The Journal of Immunology

Nucleosides from Phlebotomus papatasi Salivary GlandAmeliorate Murine Collagen-Induced Arthritis by ImpairingDendritic Cell Functions

Vanessa Carregaro,* Anderson Sa-Nunes,† Thiago M. Cunha,‡ Renata Grespan,x

Carlo J. F. Oliveira,{ Djalma S. Lima-Junior,* Diego L. Costa,* Waldiceu A. Verri, Jr.,x

Cristiane M. Milanezi,* Van My Pham,‖ David D. Brand,#,**,†† Jesus G. Valenzuela,‡‡

Joao S. Silva,* Jose M. C. Ribeiro,‖ and Fernando Q. Cunha*,‡

Among several pharmacological compounds, Phlebotomine saliva contains substances with anti-inflammatory properties. In this

article, we demonstrated the therapeutic activity of salivary gland extract (SGE) of Phlebotomus papatasi in an experimental model

of arthritis (collagen-induced arthritis [CIA]) and identified the constituents responsible for such activity. Daily administration of

SGE, initiated at disease onset, attenuated the severity of CIA, reducing the joint lesion and proinflammatory cytokine release.

In vitro incubation of dendritic cells (DCs) with SGE limited specific CD4+ Th17 cell response. We identified adenosine (ADO) and

59AMP as the major salivary molecules responsible for anti-inflammatory activities. Pharmacologic inhibition of ADO A2Areceptor or enzymatic catabolism of salivary nucleosides reversed the SGE-induced immunosuppressive effect. Importantly,

CD73 (ecto-59-nucleotidase enzyme) is expressed on DC surface during stage of activation, suggesting that ADO is also generated

by 59AMP metabolism. Moreover, both nucleosides mimicked SGE-induced anti-inflammatory activity upon DC function in vitro

and attenuated establishment of CIA in vivo. We reveal that ADO and 59AMP are present in pharmacological amounts in P.

papatasi saliva and act preferentially on DC function, consequently reducing Th17 subset activation and suppressing the auto-

immune response. Thus, it is plausible that these constituents might be promising therapeutic molecules to target immune

inflammatory diseases. The Journal of Immunology, 2011, 187: 4347–4359.

During their evolutionary process, several species of blood-feeding arthropods developed a number of sophisticatedand redundant mechanisms to overcome the hemostatic

and inflammatory/immune systems of their vertebrate hosts (1).Vasodilators, anticoagulants, inhibitors of platelet aggregation, andanti-inflammatory and immunomodulatory molecules are presentin the salivary glands and are essential to a successful blood meal(2, 3). Furthermore, these active molecules may contribute in thetransmission, as well as establishment, of arthropod-borne diseases(i.e., leishmaniasis by phlebotomines, malaria by anophelines, andLyme disease by ixodid ticks), through modulation of the hostimmune response (4, 5). Indeed, arthropod saliva has been shown

to inhibit several functions of the immune system, including acti-vation of the alternative complement pathway, phagocytosis ofpathogens, production of inflammatory cytokines by macrophagesand dendritic cells (DCs), and activity of NK cells, as well as T andB cell proliferation (6–11).In phlebotomines, it has been demonstrated that their saliva is

able to selectively inhibit several DC and macrophage functions,including Ag presentation, NO and hydrogen peroxide production,and IFN-g–induced inducible NO synthase gene expression, thusinhibiting intracellular killing by Leishmania major (9, 12). Fur-thermore, salivary proteins from certain sand fly species favordevelopment of a Th2-type immune response, either in vitro or

*Department of Biochemistry and Immunology, School of Medicine of RibeiraoPreto, University of Sao Paulo, 14049-900 Sao Paulo, Brazil; †Department of Immu-nology, Institute of Biomedical Sciences, University of Sao Paulo, 14049-900 SaoPaulo, Brazil; ‡Department of Pharmacology, School of Medicine of Ribeirao Preto,University of Sao Paulo, 14049-900 Sao Paulo, Brazil; xDepartment of Pathology,State University of Londrina, State of Parana, Brazil; {Institute of Biological andNatural Sciences, Federal University of Triangulo Mineiro, 38025-180 Minas Gerais,Brazil; ‖Section of Vector Biology, Laboratory of Malaria and Vector Research,National Institute of Allergy and Infectious Diseases/National Institutes of Health,Bethesda, MD 20892; #Research Service, Veterans Affairs Medical Center, Memphis,TN 38163; **Department of Medicine, University of Tennessee Health ScienceCenter, Memphis, TN 38163; ††Department of Molecular Sciences, University ofTennessee Health Science Center, Memphis, TN 38163; and ‡‡Vector MolecularBiology Unit, Laboratory of Malaria and Vector Research, National Institute ofAllergy and Infectious Diseases/National Institutes of Health, Bethesda, MD 20892

Received for publication October 13, 2010. Accepted for publication August 14,2011.

This work was supported by Fundacao de Amparo a Pesquisa do Estado de SaoPaulo, Comissao de Aperfeicoamento de Pessoal de Nival Superior, Conselho Nacio-nal de Pesquisas, Fundacao de Apoio ao Ensino, Pesquisa e Assistencia do Hospitaldas Clınicas da Faculdade de Medicina de Ribeirao Preto, and Instituto Nacional deCiencia e Tecnologia em Vacinas. D.D.B. was supported by a grant from the De-

partment of Veterans Affairs. J.G.V. and J.M.C.R. were supported by the IntramuralResearch Program of the Division of Intramural Research, National Institute ofAllergy and Infectious Diseases, National Institutes of Health.

Because J.G.V. and J.M.C.R. are government employees and this is a governmentwork, the work is in the public domain in the United States. Notwithstanding anyother agreements, the National Institutes of Health reserves the right to provide thework to PubMedCentral for display and use by the public, and PubMedCentral maytag or modify the work consistent with its customary practices. One can establishrights outside of the United States subject to a government use license.

Address correspondence and reprint requests to Prof. Fernando Q. Cunha or Prof.Jose M. C. Ribeiro, Department of Pharmacology, School of Medicine of RibeiraoPreto, University of Sao Paulo, Avenida Bandeirantes, 3900, Ribeirao Preto, SaoPaulo, Brazil, 14049-900 (F.Q.C.) or Section of Vector Biology, Laboratory ofMalaria and Vector Research, National Institute of Allergy and InfectiousDiseases/National Institutes of Health, Bethesda, MD 20892 (J.M.C.R.). E-mailaddresses: jribeiro@niaid.nih.gov (J.M.C.R.) and fdqcunha@fmrp.usp.br (F.Q.C.)

The online version of this article contains supplemental material.

Abbreviations used in this article: ADA, adenosine deaminase; ADO, adenosine;BMDC, bone marrow-derived cell; CIA, collagen-induced arthritis; COX, cycloox-ygenase; DC, dendritic cell; LN, lymph node; RA, rheumatoid arthritis; SGE, sali-vary gland extract; Treg, regulatory T cell.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003404

in vivo, characterized by production of high levels of IL-4 (13,14). Importantly, sand fly saliva induces release of immunomod-ulatory mediators such as IL-10 and PGE2 and inhibits productionof protective type 1 cytokines such as IL-12, IFN-g, and TNF-a,all of which enhance survival of the Leishmania parasite (15–18).We recently demonstrated that systemic pretreatment of mice

with salivary gland extract (SGE) from the Old World speciesPhlebotomus papatasi and Phlebotomus duboscqi inhibited neu-trophil migration during OVA-induced immune peritonitis (19).By exploring the specific mechanism of saliva action, we foundthat Phlebotomine saliva acts preferentially on APCs, inhibitingthe ability of DC to present Ags to T cells. These anti-inflam-matory effects seem to depend on a sequential production of PGE2

and IL-10 by DCs, which act in an autocrine manner (19).DCs are potent APCs specialized in the initiation of the im-

mune response by direct activation and differentiation of naiveT lymphocytes to specific subtypes (20). Inflamed synovia fromarthritic patients contains high numbers of both DC subsets, my-eloid and plasmacytoid, which strongly suggests a role for theseAPCs in disease perpetuation (21–23). During the Ag presentationprocess, depending on stimuli (i.e., pathogens or autoantigens),DCs that emigrate to inflamed joints produce proinflammatorymediators such as IL-1b, IL-6, IL-12p70, IL-15, IL-18, IL-23p19,and TNF-a that support expansion and differentiation of Th1 and/or Th17 cells, which play a pathologic role in arthritis (24–27).Given the ability of DCs to interact strongly with T cells, inducingand activating the lymphocyte CD4+ Th17 subset, it is plausibleto suggest that pharmacologic strategies aimed at blocking DCfunction may deserve attention as a potential therapeutic target ofautoimmune diseases.Taking into account this evidence, we examined in this article

the potential therapeutic effect of P. papatasi SGE on collagen-induced arthritis (CIA). We also identify the constituents of P.papatasi saliva that are responsible for the immunomodulatoryactivity observed.

Materials and MethodsMice

Male DBA/1J mice weighing 18–22 g were housed at the animal facility ofthe Department of Pharmacology or Immunology, School of Medicine ofRibeirao Preto, University of Sao Paulo (Brazil), in temperature-controlledrooms (22–25˚C) and received water and food ad libitum. All experimentswere conducted in accordance with National Institutes of Health guidelineson the welfare of experimental animals and with the approval of the EthicsCommittee from the School of Medicine of Ribeirao Preto.

Saliva

Salivary glands were prepared from 7- to 10-d-old laboratory-bred femalesof P. papatasi from the Laboratory of Malaria and Vector Research at theNational Institutes of Health, as previously described (28). Briefly, 50 pairsof salivary glands were dissected under sterile conditions in endotoxin-freePBS, placed in 50 ml sterile PBS buffer, and kept at 270˚C until needed.Immediately before use, the glands were disrupted by sonication usinga Sonifier 450 homogenizer (Branson). Endotoxin levels were evaluatedusing the QCL-1000(r) Chromogenic LAL Endpoint Assay kit (Lonza,Switzerland), resulting in negligible levels of endotoxin in the salivarygland supernatant.

Induction of CIA and assessment of arthritis

CIAwas elicited in mice, as previously described (29). Briefly, male DBA/1J mice (10 wk) received 200 mg diluted in acetic acid of bovine type IIcollagen (Sigma-Aldrich) emulsified in CFA (Sigma-Aldrich) by in-tradermal injection at the base of tail on day 0. Mice were boosted in-tradermally with collagen (200 mg diluted in acetic acid) emulsified inCFA (Sigma-Aldrich) on day 21. Mice were monitored daily for signs ofarthritis, as described (29). Scores were assigned based on erythema,swelling, or loss of function present in each paw on scale of 0–3, givinga maximum score of 12 per mouse. When mice reached a score of 1 for

clinical arthritis, they were treated with P. papatasi SGE (1 gland/animal)or 59AMP plus adenosine (ADO) (∼20 mM each) by i.v. route daily for 2wk. To evaluate the salivary nucleoside effectiveness, some animals wereeither treated when disease was scored 6 or the treatment was interruptedafter 1 wk. Control mice received the same volume of PBS. Alternatively,in some groups, SGE were previously incubated 6 with adenosine de-aminase (ADA; 4.3 U), an enzyme that catabolizes ADO. Scoring wasconducted in a blinded fashion. For histologic assessment, mice wereeuthanized 35 d postchallenge, and the hind limbs were removed anddemineralized thoroughly in 10% EDTA for 1–2 wk. The decalcified tis-sues were trimmed, dehydrated in graded ethanol, and embedded in par-affin. Serial sections (5 mm) were cut and mounted on glass slidesprecoated with 0.1% poly-L-lysine (Sigma-Aldrich). Histologic assessmentwas carried out following routine H&E staining. Ankle and joint sectionswere prepared and stained with H&E to study the inflammatory cell influxor using safranin-O to determine proteoglycan depletion and cartilagedestruction. Quantification of cellular infiltrate was performed by ImageJsoftware (National Institutes of Health) in 40 fields (original magnification3400) for each animal/group. To measure cytokine concentrations in theinflammatory site, articular tissues were harvested and titered in 1 ml PBScontaining protease inhibitor mixture Complete (Roche) by tissue trimmer.Articular homogenates were centrifuged, and their supernatants werecollected and stored at 270˚C for determination of IFN-g, IL-10, IL-17,and TNF-a by ELISA (BD Biosciences) and PGE2 by a RIA kit, accordingto the manufacturer’s instructions (DuPont NEN Research Products).

DC generation

BM cells were isolated from 6- to 8-wk-old DBA/1J naive mice and culturedwith murine GM-CSF (20 mg/ml; PeproTech). On day 3, half of the su-pernatant was gently removed and replaced with the same volume ofsupplemented medium. On day 6, nonadherent cells were collected andsubmitted to positive selection using anti-CD11c magnetic beads, ac-cording to the manufacturer’s instructions (Miltenyi Biotec), to eliminateresidual macrophage and granulocyte contamination. Flow cytometric eval-uation of purified DCs shows that 90% of cells express CD11cinterm or high

(marker of DC).

SGE fractionation and HPLC procedures

P. papatasi SGE (60 pairs) were diluted in 60 ml PBS and submitted tofiltration in a Microcon YM-3 (Millipore) (cutoff ,3 kDa), separatingSGE into two fractions: filtrate (,3 kDa) and retentate (.3 kDa). Mo-lecular sieving HPLC of the filtrate fraction was performed using aSuperdex 75 column (3.2 3 300 mm; Amersham Biosciences) and elutedat 0.05 ml/min with 10 mM HEPES buffer (pH 7.2), containing 0.15 MNaCl. The solvent was delivered using a CM-4100 pump (Thermo Sepa-ration Products). The eluent was monitored for UV absorbance at 220 nm(SM-4100 UV spectrophotometer; ThermoSeparation Products), withfractions being collected at 1-min intervals using a FC203-B fractioncollector (Gilson). The column was calibrated using BSA, chicken OVA,carbonic anhydrase, myoglobin, cytochrome c, angiotensin 1, tyrosine, andtryptophan. Reverse phase of filtrate was performed on a C18 column(0.5 3 150 mm; Thermo Separation Products) perfused at 25 ml/min usinga PerkinElmer ABI 1400 pump. A gradient of 60-min duration from 5–90% acetonitrile in water, containing 0.1% trifluoroacetic acid, was im-posed after sample injection. Aliquots of filtrate or these fractions weretested for cytokine-inhibitory activity after evaporation of the solvent andresuspension in RPMI 1640 media. Alternatively, filtrate, ADO (100 mM),or 59AMP (100 mM) was incubated 6 ADA (4.3 U) and then added tobone marrow-derived cell (BMDC) cultures for 3 h.

Effect of P. papatasi SGE, fractions, and nucleosides inLPS-induced BMDC maturation

BMDCs (1 3 106/ml) in RPMI 1640 supplemented with 10% FBS wereincubated with medium, total extract from P. papatasi salivary gland (16mg/ml), or salivary fractions (retentate or filtrate; 16 mg/ml each) for 12 h.In some experiments, indicated concentrations of ADO, 59AMP (3–300mM), ADO synthetic analogous P101 (100– 1000 mM), or medium wereadded to the BMDC culture. For the time-response studies, BMDCs werepretreated with ADO or 59AMP (100 mM) for 23.0, 20.5, 0.0, +0.5, and+3.0 h of LPS stimulation. In all experiments, LPS (50 ng/ml) or mediumwas added to the culture and incubated at 37˚C in 5% CO2 for 24 h in a totalvolume of 200 ml per condition. The supernatant was collected to measureTNF-a, IL-12p40, and IL-10 production by ELISA and PGE2 by RIA. Thecells were harvested, and their surface expression was characterized by flowcytometry using Abs against MHC class II, CD80, CD86, CD40, CD39,and CD73 conjugated to FITC, PE, or PerCP, as well as control isotypes.

4348 PHLEBOTOMINE SALIVA ATTENUATES COLLAGEN-INDUCED ARTHRITIS

In vivo depletion of IL-10 cytokine

The IgG1 2A5 anti-murine IL-10 was purified from ascite harvested ofpristine (Sigma-Aldrich)-primed nude backcrossed BALB/c mice injectedwith the 2A5 hybridoma cells, as described elsewhere (30). The groups ofvehicle- or nucleoside-treated arthritic mice received, at the onset of thedisease, 250 mg 2A5MAb or normal rat IgG controls by i.p. route Ab evereach 2 d, weekly during 2 wk thereafter. Scores were assessed, as de-scribed above. Draining lymph nodes (LNs) were harvested and DCmaturation was evaluated through surface expression of CD11c and MHCclass II characterized by flow cytometry using specific Abs.

T cell proliferation

To assess the influence of P. papatasi SGE treatment on Ag presentation,spleens and popliteal and inguinal LN cells harvested from arthritic micewere removed and washed twice with PBS. Tissues were minced, and thecells were filtered through a cell strainer, centrifuged at 500 3 g at 4˚C for10 min, and resuspended in RPMI 1640 medium at 2.5 3 106 cells/ml. Forintracellular staining, nonadherent cells were cultured 6 stimulation withcollagen-pulsed BMDCs pretreated with PBS or SGE (8 glands/106

BMDC) for a final ratio of 10 total cells:1 BMDC for 96 h. Brefeldin Awas added for the last 4 h of stimulation. For surface staining, cells wereincubated with anti-CD4 for 30 min at 4˚C, washed, and then fixed withBD Cytofix (BD Biosciences). Cells were permeabilized using PBS con-taining 1% FCS, 0.01% sodium azide, and 0.05% saponin and stained withanti-mouse IFN-g and anti-mouse IL-17 (all Abs from BD Biosciences),acquired on FACS Canto II (BD Biosciences), and analyzed using FlowJosoftware (Tree Star).

To purify CD4+ T cells, total cells were incubated with microbeadscoated with Abs against L3T4 (Miltenyi Biotec) and isolated using mag-netic separation. The procedures were performed in accordance withmanufacturer’s instructions. To perform in vitro coculture assay, BMDCswere preincubated with SGE, ADO, or medium for 3 h. Isolated CD4+

T cells were added to wells to a cell ratio of 10:1. In some wells, A2ARantagonist was added 2 h before SGE or ADO preincubation. In all theexperiments, C-II (5 mg/ml), plate-bound anti-CD3 (5 mg/ml), or mediumwas added to the culture and incubated for 96 h in a total volume of 200 mlper condition. Supernatants were harvested for determination of IL-17production, and cell proliferation was measured by overnight [3H]thymi-dine incorporation.

Quantitative RT-PCR

Total RNA of BMDCs preincubated with ADO (100 mM) was extract 24 hafter stimulation with LPS (50 ng/ml) using the Illustra RNAspin Mini (GEHealthcare). Gene expression was normalized to expression of the GAPDHgene. COX2, A2AR, and A2BR primer sequences are as follows: GAPDHforward, 59-TGCAGTGGCAAAGTGGAGAT-39 and reverse, 59-CGTGA-GTGGAGTCATACTGGAA-39; COX-2 forward, 59-GTGGAAAAACCT-CGTCCAGA-39 and reverse, 59-GCTCGGCTTCCAGTATTGAG-39; A2ARforward, 59-TTCTTCGCCTGCTTTGTCCT-39 and reverse, 59-ATACCC-GTCACCAAGCCATT-39; and A2B forward, 59-CTGCTCATAATGC-TGGTGATCT-39 and reverse, 59-ATCAGTTCCATGCGCTGA-39. ForCD39 and CD73 expression, total mRNA was extracted from DC cultureharvested in different periods after LPS stimuli: CD39 forward, 59-AACT-CTCTGCAATTCCGTCTCT-39 and reverse, 59-ATGTCCTTGGCCAGT-TTCTG-39; CD73 forward, 59-CAAATCCCACACAACCACTGT-39 andreverse, 59-TATCTCTTGGGCCTCCATA-39.

Statistical analysis

Data are expressed as mean 6 SEM and are representative of two to fourindependent experiments. Results of individual experiments were notcombined, as they were analyzed individually. The means from differentgroups were compared by ANOVA, followed by Tukey’s test. Statisticalsignificance was set at p , 0.05.

ResultsSystemic treatment with SGE ameliorates CIA

We first investigated the potential therapeutic effect of P. papatasiSGE on the CIA model in DBA/1J mice. The disease was elicitedas described in Materials and Methods. Mice were treated withSGE (one salivary gland/animal) by i.v. route daily for 14 d fromthe first day of clinical manifestation of disease. Collagen-immunized mice treated with PBS developed the expected dis-ease progression, showing a prominent clinical severity score of

8.0 6 2.23 (Fig. 1A) and a high number of affected paws (3.25 60.51) (Fig. 1B) compared with naive mice. In contrast, SGEtreatment ameliorated disease severity over the entire observationperiod, displaying a significant reduction of 37.5% in the clinicalscores (5.0 6 1.17) (Fig. 1A) and 38.5% in numbers of arthriticpaws (2.0 6 0.52). Histologic examination of the knees at the endof the monitoring period (14 d of treatment) revealed that un-treated mice showed severe cellular infiltration (Fig. 1C, leftcolumns, 1D, right columns) and marked reductions in matrixproteoglycan (Fig. 1C, right columns), suggesting joint cartilagedamage. In contrast, these pathologic events were profoundly re-duced in SGE-treated animals (Fig. 1C, left columns, 1D, rightcolumns). Together, these data demonstrate that P. papatasi SGEattenuated development of CIA that could be clinically detected,and this activity can prevent progression of articular damage.Taking into account that the tissue damage observed in several

autoimmune diseases (including rheumatoid arthritis [RA]) is aconsequence of proinflammatory mediators released into the in-flammatory site (31), we investigated the concentration of TNF-a,IFN-g, IL-17, and IL-15 in affected ankle joints. Paw samplesfrom arthritic mice treated with PBS contained significantly higherconcentrations of all above-mentioned inflammatory cytokinescompared with those of naive mice (Fig. 2). In contrast, micetreated with SGE showed a significant reduction in the levels ofTNF-a (Fig. 2A), IFN-g (Fig. 2B), and IL-17 (Fig. 2C), but noeffect on IL-15 levels (Fig. 2D) compared with control PBS-treated arthritic mice.We previously demonstrated that PGE2 and IL-10 are involved

in the anti-inflammatory activity of P. papatasi SGE in OVA-induced immune peritonitis (19). Thus, we attempted to addresswhether SGE treatment attenuated CIA by inducing productionof these anti-inflammatory mediators. Levels of PGE2 and IL-10were evaluated in the inflamed paws of CIA mice. Supportingour hypothesis, the treatment of CIA arthritic mice with salivaenhanced the levels of PGE2 (Fig. 2E) and IL-10 (Fig. 2F) pro-duction compared with PBS-treated arthritic mice (Fig. 2E, 2F,respectively). These data suggest that sand fly SGE triggers hostanti-inflammatory mechanisms that may control progression ofarthritis and protects the joint from extreme damage.

Blocking of DC function by SGE treatment inhibits CD4+ T cellactivation

To elucidate the mechanism(s) of successful SGE therapeutictreatment on CIA, we investigated whether blocking the DC abilityto present Ag by SGE affects CD4+ T cell activation and, con-sequently, inhibits proinflammatory cytokine release during CIA.To this purpose, nonadherent inflammatory cells from spleen anddraining LNs of arthritic mice were cultured with syngeneic bonemarrow-derived DC (BMDC) pretreated overnight with SGE (8glands/ml) or PBS, followed by collagen (5 mg/ml) or mediumstimulation for 96 h, after which the cells were phenotyped.Collagen-pulsed BMDCmarkedly triggered an increase of CD4+

T cells and expression of IL-17 and IFN-g by these cells whencompared with BMDCs cultured with PBS only. Preincubation ofcollagen-pulsed BMDC with SGE reduced proliferating CD4+

T cells (56.0% reduction) as well as IL-17 (62.0% reduction) andIFN-g–positive cells (76.0% reduction) (Fig. 3A).To obtain more evidence for the role of SGE on CD4+ T cell

activation via DC inhibition, we magnetically purified CD4+

T cells from draining LNs and cultured them with BMDCs pre-incubated with PBS or SGE (8 glands/ml) overnight, followed bystimulation with collagen (5 mg/ml), plate-bound anti-CD3 (5 mg/ml), or medium for 96 h. As expected, BMDCs pretreated withPBS and pulsed with collagen induced an intense CD4+ lym-

The Journal of Immunology 4349

phoproliferative response and IL-17 production compared withnonpulsed BMDCs (Fig. 3B, 3C). There was a significant re-duction (49.0%; p , 0.05) in Ag-specific CD4+ lymph pro-

liferation when BMDCs were pretreated with SGE (Fig. 3B).Furthermore, IL-17 production was significantly inhibited (27.5%inhibition; p , 0.05) in the supernatant of cocultures preincubated

FIGURE 2. Decreased inflam-

matory cytokines in articular joints

from SGE-treated arthritic mice.

Ankle joints fr4om naive (s) or

PBS (◊)- or SGE (♦)-treated arthritic

mice were collected for determina-

tion of TNF-a (A), IFN-g (B), IL-17

(C), IL-15 (D), and IL-10 (F) levels

by ELISA and PGE2 (E) by RIA

in the homogenate supernatants. Re-

sults are expressed as mean 6 SEM,

n = 4 (naive) and 9–10 (arthritic

groups).

FIGURE 1. SGE from P. papatasi attenuated CIA. Naive (x) or collagen-immunized and challenged DBA/1 mice were injected i.v. daily with PBS (s) or

P. papatasi SGE (1 gland/animal) (d) for 14 d. Mice were monitored for disease progression, as indicated by clinical scores (A) and number of affected

paws (B). On day 15 of SGE treatment, mice were euthanized, the articular joints were harvested, and histopathologic analysis was performed. Knee joint

sections were stained by H&E (left row) or with safranin-O (right row), a proteoglycan red marker showing profound cartilage damage in PBS-treated mice

(absence of proteoglycan) and preservation of cartilage in SGE-treated mice (C). Quantification of cellular infiltrate was performed by ImageJ software

(National Institutes of Health) in 40 fields with original magnification 3400 for each animal per group (D). Morphometric histologic examination shows

markedly less cellular infiltration in the SGE-treated than in the PBS-treated group (black bar versus striped bar). Results show the mean 6 SEM, n = 4.#p , 0.05 compared with naive group, *p , 0.05 compared with PBS-treated group.

4350 PHLEBOTOMINE SALIVA ATTENUATES COLLAGEN-INDUCED ARTHRITIS

with SGE (Fig. 3B, 3C); however, treatment of DCs with SGE didnot alter either proliferative response or IL-17 production afterpolyclonal anti-CD3 stimulation (Fig. 3D, 3E). Together, theseresults suggest that SGE suppresses the specific immune responseby acting on APCs, blocking the Ag presentation process, and, asa consequence, inhibiting proinflammatory mediator release byCD4+ T cells involved in RA.

Adenosine and adenosine monophosphate are the majoranti-inflammatory molecules in P. papatasi SGE

To identify the molecule(s) responsible(s) for the successfultherapeutic activity of P. papatasi SGE on CIA, the salivary extractwas filtered using Microcon YM3 and separated into filtrate(representing molecules with mass ,3 kDa) or retentate (com-pounds with mass .3 kDa). Filtrate, retentate, and SGE (control)samples containing ∼16 mg protein/ml (equivalent to the proteinconcentration of 8 glands diluted in 1 ml) were tested in LPS-induced DC maturation. Following LPS stimuli, BMDCs pro-duced high levels of both TNF-a and IL-12p40 compared withmedium (Fig. 4A, 4B). When cultures were preincubated overnightwith SGE, significant inhibitions of both proinflammatory medi-ators were observed (Fig. 4A, 4B). Interestingly, similar inhib-itions of TNF-a and IL-12p40 synthesis were obtained with thefiltrate, but not with the retentate, suggesting the presence of ac-tive salivary molecule(s) with ,3 kDa (Fig. 4A, 4B). Anti-inflammatory cytokines such as IL-10 are also induced by LPSand may be upregulated by P. papatasi saliva (19). In fact, LPS-induced IL-10 production was increased upon preincubationof BMDCs with SGE or filtrate, but not with retentate salivaryfraction (Fig. 4C).

To obtain further information on the biochemical nature of thelow m.w. salivary molecule(s), the filtrate fraction was submittedto reverse-phase HPLC. The absorbance of eluted fractions wasmonitored at 258 and 280 nm. The chromatogram indicated twomajor peaks at 7 and 17 min of retention, having a large 258 nmabsorption compared with absorption at 280 spectrum with ex-pected retention times of the nucleosides adenosine (ADO) andadenosine monophosphate (59AMP), respectively (Fig. 4D). Thesesubstances were previously described by our group in the salivaryglands of P. papatasi (32, 33). Other compounds eluted at 40 min,although in minor concentrations, some of them also absorbing at258 nm (Fig. 4D). In fact, the highest inhibition of TNF-a pro-duction was achieved using fractions 7 and 17 (Fig. 4E), sug-gesting that ADO and 59AMP are the major salivary constituentswith modulatory activity on DCs. Confirming this hypothesis,treatment of LPS-stimulated DCs with similar amounts of 59AMPplus ADO mimics the SGE effect, that is, inhibits TNF-a and IL-12p40 production (Fig. 4F, 4G, respectively). Moreover, treatmentof the SGE filtrate fraction or 59AMP plus ADO with ADA, anenzyme that catabolizes ADO (34), prevented the inhibitory effectof SGE and 59AMP plus ADO on TNF-a and IL-12p40 pro-duction by LPS-stimulated DCs (Fig. 4F, 4G, respectively). Inaddition, enhancement of IL-10 by SGE was also abolished byADA-treated filtrate (Fig. 4H).

ADO and 59AMP modulate DC maturation

To determine the relative contribution of ADO and 59AMP in theSGE-dependent anti-inflammatory activity of SGE, a concentra-tion-response curve and time-course inhibitory profile were con-ducted using standard commercial nucleosides.

FIGURE 3. SGE treatment inhibits Ag-specific CD4+ T cell activation. BMDCs (106 cells/ml) from DBA/1 naive mice were incubated overnight with P.

papatasi SGE (8 glands/ml) or PBS. Nonadherent cells from LNs and spleen cells from CIA mice on day 35 of disease were added to BMDC culture,

followed by stimulation with or without collagen-II (5 mg/ml) for 96 h. Percentages of CD4+CD3+, CD4+IL-17+, and CD4+IFN-g+ cells were determined by

flow cytometry (A). Purified CD4+ T cells (107 cells/ml) from arthritic mice were added to BMDC culture, and lymphoproliferation induced by C-II (5 mg/

ml) (B) or plate-bound anti-CD3 (5 mg/ml) (D) for 96 h was assessed by [3H]thymidine incorporation. Culture supernatant was harvested to measure IL-17

levels from C-II– (C) or anti-CD3–stimulated (E) cultures. The results are expressed as the mean 6 SEM obtained from two or three independent

experiments made in triplicate (n = 3 per group). #p , 0.05 when compared with medium, *p , 0.05 when compared with stimuli, *p , 0.05 compared

with saliva vehicle (PBS).

The Journal of Immunology 4351

ADO alone had no significant effect on basal cytokines releasedby BMDC in the absence of stimulus (medium); however, BMDCpretreated overnight with ADO before stimulation with LPSresulted in inhibition of TNF-a production, whereas IL-10 pro-duction was stimulated (Fig. 5A). Both effects were observed ina concentration-dependent manner (Fig. 5A). Furthermore, theimmunomodulatory effect of ADO was partially observed evenwhen it was added to the culture 3 h after LPS administration intothe culture (Fig. 5C).The impact of ADO on the maturation of DCs was evaluated by

flow cytometry. Incubation of BMDCs with ADO alone for 24 hslightly affected MHC-II, CD86, and CD40 basal expression. Asexpected, exposure to LPS enhanced the expression levels of thesesurface markers. ADO did not alter the effect of LPS on the sur-face expression of CD86, although MHC-II and CD40 upregula-tion was decreased (Supplemental Fig. 1).The anti-inflammatory effect of 59AMP present in SGE gland

could be a consequence of extracellular ADO generation. In fact,similarly to ADO, 59AMP also inhibited TNF-a production andenhanced IL-10 release in a concentration-dependent manner (Fig.5B). Whereas the kinetics of TNF-a inhibition in the presence of59AMP followed the same pattern as that displayed by ADO, IL-10 enhancement was seen only when 59AMP was added to DCculture at 23.0, 20.5, and 0 h of LPS administration, but not atlater time points (Fig. 5D), suggesting that some amount of timeis required for 59AMP to generate ADO.Extracellular generation of ADO is mediated by two sequential

enzymes, as follows: ecto-ATP disphohydrolase (CD39), which

converts ATP and ADP to AMP and, subsequently, ecto-59-nu-cleotidase (CD73), which converts 59AMP to ADO (35). A recentstudy demonstrated that regulatory T cells (Treg) express bothenzymes, and that ATP metabolic disruption by CD39 and CD73is the mechanism by which these cells mediate immunosuppres-sion (36); however, whether both nucleotidases are expressed inDCs is still unknown. Thus, we analyzed CD39 and CD73 ex-pression in LPS-matured DCs as a putative mechanism by which59AMP could generate ADO and mediate a modulatory effecton DCs. Cytometric analysis revealed that both enzymes areexpressed in BMDCs. Whereas CD39 expression seems to beconstitutive and LPS activation did not alter its expression, CD73can be upregulated by the maturation process. LPS stimulationenhanced CD73 expression by 34% compared with medium alone(Fig. 5E, 5F). Similarly, expression of CD39 transcripts has notchanged upon LPS stimulus, whereas CD73 expression signifi-cantly increased under the same conditions (Supplemental Fig. 2A,2B). Together, these results clearly demonstrate that ADO, alreadypresent in saliva and/or generated by 59AMP metabolism, mostlikely accounts for most, if not all, anti-inflammatory activitypresented by P. papatasi SGE.

ADO enhances LPS-induced PGE2 production

In a previous study, we demonstrated that the inhibitory effect ofSGE on an Ag-immune inflammation model depends on sequentialproduction of PGE2 and IL-10 by DCs, which in turn acts in anautocrine manner, reducing the Ag-presenting ability of these cells(19). Taking into account that ADO and 59AMP are the molecules

FIGURE 4. ADO and 59AMP are the

major suppressive molecules in P. papatasi

SGE. BMDCs were preincubated overnight

with SGE (16 mg/ml) or SGE microcon

YM-3 fractions: retentate (.3 kDa) and

filtrate (,3 kDa), and then stimulated with

LPS (50 ng/ml) for 24 h. TNF-a (A), IL-12

p40 (B), and IL-10 (C) production were

measured in the culture supernatants. To

characterize DC modulator(s), the filtrate

fraction was chromatographed by reverse-

phase HPLC, as described in Materials and

Methods (D). The activity of each eluted

fraction was evaluated in LPS-induced

TNF-a production by BMDCs (E). Filtrate

or ADO + 59AMP commercial standards

were previously incubated with vehicle

(DMSO) or ADA (4.3 U) for 3 h before LPS

stimulation, and cytokine production (F–H)

was measured in the culture supernatant.#p , 0.05 compared with medium, *p ,0.05 compared with LPS-stimuli, &p ,0.05 when compared with vehicle treatment.

4352 PHLEBOTOMINE SALIVA ATTENUATES COLLAGEN-INDUCED ARTHRITIS

present on saliva responsible for such anti-inflammatory effects(Fig. 4) and that PGE2 was upregulated in the articular lesions ofarthritic mice treated with SGE (Fig. 2D), we next evaluated theimpact of ADO on prostanoid production. Because PGE2 is gen-erated by the cyclooxygenase (COX) pathway, we first in-vestigated the impact of incubation of LPS-stimulated DCs withADO by measuring COX-2 mRNA levels. As expected, LPS in-duced significant expression of COX-2 mRNA in DCs comparedwith medium alone. Moreover, preincubation of these cells withADO significantly enhanced expression of COX-2 transcripts afterstimulation (Fig. 6A). In terms of the COX-2 product, PGE2 levelswere induced by LPS stimulation, which was potentiated by ADOpreincubation (Fig. 6B). ADO alone had no considerable effect onbasal prostanoid release compared with medium.To confirm the involvement of ADO in PGE2 production, cells

were preincubated with 2-phenilaminoadenosine, a synthetic an-alog of ADO, for 3 h before LPS stimulation. Potentiation ofPGE2 production could be observed by 2-phenylaminoadenosinein a concentration-dependent manner (Fig. 6C). Furthermore, aninhibitory effect on TNF-a production by DCs was also achievedby an ADO synthetic analog (Fig. 6D). Similarly to PGE2, 2-phenilaminoadenosine also potentiated LPS-induced IL-10 pro-duction in a concentration-dependent fashion (Fig. 6E). Together,these results indicate that ADO has an important impact on thecapacity of DC to generate PGE2.

ADO present in SGE affected DC function during theAg-presenting process and attenuated CIA

We investigated whether ADO inhibits DC function and de-termined which ADO receptors were expressed in BMDCs.

Transcripts for A2A and A2B receptors (A2AR and A2BR) were

detected in unstimulated cells, with A2BR being highly expressed

in resting cells (Fig. 7B). After LPS stimulation, the transcript

profile completely changed, with significant increase in A2AR

(Fig. 7A) and decrease in A2BR expression (Fig. 7B). Given that

the immunoregulatory effect of ADO is attributed to its engage-

ment to A2AR (37) and was enhanced after inflammatory stimu-

lation (Fig. 7A), the stable and selective A2AR antagonist (8,3,

clorosterylcafeine) was tested on the SGE effect on BMDC-driven

CD4+ T cell activation. The A2AR antagonist was added to DC

culture 2 h before SGE addition, and DC function was assessed by

Ag-induced lymphoproliferative response and IL-17 production.

As expected, Ag-induced proliferation and IL-17 production were

inhibited by BMDCs treated with SGE. This impaired ability of

BMDCs treated with SGE to induce proliferation and IL-17 pro-

duction by CD4+ T cells was completely prevented by A2AR

blockage, suggesting that ADO mediates the immunosuppressive

action of SGE on DCs through this receptor (Fig. 7C, 7D, re-

spectively). Corroborating this hypothesis, standard commercial

ADO also affected DC function, suppressing Ag proliferative re-

sponse and IL-17 production (Fig. 7C, 7D). Interestingly, A2AR

antagonist potentiated both proliferative response and IL-17 pro-

duction, even in the absence of SGE, suggesting the participation

of endogenous ADO in this process.We examined the effect of nucleosides on CIA. In this set of

experiments, arthritic mice were treated daily with comparable

amounts of 59AMP and ADO present in one salivary gland pair (20

mM each ones). Nucleoside treatment was as effective as SGE in

ameliorating established CIA (Fig. 7E, 7F). To confirm that ADO

FIGURE 5. Effects of ADO and

59AMP on TNF-a and IL-10 pro-

duction by LPS-stimulated DCs.

BMDCs were incubated without

(open bars) or with (shaded bars) 50

ng/ml LPS for 24 h after overnight

preincubation with increasing con-

centrations of ADO (A) or 59AMP

(B) (mM). Cytokine production was

measured in the culture supernatants

by ELISA. Time-course inhibition

of cytokine production by 100 mM

ADO (C) or 59AMP (D) in LPS-

stimulated BMDCs. The results are

expressed as the mean 6 SEM

obtained from one of three in-

dependent experiments made in

triplicate (n = 3 per group). #p ,0.05 compared with medium, *p ,0.05 compared with LPS stimuli.

Surface molecules were labeled with

FITC or PE conjugated with anti-

CD11c, anti-CD39, or anti-CD73;

representative mean fluorescence

intensity (MFI) of BMDC staining

from medium or LPS cultures is

shown in each box (E). The filled

histograms represent cells labeled

with the specific mAb; the empty

histograms represent the same cell

suspension labeled with isotypic

control mAb. F, Bars display the

relative MFI obtained from one of

four independent experiments made

in quadruplicate (n = 4). *p , 0.05

compared with medium.

The Journal of Immunology 4353

and 59AMP are the compounds of P. papatasi SGE with therapeuticeffect on CIA, we have depleted both nucleosides by treating SGEwith ADA for 3 h. Strikingly, the therapeutic effect of SGE on CIAwas completely abolished when SGE was deaminated by ADA(Fig. 7G, 7H). CIAmice that received ADA-treated SGE display nodifference in clinical scores and numbers of affected paw comparedwith control group (ADA-treated vehicle). Taken together, theseresults show that ADO and AMP are, in fact, the active constituentspresent into P. papatasi saliva responsible for anti-inflammatoryactivity and amelioration of CIA symptoms.Next, we investigated the effectiveness of nucleosides in two new

experiments with different protocols of treatment of arthritic mice.To test whether salivary nucleosides have a therapeutic effect whenthe disease is already established, arthritic mice were treated whendisease score reached 6 (representing a higher disease progression).In this condition, nucleoside treatment was not effective in reducingarthritis severity, because the clinical scores (Supplemental Fig.3A) and numbers of affected paw (Supplemental Fig. 3B) weresimilar to those of vehicle-treated mice. In the second protocol,mice were treated only during the first week after disease onset.The beneficial effect of the therapy with the nucleosides lastedonly for a few days after the treatment was stopped. Once thetreatment ended, both clinical scores (Supplemental Fig. 3C) andnumbers of affected paw (Supplemental Fig. 3D) reached the samelevel as those of vehicle-treated mice. Thus, salivary nucleosidespresent a beneficial effect on CIA only when administered ondisease onset and maintained during all periods of disease.

IL-10 released during treatment contributes toanti-inflammatory activity of nucleosides on CIA

We demonstrated that both PGE2 (Fig. 2E) and IL-10 (Fig. 2F)were increased in the paws of SGE-treated arthritic mice. To as-sess whether these mediators are responsible for SGE anti-inflammatory activity in vivo, we first tested the effect of IL-10depletion using specific Ab on treated CIA mice. Treatment ofmice with anti–IL-10 Ab initiated at the onset of disease abrogatedthe animals from protective effect of nucleosides on CIA. Theanti–IL-10 Ab concomitantly administered with ADO plus AMP

(a–IL-10/ADO + AMP) induced in arthritic mice similar evolu-tion of clinical scores as those observed in control groups (vehicle/IgG control and vehicle/anti–IL-10) (Fig. 8A). In addition, thenumbers of affected paw also were comparable to control groups(Fig. 8B). We have attempted to block endogenous PGE2 pro-duction using a COX-2 selective inhibitor, Rofecoxibe. However,mice succumbed within 1 wk of Rofecoxibe treatment, indicatinga side effect of the COX2 inhibitor (38).We further evaluated DC maturation stage in vivo after treat-

ment. Nucleosides (ADO + AMP) or vehicle (PBS) were admin-istered onset of CIA development daily for 14 d. Draining LNswere harvested, and DC maturation was analyzed by flowcytometry through CD11c and MHC-II markers on surfacemembrane. As expected, naive mice exhibited low frequency ofCD11cMHC-IIhigh, and arthritic mice treated with vehicle en-hanced the expression levels of these surface markers, suggestingthat DC was matured in vivo during disease progression (Fig. 8C,8D). Interestingly, similarly to in vitro, i.v. injection of ADO plusAMP exhibited a significant downregulation of DC maturationwith reduction of CD11cMHC-IIhigh that reached the basal levels,similar to naive group. These findings indicate that DCs exhibitselective defects induced by saliva that was associated with DCmaturation. Such inhibitory effect could be, at least in part, de-pendent of IL-10 release induced by saliva or fractions. Accord-ingly, the i.p. administration of Ab against IL-10 (anti–IL-10)during nucleoside treatment reversed the immunosuppressiveeffect of salivary fractions, potentiating the expression ofCD11cMHC-IIhigh surface markers on DC from ADO plus AMP-treated arthritic mice. Taken together, our data suggest that IL-10,and perhaps PGE2, released in the site of inflammation stronglycontributes to anti-inflammatory activity of saliva or nucleosideson CIA.

DiscussionIn the current study, we report the therapeutic effect of P. papatasiSGE in controlling the pathogenesis of CIA. We purified andidentified ADO and 59AMP as the active pharmacologic compo-nents in SGE responsible for such immunomodulatory activity.

FIGURE 6. ADO potentiates COX2 mRNA expres-

sion and PGE2 production induced by LPS stimulation.

COX2 mRNA quantification (A) and PGE2 production

(B) were quantified in BMDCs preincubated with ADO

or medium with or without LPS stimulation for 24 h.

The indicated concentration of adenosine synthetic

analogous (2-phenylaminoadenosine) was added into

BMDC culture 3 h before LPS stimuli. PGE2 (C), TNF-a

(D), and IL-10 (E) levels were detected 24 h later.

PGE2 was measured by RIA, and cytokines by ELISA.

The results are expressed as the mean 6 SEM obtained

from one of three independent experiments made in

triplicate (n = 3 per group). #p , 0.05 compared with

medium, *p , 0.05 compared with the control (LPS).

4354 PHLEBOTOMINE SALIVA ATTENUATES COLLAGEN-INDUCED ARTHRITIS

Indeed, ADO and 59AMP blocked Ag presentation by DCs, in-terfering with Th17 cell activation and consequently suppressingthe inflammatory immune response. These data indicate that P.papatasi salivary compounds could be a selective therapy focusingon DC function during the effector phase of the immune responseas well as targets to treat CIA.SGE treatment at the onset of disease attenuated the severity of

arthritis and protected joint tissue from degradation. In addition,SGE was associated with a marked decrease of CD4+ Th17 lym-phocyte activation. Importantly, IL-17 produced by CD4+ T lym-phocytes is a key cytokine implicated in human autoimmunedisease such as RA (31). In arthritis models, IL-17 promotes ac-tivation of synovial fibroblast and both leukocyte emigration andactivation, resulting in the production of several mediators in-volved in the inflammatory response and tissue lesions, includ-ing cytokines (TNF-a, IL-1b), chemokines (MIP-2/CXCL2, KC/CXCL1, IL-8/CXCL8), adhesion molecules (ICAM-1), receptoractivator of NF-kB ligand, and matrix metalloproteinases (39, 40).The therapeutic efficacy of SGE treatment in collagen-inducedrheumatoid arthritis seems to be due to blocking DC Ag pre-

sentation and consequently the CD4+ Th17 cell response sup-pression. Consistently, SGE selectively inhibits specific (i.e.,collagen), but not polyclonal stimulation, which directly stimu-lates lymphocytes, confirming the interference of P. papatasi salivain DC function. Interestingly, salivary nucleosides present abeneficial effect on CIA only when administered on disease onsetand maintained during all periods of disease. This finding furthersupports the notion that in the absence of saliva treatment, DCis constantly activated and contributed to severity of CIA.In agreement with our results, saliva from other blood-feedingarthropods, such as Ixodes scapularis ticks (41), the Lyme dis-ease vector, and Ixodes ricinus (42), also has been demonstratedto block the DC-dependent Ag-presenting process and attenuatedexperimental autoimmune encephalitis (43).During their evolutionary process, blood-sucking arthropods

developed efficient mechanisms to overcome the host immuneresponse and thus successfully complete the blood meal (2). Weidentified ADO and 59AMP as the active molecules present in thesalivary fraction of P. papatasi that inhibit DC activation. Mark-edly, the immunomodulatory effects were similarly observed with

FIGURE 7. Blocking A2AR prevents SGE immunosuppressive effect on DC function and 59AMP + ADO treatment-attenuated CIA. BMDCs (106 cells/

ml) were incubated 6 ADO (100 mM). Cells were harvested 24 h after LPS stimulation for A2AR (A) and A2BR (B) mRNA expression quantification.

Purified CD4+ T cells (107 cells/ml) from arthritic mice were added into BMDC culture overnight, pretreated with PBS, SGE (8 glands/ml), or ADO (100

mM). Specific A2AR antagonist was added into some wells 2 h before nucleoside or saliva treatment. IL-17 production (C) was measured by ELISA, and

lymphocyte proliferation by [3H]thymidine incorporation (D) was determined 96 h after C-II stimulation (5 mg/ml). #p , 0.05 when compared with

medium, *p , 0.05 compared with stimuli, &p , 0.05 compared with salivary nucleosides, %p , 0.05 compared with A2AR antagonist. Collagen-im-

munized DBA/1 mice were injected i.v. daily with salivary equimolar concentration of 59AMP + ADO (20 mM each ones) (d) or PBS (s) for 14 d after

start of disease. Mice were monitored for disease progression, as indicated by clinical scores (E) and numbers of affected paws (F). *p , 0.05 compared

with PBS-treated group. Results show the mean 6 SEM; n = 10. In some groups, salivary gland extract or vehicle (PBS) was previously incubated with

ADA (4.3 U) for 3 h. Afterward, collagen-immunized and challenged DBA/1 mice were injected i.v. daily with ADA-treated vehicle (s) or ADA-treated

P. papatasi SGE (1 gland/ animal) (d) for 14 d. Mice were monitored for disease progression, as indicated by clinical scores (G) and number of affected

paws (H). Results show the mean 6 SEM, n = 5.

The Journal of Immunology 4355

whole saliva and commercial ADO plus 59AMP. Moreover, thedeamination of ADO with ADA, an enzyme that catabolizes ADO(34), maintains the maturation of BMDCs induced by LPS. Ac-cordingly, ADO has a broad range of effects on inflammatoryleukocytes, including DCs, downregulating the production ofproinflammatory mediators and expression of costimulatorymarkers, which diminish the capacity of DCs to initiate and am-plify the inflammatory immune response (44).ADO effects are mediated by four surface receptors, named

A1R, A2AR, A2BR, and A3R, which are members of the G

protein-coupled receptor family (45). Whereas A1R and A3R are

coupled to Gi (inhibitory) proteins and mediate the inhibition of

adenylyl cyclase, A2AR and A2BR interact with Gs (stimulatory

proteins) and activate adenylyl cyclase, generating the second mes-

senger cyclic ADO monophosphate (cAMP) that downregulates

host cell activation (46). Although all four ADO receptors are

present on human and mouse peripheral blood monocytes and

mature DCs, genetic ablation or pharmacologic blocking of A2AR

or A2BR leads to overexuberant immune responses, indicating that

A2A/BR are involved in the anti-inflammatory and immunoregu-

latory role of ADO (45, 47). In this study, we show that ADO has

little or no measurable effect on cytokine production on resting

(nonstimulated) DCs; however, in the context of LPS stimulation,

ADO significantly modulates these markers, suggesting that ADO

receptors are modulated along the stages of cellular activation. In

fact, LPS-induced DC maturation results in downregulation of

A2BR expression, whereas A2AR is highly expressed. Therefore, it

seems that A2AR is the receptor responsible for ADO effects on

DCs. Consistent with our results, Panther et al. (48) demonstrated

that ADO by itself has only a very limited capacity to modulate

DCs, whereas this nucleoside provides prominent inhibition ofLPS-stimulated DCs through activation of the A2AR.Further exploring the biologic action of salivary nucleosides on

DC function, similarly to SGE, ADO showed inhibitory effects oncollagen-induced CD4+ T cell response. Interestingly, in thepresence of 8,3,cloroesterylcafeine (a selective A2AR antagonist),the inhibitory effect of SGE on CD4+ Th17 activation was pre-vented; however, such a profile was exacerbated even in the ab-sence of SGE, indicating the involvement of endogenous ADOdownregulating the inflammatory process. The exacerbation ofinflammation through blocking the A2AR has been demonstratedin a variety of inflammatory models. Treatment of mice with theselective A2AR antagonist ZM241385 potentiated liver injury andinflammation in response to Con A, Pseudomonas aeruginosa, andcarbon tetrachloride stimuli (45). Moreover, ZM241385 preventedboth anti-inflammatory effects and survival induced by low dosesof ketamine, which promotes accumulation of ADO in mice whensepsis is induced by LPS or Escherichia coli (49).The immunomodulatory effect of 59AMP present on P. papatasi

SGE is due to extracellular ADO generation. Extracellular ADOgeneration on inflammation foci is mediated via CD39, ecto-ATPdiphosphohydrolase, which rapidly converts circulating ATP andADP to 59AMP; subsequently, 59AMP is converted to extracel-lular ADO by ecto-59-nucleotidase (CD73) (36). Recent studiesshow that Tregs express high levels of CD39 and CD73 on theirsurface, and ADO generation is a mechanism by which Tregsexert their suppressive effects (50). Monocytes also express CD39and CD73, and these enzymes are active under inflammatoryconditions (51, 52). Nevertheless, the expression of both ectonu-cleotidases on the DC surface has not been demonstrated to date.Our data demonstrate that both ectonucleotidases are expressed in

FIGURE 8. Suppressive effect of salivary nucleosides in vivo is dependent of IL-10. At the onset of arthritis symptoms, naive or collagen-immunized and

challenged DBA/1 mice injected i.v. daily vehicle (PBS) or ADO + AMP were concomitantly treated (250 mg/mice; i.p. route) with normal rat IgG (IgG

control) or rat anti–IL-10 (a–IL-10) Ab three times per week. Mice were monitored for disease progression, as indicated by clinical scores (A) and number

of affected paws (B). Results show the mean 6 SEM, n = 5. *p , 0.05 compared with vehicle /IgG control. On day 15 of treatment, mice were euthanized,

the draining LNs were harvested, and DC surface molecules were labeled with anti-CD11c or anti-MHC class II mAbs conjugated with FITC and PE.

Representative dot plot of DC staining from naive, vehicle, or ADO + 59AMP-treated mice injected with a–IL-10 or rat IgG control is shown in each box

(C). D, Bars display the relative MFI, and results show the mean 6 SEM, n = 5. #p , 0.05 compared with naive mice, *p , 0.05 compared with vehicle /

IgG control, &p , 0.05 compared with vehicle/IgG control.

4356 PHLEBOTOMINE SALIVA ATTENUATES COLLAGEN-INDUCED ARTHRITIS

DCs. Although CD39 appears to be constitutive, the expressionof CD73 is increased by proinflammatory stimuli. Thus, the con-comitant expression of both enzymes on the surface of DCs helpsto generate ADO in the extracellular compartment by cleaving59AMP present in the saliva of P. papatasi, contributing to pre-vention of the tissue damage observed in CIA. Accordingly,59AMP represents a slow-release formulation of ADO, in additionto the preformed ADO present in P. papatasi saliva.Although the increased levels of cAMP seem to be due, at least in

part, to interaction and activation of A2AR, triggering a cascade ofinhibitory factors of the inflammatory response, this interactioncould also lead to an increase in expression of COX-2 (44, 53).Once activated, this enzyme converts arachidonic acid to PGG2,and subsequently to PGH2, which is rapidly metabolized by spe-cific isomerases to the potentially active prostanoids PGD2, PGE2,PGF2a, PGI2, and thromboxane A2. Our data show that eitherADO or A2R agonist was able to induce enhancement of PGE2

production by LPS-stimulated BMDCs. Prior evidence suggeststhat incubation of neutrophils with A2AR agonist increases ex-pression of COX-2, leading to production of PGE2 via cAMPstimulation (53). Furthermore, incubation of polymorphonuclearcells with ADA drastically reduces prostanoid production, withdownregulation of cAMP and COX2, establishing a direct re-lationship between ADO and PGE2 (53).A great body of evidence shows that PGE2 and IL-10 are potent

anti-inflammatory mediators, limiting tissue damage on differentexperimental autoimmune diseases, including RA (54, 55). Bothmediators were increased in the paws of SGE-treated arthriticmice, and treatment of mice with anti–IL-10 mAb blocked theprotective effect of nucleosides. Previously, we reported that SGEinhibits immune inflammation by sequential production of PGE2/IL-10 acting autocrinally on DC function (19). Thus, it is rea-sonable that both PGE2 and IL-10 contribute to the therapeuticsuccess of SGE on CIA.In addition to PGE2 and IL-10 effect, we have shown that DCs

from LNs of treated arthritic mice also present a downregulationof MHC class II molecules on their surface in vivo. Importantly,we also demonstrated that such effect was also dependent on IL-10 production. Although we have not evaluated whether the in-hibitory effect of salivary nucleosides on DC affects T cell re-sponse in vivo, we have strong evidence that its function isimpaired. We have previously published that the in vivo treatmentof OVA-immunized mice with SGE reduced the Ag-inducedproliferation of splenic T cells (19). Interestingly, saliva affectedthe proliferation of OVA-specific CD4+ T cells through DC in-hibition, whereas the incubation of OVA-CD4+ T cells with SGEhas not altered their proliferative response (19). The reduction inarthritis progression mediated by impairment of DC function hasbeen demonstrated by others. For instance, treatment of arthriticmice with type II collagen-pulsed tolerogenic DCs, which presentsa semimature phenotype (reduced the MHC-II, CD40, CD80, andCD86 costimulatory molecules on DC surface expression andreduced levels of inflammatory cytokines), decreases the pro-portion of pathogenic Th17 cells, reducing the severity and pro-gression of arthritis in these animals (56). Moreover, thehyporesponsiveness of CD4+ T or CD8+ T lymphocytes is due toimmunosuppressive mediators released by DC (i.e., IL-10, TGF-b,and PGE2) (57). Furthermore, DCs downmodulated by innateimmunity improve CIA and induced Tregs in vivo (58). Thus, it isplausible to infer that the i.v. administration of whole extract ornucleosides (adenosine and adenosine monophosphate) presentinto P. papatasi saliva ameliorates CIA by inhibiting the effector’sphase of the immune-specific response through inhibition of theDC activation.

It was not the aim of the current study to determine themechanism that SGE interferes in Ag-presenting process. None-theless, we cannot rule out the possibility that other salivarycomponents inhibit Ag degradation and/or the displacement ofCLIP peptide associated to MHC class II binding groove, pre-venting loading of antigenic peptides present on the membranesurface and subsequently affecting presentation to CD4+ T cells.In fact, it has been demonstrated that Sialostatin L, a cysteineprotease inhibitor isolated from I. scapularis tick saliva, inhibitsLPS-induced maturation of DCs, as well as it binds cathepsin Sinside lisosomal compartment, thus blocking Ag-specific T cellproliferation (43).An interesting finding of our study was that the amount of SGE

that effectively reduced RA had low amounts of both salivaryprotein and nucleosides (∼2 mg protein and 20 mM 59AMP andADO). Stressing the relevance of our finding, we demonstratedthat treatment of arthritic mice with similar amounts of each nu-cleoside reduced CIA symptoms. Interestingly, the therapeuticeffect of SGE on CIA was completely abolished when SGE wasdeaminated by ADA. Furthermore, several studies support thehypothesis that the efficacy of methotrexate on experimental andhuman RA is due to its ability to increase the levels of ADO (59).Indeed, methotrexate induces accumulation of aminoimidazo-carboxamide, a competitive inhibitor of ADA, thus allowing in-creased levels of ADO (60) and inhibition of chondrocytes (61)and synovial cells (62).In conclusion, the results presented in this article indicate that

ADO and 59AMP are present in P. papatasi SGE in pharmaco-logical concentrations and mediate the anti-inflammatory activityon RA. These constituents act through A2AR in the effector phaseof the inflammatory process, inhibiting the ability of DCs topresent Ag and thus leading to suppression of CD4+ Th17-inducedinflammatory immune response. Our data support the idea thatA2AR activation could be a novel therapeutic strategy for RA andother destructive chronic disorders.

AcknowledgmentsWe are thankful to Giuliana Bertozi for help with ELISAs, Maria Helena

Riul for help with histologic assessment, and to National Institute of Allergy

and Infectious Diseases intramural editor Brenda Rae Marshall for assis-

tance.

DisclosuresThe authors have no financial conflicts of interest.

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