IL-5 links adaptive and natural immunity specific for epitopes of oxidized LDL and protects from atherosclerosis

Abstract

During atherogenesis, LDL is oxidized, generating various oxidation-specific neoepitopes, such as malondialdehyde-modified (MDA-modified) LDL (MDA-LDL) or the phosphorylcholine (PC) headgroup of oxidized phospholipids (OxPLs). These epitopes are recognized by both adaptive T cell-dependent (TD) and innate T cell-independent type 2 (TI-2) immune responses. We previously showed that immunization of mice with MDA-LDL induces a TD response and atheroprotection. In addition, a PC-based immunization strategy that leads to a TI-2 expansion of innate B-1 cells and secretion of T15/EO6 clonotype natural IgM antibodies, which bind the PC of OxPLs within oxidized LDL (OxLDL), also reduces atherogenesis. T15/EO6 antibodies inhibit OxLDL uptake by macrophages. We now report that immunization with MDA-LDL, which does not contain OxPL, unexpectedly led to the expansion of T15/EO6 antibodies. MDA-LDL immunization caused a preferential expansion of MDA-LDL-specific Th2 cells that prominently secreted IL-5. In turn, IL-5 provided noncognate stimulation to innate B-1 cells, leading to increased secretion of T15/EO6 IgM. Using a bone marrow transplant model, we also demonstrated that IL-5 deficiency led to decreased titers of T15/EO6 and accelerated atherosclerosis. Thus, IL-5 links adaptive and natural immunity specific to epitopes of OxLDL and protects from atherosclerosis, in part by stimulating the expansion of atheroprotective natural IgM specific for OxLDL.

Figure 1

Immunization with MDA-LDL induces a specific Th2 response. C57BL/6 mice were immunized with homologous MDA-LDL in Freund’s adjuvant or remained naive. One week after the third injection, cellular and humoral immune responses were assessed. Three independent immunization studies were performed. (A) Splenocyte proliferation assay. Splenocytes of immunized (n = 6) or naive (n = 6) mice were cultured with titrated amounts of murine native LDL (open circles, immunized; open squares, naive) or murine MDA-LDL (filled circles, immunized; filled squares, naive), and antigen-specific proliferation was measured by ³[H]-thymidine uptake. (B) Increased IgG1 titers in plasma of immunized mice. Data represent titers from all mice studied (n = 14; P < 0.01, Student’s paired t test). (C) Increased frequency of MDA-LDL–specific IL-5 secreting cells in spleens of immunized mice as assessed by ELISpot assay. Splenocytes of immunized (n = 8; filled circles) and naive (n = 8; open circles) mice were incubated overnight with and without murine MDA-LDL, and the frequencies of MDA-LDL–specific IFN-γ or IL-5 spot-forming cells (SFCs) were assessed. Shown are the mean SFCs per 2 × 10⁶ cells for IFN-γ and IL-5 of individual mice from two experiments (P < 0.01, Student’s paired t test; IFN-γ vs. IL-5 SFC of immunized mice). (D) MDA-LDL–specific Th2 cytokine secretion in cultures of splenocytes from four mice incubated for 72 hours with 25 μg/ml murine native LDL (white bars) or murine MDA-LDL (black bars). Control cultures stimulated with anti-CD3 and anti-CD28 produced 11.7 ± 2.1 ng/ml IFN-γ, 156 ± 39 pg/ml IL-5, 470 ± 43 pg/ml IL-10, and 1.2 ± 0.4 ng/ml IL-13. *P < 0.05; MANOVA followed by Newman-Keuls test, comparing the stimulation with increasing amounts (0.25 [not shown], 2.5 [not shown], and 25 μg/ml) of native and MDA-LDL. All values shown in Figure 1 are mean ± SEM.

Figure 2

Decreased atherosclerosis and dominant Th2 response in immunized LDLR^–/– mice. Mice were immunized with homologous MDA-LDL (n = 10) or PBS (n = 11) in Freund’s adjuvant, and then fed a high-cholesterol diet for 13 weeks, during which they received further booster immunizations. (A) Lipoprotein profiles at time of death in pooled plasma of all mice immunized with MDA-LDL (filled circles) or PBS (open circles) as determined by FPLC. (B) Decreased atherosclerotic lesion size in cross-sections through the aortic origin in mice immunized with MDA-LDL. Values are mean ± SEM in mm²/section. (C and D) Plasma IgG1 (filled circles) and IgG2a (open circles) dilution curves of binding to MDA-LDL of mice immunized with (C) MDA-LDL or (D) PBS. Values are the mean ± SEM of all final plasma samples for each group measured in duplicate. (E and F) ELISpot assay of frequencies of MDA-LDL–specific cytokine-secreting cells in the spleens of mice immunized with (E) MDA-LDL or (F) PBS. Splenocytes were incubated overnight in the absence or presence of murine MDA-LDL with and without anti-CD28, and the frequencies of MDA-LDL–specific IFN-γ (white bars) or IL-5 (black bars) SFCs were assessed. Bars represent the mean SFCs ± SEM of 2 × 10⁶ cells of all mice for each group. P < 0.01, Student’s paired t test.

Figure 3

Antigen-specific cytokine secretion of splenocytes from cholesterol-fed LDLR^–/– mice immunized with MDA-LDL (n = 10; black bars) or PBS (n = 11; white bars). Left column: Splenocytes were cultured for 72 hours in the presence of anti-CD28 and either anti-CD3, murine native LDL, or murine MDA-LDL, and supernatants were analyzed for cytokines. Data are presented as percentage of the cytokine secretion in parallel cultures maximally stimulated with anti-CD3/CD28 (=100%). Right column: splenocytes were stimulated either alone or with indicated amounts of murine MDA-LDL (without anti-CD28). Values are mean ± SEM of splenocyte cultures of all mice from each group.

Figure 4

Increased levels of IL-5 and EO6 in the plasma of cholesterol-fed LDLR^–/– mice immunized with MDA-LDL. At time of death, plasma was obtained from mice immunized with MDA-LDL (n = 10) and PBS (n = 11). (A) IL-5 levels in plasma of MDA-LDL immunized mice are increased. (B) EO6 antibody levels in plasma of MDA-LDL–immunized mice are increased. The amount of EO6 present was determined using an ELISA based capture assay with anti–idiotypic AB1-2 as described in Methods and was calculated based on an EO6 standard curve. (C) Comparison of T15/EO6 antibody titers in LDLR^–/– mice immunized with MDA-LDL (current study) versus mice immunized with S. pneumoniae (R36a; n = 9) from a previous study (12). EO6 antibody titers were determined at a plasma dilution of 1:500 in the same assay. Purified EO6 was used as a positive control. Biot., biotinylated. (D) Circulating IgM/apoB ICs are increased in MDA-LDL–immunized mice. Results are expressed as IgM/apoB. All bars represent the mean ± SEM values of all mice from each intervention group.

Figure 5

Role of IL-5 in the production of T15/EO6 natural antibodies. (A and B) IL-5 stimulates antibody secretion in vitro. Peritoneal B-1 cells (black bars) and B-2 cells (white bars), and splenic marginal zone (MZ) B cells (dark gray bars) and follicular B cells (light gray bars) were cultured for 7 days in either medium alone, or with IFN-γ, IL-4, or IL-5 with and without anti–IL-5 mAb. Polymyxin B was added to all cultures to neutralize contaminating LPS effects. (A) IgM binding to OxLDL in culture supernatants. (B) T15/EO6 antibodies in culture supernatants, measured with the anti-idiotypic antibody AB1-2. Values are mean RLU ± SEM from duplicate determinations of triplicate cultures. This experiment was repeated three times. (C) IL-5 stimulates the production of T15/EO6 antibodies in vivo. C57BL/6 mice received daily intraperitoneal injections with recombinant mouse IL-5 (n = 6) or vehicle only (BSA; n = 4) for 7 days, and the amount of T15/EO6 antibodies was determined in the plasma. Shown is the fold increase over the baseline levels at a 1:100 plasma dilution. Bars represent mean ± SEM of triplicate determinations of individual mice. P < 0.05, Student’s unpaired t test; Welch corrected. (D) Naive IL-5^–/– mice have decreased T15/EO6 antibody levels. In IL-5^+/+ (open circles) and IL-5^–/– C57BL/6 mice (filled circles), 15–16 weeks of age (both n = 3), T15/EO6 antibody titers were determined. Shown is the binding of T15/EO6 antibodies of individual plasma samples diluted 1:100. Values are the mean RLU of triplicate determinations. (E–G) Impaired induction of T15/EO6 antibodies in IL-5^–/– mice immunized with MDA-LDL. IL-5^+/+ (open circles) and IL-5^–/– (filled circles) C57BL/6 mice were immunized with MDA-LDL and plasma antibody titers were determined. (E) IgG1 binding to MDA-LDL (F), IgM binding to OxLDL, and (G) T15/EO6 antibodies of individual plasmas at 1:250 dilution before and after immunization. Values are the mean RLU of triplicate determinations.

Figure 6

Increased atherosclerosis in IL-5–deficient LDLR^–/– mice. LDLR^–/– mice were reconstituted with bone marrow from either IL-5^–/– mice (IL-5^–/–; n = 15) or IL-5^+/+ mice (IL-5^+/+; n = 14) and fed an atherogenic diet for 16 weeks. (A) Increased extent of atherosclerosis in aortas of recipients of IL-5^–/– bone marrow (n = 15) compared to recipients of IL-5^+/+ bone marrow (n = 14; P < 0.01). Horizontal bars indicate means of each group. (B) Decreased titers of T15/EO6 antibodies in plasma of LDLR^–/– mice reconstituted with IL-5^–/– bone marrow. Data are mean ± SEM titers of all mice of each group (P < 0.05). (C) Reduced formation of circulating T15/EO6-apoB ICs in recipients of IL-5^–/– bone marrow. Results are expressed as T15/EO6 antibodies per apoB. Data are the mean ± SEM values of all mice from each intervention group at 4, 8, and 16 weeks after initiation of the atherogenic diet. P < 0.05, repeated-measures ANOVA.