BALB/c mice originally obtained from the animal facilities of Universidade Federal Fluminense (UFF) were maintained in our own facilities with water and food ad libitum. Animals aged between 6 and 8 weeks were used. All experimental protocols used in this work were approved by the Ethical Committee for Experimental Animal Use established in the Instituto de Biofísica Carlos Chagas Filho—UFRJ under reference code CEUA IBCCF 157.

The parasites used in the experiments presented in this paper were L. amazonensis (MHOM/BR/00/Josefa strain) obtained by puncture of amastigotes from the lesion of infected BALB/c mice. The promastigotes were maintained at 26°C in M199 medium (Difco, NJ, USA) containing 10% fetal bovine serum (FBS; Gibco, NM, USA) until the fifth passage in culture to perform infection experiments. Infections were carried out with stationary phase culture, between the fourth and fifth days of culture.

Bone marrow cells were obtained from tibia and femurs of five BALB/c mice (20–25 g weight, with the age of 2 months), which were anesthetized with intravenous ketamine (25 mg/kg) and xylazine (2 mg/kg). Tissues were collected, rinsed with phosphate-buffered saline (PBS), transferred to a Petri dish, and cut into small pieces (approximately 0.2–0.8 cm²). The dissected pieces were washed with PBS, cut into smaller fragments, and subsequently digested with type I collagenase (1 mg/mL in DMEM/10 mM HEPES) for 30–40 min at 37°C. Any gross remnants that persisted after collagenase digestion were poured off between 1 and 3 min, and the supernatant was transferred to a new tube containing fresh medium and centrifuged at 400 × g for 10 min at 25°C. The pellets were resuspended in 3.5 mL Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, CA, USA) containing 1% penicillin and streptomycin, with concentration of 5,000 IU/mL and 5,000 µg/mL, respectively (Gibco, NM, USA), 20% of inactivated FBS (Invitrogen, CA, USA) and 15 mM HEPES (Sigma, MO, USA), seeded in T25 flasks (4 mL per flask) and incubated at 37°C in a humidified atmosphere containing 5% CO₂. On day 3 of culture, the medium was changed, and non-adherent cells were removed. Adherent cells exhibited similar proliferation rates and, upon reaching 80% confluence, they were passaged with a 0.25% trypsin-EDTA solution (Gibco, NM, USA) and maintained in DMEM with 10% FBS (complete medium). Approximately 1 × 10⁶ cells were characterized as MSCs at the third passage according to the consensus of the International Society of Cell Therapy (16).

Mesenchymal stromal cells were further characterized by their ability to differentiate into osteocytes, chondrocytes, and adipocytes (16, 17). Osteogenic differentiation was induced by culturing MSCs for up to 3 weeks in DMEM 10% FBS and 15 mM HEPES (Sigma, MO, USA), supplemented with 10⁻⁵ mM/L dexamethasone (Sigma, MO, USA), 5 µg/mL ascorbic acid 2-phosphate (Sigma, MO, USA), and 10 mM/L β-glycerolphosphate (Sigma MO, USA) (17). To observe calcium deposition, cultures were stained with Alizarin Red S (Nuclear, SP, Brazil). To induce adipogenic differentiation, MSCs were cultured with 10⁻⁸ M dexamethasone (Sigma, MO, USA), 2.5 µg/mL insulin, and 50 µg/mL indomethacin (Sigma, MO, USA) (18). Adipocytes were easily discerned from undifferentiated cells by phase-contrast microscopy. To further confirm their identity, cells were fixed with 4% paraformaldehyde with PBS and stained with Oil Red (Sigma, MO, USA) on day 21 of adipogenic differentiation. To induce chondrogenic differentiation, MSCs were cultured in DMEM supplemented with 10 ng/mL TGF-β1 (Sigma, MO, USA), 50 nM ascorbic acid 2-phosphate (Sigma, MO, USA), and 6.25 mg/mL insulin for 3 weeks. To confirm differentiation, cells were fixed with 4% paraformaldehyde in PBS for 1 h at RT and stained with Alcian Blue pH 2.5 (19) for detecting chondroblast secreted cell matrix (with chondrogenic differentiation) (data not shown).

Flow cytometry was performed using commercially available antibodies on FACSCalibur (Becton Dickinson, San Jose, CA, USA) following standard procedures. Cells were plated in 96-well plates and then centrifuged for 4 min at 300 × g, 4°C. Afterward, 10 µL of anti-CD16/32 antibody were added in cells (Fc receptor blocker), remaining for 15 min at 4°C. At the end of incubation, the cells were washed with PBS (100 µL/well), the plate was centrifuged at 300 × g for 4 min at 4°C and the supernatant was discarded. Subsequently, the cells were incubated with 10 µL of solution containing the anti-CD11b/FITC, anti-Sca-1/APC, anti-CD44/Pecy5, anti-CD45/PE, anti-CD49/PE, and anti-CD34/EFluor660 for 30 min at 4°C under light protection. Thereafter, the cells were washed with 100 µL of PBS and again centrifuged at 300 × g for 4 min at 4°C. After discarding the supernatant, the cells were resuspended in 200 µL of 1% PFA and transferred to FACS tubes containing 100 µL of PBS. The cells were analyzed using the FACSCalibur BD flow cytometry apparatus (BectonDickenson). Data were analyzed using FlowJo software 8.7 (Figures S1A–F in Supplementary Material). The respective antibody isotypes of the abovementioned antibodies or samples of unlabeled cells were used as controls. Undifferentiated population of MSCs was used in all experiments.

Syringes containing 5 mL of RPMI (Gibco, NM, USA) medium were injected in the peritoneal cavity of BALB/c mice to obtain the macrophages. The medium containing the macrophages was withdrawn from the cavity and cells were counted with Tripan blue and transferred to a 24-well plate at the concentration of 5 × 10⁵ cells per well. The cells were incubated for 1 h at 37°C and 5% of CO₂ for adhesion of macrophages to the plate. Thereafter, the plate was washed with warm PBS three consecutive times to remove the non-adherent cells, and then, 400 µL of RPMI with 10% of FBS supplemented with glutamine, pyruvate, and non-essential amino acids were added. After 24 h, the wells were washed again with PBS to remove B1 lymphocytes and 300 µL of RPMI with 10% of supplemented FBS were added.

The peritoneal cells were plated in a concentration of 5 × 10⁵ cells per well. The infection was performed with 2.5 × 10⁶ of L. amazonensis per well (ratio of 5:1) during 4 h with stationary promastigotes of L. amazonensis. After the infection time, the wells were washed with PBS and 5 × 10⁴ MSC were added to some wells and left for 48 h at 37°C and 5% of CO₂. The plate was washed again with warm PBS three times. Finally, the plate was fixed and stained with a fast panoptic kit (Laborclin, RJ, Brazil). The infection was analyzed by optical microscopy (CX31, Olympus, Japan).

One week after being received from the UFF, the animals were infected by subcutaneous route with 20 µL of PBS containing 2 × 10⁶ promastigotes of L. amazonensis on the right rear footpad. 7 and 37 days after infection, animals were treated with 1 × 10⁵ MSCs, either intralesional (i.l.) (into the plantar cushion of the right rear footpad) or intravenously (i.v.), through the external jugular vein. Control animals received the same volume (50 µL) of PBS by i.l. or i.v. routes. MSCs (1 × 10⁵ cells) were administered in 50 µL by intravenous (i.v.) injection or 20 µL by intralesional (i.l.) injection. The animals were sedated with sevoflurane (Sevorane^®, Abbot, IL, USA) for i.v. or i.l. administration.

The lesion growth was weekly monitored with a pachymeter in millimeter scale (Mitutoyo, Japan). The measurement of the uninfected footpad was then deducted. Forty-two days after infection, mice were euthanized and the infected footpad of each animal was removed, placed in a 70% alcohol solution for 1 min, and individually weighed. Additionally, the right popliteal lymph node was removed and placed in a vial with 1 mL of M199 medium. Footpads homogenates were obtained by manual maceration of the lesions with addition of 1 mL of M199 medium and, after sedimentation of the heavier particles, 10 µL of the supernatants were recovered and resuspended in 990 µL of medium, thereby obtaining a pre-dilution of 1:100. The number of parasites in the footpads, spleens, lymph nodes, and livers was determined by the limiting dilution assay (LDA). The macerates were diluted into 96-well culture plates (Jet Biofil, China) and incubated at 26°C for 15 days. Promastigotes cultures were observed with an optical microscope (Olympus, Japan) and the last well containing promastigotes was observed and noted (limiting dilution assay).

The cells of the macerate of the popliteal lymph node were quantified by counting in a Neubauer chamber (2 × 10⁶ cells per well in 24-well plates) to characterize T cells (CD4, CD8), regulatory T cells (CD4, CD25, FoxP3), and production of cytokines (IL-10, IFN-γ). The plates were centrifuged at 300 × g for 4 min. 100 µL of FACS buffer (PBS in 10% FBS) were added to the pellet and the cells were centrifuged again.

In the plate for characterization of regulatory T cells, non-specific-labeling blockage with anti-FCR (1:100) incubation was done for 5 min, followed by surface labeling with anti-CD4/Pecy7 (1:200) and anti-CD25/APC (1:200) for additional 30 min. The volume was made up to 200 µL in FACS buffer; the cells were centrifuged, followed by fixation in a kit fixation buffer (eBioscience) during 1 h. Then, the cells were washed and 100 µL of permeabilization buffer (eBioscience) kit were added to the pellet and incubated for 1 h, followed by centrifugation. Then, the cells were incubated with anti-FCR for 20 min and intracellular labeling was performed with anti-FoxP3/AlexaFluor 488 (1:50) incubating for 30 min. The cells were washed in permeabilization buffer and resuspended in FACS buffer for flow cytometry analysis (FACSCalibur system).

In the cytokine characterization plate, the cells were stimulated during 4 h with PMA (phorbol 12-myristate 13-acetate) (20 ng/mL), ionomycin (1000 ng/mL), and monensin (5 µL/mL) to inhibit cytokines secretion. Following the stimulation, the cells were washed in FACS buffer. Blockage of non-specific labeling with anti-FCR (1:100) incubation was done for 5 min, followed by surface labeling with anti-CD4/PeCy7 (1:200), anti-CD8/FITC for 30 min. The volume was filled to 200 µL in FACS buffer; the cells were centrifuged and further washed in FACS buffer. 100 µL of 1% paraformaldehyde was added to the pellet for fixation, which was left for 1 h incubation, followed by centrifugation. 100 µL of permeabilization buffer were added to the pellet (PBS and 0.1% saponin) and left for 1 h incubation, followed by centrifugation. The cells were incubated with anti-FCR for 5 min and then intracellular labeling was performed with anti-IL-10-PE (1:60) and anti-IFN-γ/APC (1:60) incubating for 30 min. The cells were washed in permeabilization buffer and resuspended in FACS buffer for flow cytometry (FACSCalibur system). The results were analyzed using software SUMMIT V4.3 Build 2445.

Concentrations of the cytokines present in the footpad homogenate supernatants were determined by the ELISA using commercial kits (BD OptEIA) according to the datasheet instructions. Recombinant murine IL-10, IL-4, and TGF-β were used to generate the standard curves. The detection limits for these tests are 31.3 pg/mL for TGF-beta and IL-10 and 15.72 pg/mL for IL-4.

No formal sample size calculation was performed to determine the numbers of animals per group; the sample size was based on the experience of our laboratory in previous studies using this model of cutaneous leishmaniasis (20). To compare in vitro data, Student’s t-test was used. Two-way ANOVA following Bonferroni’s post hoc test was used to compare all other parameters. P-values of 0.05 or less were considered meaningful. For in vivo experiments, we compared both groups, i.l. and i.v. This comparison between i.l. and i.v. was carried out besides of the differences between the two treatment protocols, according to the reviewer suggestion. All tests were analyzed using the statistical software package GraphPad Prism v6.0 (GraphPad Software, La Jolla, CA, USA).

Mesenchymal stromal cells adhered to plastic under standard tissue culture conditions and were able to differentiate into mesenchymal lineages, including osteocytes, chondrocytes, and adipocytes under in vitro conditions (data not shown). Additionally, MSCs expressed cell surface markers, such as: CD44, CD49 and Sca-1, bud did not express CD11b, CD45 and CD34 (6). We observed that in the third passage of the culture, most cells express CD44 (95%) (Figure S1A in Supplementary Material), CD49 (88.9%) (Figure S1B in Supplementary Material), and Sca-1 (91.2%) (Figure 1C in Supplementary Material) markers, and only a small fraction expressed CD11b (0.7%) (Figure S1D in Supplementary Material), CD45 (0.6%) (Figure S1E in Supplementary Material), and CD34 (0.4%) (Figure S1F in Supplementary Material) markers. Figure S1G in Supplementary Material represents the cell profile.

We decided to evaluate the effect of MSC in vitro on infected macrophages using coculture of macrophages and MSCs. Figure 1A shows macrophages infected with L. amazonensis and Figure 1B shows macrophages infected with L. amazonensis in coculture with MSC. The presence of MSC increased the parasite load on macrophages in vitro, with a meaningful difference in terms of total number of amastigotes between infected macrophages (MΦ + Leish) and infected macrophages in coculture with MSC (MΦ + Leish + MSC) (Figure 1C). The same was observed regarding the total number of Leishmania by the amount of infected macrophages (Figure 1D). This result suggests that MSC induced an increase of susceptibility of the macrophages in the in vitro infection.

To assess the efficacy of MSC treatment in vivo, BALB/c mice were infected and treated as described in the methodology. In relation to the lesion growth, treatment with MSCs by intravenous or intralesional route did not induce protection when compared with the controls (Figure 2A). However, treatment with MSC by intravenous route increased the parasite load in the infected footpad in relation to control (PBS i.v.) and other groups (PBS i.l. and MSC i.l.) (Figure 2B). The treatment with MSC by intralesional route did not affect the parasite load in comparison with control (PBS i.v.) (Figure 2B). There is no significant difference between any groups in terms of popliteal lymph nodes parasite load (Figure 2C). These results show that the use of i.v. therapy with MSC aggravated the infection caused by L. amazonensis at the infection site.

Based on the capacity of MSC therapy act systemically, we investigated the cell and cytokine profiles in the spleen. We evaluated the percentage of effector T cells (CD4⁺ CD25⁺ FoxP3⁻) and Treg cells (CD4⁺ CD25⁺ FoxP3⁺) by quantification of CD25⁺ and FoxP3⁺ markers in CD4⁺ T cells. In both groups, the percentages of effector T cells were similar (Figure 3A). However, there were increases of Treg cells in intravenous MSC treated animals when compared to PBS i.v. group, which was not observed when comparing MSC i.l. group to its control (PBS i.l.) (Figure 3B). It could be observed, by comparing intravenous versus intralesional treatment, that intralesional groups PBS i.l. and MSC i.l. showed an increase in relation to PBS i.v.; however, no difference was noted when compared with MSC i.v. These results suggest that Treg alone was not associated to an increase of parasite load.

We evaluated the percentage of IFN-γ and IL-10 produced by CD4⁺ and CD8⁺ T cells in the spleen. We observed the increase of CD4⁺ and CD8⁺ T cells producing IL-10 in mice treated with MSC i.v. in comparison to PBS i.v., but not with MSC i.l. in comparison to PBS i.l. (Figures 4C,G). When we compared intralesional versus intravenous treatment, it was observed that IL-10 produced by T CD4⁺ in MSC i.v. was increased in relation to PBS i.l. and MSC i.l., and no difference was observed between PBS i.v. against PBS i.l. and MSC i.l. The production of IL-10 by TCD8⁺ was increased in MSC i.v. in comparison to PBS i.l., but not in relation to MSC i.l. At the same time, we observed a reduction of IFN-γ produced by TCD8⁺, but not by CD4⁺ T cells, in animals treated with MSC i.v. in comparison to PBS i.v. No difference was observed on mice treated with MSC i.l. (Figure 4). No difference was observed in terms of production of IFN-γ by TCD4⁺ between the intravenous and intralesional treatments. However, in the production of IFN-γ by TCD8⁺, we observed a decrease of production of PBS i.l. and MSC i.l. in comparison to PBS i.v. and no difference was observed when compared MSC i.v. against PBS i.l. and MSC i.l. Altogether, the production of IL-10 by T CD4⁺ and T CD8⁺ cells in the group treated with MSC by i.v. is probably the major mechanism associated to increase the susceptibility to infection.

After observing the deleterious effect of MSC by the i.v. route and the increased production of IL-10 by CD4⁺ T cells and CD8⁺ T cells, we evaluated the profile of pathogenic cytokines in the lesion by ELISA on footpad homogenate. There was a significant increase in IL-10 (Figure 5A) in the groups treated with MSC by i.v. when compared to the group treated with PBS by i.v., a fact that was not observed in the MSC i.l. group in relation to its controls (PBS i.l.). No difference was noted between intralesional and intravenous treatments (Figure 5A). This result suggests the importance of IL-10 only in i.v. treatment. There were no meaningful differences between the treated groups when compared to their controls in terms of concentration of TGF-β (Figure 5B) and IL-4 (Figure 5C).