Nathalie Boulanger

Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major.

ABSTRACT Antimicrobial peptides are major components of the innate immune response of epithelial cells. In insect vectors, these peptides may play a role in the control of gut pathogens. We have analyzed antimicrobial peptides produced by the sand fly Phlebotomus duboscqi, after challenge by injected bacteria or feeding with bacteria or the protozoan parasite Leishmania major. A new hemolymph peptide with antimicrobial activity was identified and shown to be a member of the insect defensin family. Interestingly, this defensin exhibits an antiparasitic activity against the promastigote forms of L. major, which reside normally within the sand fly midgut. P. duboscqi defensin could be induced by both hemolymph or gut infections. Defensin mRNA was induced following infection by wild-type L. major, and this induction was much less following infections with L. major knockout mutants that survive poorly in sand flies, due to specific deficiencies in abundant cell surface glycoconjugates containing phosphoglycans (including lipophosphoglycan). The ability of gut pathogens to induce gut as well as fat body expression of defensin raises the possibility that this antimicrobial peptide might play a key role in the development of parasitic infections.

Tick Saliva Represses Innate Immunity and Cutaneous Inflammation in a Murine Model of Lyme Disease

Lyme borreliosis is an arthropod-borne disease transmitted by the Ixodes tick. This spirochetal infection is first characterized by a local cutaneous inflammation, the erythema migrans. The skin constitutes a key interface in the development of the disease. During Borrelia inoculation, tick saliva affects the innate and adaptive immunity of the vertebrate host skin. Some key mediators of innate immunity such as antimicrobial peptides (cathelicidin and defensin families) have been identified as important initiators of skin inflammation. We analyzed the role of tick saliva on integumental innate immunity using different protocols of Borrelia infection, via syringe or direct tick transmission. When syringe inoculation was used, Borrelia triggered skin inflammation with induction of CRAMP, the mouse cathelicidin, and tumor necrosis factor-alpha. However, when Borrelia was transmitted directly via the tick, we observed a significant repression of inflammatory genes, suggesting a critical role of tick saliva in skin innate immunity. For all the protocols tested, a peak of intense Borrelia multiplication occurred in the skin between days 5 and 15, before bacterial dissemination to target organs. We conclude that Borrelia pathogens specifically use the tick saliva to facilitate their transmission to the host and that the skin constitutes an essential interface in the development of Lyme disease.

Antialarmin Effect of Tick Saliva during the Transmission of Lyme Disease

ABSTRACT Tick saliva has potent immunomodulatory properties. In arthropod-borne diseases, this effect is largely used by microorganisms to increase their pathogenicity and to evade host immune responses. We show that in Lyme borreliosis, tick salivary gland extract and a tick saliva protein, Salp15, inhibit in vitro keratinocyte inflammation induced by Borrelia burgdorferi sensu stricto or by the major outer surface lipoprotein of Borrelia, OspC. Chemokines (interleukin-8 [IL-8] and monocyte chemoattractant protein 1 [MCP-1]) and several antimicrobial peptides (defensins, cathelicidin, psoriasin, and RNase 7) were downregulated. Interestingly, antimicrobial peptides (AMPs) transiently inhibited bacterial motility but did not kill the organisms when tested in vitro. We conclude that tick saliva affects the chemotactic properties of chemokines and AMPs on immune cells and has an antialarmin effect on human primary keratinocytes. Alarmins are mediators that mobilize and activate antigen-presenting cells. Inhibition of cutaneous innate immunity and of the migration of immune cells to the site of the tick bite ensures a favorable environment for Borrelia. The bacterium can then multiply locally and, subsequently, disseminate to the target organs, including joints, heart, and the central nervous system.

Personal protection against biting insects and ticks.

Recent events with the first cases of local transmission of chikungunya and dengue fever virus in southern France by Aedes albopictus, adding to the nuisance and potential vectors that can be encountered when traveling in tropical or sub-tropical countries, has shown the value of a reflection on the Personal protection against vectors (PPAV). It is seen during an outbreak of vector-borne disease, or simply because of nuisance arthropods, that our fellow citizens try to protect themselves individually by using an arsenal of resources available on the market. Yet most of these means have been neither checked for effectiveness or safety tests, however, essential. Travellers, staff on mission or assignment, are looking for specific information on how to protect themselves or their families. Health workers had at their disposal so far indications that vary widely from one source to another. Therefore it seemed important to the Society of Travel Medicine (SMV) and the French Society of Parasitology (SFP) to initiate a reflection on this theme. This reflection took the form of recommendations for good practice, following the outline established by the French High Health Authority (HAS). The aim was to gather all relevant information, verified and validated and the format to be used not only by health personnel (doctors, pharmacists, nurses), but also by travel agents and individuals. This document highlights the need to take into account the risk of vector-borne diseases, some deadly, and the benefit of various methods of personal protection. The choice of methods is clearly oriented towards those whose effectiveness has been proven and potential risks assessed. The paper finally proposes two decision trees based on the transmission type (day or night) and kind of stay (short or roaming, long and steady). It concerns travellers, but also expatriates, residents and nomads.

Defensin Is Suppressed by Tick Salivary Gland Extract During the In Vitro Interaction of Resident Skin Cells with Borrelia burgdorferi

TO THE EDITOR The causative agent of Lyme disease, Borrelia burgdorferi, is a spirochete parasitizing vertebrates and transmitted by the tick, Ixodes sp. Lyme disease is the most common vector-borne disease in the Northern Hemisphere (Piesman and Gern, 2004). It manifests itself frequently with an erythema migrans rash at the site of the infection. However, the infection can progress and disseminate, affecting other skin sites, the joints, the heart, and the nervous system in humans. Control of the initial cutaneous phase may play a crucial role in the outcome of the disease, as not all patients develop disseminated clinical infection (Steere and Glickstein, 2004). The skin constitutes a complex physical barrier. First, the epidermis comprises mainly keratinocytes (KCs) and Langerhans cells. These cells possess specific Toll-like receptors (TLRs) that recognize certain defined patterns, the pathogen-associated molecular patterns, present on pathogens. The interaction between pathogen-associated molecular patterns and TLRs leads to the activation of the NF-kB pathway, with the production of inflammatory molecules including chemokines, cytokines, and antimicrobial peptides (AMPs) (Pivarcsi et al., 2004). These molecules of innate immunity are essential to the control of infection. Dermal fibroblasts (FBs) constitute a second group of resident skin cells that secrete the extracellular matrix and communicate with other cell types such as dermal dendritic cells, mast cells, macrophages, and KCs (Sorrell and Caplan, 2004). Ebnet et al. (1997) reported the activation of NF-kB upon the interaction of Borrelia with human KCs and FBs. However, no recent study has investigated the potential induction of AMPs in the interaction of B. burgdorferi with resident skin cells. We studied the induction of the proinflammatory molecule IL-8, defensins, and cathelicidin. IL-8 is a chemokine that attracts neutrophils, major cells involved in inflammation and secreting AMPs. Defensins are produced by leukocytes and various epithelial cells (Ganz, 2003). The cathelicidin LL-37, in humans, is present constitutively in neutrophil granules and is inducible in epithelial cells in response to infection. Their role in the control of several skin inflammations is well established (Nizet et al., 2001; Ong et al., 2002; Braff et al., 2005). We used ELISA to measure the secretion of IL-8 and HBD-2 (human bdefensin-2) and quantitative real-time reverse transcriptase-PCR to study the induction of HBD-2 and LL-37 mRNAs by KCs and FBs. IL-8 was induced by B. burgdorferi in a dose-dependent manner in KCs and FBs (Figure 1a and b). Regarding AMP mRNAs, defensin, but not cathelicidin, was induced in KCs (Figure 1c and f). In FBs, Borrelia did not induce HBD-2 and a weak expression of LL-37 mRNA was observed (Figure 1d and f). A kinetic study revealed that IL-8 secretion reached its highest level at 12 hours for both KCs and FBs (Figure 1g and h), whereas HBD-2 mRNA reached a peak at 6 hours in KCs (Figure 1i), also confirmed by the measure of HBD-2 secretion by ELISA (data not shown). Only very weak amounts were found to be secreted by FBs upon stimulation with Borrelia (Figure 1j). For LL-37 mRNA, only marginal expression by KCs was shown (Figure 1k), whereas Borrelia elicited a much higher expression in FBs, which peaked at 6 hours (Figure 1l). As Borrelia are transmitted by the Ixodes tick, we analyzed the effect of tick salivary gland (SG) extract on host cell inflammation. As can be seen in Figure 2, induction of IL-8 and HBD-2 was significantly inhibited by SG extract. The inhibitory effect on IL-8 Abbreviations: AMP, antimicrobial peptide; FB, fibroblast; HBD, human b-defensin; KC, keratinocyte; SG, salivary gland; TLR, Toll-like receptor

Microarray Analyses of Inflammation Response of Human Dermal Fibroblasts to Different Strains of Borrelia burgdorferi Sensu Stricto

In Lyme borreliosis, the skin is the key site of bacterial inoculation by the infected tick, and of cutaneous manifestations, erythema migrans and acrodermatitis chronica atrophicans. We explored the role of fibroblasts, the resident cells of the dermis, in the development of the disease. Using microarray experiments, we compared the inflammation of fibroblasts induced by three strains of Borrelia burgdorferi sensu stricto isolated from different environments and stages of Lyme disease: N40 (tick), Pbre (erythema migrans) and 1408 (acrodermatitis chronica atrophicans). The three strains exhibited a similar profile of inflammation with strong induction of chemokines (CXCL1 and IL-8) and IL-6 cytokine mainly involved in the chemoattraction of immune cells. Molecules such as TNF-alpha and NF-κB factors, metalloproteinases (MMP-1, -3 and -12) and superoxide dismutase (SOD2), also described in inflammatory and cellular events, were up-regulated. In addition, we showed that tick salivary gland extracts induce a cytotoxic effect on fibroblasts and that OspC, essential in the transmission of Borrelia to the vertebrate host, was not responsible for the secretion of inflammatory molecules by fibroblasts. Tick saliva components could facilitate the early transmission of the disease to the site of injury creating a feeding pit. Later in the development of the disease, Borrelia would intensively multiply in the skin and further disseminate to distant organs.

Smuggling across the Border: How Arthropod-Borne Pathogens Evade and Exploit the Host Defense System of the Skin

The skin is a critical barrier between hosts and pathogens in arthropod-borne diseases. It harbors many resident cells and specific immune cells to arrest or limit infections by secreting inflammatory molecules or by directly killing pathogens. However, some pathogens are able to use specific skin cells and arthropod saliva for their initial development, to hide from the host immune system, and to establish persistent infection in the vertebrate host. A better understanding of the initial mechanisms taking place in the skin should allow the development of new strategies to fight these vector-borne pathogens that are spread worldwide and are of major medical importance.

Update on the proteomics of major arthropod vectors of human and animal pathogens.

Vector‐borne diseases (VBDs) are defined as infectious diseases of humans and animals caused by pathogenic agents such as viruses, protists, bacteria, and helminths transmitted by the bite of blood‐feeding arthropod (BFA) vectors. VBDs represent a major public health threat in endemic areas, generally subtropical zones, and many are considered to be neglected diseases. Genome sequencing of some arthropod vectors as well as modern proteomic and genomic technologies are expanding our knowledge of arthropod–pathogen interactions. This review describes the proteomic approaches that have been used to investigate diverse biological questions about arthropod vectors, including the interplay between vectors and pathogens. Proteomic studies have identified proteins and biochemical pathways that may be involved in molecular crosstalk in BFA‐pathogen associations. Future work can build upon this promising start and functional analyses coupled with interactome bioassays will be carried out to investigate the role of candidate peptides and proteins in BFA‐human pathogen associations. Dissection of the host–pathogen interactome will be key to understanding the strategies and biochemical pathways used by BFAs to cope with pathogens.

Immunoproteomic identification of antigenic salivary biomarkers detected by Ixodes ricinus-exposed rabbit sera

Ixodes ricinus, the primary vector of tick-borne disease in Europe, is currently expanding its distribution area and its activity in many countries. Antibody responses to tick salivary antigens have been proposed as an alternative marker of exposure to tick bites. However, the identification of the I. ricinus corresponding antigens remains elusive. Using rabbits artificially exposed to I. ricinus and 2 other European tick species (Rhipicephalus sanguineus and Dermacentor reticulatus) as controls, a cross-comparison of IgG profiles was performed against protein salivary gland extracts (pSGE) from these 3 tick species using immunoblots. Immunoblot analysis highlighted a singularity in the immune patterns according to tick species exposure and pSGE antigen source. Two protein bands were detected against I. ricinus pSGE only in rabbits exposed to I. ricinus bites. An immunoproteomic approach based on a fluorescence detection method was developed to unambiguously identify corresponding antigenic spots on 2-D gels. Among the unique I. ricinus salivary antigenic proteins detected by sera from rabbits exposed to this tick species, I. ricinus calreticulin was identified. Although tick calreticulin was previously proposed as a potential antigenic marker following exposure to ticks (particularly in North American tick species), the present study suggested that Ixodes calreticulin does not appear to be cross-recognized by the 2 other tick genera tested. Additional experiments are needed to confirm the use of I. ricinus calreticulin salivary protein as a potential discriminant antigenic biomarker to Ixodes tick exposure.

Ixodes tick saliva suppresses the keratinocyte cytokine response to TLR2/TLR3 ligands during early exposure to Lyme borreliosis.

Ixodes hard tick induces skin injury by its sophisticated biting process. Its saliva plays a key role to enable an efficient blood meal that lasts for several days. We hypothesized that this feeding process may also be exploited by pathogens to facilitate their transmission, especially in the context of arthropod‐borne diseases. To test this, we used Lyme borreliosis as a model. This bacterial infection is caused by Borrelia burgdorferi sensu lato transmitted by Ixodes. We co‐incubated Borrelia with human keratinocytes in the presence of poly (I: C), a dsRNA TLR3 agonist generated by skin injury. This induced a strong cytokine response from human primary keratinocytes that was much greater than that induced by Borrelia alone. OspC, a TLR2/1 agonist and a major surface lipoprotein of Borrelia also amplified the process. Interestingly, tick saliva inhibited cytokine responses by keratinocytes to these TLR agonists. We propose that Borrelia uses the immunoprivileged site produced by tick saliva to facilitate its transmission.