TREPAR-1251; No. of Pages 9

Review

Me´nage a` trois: Borrelia, dendritic cells, and tick saliva interactions Lauren M.K. Mason1, Christiaan C. Veerman1, Teunis B.H. Geijtenbeek2, and Joppe W.R. Hovius1 1 2

Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands Department of Experimental Immunology, Academic Medical Center, Amsterdam, The Netherlands

Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis, is inoculated into the skin during an Ixodes tick bite where it is recognised and captured by dendritic cells (DCs). However, considering the propensity of Borrelia to disseminate, it would appear that DCs fall short in mounting a robust immune response against it. Many aspects of the DC-driven immune response to Borrelia have been examined. Recently, components of tick saliva have been identified that sabotage DC responses and aid Borrelia infection. In this review, we summarise what is currently known about the immune response of DCs to Borrelia and explore the mechanisms by which Borrelia manages to circumvent this immune response, with or without the help of tick salivary proteins. Lyme borreliosis Lyme borreliosis, the most prevalent vector-borne disease in the Western world [1], is caused by infection with species of spirochetes collectively referred to as Borrelia burgdorferi sensu lato. Among this group, three species, B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, account for the majority of cases, at least in Europe [2]. Borrelia is transmitted to mammals through the bite of infected Ixodes ticks. In the USA, the deer tick Ixodes scapularis transmits B. burgdorferi sensu stricto, whereas the Ixodes ricinus and Ixodes persulcatus ticks are the main vectors for B. garinii, B. afzelii, and B. burgdorferi sensu stricto in Europe and Asia. Symptoms of Lyme borreliosis typically start 1 to 2 weeks after transmission with a characteristic bull’s eye rash on the skin, called erythema migrans (see Glossary) [3]. After entering the skin, Borrelia may disseminate haematogenously [4], to the joints, the heart, or the nervous system, causing arthritis, carditis, and neuroborreliosis, respectively [5]. Antibiotic therapy is usually effective; however, a small percentage of patients suffering from Lyme arthritis do not respond to treatment [6]. Furthermore, patients may experience long-lasting Corresponding author: Mason, L.M.K. ([email protected]). Keywords: lyme borreliosis; dendritic cells; Borrelia; tick saliva. 1471-4922/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2013.12.003

aspecific symptoms even after adequate treatment [7]. At present, there is no protective vaccine available to prevent the disease in humans [8]. The potential of Borrelia to cause debilitating disease, the symptoms, which may continue even after treatment, and the lack of a suitable vaccine highlight the need to gain a better insight into the factors affecting the host immune response to infection. Borrelia is recognised by and induces various arms of the mammalian immune response, including the complement system and diverse innate immune cells [1,9]. Dendritic cells (DCs) are among the first immune cells to come into contact with Borrelia in the skin. Upon contact, they migrate to lymph nodes where they encounter T cells and induce adaptive immune responses crucial for the immunopathogenesis of Lyme borreliosis (Box 1). Different classes of DCs will be examined: (i) epidermal Langerhans cells (LCs); (ii) dermal DCs (DDCs); and (iii) in vitro generated DC models, including their role in immunity against Borrelia, and the involvement of recently discovered factors. Immunological response to Borrelia spirochetes by DCs: from pattern recognition to T cell induction An early indication that DCs serve a vital role in the immune response against Borrelia was provided almost two decades ago, when an abundance of spirochetes were revealed in the vacuoles of LCs in erythema migrans lesions of patients examined by electron microscopy [10]. Later, DDCs displayed more potency phagocytosing Borrelia than LCs [11,12], and the enrichment of mature DDCs was discovered in erythema migrans lesions [13]. Electron microscopy also showed that DCs internalise Borrelia mainly by coiling phagocytosis, in which broad pseudopods are used to engulf the bacteria. Upon phagocytosis, spirochetes are processed and presented on major histocompatibility complex (MHC) class II molecules, leading to the activation and proliferation of CD4+ T cells [12]. Both LCs and splenic DCs incubated with spirochetes in vitro were able to induce T helper (TH) cells to stimulate the production of specific antibodies by B cells against borrelial antigens. Furthermore, adoptive transfer of these splenic DCs elicited a protective immune response in vivo in mice, which provided increased resistance against tick-transmitted Borrelia [14]. These observations suggest that both DC subsets play a role in the initial immune response to Borrelia in the skin. It is not clear, however, to what extent Trends in Parasitology xx (2013) 1–9

1

TREPAR-1251; No. of Pages 9

Review Glossary Adaptive immunity: immunity acquired during life by the creation of specific immunological memory by T cells and B cells against previously encountered pathogens. Autocrine signalling: a form of cell signalling in which molecules secreted by the cell are recognised by the cell’s own receptors to bring about changes in differentiation or behaviour. Bone marrow-derived DCs: DCs generated in vitro from bone marrow precursors. Cathepsin S: an enzyme crucial for the loading of antigen onto MHC II molecules on antigen presenting cells to present to CD4+ T cells and elicit an adaptive immune response. CD4+ T cells/T helper (TH) cells: a T cell subset characterised by the expression of the surface protein CD4. These cells express receptors that recognise specific antigens presented on MHC II molecules of antigen presenting cells. They orchestrate adaptive immune responses by releasing cytokines and aiding other immune cells. Upon activation, TH cells are polarised into different effector subsets, including TH1, TH12, and TH17 cells, which are each specialised to combat different classes of pathogen. CD8+ cytoxic T cells: a T cell subset characterised by the expression of the surface protein CD8. These cells express receptors that recognise specific antigens presented on MHC I molecules of cells and identify and destroy virally infected or damaged cells. Complement system: a component of the innate immune system, composed of a number of plasma proteins, which can eradicate pathogens by cell wall lysis, enhance the action of antibodies, and attract immune cells to an infection site. DC-SIGN: a C-type lectin receptor expressed by dendritic cells. DC-SIGN recognises mannose-containing glycoproteins of various pathogens, triggering and modulating innate immune responses towards them. Dendritic cells (DCs): antigen presenting cells of the innate immune system. Immature (unstimulated) DCs recognise and engulf pathogens, after which they mature and migrate to a draining lymph node where antigen of the processed pathogen is presented to CD4+ T cells, which can subsequently launch an adaptive immune response. Dermal DCs (DDCs): a subset of DCs which patrol the dermis (lower layer) of the skin for invading pathogens. Enzootic life cycle of Borrelia: the transmission of the bacteria between ticks and their mammalian hosts. Erythema migrans: a characteristic ‘bull’s eye’ rash on the skin, typically 1–2 weeks after the tick bite, which is often the first clinical manifestation of Lyme borreliosis. Humoral immunity: a form of adaptive immunity elicited by antibodies targeting specific pathogens. Immunomodulatory cytokines: cytokines that suppress immune responses in order to prevent excessive immune reactions. Inflammasome: a multiprotein complex present in innate immune cells, which promotes the activation of several proinflammatory cytokines and other immune mechanisms. Innate immunity: the nonspecific, first line of defence against pathogens, based on the recognition of evolutionarily conserved pathogen motifs. Langerhans cells (LCs): a subset of DCs that reside in the epidermis (upper layer) of the skin. Lymph node: small organs distributed throughout the body, which contain large numbers of naı¨ve T and B cells. Antigen presenting cells migrate from peripheral tissues to lymph nodes upon pathogen uptake in order to present antigen to and activate T cells. Maturation markers: cell surface proteins that are expressed upon DC maturation and aid in T cell activation. Monocyte-derived DCs (mo-DCs): DCs generated in vitro from peripheral blood monocytes. Myeloid differentiation primary response gene 88 (MyD88): an adapter protein involved in the signalling cascade of almost all Toll-like receptors. Neuroborreliosis: a manifestation of Lyme borreliosis with involvement of the central nervous system, typically presenting in humans as a polyradiculitis (inflammation of several nerve roots) weeks to months after the tick bite. Paracrine signalling: a form of cell signalling, in which cells secrete molecules to signal to nearby cells in order to bring about changes in differentiation or behaviour. Pathogen-associated molecular patterns (PAMPs): small molecular motifs conserved within groups of pathogens, recognised by PRRs of innate immune cells. Pattern recognition receptors (PRRs): proteins expressed by innate immune cells. PRRs recognise PAMPs of microorganisms, in order to identify potential pathogens so that an innate immune response can be initiated against them. Examples of PRRs are TLRs, C-type lectin receptors, and NOD-like receptors. Proinflammatory mediators: signalling molecules, such as cytokines and chemokines, released by cells to recruit immune cells and promote inflammation. Splenic DCs: DCs resident in the spleen. These are isolated from mice for use in in vitro experiments. Synovial fluid: fluid present in the cavities of joints to reduce friction during joint movement. T regulatory (Treg) cells: a T cell subset that suppresses the function of other cells of the immune system to prevent an excessive immune response.

2

Trends in Parasitology xxx xxxx, Vol. xxx, No. x

each subset is able to recognise and mount an adaptive immune response to the pathogen. DCs recognise borrelial lipoproteins Although Borrelia does not produce the endotoxin lipopolysaccharide (LPS), in contrast to many other Gram-negative bacteria, it expresses alternative surface lipoproteins, which are recognised by DCs [15]. The outer surface proteins A and C (OspA and OspC), differentially expressed in the various environments that the spirochete encounters during its enzootic life cycle, are among the major lipoproteins recognised. These pathogen-associated molecular patterns (PAMPs) activate the innate immune system by binding Toll-like receptor (TLR)-2, which forms heterodimers with TLR-1 and TLR-6 to recognise triacylated and diacylated lipopeptides, respectively [15–18]. Additionally, TLR-10 was recently reported to form heterodimers with TLR-2 after stimulation with Borrelia, and appears to suppress TLR-2 function (M. Oosting, PhD thesis, Radboud University Nijmegen, 2013). Flagellin forms the bacterial flagellum in Borrelia, is recognised by TLR-5, and appears to be involved in detection of Borrelia and secretion of certain cytokines [19]. Although the binding of lipoproteins to TLRs elicits a robust DC response, multiple studies indicated that whole, viable Borrelia spirochetes are required to induce the complete immune reaction observed in vivo in Lyme borreliosis subjects [20–22]. This extra response requires phagocytosis and is mediated through TLR-7, TLR-8, and TLR-9 [23,24]. These intracellular TLRs traffic to phagolysosomes after phagocytosis and recognise pathogen-derived nucleic acids [25]. All TLRs involved in the recognition of Borrelia, with the exception of TLR-9, are expressed by DDCs and LCs, although the expression of TLR-2, which is generally accepted as the main TLR involved in Borrelia recognition, TLR-5, and TLR-8, were reported to be lower in LCs [26,27]. Therefore, the extent to which LCs are capable of recognising and mounting an immune response to encountered spirochetes remains unclear. Intracellularly located nuclear oligomerisation domain (NOD)-like receptors, which recognise peptides derived from bacterial cell wall peptidoglycans [28], are also involved in the recognition of Borrelia. Although NOD1 does not appear to play a role, a lack of NOD2 hampered Borrelia-induced cytokine release in in vitro studies, in NOD2-deficient mice, and in individuals with NOD2 mutations [29,30]. Unexpectedly, NOD2-deficient mice syringeinoculated with Borrelia presented robustly enhanced inflammation in joints and cardiac tissue later in infection, compared with wild type mice. The authors suggested that lack of induction of tolerance to Borrelia after prolonged NOD2 stimulation, which does occur in Borrelia-infected mice with normal NOD2 expression [29], explains this discrepancy. Of the C-type lectin receptors, which recognise foreign glycoproteins [31], thus far only the mannose receptor appears involved in recognition of Borrelia. The mannose receptor is present on both DDCs and LCs, and is proposed to function in host defence by facilitating phagocytosis; however, little is known about its role in Lyme borreliosis [32].

TREPAR-1251; No. of Pages 9

Review Box 1. DCs orchestrate adaptive immune responses in the skin Borrelia is transmitted via the bite of an infected tick into the skin of a mammalian host, where the spirochetes first encounter the immune system of the host. Several distinct types of immune cells reside in the skin, including two subsets of DCs: (i) LCs, which reside in the epidermis, the upper layer of the skin; and (ii) DDCs, which are found in the underlying dermis. DCs are key players in host defence because they form the bridge between innate and adaptive immunity. Alongside other innate immune cells, they are an integral part of the first line of defence against threatening pathogens, which is imperative in a compartment that forms a barrier to the outside world, such as the skin. At the same time, they are specialised in the presentation of antigen to T lymphocytes and dictate the subsequent adaptive immune response mounted [89]. In the skin, immature DCs sense and internalise pathogens by recognising PAMPs via PRRs, such as TLRs, NOD-like receptors, and C-type lectin receptors [90]. These PRRs are differentially expressed in DDCs and LCs, so that each subset is specialised to orchestrate an immune response against a specific set of pathogens. For example, LCs are believed to elicit strong immunogenic activity against viruses, while serving a more tolerogenic role against bacteria. This may reflect the need for LCs to tolerate the innocuous skin flora [26]. Upon internalisation by DCs, pathogens are processed into antigens and presented on MHC class II molecules. Simultaneously, DCs mature and migrate to draining lymph nodes, where they encounter naı¨ve T cells. These T cells are subsequently primed to launch an adaptive immune response tailored to target the specific pathogen encountered [52]. Cytokines produced by DCs dictate the fate of CD4+ T cells and their polarisation into different effector T cell subsets such as TH1, TH2, TH17, and Treg cells [91,92].

Borrelia triggers DC activation TLR-mediated recognition of Borrelia leads to the myeloid differentiation primary response gene 88 (MyD88)-dependent activation of the nuclear factor kB (NF-kB) signalling cascade, which triggers the transcription of a large set of genes by DCs. These include endocytosis-associated genes, chemokines, many apoptosis inhibitors, matrix metalloproteases, adhesion molecules, and a large subset of cytokines, including proinflammatory mediators, neutrophil attractants, and immunomodulatory cytokines [33–35]. Furthermore, the expression of the co-stimulatory molecules CD80 and CD86 is induced, both of which interact with CD4+ T cells to trigger clonal expansion. CD40, CD83, and MHC II molecules are also upregulated upon recognition of Borrelia. These surface markers characterise maturation of DCs and enable presentation of antigen to CD4+ T cells [11,21]. The inflammasome is invoked during the TLR-mediated host response to Borrelia because caspase 1 is activated by apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), initiating cleavage of pro-interleukin-1b (pro-IL-1b) into its active counterpart. However, the significance of this process in immunity to Borrelia is questionable because caspase 1-deficient mice and ASC-deficient syringe-inoculated mice did not have a greatly altered Borrelia burden or course of Lyme borreliosis [36], although ASC- and caspase 1-dependent IL-1b production was crucial for arthritis induction in mice after intra-articular injection of Borrelia in a separate study [37]. In contrast to TLR2/1, TLR2/6, and TLR5 activation, signalling through TLR7, 8, and 9 also results in the activation of interferon regulatory factor 7 (IRF7), a transcription factor necessary for the expression of type 1 interferons (IFNs),

Trends in Parasitology xxx xxxx, Vol. xxx, No. x

IFN-a and IFN-b. Production of type 1 IFNs is upregulated in human monocytes after phagocytosis of Borrelia [23,24,38]. These induce transcription of IFN-stimulated genes via the Janus-activated kinase (JAK)/STAT pathway in an autocrine manner. Among these are various genes considered key for DC maturation, expression of co-stimulatory molecules, and antigen cross-presentation [39]. Type I IFNs were also implicated in the development of arthritis in syringe-inoculated mice [40]. MyD88-dependent signalling is vital for the efficient clearance of Borrelia; however, inflammation of the joints and heart was unaltered in syringe-inoculated MyD88 knockout mice, highlighting the importance of alternative signalling pathways in Lyme borreliosis immunopathogenesis, such as signalling via NOD2 [41]. Crosstalk between the distinct signalling pathways probably occurs, modulating the outcome and tailoring a more Borrelia-specific response [28]. In addition to the upregulation of MHC II for antigen display, upon infection with Borrelia, LCs and DDCs express type 1 CD1 molecules, CD1b and CD1c, both in vivo and ex vivo [42]. CD1 proteins are structurally related to MHC proteins and contain an antigen-binding groove, which binds self or foreign lipid antigen for display at T cells; they might aid in the distinction between infected and uninfected states [43]. The expression of CD1 on DCs upon stimulation with Borrelia takes several days to reach its maximum and is initiated in an autocrine and paracrine manner through the TLR-2-mediated release of IL-1b and granulocyte–macrophage colony-stimulating factor (GMCSF) [42]. Although a specific borrelial lipid antigen, which binds to CD1 proteins, has yet to be identified, CD1b or CD1c mediate IFN-g secretion by T cells in neuroborreliosis patients, implying that antigens for these CD1 proteins do exist [44]. Various effector T cell subsets are deployed to combat Borrelia infection Following antigen presentation and cytokine production, DCs prime CD4+ T cells into effector TH cells in the lymph nodes. Various TH subsets exist, specialised in activating separate components of the immune system, depending on the type of invading pathogen detected. TH1 cells induce cell mediated immunity against intracellular pathogens, whereas TH2 cells are instrumental in producing humoral responses and mediate immunity to helminths, for example [45]. The recently characterised TH17 cells appear to be particularly effective in combating extracellular pathogens by mobilising and activating neutrophils [46]. In mice infected with viable Borrelia by means of syringe inoculation, both the TH1 cytokine IFN-g and the TH2 cytokine IL4 were produced in the lymph nodes, indicating a mixed TH1/TH2 response [47], whereas in the joints and skin of Lyme borreliosis patients a strong TH1 response was observed [13,48,49]. In human studies, the amount of IFN-g produced correlates with the severity of symptoms, suggesting that the TH1 response may be a causative factor of Lyme borreliosis symptoms. Production of the TH17 cytokine, IL-22, was observed in vitro using peripheral blood mononuclear cells stimulated with live Borrelia and was also detected in erythema migrans lesions of 3

TREPAR-1251; No. of Pages 9

Review dermatoborreliosis patients; however, the presence of the hallmark TH17 cytokine, IL-17, could not be demonstrated [46]. Conversely, suppression of IL-17 prevented the development of arthritis in a mouse model of Borrelia infection [50], and IL-17 was elevated in the cerebrospinal fluid of 49% of neuroborreliosis patients [51]. Therefore, it is clear that the role of this subset in Lyme borreliosis needs further exploration and the role of DCs in the activation of these cells in Lyme borreliosis remains to be elucidated. T regulatory (Treg) cells dampen immune responses and thus prevent excessive or inappropriate immune responses to occur [52]. A lack of these cells may be a key component in Lyme arthritis resistant to antibiotics, which in many aspects resembles autoimmune disease. Indeed, patients suffering from antibiotic-refractory Lyme arthritis often have fewer FoxP3 positive cells (Treg cells) in synovial fluid than patients with antibiotic-responsive arthritis [53]. Furthermore, having a higher proportion of Treg cells in synovial fluid was correlated with a faster resolution of symptoms. Patients with low percentages of these cells were more prone to persistent complaints [54]. How the population of Treg cells is shaped by DCs during a Borrelia infection and what the underlying causes are of interindividual variability remain to be clarified. Apart from being degraded in DC phagosomes for presentation to CD4+ T cells, live Borrelia can also be detected in the cytoplasm [10,12]. Consequently, antigens of these microbes may be treated as self (or viral), and thus presented on MHC class I molecules, leading to activation of CD8+ cytotoxic T cells. Indeed, in in vitro studies a robust CD8+ T cell response has been observed, primed by DCs containing cytosolic borrelial lipoproteins. Furthermore, CD8+ T cells are abundantly present in skin biopsies of erythema migrans lesions [13,55]. It is not clear, however, whether CD8+ T cells play a role in combating Borrelia infection in vivo. Flaws and suppression of the DC response to Borrelia Borrelia induce a less robust DC response compared with other Gram-negative bacteria. Despite the fact that DCs phagocytose Borrelia, express a large subset of cytokines, and elicit an adequate humoral immune reaction against borrelial proteins, this response is apparently inadequate to prevent infection in the sizable group of individuals that suffer from disseminated Lyme borreliosis. This may be partly attributed to the lack of LPS expression by Borrelia, in contrast to other Gram-negative bacteria, for example, Escherichia coli. A microarray analysis of DCs challenged with LPS in vitro revealed a more robust transcriptional reaction compared with that of DCs challenged with Borrelia [34]. The most striking differences were observed in the mRNA expression of CD38 and C–C chemokine receptor type 7 (CCR7), both considered crucial for DC migration to the lymph nodes [56]. DCs stimulated with Borrelia migrated at a lower rate than LPS-stimulated DCs in vitro, and the migratory capacity of DCs to murine lymph nodes upon challenge with Borrelia was only half of that of DCs challenged with E. coli in vivo [57]. These results suggest that DCs respond less efficiently to Borrelia than to Gramnegative bacteria; however, the comparison of isolated LPS 4

Trends in Parasitology xxx xxxx, Vol. xxx, No. x

to Borrelia in vitro does not take into account the complex response in vivo that may occur when DCs are stimulated with viable bacteria, involving stimulation of multiple pattern recognition receptors (PRRs) and thus crosstalk, which may nuance the immune response. Tick saliva inhibits DC functions In addition to the less robust immune response elicited by DCs stimulated with Borrelia, a significant role in the suppression of innate immunity during a Borrelia infection may be attributed to tick saliva. This is secreted into the skin of the host during a blood meal and exhibits a wide range of vasodilatory, antihaemostatic, and anti-inflammatory effects, providing a survival advantage for both ticks and tick-borne pathogens [58]. The specific effects on DCs have been examined in several studies and are numerous (Figure 1 and Table 1). Experiments using a variety of DC subtypes stimulated with various stimuli, including a range of TLR ligands and viable Borrelia spirochetes, show that components of tick saliva are able to modulate diverse aspects of DC functionality. Phagocytosis, one of the most pivotal functions for pathogen recognition and clearing, was inhibited in murine splenic DCs [59]. Furthermore, its presence was shown to inhibit the expression of co-stimulatory molecules CD40, CD80, and CD86 in response to a number of stimuli [60–65]. DC secretion of proinflammatory cytokines IL-12, IL-6, tumour necrosis factor a (TNF-a), and IL-1b was diminished in multiple studies, whereas the production of the anti-inflammatory cytokine IL-10 was enhanced [33,59,61,65]. Moreover, an inhibitory effect on early DC migration to the lymph nodes [60] and immature DC turnover in the skin were both reported [66]. Finally, tick saliva impairs antigen presentation to T cells, retarding Borrelia-targeted T cell proliferation [33,59–62]. The mechanisms responsible for these effects have only been partially elucidated. Tick saliva interferes with several signalling pathways, which may account for many of the inhibitory effects on DCs. For example, the inhibition of B. afzelii-induced TNF-a production was mediated to some degree through suppression of extracellular signal-regulated kinase 1/2 (ERK1/2), NF-kB, and phosphoinositide 3-kinase (PI3K) signalling pathways [67]. In addition, suppression of IFN-dependent activation of signal transducer and activation of transcription 1 (STAT-1) in murine splenic DCs in response to B. afzelii or LPS was noted [68]. Impaired DC migration may be attributed to chemokine neutralising properties of tick saliva. The saliva of the Rhipicephalus sanguineus tick, a member of the Ixodidae tick family, although not a vector of Borrelia, inhibits macrophage inflammatory protein (MIP)-1a directly, blocking migration of immature DCs from blood to peripheral tissues in vivo [66]. Furthermore, the expression of the C–C chemokine receptor type 5 (CCR-5), the receptor which binds the chemokines MIP-1a, MIP-1b, and chemokine ligand 5 (CCL-5) is downregulated in immature DCs. Salivary gland extract of I. ricinus also possesses similar chemokine-blocking properties [69]. Thus, tick saliva interferes with multiple DC functions, impairing the DC-driven immune response on various levels.

TREPAR-1251; No. of Pages 9

Review

Trends in Parasitology xxx xxxx, Vol. xxx, No. x

Epidermis

LC

Borrelia

DDC

Dermis

Tick salivary compounds

↓ Maturaon

↓ Phagocytosis

Maturaon markers

Cytokines

Lymph node

Mature DC

MHC II

↓ Migraon

↓ IL-6, IL-12 TNF-α, IL-1β ↑ IL-10

Tnaive

↓ Angen presentaon

Teffector

↓ Tcell proliferaon ↓ Th1 polarisaon ↑ Th2 polarisaon

TRENDS in Parasitology

Figure 1. Compounds in tick saliva impair DC functions on various levels. Borrelia-infected ticks secrete saliva as well as Borrelia into the skin during a blood meal. Borrelia is recognised and phagocytosed by skin dendritic cells (DCs), dermal DCs (DDCs), and Langerhans cells (LCs). However, certain immunomodulatory compounds in tick saliva (in orange boxes) have been reported to interfere with this interaction and impair the DC response to Borrelia on various levels. Studies have shown that tick saliva impairs phagocytosis by DCs [59] and inhibits upregulation of maturation markers, proteins expressed upon DC maturation which aid in T cell activation, and MHC class II [60–62,64,65]. Furthermore, there is less production of proinflammatory cytokines IL-6, IL-12, TNF-a, and IL-1b, and greater IL-10 production [33,59,61–65]. Tick saliva was reported to impair migration of DCs from the skin to lymph nodes [60]. Finally, DC–T cell interactions are impeded as antigen presentation and DC-induced T cell proliferation are affected [33,59–62], and there is a shift towards T helper (TH)2 polarisation [60,79,80].

Specific components in tick saliva that interfere with DC activity Several specific compounds have been identified in tick saliva, which interfere with the functioning and activity of DCs. Prostaglandin E2 (PGE2) in I. scapularis and R. sanguineus saliva was identified as an inhibitor of IL-12 and TNF-a production, while promoting IL-10 production by bone marrow-derived DCs (BMDCs) in response to TLR2, TLR-4, and TLR-9 ligands [61,64]. The purine nucleoside adenosine was also identified as an anti-inflammatory element of R. sanguineus saliva, which, together with PGE2, produced an additive effect on cytokine production and diminished CD40 expression. The effects of both compounds are partly initiated via production of cyclic

adenosine monophosphate (cAMP), which activates protein kinase A (PKA), an enzyme that plays a role in cytokine modulation [70]. PKA was also recently found to be responsible for an Ixodes saliva-mediated increase in IL-10 production in murine splenic DCs [67]. Another important component, which has been observed to hinder DC function, is an Ixodes salivary gland protein of 15 kDa, Salp15. Salp15 was discovered in I. scapularis saliva, whereas I. ricinus secretes three homologues [71]. This protein was also identified as an immunosuppressive component, which binds to the outer surface protein C (OspC) of Borrelia, preventing antibody mediated killing by the immune host [72]. In addition, Salp15 interacts with the CD4 receptor, interfering with T 5

TREPAR-1251; No. of Pages 9

Review

Trends in Parasitology xxx xxxx, Vol. xxx, No. x

Table 1. Tick saliva–DC interactions Cell type

Stimulus

Human mo-DCs

B. burgdorferi/LPS/LTA

Tick saliva/ component Salp15

Murine splenic DCs

B. afzelii (viable)

Saliva (I. ricinus)

Murine splenic DCs

Poly I:C, imiquimod, CpG

Saliva (I. ricinus)

Murine splenic DCs Murine splenic DCs

B. afzelii (viable)/LPS B. afzelii (viable)/LTA

Saliva (I. ricinus) Saliva (I. ricinus)

Murine splenic DCs Murine BM-derived DCs

– LPS, PGN, CpG

Saliva (I. ricinus) Saliva (I. scapularis)/PGE2

Murine BM-derived DCs

LPS

Sialostatin L

Murine BM-derived DCs

LPS, LTA, Poly(I:C), CpG

Murine BM-derived DCs Murine BM-derived DCs

LPS, PGN, CpG LPS

Saliva (R. sanguineus)/ PGE2/Ado Saliva (R. sanguineus) Saliva (R. sanguineus)

Murine BM-derived DCs

–

Saliva (R. sanguineus)

Murine skin DCs (in vivo)

Ear paint

Saliva (I. ricinus)

LC-deficient mice

B. burgdorferi (viable)

Saliva (I. scapularis)

Effect

Refs

Inhibited cytokine production and T cell proliferation Inhibited phagocytosis, cytokine production, and T cell proliferation Inhibited maturation and T cell proliferation, promoted TH2 skew Inhibited IFN signalling Inhibited NF-kB, p65, PI3K/Akt and Erk1/2 signalling pathways Promoted TH2 skew Inhibited maturation, cytokine production, and T cell proliferation Inhibited maturation, cytokine production, and T cell proliferation Inhibited maturation and cytokine production

[32]

Inhibited cytokine production Inhibited maturation and cytokine production, T cell proliferation not affected Inhibited migration of immature DCs and production of T cell cytokines Inhibited maturation, migration, and T cell proliferation, promoted TH2 skew Impaired TH1 response (via LCs)

[57] [58] [66] [65] [76] [59] [60] [61] [62] [63] [64] [58] [77]

Abbreviations: DC, dendritic cell; mo-DC, monocyte-derived DC; LC, Langerhans cell; BM, bone marrow; LPS, lipopolysaccharide; LTA, lipoteichoic acid; Poly I:C, polyinosinic–polycytidylic acid; CpG, unmethylated cytosine–phosphate–guanine site; PGN, peptidoglycan; PGE2, prostaglandin E2; Ado, adenosine.

cell activation [72–74]. However, Salp15 also binds to the C-type lectin receptor DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin), which is involved in pathogen recognition and is highly expressed by DDCs [75]. The interaction of Salp15 with DC-SIGN in vitro triggers a Raf-1 MEK1/MEK2dependent signalling cascade independent of Erk, which inhibits the production of IL-12p70, IL-6, IL-1b, and TNFa by immature DCs activated with TLR ligands or viable Borrelia, whereas IL-10 production is elevated. Finally, the presence of Salp15 attenuates DC-induced T cell activation. Production of IL-6 and TNF-a was inhibited due to decreased mRNA stability, whereas IL-12p70 expression was diminished at the transcriptional level by impaired nucleosome remodelling at the promoter region [33]. Sialostatin L, a cysteine protease inhibitor secreted from I. scapularis salivary glands, acts in a distinct way to inhibit the functions of DCs [62]. The enzyme inhibits production of the cytokines IL-12 and TNF-a, upregulation of CD80 and CD86, and interferes with antigen presentation by binding the enzyme cathepsin S, which is considered to be crucial in antigen processing and cleavage of the invariant chain on MHC II molecules [76]. Inhibition of cathepsin S leads to defective antigen loading onto MHC II molecules. In vivo, Sialostatin L decreases antigen-specific T cell proliferation in a cathepsin S-dependent manner. Silencing of Sialostatin L in combination with Sialostatin L2, a related tick salivary protein, by RNA interference was associated with an elevated host immune response and failure of the ticks to finish the blood meal [77]. In addition, recombinant Sialostatin L2, but not Sialostatin L, facilitated transmission of Borrelia to mice in vivo [78]. 6

Tick saliva induces TH2 skewing The cytokine profile mediated by tick saliva in virtually all studies corresponded with that of a TH2 polarised system: low levels of IL-12 and IFN-g and enhanced IL-4 [60,79,80]. Furthermore, mice inoculated with Borrelia via tick infestation developed a TH2 response, with higher levels of IL-4 and IgE and lower levels of IgG2a in sera than syringeinoculated mice [47]. This might be beneficial for ticks because tick resistance, the acquired immunity to ticks seen after repeated tick infestations, is associated with a delayed-type hypersensitivity reaction, which is mediated through a TH1 response [81]. Regarding spirochetes, the advantages are more evident. In murine models, resistance to Borrelia infection is acquired when TH1-associated cytokines are reconstituted during tick feeding or when TH2 cytokines are suppressed [82,83]. Moreover, transfer of IFN-g producing T cells promoted resolution of Lyme carditis [84]. Finally, the T cell response in C3H/HeJ mice, which are susceptible to Lyme borreliosis, was strongly skewed towards a TH2 response after Borrelia-infected tick feeding, whereas the Lyme-resistant strain BALB/c had a pronounced TH1 profile [85]. LCs may be instrumental in the saliva-mediated suppression of TH1 skewing, because mice deficient in these cells demonstrated a more robust TH1 response in lymph nodes compared with wild type mice after infestation with infected ticks [80]. As mentioned earlier, a TH1 response predominates in patients with Lyme borreliosis [13,48,49]; although the TH1 response might cause symptoms, it results in clearance of the pathogen. Hypothetically, the early TH2 profile induced by tick saliva enables the spirochetes to disseminate and survive in the host, whereas, later, when the tick

TREPAR-1251; No. of Pages 9

Review has left the host, the primary response to Borrelia (i.e., production of TH1 cytokines) can be initiated, causing local inflammation and promoting Lyme borreliosis symptoms. Concluding remarks DCs recognise B. burgdorferi and employ various strategies to induce an adaptive immune response against these spirochetes in vitro. Furthermore, when stimulated with Borrelia, DCs induce a protective response in vivo [14]. However, this response appears to be less robust than that of DCs towards other (LPS-bearing) bacteria, such as E. coli, and is further impeded because Borrelia exploits the immunomodulatory mechanisms of the tick in order to circumvent the immune response orchestrated by DCs and achieve dissemination. As shown in multiple studies, tick saliva directly modulates the cytokine production and phagocytosing capabilities of DCs and also appears to have striking effects on the expression of co-stimulatory molecules and polarisation of T cells. This highlights an important point: infection models in which Borrelia is inoculated artificially by syringe lack a major component, which has consequences for the immunopathogenesis of Lyme borreliosis. However, the results of experiments performed using tick saliva in vitro using stimuli other than Borrelia should be cautiously interpreted because other stimuli trigger different pathways and potentially lead to dissimilar immune responses. Moreover, although DCs of the skin are the primary responders in the early stages of a Borrelia infection, BMDCs or splenic DCs, which are known to express qualitatively different membrane receptors and are likely to have different functions in infection, are often used in experiments [86,87]. The role that LCs play in early Borrelia infection remains largely unexplored. Finally, because human DDCs are difficult to obtain, murine DCs are used extensively, but the differences between human and murine DCs are numerous, and the course of Lyme borreliosis also differs between the two [88]. Therefore, models are needed that more closely resemble the physiological state of DCs in human Lyme borreliosis. Given the instrumental role that DCs play in providing a bridge between innate and adaptive immunity, it is essential to examine their role in the early pathology of Lyme borreliosis. In addition, targeting the mechanisms by which the human immune system fails to eradicate Borrelia spirochetes, particularly the interference of tick salivary proteins, could restore protective DC function and may provide novel therapeutic and vaccination possibilities. Acknowledgments J.W.H. is a recipient of a VENI stipend (91611065) from the Netherlands Organisation for health research and development (ZonMw).

References 1 Radolf, J.D. et al. (2012) Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat. Rev. Microbiol. 10, 87–99 2 Stanek, G. et al. (2012) Lyme borreliosis. Lancet 379, 461–473 3 Wormser, G.P. et al. (2006) The clinical assessment, treatment, and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clin. Infect. Dis. 43, 1089–1134 4 Wormser, G.P. et al. (2005) Brief communication: hematogenous dissemination in early Lyme disease. Ann. Intern. Med. 142, 751–755

Trends in Parasitology xxx xxxx, Vol. xxx, No. x

5 Stanek, G. et al. (2011) Lyme borreliosis: clinical case definitions for diagnosis and management in Europe. Clin. Microbiol. Infect. 17, 69–79 6 Steere, A.C. (2001) Lyme disease. N. Engl. J. Med. 345, 115–125 7 Marques, A. (2008) Chronic Lyme disease: a review. Infect. Dis. Clin. North Am. 22, 341–360 8 Schuijt, T.J. et al. (2011) Lyme borreliosis vaccination: the facts, the challenge, the future. Trends Parasitol. 27, 40–47 9 de Taeye, S.W. et al. (2013) Complement evasion by Borrelia burgdorferi: it takes three to tango. Trends Parasitol. 29, 119–128 10 Hulinska, D. et al. (1994) Electron microscopy of Langerhans cells and Borrelia burgdorferi in Lyme disease patients. Zentralbl. Bakteriol. 280, 348–359 11 Suhonen, J. et al. (2003) Interaction between Borrelia burgdorferi and immature human dendritic cells. Scand. J. Immunol. 58, 67–75 12 Filgueira, L. et al. (1996) Human dendritic cells phagocytose and process Borrelia burgdorferi. J. Immunol. 157, 2998–3005 13 Salazar, J.C. et al. (2003) Coevolution of markers of innate and adaptive immunity in skin and peripheral blood of patients with erythema migrans. J. Immunol. 171, 2660–2670 14 Mbow, M.L. et al. (1997) Borrelia burgdorferi-pulsed dendritic cells induce a protective immune response against tick-transmitted spirochetes. Infect. Immun. 65, 3386–3390 15 Hirschfeld, M. et al. (1999) Cutting edge: inflammatory signaling by Borrelia burgdorferi lipoproteins is mediated by Toll-like receptor 2. J. Immunol. 163, 2382–2386 16 Takeuchi, O. et al. (2001) Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int. Immunol. 13, 933–940 17 Takeuchi, O. et al. (2002) Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J. Immunol. 169, 10–14 18 Aliprantis, A.O. et al. (1999) Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2. Science 285, 736–739 19 Shin, O.S. et al. (2008) Distinct roles for MyD88 and Toll-like receptors 2, 5, and 9 in phagocytosis of Borrelia burgdorferi and cytokine induction. Infect. Immun. 76, 2341–2351 20 Salazar, J.C. et al. (2005) Lipoprotein-dependent and -independent immune responses to spirochetal infection. Clin. Diagn. Lab. Immunol. 12, 949–958 21 Moore, M.W. et al. (2007) Phagocytosis of Borrelia burgdorferi and Treponema pallidum potentiates innate immune activation and induces gamma interferon production. Infect. Immun. 75, 2046– 2062 22 Cruz, A.R. et al. (2008) Phagocytosis of Borrelia burgdorferi, the Lyme disease spirochete, potentiates innate immune activation and induces apoptosis in human monocytes. Infect. Immun. 76, 56–70 23 Petzke, M.M. et al. (2009) Recognition of Borrelia burgdorferi, the Lyme disease spirochete, by TLR7 and TLR9 induces a type I IFN response by human immune cells. J. Immunol. 183, 5279–5292 24 Cervantes, J.L. et al. (2011) Phagosomal signaling by Borrelia burgdorferi in human monocytes involves Toll-like receptor (TLR) 2 and TLR8 cooperativity and TLR8-mediated induction of IFN-b. Proc. Natl. Acad. Sci. U.S.A. 108, 3683–3688 25 Kawai, T. and Akira, S. (2007) TLR signaling. Semin. Immunol. 19, 24– 32 26 van der Aar, A.M. et al. (2007) Loss of TLR2, TLR4, and TLR5 on Langerhans cells abolishes bacterial recognition. J. Immunol. 178, 1986–1990 27 Flacher, V. et al. (2006) Human Langerhans cells express a specific TLR profile and differentially respond to viruses and Gram-positive bacteria. J. Immunol. 177, 7959–7967 28 Strober, W. et al. (2006) Signalling pathways and molecular interactions of NOD1 and NOD2. Nat. Rev. Immunol. 6, 9–20 29 Petnicki-Ocwieja, T. et al. (2011) Nod2 suppresses Borrelia burgdorferi mediated murine Lyme arthritis and carditis through the induction of tolerance. PLoS ONE 6, e17414 30 Oosting, M. et al. (2010) Recognition of Borrelia burgdorferi by NOD2 is central for the induction of an inflammatory reaction. J. Infect. Dis. 201, 1849–1858 31 Robinson, M.J. et al. (2006) Myeloid C-type lectins in innate immunity. Nat. Immunol. 7, 1258–1265 32 Cinco, M. et al. (2001) Evidence of involvement of the mannose receptor in adhesion of Borrelia burgdorferi to monocyte/macrophages. Infect. Immun. 69, 2743–2747 7

TREPAR-1251; No. of Pages 9

Review 33 Hovius, J.W. et al. (2008) Salp15 binding to DC-SIGN inhibits cytokine expression by impairing both nucleosome remodeling and mRNA stabilization. PLoS Pathog. 4, e31 34 Hartiala, P. et al. (2007) Transcriptional response of human dendritic cells to Borrelia garinii – defective CD38 and CCR7 expression detected. J. Leukoc. Biol. 82, 33–43 35 Berende, A. et al. (2010) Activation of innate host defense mechanisms by Borrelia. Eur. Cytokine Netw. 21, 7–18 36 Liu, N. et al. (2009) The caspase 1 inflammasome is not required for control of murine Lyme borreliosis. Infect. Immun. 77, 3320–3327 37 Oosting, M. et al. (2012) Murine Borrelia arthritis is highly dependent on ASC and caspase-1, but independent of NLRP3. Arthritis Res. Ther. 14, R247 38 Salazar, J.C. et al. (2009) Activation of human monocytes by live Borrelia burgdorferi generates TLR2-dependent and independent responses which include induction of IFN-b. PLoS Pathog. 5, e1000444 39 Montoya, M. et al. (2002) Type I interferons produced by dendritic cells promote their phenotypic and functional activation. Blood 99, 3263– 3271 40 Miller, J.C. et al. (2008) A critical role for type I IFN in arthritis development following Borrelia burgdorferi infection of mice. J. Immunol. 181, 8492–8503 41 Liu, N. et al. (2004) Myeloid differentiation antigen 88 deficiency impairs pathogen clearance but does not alter inflammation in Borrelia burgdorferi-infected mice. Infect. Immun. 72, 3195–3203 42 Yakimchuk, K. et al. (2011) Borrelia burgdorferi infection regulates CD1 expression in human cells and tissues via IL1-b. Eur. J. Immunol. 41, 694–705 43 Moody, D.B. (2006) TLR gateways to CD1 function. Nat. Immunol. 7, 811–817 44 Ekerfelt, C. et al. (2003) Phenotypes indicating cytolytic properties of Borrelia-specific interferon-g secreting cells in chronic Lyme neuroborreliosis. J. Neuroimmunol. 145, 115–126 45 de Jong, E.C. et al. (2005) Dendritic cell-mediated T cell polarization. Springer Semin. Immunopathol. 26, 289–307 46 Bachmann, M. et al. (2010) Early production of IL-22 but not IL-17 by peripheral blood mononuclear cells exposed to live Borrelia burgdorferi: the role of monocytes and interleukin-1. PLoS Pathog. 6, e1001144 47 Christe, M. et al. (2000) Cytokines (IL-4 and IFN-g) and antibodies (IgE and IgG2a) produced in mice infected with Borrelia burgdorferi sensu stricto via nymphs of Ixodes ricinus ticks or syringe inoculations. Parasitol. Res. 86, 491–496 48 Yssel, H. et al. (1991) Borrelia burgdorferi activates a T helper type 1like T cell subset in Lyme arthritis. J. Exp. Med. 174, 593–601 49 Gross, D.M. et al. (1998) T helper 1 response is dominant and localized to the synovial fluid in patients with Lyme arthritis. J. Immunol. 160, 1022–1028 50 Burchill, M.A. et al. (2003) Inhibition of interleukin-17 prevents the development of arthritis in vaccinated mice challenged with Borrelia burgdorferi. Infect. Immun. 71, 3437–3442 51 Henningsson, A.J. et al. (2011) Indications of Th1 and Th17 responses in cerebrospinal fluid from patients with Lyme neuroborreliosis: a large retrospective study. J. Neuroinflammation 8, 36 52 Kapsenberg, M.L. (2003) Dendritic-cell control of pathogen-driven Tcell polarization. Nat. Rev. Immunol. 3, 984–993 53 Vudattu, N.K. et al. (2013) Dysregulation of CD4+CD25(high) T cells in the synovial fluid of patients with antibiotic-refractory Lyme arthritis. Arthritis Rheum. 65, 1643–1653 54 Shen, S. et al. (2010) Treg cell numbers and function in patients with antibiotic-refractory or antibiotic-responsive Lyme arthritis. Arthritis Rheum. 62, 2127–2137 55 Beermann, C. et al. (2000) Lipoproteins from Borrelia burgdorferi applied in liposomes and presented by dendritic cells induce CD8+ T-lymphocytes in vitro. Cell. Immunol. 201, 124–131 56 Randolph, G.J. et al. (2008) Migration of dendritic cell subsets and their precursors. Annu. Rev. Immunol. 26, 293–316 57 Hartiala, P. et al. (2010) TLR2 utilization of Borrelia does not induce p38- and IFN-b autocrine loop-dependent expression of CD38, resulting in poor migration and weak IL-12 secretion of dendritic cells. J. Immunol. 184, 5732–5742 8

Trends in Parasitology xxx xxxx, Vol. xxx, No. x

58 Ribeiro, J.M. et al. (1985) Antihemostatic, antiinflammatory, and immunosuppressive properties of the saliva of a tick, Ixodes dammini. J. Exp. Med. 161, 332–344 59 Slamova, M. et al. (2011) Effect of tick saliva on immune interactions between Borrelia afzelii and murine dendritic cells. Parasite Immunol. 33, 654–660 60 Skallova, A. et al. (2008) Tick saliva inhibits dendritic cell migration, maturation, and function while promoting development of Th2 responses. J. Immunol. 180, 6186–6192 61 Sa-Nunes, A. et al. (2007) Prostaglandin E2 is a major inhibitor of dendritic cell maturation and function in Ixodes scapularis saliva. J. Immunol. 179, 1497–1505 62 Sa-Nunes, A. et al. (2009) The immunomodulatory action of sialostatin L on dendritic cells reveals its potential to interfere with autoimmunity. J. Immunol. 182, 7422–7429 63 Oliveira, C.J. et al. (2010) Tick saliva induces regulatory dendritic cells: MAP-kinases and Toll-like receptor-2 expression as potential targets. Vet. Parasitol. 167, 288–297 64 Oliveira, C.J. et al. (2011) Deconstructing tick saliva: non-protein molecules with potent immunomodulatory properties. J. Biol. Chem. 286, 10960–10969 65 Cavassani, K.A. et al. (2005) Tick saliva inhibits differentiation, maturation and function of murine bone-marrow-derived dendritic cells. Immunology 114, 235–245 66 Oliveira, C.J. et al. (2008) Tick saliva inhibits the chemotactic function of MIP-1a and selectively impairs chemotaxis of immature dendritic cells by down-regulating cell-surface CCR5. Int. J. Parasitol. 38, 705– 716 67 Lieskovska, J. and Kopecky, J. (2012) Effect of tick saliva on signalling pathways activated by TLR-2 ligand and Borrelia afzelii in dendritic cells. Parasite Immunol. 34, 421–429 68 Lieskovska, J. and Kopecky, J. (2012) Tick saliva suppresses IFN signalling in dendritic cells upon Borrelia afzelii infection. Parasite Immunol. 34, 32–39 69 Vancova, I. et al. (2010) Anti-chemokine activities of ixodid ticks depend on tick species, developmental stage, and duration of feeding. Vet. Parasitol. 167, 274–278 70 Su, Y. et al. (2008) Cooperation of adenosine and prostaglandin E2 (PGE2) in amplification of cAMP–PKA signaling and immunosuppression. Cancer Immunol. Immunother. 57, 1611–1623 71 Hovius, J.W. et al. (2007) Identification of Salp15 homologues in Ixodes ricinus ticks. Vector Borne Zoonotic Dis. 7, 296–303 72 Ramamoorthi, N. et al. (2005) The Lyme disease agent exploits a tick protein to infect the mammalian host. Nature 436, 573–577 73 Garg, R. et al. (2006) Cutting edge: CD4 is the receptor for the tick saliva immunosuppressor, Salp15. J. Immunol. 177, 6579–6583 74 Anguita, J. et al. (2002) Salp15, an Ixodes scapularis salivary protein, inhibits CD4+ T cell activation. Immunity 16, 849–859 75 Geijtenbeek, T.B. et al. (2009) Pathogen recognition by DC-SIGN shapes adaptive immunity. Future Microbiol. 4, 879–890 76 Zavasnik-Bergant, T. and Turk, B. (2006) Cysteine cathepsins in the immune response. Tissue Antigens 67, 349–355 77 Kotsyfakis, M. et al. (2007) Selective cysteine protease inhibition contributes to blood-feeding success of the tick Ixodes scapularis. J. Biol. Chem. 282, 29256–29263 78 Kotsyfakis, M. et al. (2010) The crystal structures of two salivary cystatins from the tick Ixodes scapularis and the effect of these inhibitors on the establishment of Borrelia burgdorferi infection in a murine model. Mol. Microbiol. 77, 456–470 79 Mejri, N. and Brossard, M. (2007) Splenic dendritic cells pulsed with Ixodes ricinus tick saliva prime naive CD4+T to induce Th2 cell differentiation in vitro and in vivo. Int. Immunol. 19, 535–543 80 Vesely, D.L. et al. (2009) Langerhans cell deficiency impairs Ixodes scapularis suppression of Th1 responses in mice. Infect. Immun. 77, 1881–1887 81 Ferreira, B.R. and Silva, J.S. (1999) Successive tick infestations selectively promote a T-helper 2 cytokine profile in mice. Immunology 96, 434–439 82 Zeidner, N.S. et al. (2008) Suppression of Th2 cytokines reduces ticktransmitted Borrelia burgdorferi load in mice. J. Parasitol. 94, 767–769 83 Zeidner, N. et al. (1996) Suppression of acute Ixodes scapularis-induced Borrelia burgdorferi infection using tumor necrosis factor-a, interleukin-2, and interferon-g. J. Infect. Dis. 173, 187–195

TREPAR-1251; No. of Pages 9

Review 84 Bockenstedt, L.K. et al. (2001) CD4+ T helper 1 cells facilitate regression of murine Lyme carditis. Infect. Immun. 69, 5264–5269 85 Zeidner, N. et al. (1997) Effects of Ixodes scapularis and Borrelia burgdorferi on modulation of the host immune response: induction of a TH2 cytokine response in Lyme disease-susceptible (C3H/HeJ) mice but not in disease-resistant (BALB/c) mice. Infect. Immun. 65, 3100–3106 86 Shortman, K. and Liu, Y.J. (2002) Mouse and human dendritic cell subtypes. Nat. Rev. Immunol. 2, 151–161 87 Kushwah, R. and Hu, J. (2011) Complexity of dendritic cell subsets and their function in the host immune system. Immunology 133, 409–419

Trends in Parasitology xxx xxxx, Vol. xxx, No. x

88 Barthold, S.W. et al. (1991) Kinetics of Borrelia burgdorferi dissemination and evolution of disease after intradermal inoculation of mice. Am. J. Pathol. 139, 263–273 89 Banchereau, J. and Steinman, R.M. (1998) Dendritic cells and the control of immunity. Nature 392, 245–252 90 Janeway, C.A., Jr and Medzhitov, R. (2002) Innate immune recognition. Annu. Rev. Immunol. 20, 197–216 91 Mills, K.H. (2004) Regulatory T cells: friend or foe in immunity to infection? Nat. Rev. Immunol. 4, 841–855 92 Liew, F.Y. (2002) TH1 and TH2 cells: a historical perspective. Nat. Rev. Immunol. 2, 55–60

9