Increased Natural Killer (NK)-cell cytotoxicity and Trypanosoma cruzi-specific memory B cells in subjects with discordant serology for Chagas disease.

The presence of memory T cell specific for Trypanosoma cruzi in subjects with discordant serology for Chagas disease supports a cleared infection in these subjects. Using high-dimensional flow cytometry, ELISPOT assays and quantitative PCR, antibody-secreting cells and memory B cells specific for T. cruzi, total B-cell phenotypes, innate immune responses and parasite DNA were evaluated in serodiscordant, seropositive and seronegative subjects for T. cruzi infection. T. cruzi-specific memory B cells but no antibody-secreting cells specific for T. cruzi, increased proportion of nonclassical monocytes and increased levels of polyfunctional NK cells were found in serodiscordant compared with seropositive subjects. None of the serodiscordant subjects evaluated showed detectable parasite DNA, most of them did not show cardiac abnormalities and a group of them had had confirmed positive serology for Chagas disease. The unique immune profiles in serodiscordant subjects support that T. cruzi infection was cleared or profoundly controlled in these subjects.

Mixed T Helper1/T Helper2/T Cytotoxic Profile in Subjects with Chronic Chagas Disease with Hypersensitivity Reactions to Benznidazole.

Dermatitis is the most common adverse event during treatment with benznidazole in chronic Chagas disease and is probably mediated by T cells. A set of molecules representative of the different type IV hypersensitivity reactions was evaluated in the circulation and skin biopsies of Trypanosoma cruzi-infected subjects presenting dermatitis during benznidazole administration. Through cytometric bead assays and enzyme-linked immunosorbent assay capture techniques, the serum levels of cytokines, chemokines, proapoptotic molecules, and mediators of the activation and migration of eosinophils and T cells were measured in subjects infected with Trypanosoma cruzi who exhibited skin adverse events (n = 22) and compared with those without adverse events (n = 37) during benznidazole therapy. Serum levels of interleukin- 5 (IL-5), soluble Fas cell surface death receptor ligand (FAS-L), and interferon γ-induced protein (IP-10) significantly increased at 7 to 30 days posttreatment with benznidazole and decreased thereafter in subjects with dermatitis but not in those without dermatitis. Circulating eotaxin levels were lower in subjects with dermatitis than in those without. Two patterns emerged in the skin biopsies: a T helper 1/T cytotoxic profile and a T helper 2/T cytotoxic profile with the presence of CD4+ and CD8+ T cells. Increased low-density lipoprotein (LDL), glutamic-oxaloacetic transaminase (GOT), uremia, and T cell activation emerged as risk factors for the development of dermatitis during benznidazole administration. These results support a delayed-type hypersensitivity reaction to benznidazole, involving CD4+ and CD8+ T cells and eosinophils, and a mixed cytokine profile. This study provides new insights for better management of adverse drug reactions to benznidazole. IMPORTANCE This study identified the risk factors for the development of adverse reactions to benznidazole and identified a set molecule to monitor the appearance of these reactions. This knowledge might improve the safety of benznidazole administration.

Reduced Trypanosoma cruzi-specific humoral response and enhanced T cell immunity after treatment interruption with benznidazole in chronic Chagas disease.

BACKGROUND: Interruption of benznidazole therapy due to the appearance of adverse effects, which is presumed to lead to treatment failure, is a major drawback in the treatment of chronic Chagas disease. METHODS: Trypanosoma cruzi-specific humoral and T cell responses, T cell phenotype and parasite load were measured to compare the outcome in 33 subjects with chronic Chagas disease treated with an incomplete benznidazole regimen and 58 subjects treated with the complete regimen, during a median follow-up period of 48 months. RESULTS: Both treatment regimens induced a reduction in the T. cruzi-specific antibody levels and similar rates of treatment failure when evaluated using quantitative PCR. Regardless of the regimen, polyfunctional CD4+ T cells increased in the subjects, with successful treatment outcome defined as a decrease of T. cruzi-specific antibodies. Regardless of the serological outcome, naive and central memory T cells increased after both regimens. A decrease in CD4+ HLA-DR+ T cells was associated with successful treatment in both regimens. The cytokine profiles of subjects with successful treatment showed fewer inflammatory mediators than those of the untreated T. cruzi-infected subjects. High levels of T cells expressing IL-7 receptor and low levels of CD8+ T cells expressing the programmed cell death protein 1 at baseline were associated with successful treatment following benznidazole interruption. CONCLUSIONS: These findings challenge the notion that treatment failure is the sole potential outcome of an incomplete benznidazole regimen and support the need for further assessment of the treatment protocols for chronic Chagas disease.

Correction: Trypanosoma cruzi-specific IFN-γ-producing cells in chronic Chagas disease associate with a functional IL-7/IL-7R axis.

[This corrects the article DOI: 10.1371/journal.pntd.0006998.].

Trypanosoma cruzi-specific IFN-γ-producing cells in chronic Chagas disease associate with a functional IL-7/IL-7R axis.

BACKGROUND: The severity of cardiac disease in chronic Chagas disease patients is associated with different features of T-cell exhaustion. Here, we assessed whether the ability of T cells to secrete IFN-γ in response to T. cruzi was linked to disruption in immune homeostasis and inflammation in patients with chronic Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: PBMCs from chronic Chagas disease patients and uninfected controls were examined for frequencies of T. cruzi-responsive IFN-γ-producing cells by ELISPOT and cellular expression and function of IL-7R using flow cytometry. Serum levels of IL-7, IL-21, IL-27, soluble IL-7R, and inflammatory cytokines were also evaluated by ELISA or CBA techniques. Patients possessing T. cruzi-specific IFN-γ-producing cells (i.e. IFN-γ producers) had higher levels of memory T cells capable of modulating the alpha chain of IL-7R and an efficient response to IL-7 compared to that in patients lacking (i.e. IFN-γ nonproducers) parasite-specific T-cell responses. IFN-γ producers also showed low levels of soluble IL-7R, high basal expression of Bcl-2 in T cells and low basal frequencies of activated CD25+ T cells. Modulation of IL-7R was inversely associated with serum IL-6 levels and positively associated with serum IL-8 levels. Circulating IL-21 and IL-27 levels were not associated with the frequency of IFN-γ producing cells but were reduced in less severe clinical forms of the disease. In vitro stimulation of PBMCs with IL-7 or IL-27 enhanced IFN-γ production in IFN-γ producers but not in IFN-γ nonproducers. CONCLUSIONS/SIGNIFICANCE: Alterations of the IL-7/IL-7R axis and in the levels of inflammatory cytokines were linked to impaired T. cruzi-specific IFN-γ production. These alterations might be responsible of the process of immune exhaustion observed in chronic Chagas disease.

Distinct monocyte subset phenotypes in patients with different clinical forms of chronic Chagas disease and seronegative dilated cardiomyopathy.

BACKGROUND: Chronic infection with Trypanosoma cruzi leads to a constant stimulation of the host immune system. Monocytes, which are recruited in response to inflammatory signals, are divided into classical CD14hiCD16-, non-classical CD14loCD16+ and intermediate CD14hiCD16+ subsets. In this study, we evaluated the frequencies of monocyte subsets in the different clinical stages of chronic Chagas disease in comparison with the monocyte profile of seronegative heart failure subjects and seronegative healthy controls. The effect of the anti-parasite drug therapy benznidazole on monocyte subsets was also explored. METHODOLOGY/PRINCIPAL FINDINGS: The frequencies of the different monocyte subsets and their phenotypes were measured by flow cytometry. Trypanosoma cruzi-specific antibodies were quantified by conventional serological tests. T. cruzi-infected subjects with mild or no signs of cardiac disease and patients suffering from dilated cardiomyopathy unrelated to T. cruzi infection showed increased levels of non-classical CD14loCD16+ monocytes compared with healthy controls. In contrast, the monocyte profile in T. cruzi-infected subjects with severe cardiomyopathy was skewed towards the classical and intermediate subsets. After benznidazole treatment, non-classical monocytes CD14loCD16+ decreased while classical monocytes CD14hiCD16-increased. CONCLUSIONS/SIGNIFICANCE: The different clinical stages of chronic Chagas disease display distinct monocyte profiles that are restored after anti-parasite drug therapy. T. cruzi-infected subjects with severe cardiac disease displayed a profile of monocytes subsets suggestive of a more pronounced inflammatory environment compared with subjects suffering from heart failure not related to T. cruzi infection, supporting that parasite persistence might also alter cell components of the innate immune system.

Distinct Treatment Outcomes of Antiparasitic Therapy in Trypanosoma cruzi-Infected Children Is Associated With Early Changes in Cytokines, Chemokines, and T-Cell Phenotypes.

Background: In contrast to adults, Trypanosoma cruzi-infected children have more broadly functional Trypanosoma cruzi-specific T cells, and the total T-cell compartment exhibits fewer signs of immune exhaustion. However, not much is known about the link between immunocompetence and the treatment efficacy for human Chagas disease. Methods: Using cytokine enzyme-linked immunosorbent spot (ELISPOT) polychromatic flow cytometry, cytometric bead assay, multiplex serological assays and quantitative PCR, we evaluated T. cruzi-specific T-cell and antibody immune responses, T-cell phenotypes and parasitemia in children in the early chronic phase of Chagas disease undergoing anti-Trypanosoma cruzi treatment. Results: Treatment with benznidazole or nifurtimox induced a decline in T. cruzi-specific IFN-γ- and IL-2-producing cells and proinflammatory cytokines and chemokines. T-cell responses became detectable after therapy in children bearing T-cell responses under background levels prior to treatment. The total frequencies of effector, activated and antigen-experienced T cells also decreased following anti-T. cruzi therapy, along with an increase in T cells expressing the receptor of the homeostatic cytokine IL-7. Posttreatment changes in several of these markers distinguished children with a declining serologic response suggestive of successful treatment from those with sustained serological responses in a 5-year follow-up study. A multivariate analysis demonstrated that lower frequency of CD4+CD45RA-CCR7-CD62L- T cells prior to drug therapy was an independent indicator of successful treatment. Conclusions: These findings further validate the usefulness of alternative metrics to monitor treatment outcomes. Distinct qualitative and quantitative characteristics of T cells prior to drug therapy may be linked to treatment efficacy.

The Significance of Discordant Serology in Chagas Disease: Enhanced T-Cell Immunity to Trypanosoma cruzi in Serodiscordant Subjects.

BACKGROUND: Subjects are considered infected with Trypanosoma cruzi when tested positive by at least two out of three serological tests, whereas a positive result in only one of up to three tests is termed "serodiscordant" (SD). Assessment of parasite-specific T-cell responses may help discriminate the uninfected from infected individuals among SD subjects. METHODS: Peripheral blood mononuclear cells from SD and seropositive (SP) subjects, who were born in areas endemic for T. cruzi infection but living in Buenos Aires city, Argentina, at the time of the study, and seronegative unexposed subjects were included for analysis. The function and phenotype of T cells were assessed by interferon-γ (IFN-γ) and interleukin (IL)-2 enzyme-linked immunospot assay and multiparameter flow cytometry. T. cruzi-specific antibodies were quantified by conventional serology and a multiplex assay format. RESULTS: SD subjects exhibited immunity cell responses to T. cruzi but in contrast to SP subjects, T cells in SD subjects more often display the simultaneous production of IFN-γ and IL-2 in response to T. cruzi antigens and have a resting phenotype. SD individuals also have higher IFN-γ spot counts, polyfunctional CD4+ T-cells enriched in IL-2 secreting cells and low levels of antibodies specific for a set of T. cruzi-derived recombinant proteins compared with the SP group. Long-term follow-up of SD individuals confirmed that humoral and T-cell responses fluctuate but are sustained over time in these subjects. T cells in SD subjects for T. cruzi infection did not recognize Leishmania antigens. CONCLUSION: Both T-cell and humoral responses in most subjects assessed by conventional tests as SD for T. cruzi infection indicate prior exposure to infection and the establishment of immunological memory suggestive of a resolved infection.