Mucosal Administration of Collagen V Ameliorates the Atherosclerotic Plaque Burden by Inducing Interleukin 35-dependent Tolerance

Abstract

We have shown previously that collagen V (col(V)) autoimmunity is a consistent feature of atherosclerosis in human coronary artery disease and in the Apoe(-/-) mouse model. We have also shown sensitization of Apoe(-/-) mice with col(V) to markedly increase the atherosclerotic burden, providing evidence of a causative role for col(V) autoimmunity in atherosclerotic pathogenesis. Here we sought to determine whether induction of immune tolerance to col(V) might ameliorate atherosclerosis, providing further evidence for a causal role for col(V) autoimmunity in atherogenesis and providing insights into the potential for immunomodulatory therapeutic interventions. Mucosal inoculation successfully induced immune tolerance to col(V) with an accompanying reduction in plaque burden in Ldlr(-/-) mice on a high-cholesterol diet. The results therefore demonstrate that inoculation with col(V) can successfully ameliorate the atherosclerotic burden, suggesting novel approaches for therapeutic interventions. Surprisingly, tolerance and reduced atherosclerotic burden were both dependent on the recently described IL-35 and not on IL-10, the immunosuppressive cytokine usually studied in the context of induced tolerance and amelioration of atherosclerotic symptoms. In addition to the above, using recombinant protein fragments, we were able to localize two epitopes of the α1(V) chain involved in col(V) autoimmunity in atherosclerotic Ldlr(-/-) mice, suggesting future courses of experimentation for the characterization of such epitopes.

Keywords: IL-35; atherosclerosis; autoimmune disease; autoimmunity; collagen type V; immunotherapy; vascular smooth muscle cells.

FIGURE 1.

Mucosal administration induces tolerance to col(V) in Ldlr^−/− mice on a Western diet. A, schematic relating the beginning of col(V) inoculation of Ldlr^−/− mice on normal chow with placing the mice on a Western diet, injections with anti-Ebi3 antibodies, and times of sacrifice. B–D, Ldlr^−/− mice were fed a Western diet for 14 weeks before experimentation. All mice were immunized with TT/DT 2 weeks before the assays. TV-DTH measured cellular responses to TT/DT, col(V), and col(I) by splenocytes from mice treated nasally with PBS (B, n = 8 mice/assay), col(I) (C, n = 6 mice/assay), and col(V) (D, n = 8 mice/assay). Data are shown as mean ± S.E. ***, p ≤ 0.0005; NS, not significant.

FIGURE 2.

Induced col(V) tolerance is regulated by IL-35 but not by IL-10 or TGF-β1. Ldlr^−/− mice were fed a Western diet for 14 weeks and were immunized with TT/DT 2 weeks before the assays. TV-DTH assays compared T cell responses between splenocytes injected with col(V) alone or in the presence of neutralizing antibodies to cytokines. A and B, TV-DTH responses were compared for splenocytes from PBS-treated mice (A, n = 8 mice/group) and from col(I)-treated mice (B, n = 6 mice/group) injected with col(V) alone or together with neutralizing antibodies to IFN-γ or IL-17. C, splenocytes from col(V)-treated Ldlr^−/− mice kept on a Western diet for 14 weeks were injected with TT/DT, with col(V) or col(I) alone or together with control IgG, or with neutralizing antibodies to TGF-β or IL-10 (n = 8 mice/group). D, splenocytes from col(V)-treated Ldlr^−/− mice kept on a Western diet for 14 weeks were injected with TT/DT, or with col(V) alone (n = 6) or together with neutralizing antibodies to p28 (a subunit of IL-27 but not IL-35), p35 (a subunit of both IL-35 and IL-12), or Ebi3 (a subunit of both IL-35 and IL-27), or together with both p35 and Ebi3 (n = 6 mice/group). E, quantitative analysis of the percent of plaque area in ORO-stained en face preparations of the descending aortas of Ldlr^−/− mice treated nasally treated with PBS, col(I), or col(V) and maintained on a Western diet for 14 weeks (for each group, n = 8 mice). Data are shown as mean ± S.E. *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005; NS, not significant.

FIGURE 3.

Levels of the IL-35 subunits Ebi3 and p35 are increased in the inguinal lymph nodes of col(V)-treated mice. A, representative immunoblots of inguinal lymph nodes probed with antibodies to Ebi3, p35, p28, p40, and tubulin, the latter as a loading control. B and C, quantification of the intensity of bands for Ebi3 (B) and p35 (C) for three immunoblots of inguinal lymph nodes from three different mice. *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005.

FIGURE 4.

Ldlr^−/− mice on a Western diet for 24 weeks have IL-17-dependent immune reactivity to col(V). A and B, TV-DTH assays of splenocytes from PBS-treated (A) and col(I)-treated (B) mice on a Western diet for 24 weeks and then injected into the footpads with col(V) alone or in the presence of neutralizing antibodies to cytokines showed IL-17-dependent and, to a lesser degree, IFN-γ-dependent immune reactivity to col(V). Data are shown as mean ± S.E.

FIGURE 5.

Amelioration of the atherosclerotic burden by induced col(V) tolerance is dependent on IL-35. A, in TV-DTH assays, splenocytes from col(V)-treated Ldlr^−/− mice subjected to injections with IgG and kept for 24 weeks on a Western diet were injected with TT/DT (n = 7), col(I) plus IgG (n = 7), or col(V) plus IgG (n = 7) or plus neutralizing antibodies to p28 (a subunit of IL-27 but not IL-35) (n = 6), p35 (a subunit of both IL-35 and IL-12, n = 5), or Ebi3 (a subunit of both IL-35 and IL-27, n = 5), or plus both p35 and Ebi3 (n = 6). B, in TV-DTH assays, splenocytes from col(V)-treated Ldlr^−/− mice subjected to injections with neutralizing antibodies to Ebi3 and kept for 24 weeks on a Western diet were injected with TT/DT, with col(I) or with col(V) alone, or plus neutralizing antibodies to IFN-γ or IL-17 (n = 6 in each case). C, quantitative analysis of the percent plaque area in ORO-stained en face preparations of descending thoracic aortas of Ldlr^−/− mice treated nasally with PBS or col(I) or treated nasally with col(V) and then subjected to injections with IgG or neutralizing antibody to Ebi3. D, representative ORO-stained en face preparations of descending thoracic aortas. E, immunoblotting with anti-MCP-1 antibody or with antibody to α-tubulin (as a loading control) of descending thoracic aortic extracts from mice treated nasally with PBS (P) or col(I) (I) or treated nasally with col(V) and then subjected to injections with IgG or neutralizing antibody to Ebi3. F, representative flow plots (left panels) and flow cytometric quantification (right panels) of the percentage of CD11b⁺ monocytes/macrophages (top panels) or of Ly-6C^high inflammatory CD11b⁺ monocytes/macrophages (bottom panels) in the aortas of Ldlr^−/− mice treated nasally with PBS (n = 9) or with col(V) and the latter subjected to injections with IgG (n = 8) or neutralizing antibodies to Ebi3 (n = 5) and maintained for 24 weeks on a Western diet. Data are shown as mean ± S.E. *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005; NS, not significant.

FIGURE 6.

Epitopes underlying Col(V) autoimmunity in Ldlr^−/− mice are localized to the N-terminal half of the α1(V) triple-helical domain. A, schematic aligning the six recombinant fragments with the triple-helical region of the α1(V) chain. B, Coomassie-stained SDS-PAGE gel of purified α1(V) recombinant fragments. Fragments 1–4 ran with the expected mobilities, whereas fragments 5 and 6 had delayed mobilities, presumably because of extensive posttranslational modifications. Molecular masses (in kilodalton) are indicated for protein standards. C, TV-DTH measured T cell responses to α1(V) recombinant fragments 1–6 and 15-mer peptides p599 and p909 (34) using splenocytes from Col5a2^loxP/loxP:Ldlr^−/− mice that had been on a Western diet for 24 weeks. Data are shown as mean ± S.E.

FIGURE 7.

Amino acid sequences of the six recombinant fragments spanning the α1(V) COL1 domain. Not shown is the amino acid sequence (MRAWIFFLLCLAGRALAAPLA) of the signal peptide at the N terminus of each fragment. Each fragment had at its C terminus the pro-α1(V) C-propeptide to enable chain association and the formation of triple-helical molecules (necessary for efficient secretion), followed by a His tag (HHHHHH) for purification.