Evaluation of the immune response against Trypanosoma cruzi cytosolic tryparedoxin peroxidase in human natural infection

Abstract

Trypanosoma cruzi, the aetiological agent of Chagas disease, has a highly efficient detoxification system to deal with the oxidative burst imposed by its host. One of the antioxidant enzymes involved is the cytosolic tryparedoxin peroxidase (c-TXNPx), which catalyses the reduction to hydrogen peroxide, small-chain organic hydroperoxides and peroxynitrite. This enzyme is present in all parasite stages, and its overexpression renders parasites more resistant to the oxidative defences of macrophages, favouring parasite survival. This work addressed the study of the specific humoral and cellular immune response triggered by c-TXNPx in human natural infection. Thus, sera and peripheral blood mononuclear cells (PBMC) were collected from chronically infected asymptomatic and cardiac patients, and non-infected individuals. Results showed that levels of IgG antibodies against c-TXNPx were low in sera from individuals across all groups. B-cell epitope prediction limited immunogenicity to a few, small regions on the c-TXNPx sequence. At a cellular level, PBMC from asymptomatic and cardiac patients proliferated and secreted interferon-γ after c-TXNPx stimulation, compared with mock control. However, only proliferation was higher in asymptomatic patients compared with cardiac and non-infected individuals. Furthermore, asymptomatic patients showed an enhanced frequency of CD19⁺ CD69⁺ cells upon exposure to c-TXNPx. Overall, our results show that c-TXNPx fails to induce a strong immune response in natural infection, being measurable only in those patients without any clinical symptoms. The low impact of c-TXNPx in the human immune response could be strategic for parasite survival, as it keeps this crucial antioxidant enzyme activity safe from the mechanisms of adaptive immune response.

Keywords: B-cell epitope prediction; T-cell and B-cell response; chronic Chagas disease; peroxiredoxin.

Figure 1

Humoral response against cytosolic tryparedoxin peroxidase (c‐TXNPx) in chronic Chagas disease patients. Anti‐c‐TXNPx (a) and anti‐Trypanosoma cruzi (b) antibody titration was carried out in sera from patients with chronic asymptomatic (AS; n = 14) and chronic cardiac (CCC; n = 17) Chagas disease, and non‐infected individuals (NI; n = 15) by ELISA. The titre of antibodies from each individual was expressed as the sera dilution factor that corresponds to the IC ₅₀ value of the adjusted curve, which was calculated by non‐linear regression analysis. Each dot corresponds to the value from a single individual, and the horizontal line shows the median value for each group. Statistical analysis was performed using the non‐parametric Kruskal–Wallis test. Statistically significant differences are indicated (***P < 0·001).

Figure 2

B‐cell epitope prediction within cytosolic tryparedoxin peroxidase (c‐TXNPx) protein sequence. (a) BepiPred linear B‐cell epitope prediction. The x‐axis shows the amino acid position in c‐TXNPx sequence, and the y‐axis shows the score value for each residue. The horizontal red line indicates the threshold score value of 0·53, from which positive epitopes are considered. (b) DiscoTope conformational B‐cell epitope prediction. The x‐axis shows the amino acid position in c‐TXNPx sequence, and the y‐axis shows the score value for each residue. The horizontal red line indicates the threshold value of −3·7 (default setting), from which positive epitopes are considered. (c) Graphical three‐dimensional representation of predicted epitopes within c‐TXNPx structure. Linear (red) and conformational (blue) epitopes and regions of overlapping (yellow) are specified.

Figure 3

Cellular response of peripheral blood mononuclear cells (PBMC) from chronic Chagas disease patients triggered by cytosolic tryparedoxin peroxidase (c‐TXNPx): 2 × 10⁵ PBMC/well from asymptomatic (AS; n = 11) and cardiac (CCC; n = 11) Chagas patients and non‐infected donors (NI; n = 8) were incubated with Trypanosoma cruzi lysate, recombinant c‐TXNPx and no‐antigen (culture medium only) or mock as controls for 5 days in complete RPMI medium. Each condition was performed in triplicate. (a, c) Cells were pulsed with [³H]thymidine for the last 18 hr of incubation, and PBMC proliferation was determined by [³H]thymidine uptake. (b, d) Culture supernatants were collected after 5 days of stimulation with c‐TXNPx, mock or T. cruzi lysate, and interferon‐γ (IFN‐γ) secretion was quantified by ELISA. Box and whisker plots (min to max) show the stimulation index (SI), calculated for each readout as the mean value for triplicate stimulated cultures of each individual divided by the mean value of triplicate non‐stimulated cultures (medium only). Each dot represents data from a single individual. Statistical analysis was performed using linear mixed‐effects models fitted by maximum likelihood and Tukey honest significant difference for post hoc comparisons. Statistically significant differences between groups are indicated (***P < 0·001, **P < 0·01, *P < 0·05).

Figure 4

Expression of CD69 and HLA‐DR activation marker on CD19⁺, CD4⁺ and CD8⁺ lymphocytes from patients with chronic Chagas disease stimulated with cytosolic tryparedoxin peroxidase (c‐TXNPx). Peripheral blood mononculear cells (PBMC) (1 × 10⁶ cells/well) from asymptomatic (AS; n = 5) and cardiac (CCC; n = 5) Chagas patients and non‐infected donors (NI; n = 7) were incubated with Trypanosoma cruzi lysate, recombinant c‐TXNPx and no‐antigen (culture medium only) or mock as controls for 5 days in complete RPMI medium in a 48‐well plate. After the incubation period, cells were harvested, washed with phosphate‐buffered saline and stained with fluorescence‐labelled monoclonal antibodies as detailed in Table 2. One hundred thousand events within lymphocyte population were acquired in a FACSCanto II Flow cytometer. Dead cells were excluded by forward‐ versus side‐scatter (FSC/SSC) gating. Doublets were excluded by FSC‐A versus FSC‐H. Singlet cells were then determined according to the gating strategy detailed in the Supplementary material (Fig. S1). Fold change of CD19⁺ CD69⁺ (a and g, CD4⁺ CD69⁺ (b and h), CD8⁺ CD69⁺ (c and i), CD19⁺ HLA‐DR ⁺ (d and j), CD4⁺ HLA‐DR ⁺ (e and k), CD8⁺ HLA‐DR ⁺ (f and l) upon stimulation with c‐TXNPx or mock and T. cruzi lysate, respectively. Statistical analysis was performed using linear mixed‐effects models fitted by maximum likelihood and Tukey honest significant difference for post hoc comparisons. Statistically significant differences between groups are indicated (*P < 0·05).