Deficient control of Trypanosoma cruzi infection in C57BL/6 mice is related to a delayed specific IgG response and increased macrophage production of pro-inflammatory cytokines
Introduction
Chagas' disease is caused by the intracellular protozoan Trypanosoma cruzi. About 20 million people in Latin America are infected, and 40 million are at risk of acquiring the infection (WHO, 2000).
Control of T. cruzi infection is achieved through the coordinated activation of several immune effector mechanisms. Experimental studies indicate that antibodies and macrophages play an essential role in destroying circulating trypomastigotes and killing intracellular forms, respectively (Lages-Silva et al., 1987, Umekita and Mota, 2000, Pascutti et al., 2003). Macrophage trypanocidal properties involve, in part, the expression of inducible NO synthase (iNOS), which is mostly stimulated by interferon gamma (IFN-γ) and tumour necrosis factor alpha (TNFα) leading to the subsequent nitric oxide production (NO) by these cells (Reed, 1988, Torrico et al., 1991, Revelli et al., 1995, Black et al., 1989, Golden and Tarleton, 1991, Muñoz Fernandez et al., 1992). However, NO, along with cytokines promoting its production, i.e., TNFα, may act as mediators of tissue damage when produced in excessive amounts (Moncada and Higgs, 1993, Aggarwal and Natarajan, 1996).
Experimental murine models of Chagas' disease have been helpful in getting a better insight on the course of infection and disease pathology. Earlier work by infecting C57BL/6 and BALB/c mice with the Tulahuen strain of T. cruzi revealed an acute disease, which was lethal in the former group, with two thirds of BALB/c mice being recovered from such episode. Fatal disease outcome in C57BL/6 mice was accompanied by a substantial imbalance between pro- and anti-inflammatory cytokine levels in sera, compatible with an excessive inflammatory reaction arising from an unfavourable parasite–host relationship (Roggero et al., 2002). Reinforcing this view, challenge with LPS during infection of C57BL/6 mice was followed by a more pronounced increase of TNFα levels in serum.
Partly because LPS desensitisation was shown to protect mice from harmful effects of LPS challenge (Erroi et al., 1993) and given that LPS tolerance conferred an increased resistance to systemic infection with Cryptococcus neoformans or Salmonella enterica (Rayhane et al., 2000, Lehner et al., 2001) we further analysed the influence of LPS-pretreatment on acutely infected C57BL/6 mice. By applying some modification to the classical protocol (desensitisation followed by a sublethal LPS dose) pretreatment with LPS prolonged survival time of C57BL/6 mice. In view that pentoxifylline (PTx) affected in vitro LPS tolerance, experiments by administering PTx in combination with the tolerance-inducing LPS doses were also performed. Such schedule was shown to partly interfere with tolerance induction causing a significantly reduced mortality accompanied by decreased TNFα and IL-6 serum concentrations (Roggero et al., 2004). It follows, that a finely tuned balance of the anti-parasite immune response was required for the host to clear the infection without excessive pathological consequences.
The more detrimental disease of C57BL/6 mice may be related to a deficient ability of parasite handling by macrophages, that turns out to be more harmful than beneficial. Initial studies in peritoneal macrophages—PMs—from C57BL/6 and BALB/c mice revealed no major differences in the amount of intracellular forms, while showing an increased TNFα production in cells from the former group. Whether this phenomenon extends to the production of other mediators deserves further exploration. On the other hand, since LPS tolerance can be reproduced in macrophages (Ziegler-Heitbrock et al., 1992), in vitro infection of macrophages from LPS-pretreated mice would provide a better understanding on the mechanisms by which LPS-pretreatment protected C57BL/6 mice.
Accordingly, PMs from both mouse strains and also from LPS-pretreated C57BL/6 mice were analysed for the in vitro features of T. cruzi infection, that is parasite replication and cytokine production, at different infection schedules. Given that humoral responses are mounted to contain infection, the in vivo quantitative production of specific IgG antibodies was also investigated for a better evaluation of the host response.
Section snippets
Mice and parasites
Male BALB/c and C57BL/6 mice (6–8 weeks of age) were bred in the animal facilities at the School of Medicine of Rosario. Animals had access to food and water ad libitum, and were handled according to institutional guidelines for their handling. Trypomastigotes of the Tulahuen strain of T. cruzi, were maintained by serial passages in CBi suckling mice. Heparinised blood obtained from infected mice was diluted in physiological solution and washed twice. Live blood parasites were counted using a
Studies in C57BL/6 and BALB/c mice
The first set of studies is referred to experiments carried out in the two strains of mice undergoing no in vivo interventions, before collecting PM.
Characteristics of the in vitro infection
We initially analysed the features of the in vitro infection in PMs (the visualisation of intracellular parasites is depicted in Fig. 1). Table 1 shows the number of amastigotes of 500 cells/well and the number of amastigotes/infected cell at the 1 : 1 PM : parasite ratio, 24 and 48 h following exposure. Intracellular parasite counts revealed no
Discussion
Our preliminary study in BALB/c and C57BL/6 mice showed that these strains were an appropriate model to investigate the mechanisms accounting for the divergent disease outcome during acute T. cruzi infection. In fact, inoculation with Tulahuen trypomastigotes led to an acute disease, of lethal course in C57BL/6 mice, whereas most of the BALB/c mice recovered from the acute episode (Roggero et al., 2002). In view that macrophages harbour the intracellular multiplication of parasites and at the
Conclusions
Extending our former demonstration on the failure of C57BL/6 mice to control acute T. cruzi we now add evidence that such deficit might be also related with their inability to mount a satisfactory parasite-specific IgG response, coupled with an increased TNFα production by PMs. LPS-pretreatment promoted a better balance between pro- and anti-inflammatory cytokine syntheses by PMs, but impaired their ability to control intracellular parasite growth, implying that additional mechanisms are
Acknowledgements
The study was supported by a research grant from FONCYT (BID 1201/OC-AR, 05–06412). We thank Jeanne Wietzerbin, Ivan Marcipar and Maria J. Svetaz for their assistance and contributions.
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