New Biomarker in Chagas Disease: Extracellular Vesicles Isolated from Peripheral Blood in Chronic Chagas Disease Patients Modulate the Human Immune Response

Abstract

Chagas disease, a neglected tropical disease (NTD) caused by the flagellated protozoan Trypanosoma cruzi (T. cruzi), is a major public health problem. It was initially restricted to Latin America, but it is now expanding globally. Host and pathogen interactions are crucial in the establishment of disease, and since 1970, it has been known that eukaryotic cells release extracellular vesicles (EVs), which in turn have an important role in intercellular communication in physiological and pathological conditions. Our study proposed to characterize and compare circulating EVs isolated from the plasma of chronic Chagas disease (CCD) patients and controls. For this, peripheral blood was collected from patients and controls, and mononuclear cells (PBMCs) were isolated and stimulated with parasite EVs, showing that patient cells released fewer EVs than control cells. Then, after plasma separation followed by EV total shedding enrichment, the samples were subjected to ultracentrifugation to isolate the circulating EVs, which then had their size and concentration characterized by nanoparticle tracking analysis (NTA). This showed that patients had a lower concentration of circulating EVs while there were no differences in size, corroborating the in vitro data. Additionally, circulating EVs were incubated with THP-1 cells (macrophages) that, after the interaction, had their supernatant analyzed by ELISA for cytokine detection. In relation to their ability to induce cytokine production, the CCD patient EVs were able to induce a differential production of IFN-γ and IL-17 in relation to controls, with differences being more evident in earlier/less severe stages of the disease. In summary, a decreased concentration of circulating EVs associated with differential activation of the immunological system in patients with CCD is related to parasite persistence and the establishment of chronic disease. It is also a potential biomarker for monitoring disease progression.

Figure 1

CCD patients' clinical data analysis. Frequency of patients by the stage of cardiac burden in relation to the NYHA functional classification.

Figure 2

Scanning electron microscopy (SEM) of CCD PBMCs releasing EVs after 24 and 48 h of parasite EV stimulus. 24 h control in (a, b), 48 h control in (e, f), 24 h parasite EV stimulus in (c, d), and 48 h parasite EV stimulus in (g, h).

Figure 3

Comparison of EV profiles from CCD and CTRL PBMCs after a 24 h incubation. Concentration/size distribution (top), mean size ± 95%CI (center) and mean concentration ± 95%CI (bottom) after (a) culture medium (^∗p = 0.0326 and ^∗∗∗∗p < 0.0001), (b) T. cruzi extract (^∗∗∗p = 0.0003 and ^∗∗∗∗p = 0.0002), or (c) T. cruzi EV stimuli (^∗∗∗∗p < 0.0001).

Figure 4

Comparison of circulating EV profiles in CCD patients in relation to CTRL. (a) Concentration (particles/mL) × size (nm) profile. (b) Frequency of EV concentration among different degrees of cardiac burden. (c) Frequency of EV size among different stages of cardiac burden.

Figure 5

Cytokine production by THP-1 cells (macrophages) quantified in the supernatant by ELISA after 24 h of stimulation with CCD or CTRL EVs. (^∗p = 0.0438 and ^∗∗∗p = 0.0002).

Figure 6

Cytokine production by THP-1 cells (macrophages) quantified in the supernatant by ELISA after 24 h of stimulation with CCD or CTRL EVs among patients grouped based on the clinical stage of the cardiac burden and controls. (a) IFN-γ concentration in pg/mL (^∗p = 0.0420, ^∗∗p = 0.0291, and ^∗∗∗p = 0.0118). (b) Frequency of IFN-γ concentration values. (c) IL-17 concentration in pg/mL. (d) Frequency of IL-17 concentration values.

Figure 7

Cytokine production by THP-1 cells (macrophages) quantified in the supernatant by ELISA after 24 h of stimulation with CCD or CTRL EVs among patients grouped based on NYHA functional classification and controls. (a) IFN-γ concentration in pg/mL (^∗∗∗∗p = 0.0001). (b) Frequency of IFN-γ concentration values (0: CTRL). (c) IL-17 concentration in pg/mL (^∗p = 0.0125). (d) Frequency of IL-17 concentration values (0: CTRL).

Figure 8

Correlation of age (X axis), cytokine production by THP-1 cells (macrophages) quantified in the supernatant by ELISA after 24 h of stimulation with CCD or CTRL EVs (Y axis) and sex (•: female; X: male). (a) IFN-γ (r = 0.34). (b) IL-17 (r = −0.15).