B-1 lymphocytes are murine cellular subpopulations that have their development and other characteristics that are distinct from the conventional B-2 cell populations. The B-1 cell population is a minor B-cell compartment that has been identified in mice and is involved primarily in innate immune responses. The interest in this cellular population has increased considerably due to its relationship with leukemia, autoimmunity and auto-reactivity[3-6]. Recently, several manuscripts demonstrated that B-1 cells are able to transform into a type of phagocyte called a B-1 cell-derived phagocyte (B-1CDP) and play a role as antigen presenting cells (APCs)[7-10]. Borrello and Phipps demonstrated that B-1 lymphocytes from the peritoneum of mice differentiate into phagocytic cells that are similar to macrophages.
Leishmania major (L. major) is the causative agent of cutaneous leishmaniasis (CL) in the Old World and is transmitted by the bite of the female phlebotomine sandfly, which injects the infective metacyclic promastigote forms into the dermis of the host. Cutaneous leishmaniasis is characterized by an ulcerative lesion that appears on the skin at the site of the sandfly bite and generally heals spontaneously. In experimental models of susceptibility, L. major infection induces a Th2-type immune response. In models of susceptibility to L. major infection, there is a production of anti-inflammatory mediators, which negatively modulate the response of the vertebrate host, favoring the establishment of infection[13-15]. Graf and collaborators demonstrated that B-1 cells express COX-1 and up-regulate COX-2 and prostaglandin production in response to inflammatory signals. Our group recently demonstrated that B-1 CDP cells are easily infected by L. major and exhibit high susceptibility to infection and that this mechanism is dependent on prostaglandin E2 (PGE2)/interleukin-10 (IL-10) production.
Based on these data, we investigated the interaction between B-1 cells and L. major-infected macrophages from BALB/c mice and BALB/c XID mice (a lineage that is genetically depleted of B-1 cells) to elucidate the possible influence of this minor B-cell population on the progression of infection in vitro. We performed experiments to investigate whether B-1 cells could mediate any effect on macrophage susceptibility to infection based on cell direct interaction or cytokine-driven modulation.
MATERIALS AND METHODS
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (United States). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Health Science Center of the Federal University of Rio de Janeiro (CEUA-CCS, Permit Number: IBCCF 062/14), and all efforts were made to minimize suffering.
Mice and parasites
BALB/c and BALB XID (a lineage that is genetically depleted of B-1 cells) mice of both sexes, aged 6-8 wk, were obtained from the Oswaldo Cruz Institute Animal Care Facility (Fiocruz, Rio de Janeiro, Brazil). C57BL/6 IL-10 knockout (KO) and C57BL/6 wild-type mice were kindly donated by Professor João Santana Silva from the Department of Pharmacology, School of Medicine, USP, Ribeirão Preto. L. major strain LV39 (MRHO/Sv/59/P) was isolated each month from the footpads of infected BALB/c mice and maintained in vitro as proliferating promastigotes. The parasites were maintained in Schneider’s medium (Gibco, Life Technologies) supplemented with 10% FCS, 1% glutamine and 2% human urine maintained in the animal facility at the Federal University of Rio de Janeiro (UFRJ).
B-1 cells were isolated from BALB/c mice using the protocol described by Abrahão et al. Briefly, macrophages were harvested via peritoneal lavage of BALB/c mice using cold DMEM medium (Gibco, Life Technologies). The total population of cells from the peritoneum was plated into 25 cm2 tissue culture flasks (Corning) and incubated at 37 °C in a 5% CO2 atmosphere for 120 min. The non-adherent cells were discarded, and DMEM medium containing 2 mmol/L glutamine, 50 μmol/L 2-ME, 10 μg/mL of gentamicin, 1 mmol/L sodium pyruvate, and 100 µmol/L MEM nonessential amino acids plus 10% fetal calf serum (FCS) was added to the adherent monolayer. The cultures were maintained for 5 d without changing the medium under the conditions described above. The non-adherent cell population was composed primarily of B-1 cells, as indicated by flow cytometry (results not shown), whereas the adherent cells represented an enriched macrophage population.
Macrophages and infection
Primary BALB/c or BALB/c XID peritoneal macrophages (2 × 105 cells/well) were cultured in 24-well plates (Corning) containing sterile round glass coverslips (13 mm) and allowed to attach for 2 h at 37 °C in 5% CO2. The adherent macrophages were infected for 24 h with stationary phase L. major promastigotes at a multiplicity of infection of 10:1 (parasite/macrophage) and were then incubated at 37 °C in 5% CO2. After 24 h, the monolayers were washed extensively with warm HBSS (Gibco, Life Technologies) to remove extracellular parasites. All cultures were maintained in medium containing 1% Nutridoma-SP (Roche, Basel, Switzerland) instead of FCS. B-1 cells were added at a 10:1 ratio (B-1 cell/macrophage) in the presence or absence of antibodies, solvents and reagents. After 3 d, infected macrophages monolayers were extensively washed to remove non-phagocytosed promastigotes, and medium was replaced by Schneider medium (Life Technologies), supplemented with 20% FCS and 2% human urine. Infected monolayers were cultured at 26 °C for additional 3 d. The number of motile promastigotes released into the cellular supernatant was evaluated using a Neubauer chamber.
Assessment of the intracellular load of L. major
The relative intracellular load of L. major was assessed by counting the number of motile extracellular promastigotes released in each well. Infected peritoneal macrophages cultured on glass coverslips in the presence or absence of B-1 cells for 3 d at 37 °C. After this time the cultures were washed and stained with May-Grunwald Giemsa (Sigma-Aldrich), and intracellular amastigotes were counted in 100 infected macrophages. The results are shown as the number of intracellular amastigotes per macrophages and as the percentage of infected macrophages. All results are presented as the mean and SE of triplicate cultures.
Antibodies, cytokines and inhibitors
Peritoneal macrophage monolayers were treated with 10 μg/mL of aspirin (Sigma-Aldrich). Aspirin acts as an inhibitor of PGE2 production[17,18]. Neutralizing anti-transforming growth factor) (TGF)-β and normal chicken IgY (R and D System), anti-IL-10 and rat IgG1 isotype control (BioSource Europe, Nivelles) antibodies were used at a concentration of 10 µg/mL.
Cytokine and PGE2 measurement
The concentrations of cytokines in the supernatants obtained from infected cell cultures were quantified after 24 h of incubation using the sandwich immunoassay (ELISA) method according to the methodology recommended by the manufacturer (R and D). The optical density was obtained by reading the absorbance in a plate spectrophotometer (Versamax Microplates Reader Molecular Devices, United States), with a filter of 405 nm. The concentrations of cytokines were calculated from a standard curve of recombinant cytokines. PGE2 was quantified using a PGE2-specific EIA kit, according to the methodology recommended by the manufacturer (Cayman Chemical, Ann Arbor, MI).
Lipid body evaluation
To observe lipid bodies (LBs), macrophages were fixed with 3.7% formaldehyde for 10 min and stained by osmium tetroxide. The morphology of fixed cells was observed, and osmium-stained lipid bodies were counted by light microscopy with a 100 × objective lens in 50 consecutively scanned leukocytes.
Statistical analysis was performed using the program GraphPad InStat version 3.01 (San Diego, CA, United States). The data were analyzed using a t-test. Differences with a P value of 0.05 or lower were considered to be significant.
Macrophage interactions with B-1 cells induce the growth of L. major
In this study, we analyzed the role of B-1 lymphocytes in modulating the replication of L. major in infected peritoneal macrophages in vitro. B-1 cells obtained from wild-type BALB/c mice and co-cultured with peritoneal macrophages from BALB/c and BALB XID mice infected with L. major promastigotes induced not only accentuated intracellular amastigote growth (Figure 1A) but also an increase in the percentage of infected cells (Figure 1B). The results obtained by counting the number of amastigotes in infected macrophages were confirmed via the quantification of motile promastigote forms in the supernatants after 5 d of interaction between B-1 cells and L. major-infected macrophages (Figure 1C). It is important to mention that XID macrophages were more resistant to infection with L. major than BALB/c macrophages (Figure 1).
Figure 1 Effect of B-1 cells co-cultured with macrophages infected with Leishmania major.
B-1 cells and peritoneal macrophages from BALB/c mice (white bars) or XID mice (black bars) were cultured (105/well) and infected with metacyclic promastigotes of L. major. After 24 h, the cell culture was washed and phagocytes were cultured for another 3 d with DMEM supplemented with 10% FBS at 37 °C. After this period, cells were stained and amastigotes inside the phagocytes were counted under the light microscope (A) and set the percentage of infected cells (B). To quantify promastigotes forms in the supernatants, the cells were cultures and infected with L. major. After 24 h of infection, so were washed and cultured at a temperature of 27 °C in Schneider medium 20% FBS during 5 d. After this period, the promastigotes were quantified in the supernatant of the cultures of infected phagocytes (C). All cultures were performed in triplicate and bars show the mean ± SD. Statistical analysis were performed by t-test from representative results of three different experiments. aP < 0.05, bP < 0.005 and dP < 0.0001.
The immunomodulatory factor of B-1 cells is not dependent on physical contact
We then assessed whether B-1/macrophage physical contact was required for the increased intracellular amastigote replication that was observed in our co-cultures. Increasing T. cruzi replication in macrophages requires recognition with apoptotic cells, thus directing the process of adhesion and phagocytosis[19-21]. However, when using a Trans-well system in our model of intracellular L. major growth, we found that parasite multiplication was independent of cell contact between B-1 cells and infected macrophages from both types of mice (Figure 2). These results indicate that in our system, the observed parasite growth is mediated by a soluble factor(s) that is released into the cultures.
Figure 2 The immunomodulatory effect of co-cultured infected macrophages and B-1 cells is independent of contact.
Peritoneal macrophages from BALB/c (A) and XID (B) mice were plated in 24 wells vessels at 105 cells/well in complete culture medium and infected with L. major (106/well). After 24 h of infection, cells were washed and B-1 cells were added at same compartment (Unsep), or separated (Sep) by a cell-impermeable culture insert. The cells were cultured in Schneider medium for 5 d at a temperature of 27 °C. After this period, the promastigotes were quantified in the supernatant of the cultures of infected phagocytes. All cultures were performed in triplicate and bars show the mean ± SD. Statistical analysis were performed by t-test from representative results of three different experiments. aP < 0.05 and bP < 0.005.
IL-10 released into the culture medium by B-1 cells controls L. major growth
B-1 cells are known to produce high levels of IL-10[13,22,23]. However, the importance of B-1-derived IL-10 in infected macrophages must be clarified. Recently, our group demonstrated that the IL-10 produced by B-1 CDP cells is important to facilitate intracellular infection and increase the number of motile L. major promastigotes in the supernatants. Based on this information, we investigated whether B-1 cell-derived IL-10 should be related in the susceptibility of macrophages to intracellular infection.
Our data show that the amount of IL-10 in the supernatants of macrophages infected with L. major is increased in the presence of B-1 cells (Figure 3A). The cytokine TGF-β, which is another important mediator involved in the modulation of macrophages infected with intracellular parasites[19-20], was not detected in the present study model (data not shown).
Figure 3 Interleukin-10 is a determinant factor for the increased parasite load in macrophages.
Peritoneal macrophages from BALB/c or XID mice co-cultured with B-1 cells were cultured in the presence or absence of L. major (MOI 10:1). After 24 h, the supernatant was collected and IL-10 was measured by ELISA (A). All cultures were performed in triplicate and bars show the mean ± SD. Representative result of three different experiments. aP < 0.05, bP < 0.005 and dP < 0.0001. Co-cultures of B-1 cells and infected macrophages from BALB/c or XID mice were treated or not with doses of monoclonal neutralizing anti-IL-10 or control isotype. Once were infected with L. major, after 24 h, the cell cultures were washed with DMEM and incubated 3 d and then passed to Schneider medium. After 5 d in medium Schneider, promastigotes were counted in the supernatant (B). Statistical analysis were performed by t-test from representative results of three different experiments and bars show the mean ± SD. aP < 0.05 and bP < 0.005.
To determine the real importance of B-1-derived IL-10 in L. major-infected macrophages, we added a neutralizing anti-IL-10 antibody to the cell cultures. Our results demonstrated that the neutralization of IL-10 induced an important reduction in the number of promastigotes released into the supernatant (Figure 3B and C). The data previously described demonstrate that the production of this cytokine by B-1 lymphocytes is relevant for increasing parasite growth in peritoneal macrophages.
The presence of B-1 cells induced lipid body formation and PGE2 production in infected macrophages
Lipid bodies (LBs) are organelles that are related in arachidonic acid metabolism and produce lipid mediators in response to inflammatory stimulus[24,25]. Infection with intracellular pathogens alters and increases the number of the lipid bodies[26,27] in infected cells. In recent years, we have described a progress of the number of lipid bodies in phagocytic cells infected with Trypanosoma cruzi and L. major and evidenced the role of these lipid bodies in disease development[13,26].
The results shown in Figure 4 demonstrate that infected macrophages co-cultured with B-1 cells contain a large number of lipid bodies in comparison to infected macrophages cultivated without B-1 cells (Figure 4). Our data demonstrate that macrophages from BALB/c mice contain more lipid bodies than XID macrophages, even in absence of a stimulus (Figure 4).
Figure 4 Presence of B-1 cells increased numbers of lipid bodies in infected macrophages.
Peritoneal macrophages from BALB/c or XID mice were incubated with glass coverslips; some cultures were infected with L. major. Stained with Osmium tetroxide, the slides were washed and stained with DAPI (Sigma). The morphology of fixed cells was observed, and Nile red LBs were counted by light microscopy with a 100 × objective lens in 50 consecutively scanned leukocytes (A). Representative images of lipid body formation in infected macrophages from BALB/c mice (B), infected macrophages from BALB/c mice cultured in the presence of B-1 cells (C), infected macrophages from XID mice (D) and infected macrophages from XID mice co-cultured with B-1 cells (E). Black arrows pointing to the LB. Statistical analysis were performed by t-test from representative results of three different experiments. aP < 0.05 and bP < 0.005.
Production of PGE2
The mechanism of LB formation and PGE2 synthesis during L. major infection in co-cultures of macrophages and B-1 cells was investigated. Phagocytes derived from B-1 cells (B-1CDP cells) constitutively express COX enzymes and produce large amounts of PGE2 in response to inflammatory signals or infection with Leishmania spp.. However, the production of the lipid mediator PGE2 by B-1 cells has not yet been described. Based on this information, we investigated the production of PGE2 by L. major-infected peritoneal macrophages obtained from BALB/c and XID mice. As described in Figure 5, the presence of B-1 cells induced considerable amounts of PGE2 in both types of infected macrophages. Our data also demonstrate that infected macrophages from XID mice produce less PGE2 than macrophages from BALB/c mice, even in the absence of B-1 cells (Figures 5 and 6).
Figure 5 Infected macrophages co-cultured with B-1 cells secret high levels of prostaglandin E2.
Infected macrophages from BALB/c or XID mice were incubated in the presence or absence of L. major (MOI 10:1). After 24 h of infection the supernatant was collected, and B-1 cells were added to the culture. After 24 h the level of PGE2 secreted was measured by EIA. All cultures were performed in triplicate and bars show the mean ± SD. Statistical analysis were performed by t-test from representative results of three different experiments and bars show the mean ± SD. aP < 0.05 and bP < 0.005.
To characterize the importance of PGE2 for parasite load, we added two different non-steroidal anti-inflammatory drugs (NSAIDs) to the B-1 cell/macrophage cultures: aspirin and indomethacin. These two drugs are recognized as potent inhibitors of PGE2 production[19,20]. Our data demonstrate that the blockade of PGE2 production affected the parasite burden inside the macrophages (Figure 6).
Figure 6 Blockage of the cyclooxygenase pathway induces protective phenotype in the Leishmania major infection.
Infected macrophages from BALB/c or XID mice macrophages were incubated in the presence or absence of L. major, 24 h after the cellular cultures were washed and B-1 cells were added. The co-culture was treated or not with aspirin (AAS) (10 mg/mL). After 24 h of incubation, the cells were washed and incubated again for 3 d. After this time, the promastigotes were counted in the culture supernatant. All cultures were performed in triplicate and bars show the mean ± SD. Statistical analysis were performed by t-test from representative results of three different experiments and bars show the mean ± SD. aP < 0.05, bP < 0.005 and dP < 0.0001.
Blockade of PGE2 by NSAIDs reduced IL-10 production by infected macrophages
Following the rationale that the inhibition of PGE2 decreased the release of promastigotes into the supernatant of L. major-infected macrophages co-cultured with B-1 cells, we investigated whether that lipid mediator has an effect on IL-10 production. The results shown in Figure 7 reveal that an important reduction of IL-10 production occurred when the cultures were treated with NSAIDs (Figure 7).
Figure 7 Effect of prostaglandin E2 inhibition on interleukin-10 production.
Infected peritoneal macrophages from BALB/c or XID mice were co-cultured with B-1 cells and incubated with or without aspirin (AAS) (10 μg/mL). After 24 h of incubation, the supernatant was collected and interleukin-10 measured by enzyme-linked immunosorbent assay. All cultures were performed in triplicate and bars show the mean ± SD. Statistical analysis were performed by t-test from representative results of three different experiments and bars show the mean ± SD. bP < 0.005 and dP < 0.0001.
B-1 cells isolated from IL-10 KO mice do not stimulate L. major growth
To demonstrate that the presence of the soluble cytokine IL-10 was essential for favoring intracellular infection, we used murine B-1 lymphocytes from IL-10 KO animals. When L. major-infected macrophages were co-cultured with B-1 cells from wild-type mice, the number of intracellular amastigotes (Figure 8A), the percentage of infected macrophages (Figure 8B) and the number of promastigotes released (Figure 8C) all increased. On the other hand, when infected macrophages were co-cultivated with B-1 cells from IL-10 KO mice, the modulatory effect that favors the parasite was not observed. These results confirm the importance of IL-10 released by B-1 cells in the modulation of macrophage infection by L. major.
Figure 8 B-1 cells from interleukin-10 deficient mice are more competent to control Leishmania major infection in macrophages.
To confirm the production of IL-10 is involved in the susceptibility to infection by L. major, we used B-1 cells from BALB/c (white bars) and from IL-10 KO mice (black bars). Our data demonstrate decreased in the number of intracellular amastigotes (A) and percentage of infected cells (B). We also observed the significant decrease in the liberated promastigotes forms by infected macrophages co-cultured with B-1 cells from IL-10 KO mice (C). Statistical analysis was performed by t-test from representative results of three different experiments and bars show the mean ± SD. bP < 0.005 and dP < 0.0001.
The findings reported in this manuscript are strengthened by experimental data. We co-cultivated B-1 cells with L. major-infected peritoneal macrophages from BALB/c mice and BALB XID mice, which are devoid of B-1 cells.
B-1 cells are of interest due to the immunomodulatory effects that these cells have exerted in different models of infection and inflammation[2,13,23,28-31]. B-1 cells from the peritoneal cavity migrate to inflammatory and/or infectious sites and release a series of immunomodulatory factors into the environment and also have the ability to differentiate into phagocytes[2,32]. Our group recently demonstrated a role for B-1 cell-derived phagocytes (B-1CDP) in L. major infection in vitro.
In the present study, we demonstrate that B-1 lymphocytes are efficient at inducing parasite replication when co-cultured with L. major-infected macrophages. The increased number of amastigotes observed in infected macrophages co-cultured with B-1 cells in our system is related to the number of motile promastigotes in the supernatants. Peritoneal macrophages isolated from XID mice exhibit resistance to infection with L. major when compared to BALB/c mice. However, when B-1 lymphocytes from BALB/c were added to the cultures of infected XID peritoneal macrophages in vitro, the cells became susceptible to infection, in comparison to co-cultures without B-1 cells. B-1 cells are known to produce large amounts of cytokines that could modulate macrophages and promote the intracellular infection of macrophages[13,23]. To determine if cell-cell physical contact was responsible for macrophage infection and parasite replication, we used a cell-impermeable membrane and observed that macrophage infection occurred in the absence of cell contact. This result demonstrates that soluble factors were being secreted into the medium by B-1 cells.
As reported in several studies, B-1 cells can produce large amounts of IL-10, which plays an important immunomodulatory action in different cell types[13,23,33]. IL-10 is an important mediator, which when released acts directly on different cell populations and is known to be an inhibitor of different types of cytokines, which are important factors for the activation of phagocytes[34-37] and consequently suppress the secretion of nitric oxide (NO). NO is an important factor that has leishmanicidal activity and favors the arginase pathway, which is involved in the activity of ornithine decarboxylase[19,38-40]. Arginase would facilitate the intracellular growth of pathogens[19,20].
In addition to IL-10, TGF-β is another cytokine that is important in modulating the intracellular growth of parasites[19,20,26]. TGF-β has been implicated in increased parasite replication in different in vivo and in vitro models of experimental Leishmania infection[41-44]. The results reported in this manuscript strongly suggest that macrophage susceptibility to infection with L. major was related to the production of IL-10 by B-1 cells. Our data also indicated that the TGF-β was not related in the increased intracellular infection (data not shown). The importance of IL-10 produced by B-1 cells was confirmed in our study when we added a neutralizing antibody anti-IL-10 and observed a marked decrease in the number of parasites released into the culture supernatants.
We also found a large number of lipid bodies in the cytoplasm of L. major-infected macrophages co-cultured with B-1 cells. Lipid bodies are lipid-rich cytoplasmic organelles that control the accumulation and hydrolysis of neutral lipids and are found primarily in adipocytes[45,46]. Macrophages were previously reported to have few lipid bodies in their cytoplasm, but the number of lipid bodies in these phagocytic cells can increase after stimulation[13,26]. T. cruzi and Leishmania are able to induce lipid bodies in the cytoplasm of infected macrophages[13,26]. It is already known that lipid bodies can be used as lipid sources that favor parasite replication or contribute to the production of soluble mediators involved in inflammation. Lipid bodies store arachidonic acid, which is important for the production of lipid mediators.
Our data demonstrate that the number of lipid bodies in the cytoplasm of infected macrophages increased when these cells were co-cultured with B-1 cells. In addition to the increased number of lipid bodies, we also observed strong production of PGE2 in the co-cultures. Our data also show that the inhibition of PGE2 modulated the infection, resulting in a decrease in the number of promastigote forms released in co-cultures of infected macrophages and B-1 cells. PGE2 is a lipid mediator that plays an important role in the production of factors, such as TGF-β and IL-10.
Our group recently described the production of anti-inflammatory mediators in experimental T. cruzi and Leishmania models, resulting in the inhibition of leishmanicidal products[13,19].
PGE2 is an important modulator of the T lymphocytes activation and the production of NO by phagocytes and favors the infection of intracellular parasites[19,21]. In addition, PGE2 is essential for increasing parasite growth in macrophages that have ingested apoptotic cells[19,21]. The presence of the lipid mediator PGE2 acts as a potent inhibitor of not only the immune response mediated by T lymphocytes but also the production of microbicidal factors by macrophages, favoring the growth of intracellular parasites[13,19,21]. In addition, PGE2 is a key lipid mediator that has been implicated in T. cruzi amastigote proliferation inside macrophages that have phagocytosed apoptotic bodies[19-21,26].
Our results demonstrate that the production of IL-10 by B-1 cells is involved in the immunomodulatory mechanism in infected macrophages and consequently favors the replication of intracellular parasites. This result clarifies the involvement of IL-10 produced by B-1 cells as the key factor for the modulation of macrophage activation. A similar result was obtained by Arcanjo et al, who used B-1CDP phagocytes from IL-10 KO mice and observed that intracellular infection with L. major was impaired. Another elegant work reported similar data in the context of in vitro infection by Coxiella burnetii. The authors observed that the macrophages of XID mice, which present a defect in the production of B-1 lymphocytes, have a better resistance to intracellular infection by C. burnetti when compared to the macrophages of wild-type mice.
In summary, our work demonstrates that one role of the B-1 cell population is to produce an anti-inflammatory cytokine and a lipid mediator that exert their effects on macrophages of the innate immune system, contributing to L. major growth and replication in vitro, probably by inhibition of NO and ROS production. Further studies should be addressed to investigate the importance of B-1 cells in the lesion sites highlighting a possible clinical significance of these cells in the infection.