50, 75, 100, 250, 500, 750, 1000 ng/ml) of a mixture of unlabeled linoleic acid, arachidonic acid, HODEs, and HETEs (13-HODE, 9-HODE, 15-HETE, 5-HETE, 12-HETE) was also run for quantification of individual fatty acid and HODE/HETE species. Each point of this standard curved was spiked with deuterated linoleic acid, arachidonic acid, and HODEs/HETEs standards, exactly as described above for samples (20 ng each deuterated fatty acid and HODE/HETE/standard curve point). The transitions monitored were the following mass-to-charge ratios (m/z): m/z 295.1→194.8 for 13-HODE; 295.0→171.0 for 9-HODE; 319.1→219.0 for 15-HETE; 319.1→115.0 for 5-HETE; 319.1→179.0 for 12-HETE; 299.0→197.9 for 13(S)-HODE-d4; 299.1→172.0 for 9(S)-HODE-d4; 327.1→226.1 for 15(S)-HETE-d8; 327.1→116.0 for 5(S)-HETE-d8; 327.1→184.0 for 12(S)-HETE-d8, 303.0→59.0 for arachidonic acid; 311.0→267.0 for arachidonic acid-d8; 279.0→261.0 for linoleic acid; and 283.0→265.0 for linoleic acid-d4.
Statistics
Statistical analysis was performed using SigmaPlot 11 (Systat Software, San Jose, CA). One-way ANOVA was used for multiple comparisons, and the Student t test was used for single comparisons. For all statistical analyses a p value < 0.05 was considered significant.
Results
Transgenic expression of ApoA-I suppresses lymphocyte activation and autoimmunity
We transplanted BM from Ly5.2 SLE-prone Sle123 mice into 6-wk-old Ly5.1 WT mice, Ly5.1 ApoA-I−/− mice, or Ly5.1 human ApoA-Itg mice (Sle123→WT, Sle123→ApoA-I−/−, and Sle123→ApoA-Itg). Mice that received BMT were subsequently bled at intervals and analyzed at 38 wk of age (32 wk post-BMT) to measure autoimmune phenotypes and glomerulonephritis (GN). Groups of Ly5.1 WT, Ly5.1 ApoA-I−/−, and Ly5.1 ApoA-Itg mice were also transplanted with Ly5.2 WT BM (WT→WT, WT→ApoA-I−/−, and WT→ApoA-Itg control BMT groups) to control for effects of lethal irradiation and hematopoietic reconstitution. A schematic representation is shown in Fig. 1A. Myeloid and lymphoid repopulation was equivalent among WT, ApoA-I−/−, and ApoA-Itg mice transplanted with Sle123 BM (Table I), such that any differences in autoimmune phenotypes could not be ascribed to differences in hematolymphoid reconstitution with Sle123 cells.
(A) BMT scheme used to generate chimeric autoimmune mice (animals were transplanted with WT or Sle123 BM at 6 wk of age). (B) Plasma HDL-C levels in the indicated control, control WT BMT, and Sle123 BMT mice (38 wk of age, 32 wk post-BMT, n = 8). (C) Plasma PON-1 activity against paraoxon substrate in the indicated control, control WT BMT, and Sle123 BMT mice (38 wk of age, 32 wk post-BMT, n = 8). Note autoimmune-mediated reductions in HDL-C and HDL-associated PON-1 activity in Sle123→WT mice (dashed box). (D) Plasma levels of mApoA-I and human ApoA-I (hApoA-I) in four control and four Sle123 BMT mice (38 wk of age, 32 wk post-BMT). (E) Graph showing band intensities for plasma mApoA-I. Note the reduction in plasma mApoA-I in Sle123→WT mice compared with their control WT counterparts. ApoA-Itg animals have markedly lower endogenous mApoA-I levels, as previously reported (19). All data are mean ± SD. *p < 0.05 within WT, ApoA-I−/−, or ApoA-Itg group, one-way ANOVA, ‡p < 0.05 versus control group, t test.
As expected, because BM-derived cells do not express ApoA-I (30), and ApoA-I is important for stabilizing PON-1 activity on HDL particles (31), plasma levels of HDL-C and HDL-associated PON-1 activity were decreased in transplanted and control non-BMT ApoA-I−/− mice and increased in transplanted and control non-BMT ApoA-Itg animal groups (Fig. 1B, 1C, Table II). As previously reported (32), plasma LDL-C and triglyceride levels were increased (Table II) and endogenous mouse ApoA-I (mApoA-I) levels were significantly reduced by posttranscriptional mechanisms in ApoA-Itg animals (19) (Fig. 1D, 1E). Despite these reductions in endogenous mApoA-I, total ApoA-I levels (mApoA-I plus human transgenic ApoA-I) are increased ∼2-fold in ApoA-Itg mice (19).
Plasma levels of anti-dsDNA IgG autoantibodies were significantly lower in Sle123→ApoA-Itg mice compared with their Sle123→WT and Sle123→ApoA-I−/− counterparts at 32 and 38 wk of age (26 and 32 wk post-BMT, respectively) (Fig. 2A). However, Sle123→ApoA-I−/− animals had comparable levels of autoantibodies as Sle123→WT animals at all time points measured (Fig. 2A). Although control WT BMT animals had reduced numbers of lymph node B cells and CD4+ T cells compared with their control non-BMT counterparts, presumably due to lethal irradiation and transplantation, autoimmune-associated increases in lymphocytes were evident in WT mice transplanted with Sle123 BM (Figs. 2B, 3B). B cell numbers and activation were reduced in lymph nodes of Sle123→ApoA-Itg mice by 41 and 57%, respectively (Fig. 2B). Lymph node CD4+ (but not CD8+) T cell numbers and activation were reduced in Sle123→ApoA-Itg mice by 38 and 43%, respectively (Fig. 3). Similar suppressive effects of transgenic ApoA-I expression were observed in splenic B and CD4+ T cells from Sle123→ApoA-Itg mice (B cells were reduced by 47%, CD4+ T cells were reduced by 57%, CD8+ T cells were not reduced). ApoA-I deficiency did not result in opposite effects, demonstrating that, unlike in hypercholesterolemic SLE models (8, 10, 11, 15), ApoA-I deficiency does not exacerbate autoimmunity under normal (normocholesterolemic) conditions in mice.
Transgenic ApoA-I expression reduces B cell activation and anti-ds DNA IgG autoantibodies in Sle123→ApoA-Itg mice. (A) Anti-dsDNA autoantibody levels in control (n = 7), WT BMT control (n = 7), and Sle123 BMT (n = 10) groups. Ages of animals are shown on x-axis. (B) Flow cytometric analysis of lymph node B220+ B lymphocyte activation (measured by CD69 expression) in 38-wk-old control (n = 12–15), control WT BMT (n = 10–12), and Sle123 BMT (n = 12–15) mice. Reduced numbers of B cells and lower B cell activation occurred in Sle123→ApoA-Itg mice. All data are mean ± SD. *p < 0.05 within control, control WT BMT, or Sle123 BMT group, one-way ANOVA, ‡p < 0.05 versus control, t test.
Reduced numbers and activation of CD4+ T lymphocytes in Sle123→ApoA-Itg mice. (A) Flow cytometric analysis (showing gates) of lymph node CD4+ T lymphocyte activation (measured by CD69 and CD44highCD62Llow expression) in 38-wk-old control, control WT BMT, and Sle123 BMT mice. (B) Numbers of CD4+ T lymphocytes in lymph nodes of the indicated mice (n = 12–15 control, n = 10–12 control WT BMT, n = 10–12 Sle123 BMT mice). Percentage (C) and absolute numbers (D) of CD69-expressing CD4+ T lymphocytes in lymph nodes of indicated mice. (E) CD44highCD62Llow CD4+ T lymphocytes in lymph nodes of indicated mice. Note that ApoA-I deficiency in this normocholesterolemic setting does not augment T cell activation. All data are mean ± SD. *p < 0.05 within control, control WT BMT, or Sle123 BMT groups, one-way ANOVA, ‡p < 0.05 versus control, t test.
Consistent with fewer activated CD4+ T cells in Sle123→ApoA-Itg mice, the numbers of proliferating CD4+ (but not CD8+) T cells were also reduced (by 58%) (Fig. 4A). Importantly, this was not associated with a decreased rate of proliferation, because the level of BrdU incorporation into CD4+ T cells, as measured by BrdU mean fluorescence intensity, was equivalent among Sle123→WT, Sle123→ApoA-I−/−, and Sle123→ApoA-Itg mice (59 ± 9, 56 ± 14, and 51 ± 11, respectively). Thus, unlike in hypercholesterolemic mice, in which the proliferative rate of T lymphocytes was increased by ApoA-I deficiency (8), loss or transgenic expression of ApoA-I in our normocholesterolemic Sle123 BMT model had no significant effect on the rate of T lymphocyte proliferation.
Reduced frequency of proliferating CD4+ T cells and lower IFN-γ–secreting Th1 (but not IL-17–secreting Th17) and Tfh cells in Sle123→ApoA-Itg mice. (A) Flow cytometric analysis of in vivo BrdU incorporation into lymph node CD4+ T cells 6 h following i.p. BrdU injection (n = 8). ‡p < 0.05, versus control non-BMT, t test, *p < 0.05, within control, control WT BMT, or Sle123 BMT groups, one-way ANOVA. (B) Numbers of IFN-γ–secreting and IL-17–secreting CD4+ T cells in lymph nodes from the indicated 38-wk-old mice (representative flow cytometric plots are shown). CXCR5+PD-1+BCL-6+ Tfh cells (C) and CD19+CD95+PNA+ GC B cells (D) in 38-wk-old WT, WT→WT, Sle123→WT, Sle123→ApoA-I−/−, and Sle123→ApoA-Itg mice (n = 6). All data are mean ± SD. ‡p < 0.05 versus WT and WT→WT control groups, one-way ANOVA, *p < 0.05 within Sle123 BMT group, one-way ANOVA.
Similarly to a previous study in SLE-prone gld mice (18), plasma HDL-C levels, mApoA-I, and PON-1 activity were reduced as a result of autoimmunity in Sle123→WT mice (Fig. 1B–D, Table II, Supplemental Fig. 1A, 1B). Indeed, reductions in plasma HDL-C in autoimmune Sle123→WT mice were inversely related to certain parameters of autoimmunity, including anti-dsDNA autoantibodies and CD4+ T cell activation (Supplemental Fig. 1D). Autoimmune-mediated reduction in endogenous mApoA-I was also seen in Sle123→ApoA-Itg animals (Fig. 1D, 1E, Supplemental Fig. 1A, 1B).
Interestingly, we observed the effects of ApoA-I deficiency in control nonautoimmune WT BMT mice, including elevations in plasma PON-1 activity (increased 57%) (Fig. 1C, Table II) and an ∼5-fold increase in the numbers of lymph node B cells (Fig. 2B). CD4+ T cells were increased in WT→ApoA-I−/− mice (3-fold) and non-BMT ApoA-I−/− mice (2-fold) (Fig. 3B) compared with their WT counterparts, suggesting that this was an intrinsic property of ApoA-I−/− mice. However, CD4+ lymphocyte expansion associated with the development of autoimmunity obscured this phenotype in Sle123 BMT mice (Fig. 3B). In WT→ApoA-Itg animals, we observed slightly increased plasma HDL-C (increased 14%) and higher PON-1 activity (increased 52%) compared with their control non-BMT ApoA-Itg counterparts (Fig. 1B, 1C, Table II).
Reduced Th1 and follicular helper T cells in Sle123→ApoA-Itg mice
Because the reduction in CD4+ T cells in Sle123→ApoA-Itg mice was relatively mild (38% decrease), we reasoned that it might reflect a suppressive effect on the activation and/or differentiation of select CD4+ T cell populations, as opposed to a generalized reduction in all CD4+ cells. IFN-γ–secreting Th1 cells, but not IL-17–secreting Th17 cells, were reduced by half in Sle123→ApoA-Itg mice (Fig. 4B). CD4+CXCR5+PD1+BCL6+ follicular helper T (Tfh) cells provide help to B cells for GC B cell formation and Ab production in response to foreign Ags, as well as for pathogenic autoantibody production in SLE (33). Tfh cells were also significantly reduced in Sle123→ApoA-Itg mice (65% decrease) (Fig. 4C). Consistent with their role in GC formation (33), reduced Tfh cells resulted in lower numbers of GC B cells (47% decrease) (Fig. 4D). Again, ApoA-I deficiency did not result in opposite effects to those in ApoA-Itg mice. In addition, immune-suppressive effects of ApoA-I were mediated independently of changes in the numbers of CD4+Foxp3+ regulatory T cells (data not shown). Finally, although ApoA-I was shown to modulate CD11c+ DC lymph node migration in hypercholesterolemic settings, as measured by a FITC skin painting assay (34), we did not observe any difference in CD11c+ DC migration among WT, ApoA-I−/−, and ApoA-Itg mice using the same assay (Supplemental Fig. 2A).
Immune-suppressive action of ApoA-I is associated with modulation of cellular oxidized lipids but not cholesterol
ApoA-I deficiency in hypercholesterolemic mouse models causes systemic autoimmunity by promoting lymphocyte cholesterol accumulation, resulting in modulation of lipid raft–dependent AgR-mediated activation (2, 8, 10, 11, 35). Conversely, increasing ApoA-I in hypercholesterolemic mice may regulate AgR-mediated lymphocyte activation by reducing cellular cholesterol to modulate lipid rafts and AgR activation in the opposite direction (2, 8, 35). However, cholesterol levels in lymphocytes from autoimmune and control WT, ApoA-I−/−, and ApoA-Itg mice were not significantly different, as measured by GC-MS (Fig. 5A). Thus, immune suppression by ApoA-I in normocholesterolemic SLE mice was independent of changes in lymphocyte cholesterol homeostasis.
(A) Immune suppression in Sle123→ApoA-Itg mice is not associated with reduced lymphocyte cholesterol content. Total (free plus esterified) and free cholesterol content of lymphocytes from the indicated control, WT BMT, and Sle123 BMT mice (mean ± SD, n = 5). (B) Levels of the indicated HODEs and linoleic acid in lymphocytes from the indicated WT BMT and Sle123 BMT mice. All data are mean ± SD (n = 5). Note that increases in lymphocyte 13-HODE and 9-HODE levels in Sle123→ApoA-Itg mice are not simply due to elevated levels of its precursor, linoleic acid. ‡p < 0.05 versus control non-BMT, t test, *p < 0.05 within Sle123 BMT group, one-way ANOVA.
HODEs and HETEs derived from 12/15-LO enzymatic– and reactive oxygen species–mediated oxidation of linoleic and arachidonic acids, respectively, have been widely used as measures of oxidative stress (36, 37), and the reduction in their levels in plasma and tissues has been used as a surrogate measure of the anti-inflammatory action of ApoA-I in hypercholesterolemic rodent models (5, 7). Surprisingly, ApoA-I–mediated immune suppression in Sle123→ApoA-Itg mice was associated with increased lymphocyte levels of 13-HODE and 9-HODE (Fig. 5B). Linoleic acid was not significantly different among WT, ApoA-I−/−, and ApoA-Itg animals (Fig. 5B), suggesting that the increases in 13-HODE and 9-HODE were not simply due to higher precursor fatty acid levels. Lymphocyte levels of 5-HETE, 12-HETE, 15-HETE, and their precursor, arachidonic acid, were not significantly different among WT, ApoA-I−/−, and ApoA-Itg mice (data not shown).
ApoA-I improves GN in Sle123 BMT mice
GN was significantly improved in autoimmune Sle123→ApoA-Itg mice but was not exacerbated in their Sle123→ApoA-I−/− counterparts (Fig. 6). Histological analysis of glomeruli from control and BMT groups showed significant increases in glomerular area and overall GN activity scores in Sle123 BMT mice compared with their control non-BMT and control WT BMT counterparts (Fig. 6A, 6B). These measures of GN were significantly improved in Sle123→ApoA-Itg mice, whereas ApoA-I deficiency had no significant effect on GN (Fig. 6A, 6B). GN scores were also moderately increased in control WT BMT groups compared with their control non-BMT counterparts (Fig. 6B), reflecting deleterious effects of lethal irradiation and/or BM reconstitution on renal pathology. Albuminuria in Sle123→ApoA-Itg mice was slightly reduced, but among the three Sle123 BMT groups this was only statistically significant in a pairwise comparison with Sle123→WT animals (p = 0.052, one-way ANOVA) (Fig. 6C). Improvement of GN in Sle123→ApoA-Itg mice was associated with marked reductions in glomerular IgG deposition (Fig. 7A), consistent with the lower plasma autoantibodies in these animals (Fig. 2A). The numbers of kidney-infiltrating CD4+ T cells (Fig. 7B), CD11b+ cells (Fig. 8A), and NIMPR4+ neutrophils (Fig. 8B) were also reduced in Sle123→ApoA-Itg mice, consistent with reduced renal inflammation.
Protection against GN in Sle123→ApoA-Itg mice. (A) PAS-stained kidney sections showing representative glomeruli from the indicated 38-wk-old non-BMT control, control WT BMT, and Sle123 BMT mice. (B) Average glomerular area (upper panel) and overall GN histological scores (lower panel) in the indicated control (n = 5), control BMT (n = 5), and Sle123 BMT (n = 6) groups (nine glomeruli scored per mouse). ‡p < 0.05, versus indicated control, t test, *p < 0.05, within Sle123 BMT group, one-way ANOVA. (C) Increases in urine albumin/creatinine ratio in the indicated 38-wk-old control (n = 4), control BMT (n = 4), and Sle123 BMT (n = 6–8) animals. All data are mean ± SD. *p = 0.052, one-way ANOVA, ‡p = 0.049, versus Sle123→WT, t test.
Reduced glomerular IgG deposition and renal CD4+ T lymphocyte infiltration in Sle123→ApoA-Itg mice. Representative anti-IgG (A) and CD4 (B) kidney sections from the indicated 38-wk-old control (n = 5), control WT BMT (n = 5), and Sle123 BMT (n = 7–8) mice. Graphs show glomerular IgG immunofluorescence staining intensity and CD4+ T cell numbers in the indicated mice. Each data point represents the average of nine glomeruli/mouse. *p < 0.05 within its control or BMT group, one-way ANOVA, ‡p < 0.05 versus both non-BMT and BMT control groups of the same host genotype or non-BMT control group, t test.
Reduced renal CD11b+ (A) and NIMPR14+ neutrophil (B) inflammatory cell infiltration in Sle123→ApoA-Itg mice. Representative CD11b and NIMPR14 stained kidney cryosections from the indicated 38-wk-old control, control WT BMT, and Sle123 BMT mice. Graphs show CD11b staining intensity and NIMPR14+ neutrophil numbers in the indicated mice. Each data point represents the average of nine glomeruli/mouse. *p < 0.05 within its control or BMT group, one-way ANOVA, ‡p < 0.05 versus non-BMT control mice of the same host genotype, t test.
The lack of a potentiating effect of ApoA-I deficiency on autoimmunity may be due to compensatory increases in HDL ApoE
We reasoned that the absence of an exacerbatory effect of ApoA-I deficiency on autoimmunity in Sle123→ApoA-I−/− mice might be due to compensatory modifications of other major Apos on HDL in the absence of ApoA-I (29, 38). Like ApoA-I, ApoE can bind cholesterol and oxidized lipids and promote cholesterol transport (39). Plasma levels of ApoE (Supplemental Fig. 1A, 1C) and ApoE levels on FPLC-separated HDL fractions (Fig. 9B) were significantly increased in ApoA-I−/− mice. In contrast, ApoA-II was present at low levels in all mice (Supplemental Fig. 1A, 1C), consistent with the very low levels of this Apo in the C57BL/6J strain (40). Thus, compositional changes in HDL in ApoA-I−/− mice that compensate for the loss of those functions of ApoA-I that mediate immune suppression include increased ApoE.
(A) Cholesterol content of FPLC-separated plasma lipoproteins from the indicated 38-wk-old mice (FPLC fraction number on x-axis). (B) Representative anti- mApoA-I, anti-mouse ApoE (mApoE), and anti-human ApoA-I (hApoA-I) Western blots of FPLC HDL fractions. Note the reduction in HDL mApoA-I in Sle123→WT mice compared with their control WT counterparts (B, left panels). Also note the marked increases in HDL mApoE content in ApoA-I−/− mice and the reductions in HDL mApoA-I content in ApoA-Itg mice. Data are representative of three independent experiments.
Discussion
Using a normocholesterolemic murine SLE BM transfer model, we found that increasing ApoA-I levels through transgenic human ApoA-I expression suppressed lymphocyte activation, reduced pathogenic IFN-γ–secreting CD4+ Th1 cells and Tfh cells, lowered GC B cell numbers and autoantibody production, and improved GN. However, it did not reduce the effects of Sle123-induced autoimmunity to zero. Protection against SLE-mediated GN in Sle123→ApoA-Itg mice was associated with reductions in kidney-infiltrating CD4+ T cells (Fig. 7B), as well as lower CD11b+ monocytic and NIMPR14+ neutrophil infiltrates (Fig. 8). Although reduced levels of autoantibodies leading to less glomerular IgG deposition could be responsible for this reduced renal inflammation (Figs. 2A, 7A), the direct anti-inflammatory action of ApoA-I may also be a major contributing mechanism, a possibility that could be addressed using a nonautoimmune antiglomerular basement membrane Ab–mediated nephritis model (41).
Other immune processes previously shown to be modified by ApoA-I in hypercholesterolemic mice, such as CD4+Foxp3+ regulatory T cell generation (42) (data not shown) or CD11c+ DC migration (Supplemental Fig. 2A) (34), were unaffected in this normocholesterolemic model. Unlike in hypercholesterolemic mice (8), ApoA-I deficiency did not result in opposite effects on autoimmunity, and modulation of ApoA-I did not affect immune cell cholesterol levels (Fig. 5A). Taken together, these results show that potentiating effects of hypercholesterolemia on lymphocyte activation (and possibly other immune mechanisms) are required for ApoA-I to become important for preventing systemic autoimmunity (8) and that a function of ApoA-I, other than cholesterol transport, is the major mechanism accounting for its immune-suppressive action in normal mice. However, it is possible that spatial, rather than quantitative, changes in cholesterol in lymphocyte membrane rafts play a role.
Other than cholesterol transport and maintenance of cellular cholesterol homeostasis, the antioxidant properties of HDL involving ApoA-I–mediated removal and PON-1–mediated hydrolysis of atherogenic oxidized lipids contribute to its anti-inflammatory action in hypercholesterolemic atherosclerosis models (1, 43). The major classes of oxidized lipids bound by ApoA-I include the oxidized metabolites of linoleic acid and arachidonic acid (HODEs and HETEs, respectively) (7, 44). Although several HODE and HETE species can recapitulate proinflammatory effects of oxidized LDL on vascular cells (45–47), they may have different properties depending on the cell/tissue context and disease setting (48–50). Indeed, we did not observe increases in lymphocyte HODE and HETE levels with the development of autoimmunity in Sle123→WT mice or their reduction with immune suppression by ApoA-I in Sle123→ApoA-Itg mice (Fig. 5B) (data not shown for arachidonic acid and HETEs). Surprisingly, however, levels of the major oxidative metabolites of linoleic acid, 13-HODE and 9-HODE, were significantly higher in lymphocytes from autoimmune Sle123→ApoA-Itg animals, and this was not due to higher precursor linoleic acid levels (Fig. 5B). Concentrations of 13-HODE and 9-HODE in the plasma were not significantly different between Sle123→WT and Sle123→ApoA-Itg mice (Supplemental Fig. 2C), suggesting that the higher levels of these oxidized fatty acids in immune cells from autoimmune Sle123→ApoA-Itg animals were probably not due to an increased circulating plasma pool in ApoA-Itg animals. Further work using flow-sorted immune cell preparations in conjunction with chiral phase LC-MS, liquid chromatography mass spectrometry approaches to distinguish (R)-HODEs versus (S)-HODEs will be useful to determine how augmenting ApoA-I levels increases lymphocyte 13-HODE and 9-HODE levels in SLE and why it does so only in the context of autoimmunity (control ApoA-Itg animals did not have the same increases; Fig. 5B). Nevertheless, it is noteworthy that 13-HODE and 9-HODE are agonists for peroxisome proliferator–activated receptor γ (PPARγ), a ligand-dependent nuclear receptor expressed in multiple cell types, including T cells, B cells, macrophages, and DCs (51, 52). Anti-inflammatory activities have been attributed to ligand-activated PPARγ, including suppression of T cell activation, proliferation, Th1 polarization, and autoimmunity (51, 53–56). For example, 13-HODE can regulate T lymphocyte function by blocking T cell IL-2 production through PPARγ, reducing T cell differentiation and proliferation (53). Higher levels of PPARγ were reported in SLE lymphocytes (57), and PPARγ agonist treatment of T cells from SLE patients induces transcriptional repression of genes involved in T cell activation, with those related to Th1 differentiation most affected (58). Mice lacking PPARγ only in CD4+ T cells develop an autoimmune phenotype characterized by expansion of Tfh cells, resulting in increased GC B cells, production of anti-DNA autoantibodies, and glomerular inflammation (59). Based on our finding that Th1 and Tfh cells are reduced in Sle123→ApoA-Itg mice (Fig. 4B, 4C), it is possible that PPARγ activation by 13-HODE and/or 9-HODE in lymphocytes may be a mechanism for ApoA-I–mediated immune suppression, reducing T cell activation and subsequent expansion of Th1 and Tfh CD4+ T cells. Indeed, effects of ApoA-I on the lipid environment of CD4+ T lymphocytes and other immune cell types in SLE may have a significant influence over the repertoire of transcription factors and cytokine signals that control CD4+ T cell differentiation into Th and effector subsets (33, 60). Therefore, it is important to determine whether 13-HODE and 9-HODE are generated intrinsically by the lymphocytes themselves or whether they are derived from nearby innate immune cells, such as macrophages, which were reported to produce these oxylipids in response to inflammatory stimuli through 12/15-LO enzyme activation to inhibit T cell activation and differentiation through PPARγ (53).
Immune suppression by ApoA-I occurred despite the predisposition of animals to develop reduced HDL-C and ApoA-I levels (see Sle123→WT in Figs. 1B, 1D, 1E, 9B, Supplemental Fig. 1A, 1B) and lower HDL-associated PON-1 activity (see Sle123→WT in Fig. 1C). Indeed, there was a significant inverse correlation between HDL-C levels in autoimmune Sle123→WT mice and certain parameters of autoimmunity, including anti-dsDNA autoantibodies and CD4+ T lymphocyte activation (Supplemental Fig. 1A, 1D), reflecting autoimmune-mediated HDL reduction, as previously described in normocholesterolemic gld mice (18). This is an important finding because it suggests that the predisposition of SLE to lower HDL/ApoA-I levels and PON-1 activity (16) may not necessarily make it refractory to immune-suppressive or anti-inflammatory therapeutic strategies targeting ApoA-I, including ApoA-I mimetic peptides (14). Furthermore, autoimmune-mediated reductions in ApoA-I, such as those observed in Sle123→WT animals, are considered to contribute to premature atherosclerosis in lupus patients (61, 62), as well as to amplify lymphocyte activation and autoimmunity itself (8, 10). However, unlike in the setting of hypercholesterolemia (8), we found no evidence for an exacerbatory effect of ApoA-I deficiency on any parameter of autoimmunity (something that would be expected if autoimmune-mediated HDL reduction and dysfunction amplified autoimmunity itself). Similarly, because ApoA-I is important for stabilizing PON-1 activity on HDL particles (31), PON-1 activity was ∼3-fold lower in ApoA-I−/− mice compared with their WT counterparts (Fig. 1C), yet this did not affect autoimmunity. Therefore, our cumulative data suggest that autoimmune-mediated reductions in ApoA-I (HDL) levels and PON-1 activity in SLE patients (61, 63) may not necessarily amplify autoimmunity and inflammation. However, it remains to be determined to what extent the higher PON-1 activity in ApoA-Itg mice contributed to the observed immune-suppressive and anti-inflammatory effects of increased ApoA-I in our study.
ApoA-I deficiency is proinflammatory and can promote systemic autoimmunity in hypercholesterolemic mouse models by deregulating lymphocyte cholesterol homeostasis to promote lymphocyte activation (8, 10, 11, 15). Therefore, the absence of a significant effect of ApoA-I deficiency on any parameter of autoimmunity measured (Figs. 2–4) may seem surprising based on the immune-suppressive effects of transgenic human ApoA-I expression that we observed in the same mouse SLE model system. Although it will be important to rule out possible species differences in mouse versus human ApoA-I in this and other autoimmune models, it is noteworthy that compositional alterations compensating for ApoA-I deficiency may have contributed to a lack of any potentiating effect of ApoA-I deficiency on autoimmunity. For example, we found marked increases in the ApoE content of HDL in ApoA-I−/− mice (Fig. 9B, Supplemental Fig. 1A, 1C), which may have compensated for the loss of important ApoA-I functions involved in immune-suppression, such as lipid binding, but not maintenance of PON-1 activity on HDL (31), considering the markedly lower HDL-associated PON-1 activity in ApoA-I−/− animals (Fig. 1C).
Bioactive lipid mediators with established immune-regulatory roles that are bound and carried by HDL, such as sphingosine-1-phosphate, also warrant investigation with respect to mechanisms of ApoA-I–mediated immune suppression and anti-inflammatory action in SLE (2, 64, 65). Furthermore, the ability of HDL to modulate lipid homeostasis can affect immunity and renal inflammation through TLRs (66), perhaps even those mediating aberrant recognition of nucleic acids leading to pathogenic autoantibody production in SLE, like TLR7 and TLR9 (67). Therefore, it will be important to perform broader lipidomic profiling to identify lipid metabolites that are associated with immune-suppressive and anti-inflammatory actions of ApoA-I, because some of these are likely to be viable targets for therapeutic development. In this respect, it will be important to determine whether oxidative changes to which ApoA-I is inherently susceptible in chronic inflammatory settings, such as SLE, may be limiting its immunosuppressive potential, such that using mutant forms of ApoA-I resistant to oxidative loss of function (68) may provide the most robust platform for these lipidomic approaches. Therefore, future studies characterizing the lipidome in tissues from autoimmune animal models with experimentally manipulated ApoA-I levels, HDL composition, or treated with Apo mimetic peptides could be used to identify novel lipid-based mechanisms of immune suppression and anti-inflammatory action in SLE.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We thank the UAB Animal Resources Program Comparative Pathology Laboratory for histological slide preparation, the UAB-University of California, San Diego O’Brien Center for Acute Kidney Injury Research (P30 DK079337) for creatinine measurements, and Dr. Mike Thomas of the Mass Spectrometer Facility of the Comprehensive Cancer Center of Wake Forest School of Medicine for lymphocyte cholesterol analysis. We thank Dr. Srinu Reddy (Department of Medicine, University of California, Los Angeles) for helpful advice on HODE/HETE LC-MS/MS and Dr. Keiichi Higuchi (Shinshu University) for generously providing the anti–ApoA-II serum. We are also grateful to Dr. Beatriz Leon-Ruiz (Department of Microbiology, UAB) for advice on Tfh cell and GC B cell flow cytometric analyses and Dr. David Redden (Department of Biostatistics, UAB) for assistance with statistical analyses.
Footnotes
This work was supported by a Novel Research Project in Lupus from the Lupus Research Institute (to J.H.K.), National Institutes of Health Grant T32 HL007918 (to L.L.B.), and the University of Alabama at Birmingham/University of California, San Diego O’Brien Core Center for Acute Kidney Injury Research (National Institutes of Health Grant P30 DK 079337). The AB Sciex 4000 QTRAP mass spectrometer was purchased with funds from National Institutes of Health Shared Instrumentation Grant S10RR19231 (to S.B.). The Mass Spectrometer Facility of the Comprehensive Cancer Center of Wake Forest School of Medicine is supported in part by National Cancer Institute Grant 5P30CA12197, and the Finnigan TSQ Quantum XLS mass spectrometer was funded by National Institutes of Health Shared Instrumentation Grant 1S10RR027940.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- Apo
- apolipoprotein
- BM
- bone marrow
- BMT
- BM transplantation
- DC
- dendritic cell
- FPLC
- fast-performance LC
- GC
- germinal center
- GC-MS
- gas chromatography–mass spectrometry
- GN
- glomerulonephritis
- HDL
- high-density lipoprotein
- HDL-C
- HDL cholesterol
- HETE
- hydroxyeicosatetraenoic acid
- HODE
- hydroxyoctadecadienoic acid
- LC
- liquid chromatography
- LC-MS/MS
- liquid chromatography-tandem mass spectrometry
- LDL
- low-density lipoprotein
- LDL-C
- LDL cholesterol
- LO
- lipoxygenase
- mApoA-I
- mouse ApoA-I
- PAS
- periodic acid–Schiff
- PBST
- PBS containing 0.5% Tween 20
- PON-1
- paraoxonase-1
- PPARγ
- peroxisome proliferator–activated receptor γ
- SLE
- systemic lupus erythematosus
- Tfh
- follicular helper T
- UAB
- University of Alabama at Birmingham
- WT
- wild-type.
- Received April 8, 2015.
- Accepted September 21, 2015.
- Copyright © 2015 by The American Association of Immunologists, Inc.
References
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