Leukotriene B4 Modulates P2X7 Receptor–Mediated Leishmania amazonensis Elimination in Murine Macrophages

Mariana M. Chaves, Camila Marques-da-Silva, Ana Paula T. Monteiro, Cláudio Canetti and Robson Coutinho-Silva
J Immunol May 15, 2014, 192 (10) 4765-4773; DOI: https://doi.org/10.4049/jimmunol.1301058

Abstract

ATP is an important signaling molecule in the immune system, and it is able to bind the P2X7 purinergic receptor. Recently, our group showed that ATP-treated macrophages eliminate Leishmania amazonensis. It has been reported that leukotriene B4 (LTB4) reduces the parasitic load of infected macrophages. Additionally, it has been demonstrated that the P2X7 receptor can induce PLA2 activation and arachidonic acid mobilization. Based on these findings, we investigated whether LTB4 is produced upon P2X7 receptor activation and examined whether LTB4 modulates parasite elimination. Using macrophages lacking the P2X7 receptor, we observed that ATP was not able to reduce L. amazonensis load. This result suggests a role of the P2X7 purinergic receptor in parasite elimination. In addition, ATP was sufficient to induce LTB4 release from infected control macrophages but not from macrophages lacking the P2X7 receptor. Moreover, we found that ATP failed to decrease the parasitic load in 5-lipoxygenase (LO)–deficient macrophages. Treatment with the 5-LO inhibitor AA861 also impairs the ATP effect on parasitic loads. Furthermore, macrophages from 5-LO knockout mice eliminated L. amazonensis in the presence of exogenous LTB4, and macrophages obtained from P2X7 receptor knockout mice eliminated L. amazonensis when incubated with ionomycin. Finally, we demonstrated that in the presence of CP105696, an antagonist for LTB4 high-affinity receptor, ATP was not able to reduce parasitic load. These results indicate that P2X7 receptor activation leads to LTB4 formation, which is required for L. amazonensis elimination.

Introduction

Infection by the protozoan parasite Leishmania spp. leads to clinical presentations that can range from skin lesions to fatal visceral injuries. Leishmaniasis can be caused by at least 17 different subtypes of parasites and affects >12 million people worldwide in tropical and subtropical areas (1). Leishmania are organisms that have two forms in their life cycle and preferentially infect macrophages in their mammalian hosts. The natural infective form is promastigote, which, once inside macrophages, transforms itself into amastigotes within a period of 24–72 h postinfection (2). Many cases of leishmaniasis in Latin America are caused by L. amazonensis species, and this infection is considered a public health problem (3). Thus, understanding the infection caused by L. amazonensis and identifying approaches to minimize infection are of the utmost importance.

During an infection, inflammation is initiated by invading microorganisms through a small repertoire of conserved determinants usually called pathogen-associated molecular patterns (4). However, it is known that inflammation may also be initiated in the absence of pathogens (sterile inflammation) in cases in which there is cellular stress or tissue damage by molecules termed damage-associated molecular patterns (5). The presence of extracellular ATP (ATPe) is widely considered a type of damage-associated molecular pattern when it reaches high levels in the extracellular environment. This is caused by P2X7 receptor activation that induces inflammasome assembly and activation to generate proinflammatory mediators such as IL-1β and IL-18 (6, 7).

P2 receptors are activated by extracellular nucleotides such as ATP, and these receptors are regarded as the earliest factors supporting a wide range of biological effects in the short and long term (8). In the extracellular environment, ATP acts as a danger signal by binding to membrane receptors of the P2X and P2Y families. P2X receptors belong to the family of ionotropic receptors, which include seven homomeric receptor subtypes. The P2X1 to P2X7 receptors have been cloned in mammals (9). These receptor-dependent mechanisms were described by our group as modulators of infections caused by bacterial pathogens and protozoa (10, 11). Our group showed that ATPe was able to decrease the parasite load of macrophages infected with L. amazonensis and that these macrophages overexpress P2X7 receptor (12). Furthermore, P2 receptors have been associated with the modulation of lipid metabolism (13). Several studies have demonstrated the involvement of the P2X7 receptor in the synthesis of leukotrienes (LTs) through the activation of phospholipase A2 (PLA2) and arachidonic acid mobilization (14–16).

LTs are chemical messengers that transmit signals from cells of the immune system to virtually all cell types in the surrounding tissue (17). LTs are divided into two large families, LTB4 and cysteinyl-LTs, which activate distinct receptors in immune cells. The biosynthesis of LTs is triggered by stimuli such as Ags, cytokines, and immune complexes (18). The activation of LTs synthesis occurs during bacterial, fungal, viral, and protozoa infection in animal and humans models (19). LTB4 is essential for controlling infection with L. amazonensis (20). Thus, we hypothesized that P2X7 receptor activation by ATPe might cause LTB4 synthesis and release and cause the elimination of L. amazonensis from macrophages.

Materials and Methods

Chemical

DMEM, 199 medium, FBS, penicillin, and penicillin/streptomycin were obtained from Life Technologies/BRL (São Paulo, Brazil). ATP, hemin, and ionomycin were purchased from Sigma-Aldrich (St. Louis, MO). LTB4 was obtained from Cayman Chemical (Ann Arbor, MI). AA861 was purchased from Biomol Research Laboratories (Plymouth Meeting, PA), and CP 105696 was a gift from Pfizer Laboratories (Groton, CT).

Mice

Male or female BALB/c, C57BL/6, SV129, P2X7 receptor–deficient, and 5-lipoxygenase (5-LO)–deficient mouse strains aged 8–16 wk were used to obtain peritoneal macrophages. The animals were housed in temperature-controlled rooms and received water and food ad libitum. The transgenic animals were held in a special room designed for handling this type of mouse. The use of animals in this research project is authorized by Comissão de Ética no Uso de Animais–Universidade Federal do Rio de Janeiro under number IBCCF154.

Parasites

L. amazonensis (strain MHOM/BR/75/Josefa) amastigotes were obtained from popliteal lymph nodes of infected BALB/c mice. Axenic promastigotes were transformed at 27°C in 199 medium supplemented with 100 U/ml penicillin/streptomycin, 0.25% hemin, 2 mM l-glutamine, and 10% inactivated FBS.

Macrophage culture and infection

Macrophages were obtained from peritoneal cavity lavage of BALB/c, C57BL/6, P2X7 receptor–deficient, SV129, and 5-LO–deficient mice with 8 ml PBS. The cells were plated at a density of 2 × 105 cells/well in 24-well plates with 13-mm laminules or in 96-well plates. Nonadherent cells were removed by washing twice with PBS. Macrophages were cultured in DMEM medium supplemented with 2 mM l-glutamine, 100 U/ml penicillin/streptomycin, and 10% inactivated FCS. The macrophage monolayer was infected with 2 × 106 L. amazonensis promastigotes for 4 h at 37°C in 5% CO2. Noninternalized parasites were removed by washing twice with PBS. The infected macrophages were maintained for an additional 48 h at 37°C in 5% CO2.

Infection index

The infection index was obtained by the direct counting of infected cells using an optical microscope (Primo Star; Zeiss). After 48 h of infection, macrophages were fixed, stained with May-Grünwald-Giemsa (Panótico Rápido; Laborclin), and mounted on glass slides for analysis with an optical microscope. The infection index was measured by counting at least 100 cells in a total of five fields. The infected macrophages and average number of parasites per macrophage were counted. The results were expressed as the normalized infection index, which is the percentage of infected macrophages multiplied by the average number of amastigotes per macrophage (21). The index value is normalized to controls because L. amazonensis infectivity affects the infection index.

ATP measurements

The amount of released ATP was measured using an ATP bioluminescence assay (Sigma-Aldrich) kit according to the manufacturer's instructions. The supernatants (500 μl) from L. amazonensis–infected macrophages were examined with a bioluminometer (SpectraMax M5; Molecular Devices). ATP concentrations were determined through a standard curve fit.

LTB4 quantification

To measure the LTB4 released in the supernatant of cells, we performed enzyme immunoassays. The macrophages from BALB/c, C57BL/6, and knockout (KO) P2X7 receptor mice were plated in 96-well plates and infected or left untreated. Forty-eight hours postinfection, the supernatant was removed, and new medium without FBS was added. The cultures were then treated with ATP for 30 min. The culture supernatants were collected for detection using the LTB4 enzyme immunoassay (Cayman Chemical). The results were measured in an ELISA plate reader at a wavelength between 405 and 420 nm. The supernatants were stored at −80°C until analysis.

Image acquisition and analysis

Data were analyzed using GraphPad InsTat software (version 5.0; GraphPad Software). The results were expressed as the means ± SEM. The statistical analysis was performed by one-way ANOVA, followed by a Tukey multiple-range post hoc test and Student t test. Statistically significant differences between groups were considered for p < 0.05. Micrographs were digitalized using the AxioCam program.

Results

P2X7 receptor is involved in L. amazonensis elimination mediated by ATPe

We and others have suggested the participation of P2X7 receptors in ATP-induced control of many intracellular parasites (10, 11), including in L. amazonensis–infected macrophages (12). To test the involvement of the P2X7 receptor in the elimination mechanism of L. amazonensis, we used macrophages from P2X7 receptor–deficient mice. After stimulation with 500 μM ATPe, the P2X7 receptor–deficient macrophages did not exhibit decreases in parasite load (Fig. 1B, 1D, 1E). However, wild-type (WT)–infected macrophages showed decreased parasite load when exposed to ATPe (Fig. 1A, 1C, 1E). These results suggest a key role for this receptor in the elimination process.

FIGURE 1.
FIGURE 1.

P2X7 receptor is important in the elimination of L. amazonensis mediated by ATP. (A–D) Infected macrophages from WT or P2X7 receptor KO mice were treated with or without 500 μM ATP for 30 min. After 24 h, cells were May-Grünmwald-Giemsa stained, and the infection index was determined by direct counting with an optical microscope (E). (F) Supernatants of infected macrophages from WT mice were tested with a bioluminescent ATP detection assay. Data correspond to the mean ± SEM of n = 3 from three independent experiments performed in triplicate with pooled cells from three to four animals. Arrows represent vacuoles with L. amazonensis and asterisks correspond to empty vacuoles. #p < 0.05.

Previous studies have shown the ability of infecting pathogens to cause ATP release in a physiological form (22, 23). Thus, we measured the ATP release during infection with L. amazonensis at different time points. The results indicate that this pathogen leads to ATP release into the extracellular medium (Fig. 1F) and suggest that ATP released during infection by L. amazonensis is likely to act by physiologically controlling the infection. Moreover, the elimination of L. amazonensis via the P2X7 receptor does not involve apoptosis (data not shown).

ATP induces release of LTB4 in macrophages after P2X7 receptor activation

Previous studies have demonstrated that the P2X7 receptor regulates events in the LTs production cascade, such as PLA2 activation and arachidonic acid mobilization (14–16). Other works have shown that apoptosis triggered after activation of the P2X7 receptor is dependent on PLA2 and 5-LO (24). Based on these findings and on data indicating that LTB4 reduces the parasitic load of infected macrophages (20), we examined whether the ATPe-induced release of LTB4 requires the P2X7 receptor. Our data demonstrate that ATP was able to induce LTB4 production. However, infected macrophages had decreased LTB4 release compared with uninfected macrophages exposed to ATP. Infected macrophages had significantly higher LTB4 release than both infected and uninfected nonstimulated macrophages (Fig. 2A). We then analyzed whether the P2X7 receptor was involved in ATP-mediated LTB4 release. As shown in Fig. 2B, uninfected P2X7 receptor–deficient macrophages produced less LTB4 than WT macrophages when stimulated with ATPe. Moreover, infected P2X7 receptor–deficient macrophages did not induce LTB4 secretion upon ATPe stimulation and had levels similar to the control group (unstimulated macrophages). These results demonstrate the requirement of this receptor in ATP-induced LTB4 liberation in infected and uninfected cells. Those data suggest that ATPe leads to the release of LTB4 in different strains of mice.

FIGURE 2.
FIGURE 2.

P2X7 receptor–dependent LTB4 release in macrophages infected with L. amazonensis induced by ATP. BALB/c (A) and C57BL/6 or P2X7 receptor (B) KO macrophages infected or uninfected were treated with or without 500 μM ATP for 30 min, and the release of LTB4 was quantified by enzyme immunoassay. Data correspond to the mean ± SEM of n = 3 from three independent experiments performed in triplicate with pooled cells from three to four animals. #p < 0.05.

5-LO is important in the decrease of parasite load mediated by ATPe

The P2X7 receptor participates in the elimination of L. amazonensis and leads to LTB4 release. Serezani and coworkers (20) demonstrated that 5-LO participates in the elimination of L. amazonensis. Therefore, we investigated the importance of 5-LO in reducing the parasite load triggered by ATPe. To address this question, we used macrophages from WT (SV129 mice) and 5-LO KO mice. Infected macrophages derived from WT mice showed a decrease in parasite burden after ATP treatment (Fig. 3A, 3C, 3E). However, macrophages obtained from 5-LO KO mice did not show any difference in parasite load following ATPe treatment (Fig. 3B, 3D, 3E). Furthermore, pretreatment of macrophage cultures from BALB/c with a specific pharmacological inhibitor of 5-LO, AA861, also blocked the parasitic load decrease induced ATPe (Fig. 4). These data suggest that 5-LO is involved in the elimination of L. amazonensis mediated by ATPe. These data suggest that ATP eliminates L. amazonensis in macrophages from C57BL/6, SV129, and BALB/c. As control, we performed permeabilization experiments in the presence of AA861 and found no decrease of permeabilization in infected macrophages mediated by ATP. Thus, we can verify that this drug does not interfere with the direct functionality of P2X7 receptor (data not shown).

FIGURE 3.
FIGURE 3.

5-LO contributes to L. amazonensis elimination mediated by ATP. (A–D) Macrophages from WT or 5-LO KO mice were infected and treated or with or without 500 μM ATP for 30 min. After 24 h, cells were stained with May-Grünwald-Giemsa, and the infection index was determined by direct counting with an optical microscope (E). (F) Macrophages from WT mice were infected and immediately stained with May-Grünwald-Giemsa to determinate the infection index. Data correspond to the mean ± SEM of n = 3 from three independent experiments performed in triplicate with pooled cells from three to four animals. Arrows represent vacuoles with L. amazonensis and asterisks correspond to empty vacuoles. #p < 0.05.

FIGURE 4.
FIGURE 4.

Inhibition of 5-LO prevents ATP antiparasitic action. Infected macrophages derived from BALB/c mice were treated with (B) or without (A, C) 10 μM of AA861 for 30 min and then treated with 500 μM ATP for 30 min. After 24 h, the infection rate was determined as described in Materials and Methods (D). Data correspond to the mean ± SEM of n = 3 from three independent experiments performed in triplicate with pooled cells from three to four animals. Arrows represent vacuoles with L. amazonensis. #p < 0.05 compared with control macrophages.

Unexpectedly, we found that the infection index in cells derived from 5-LO KO mice was lower after 48 h of infection. We hypothesized that there was less Leishmania parasites phagocytosis by 5-LO KO cells or that the cells are able to control the infection. To test our hypothesis, macrophages were fixed after 4 h of infection. Fig. 3F shows that the input of L. amazonensis was decreased early in infection and suggests that macrophages derived from 5-LO KO mice have a reduced phagocytic capacity of L. amazonensis.

LTB4 causes the elimination of L. amazonensis in macrophages from 5-LO KO mice

To demonstrate the role of LTB4 in the elimination of L. amazonensis, we treated macrophages from 5-LO KO mice with exogenous LTB4 and evaluated whether the treatment reduced parasite load. Fig. 5 indicates that exogenous LTB4 significantly decreased parasite burden in macrophages from 5-LO KO mice. Ionomycin is a molecule widely used as an inducer of LTB4 release. We found that ionomycin treatment lead to elimination of L. amazonensis in macrophages from P2X7 receptor KO mice. This effect was reversed by pretreatment with an LTB4 receptor antagonist (CP105696; Fig. 6). These data reinforce the involvement of the LTB4–BLT1 axis during parasite elimination.

FIGURE 5.
FIGURE 5.

Elimination of L. amazonensis in 5-LO KO macrophages is restored by LTB4. Infected macrophages from 5-LO KO were treated or not with 100 nM LTB4 for 30 min (A, B). After 24 h, cells were fixed with May-Grünwald-Giemsa, and the infection index was determined by direct counting under microscope optical (C). Data correspond to the mean ± SEM of n = 3 from three independent experiments performed in triplicate with pool cells of the three to four animals. Arrows represent vacuoles with L. amazonensis, and asterisks correspond to empty vacuoles. #p < 0.05.

FIGURE 6.
FIGURE 6.

Ionomycin decreases parasite load in macrophages from P2X7 receptor KO mice infected with L. amazonensis. Infected macrophages obtained from P2X7 receptor KO mice were treated with or without 10 μM CP105696 for 30 min and then 10 μM ionomycin for 30 min (A–C). Twenty-four hours later, the infection rate was determined as described in Materials and Methods (D). Data correspond to the mean ± SEM of n = 3 from three independent experiments performed in triplicate with pooled cells from three to four animals. Arrows represent vacuoles with L. amazonensis, and asterisks correspond to empty vacuoles. #p < 0.05 compared with control macrophages.

BLT1 is needed to decrease the parasite load induced by ATPe

To investigate the necessity of activating the LTB4 high-affinity receptor BLT1 to eliminate L. amazonensis induced by ATPe, we used the specific BLT1 antagonist CP105696 in macrophage cultures from BALB/c mice. As previously observed, ATPe was able to decrease the parasitic load in macrophages from BALB/c, and CP105696 treatment impaired the effect of ATPe in parasite burden. This result suggests receptor involvement in the death of Leishmania after activation of the P2X7 receptor (Fig. 7). However, our data also show that infected macrophages treated with only CP105696 increase parasitic burden (Fig. 7D). CP105696 did not effect ATP-induced permeabilization in infected macrophages (data not shown), indicating that this inhibitor did not directly impair P2X7 activation.

Taken together, these data demonstrate that LTB4, via BLT1 receptors, are involved in the mechanism controlling L. amazonensis infection mediated by P2X7 receptors (Fig. 8).

FIGURE 7.
FIGURE 7.

BLT1 is necessary to decrease the parasitic load of macrophages infected with L. amazonensis. Infected macrophages derived from BALB/c mice were treated with or without 10 μM CP105696 for 30 min and then treated with 500 μM ATP for 30 min (A–C). Twenty-four hours later, the infection rate was determined as described in Materials and Methods (D). Data correspond to the mean ± SEM of n = 3 from three independent experiments performed in triplicate with pooled cells from three to four animals. Arrows represent vacuoles with L. amazonensis, and asterisks correspond to empty vacuoles. #p < 0.05 compared with control macrophages.

FIGURE 8.
FIGURE 8.

Elimination of L. amazonensis via P2X7 receptor is mediated by LTB4. During infection with L. amazonensis, ATP is released into the extracellular medium. This ATP is then able to activate the P2X7 receptor, which causes activation of the LTB4 production cascade. LTB4 is also released and activates specific receptors such as BLT1 receptor. In a manner not yet known, BLT1 receptor activation leads to elimination of L. amazonensis in macrophages. VP La, L. amazonensis parasitophorous vacuole.

Discussion

Our group has recently demonstrated the involvement of extracellular nucleotides such as ATP and UTP in the elimination of L. amazonensis (12, 25). We demonstrated that the elimination of L. amazonensis induced by ATPe is independent of NO production but does involve increased apoptosis of infected cells. However, the processes leading to L. amazonensis death have not yet been fully elucidated. Moreover, the literature data show that apoptosis triggered by the P2X7 receptor involves the participation of 5-LO (24). In 2006, Serezani and coworkers (20) demonstrated that LTB4 caused L. amazonensis elimination of infected macrophages in vitro and that 5-LO KO mice were more susceptible to infection. Our data suggest that the activation of the P2X7 receptor leads to the release of LTs, particularly LTB4, which causes the elimination of L. amazonensis.

The role of ATPe during protozoan infection has also been studied. It has been demonstrated that the P2X7 receptor participates in the elimination of many intracellular parasites (26). The role of the P2X7 receptor via ATPe has been demonstrated in Toxoplasma gondii infection. Many studies have shown that activation of the P2X7 receptor leads to death of this parasite. Correa and coworkers (11) showed that the elimination mechanism involves the production of reactive oxygen species and phagolysosomal fusion. Other studies have shown that the involvement of host cell apoptosis triggered by the P2X7 receptor is another elimination mechanism (27). The elimination of T. gondii and the role of the P2X7 receptor have also been shown using in vivo models (27).

This present study demonstrated that the involvement of LTB4 in L. amazonensis elimination requires the activation of the P2X7 receptor by ATPe. We observed that ATPe leads to an increased release of LTB4 in infected and uninfected macrophages relative to their respective controls and corroborates data from Barberà-Gremades and coworkers (28). Studies have shown the participation of the P2X7 receptor not only in the production of LTs by acting on the enzyme PLA2 (14), but also in the actual release of cysteinyl LTs in astrocytes (29). To demonstrate the involvement of the P2X7 receptor in the release of LTB4 mediated by ATP in control macrophages or macrophages infected with L. amazonensis, we measured LTB4 released from P2X7 receptor KO mouse macrophages. These macrophages had dramatically decreased LTB4 release and demonstrated that P2X7 receptor activation is the primary P2 receptor responsible in the LTB4 production induced by ATPe in macrophages. However, a small release was observed in uninfected macrophages, suggesting the activation of other purinergic receptors by ATP. Many studies in the literature have indicated the activation of purinergic receptors in the activation cascade of eicosanoid production. In 1983, Ochi and colleagues (30) reported that a 5-LO–dependent calcium reaction is stimulated by ATP. In 1998, Alzola and collaborators (14) showed that activation of the P2X7 receptor by ATP in cells of the submandibular gland in rats leads to activation of cytosolic PLA2 (cPLA2) and calcium-independent PLA2 and consequent secretion of kallikrein. Another report showed a decrease in arachidonic acid mobilization in the presence of P2X7 receptor antagonists such as oxidized ATP in P388D1 macrophages stimulated with LPS (15). Moreover, other studies have shown that lipid mediators can interact with P2Y receptors. Recently, it was reported that PGE2 selectively impairs P2Y receptor–mediated Ca2+ mobilization (31). Other works have demonstrated that pulmonary inflammation mediated by LTE4 requires P2Y12 receptor (32).

Notably, our results also showed that infected macrophages have a decreased LTB4 synthetic capacity compared with uninfected macrophages. This finding suggests that L. amazonensis inhibits the LT synthesis pathway as a prosurvival mechanism. It is already known that when activated, the P2X7 receptor causes two distinct mechanisms to uptake molecules <900 Da in the cell membrane. One of the mechanisms allows the entry of cations, and another allows the entry of anions (33). Marques-da-Silva and coworkers (34) showed that Leishmania inhibits the opening of cationic pores in infected macrophages when they are exposed to ATPe. It is already well known that Ca2+ is required for the activation of cPLA2 and 5-LO (35, 36). It is possible that the inhibition of cation pores by L. amazonensis decreases the entry of Ca2+ and leads to a decrease in the activity of cPLA2 and 5-LO enzymes, which reduces LTB4 release. A detailed investigation of this process is still required for a better understanding of how infection affects the production of LTB4 induced by ATP. Other studies in the literature have shown the ability of some pathogens to cleave certain types of lipid mediators. This result suggests the adaptation of parasites to hosts and reinforces the importance of these molecules as mediators of parasite control. Aliberti and coworkers (37) showed that T. gondii has the ability to cleave LTA4, a precursor for all LT classes in lipoxin A4, which is a lipid mediator with anti-inflammatory properties. The anti-inflammatory molecules prevent an immune response of Th1 cells. Furthermore, it has been shown that L. major produces PG from arachidonic acid using the enzyme PGF2α synthase encoded by the genome (38). Several possibilities exist to explain the lower LTB4 release by macrophages infected with L. amazonensis when stimulated with ATPe. For example, the existence of an enzyme that cleaves LTs expressed by L. amazonensis or a direct inhibitory effect on the key enzymes involved such as PLA2, 5-LO, and 5-LO–activating protein would explain our results.

The results showed that infected macrophages treated with CP105696, an antagonist for the LTB4 high-affinity receptor, have increased parasitic burden. However, infected macrophages from P2X7 receptor–deficient mice treated with CP105696 do not show increased infection. This result can be explained because P2X7 receptors modulate ATPe levels (39). The data suggest that P2X7 receptor activation leads to pannexin 1 channel opening and allows the secretion of ATP into the extracellular space. Thus, endogenous ATP could be leading to LTB4 basal release and endogenous infection control. However, in the absence of the P2X7 receptor, this does not occur.

After evaluating the parasitic load in macrophages from P2X7 receptor KO mice and the ability to promote LTB4 release by P2X7 receptor, we examined whether the enzyme 5-LO is important in reducing the parasitic load of macrophages infected with L. amazonensis. Our results showed that this enzyme participates in the L. amazonensis elimination mediated by ATPe. There are currently no studies showing the involvement of LTs in the elimination cascade of intracellular parasites mediated by nucleotides. To confirm the involvement of LTB4 in L. amazonensis elimination by infected macrophages, we treated macrophages from 5-LO KO mice with exogenous LTB4. The LTB4 treatment enhanced the elimination of Leishmania and is consistent with data previously shown by Serezani and coworkers (19).

It is already known that LTs participate in the elimination of pathogens such as Trypanosoma (40). However, the mechanism triggered for the deletion process is not well understood. Trypanosoma are also trypanosomatids like Leishmania. Thus, it is reasonable that the elimination mechanisms are similar for these two organisms. Many studies have shown the involvement of 5-LO during murine Chagas’ disease. Literature data show that LTB4 leads to an increase in NO synthesis, which participates in the resistance to the infection (41). However, Clark and coworkers (36) demonstrated in vivo that 5-LO KO mice have increased NO and IL-6 levels in plasma with a concomitant decrease in the expression of iNOS in cardiac tissue 12 d postinfection with T. cruzi. Another study demonstrated the release 5-LO metabolites triggered cell migration and caused severe inflammation in response to parasitized tissue. The inflammation generates myocarditis that is deleterious to the host. However, 5-LO KO mice have an increase in parasitemia (42). Thus, the data on the mechanism induced by 5-LO to eliminate T. cruzi are controversial and have not been confirmed.

In this study, we have demonstrated that Leishmania infection induces ATP release from infected cells. It is interesting to note that there are other endogenous P2X7 receptor ligands, such as cationic antimicrobial peptide LL-37 (43, 44). LL-37 is a cathelicidin-type peptide that destabilizes the surface of the pathogen membrane. LL-37 directly kills parasites and modulates innate and adaptive immune responses (45). In particular, LL-37 has been shown to kill Leishmania infections both in vitro (46, 47) and in vivo (48). Additionally, LTB4 induces the release of LL-37 in some cell types (49). Thus, it tempting to hypothesize that LL-37 may be acting in L. amazonensis–infected macrophages by favoring the innate immunity through paracrine activation of the P2X7 receptor. The possibility of LL-37 being released and participating in ATP-activated P2X7 receptor purinergic signaling deserves further investigation, and it is one focus of our future work.

This work demonstrated that P2X7 receptor activation by ATP eliminates L. amazonensis by activating the LTB4 cascade (Fig. 8). Based on this information, we suggest an involvement of LTB4 in the process of L. amazonensis elimination mediated by ATPe. More details of the process must be elucidated to better understand the general mechanisms of elimination mediated by extracellular ATP and its immunological role during infection by L. amazonensis. These results are important for the development of new drugs targeting leishmaniasis.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Pryscilla Braga for technical assistance and Gladys Corrêa for help in preparing the final figures.

Footnotes

  • This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, Programa de Núcleos de Excelência–Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Instituto Nacional de Pesquisa Translacional da Região Amazônica.

  • Abbreviations used in this article:

    ATPe
    extracellular ATP
    BLT1
    leukotriene B4 receptor 1
    cPLA2
    cytosolic phospholipase A2
    KO
    knockout
    5-LO
    5-lipoxygenase
    LTB4
    leukotriene B4
    PLA2
    phospholipase A2
    WT
    wild-type.

  • Received April 19, 2013.
  • Accepted March 10, 2014.

References

  1. ↵
    1. Natera S.,
    2. C. Machuca,
    3. M. Padrón-Nieves,
    4. A. Romero,
    5. E. Díaz,
    6. A. Ponte-Sucre
    . 2007. Leishmania spp.: proficiency of drug-resistant parasites. Int. J. Antimicrob. Agents 29: 637–642.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Ruhland A.,
    2. N. Leal,
    3. P. E. Kima
    . 2007. Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis. Cell. Microbiol. 9: 84–96.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Schwartz E.,
    2. C. Hatz,
    3. J. Blum
    . 2006. New world cutaneous leishmaniasis in travellers. Lancet Infect. Dis. 6: 342–349.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Janeway C. A., Jr..,
    2. R. Medzhitov
    . 2002. Innate immune recognition. Annu. Rev. Immunol. 20: 197–216.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Meylan E.,
    2. J. Tschopp,
    3. M. Karin
    . 2006. Intracellular pattern recognition receptors in the host response. Nature 442: 39–44.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Sanz J. M.,
    2. F. Di Virgilio
    . 2000. Kinetics and mechanism of ATP-dependent IL-1 beta release from microglial cells. J. Immunol. 164: 4893–4898.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Ferrari D.,
    2. C. Pizzirani,
    3. E. Adinolfi,
    4. R. M. Lemoli,
    5. A. Curti,
    6. M. Idzko,
    7. E. Panther,
    8. F. Di Virgilio
    . 2006. The P2X7 receptor: a key player in IL-1 processing and release. J. Immunol. 176: 3877–3883.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Burnstock G.
    2006. Purinergic signalling—an overview. Novartis Found. Symp. 276: 26–48, discussion 48–57, 275–281.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Donnelly-Roberts D. L.,
    2. M. F. Jarvis
    . 2007. Discovery of P2X7 receptor-selective antagonists offers new insights into P2X7 receptor function and indicates a role in chronic pain states. Br. J. Pharmacol. 151: 571–579.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Coutinho-Silva R.,
    2. L. Stahl,
    3. M. N. Raymond,
    4. T. Jungas,
    5. P. Verbeke,
    6. G. Burnstock,
    7. T. Darville,
    8. D. M. Ojcius
    . 2003. Inhibition of chlamydial infectious activity due to P2X7R-dependent phospholipase D activation. Immunity 19: 403–412.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Corrêa G.,
    2. C. Marques da Silva,
    3. A. C. de Abreu Moreira-Souza,
    4. R. C. Vommaro,
    5. R. Coutinho-Silva
    . 2010. Activation of the P2X(7) receptor triggers the elimination of Toxoplasma gondii tachyzoites from infected macrophages. Microbes Infect. 12: 497–504.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Chaves S. P.,
    2. E. C. Torres-Santos,
    3. C. Marques,
    4. V. R. Figliuolo,
    5. P. M. Persechini,
    6. R. Coutinho-Silva,
    7. B. Rossi-Bergmann
    . 2009. Modulation of P2X(7) purinergic receptor in macrophages by Leishmania amazonensis and its role in parasite elimination. Microbes Infect. 11: 842–849.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Costa-Junior H. M.,
    2. C. Marques-da-Silva,
    3. F. S. Vieira,
    4. L. C. Monção-Ribeiro,
    5. R. Coutinho-Silva
    . 2011. Lipid metabolism modulation by the P2X7 receptor in the immune system and during the course of infection: new insights into the old view. Purinergic Signal. 7: 381–392.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Alzola E.,
    2. A. Pérez-Etxebarria,
    3. E. Kabré,
    4. D. J. Fogarty,
    5. M. Métioui,
    6. N. Chaïb,
    7. J. M. Macarulla,
    8. C. Matute,
    9. J. P. Dehaye,
    10. A. Marino
    . 1998. Activation by P2X7 agonists of two phospholipases A2 (PLA2) in ductal cells of rat submandibular gland. Coupling of the calcium-independent PLA2 with kallikrein secretion. J. Biol. Chem. 273: 30208–30217.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Balboa M. A.,
    2. J. Balsinde,
    3. C. A. Johnson,
    4. E. A. Dennis
    . 1999. Regulation of arachidonic acid mobilization in lipopolysaccharide-activated P388D(1) macrophages by adenosine triphosphate. J. Biol. Chem. 274: 36764–36768.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. El Ouaaliti M.,
    2. M. Seil,
    3. J. P. Dehaye
    . 2012. Activation of calcium-insensitive phospholipase A(2) (iPLA(2)) by P2X(7) receptors in murine peritoneal macrophages. Prostaglandins Other Lipid Mediat. 99: 116–123.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Flamand N.,
    2. P. Mancuso,
    3. C. H. Serezani,
    4. T. G. Brock
    . 2007. Leukotrienes: mediators that have been typecast as villains. Cell. Mol. Life Sci. 64: 2657–2670.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Okunishi K.,
    2. M. Peters-Golden
    . 2011. Leukotrienes and airway inflammation. Biochim. Biophys. Acta 1810: 1096–1102.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Peters-Golden M.,
    2. C. Canetti,
    3. P. Mancuso,
    4. M. J. Coffey
    . 2005. Leukotrienes: underappreciated mediators of innate immune responses. J. Immunol. 174: 589–594.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Serezani C. H.,
    2. J. H. Perrela,
    3. M. Russo,
    4. M. Peters-Golden,
    5. S. Jancar
    . 2006. Leukotrienes are essential for the control of Leishmania amazonensis infection and contribute to strain variation in susceptibility. J. Immunol. 177: 3201–3208.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Lonardoni M. V.,
    2. M. Russo,
    3. S. Jancar
    . 2000. Essential role of platelet-activating factor in control of Leishmania (Leishmania) amazonensis infection. Infect. Immun. 68: 6355–6361.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Park E.,
    2. H. S. Na,
    3. Y. R. Song,
    4. S. Y. Shin,
    5. Y. M. Kim,
    6. J. Chung
    . 2014. Activation of NLRP3 and AIM2 inflammasomes by Porphyromonas gingivalis infection. Infect. Immun. 82: 112–123.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Pettengill M. A.,
    2. C. Marques-da-Silva,
    3. M. L. Avila,
    4. S. d’Arc dos Santos Oliveira,
    5. V. W. Lam,
    6. I. Ollawa,
    7. A. A. Abdul Sater,
    8. R. Coutinho-Silva,
    9. G. Häcker,
    10. D. M. Ojcius
    . 2012. Reversible inhibition of Chlamydia trachomatis infection in epithelial cells due to stimulation of P2X(4) receptors. Infect. Immun. 80: 4232–4238.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Costa-Junior H. M.,
    2. A. N. Mendes,
    3. G. H. Davis,
    4. C. M. da Cruz,
    5. A. L. Ventura,
    6. C. H. Serezani,
    7. L. H. Faccioli,
    8. A. Nomizo,
    9. C. G. Freire-de-Lima,
    10. Rda. C. Bisaggio,
    11. P. M. Persechini
    . 2009. ATP-induced apoptosis involves a Ca2+-independent phospholipase A2 and 5-lipoxygenase in macrophages. Prostaglandins Other Lipid Mediat. 88: 51–61.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Marques-da-Silva C.,
    2. M. M. Chaves,
    3. S. P. Chaves,
    4. V. R. Figliuolo,
    5. J. R. Meyer-Fernandes,
    6. S. Corte-Real,
    7. C. Lameu,
    8. H. Ulrich,
    9. D. M. Ojcius,
    10. B. Rossi-Bergmann,
    11. R. Coutinho-Silva
    . 2011. Infection with Leishmania amazonensis upregulates purinergic receptor expression and induces host-cell susceptibility to UTP-mediated apoptosis. Cell. Microbiol. 13: 1410–1428.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Coutinho-Silva R.,
    2. D. M. Ojcius
    . 2012. Role of extracellular nucleotides in the immune response against intracellular bacteria and protozoan parasites. Microbes Infect. 14: 1271–1277.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Miller C. M.,
    2. A. M. Zakrzewski,
    3. R. J. Ikin,
    4. N. R. Boulter,
    5. M. Katrib,
    6. M. P. Lees,
    7. S. J. Fuller,
    8. J. S. Wiley,
    9. N. C. Smith
    . 2011. Dysregulation of the inflammatory response to the parasite, Toxoplasma gondii, in P2X7 receptor-deficient mice. Int. J. Parasitol. 41: 301–308.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Barberà-Cremades M.,
    2. A. Baroja-Mazo,
    3. A. I. Gomez,
    4. F. Machado,
    5. F. Di Virgilio,
    6. P. Pelegrín
    . 2012. P2X7 receptor-stimulation causes fever via PGE2 and IL-1β release. FASEB J. 26: 2951–2962.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Ballerini P.,
    2. R. Ciccarelli,
    3. F. Caciagli,
    4. M. P. Rathbone,
    5. E. S. Werstiuk,
    6. U. Traversa,
    7. S. Buccella,
    8. P. Giuliani,
    9. S. Jang,
    10. E. Nargi,
    11. et al
    . 2005. P2X7 receptor activation in rat brain cultured astrocytes increases the biosynthetic release of cysteinyl leukotrienes. Int. J. Immunopathol. Pharmacol. 18: 417–430.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Ochi K.,
    2. T. Yoshimoto,
    3. S. Yamamoto,
    4. K. Taniguchi,
    5. T. Miyamoto
    . 1983. Arachidonate 5-lipoxygenase of guinea pig peritoneal polymorphonuclear leukocytes. Activation by adenosine 5′-triphosphate. J. Biol. Chem. 258: 5754–5758.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Través P. G.,
    2. M. Pimentel-Santillana,
    3. L. M. Carrasquero,
    4. R. Pérez-Sen,
    5. E. G. Delicado,
    6. A. Luque,
    7. M. Izquierdo,
    8. P. Martín-Sanz,
    9. M. T. Miras-Portugal,
    10. L. Boscá
    . 2013. Selective impairment of P2Y signaling by prostaglandin E2 in macrophages: implications for Ca2+-dependent responses. J. Immunol. 190: 4226–4235.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Paruchuri S.,
    2. H. Tashimo,
    3. C. Feng,
    4. A. Maekawa,
    5. W. Xing,
    6. Y. Jiang,
    7. Y. Kanaoka,
    8. P. Conley,
    9. J. A. Boyce
    . 2009. Leukotriene E4-induced pulmonary inflammation is mediated by the P2Y12 receptor. J. Exp. Med. 206: 2543–2555.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Schachter J.,
    2. A. P. Motta,
    3. A. de Souza Zamorano,
    4. H. A. da Silva-Souza,
    5. M. Z. Guimarães,
    6. P. M. Persechini
    . 2008. ATP-induced P2X7-associated uptake of large molecules involves distinct mechanisms for cations and anions in macrophages. J. Cell Sci. 121: 3261–3270.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Marques-da-Silva C.,
    2. M. M. Chaves,
    3. J. C. Rodrigues,
    4. S. Corte-Real,
    5. R. Coutinho-Silva,
    6. P. M. Persechini
    . 2011. Differential modulation of ATP-induced P2X7-associated permeabilities to cations and anions of macrophages by infection with Leishmania amazonensis. PLoS ONE 6: e25356.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Sharp J. D.,
    2. D. L. White,
    3. X. G. Chiou,
    4. T. Goodson,
    5. G. C. Gamboa,
    6. D. McClure,
    7. S. Burgett,
    8. J. Hoskins,
    9. P. L. Skatrud,
    10. J. R. Sportsman,
    11. et al
    . 1991. Molecular cloning and expression of human Ca(2+)-sensitive cytosolic phospholipase A2. J. Biol. Chem. 266: 14850–14853.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Clark J. D.,
    2. L. L. Lin,
    3. R. W. Kriz,
    4. C. S. Ramesha,
    5. L. A. Sultzman,
    6. A. Y. Lin,
    7. N. Milona,
    8. J. L. Knopf
    . 1991. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP. Cell 65: 1043–1051.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Aliberti J.,
    2. C. Serhan,
    3. A. Sher
    . 2002. Parasite-induced lipoxin A4 is an endogenous regulator of IL-12 production and immunopathology in Toxoplasma gondii infection. J. Exp. Med. 196: 1253–1262.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Kabututu Z.,
    2. S. K. Martin,
    3. T. Nozaki,
    4. S. Kawazu,
    5. T. Okada,
    6. C. J. Munday,
    7. M. Duszenko,
    8. M. Lazarus,
    9. L. W. Thuita,
    10. Y. Urade,
    11. B. K. Kubata
    . 2003. Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania. Int. J. Parasitol. 33: 221–228.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Brandao-Burch A.,
    2. M. L. Key,
    3. J. J. Patel,
    4. T. R. Arnett,
    5. I. R. Orriss
    . 2012. The P2X7 Receptor is an Important Regulator of Extracellular ATP Levels. Front. Endocrinol. (Lausanne) 3: 41.
    OpenUrlPubMed
  40. ↵
    1. Panis C.,
    2. T. L. Mazzuco,
    3. C. Z. Costa,
    4. V. J. Victorino,
    5. V. L. Tatakihara,
    6. L. M. Yamauchi,
    7. S. F. Yamada-Ogatta,
    8. R. Cecchini,
    9. L. V. Rizzo,
    10. P. Pinge-Filho
    . 2011. Trypanosoma cruzi: effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on levels of cytokines, nitric oxide and iNOS expression in cardiac tissue in the acute phase of infection in mice. Exp. Parasitol. 127: 58–65.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Talvani A.,
    2. F. S. Machado,
    3. G. C. Santana,
    4. A. Klein,
    5. L. Barcelos,
    6. J. S. Silva,
    7. M. M. Teixeira
    . 2002. Leukotriene B(4) induces nitric oxide synthesis in Trypanosoma cruzi-infected murine macrophages and mediates resistance to infection. Infect. Immun. 70: 4247–4253.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Pavanelli W. R.,
    2. F. R. Gutierrez,
    3. F. S. Mariano,
    4. C. M. Prado,
    5. B. R. Ferreira,
    6. M. M. Teixeira,
    7. C. Canetti,
    8. M. A. Rossi,
    9. F. Q. Cunha,
    10. J. S. Silva
    . 2010. 5-lipoxygenase is a key determinant of acute myocardial inflammation and mortality during Trypanosoma cruzi infection. Microbes Infect. 12: 587–597.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Chotjumlong P.,
    2. J. G. Bolscher,
    3. K. Nazmi,
    4. V. Reutrakul,
    5. C. Supanchart,
    6. W. Buranaphatthana,
    7. S. Krisanaprakornkit
    . 2013. Involvement of the P2X7 purinergic receptor and c-Jun N-terminal and extracellular signal-regulated kinases in cyclooxygenase-2 and prostaglandin E2 induction by LL-37. J. Innate Immun. 5: 72–83.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Elssner A.,
    2. M. Duncan,
    3. M. Gavrilin,
    4. M. D. Wewers
    . 2004. A novel P2X7 receptor activator, the human cathelicidin-derived peptide LL37, induces IL-1 beta processing and release. J. Immunol. 172: 4987–4994.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Doss M.,
    2. M. R. White,
    3. T. Tecle,
    4. K. L. Hartshorn
    . 2010. Human defensins and LL-37 in mucosal immunity. J. Leukoc. Biol. 87: 79–92.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    1. Kulkarni M. M.,
    2. W. R. McMaster,
    3. W. Kamysz,
    4. B. S. McGwire
    . 2009. Antimicrobial peptide-induced apoptotic death of leishmania results from calcium-de pend ent, caspase-independent mitochondrial toxicity. J. Biol. Chem. 284: 15496–15504.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Kulkarni M. M.,
    2. W. R. McMaster,
    3. E. Kamysz,
    4. W. Kamysz,
    5. D. M. Engman,
    6. B. S. McGwire
    . 2006. The major surface-metalloprotease of the parasitic protozoan, Leishmania, protects against antimicrobial peptide-induced apoptotic killing. Mol. Microbiol. 62: 1484–1497.
    OpenUrlCrossRefPubMed
  48. ↵
    1. Kulkarni M. M.,
    2. J. Barbi,
    3. W. R. McMaster,
    4. R. L. Gallo,
    5. A. R. Satoskar,
    6. B. S. McGwire
    . 2011. Mammalian antimicrobial peptide influences control of cutaneous Leishmania infection. Cell. Microbiol. 13: 913–923.
    OpenUrlCrossRefPubMed
  49. ↵
    1. Rivas-Santiago B.,
    2. R. Hernandez-Pando,
    3. C. Carranza,
    4. E. Juarez,
    5. J. L. Contreras,
    6. D. Aguilar-Leon,
    7. M. Torres,
    8. E. Sada
    . 2008. Expression of cathelicidin LL-37 during Mycobacterium tuberculosis infection in human alveolar macrophages, monocytes, neutrophils, and epithelial cells. Infect. Immun. 76: 935–941.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section