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Ixodes ricinus tick saliva modulates tick-borne encephalitis virus infection of dendritic cells
Microbes and Infection 12 (2010) 580e585 www.elsevier.com/locate/micinf
Short communication
Ixodes ricinus tick saliva modulates tick-borne encephalitis virus infection of dendritic cells Anna Fialova´ a,*, Zdenek Cimburek b, Giandomenica Iezzi c, Jan Kopecky´ a a
´ Budejovice, Czech Republic Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branisovska´ 31, CZ-37005 Ceske b Center of Flow Cytometry, Institute of Microbiology, Academy of Science of the Czech Republic, Vı´denska´ 1083, CZ-14220 Prague 4, Czech Republic c Institute of Surgical Research, Basel University Hospital, Hebelstrasse 20, CH-4031 Basel, Switzerland Received 26 January 2010; accepted 30 March 2010 Available online 8 April 2010
Abstract Tick-borne encephalitis virus is an important human pathogen, naturally delivered into host skin via a tick bite. To examine the effects of the virus on dendritic cell biology, we cultured dendritic cells with two tick-borne encephalitis virus strains of different virulence in the presence of Ixodes ricinus tick saliva. Tick saliva treatment increased proportion of virus-infected cells, led to a decrease in virus-induced TNF-a and IL-6 production and to reduced virus-induced apoptosis. Our data indicate that tick saliva modulate virus-mediated alterations in dendritic cells, thus probably being involved in the early infection process in the host. Ó 2010 Elsevier Masson SAS. All rights reserved. Keywords: Tick-borne encephalitis virus; Dendritic cell; Tick saliva; Ixodes ricinus
1. Introduction Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus within the family Flaviviridae, is according to the World Health Organization the most important arthropod-borne virus transmitted by ticks in Europe causing severe human infections [1]. The endemic area of TBEV ranges from Western Europe to China and the virus is taxonomically classified into three subtypes, namely European, Siberian and Far Eastern [2]. Typically, tick-borne encephalitis caused by European strains is a biphasic disease with the first stage accompanied by non-specific symptoms, including fever and fatigue. During the second stage of infection, 20e30% of patients develop neurological symptoms ranging from mild meningitis to severe encephalitis. The mortality rates are subtype-dependent and vary from 1 to 5% for European strains up to 60% for Far Eastern strains [3]. * Corresponding author. Department of Immunology, 2nd School of ´ valu 84, CZ-15006 Prague 5, Czech Medicine, Charles University, V U Republic. Tel.: þ420 224 435 950; fax: þ420 224 435 962. E-mail address: [email protected] (A. Fialova´). 1286-4579/$ - see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2010.03.015
TBEV is naturally delivered into host skin during blood sucking by an infected tick. It is supposed that in the early phase of infection, the virus replicates within the dermis and subsequently in the skin draining lymph nodes [4]. However, the route by which the virus enters the lymph nodes remains unknown. It is supposed that immunomodulatory activities of the tick saliva can be exploited by tick-borne pathogens to facilitate their transmission and replication in the host. According to Labuda et al. [5], the probability of TBEV transmission from infected guinea-pigs to uninfected feeding ticks was significantly enhanced if the virus inoculum was supplemented with salivary gland extract derived from adult female ticks. Simultaneously, development of viraemia was observed in markedly higher proportion of guinea-pigs inoculated with virus and tick salivary gland extract, in comparison with guinea-pigs inoculated with virus alone. However, systemic viraemia is not necessary for virus transmission. Surprisingly, TBEV transmission has been reported to be even more efficient among ticks co-feeding on a non-viraemic host in comparison to a host with high amount of the virus in blood [6]. In their following study, Labuda et al. [7] have demonstrated that even hosts with neutralizing antibodies to TBEV
A. Fialova´ et al. / Microbes and Infection 12 (2010) 580e585
are still able to support virus transmission between infected and uninfected ticks provided they feed close together. In case of the non-viraemic transmission, TBEV has only been detected in the skin at site of the tick bite, but not in the uninfested skin. This data indicates that the infested skin may be essential for TBEV replication early after virus transmission by ticks [8]. Viruses delivered into the skin encounter different cell types, including rich dendritic cell (DC) networks, which are intended for pathogen elimination in the early stages of infection [9]. On the one hand, DCs are the key players in the induction of protective immunity to viral infection; on the other hand, they can be also exploited by viruses to circumvent host immune response [10]. Similarly to other arthropod-borne viruses, DCs are known to be permissive for TBEV [8]; however, the effect of TBEV infection on DC phenotype and function has not been evaluated so far. Similarly, whether or not the tick saliva modulates TBEV-induced alterations in DC biology also remains to be addressed. In this study, we examined the effects of infection with two different TBEV strains on DC maturation, cytokine production and apoptosis in the presence or absence of Ixodes ricinus tick saliva. 2. Materials and methods
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suckling mouse brain passages. The prototype strain Neudoerfl (kindly provided by Professor F. X. Heinz, Medical University of Vienna, Austria; GenBank accession no. U27495) was passaged five times in suckling mice. Purified spleen DCs were exposed to TBEV at indicated multiplicities of infection (MOIs) in the presence or absence of tick saliva (final concentration ¼ 10 mg/ml). The saliva was added to the DC culture either 4 h prior to the virus (virus þ saliva 4 h) or simultaneously with the virus (virus þ saliva). A corresponding volume of 115 mM pilocarpine was included as an additional control treatment. In all experiments, the appropriate volume of a virus-free mouse brain suspension was used as a negative control (mock infection). After 4 h of incubation, cells were washed to remove cell-free virus and cultured in complete IMDM. Cells were harvested and labelled for subsequent FACS analysis 24 h after exposure to the virus. 2.5. Cytokine measurements Cell-free culture supernatants were harvested 24 and 48 h after TBEV exposure and stored at 20 C until further analysis. Samples were tested for IFN-b, TNF-a, IL-6, IL-12p70, and IL-10, using mouse IFN-beta ELISA Kit (PBL Biomedical Laboratories) and Ready-SET-Go! ELISA Sets (eBioscience) following the manufacturer’s instructions.
2.1. Animals Female C57BL/6 mice (10 weeks of age) were obtained from Charles River Laboratories. Guinea-pigs used for tick ´ feeding were bred at the Institute of Parasitology, Ceske Budejovice. All experiments were performed by permission of the Czech animal ethics committee. 2.2. Tick saliva collection TBEV-free adult I. ricinus ticks from the colony maintained ´ Budejovice were at the Institute of Parasitology in Ceske allowed to feed on guinea-pigs. After 6 days, partially engorged female ticks were removed and the saliva was collected and processed as previously described [11].
2.6. Flow cytometry Cells were stained with Alexa FluorÒ 488-labelled antiCD11c, APC-labelled anti-B7-2, FITC-labelled anti-MHC I, and Alexa FluorÒ 700-labelled anti-I-Ab (all from eBioscience). Surface labelled cells were fixed with 2% formaldehyde solution, permeabilized with 0.5% saponin and stained for intracellular expression of viral envelope protein using anti-TBEV monoclonal antibody 19/75, kindly provided by Prof. Matthias Niedrig, Robert Koch-Institute, Germany. The antibody was PE-conjugated using ZenonÒ R-Phycoerythrin Mouse IgG1 Labeling Kit (Molecular Probes). Cells were analyzed using BD LSR II cell analyzer (BD Biosciences) and FlowJo software (Tree Star).
2.3. DC isolation and purification 2.7. Analysis of apoptotic cells Isolated mouse spleens were minced with scissors, digested in IMDM containing 1 mg/ml collagenase D (Roche), and passed through a nylon cell strainer (BD Falcon). DCs were isolated using magnetic beads conjugated with anti-CD11c Ab and MACS Column separation following the manufacturer’s instructions (Miltenyi Biotec). Purity of isolated DCs (>87% CD11cþ cells) was determined by FACS analysis.
Cells were harvested 48 h after TBEV exposure, fixed with 2% formaldehyde solution, permeabilized with 0.5% saponin, stained for active caspase-3 using PE-labelled anti-active caspase-3 mAb (BD Pharmingen) and analyzed by flow cytometry.
2.4. TBEV infection
2.8. Statistical analysis
Two TBEV strains of different virulence were used in the experiments. The highly virulent strain Hypr (GenBank accession no. U39292) was propagated through numerous
Statistical analyses were performed by ANOVA followed by Tukey Post Hoc test, using StatisticaÒ v.7.1 software. A p value <0.05 was considered as significant.
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3. Results 3.1. Tick saliva increases percentage of TBEV-infected cells To assess the effect of tick saliva on the proportion of infected cells, DCs were exposed to the TBEV at MOI of 1 and 10 in the presence or absence of the saliva. Whereas the Hypr strain at MOI of 10 infected 40.1 1.9% of DCs in saliva-free cultures, only 1.4 0.1% of DCs were Neudoerfl-positive. The tick saliva significantly increased the percentage of TBEV-infected cells in the cultures exposed to the high MOI of both virus strains to 47.6% and 2.4%, respectively (Fig. 1A and B). No significant enhancement was observed in DCs cultured in the presence of pilocarpine.
3.3. Tick saliva diminishes levels of TNF-a and IL-6 in TBEV-infected DC cultures To evaluate whether tick saliva impairs virus-induced cytokine production we assessed levels of TNF-a, IFN-b, IL-6, IL-10 and IL-12p70 in cell culture supernatants. We detected significant amounts of TNF-a (Fig. 2A and B) and IFN-b (Fig. 2E and F), and high amounts of IL-6 (Fig. 2C and D), but only minimal content of IL-12p70 and IL-10 (data not shown) in TBEV-infected cultures as compared to mockinfected cultures. The presence of tick saliva markedly reduced the enhanced levels of TNF-a and IL-6 but did not affect IFN-b level in both Neudoerfl and Hypr-exposed cultures. Pilocarpine did not exert any significant effect. 3.4. Tick saliva mitigates TBEV-induced apoptosis in DCs
3.2. Tick saliva modulates Neudoerfl-induced DC maturation Simultaneously, we analyzed the phenotype of virusinfected and bystander cells in the cultures. Both of the TBEV strains used induced strong up-regulation of MHC class II and B7-2 molecules in infected cells. On the contrary, bystander uninfected cells kept immature phenotype (Fig. 1C and D). The tick saliva significantly impaired Neudoerfl-induced upregulation of MHC class II, but did not affect maturation induced by the Hypr strain (data not shown).
Additionally, we studied the effect of TBEV and tick saliva on DC viability. As indicated by the higher percentage of active caspase-3 positive cells 48 h following virus exposure, both of the TBEV strains used significantly increased apoptosis in DC cultures (Fig. 3). In the Hypr-treated cultures the proportion of apoptotic cells was significantly decreased in the presence of the tick saliva, whereas pilocarpine did not affect DC viability. However, the tick saliva seemed to have no significantly negative effect on DC apoptosis in Neudoerflexposed cultures.
Fig. 1. Percentage of TBEV-infected cells is enhanced in the presence of tick saliva. TBEV-infected cells significantly up-regulate MHC class I, MHC class II, and B7-2 molecules, whereas bystander cells keep immature phenotype. A, Percentage of Hypr-positive DCs 24 h after virus exposure. B, Percentage of Neudoerflpositive DCs 24 h after virus exposure. Data are expressed as the mean proportion of infected cells þ SEM. *, effect of tick saliva, p < 0.05, ANOVA followed by Tukey Post Hoc test. Tick saliva was added to cultures either 4 h prior to the virus (Virus þ saliva 4 h), or together with the virus (Virus þ saliva). C, Median of MHC class I, MHC class II, and B7-2 fluorescence in DCs exposed to Hypr at MOI of 10. D, Median of MHC class I, MHC class II, and B7-2 fluorescence in DCs exposed to Neudoerfl at MOI of 10. Data are expressed as median þ SEM. þ, effect of TBEV, p < 0.05; ANOVA followed by Tukey Post Hoc test. Data are representative of four repeat experiments.
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Fig. 2. Tick saliva diminishes levels of TNF-a (A, B) and IL-6 (C, D), bud did not markedly affect level of IFN-b (E, F) produced by TBEV-exposed DC cultures. Data are expressed as the mean cytokine concentration in culture supernatants 24 h and 48 h after virus exposure þ SEM. *, effect of tick saliva, p < 0.05; þ, effect of TBEV, p < 0.05; ANOVA followed by Tukey Post Hoc test. Tick saliva was added to cultures either 4 h prior to the virus (Virus þ saliva 4h), or together with the virus (Virus þ saliva). Data are representative of four repeat experiments.
4. Discussion It is known that many viruses have evolved mechanisms to avoid their recognition by the immune system via modification of the DC maturation programme, efficiency of migration, antigen presentation capacity and the outcome of DC/T cell interactions [10,12]. However, effects exerted by the TBEV on DCs have not been evaluated so far. Here we have described for the first time the effects mediated by TBEV on DC and, additionally, we have analyzed their possible modulation by tick saliva. We have found that DC exposure to TBEV resulted in DC infection, maturation, release of inflammatory cytokines such as TNF-a, IFN-b and IL-6 and, eventually, in apoptosis of infected cells. Importantly, these effects were to various extents modulated by the presence of tick saliva. Indeed, DC exposure to tick saliva resulted in higher percentages of infected cells and lower fractions of apoptotic cells. Furthermore, tick saliva inhibited TBEV-induced release of inflammatory cytokines, namely TNF-a and IL-6. By using spleen-derived DCs and two TBEV strains of different virulence we have demonstrated that although DCs are permissive for TBEV, their permissiveness markedly depends on the virus strain used. In the presence of tick saliva DCs have demonstrated greater permissiveness to both TBEV strains used, while the extent of the saliva effect negatively correlated with the virulence of the virus strain. Thus, it seems that the presence of saliva might be more beneficial for lowvirulence strains such as Neudoerfl. In the Neudoerfl-exposed cultures, the effect of the tick saliva was more pronounced, if the saliva were added 4 h prior to the virus. This suggests that
the increase in DC permissivity might be due to the effect of saliva on the cells rather than on the virus itself. However, this effect was not observed in Hypr-exposed DCs. Studies focused on the mosquito-borne member of the flavivirus family, the Dengue virus (DV), showed that immature but not mature DCs are permissive to DV infection [13,14]. Similarly, we have also observed decreased, albeit not completely abrogated percentage of TBEV-infected cells upon CpG stimulation (data not shown). Our previous study demonstrated that the tick saliva inhibits DC maturation, upon TLR3, TLR7, TLR9, or CD40 ligation [11]. This might suggest that in the presence of the tick saliva, DCs keep a less mature phenotype and therefore remain permissive for the virus, however; no significant effect of the tick saliva on virus-induced upregulation of MHC class II and B7-2 molecules has been observed in this study, with the exception of the expression of MHC class II molecules induced by the Neudoerfl strain. Human DCs exposed to dengue virus have been reported to secrete TNF-a, but little or no IL-10 and IL-12p70 [14]. Similarly, our study has shown that DC infection by both TBEV strains used resulted in significant production of TNF-a and IL6, whereas IL-10 and IL-12p70 production was undetectable. The virulent Hypr strain induced higher levels of cytokines compared to the Neudoerfl strain. We suppose that cytokines are presumably produced by infected rather than bystander DCs, since bystander cells kept immature phenotype as assessed by low expression of B7-2 and MHC class II molecules. In addition, we have demonstrated the significant impact of tick saliva on the levels of TNF-a and IL-6. Our findings are in accordance with other authors who have reported tick saliva-mediated
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Fig. 3. Tick saliva diminishes Hypr-induced apoptosis of DCs. A, Percentage of active caspase-3 positive cells in DC cultures infected by Hypr at MOI of 10. B, Percentage of active caspase-3 positive cells in DC cultures infected by Neudoerfl at MOI of 10. Data are expressed as the mean proportion of apoptotic cells þ SEM. *, effect of tick saliva, p < 0.05; þ, effect of TBEV, p < 0.05; ANOVA followed by Tukey Post Hoc test. Tick saliva was added to cultures either 4 h prior to the virus (Virus þ saliva 4 h), or together with the virus (Virus þ saliva). Histograms illustrate changes in active caspase-3 expression in DCs exposed to Hypr at MOI of 1 (C), Hypr at MOI of 10 (D), and Neudoerfl at MOI of 10 (E) in the presence or absence of the tick saliva. Gating was performed on CD11cþ cells. Experiments were performed three times, and representative data are shown.
decrease of TLR ligand-induced IL-6 and TNF-a production [15,16]. Together with IL-1, TNF-a is known to promote DC migration from the skin into regional lymph nodes [17]. Indeed, in our previous study [11], we have shown that tick saliva impaired early migration of skin DCs from inflamed skin. Additionally, saliva reduced TBEV-mediated DC apoptosis. These observations may suggest that in the presence of tick saliva infected DCs might stay in the skin for a prolonged time as a further source of the virus. This would be in accordance with Labuda et al. [8], who predicated that the skin at the tick bite site may be crucial for TBEV replication and following transmission to uninfected feeding ticks. Our findings suggest that tick saliva may facilitate the spread of TBEV through several mechanisms e firstly by increasing viral infectivity, especially that of low-virulence strains and by simultaneous attenuation of inflammatory response elicited by the infection. Further, tick saliva prolongs the survival of infected DCs which may, together with the saliva-induced impairment of DC migration observed previously, enhance chances of TBEV transmission among infected and uninfected
ticks co-feeding on the same host. Nevertheless, whether or not the interactions among DCs, TBEV and the saliva of its tick vector could significantly influence viral accessibility for feeding ticks in vivo is a matter for further examination. Acknowledgements The project was supported by grant IAA600960811 from the Grant Agency of the Academy of Sciences of the Czech Republic, the project of the Basic Research Centre LC06009 from the Czech Ministry of Education, and the research projects of the Institute of Parasitology Z60220518 and of the ´ Faculty of Science, University of South Bohemia, Ceske Budejovice MSM 6007665801. References [1] WHO Regional Office for Europe (WHO/EURO), The vector-borne human infections of Europe: their distribution and burden on public health, Copenhagen, Denmark, 2004, pp. 37e53.
A. Fialova´ et al. / Microbes and Infection 12 (2010) 580e585 [2] M. Ecker, S.L. Allison, T. Meixner, F.X. Heinz, Sequence analysis and genetic classification of tick-borne encephalitis viruses from Europe and Asia. J. Gen. Virol. 80 (1999) 179e185. [3] T.S. Gritsun, V.A. Lashkevich, E.A. Gould, Tick-borne encephalitis. Antiviral. Res. 57 (2003) 129e146. [4] P. Albrecht, Pathogenesis of neurotropic arbovirus infections. Curr. Top. Microbiol. Immunol. 43 (1968) 44e91. [5] M. Labuda, L.D. Jones, T. Williams, P.A. Nuttall, Enhancement of tickborne encephalitis virus transmission by tick salivary gland extracts. Med. Vet. Entomol. 7 (1993) 193e196. [6] M. Labuda, P.A. Nuttall, O. Kozuch, E. Eleckova´, T. Williams, E. Zuffova´, A. Sabo´, Nonviraemic transmission of tick-borne encephalitis virus: a mechanism for arbovirus survival in nature. Experientia 49 (1993) 802e805. [7] M. Labuda, O. Kozuch, E. Zuffova, E. Eleckova, R.S. Hails, P.A. Nuttall, Tick-borne encephalitis virus transmission between ticks cofeeding on specific immune natural rodent hosts. Virology 235 (1997) 138e143. [8] M. Labuda, J.M. Austyn, E. Zuffova, O. Kozuch, N. Fuchsberger, J. Lysy, P.A. Nuttall, Importance of localized skin infection in tick-borne encephalitis virus transmission. Virology 219 (1996) 357e366. [9] C. Debenedictis, S. Joubeh, G. Zhang, M. Barria, R.F. Ghohestani, Immune functions of the skin. Clin. Dermatol. 19 (2001) 573e585. [10] D. Kaiserlian, B. Dubois, Dendritic cells and viral immunity: friends or foes? Semin. Immunol. 13 (2001) 303e310. [11] A. Skallova´, G. Iezzi, F. Ampenberger, M. Kopf, J. Kopecky, Tick saliva inhibits dendritic cell migration, maturation, and function while
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promoting development of Th2 responses. J. Immunol. 180 (2008) 6186e6192. C. Pohl, J. Shishkova, S. Schneider-Schaulies, Viruses and dendritic cells: enemy mine. Cell Microbiol. 9 (2007) 279e289. S.J. Wu, G. Grouard-Vogel, W. Sun, J.R. Mascola, E. Brachtel, R. Putvatana, M.K. Louder, L. Filgueira, M.A. Marovich, H.K. Wong, A. Blauvelt, G.S. Murphy, M.L. Robb, B.L. Innes, D.L. Birx, C.G. Hayes, S. S. Frankel, Human skin Langerhans cells are targets of dengue virus infection. Nat. Med. 6 (2000) 816e820. D.H. Libraty, S. Pichyangkul, C. Ajariyakhajorn, T.P. Endy, F.A. Ennis, Human dendritic cells are activated by dengue virus infection: enhancement by gamma interferon and implications for disease pathogenesis. J. Virol. 75 (2001) 3501e3508. A. Sa´-Nunes, A. Bafica, D.A. Lucas, T.P. Conrads, T.D. Veenstra, J.F. Andersen, T.N. Mather, J.M. Ribeiro, I.M. Francischetti, Prostaglandin E2 is a major inhibitor of dendritic cell maturation and function in Ixodes scapularis saliva. J. Immunol. 179 (2007) 1497e1505. J.W. Hovius, M.A. de Jong, J. den Dunnen, M. Litjens, E. Fikrig, T. van der Poll, S.I. Gringhuis, T.B. Geijtenbeek, Salp15 binding to DC-SIGN inhibits cytokine expression by impairing both nucleosome remodeling and mRNA stabilization. PLoS Pathog. 4 (2008) e31. M. Cumberbatch, R.J. Dearman, I. Kimber, Langerhans cells require signals from both tumour necrosis factor-a and interleukin-1b for migration. Immunology 92 (1997) 388e395.
Short communication
Ixodes ricinus tick saliva modulates tick-borne encephalitis virus infection of dendritic cells Anna Fialova´ a,*, Zdenek Cimburek b, Giandomenica Iezzi c, Jan Kopecky´ a a
´ Budejovice, Czech Republic Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branisovska´ 31, CZ-37005 Ceske b Center of Flow Cytometry, Institute of Microbiology, Academy of Science of the Czech Republic, Vı´denska´ 1083, CZ-14220 Prague 4, Czech Republic c Institute of Surgical Research, Basel University Hospital, Hebelstrasse 20, CH-4031 Basel, Switzerland Received 26 January 2010; accepted 30 March 2010 Available online 8 April 2010
Abstract Tick-borne encephalitis virus is an important human pathogen, naturally delivered into host skin via a tick bite. To examine the effects of the virus on dendritic cell biology, we cultured dendritic cells with two tick-borne encephalitis virus strains of different virulence in the presence of Ixodes ricinus tick saliva. Tick saliva treatment increased proportion of virus-infected cells, led to a decrease in virus-induced TNF-a and IL-6 production and to reduced virus-induced apoptosis. Our data indicate that tick saliva modulate virus-mediated alterations in dendritic cells, thus probably being involved in the early infection process in the host. Ó 2010 Elsevier Masson SAS. All rights reserved. Keywords: Tick-borne encephalitis virus; Dendritic cell; Tick saliva; Ixodes ricinus
1. Introduction Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus within the family Flaviviridae, is according to the World Health Organization the most important arthropod-borne virus transmitted by ticks in Europe causing severe human infections [1]. The endemic area of TBEV ranges from Western Europe to China and the virus is taxonomically classified into three subtypes, namely European, Siberian and Far Eastern [2]. Typically, tick-borne encephalitis caused by European strains is a biphasic disease with the first stage accompanied by non-specific symptoms, including fever and fatigue. During the second stage of infection, 20e30% of patients develop neurological symptoms ranging from mild meningitis to severe encephalitis. The mortality rates are subtype-dependent and vary from 1 to 5% for European strains up to 60% for Far Eastern strains [3]. * Corresponding author. Department of Immunology, 2nd School of ´ valu 84, CZ-15006 Prague 5, Czech Medicine, Charles University, V U Republic. Tel.: þ420 224 435 950; fax: þ420 224 435 962. E-mail address: [email protected] (A. Fialova´). 1286-4579/$ - see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2010.03.015
TBEV is naturally delivered into host skin during blood sucking by an infected tick. It is supposed that in the early phase of infection, the virus replicates within the dermis and subsequently in the skin draining lymph nodes [4]. However, the route by which the virus enters the lymph nodes remains unknown. It is supposed that immunomodulatory activities of the tick saliva can be exploited by tick-borne pathogens to facilitate their transmission and replication in the host. According to Labuda et al. [5], the probability of TBEV transmission from infected guinea-pigs to uninfected feeding ticks was significantly enhanced if the virus inoculum was supplemented with salivary gland extract derived from adult female ticks. Simultaneously, development of viraemia was observed in markedly higher proportion of guinea-pigs inoculated with virus and tick salivary gland extract, in comparison with guinea-pigs inoculated with virus alone. However, systemic viraemia is not necessary for virus transmission. Surprisingly, TBEV transmission has been reported to be even more efficient among ticks co-feeding on a non-viraemic host in comparison to a host with high amount of the virus in blood [6]. In their following study, Labuda et al. [7] have demonstrated that even hosts with neutralizing antibodies to TBEV
A. Fialova´ et al. / Microbes and Infection 12 (2010) 580e585
are still able to support virus transmission between infected and uninfected ticks provided they feed close together. In case of the non-viraemic transmission, TBEV has only been detected in the skin at site of the tick bite, but not in the uninfested skin. This data indicates that the infested skin may be essential for TBEV replication early after virus transmission by ticks [8]. Viruses delivered into the skin encounter different cell types, including rich dendritic cell (DC) networks, which are intended for pathogen elimination in the early stages of infection [9]. On the one hand, DCs are the key players in the induction of protective immunity to viral infection; on the other hand, they can be also exploited by viruses to circumvent host immune response [10]. Similarly to other arthropod-borne viruses, DCs are known to be permissive for TBEV [8]; however, the effect of TBEV infection on DC phenotype and function has not been evaluated so far. Similarly, whether or not the tick saliva modulates TBEV-induced alterations in DC biology also remains to be addressed. In this study, we examined the effects of infection with two different TBEV strains on DC maturation, cytokine production and apoptosis in the presence or absence of Ixodes ricinus tick saliva. 2. Materials and methods
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suckling mouse brain passages. The prototype strain Neudoerfl (kindly provided by Professor F. X. Heinz, Medical University of Vienna, Austria; GenBank accession no. U27495) was passaged five times in suckling mice. Purified spleen DCs were exposed to TBEV at indicated multiplicities of infection (MOIs) in the presence or absence of tick saliva (final concentration ¼ 10 mg/ml). The saliva was added to the DC culture either 4 h prior to the virus (virus þ saliva 4 h) or simultaneously with the virus (virus þ saliva). A corresponding volume of 115 mM pilocarpine was included as an additional control treatment. In all experiments, the appropriate volume of a virus-free mouse brain suspension was used as a negative control (mock infection). After 4 h of incubation, cells were washed to remove cell-free virus and cultured in complete IMDM. Cells were harvested and labelled for subsequent FACS analysis 24 h after exposure to the virus. 2.5. Cytokine measurements Cell-free culture supernatants were harvested 24 and 48 h after TBEV exposure and stored at 20 C until further analysis. Samples were tested for IFN-b, TNF-a, IL-6, IL-12p70, and IL-10, using mouse IFN-beta ELISA Kit (PBL Biomedical Laboratories) and Ready-SET-Go! ELISA Sets (eBioscience) following the manufacturer’s instructions.
2.1. Animals Female C57BL/6 mice (10 weeks of age) were obtained from Charles River Laboratories. Guinea-pigs used for tick ´ feeding were bred at the Institute of Parasitology, Ceske Budejovice. All experiments were performed by permission of the Czech animal ethics committee. 2.2. Tick saliva collection TBEV-free adult I. ricinus ticks from the colony maintained ´ Budejovice were at the Institute of Parasitology in Ceske allowed to feed on guinea-pigs. After 6 days, partially engorged female ticks were removed and the saliva was collected and processed as previously described [11].
2.6. Flow cytometry Cells were stained with Alexa FluorÒ 488-labelled antiCD11c, APC-labelled anti-B7-2, FITC-labelled anti-MHC I, and Alexa FluorÒ 700-labelled anti-I-Ab (all from eBioscience). Surface labelled cells were fixed with 2% formaldehyde solution, permeabilized with 0.5% saponin and stained for intracellular expression of viral envelope protein using anti-TBEV monoclonal antibody 19/75, kindly provided by Prof. Matthias Niedrig, Robert Koch-Institute, Germany. The antibody was PE-conjugated using ZenonÒ R-Phycoerythrin Mouse IgG1 Labeling Kit (Molecular Probes). Cells were analyzed using BD LSR II cell analyzer (BD Biosciences) and FlowJo software (Tree Star).
2.3. DC isolation and purification 2.7. Analysis of apoptotic cells Isolated mouse spleens were minced with scissors, digested in IMDM containing 1 mg/ml collagenase D (Roche), and passed through a nylon cell strainer (BD Falcon). DCs were isolated using magnetic beads conjugated with anti-CD11c Ab and MACS Column separation following the manufacturer’s instructions (Miltenyi Biotec). Purity of isolated DCs (>87% CD11cþ cells) was determined by FACS analysis.
Cells were harvested 48 h after TBEV exposure, fixed with 2% formaldehyde solution, permeabilized with 0.5% saponin, stained for active caspase-3 using PE-labelled anti-active caspase-3 mAb (BD Pharmingen) and analyzed by flow cytometry.
2.4. TBEV infection
2.8. Statistical analysis
Two TBEV strains of different virulence were used in the experiments. The highly virulent strain Hypr (GenBank accession no. U39292) was propagated through numerous
Statistical analyses were performed by ANOVA followed by Tukey Post Hoc test, using StatisticaÒ v.7.1 software. A p value <0.05 was considered as significant.
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3. Results 3.1. Tick saliva increases percentage of TBEV-infected cells To assess the effect of tick saliva on the proportion of infected cells, DCs were exposed to the TBEV at MOI of 1 and 10 in the presence or absence of the saliva. Whereas the Hypr strain at MOI of 10 infected 40.1 1.9% of DCs in saliva-free cultures, only 1.4 0.1% of DCs were Neudoerfl-positive. The tick saliva significantly increased the percentage of TBEV-infected cells in the cultures exposed to the high MOI of both virus strains to 47.6% and 2.4%, respectively (Fig. 1A and B). No significant enhancement was observed in DCs cultured in the presence of pilocarpine.
3.3. Tick saliva diminishes levels of TNF-a and IL-6 in TBEV-infected DC cultures To evaluate whether tick saliva impairs virus-induced cytokine production we assessed levels of TNF-a, IFN-b, IL-6, IL-10 and IL-12p70 in cell culture supernatants. We detected significant amounts of TNF-a (Fig. 2A and B) and IFN-b (Fig. 2E and F), and high amounts of IL-6 (Fig. 2C and D), but only minimal content of IL-12p70 and IL-10 (data not shown) in TBEV-infected cultures as compared to mockinfected cultures. The presence of tick saliva markedly reduced the enhanced levels of TNF-a and IL-6 but did not affect IFN-b level in both Neudoerfl and Hypr-exposed cultures. Pilocarpine did not exert any significant effect. 3.4. Tick saliva mitigates TBEV-induced apoptosis in DCs
3.2. Tick saliva modulates Neudoerfl-induced DC maturation Simultaneously, we analyzed the phenotype of virusinfected and bystander cells in the cultures. Both of the TBEV strains used induced strong up-regulation of MHC class II and B7-2 molecules in infected cells. On the contrary, bystander uninfected cells kept immature phenotype (Fig. 1C and D). The tick saliva significantly impaired Neudoerfl-induced upregulation of MHC class II, but did not affect maturation induced by the Hypr strain (data not shown).
Additionally, we studied the effect of TBEV and tick saliva on DC viability. As indicated by the higher percentage of active caspase-3 positive cells 48 h following virus exposure, both of the TBEV strains used significantly increased apoptosis in DC cultures (Fig. 3). In the Hypr-treated cultures the proportion of apoptotic cells was significantly decreased in the presence of the tick saliva, whereas pilocarpine did not affect DC viability. However, the tick saliva seemed to have no significantly negative effect on DC apoptosis in Neudoerflexposed cultures.
Fig. 1. Percentage of TBEV-infected cells is enhanced in the presence of tick saliva. TBEV-infected cells significantly up-regulate MHC class I, MHC class II, and B7-2 molecules, whereas bystander cells keep immature phenotype. A, Percentage of Hypr-positive DCs 24 h after virus exposure. B, Percentage of Neudoerflpositive DCs 24 h after virus exposure. Data are expressed as the mean proportion of infected cells þ SEM. *, effect of tick saliva, p < 0.05, ANOVA followed by Tukey Post Hoc test. Tick saliva was added to cultures either 4 h prior to the virus (Virus þ saliva 4 h), or together with the virus (Virus þ saliva). C, Median of MHC class I, MHC class II, and B7-2 fluorescence in DCs exposed to Hypr at MOI of 10. D, Median of MHC class I, MHC class II, and B7-2 fluorescence in DCs exposed to Neudoerfl at MOI of 10. Data are expressed as median þ SEM. þ, effect of TBEV, p < 0.05; ANOVA followed by Tukey Post Hoc test. Data are representative of four repeat experiments.
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Fig. 2. Tick saliva diminishes levels of TNF-a (A, B) and IL-6 (C, D), bud did not markedly affect level of IFN-b (E, F) produced by TBEV-exposed DC cultures. Data are expressed as the mean cytokine concentration in culture supernatants 24 h and 48 h after virus exposure þ SEM. *, effect of tick saliva, p < 0.05; þ, effect of TBEV, p < 0.05; ANOVA followed by Tukey Post Hoc test. Tick saliva was added to cultures either 4 h prior to the virus (Virus þ saliva 4h), or together with the virus (Virus þ saliva). Data are representative of four repeat experiments.
4. Discussion It is known that many viruses have evolved mechanisms to avoid their recognition by the immune system via modification of the DC maturation programme, efficiency of migration, antigen presentation capacity and the outcome of DC/T cell interactions [10,12]. However, effects exerted by the TBEV on DCs have not been evaluated so far. Here we have described for the first time the effects mediated by TBEV on DC and, additionally, we have analyzed their possible modulation by tick saliva. We have found that DC exposure to TBEV resulted in DC infection, maturation, release of inflammatory cytokines such as TNF-a, IFN-b and IL-6 and, eventually, in apoptosis of infected cells. Importantly, these effects were to various extents modulated by the presence of tick saliva. Indeed, DC exposure to tick saliva resulted in higher percentages of infected cells and lower fractions of apoptotic cells. Furthermore, tick saliva inhibited TBEV-induced release of inflammatory cytokines, namely TNF-a and IL-6. By using spleen-derived DCs and two TBEV strains of different virulence we have demonstrated that although DCs are permissive for TBEV, their permissiveness markedly depends on the virus strain used. In the presence of tick saliva DCs have demonstrated greater permissiveness to both TBEV strains used, while the extent of the saliva effect negatively correlated with the virulence of the virus strain. Thus, it seems that the presence of saliva might be more beneficial for lowvirulence strains such as Neudoerfl. In the Neudoerfl-exposed cultures, the effect of the tick saliva was more pronounced, if the saliva were added 4 h prior to the virus. This suggests that
the increase in DC permissivity might be due to the effect of saliva on the cells rather than on the virus itself. However, this effect was not observed in Hypr-exposed DCs. Studies focused on the mosquito-borne member of the flavivirus family, the Dengue virus (DV), showed that immature but not mature DCs are permissive to DV infection [13,14]. Similarly, we have also observed decreased, albeit not completely abrogated percentage of TBEV-infected cells upon CpG stimulation (data not shown). Our previous study demonstrated that the tick saliva inhibits DC maturation, upon TLR3, TLR7, TLR9, or CD40 ligation [11]. This might suggest that in the presence of the tick saliva, DCs keep a less mature phenotype and therefore remain permissive for the virus, however; no significant effect of the tick saliva on virus-induced upregulation of MHC class II and B7-2 molecules has been observed in this study, with the exception of the expression of MHC class II molecules induced by the Neudoerfl strain. Human DCs exposed to dengue virus have been reported to secrete TNF-a, but little or no IL-10 and IL-12p70 [14]. Similarly, our study has shown that DC infection by both TBEV strains used resulted in significant production of TNF-a and IL6, whereas IL-10 and IL-12p70 production was undetectable. The virulent Hypr strain induced higher levels of cytokines compared to the Neudoerfl strain. We suppose that cytokines are presumably produced by infected rather than bystander DCs, since bystander cells kept immature phenotype as assessed by low expression of B7-2 and MHC class II molecules. In addition, we have demonstrated the significant impact of tick saliva on the levels of TNF-a and IL-6. Our findings are in accordance with other authors who have reported tick saliva-mediated
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Fig. 3. Tick saliva diminishes Hypr-induced apoptosis of DCs. A, Percentage of active caspase-3 positive cells in DC cultures infected by Hypr at MOI of 10. B, Percentage of active caspase-3 positive cells in DC cultures infected by Neudoerfl at MOI of 10. Data are expressed as the mean proportion of apoptotic cells þ SEM. *, effect of tick saliva, p < 0.05; þ, effect of TBEV, p < 0.05; ANOVA followed by Tukey Post Hoc test. Tick saliva was added to cultures either 4 h prior to the virus (Virus þ saliva 4 h), or together with the virus (Virus þ saliva). Histograms illustrate changes in active caspase-3 expression in DCs exposed to Hypr at MOI of 1 (C), Hypr at MOI of 10 (D), and Neudoerfl at MOI of 10 (E) in the presence or absence of the tick saliva. Gating was performed on CD11cþ cells. Experiments were performed three times, and representative data are shown.
decrease of TLR ligand-induced IL-6 and TNF-a production [15,16]. Together with IL-1, TNF-a is known to promote DC migration from the skin into regional lymph nodes [17]. Indeed, in our previous study [11], we have shown that tick saliva impaired early migration of skin DCs from inflamed skin. Additionally, saliva reduced TBEV-mediated DC apoptosis. These observations may suggest that in the presence of tick saliva infected DCs might stay in the skin for a prolonged time as a further source of the virus. This would be in accordance with Labuda et al. [8], who predicated that the skin at the tick bite site may be crucial for TBEV replication and following transmission to uninfected feeding ticks. Our findings suggest that tick saliva may facilitate the spread of TBEV through several mechanisms e firstly by increasing viral infectivity, especially that of low-virulence strains and by simultaneous attenuation of inflammatory response elicited by the infection. Further, tick saliva prolongs the survival of infected DCs which may, together with the saliva-induced impairment of DC migration observed previously, enhance chances of TBEV transmission among infected and uninfected
ticks co-feeding on the same host. Nevertheless, whether or not the interactions among DCs, TBEV and the saliva of its tick vector could significantly influence viral accessibility for feeding ticks in vivo is a matter for further examination. Acknowledgements The project was supported by grant IAA600960811 from the Grant Agency of the Academy of Sciences of the Czech Republic, the project of the Basic Research Centre LC06009 from the Czech Ministry of Education, and the research projects of the Institute of Parasitology Z60220518 and of the ´ Faculty of Science, University of South Bohemia, Ceske Budejovice MSM 6007665801. References [1] WHO Regional Office for Europe (WHO/EURO), The vector-borne human infections of Europe: their distribution and burden on public health, Copenhagen, Denmark, 2004, pp. 37e53.
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