Deficiency of antigen-presenting cell invariant chain reduces atherosclerosis in mice

Abstract

Background: Adaptive immunity and innate immunity play important roles in atherogenesis. Invariant chain (CD74) mediates antigen-presenting cell antigen presentation and T-cell activation. This study tested the hypothesis that CD74-deficient mice have reduced numbers of active T cells and resist atherogenesis.

Methods and results: In low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice, CD74 deficiency (Ldlr(-/-)Cd74(-/-)) significantly reduced atherosclerosis and CD25(+)-activated T cells in the atheromata. Although Ldlr(-/-)Cd74(-/-) mice had decreased levels of plasma immunoglobulin (Ig) G1, IgG2b, and IgG2c against malondialdehyde-modified LDL (MDA-LDL), presumably as a result of impaired antigen-presenting cell function, Ldlr(-/-)Cd74(-/-) mice showed higher levels of anti-MDA-LDL IgM and IgG3. After immunization with MDA-LDL, Ldlr(-/-)Cd74(-/-) mice had lower levels of all anti-MDA-LDL Ig isotypes compared with Ldlr(-/-) mice. As anticipated, only Ldlr(-/-) splenocytes responded to in vitro stimulation with MDA-LDL, producing Th1/Th2 cytokines. Heat shock protein-65 immunization enhanced atherogenesis in Ldlr(-/-) mice, but Ldlr(-/-) Cd74(-/-) mice remained protected. Compared with Ldlr(-/-) mice, Ldlr(-/-)Cd74(-/-) mice had higher anti-MDA-LDL autoantibody titers, fewer lesion CD25(+)-activated T cells, impaired release of Th1/Th2 cytokines from antigen-presenting cells after heat shock protein-65 stimulation, and reduced levels of all plasma anti-heat shock protein-65 Ig isotypes. Cytofluorimetry of splenocytes and peritoneal cavity cells of MDA-LDL- or heat shock protein-65-immunized mice showed increased percentages of autoantibody-producing marginal zone B and B-1 cells in Ldlr(-/-)Cd74(-/-) mice compared with Ldlr(-/-) mice.

Conclusions: Invariant chain deficiency in Ldlr(-/-) mice reduced atherosclerosis. This finding was associated with an impaired adaptive immune response to disease-specific antigens. Concomitantly, an unexpected increase in the number of innate-like peripheral B-1 cell populations occurred, resulting in increased IgM/IgG3 titers to the oxidation-specific epitopes.

Figure 1

Atherosclerotic lesion characterizations in Ldlr^−/− and Ldlr^−/−Cd74^−/− mice that consumed an atherogenic diet for 12 and 26 weeks, including aortic arch lesion grade (A), intima area (B), thoracic-abdominal aorta lipid deposition with representative aortas shown to the right panels (C), lesion Mac-3⁺ macrophage-positive area (D), NK-1.1⁺ T cell number (E), CD4⁺ T cell number (F), CD1d-positive area (G), CD8⁺ cell number (H), CD11c⁺ dendritic cell number (I), and CD25⁺ activated T cell number (J). Mann-Whitney U test. P<0.05 was considered statistical significant. The number of mice in each group is indicated in parentheses.

Figure 2

Anti-MDA-LDL antibody titers (mean ± SE) in Ldlr^−/−Cd74^−/− and Ldlr^−/− mice that consumed an atherogenic diet for 12 and 26 weeks. The number of mice in each group is indicated in parentheses. No statistical test was performed.

Figure 3

Plasma IgG1 (A) and IgM (B) titers that bound to MDA-LDL in Ldlr^−/−Cd74^−/−and Ldlr^−/− mice pre- and post-immunization with MDA-LDL, IFA, or PBS. Each data point is mean ± SE of six mice. No statistical test was performed.

Figure 4

Culture medium cytokine levels after stimulation with MDA-LDL or native LDL in splenocytes from MDA-LDL-immunized Ldlr^−/−Cd74^−/− and Ldlr^−/− mice. Each data point is mean ± SE of six mice. No statistical test was performed.

Figure 5

Aortic arch lesion grade (A), intima area (B), lesion CD4⁺ T cell number (C), and CD25⁺ activated T cell number (D) in Ldlr^−/− and Ldlr^−/−Cd74^−/− mice immunized with PBS or HSP65 while consuming an atherogenic diet for 12 weeks. The number of mice in each group is indicated in parentheses. Mann-Whitney U test followed with Bonferroni post-hoc correction. P < 0.016 was considered statistical significant.

Figure 6

Antibody levels to HSP65 in Ldlr^−/−Cd74^−/− (n=6) and Ldlr^−/− (n=11) mice pre-(8 weeks old) and post-HSP65 immunization (mean ± SE). All mice consumed an atherogenic diet for 12 weeks after initial immunization and before harvesting. No statistical test was performed.

Figure 7

HSP65 antigen recall assays of splenocytes and lymph node cells from HSP65-immunized Ldlr^−/−Cd74^−/− (n=6) and Ldlr^−/− (n=10) mice. Data are presented as mean ± SE. Unpaired student’s t test followed with Bonferroni post-hoc correction. P < 0.0125 was considered statistical significant.

Figure 8

Flow cytometry analysis for CD19⁺ splenocytes and peritoneal cavity cells from Ldlr^−/− and Ldlr^−/−Cd74^−/− mice immunized with PBS, MDA-LDL, or HSP65. A. Percentages of IgM⁺CD19⁺ CD43⁺ B-1 cells, CD19⁺CD21⁺CD23⁻ marginal zone-B cells (MZ-B), and CD19⁺CD21⁻CD23⁺ follicular-B cells (FO-B) in spleen. B. Representative flow cytometry analysis of splenocytes from MDA-LDL-immunized Ldlr^−/− and Ldlr^−/−Cd74^−/− mice. C. Percentage of CD19⁺ cells in peritoneal cavity: CD11b⁺ B-1 and CD11b⁻ B-2 cells. The number of mice in each group is indicated inside the bar, and data are mean±SE. Student t test, P<0.05 was considered statistical significant. D. Representative flow cytometry scans of peritoneal cavity cells from PBS-immunized Ldlr^−/− and Ldlr^−/−Cd74^−/− mice. Total B-1 cells (CD5⁺ (B-1a) and CD5⁻ (B-1b)) are counted.