Heat-killed Trypanosoma cruzi induces acute cardiac damage and polyantigenic autoimmunity

Abstract

Chagas heart disease, caused by the protozoan parasite Trypanosoma cruzi, is a potentially fatal cardiomyopathy often associated with cardiac autoimmunity. T. cruzi infection induces the development of autoimmunity to a number of antigens via molecular mimicry and other mechanisms, but the genesis and pathogenic potential of this autoimmune response has not been fully elucidated. To determine whether exposure to T. cruzi antigens alone in the absence of active infection is sufficient to induce autoimmunity, we immunized A/J mice with heat-killed T. cruzi (HKTC) emulsified in complete Freund's adjuvant, and compared the resulting immune response to that induced by infection with live T. cruzi. We found that HKTC immunization is capable of inducing acute cardiac damage, as evidenced by elevated serum cardiac troponin I, and that this damage is associated with the generation of polyantigenic humoral and cell-mediated autoimmunity with similar antigen specificity to that induced by infection with T. cruzi. However, while significant and preferential production of Th1 and Th17-associated cytokines, accompanied by myocarditis, develops in T. cruzi-infected mice, HKTC-immunized mice produce lower levels of these cytokines, do not develop Th1-skewed immunity, and lack tissue inflammation. These results demonstrate that exposure to parasite antigen alone is sufficient to induce autoimmunity and cardiac damage, yet additional immune factors, including a dominant Th1/Th17 immune response, are likely required to induce cardiac inflammation.

Figure 1. Immunization with heat-killed T. cruzi causes acute cardiac damage but not myocarditis.

A/J mice were infected with T. cruzi, immunized with heat-killed T. cruzi epimatigotes (HKTC), T. cruzi tissue-culture trypomastigotes (HKTC_tct) T. cruzi cultured metacyclic trypomastigotes (HKTC_cmt) or injected with PBS. (A) One representative image from each group at 21 d.p.i. is shown. (B) The mean histopathology score for inflammation is indicated. For 7, 14, and 21 d.p.i., n = 10 for all groups; for 28 d.p.i. n = 5 for HKTC-immunized, heat-killed L. amazonensis (HKLA)-immunized, and PBS-immunized groups. (C) The mean serum cTnI level is indicated, n = 4 for T. cruzi-infected and PBS-immunized groups; n = 6 for the HKTC-immunized group. (B and C) Error bars indicate SEM. * P<0.05 compared to the PBS control group. † T. cruzi-infected mice were not analyzed at this time point because they do not survive past 25 d.p.i.

Figure 2. Development of humoral autoimmunity in mice immunized with HKTC is of similar antigen specificity as in T. cruzi infected mice.

A/J mice were infected with T. cruzi (n = 7), immunized with HKTC (n = 7), or injected with PBS (n = 3). ELISA analysis was used to test individual serum samples for IgG reactivity to T. cruzi as well as to a panel of cardiac antigens (actin, Cha antigen, desmin, laminin, myoglobin, myosin, tropomyosin) and non-cardiac antigens (elastin, mucin) or the immunologically irrelevant antigen BSA at 21 d.p.i. Each data point represents the mean antibody titer of each cohort above the background response to BSA. Error bars indicate SEM. Data are representative of two independent experiments. * P<0.05.

Figure 3. T. cruzi-infected and HKTC-immunized mice develop DTH specific for a number of cardiac and non-cardiac proteins.

A/J mice were infected with T. cruzi, immunized with HKTC, immunized with HKLA, or injected with PBS. (A) At 21 d.p.i., antigen-specific DTH responses to a panel of cardiac and non-cardiac antigens were measured by a standard 24-hour ear swelling assay. (B) DTH responses specific for HKTC and myosin were measured in A/J mice immunized heat-killed T. cruzi epimatigotes (HKTC), T. cruzi tissue-culture trypomastigotes (HKTC_tct) T. cruzi cultured metacyclic trypomastigotes (HKTC_cmt), supernatant from T. cruzi-infected myoblast cultures (supernatant), or injected with PBS. Data represent the net ear swelling response over the basal response to an irrelevant antigen (BSA). Error bars indicate SEM (n = 4). Data are representative of two independent experiments. * P<0.05 compared to the PBS control group.

Figure 4. Splenocytes from T. cruzi-infected mice produce a different cytokine profile when stimulated with cardiac autoantigens than those from HKTC-immunized mice.

A/J mice were infected with T. cruzi, or immunized with HKTC, HKLA, or PBS. At 21 d.p.i., splenocytes were isolated and combined from five mice per group and stimulated with the indicated antigens. (A) IFN-γ, (B) IL-4, and (C) IL-17 production were measured by ELISPOT. Data are representative of four independent experiments. Error bars indicate SEM (n = 4). * P<0.05 compared to the PBS control group.

Figure 5. T. cruzi-infection but not HKTC immunization induces robust autoreactive Th1 and Th17 responses.

A/J mice were infected with T. cruzi (INF), or immunized with HKTC (HKTC), or PBS (PBS). At 21 d.p.i., splenocytes were isolated and stimulated with HKTC or Myo 4. Intracellular cytokine staining of CD90+CD4+ splenocytes stimulated with anti-CD3 for IFN-γ, IL-4, and IL-17 was analyzed via flow cytometry. (A) The mean percentage of cytokine-positive cells is shown. Error bars indicate SEM (n = 4 for INF and HKTC; n = 3 for PBS). Data are representative of two independent experiments. (B) Representative plots of the cells quantified in panel A are shown. The mean percentage and SEM of IFN-γ+IL-17-, IFN-γ+IL-17+, and IFN-γ−IL-17+ cells are indicated.

Figure 6. T. cruzi-infected mice have higher serum IL-12 than do HKTC-immunized mice.

A/J mice were infected with T. cruzi (INF), immunized with epimastigote HKTC (HKTC), trypomastigote HKTC (HKTC_tct) or PBS (PBS). At 21 d.p.i., sera was collected and IL-12 levels were measured via ELISA. The mean concentration of serum IL-12 is shown. Error bars indicate SEM (n = 8 for INF; n = 7 for other groups). * P<0.05 compared to the PBS control group.