Host skin immunity to arthropod vector bites: from mice to humans

Infections caused by vector-borne pathogens impose a significant burden of morbidity and mortality in a global scale. In their quest for blood, hematophagous arthropods penetrate the host skin and may transmit pathogens by the bite. These pathogens are deposited along with saliva and a complex mixture of vector derived factors. Hematophagous arthopod vectors have evolved a complex array of adaptations to modulate the host immune response at the bite site with the primary goal to improve blood feeding, which have been exploited throughout evolution by these pathogens to enhance infection establishment in the host. While this paradigm has been firmly established in mouse models, comparable data from human studies are scarce. Here we review how the host skin immune response to vector bites in animal models is hijacked by microbes to promote their pathogenesis. We mainly explored four distinct vector-pathogen pairs of global health importance: sand flies and Leishmania parasites, Ixodes scapularis ticks and Borrelia burgdorferi, Aedes aegypti mosquitoes and arboviruses, and Anopheles gambiae mosquitos and Plasmodium parasites. Finally, we outline how critical it is for the field of vector biology to shift from rodent models to clinical studies focused on the interface of vector-pathogen-host immune system to push further the frontiers of knowledge of the field

Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites

Sand flies bite mammalian hosts to obtain a blood meal, driving changes in the host inflammatory response that support the establishment of Leishmania infection. This effect is partially attributed to components of sand fly saliva, which are able to recruit and activate leukocytes. Our group has shown that heme oxygenase-1 (HO-1) favors Leishmania survival in infected cells by reducing inflammatory responses. Here, we show that exposure to sand fly bites is associated with induction of HO-1 in vivo. Histopathological analyses of skin specimens from human volunteers experimentally exposed to sand fly bites revealed that HO-1 and Nrf2 are produced at bite sites in the skin. These results were recapitulated in mice ears injected with a salivary gland sonicate (SGS) or exposed to sand fly bites, indicating that vector saliva may be a key factor in triggering HO-1 expression. Resident skin macrophages were the main source HO-1 at 24–48 h after bites. Additionally, assays in vivo after bites and in vitro after stimulation with saliva both demonstrated that HO-1 production by macrophages was Nrf2-dependent. Collectively, our data demonstrates that vector saliva induces early HO-1 production at the bite sites, representing a major event associated with establishment of naturally-transmitted Leishmania infections

Molecular signatures of neutrophil extracellular traps in human visceral leishmaniasis

Abstract Background Infections with parasites of the Leishmania donovani complex result in clinical outcomes that range from asymptomatic infection to severe and fatal visceral leishmaniasis (VL). Neutrophils are major players of the immune response against Leishmania, but their contribution to distinct states of infection is unknown. Gene expression data suggest the activation of the NETosis pathway during human visceral leishmaniasis. Thus, we conducted an exploratory study to evaluate NET-related molecules in retrospective sera from VL patients, asymptomatic individuals and uninfected endemic controls. Results We demonstrate that VL patients and asymptomatic individuals exhibit differential regulation of molecules associated with neutrophil extracellular traps (NET). These differences were observed at the transcriptional level of genes encoding NET-associated proteins; in quantifications of cell free DNA and metalloproteinase 9; and in enzymatic activity of DNAse and elastase. Moreover, multivariate analysis resulted in class-specific signatures, and ROC curves demonstrate the ability of these molecules in discriminating asymptomatic infection from uninfected controls. Conclusion Molecules that are associated with NETs are differentially regulated between distinct states of infection with L. infantum, suggesting that NETs might have distinct roles depending on the clinical status of infection. Although unlikely to be exclusive for VL, these signatures can be useful to better characterize asymptomatic infections in endemic regions of this disease

Immunity to LuloHya and Lundep, the salivary spreading factors from <i>Lutzomyia longipalpis</i>, protects against <i>Leishmania major</i> infection

<div><p>Salivary components from disease vectors help arthropods to acquire blood and have been shown to enhance pathogen transmission in different model systems. Here we show that two salivary enzymes from <i>Lutzomyia longipalpis</i> have a synergist effect that facilitates a more efficient blood meal intake and diffusion of other sialome components. We have previously shown that Lundep, a highly active endonuclease, enhances parasite infection and prevent blood clotting by inhibiting the intrinsic pathway of coagulation. To investigate the physiological role of a salivary hyaluronidase in blood feeding we cloned and expressed a recombinant hyaluronidase from <i>Lu</i>. <i>longipalpis</i>. Recombinant hyaluronidase (LuloHya) was expressed in mammalian cells and biochemically characterized <i>in vitro</i>. Our study showed that expression of neutrophil CXC chemokines and colony stimulating factors were upregulated in HMVEC cells after incubation with LuloHya and Lundep. These results were confirmed by the acute hemorrhage, edema and inflammation in a dermal necrosis (dermonecrotic) assay involving a massive infiltration of leukocytes, especially neutrophils, in mice co-injected with hemorrhagic factor and these two salivary proteins. Moreover, flow cytometry results showed that LuloHya and Lundep promote neutrophil recruitment to the bite site that may serve as a vehicle for establishment of <i>Leishmania</i> infection. A vaccination experiment demonstrated that LuloHya and Lundep confer protective immunity against cutaneous leishmaniasis using the <i>Lu</i>. <i>longipalpis—Leishmania major</i> combination as a model. Animals (C57BL/6) immunized with LuloHya or Lundep showed minimal skin damage while lesions in control animals remained ulcerated. This protective immunity was abrogated when B-cell-deficient mice were used indicating that antibodies against both proteins play a significant role for disease protection. Rabbit-raised anti-LuloHya antibodies completely abrogated hyaluronidase activity <i>in vitro</i>. Moreover, <i>in vivo</i> experiments demonstrated that blocking LuloHya with specific antibodies interferes with sand fly blood feeding. This work highlights the relevance of vector salivary components in blood feeding and parasite transmission and further suggests the inclusion of these salivary proteins as components for an anti-<i>Leishmania</i> vaccine.</p></div

Biochemical characterization of the recombinant protein LuloHya.

<p><b>(A)</b> Purification of LuloHya by size exclusion chromatography using Superdex 200 Increase 10/300 GL column. <b>(B)</b> Coomassie-stained gel electrophoresis of LuloHya (1 μg). Mouse anti-LuloHya antibodies (1:5,000) recognized LuloHya (100 ng) and a single band in the SGE (5 pairs of SG) by Western blot. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). <b>(C)</b> NuPAGE Novex 4–12% Bis-Tris protein gel shows differences in electrophoretic pattern of LuloHya (1 μg) and its deglycosylated form (deLuloHya: 1 μg) which runs at the expected molecular weight (42.3 kDa). M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). <b>(D)</b> Hyaluronidase activity of 10 nM LuloHya and its deglycosylated form (deLuloHya). As negative controls, 4 micrograms of HA were incubated with TBS instead of recombinant protein or the deglycosylation enzyme mix (DeglycoMx Kit, QABio) without protein. <b>(E)</b> Turbidimetric assay showed a clear pH dependence of hyaluronidase activity of LuloHya. Reaction mixtures were prepared with solutions containing 25 mM buffer (described in Methods), 100 mM NaCl, 0.1% BSA, and different pH values (4–12.5; adjusted with a pHmeter 430, Corning). <b>(F)</b> Ionic strength dependency was analyzed in reaction mixtures of 25 mM HEPES, 0.1% BSA, pH 7.3 with variable NaCl concentration (25–1000 mM). Hyaluronidase activity is inversely expressed as the remaining HA (%) after treatment of 4 μg of HA with 10 nM enzyme during 1 h at 37°C. Biological triplicates and technical duplicates were assessed. Multiple comparisons were done by one-way ANOVA (****: p<0.0001). Bars indicate SEM.</p

Validation of gene expression of cytokines and chemokines from HMVEC cells in the presence of LuloHya, Lundep or SGE.

<p>RT-PCR for validation of gene expression results obtained with the Human Cytokines & Chemokines RT² Profiler PCR Array PAHS-150ZD. Specific set of primers were used to amplify <b>(A)</b> CSF2; <b>(B)</b> CSF3; <b>(C)</b> LIF; <b>(D)</b> CXCL1; <b>(E)</b> CXCL2 and <b>(F)</b> CXCL8. Biological triplicates and technical duplicates were analyzed. Negative controls consisted of cDNA isolated from HMVEC cells incubated with incomplete medium. Results are expressed as the fold change of the gene expression normalized against the standard gene HPRT1 (NM_000194). Multiple comparisons were done by one-way ANOVA (****: p<0.0001; **: p<0.01; *: p<0.05). Bars indicate the SEM.</p

Anti-LuloHya antibodies block the hyaluronidase activity of SGE and LuloHya.

<p>Hyaluronidase activity of <i>Lu</i>. <i>longipalpis</i> SGE and 10 nM LuloHya in the presence of 5 μg of rabbit IgG-control (pre-immune IgG) or 5 μg of rabbit anti-LuloHya IgG. As negative controls, TBS was added instead of SGE or recombinant protein. Biological triplicates were tested. Bars indicate SEM.</p

LuloHya and Lundep enhance dermonecrotic lesions caused by HF injection.

<p><b>(A)</b> Ears of animals injected intradermally with 3 μg of HF along with SGE (equivalent to 2 SG), 10 μg of LuloHya, 10 μg of Lundep or a combination of 5 μg of LuloHya and Lundep (HF column) showed larger lesions than ears injected with HF and PBS. Left column pictures (PBS) show mice ears injected with SGE, LuloHya, Lundep and the combination of both proteins in the absence of HF. Two hours after injection, ears were excised and measurements of the hemorrhage area were taken. <b>(B)</b> Lesion area in all groups injected along HF was significantly greater than control (mice ears injected with PBS and HF). The lesion area (mm) was calculated as follows, Area = where <i>D</i> is the longest and <i>d</i> the smallest lesion diameter. Multiple comparisons were done by one-way ANOVA (****: p<0.0001). Bars indicate SEM. <b>(C)</b> Histological sections of mice ears inoculated with HF were characterized by moderate expansion of the sub-epithelial tissues by a mixture of edema fluid and infiltrating leukocytes. Ear sections inoculated with HF and SGE, LuloHya or Lundep showed acute hemorrhage and inflammation. Mice ears from control groups (without HF) were within normal histological limits. <b>(D)</b> Histological ear sections from mice inoculated with HF and both recombinant proteins LuloHya and Lundep showed greater areas of hemorrhage and edema than the inoculation of HF with the recombinant proteins separately. Bar scales indicate 50 μm.</p

Vaccination studies with LuloHya and Lundep against <i>L</i>. <i>major</i> infection.

<p><b>(A)</b> Lesion size of C57BL/6 mice due to <i>L</i>. <i>major</i> infection steadily increased from the second week of the follow-up period until it stabilized or started to decrease after week 7 in all animals. For control animals (only immunized with Magic Mouse Adjuvant) lesion size was higher than vaccinated groups from week 4 onwards. The symbols represent the lesion size mean of 10 ears ± SEM analyzed by analysis of variance. <b>(B)</b> Lesion size values of C57BL/6 mice were converted to the area under the curve (AUC) showing that mice immunized against either LuloHya and Lundep presented significantly reduced lesions. <b>(C)</b> Parasite load of ears from C57BL/6 mice vaccinated with LuloHya or Lundep was significantly lower than control group (P<0.05). <b>(D)</b> <i>L</i>. <i>major</i> lesion size in B-cell-deficient B6.129S2-<i>Ighm</i>^{<i>tm1Cgn</i>}/J mice. <b>(E)</b> There are no statistical significant differences in lesion size of B6.129S2-<i>Ighm</i>^{<i>tm1Cgn</i>}/J mice immunized with either LuloHya, Lundep or adjuvant. <b>(F)</b> Parasite load of ears from B6.129S2-<i>Ighm</i>^{<i>tm1Cgn</i>}/J mice vaccinated with LuloHya or Lundep showed no differences with the control group. Multiple comparisons were done by one-way ANOVA (****: p<0.0001; ***: p<0.001; **: p<0.01; *: p<0.05; ns: non-significant). Bars indicate SEM.</p

Specificity of hyaluronidase activity of LuloHya.

<p><b>(A)</b> Five micrograms of high molecular weight HA (H), chondroitin sulfate B (CS), dextran sulfate (DS) and heparin (Hep) were fractionated on a 1.2% agarose gel alone or after incubation with LuloHya or <i>Lu</i>. <i>longipalpis</i> SGE. As molecular weight markers, Select-HA HiLadder, Select-HA 250k (Hyalose) and GeneRuler 1kb DNA ladder were used (Thermo Scientific). <b>(B)</b> Five micrograms of high, medium and low molecular weight HA (H, M and L, respectively) were separated on a 1.2% agarose gel alone of after incubation with LuloHya, bovine hyaluronidase (BovHya), <i>Streptomyces hyalurolyticus</i> hyaluronidase (BactHya) and <i>Lu</i>. <i>longipalpis</i> SGE.</p