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DECONSTRUCTING TICK SALIVA: NON-PROTEIN MOLECULES

WITH POTENT IMMUNOMODULATORY PROPERTIES Carlo José F. Oliveira

1, Anderson Sá-Nunes

2, Ivo M. B. Francischetti

3,

Vanessa Carregaro 1, Elen Anatriello

1, João S. Silva

1, Isabel K. F. de

Miranda Santos 1, José M. C. Ribeiro

3, and Beatriz R. Ferreira

1,4,*

From the Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto,

University of São Paulo, São Paulo, SP, Brazil,1 Department of Immunology, Institute of

Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil,2 Laboratory of Malaria and

Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of

Health, Bethesda, MD, USA,3 and Department of Maternal-Child Nursing and Public Health,

School of Nursing of Ribeirão Preto, Ribeirão Preto, SP, Brazil 4

Running head: Ado and PGE2 from tick saliva are primary inhibitors of DCs

Address correspondence to: Beatriz R. Ferreira, School of Nursing of Ribeirão Preto, SP,

University of São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14040-902, Brazil.

Tel.: +55 016 3602 0537; Fax: +55 016 3602 0518; E-mail: brferrei@usp.br

Dendritic cells (DCs) are powerful

initiators of innate and adaptive immune

responses. Ticks are blood-sucking

ectoparasite arthropods that suppress host

immunity by secreting immunomodulatory

molecules in their saliva. Here, compounds

present in Rhipicephalus sanguineus tick

saliva with immunomodulatory effects on

DC differentiation, cytokine production, and

costimulatory molecule expression

were

identified. R. sanguineus tick saliva inhibited

IL-12p40 and TNF-α, while potentiating IL-

10 cytokine production by bone marrow

(BM)-derived DCs stimulated by Toll-like

receptor (TLR)-2, -4, and -9 agonists. In

order to identify the molecules responsible

for these effects, we fractionated the saliva

through microcon filtration and reversed-

phase high performance liquid

chromatography (RP-HPLC) and tested

each fraction for DC maturation. Fractions

with proven effects were analyzed by micro-

HPLC tandem mass spectrometry or

competition ELISA assay. Thus, we

identified for the first time in tick saliva, the

purine nucleoside adenosine (Ado;

concentration ~ 110 pmoles/µL) as a potent

antiinflammatory salivary inhibitor of DC

cytokine production. We also found

prostaglandin-E2 (PGE2 ~ 100 nM) with

comparable effects in modulating cytokine

production by DCs. Both Ado and PGE2

inhibited cytokine production by inducing

cAMP-PKA signaling in DCs. Additionally,

both Ado and PGE2 were able to inhibit

expression of CD40 in mature DCs. Finally,

flow cytometry analysis revealed that PGE2,

but not Ado, is the differentiation inhibitor

of BM-derived DCs. The presence of non-

protein molecules adenosine and PGE2 in

tick saliva indicates an important

evolutionary mechanism used by ticks to

subvert host immune cells and allow them to

successfully complete their blood meal and

life cycle.

Ticks are phylogenetically distant from

their hosts but, in general, these ectoparasites

have developed, through their evolution,

measures for adapting to host defense

strategies. The most well studied approaches

that ticks employ to evade host responses are

the refined cocktails of proteic and non-protein

molecules present in their saliva with

anticlotting, antiinflammatory, or

immunomodulatory activities. As a result, a

number of authors have demonstrated the

suppression of cell- and humoral-mediated

immune responses upon in vitro assays as well

as following experimental models of tick

infestations or naturally infested hosts (1–8).

In the last four decades, many of these

proteic and non-protein molecules have been

characterized and their specific functions

identified. To date, a diversity of proteic (e.g.,

chitinases, mucins, ixostatins, cystatins,

defensins, hyaluronidases, Kunitz, lectins, and

lipocalins) and non-protein molecules (e.g.,

http://www.jbc.org/cgi/doi/10.1074/jbc.M110.205047The latest version is at JBC Papers in Press. Published on January 26, 2011 as Manuscript M110.205047

Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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prostaglandins and endocannabinoids) have

been characterized (9–14). Regarding anti-tick

immunity, molecules present in tick saliva have

been associated with modulation of various

steps of host immune responses. For example,

Sialostatin L and PGE2, inhibit maturation of

dendritic cells (DCs) and prevent antigen

presentation (13,15); DAP-36 and SALP15,

inhibit T cell proliferation and activation

(16,17); ISL 929 and ISL 1373, reduce

recruitment of neutrophils (18); IgG-binding

proteins, theoretically decrease antibody

functions (3,19); ISAC, SALP-20, and OmCI,

inhibit alternative and/or classical pathways of

the complement system (20,21); and EVASIN-

1, -3 and -4, bind chemokines and hamper cell

migration (22,23).

Despite the characterization of these

molecules, most compounds in tick saliva

thought to interfere in numerous other

immunologic events described in the literature

remain to be identified. In previous works, we

have demonstrated the modulatory effects of

saliva from Rhipicephalus sanguineus ticks on

differentiation, migration, and maturation of

DCs (24–26). Despite our wealth of

knowledge, the molecules responsible for such

effects have not yet been elucidated. In this

study, we have identified for the first time the

purine nucleoside adenosine (Ado), isolated

directly from tick saliva, as an inhibitor of

production of pro-inflammatory IL-12p40 and

TNF-α cytokines and stimulator of production

of anti-inflammatory IL-10 by murine DCs

activated with Toll-like receptor (TLR)

agonists. We have also identified

prostaglandin-E2 (PGE2) in R. sanguineus tick

saliva, which presented similar effects of Ado

on cytokine production by DCs and

additionally suppressed the differentiation of

DCs from blood cell precursors. Our results

also demonstrate that both, Ado and PGE2,

exert their modulatory effects on cytokine

production by inducing a common cAMP-PKA

signaling pathway. Furthermore, both Ado and

PGE2 were able to inhibit expression of CD40

in mature DCs, and presented additive effects

when administered together. Thus, the present

report demonstrates the central involvement of

tick salivary Ado and PGE2 in modulation of

the host inflammatory/immune responses.

Moreover, the data presented herein provide

important insight to the evolution of host-tick

interaction and provide a foundation for future

pharmaceutical interventions that target non-

protein molecules used by ticks to permit their

blood feeding.

Experimental Procedures

Experimental animals- Female C57BL/6 mice

(6 to 10 weeks old) were purchased from

Taconic Farms (Germantown, NY). Mice were

bred and maintained at an American

Association of Laboratory Animal Care-

accredited facility at the National Institute of

Allergy and Infectious Diseases (NIAID),

National Institutes of Health. All experiments

with mice were evaluated and approved by the

Experimental Animal Ethics Committee of the

National Institutes of Health. The experiments

with dogs were evaluated and approved by the

Ethics Committee on Animal Use of the School

of Medicine of Ribeirão Preto (USP)/Brazil and

are in line with the Guidelines for Animal

Users as issued by the National Institute of

Health.

Saliva collection– R. sanguineus ticks were

laboratory reared, as previously described by

Ferreira and Silva (27). To obtain engorged

ticks for saliva collection, dogs (N = 10) were

infested with 70 pairs of adult R. sanguineus

ticks restricted by plastic feeding chambers

fixed to their backs. The saliva collection

procedure was performed in partially engorged

female ticks (after 5–7 days of feeding) by

inoculation of 10 μl of a 0.2% (v/v) solution of

dopamine in phosphate-buffered saline, pH 7.4,

into the haemocoele using a 12.7 × 0.33-mm

needle (Becton-Dickinson, Franklyn Lakes,

NJ). Saliva was harvested using a micropipette,

kept on ice, pooled, filtered through a 0.22 μm

pore filter (Costar-Corning Inc., Cambridge,

MA), and stored at –70oC for further use.

Saliva protein concentration (~ 970 µg/mL)

was determined by molecular sieving using

absorbance at 280 nm.

Separation of tick saliva fractions by microcon

filtration– To determine the approximate mass

of the active component(s) of R. sanguineus

tick saliva, samples were initially fractionated

by microcon centrifugal filters (Millipore,

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Bedford, MA) following manufacturer’s

instructions to provide fractions with apparent

molecular weights of < 5 and 5–100 kDa.

Reagents and chemicals– Ultrapure

Escherichia coli 0111:B4 LPS, Staphylococcus

aureus peptidoglycan (PGN), and

oligonucleotide CpG-1826 (CpG ODN-1826)

were purchased from InvivoGen (San Diego,

CA). Ado, Ado deaminase (ADA), H-89

(B1427; PKA inhibitor), PGE2, and

recombinant murine GM-CSF were obtained

from Sigma Aldrich (St. Louis, MO). Doses of

each TLR ligand or reagent were determined

based on manufacturer's recommendations

and/or our own concentration–response studies

(data not shown). Cytokines were determined

by BD OptEIATM

ELISA sets from BD

Biosciences (San Diego, CA). Antibodies for

flow cytometry were also purchased from BD

Biosciences.

Generation of bone marrow (BM)-derived

DCs– DCs were generated according to the

method of Inaba et al. (28), with modifications

(13). Briefly, BM cells from femurs of

C57BL/6 mice were cultured in complete

medium (RPMI 1640 medium with 10% heat-

inactivated FBS, 2 mM L-glutamine, 100 U/

mL penicillin, 100 µg/mL streptomycin, 0.05

mM 2-ME) and 20 ng/mL GM-CSF. At day 0,

cells were seeded at 106 per 100-mm petri dish

(Falcon 1029 plates; BD Discovery Labware,

Bedford, MA) in 10 mL of medium. At days 3

and 6, another 10 mL of complete medium

containing 20 ng/mL GM-CSF was added to

the plates. The differentiated cells were

harvested on days 6–7 of culture for flow

cytometry analysis and cytokine production

assays.

Flow cytometry- For experiments on the effect

of saliva on DC differentiation, BM-derived

DCs were cultured as described above in the

presence of the indicated concentrations of tick

saliva or saliva fractions and incubated with

fluorochrome labeled antibodies. Percentage of

CD11c+ cells (DC-restricted marker for mice)

and CD11b+ cells (a myeloid cell marker) was

evaluated.

In another set of experiments described below,

DCs were also collected to evaluate the

expression of co- and stimulatory molecule

expression. To that end, cells were stained with

fluorochrome labeled antibodies against

CD11c, CD40, CD86, and MHC class II (I-A/I-

E) molecules. Data were acquired using a

FACSCalibur (BD Immunocytometry Systems)

with CellQuest (BD Biosciences) and analyzed

with FlowJo software (Tree Star Inc., Ashland,

OR).

Cytokine production assay- Six- to 7-day

cultured BM-derived DCs were gently

collected, washed twice, and resuspended at 106

cells/mL in complete medium. Cells were then

cultured at 105 cells/well in round-bottom 96-

well cluster plates (Costar, Cambridge, MA)

and incubated, depending on the experiments,

with medium, saliva, saliva filtrates or saliva

fractions for 30 minutes before addition of

TLR-2 (PGN, 10 µg/mL), TLR-4 (LPS, 100

ng/mL), or TLR-9 (CpG ODN-1826, 0.15 µM)

ligands. Following overnight incubation (18

hrs) at 37°C and 5% CO2, cell-free supernatants

were collected and levels of IL-12p40, TNF-α,

and IL-10 determined.

HPLC procedures– R. sanguineus saliva (1.5

mL) was centrifuged through a YM-5 microcon

device (Millipore, Bedford, MA). The filtered

5 kDa fraction was submitted to a reversed-

phase HPLC on a C18 column (4.3 × 150 mm;

ThermoSeparation Products, Riviera Beach,

FL) perfused at 0.5 mL/min using a CM-4100

pump (Thermo Separation Products, Riviera

Beach, FL).The eluent was monitored at 220-

500 nm using a diode array detector (model

SPD M10AV, Shimadzu, Columbia, MD) A

gradient of 80-minutes duration from 5 to 80%

acetonitrile in water, containing 0.1%

trifluoroacetic acid, was imposed after injection

of the sample. Aliquots of these fractions were

dried in 96-well plates and tested for cytokine

inhibitory activity and co- and stimulatory

molecule expression on DCs at dilutions

compatible for those employed for crude saliva.

Mass spectrometry– Selected fractions from the

RP-HPLC experiment were analysed by mass

spectrometry by direct injection (5 µl) of the

fractions into a Thermo-Finnigan LCQ Deca

XP Ion Trap Mass Spectrometer, through an

ion-spray interface supplied with a continuous

flow of 50% methanol in water containing

0.1% acetic acid. Fragmentation of selected

ions was performed with 30% maximum

intensity. Calibration curves using commercial

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adenosine were performed in order to estimate

salivary adenosine concentration. PGE2 concentration in saliva, filtrate, and

fractions– The amount of PGE2 in saliva

samples as well as its concentration in the

filtrate and fractions used for cell culture was

determined by competition ELISA kit (R&D

Systems, Minneapolis, MN), according to

manufacturer's instructions. The detection limit

for the assay was (~ 42 pM).

Ado determination in 5 kDa filtered saliva

fractions– The amount of Ado in saliva

fractions used for cell culture was determined

by mass spectrometry of the molecular ions

present in fraction 11 (F11). To further confirm

functional activity of Ado in the saliva, we also

carried out experiments incubating the saliva or

saliva fractions with ADA (3.0 U for 1 hr at

25oC) and testing each one for maturation of

DCs.

Measurement of cAMP in DCs– DCs exposed

to saliva or saliva fractions were used to

measure production of intracellular levels of

cAMP.

Cells (106) were seeded in 96-well

round-bottom tissue culture plates and treated

with saliva or saliva fractions for 15 minutes.

Cells were then lysed with 0.1 M HCl/0.1%

Triton X-100, and intracellular cAMP was

measured by commercial enzyme immunoassay

kits (Cayman Chemicals, Ann Arbor, MI)

according to manufacturer's instructions.

Data analysis– Data are shown as mean ±

SEM. Statistical differences were analyzed by

ANOVA followed by Tukey-Kramer post-hoc

analysis test (INSTAT software; GraphPad,

San Diego, CA). A P value of 0.05 or less was

considered statistically significant.

RESULTS

R. sanguineus tick saliva inhibits

production of IL-12p40 and TNF-α while

increasing IL-10 cytokine production by DCs

triggered by different TLR agonists– We first

tested whether R. sanguineus tick saliva could

modulate cytokine production by DCs matured

with PGN, LPS, and CpG (TLR-2, -4, and -9

agonists, respectively). As expected, treatment

of DCs for 18 hrs with these TLR agonists

increased the levels of IL-12p40 and TNF-α

(Fig. 1A, B); however, when cell cultures were

pre-incubated with whole saliva (dilution 1:20

v:v) for 30 minutes, significant inhibition (P <

0.05) of PGN-, LPS-, or CPG-induced IL-

12p40 and TNF-α production by DCs was seen

(Fig. 1A, B). Conversely, pre-incubation with

saliva (1:20 v:v) enhanced PGN-, LPS-, or

CPG-induced IL-10 production by these cells

(Fig. 1C) (P < 0.05). Levels of IL-12p40, TNF-

α, and IL-10 were not changed in cultures

incubated

with saliva alone, suggesting that

saliva lacks either contaminants or TLR ligands

(Fig. 1).

To determine the closest molecular weight

(MW) of the component(s) in the saliva

responsible for modulation of cytokine

production in DCs, 500 µL of saliva were

sequentially centrifuged through microcon

devices. The filtrate (equivalent volume to

saliva diluted 1:20) with MW lower than 5 kDa

or between 5 and 100 kDa were tested in the

LPS-induced cytokine production assay (Fig.

1D–F). The data indicate that when LPS-treated

DCs were pre-incubated with the fraction of

saliva containing molecules with MW lower

than 5 kDa their competence to produce IL-

12p40 and TNF-α cytokines was repressed

(P < 0.05). The inhibition was similar to that

seen with non-fractionated saliva (Fig. 1D, E).

Saliva fractions containing molecules with MW

lower than 5 kDa also additively up-regulated

production of IL-10 by LPS-treated DCs (Fig.

1F). Fractions containing molecules with MW

between 5 and 100 kDa did not modify

production of these cytokines (Fig. 1D–F).

Analysis of tick saliva fractionated by RP-

HPLC indicates that it contains two active

components lower than 5 kDa- To identify the

component(s) responsible for the effects of tick

saliva on DCs, we further fractionated 1.5 mL

of YM-5 filtrate by reverse-phase HPLC and

tested the fractions for production of the

cytokines in LPS-treated DCs. Fig. 2A depicts

an HPLC fingerprint of the 80 fractions 5

kDa saliva filtrate at the absorbance of 220 nm.

The 80 fractions were then assembled into 10

pools (comprising eight fractions each) that

were tested independently for production of IL-

12p40 and TNF-α on DCs stimulated with LPS.

When DCs were pre-incubated for 30 minutes

with pools 2 (containing fractions 9-16) and 7

(containing fractions 49–56), but not the

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remaining pools, LPS-induced IL-12p40 and

TNF-α production was diminished (Fig. 2B, C).

Next, we tested individually each of the

fractions of pools 2 and 7 in a similar assay. As

shown in Fig. 2 (D, E and F, G, respectively)

only fractions 11 (F11, from pool 2) and 51

(F51, from pool 7) presented a positive

inhibitory effect on production of IL-12p40 and

TNF-α by DCs.

Ado (F11) and PGE2 (F51) are the main

modulators of IL-12p40, TNF-α, and IL-10

cytokine production in R. sanguineus saliva–

To evaluate the chemical composition of the

molecules present in the two subfractions (F11

and F51) with positive inhibitory effects on

DCs, we employed different assays. For F11,

we used a combination of two different

apparatus: molecule absorbance analysis and

reverse-phase HPLC MS/MS. Absorbance

measurement of F11, but not F10 or F12,

showed a high concentration of molecules with

258 nm of absorbance, suggestive of the

presence of adenosine nucleosides (Fig. 3A).

The mass spectra of the molecular ions present

in F11 were informative and analogous to Ado

(Fig 3B). Fragmentation of the 268 mass ion

leads to production of an ion having m/z 136,

as expected for adenine (55) (Fig. 3C). Finally,

MS showed the presence of approximately 110

pmoles/µL of Ado in F11 (Fig. 3D).

It has been demonstrated by Sá-Nunes and

collaborators that PGE2 was present at the end

of the gradient of a micro-HPLC from Ixodes

scapularis saliva (13). Because this eicosanoid

had already been also described in the saliva of

R. sanguineus ticks (28), as well as in the saliva

of many other tick species (29-31), we asked

whether F51 from R. sanguineus tick saliva—

also found at the end of a micro-HPLC

gradient—could be PGE2. Using a PGE2-

specific competition ELISA, we found that

saliva, 5 kDa saliva filtrate, and F51 indeed

contained PGE2, and that the concentration of

both samples was ~ 100 nM (Fig. 3E).

F11 loses activity on DCs when exposed to

the enzyme ADA- To evaluate whether the Ado

identified in F11 would have similar activity to

synthetic Ado, we treated saliva or F11 with

ADA, which degrades Ado into inosine, and

tested it on DCs. As previously shown, R.

sanguineus saliva (dilution 1:20) presented a

potent inhibitory effect on LPS-induced

production of IL-12p40 and TNF-α by DCs.

When this saliva was treated with ADA, its

effects were partially impaired (P < 0.05) (Fig.

4A, B). Furthermore, when F11 was treated

with ADA, this fraction entirely lost its

cytokine modulating outcome (P < 0.05) (Fig.

4A, B). Intriguingly, while F11 treated with

ADA also lost its ability to enhance LPS-

induced production of IL-10 (P < 0.05), this did

not happen to ADA-treated saliva (Fig. 4C).

The rationale for this is unknown; however, as

saliva also contains PGE2, possibly this

component alone is enough to sustain

production of IL-10 independently of Ado.

Indeed, when DCs were pre-cultured with F51

(which contains PGE2), a similar enhancement

of IL-10 production induced by LPS was seen

in comparison with DCs pre-cultured with total

saliva (Fig. 4C). As expected, ADA treatment

did not alter the effect of F51 (which contains

PGE2, not Ado) on production of cytokines by

LPS-stimulated DCs (Fig. 4A, B).

It has been shown that inosine is able to

modulate the effect of IL-12 and TNF-α of

murine macrophages and splenic cells (32). To

prove that inosine generated in the above

experiment by the action of Ado degradation

does not exert modulatory effects on murine

DCs, we pre-treated DCs with inosine and

stimulated them overnight with LPS. The

results showed that even at a concentration of

100 µM, standard inosine was not able to

modulate production of IL-12 and TNF-α by

murine LPS-matured DCs (data not shown).

F11 and F51 from R. sanguineus saliva

cooperate in the amplification of cAMP-PKA

signaling– Because initiation of the signaling

pathway used by Ado and PGE2 to modulate

cytokine is via production of cAMP (33, 34),

we evaluated whether F11 and F51 from R.

sanguineus tick saliva increases production of

cAMP in DCs. As depicted in Fig. 5A, when

F11 or F51 was added separately, it induced a

significant (P < 0.05) production of cAMP.

Further, when added together, they showed an

additive effect – comparable to whole saliva -

on the amplification of cAMP (Fig. 5A). As a

positive control, DCs were also incubated with

forskolin, a drug known to induce cAMP

production in different cell types, and massive

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production (P < 0.05) of cAMP was seen (Fig.

5A).

A downstream intracellular molecule

activated by cAMP is the protein kinase A

(PKA), an essential enzyme for cell signaling,

that among other effects plays an important role

in modulation of cytokines induced by both

Ado and PGE2 (33–35). Having shown that F11

and F51 induce cAMP production, we further

evaluated whether these fractions had their

immunosuppressive effects blocked by H-89, a

PKA inhibitor. To answer this question, DCs

were treated with H-89 (3 µM), and the ability

of F11 and F51 to modulate LPS-induced IL-

12p40, TNF-α, and IL-10 cytokine production

was tested. Our results demonstrate that H-89

reversed significantly (P < 0.05) but not

completely the inhibitory effect of F11and F51

(Fig. 5B–D). When DCs were pre-incubated

with H-89, the competence of F11 or F51 alone

or F11 plus F51 to inhibit IL-12p40 and TNF-α

production stimulated by LPS was significantly

reversed (Fig. 5B, C). Further, production of

IL-10 induced by F11 or F51 in the presence of

LPS was completely restored after H-89

treatment, while IL-10 production induced by

F11 plus F51 was restored only partially (Fig.

5D). No difference in cytokine production was

seen when H-89 was added alone to DCs

stimulated by LPS (data not shown). These

results suggest F11 and F51 modulate only

partially LPS-induced cytokine production in

DCs by cooperating in amplification of cAMP-

PKA signaling.

F11 and F51 inhibit LPS-induced CD40

expression in DCs– Previous results published

by our group demonstrated that saliva from

R. sanguineus ticks inhibits expression of

CD40 and CD86 molecules on the surface of

murine BM–derived DCs (24). To evaluate

whether PGE2 and/or Ado are responsible for

these effects, BM-derived DCs were stimulated

with LPS in the presence of medium, whole

saliva (1:20), 5 kDa and 5–100 kDa filtrates,

F11, F51, or F11 plus F51 and the percentage

and medium intensity of expression of CD40

and CD86 in CD11c+/I-A/I-E

+ DCs was

analyzed by flow cytometry. Following

treatment with purified TLR agonists PGN

(TLR2 ligand), LPS (TLR4 ligand), or CpG

1826 (TLR9 ligand), CD11c+/I-A/I-E

+ DCs

increased the intensity of expression of CD40

and CD86 (Fig. 6A, B). When DC cultures were

pre-incubated with whole saliva for 30 minutes

at 1:20 dilution, significant inhibition (P <

0.05) of the percentage of PGN-, LPS-, or

CPG-induced CD40 (Fig. 6A) and CD86 (Fig.

6B) expression was observed. Pre-incubation

with the filtrate of 0–5 kDa but not 5–100 kDa

also inhibited LPS-induced expression of CD40

and CD86 (Fig. 6C,D).

To further investigate whether the effects

of saliva or filtrate are mediated by F11, F51,

or both, DCs were pre-incubated with F11, F51

or F11 plus F51 for 30 minutes and

subsequently stimulated with LPS, and

expression of CD40 and CD86 molecules was

evaluated 18 hrs later. As shown in Fig. 6E,

pre-incubation with F11 or F51 inhibited (P <

0.05) LPS-induced expression of CD40.

Moreover, F11 and F51 demonstrated an

additive inhibitory effect (Fig. 6E). On the

other hand, F11, F51 or F11 plus F51 did not

affect expression of CD86 molecules (data not

shown). We attempted to analyze the different

fractions from the filtrate of 0–5 kDa on CD86

expression, but following F11 and F51, no

fractions were found with those properties (data

not shown). These findings suggest that

molecules present in saliva or 0–5 kDa filtrate

only suppress LPS-induced CD86 expression

when these compounds act together on DCs.

Further studies must be done to characterize

which molecules can account for inhibition of

expression of CD86 by saliva.

F51 impairs differentiation of BM-derived

DCs– A previous work demonstrated that

saliva from R. sanguineus ticks inhibits

differentiation of DCs (24). To determine

whether saliva-impaired differentiation of DCs

was caused by the presence of saliva Ado,

PGE2, or both, BM-derived DCs were

developed with GM-CSF in the presence of

saliva, F11, F51, or both. Within 6–7 days of

differentiation, the percentage of CD11c+

CD11b+

was determined by flow cytometry.

Fig. 7A and B show that R. sanguineus saliva

significantly inhibited differentiation of BM

precursors into CD11c+/CD11b

+ DCs (P <

0.05). Similarly to saliva, when F51 was added

to cultures there was a significant inhibition of

DC differentiation (P < 0.05). F11 alone or

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together to F51 did not impair DC

differentiation (Fig. 7A, B). More important,

even when tested in a high concentration (100

µM), F11 did not hamper DC differentiation

(data not shown).

DISCUSSION

The discovery of new molecules produced

by parasites has become an important venue of

many researchers who are trying to

comprehend host-parasite interactions. This

tendency is growing because information about

the biological activities, structure, ligand-

binding affinities, and concentration of these

molecules open to a greater extent the array of

possibilities for find out how these organisms

evade hemostatic, inflammatory, and

immunologic host responses.

Our group has worked to identify the

molecules present in the saliva of the dog tick

R. sanguineus with modulatory effects on

diverse immune cells. Here, we indentify for

the first time in tick saliva the nucleoside Ado

as a potent immunosuppressor of DCs. This

report also demonstrates PGE2 from saliva of R.

sanguineus ticks with comparable modulatory

effects. Our data demonstrate clearly that both

Ado and PGE2 cooperate in modulating the

production of IL-12p40, TNF-α, and IL-10

cytokines and inhibiting expression of the

stimulatory molecule CD40 on DCs activated

with TLR agonists. The additive effect induced

by Ado and PGE2 probably results from the

capacity of both molecules to cooperate in

induction of cAMP-PKA signaling in DCs.

First we demonstrated that salivary

molecules from R. sanguineus ticks with MW

below 5 kDa downregulates production of IL-

12p40 and TNF-α and upregulates production

of IL-10 by DCs stimulated with TLR-2, -4,

and -9 agonists. These findings are of great

interest, as IL-12p40 and TNF-α can induce

functional maturation of DCs and could

selectively stimulate their ability to induce

Th1-type responses against ticks and tick-borne

pathogens, while IL-10 is well known as an

immunosuppressive cytokine (36–38).

Using microcon filtration and reverse-

phase HPLC MS/MS, we found the nucleoside

Ado in the saliva of R. sanguineus ticks. The

saliva fraction that contained Ado presented a

relevant effect on the biology of DCs, which

may have important implications for our

understanding of tick-host interactions. It is

known that Ado is an endogenous purine

nucleoside that modulates a wide variety of

immunologic functions in antigen-presenting

cells (39–42). It impairs TNF-α- and IL-12p40-

mediated responses to LPS in human and

murine DCs while enhancing production of IL-

10 (43,44). Ado also suppresses production of

IL-12 and TNF-α in monocytes and

macrophages (33, 45-46). In agreement with

our results, others have shown that Ado-

induced inhibition of TNF-α production by

macrophages is not restricted to TLR-4-

mediated activation, because Ado also

downmodulates TNF-α production when cells

are activated by TLR-2, -3, -7, and -9 agonists

(47).

Besides blocking LPS-induced TNF-

production, Ado markedly induces heme

oxygenase 1 (HO-1) in inflamed tissues (48).

This is noteworthy because ticks are blood

feeders and abundant quantities of heme, a

potent pro-oxidant and pro-inflammatory agent,

are probably released at the tick feeding site.

HO-1 removes heme and, further, generates

three metabolites—carbon monoxide (CO),

ferrous iron, and biliverdin—all of which have

an immune protective effect (49). A positive

feedback loop exists among Ado, HO-1, and

CO and resolves the inflammatory response

(48). Blocking of HO-1 by RNA interference

abrogates the effects of adenosine on TNF-

and HO-1 in macrophages. Another venue in

which tick salivary Ado may disrupt host

defenses is the migration of phagocytic cells to

the site of tissue damage caused by tick

feeding. Signaling by means of purinergic

receptors has been recently shown to be

essential for chemotaxis of macrophages in a

gradient of C5a (50). This requires sequential

hydrolysis of nucleotides ending in Ado, and an

excess of Ado provided by tick saliva may

disrupt the sequence of events resulting in

correct chemotactic navigation.

Components from tick saliva and salivary

glands, including Ado, inhibit the biology of

not only DCs but that of many other immune

cells including neutrophils, mast cells, NK

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cells, T and B cells (42, 51-55). This can be

particularly relevant, as the tick Ado identified

in this work could be responsible for the effects

described above. Further studies must be

performed to test this hypothesis. Furthermore,

Ado possibly has an important role in evolution

as an evasion mechanism for blood-sucking

parasites. Indeed, salivary glands of other

classes of hematophagous arthropods, notably

sand flies Phlebotomus argentipes and

Phlebotomus papatasi (55,56), also produce

Ado; however, it has not yet been tested for its

immunomodulatory properties.

We found another fraction of R.

sanguineus tick saliva that had a modulatory

effect on cytokine production by DCs. Using a

competition ELISA we ascertained that this

fraction contained PGE2 in a considerable

amount. Similarly to data described here, saliva

of many tick species has been shown to contain

PGE2; moreover, it has been associated with the

saliva effect on suppression of host immune

responses (13, 57-58). As we reported with

PGE2 of the saliva, synthetic PGE2 inhibits the

ability of monocytes, macrophages, and DCs to

secrete cytokines such as IL-12 and TNF-α

(59–63) and may shift the balance in favor of a

Th2 immune response. PGE2 can also act

directly on T cells by impairing production of

IL-2 and IFN-γ (64), which also could induce a

Th2-type response. In support of our findings,

PGE2 isolated from I. scapularis saliva is the

major inhibitor of IL-12 and TNF-α induced by

LPS-stimulated DCs (13). However, PGE2

concentrations in I. scapularis saliva were 10-

to 100-fold higher than that found in our study

(Sá-Nunes, personal communication). This

might explain why R. sanguineus saliva needs

an additional molecule – Ado – to reach

modulatory activities in pharmacological

levels.

Related to Ado and PGE2-induced

intracellular cascade, we observed as expected

that both molecules in the saliva fractions were

able to induce production of cAMP by DCs;

furthermore, when combined they showed an

additive effect. Interestingly, Ado or PGE2 used

individually or in combination lose their

modulatory activity when DCs are pre-

incubated with H-89, a PKA inhibitor. This

result is important because we demonstrate that

both molecules affect production of cytokines

by DCs by a common signal transduction

pathway, linking increased production of

cAMP and PKA activation. At the same line, it

was demonstrated that Ado and PGE2 produced

by human regulatory T cells have additive

effects in downregulating functions of immune

cells through intracellular cAMP-PKA

signaling (65). It is essential to mention that the

treatment of DCs with the PKA inhibitor did

not restore completely the production of IL-

12p40 and TNF-α, suggesting that Ado and

PGE2 may modulate DCs via signaling

pathways other than cAMP-PKA. Our data are

in accordance with those of Vassiliou, et al.

(60), who reported that inhibition of LPS-

stimulated TNF-α production in murine DCs by

PGE2 was only partially prevented by the PKA

inhibitor H-89. In support of this possibility, a

recent work has revealed that alternative

cAMP-binding targets, such as the guanine

nucleotide exchange protein (Epac-1), can be

also responsible for inhibition of TNF-α

cytokine production in DCs (66).

Enhancement of co- and stimulatory

molecules on DCs' surface is fundamental to

induce a proper T cell response. Here, we

demonstrate that tick saliva suppressed

expression of CD40 and CD86 in DCs.

Blocking binding of CD40 with its ligand

CD40L (CD154) prevents T cell dependent

antibody production and reduces CD4+ T cell

priming and expansion, which can impair the

development of a protective host response to

parasitic infections (67–70). Indeed, it was

shown that the blockage of CD40-CD154 and

CD86-CD28 interactions could result in T cell

anergy and high IL-10 production (71). This

aspect is relevant because the modulatory effect

on cytokine production, together with reduction

in expression of CD40 and CD86, could be an

important "double-escape" mechanism used by

ticks to induce regulatory T cells, T cell anergy,

or to inhibit antigen presentation. These host

conditions could probably help blood-feeding

arthropods to complete their meal and,

moreover, could facilitate transmission of

vector-borne pathogens during the parasitic

stage. Interestingly, only CD40 but not CD86

expression was downregulated by saliva

fractions containing Ado and/or PGE2. The

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explanation for why crude tick saliva and saliva

filtrate impair CD86 expression but saliva

fractions individually do not is unknown.

Possibly a combination of saliva fractions is

required. Additional studies must be done to

identify these molecules.

Finally, we showed that PGE2 from saliva

dramatically inhibits differentiation of BM–

derived DCs. These results are in accordance

with previous results showing that PGE2

inhibits differentiation of murine and human

DCs (72,73). The effect of R. sanguineus saliva

in inhibiting differentiation of DCs was

previously demonstrated by Cavassani et al.

(24); however, only now has the molecule

responsible for this effect been identified.

Saliva fractions that contain Ado showed no

effect on DC differentiation, possibly explained

by work demonstrating that Ado is related to

macrophage inhibition and DC differentiation

in human (74,75) but not in murine (76) cells.

Modulation of BM cells by PGE2 during

differentiation to DCs is vitally important

because in addition to inhibiting of the number

of DCs differentiated, the DCs differentiated in

the presence of PGE2 produce high levels of

anti-inflammatory cytokines and induce a Th2-

type immune response (76), a phenotype

associated with immune suppression and

tolerance to ticks.

In conclusion, we describe for the first

time Ado as one of the most important

immunomodulatory molecules identified to

date in a tick saliva. This molecule, produced

and secreted in high concentration, is able to

modulate almost all cells of the immune system

(42), in addition to being vasodilatory and

inhibiting platelet aggregation (77,78).

Moreover, we also found PGE2 in R.

sanguineus saliva, which combined with Ado

may augment anti-inflammatory and

immunologic responses of their hosts. It is

important to highlight that both salivary Ado

and PGE2 are non-protein endogenous small

host molecules and accordingly no host

immune responses can be directed against

them. Taken together, our results support the

concept that the biological activities of Ado and

PGE2 counteract most of the host defenses and

should help the tick to feed and reproduce.

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FOOTNOTES

This work was supported in part by the Fundação de Amparo a Pesquisa do Estado de São Paulo

(FAPESP - 06/54985-4 and 07/00035-8), Coordenação de Aperfeiçoamento de Pessoal de Nível

Superior (CAPES), and the Millennium Institute for Vaccine Development and Technology

(Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq - 420067/2005-1). We

thank Cristiane M. Milanezi and the D.V.M. José Januário das Neves for excellent technical

assistance.

This work was also supported by the Intramural Research Program of the Division of Intramural

Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. We

thank NIAID intramural editor Brenda Rae Marshall for assistance.

Because IMBF and JMCR are government employees and this is a government work, the work

is in the public domain in the United States. Notwithstanding any other agreements, the NIH

reserves the right to provide the work to PubMedCentral for display and use by the public, and

PubMedCentral may tag or modify the work consistent with its customary practices. You can

establish rights outside of the U.S. subject to a government use license.

The abbreviations used are: ADA, Ado deaminase; Ado, adenosine; BM, bone marrow; DCs,

dendritic cells; Filt, filtrate; HO-1, heme oxygenase 1; RP-HPLC, reversed-phase high performance

liquid chromatography; MS, mass spectrometry; MS/MS tandem MS; MW, molecular weight;

PGE2, prostaglandin-E2; PGN, peptidoglycan; PKA, protein kinase A; TLR, Toll-like receptor.

FIGURE LEGENDS

Fig. 1. Tick saliva modulates cytokine production induced by diverse Toll-like ligands by use

of molecules with MW< 5 kDa. DCs from C57BL/6 mice were produced from BM cells cultured

with GM-CSF (20 ng/mL) for 6–7 days. Next, cells were washed and pre-incubated with medium (-

), saliva (Sal; dilution 1:20) (A–C) or saliva (1:20) and saliva filtrates (filtrates < 5 kDa or 5–100

kDa (1:20)) (D–F). After 30 minutes, cells were stimulated overnight with TLR-2 (PGN; 10

µg/mL), TLR-4 (LPS, 100 ng/mL) and TLR-9 (CPG, 150 nM) ligands. After 18 hrs of incubation,

cytokine levels in culture supernatants were measured by ELISA. The results are expressed as the

mean standard error of the mean (SEM) obtained from one of three independent experiments

performed in triplicate (N = 3). *: P < 0.05 vs. respective LPS, CPG, and PGN groups without

saliva or saliva filtrates.

Fig. 2. Tick saliva contains two fractions with MW < 5 kDa that inhibit IL-12p40 and TNF-.

DCs from C57BL/6 mice were produced from BM cells cultured with GM-CSF (20 ng/mL) for 6–7

days. To isolate the molecules from the 5 kDa saliva filtrate related to DC modulation, saliva was

filtered using an YM-5 (cut-off 5,000 Da) membrane and the filtrate was fractionated in 80

fractions by reverse-phase HPLC using the conditions described in Experimental Procedures. An

HPLC chromatogram of the 5 kDa filtrate at 220 nm is demonstrated in A. B and C demonstrate by

ELISA the production of IL-12p40 and TNF-α from the supernatant of DCs that were pre-incubated

with different pools (pools containing eight fractions each, according to the eluting time,

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successively) from the separated filtrate and 30 minutes later stimulated for 18 hrs with LPS (100

ng/mL). D–G show production of IL-12p40 and TNF-α from the supernatant of DCs pre-incubated

with fractions 9-16 (D,E) and 49–56 (F,G), for 30 minutes and subsequently stimulated for 18 hrs

with LPS (100 ng/mL). Arrows indicate the pools and the isolated fractions with strongest

inhibitory effect for each assay. Results are expressed as the mean SEM obtained from one of

two independent experiments performed in triplicate (N = 3).

Fig. 3. Ado (fraction 11 – F11) and PGE2 (fraction F51 – F51) are the major modulators of R.

sanguineus tick saliva. UV spectrum of fraction F11 as well as the adjacent fractions (fractions 10

[F10] and 12 [F12]) is demonstrated (A). Mass spectrogram of F11 (B). Mass spectrogram deriving

from the fragmentation of the m/z ion 268 with 30% maximum collision intensity (C). The

concentration of the molecule present in F11 was also determined by MS (D). The concentration of

the molecule present on F51 was measured using a standard commercial ELISA kit and compared

with the concentration obtained with a given dilution of saliva (Sal), YM-5 saliva filtrate (Filt 0-5

kDa), fractions 10, 11, and 12, and the 51 adjacent fractions (50 and 52) (F).

Fig. 4. Molecules from F11 lose activity when exposed to the enzyme ADA. DCs from

C57BL/6 mice were produced from BM cells cultured with GM-CSF (20 ng/mL) for 6–7 days.

Saliva (Sal), F11, or F51 (all diluted 1:20) were exposed or not with ADA (3.0 U) for 1 hr and

subsequently added to the DC culture. Thirty minutes later, cells were stimulated with LPS (100

ng/mL). After 18 hrs of incubation with LPS, cytokine levels in culture supernatants were measured

by specific ELISA for IL-12p40 (A), TNF-α (B), and IL-10 (C) according to manufacturer's

instructions. *, P < 0.05 vs. LPS group without saliva or saliva fractions; &, P < 0.05 vs. LPS +

saliva; #, P < 0.05 vs. LPS + F11.

Fig. 5. F11 and F51 have complementary immunosuppressive effects on DCs by amplification

of the production of cAMP and activation of the enzyme PKA. DCs were harvested on day 6–7 of

culture, washed with phosphate-buffered saline, and placed in a RPMI fresh culture medium. For

evaluation of cAMP production, DCs were incubated for 15 minutes with medium (-), F11, F51,

F11 + F51, saliva (all diluted 1:20), or Forskolin (positive control) and the cAMP levels measured

by competition ELISA according to manufactures' instructions (A). *, P < 0.05 vs. medium alone; &, P < 0.05 vs. F11 or F51;

#, P < 0.05 vs. F11, F51, F11 + F51, or saliva. To evaluate the effect of

F11 and F51 on activation of the enzyme PKA, DCs were exposed to H-89 (inhibitor of PKA; 3

µM) for 45 minutes and subsequently incubated with F11, F51, or F11 + F51 for 30 minutes, after

which DCs were stimulated with medium (-) or LPS (100 ng/mL). After 18 hrs of incubation with

LPS, cytokine levels in culture supernatants were measured by specific ELISA for IL-12p40 (B),

TNF-α (C), and IL-10 (D) according to manufacturer's instructions. *, P < 0.05 vs. LPS group

without F11, F51, or F11 + F51; &, P < 0.05 vs. LPS + F11, LPS + F51, and LPS + F11 + F51. The

results are expressed as the mean SEM obtained from one of two independent experiments

performed in triplicate (N = 3).

Fig. 6. F11 and F51 cooperate in inhibition of CD40 expression in LPS-matured DCs. DCs

from C57BL/6 mice were produced from BM cells cultured with GM-CSF (20 ng/mL) for 6-7 days.

Next, cells were washed and pre-incubated with medium (-), saliva (Sal), saliva filtrates (Filt) (Filt

0-5 kDa and Filt 5-100 kDa), F11, F51, or F11 +F51 (all diluted 1:20). After 30 minutes, cells were

stimulated by 18 hrs with TLR-2 (PGN; 10 µg/mL), TLR-4 (LPS; 100 ng/mL), or TLR-9 (CPG;

150 nM) agonists, depending on the experiment. CD11c+/ I-A/I-E

+ cells were gaited for expression

of CD40 and CD86 on their surface. It was demonstrated that saliva inhibits expression of CD40

and CD86 in PGN-, LPS-, and CPG-stimulated DCs (A and B). C and D show that inhibition of

CD40 and CD86 in CD11c+/I-A/I-E

+ DCs is mediated by molecule(s) presents on the filtrate lower

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than 5 kDa. Results are expressed as the mean SEM obtained from one of two independent

experiments performed in triplicate (N = 3 per group). Representative dot plots of each treatment

(medium, LPS, LPS + F11, LPS + F51, or LPS + F11 + F51) are also shown (E). ―*, P < 0.05 vs.

LPS, CPG, and PGN groups without saliva or saliva filtrates.

Fig. 7. F51 from tick saliva inhibits differentiation of BM-derived DCs. BM-derived cells from

C57BL/6 mice were cultured with GM-CSF (20 ng/mL) in the presence of tick saliva, F11, F51, or

F11 + F51 as indicated. Cells were harvested on day 6–7, labeled with the designated monoclonal

antibodies (mAbs), and analyzed by flow cytometry. In A, representative dot plots for CD11c

and CD11b markers, in 7-day differentiated cells, are demonstrated. B shows the mean

percentage ± SEM of CD11c+ CD11b

+ cells 7 days post differentiation. The data are representative

of two independent experiments. ―*, P < 0.05 compared with cells cultured with Medium only

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Beatriz R. FerreiraElen Anatriello, João S. Silva, Isabel K. F. de Miranda Santos, José M.C. Ribeiro and

Carlo José F. Oliveira, Anderson Sá-Nunes, Ivo M. B. Francischetti, Vanessa Carregaro,properties

Deconstructing tick saliva: non-protein molecules with potent immunomodulatory

published online January 26, 2011J. Biol. Chem. 

  10.1074/jbc.M110.205047Access the most updated version of this article at doi:

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