Immunology and Cell Biology (2010) 0, 19 & 2010 Australasian Society for Immunology Inc.

All rights reserved 0818-9641/10 $32.00

www.nature.com/icb

ORIGINAL ARTICLE

Recognition by toll-like receptor 2 induces antigenpresenting cell activation and Th1 programming during infection by Neospora caninum
Tiago WP Mineo1,2, Carlo JF Oliveira1,3, Fredy RS Gutierrez1 and Joao S Silva1
Neospora caninum is an apicomplexan parasite responsible for major economic losses due to abortions in cattle. Toll-like receptors (TLRs) sense specic microbial products and direct downstream signaling pathways in immune cells, linking innate, and adaptive immunity. Here, we analyze the role of TLR2 on innate and adaptive immune responses during N. caninum infection. Inammatory peritoneal macrophages and bone marrow-derived dendritic cells exposed to N. caninum-soluble antigens presented an upregulated expression of TLR2. Increased receptor expression was correlated to TLR2/MyD88-dependent antigen-presenting cell maturation and pro-inammatory cytokine production after stimulation by antigens. Impaired innate responses observed after infection of mice genetically decient for TLR2(/) was followed by downregulation of adaptive T helper 1 (Th1) immunity, represented by diminished parasite-specic CD4+ and CD8+ T-cell proliferation, IFN-c:interleukin (IL)-10 ratio, and IgG subclass synthesis. In parallel, TLR2/ mice presented higher parasite burden than wild-type (WT) mice at acute and chronic stages of infection. These results show that initial recognition of N. caninum by TLR2 participates in the generation of effector immune responses against N. caninum and imply that the receptor may be a target for future prophylactic strategies against neosporosis. Immunology and Cell Biology (2010) 0, 000000. doi:10.1038/icb.2010.52 Keywords: dendritic cells; MyD88; neosporosis; T helper 1 immune response; TLR2

1Department of Biochemistry and Immunology, School of Medicine of Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Brazil; 2Institute of Biomedical Sciences, Universidade Federal de Uberlandia, Uberlandia, Brazil and 3Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Correspondence: Dr TWP Mineo and Dr JS Silva, Department of Biochemistry and Immunology, Immunoparasitology Laboratory, FMRP/USP, Av. Bandeirantes, 3900 Ribeirao Preto, SP 14.049-900, Brazil. E-mail: [email protected] or [email protected] Received 30 September 2009; revised 17 March 2010; accepted 22 March 2010

Neospora caninum, an obligatory intracellular protozoan, has an economic impact in cattle-raising farms due to reproductive disorders and is the major identiable cause of bovine abortions.1 Dogs and coyotes have been implicated as its denitive hosts, due to fecal oocyst shedding.2,3 Questions have been raised as to whether this parasite is able to infect humans, as divergent serological evidence has been found in various populations worldwide.4,5 Environment-contaminating oocysts are responsible for parasite dissemination, and infection is maintained in herds through endogenous or exogenous transplacental infections, with infection rates of up to 50% within affected herds.6 Although no specic studies have estimated global economic impact generated by neosporosis in cattle, sparse regional data indicate that nancial losses from costs of miscarriages and veterinary medical assistance might reach several hundred million dollars per year.7 Control strategies for N. caninum require complex approaches, as this parasite may invade a large variety of host cells, presents evolved mechanisms of immune modulation, and induces latent infections through stage conversion.8 N. caninum infection requires a T helper 1 (Th1)-type immune response to cease parasite replication, mainly established by early IFN-g production dependent on lymphocyte

EC

TE

PR

priming by interleukin (IL)-12. IL-12, a key cytokine produced by antigen-presenting cells (APCs), is involved in the commitment of naive CD4+ lymphocytes to the Th1 subset. In the absence of IL-12 and IFN-g, mice presented higher mortality to N. caninum acute infection due to inactivation of peritoneal macrophages, showing the key role of these cytokines in protection of the host.9,10 Professional APCs such as dendritic cells (DCs) have a critical function in modulation of the primary immune response to intracellular pathogens. Mature APCs present antigens and activate lymphocytes, leading to antigen-specic acquired immunity along with immunologic memory, which will enable the host to overcome re-exposure to the same agent.11 These events are triggered by recognition of conserved pathogen-associated molecular patterns by innate immune receptors such as the toll-like receptors (TLRs), largely expressed in APCs. TLRs are able to recognize several classes of pathogens, including members of the phylum Apicomplexa, such as Eimeria sp., Plasmodium sp., and Toxoplasma gondii.1214 Our group has recently shown that DCs, and macrophages to a minor extent, are the main cell lineages to produce IL-12 after in vivo exposure to N. caninum and mice genetically decient in MyD88, a crucial adaptor

TLR2/MyD88-dependent immunity to N. caninum TWP Mineo et al 2

protein for the IL-1 receptor and most of the TLRs, lack overall IL-12 synthesis and early IFN-g production in response to infection by the parasite, which results in increased mortality.15 The role of DCs in the immune response to N. caninum has recently been debated by different research groups;1517 however, it remains unclear which TLRs are able to recognize N. caninum and how this initial recognition may modulate adaptive immune responses. Here, we show that TLR2 is critical for DC activation, proper T-cell response, and antibody production during N. caninum infection, which are directly associated with reduction of parasite loads during acute and chronic phases of infection. RESULTS N. caninum increases TLR2+ inammatory macrophage population TLR2 is the innate receptor with the broadest range of recognition, being able to identify diverse pathogen compounds.18 To unravel its function in immunity against N. caninum, we rst evaluated the expression of TLR2 in mouse inammatory peritoneal macrophages (MF) exposed to N. caninum antigens. Exposure to tachyzoite suspension supernatant (soluble antigensNLA) induced an increase in the percentage of cells expressing the surface receptor (Figure 1), shown to be compatible with 24 h incubation with lipopolysaccharide (LPS). It is noteworthy that untreated MF presented heterogeneous expression of surface TLR2, whereas cells exposed to antigenic stimuli presented homogeneous expression of the receptor. Exposure of bone marrow-derived DCs (BMDCs) to NLA and live tachyzoites also induced TLR2 upregulation (data not shown). N. caninum triggers TLR2-dependent APC activation TLRs are required for DC maturation during infectious processes.19 We, therefore, assessed APC maturation during N. caninum infection. BMDCs obtained from wild-type (WT) mice incubated for 24 h with live N. caninum tachyzoites demonstrated increased major histocompatibility complex (MHC)-II and CD86 expression. Expression of other costimulatory molecules, such as CD40 and CD80, was relatively unaffected by incubation with N. caninum (data not shown). Next, we examined whether TLR2 is required during

N. caninum-induced DC maturation. The upregulation of MHC-II and CD86 surface molecules in response to N. caninum in WT BMDCs (Figure 2a) was not observed in MyD88/ or TLR2/ cells, as the expression of the antigen presentation and costimulatory markers were unchanged after stimulation with live parasites. It is noteworthy that MyD88/ BMDCs presented high expression of both surface molecules, even in the absence of parasite stimulus. The role of TLR2 in cytokine production by APCs during infection with N. caninum was also assessed, and culture supernatants of BMDCs generated from WT, MyD88/, and TLR2/ mice exposed to live tachyzoites, NLA, and LPS were examined. BMDCs from WT mice cultivated in the presence of LPS or live parasites secreted high concentrations of IL-12p40. Although control BMDCs produced only basal levels of the cytokine, cells exposed to N. caninum tachyzoites secreted nearly 3000 pg ml1 of IL-12p40 after 24 h. In contrast, MyD88/ and TLR2/ BMDCs failed to produce IL-12p40 after incubation with live parasites and antigens (Figure 2b), and MyD88decient cells were also unable to produce this key cytokine after LPS stimulation. Moreover, BMDCs from TLR2/ and MyD88/ mice signicantly increased IL-10 secretion after exposure to NLA in vitro compared with those from WT mice. TNF-a production followed the same pattern as that observed for IL-12, being elicited in WT BMDCs, in contrast to TLR2/ and MyD88/ cells (data not shown). TLR2/MyD88 is required for induction of adaptive Th1 responses against N. caninum Costimulation, antigen presentation, and pro-inammatory cytokine production by APCs are key factors for appropriate T-cell priming, and TLR2, along with other TLRs, is known to be involved in these processes.20 To evaluate whether the results gathered from BMDCs and MF in vitro complied with infection in vivo, we investigated activation of spleen DCs from WT mice, by comparing with cells from TLR2/ and MyD88/ mice, infected with CFSE-labeled N. caninum tachyzoites. As observed 7 days post-infection (p.i.), upregulation of intracellular IL-12 was detected with higher intensity in WT DCs associated with parasite forms (CFSE+) and bystander cells were also able to produce the cytokine (Figure 3a). In contrast, surface MHC-II
RPMI

Isotype IgG

TE

PR
95.4% 5 g/ml 84.3% TLR2

O
81.1%

LPS 13.1% 64.5%

R
100 101 102 103 104 IgG NLA 1 g/ml 60.5% CD11b 10 g/ml TLR2 20 g/ml 51.2%

C
Immunology and Cell Biology

Figure 1 Expression of surface TLR2 is altered after exposure to N. caninum. MF stimulated with RPMI alone, LPS, and NLA for 24 h were checked for population positivity to surface TLR2. As reaction controls, MF were incubated with isotype-matched controls. Results are represented as percentage of TLR2+ cells in density plots, where warmer colors indicate higher cell densities.

CD11b+ FSC-H

CD11b+ FSC-H

EC

1.2%

TLR2/MyD88-dependent immunity to N. caninum TWP Mineo et al 3

RPMI 613 WT

Nc 1050

NLA 1110

RPMI 85

Nc 181

NLA 89

892 MyD88 -/-

637

874

166

166

167

647 TLR2
-/-

541

510

73

77

84

100 101 102 103 104

100 101 102 103 104

MHC-II 8000 IL-12p40 production (pg/ml) 4000 3000 2000 * 1000 0 RPMI * LPS Nc NLA * IL-10 production (pg/ml)

CD86 2000 1500 1000 500 0 RPMI

WT MyD88 -/TLR2 -/-

F
LPS

Nc

NLA

and CD86 (Figure 3b) presented higher expression strictly on splenic WT CD11c+CFSE+ cells. Upregulation of both IL-12p40 and cell surface markers were impaired in genetically decient mice, being completely abolished in cells from MyD88/ mice. TLR2/ spleen CD11c+CFSE+ cells presented a slight increase in IL-12p40 expression, whereas MHC-II and CD86 expression remained unaltered, differently from the observed for WT cells (Figure 3). To evaluate whether impaired APC maturation in genetically decient mice was correlated with defective adaptive immune responses, spleen cells from WT, MyD88/, and TLR2/-infected mice were cultured ex vivo under specic and mitogenic stimulation. As veried by the percentage of cells with CFSE decay, WT T cells increased proliferation rates after stimulation with NLA, indicating responsiveness to the antigenic stimuli (Figure 4a). It is noteworthy that WT T cells presented a background proliferation, which may be explained by the presence of N. caninum antigens and/or viable parasites already present in spleen cell preparations. Although background proliferation was diminished in both decient mice groups, TLR2/ mice presented spleen cell division after ex vivo stimulation with NLA. The impaired immune response generated by TLR2/ mice was also observed in the analysis of IgG isotype production during the course of infection. WT mice showed a sustained production of IgG2a (Figure 4b) and IgG1 (Figure 4c) subclasses, with higher levels of IgG2a-specic antibody kinetics. Differently, TLR2/ mice presented an overall decit in IgG production. Samples obtained from convalescent MyD88/ mice between 11 and 14 days p.i. were also analyzed, and presented IgG2a levels comparable to TLR2/ mice and IgG1 production similar to WT mice (data not shown).

EC

TE

PR

IFN-g and IL-10 production by spleen cells were also assessed in infected WT, TLR2/, and MyD88/ mice through ELISA and intracellular cytokine staining. T cells from all mouse groups were strongly stimulated to produce IFN-g after incubation with NLA and tachyzoite suspension precipitate (particulate antigensNPA) (Figure 5a). Conversely, culture supernatants from both decient hosts revealed elevated concentrations of IL-10 compared with WT mice (Figure 5b). Intracellular cytokine staining conrmed that both CD4+ and CD8+ T-cell subsets from WT mice synthesized higher levels of IFN-g after ex vivo stimulation with NLA compared with IL-10. On the other hand, CD3+CD4+ and CD3+CD8+ cells from TLR2/ and MyD88/ mice developed a pronounced change in cytokine prole, presenting unaltered IFN-g expression and increased IL-10 intracellular staining after antigenic recall, evidenced in the shift in IFN-g:IL-10 mean intensity of uorescence ratio toward the antiinammatory cytokine (Figure 5c).

Immune responses generated by TLR2 confer protection against parasite replication Induction of early Th1 responses is critical for control of the infection by N. caninum.15 To assess whether TLR2-dependent immune responses are effective in the control of parasite replication, parasite burden was evaluated in different compartments and at distinct phases of in vivo infection of WT, TLR2/, and MyD88/ mice. Compared with WT, Tlr2/ and MyD88/ mice showed increased parasitism in cells from peritoneal exudate (Figure 6a) and spleen CD11chigh DCs (Figure 6b) at 7 days p.i. MyD88/ mice were shown to be even more susceptible than the TLR2/ single receptor-decient mice. Notably, a vefold increase in chronic-phase parasite burden was observed in
Immunology and Cell Biology

Figure 2 Neospora caninum triggers TLR2-dependent dendritic cell activation. BMDCs obtained from WT, and TLR2/ mice exposed to culturederived N. caninum tachyzoites (Nc) and NLA were submitted to (a) ow cytometry of surface MHC-II and CD86 expression and (b) measurement of secreted IL-12p40 and IL-10. Numbers in the upper right of histograms indicate the mean intensity of uorescence for each condition. *Statistical differences (Po0.05) between BMDCs obtained from WT and genetically decient mice lineages for the same stimuli.

MyD88/,

TLR2/MyD88-dependent immunity to N. caninum TWP Mineo et al 4

Isotype control 1.3%

Nave WT 2.9%

b
5000 4000
MHC-II (MIF)

WT MyD88 -/TLR2 -/-

100 101 102 103 104 CFSE 56.1% WT

3000 2000 1000 0 1000

CFSE + 89.7%

*
2.2% MyD88 -/7.8%
CD86 (MIF)

800 600 400 200 0 Nave NcCFSE NcCFSE +

8.1% TLR2 -/-

22.3%

IL-12p40

DISCUSSION The description of TLRs by Janeway and colleagues21 in the late 1990s has revolutionized the concept of microbial recognition by the immune system. We now know that these innate immune receptors are responsible for triggering the assembly of a harsh inammatory environment against pathogens and that stimulation of TLR causes an immediate protective response, including production of diverse antimicrobial peptides and cytokines such as IL-12.22 We have shown here that the TLR2/MyD88 pathway is required by DCs to efciently prime adaptive immune responses against the protozoan N. caninum, which consequently leads to a reduced parasite burden during the acute and chronic phases of infection. Although MyD88 has been recently described as a crucial protein for resistance against the acute phase of infection,15 and CpG-ODNa TLR9 ligandhas been shown to be a promising adjuvant candidate,23 this is the rst work to describe the direct role of a specic TLR in the immune response against N. caninum. Of all TLRs described, TLR2 is the pathogen recognition receptor capable of detecting the widest pathogen-associated molecular pattern array and recognizes antigens from viruses, bacteria, fungi, and parasites.24 A possible explanation for this promiscuity is the ability of TLR2 to form receptor clusters in response to different microbial ligands. To date, it is known to combine with other TLRs, as preexisting heterodimers of TLR1/TLR2 and TLR2/TLR6, as well as ligand-induced association between TLR2/TLR6 with CD36.25
Immunology and Cell Biology

EC

TE

the brain samples of TLR2/ mice (Figure 6c). Moreover, although only MyD88/ mice succumbed to the infection by 14 days p.i. with a regular parasite inoculum, 100% of WT and Tlr2/ mice survived infection with 10-fold higher inoculum doses (Figure 6d).

PR

TLR clustering including TLR2 is required for the efcient priming of adaptive immune, although TLR2/ do not succumb to infection as the Myd88/ mice, they have impaired DC function. MyD88/ mice were included in the experiments as controls, as TLR2 signaling is completely dependent on the adaptor protein.26 TLR2 has been previously shown to trigger pro-inammatory signaling cascades after recognition of pathogen-associated molecular patterns from different protozoa.27 Exposure to N. caninum induced the increase of TLR2+ populations of BMDCs and MF, and this result could be interpreted to mean that the receptor behaves as an unspecic APC activation marker; however, it was clear that APCs required TLR2 for complete activation after exposure to N. caninum. LPS used as controls of the in vitro experiments yielded expected results toward cytokine production; however, some TLR2-dependent effects in cell surface marker expression were observed, perhaps due to the fact that the LPS used was not ultrapure, and other microbial ligands could be present in the commercial preparation. Development of an adequate Th1 immune response is considered a keystone to protection against intracellular parasites such as N. caninum. DCs can inuence the extent of the immune response generated, by acting through efcient antigen presentation, crosspriming, downregulation of inammatory responses, immunologic memory, and tolerance.28 Our data provide evidence that infection of DCs by N. caninum induces a TLR2-dependent pro-inammatory IL-12p40 cytokine production, which has a pivotal role in the initiation of effective Th1 response against the parasite; however, the production of anti-inammatory IL-10 during infection by the protozoan has not been properly assessed. IL-10 has known regulatory properties and is ubiquitously produced.29 It is clear that the absence of TLR2 and MyD88 leads to signicantly increased production of

Figure 3 Splenic DCs also require TLR2 for complete activation after in vivo infection with N. caninum. Gated splenic CD11c+ cells obtained from WT, MyD88/, and TLR2/ mice infected with CFSE-stained N. caninum tachyzoites (7 days p.i., NcCFSE) were divided into NcCFSE and NcCFSE+ and analyzed for expression of (a) intracellular IL-12p40 and (b) surface MHC-II and CD86 expression. Additional naive group of mice were added in MHC-II and CD86 analysis. *Statistical signicance (Po0.05) between the different mouse lineages for each of the parameters analyzed.

TLR2/MyD88-dependent immunity to N. caninum TWP Mineo et al 5

CD3+CD4+ RPMI NLA RPMI

CD3+CD8+ NLA

WT

14.8%

23.4%

13.9%

28.4%

MyD88 -/7.9% 5.2% 5.0% 4.0%

TLR2 -/CD4+

4.7%
100 101 102 103 104

14.0%

CD8+

3.6%
100 101 102 103 104

13.2%

CFSE 8 Anti- N. caninum IgG2a (EI) 6 4 * 2 0 0 15 30 Days post-infection * Anti- N. caninum IgG1 (EI) 8 6 4 2 0 WT TLR2-/-

O
0

F
* 15 30 Days post-infection
Immunology and Cell Biology

EC

Figure 4 Spleen cell proliferation and IgG subclass production is partially impaired in the absence of TLR2. (a) Total spleen cells obtained from WT, MyD88/, and TLR2/ mice infected with N. caninum after 7 days p.i. were pre-stained with CFSE and cultured in the presence of RPMI alone and NLA for 72 h. From the total pool of cells, CD3+CD4+ and CD3+CD8+ cells within the lymphocyte gate were analyzed for CFSE decay. In addition, serum samples obtained from WT and TLR2/ mice inoculated with 1106 N. caninum tachyzoites after 0, 15, and 30 days of infection were assayed for the presence of specic (b) IgG2a and (c) IgG1 against NLA. Results were expressed as ELISA Index (EI). *Statistical signicance (Po0.05) between the different mouse lineages for each assayed date of infection.

TE

IL-10 by DCs and lymphocytes directly stimulated by live parasites and its antigens. In addition, upregulation of antigen presentation and costimulatory molecules was determined in vivo and in vitro in the presence of live parasites. These events are essential for appropriate T-cell priming and are driven by NF-kB activation after pathogen recognition by innate immune cells through TLR-dependent mechanisms.19 The quality of expression of these molecules is a determinant in DC maturation, which will lead to efcient DClymphocyte interactions. Recent work has shown that failure in the maturation process of APCs may lead to defective adaptive responses and induction of regulatory T-cell clones.30 DCs and their surface markers should be taken into account for future studies on protein immunogenicity and potential adjuvants for neosporosis. IFN-g is an essential cytokine for driving such responses, whereas IL-10 exerts an opposite role in controlling the development of Th1biased immunity. IFN-g is required for host resistance to N. caninum,

PR
inducing effector mechanisms as apoptosis of infected cells and nitric oxide production,31 and has been previously shown to rescue MyD88/ from fatal N. caninum infection.15 Lymphocyte proliferation and cytokine prole after infection revealed that TLR2 partially dictates the development of adaptive immune responses against N. caninum, whereas the absence of MyD88 abolished T-cell priming to Th1-type responses. The mechanisms underlying IL-10 production during N. caninum infection and the involvement of TLR in inhibiting its expression are still unknown. The regulation of IFN-g-mediated responses exerted by IL-10 in this process requires further investigation and represents a potential therapeutic target for neosporosis. It is noteworthy that TLR2/ mice were not able to produce adequate amounts of anti-N. caninum IgG antibodies, as has already been described for bacterial agents32 but not for protozoa. Further elucidation of the role of TLR and other innate recognition receptors in antibody induction for parasitic infections is needed for vaccine and diagnosis strategy design.

TLR2/MyD88-dependent immunity to N. caninum TWP Mineo et al 6

10000 IFN- production (pg/ml) * 8000 6000 4000 2000 0 WT 1000 RPMI IL-10 production (pg/ml) 800 600 400 200 0 WT 2.00 WT 1.75 TLR2-/MyD88-/MyD88 -/TLR2 -/NLA NPA * * * MyD88 -/TLR2 -/* * * * *

IFN-:IL-10 ratio (MIF)

1.50

1.00

EC

0.75

TE O R R
CD3+CD4+ CD3+CD8+

1.25

0.50

Figure 5 Th1 cytokine prole is dependent on intact TLR2 signaling after N. caninum infection in vivo. Total spleens cells were obtained from WT, MyD88/, and TLR2/ mice infected with 1106 N. caninum tachyzoites 7 days p.i. (a) IFN-g and (b) IL-10 were determined in supernatants from cells cultured in the presence of RPMI alone, NLA (10 mg ml1), and NPA (10 mg ml1), and culture after 72 h of ex vivo stimulation. *Statistical signicance (Po0.05) between cells stimulated only with RPMI in relation to specic antigenic stimuli. (c) Intracellular staining was performed in spleen cells cultured ex vivo for 48 h in the presence of RPMI alone and NLA. Results were analyzed as IFN-g:IL-10 mean intensity of uorescence ratio.

Similarly to cytokine production and T-cell priming, parasitism in TLR2/ mice was found to be intermediary when compared with WT and MyD88/ mice, which indicates that TLR2 contributes partially to this process. MyD88 is an adaptor protein for most TLRs and also participates in IL-1 and/or IL-18 receptor signaling,19
Immunology and Cell Biology

PR

METHODS Parasite

N. caninum tachyzoites (NC-1 isolate) were maintained by serial passages in Vero cells, cultured in RPMI 1640 medium supplemented with 2 mM glutamine, 100 U penicillin ml1 and 100 mg streptomycin ml1 (Invitrogen, Carlsbad, CA, USA) in a 5% CO2 atmosphere, at 37 1C. Tachyzoites were harvested by scraping off the cell monolayer 23 days p.i., passed through a 26-gauge needle for host cell lysis, and centrifuged at low speed (45 g) for 1 min at 4 1C to remove host cell debris. The supernatant containing parasite suspension was collected and then washed twice (1000 g, 10 min, 4 1C) in phosphatebuffered saline (pH 7.2), and the resulting pellet was resuspended in phosphate-buffered saline for experimental infections and antigen preparation.

Antigen preparation
N. caninum antigens were prepared with parasites obtained from infected monolayers as described above. Antigens were prepared by repeated freeze-thaw cycles (10 cycles). The tachyzoite suspension was then centrifuged (10 000 g, 30 min, 4 1C), and NPA and NLA were collected and both antigenic preparations were ltered through a 0.22-mm membrane (Millipore, Billerica, MA, USA) and its protein concentration determined by bicinchoninic acid assay (Sigma-Aldrich, St Louis, MO, USA). Aliquots were stored at 80 1C until use. Antigen integrity was followed by routine electrophoretic analysis of protein content.

Animals
Six- to 8-week-old C57BL/6 background mice (WT) and mice genetically decient in TLR2 (TLR2/) and MyD88 (MyD88/) were bred and maintained at the animal facilities of the Department of Biochemistry and Immunology, School of Medicine of Ribeirao Preto, USP (Ribeirao Preto, Brazil) with food and water ad libitum. WT littermates were acquired at a commercial supplier (The Jackson Laboratory, Bar Harbor, ME, USA). Genetically decient mice were screened for their respective gene depletion and mated along with decient siblings to preserve their lineage characteristics. For in vivo experiments, mice (n5) were infected with 1106 tachyzoites by the

so it is expected that MyD88/ mice would present a stronger phenotype towards N. caninum infection. Unlike MyD88/ mice, which uniformly succumbed to N. caninum infection, WT and TLR2/ mice were completely resistant to fatal disease. These results are consistent with those obtained related to the role of TLR11 in the T. gondii model of infection.33 Although the absence of TLR2 does not affect IL-12 production to T. gondii stimulation,14 TLR11/ mice are unable to produce IL-12 and prime-specic CD4+ lymphocytes,34 adding further evidence to the discussion concerning the marked differences between both closely related parasites;35 however, Prolinthe TLR11 ligand present in T. gondiiis also found in N. caninum with high-sequence homology.33 In that sense, it is possible to speculate that N. caninum prolin may trigger similar events through TLR11, which would help to explain the partial role of TLR2 in the induction of proper immune responses against N. caninum infection. We are seeking the TLR2-specic N. caninum agonist; once isolated and sequenced, it may be used in support therapy for neosporosis and other pathologic processes where TLR2 activation is desirable. Together, the presented data show that Th1 responses elicited for parasite control are induced during primary interactions between the parasite and the host innate immune cell receptors. We have shown that the TLR2/MyD88 innate recognition pathway triggers APC overexpression of costimulatory and antigen-presentation molecules, T lymphocyte programming, and pro-inammatory cytokine production and is also required for proper IgG synthesis against N. caninum, which makes this innate receptor a relevant target for vaccine and therapy design against neosporosis.

TLR2/MyD88-dependent immunity to N. caninum TWP Mineo et al 7

3%

b
Splenic CD11c+CFSE+ cells (%)

c
80 * * N. caninum DNA (pg/20mg tissue) 60 40 20 0 WT MyD88 -/- TLR2 -/15
15 dpi 30 dpi

WT

10

100 101 102 103

104

12%

0 WT TLR2 -/-

MyD88

-/-

d
Survival (%)

100 80 60 40 20 0 0 5

WT 106 107 TLR2 -/- 106, 107

8%

TLR2 -/-

MyD88 -/- 106 10 15 20 25 Days post-infection 30

FSC-H

CFSE

Peritoneal MU isolation

MF were collected from WT mice 72 h after i.p. injection of 1.5 ml of Brewer thioglycolate solution at 2% (Sigma-Aldrich). Cells were harvested by peritoneal washing with 5 ml of cold sterile phosphate-buffered saline, centrifuged at 450 g for 10 min at 4 1C, suspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, and added to 24-well tissue culture microplates at 1106 cells per well. To eliminate other cell types, wells were washed thoroughly to remove nonadherent cells after a 3-h incubation period at 37 1C. MF purity was assessed by ow cytometry, and the cell isolation protocol was considered satisfactory with over 95% of CD11bhigh expression. MF were exposed to crescent concentrations of NLA (1, 5, 10, and 20 mg ml1) and LPS (1 mg ml1; Sigma-Aldrich) to observe the expression of TLR2, MHC-II, and costimulatory molecules by ow cytometry.

EC

TE

intraperitoneal (i.p.) route. Survival assays were performed with the same inoculum in the different mice groups tested (n7), with the exception of additional WT and TLR2/ groups infected with 1107 tachyzoites. Maintenance and care of these animals complied with guidelines of the Laboratory Animal Ethics Committee from the FMRP/USP. Animal euthanasia was performed in accordance with international welfare standards.36

Generation of BMDCs
BMDCs were generated from WT, TLR2/, and MyD88/ mice as described earlier.37 On day 8, nonadherent cells were removed and analyzed by ow cytometry, and cell differentiation was followed by major CD11chigh expression (over 80%). BMDCs were plated at 7105 cells in 400 ml of RPMI supplemented with 10% FBS (Gibco-BRL, Gaithersburg, MD, USA) and exposed to NLA (10 mg ml1), N. caninum tachyzoites (MOI 1), or LPS (1 mg ml1; SigmaAldrich) to observe the expression of TLR2 as well as costimulatory and antigen-presentation markers. Supernatants were collected after 18 h for cytokine measurements.

PR

Spleen cell activation

For measurement of ex vivo cytokine production and expression, single-cell suspensions were prepared from spleens of WT, TLR2/, and MyD88/ mice 7 days p.i. Spleen cells were cultured at 2105 cells per well in 200 ml medium and incubated 72 h for IFN-g and IL-10 measurements after stimulation with medium, NLA (10 mg ml1), or NPA (10 mg ml1). To access intracellular cytokine production and surface antigen expression, spleen cells were obtained from infected mice and cultured in the presence or absence of NLA (10 mg ml1); 4 h before cytokine staining, GolgiStop (BD, Franklin Lakes, NJ, USA) was added to halt protein secretion. Ex vivo T-lymphocyte proliferation was determined by cell division using CFSE staining. Spleen cells collected from infected WT, MyD88/, and TLR2/ mice (7 days p.i.) were labeled with CFSE (2.5 mM; Invitrogen) for 8 min at 37 1C and cultured in the presence of Concanavalin A (2.5 mg ml1), NLA (10 mg ml1), or medium alone for 72 h. Cells were collected, phenotyped, and analyzed by ow cytometry. The intensity of CFSE uorescence was detected in the FL-1 channel, within the lymphocyte gate. Frequencies of a given cell subset within each division cycle was determined by drawing regions that represented increments of twofold reduction in CFSE intensity relative to that in the undivided cells.

Flow cytometric analysis

For immunostaining, biotinylated, APC-, FITC-, PE-, PE-Cy7-, and PerCPconjugated antibodies against murine CD3, CD4, CD8, CD11c, CD40, CD80, CD86, IFN-g, IL-10, IL-12p40, MHC-II, TLR2, TNF-a, and respective mouse and rat isotype controls were used. CD3 (clone 145-2C11), CD4 (L3T4), and TLR2 (6C2) were purchased from eBiosciences (San Diego, CA, USA). CD8 (Ly-2) and CD11c (HL3) were acquired from BD. CD40 (1C10), CD80 (1G10), CD86 (GL1), and MHC-II (NIMR4) were purchased from Southern Biotechnologies (Birmingham, AL, USA). Biotinylated antibodies used for intracellular cytokine staining were anti-IL-10, anti-TNF-a (R&D Systems, Minneapolis, MN, USA), anti-IL-12p40, and anti-IFN-g (BD). Flow cytometry protocols were performed as described earlier.15 Intracellular cytokine production was performed with a commercially available kit (BD) used according to
Immunology and Cell Biology

Figure 6 TLR2 is required for resistance against parasite replication to acute and chronic phases of infection. Cells from total peritoneal exudate (a) and spleen cells (b) obtained from WT, MyD88/, and TLR2/ mice infected with CFSE-stained N. caninum tachyzoites (NcCFSE) were checked for parasite positivity 7 days p.i. (c) Central nervous system parasite burden quantied by qPCR in WT and TLR2/ mice 15 and 30 days p.i. (d) Survival curves for WT and TLR2/ mice infected with 1106 and 1107 N. caninum tachyzoites and MyD88/ mice infected with 1106 parasites. *Statistical difference (Po0.05) between WT mice and the decient mouse lineages tested.

TLR2/MyD88-dependent immunity to N. caninum TWP Mineo et al 8 the manufacturers instructions. Results were represented as percentage of positive cells or by mean intensity of uorescence, which quanties the staining density for the target molecule within individual-gated cells. Data analysis was carried out with FlowJo 8.7 software (Tree Star, Ashland, OR, USA).

Cytokine ELISA
Concentrations of murine IL-12p40, IL-10, IFN-g, and TNF-a in culture supernatants were measured by a sandwich ELISA using paired cytokinespecic monoclonal antibodies according to the manufacturers instructions (BD). Known concentrations of recombinant cytokines were added to each reaction, and estimated results were obtained by four parameters logistic (4PL) extrapolation. Detection limits for each assay were: IL-12p40 and TNFa15.6 pg ml1; IFN-g and IL-1031.3 pg ml1. None of the tested samples was thawed more than once.

IgG isotype ELISA

N. caninum-specic IgG1 and IgG2a antibodies were measured by ELISA as described elsewhere.23 Briey, high-afnity microtiter plates were coated with NLA (0.5 mg per well) and blocked for unspecic binding with 1% BSA. Serum samples were diluted 1:25 in blocking buffer and incubated for 2 h at 37 1C. After washing, biotinylated anti-mouse IgG1 (1:4000) or anti-mouse IgG2a (1:2000) antibodies (Caltag Laboratories, San Francisco, CA, USA) were added and incubated for 1 h at 37 1C, followed by an incubation step with streptavidin peroxidase (1:1000; Sigma-Aldrich). The assays were developed with 0.01 M2,2/-azino-bis-3-ethyl-benzthiazoline sulfonic acid (Sigma-Aldrich) and 0.03% H2O2, and absorbance was determined in a plate reader at 405 nm. Antibody titers were arbitrarily expressed as ELISA Indexes (EI) according to Silva et al.38 following the formula: EI ODsample =ODcutoff ; where values of EIX1 were considered positive. The cutoff for a positive test was determined as the mean OD for the negative control plus 5 s.d.

Parasite detection

Statistical analysis

The authors declare no conict of interest.

ACKNOWLEDGEMENTS
We thank Cristiane M Milanezi, Dr Deise AO Silva, and Walter M Turato for technical assistance and Dr Neide M Silva, Djalma S Lima Jr, and Luciana Benevides for helpful suggestions. We also thank NIAID intramural editor Brenda Rae Marshall for assistance. TLR2/ and MyD88/ mice were originally provided by Dr Shizuo Akira (Osaka University, Osaka, Japan). This work was supported by the Brazilian funding agencies CNPq (473178/2007-9) and FAPESP (2006/06803-4).
Immunology and Cell Biology

CONFLICT OF INTEREST

Statistical signicance of the obtained results was calculated using two-way analysis of variance followed by Bonferroni post-tests. Survival curves were compared using KaplanMeier survival analysis through log-rank MantelCox test. Values for Po0.05 were considered signicant. In vitro experiments were performed with 35 replicates at each experiment, from cell preparations obtained from at least three mice. In addition, independent in vitro assays were performed at least three times to conrm the obtained results. Data were analyzed with GraphPad Prism software (GraphPad, La Jolla, CA, USA).

EC

TE

In vivo parasite propagation was accessed by detection of CFSE-labeled tachyzoites in peritoneal exsudate and spleen samples at 7 days p.i. Parasites were labeled as described earlier39; ow cytometry analysis was performed considering that CFSE+ cells were infected or had tachyzoites attached to their surface. Brain parasite burden was determined at 15 and 30 days p.i. in WT and TLR2/ mice by quantitative real-time PCR18 using primer pairs (sense 3GCTGAACACCGTATGTCGTAAA-5; antisense 3-AGAGGAATGC CACATAGAAGC-5) designed to detect the N. caninum Nc-5 sequence through the SYBR green detection system (Invitrogen).

1 Reichel MP, Ellis JT. If control of Neospora caninum infection is technically feasible does it make economic sense? Vet Parasitol 2006; 142: 2334. 2 McAllister MM, Dubey JP, Lindsay DS, Jolley WR, Wills RA, McGuire AM. Dogs are denitive hosts of Neospora caninum. Int J Parasitol 1998; 28: 14731478. 3 Gondim LF, McAllister MM, Pitt WC, Zemlicka DE. Coyotes (Canis latrans) are denitive hosts of Neospora caninum. Int J Parasitol 2004; 34: 159161. 4 Lobato J, Silva DA, Mineo TW, Amaral JD, Segundo GR, Costa-Cruz JM et al. Detection of immunoglobulin G antibodies to Neospora caninum in humans: high seropositivity rates in patients who are infected by human immunodeciency virus or have neurological disorders. Clin Vaccine Immunol 2006; 13: 8489. 5 McCann CM, Vyse AJ, Salmon RL, Thomas D, Williams DJ, McGarry JW et al. Lack of serologic evidence of Neospora caninum in humans, England. Emerg Infect Dis 2008; 14: 978980. rkman C, Trees AJ. Endogenous and exogenous transpla6 Williams DJ, Hartley CS, Bjo cental transmission of Neospora caninumhow the route of transmission impacts on epidemiology and control of disease. Parasitology 2009; 20: 16. 7 Trees AJ, Davison HC, Innes EA, Wastling JM. Towards evaluating the economic impact of bovine neosporosis. Int J Parasitol 1999; 29: 11951200. 8 Hemphill A, Vonlaufen N, Naguleswaran A. Cellular and immunological basis of the host-parasite relationship during infection with Neospora caninum. Parasitology 2006; 133 (Part 3): 261278. 9 Khan IA, Schwartzman JD, Fonseka S, Kasper LH. Neospora caninum: role for immune cytokines in host immunity. Exp Parasitol 1997; 85: 2434. 10 Nishikawa Y, Tragoolpua K, Inoue N, Makala L, Nagasawa H, Otsuka H et al. In the absence of endogenous gamma interferon, mice acutely infected with Neospora caninum succumb to a lethal immune response characterized by inactivation of peritoneal macrophages. Clin Diagn Lab Immunol 2001; 8: 811816. 11 OGarra A, Murphy KM. From IL-10 to IL-12: how pathogens and their products stimulate APCs to induce T(H)1 development. Nat Immunol 2009; 10: 929932. 12 Kim CH, Lillehoj HS, Bliss TW, Keeler Jr CL, Hong YH, Park DW et al. Construction and application of an avian intestinal intraepithelial lymphocyte cDNA microarray (AVIELA) for gene expression proling during Eimeria maxima infection. Vet Immunol Immunopathol 2008; 124: 341354. 13 Adachi K, Tsutsui H, Kashiwamura S, Seki E, Nakano H, Takeuchi O et al. Plasmodium berghei infection in mice induces liver injury by an IL-12- and Toll-like receptor/ myeloid differentiation factor 88-dependent mechanism. J Immunol 2001; 167: 59285934. 14 Scanga CA, Aliberti J, Jankovic D, Tilloy F, Bennouna S, Denkers EY et al. MyD88 is required for resistance to Toxoplasma gondii infection and regulates parasite-induced IL-12 production by dendritic cells. J Immunol 2002; 168: 59976001. 15 Mineo TW, Benevides L, Silva NM, Silva JS. Myeloid differentiation factor 88 is required for resistance to Neospora caninum infection. Vet Res 2009; 40: 32. 16 Strohbusch M, Muller N, Hemphill A, Margos M, Grandgirard D, Leib S et al. Neospora caninum and bone marrow-derived dendritic cells: parasite survival, proliferation, and induction of cytokine expression. Parasite Immunol 2009; 31: 366372. 17 Teixeira L, Botelho AS, Mesquita SD, Correia A, Cerca F, Costa R et al. Plasmacytoid and conventional dendritic cells are early producers of IL-12 in Neospora caninuminfected mice. Immunol Cell Biol 2009; 88: 7986. 18 Jin MS, Lee JO. Structures of the Toll-like receptor family and its ligand complexes. Immunity 2008; 29: 182191. 19 Kawai T, Akira S. Innate immune recognition of viral infection. Nat Immunol 2006; 7: 131137. 20 Wetzler LM. The role of Toll-like receptor 2 in microbial disease and immunity. Vaccine 2003; 21(Suppl 2): S55S60. 21 Medzhitov R, Preston-Hurlburt P, Janeway Jr CA. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997; 388: 394397. 22 Coban C, Ishii KJ, Horii T, Akira S. Manipulation of host innate immune responses by the malaria parasite. Trends Microbiol 2007; 15: 271278. 23 Ribeiro DP, Freitas MM, Cardoso MR, Pajuaba AC, Silva NM, Mineo TW et al. CpG-ODN combined with Neospora caninum lysate, but not with excreted-secreted antigen, enhances protection against infection in mice. Vaccine 2009; 27: 25702579. 24 Texereau J, Chiche JD, Taylor W, Choukroun G, Comba B, Mira JP. The importance of Toll-like receptor 2 polymorphisms in severe infections. Clin Infect Dis 2005; 41(Suppl 7): S408S415. 25 Triantalou M, Gamper FG, Haston RM, Mouratis MA, Morath S, Hartung T et al. Membrane sorting of Toll-like receptor (TLR)-2/6 and TLR2/1 heterodimers at the cell surface determines heterotypic associations with CD36 and intracellular targeting. J Biol Chem 2006; 281: 3100231011. 26 Monteiro AC, Schmitz V, Svensjo E, Gazzinelli RT, Almeida IC, Todorov A et al. Cooperative activation of TLR2 and bradykinin B2 receptor is required for induction of type 1 immunity in a mouse model of subcutaneous infection by Trypanosoma cruzi. J Immunol 2006; 177: 63256335. 27 Takeuchi O, Kaufmann A, Grote K, Kawai T, Hoshino K, Morr M et al. Cutting edge: preferentially the R-stereoisomer of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 activates immune cells through a toll-like receptor 2- and MyD88dependent signaling pathway. J Immunol 2000; 164: 554557. 28 Scott P, Hunter CA. Dendritic cells and immunity to leishmaniasis and toxoplasmosis. Curr Opin Immunol 2002; 14: 466470. 29 Couper KN, Blount DG, Riley EM. IL-10: the master regulator of immunity to infection. J Immunol 2008; 180: 57715777.

PR

TLR2/MyD88-dependent immunity to N. caninum TWP Mineo et al 9

30 Eljaafari A, Li YP, Miossec P. IFN-g, as secreted during an alloresponse, induces differentiation of monocytes into tolerogenic dendritic cells, resulting in FoxP3+ regulatory T cell promotion. J Immunol 2009; 183: 29322945. 31 Tanaka T, Nagasawa H, Fujisaki K, Suzuki N, Mikami T. Growth-inhibitory effects of interferon-g on Neospora caninum in murine macrophages by a nitric oxide mechanism. Parasitol Res 2000; 86: 768771. n 32 Cervantes-Barraga L, Gil-Cruz C, Pastelin-Palacios R, Lang KS, Isibasi A, Ludewig B et al. TLR2 and TLR4 signaling shapes specic antibody responses to Salmonella typhi antigens. Eur J Immunol 2009; 39: 126135. 33 Yarovinsky F, Zhang D, Andersen JF, Bannenberg GL, Serhan CN, Hayden MS et al. TLR11 activation of dendritic cells by a protozoan proling-like protein. Science 2005; 308: 16261629. 34 Yarovinsky F, Kanzler H, Hieny S, Coffman RL, Sher A. Toll-like receptor recognition regulates immunodominance in an antimicrobial CD4+ T cell response. Immunity 2006; 25: 655664. 35 Innes EA, Mattsson JG. Neospora caninum emerges from the shadow of Toxoplasma gondii. Trends Parasitol 2007; 23: 4344. 36 AVMA Panel on Euthanasia. American Veterinary Medical Association. 2000 Report of the AVMA Panel on Euthanasia. J Am Vet Med Assoc 2001; 218: 669696. 37 Mineo TW, Oliveira CJ, Silva DA, Oliveira LL, Abatepaulo AR, Ribeiro DP et al. Neospora caninum excreted/secreted antigens trigger CC-chemokine receptor 5-dependent cell migration. Int J Parasitol 2010. (in press). 38 Silva DA, Silva NM, Mineo TW, Pajuaba Neto AA, Ferro EA, Mineo JR. Heterologous antibodies to evaluate the kinetics of the humoral immune response in dogs experimentally infected with Toxoplasma gondii RH strain. Vet Parasitol 2002; 107: 181195. 39 Chang HK, Thalhofer C, Duerkop BA, Mehling JS, Verma S, Gollob KJ et al. Oxidant generation by single infected monocytes after short-term uorescence labeling of a protozoan parasite. Infect Immun 2007; 75: 10171024.

Q1

EC

TE

PR

O
Immunology and Cell Biology