Complete Freund's adjuvant induces experimental autoimmune myocarditis by enhancing IL-6 production during initiation of the immune response

Abstract

Introduction: Complete Freund's Adjuvant (CFA) emulsified with an antigen is a widely used method to induce autoimmune disease in animal models, yet the contribution of CFA to the immune response is not well understood. We compared the effectiveness of CFA with Incomplete Freund's Adjuvant (IFA) or TiterMax Gold Adjuvant (TMax) in experimental autoimmune myocarditis (EAM) in male mice.

Methods: EAM was induced in A/J, BALB/c, and IL6KO BALB/c male mice by injection of the myocarditogenic peptide in CFA, IFA, or TMax on days 0 and 7. EAM severity was analyzed by histology on day 21. In addition, specific flow cytometry outcomes were evaluated on day 21.

Results: Only mice immunized with CFA and myocarditogenic peptide on both days 0 and 7 developed substantial myocarditis as measured by histology. We observed a significantly increased level of IL6 in the spleen 3 days after CFA immunization. In the spleen and heart on day 21, there was an expansion of myeloid cells in CFA-immunized mice, as compared to IFA or TMax-immunized animals. Recombinant IL-6 at the time of IFA immunization partially restored susceptibility of the mice to EAM. We also treated EAM-resistant IL-6 knockout mice with recombinant IL-6 around the time of the first immunization, on days -1 to 2, completely restoring disease susceptibility, showing that the requirement for IL-6 coincides with primary immunization. Examining APC populations in the lymph node draining the immunization site evidenced the contribution of IL-6 to the CFA-dependence of EAM was through controlling local dendritic cell (DC) trafficking.

Conclusions: CFA used with myocarditogenic peptide twice is required to induce EAM in both A/J and Balb/c mice. Although IFA and TiterMax induce antibody responses, only CFA preferentially induced autoantigen-specific responses. CFA expands monocytes in the heart and in the spleen. IL-6 signaling is required during short window around primary immunization to induce EAM. In addition, IL-6 deficient mice resistance to EAM could be reversed by injecting IL-6 around first immunization. IL-6 expands dendritic cell and monocytic populations and ultimately leads to a robust T-cell driven immune response in CFA immunized mice.

Keywords: Autoimmunity; adjuvant; myocarditis.

Figure 1

CFA is required for EAM induction. A/J mice were immunized on days 0 and 7 with myocarditogenic peptide emulsified in CFA or IFA or TiterMax. Mice were sacrificed 21 days post‐immunization. Data points represent individual mice. EAM was assessed by histopathology (A) as well as by heart weight/body weight ratio (B) Representative hematoxylin and eosin‐staining of cardiac sections from A/J mice immunized with CFA/CFA, CFA/IFA, IFA/IFA/, and TiterMax/TiterMax (C). All data are analyzed by one‐way ANOVA followed by Tukey's post‐test. * denotes p < 0.05, ** denotes p < 0.01 by Tukey post‐test for the groups indicated.

Figure 2

Total IgG (A), IgG1 (B), IgG2a (C), and ratio of total IgG to MyHCα_339–352 IgG (D) were measured in all groups (CFA/CFA, CFA/IFA, IFA/IFA/, and TiterMax/TiterMax). All values are in ng/ml. Bars represent mean. N = 4–5 mice/group. All data analyzed by one‐way ANOVA followed by Tukey's post‐test. * denotes p < 0.05 by Tukey post‐test for the groups indicated.

Figure 3

CFA expands the myeloid population in the spleen at day 21. A/J mice were immunized on day 0 and 7 with myocarditogenic peptide emulsified in CFA or IFA or TiterMax. Total number of CD11b⁺ monocytes (A), CD11c⁺ dendritic cells (B), CD4⁺ T cells (C), CD19⁺ B cells (D), DX5⁺ NK cells (E), CD8⁺ T cells (F), or CD4⁺CD25⁺FoxP3⁺ Tregs (G) in the spleens on day 21 of EAM is shown. Spleens from naive animals were used as controls. Data points represent individual mice. Bars represent mean. N = 4–5 mice/group. All data are analyzed by one‐way ANOVA followed by Tukey's post‐test. * denotes p < 0.05 by Tukey post‐test for the groups indicated.

Figure 4

CFA expands the myeloid population in the heart at day 21. BALB/c mice were immunized on days 0 and 7 with myocarditogenic peptide emulsified in CFA or IFA or TiterMax. Cell population were analyzed from their hearts on day 21 of EAM. Total number of neutrophils Ly6G^hiCD11b⁺ (A), macrophages CD64⁺F4/80⁺CD11b⁺ (B), inflammatory monocytes Ly6C^hiCD11b⁺ (C), and non‐inflammatory monocytes Ly6C^loCD11b⁺ (D) is shown. Representative flow cytometry gating of these myeloid populations is shown (E). Data points represent individual mice. Bars represent mean. N = 4–5 mice/group. All data are analyzed by one‐way ANOVA followed by Tukey's post‐test. * denotes p < 0.05 by Tukey post‐test for the groups indicated.

Figure 5

CFA stimulates an inflammatory environment in the spleen at day 3. A/J mice were immunized on day 0 with myocarditogenic peptide emulsified in CFA or IFA or TiterMax. Total number of CD11b⁺CD11c^−/lo monocytes in the spleen 3 days after the first injection with the shown adjuvant (A). CD11b⁺CD11c^−/lo monocytes production of IL‐10 (B), expression of PD‐L1 (C). Total numbers of conventional CD8α⁺CD11c⁺ dendritic cells (D) IL‐1β production by CD8α⁺CD11c⁺ dendritic cells (E). IL‐6 production was analyzed from spleens of Balb/c mice after on day 3 of immunization with the appropriate adjuvant (F). Spleens from naive animals were used as controls. Data points represent individual mice. Bars represent mean. N = 4–5 mice/group. All data are analyzed by one‐way ANOVA followed by Tukey's post‐test. * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001 by Tukey post‐test for the groups indicated.

Figure 6

IL‐6 is required for the initial response to immunization in order to induce EAM. EAM was induced in WT A/J mice immunized with IFA or CFA, alone or with recombinant IL6 treatment on the day indicated (x‐axis). Mice were sacrificed 21 days post‐immunization. Myocarditis severity was assessed by histopathology (A). N = 4 mice/group. EAM was induced in WT Balb/c mice or IL‐6KO mice on Balb/c background. On days −1, 0, 1, 2 mice were treated with either PBS or 50 ng recombinant IL‐6 iv. Mice were sacrificed 21 days post‐immunization (B). Myocarditis severity was assessed by histopathology. N = 6 mice/group. Data points represent individual mice. Bars represent mean. Data are analyzed by one‐way ANOVA followed by Tukey's post‐test. * denotes p < 0.05 by Tukey post‐test for the groups indicated.

Figure 7

IL6 is required in order to induce inflammatory DC trafficking to the draining LN following immunization. EAM was induced in WT Balb/c mice treated with isotype control IgG2b antibodies or anti‐IL‐6‐receptor antibodies on days −3 and 0. Mice were sacrificed 3 days post‐immunization. Response to immunization was assessed in the draining lymph node of the immunization site by flow cytometry. Total number of CD11c⁺ dendritic cells in the inguinal lymph nodes draining sites of immunization (A). Expression of MHC Class II by CD11c⁺ dendritic cells (B). Expression of CCR7 by CD11c⁺ dendritic cells (C). Data were analyzed by Student's t‐test. Data are representative of three independent experiments. Total lymph node homogenates were assessed for CCL21 levels by ELISA (D). Data are analyzed by Student's t‐test. N = 7 mice/group. Data points represent individual mice. Bars represent mean. * denotes p < 0.05