Promastigotes of L. (L.) amazonensis LV79 strain (MPRO/BR/72/M1841) were cultured at 24°C in M199 medium supplemented with 10% fetal calf serum (FCS). Parasites were subcultured every 7 days at an initial inoculum of 2 × 10⁶/mL.

When indicated, promastigotes were incubated for 2 h with 200 ng/ml sCD100 in M199 at 24°C, centrifuged at 2,500 × g for 10 min and resuspended in RPMI 1640 medium supplemented with 10% FBS for subsequent infection of peritoneal macrophages.

All animals were used according to the Brazilian College of Animal Experimentation guidelines, and the protocols were approved by the Institutional Animal Care and Use Committee (CEUA) of the University of São Paulo (protocol number 001/2009).

sCD100 protein fused to Fc portion of IgG was produced in HEK (human embryonic kidney) 293T cells transfected with CD100-Fc plasmid, kindly given by Kumanogoh et al. (2000). Briefly, 7,5 × 10⁶ HEK cells were plated in DMEM with 5% serum “low-IgG” (Life Technologies) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate and 1× antibiotic-antimycotic solution (Life Technologies). Ten micrograms of CD100-Fc plasmid were mixed with 1 mL of a 150 mM NaCl solution and then with 100 μL of a polyethylenimine solution (PEI, Sigma) at 0.45 mg/mL. The total volume was added slowly to each dish, which was then incubated at 37°C with 5% CO2 for 7 days. After this period, supernatants of cell cultures were collected, filtered through a 0.22 μm membrane and centrifuged at 7,500 × g for 10 min. PMSF to 100 μM was added to the supernatant and proteins were precipitated with 60% w/v ammonium sulfate under slow stirring at 4°C for 24 h. Two successive centrifugations at 10,000 × g for 45 min were performed, precipitates were resuspended in PBS and centrifuged. The supernatant was incubated with protein G beads (1 mL Protein G Sepharose 4FF GE beads/50 mL supernatant) under rotation at 4°C for 24 h. The suspension was then centrifuged at 800 × g for 5 min and the beads were transferred to a chromatography column (Bio-Rad). The column was washed twice with 5 mL ice cold PBS and protein was eluted in aliquots of 500 μL using 0.1 M glycine buffer, pH 3.0, neutralized by 50 μl 1 M Tris buffer pH 8.0. Protein concentrations were determined by Bradford (Bio-Rad) and sCD100 was analyzed by SDS–PAGE and Western blot.

Peritoneal macrophages were isolated as previously described (Velasquez et al., 2016). We then transferred 8 × 10⁵ cells in RPMI 1640 pH 7.2 to each well of 24 well plates laid with 13 mm circular coverslips. After 2 h of incubation at 37°C in 5% CO₂, the medium was changed to RPMI with 10% FCS with or without 100 or 200 ng/mL sCD100 and cells were incubated until the next day. Infection was performed with L. (L.) amazonensis promastigotes at the beginning of stationary phase (day four) using a multiplicity of infection (MOI) of 5:1 for 4 h. After removing the non-internalized parasites, cells were further incubated for 24, 48, and 72 h with or without (control) sCD100. Control experiments for the effect of sCD100 Fc portion on Leishmania infection were performed using recombinant IgG1 and FcR blocker, and are described latter. Zymosan particles were incubated with macrophages at a ratio of 1:1 for 1 h to analyze the phagocytosis index. In Leishmania and zymosan experiments cells were fixed with methanol, stained with Giemsa and mounted with Entellan (Merck). One hundred macrophages were analyzed per glass slide to determine the proportion of infected cells (IM), the total number of amastigotes (AMA), amastigotes/infected macrophage and Infection Index or Phagocytosis Index (II = IM x AMA). Three coverslips were prepared for each condition.

Experimental procedures were carried as previously described (Teixeira A. A. et al., 2015). Briefly, peritoneal macrophages were seeded in 24 well plates laid with 13 mm circular coverslips as described above, with or without 200 ng/mL sCD100. Cells were then infected with tissue culture-derived trypomastigotes (Y strain) at MOI = 10 with or without 200 ng/mL CD100 (N = 3 for each condition) for 2 h at 37°C. The cells were then washed 10 times with PBS and further cultivated for 24 h to allow parasite differentiation. Macrophages were fixed with 4% paraformaldehyde, stained with anti-T. cruzi polyclonal antibody and propidium iodide and photographed with an epifluorescence microscope. Quantification was performed by counting the number of total and infected cells in at least 4 different fields (20 × magnification) for each replicate.

To monitor the effect of sCD100 Fc portion on Leishmania infection, incubations with L. (L.) amazonensis were performed in the presence of sCD100 and FcR blocker, and in the presence of human recombinant Fc portion of IgG1. To evaluate the role of CD72 in sCD100 effects, infections were done in the presence of anti-CD72. For FcR blocking: macrophages were plated and incubated with Fc receptor blockers (CD16 and CD32- BD Biosciences) at 0.1 ug/mL for 10 min, and then 200 ng/mL of sCD100 was added. Leishmania was added in the following day in the presence of FcR blocker and sCD100 for 4 and 24 h. For IgG1, macrophages were plated and incubated with 70 ng/mL of human recombinant IgG1 Fc (R&D Systems) and 200 ng/mL sCD100, and infected in the following day in the presence of the same proteins. For anti-CD72, plated macrophages were incubated with 10 μg/mL of anti-CD72 H-96 (Santa Cruz Biotechnology) and 1 h later sCD100 was added to 200 ng/mL. Infection was performed in the following day in the presence of both proteins. Control conditions included incubation only with sCD100 and with each treatment (Fc blocker or Fc-IgG1) separately, as well as untreated infected macrophages. The analysis and comparisons were based on infection rates.

Resident peritoneal macrophages from BALB/c mice were plated on glass slides as described and incubated with no stimulus or with 100 or 200 ng/mL of sCD100, or 100 or 200 ng/mL of BSA overnight.

For phagocytosis and for F-actin and CD100 labeling: Promastigotes of L. (L.) amazonensis were added in the proportion of 10 parasites per cell in the presence or absence of sCD100 or BSA and the plate was kept on ice for 2 h, and at 33°C with 5% CO₂ for different periods. After washing, macrophages were fixed with 4% paraformaldehyde for 10 min, washed with PBS 1X with 2% FBS, incubated for 30 min in 50 mM ammonium chloride and washed in PBS 1X with 2% FBS.

For labeling of CD100 and actin, cells were permeabilized with TBS containing 1% BSA and 0.1% Triton for 10 min. Slides were washed and incubated overnight with anti-CD100 (eBioscience) at 1:50 dilution, and after washing were incubated with anti-Rat IgG Alexa Fluor 568 (Thermo Scientific) diluted 1: 1000 and DAPI 1: 600 for 1 h. Alternatively, cells were incubated with phalloidin Texas Red (Molecular Probes) 1: 500 and DAPI 1: 600 for 1 h. In both cases, coverslips were washed five times with PBS and mounted in ProLong (Molecular Probes).

For analysis of phagocytosis glass slides were incubated overnight with anti-Leishmania serum diluted 1:75 in PBS 1X, washed five times with PBS and incubated for 1 h with a mix containing anti-mouse IgG Alexa Fluor 488 (Thermo Scientific) 1: 1000. After washing, permeabilization with TBS containing 1% BSA and 0.1% Triton for 10 min and further washing, phalloidin Texas Red (Molecular Probes) 1: 500 and DAPI 1: 600 were added for 1 h. After five washes, cells were mounted in ProLong (Molecular Probes). For calculation of the phagocytosis index, 500 macrophages were analyzed and promastigotes were quantified as attached (labeled in green and blue) or internalized (labeled only with blue- DAPI).

For CD72 labeling, slides containing non-infected macrophages were fixed as described. After washing they were incubated with anti-CD72 antibody M-96 (Santa Cruz Biotechnologies) 1:75 overnight, washed and incubated for 2 h with DAPI at 1: 600 and anti-rabbit Alexa Fluor 488 (Thermo Scientific) 1: 100. After labeling, coverslips were washed three times in PBS 1X with 2% FBS and cells were mounted in ProLong (Molecular Probes).

For CD100 quantification, images were acquired in a DMI6000B/AF6000 (Leica) fluorescence microscope coupled to a digital camera system (DFC 365 FX) and analyzed with the Image J program.

For actin and CD72 labeling and for the phagocytosis assay, images were captured using a Zeiss LSM 780 confocal laser scanning inverted microscope (Carl Zeiss, Germany) in a 1024 × 1024 pixel format. Image stacks comprised 8 images captured with a Plan-Apochromat 63×/1.4 DIC Oil M27 objective (Zeiss), applying a zoom factor of 1.0. Step intervals along the Z-axis ranged from 450 nm. Image acquisition and processing were performed using the Zen 2011 software (Zeiss, version 11.00.190).

SDS–PAGE (running gels with 10% acrylamide: bisacrylamide) and Western blots were performed as we previously described (Teixeira P. C. et al., 2015), using the following antibodies and incubation conditions: anti β-actin (Imuny, Brazil) 1: 1000 overnight and anti-rabbit IgG (H + L) (Imuny, Brazil) 1: 2000 for 1 h; anti-CD72 H-96 (Santa Cruz Biotechnology) 1: 200 overnight and anti-rabbit HRP (Imuny) 1: 1000 for 1 h, anti-GAPDH (Sigma-Aldrich) 1: 10000 overnight and anti-rabbit HRP (Imuny) 1: 1000 for 1 h. Normalizations for CD72 were done using anti-GAPDH while actin polymerization was estimated by F/G ratio.

Statistical analyses were done using t-test or one-way ANOVA followed by Tukey’s multiple comparison test, depending on the number of samples. Data were considered statistically different (^∗) when p < 0.05.

Although CD100 expression in macrophages has already been documented (Kikutani and Kumanogoh, 2003; Nkyimbeng-Takwi and Chapoval, 2011), its expression and effects during parasitic infection were never explored. Thus, we have performed labeling and quantification of CD100 protein in peritoneal BALB/c macrophages under controlled conditions and at different time points following infection with L. (L.) amazonensis promastigotes at a MOI (multiplicity of infection) of 10 parasites per macrophage. We observed that CD100 protein levels are altered following infection in a time dependent manner (Figure 1A): intracellular CD100 increases between 5 and 30 min after infection and then returns to basal levels within 4 h (Figure 1B).

After demonstrating that Leishmania infection increases macrophage CD100 endogenous levels, we evaluated whether the host cell infection by the parasite was affected by soluble CD100 (sCD100) added to the media. In the Leishmania lesion environment, sCD100 may be shed by macrophages, which express low levels of the protein (Sierra et al., 2008; Li et al., 2009; Luque et al., 2013), or by other cells, mainly activated T cells, which are known to release sCD100 (Wang et al., 2001). We thus analyzed the role of exogenous sCD100 on macrophage infection by Leishmania. We produced sCD100-Fc recombinant protein (from now on named as sCD100) in HEK293T and incubated macrophages with sCD100 before and together with L. (L.) amazonensis for different times.

Our results show that the number of amastigotes per cell (Figure 2A), and consequently infection rates (calculated as a product of the proportion of infected cells and the number of amastigotes) (Figure 2C), increase significantly in the presence of sCD100 in 4, 24, 48, and 72 h of infection. On the other hand, the percentages of infected macrophages (Figure 2B) do not change significantly over time with or without sCD100.

A possible explanation for the increase of infection by L. (L.) amazonensis induced by sCD100 could be the binding of this molecule to the parasite, promoting its adhesion to a CD100 receptor present on the macrophage or to Fc receptors, as sCD100 protein is fused to human Fc region of IgG1 (Kumanogoh et al., 2000). To verify the first hypothesis, we pre-incubated promastigotes with sCD100 prior to macrophage infection. Again, infection was performed for 4, 24, 28, and 72 h. No significant difference in the number of macrophages infected with L. (L.) amazonensis or in the number of amastigote forms inside individual infected cells was observed by pre-incubation with sCD100 relative to controls at all time points (Figure 3). These data indicate that sCD100 does not increase infection by direct contact/interaction with the parasite. On the other hand, when macrophages are pre-incubated with sCD100, the infection rate increases significantly (Figures 3A–D, control vs. sCD100).

Because the recombinant sCD100 that we used is produced as fusion between sCD100 and the Fc portion of IgG1, we next analyzed whether its effect on phagocytosis could be due to an interaction of the Fc IgG1 portion with the macrophage Fc receptor (FcR). To evaluate this possibility, macrophage infection assays were repeated in the presence of recombinant Fc region of human IgG1. The same control protein has been used in different studies, including one with Leishmania infection (Cortez et al., 2011). As expected, sCD100 alone increased macrophage infection by L. (L.) amazonensis while soluble IgG1 had no effect (Figure 4). Similar results were obtained when we blocked Fc receptor using the commercial blockers CD16 and CD32 (Supplementary Figure 1). Taken together, these results demonstrate that the increase in infection is directly mediated by sCD100.

CD72 is considered the main receptor for CD100 in macrophages (Kumanogoh et al., 2000; Ishida et al., 2003; Smith et al., 2011), but its expression has not been analyzed in murine peritoneal macrophages. We thus verified the expression of this receptor in these cells. By Western blot we observed the reactivity of CD72 immunospecific antibodies with a 45 kDa protein, the expected molecular weight for this receptor (Figure 5A, lane 1). Its expression is not altered upon incubation of the macrophages with sCD100, since we observed similar levels (ratios of CD72/GAPDH, the endogenous control used) in macrophages plated in the presence or not of sCD100 (Figure 5A, lane 2). An unrelated cell line (L929 fibroblast), which does not express CD72, was used as negative control (Figure 5A, lane 3). By immunofluorescence, we observed CD72 labeling in peritoneal macrophages (Figure 5B), confirming the presence of the receptor in these cells.

Next, we evaluated whether blockage of CD72 would affect sCD100-induced increase in macrophage infection. Macrophages were preincubated with anti-CD72 antibodies and infected with Leishmania. We observed that anti-CD72 antibodies alone have no effect on macrophage infection (Figure 5C, lane 2), as the number of infected cells is similar to control, and that sCD100 increases macrophage infection, as expected. However, when anti-CD72 antibody is added before sCD100, infection levels returned to basal levels (Figure 5C). These results indicate that sCD100 increases L. (L.) amazonensis infection in a CD72 dependent manner.

We have shown that sCD100 increases infection of macrophages after 4 h of incubation with Leishmania, suggesting that the molecule could participate in the initial steps of phagocytosis. To test this hypothesis, we performed a phagocytosis assay in which macrophages were plated (overnight) in the presence or absence of sCD100 and then incubated with promastigotes in the presence or abscence of sCD100 for only 5 min. Promastigotes attached to the host cell were visualized by fluorescent microscopy in green and blue (anti-Leishmania and DAPI, respectively), while internalized parasites were labeled only in blue (DAPI) (Supplementary Figure 2). Phagocytosis of Leishmania was assessed quantitatively by the percentage of infected macrophages and the total number of promastigotes phagocytosed. Phagocytosis of zymosan was also analyzed. An increase in infected macrophages and internalized promastigotes (Figures 6A,B) was observed in the presence of sCD100, indicating that it affects the initial steps of phagocytosis. Besides, there was a significant increase in the phagocytosis of zymosan (represented by the phagocytosis index) at both sCD100 concentrations tested (Figure 6C), indicating that this protein augments phagocytosis in general and not specifically of Leishmania parasites.

Since Leishmania is internalized in an actin-dependent process, we analyzed whether sCD100 modulated actin polymerization. Actin was analyzed in macrophages plated in the presence or abscence of sCD100 and infected with promastigotes in the presence or abscence of sCD100 for different periods. Figure 7A shows representative confocal images of immunofluorescence of macrophages incubated with sCD100 and infected for 30 min and 4 h, as well as the same conditions with no sCD100. The analysis of multiple fields at 4 h of infection showed that F-actin (polymerized actin, labeled with Texas Red Phalloidin) has a different organization in macrophages stimulated with sCD100. In fact, while a cortical distribution (arrowhead) of F-actin is shown in the absence of sCD100 (Figure 7A, image 3), the presence of sCD100 leads to a more heterogeneous and cytoplasmic distribution of polymerized actin (Figure 7A, image 4). A cytoplasmic pattern of F-actin was also visualized at 30 min of infection, in both conditions (Figure 7A, images 1, 2). Confirming our previous results, the presence of sCD100 at 4 h of infection increased the number of promastigotes attached (arrows) and internalized by macrophages (Figure 7A, images 6,5, respectively), suggesting a correlation between the effect of sCD100 on F-actin dynamics and Leishmania infection.

Since small differences in F-actin polymerization may not be perceived by immunofluorescence, we evaluated actin polymerization in control macrophages and in macrophages treated with sCD100, infected or not with L. (L.) amazonensis by Western blot (Figure 7B). The experiment was based on the separation of F-actin (insoluble) and G-actin (soluble) by ultracentrifugation, followed by blotting for actin in the two fractions. As a technical control, we employed extracts of macrophages treated with Latrunculin A, which blocks actin polymerization (Oliveira et al., 1996). Under all conditions F-actin band is about two times more intense than G-actin band, irrespective of sCD100 stimulus or infection by L. (L.) amazonensis (Figure 7B, representative experiment). Thus, no clear correlation between sCD100 and actin polymerization can be drawn. The control with Latrunculin A shows a clear decrease in the F-actin band, indicating that the separation of soluble and insoluble actin was efficient using this method.

We have here shown that sCD100 binds to macrophage CD72 and increases Leishmania phagocytosis. We have also shown that treatment of macrophages with sCD100 increases phagocytosis of zymosan, indicating that this effect is not specific for Leishmania. Therefore, we decided to investigate whether cCD100 could modulate infection of macrophage by other parasites.

Trypanosoma cruzi has several developmental stages, and the infective trypomastigotes can enter almost all nucleated cells of the vertebrate host, phagocytic or non-phagocytic. Tissue resident macrophages are importante targets for early infection, and parasite entry into these cells can occur by a phagocytic-like or a non-phagocytic mechanism (Epting et al., 2010; Barrias et al., 2013). We thus analyzed whether sCD100 could also affect macrophage infection by T. cruzi trypomastigotes. Trypomastigotes were incubated with macrophages in the presence or absence of sCD100 and the percentage of infected cells was quantified. Similar to Leishmania, we observed that sCD100 promoted a significant increase in cell infection by T. cruzi (Figure 8). These results suggest that CD100 may play an important role in macrophage infection by different trypanosomatid parasites.