Int. J. Exp. Path. (2014)

ORIGINAL ARTICLE

Salivary gland homogenates from wild-caught sand flies Lutzomyia flaviscutellata and Lutzomyia (Psychodopygus) complexus showed inhibitory effects on Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis infection in BALB/c mice Fernanda C. Francesquini*, Fernando T. Silveira†,‡, Luiz Felipe D. Passero*, Thaise Y. Tomokane*, Ana Kely Carvalho*, Carlos Eduardo P. Corbett* and Marcia D. Laurenti* *Laboratory of Pathology of Infectious Diseases LIM-50, Medical School, University of S~ ao Paulo, S~ ao Paulo, S~ ao Paulo State, a State, Brazil, †Laboratory of Leishmaniasis ‘Prof. Dr. Ralph Lainson’, Evandro Chagas Institute, Ministry of Health, Belem, Par a, Belem, Par a State, Brazil Brazil and ‡Tropical Medicine Nucleus, Federal University of Par

INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY

SUMMARY

During the natural transmission of Leishmania parasites, the infected sand fly female regurgitates promastigotes into the host’s skin together with its saliva. It has been reported that vector saliva contains immunomodulatory molecules that facilitate the establishment of infection. Thus, the main objective of this study was to evaluate the specificity of Lutzomyia (Lu.) flaviscutellata and Lu. (Psychodopygus) complexus salivas on the infectivity of Leishmania (L.) (Leishmania) amazonensis and L. (Viannia) doi: 10.1111/iep.12104 braziliensis, respectively. BALB/c mice were inoculated into the skin of hind footpad with L. (L.) amazonensis and L. (V.) braziliensis promastigotes in the absence or presence of Lu. flaviscutellata and Lu. (P.) complexus salivary gland homogenates (SGHs). The evolution of the infection was evaluated by lesion size, histopathological analysis and determination of the parasite load in the skin biopsies collected from the site of infection at 4 and 8 weeks PI. The lesion size and the parasite load of both groups of mice infected in the presence of SGHs were smaller than the control groups. The histopathological features showed that the inflammatory reaction was less promiReceived for publication: 8 April 2014 nent in the groups of mice infected in the presence of both SGHs when compared to Accepted for publication: 28 the control group. The results showed that the presence of SGHs of Lu. flaviscutellata September 2014 and Lu. (P.) complexus led to induction of processes that were disadvantageous to Correspondence: parasite establishment during infection by L. (L.) amazonensis and L. (V.) braziliensis. M arcia D. Laurenti An inhibitory effect on Leishmania infection could be observed in both groups inocuDepto Patologia, Faculdade de lated with SGHs, especially when the SGH from Lu. (P.) complexus was used. Medicina, Universidade de S~ao Paulo Av. Dr. Arnaldo, 455 – 1° andar – sala 1209 CEP: 01246-903, Cerqueira Cesar, S~ ao Paulo (SP), Brasil Tel./Fax: +55 11 30618339 E-mail: [email protected]

Keywords BALB/c mice, experimental cutaneous leishmaniasis, Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, Lutzomyia (Psychodopygus) complexus, Lutzomyia flaviscutellata, salivary gland homogenate

Diseases transmitted by arthropod bites are considered a serious public health problem worldwide, as they greatly impact the animal and human populations living in tropical and subtropical areas. During blood feeding the active components of arthropod saliva interact with components of the extracellular matrix and blood of hosts, frequently leading to

the initiation of processes advantageous to vectorborne pathogens (Ribeiro 1987, 1989; Champagne 1994; Wikel 1999). During its blood meal, the vector of Leishmania parasites regurgitates a large part of its saliva, which contains promastigote forms of Leishmania, into the host’s skin (Roberts 2006). Phlebotomine saliva, as well as its

© 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology

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components, has been studied often with respect to the disease outcome caused by different species of Leishmania parasites. In this way, Titus and Ribeiro (1988) described the biological effect of the saliva of Lutzomyia (Lu.) longipalpis on the experimental infections caused by Leishmania (L.) major in CBA mice, which is a murine model of Leishmania resistance. The CBA mice innoculated with parasites together with saliva had larger lesions and higher skin parasitism when compared to the control mice inoculated with parasite alone, even when a small number of parasites were used to infect the mice (Titus & Ribeiro 1990). Other studies have also demonstrated that Lu. longipalpis saliva exacerbated experimental infections caused by L. (L.) amazonensis and L. (V.) braziliensis (Samuelson et al. 1991; Theodos et al. 1991). The effect of phlebotomine saliva, which aids in the establishment of infection, was also described for Phlebotomus papatasi during the development of experimentally cutaneous leishmaniasis caused by L. major (Belkaid et al. 1998). The proposed mechanism for this effect was related to the immunomodulatory properties of the saliva and its components (Theodos et al. 1991; Kamhawi 2000; Andrade et al. 2007). The effect probably occurred because the Lu. longipalpis salivary gland lysate decreases the ability of murine macrophages to present antigens, thus compromising the activation of specific T lymphocytes. In addition, macrophages did not respond to interferon (IFN)-c activation, and they drastically reduced the production of hydrogen peroxide and nitric oxide (Titus & Ribeiro 1990; Theodos & Titus 1993; Hall & Titus 1995). Additionally, mice co-injected with parasites and salivary gland extracts displayed higher levels of interleukin (IL)-10 messenger (m)RNA in the tissue, as well as higher amounts of the bioactive protein in the supernatant of draining lymph node cells than did the mice injected only with parasites, suggesting that saliva components can upregulate the mechanisms associated with parasite evasion (Norsworthy et al. 2004). However, studies have proposed that the effect of phlebotomine saliva on the outcome of Leishmania infection depends on parasite and vector characteristics, as well as on the host’s genetic and immunological background (Locksley et al. 1991). Recently, it was demonstrated that Lu. longipalpis saliva exacerbates the infection caused by L. (L.) amazonensis in C57BL/6 mice, but this effect was more prominent when parasites were co-injected with laboratoryreared Lu. longipalpis saliva, as compared to wild-caught vector saliva (Laurenti et al. 2009a,b). In addition, L. (L.) infantum chagasi infection in the presence of Lu. longipalpis saliva did not lead to early amastigote detection, nor did it alter the evolution of infection in dogs (Paranhos-Silva et al. 2003). Similarly, Gomes et al. (2008) did not find any alteration in disease outcomes in hamsters that were infected with L. (L.) infantum chagasi in the presence of Lu. longipalpis saliva when compared to hamsters infected only with parasites. Controversially, the role of phlebotomine saliva on the protection of Leishmania infection was also described

(Kamhawi et al. 2000). Previously, it was demonstrated that mice pre-exposed to P. papatasi salivary gland lysate could restrain the spreading of L. major (Belkaid et al. 1998). This protective effect elicited by the components of vector saliva appears to be parasite/vector specific (Thiakaki et al. 2005). A recent study showed that the immunization of hamsters with the salivary protein of Lu. longipalpis conferred protection against the fatal evolution of visceral leishmaniasis caused by L. chagasi, as these animals presented with low levels of parasitism, and this correlated with increased IFNc and inducible nitric oxide synthase (iNOS) mRNA copies in the spleen and liver, reinforcing the concept of using phlebotomine saliva components in vaccine strategies (Gomes et al. 2008). In addition to parasite/vector specificities in the development of effective immunity against leishmaniasis, the relative amount of specific proteins present in phlebotomine saliva can determine the host’s immunity profile, which will be induced. In this case, Oliveira et al. (2008) demonstrated that PpSP15 and PpSP44 recombinant proteins from P. papatasi induced immunological responses associated with resistance and susceptibility to Leishmania major infection, respectively (Oliveira et al. 2008). In addition, DNA plasmids coding for ten selected transcripts of Lu. intermedia were constructed and their immunogenicity was evaluated. One of them, coding for a 4.5-kDa protein, induced a cellular immune response and conferred protection against L. braziliensis infection. This protection correlated with a decreased parasite load and an increased frequency of IFNc-producing cells (de Moura et al. 2013). Despite many studies on the exacerbating and protective effects of phlebotomine salivas in Leishmania infection, few studies were conducted on the natural parasite/vector relationship specially using salivary gland homogenate (SGH) from wild-caught sand flies. Thus, the main objective of this study was to evaluate the course of experimental cutaneous infections caused by L. (L.) amazonensis and L. (V.) braziliensis in the presence of SGHs from their specific vectors, Lu. flaviscutellata and Lu. (Psychodopygus) complexus, respectively.

Materials and methods Mice Eight-week-old BALB/c mice obtained from the Animal Facility of the S~ ao Paulo University, Medical School, Brazil, were maintained in our laboratory during the experiments, according to the guidelines of the institutional rules regarding the welfare of experimental animals and with the approval of the Animal Ethics Committee of S~ ao Paulo University (protocol number 120/11).

Ethical approval These strains were provided by Prof. Fernando Tobias Silveira, our colaborator. They belong to criobank under his supervision. International Journal of Experimental Pathology

Sand fly saliva in Leishmania infection

Parasites Leishmania. (L.) amazonensis (MHOM/BR/1973/M2269) and L. (V.) braziliensis (MHOM/BR/1995/M15280) parasites were isolated from patients with anergic diffuse cutaneous and mucocutaneous leishmaniasis, respectively, from Par a State, north Brazil. The parasites were identified using monoclonal antibodies (Shaw et al. 1989) and isoenzyme electrophoretic profiles (Miles et al. 1980) at the Laboratory of Leishmaniasis, Evandro Chagas Institute (Belem, Para State, Brazil). Leishmania (L.) amazonensis was maintained in the footpads of BALB/c mice, isolated and grown in Roswell Park Memorial Institute (RPMI)1640 medium (Gibcoâ; Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% heat-inactivated foetal bovine serum (FBS), 10 lg/ml of gentamicin and 1,000 UI/ml of penicillin at 25 °C. Leishmania (V.) braziliensis has been maintained in hamster footpad, isolated and grown in Schneider’s Drosophila medium (SigmaAldrich Co., St. Louis, MO, USA), supplemented with 10% heat-inactivated FBS, 10 lg/ml of gentamicin and 100 UI/ml of penicillin at 25 °C. On the sixth day of culture, promastigote forms from the stationary phase of culture growth were centrifuged (1,620 g, for 10 min) in phosphate-buffered saline (PBS) solution, pH 7.4, and they were used for mice infection.

Sand flies and SGH production Wild-caught sand flies of Lu. flaviscutellata, Lu. (P.) complexus and Lu. longipalpis were collected in the municipalities of Tracateua and Cameta, both located at the north and north-eastern regions of Para State, Brazil, respectively. The SGHs were obtained from the female phlebotomines that were immobilized in 20 °C for about 2 min. The dissected salivary glands were collected in PBS, pH 7.2, and stored at 80 °C. At the time of the experiments, the salivary glands were disrupted by freeze–thawing and vortexed to homogenate the lysate. The equivalent of a half-pair of salivary glands was used for each inoculation point.

BALB/c mice infection Ninety BALB/c mice (six/group) were used for each experiment. Twenty-four mice were infected subcutaneously into the hind footpad with 106 promastigote forms from the stationary phase, either with L. (L.) amazonensis from a low in vitro passage (≤6 passages) in 50 ll of PBS in the presence of the equivalent of a half-pair of SGH of Lu. flaviscutellata (n = 12) or with Lu. (P.) complexus (n = 12), and 24 mice were infected with L. (V.) braziliensis in the presence of the equivalent of a half-pair of SGH of Lu. flaviscutellata (n = 12) or Lu. (P.) complexus (n = 12). As with the control groups, mice were inoculated only with the parasite, L. (L.) amazonensis (n = 12) and L. (V.) braziliensis (n = 12) in the absence of SGH,

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and only with PBS (n = 12). Mice infected with L. (L.) amazonensis in the presence of SGH of Lu. longipalpis (n = 6) were also used as a positive control. The experiment was repeated three times.

Macroscopic evaluation of infection The hind footpad swelling in each infected mouse was monitored weekly for 8 weeks by measuring the thickness of the infected footpad with a metric calliper and subtracting the thickness of the uninfected control footpad.

Evaluation of the parasite load The parasite load in the skin lesion was determined using the quantitative limiting-dilution assay, as previously described (Passero et al. 2010). Briefly, the infected footpads were aseptically excised at the fourth and eighth weeks PI and homogenized in Schneider’s medium. The cellular suspension was subjected to 12 serial dilutions with four replicate wells. The number of viable parasites was determined from the highest dilution that promastigotes could be grown after 10 days of incubation at 25 °C.

Histopathology analysis At 4 and 8 weeks PI, biopsies from the hind footpads were collected to evaluate the histopathological changes in the skin. The tissue fragments were collected with a 4-mm punch and fixed in 10% formalin, pH 7.2, and processed by the usual techniques for optical microscopy. Qualitative analysis was performed to evaluate the main histological changes observed in the dermis and epidermis, and a semiquantitative analysis was performed to characterize the cells involved in the dermal inflammatory infiltrate, such as polymorphonuclear (PMN), mononuclear and lymphocyte cells, as well as parasitism. The parameters were scored as (0) negative, (1) mild, (2) moderate, (3) intense and (4) severe for each animal. Skin sections of the control mice (injected only with PBS) served as the pattern for normal skin with the absence of an inflammatory process.

Statistical analysis The GRAPHPAD PRISM version 5.0 software for Windows (GraphPad Software, Inc., La Jolla, CA, USA) was used to analyse the results. Differences in lesion size and skin parasitism were assessed by the analysis of variance (ANOVA) parametric test, followed by Bonferroni’s test for multiple comparisons. The parametric t-test was used to evaluate differences among the experimental groups for the semiquantitative studies of histological sections. Significant differences were considered when P < 0.05. The results were expressed as the mean  SD. The experiment was repeated three times.

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Results Macroscopic evaluation of infection Leishmania (L.) amazonensis induced the progressive growth of skin lesions in all groups of BALB/c mice during the time of infection. However, mice infected with L. (L.) amazonensis plus Lu. flaviscutellata SGH presented with smaller lesion sizes than did the control group since the sixth week PI (P < 0.05); moreover, mice infected with L. (L.) amazonensis plus Lu. (P.) complexus SGH showed significantly lower lesion sizes from the third week until the eighth week PI, as compared to the control group (P < 0.05). As was expected, mice infected with L. (L.) amazonensis in the presence of Lu. longipalpis SGH showed higher lesion sizes than did the control mice, which were infected only with parasite, since the first week PI (P < 0.05) (Figure 1a). On the other hand, L. (V.) braziliensis-infected BALB/c mice showed a slight evolution of lesions, with development of small lesion at the fifth week PI followed by decreases in the size of lesions. Mice infected with L. (V.) braziliensis plus Lu. flaviscutellata SGH showed lower lesion sizes at the second, fourth, fifth, seventh and eighth weeks PI as compared to the control group (P < 0.05), but the sizes were greater (P < 0.05) than those in the group infected with L. (V.) braziliensis plus Lu. (P.) complexus SGH at the third, fifth, sixth and seventh weeks PI (Figure 1b), which did not show macroscopic alterations.

Parasite load BALB/c mice infected with L. (L.) amazonensis plus Lu. (P.) complexus SGH showed lower (P < 0.05) parasite load than did the animals infected with L. amazonensis alone, and (a)

also when compared to the mice infected with L. (L.) amazonensis plus Lu. flaviscutellata SGH at the fourth week PI. At the eighth week PI, the parasite load of both groups of mice infected with L. (L.) amazonensis plus Lu. (P.) complexus or Lu. flaviscutellata SGHs was lower (P < 0.05) than that of the control group. Regarding the evolution of the infection, a significant increase in the number of viable parasites was observed at the inoculation site in the control group (mice infected only with the parasites), while no difference was observed in both groups infected with Lu. flaviscutellata or Lu. (P.) complexus SGHs (Figure 2a). In general, BALB/c mice infected with L. (V.) braziliensis presented with lower levels (P < 0.05) of skin parasitism when compared to animals infected with L. (L.) amazonensis. At the fourth week PI, both groups of mice infected with L. (V.) braziliensis plus Lu. (P.) complexus or Lu. flaviscutellata SGHs showed lower (P < 0.05) numbers of viable parasites in the skin than did the animals infected with L. (V.) braziliensis alone. At the eighth week, no difference was observed in the parasite load among groups. During the evolution of the infection, the control group presented with a significant decrease (P < 0.05) in the number of parasites at the inoculation site, while no difference was observed in both groups infected with Lu. flaviscutellata or Lu. (P.) complexus SGHs (Figure 2b).

Histopathological analysis Qualitative histopathological analysis showed that skin lesions of all groups of BALB/c mice infected with L. amazonensis in the presence or absence of SGHs presented with similar histological features at the fourth week PI (Figure 3a, c and e), characterized by moderate mononuclear inflammatory infiltrate in the dermis (blue arrows), with the presence of parasitized macrophages (black (b)

Figure 1 BALB/c mice were infected in the hind footpad with 106 promastigote forms of Leishmania (L.) amazonensis (a) or L. (V.) braziliensis (b) in the stationary phase of growth in the presence or absence of salivary gland homogenates (SGHs) from Lutzomyia flaviscutellata, Lu. (P.) complexus or Lu. longipalpis, and the evolution of the lesion size was monitored weekly for 8 weeks PI. *P < 0.05 between mice infected in the presence of Lu. flaviscutellata SGH and control mice (infected in the absence of SGH); §P < 0.05 between mice infected in the presence of Lu. (P.) complexus SGH and the control mice; #P < 0.05 between mice infected in the presence of Lu. longipalpis SGH and the control mice; ♦ P < 0.05 between mice infected in the presence of Lu. flaviscutellata SGH and the mice infected in the presence of Lu. (P.) complexus SGH. Values represent the mean  SD of the three independent experiments. International Journal of Experimental Pathology

Sand fly saliva in Leishmania infection (a)

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Figure 2 Fragments of hind footpads from animals infected with Leishmania (L.) amazonensis (a) or L (V.) braziliensis (b) in the presence or absence of salivary gland homogenate (SGH) from Lutzomyia flaviscutellata and Lu. (P.) complexus were macerated, and the parasite loads were determined through limiting-dilution assay. *P < 0.05 between mice infected in the presence of Lutzomyia flaviscutellata SGH and the control mice (infected in the absence of SGH) at the same time of infection; §P < 0.05 between the mice infected in the presence of Lu. (P.) complexus SGH and the control mice at the same time of infection; #P < 0.05 in the evolution of the infection, between the fourth and eighth week PI. Values represent the mean  SD of the three independent experiments.

Figure 3 Histological sections of the skin from BALB/c mice hind footpads infected with Leishmania (L.) amazonensis, L (L.) amazonensis plus Lutzomyia flaviscutellata salivary gland homogenate (SGH) and L. (L.) amazonensis plus Lu. (P.) complexus SGH at 4 (a, c and e, respectively) and 8 weeks PI (b, d and f, respectively), showing mononuclear inflammatory infiltrate in the dermis (blue arrow) with many parasites (black arrow) and focal areas of necrosis (red arrow). (haematoxylin and eosin; 209: a, c, e and 409: b, f) International Journal of Experimental Pathology

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arrows), few lymphocytes and focal areas of necrosis (red arrows). At the eighth week PI, the lesions showed an intense mononuclear inflammatory process, with a predominance of heavily parasitized vacuolated macrophages (black arrows), which invaded the muscle layers. Evident necrotic areas (red arrows) were also observed (Figure 3b, d and f). The skin lesions in all groups of BALB/c mice infected with L. braziliensis in the presence or absence of SGHs showed a discrete mononuclear inflammatory process in the dermis (blue arrow) that was composed of rare parasitized macrophages (black arrows) and a few lymphocytes at the fourth week PI (Figure 4a, c and e). At the eighth week PI, the cellular infiltrate varied from discrete to moderate without evidence of the amastigote forms in the macrophages. The mice infected in the presence of Lu. (P.) complexus SGH showed a total regression of the inflammatory process at the end of the experiment (Figure 4b, d and f). No changes were observed in the epidermis of mice infected with L. (L.) amazonensis or with L. (V.) braziliensis in the absence or presence of SGHs from both phlebotomine sand flies (data not shown). A semiquantitative histopathological analysis showed similar skin parasitism among the experimental groups of BALB/c mice infected with L. (L.) amazonensis at the fourth and eighth weeks PI (Figure 5a). Regarding the L. (V.) bra(a)

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ziliensis infection, it was noted that BALB/c mice infected with L. (V.) braziliensis plus Lu. (P.) complexus SGH presented with significantly lower (P < 0.05) tissue parasitism than did the animals infected with L. (V.) braziliensis plus Lu. flaviscutellata SGH and the control group at the fourth week PI. However, at the eighth week PI, the parasitism scores were low and similar among the experimental groups infected with L. (V.) braziliensis. (Figure 5a). Concerning PMN cells (Figure 5b), in the skin lesions of BALB/c mice infected with L. (L.) amazonensis in the absence or presence of Lu. flaviscutellata or Lu. (P.) complexus SGHs, similar scores of PMN cells were obtained either at the fourth week or at the eighth week PI. Animals infected with L. (V.) braziliensis plus Lu. (P.) complexus SGH presented with lower scores of PMN cells when compared to those mice infected with the parasite plus Lu. flaviscutellata SGH, and also those infected with the parasite alone (P < 0.05) at the fourth week PI. At the eighth week PI, the score of PMN cells was similar among all of the experimental groups of mice infected with L. (V.) braziliensis (Figure 5b). Animals infected with L. (L.) amazonensis with or without SGHs did not show alterations in macrophage scores at the fourth and eighth weeks PI. Conversely, animals infected with L. (V.) braziliensis plus Lu. (P.) complexus SGH pre-

Figure 4 Histological sections of the skin from BALB/c mice hind footpads infected with Leishmania (V.) braziliensis, L (L.) braziliensis plus Lutzomyia flaviscutellata salivary gland homogenate (SGH) and L. (L.) braziliensis plus Lu. (P.) complexus SGH at 4 (a, c and e, respectively) and 8 weeks PI (b, d and f, respectively), showing inflammatory reactions in the dermis (blue arrow), composed of polymorphonuclear and mononuclear cells, with few parasites inside the macrophages (black arrow) (haematoxylin and eosin; 209). International Journal of Experimental Pathology

Sand fly saliva in Leishmania infection (a)

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Figure 5 Semiquantitative histopathological analysis performed in the skin sections of BALB⁄c mice infected with Leishmania (L.) amazonensis or L (V.) braziliensis in the presence or absence of Lutzomyia flaviscutellata and Lu. (P.) complexus salivary gland homogenate (SGH). The following scores were used: (0) negative, (1) mild, (2) moderate, (3) intense and (4) very intense, according to the intensity of tissue parasitism and the presence of inflammatory cells. The values represent the mean  SD of the three independent experiments of parasitism (a), polymorphonuclear cells (b), macrophages (c) and lymphocytes (d). *P < 0.05 between the mice infected in the presence of Lu. flaviscutellata SGH and the control mice (infected in the absence of SGH) at the same time of infection; §P < 0.05 between the mice infected in the presence of Lu. (P.) complexus SGH and the control mice at the same time of infection; ♦P < 0.05 between the mice infected in the presence of Lu. flaviscutellata SGH and the mice infected in the presence of Lu. (P.) complexus SGH at the same time of infection.

sented with lower scores of macrophages than in the lesions from the control mice and the mice infected with L. (V.) braziliensis plus Lu. flaviscutellata SGH (P < 0.05). At the eighth week PI, the scores of macrophages were low and similar among all the experimental groups of mice infected with L. (V.) braziliensis (Figure 5c). With respect to the lymphocytes, the number of these cells in the skin lesions of BALB/c mice infected with L. (L.) amazonensis in the absence or presence of both SGHs was similar among all experimental groups at the fourth and eighth week PI. However, a significantly lower number of lymphocytes were observed in the skin of BALB/c mice infected with L. (V.) braziliensis plus Lu. (P.) complexus SGH compared to those infected with L. (V.) braziliensis plus Lu. flaviscutellata SGH at the fourth week PI (P < 0.05). Similarly, at the eighth week PI, mice infected with L. (V.) braziliensis plus Lu. (P.) complexus SGH presented with significantly fewer lymphocytes in the lesions when compared to those mice infected with L. (V.) braziliensis plus Lu. flaviscutellata SGH and the control group (P < 0.05) (Figure 5d).

International Journal of Experimental Pathology

Discussion There are several reports concerning the effects of phlebotomine saliva on the enhancement of Leishmana infection. However, most of them were performed with laboratorycolonized vectors; this implies that some artificial factors certainly differ from the natural environmental conditions where Leishmania transmission occurs. Therefore, the outcome of the infection can be altered. Recently, it was demonstrated that the saliva from laboratory-reared and wildcaught sand flies significantly differs in terms of composition and the amount of salivary proteins, resulting in different biological effects in the experimental hosts, thus altering the outcome of Leishmania infection (Laurenti et al. 2009a,b). Furthermore, studies on the infection of Leishmania in terms of the natural association between vectors and parasites are rare and controversial. In this regard, the present study aimed to shed light on the effect of the saliva from the wildcaught sand flies, Lu. flaviscutellata and Lu. (P.) complexus, the main vectors of L. (L.) amazonensis and L. (V.) braziliensis, respectively, in the Amazonian region of Brazil.

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In spite of some reports that have been shown that Lu. longipalpis SGHs are able to enhance the infection caused by L. major (Belkaid et al. 1998), L. (V.) braziliensis (Theodos et al. 1991) and L. amazonensis (Laurenti et al. 2009a), there is a study that has demonstrated that Lu. longipalpis saliva did not alter the infection caused by L. infantum chagasi in hamsters, which is a natural parasite/ vector relationship in Latin America (Gomes et al. 2008). Likewise, beagle dogs injected with L. infantum chagasi alone presented with similar clinical, parasitological and immunological outcomes as those infected with the parasite plus Lu. longipalpis saliva (Paranhos-Silva et al. 2003). Therefore, the association between vector saliva and parasite is not always detrimental to the host, as has been previously documented. In this regard, the results of this study have clearly demonstrated that BALB/c mice inoculated with L. (L.) amazonensis plus the SGH of its natural phlebotomine vector, wild-caught Lu. flaviscutellata or Lu. (P.) complexus, which is the natural phlebotomine vector of L. (V.) braziliensis, presented with smaller lesion sizes and less skin parasitism when compared to the control group. Similarly, mice infected with L. (V.) braziliensis plus Lu. (P.) complexus or Lu. flaviscutellata SGHs also presented with smaller lesion sizes and less skin parasitism when compared to L. (V.) braziliensis-infected BALB/c mice. In addition to the parasite and vector specificity in the development of Leishmania infection, the different proteins present in phlebotomine saliva must also be considered, as distinct proteins are able to induce different immunological profiles that could be correlated with infection resistance or susceptibility (Oliveira et al. 2008). Recently, it was reported that one in ten DNA plasmids coding for transcripts of Lu. intermedia used to immunize mice was able to induce a cellular immune response that conferred protection against L. braziliensis infection, as sensitized animals presented with less parasitism and a higher frequency of IFN-cproducing cells when compared to non-immunized animals (de Moura et al. 2013). Although the present study has used whole SGHs, the results suggest that wild-caught Lu. flaviscutellata and Lu. (P.) complexus SGHs should contain compounds that are able to stimulate the mechanisms associated with cellular immunity, thus favouring a decrease in the disease that was caused by both parasites at the skin site of infection. Moreover, it has been reported that different inoculums of salivary gland extract can markedly modify the cellular immune response, which is reflected in the pattern of susceptibility or resistance to Leishmania infection (Carregaro et al. 2013). Additionally, a histological analysis of the dermal site of infection showed that a favourable histological process was found mainly in L. (V.) braziliensis infection in the presence of Lu. (P.) complexus SGH, at the fourth week PI, where the inflammatory infiltrate was mild in the dermis, and there was participation of a few macrophages and lymphocytes. At this point, mice infected with L. (V.) braziliensis plus Lu. flaviscutelatta SGH already presented with similar histological features to those observed in the

mice infected with L. (V.) braziliensis alone; however, there were fewer parasites within the macrophages. At the eighth week PI, the parasitism drastically decreased, yielding only a few macrophages and PMN cells in the lesions of all groups infected with L. (V.) braziliensis. Nevertheless, lymphocytes could be detected in the control and L. (V.) braziliensis plus Lu. flaviscutellata SGH groups. It has been well documented that L. (V.) braziliensis causes a mild infection in BALB/c mice with the presence of a few infected macrophages, neutrophils and high numbers of lymphocytes (DeKrey et al. 1998; Maioli et al. 2004), ultimately resulting in parasite destruction. Moreover, selfhealing lesions are frequently noted (Donnelly et al. 1998; de Moura et al. 2005), as demonstrated in the present study. Conversely, animals infected with L. (L.) amazonensis in the absence or presence of both phlebotomine SGHs presented with intense parasitism and moderate inflammatory infiltrates, which are composed of mononuclear and PMN cells at the fourth week PI. However, with the evolution of the infection (at the eighth week PI), the inflammatory infiltrate of infected mice with or without both vector SGHs was basically monomorphic, showing high numbers of macrophages that were full of amastigotes and with scarce or absent lymphocytes. Similar findings confirm that L. (L.) amazonensis parasites can rapidly proliferate inside BALB/c macrophages, and the absence of lymphocytes in the lesion can be a factor that enables parasite spreading (Qi et al. 2004; Carvalho et al. 2012). In the animals infected with L. (L.) amazonensis plus Lu. (P.) complexus SGH, high numbers of lymphocytes were observed in the skin lesions in relation to the other groups; this could be related to low parasitism, as verified by the limiting-dilution assay in this experimental group. In conclusion, the present results indicate that the SGHs of wild-caught sand flies lead to the initiation of disadvantageous processes to parasite establishment during experimental infections caused by L. (L.) amazonensis and L. (V.) braziliensis, as small lesion sizes and low skin parasitism were detected in relation to infection with Lu. flaviscutellata and Lu. (P.) complexus SGHs. It should be highlighted that the inhibitory effect could be observed in both groups inoculated with SGHs, especially when the SGH from Lu. (P.) complexus was used. Further studies concerning the immune response and salivary gland composition are necessary to elucidate the role of Lu. flaviscutellata and Lu. (P.) complexus SGHs in the outcome of L. (L.) amazonensis and L. (V.) braziliensis infections.

Acknowledgements This project was supported by LIM50 HC-FMUSP. We would like to thank Iorlando Barata, Roberto Carlos Brand~ ao, Jose Aprıgio Lima, Maria Suely Pinheiro, Edna Le~ ao, Luciene Aranha Santos and Dr. Thiago Vasconcelos for their entomological assistance. Fernanda C. Francesquini received a Master Scholarship from CAPES, Brazil. M arcia D. Laurenti is a Research Fellow from CNPq, Brazil. International Journal of Experimental Pathology

Sand fly saliva in Leishmania infection

References Andrade B.B., de Oliveira C.I., Brodskyn C.I., Barral A. & Barral-Netto M. (2007) Role of sand fly saliva in human and experimental leishmaniasis: current insights. Scand. J. Immunol. 66, 122–127. Belkaid Y., Kamhawi S., Modi G. 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. Carregaro V., Costa D.L., Brodskyn C. et al. (2013) Dual effect of Lutzomyia longipalpis saliva on Leishmania braziliensis infection is mediated by distinct saliva-induced cellular recruitment into BALB/c mice ear. BMC Microbiol. 13, e102. Carvalho A.K., Silveira F.T., Passero L.F., Gomes C.M., Corbett C.E. & Laurenti M.D. (2012) Leishmania (V.) braziliensis and L. (L.) amazonensis promote differential expression of dendritic cells and cellular immune response in murine model. Parasite Immunol. 34, 395–403. Champagne D. (1994) The role of salivary vasodilators in bloodfeeding and parasite transmission. Parasitol. Today 10, 430–433. de Moura T.R., Novais F.O., Oliveira F. et al. (2005) Toward a novel experimental model of infection to study American cutaneous leishmaniasis caused by Leishmania braziliensis. Infect. Immun. 73, 5827–5834. de Moura T.R., Oliveira F., Carneiro M.W. et al. (2013) Functional transcriptomics of wild-caught Lutzomyia intermedia salivary glands: identification of a protective salivary protein against Leishmania braziliensis infection. PLoS Negl. Trop. Dis. 7, e2242. DeKrey G.K., Lima H.C. & Titus R.G. (1998) Analysis of the immune responses of mice to infection with Leishmania braziliensis. Infect. Immun. 66, 827–829. Donnelly K.B., Lima H.C. & Titus R.G. (1998) Histologic characterization of experimental cutaneous leishmaniasis in mice infected with Leishmania braziliensis in the presence or absence of sand fly vector salivary gland lysate. J. Parasitol. 84, 97–103. Gomes R., Teixeira C., Teixeira M.J. 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. USA 105, 7845–7850. Hall L.R. & Titus R.G. (1995) Sand fly vector saliva selectively modulates macrophage functions that inhibit killing of Leishmania major and nitric oxide production. J. Immunol. 155, 3501–3506. Kamhawi S. (2000) The biological and immunomodulatory properties of sand fly saliva and its role in the establishment of Leishmania infections. Microbes Infect. 2, 1765–1773. Kamhawi S., Belkaid Y., Modi G., Rowton E. & Sacks D. (2000) Protection against cutaneous leishmaniasis resulting from bites of uninfected sand flies. Science 17, 1351–1354. Laurenti M.D., Silveira V.M.S., Secundino N.F.C., Corbett C.E.P. & Pimenta P.F. (2009a) Saliva of laboratory-reared Lutzomyia longipalpis exacerbates Leishmania (Leishmania) amazonensis infection more potently that saliva of wild-caught Lutzomyia longipalpis. Parasitol. Int. 58, 220–226. Laurenti M.D., da Matta V.L., Pernichelli T. et al. (2009b) Effects of saliva gland homogenate from wild-caught and laboratory-reared Lutzomyia longipalpis on the evolution and immunomodulation of Leishmania (leishmania) amazonensis infection. Scand. J. Immunol. 70, 389–395.

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Locksley R.M., Heinzel F.P., Holaday B.J., Mutha S.S., Reiner S.L. & Sadick M.D. (1991) Induction of Th1 and Th2 CD4+ subsets during murine Leishmania major infection. Res. Immunol. 142, 28–32. Maioli T.U., Takane E., Arantes R.M., Fietto J.L. & Afonso L.C. (2004) Immune response induced by New World Leishmania species in C57BL/6 mice. Parasitol. Res. 94, 207–212. Miles M.A., P ovoa M.M., de Souza A.A., Laison R. & Shaw J.J. (1980) Some methods for the enzymic characterization of Latin-America Leishmania with particular reference to Leishmania mexicana amazonensis and subspecies of Leishmania hertigi. Trans. R. Soc. Trop. Med. Hyg. 74, 243–252. Norsworthy N.B., Sun J., Elnaiem D., Lanzaro G. & Soong L. (2004) Sand fly saliva enhances Leishmania amazonensis infection by modulating interleukin-10 production. Infect. Immun. 72, 1240–1247. Oliveira F., Lawyer P.G., Kamhawi S. & Valenzuela J.G. (2008) Immunity to distinct sand fly salivary proteins primes the anti-Leishmania immune response towards protection or exacerbation of disease. PLoS Negl. Trop. Dis. 2, e226. Paranhos-Silva M., Oliveira G.G., Reis E.A. et al. (2003) A follow-up of Beagle dogs intradermally infected with Leishmania chagasi in the presence or absence of sand fly saliva. Vet. Parasitol. 114, 97–111. Passero L.F.D., Marques C., Vale-Gato I., Corbett C.E.P., Laurenti M.D. & Santos Gomes G. (2010) Exacerbation of Leishmania (Viannia) shawi infection in BALB/c mice after immunization with soluble antigen from amastigote forms. APMIS 118, 973–981. Qi H., Ji J., Wanasen N. & Soong L. (2004) Enhanced replication of Leishmania amazonensis amastigotes in gamma interferon-stimulated murine macrophages: implications for the pathogenesis of cutaneous leishmaniasis. Infect. Immun. 72, 988–995. Ribeiro J.M. (1987) Role of saliva in blood-feeding by arthropods. Ann. Rev. Entomol. 32, 463–478. Ribeiro J.M. (1989) Vector saliva and its role in parasite transmission. Exp. Parasitol. 69, 104–106. Roberts M.T. (2006) Current understandings on the immunology of leishmaniasis and recent developments in prevention and treatment. Br. Med. Bull. 75–76, 115–130. Samuelson E., Lerner R., Tesh R. & Titus R. (1991) A mouse model of Leishmania braziliensis infection produced by coinjection with sand fly saliva. J. Exp. Med. 173, 49–54. Shaw J.J., Ishikawa E.A. & Laison R. (1989) A rapid and sensitive method for the identification of Leishmania with monoclonal antibodies using fluorescein labelled–avidin. Trans. R. Soc. Trop. Med. Hyg. 83, 783–784. Theodos C.M. & Titus R.G. (1993) Salivary gland material from the sand fly Lutzomyia longipalpis has an inhibitory effect on macrophage function in vitro. Parasite Immunol. 15, 481–487. Theodos C.M., Ribeiro J.M. & Titus R.G. (1991) Analysis of enhancing effect of sand fly saliva on Leishmania infection in mice. Infect. Immun. 59, 1592–1598. Thiakaki M., Rohousova I., Volfova V., Volf P., Chang K.P. & Soteriadou K. (2005) Sand fly specificity of saliva-mediated protective immunity in Leishmania amazonensis-BALB/c mouse model. Microbes Infect. 7, 760–766. Titus R.G. & Ribeiro J.M. (1988) Salivary gland lysates from the sand fly Lutzomyia longipalpis enhance Leishmania infectivity. Science 239, 1306–1308. Titus R.G. & Ribeiro J.M. (1990) The role of vector saliva in transmission of arthropod-borne diseases. Parasitol. Today 6, 157– 160. Wikel S.K. (1999) Tick modulation of host immunity: an important factor in pathogen transmission. Int. J. Parasitol. 29, 851–859.

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During the natural transmission of Leishmania parasites, the infected sand fly female regurgitates promastigotes into the host's skin together with it...
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