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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: [email protected]
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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Oliveira 14
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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