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.

Isolation and molecular characterization of circulating extracellular vesicles from blood of chronic Chagas disease patients

“…In the field of Chagas disease and EVs, most scientific works has focused on the identification of new molecules from vesicles released by different stages of T. cruzi parasites cultured in vitro (Bautista‐López et al 2017; Bayer‐Santos et al, 2013; Ribeiro et al, 2018). Only two papers have reported analyses on the purification and characterization of circulating EVs, in blood from patients with CCD, using SEC (Jung et al, 2012) or ultracentrifugation for purifying EVs (Madeira et al, 2021) as stand‐alone methodologies. In the present study and following recent recommendations (Coumans et al, 2017; Thery et al, 2018), we have combined ultracentrifugation followed by SEC to purify circulating EVs from CCD patients.…”

“…Three milliliters of plasma or serum were centrifuged for 18 h at 100,000 g (Thermo Scientific™ Sorvall™ WX100 Ultra Centrifuge) using a fixed angle rotor (Thermo Scientific™ T‐8100 Fixed Angle Rotor). The supernatant was discarded, and the pellet was resuspended in 100 µl of 0.45 µm filtered phosphate‐buffered saline (PBS), as described (Madeira et al, 2021).…”

“…The presence of parasite is hardly detected in chronic patients. Only two studies have been performed to assess circulating EVs from patients with CCD: One concerning a single patient with reactivation after a heart transplant (Cortes‐Serra et al, 2020) and another that assessed immune responses and disease stage stratification but did not focus on the isolation method per se (Madeira et al, 2021). Therefore, the aim of this study is to describe the effect of different anticoagulants and the combination of two purification methods for isolation and characterization of circulating EVs from blood of CCD patients according to current ISEV guidelines (Madeira et al, 2021; Théry et al, 2018).…”

Impact of the Extracellular Vesicles Derived From Trypanosoma cruzi: A Paradox in Host Response and Lipid Metabolism Modulation

“…Recently, Madeira et al, 2021, observed a lower concentration of circulating EVs associated with differential activation of the immunological system in patients with chronic Chagas disease, with increased production of IFN-g, when compared with uninfected healthy controls (Madeira et al, 2021). This data was associated with parasite persistence suggesting that the EVs can be potential candidates as biomarkers during the course of Chagas disease.…”

Extracellular Vesicles: Potential Role in Remote Signaling and Inflammation in Trypanosoma cruzi-Triggered Disease

“…Circulating EVs of chronic CD patients are similar in size but have lower concentrations than those of healthy individuals. Additionally, stratification of CD patients revealed that a reduction in EV concentration is associated with the severity of cardiac clinical parameters (Madeira et al, 2021). These differences may be related to differential release or retention of EVs in the tissues during disease progression, which may be a marker or contributor to CD pathogenesis.…”