Stella M. González Cappa

Trypanosoma cruzi Induces Regulatory Dendritic Cells In Vitro

ABSTRACT A main feature of acute infection with Trypanosoma cruzi is the presence of immunological disorders. A previous study demonstrated that acute infection with the virulent RA strain downregulates the expression of major histocompatibility complex class II (MHC-II) on antigen-presenting cells and impairs the T-cell stimulatory capacity of splenic dendritic cells (DC). In the present work, we assessed the ability of trypomastigotes (Tp) to modulate the differentiation stage and functionality of bone marrow-derived DC in vitro. We observed that the Tp stage of T. cruzi failed to activate DC, which preserved their low expression of MHC-II and costimulatory molecules, as well as their endocytic activity. We also show that Tp induced transforming growth factor β (TGF-β) secretion by DC and enhanced the gap between interleukin-10 (IL-10) and IL-12p70 production, showing a higher IL-10/IL-12p70 ratio upon lipopolysaccharide (LPS) treatment. In addition, we observed that Tp prevented DC full activation induced by LPS, thereby downregulating their MHC-II surface expression and inhibiting their capacity to stimulate lymphocyte proliferation. In vitro IL-10 neutralization during the differentiation process of DC with Tp+LPS showed a reversion of their inhibitory effect during mixed lymphocyte reaction. In contrast, only simultaneous neutralization of IL-10 and TGF-β, after DC differentiation, was involved in the partial restitution of lymphocyte proliferation. Since both TGF-β and IL-10 are immunosuppressive cytokines essential in the modulation of the immune response and important in the induction of tolerance, our results suggest for the first time that Tp are responsible for the generation of regulatory DC in vitro.

PGE2 involvement in experimental infection with Trypanosoma cruzi subpopulations

PGE2 involvement in experimental Trypanosoma cruzi infection depends on the lethal capacity of the parasite subpopulation used. Mice acutely infected with non-lethal K98 displayed an enhancement in PGE2 serum levels during the acute period, while those infected with lethal T. cruzi subpopulations (RA or K98-2) showed levels not different from normal mice. The enhancement detected in K98 group could be related both to an increased number of CD8+ T cell number and to enhanced PGE2 release per cell by CD8+; values of PGE2 release by adherent cells were not altered in this group. Treatment with cyclooxygenase inhibitors enhanced mortality rates of mice infected with K98, and administration of 16,16-dimethyl PGE2 (dPGE) reversed this effect. However, mice infected with RA did not reduce their mortality rates by administration of diverse doses of dPGE. These findings suggest that PGE2 could play a role in resistance in mice infected with K98.

Alterations in cardiac beta-adrenergic receptors in chagasic mice and their association with circulating beta-adrenoceptor-related autoantibodies

OBJECTIVE Cardiac tissue from chagasic mice was studied to evaluate the expression and biological activity of beta-adrenoceptors in association with circulating beta-adrenoceptor-related autoantibodies. METHODS BALB/c inbred mice that were either treated or not treated with atenolol (2.5 mg/kg) and infected or not infected with 1 x 10(4) trypomastigotes (CA-1 strain) were sacrificed weekly up to week nine. Morphological, binding and contractility studies were performed on the four different groups of animals. The effect of their serum antibodies was also assayed in binding and contractility studies on normal heart preparations. RESULTS Hearts from chagasic myocarditis mice showed a beta-adrenoceptor-related dysfunction, with a decrease in heart contractility, impaired response to exogenous beta-adrenoceptor agonist and a significant reduction in beta-adrenergic binding sites. Those effects were maximum at eight-nine weeks post-infection and were improved by treating infected mice with atenolol. In addition, serum or IgG from chagasic myocarditis mice was capable of interacting with cardiac beta-adrenoceptors, reducing the number of binding sites and inhibiting the contractile response to exogenous norepinephrine. IgG effects that were observed in normal myocardium, were highest in sera from mice eight-nine weeks post-infection and correlate with the degree of myocarditis. Moreover, chagasic autoantibodies from infected mice recognized a peptide corresponding to the sequence of the second extracellular loop of the human beta 1-adrenoceptor. CONCLUSIONS (1) The development of alterations in beta-adrenergic receptors, related to cardiac dysfunction, may be associated with the presence of circulating antibodies against these receptors and (2) it is possible that the chronic deposits of these autoantibodies in cardiac beta-adrenoceptors could lead to a progressive blockade with sympathetic denervation, a phenomenon that has been described in the course of chagasic myocarditis.

TRYPANOSOMA CRUZI: EFFECT OF PARASITE SUBPOPULATION ON MURINE PREGNANCY OUTCOME

C3H/HeN female mice infected with distinct Trypanosoma cruzi subpopulations (RA strain [pantropic/reticulotropic] and K98 clone of the CA-I strain [myotropic]) show differences both in inflammatory compromise of the genital tract and in the outcome of pregnancy. The group of mice infected with the K98 clone show lymphomononuclear infiltrates in pelvian fat and in uterus interstitium, coexisting with the presence of T. cruzi DNA, and show moderate oophoritis, perioophoritis, and vasculitis. However, neither parasite DNA nor inflammatory foci were detected in the uterus, and only mild oophoritis was observed among RA-infected mice at mating time. Independently from the parasite subpopulation, females developed estrous 30 days postinoculation (PI), and at the same time, parasite counts were similar for K98 and for RA-infected mice. However, fertility was significantly diminished in K98-infected females. On day 14 of gestation, fetal resorptions increased in this group and cannot be attributed to hormonal disbalance because similar serum progesterone levels were found in all groups. At this time (44 days PI), parasitemia was higher in K98- than in RA-infected mice. However, resorptions were not triggered by massive infection because polymerase chain reaction failed to prove parasite DNA in resorbing fetuses. In contrast with K98 females, RA-infected mice delivered T. cruzi–infected newborns.

Trypanosoma cruzi: Isolate dependence in the induction of lytic antibodies in the mouse and rabbit

Lytic antibodies able to interact with the live trypomastigotes of Trypanosoma cruzi have been associated with both protection and active infection. There are T. cruzi isolates unable to induce lytic antibodies despite their capacity in eliciting agglutinins and precipitins for immunofluorescent labeling. The hosts spontaneous cure was ruled out as being responsible for negative results. The test performed either at 4 C or in the presence of sodium azide proved that negative lytic assays could not be attributed to capping phenomena. The classification of the parasites as T. cruzi was confirmed by their behavior in tissue culture and in the vector, as well as by the cross-protection exhibited by chronically infected mice against other lethal T. cruzi isolates. Cross-resistance achieved by these mice also suggests that the hosts protection during chronic infection is independent of the lytic antibody titer.

Induction of macrophage activation and opsonizing antibodies by Trypanosoma cruzi subpopulations

Summary Macrophage activation and production of opsonizing antibodies were studied in mice either infected with a lethal and reticulotropic Trypanosoma cruzi strain, RA, or with a non lethal and myotropic strain, CA‐I, as well as with a clone, K98 (derived from CA‐I), similar to the parental strain. Measurement of macrophage respiratory burst by chemiluminiscence disclosed that T. cruzi infection induced an enhancement of the respiratory burst, no matter the parasite subpopulation employed. But, while in mice surviving RA infection the respiratory burst was higher than during the acute period and parasitaemia was efficiently controlled, in mice infected with K98 enhanced respiratory burst coexisted with measurable levels of parasitaemia either at acute or chronic infection periods. Macrophage activation was also proved by enhanced trypanocidal activity in macrophages derived from mice infected with any of the parasite subpopulations. Sera from RA mice opsonized and lysed T. cruzi bloodstream forms efficiently, whereas sera from CA‐I or K98 mice neither lysed nor opsonized this parasite stage. All three subpopulations assayed here showed IgG bound to their membranes in vivo and similar capping kinetics, but only antibodies bound to RA parasites invariably triggered lysis. Therefore, the role played by macrophage activation in resistance and control of Pm levels is related to some features of each T. cruzi subpopulation, such as its capacity to invade macrophages and to elicit opsonizing antibodies.

In vivo macrophage function in experimental infection with Trypanosoma cruzi subpopulations

The macrophage function was investigated in mice infected with Trypanosoma cruzi. Two subpopulations of the parasite were utilized, RA and K98. Strain RA is efficiently internalized by macrophages and is lethal for mice, and clone K98 is poorly phagocytosed by macrophages and is not lethal. Treatment with silica enhanced parasitemia and mortality in mice infected with both parasite subpopulations. Parasitemia kinetics, however, were affected only in mice infected with RA, which suggests that macrophage effector mechanisms may play a more relevant role in this experimental group than in mice infected with K98. Resistance to Salmonella typhimurium infection and bactericidal activity of macrophages depended upon the T. cruzi subpopulation utilized and the infection period. Infection with K98 induced only a trend towards enhanced resistance to bacterial challenge during both the acute and chronic phases, whereas a significantly enhanced bactericidal activity of spleen and liver phagocytes was observed. Mice acutely infected with RA showed significantly enhanced susceptibility to S. typhimurium infection and lower bactericidal activity. Mice surviving infection with this aggressive strain, however, showed significantly enhanced resistance and bactericidal activities. Mice acutely infected with the RA strain displayed a dissociation between macrophage capacities to control S. typhimurium and T. cruzi. A similar phenomenon was also observed in other parasitoses (schistosomiasis, African trypanosomiasis). This fact may be due to differences in the lethal mechanisms through which macrophages control these parasites and S. typhimurium.

Central role of extracellular signal-regulated kinase and Toll-like receptor 4 in IL-10 production in regulatory dendritic cells induced by Trypanosoma cruzi

Several Trypanosoma cruzi molecules that stimulate macrophages activity were described as Toll-like receptor 2 (TLR2) ligands. Besides, the models of dendritic cells (DC) are poorly characterised. We have previously demonstrated that live-trypomastigotes (Tp) plus lipopolysaccharide (LPS) induce DC with tolerogenic properties that produce high levels of interleukin (IL)-10 and an impaired capacity to induce lymphoproliferation. Here, we show that the regulatory phenotype was observed with heat-killed trypomastigotes (Tphk) stimulation, ruling out DC infection. T. cruzi induced a particular DC activation state increasing LPS-activation of extracellular regulated kinase (ERK) 1/2 and signal transducer and activator of transcription (STAT) 3. Inhibition of ERK down-regulated IL-10 production and restored DC stimulatory capacity, showing the importance of this pathway in the DC modulation. A recent work shows that signalling via TLR4 and TLR2 induces a synergism in anti-inflammatory cytokine production in murine DC. Upon TLR2 and TLR4 stimulation using Pam(3)Cys or LPS and Tphk in DC from TLR2 knock out (KO) or TLR4-mutant mice, we showed that high levels of IL-10 were independent of TLR2 but associated with TLR4 and NF-kappaB signallization. Although sialic acid has been described as a molecule responsible of DC inhibition, we determine that it is not associated with T. cruzi-IL-10 modulatory response. In conclusion, all these findings demonstrate a key role of ERK and TLR4 in association with NF-kappaB in IL-10 modulation induced by T. cruzi and suggest that this regulatory effect involves parasite-DC interactions not described yet.

T lymphocytes from T. cruzi-infected mice alter heart contractility: Participation of arachidonic acid metabolites

T lymphocytes from T. cruzi infected mice susceptible to the development of myocarditis altered the contractility of normal mouse atria in vitro. While lymphocytes obtained from normal mice had no effect, lymphocytes from T. cruzi-infected mice cultured with normal atria induced negative or positive inotropic effects depending upon the post-infection period; negative inotropism was induced by lymphocytes obtained from animals at 1 to 4 weeks post-infection, and positive inotropism was induced by lymphocytes taken at 7 to 14 weeks post-infection. These effects were mediated by soluble factors as evidenced by the ability of lymphocyte culture supernatants to alter contractility. Cell enrichment experiments indicated that T lymphocytes rather than B lymphocytes were responsible for these inotropic effects. Lyt(2+)-enriched T lymphocytes were found to be responsible for triggering the negative inotropic effect at 3 weeks post-infection when myocarditis was less intense, whereas Lyt1(+)-enriched T lymphocytes induced the positive inotropic effect at 8 weeks after T. cruzi infection when myocarditis was severe. Furthermore, inhibitors of the cyclooxygenase pathway of arachidonic acid metabolism blunted the negative inotropic effect while inhibitors of lipoxygenase pathway inhibited the positive inotropic effect. PGE2 was found to be spontaneously released by Lyt(2+)-enriched T cells obtained at 3 weeks post-infection while LTC4 was released by atria cultured in the presence of Lyt 1+ T cells obtained at 8 weeks post-infection. In conclusion, these findings suggest that infiltrating T lymphocytes may contribute to myocardial dysfunction during T. cruzi infection by releasing or inducing the release of harmful arachidonic acid metabolites such as PGE2 and LTC4 which alter normal cardiac function.

Characterization of Biomphalaria orbignyi, Biomphalaria peregrina and Biomphalaria oligoza by polymerase chain reaction and restriction enzyme digestion of the internal transcribed spacer region of the RNA ribosomal gene

The correct identification of Biomphalaria oligoza, B. orbignyi and B. peregrina species is difficult due to the morphological similarities among them. B. peregrina is widely distributed in South America and is considered a potential intermediate host of Schistosoma mansoni. We have reported the use of the polymerase chain reaction and restriction fragment length polymorphism analysis of the internal transcribed spacer region of the ribosomal DNA for the molecular identification of these snails. The snails were obtained from different localities of Argentina, Brazil and Uruguay. The restriction patterns obtained with MvaI enzyme presented the best profile to identify the three species. The profiles obtained with all enzymes were used to estimate genetic similarities among B. oligoza, B. peregrina and B. orbignyi. This is also the first report of B. orbignyi in Uruguay.