Short treatment with the tumour necrosis factor-alpha blocker infliximab diminishes chronic chagasic myocarditis in rats without evidence of Trypanosoma cruzi reactivation

Abstract

Tumour necrosis factor (TNF)-alpha is crucial for resistance to Trypanosoma cruzi acute infection, but there is scant information on its role during the chronic phase. To address this issue, we analysed whether a short treatment with a TNF-alpha blocker affected the course and characteristics of chronic disease in a rat experimental model of T. cruzi infection. An anti-TNF-alpha agent (infliximab) was administered during the chronic phase for a period of 4 weeks (3 mg/kg/week), while control infected rats were inoculated with saline physiological solution. Search for parasites yielded non-successful results in all infected groups, irrespective of treatment. Nevertheless, the presence of T. cruzi kDNA in heart tissue was detected in infected and infected plus treated animals. Because infliximab might induce changes in the anti-parasite cytokine response, circulating levels of interleukin (IL)-10, interferon-gamma and nitric oxide were evaluated. An increase in IL-10 levels was observed only in the infected group treated with the anti-TNF-alpha blocker compared to the remaining groups (P < 0.05). A clear attenuation of histological damage associated with a diminution of cardiac TNF-alpha mRNA expression was observed in the infected and treated animals compared to the infected and non-treated group. Blocking of TNF-alpha during a relatively short period in chronically infected rats did not lead to evident parasite reactivation but reduced myocarditis severity significantly, indicating a role of this cytokine in the pathogenesis of chronic myocardial damage.

Fig. 1

Detection of kDNA in chronic infected heart tissues. Representative agarose gel electrophoresis results for kDNA by heart tissue homogenates from rats after infliximab treatment (120 days post-infection) analysed by polymerase chain reaction. Lane 1: molecular weight marker [M_WM 500 base pairs (bp) ladder]; lane 2: heart tissue extracted from a rat with acute infection; lanes 3 and 4: heart tissue from non-infected and physiological saline-treated rats; lanes 5–7: heart tissue from chronic infected rats; lanes 8 and 9: heart tissue from non-infected but infliximab-treated rats; lanes 10–12: heart tissue from infected and infliximab-treated rats. Arrow indicates M_WM and Trypanosoma cruzi-specific products of 330 bp. Each line is a sample of a single animal.

Fig. 2

Immunoglobulin (Ig)-specific levels after infliximab therapy. IgM and IgG Trypanosoma cruzi-specific levels were evaluated by enzyme-linked immunosorbent assay after infliximab treatment (120 days post-infection). Detectable amounts of specific IgM (1/20) and IgG (1/200 dilution) were seen in both groups of chronically infected rats, without significant differences between them. Tc and Tc + infliximab groups significantly different from their non-infected counterparts (P < 0·001, both immunoglobulin isotypes). Values represent means ± standard error of the mean of optical density unit of six to nine rats/group. A representative experiment of two independent rounds.

Fig. 3

Tumour necrosis factor (TNF)-α mRNA expression in myocardial tissue. TNF-α mRNA expression in myocardial tissue was evaluated by semiquantitative reverse transcription–polymerase chain reaction after infliximab treatment (120 days post-infection). β-actin mRNA was used to normalize TNF-α mRNA among samples. Relative values were calculated considering TNF-α/β-actin ratio as 1 for infected but non-treated animals. No expression of TNF-α mRNA was seen in both non-infected groups, Normalized results correspond to two experimental series of four to six animals/group (media of relative value of sample corresponding to an individual animal ± standard error of the mean).

Fig. 4

Histopathology evaluation of chronic myocarditis. (a) Intense inflammatory foci in a chronically infected rat; (b) mild inflammatory foci observed in chronically infected and infliximab-treated rat; (c) CD8⁺ cells in heart tissue of infected animals (mild foci); (d) CD8⁺ cells in the myocardium of chronically infected and infliximab-treated animals (mild foci). Magnification panels 20×. In all cases, the respective non-infected control groups are showed in the small cages.