An L. braziliensis isolate (MHOM/BR/LTCP11245) previously obtained from a CL patient’s skin lesion was initially cultivated in biphasic medium (NNN). After isolation, parasites were identified by multilocus enzyme electrophoresis and cryopreserved in liquid nitrogen. Following selection, parasites were expanded and cultivated in Schneider’s culture medium (SIGMA) supplemented with 20% inactive fetal bovine serum (FBS) (GIBCO), 1% L-glutamine and antibiotics.

L. braziliensis promastigotes were cultured at 37°C under 5% CO₂ for 4 hours to optimize protein and vesicle secretion (31). Supernatants collected from in vitro cultures were centrifuged (2500rpm) and filtered (0.22 µm), after which exosomes were isolated through sequential ultracentrifugation (10 x 10⁵g). The EV particle-size distribution was determined by diffraction analysis using a NS300 particle-size tracker and Nanosight NTA 3.0 software using light scatter mode (Malvern Instruments Ltd., Technologies, Malvern, UK) ( Figure 1 ) (33–35).

Peripheral blood mononuclear cells (PBMC) were obtained from heparinized venous blood from healthy subjects by Ficoll-paque density gradient centrifugation (GE Healthcare). Cells were washed twice in saline, and monocytes were isolated from PBMCs using magnetic beads in accordance with the manufacturer’s protocol (Dynabeads untouched human monocytes; Invitrogen Dynal AS, Oslo, Norway). This process was performed twice, after which cells were washed in phosphate-buffered saline (PBS), then adjusted to the desired concentration, and resuspended in RPMI 1640 (GIBCO BRL., Grand Island, NY USA) supplemented with 10% FBS (GIBCO BRL., Grand Island, NY USA) and antibiotics. Monocytes were then allowed to adhere to polystyrene culture plates and incubated for 6 days at 37°C under 5% CO₂ until differentiation into macrophages, as previously described (36).

Monocyte-derived macrophages (3 x 10⁵) were stimulated, using a transwell membrane, with soluble factors from L. braziliensis for 24 hours at 37 °C, 5% CO₂ in RPMI ( Figure 2A ). Following stimulation, cells were washed twice in saline and infected or not with L. braziliensis (MOI 5:1) for 2 hours. Next, the remaining promastigotes were washed out, and cells were reincubated for another 24 hours at 37 °C, 5% CO₂ in RPMI. The slides were subsequently submitted to panoptic staining for posterior quantification of infected macrophages and amastigotes per 100 cells via optical microscopy.

Monocyte-derived macrophages (3x10⁵) were stimulated with exosomes from L. braziliensis (300 vesicles/macrophage) for 24 hours at 37°C under 5% CO₂ in RPMI. After stimulation, cells were washed twice in saline and infected or not with L. braziliensis (MOI 5:1) for 2 hours. The remaining promastigotes were washed out, then cells were incubated for another 24 hours (37 °C, 5% CO₂) in RPMI.

For some experiments involving the inhibition of exosome/vesicle generation, 1x10⁷ Leishmania/mL were treated with GW4869 (20ng/ml), a vesicle generation inhibitor, (https://www.sigmaaldrich.com/BR/pt/product/sigma/d1692) for 30 minutes at room temperature, then washed three times to prevent contact between the inhibitor and human cells during stimulation/infection protocols. To assess GW4869 toxicity against Leishmania parasites, we evaluated L. braziliensis viability after treatment with GW4869 using propidium iodide as cell death marker, by flow cytometry.

For the blockade of the NLRP3 inflammasome, monocyte-derived macrophages (3x10⁵) were first treated with the NLRP3 inhibitor glibenclamide (100µM) for 2 hours, then stimulated with L. braziliensis exosomes (300:1) for 24 hours. Cells were then washed twice and infected or not with L. braziliensis (MOI 5:1) for 2 hours, washed again to remove any remaining promastigotes, and finally incubated for 24 hours at 37 °C under 5% CO₂in RPMI. Unstimulated macrophages and untreated stimulated cells were used as controls.

To evaluate infection rate, 3x10⁵ macrophages/well were plated on Nunc^® Labtek^® plates and stimulated with exosomes from L. braziliensis (300 vesicles/macrophage), then incubated for 24 hours at 37° C with 5% CO₂. Cells were then washed twice and infected or not with L. braziliensis (MOI 5:1) for 2 hours, washed again to remove any remaining promastigotes, and finally incubated for 24 hours at 37 °C under 5% CO₂ in RPMI. The slides were submitted to panoptic staining for the later quantification of infected macrophages and the number of amastigotes per 100 cells, performed via optical microscopy.

Following stimulation and/or infection protocols, the supernatants from cultures were collected for cytokine (IL-1β, TNF, IL-6 and IL-10) quantification via Luminex (Bio-Plex Pro Human Cytokine 27-plex Assay) or ELISA.

Monocyte-derived macrophages from healthy subjects were stimulated with L. braziliensis exosomes for 24 hours and infected or not with L. braziliensis for another 24 hours, as described above. After the final incubation, cells were collected and placed in 5mL polystyrene FACS tubes (BD Biosciences Falcon™ 352052) for cell labeling with conjugated antibodies αCD14 (APC) and αHLA-DR (PerCP) to determine cell populations of interest, as well as αNLRP3 (PE). Events were acquired on a flow cytometer (BD LSRFortessa™ Cell analyzer) and data were analyzed using Flowjo^® software.

To evaluate reactive oxygen species (ROS) production, macrophages were stimulated as described in “Exosome Stimulation and Macrophage infection with L. braziliensis” section, then treated with dihydrorhodamine-123 at 10ng/mL (Cayman Chemical Company) for 10 minutes. Cells were subsequently labeled with αHLA-DR and αCD14 to evaluate fluorescence intensity by flow cytometry, with data analysis performed via FlowJo^® software.

Cells stimulated with exosomes and infected or not with L. braziliensis, followed by incubation at 37°C under 5% CO₂ for 2 hours, were harvested in TRIzol Reagent (Invitrogen). RNA extraction was performed using TRIzol RNA isolation, according to manufacturer’s instructions. RNA concentration and integrity were determined by spectrophotometric optical density measurements (260 and 280 nm). Gene expression was analyzed performed as previously described (37).

C57BL/6 mice, both wild-type and others genetically deficient for NLRP3^−/−, were obtained as previously described (38). All animals were maintained at the UFMG Animal Facility and used for experimentation at 6–8 weeks of age. Bone marrow-derived macrophages (BMDM) were prepared and infected as previously described (39, 40). Briefly, bone marrow cells were isolated from the femurs and tibias of the animals, cultured in RPMI 1640 supplemented with 30% L929 cell-conditioned medium and 20% FBS for 7 days. BMDM (0.5×10⁶) were treated or not with lipopolysaccharide (LPS) for 6 hours (500 ng/ml) and stimulated or not with L. braziliensis exosomes (300:1), followed by infection with stationary phase Leishmania braziliensis (MOI 10:1) for 24 hours. After 48 hours, supernatants were harvested and IL-1β, TNF and IL-6 concentrations were detected by ELISA.

Statistical analysis was performed using the Wilcoxon test for paired variables and Mann-Whitney rank test for unpaired measurements (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). All experimental data were analyzed using Prism GraphPad^® 8.0.2, which was also used for graphical data representations.

Macrophages can be “trained” to enhance response to infection (41). For instance, macrophages exposed to Saccharomyces cerevisiae demonstrated an increased ability to produce TNF in response to TLR ligands, such as LPS (42). To investigate whether soluble factors from L. braziliensis would interfere in cytokine production, human macrophages were cultured with L. braziliensis separated by a membrane, which allowed only soluble factors/small molecules to cross the barrier. Macrophages exposed to L. braziliensis products through the membrane produced more IL-1β upon infection than those that were not previously exposed to parasite factors ( Figure 2B ). Moreover, exposure to L. braziliensis soluble factors was also shown to induce the production of inflammatory mediators TNF, IL-6 and IL-1β by uninfected macrophages ( Figure 2C ). This data provides evidence that exposure to L. braziliensis modulates immune response prior to the establishment of cellular infection.

We first investigated whether L. braziliensis exosomes induced an inflammatory response in macrophages, and then assessed the effects of stimulating macrophages with these vesicles prior to infection with L. braziliensis. Compared to unstimulated cells, macrophages stimulation with EVs were found to induce IL-1β, TNF, IL-10 and IL-6 production ( Figure 3 ). Pre-sensitization of macrophages with exosomes prior to infection was shown to increase IL-1β and IL-6 levels ( Figure 3 ). Similarly, pre-sensitization with exosomes produced no influence on infection rate, nor the number of parasites internalized (data not shown).

To further ascertain the role of exosomes in inducing inflammatory cytokine production, we infected human macrophages with L. braziliensis previously treated with GW4869, and then compared cytokine expression with other untreated Leishmania-infected cells. As in the experiments above, our data shows that cells infected with exosome-free L. braziliensis produced significantly less IL-1β, TNF, IL-10 and IL-6 upon infection ( Figure 4A ). Although no differences were observed in parasite internalization by macrophages pre-sensitized with exosomes compared to unstimulated cells (data not shown), the cells infected with exosome-free L. braziliensis presented less infectivity, as evidenced by fewer numbers of infected macrophages and lower numbers of amastigotes within the cells ( Figure 4B ). As treatment with GW4869 could have interfered with L. braziliensis survival, we performed a dose-response curve with different GW4869 concentrations. Our experiments show no toxicity of GW4869 over L. braziliensis ( Figure 4C ).

Most of the data presented in this study demonstrate the important association between L. braziliensis exosomes and IL-1β production. Consequently, the participation of NLRP3 in IL-1β production was investigated by evaluating both the expression of NLRP3 protein by cultured macrophages ( Figure 5A ) and the production of cytokines by cells stimulated with exosomes after the blockade of NLRP3 through glibenclamide treatment ( Figure 5B ). Our results indicate lower NLRP3 protein expression in cells stimulated with exosomes for 24 hours compared to basal level cells, while exosome stimulation in macrophages prior to infection induced more consumption, and probably further expression regulation, of this inflammasome than unsensitized cells ( Figure 5A ), which corroborates our previous results. Moreover, glibenclamide-treated cells were found to secrete less IL-1β, TNF, IL-6 and IL-10 than untreated cells, regardless of L. braziliensis infection or exosome stimulation. ( Figure 5B ), which suggests that cytokine expression induced by exosome stimulation is dependent on NLRP3.

The secretion of IL-1β may be dependent on inflammasome activation (8, 14, 39). NLRP3, the main inflammasome receptor responsible for inducing IL-1β production, forms a complex with ASC and Caspase-1 for the processing and secretion of IL-1β and IL-18 (43, 44). Since our results showed that L. braziliensis exosome stimulation followed by infection induced high levels of IL-1β secretion by human macrophages, we decided to investigate whether NLRP3 activation is required to drive IL-1β production upon L. braziliensis infection in a murine model.

Bone marrow-derived macrophages (BMDM) from C57BL/6 WT mice and mice deficient for NLRP3 were infected with L. braziliensis after previous stimulation with L. braziliensis exosomes. Levels of IL-1β, IL-6 and TNF were quantified by ELISA in cell supernatants. IL-1β production in mice was found to be dependent on the NLRP3 inflammasome, as IL-1β production was completely abrogated in cells from NLRP3^-/- mice, regardless of stimulation. However, NLRP3 did not appear to be involved in the production of the other cytokines evaluated, as levels did not differ between WT and NLRP3^-/- mouse cells ( Figure 6 ).

Our group previously showed that reactive oxygen species (ROS) constitute a major endogenous factor in Leishmania killing (13, 45). Furthermore, TNF production is directly associated with ROS production through the activation of the NF-κB signaling pathway. Therefore, to investigate the role of L. braziliensis exosomes on ROS production, ROS expression ( Figure 7 ) was evaluated in macrophages stimulated with exosomes further infected with L. braziliensis. Our data show that while L. braziliensis exosomes induced ROS production by uninfected macrophages, pre-sensitization did not interfere in ROS production following in vitro macrophage infection ( Figure 7 ).

Herein L. braziliensis exosomes were found to induce TNF and ROS production by human macrophages. It has been shown that NF-kB activation induces TNF production through the activation of TLRs 2-4 upon L. braziliensis infection (18). To determine the role of L. braziliensis exosomes in TLR2 and NFkB expression, human macrophages were stimulated with EVs for 2 hours and infected or not with L. braziliensis for another 2 hours. Uninfected macrophages stimulated with exosomes were found to induce higher expression of NF-κB and TLR2 ( Figure 8 ), yet infected cells previously stimulated with EVs demonstrated increased TLR2 expression, yet no effect on NF-κB expression ( Figure 8 ). No differences were observed among the groups with regard to TLR4 expression. These data corroborate with our findings on exosome-induced TNF and ROS production, as these molecules are formed as a result of the NF-κB pathway.