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Infection and Immunity, December 2008, p. 5543-5552, Vol. 76, No. 12
0019-9567/08/$08.00+0     doi:10.1128/IAI.00683-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Trypanosoma cruzi Infection Is Enhanced by Vector Saliva through Immunosuppressant Mechanisms Mediated by Lysophosphatidylcholine

Rafael D. Mesquita ,^1,, Alan Brito Carneiro,^1, André Bafica,² Felipe Gazos-Lopes,¹ Christina M. Takiya,³ Thaís Souto-Padron,⁴ Danielle P. Vieira,⁴ Antônio Ferreira-Pereira,⁴ Igor C. Almeida,⁵ Rodrigo T. Figueiredo,⁴ Bárbara N. Porto,⁴ Marcelo T. Bozza,⁴ Aurélio V. Graça-Souza,¹ Angela H. C. S. Lopes,⁴ Geórgia C. Atella,¹ and Mário A. C. Silva-Neto^1*

Instituto de Bioquímica Médica,¹ Instituto de Ciências Biomédicas, Departamento de Histologia e Embriologia,³ Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, UFRJ, 21940-590 Rio de Janeiro, Rio de Janeiro, Brazil,⁴ Divisão de Imunologia, Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brasil,² Department of Biological Sciences, The Border Biomedical Research Center, University of Texas at El Paso, El Paso, Texas 79968⁵

Received 30 May 2008/ Returned for modification 12 July 2008/ Accepted 4 September 2008

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Trypanosoma cruzi, the etiological agent of Chagas disease, is transmitted by bug feces deposited on human skin during a blood meal. However, parasite infection occurs through the wound produced by insect mouthparts. Saliva of the Triatominae bug Rhodnius prolixus is a source of lysophosphatidylcholine (LPC). Here, we tested the role of both triatomine saliva and LPC on parasite transmission. We show that vector saliva is a powerful inducer of cell chemotaxis. A massive number of inflammatory cells were found at the sites where LPC or saliva was inoculated into the skin of mice. LPC is a known chemoattractant for monocytes, but neutrophil recruitment induced by saliva is LPC independent. The preincubation of peritoneal macrophages with saliva or LPC increased fivefold the association of T. cruzi with these cells. Moreover, saliva and LPC block nitric oxide production by T. cruzi-exposed macrophages. The injection of saliva or LPC into mouse skin in the presence of the parasite induces an up-to-sixfold increase in blood parasitemia. Together, our data suggest that saliva of the Triatominae enhances T. cruzi transmission and that some of its biological effects are attributed to LPC. This is a demonstration that a vector-derived lysophospholipid may act as an enhancing factor of Chagas disease.

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Chagas disease is caused by the trypanosomatid protozoon Trypanosoma cruzi. The disease was described in the early twentieth century by Carlos Chagas, who reported the pathophysiological aspects and also its mechanism of transmission in a series of studies published in 1909 (16-18). Unfortunately, almost 100 years later, specific and efficient methods to block the transmission of this parasite or treat this condition still remain controversial (64). The current number of infected patients is 11 million, with 200,000 new cases and at least 21,000 associated deaths each year (22). The mammalian host cycle starts with the feeding of an infected triatomine bug, which eliminates infective metacyclic trypomastigotes in its feces near the insect bite injury. The host eventually scratches the region, resulting in contamination of the bite wound with the bug's feces. Following entrance into the bloodstream, parasites initially invade mononuclear cells, where they differentiate into amastigote forms that multiply and differentiate into intracellular trypomastigotes. In fact T. cruzi can ultimately infect any nucleated cell of the body. This leads to lysis of invaded cells and initiates several cycles of cellular invasion by extracellular trypomastigotes. In the chronic stage of the disease, blood parasitemia gradually decreases to minimum levels due to the host immune response. However, the parasite levels are high enough to allow the infection of triatomine bugs that may eventually feed on the same host. In the infected vector, trypomastigotes differentiate into epimastigotes and undergo another round of differentiation in which metacyclic trypomastigotes are generated and then released in the feces in the next blood-feeding cycle (5, 32). Despite the fact that two bug-derived biological fluids (feces and saliva) are inoculated into the bite wound, no vector-derived molecules have been described as playing a role in the mechanism of T. cruzi infection of vertebrate hosts. Previously, we demonstrated the presence of phospholipids in the saliva of a blood-sucking insect, Rhodnius prolixus, one of the main vectors of Chagas disease in South America (24). Also in that study, we showed that lysophosphatidylcholine (LPC) is a component of this insect's salivary secretion and may act as an antihemostatic molecule during blood feeding. To that end, we demonstrated that it blocks platelet aggregation and induces nitric oxide production in cultured endothelial cells.

LPC is a phospholipid produced by the hydrolysis of phosphatidylcholine (PC) by phospholipase A₂ (PLA₂). Its role as a powerful modulator of vascular biology was first indicated by the finding that endothelium-dependent arterial relaxation occurred due to the presence of LPC in oxidized low-density lipoprotein (LDL) (33). Two-thirds of LDL is composed of PC, although half of this may be converted to LPC during oxidative events (39). An increase in the LPC concentration in oxidized LDL results from the oxidation of PC sn-2 residues, followed by the hydrolysis of the fatty acyl chain by several enzymes in the plasma, such as secretory PLA₂ (sPLA₂), platelet-activating factor (PAF)-acetylhydrolase, lecithin:cholesterol acyltransferase, and paraoxonase (28, 39). Therefore, human plasma's concentration of LPC is high (140 to 150 µM) and is able to trigger several steps of the atherogenic process. LPC carries out this function mainly through its interactions with different cells. The receptors involved in these interactions have not been thoroughly characterized (19, 42). LPC has long been recognized as a potent inducer of human monocyte and T-lymphocyte chemotaxis (40, 48, 50). T-cell migration was shown to depend upon the ability of LPC to interact with the G2A receptor, which induces its redistribution to the cell surface. However, the intracellular signaling events that mediate this redistribution are still being elucidated (50). LPC also induces the expression of adhesion molecules on endothelial cells, which further promotes monocyte recruitment (67). Recently, it was found that apoptotic cells promote their own phagocytosis by generating extracellular LPC, which guides professional phagocytes to the inflammation site (34). This finding raised the possibility that extracellular LPC may be generated from an intracellular pool and through a lipoprotein-independent pathway.

LPC acts as a chemotactic molecule when it is released from apoptotic cells. It induces the migration of phagocytes, which ensures the appropriate removal of such cells and avoids secondary necrosis (34). Defects in this system may result in impairment of apoptotic cell clearance and trigger autoimmune diseases (6). Several stimulatory effects of LPC have been reported for immune cells, including macrophages. It inhibits the production of several cytokines, such as tumor necrosis factor alpha (TNF-), interleukin-12 (IL-12), and IL-6, resulting in lower levels in the peritoneal-lavage fluid of mice after cecal ligation and puncture (68). In addition, it enhances bacterial clearance in vivo by modulating neutrophil bactericidal activity. Proinflammatory and effector molecules, such as IL-12 and nitric oxide (NO), are known to be critical for the control of T. cruzi infection (15, 46).

In human skin, the extracellular concentration of lysophospholipid is likely very low due to the absence of true lipoprotein carriers, such as LDL. Even so, the presence of sPLA₂ may result in the release of considerable amounts of LPC, which triggers signaling cascades in several cell types present in human skin (60). Due to the many effects of LPC on vascular biology, we tested the abilities of Rhodnius bug saliva and LPC to elicit cell responses and contribute to the transmission of T. cruzi. Here, we show that vector-derived saliva and LPC are powerful inducers of pathogen infection and Chagas disease transmission.

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Chemicals. EDTA, EGTA, NaN₃, culture medium RPMI 1640, liver infusion tryptose, Dulbecco's modified Eagle's medium, Giemsa and trypan blue stains, molecular-weight standards, a cocktail of protease inhibitors, Tris, glycine, acrylamide, bisacrylamide, N,N,N',N''-tetramethylethylenediamine (TEMED), dimethyl sulfoxide, B-type bovine gelatin, folin reagent, PC, phosphatidylethanolamine, and phosphatidylinositol were purchased from Sigma Chemical Company (St. Louis, MO). Whenever indicated, LPS from Escherichia coli was used in the experiments. Bovine serum albumin (fraction V) was purchased from Calbiochem (La Jolla, CA). Sodium fluoride, Na⁺-K⁺-bitartrate, and trichloroacetic acid were purchased from Reagen (Rio de Janeiro, Rio de Janeiro, Brazil). Sterile apirogenic saline (0.9% NaCl) was purchased from Darrow Laboratory S/A (Rio de Janeiro, Rio de Janeiro, Brazil). Ficoll (Lymphoprep) was acquired from Axis-Shield (Oslo, Norway).

Experimental animals and insects. BALB/c mice weighing 20 to 25 g were obtained from the animal facility of Instituto de Microbiologia, Prof. Paulo de Góes, UFRJ (Rio de Janeiro, Brazil). These animals were used throughout this study. The animals were maintained in a room with constant temperature and humidity and had free access to a pelleted diet and water. In some experiments, Toll-like receptor 2-deficient (TLR2^–/–) animals, obtained from S. Akira (Osaka University, Osaka, Japan) via D. Golenbock (University of Massachusetts Medical School, Worcester, MA), and their controls, wild-type C57BL/6 mice (Taconic Farms, Germantown, NY), were used. These animals were bred and maintained at an American Association of Laboratory Animal Care-accredited facility at the National Institute of Allergy and Infectious Diseases, National Institutes of Health. Insects were unfed fifth-instar nymphs taken from a colony of R. prolixus maintained at 28°C and 70% relative humidity in Hatisaburo Masuda's laboratory at UFRJ, Rio de Janeiro, Brazil. In this study, the mice used as donors of blood or peritoneal macrophages or for in vivo studies were handled according to the federal guidelines and institutional policies for the treatment of vertebrate animals.

Preparations of saliva and LPC from R. prolixus LPC. The insects were cleaned with water and ethanol. The salivary glands were dissected in sterile apyrogenic saline (0.9% NaCl) at a final concentration of three pairs of salivary glands per 10 µl. Sterile saliva was collected by gentle disruption of the salivary glands with forceps and centrifugation at 11,000 x g for 5 min. A salivary gland pair typically yields 100 µg of highly concentrated saliva by this technique (41). This procedure also allows for easy quantification of the amount of saliva used and for simple serial dilutions of saliva in all experiments. The protein content of the samples was determined by using Lowry's method (36); bovine serum albumin served as the protein standard. LPC was obtained by the extraction of total lipids from salivary glands and feces. It was purified by thin-layer chromatography as described previously (24). R. prolixus LPC from saliva is henceforth designated RpLPC. By accounting for the losses that occur during RpLPC isolation, 100 µg of collected saliva generally yields 0.15 µg of RpLPC and 0.9 µg of R. prolixus PC. Assuming that the volume of the salivary gland lumen is approximately 2 µl, the concentrations of RpLPC and R. prolixus PC inside the gland are 150 µM and 900 µM, respectively (24). These data indicate that the maximum amount of RpLPC injected at once would be 300 pmol. Values close to those estimates (300 pmol and 150 µM) were used as the basis for most of the experiments. In addition to RpLPC, we used commercial LPC with the same fatty acid composition obtained from Sigma Chemical Company (St. Louis, MO) in some experiments for this study.

Chemotaxis assays. (i) Neutrophil in vitro chemotaxis. Neutrophil purification and the chemotactic assay were performed as previously described (47). Briefly, blood from healthy volunteers was drawn into a syringe containing heparin. The blood was diluted twofold with Hanks saline (HBBS), 20 ml was layered over a 10-ml Ficoll gradient (Lymphoprep; Axis-Shield), and the gradient was centrifuged (45 min at 500 x g). The neutrophil pellet was suspended in 12 ml of ice-cold 0.15 M KCl for red blood cell hemolysis, followed by another centrifugation (10 min at 500 x g). The neutrophils were suspended in RPMI 1640 without fetal calf serum (FCS), and their purity and viability were always between 97 and 99%. Chemotaxis was tested in vitro in ChemoTx system 96-well plates (NeuroProbe), using either saliva or LPC dilutions in the lower chamber (300 µl final volume) and 5 x 10⁵ neutrophils in 50 µl in the upper chamber. The final amounts of LPC or estimated amounts of RpLPC in whole saliva are indicated in figures. The system was then incubated for 2 h at 37°C in a 5% CO₂ atmosphere. The number of neutrophils in the lower chamber was counted. This value was normalized by dividing the number of translocated cells following the stimulus by the number that translocated by random migration (RPMI only). The chemotaxis index was represented as a percentage of that of the positive control (10 nM leukotriene B₄). Blood from healthy volunteers was drawn according to the federal guidelines and institutional policies for the treatment of human beings.

(ii) In vivo chemotaxis on mouse skin. Four-week-old male BALB/c mice were previously cleaned with a 3.33 g/liter solution of neguvon/assuntol every other day for 1 week in order to eliminate ectoparasites. Ten days after the last cleaning procedure, mice were anesthetized with an intramuscular injection of ketamine (100 mg/kg of body weight) in the leg. In order to determine the field for inoculation in mouse skin, animals were tricotomized in a circular area with a 0.5-cm radius. Each animal was inoculated subcutaneously with three injections of 10 µl each by using a 10-µl Hamilton microsyringe. The injections contained 200 µg/ml of saliva (total protein 2 µg) or 150 µM LPC (0.75 µg of commercial LPC) in 0.9% sterile saline. Parallel controls were run in which each of the animals received a 10-µl injection of sterile saline. Twenty-four hours later, the animals were killed by cervical disruption and skin biopsies were carried out as follows. Briefly, defined skin fragments were removed and immediately fixed in Bouin's solution. Eight hours later, the skin fragments were dehydrated with increasing concentrations of ethanol in water, clarified in xylene, and blocked in paraffin. Five-micrometer-thick slices were obtained, stained with hematoxylin and eosin (H&E), and observed under an optical microscope (Nikon Eclipse E800, Japan).

RpLPC purification and MS analysis. RpLPC dissolved in chloroform/methanol (1:1, vol/vol) with 0.1% formic acid was analyzed by electrospray ionization-mass spectrometry (ESI-MS) using a Finnigan LCQ DUO ion trap mass spectrometer (Thermo Fisher, San Jose, CA). This solution was injected (5 µl/min flow rate) into the ESI-MS source by using a fused-silica capillary (75 µm by 250 µm). In all analyses, the capillary temperature was kept at 200°C and the voltage at –47 eV. Spectra were collected in the positive-ion mode within the 200 to 2,000 mass/charge (m/z) range, with scan intervals of 3 s. Parent ion fragmentation (tandem ESI-MS) experiments were carried out with 30 to 40% (1.5 to 2.0 eV) relative collision energy. The parameters were optimized by using a commercial standard of PCs (Avanti Polar Lipids, Inc.) dissolved in chloroform/methanol and analyzed at 7 to 70 pmol/µl.

Cell populations. Murine peritoneal macrophages from BALB/c mice were obtained 4 days after the injection of 4% thioglycolate. Bone marrow-derived macrophages (BMM) were generated as previously described (10, 37). Briefly, BMM cells were removed from femurs and tibias of mice. The cells were washed and resuspended in Dulbecco's modified Eagle's medium containing glucose and supplemented with 2 mM L-glutamine, 10% FCS, 10 mM HEPES, 100 µg/ml streptomycin, 100 U/ml penicillin (all from Sigma-Aldrich, Saint Louis, MO), and 20 to 30% L929-cell-conditioned medium (as a source of macrophage colony-stimulating factor [M-CSF]). The cells were then incubated for 7 days at 37°C in 5% CO₂. Bone marrow-derived dendritic cells (BMDC) were generated as described elsewhere (10, 37). Briefly, BMDC were cultured in RPMI 1640 (Invitrogen, Grand Island, NY) supplemented with 2 mM L-glutamine, 10% heat-inactivated FCS, 100 µg/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-mercaptoethanol (all from Sigma-Aldrich, St. Louis, MO) (complete medium), and granulocyte-M-CSF (Invitrogen, 20 ng/ml). On days 3 and 6, complete medium containing granulocyte-M-CSF (10 ng/ml) was added. BMDC were used at day 6 or 7 of culture.

In vitro infection with T. cruzi. Trypanosoma cruzi (clone Dm28c) culture and in vitro metacyclogenesis were performed as described previously (13, 21). The macrophage infection assay was performed with modifications to the previously described protocol (54). Briefly, resident peritoneal macrophages from BALB/c mice were collected in 0.9% saline and allowed to adhere to coverslips. The coverslips were placed in a 24-well culture plate and were incubated for 30 min at 37°C in a 5% CO₂ atmosphere. Macrophages were preincubated with different dilutions of Rhodnius saliva or RpLPC for 30 min in RPMI medium, washed, and allowed to interact with the parasites (1:10 host cell/parasite ratio) for 2 h. Then, the cells were washed and again incubated with the parasites under the same conditions. Finally, the coverslips were fixed and stained with Giemsa, and the percentage of infected macrophages was determined by counting 600 cells on triplicate coverslips, as described previously (25). The association index was determined by multiplying the percentage of infected macrophages by the mean number of parasites per cell. This index indicates the intensity (average number of parasites in each cell) and the extent of cell invasion (number of cells with at least one associated or intracellular parasite).

Determination of intracellular calcium concentrations in macrophages. For calcium assays, cells were collected in sterile saline and washed twice in 20 mM HEPES, pH 7.4, 103 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 0.5 mM CaCl₂, 25 mM NaHCO₃, 15 mM glucose, and 0.05% fatty acid-free bovine serum albumin. Cells were then incubated for 5 min with 5 µM fura 2-acetoxymethyl ester. After incubation, the cells were washed twice and resuspended in the same buffer in several aliquots of 1 x 10⁶ to 5 x 10⁶ cells. Control peritoneal macrophages were treated with 1 µM PAF, a positive modulator of intracellular calcium release. Alternatively, cells were treated with different concentrations of diluted saliva or LPC. The amount of [Ca²⁺]_i was estimated from the change in the fluorescence of fura 2-loaded cells. The fluorescence emission at 510 nm from an excitation wavelength of 380 nm was measured. The intracellular calcium concentration was evaluated by fluorescence exchange by excitation every 0.2 s in a Jasco FP-6300 fluorescence spectrophotometer. Further experimental conditions are described elsewhere (35, 45).

NO and cytokine production. Briefly, peritoneal macrophages, which were obtained as described above, were plated in 24-well culture plates for 30 min at 37°C in a 4% CO₂ atmosphere. Adherent cells were cultured in RPMI 1640 medium supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were stimulated with either LPS (2 µg/ml) or T. cruzi (clone Dm28c; 1:10 host cell/parasite ratio) in the presence or absence of saliva or different concentrations of LPC and 37.5 µM fatty acid-free bovine serum albumin. Twenty-four hours later, the level of nitrite production was determined by using Griess reagent (4). In a set of experiments, BMM were exposed to T. cruzi (Y strain) trypomastigotes in the presence or absence of LPC, and nitrite was measured in the supernatants of cells after 48 h of stimulation. Cytokine production (IL-12 and TNF-) was assessed by using peritoneal macrophages, BMM, or BMDC. The supernatants from peritoneal macrophages evaluated for NO production as described above were also evaluated for IL-12 (antibody against IL-12p40) and TNF- production. BMM and BMDC (10⁶/ml) were infected in vitro with Y-strain T. cruzi trypomastigotes in the presence or absence of LPC for 20 h. IL-12 and TNF- were detected in the supernatant by enzyme-linked immunosorbent assay (ELISA), as described previously (9). In some experiments, BMM cells obtained from either wild-type mice or TLR2^–/– mice were also evaluated for cytokine production.

In vivo infection with T. cruzi. Trypanosoma cruzi (clone Dm28c) trypomastigote forms were obtained from LLC-MK2 cells infected with tissue culture trypomastigotes as previously described (11). A total of 45 animals were used in three different experiments. BALB/c mice (5 animals for each condition for each experiment) were anesthetized with intramuscular ketamine injections (100 mg/kg) in the leg. The mice were pretreated for 15 min after subcutaneous injection of 100 µl of sterile saliva (70 µg total protein) or RpLPC (0.1 µg) in the back. The control group received a subcutaneous injection of 100 µl of sterile PBS. The needle was kept in the back of the animal during the preincubation period and was used again to deliver the parasites (5 x 10⁵ in 100 µl of saline). Blood parasitemia was measured three times per week after the seventh day of infection until three consecutive null counts were recorded. Blood was obtained from a small cut at the end of the tail. The blood was diluted fivefold in red blood cell lysis buffer (150 mM NH₄Cl, 0.1 mM EDTA, and 10 mM KHCO₃, pH 7.4), and parasitemia was measured in a Neubauer chamber.

Statistical analysis. Infection time course data were analyzed in the GraphPad Prism software 3.0 statistical package using two-tailed analysis of variance. The results for each treatment condition were analyzed in comparison to the results for the control during the course of the infection, and those treatments with a P value of <0.05 were considered significant. Also, individual time points under each condition were analyzed in the GraphPad Prism software 3.0 statistical package using the two-tailed unpaired t test. The result for each point was analyzed in comparison to the result for the related control point, and those points with a P value of <0.05 were considered significant.

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MS analysis of purified RpLPC. LPC is widely described as a potent inducer of cell chemotaxis during inflammatory responses (40, 48, 50). This molecule is particularly abundant in Rhodnius saliva, where it reaches micromolar concentrations (24); conversely, it is barely present on vertebrate skin and interstitial fluid in normal conditions (67). Thus, LPC is a candidate chemotactic molecule for inflammatory cells following the injection of Rhodnius saliva into the mammalian host bloodstream. Therefore, we isolated RpLPC from Rhodnius saliva by thin-layer chromatography, as previously described (24), and carried out structural characterization by MS. The MS1 spectrum profile of purified RpLPC showed a major, singly charged ion species at m/z 518.3, which most likely corresponds to the sodium adduct of an LPC with a C_16:0-acyl chain (Fig. 1A). Two other ion species at m/z 478.6 and 496.4 were tentatively assigned to C_16:0-acyl-LPC species (related to the major ion species at m/z 518.3) without the sodium adduct (m/z 496.4) or in its dehydrated form (m/z 478.6). The remaining ion species observed in the spectrum were considered contaminants, since they were also detected in the analysis of the RpLPC-equivalent silica region of a mock sample (without RpLPC) (data not shown). To confirm the tentative assignment of the major LPC species at m/z 518.3, this ion was subjected to collision-induced dissociation (MS2 fragmentation) in the ion trap (Fig. 1B). Two major daughter ions were observed at m/z 459 and 184, which were respectively assigned as [M–trimethylamine+H–H+Na⁺]⁺ and [phosphocholine+H]⁺ (Fig. 1B). To further confirm our assignments, the daughter ion at m/z 459 was further analyzed by MS3 fragmentation. Two ion species were generated at m/z 313 and 147, corresponding to [M–phosphocholine+H]⁺ and [ethylphosphate+H–H+Na⁺]⁺, respectively (Fig. 1C). Consequently, our MS data strongly suggest that palmitic acid is the major (if not only) acyl chain present on RpLPC. A similar composition was found for the commercial LPC that is commonly used for vascular biology studies (34, 50).

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FIG. 1. Characterization of RpLPC. RpLPC was purified from saliva by thin-layer chromatography and extracted from silica by using organic solvents. Mass spectra were acquired in the positive-ion mode by using an ESI-ion trap-MS. (A) MS1 spectrum of RpLPC purified from insect saliva. Major singly charged ion species of RpLPC corresponding to [M+H–H2O]+, [M+H]+, and [M+H–H+Na+]+ are observed at m/z 478.6, 496.4, and 518.3, respectively. (B) MS2 spectrum of parent-ion species at m/z 518.3. (C) MS3 spectrum of fragment ion at m/z 459.1 observed in spectrum B. RpLPC ion fragments corresponding to phosphocholine and ethylphosphate are shown in spectra A and B, respectively.

Saliva and LPC-induced cell chemotaxis. The presence of chemotactic molecules in triatomine saliva has not been previously examined. Previous experiments suggested that bug saliva induces cell migration in a dose-dependent manner. Different cell types, such as neutrophils, monocytes, and eosinophils, responded to this stimulus in the pleurisy assay (data not shown). LPC has long been recognized as a potent inducer of human monocyte chemotaxis (40, 48, 50). However, concentrated saliva, which contains several antihemostatic molecules, produced hemorrhages when injected into the thoraces of mice during previous pleurisy assays of cell chemotaxis. Therefore, in order to determine whether neutrophil chemotaxis could be induced by LPC, a dose-response curve was conducted using Boyden chambers. The results in Fig. 2A show that highly (1,000-fold) diluted bug saliva induced a significant level of cell chemotaxis. Nevertheless, the R. prolixus sialome, i.e., the collection of proteins present in spit saliva, is not known to contain any gene product that could account for such chemotactic activity (51). The results in Fig. 2B show that commercial LPC is also able to induce neutrophil chemotaxis. However, the maximum amount of saliva-derived LPC (RpLPC) injected into mouse skin at once would be 300 pmol. The results in Fig. 2A show that 1,000-fold-diluted saliva which may contain 0.045 nmol of RpLPC induces cell chemotaxis at levels close to those induced by 180 nmol of commercial LPC, as seen in Fig. 2B.

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FIG. 2. Rhodnius saliva induces cell chemotaxis. (A) Human neutrophils were purified from healthy volunteers' blood by using Ficoll gradients. Neutrophil chemotaxis was tested in vitro in ChemoTx system 96-well plates (NeuroProbe) using different saliva dilutions in RPMI medium, as indicated. Chemotaxis index was calculated as the percentage of positive-control chemotaxis obtained with 10 nM leukotriene TB4. Control (C) cells were grown in RPMI medium, and their migration was considered to be random. Numbers above the bars indicate the final amount of RpLPC or LPC under each treatment condition. (B) The same experiment as described for panel A, but commercial LPC was used as the inducer of cell chemotaxis. Results for panels A and B are presented as the means ± standard errors of triplicate measurements. Data shown are representative of two independent experiments. (C to E) Skin histology. (C) Saline-injected skin sections stained with H&E. Subcutaneous layer demonstrating a minimal amount of interstitial edema (*) associated with rare inflammatory cells. (D) Photomicrograph of mouse skin section (H&E) after saliva injection showing inflammatory cells (arrowhead) recruited to subcutaneous tissue. (E) An intense inflammatory reaction (arrow) and interstitial edema were observed in subcutaneous and reticular dermis after LPC injections (H&E-stained section). LPC used in these experiments has the same fatty acid composition as RpLPC. Tissue layers indicated in panels C, D, and E are subcutaneous tissue (ST) and muscle tissue (M). Scale bars, 25 µm.

In order to assess the ability of bug saliva and LPC to induce cell chemotaxis in vivo, BALB/c mice were subcutaneously injected with either saliva or LPC, and skin biopsies were conducted 24 h later. The results in Fig. 2C show that a fair number of cells are induced to migrate to the lesion by the injection of sterile saline alone. However, the injection of only 2 µg (6 pmols RpLPC) of Rhodnius saliva induced the migration of a massive number of cells, including mono- and polymorphonuclear cells, as visualized in Fig. 2D. In fact, this treatment caused a significant leukocyte infiltration, predominantly comprised of polymorphonuclear cells migrating close to the muscle fibers (Fig. 2D). In control injection sites, we noticed only a slight infiltration of cells close to the blood vessel (Fig. 2C). Such results were obtained with a wide range of saliva injection doses (75 to 300 µg/ml, data not shown). Similar results were not observed with similar doses of commercial LPC injections (data not shown). The minimum level of LPC able to induce significant chemotaxis in mouse skin was 1.5 nmol (Fig. 2E). Therefore, the above set of results indicates that, while host cell chemotaxis is stimulated by vector-derived saliva, the salivary constituent RpLPC is not the inducer of neutrophil chemotaxis. T. cruzi infection occurs by two different routes: the wound produced by insect mouthparts during feeding and ocular mucosa (59). In both cases, the feces of the insect are the vehicle for parasite transmission. Triatomine intestine perimicrovillar membranes massively incorporate phospholipids during the blood digestion process (8). Therefore, bug feces also contain significant amounts of PC and LPC (data not shown). This suggests that vector-derived LPC should be present at the site of inoculation by either route of T. cruzi infection, i.e., host skin or ocular mucosa.

Rhodnius saliva and RpLPC increase the association of T. cruzi with macrophages. To investigate the role of triatomine saliva and RpLPC in parasite transmission, murine peritoneal macrophages were allowed to adhere to 24-well plates and were incubated with different concentrations of either saliva or RpLPC for 30 min. The results in Fig. 3A show an increase in the association index of the parasite with macrophages in the presence of both saliva and RpLPC. The same results were obtained when macrophages were preincubated with either saliva or RpLPC before the parasites were added to the macrophage culture, as well as with macrophages obtained from Swiss mice (data not shown). The invasion of host cells by T. cruzi involves the subversion of numerous signaling pathways, which seems to result in an increase in intracellular calcium (12). The results in Fig. 3B and C show that both Rhodnius saliva and commercial LPC triggered an increase in peritoneal macrophage intracellular calcium in a dose-dependent manner, which may promote parasite invasion. The intracellular calcium released was of the same magnitude as the release from the controls treated with PAF but had a different profile (data not shown). It is interesting to note that highly diluted saliva, up to a 500-fold dilution, still led to a significant enhancement of the association index between the parasite and macrophages and it also leads to an increase in intracellular free calcium concentrations (Fig. 3A and B). In conclusion, low concentrations of saliva allow host cell invasion, but high concentrations of commercial LPC are required to mimic the effects of whole saliva on intracellular calcium. Therefore, it is likely that highly diluted saliva possesses another compound(s) able to trigger intracellular calcium in invaded macrophages. Such an effect is probably not triggered by RpLPC present in the whole bug saliva.

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FIG. 3. Saliva and RpLPC enhance in vitro infection of macrophages by T. cruzi and increase host cell free intracellular calcium. (A) Peritoneal macrophages from BALB/c mice were pretreated in vitro with different saliva dilutions or with 1 µM RpLPC. Then, the macrophages were incubated with T. cruzi at a 1:10 host cell/parasite ratio.The association index was calculated as described in Materials and Methods. C, control. Error bars show standard errors. (B) Representative tracing of intracellular calcium response induced by R. prolixus saliva. (C) Representative tracing of intracellular calcium oscillation induced by LPC. LPC used in these experiments has the same fatty acid composition as RpLPC.

Saliva LPC attenuates proinflammatory cytokine and NO production triggered by T. cruzi. A number of innate-immunity-associated pathways involved in the recognition of T. cruzi have been described (15, 62). TLRs are a major family of receptors involved in pathogen recognition. Following the activation of pattern recognition receptors by T. cruzi, host cells produce TNF-, IL-12, and NO. In addition to MyD88, TLR2, -4, and -9 have been implicated in the immune signaling network used by a mammalian host to control T. cruzi infection (9, 14, 15, 31, 62). TLR2 expression is essential for the induction of IL-12, TNF-, and NO production by parasite molecules, such as glycosylphosphatidylinositol anchors, which have been isolated from the surface of T. cruzi trypomastigotes (3). Saliva abolishes NO (Fig. 4A) but not IL-12 (Fig. 4B) and TNF- (data not shown) production by T. cruzi-exposed macrophages. In addition, LPC impairs nitrite production by macrophages stimulated by LPS, T. cruzi, or LPS in the presence of IFN- in vitro (Fig. 5A and B). Consistent with previously published data, the results shown in Fig. 6A demonstrate that IL-12 production in T. cruzi-exposed BMM is largely dependent on TLR2. T. cruzi-stimulated TNF- production by BMM is not abolished by LPC treatment (data not shown). In contrast, parasite-induced IL-12 synthesis is completely blocked by LPC in a TLR2-independent fashion (Fig. 6A). Furthermore, IL-12 production by BMDC is also regulated by LPC (Fig. 6B). LPC was found to inhibit IL-12 synthesis at doses of 0.1 to 10 µM, and no effect was observed when a higher dose of 100 µM was used (Fig. 6B). These data suggest that LPC regulates the proinflammatory mediators NO and IL-12 by different mechanisms in different cell populations.

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FIG. 4. Saliva from R. prolixus inhibits NO but not IL-12 production by T. cruzi-exposed macrophages in vitro. (A) Peritoneal macrophages were exposed to live T. cruzi (1:10 parasite/host cell ratio) in the presence (+) or absence (–) of saliva (200 µg/ml) or LPS (2 µg/ml). Cells were stimulated as indicated for 48 h. The supernatant of each culture was treated with Griess reagent to determine the nitrite concentration. Data are expressed as the means ± standard errors of the results of two independent experiments with triplicate cultures. (B) Supernatants obtained in the experiments described for panel A were also used to measure IL-12 by ELISA. Data shown are representative of two independent experiments.

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FIG. 5. LPC inhibits NO production by T. cruzi-exposed macrophages in vitro. (A) Peritoneal macrophages were stimulated with 2 µg/ml LPS and different concentrations of LPC as indicated for 24 h. The supernatant of each macrophage culture was treated with Griess reagent to determine the nitrite concentration. Data are expressed as the means ± standard errors of the results of two independent experiments with triplicate cultures. (B) BMM (106/ml) were exposed to live T. cruzi trypomastigotes (1 parasite per cell) or LPS (100 ng/ml) plus IFN- (500 U/ml) in the presence or absence of different concentrations of LPC as indicated for 48 h. Nitrite was measured in culture supernatants by the Griess reaction. Results are expressed as the means ± standard errors of the results of triplicate measurements. Data shown are representative of two independent experiments. LPC used in these experiments has the same fatty acid composition as RpLPC. +, present; –, absent.

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FIG. 6. LPC modulates IL-12 production by T. cruzi-exposed antigen-presenting cells in vitro. (A) BMM cells (106/ml) from wild-type (WT) C57BL/6 or TLR2–/– mice were exposed to live T. cruzi (1:1 parasite/host cell ratio) in the presence or absence of different concentrations of LPC (10 or 100 µM) for 20 h. IL-12 was measured in culture supernatants of BMM cells by ELISA. Results are presented as the means ± standard errors of the results of triplicate measurements. (B) BMDC (106/ml) from wild-type C57BL/6 mice were exposed to live T. cruzi (1:1 parasite/host cell ratio) in the presence or absence of indicated concentrations of LPC for 20 h. IL-12 was measured in culture supernatants of BMDC cells by ELISA. Results are presented as the means ± standard errors. Data shown are representative of two independent experiments. LPC used in these experiments has the same fatty acid composition as RpLPC. +, present; –, absent.

RpLPC and Rhodnius saliva promote an increase in blood parasitemia. The above set of results suggests that LPC interferes with several aspects of T. cruzi infection in vitro. To further assess the effect of Rhodnius saliva, as well as RpLPC, on T. cruzi infection in vivo, groups of BALB/c mice were infected with the parasite and blood parasitemia was evaluated for a few weeks following infection. The inoculation of either saliva or RpLPC into the skin induced higher levels of blood parasitemia during the course of infection, which ranged from a two- to sixfold increase depending on the day of observation (Fig. 7). These results suggest that saliva and the salivary constituent LPC could play an important role in T. cruzi infection in vivo.

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FIG. 7. Rhodnius saliva and RpLPC enhance in vivo T. cruzi infection. BALB/c mice were injected subcutaneously with 70 µg of saliva and 0.1 µg of RpLPC. Fifteen minutes later, T. cruzi trypomastigotes (105 parasites) were injected at the same site. Blood parasitemia was measured from week 2 until week 5 following the infection. , saliva or RpLPC time courses showed statistically significant difference from control time course (analysis of variance; P < 0.01). Parasitemia at individual time points for each treatment condition showed statistically significant differences from parasitemia at related points for control (t test; *, P < 0.05, and **, P < 0.01). The data are presented as the means ± standard errors of the results of three independent experiments.

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LPC is now recognized as a regulator of several aspects of vascular biology. Since the saliva of R. prolixus is the only natural source of LPC that is injected into human skin during hematophagy, we hypothesized that saliva and LPC may favor T. cruzi transmission. In the present study, we have shown that bug saliva (i) increases cell concentration, likely due to chemotaxis to the site of insect feeding; (ii) increases the association between the injected parasite and target macrophages, probably by increasing intracellular calcium concentrations; (iii) blocks NO production; and (iv) increases blood parasitemia in T. cruzi-injected animals. In this context, LPC was able to attenuate T. cruzi-induced immune responses by macrophages and DC. This later effect is specifically attributed to downregulation of the production of both IL-12 and NO, two important mediators involved in the control of parasite infection (2, 65). Of note, some of the processes observed with saliva are potentially mimicked by RpLPC or commercial LPC, especially the increase in blood parasitemia when LPC is injected into mouse skin in the presence of the parasite.

Skin biopsy showed that intracutaneous LPC injection promotes cell chemotaxis (Fig. 2). The role of intracutaneously injected LPC in the production of a proinflammatory cell infiltrate was investigated in human volunteers (55). Our results demonstrated that only high concentrations of LPC may act as a powerful chemotaxis stimulus for neutrophils. Therefore, salivary LPC may act as a molecule able to increase the number of monocytes and macrophages at the lesion site that are prone to parasite infection. Furthermore, it was previously demonstrated that neutrophil depletion affects the outcome of experimental Chagas disease infections. The disease is exacerbated in BALB/c mice, and a degree of host protection is conferred in C57BL/6 mice (20). These different effects are probably due to the role of neutrophils in the Th1 and Th2 dichotomy. Interestingly, it was demonstrated that LPC may play a role in neutrophil apoptosis and also that the uptake of apoptotic cells by infected macrophages modulates the growth of T. cruzi in infected macrophages (23). Therefore, saliva-induced neutrophil chemotaxis could be a chief event that determines the outcome of infection. Mononuclear cell chemotaxis is induced by saliva and, as reported in the literature, lysophospholipids may contribute to the success of parasite infection, since the number of such cells at the lesion is increased severalfold. Also, it was previously shown that LPC-induced production of monocyte chemoattractant protein 1 by endothelial cells accounts for the increase in cell migration that may facilitate parasite entry at the injection site (61).

It is noteworthy that the injection of RpLPC followed by parasite inoculation promoted a two- to sixfold increase in blood parasitemia (Fig. 7). However, we estimated that a pair of salivary glands contains only 0.15 µg of RpLPC. Nevertheless, most of the saliva-related effects reported here, such as the induction of cell chemotaxis in vitro and in vivo (Fig. 2), an increase in the association index (Fig. 3A), and an enhancement of the free-intracellular-calcium concentration (Fig. 3B), were promoted by saliva that was diluted at least 50-fold. This evidence suggests that LPC locally generated in injected saliva while the bug feeds on human skin is not solely responsible for most of the effects unless its concentrations are further increased during the biological process. The production of lipid mediators by sPLA₂ in activated macrophages is a remarkable feature of chronic inflammation. Such enzymes differ from cytosolic PLA₂ with respect to their low molecular mass and absolute requirement for millimolar calcium concentrations for catalysis (56). The results in Fig. 3B and C show that the treatment of peritoneal macrophages with either saliva or LPC triggers intracellular calcium waves. Therefore, it is likely that the first round of cells that migrate toward injected saliva may secrete PLA₂ and contribute locally to the hydrolysis of remaining PC from the injected saliva. Two additional classes of PLA₂ (constitutive PLA₂ and inducible PLA₂) also are able to generate LPC and induce monocyte chemotaxis into sites of T. cruzi infection as has been suggested previously for monocyte recruitment to apoptotic cells (34). This would increase the concentration of this lipid and induce the migration of another round of cells to the wound site. Also, LPC may be reintroduced from the bug feces deposited in the wound by scratching of the wound site on the skin. Finally, the invading parasite is also able to shed vesicular bodies containing LPC (1). Furthermore, it was recently shown that thromboxane A₂ (TXA₂) is a regulator of pathogenesis during T. cruzi infection (7). Therefore, either the lysophospholipid or the release of fatty acyl chains in the presence of the appropriate processing enzyme from the eicosanoid pathway could lead to the generation of molecules that potently establish the infection. Thus, the introduction of LPC from different sources may allow for a final concentration in the wound that is sufficient to drive several of the mechanisms described in the present study.

Intracellular signaling in macrophages is manipulated by T. cruzi. Some of the key signaling events have been described in the literature. The release of intracellular calcium stores, which is concomitant with the activation of phosphatidylinositol 3-kinase and Akt, is generally involved in T. cruzi-mediated alterations in cell signaling. However, the downstream targets of such pathways have not yet been demonstrated (12). An important feature of this altered cell signaling is the inhibition of an inflammatory state at the site of parasite inoculation. In this regard, LPC and saliva were able to counteract LPS and T. cruzi-induced NO production in the present study (Fig. 4A and 5). LPC has been demonstrated to activate protein kinase C in endothelial and smooth muscle cells, which could account for the inhibition of NO production by macrophages (19, 27).

Our data suggest that LPC (Fig. 6) but not saliva (Fig. 4B) regulates proinflammatory molecules such as IL-12, which is known to be critical for the control of T. cruzi infection in antigen-presenting-cell populations (macrophages and DC) in vitro. The ability of saliva to suppress IL-12 production by T. cruzi-exposed cells was only evaluated with peritoneal macrophages in the present study. Thus, it is hard to define if the absence of the immunosuppressive effect in whole saliva is due to differences in the cell type. Also, it remains to be determined whether LPC regulates the innate recognition of T. cruzi in vivo. The results in Fig. 6 show that LPC suppresses IL-12 production by macrophages and DC but with different dose-response profiles. This is an interesting observation that suggests a potential difference in lipid metabolism, lipid signaling, or both in these two closely related cell types. Macrophages usually express more TLR2 and similar levels of TLR4 mRNA transcripts compared to the expression of these TLRs in DC. No matter that both cell types respond to several TLR ligands through the production of TNF, the level of IL-12 produced by DC is higher than the level produced by macrophages. On the other hand, macrophages produce higher levels of IL-10 than DC. Such results indicate several steps in intracellular signaling that may differentiate between these cells, but evidence from the literature suggests that most of the differences may rely on different subsets of protein kinase C isoforms and the routes for activation of extracellular signal-regulated kinases (66).

The modulation of murine lymphocytes by R. prolixus saliva was shown previously, but the molecule involved remains to be identified (29). The saliva of Aedes aegypti modulates the host immune response by blocking the production of proinflammatory cytokines and potentiates subsequent infection by Sindbis virus (58). Products of the eicosanoid pathways present in tick saliva were implicated in a long-term immunosuppression process (57). Recently, it was shown that tick saliva blocks host cell-mediated phagocytosis of Borrelia burgdorferi through the inhibition of adhesion, but not chemotaxis (43). The saliva of members of the Phlebotominae increases infection by Leishmania in mice (26, 63), and the immunomodulatory activities of these vectors are considered the hallmark of successful host infection (30, 38, 44, 49). It is important to note that most of these effects are due to the activity of the insect-derived polypeptide maxadillan that was recently shown to modulate the feeding behavior of the sand fly and to enhance the transmission of Leishmania (53).

The surge of lysophospholipids in the secretions of predatory organisms may occur in several instances. In fact, such molecules are present in snake venom, where they paralyze the neuromuscular junction and facilitate feeding. Therefore, wide-array screening of lipid and lipid-derived molecules in animal secretions may provide interesting data to serve as the starting point for a pharmacological review of the effects usually attributed to proteic molecules (52). Parasite-derived TXA₂, such as that synthesized by T. cruzi, modulates disease pathogenesis in the absence of host-derived TXA₂. These results indicate that TXA₂ controls the proliferation of parasites and the resulting inflammatory response to infection by T. cruzi (7). The parasite also synthesizes a PAF-like lipid that induces the aggregation of rabbit platelets, stimulates the infection of mouse macrophages, and triggers the differentiation of epimastigotes into metacyclic trypomastigotes (25). Since TXA₂, PAF, and LPC are well-known mammalian lipid mediators that share common chemical structures, it is interesting that insect-derived LPC also interacts with the mammalian host as an enhancer of Chagas disease. Our data, therefore, may represent one of the rare examples of coevolution of a parasite, a vector, and a mammalian host, all leading to the synthesis of the same biochemical modulator which then favors simultaneous blood feeding and parasite transmission. The identification of the mammalian LPC receptors may permit the design of specific experiments with transgenic animals to further elucidate the LPC-dependent pathway of T. cruzi transmission.

 
We thank Heloísa L. Coelho, Lilian Gomes, and Cleuza Alexandre Silva for technical assistance. We are indebted to Alan Sher (NIAID/NIH) for insightful scientific advice and Pat Casper (NIAID/NIH) for technical assistance.

This work was supported by two grants provided by the International Foundation for Science (IFS) to G. C. Atella (F/3619-1) and to M. A. C. Silva-Neto (F/2887-3), by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and by Fundação de Amparo a Pesquisa Carlos Chagas Filho do Estado do Rio de Janeiro (FAPERJ-PENSA RIO, E-26/110.401/2007). I.C.A. is supported by NIH/NCRR grants 5G12RR008124, 1R01AI070655 and 2506GM00812-37 (to the Border Biomedical Research Center/University of Texas at El Paso). We are grateful to the Biomolecule Analysis Core Facility/BBRC/UTEP, supported by NIH/NCRR grant 5G12RR008124.

 
* Corresponding author. Mailing address: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, UFRJ, 21940-590, Rio de Janeiro, RJ, Brazil. Phone: 5521-2562-6786. Fax: 5521-2270-8647. E-mail: maneto{at}bioqmed.ufrj.br

Published ahead of print on 15 September 2008.

Editor: W. A. Petri, Jr.

R.D.M. and A.B.C. contributed equally to this study.

Permanent address: CEFET-Química, Rio de Janeiro, RJ, Brazil.

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Infection and Immunity, December 2008, p. 5543-5552, Vol. 76, No. 12
0019-9567/08/$08.00+0     doi:10.1128/IAI.00683-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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