African Trypanosomiasis: Extracellular Vesicles Shed by Trypanosoma Brucei Brucei Manipulate Host Mononuclear Cells

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Article African Trypanosomiasis: Extracellular Vesicles Shed by Trypanosoma brucei brucei Manipulate Host Mononuclear Cells

Tatiana Dias-Guerreiro 1,†, Joana Palma-Marques 1,†, Patrícia Mourata-Gonçalves 1, Graça Alexandre-Pires 2 , Ana Valério-Bolas 1 , Áurea Gabriel 1 , Telmo Nunes 3, Wilson Antunes 4, Isabel Pereira da Fonseca 2, Marcelo Sousa-Silva 1,5 and Gabriela Santos-Gomes 1,*

1 Global Health and Tropical Medicine (GHTM), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), 1349-008 Lisboa, Portugal; [email protected] (T.D.-G.); [email protected] (J.P.-M.); [email protected] (P.M.-G.); [email protected] (A.V.-B.); [email protected] (Á.G.); [email protected] (M.S.-S.) 2 Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477 Lisboa, Portugal; [email protected] (G.A.-P.); [email protected] (I.P.d.F.) 3 Microscopy Center, Faculty of Sciences, University of Lisbon, Campo Grande, 1749-016 Lisboa, Portugal; [email protected] 4 Unidade Militar Laboratorial de Defesa Biológica e Química (UMLDBQ), Laboratório de Imagem Nano-Morfológica e Espectroscopia de Raios-X, 1100-471 Lisboa, Portugal; [email protected] 5 Centro de Ciências da Saúde, Departamento de Analises Clínicas e Toxicológicas, Universidade Federal do Rio Grande do Norte, Natal 59078-970, Brazil Citation: Dias-Guerreiro, T.; * Correspondence: [email protected]; Tel.: +351-21-365-26-00; Fax: +351-21-363-21-05 Palma-Marques, J.; † These two authors contributed equally to this study and are considered co-ﬁrst authors. Mourata-Gonçalves, P.; Alexandre-Pires, G.; Valério-Bolas, A.; Abstract: African trypanosomiasis or sleeping sickness is a zoonotic disease caused by Trypanosoma Gabriel, Á.; Nunes, T.; Antunes, W.; brucei, a protozoan parasite transmitted by Glossina spp. (tsetse ﬂy). Parasite introduction into mam- Fonseca, I.P.d.; Sousa-Silva, M.; et al. mal hosts triggers a succession of events, involving both innate and adaptive immunity. Macrophages African Trypanosomiasis: (MΦ) have a key role in innate defence since they are antigen-presenting cells and have a micro- Extracellular Vesicles Shed by bicidal function essential for trypanosome clearance. Adaptive immune defence is carried out by Trypanosoma brucei brucei Manipulate lymphocytes, especially by T cells that promote an integrated immune response. Like mammal cells, Host Mononuclear Cells. Biomedicines T. b. brucei parasites release extracellular vesicles (TbEVs), which carry macromolecules that can be 2021, 9, 1056. https://doi.org/ biomedicines9081056 transferred to host cells, transmitting biological information able to manipulate cell immune response. However, the exact role of TbEVs in host immune response remains poorly understood. Thus, the cur-

Academic Editor: Maria Pereira rent study examined the effect elicited by TbEVs on MΦ and T lymphocytes. A combined approach of microscopy, nanoparticle tracking analysis, multiparametric flow cytometry, colourimetric assays Received: 16 July 2021 and detailed statistical analyses were used to evaluate the influence of TbEVs in mouse mononuclear Accepted: 16 August 2021 cells. It was shown that TbEVs can establish direct communication with cells of innate and adaptative Published: 20 August 2021 immunity. TbEVs induce the differentiation of both M1- and M2-MΦ and elicit the expansion of MHCI+, MHCII+ and MHCI+MHCII+ MΦ subpopulations. In T lymphocytes, TbEVs drive the Publisher’s Note: MDPI stays neutral overexpression of cell-surface CD3 and the nuclear factor FoxP3, which lead to the differentiation of with regard to jurisdictional claims in regulatory CD4+ and CD8+ T cells. Moreover, this study indicates that T. b. brucei and TbEVs seem published maps and institutional affil- to display opposite but complementary effects in the host, establishing a balance between parasite iations. growth and controlled immune response, at least during the early phase of infection.

Keywords: African trypanosomiasis; Trypanosoma brucei brucei; exosomes; macrophages; T lymphocytes; regulatory T cells Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and 1. Introduction conditions of the Creative Commons Attribution (CC BY) license (https:// African trypanosomiasis (AT), also known as sleeping sickness in humans and Nagana creativecommons.org/licenses/by/ in cattle, is a vector-borne disease caused by an extracellular kinetoplastida parasite of 4.0/). the Trypanosomatidae family, genus Trypanosoma, and species Trypanosoma brucei, which

Biomedicines 2021, 9, 1056. https://doi.org/10.3390/biomedicines9081056 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 1056 2 of 17

is transmitted by the hematophagous dipteran Glossina spp. (tsetse fly). This parasitosis is considered a neglected tropical disease restricted to the intertropical region of Africa following the geographical distribution of its vector. Parasite transmission to mammals occurs by the inoculation of metacyclic trypomastigotes forms during the insect blood meal. After being introduced into the host dermis, parasites start to replicate, giving origin to the initial lesion or the inoculation chancre [1,2]. After two or three weeks, the chancre tends to disappear, and the disease can evolve in two distinct successive phases [2]: Phase I or the hemolymphatic stage is characterized by successive waves of invasion of the blood and lymphatic system by trypanosomes, which causes intermittent fever [2,3] and phase II or meningoencephalitis stage, where the typical symptoms of sleeping sickness (dementia, cachexia, coma, and death) may become evident [2–4]. Trypanosome parasites have a dense surface coat constituted by the variant surface glycoprotein (VSG). These immunogenic coats present at the cell surface as homodimers and anchored in the membrane through gly- cosylphosphatidylinositol constitute the pathogen-associated molecular patterns (PAMPs) that are recognized by pattern-recognition receptors (PPRs) of innate immunity [2,4]. The innate immune response includes macrophages (MΦ), which can engulf foreign antigens through endocytosis. When soluble parasite factors and VSG interacts with MΦ, they became classically activated (M1-MΦ) and synthesize reactive oxygen inter- mediates (ROI), produce nitric oxide (NO), and release proinflammatory cytokines, as is the case of tumor necrosis factor (TNF)-α and interleukin (IL)-6 (type I cytokines) [2,5]. To reduce inflammation, trypanosome blood forms release some components, such as Trypanosoma brucei-derived kinesin-heavy chain (TbKHC-1) and adenylate cyclase (ADC), which upregulate the IL-10 production that prevents TNF−α expression [6,7], leading to the differentiation of alternative activate MΦ (M2-MΦ) and production of anti-inflammatory cytokines [5]. The inflammatory response is critical for parasite control in the early stage of infection [5] and the switch from M1-MΦ to M2-MΦ seems to occur four weeks after infection when the patient is in the late stage of disease [8]. Together with dendritic cells, MΦ are antigen-presenting cells (APC), establishing a bridge with adaptive immunity. Parasite antigens complexed with class I molecules of major histocompatibility complex (MHCI) are presented to CD8+ T cells and parasite antigen bound to class II molecules of major histocompatibility (MHCII) complex are recognized by CD4+ T cells. Both CD4+ and CD8+ T cells are crucial in the orchestration of host adaptive immune response against T. b. brucei parasites. However, the successive peaks of parasitemia exhibiting specific VSG induce a continuous activation of T cell clones, which leads to cell exhaustion and then to immunosuppression, and consequently impaired parasite control [9]. Like mammal cells, trypanosomes also secrete extracellular vesicles (EV), which comprise nanovesicles that differ in size and carry parasite molecular components, such as proteins, lipids, and nucleic acids [10]. The parasite T. brucei releases EV (TbEVs) that seems to play a role in both parasite-parasite and parasite-host interactions, influencing host immune response [11]. Moreover, TbEVs appear to incorporate different flagellar proteins, acting like virulence factors, and be highly enriched in oligomers, such as tetraspanins (a broadly expressed superfamily of transmembrane glycoproteins), known to be involved in antigen presentation, T cell signalization and activation, and in MHCI and MHCII generation. Also, it was demonstrated that upon fusion with erythrocytes, these TbEVs are responsible for the removal of red cells from the host bloodstream [12]. For parasite survival in the mammal host and success in being transmitted to the insect vector, ensuring the completion of the T. brucei life cycle, the infected host must develop a balanced immune response able to prevent the host killing and allow parasite replication. Despite all the studies done to understand the immune mechanisms associated with this parasitic disease, the exact role played by TbEVs in host immune response is still evasive. Thus, this work explores the effect of TbEVs in the immune activation of mouse mononuclear cells. Biomedicines 2021, 9, 1056 3 of 17

2. Materials and Methods 2.1. Experimental Design To explore the effect of TbEVs on host immune response an experimental design comprising three main steps was established. The first step aims to examine the shape, size, and density of purified TbEVs using electron microscopy and nanoparticle tracking analysis. In the second step, the aim was to investigate the innate activity of MΦ when stimulated by TbEVs, including their microbicide and antigenic presentation potential, and the last goal step aims to assess the differentiation of T cell subsets related to effector and regulatory adaptive immune response. Mouse MΦ-like cells were exposed to T. b. brucei trypomastigotes and stimulated by TbEVS and BALB/c mice peripheral blood mononuclear cells (PBMC) were exposed to T. b. brucei trypomastigotes and stimulated by TbEVS and parasite antigen (Ag). Type I and type II MΦ activity was analyzed by NO and urea production using colourimetric assays. The potential of MΦ to present parasite antigens to lymphocytes was indirectly evaluated by the expression and density of surface molecules MHCI and MHCII by flow cytometry. T lymphocyte subsets were immunophenotyped by flow cytometry through surface expression and density of CD3 and CD25 molecules and intracellular expression of FoxP3. In parallel, resting cells, MΦ stimulate by phorbol myristate acetate (PMA) and PBMC stimulated by concanavalin A (ConA) were also evaluated. Furthermore, obtained data were subject to statistical procedures.

2.2. BALB/c Mice Six to eight-week-old male BALB/c Mus musculus mice were purchased from the Instituto Gulbenkian de Ciências (IGC, Lisbon, Portugal) and maintained in the IHMT animal facility, in sterile cabinets with sterile food and water ad libitum. Mice were used to recover Trypanosome blood forms and to isolate PBMC. The animals were handled according to the Portuguese National Authority for Animal Health (Ref. 0421/000/000/2020, 23 September 2020, DGAV—Direção Geral de Alimentação e Veterinária), in conformity with the institutional guidelines and the experiments performed in compliance with Por- tuguese law (Decree-Law 113/2013), EU requirements (2010/63/EU), and following the recommendations of the Federation of European Laboratory Animal Science Associations (FELASA).

2.3. Trypanosoma brucei brucei Parasites Peripheral blood of BALB/c mice infected with T. brucei brucei strain G.V.R. 35 was collected (Supplementary Videos S1 and S2) and treated with ammonium-chloride-potassium lysis buffer. Trypanosoma blood forms were then purified using diethyl aminoethyl (DEAE)- cellulose columns [13] and maintained in Schneider Drosophila medium (Sigma-Aldrich, Hamburg, Germany) supplemented with 10% (v/v) of heat-inactivated fetal bovine serum (hiFBS) free of extracellular vesicles (FBS-exofree, Thermo Fisher Scientific, Waltham, MA, USA) at 24 ◦C. Parasite morphology and motility were checked every day by direct microscopy and, parasite topography was evaluated by scanning electron microscopy (SEM, SEM-UR- LBDB Hitachi SU8010 High-Technologies Corporation, Ibaraki, Japan). TbEVs were purified from the supernatant of cultures exhibiting motile trypomastigotes.

2.4. T. b. brucei Extracellular Nanovesicles and Parasite Crude Antigen Trypomastigotes harvested from cultures by centrifugation at 2000× g for 10 min at 4 ◦C were used to produce parasite antigen (Ag), and TbEVs were obtained from culture supernatants. Parasites were washed twice in phosphate-buffered saline (PBS) 2 mM ethylenediaminetetraacetic acid (EDTA), resuspended in PBS, and then disrupted by eight freeze-thawing cycles ranging from −20 ◦C to room temperature. After centrifugation, protein content (mg·mL−1) was determined using a Nanodrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), and Ag was preserved at −20 ◦C. Supernatants of trypomastigote culture incubated for 48 h were filtered through 0.2 mm syringe filters Biomedicines 2021, 9, 1056 4 of 17

(VWR International, Radnor, PE, USA), and TbEVs were puriﬁed using Exosome Spin Columns (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Isolated TbEVs were examined by SEM, single particle size and concentration analyzed by Nanoparticle Tracking Analysis (NanoSight NTA 3.2), and protein content was estimated by spectrophotometry.

2.5. Morphology of T. b. brucei and Parasite Extracellular Vesicles Cultured T. b. brucei parasites deposited on glass slides were observed under an optical microscope to evaluate parasite motility and morphology. The topography of TbEVs and morphology of cultured-derived parasites were examined by SEM. Round glass coverslips were immerged into poly-D-lysine (Sigma-Aldrich, Burlington, MA, USA) overnight to increase adherence and later placed in a 24-well plate. Then parasites were allowed to adhere to the coverslips and were fixed with PBS 4% paraformalde- hyde (Merck, Rahway, NJ, USA) for 30 min at 4 ◦C. Coverslips were rinsed three times with distilled water, treated with 0.5% osmium tetroxide (Sigma-Aldrich), and washed again. Then, plates were incubated with a fixative solution of 1% tannic acid (Sigma-Aldrich) for 30 min. TbEVs were fixed to coverslips with 2.5% glutaraldehyde, 0.1 M sodium ca- codylate buffer, pH 7.4 for 2 h at 4 ◦C. Afterwards, both parasites- and TbEV-coverslips were washed and then dehydrated by sequential addition of 30%, 50%, 70%, 80%, and 90% ethanol for 5 min each. Coverslips were immersed in 100% ethanol and then treated with hexamethyldisilazane solvent (Sigma-Aldrich), coated with gold-palladium, mounted on stubs to be observed under an ultra-high resolution scanning electron microscope. The surface area and perimeter of TbEVs were quantified from acquired images using Image J software.

2.6. Macrophage Cell Line and Primary Mononuclear Cells PMBC were isolated from healthy (non-infected) BALB/c mice by density gradi- ent [14]. Brieﬂy, cells at Hystopaque-1077 (Sigma-Aldrich) and plasma interface were removed and washed three times in PBS by centrifugation at 370× g for 10 min at 4 ◦C. Then, the supernatant was discarded and the PMBC-containing pellet resuspended in PBS. Cell viability was evaluated using the trypan blue staining method and cell concentration estimated in a Neubauer-counting chamber by direct microscopy. A macrophage (MΦ)-like cell line (P388D1, ATCC, Bird Park, Hawaii, USA) previously isolated from a mouse lymphoma was expanded in RPMI-1640 (Lonza, Basel, Switzerland) supplemented with 10% (v/v) of hiFBS, 2 mM L-glutamine (Merck) (complete RPMI ◦ medium) at 37 C in a humidiﬁed atmosphere with 5% CO2.

2.7. Stimulation of Macrophages and PBMC by TbEVs MΦ (2 × 106 cells per well) and PBMC (1 × 105 cells per well) were plated in a sterile 96-well plate with complete RPMI medium and separately exposed to viable parasites (3 parasites per cell) and stimulated with TbEVs (10 µg·mL−1). PBMC also was stimulated by T. b. brucei soluble antigen (10 µg·mL−1). MΦ plates were incubated for 24 h and PBMC ◦ plates for 72 h at 37 C in a humidiﬁed atmosphere with 5% CO2. In parallel, unstimulated cells, used as the negative control, and PMA (Promega, Madison, WI, USA) stimulated MΦ and ConA (Sigma-Aldrich)-stimulated lymphocytes, used as the positive controls, were also incubated.

2.8. Nitric Oxide and Urea Production by Macrophages Supernatants of MΦ exposed to viable parasites, stimulated by TbEVs and PMA, and non-stimulate (resting MΦ) were collected and used for quantiﬁcation of urea by the commercial kit QuantiChrom™ Urea Assay Kit-DIUR-100 (BioAssay System, Hayward, − CA, USA) and the indirect measurement of NO levels through the detection of NO2 and − NO3 using the Nitrate/Nitrite Colorimetric Assay (Abnova, Walnut, CA, USA). Both assays were performed according to the manufacturer’s instructions. Biomedicines 2021, 9, 1056 5 of 17

2.9. Macrophage and Lymphocyte Immunophenotyping Cells (MΦ and PBMC) exposed to motile parasites and stimulated were harvested from plates and washed with PBS. MΦ were labelled with mouse anti-MHCI (H-2Kb) monoclonal antibody FITC directly conjugated and mouse anti-MHCII (I-A/I-E) monoclonal antibody PE directly conjugated (BioLegend, San Diego, CA, USA). PBMC were resuspended in PBS 0.5% hiFBS 2 mM EDTA and were added magnetic microbeads coated with mouse anti-CD8a (Ly-2) monoclonal antibody (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were incubated for 15 min at 4 ◦C protected from light and then washed. CD8+ cells were sorted by positive selection, using a MACS® system (Miltenyi Biotec) while CD8− cells were eluted. + − Sorted CD8 and CD8 cell fractions were washed with PBS 2% hiFBS 0.01% NaN3 and incubated for 30 min with mouse anti-CD3 monoclonal antibody FITC-directly conjugated and mouse anti-CD25 monoclonal antibody PerCP-cy5.5 directly conjugated (BioLe- gend). After two washes in PBS, cell suspensions were fixed with PBS 2% formaldehyde. Cells were then resuspended in permeabilization buffer (PBS 1% FBS, 0.1% NaN3, 0.5% Triton-X, pH 7.4–7.6) and incubated for 20 min, washed and labelled with mouse anti-FoxP3 monoclonal antibody PE directly conjugated (BioLegend). Cell acquisition was performed in a 4-colour flow cytometer (BD FACSCalibur, BD Biosciences, USA) and data were analyzed using Flowjo V10 (Tree Star Inc., Ashland, OR, USA). FSC-H vs. SSC-H gate was used to remove debris and pyknotic cells in the lower left quadrant of the plot as well as the large (off-scale) debris found in the upper right quadrant. Singlet gate was used to define the non-clumping cells based on pulse geometry FSC-H vs. FSC-A, eliminating the doublets. CD3+, CD25+, and FoxP3+ cell subsets were defined using fluorescence minus one control (FMOs). To validate the composition of magnetically separated cell fractions, samples of unprimed CD8+ and CD8− cell fractions were stained with the anti-CD3 monoclonal antibody and anti-CD4 monoclonal antibody PerCp directly conjugated (BioLegend) and evaluated by flow cytometry. In CD8+ cell fraction, less than 20% of cells presented a CD3+CD8− phenotype, indicating that this cell fraction mainly was constituted by CD8+ T cells and in the CD8− cell fraction ≈85% of the cells evidenced a CD3+CD4+ phenotype, which is consistent with the predominance of CD4+ T cells.

2.10. Statistical Analysis Data analysis was performed using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA). After verification by a Kolmogorov–Smirnov test that the data of the current study do not evidence a normal distribution, significant differences were determined using the non-parametric Wilcoxon matched-pair signed-rank test. A 5% (p < 0.05) significance level was used to evaluate statistical significance. The surface and perimeter of TbEVs are represented by violin plots (median, interquartile ranges and distribution). Cell results of at least three independent experiments evaluated in triplicate are expressed by whiskered box-plots, indicating the median, maximum and minimum values or by graph bars (mean and standard error). The relative importance of TbEVs in cell activity was assessed by the principal component analysis (PCA) of exploratory multivariate statistical analysis, using Past4.03 (Natural History Museum, University of Oslo, Oslo, Norway). PCA analysis organizes data in principal components, which can be visualized graphically with a minimal loss of information, making visible the differences between the activation of cells exposed to T. b. brucei parasites, TbEVs and Ag stimulated cells. K-means cluster analysis also was used for cluster validation.

3. Results 3.1. T. b. brucei Trypomastigote Forms Release Extracellular Vesicles Cultured-derived T. b. brucei parasites observed by direct microscopy and by scanning electronic microscopy showed an elongated body, a ﬂagellum (Figure1A), and an undulating membrane (Figure1B), which are recognised as the morphological characteristic of the Biomedicines 2021, 9, x FOR PEER REVIEW 6 of 18

cell activity was assessed by the principal component analysis (PCA) of exploratory Biomedicines 2021, 9, x FOR PEER REVIEW 6 of 18 multivariate statistical analysis, using Past4.03 (Natural History Museum, University of Oslo, Oslo, Norway). PCA analysis organizes data in principal components, which can be visualized graphically with a minimal loss of information, making visible the differences betweencell activity the activation was assessed of cells by exposed the principal to T. b. componentbrucei parasites, analysis TbEVs (PCA) and Agof stimulatedexploratory cells.multivariate K-means statistical cluster analysis analysis, also using was usedPast4.03 for cluster(Natural validation. History Museum, University of Oslo, Oslo, Norway). PCA analysis organizes data in principal components, which can be 3.visualized Results graphically with a minimal loss of information, making visible the differences 3.1.between T. b. brucei the activation Trypomastigote of cells Forms exposed Release to T.Extracellular b. brucei parasites, Vesicles TbEVs and Ag stimulated cells.Cultured-derived K-means cluster analysisT. b. brucei also wasparasites used forobserved cluster validation.by direct microscopy and by scanning electronic microscopy showed an elongated body, a flagellum (Figure 1A), and an3. Resultsundulating membrane (Figure 1B), which are recognised as the morphological characteristic3.1. T. b. brucei of Trypomastigote the trypomastigote Forms Releaseform. Besides, Extracellular cultured-derived Vesicles parasite exhibited Biomedicines 2021, 9, 1056 6 of 17 nanovesiclesCultured-derived that seems toT. budb. bruceifrom theparasites cell surface observed and flagellum by direct (Figure microscopy 2A,B). and by scanning electronic microscopy showed an elongated body, a flagellum (Figure 1A), and an undulating membrane (Figure 1B), which are recognised as the morphological trypomastigotecharacteristic of form. the trypomastigote Besides, cultured-derived form. Besides, parasite cultured-derived exhibited nanovesicles parasite that exhibited seems tonanovesicles bud from the that cell seems surface to bud and from ﬂagellum the cell (Figure surface2A,B). and flagellum (Figure 2A,B).

Figure 1. Trypomastigote forms of Trypanosoma brucei brucei. Culture-derived parasites were morphologically evaluated under an inverted light microscope (A, size bar—10 μm, ×400 magnification) and after metalization topographically analyzed by SEM (B). Image B was artificially coloured using the GIMP2.10.0 software. Figure 1.1. Trypomastigote forms ofTrypanosoma Trypanosoma brucei brucei brucei brucei. Culture-derived parasites were SuspensionTrypomastigote of purified forms nanovesicles of derived from trypomastigote. Culture-derived forms parasites of T. were b. brucei mor- phologicallymorphologically evaluated evaluated under anunder inverted an inverted light microscope light microscope (A, size bar—10 (A, µsizem, ×bar—10400 magniﬁcation) μm, ×400 evidenced a mean protein concentration of 5.107 μg·mL−1 (≈35.28) and NTA analysis andmagnification) after metalization and after topographically metalization topographically analyzed by SEM analyzed (B). Image by SEM B was (B artiﬁcially). Image B colouredwas artificially using revealedcoloured thatusing the the size GIMP2.10.0 of TbEVs software. was within a range of 50 nm and 350 nm. the GIMP2.10.0 software. Suspension of purified nanovesicles derived from trypomastigote forms of T. b. brucei evidenced a mean protein concentration of 5.107 μg·mL−1 (≈35.28) and NTA analysis revealed that the size of TbEVs was within a range of 50 nm and 350 nm.

FigureFigure 2. 2. ReleaseRelease of of extracellular extracellular nanovesicles nanovesicles by by T.T. b. b. brucei brucei andand size size and and concentration concentration of of purified purified TbEVs.TbEVs. RepresentativeRepresentative images images (A ,B()A of,B culture-derived) of culture-derived parasites parasites shedding nanovesiclesshedding nanovesicles (arrowheads) (arrowheads)were acquired were by SEM. acquired TbEVs by harvested SEM. TbEVs from harvested supernatants from of supernatantsT. b. brucei cultures of T. b. were brucei analyzed cultures by wereNTA analyzed (C). Mean by values NTA ( (blue)C). Mean + SD values (red) are(blue) represented + SD (red) by ar averagee represented finite trackby average length finite adjustment track length(FTLA) adjustment concentration/size (FTLA) concentration/size graph. graph. Figure 2. Release of extracellular nanovesicles by T. b. brucei and size and concentration of purified TbEVs. Representative images (A,B) of culture-derived parasites shedding nanovesicles Suspension of purified nanovesicles derived from trypomastigote forms of T. b. brucei (arrowheads) were acquired by SEM. TbEVs harvested from supernatants of T. b. brucei cultures evidenced a mean protein concentration of 5.107 µg·mL−1 (≈35.28) and NTA analysis were analyzed by NTA (C). Mean values (blue) + SD (red) are represented by average finite track revealedlength adjustment that the (FTLA) size of concentration/size TbEVs was within graph. a range of 50 nm and 350 nm. Two peaks of higher concentration of TbEVs with sizes around 100 and 170 nm were detected, while TbEVs bigger than 200 nm seem to be rare (Figure2C). The topographic analysis place in evidence vesicles with a spherical shape and a smooth surface (Figure3A ), with perimeter ranging between 335 and 3311 nm (mean 1634 nm ± 94.07) (Figure3B) and surface area between 9 and 872 nm (mean 255.2 nm ± 25.40) (Figure3C). These data also indicate that cultured-derived T. b. brucei parasites release two main classes of TbEVs: small TbEVs (with perimeter and surface area around 65 nm and 905 nm, respectively) and large TbEVs (with perimeter and surface area around 346 nm and 2088 nm, respectively). Biomedicines 2021, 9, x FOR PEER REVIEW 7 of 18

Two peaks of higher concentration of TbEVs with sizes around 100 and 170 nm were detected, while TbEVs bigger than 200 nm seem to be rare (Figure 2C). The topographic analysis place in evidence vesicles with a spherical shape and a smooth surface (Figure 3A), with perimeter ranging between 335 and 3311 nm (mean 1634 nm ± 94.07) (Figure 3B) and surface area between 9 and 872 nm (mean 255.2 nm ± 25.40) (Figure 3C). These data also indicate that cultured-derived T. b. brucei parasites release two main classes of TbEVs: small TbEVs (with perimeter and surface area around 65 nm and 905 nm, respectively) Biomedicines 2021, 9, 1056 and large TbEVs (with perimeter and surface area around 346 nm and 20887 of nm, 17 respectively).

FigureFigure 3. 3.Topography Topography and and dimensions dimensions of ofT. T. b. b. brucei bruceiextracellular extracellular nanovesicles. nanovesicles. TbEVs TbEVs puriﬁed purified from from culture-derived culture-derived parasitesparasites were were analyzed analyzed by SEMby SEM and and images images were were acquired. acquired. A representative A representa imagetive image of TbEVs of TbEVs (A) artiﬁcially (A) artificially coloured, coloured, using theusing GIMP2.10.0 the GIMP2.10.0 software software is shown. is shown. Perimeter Perimeter (B) and (B surface) and surface area (C area) of ( TbEVsC) of TbEVs estimated estimated by Image by Image J are representedJ are represented by violinby violin plots, plots, indicating indicating median median (thick (thick blue bars),blue ba interquartilers), interquartile range range (blue (b dots)lue dots) and distribution. and distribution.

3.2.3.2. T. T. b. b. brucei brucei and and TbEVs TbEVs Induced Induced Mouse Mouse M MΦΦto to Produce Produce NO NO and and Urea Urea Biomedicines 2021, 9, x FOR PEER REVIEW 8 of 18 MMΦΦactivity activity after after stimulation stimulation with with TbEVs TbEVs was was examined examined by by the the ability ability of of cells cells to to metabolizemetabolize arginine arginine (Figure (Figure4). 4). Resting-MΦ incubated for 24 h (negative control) exhibited a residual NO synthesis (Figure 4B). However, after PMA-stimulation (positive control), cells revealed a significant NO increase (p = 0.0078), thus confirming the viability and functionality of these cells that could transform arginase into NO through the enzymatic activity of NOS2. MΦ stimulated by TbEVs (p = 0.0313) or T. b. brucei parasites (p = 0.0078) (Figure 4A) showed significant increases in NO levels when compared with resting MΦ. MΦ exposed to parasites exhibited the higher NO production that was significantly different from TbEVs exposed-MΦ (p = 0.0313). In resting-MΦ were detected low levels of urea (Figure 4C). However, PMA stimulated cells showed a significant increase in urea production (p = 0.0156), indicating that these cells can convert arginine into urea through arginase enzymatic activity. MΦ stimulated by TbEVs or exposed to parasites also exhibited significant high levels of urea

when compared with resting-MΦ (p = 0.0313). FigureFigure 4. 4.Production Production of of nitric nitric oxide oxideAlthough andand ureaurea byby both macrophages T. b. brucei exposed exposed parasites to to TbEVs. TbEVs. and MTbEVs MΦ Φ(arrows)(arrows) promote exposed exposed mouse to trypomastigote to trypomastigoteMΦ to produce NO forms were observed under an inverted light microscope (A, size bar—20 μm,µ ×100× magnification). NO (B) and urea (C) forms were observed under anand inverted release light urea, microscope parasites ((elicitA), size the bar—20highest secretion.m, 100 magnification). NO (B) and urea (C)production production were were evaluated evaluated in inM MΦ Φexposedexposed to toT. T.b. b.brucei brucei (Tbb(Tbb) parasites,) parasites, in inMΦ M stimulatedΦ stimulated by byTbEVs TbEVs and and PMA, PMA, and and in in resting-MΦ (negative control, NC). Results of three independent experiments (n = 12) and two replicates per sample are resting-MΦ (negative control, NC). Results of three independent experiments (n = 12) and two replicates per sample are represented by whiskered box plots, including median, minimum, and maximum values. The non-parametric Wilcoxon representedmatched-pairs by whiskered signed-rank box test plots, was used including for statistical median, comparisons minimum, (p and < 0.05). maximum * (p < 0.05) values. and ** The (p < non-parametric0.01) indicate statistical Wilcoxon matched-pairssignificance. signed-rank test was used for statistical comparisons (p < 0.05). * (p < 0.05) and ** (p < 0.01) indicate statistical significance. 3.3. TbEVs Direct the Differentiation of MHCI+, MHCII+ and MHCI+MHCII+ Macrophage SubsetsResting-M Φ incubated for 24 h (negative control) exhibited a residual NO synthesis (FigureTo4 B).indirectly However, evaluate after PMA-stimulationthe possible presentation (positive of control),parasite cellsantigens revealed by mouse a significant MΦ, NOthe expression increase (p of= MHC 0.0078), molecules thus confirming by MΦ exposed the viability to TbEVs and for 24 functionality h and the frequency of these of cells thatMHCI could+ and transform MHCII+ MΦ arginase subsets were into NOanalyzed through by flow the cytometry enzymatic (Supplementary activity of NOS2. Figure M Φ S1 and Figure 5).

A B C

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Figure 5. MHCI and MHCII surface expression by MΦ exposed to TbEVs. After 24 h of incubation, the frequency of MHCI+ (A), MHCII+ (B), and MHCI+MHCII+ cells (C) was evaluated in MΦ exposed to T. b. brucei (Tbb) parasites, in MΦ stimulated by TbEVs and PMA, and in resting-MΦ (negative control, NC). Results of at least three independent experiments (n = 32) and at two replicates per sample are represented by whisker box-plot, median, minimum, and maximum values. The non- parametric Wilcoxon matched-pairs signed-rank test was used for statistical comparisons. * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001) indicate statistically significant values.

A significant high frequency of MHCI+ (Figure 5A), MHCII+ (p = 0.0001) (Figure 5B), and MHCI+MHCII+ MΦ (p = 0.0313) was observed in cells stimulated by PMA (Figure 5C) when compared with resting cells. MHCI+ MΦ (p = 0.01) and MHCII+ MΦ (p = 0.0039) subsets were inhibited after T. b. brucei exposure. On the other hand, TbEVs elicited a significantly high expression of MHCI+ (p = 0.0003) and MHCII+ MΦ (p = 0.0004) in comparison to resting cells and cells exposed to parasites (p MHCI+ MΦ <0.0001, p MHCII+ MΦ = 0.0039). TbEVs seemed to be responsible for a significative expansion of MHCI+MHCII+ MΦ subset (p = 0.0313) (Figure 5C). Also, in this case, the exposure to parasites caused a Biomedicines 2021, 9, x FOR PEER REVIEW 8 of 18

Biomedicines 2021, 9, 1056 8 of 17

stimulated by TbEVs (p = 0.0313) or T. b. brucei parasites (p = 0.0078) (Figure4A) showed significant increases in NO levels when compared with resting MΦ.MΦ exposed to parasites exhibited the higher NO production that was significantly different from TbEVs exposed-MΦ (p = 0.0313). Figure 4. Production of nitric oxideIn resting-M and urea Φbywere macrophages detected exposed low levels to TbEVs. of urea M (FigureΦ (arrows)4C). exposed However, to trypomastigote PMA stimulated forms were observed undercells an showedinverted light a significant microscope increase (A, size in bar—20 ureaproduction μm, ×100 magnification). (p = 0.0156), NO indicating (B) and urea that (C these) production were evaluatedcells in M canΦ exposed convert to arginine T. b. brucei into ( ureaTbb) parasites, through arginasein MΦ stimulated enzymatic by activity.TbEVs and MΦ PMA,stimulated and in by resting-MΦ (negative control,TbEVs NC). or Results exposed of tothree parasites independent also exhibited experiments significant (n = 12) highand two levels replicates of urea per when sample compared are represented by whiskered box plots, including median, minimum, and maximum values. The non-parametric Wilcoxon with resting-MΦ (p = 0.0313). matched-pairs signed-rank test was used for statistical comparisons (p < 0.05). * (p < 0.05) and ** (p < 0.01) indicate statistical Although both T. b. brucei parasites and TbEVs promote mouse MΦ to produce NO significance. and release urea, parasites elicit the highest secretion. 3.3. TbEVs Direct the Differentiation of MHCI+, MHCII+ and MHCI+MHCII+ Macrophage 3.3. TbEVs Direct the Differentiation of MHCI+, MHCII+ and MHCI+MHCII+ MacrophageSubsets Subsets ToTo indirectly indirectly evaluate evaluate the the possible possible presentation presentation of parasite of parasite antigens antigens by mouse by mouse MΦ, theMΦ, expressionthe expression of MHC of MHC molecules molecules by MΦ byexposed MΦ exposed to TbEVs to forTbEVs 24 h andfor 24 the h frequencyand the frequency of MHCI +of andMHCI MHCII+ and+ MMHCIIΦ subsets+ MΦ weresubsets analyzed were analyzed by flow cytometryby flow cytometry (Supplementary (Supplementary Figure S1 Figureand FigureS1 and5). Figure 5).

A B C

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Figure 5. MHCI and MHCII surface expression by MΦ exposed to TbEVs. After 24 h of incubation, the frequency of Figure 5. MHCI and MHCII surface expression by MΦ exposed to TbEVs. After 24 h of incubation, the frequency of MHCI+ MHCI+ (A), MHCII+ (B), and MHCI+MHCII+ cells (C) was evaluated in MΦ exposed to T. b. brucei (Tbb) parasites, in MΦ (A), MHCII+ (B), and MHCI+MHCII+ cells (C) was evaluated in MΦ exposed to T. b. brucei (Tbb) parasites, in MΦ stimulated stimulatedby TbEVs by and TbEVs PMA, and and PMA, in resting-M and in resting-MΦ (negativeΦ (negative control, control,NC). Results NC). Resultsof at least of atthree least independent three independent experiments experiments (n = 32) (nand= 32) at andtwo atreplicates two replicates per sample per sample are represented are represented by whisker by whisker box-plot, box-plot, median, median, minimum, minimum, and maximum and maximum values. The values. non- Theparametric non-parametric Wilcoxon Wilcoxon matched-pairs matched-pairs signed-rank signed-rank test was test used was for used statistical for statistical comparisons. comparisons. * (p < 0.05), * (p < ** 0.05), (p <** 0.01), (p < *** 0.01), (p < ***0.001), (p < 0.001), and **** and (p **** < 0.0001) (p < 0.0001) indicate indicate statistically statistically significant signiﬁcant values. values.

+ + AA significant significant high high frequency frequency of of MHCI MHCI(Figure+ (Figure5A), 5A), MHCII MHCII(+p (p= = 0.0001) 0.0001) (Figure (Figure5B), 5B), + + andand MHCI MHCIMHCII+MHCII+M MΦΦ ((pp == 0.0313)0.0313) waswas observedobservedin in cells cells stimulated stimulated by by PMA PMA ( Figure(Figure5C 5C)) + + whenwhen compared compared with with resting resting cells. cells. MHCI MHCI+M MΦΦ( p(p= = 0.01) 0.01) and and MHCII MHCII+M MΦΦ( p(p= = 0.0039) 0.0039) subsetssubsets were were inhibited inhibited after afterT. T. b. b. bruceibrucei exposure.exposure. On the other hand, TbEVs elicited elicited a + + asignificantly significantly high high expression of MHCIMHCI+ ((p == 0.0003)0.0003) andand MHCIIMHCII+ MMΦΦ (p(p= = 0.0004) 0.0004) in in comparison to resting cells and cells exposed to parasites (p + <0.0001, p + comparison to resting cells and cells exposed to parasites (pMHCI MHCI+ MMΦΦ <0.0001, p MHCIIMHCII+ MΦ = M0.0039).Φ = 0.0039). + + TbEVsTbEVs seemed seemed to to be be responsible responsible for fo ar significativea significative expansion expansion of of MHCI MHCIMHCII+MHCIIM+ MΦΦ subsetsubset ( p(p= = 0.0313) 0.0313) (Figure (Figure5C). 5C). Also, Also, in in this this case, case, the the exposure exposure to to parasites parasites caused caused a a significant reduction of this cell subpopulation when comparing with resting-MΦ and TbEVs stimulated MΦ (p = 0.0313). When analyzing the levels of fluorescence intensity, no differences were observed between resting-MΦ,MΦ exposed to parasites and TbEVS stimulated MΦ. Only PMA- stimulated MΦ evidence a considerable augment of MHCI (p = 0.0313). Altogether the results indicated that TbEVs enhance the expression of MHCII class I and class II in MΦ, favouring antigen presentation, but did not increase the surface density of these molecules on the cell surface. On the other hand, T. b. brucei parasites promote Biomedicines 2021, 9, x FOR PEER REVIEW 9 of 18

significant reduction of this cell subpopulation when comparing with resting-MΦ and TbEVs stimulated MΦ (p = 0.0313). When analyzing the levels of fluorescence intensity, no differences were observed between resting-MΦ, MΦ exposed to parasites and TbEVS stimulated MΦ. Only PMA- stimulated MΦ evidence a considerable augment of MHCI (p = 0.0313). Altogether the results indicated that TbEVs enhance the expression of MHCII class I and class II in MΦ, favouring antigen presentation, but did not increase the surface density of these molecules on the cell surface. On the other hand, T. b. brucei parasites promote a Biomedicines 2021, 9, 1056 9 of 17 reduction of MHCI+, MHCII+ and MHCI+MHCII+ MΦ subsets, possible avoiding the antigen presentation and activation of cytotoxic and helper T cells.

3.4.a reduction TbEVs Promoted of MHCI Specific+, MHCII Mouse+ and Macrophage MHCI+MHCII Activation+ M Φ subsets, possible avoiding the antigenTo identify presentation correlations and activation between of the cytotoxic influence and of helper TbEVs T and cells. T. b. brucei parasites on mouse MΦ, data were analysed by PCA. This statistical analysis indicated that the overall 3.4. TbEVs Promoted Specific Mouse Macrophage Activation influence of TbEVs and PMA on MΦ activity is correlated (Figure 6A). On contrary, T. b. bruceiTo effects identify on mouse correlations MΦ were between distinct the from influence TbEVs ofand TbEVs resting-M andΦT.. These b. brucei resultsparasites were confirmedon mouse by M Φcluster, data analysis, were analysed which aggregates by PCA. This in the statistical same cluster analysis (cluster indicated 3) the that effects the ofoverall TbEVs influence and PMA of TbEVson MΦ. and MΦ PMA activity on McausedΦ activity by parasite is correlated exposure (Figure was6A). found On contrary,through clustersT. b. brucei 2 andeffects 3, onwith mouse most M ofΦ thewere effects distinct in fromcluster TbEVs 2 whereas and resting-M resting-MΦ. TheseΦ is mainly results localizedwere confirmed in cluster by 1 cluster(Figure analysis, 6B). which aggregates in the same cluster (cluster 3) the effectsTherefore, of TbEVs this and analysis PMA on highlights MΦ.MΦ activitythat TbEVs caused activation by parasite of exposurerodent M wasΦ can found be through clusters 2 and 3, with most of the effects in cluster 2 whereas resting-MΦ is mainly different from cell activation induced by T. b. brucei. localized in cluster 1 (Figure6B).

FigureFigure 6. 6. InfluenceInﬂuence of of TbbEVs TbbEVs on on mice mice macrophages. macrophages. The The relationship relationship between between the the effect effect of of TbbEVs TbbEVs (black (black dots), dots), PMA PMA (positive(positive control, control, pink pink dots), dots), and and T.T. b. b. brucei brucei parasitesparasites (brown (brown dots) dots) in in MØ, MØ, includin includingg urea urea and and NO NO production production and and density density ofof MHCI MHCI and and MHCII, MHCII, is is represented represented by by a a variable variable correlation correlation plot plot ( (AA).). Resting Resting MØ MØ (negative (negative control) control) are are indicated indicated by by greengreen dots. dots. Cluster Cluster distribution distribution of of the the e effectffect TbEVs, TbEVs, PMA, PMA, and and parasites parasites (Tb) (Tb) in in MØ MØ compared compared with with resting resting cells cells (NC) (NC) are are represented by heat map (B). represented by heat map (B).

3.5. TbEVsTherefore, Favored this the analysis Differentiation highlights of CD4 that+ TbEVsT Cellsactivation and Increment of rodent the Surface MΦ can Expression be different of CD3from Molecules cell activation induced by T. b. brucei. The frequency of CD3+ cells (Figure 7) and expression level of CD3 molecules were + examined3.5. TbEVs in Favored mononuclear the Differentiation blood cells of CD4exposTed Cells to parasites and Increment or stimulated the Surface by Expression TbEVs or of CD3 Molecules parasite Ag (supplementary Figure 2). + InThe both frequency CD4+ (Figure of CD3 7A)cells and(Figure CD8+ (Figure7) and expression7B) cell fractions, level of the CD3 frequency molecules of CD3 were+ cellsexamined were significantly in mononuclear lower blood in cells cells exposed exposed to toT. parasitesb. brucei in or comparison stimulated to by unprimed TbEVs or cellsparasite (p = Ag0.0078). (Supplementary However, ConA Figure stimulation S2). caused a significant increase in CD3+ cells + + + in bothIn cell both fractions CD4 (Figure (p = 0.0078).7A) and In CD8both cell(Figure fractions,7B) cell cells fractions, exposed the to parasites frequency were of CD3 also statisticallycells were significantlydifferent from lower TbEVs in cells and exposed Ag stimulated to T. b. brucei cells in(p comparison = 0.0313). TbEVs to unprimed were cells (p = 0.0078). However, ConA stimulation caused a significant increase in CD3+ cells in both cell fractions (p = 0.0078). In both cell fractions, cells exposed to parasites were also statistically different from TbEVs and Ag stimulated cells (p = 0.0313). TbEVs were responsible for a significant increase of CD3+ cell subset in the CD4+ cell fraction (p = 0.0078) in comparison with unprimed and parasite exposed cells. The fluorescence intensity of CD3-labelled cells in both cell fractions significantly increased (p = 0.0313) in Ag and TbEVs stimulated PBMC when compared to unprimed cells. Stimulation by ConA also induced a significant increase (p = 0.0313) in CD3-fluorescence intensity of CD4+ cell fraction. On the other hand, when compared to unprimed cells and TbEVs and Ag-stimulated cells, exposure to T. b. brucei cultured-derived parasites weakened CD3 fluorescence in CD4+ and CD8+ cell fractions (p = 0.0313). Altogether, these results indicated that TbEVs stimulation seemed to trigger the expansion of the CD4+ (CD3hi) T cell subset and induce the expression of CD3 molecules BiomedicinesBiomedicines2021 2021, ,9 9,, 1056 x FOR PEER REVIEW 1010 ofof 1718

onresponsible CD8+ T cells. for a In significant contrast, T. increase b. brucei parasitesof CD3+ cell impaired subset the in differentiationthe CD4+ cell offraction CD8+ and(p = CD40.0078)+ T in cells comparison and promoted with theunprimed down expression and parasite of CD3exposed molecules cells. at the cell surface.

FigureFigure 7.7.Frequency Frequency ofof CD3 CD3++ cellscells afterafter exposureexposure ofof mononuclearmononuclear cellscells to to TbEVs. TbEVs. CD4 CD4++ ((AA)) andand CD8 CD8++ ((BB)) cellcell fractionsfractions exposedexposed to toT. T. b. b. bruceibruceiparasites parasites( (TbbTbb),), stimulated stimulated by by parasite parasite antigen antigen (Ag), (Ag), TbEVs, TbEVs, and and ConA, ConA, and and unprimed unprimed cells cells (negative (negative control,control, NC)NC) werewere labelledlabelled withwith CD3-monoclonalCD3-monoclonal antibodyantibody andand evaluatedevaluated byby flowflow cytometry.cytometry. ResultsResults ofof atat leastleast threethree independent experiments (n = 20) performed in duplicate are represented by whiskered box-plot, median, minimum, and independent experiments (n = 20) performed in duplicate are represented by whiskered box-plot, median, minimum, maximum values. The non-parametric Wilcoxon matched-pair signed-rank test was used for statistical comparisons. * (p and maximum values. The non-parametric Wilcoxon matched-pair signed-rank test was used for statistical comparisons. < 0.05) and ** (p < 0.01) indicate significant differences. *(p < 0.05) and ** (p < 0.01) indicate significant differences. The fluorescence intensity of CD3-labelled cells in both cell fractions significantly 3.6. TbEVs Led the Expansion of Regulatory CD4+ T Cells and FoxP3+CD4+ T Cell Subset and Enhancedincreased Surface (p = 0.0313) CD25 +inand Ag Intracellular and TbEVs FoxP3 stimulated+ Molecules PBMC when compared to unprimed cells. Stimulation by ConA also induced a significant increase (p = 0.0313) in CD3- To assess the effect of TbEVs on CD4+ and CD8+ T cell subsets, mononuclear blood fluorescence intensity of CD4+ cell fraction. On the other hand, when compared to cells were exposed to cultured parasites and stimulated by TbEVs or parasite Ag. The unprimed cells and TbEVs and Ag-stimulated cells, exposure to T. b. brucei cultured- frequency of CD4+ T cells expressing CD25 (Figure8) molecules was evaluated as well as derived parasites weakened CD3 fluorescence in CD4+ and CD8+ cell fractions (p = 0.0313). the density of CD25 molecules on the cell surface and intracellular FoxP3 molecules on Altogether, these results indicated that TbEVs stimulation seemed to trigger the stimulated cells. Since parasite-exposed cells evidence a low frequency of CD3+ cells only expansion of the CD4+ (CD3hi) T cell subset and induce the expression of CD3 molecules expression and density CD25 were examined. on CD8+ T cells. In contrast, T. b. brucei parasites impaired the differentiation of CD8+ and T. b. brucei parasites induced a significant expansion of the CD4+ T cell subset ex- CD4+ T cells and promoted the down expression of CD3 molecules at the cell surface. pressing CD25 comparing to unprimed cells, TbEVs, and Ag stimulated cells (p = 0.0313) (Figure8C). Parasite Ag and TbEVs caused a high expansion of FoxP3 + CD4+ (Figure8D) 3.6. TbEVs Led the Expansion of Regulatory CD4+ T Cells and FoxP3+CD4+ T Cell Subset and and FoxP3+CD25+ CD4+ T cells (p = 0.0313) (Figure8A) in comparison to unprimed cells. Enhanced Surface CD25+ and Intracellular FoxP3+ Molecules Stimulation of PBMC by ConA led to a significant expansion (p = 0.0313) of CD4+ T cell subsetTo assess (Figure the8 effectB). On of the TbEVs other on hand, CD4 parasites,+ and CD8 TbEVs+ T cell or subsets, Ag did mononuclear not seem to affect blood thecells CD4 were+ (FoxP3 exposed− CD25 to cultured−) T cell parasites subset. and stimulated by TbEVs or parasite Ag. The frequencyTbEVs of and CD4 parasites+ T cells promotedexpressing a CD25 higher (Figure fluorescence 8) molecules intensity was of evaluated CD25-labeled as well cells as whenthe density compared of CD25 with molecules unprimed on lymphocytes the cell surface (p =and 0.0313). intracellular Furthermore, FoxP3 moleculesT. b. brucei on parasitesstimulated induced cells. Since a higher parasite-exposed expression of CD25cells evidence molecules a low in comparison frequency of to TbEVsCD3+ cells and only Ag stimulatedexpression cellsand density (p = 0.0313). CD25 Significant were examined. overexpression of intracellular FoxP3 molecules was alsoT. b. found brucei in parasites CD4+ T cells induced after stimulationa significant with expansion TbEVs or of Ag the (p =CD4 0.0331).+ T cell subset expressingThese resultsCD25 comparing indicated that to unprimed TbEVs and cells, parasite TbEVs, Ag and favoured Ag stimulated expansion cells of regulatory(p = 0.0313) CD4(Figure+ T cell8C). subset Parasite (FoxP3 Ag and+CD25 TbEVs+CD3 caused+ CD4 +a, high T reg expansion cells) and of FoxP3 FoxP3+CD4+ CD4+ T+ cells.(Figure More- 8D) over,and FoxP3T. b. brucei+CD25parasites+ CD4+ T enhancedcells (p = 0.0313) the frequency (Figureof 8A) CD25 in comparison+CD4+ T cells. to unprimed Among CD4 cells.+ T cells,Stimulation the overexpression of PBMC of byα-chain ConA of led IL2-receptor to a significant (CD25) expansion was induced (p = 0.0313) by parasites of CD4 and+ T TbEVs,cell subset and (Figure the increased 8B). On expression the other ofhand, FoxP3 pa wasrasites, elicited TbEVs by or TbEVs Ag did and not Ag. seem to affect the CD4+ (FoxP3− CD25−) T cell subset. + + + 3.7. TbEVsTbEVs Direct and parasites the Expansion promoted of Effector a higher CD8 fluoT Cellsrescence and CD8 intensityT Cells of ExpressingCD25-labeled FoxP3 cells Phenotype and Increase Surface CD25 and Intracellular FoxP3 Molecules when compared with unprimed lymphocytes (p = 0.0313). Furthermore, T. b. brucei + + parasitesCD8 inducedT cell subsets a higher were expression examined of CD25 by estimating molecules thein comparison frequency to of TbEVs CD8 Tand cells Ag expressingstimulated CD25cells (p in = mononuclear0.0313). Significant blood overexpression cells exposed toof intracellular parasites and FoxP3 stimulated molecules by TbEVswas also or found parasite in CD4 Ag (Figure+ T cells9 ).after The stimulation expression with of FoxP3 TbEVs was or Ag evaluated (p = 0.0331). in stimulated cells.These Furthermore, results indicated the potential that TbEVs of these and cellsparasi tote recognize Ag favoured IL-2 expansion and regulate of regulatory the cell CD4+ T cell subset (FoxP3+CD25+CD3+ CD4+, T reg cells) and FoxP3+CD4+ T cells. Moreover, Biomedicines 2021, 9, x FOR PEER REVIEW 11 of 18

Biomedicines 2021, 9, 1056 11 of 17 T. b. brucei parasites enhanced the frequency of CD25+CD4+ T cells. Among CD4+ T cells, the overexpression of α-chain of IL2-receptor (CD25) was induced by parasites and TbEVs,immune and activity the increased was indirectly expression assessed of by FoxP3 the density was of elicited surface CD25by TbEVs and intracellular and Ag. FoxP3 molecules.

A B *

* * *

* * * * *

Figure 8. Differentiation of CD25+ and FoxP3+ CD4+ T cell subsets induced by T. b. brucei EVs. CD4+ cell fraction exposed + + + + Figure 8.to DifferentiationT. b. brucei parasites of (CD25Tbb), stimulated and FoxP3 by Ag, CD4 TbbEVs, T cell and ConA,subsets and induced unprimed by cells T. (negative b. brucei control, EVs. CD4 NC) were cell labelled fraction exposed to T. b. bruceiwith CD3, parasites CD25and (Tbb FoxP3), stimulated monoclonal by antibodies Ag, TbbEVs, and evaluated and ConA, by flow and cytometry. unprimed CD3 +cellscells (negative were gated control, and the NC) were labelled withfrequency CD3, of CD25 CD25+ FoxP3and FoxP3+ (A), CD25 monoclonal−FoxP3− ( Banti) CD25bodies+FoxP3 and− ( evaluatedC) and CD25 by−FoxP3 flow+ cytometry.(D) cell subsets CD3 were+ cells estimated. were gated and the frequencyIn consequence of CD25 of+FoxP3 the low+ (A levels), CD25 of CD3−FoxP3+ cells,− (B the) CD25 frequency+FoxP3 and− ( densityC) and of CD25 FoxP3−FoxP3 were not+ (D considered) cell subsets in T. b.were brucei estimated. In consequenceexposed-PBMC. of the low Results levels ofof three CD3 independent+ cells, the frequency experiments and (n = density 18) performed of FoxP3 in duplicate were not are considered represented by in whiskered T. b. brucei exposed- PBMC. Resultsbox-plot, of median, three minimum,independent and maximumexperiments values. (n The = 18) non-parametric performed Wilcoxon in duplicate matched-pair are represented signed-rank by test whiskered was used box-plot, median, forminimum, statistical comparisons.and maximum * (p < 0.05)values. indicated The significantnon-parametric differences. Wilcoxon matched-pair signed-rank test was used for statistical comparisons. * (p < 0.05) indicatedWhen compared significant to unprimeddifferences. CD3 +CD8+ cells, it was observed that ConA stimulation caused a significant high frequency of CD8+ T cell subsets expressing CD25+ and + + + ++ + + + 3.7. FoxP3TbEVs (Directp CD25 theFoxP3 Expansion= 0.0313, of Figure Effector9A; pCD8CD25 T= Cells 0.0078, and Figure CD89C) T as Cells well Expressing as CD8 FoxP3 Phenotype(CD25− andFoxP3 Increase−) T cells Surface (p = 0.0516, CD25 Figure and Intracellular9B). Cells stimulated FoxP3 Molecules by TbEVs ( p = 0.0156) showed significant high frequencies of CD25+FoxP3+CD8+ T cells (Figure9A). + + CD8TbEVs T cell and Agsubsets induced were a significant examined expansion by estimating of CD8+ (CD25 the−FoxP3 frequency−) T cell subsetof CD8 T cells expressing(p = 0.0156) CD25 and promotedin mononuclear the retraction blood of CD25 cell−sFoxP3 exposed+ CD8 +toT cellparasites subset ( pand= 0.0313) stimulated by TbEVs(Figure or 9parasiteB). However, Ag when(Figure compared 9). The to expression unprimed cells, of T.FoxP3 b. brucei wasparasites evaluated caused in a stimulated + cells.significant Furthermore, restrain ofthe CD8 potentialT cells. of these cells to recognize IL-2 and regulate the cell CD8+ T cells exposed to T. b. brucei parasites or stimulated by ConA, TbEVs, and immuneparasite activity Ag showed was a indirectly significant increase assessed of CD25 by the molecules density (p of= 0.0313) surface when CD25 comparing and intracellular FoxP3with molecules. unprimed cells. Furthermore, FoxP3 fluorescent intensity also increased in CD8+T cellsWhen stimulated compared by TbEVs to unprimed (p = 0.0313). CD3+CD8+ cells, it was observed that ConA stimulation caused aAltogether significant the resultshigh frequency indicated that of TbEVs CD8 mainly+ T cell triggered subsets the expressing expansion of CD25 effector+ and FoxP3+ CD8+T cells and CD8+ T cell subsets expressing FoxP3 associated with a higher density (p CD25+ FoxP3+ = 0.0313, Figure 9A; p CD25+ = 0.0078, Figure 9C) as well as CD8+ (CD25−FoxP3−) T cells (p = 0.0516, Figure 9B). Cells stimulated by TbEVs (p = 0.0156) showed significant high frequencies of CD25+FoxP3+CD8+ T cells (Figure 9A). TbEVs and Ag induced a significant expansion of CD8+ (CD25−FoxP3−) T cell subset (p = 0.0156) and promoted the retraction of CD25−FoxP3+ CD8+ T cell subset (p = 0.0313) (Figure 9B). However, when compared to unprimed cells, T. b. brucei parasites caused a significant restrain of CD8+T cells. CD8+ T cells exposed to T. b. brucei parasites or stimulated by ConA, TbEVs, and parasite Ag showed a significant increase of CD25 molecules (p = 0.0313) when comparing BiomedicinesBiomedicines2021 2021, 9, 9 1056, x FOR PEER REVIEW 1212 of of 17 18

ofwith FoxP3 unprimed molecules. cells. The Furthermore, overexpression FoxP3 of fluorescentα-chain of IL2-receptorintensity also (CD25) increased induced in CD8 by+T parasitescells stimulated can be mainly by TbEVs associated (p = 0.0313). with the expansion of CD25+ CD8+ T cell subset.

A B * * * * *

C D * ** * * * *

Figure 9. CD25+ and FoxP3+ CD8+ T cell subsets induced by T. b. brucei EVs. Mouse lymphocytes exposed to T. b. Figure 9. CD25+ and FoxP3+ CD8+ T cell subsets induced by T. b. brucei EVs. Mouse lymphocytes exposed to T. b. brucei brucei parasites (Tbb), stimulated by Ag, TbEVs, and ConA, and unprimed cells (negative control, NC) were labelled parasites (Tbb), stimulated by Ag, TbEVs, and ConA, and unprimed cells (negative control, NC) were labelled with CD3, + withCD25 CD3, and CD25 FoxP3 and monoclonal FoxP3 monoclonal antibodies antibodies and evaluated and evaluatedby flow cytometry. by ﬂow cytometry. CD3+ cells CD3were gatedcells wereand the gated frequency and the of + + − − + − − + frequencyCD25+FoxP3 of CD25+ (A), FoxP3CD25−FoxP3(A), CD25− (B), CD25FoxP3+FoxP3(B),− CD25(C), andFoxP3 CD25(−CFoxP3), and+ ( CD25D) cell FoxP3subsets( wereD) cell estimated. subsets were In consequence estimated. Inof consequence the low levels of theof CD3 low+ levelscells, the of CD3 frequency+ cells, and the frequencydensity of andFoxP3 density were ofnot FoxP3 considered were not in T. considered b. brucei exposed-PBMC. in T. b. brucei exposed-PBMC.Results of (n = Results6) at least of three (n = 6)independent at least three experiments independent performe experimentsd in duplicate performed are represented in duplicate by are whiskers represented box-plot, by whiskersmedian, box-plot, minimum median, and maximum minimum values. and maximum The non-parametric values. The non-parametric Wilcoxon matched-pair Wilcoxon matched-pairsigned-rank test signed-rank was used test for wasstatistical used for comparisons. statistical comparisons. * (p < 0.05) and * (p <** 0.05)(p < 0.01) and **indicate (p < 0.01) significant indicate differences. signiﬁcant differences.

3.8. TbEVsAltogether and Parasite the Agresults Triggered indicated Related that Influence TbEVs on themainly Phenotype triggered of BALB/c the Miceexpansion T Cells of effector CD8+T cells and CD8+ T cell subsets expressing FoxP3 associated with a higher PCA analysis of CD4+ and CD8+ T cells indicated a positive correlation between the density of FoxP3 molecules. The overexpression of α-chain of IL2-receptor (CD25) effect of TbEVs and parasite Ag on T cells (Figure 10A), which contrasted with T. b. brucei induced by parasites can be mainly associated with the expansion of CD25+ CD8+ T cell effects. These results were confirmed by cluster analysis, which showed the effects of Ag subset. and TbEVs grouped in cluster 3. On the other hand, the influence of parasites in T cells was grouped at cluster 1. Furthermore, ConA stimulation outcomes were localized in cluster 3.8. TbEVs and Parasite Ag Triggered Related Influence on the Phenotype of BALB/c Mice T 2 and the intrinsic activity of unprimed T cells were mainly within cluster 3 (Figure 10B). Cells Overall, the effect of TbEVs on BALB/c mice T cells seemed to be similar to cell stimulation causedPCA by parasite analysis Ag of CD4 and+ slightly and CD8 different+ T cells of indicated unprimed a positive cells, but correlation completely between different the fromeffectT. of b. TbEVs brucei parasites. and parasite Ag on T cells (Figure 10A), which contrasted with T. b. brucei effects. These results were confirmed by cluster analysis, which showed the effects of Ag and TbEVs grouped in cluster 3. On the other hand, the influence of parasites in T cells was grouped at cluster 1. Furthermore, ConA stimulation outcomes were localized in cluster 2 and the intrinsic activity of unprimed T cells were mainly within cluster 3 (Figure 10B). Overall, the effect of TbEVs on BALB/c mice T cells seemed to be similar to cell Biomedicines 2021, 9, x FOR PEER REVIEW 13 of 18

Biomedicines 2021, 9, 1056 13 of 17 stimulation caused by parasite Ag and slightly different of unprimed cells, but completely different from T. b. brucei parasites.

Figure 10. The effect TbEVs on mice T cells. Relati Relationshiponship between between the the effect effect of of TbEVs TbEVs (bla (blackck dots), dots), parasite parasite Ag Ag (blue (blue dots), dots), ConA (pink dots) and viable T.T. b.b. bruceibrucei trypomastigotestrypomastigotes (brown(brown dots)dots) in in CD3 CD3++ cells,cells, includingincluding frequencyfrequency ofof CD25CD25++,, FoxPFoxP+,, and CD25CD25++FoxP3+ subsetssubsets and thethe densitydensityof of CD3, CD3, CD25, CD25, and and FoxP3 FoxP3 molecules molecules are are represented represented by aby variable a variable correlation correlation plot (plotA). In(A consequence). In consequence of the of low the levels low levels of CD3 of+ CD3cells,+ cells, the frequency the frequency and density and density of FoxP3 of FoxP3 were notwere considered not considered in T. b. in brucei T. b. exposed-PBMC.brucei exposed-PBMC. Unprimed Unprimed cells (negative cells (negative control) control) are indicated are indicated by green by dots.green Cluster dots. Cluster distribution distribution of the effectof the TbEVs, effect parasiteTbEVs, parasite Ag, ConA, Ag, andConA, trypomastigotes and trypomastigotes (Tbb) on ( TTbb cells) on compared T cells compared with unprimed with unprimed cells (NC) cells are (NC) represented are represented by the heat by the heat map (B). map (B).

4. Discussion Discussion African trypanosomes have have been extensivel extensivelyy studied since they are responsible for severe diseases in both medical and veterinaryveterinary contexts.contexts. To To survive, this extracellular protozoan affords several mechanisms that that use to evade the mammals’ immune system. system. Also, to ensure the completion of its life cycle,cycle, the parasite needs to avoid host mortality during thethe hemolymphatic hemolymphatic phase. phase. Therefore, Therefore, a balance a betweenbalance infectionbetween level infection (parasitemia) level (parasitemia)and the intensity and of the the intensity inflammatory of the immune inflammatory response immune must be response achieved must in the be host. achieved Since insafe the and host. efficient Since anti-trypanosomal safe and efficient drugsanti-trypanosomal and vaccines aredrugs lacking, and vaccines many are are the lacking, studies manyperformed are the to understandstudies performed the mechanisms to understand associated the mechanisms with the host associated immune response with the direct host immuneagainst T. response brucei infection. direct against Although T. brucei different infection. approaches Although were different applied, approaches and numerous were applied,findings and reported, numerous some findings questions reported, remain so unanswered,me questions andremain some unanswered, mechanisms and are some not mechanismsentirely understood are not orentirely still controversial. understood or still controversial. Since EVsEVs releasedreleased by by eucaryotic eucaryotic cells cells seem s toeem play to a roleplay in a intercellular role in intercellular communi- communication,cation, interfering interfering with several with cellular several processes, cellular suchprocesses, as the such activation as the of activation microbicide of microbicideprocesses and processes generation and of generation immune mediators of immune by mediators changing geneby changing expression gene and expression affecting andsignalling affecting pathways signalling the potentialpathways of trypomastigotesthe potential of derivedtrypomastigotes EVs in influencing derived EVs the im-in influencingmune activity the of immune innate and activity adaptive of immuneinnate and cells adaptive was explored. immune Findings cells was of the explored. current Findingsstudy indicate of the thatcurrent TbEVs study shed indicate by trypomastigotes that TbEVs shed is enriched by trypomastigotes in proteins is and enriched are a het- in proteinserogeneous and population are a heterogeneou of sphericals population vesicles mainly of spherical constituted vesicles by smallermainly (<0.17constituted nm) and by smallerbiggest (>0.17(<0.17 nm)nm) EVs,and biggest which is (>0.17 in line nm) with EVs, previous which findings is in line [12 with]. previous findings [12]. Since the expansion in different organs and tissues of the MΦ population, as is the caseSince of the the liver, expansion spleen, and in different the bone marroworgans and was tissues described of the in T.M bruceiΦ population,infected mice,as is the intercommunication of TbEVs with innate immune cells were examined. It is reported that case of the liver, spleen, and the bone marrow was described in T. brucei infected mice, the inducible nitric oxide synthase (iNOS) peaked six days post-infection and that oxidized intercommunication of TbEVs with innate immune cells were examined. It is reported that L-arginine generates L-citrulline and NO [15]. In the current study, TbEVs trigger mouse inducible nitric oxide synthase (iNOS) peaked six days post-infection and that oxidized L- MΦ to produce NO as well as T. brucei cultured parasites, suggesting a role for TbEVs at arginine generates L-citrulline and NO [15]. In the current study, TbEVs trigger mouse the early stage of sleeping sickness. NO is a highly regulated effector molecule, which MΦ to produce NO as well as T. brucei cultured parasites, suggesting a role for TbEVs at participates in several physiological and immune processes and can inactivate pathogens the early stage of sleeping sickness. NO is a highly regulated effector molecule, which including T. brucei parasites [15]. However, high NO levels can be a disadvantage to the participates in several physiological and immune processes and can inactivate pathogens host, given the large spectra of cell disorders that are associated with NO activity. In sleeping sickness, the persistence of M1-MΦ is related to anaemia and systemic immune response [16–19]. Biomedicines 2021, 9, 1056 14 of 17

To counteract the possible harmful effect of NO in the host and perpetuate parasite replication, African trypanosomes induce the differentiation of M2-MΦ. L-Arginine can be hydrolyzed by arginase, generating ornithine, urea, proline and polyamines [20]. A previous study performed in T. b. brucei-infected mice indicates that M1-MΦ predominate in the early stage of infection while M2-MΦ prevail at a later infection stage (1–4 weeks) [18]. In the current study was found that TbEVs and cultured parasites induced urea and NO production by mouse MΦ. These findings reinforce the role of TbEVs in the early stage of infection as a MΦ modulator. By inducing the differentiation of mouse MΦ expressing M1 and M2 phenotypes, TbEVs seem to strengthen parasite activity in sustaining a balance between pro-inflammatory and anti-inflammatory responses. Moreover, by simultaneously ensuring the supported production of polyamines, which are essential nutrients for parasite survival and replication [7,21], TbEVs can facilitate the establishment of a chronic infection. The kinesin-1 heavy chain (TbKHC-1) released by African trypanosomes has been described as an activator of the arginine pathway, leading to the production of polyamines [7]. Taking into account the above considerations, TbKHC-1 may form part of TbEVs’ cargo. Contrary to T. b. brucei parasites that cause a marked reduction in the frequency of MHCI+ and MHCII+ MΦ, affecting the recognition of parasite antigens by T lymphocytes and compromising the adaptive immune response, TbEVs induce differentiation of MHCI+ and MHCII+ MΦ and double-positive MΦ (MHCI+ MHCII+), indicating that these cells can establish a link with acquired immunity by presenting parasite antigens to helper and cytotoxic T cells. Even so, the density of MHCI and MHCII molecules on the membrane surface of MΦ remained similar to resting MΦ. Though not fully understood similar inhibition of MHC expression avoiding antigen presentation was mentioned in a study carried out on T. cruzi, which is an intracellular parasite responsible for Chagas’ disease or American trypanosomiasis [22]. Therefore, TbEVs that appear to exert an action contrary to the parasite may cause the activation of adaptive immunity at least to some point, activating both CD4+ and CD8+ T cells. During the course of infection, T. brucei releases the stumpy induction factor (TSIF) that has been associated with the impairment of T cell proliferation [23]. A previous study performed in patients infected with T. b. gambiense has shown that T cells were considerably lower when compared with controls [24]. Thus, considering the high importance of cellular activation in orchestrating an integrated host immune response against sleeping sickness, the communication established by TbEVs on T cell expansion was also examined. In agree- ment with previous findings, a marked reduction of both CD3+CD4+ and CD3+CD8+ T cells caused by T. b. brucei parasites was found in the current study. However, TbEVs favour the expansion of CD4+ T cell subpopulation and induce the increase of CD3 expression in both CD4+ and CD8+ subsets (CD3hi CD4+ and CD3hi CD8+ T cells). CD3 complex is a T cell co-receptor responsible for intracellular signalling. Altogether, these findings indicate that, contrary to T. b. brucei, TbEVs may have the necessary conditions to stimulate T cells. However, in a previous study, Boda and colleagues [25] reported that effector CD8+ T cells were significantly lower in patients with African trypanosomiasis which corroborates our findings. In the current study, T. b. brucei parasites induce the expansion of CD4+ and CD8+ T cell subpopulations expressing a CD25 phenotype and favour the expression of CD25 molecules (CD25hiCD4+ and CD25hiCD8+ T cells). CD25 is the α chain of the IL-2 receptor, which is expressed by regulatory T (Treg) cells. CD25 is required for interleukin (IL)-2 signalling that mediates T cell activation and proliferation. To become fully activated, CD8+ T cells require the help of CD4+ T cells mediated by IL-2. However, Kalia et al. [26] have found that CD25hiCD8+ T cells can perceive proliferation signals (IL-2) and differentiate into short-lived effector cells and Olson et al. [27] reported that lymphocyte triggering factor (TLTF) released by T. b. brucei specifically elicit CD8+ T cells to generate IFN-γ. Moreover, it was reported that activated CD4+ T cells secrete IFN-γ and is recognised that this cytokine functions as a T. b. brucei growth factor [28]. Thus, T. b. brucei parasites may favour the expansion of CD25+ T cells to ensure their survival. Biomedicines 2021, 9, 1056 15 of 17

FoxP3 nuclear factor is a regulator of gene expression that suppresses the function of NFAT and NF-κB nuclear factors, avoiding the expression of pro-inflammatory cytokine genes, including IL-2. CD4+ Treg cells which constitutively express FoxP3 can suppress leukocyte effector activity, contributing to the maintenance of immune homeostasis. These regulatory cells also exhibit a high expression of CD25 molecules. In African trypanosomiasis, the expansion of Treg cells seems to occur from the parasite establishment to the chronic stage, being associated with parasite tolerance (reviewed in [29]) and in trypanotolerant C57BL/6 mice, the expansion of CD25+ FoxP3+ CD4+ T cell subset was demonstrated after the first peak of parasitemia [30]. On the other hand, the lack of expansion of Treg cells is associated with tissue damage and impaired survival of infected mice [31]. In the current study, TbEVs promote the expansion of CD4+ and CD8+ Treg cells and FoxP3+ CD4+ T cell subset. According to Zelenay et al. [32], CD4+ T cells expressing FoxP3+ phenotype are committed Treg cells able to regain CD25 expression. Thus, TbEVs seem to induce the differentiation of CD4+ and CD8+ Treg cells and unconventional CD4+ Treg cells. In previous studies, it was reported that unconventional-Treg cells also can exert regulatory functions [33]. During chronic parasite infection, uncontrolled inflammation is one of the most clinical features noticed that can become lethal if not controlled by Treg cells [34,35]. Therefore, during infection TbEVs can lead to the establishment of a pool of T cells with a regulatory phenotype able to balance excessive inflammation. Altogether, these findings indicate that T. b. brucei parasites can activate the me- tabolization of arginine by MΦ and [36] modulate T cells, ensuring the production of polyamines and IFN-γ, both critical for parasite survival. At the same time, this parasite avoids antigen presentation by MΦ and T cells activation. However, the findings of the present study place in evidence that TbEVs can establish direct communication with cells of innate and adaptive immunity, and the effect of TbEVs on these cells is different from the parasite itself. TbEVs can elicit parasite antigenic presentation to CD4+ and CD8+ T cells possible leading to the activation of the cellular immune response in addition to the bidirectional activation of mouse MΦ, contributing to the release of factors essential to parasite growth and, also, to parasite destruction. Moreover, TbEVs also seems to have a direct effect on CD4+ T lymphocytes, triggering the expression of T cell co-receptors, which are key players in intracellular signalling. The expression of the nuclear factor FoxP3 was also stimulated by TbEVs, guiding the differentiation of CD4+ and CD8+ Treg cells. imposing regulation on host inflammatory immune response which is a hallmark of sleeping sickness. Since an unbalanced inflammatory response against the parasite can become lethal to the host, the role of Treg cells in protecting from collateral tissue damage is crucial. On the other hand, TbEVs induced the differentiation of effector CD8+ T cells and drive the overexpression of FoxP3 molecules in CD4+ T cells which are involved in maintaining immune homeostasis. Therefore, parasites and TbEVs seem to display complementary effects in the host immune response that can ensure parasite survival, delaying disease severity and the eventual death of the host. Furthermore, TbEVs represent a source of biomarkers that can open new avenues for a better diagnosis of sleeping sickness and the development of prophylactic measures to control the disease caused by T. brucei in Subsaharan Africa.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/biomedicines9081056/s1, Supplementary Figures S1 and S2, Supplementary videos: https: //drive.google.com/file/d/0B7h7gNscHF0TWmZNRl9sZWpQUDg/view?usp=sharing. Supple- mentary Video S1. Blood infected with T. b. brucei. Blood of infected BALB/c mice was directly observed at ×100 magnification under an optical microscope and video recorded. https: //drive.google.com/file/d/1oET-8Ehh3mLJIz6FoFlCoO_qEac4pwPV/view?usp=sharing. Supple- mentary Video S2. T. b. brucei blood forms. Parasites were directly observed in the blood of infected BALB/c mice at ×400 magnification under an optical microscope and video recorded. Author Contributions: Conceptualization, formal analysis, supervision, G.S.-G.; investigation, T.D.-G. and J.P.-M.; formal analysis, writing—review and editing, G.A.-P.; resource, W.A. and T.N.; resources, writing—review and editing, A.V.-B.; writing—review and editing, P.M.-G., Á.G. and Biomedicines 2021, 9, 1056 16 of 17

I.P.d.F. resources—supervision, M.S.-S. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by FCT—Foundation for Science and Technology, I.P., through research grant PTDC/CVT-CVT/28908/2017 and by national funds within the scope of Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA, UIDB/00276/2020) and Global Health and Tropical Medicine (GHTM, UID/04413/2020). Institutional Review Board Statement: This study was approved by the IHMT’s ethics committee and the Portuguese National Authority for Animal Health (DGAV—Direção Geral de Alimentação e Veterinária)—Ref. 0421/000/000/2020, 23 September 2020. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author (G.S.-G.). The data are not publicly available due to confidentiality. Conflicts of Interest: The authors declare no conflict of interest.

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