Silvia Revelli

Differential susceptibility to acute Trypanosoma cruzi infection in BALB/c and C57BL/6 mice is not associated with a distinct parasite load but cytokine abnormalities

Inoculation of Trypanosoma cruzi, Tulahuén strain, into C57BL/6 and BALB/c mice led to an acute infection characterized by marked parasitaemia, myocardial inflammation and thymocyte depletion. While C57BL/6 mice showed a progressive and lethal disease, BALB/c mice partly recovered. To characterize these murine models more effectively, we studied the parasite burden, serum levels of major infection outcome‐related cytokines, the in vitro features of T. cruzi infection in peritoneal macrophages and the immunophenotype of thymic cells. The greater disease severity of T. cruzi‐infected C57BL/6 mice was not linked to an increased parasite load, as parasitaemia, myocardial parasite nests and amastigote counts in peritoneal macrophages were not different from those in BALB/c mice. Cortical thymocyte loss was accompanied by the presence of apoptotic bodies and fragmented nuclear DNA, whereas fluorocytometric analysis at 17 days postinfection (p.i.) revealed a more pronounced loss of CD4+ CD8+ cells in C57BL/6 mice. This group displayed higher levels of TNF‐α on days 14 and 21 p.i., in the presence of lower IL‐1β and IL‐10 concentrations by days 14 and 21, and days 7 and 14 p.i., respectively. Day‐21 evaluation showed higher concentrations of nitrate and TNF‐α soluble receptors in C57BL/6 mice with no differences in IFN‐γ levels, with respect to the BALB/c group. Increased morbidity of C57BL/6 T. cruzi‐infected mice does not seem to result from an aggravated infection but from an unbalanced relationship between pro‐ and anti‐inflammatory mediators.

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

Tumour necrosis factor (TNF)‐α 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‐α blocker affected the course and characteristics of chronic disease in a rat experimental model of T. cruzi infection. An anti‐TNF‐α 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‐α blocker compared to the remaining groups (P < 0·05). A clear attenuation of histological damage associated with a diminution of cardiac TNF‐α mRNA expression was observed in the infected and treated animals compared to the infected and non‐treated group. Blocking of TNF‐α 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.

Trypanocidal Drug Benznidazole Impairs Lipopolysaccharide Induction of Macrophage Nitric Oxide Synthase Gene Transcription Through Inhibition of NF-κB Activation

In murine macrophages, inducible NO synthase II (NOSII) gene expression is promoted at a transcriptional level by LPS and/or IFN-γ with benznidazole (BZL), a trypanocidal drug, acting to down-regulate NOSII gene induction and hence inhibiting NO production. By performing transient transfection experiments, we now report that BZL also inhibited the expression of NOSII gene promoter or multimerized NF-κB binding site controlled reporter genes. By contrast, no effect was observed on the expression of a reporter gene under the control of the NOSII promoter-derived IFN regulatory factor element. EMSAs demonstrated that BZL inhibited the nuclear availability of NF-κB in stimulated macrophages. NF-κB is activated in macrophages by phosphorylation, ubiquitination, and subsequent proteolysis of IκB. Within this setting, Western blot was also performed to show that BZL blocked IκBα degradation. Collectively, these results demonstrate that BZL is able to specifically inhibit macrophage NF-κB activation after LPS plus IFN-γ stimulation.

Acute and Chronic Experimental Trypanosoma cruzi Infection in the Rat. Response to Systemic Treatment with Recombinant Rat Interferon-Gamma

We examined the effects of recombinant rat inteferon‐gamma (IFN‐γ) injections on the parasitologic, serologic, immunologic and histopathologic features of acute and chronic experimental Trypanosoma cruzi (T. cruzi) infections in “l” rats. Upon infection at weaning, two rat groups were allocated to receive a 20‐day cycle of IFN‐γ injections, 20,000 IU/rat each, which started at 1, and 7 days post‐infection (pi). Treatment with IFN‐γ, initiated at either 1 or 7 days pi, resulted in comparatively lower peak parasitemias (P<0.02) but in similar levels of anti‐T. cruzi circulating antibodies and serum IFN‐γ activities. The latter appeared significantly increased during acute infection whereas biologically active tumor necrosis factor was virtually undetectable in serum from infected rats regardless of whether they had been given IFN‐γ or not. The prevalence of chronic focal myocarditis in IFN‐γ‐treated infected rats showed no differences with respect to the one recorded in control‐infected counterparts. The inverse CD4/CD8 ratio of spleen and lymph node T cells that usually accompanies chronic infection was reversed by IFN‐γ. Mononuclear cells carrying class III‐A and I‐E molecules, that were found to have increased at both compartments, appeared also modified upon IFN‐γ treatment with an overincrease of I‐A‐positive cells, and a normalization of I‐E‐bearing cells.

Age‐Related Increase in Resistance to Acute Trypanosoma cruzi Infection in Rats is Associated with an Appropriate Antibody Response

Inoculation at weaning with Trypanosoma cruzi in inbred ‘l’ rats resulted in a self‐resolving acute infection characterized by marked parasitaemias, whereas challenge to adult rats revealed a mild disease with extremely low parasitaemias. To explore the mechanisms underlying such age‐associated differences in disease outcome, we analysed the in vitro replication of T. cruzi, nitric oxide and tumour necrosis factor‐α (TNF‐α) production in peritoneal macrophages (PMs), the serum concentrations of the specific immunoglobulins (Igs) IgM and IgG, antibodies exhibiting lytic activity against bloodstream forms of T. cruzi and circulating levels of nitrate, TNF‐α and interferon‐γ (IFN‐γ). Macrophages from young rats were as effective as their adult counterparts for restraining intracellular parasite replication. When stimulated with IFN‐γ, culture supernatants from young PMs contained higher amounts of nitrite and TNF‐α. Serum samples from 4 and 7 days post infection revealed easily detectable amounts of nitrate, with values being further augmented by day 7 post infection and significantly higher in the young group. TNF‐α levels were only detected in the young group by day 7 post infection. Both groups had increased amounts of IFN‐γ in their sera, although in adult rats, this trend was followed by a significant drop at day 7, with young rats showing values still higher by the same time point evaluation. In contrast, young rats presented significantly lower levels of IgM and IgG antibodies during the first week of infection. Increased resistance in adult rats seems to be the result of a more appropriate antibody production.

Antibody Maturation in Trypanosoma cruzi-Infected Rats

ABSTRACT The study of antibody avidity changes during infection has improved the understanding of the pathologic processes involved in several infectious diseases. In some infections, like toxoplasmosis, this information is being used for diagnostic purposes. Results of the evolution of antibody avidity for different specific antigens inTrypanosome cruzi-infected rats are presented. A Western blotting technique, combined with avidity analysis to identify antigens that elicit high-avidity antibodies, is suggested. In this system, antibodies showed high avidity values only during the chronic phase of infection and only in relation to antibodies against 21-, 33-, 41-, 42-, 56-, 58-, 66-, and 72-kDa antigens. Finally, a 97-kDa T. cruzi antigen, which was recognized by high-avidity antibodies and occurred in noninfected rats, was identified. These results allow us to evaluate the different antigens in chagasic infection. Our results show that with the correct choice of antigen it is possible to detect differences in maturation of antibodies and to discriminate, in an experimental model, between recent (acute) and chronic infections.

Benznidazole blocks NF-κB activation but not AP-1 through inhibition of IKK

Previously, we demonstrated that benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, modulates pro-inflammatory cytokines and NO release in macrophages by inhibiting NF-kappaB. We now proceeded to elucidate the molecular mechanisms by which BZL exerts its inhibitory action on NF-kappaB. We demonstrated that the inhibitory effect of BZL is not extended to other macrophage responses, since it did not inhibit other typical hallmarks of macrophage activation such as phagocytosis, MHC-II molecules expression or production of reactive oxygen species (ROS) by NADPH oxidase. BZL was able to interfere specifically with the activation of NF-kappaB pathway without affecting AP-1 activation in RAW 264.7 macrophages, not only in LPS-mediated activation, but also for other stimuli, such as pro-inflammatory cytokines (IL-1beta, TNF-alpha), PMA or H(2)O(2). Also, BZL delayed the activation of p38 MAPK, but not that of ERK1/2 and JNK. Finally, treatment with BZL inhibited IkappaBalpha phosporylation and hence its degradation, whereas it did not block IkappaB kinase (IKK) alpha/beta phosphorylation. Collectively, BZL behaves as a broad range specific inhibitor of NF-kappaB activation, independently of the stimuli tested.

Levels of tumor necrosis factor alpha, gamma interferon, and interleukins 4, 6, and 10 as determined in mice infected with virulent or attenuated strains of Trypanosoma cruzi

Abstract Inoculation of BALB/c mice with the virulent Tulahuen (TUL) strain of T. cruzi was shown to lead to progressive and eventually lethal infections, whereas infection with an attenuated strain (TCC) resulted in a hardly noticeable experimental disease producing no tissue damage. To determine whether differences in such infection outcome are associated with a particular pattern of cytokine response, a study was undertaken to investigate the serum levels of TNF-α, IFN-γ, IL-4, IL-6, and IL-10 by using an enzime-linked immunosorbent assay. Mice from both infected groups were bled at 5, 9, 15, 22, 30 and 48 days post-infection (pi), with the same interval being applied for obtention of serum samples in age-matched uninfected mice, a group that yielded negative results in all cases. Infection with the TUL strain of T. cruzi was accompanied by a significant increase of TNF-α serum concentrations at day 5 pi, and detectable amounts of IFN-γ by day 15 pi, which were exclusively recorded in this group. Serum IL-4 was mostly present in TCC mice with values at day 15 pi being statistically significant in relation to TUL-infected mice. IL-10 was firstly detected at 3 weeks after infection, and showed higher levels in the TCC group, although comparisons with TUL-infected group were not significant. At our limit of detection, no samples were found to contain IL-6 serum concentrations. Infection with virulent parasites seems to be associated with presence of Th1-type cytokines, whereas challenge with the attenuated TCC strain appears as being related to a Th2-type profile.

Tumor necrosis factor alpha induced by Trypanosoma cruzi infection mediates inflammation and cell death in the liver of infected mice

Trypanosoma cruzi (T. cruzi) infected C57BL/6 mice developed a progressive fatal disease due to an imbalance in the profile of circulating related compounds accompanying infection like tumor necrosis factor alpha (TNFalpha). TNFalpha has been proposed as an important effector molecule in apoptosis. In this work, we evaluate inflammation and the proteins involved in apoptotic process in liver of infected mice and the role of TNFalpha. C57BL6/mice were infected subcutaneously with 100 viable trypomastigotes of Tulahuén strain of T cruzi. One set of these animals were treated with 375 microg of antihuman TNFalpha blocking antibody. Animals were sacrificed at 14 days post-infection (p.i).The analyses of Bcl-2 family proteins revealed an increase of the pro-apoptotic proteins Bax and tBid in T. cruzi-infected mice. Compared with control animals, cytochrome c release was increased. Apoptosis was also induced in infected mice. Anti-TNFalpha treatment decreases hepatic apoptosis. Our results suggest that T. cruzi infection induces programmed cell death in the host liver by increase of TNFalpha production, associated with TNF-R1 over-expression, that set in motion the Bid cleavage and mitochondrial translocation, Bax mitochondrial translocation, cytochrome c release, and ultimately apoptosis induction.

Enhanced myocardial lesions in chronically Trypanosoma cruzi-infected rats subjected to adult thymectomy

Control animals and rats infected 90 days earlier, by inoculation of 1 x 10(6) trypomastigotes of Trypanosoma cruzi at weaning, were subjected to adult thymectomy (ATx) or sham operation (S-ATx) and assessed 3 months later for the presence of myocardial lesions and levels of lymph node and spleen T-cell populations. Chronic focal myocarditis (CFM) developed in 78% and 84% of S-ATx or ATx infected rats, respectively. While the two groups of infected rats did not differ as to the occurrence of myocardial lesions, large foci of CFM were more prevalent in ATx infected rats. Chronic T. cruzi (Tc) infection resulted in decreased CD4+ and increased CD8+ lymph node and spleen cell, with CD8+ lymphocytes being lowered to normal values in the spleen of the ATx infected group. It is suggested that ATx might act by interfering with a down-regulating immunoregulatory mechanism, leading to an exacerbation of autoimmune reactions believed to be involved in the generation of myocardial damage.