T-bet deficiency reduces atherosclerosis and alters plaque antigen-specific immune responses

Abstract

The influence of the immune system on atherosclerosis involves both helper T (Th) cell and antibody responses to plaque antigens. These responses may have proatherogenic and protective effects. T-bet is a transcription factor required for Th1 differentiation and regulates the balance between Th1 and Th2 responses in inflammatory diseases. To clarify how helper T cell subset differentiation influences atherosclerosis, we compared lesion development and immune responses to plaque antigens in low-density lipoprotein receptor-deficient (Ldlr-/-) mice with or without functional T-bet genes. Atherosclerosis was significantly reduced in T-bet-deficient Ldlr-/- mice compared with Ldlr-/- controls, and the lesions that did develop in the absence of T-bet had less smooth muscle cell content. Furthermore, T-bet deficiency caused a Th2 switch in the response to the atherosclerosis-associated antigen heat shock protein-60, and a change in T-dependent isotypes of oxidized LDL-specific antibodies. Of particular significance, T-bet deficiency caused a >250% increase in the titer of E06 antibodies, which are known to be atheroprotective and whose production by B-1 B cells is enhanced by IL-5. These findings establish that T cell subset differentiation influences both T cell and antibody responses that modulate atherosclerosis, and validate the therapeutic goal of skewing T responses to atherosclerosis-associated antigens.

Fig. 1.

Reduced aortic atherosclerosis in T-bet-deficient Ldlr^-/- mice. Quantification of aortic lesions was performed on digitally analyzed specimen images, as described in Methods. (A and B) Lipid and intimal positive areas, respectively, of aortic arch sections, are shown (n = 16 per group: 8 males and 8 females). (C) En face lipid positive areas of descending aorta are shown (n = 12 per group: 6 males and 6 females). Horizontal bars represent means. P values <0.05 for intergroup comparisons are shown.

Fig. 2.

Phenotype of aortic arch atherosclerotic lesions in T-bet ^-/-Ldlr^-/- and T-bet^+/+Ldlr^-/- mice. Frozen sections of aortic arches taken from mice after 8 weeks of proatherogenic diet were stained with antibodies specific for macrophages (Mac3), smooth muscle cells (actin), and class II MHC, as described in Methods. Sections stained with isotype control antibodies were negative (data not shown). See Table 1 for quantification of stained areas.

Fig. 3.

Differences in cytokines production by CD4⁺ cells from T-bet ^-/-Ldlr^-/- and T-bet^+/+Ldlr^-/- mice. CD4⁺ T cells were isolated from six T-bet^-/-Ldlr^-/- or T-bet^+/+Ldlr^-/- mice after 8 weeks of diet as described in Methods. The cells were stimulated in cultures with plate-bound anti-CD3ε or with recombinant murine hsp60 (mhsp60) plus syngeneic mitomycin C-treated spleen cells. Medium alone (No antigen) and ovalbumin (data not shown) were used as controls for nonspecific cytokine production. Culture supernatants were removed at 48 h and analyzed by ELISA for IFN-γ (A), IL-4 (B), IL-5 (C), and IL-10 (D). Data represent mean ± SEM of each cytokine determination. *, P < 0.05 for T-bet^-/-Ldlr^-/- versus T-bet^+/+Ldlr^-/- comparisons; #, P < 0.05 for no antigen versus murine hsp60 stimulation.

Fig. 4.

Differences in atherosclerotic antigen-specific serum immunoglobulins between T-bet^-/-Ldlr^-/- and T-bet^+/+Ldlr^-/- mice. Titers of total IgG1, total IgG2a, MDA-LDL-specific, and CuOx-LDL-specific IgM, IgG1, and IgG2a, and EO6 idiotype IgM were detected by specific ELISA from serum collected from 10 animals for each study group, after 8 weeks of diet, as described in Methods. Data represent mean ± SEM of each Ig type determination. *, P < 0.05 for T-bet^-/-Ldlr^-/- versus T-bet^+/+Ldlr^-/- comparisons. RLU, relative light units.