Cardiac myosin autoimmunity in acute Chagas' heart disease

Abstract

Infection with Trypanosoma cruzi, the agent of Chagas' disease, may induce antibodies and T cells reactive with self antigens (autoimmunity). Because autoimmunity is generally thought to develop during the chronic phase of infection, one hypothesis is that autoimmunity develops only after long-term, low-level stimulation of self-reactive cells. However, preliminary reports suggest that autoimmunity may begin during acute T. cruzi infection. The goal of the present study was to investigate whether cardiac autoimmunity could be observed during acute T. cruzi infection. A/J mice infected with the Brazil strain of T. cruzi for 21 days developed severe myocarditis, accompanied by humoral and cellular autoimmunity. Specifically, T. cruzi infection induced immunoglobulin G (IgG) autoantibodies and delayed type hypersensitivity (DTH) to cardiac myosin. This autoimmunity resembles that which develops in A/J mice immunized with myosin in complete Freund's adjuvant in that myosin-specific antibodies and DTH responses both develop by 21 days postinfection or postimmunization. While the levels of myosin IgG in T. cruzi-infected mice were slightly lower than those in myosin-immunized mice, the magnitude of myosin DTH in the two groups was statistically equivalent. In contrast, C57BL/6 mice, which are resistant to myosin-induced myocarditis and its associated autoimmunity, developed undetectable or low levels of myosin IgG and did not exhibit myosin DTH or myocarditis upon T. cruzi infection. Therefore, humoral and cellular cardiac autoimmunity can develop during acute T. cruzi infection in the genetically susceptible host.

FIG. 1

A/J mice infected with T. cruzi or immunized with cardiac myosin develop severe myocarditis. A/J mice were infected with 10⁴ T. cruzi trypomastigotes (infected) or immunized with 300 μg of myosin in CFA (immunized). Control mice were injected with PBS (uninfected) or immunized with CFA-PBS (unimmunized). At 21 d.p.i., cardiac sections from these mice were stained with hematoxylin and eosin. All infected (n = 5) and immunized (n = 5) mice developed severe myocarditis, with myocyte necrosis and mononuclear cellular infiltration. No myocarditis was observed in uninfected (n = 5) or unimmunized mice (n = 5). Arrowhead indicates the pseudocyst magnified in the inset. Bars, 50 μm.

FIG. 2

A/J mice produce antibodies specific for cardiac proteins after infection with T. cruzi or immunization with myosin. Serum samples from four groups of A/J mice (five mice per group) obtained 21 d.p.i. with PBS (uninfected group), with PBS-CFA (unimmunized group), with 10⁴ T. cruzi trypomastigotes (infected group), or with myosin-CFA (immunized) were pooled for use in Western blot and ELISA analyses. (A) A/J mouse heart homogenate (top) or purified cardiac myosin (bottom) was resolved by SDS-PAGE, transferred to nitrocellulose, and blotted with 1:100 dilutions of sera from groups 1 to 4. The arrowhead indicates the position of cardiac myosin heavy chain. (B) Purified regulatory myosin light-chain kinase (MLCK) and myelin basic protein (MBP) were resolved by SDS-PAGE, transferred to nitrocellulose, and blotted with 1:100 dilutions of sera from the groups. A Coomassie blue-stained gel is also shown. (C) Sera from the four groups were tested for myosin-specific IgG by ELISA. Each datum point represents the average for three wells (standard error of the mean, <0.09).

FIG. 3

T. cruz-infected mice and myosin-immunized mice display myosin-specific cellular autoimmunity. Myosin-specific DTH was determined by a 24-h ear swelling assay. Error bars indicate standard errors of the means. (A) Time-course of myosin-specific DTH in T. cruzi-infected mice. Myosin-specific DTH was measured in eight mice infected with 10⁴ T. cruzi trypomastigotes (infected) and in five mice injected with PBS (uninfected) at each d.p.i. For comparison, at 21 d.p.i., myosin-specific DTH was measured in eight myosin-immunized (immunized) mice and five PBS-CFA-immunized (unimmunized) control mice. T. cruzi-infected mice developed significant myosin-specific DTH as early as 7 d.p.i. (B) T. cruzi-specific DTH was also measured in eight infected mice and five uninfected mice at 21 d.p.i. ∗, P of <0.001; †, P = 0.002 compared to the respective controls.

FIG. 4

A/J mice develop stronger myosin autoimmunity than do C57BL/6 mice both upon T. cruzi infection and upon myosin immunization. Groups of A/J or C57BL/6 mice were infected with 10⁴ T. cruzi trypomastigotes (infected), injected with PBS (uninfected), immunized with myosin (immunized), or immunized with PBS-CFA (unimmunized). (A) Myosin autoantibody production. The infected and immunized groups contained 10 mice each while uninfected and unimmunized groups contained four mice each. At 21 d.p.i., serum from each mouse was individually tested for myosin-specific IgG in a myosin ELISA. Each datum point represents the mean OD₄₅₀ value of the mice in each group, and the error bars indicate the standard errors of the means. All experimental groups were significantly different (P < 0.001) from their respective controls. Higher levels of cardiac myosin-specific IgG were detected in infected A/J than in infected C57BL/6 mice (P < 0.001) and in myosin-immunized A/J than in myosin-immunized C57BL/6 mice (P = 0.002). (B) Myosin and T. cruzi-specific DTH was determined by a 24-h ear swelling assay at 21 d.p.i. Myosin DTH was measured in mice as described for panel A. T. cruzi DTH was measured in five infected A/J and five infected C57BL/6 mice. Error bars indicate standard errors of the means. ∗, P of <0.001 compared to the C57BL/6 group.

FIG. 5

A/J mice, but not C57BL/6 mice, develop myocarditis upon both T. cruzi infection and myosin immunization. A/J and C57BL/6 mice were infected with 10⁴ T. cruzi trypomastigotes (infected) or immunized with 300 μg of myosin in CFA (immunized). At 21 d.p.i., cardiac sections from these mice were stained with hematoxylin and eosin. Representative sections are shown. Infected (five of five) and immunized (four of five) A/J mice developed myocarditis while no disease was observed in infected mice (n = 5), immunized C57BL/6 mice (n = 5), or any of the controls, A/J and C57BL/6 mice injected with PBS or immunized with PBS-CFA (four mice per control group). Arrowheads indicate pseudocysts. Bars, 50 μm.