During blood feeding, sand flies inject Leishmania parasites in the presence of saliva. The types and functions of cells present at the first host-parasite contact are critical to the outcome on infection and sand fly saliva has been shown to play an important role in this setting. Herein, we investigated the in vivo chemotactic effects of Lutzomyia intermedia saliva, the vector of Leishmania braziliensis, combined or not with the parasite.
Methods and Findings
We tested the initial response induced by Lutzomyia intermedia salivary gland sonicate (SGS) in BALB/c mice employing the air pouch model of inflammation. L. intermedia SGS induced a rapid influx of macrophages and neutrophils. In mice that were pre-sensitized with L. intermedia saliva, injection of SGS was associated with increased neutrophil recruitment and a significant up-regulation of CXCL1, CCL2, CCL4 and TNF-α expression. Surprisingly, in mice that were pre-exposed to SGS, a combination of SGS and L. braziliensis induced a significant migration of neutrophils and an important modulation in cytokine and chemokine expression as shown by decreased CXCL10 expression and increased IL-10 expression.
These results confirm that sand fly saliva modulates the initial host response. More importantly, pre-exposure to L. intermedia saliva significantly modifies the host's response to L. braziliensis, in terms of cellular recruitment and expression of cytokines and chemokines. This particular immune modulation may, in turn, favor parasite multiplication.
Transmission of Leishmania parasites occurs during blood feeding, when infected female sand flies inject humans with parasites and saliva. Chemokines and cytokines are secreted proteins that regulate the initial immune responses and have the potential of attracting and activating cells. Herein, we studied the expression of such molecules and the cellular recruitment induced by salivary proteins of the Lutzomyia intermedia sand fly. Of note, Lutzomyia intermedia is the main vector of Leishmania braziliensis, a parasite species that causes cutaneous leishmaniasis, a disease associated with the development of destructive skin lesions that can be fatal if left untreated. We observed that L. intermedia salivary proteins induce a potent cellular recruitment and modify the expression profile of chemokines and cytokines in mice. More importantly, in mice previously immunized with L. intermedia saliva, the alteration in the initial inflammatory response was even more pronounced, in terms of the number of cells recruited and in terms of gene expression pattern. These findings indicate that an existing immunity to L. intermedia sand fly induces an important modulation in the initial immune response that may, in turn, promote parasite multiplication, leading to the development of cutaneous leishmaniasis.
Citation: de Moura TR, Oliveira F, Rodrigues GC, Carneiro MW, Fukutani KF, Novais FO, et al. (2010) Immunity to Lutzomyia intermedia Saliva Modulates the Inflammatory Environment Induced by Leishmania braziliensis. PLoS Negl Trop Dis 4(6): e712. https://doi.org/10.1371/journal.pntd.0000712
Editor: Genevieve Milon, Institut Pasteur, France
Received: December 22, 2009; Accepted: April 22, 2010; Published: June 15, 2010
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This work was supported by grants from FAPESB, PAPES/FIOCRUZ, and CNPq. TRdM was supported by a CNPq fellowship. MB-N, CB, AB, and CIdO are senior investigators from CNPq. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The intracellular protozoan parasites of the Leishmania species are transmitted to vertebrate host through the bites of sand flies. Within the vertebrate host, Leishmania parasites reside in phagocytes and induce a spectrum of diseases ranging from a single self-healing cutaneous lesion to the lethal visceral form. It is currently estimated that leishmaniasis affects two million people per year worldwide .
Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis (CL) in Brazil, can be transmitted to the human host by the bite of the sand fly Lutzomyia intermedia. , . Several studies have shown that pre-exposure to saliva or to bites from uninfected sand flies results in protection against subsequent infection with Leishmania major –, Leishmania. amazonensis , and Leishmania chagasi . On the contrary, pre-exposure to Lutzomyia intermedia saliva enhanced infection with L. braziliensis in the mouse model; disease exacerbation was correlated with generation of a Th2 response evidenced by a reduction in the IFN-γ/IL-4 ratio . Importantly, individuals with active CL showed higher humoral immune responses to L. intermedia saliva compared with control subjects, a finding also demonstrated with Old World CL  . These data indicate an association between disease and immune response to L. intermedia saliva in humans.
In the case of L. intermedia, the lack of protection observed following pre-exposure to saliva in the murine model may be related to differences in the initial inflammatory response induced by the salivary proteins. Several studies have shown the potential of salivary antigens from Lutzomyia longipalpis, Phlebotomus duboscqi, Phlebotomus papatasi and Phlebotomus ariasi to modulate cell recruitment and production of immune response mediators – however, little is known regarding these effects when using L. intermedia saliva. Our group has previously shown that pre-treatment of human monocytes with L. intermedia followed by L. braziliensis infection led to a significant increase in TNF-α, IL-6, and IL-8 production , indicating the ability of L. intermedia saliva to alter the inflammatory milieu. To gain further information regarding the events associated with the initial host response to L. intermedia saliva, we employed the air pouch model of inflammation. This model simulates inoculation of the sand fly in a closed environment and allows for subsequent analysis of inflammatory parameters and mediators induced in vivo by distinct stimuli . Using this model, we showed that saliva from L. longipalpis rapidly induced CCL2 expression and macrophage recruitment, in synergy with L. chagasi parasites, in BALB/c mice . Here we describe the ability of L. intermedia salivary gland sonicate (SGS) to modulate the host immune response in naïve and in SGS-sensitized mice. We have demonstrated that L. intermedia salivary proteins induce neutrophil recruitment and modulate cytokine and chemokine expression. Crucially, a downregulation in CXCL10 paralleled by an increase in IL-10 expression was observed in SGS-sensitized mice stimulated with saliva+L. braziliensis. This correlates with disease exacerbation previously observed in mice immune to L. intermedia SGS and challenged with L. braziliensis .
Leishmania braziliensis promastigotes (strain MHOM/BR/01/BA788 ) were grown in Schneider medium (Sigma Chemical Corporation, St. Louis, MO, USA) supplemented with 100 U/ml of penicillin, 100 µg/ml of streptomycin, 10% heat-inactivated fetal calf serum (all from Invitrogen, San Diego, CA, USA), and 2% sterile human urine. Stationary-phase promastigotes from second passage culture were used in all experiments.
Female BALB/c mice (6–8 weeks of age) were obtained from CPqGM/FIOCRUZ Animal Facility where they were maintained under pathogen-free conditions. All procedures involving animals were approved by the local Ethics Committee on Animal Care and Utilization (CEUA—CPqGM/FIOCRUZ).
Sand flies and preparation of SGS
Adult Lutzomyia intermedia sand flies were captured in Corte de Pedra, Bahia, and were used for dissection of salivary glands. Salivary glands were stored in groups of 20 pairs in 20 µl NaCl (150 mM)-Hepes buffer (10 mM; pH7.4) at −70°C. Immediately before use, salivary glands were disrupted by ultrasonication in 1.5-ml conical tubes. Tubes were centrifuged at 10,000×g for two minutes, and the resultant supernatant—salivary gland sonicate (SGS)—was used for the studies. The level of lipopolysaccharide (LPS) contamination of SGS preparations was determined using a commercially available LAL chromogenic kit (QCL-1000; Lonza Biologics, Portsmouth, NH, USA); LPS concentration was <0.1 ng/ml.
Sand fly saliva immunization
BALB/c mice (groups of five to six) were immunized three times with SGS (equivalent to one pair of salivary glands) in 10 µl of PBS in the dermis of the right ear using a 27.5 G needle. Immunizations were performed at two-week intervals. Control mice were injected with PBS. Development of an immune response against L. intermedia saliva was confirmed by ELISA as previously described .Immune sera were pooled from SGS-immunized mice and employed in neutralization experiments. Immune mice were employed in air pouch experiments.
In vivo cell recruitment into the air pouch
Air pouches were raised on the dorsum of anesthetized BALB/c mice (groups of five to six) by injection of 3 ml of air, as described elsewhere . Air pouches were inoculated with either one of the following stimuli: L. intermedia SGS (equivalent to one pair of salivary glands/animal); L. intermedia SGS pre-incubated with a pool of anti-SGS immune sera (SGS+50 µl of immune serum pre-incubated for one hour at 37°C); a pool of anti-SGS immune sera alone; stationary-phase L. braziliensis promastigotes (105 parasites); or L. braziliensis+SGS. Air pouches in control mice were injected with endotoxin-free saline (negative control) or with LPS (Calbiochem, San Diego, CA, USA) (20 µg/ml; positive control). After twelve hours, animals were euthanized and pouches washed with 5 ml of endotoxin-free saline for collection of exudates containing leukocytes. Lavage fluids were washed, and cell pellets were resuspended in saline, stained in Turk's solution, and counted in a Neubauer hemocytometer. Cells were cytoadhered to glass slides using Shandon cytospin2 and stained with hematoxylin and eosin to determine proportions of monocytes/macrophages, neutrophils, lymphocytes, basophils, and eosinophils. Air pouch lining tissue was placed in 5–10 volumes of RNAlater (Ambion Inc., Austin, TX, USA), and samples were stored at −80°C.
RNA isolation and real-time PCR
Total RNA was extracted from the air pouch lining tissue using the RNeasy Protect Mini Kit (Qiagen, Inc., Santa Clara, CA, USA) according to manufacturer's instructions. The resulting RNA was resuspended in 20 µl diethyl pyrocarbonate (DEPC)-treated water and stored at −80°C until use. cDNA synthesis for detection of cytokine mRNA was performed after reverse transcription (Im Prom-II™ reverse transcription system). Real-time PCR was performed in triplicate on the Abi Prism 7500 (Applied Biosystems, Inc., Fullerton, CA, USA); thermal cycle conditions consisted of a two-minute initial incubation at 50°C followed by ten-minute denaturation at 95°C and 50 cycles at 95°C for 15 seconds and 60°C for one minute each. Each sample and the negative control were analyzed in triplicate for each run. The comparative method was used to analyze gene expression. Chemokine or cytokine cycle threshold (Ct) values were normalized to GAPDH expression as determined by ΔCt = Ct (target gene)−Ct (GAPDH gene). Fold change was determined by 2−ΔΔCt, where ΔΔCt = ΔCt (target)−ΔCt (saline) . The following primers were employed: GAPDH (Forward: 5′-TGTGTCCGTCGTGGATCT GA-3′; Reverse: 5′-CCTGCTTCACCACCTTCTTGA-3′); CCL2 (Forward: 5′-CAGGTC CCTGTCATGCTTCTG-3′; Reverse: 5′-GAGCCAACACGTGGATGCT-3′) ; CCL3 (Forward: 5′-TCTTCTCAGCGCCATATGGA-3′; Reverse: 5′-CGTGGAATCTTCCGG CTGTA-3′); CCL4 (Forward: 5′-TGCTCGTGGCTGCCTTCT-3′; Reverse: 5′-CAGGAA GTGGGAGGGTCAGA-3′); CXCL1: (Forward: 5′-CCGAAGTCATAGCCACACTCAA-3′; Reverse: 5′-AATTTTCTGAACCAAGGGAGCTT-3′); CXCL10: (Forward: 5′-GGACGG TCCGCTGCAA-3′; Reverse: 5′-CCCTATGGCCCTCATTCTCA-3′); IFN-γ (Forward: 5′-CTACACACTGCATCTTGGCTTTG-3′; Reverse: 5′-TGACTGCGTGGCAGTA-3′); TNF-α (Forward: 5′-GGTCCCCAAAGGGATGAGAA-3′; Reverse: 5′-TGAGGGTCT GGGCCATAGAA-3′); and IL-10 (Forward: 5′-CAGCCGGGAAGACAATAACTG-3′; Reverse: 5′-CGCAGCTCTAGGAGCATGTG-3′). Primers were designed using Primer Express Software (Applied Biosystems).
Histology and immunohistochemistry
BALB/c mice (n = 5) were intradermally immunized with L. intermedia SGS (equivalent to one pair of salivary glands) or injected with PBS three times in the right ear at two-week intervals. After the third injection, pre-sensitized or control animals were intradermally inoculated with L. intermedia SGS, in the opposite (left) ear dermis. Twenty-four and forty-eight hours after SGS injection, animals were euthanized and the ear was biopsied and stored in 10% neutral buffered formalin. Ears were mounted in paraffin blocks, sectioned at 5-µm intervals, and stained with hematoxylin and eosin for histologic analysis. Paraffin-embedded sections of ears fixed in 10% neutral buffered formalin were used for immunohistochemistry. Myeloperoxidase rabbit anti-mouse (Dako, Carpenteria, CA, USA) was used at 1∶1000 dilution. A secondary biotinylated goat anti-rabbit antibody was used at 1∶500 for 15 minutes (Vector Laboratories, Burlingame, CA, USA) and detected by R.T.U. Vectastin Elite ABC reagent (Vector Laboratories) and DAB chromagen.
Data are presented as the mean with 95%CI. The significance of the results was calculated using nonparametric statistical tests: two-sided Mann-Whitney for comparisons between two groups; Kruskal-Wallis followed by Dunn's multiple comparison test for comparisons between three groups. Analyses were conducted using Prism (GraphPad Software Inc., San Diego, CA, USA); a P-value of <0.05 was considered significant.
In vivo effect of L. intermedia SGS on leukocyte recruitment
We initially studied the cellular recruitment induced by L. intermedia SGS inoculation. Air pouches were induced in BALB/c mice and subsequently probed with different stimuli: endotoxin-free saline; L. intermedia SGS; or LPS. L. intermedia SGS induced a significant increase in leukocyte accumulation in the air pouch compared with saline injection (Figure. 1A). Most cells recruited by inoculation of L. intermedia SGS into air pouches were neutrophils, followed by monocytes (Figure 1B). LPS inoculation was used as a positive control for cell recruitment and, as expected, led to a predominant recruitment of neutrophils (Figure 1B). Moreover, inoculation of L. intermedia SGS did not lead to significant changes in either eosinophil or lymphocyte recruitment.
Air pouches were raised on BALB/c mice (five to six per group) and were inoculated with either endotoxin-free saline, L. intermedia SGS, or LPS. Exudates were collected twelve hours later. Leukocytes were enumerated microscopically. (A) Total number of leukocytes and (B) total number of neutrophils, monocytes, eosinophils, and lymphocytes accumulated in air pouches. The data are representative of three independent experiments. (** P<0.01; *** P<0.001).
Anti-SGS antibodies inhibit leukocyte recruitment induced by L. intermedia SGS
To confirm that the effect of L. intermedia SGS on leukocyte accumulation within air pouches was specific, we pre-incubated SGS with anti-SGS immune sera obtained from mice immunized with L. intermedia SGS (data not shown,  ). Pre-incubation of L. intermedia SGS with anti-SGS immune sera inhibited leukocyte accumulation induced by L. intermedia SGS by 56% (Figure 2A), whereas air-pouch inoculation with immune sera alone led to a cellular recruitment similar to that induced by saline (Figure 2A). Notably, the significant decrease in cellular recruitment following incubation of L. intermedia SGS with antisera was associated with a significant reduction (81%) in the number accumulating neutrophils (Figure 2B). Recruitment of monocytes, lymphocytes, and eosinophils, however, remained unchanged (Figure 2B).
Air pouches were raised on BALB/c mice (five to six per group) and were inoculated with either endotoxin-free saline, L. intermedia SGS, L. intermedia SGS+immune sera (SGS+α-SGS), or immune sera alone (α-SGS). Exudates were collected twelve hours after initial stimulation. Leukocytes were enumerated microscopically. (A) Total number of leukocytes and (B) total number of neutrophils, monocytes, eosinophils, and lymphocytes accumulated in air pouches. The data shown are from a single experiment representative of three independent experiments. (* P<0.05; ** P<0.01).
Enhanced neutrophil recruitment in mice immunized with L. intermedia saliva
L. intermedia SGS was able to induce a significant increase in leukocyte recruitment in the air-pouch model of inflammation when compared with saline (Figure 1). This effect was particularly powerful on neutrophil migration and was abrogated when SGS was pre-incubated with anti-SGS-specific antiserum (Figure 2B). We then investigated the initial inflammatory response in mice that had been previously immunized with L. intermedia SGS. Air pouches were raised on the back of immune mice, and pouches were stimulated with L. intermedia SGS. Control mice were injected with endotoxin-free PBS. Mice immunized with L. intermedia SGS showed a significant increase in the total number of leukocytes (Figure 3A) accumulating in the air pouch compared with control mice injected with PBS. Surprisingly, this increase was associated with an accumulation of neutrophils (53%) migrating to the air pouch (Figure 3B), whereas migration of monocytes, eosinophils, and lymphocytes remained unaltered in SGS-immunized mice compared with control mice injected with PBS. Because chemokines, together with adhesion molecules, are key controllers of leukocyte migration, we tested for chemokine expression in the pouch lining tissue. CXC-class chemokines act mainly on neutrophils, whereas CC-class chemokines act on a larger group of cells including monocytes, eosinophils, and lymphocytes. Additionally, cytokines have long been recognized as key elements in the host response against Leishmania (reviewed in . As shown in Figure 3C, expression of CXCL1, CCL2, and CCL4 was significantly upregulated in SGS-immunized mice compared with control mice injected with PBS. Moreover, SGS-immune mice also displayed a significant increase in TNF-α expression without significant modulation in expression of IL-10 or IFN-γ (Figure 3D).
BALB/c mice (five to six per group) received three inoculations with endotoxin-free PBS or L. intermedia SGS. Fifteen days after the last immunization, air pouches were raised and inoculated with L. intermedia SGS. Exudates and air pouch lining tissue were collected twelve hours later. Leukocytes were enumerated microscopically. (A) Total number of recruited leukocyte and (B) total number of neutrophils, monocytes, eosinophils, and lymphocytes accumulated in air pouches. Relative expression of chemokines (C) and cytokines (D) in the air pouch lining tissue was determined by real-time PCR. The data shown are from a single experiment representative of two independent experiments. (* P<0.05; ** P<0.01).
We then investigated whether the neutrophil accumulation effect observed in air pouches raised in SGS-immune mice and stimulated with SGS could be replicated in the ear dermis. As shown in Figure 4, ear sections from control mice injected with PBS showed very few inflammatory cells at either 24 or 48 hours after SGS challenge. In contrast, ear sections from SGS-immunized mice displayed, 24 hours after SGS-challenge, numerous polymorphonuclear and few mononuclear cells (Figure 4); at 48 hours, the inflammatory infiltrate was further increased. Presence of neutrophils was confirmed by myeloperoxidase staining and was not observed in control mice injected with PBS.
Ears of BALB/c mice (five per group) were injected with PBS (control) or were immunized with L. intermedia SGS (five per group). Both groups were challenged in the contra lateral ear with L. intermedia SGS. Ear sections were obtained twenty-four and forty-eight hours after challenge and stained with H&E. Neutrophils were detected by myeloperoxidase staining at forty-eight hours after challenge with SGS. Sections were analyzed by optical microscopy under 200× and 400× magnifications (insert). Sections from one representative experiment are shown.
In vivo effect of L. intermedia SGS on leukocyte recruitment induced by L. braziliensis alone or in combination with SGS
Because SGS-immune mice displayed enhanced neutrophil recruitment, we investigated whether the presence of L. braziliensis, the parasite transmitted by L. intermedia sand flies, would exert any effect in this outcome. Therefore, air pouches were raised on the back of either naïve or SGS-immunized mice and pouches were stimulated with L. braziliensis (Lb) or L. braziliensis+L. intermedia SGS (Lb+SGS). In naïve mice, we did not detect significant differences in the number of accumulating leukocytes (Figure 5A) or in the recruited cell subsets (Figure 5B) following inoculation with Lb or Lb+SGS (Figure 5B). On the contrary, in SGS-immunized mice, Lb+SGS led to a robust and significant increase in the number of accumulating leukocytes compared with Lb alone (Figure 5C). The increase in the number of leukocytes was due to accumulation of neutrophils in the pouches upon inoculation of Lb+SGS (Figure 5D). There was no significant modulation in the recruitment of monocytes, eosinophils, or lymphocytes in naïve or SGS-immunized mice upon inoculation of Lb or Lb+SGS (Figure 5B and 5D, respectively).
BALB/c mice (five to six per group) received three immunizations with L. intermedia SGS. Fifteen days after the last immunization, air pouches were raised in naïve mice and in mice immunized with L. intermedia SGS. Air pouches were inoculated with L. braziliensis alone (Lb) or with L. braziliensis+L. intermedia saliva (Lb+SGS). Exudates were collected twelve hours later. Leukocytes were enumerated microscopically. (A) Total number of leukocytes accumulated in air pouches and (B) total number of neutrophils, monocytes, eosinophils, and lymphocytes accumulated in air pouches of naïve mice. (C) Total number of leukocytes accumulated in air pouches and (D) total number of neutrophils, monocytes, eosinophils, and lymphocytes accumulated in air pouches of SGS-immunized mice The data shown are from a single experiment representative of three independent experiments. (* P<0.05).
In vivo effect of L. intermedia SGS on chemokine and cytokine expression induced by L. braziliensis alone or in combination with SGS
We then investigated the modulation in cytokine and chemokine expression induced by L. braziliensis alone or in the presence of saliva in naïve and in SGS-immunized mice. In naïve mice, pouch stimulation with Lb+SGS induced a significant increase in CXCL10 and CCL2 expression compared with pouch inoculation with Lb alone (Figure 6A). In SGS-immunized mice, chemokine expression was over two-fold higher compared with naïve mice (Figure 6B). More important, pouch inoculation with Lb+SGS led to a different pattern of chemokine expression as indicated by a significant upregulation in expression of CXCL1, CCL3, and CCL4 compared with inoculation of Lb alone (Figure 6B). Of note, in SGS-immunized mice, pouch inoculation with Lb+SGS led to a significant decrease in CXCL10 expression (Figure 6B) as opposed to naïve mice, in which pouch inoculation with Lb+SGS led to upregulation in CXCL10 expression (Figure 6A). Regarding cytokine expression, naïve mice displayed augmented expression of both TNF-α and IL-10 upon pouch inoculation with Lb+SGS (Figure 6C) compared with inoculation with Lb alone. In SGS-immunized mice, stimulation with Lb+SGS led to specific increase in IL-10 expression (Figure 6D). In this same group, inoculation of Lb+SGS was not capable of significantly decreasing expression of IFN-γ and TNF-α (Figure 6D).
BALB/c mice (five to six per group) received three immunizations with L. intermedia SGS. Fifteen days after the last immunization, air pouches were raised in naïve mice and mice immunized with L. intermedia SGS. Air pouches were inoculated with L. braziliensis alone (Lb) or with L. braziliensis+L. intermedia saliva (Lb+SGS). Air pouch lining tissue was submitted to real-time PCR for relative quantification of chemokines and cytokines. Chemokine expression in (A) naïve and (B) SGS-immunized mice. Cytokine expression in (C) naïve and (D) SGS-immunized mice. Bars represent the means and standard errors of the means of five mice per group. The data shown are from a single experiment representative of two independent experiments. (* P<0.05; ** P<0.01).
Sand flies use saliva to manipulate host homoeostasis, favoring the acquisition of a blood meal. These sand fly salivary molecules modify the skin microenvironment and this, in turn, may favor infection by Leishmania parasites (rev. in ). Indeed, we previously observed that L. intermedia SGS-immune mice show a higher disease burden when challenged with L. braziliensis . To gain understanding of the early events associated with inoculation of L. intermedia sand fly saliva, we evaluated leukocyte migration and chemokine/cytokine expression induced in the air-pouch model of inflammation. Importantly, the L. intermedia sand fly is the vector of L. braziliensis , , the main etiologic agent of cutaneous leishmaniasis.
Injection of L. intermedia SGS into air pouches led to a significant increase in the recruitment of neutrophils and monocytes, corroborating previous findings that both of these cell populations are recruited to the site of saliva inoculation , , , , , . Indeed, the initial events following saliva inoculation have recently been explored by in vivo live imaging . It was shown that sand fly biting leads to potent neutrophil migration and that these cells are efficiently infected by L. major, indicating that neutrophils may serve as host cells for Leishmania in the early phase of infection, as previously suggested , . Differently from L. longipalpis saliva , L. intermedia did not lead to accumulation of eosinophils, which are strongly related to mosquito bites and allergies. This distinction in the cellular recruitment induced by L. intermedia vs. L. longipalpis saliva may be explained by variation in the salivary components within sand flies, such as maxadilan, present only in L. longipalpis , and hyaluronidase, present in both L. longipalpis and various species within the genus Phlebotomus , .
Pre-incubation of L. intermedia SGS with specific antisera was able to partially neutralize the leukocyte-recruiting effects of SGS, mainly decreasing the number of accumulating neutrophils, without a significant effect on monocytes. Similarly, Belkaid et al. showed that anti-SGS antibodies could neutralize the ability of P. papatasi SGS to enhance L. major infection in BALB/c mice ; however, SGS-immune mice showed an enhanced neutrophil recruitment upon stimulation with SGS in pre-sensitized animals. The actual levels of anti-saliva antibodies into the pouch exudates are unknown and may not be sufficient to neutralize the in vivo effects of the saliva. Another possibility for the in vivo findings is that salivary molecules are able to trigger cytokine/chemokine expression, despite the presence of neutralizing antibodies, leading to enhanced neutrophil recruitment.
Leukocyte recruitment to sites of inflammation is a key event in both innate and adaptive immunity, and chemokines are major players that regulate the sequential steps of leukocyte rolling, firm adherence, and transmigration. In this sense, we tested for CXC-class chemokines, that act mainly on neutrophils, and CC-class chemokines that act on a larger group of cells including monocytes, eosinophils, and lymphocytes. In mice sensitized and stimulated with L. intermedia SGS, we saw increased neutrophil recruitment and significant upregulation in the expression of CXCL1, CCL2, and CCL4. Indeed, CXC chemokines, such as CXCL1, are critical molecules for neutrophil recruitment , and CXCL1 is also a dominant chemokine in murine inflammatory responses . CCL2 mediates neutrophil adherence and transmigration, a process dependent on activation of mast cells and release leukotrienes and PAF , and CCL4 expression has been associated with a type 1 immune response . Therefore, the enhanced neutrophil chemotaxis in SGS-immunized mice may result from a concomitant upregulation in CXCL1 and CC chemokines (CCL2 and CCL4) and may be further amplified by upregulation in TNF-α, favoring a pro-inflammatory environment as shown by upregulation in CCL4 expression. Indeed, OVA-immunized mice displayed increased neutrophil migration upon antigen stimulation ; this effect was dependent on the release of TNF-α, and leukotriene B(4)  and mediated by CCL3  . Increased neutrophil recruitment was also observed when SGS immunization was conducted in the ear dermis: SGS challenge led to development of an inflammatory reaction characterized by the presence of numerous neutrophils, confirming previously published results . Similarly, exposure of mice to the bites of uninfected L. longipalpis, the vector of L. chagasi, induced an analogous effect . In addition, it has been shown that PSG, the proteophosphoglycan-rich gel secreted by L. mexicana, also leads to potent neutrophil and macrophage recruitment .
In naïve mice, sand fly saliva , , – and fPPG, a component in PSG ,favor the initial establishment of Leishmania infection. In naïve mice, pouch stimulation with L. braziliensis+SGS was unable to alter the cellular recruitment induced by L. braziliensis alone (Figure 5A), as opposed to previous studies conducted with L. longipalpis SGS+L. chagasi  or with L. major+L. longipalpis SGS ; however, pouch stimulation with Lb+SGS induced significant upregulation in the expression of CCL2, CXCL10, TNF-α, and IL-10 (Figure 6A). Accordingly, experimental infection with L. braziliensis leads to increased leukocyte recruitment, CCL2 and CXCL10 expression , and production of IL-10 . More recently, increase CXCL10 and IL-10 expression were observed upon infection of human monocytes with L. braziliensis . Therefore, we can suggest that, although presence of sand fly saliva does not add to the cellular recruitment induced by L. braziliensis, salivary antigens modulate the microenvironment, which may favor parasite establishment as previously suggested . Here we were unable to determine parasite load in cellular exudates obtained from stimulated pouches; however, earlier work from our group also showed that pre-treatment of human monocytes with L. intermedia SGS followed by L. braziliensis infection led to a significant increase in TNF-α production without significant augmentation in the parasite load .
Pre-exposure to L. longipalpis  or P. papatasi saliva  or to bites from uninfected P. papatasi  results in protection against leishmaniasis; however, pre-exposure to L. intermedia saliva does not generate a protective effect upon a challenge infection with L. braziliensis+L. intermedia SGS  although SGS immunized mice do show a significantly lower initial parasite burden after challenge with L. braziliensis+SGS. We hypothesized that this early control in parasite load could be exerted by inflammatory cells (mono and polymorphonuclear cells) that are recruited following stimulation with saliva . Indeed, the results herein show that SGS-immune mice displayed increased leukocyte recruitment, with a marked neutrophil influx (Figure 3) and a similar finding was observed upon inoculation of Lb+SGS (Figure 5). We have recently shown that macrophages and neutrophils collaborate towards L. braziliensis elimination from infected macrophages . Therefore, the current results support our previous hypothesis that an initial inflammatory environment may account for the early control of parasite load in SGS-immunized mice upon challenge with Lb+SGS. This control, however, is limited and L. braziliensis multiplication is later on observed, probably resulting from the pathogen favorable immune response (lower IFN-γ to IL-4 ratio) developed in SGS-immunized mice . Indeed, in the present work, SGS-immunized mice stimulated with Lb+SGS showed decreased CXCL10 expression paralleled with an increased IL-10 expression. Presence of CXCL10 is seen in many Th1-type inflammatory diseases, where it is thought to play an important role in recruiting activated T cells into sites of tissue inflammation . IL-10, on the contrary, is associated with a non-healing L. major infection  and L. major persistence . Consequently, lack of CXCL10 and presence of IL-10 may create a de-activating environment, favoring L. braziliensis expansion in the context of SGS-immunized mice.
We cannot exclude that the increased neutrophil recruitment observed in SGS-immunized mice may also be relevant to the “Trojan horse” model, as documented for L. major infection , in which parasites within neutrophils are silently transferred to macrophages and successfully establish infection. Indeed, the early influx and persistence of neutrophils after sand fly transmission of L. major appears critical for the development of cutaneous disease . Additionally, L. major internalization delays the neutrophil apoptotic death program and induces CCL4 release, which recruits macrophages to the infection site , . Indeed, increased CCL4 expression was observed upon inoculation of Lb+SGS.
Collectively, our data show that in naïve mice, inoculation of L. intermedia saliva plus L. braziliensis modifies the initial inflammatory environment as seen by increased neutrophil recruitment and IL-10 and TNF-α expression. Crucially, in mice sensitized with L. intermedia saliva and stimulated with L. braziliensis, these initial events are further modulated, as seen by a specific decrease in CXCL10 and a persistently increased IL-10 expression. We can speculate that the resulting effects leads to the higher disease burden as previously documented . This study again shows important effects of the L. intermedia sand fly and L. braziliensis interaction. More important, it emphasizes how the immune response to sand fly may exert an under-appreciated role in endemic areas. We are currently characterizing L. intermedia salivary antigens to further identify the components that may induce the effects described here.
We thank Edvaldo Passos for technical assistance Larry Faucette, HT, ASCP, for immunohistochemistry technical support and NIAID intramural editor Brenda Rae Marshall for assistance.
Conceived and designed the experiments: TRdM CIdO. Performed the experiments: TRdM GCR MWC KFF FON. Analyzed the data: TRdM FO GCR MWC KFF MBN CB CIdO. Contributed reagents/materials/analysis tools: JCM AB. Wrote the paper: TRdM FO CIdO.
- 1. WHO web site (2010) Available: http://www.who.int/leishmaniasis/disease_epidemiology/en/index.html.
- 2. Lainson R, Shaw JJ (2005) New World Leishmaniasis;. In: Cox FEG, Wakelin D, Gillespie SH, Despomminer DD, editors. London: ASM Press. pp. 313–349.
- 3. Rangel EF, Lainson R (2003) Ecologia das Leishmanioses: transmissores de leishmaniose tegumentar americana. In: Rangel EF, R. L, editors. Rio de Janeiro: FIOCRUZ. pp. 291–310.
- 4. Titus RG, Ribeiro JM (1988) Salivary gland lysates from the sand fly Lutzomyia longipalpis enhance Leishmania infectivity. Science 239: 1306–1308.
- 5. Belkaid Y, Kamhawi S, Modi G, Valenzuela J, Noben-Trauth N, et al. (1998) Development of a natural model of cutaneous leishmaniasis: powerful effects of vector saliva and saliva preexposure on the long-term outcome of Leishmania major infection in the mouse ear dermis. J Exp Med 188: 1941–1953.
- 6. Belkaid Y, Mendez S, Lira R, Kadambi N, Milon G, et al. (2000) A natural model of Leishmania major infection reveals a prolonged “silent” phase of parasite amplification in the skin before the onset of lesion formation and immunity. J Immunol 165: 969–977.
- 7. Belkaid Y, Valenzuela JG, Kamhawi S, Rowton E, Sacks DL, et al. (2000) Delayed-type hypersensitivity to Phlebotomus papatasi sand fly bite: An adaptive response induced by the fly? Proc Natl Acad Sci U S A 97: 6704–6709.
- 8. Thiakaki M, Rohousova I, Volfova V, Volf P, Chang KP, et al. (2005) Sand fly specificity of saliva-mediated protective immunity in Leishmania amazonensis-BALB/c mouse model. Microbes Infect 7: 760–766.
- 9. Gomes R, Teixeira C, Teixeira MJ, Oliveira F, Menezes MJ, et al. (2008) Immunity to a salivary protein of a sand fly vector protects against the fatal outcome of visceral leishmaniasis in a hamster model. Proc Natl Acad Sci U S A 105: 7845–7850.
- 10. de Moura TR, Oliveira F, Novais FO, Miranda JC, Clarencio J, et al. (2007) Enhanced Leishmania braziliensis Infection Following Pre-Exposure to Sandfly Saliva. PLoS Negl Trop Dis 1: e84.
- 11. Rohousova I, Ozensoy S, Ozbel Y, Volf P (2005) Detection of species-specific antibody response of humans and mice bitten by sand flies. Parasitology 130: 493–499.
- 12. Silva F, Gomes R, Prates D, Miranda JC, Andrade B, et al. (2005) Inflammatory cell infiltration and high antibody production in BALB/c mice caused by natural exposure to Lutzomyia longipalpis bites. Am J Trop Med Hyg 72: 94–98.
- 13. Costa DJ, Favali C, Clarencio J, Afonso L, Conceicao V, et al. (2004) Lutzomyia longipalpis salivary gland homogenate impairs cytokine production and costimulatory molecule expression on human monocytes and dendritic cells. Infect Immun 72: 1298–1305.
- 14. Rogers KA, Titus RG (2003) Immunomodulatory effects of Maxadilan and Phlebotomus papatasi sand fly salivary gland lysates on human primary in vitro immune responses. Parasite Immunol 25: 127–134.
- 15. Anjili CO, Mbati PA, Mwangi RW, Githure JI, Olobo JO, et al. (1995) The chemotactic effect of Phlebotomus duboscqi (Diptera: Psychodidae) salivary gland lysates to murine monocytes. Acta Trop 60: 97–100.
- 16. Titus RG (1998) Salivary gland lysate from the sand fly Lutzomyia longipalpis suppresses the immune response of mice to sheep red blood cells in vivo and concanavalin A in vitro. Exp Parasitol 89: 133–136.
- 17. Oliveira F, Kamhawi S, Seitz AE, Pham VM, Guigal PM, et al. (2006) From transcriptome to immunome: identification of DTH inducing proteins from a Phlebotomus ariasi salivary gland cDNA library. Vaccine 24: 374–390.
- 18. Menezes MJ, Costa DJ, Clarencio J, Miranda JC, Barral A, et al. (2008) Immunomodulation of human monocytes following exposure to Lutzomyia intermedia saliva. BMC Immunol 9: 12.
- 19. Yoshino S, Cromartie WJ, Schwab JH (1985) Inflammation induced by bacterial cell wall fragments in the rat air pouch. Comparison of rat strains and measurement of arachidonic acid metabolites. Am J Pathol 121: 327–336.
- 20. Teixeira CR, Teixeira MJ, Gomes RB, Santos CS, Andrade BB, et al. (2005) Saliva from Lutzomyia longipalpis induces CC chemokine ligand 2/monocyte chemoattractant protein-1 expression and macrophage recruitment. J Immunol 175: 8346–8353.
- 21. de Moura TR, Novais FO, Oliveira F, Clarencio J, Noronha A, et al. (2005) Toward a novel experimental model of infection to study American cutaneous leishmaniasis caused by Leishmania braziliensis. Infect Immun 73: 5827–5834.
- 22. Matte C, Olivier M (2002) Leishmania-induced cellular recruitment during the early inflammatory response: modulation of proinflammatory mediators. J Infect Dis 185: 673–681.
- 23. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408.
- 24. Sacks D, Noben-Trauth N (2002) The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol 2: 845–858.
- 25. Andrade BB, de Oliveira CI, Brodskyn CI, Barral A, Barral-Netto M (2007) Role of sand fly saliva in human and experimental leishmaniasis: current insights. Scand J Immunol 66: 122–127.
- 26. Valenzuela JG, Belkaid Y, Garfield MK, Mendez S, Kamhawi S, et al. (2001) Toward a defined anti-Leishmania vaccine targeting vector antigens: characterization of a protective salivary protein. J Exp Med 194: 331–342.
- 27. Peters NC, Egen JG, Secundino N, Debrabant A, Kimblin N, et al. (2008) In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science 321: 970–974.
- 28. Aga E, Katschinski DM, van Zandbergen G, Laufs H, Hansen B, et al. (2002) Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major. J Immunol 169: 898–905.
- 29. van Zandbergen G, Klinger M, Mueller A, Dannenberg S, Gebert A, et al. (2004) Cutting edge: neutrophil granulocyte serves as a vector for Leishmania entry into macrophages. J Immunol 173: 6521–6525.
- 30. Warburg A, Saraiva E, Lanzaro GC, Titus RG, Neva F (1994) Saliva of Lutzomyia longipalpis sibling species differs in its composition and capacity to enhance leishmaniasis. Philos Trans R Soc Lond B Biol Sci 345: 223–230.
- 31. Cerna P, Mikes L, Volf P (2002) Salivary gland hyaluronidase in various species of phlebotomine sand flies (Diptera: psychodidae). Insect Biochem Mol Biol 32: 1691–1697.
- 32. Volfova V, Hostomska J, Cerny M, Votypka J, Volf P (2008) Hyaluronidase of bloodsucking insects and its enhancing effect on leishmania infection in mice. PLoS Negl Trop Dis 2: e294.
- 33. Kobayashi Y (2008) The role of chemokines in neutrophil biology. Front Biosci 13: 2400–2407.
- 34. Bozic CR, Kolakowski LF Jr, Gerard NP, Garcia-Rodriguez C, von Uexkull-Guldenband C, et al. (1995) Expression and biologic characterization of the murine chemokine KC. J Immunol 154: 6048–6057.
- 35. Reichel CA, Rehberg M, Lerchenberger M, Berberich N, Bihari P, et al. (2009) Ccl2 and Ccl3 Mediate Neutrophil Recruitment via Induction of Protein Synthesis and Generation of Lipid Mediators. Arterioscler Thromb Vasc Biol.
- 36. Schrum S, Probst P, Fleischer B, Zipfel PF (1996) Synthesis of the CC-chemokines MIP-1alpha, MIP-1beta, and RANTES is associated with a type 1 immune response. J Immunol 157: 3598–3604.
- 37. Klein A, Cunha FQ, Ferreira SH (1995) The role of lymphocytes in the neutrophil migration induced by ovalbumin in immunized rats. Immunology 84: 577–584.
- 38. Canetti C, Silva JS, Ferreira SH, Cunha FQ (2001) Tumour necrosis factor-alpha and leukotriene B(4) mediate the neutrophil migration in immune inflammation. Br J Pharmacol 134: 1619–1628.
- 39. Ramos CD, Canetti C, Souto JT, Silva JS, Hogaboam CM, et al. (2005) MIP-1alpha[CCL3] acting on the CCR1 receptor mediates neutrophil migration in immune inflammation via sequential release of TNF-alpha and LTB4. J Leukoc Biol 78: 167–177.
- 40. Rogers M, Kropf P, Choi BS, Dillon R, Podinovskaia M, et al. (2009) Proteophosophoglycans regurgitated by Leishmania-infected sand flies target the L-arginine metabolism of host macrophages to promote parasite survival. PLoS Pathog 5: e1000555.
- 41. Samuelson J, Lerner E, Tesh R, Titus R (1991) A mouse model of Leishmania braziliensis braziliensis infection produced by coinjection with sand fly saliva. J Exp Med 173: 49–54.
- 42. Lima HC, Titus RG (1996) Effects of sand fly vector saliva on development of cutaneous lesions and the immune response to Leishmania braziliensis in BALB/c mice. Infect Immun 64: 5442–5445.
- 43. Theodos CM, Ribeiro JM, Titus RG (1991) Analysis of enhancing effect of sand fly saliva on Leishmania infection in mice. Infect Immun 59: 1592–1598.
- 44. Rogers ME, Ilg T, Nikolaev AV, Ferguson MA, Bates PA (2004) Transmission of cutaneous leishmaniasis by sand flies is enhanced by regurgitation of fPPG. Nature 430: 463–467.
- 45. Monteiro MC, Lima HC, Souza AA, Titus RG, Romao PR, et al. (2007) Effect of Lutzomyia longipalpis salivary gland extracts on leukocyte migration induced by Leishmania major. Am J Trop Med Hyg 76: 88–94.
- 46. Teixeira MJ, Fernandes JD, Teixeira CR, Andrade BB, Pompeu ML, et al. (2005) Distinct Leishmania braziliensis Isolates Induce Different Paces of Chemokine Expression Patterns. Infect Immun 73: 1191–1195.
- 47. Vargas-Inchaustegui DA, Hogg AE, Tulliano G, Llanos-Cuentas A, Arevalo J, et al. (2009) CXCL10 production by human monocytes in response to Leishmania braziliensis infection. Infect Immun.
- 48. Ribeiro JM (1995) Blood-feeding arthropods: live syringes or invertebrate pharmacologists? Infect Agents Dis 4: 143–152.
- 49. Kamhawi S, Belkaid Y, Modi G, Rowton E, Sacks D (2000) Protection against cutaneous leishmaniasis resulting from bites of uninfected sand flies. Science 290: 1351–1354.
- 50. Novais FO, Santiago RC, Bafica A, Khouri R, Afonso L, et al. (2009) Neutrophils and Macrophages Cooperate in Host Resistance against Leishmania braziliensis Infection. J Immunol.
- 51. Dufour JH, Dziejman M, Liu MT, Leung JH, Lane TE, et al. (2002) IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. J Immunol 168: 3195–3204.
- 52. Kane MM, Mosser DM (2001) The role of IL-10 in promoting disease progression in leishmaniasis. J Immunol 166: 1141–1147.
- 53. Belkaid Y, Hoffmann KF, Mendez S, Kamhawi S, Udey MC, et al. (2001) The role of interleukin (IL)-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti-IL-10 receptor antibody for sterile cure. J Exp Med 194: 1497–1506.
- 54. Muller K, van Zandbergen G, Hansen B, Laufs H, Jahnke N, et al. (2001) Chemokines, natural killer cells and granulocytes in the early course of Leishmania major infection in mice. Med Microbiol Immunol 190: 73–76.