research article kaurenoic acid possesses leishmanicidal...

Research ArticleKaurenoic Acid Possesses Leishmanicidal Activity by Triggeringa NLRP12IL-1120573cNOSNO Pathway

Milena Menegazzo Miranda1 Carolina Panis2 Suelen Santos da Silva1

Juliana Aparecida Macri1 Natalia Yoshie Kawakami1 Thiago Hideki Hayashida3

Tiago Bervelieri Madeira3 Vinicius Ricardo Acquaro Jr3 Suzana Lucy Nixdorf3

Luciana Pizzatti4 Seacutergio Ricardo Ambroacutesio5 Rubens Cecchini1 Nilton Syogo Arakawa3

Waldiceu Aparecido Verri Jr1 Ivete Conchon Costa1 and Wander Rogeacuterio Pavanelli1

1Department of Pathological Sciences Center of Biological Sciences State University of Londrina 86057-970 Londrina PR Brazil2Laboratory of Inflammatory Mediators State University of Western Parana 85605-010 Francisco Beltrao PR Brazil3Department of Chemistry Center of Exact Sciences State University of Londrina 86057-970 Londrina PR Brazil4Department of Biochemistry Federal University of Rio de Janeiro 21941-909 Rio de Janeiro RJ Brazil5Nucleus of Research in Exact and Technological Sciences University of Franca 14404-600 Franca SP Brazil

Correspondence should be addressed to Wander Rogerio Pavanelli wanderpavanelliyahoocombr

Received 9 December 2014 Accepted 24 April 2015

Academic Editor Nina Ivanovska

Copyright copy 2015 Milena Menegazzo Miranda et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Leishmania amazonensis (L amazonensis) infection can cause severe local and diffuse injuries in humans a condition clinicallyknown as American cutaneous leishmaniasis (ACL) Currently the therapeutic approach for ACL is based on Glucantime whichshows high toxicity and poor effectiveness Therefore ACL remains a neglected disease with limited options for treatment Hereinthe in vitro antiprotozoal effect and mechanisms of the diterpene kaurenoic acid [ent-kaur-16-en-19-oic acid] (KA) against Lamazonensis were investigated KA exhibited a direct antileishmanial effect on L amazonensis promastigotes Importantly KAalso reduced the intracellular number of amastigote forms and percentage of infected peritoneal macrophages of BALBc miceMechanistically KA treatment reestablished the production of nitric oxide (NO) in a constitutive NO synthase- (cNOS-) dependentmanner subverting the NO-depleting escape mechanism of L amazonensis Furthermore KA induced increased production of IL-1120573 and expression of the inflammasome-activating component NLRP12 These findings demonstrate the leishmanicidal capabilityof KA against L amazonensis in macrophage culture by triggering a NLRP12IL-1120573cNOSNO mechanism

1 Introduction

American cutaneous leishmaniasis (ACL) is a devastatingillness caused by the protozoa Leishmania spp ACL displaysdistinct clinical manifestations depending on both the para-site strain and the capability of the host to mount an effectiveimmune response Therefore this disease may clinicallyappear in the host as cutaneous mucocutaneous or diffuseforms [1]

The treatment of ACL is based on a highly toxicchemotherapy with the antimonials sodium stibogluconate(Pentostam) and antimonate N-methyl-glucamine (Glucan-time) In case of lack of response second-line drugs such

as amphotericin B or pentamidines are used [2] Howeverthese drugs frequently exhibit high toxicity which has beenrelated to its restricted use and resistance resulting in morerestrictions in chemotherapy [2ndash5] These considerationsreveal the urgency to develop new therapeutic agents for thetreatment of this disease

Therefore the search for more effective and less toxicchemotherapeutic agents for the treatment of ACL is increas-ing There are various reported studies of synthetic com-pounds and natural products as potential sources of leish-manicidal activity [6 7] An interesting molecule towardsthis aim is kaurenoic acid [ent-kaur-16-en-19-oic acid] (KA)

Hindawi Publishing CorporationMediators of InflammationVolume 2015 Article ID 392918 10 pageshttpdxdoiorg1011552015392918

2 Mediators of Inflammation

a diterpene obtained from various Brazilian plants [8 9]Thismolecule has been reported as showing a wide variety of bio-logical activities such as antiprotozoal [10 11] antimicrobial[12] antinociceptive [13] vasorelaxant hypotensive [14 15]and anti-inflammatory [13 16 17] activities Moreover someimmunomodulatory properties of KA have been reported inthese models

The antiprotozoal activity of this diterpene involves itsdirect action in altering cell membrane integrity and mito-chondrial membrane depolarization in promastigote andamastigote forms of L amazonensis [18] and epimastigoteforms of Trypanosoma cruzi [19] respectively This directactivity of KA against the protozoa is not enough to reflectits overall potential as a therapeutic leishmanicidal drugsince Leishmania spp parasites replicate intracellularly inmacrophages and have several escape mechanisms againstmicrobicidal molecules that are not dependent on the directaction of antileishmanial drugs [20ndash23]

Members of the Nod-like receptor (NLR) family ofproteins have emerged as important innate immune sen-sors of pathogen-associated molecular patterns (PAMPS)and damage-associated molecular patterns (DAMPS) [24]NLRs are the key components of the inflammasome thatregulate the maturation of the potent inflammatory cytokineinterleukin- (IL-) 1120573 [25]

Accordingly Lima-Junior et al found that the NLRP-3inflammasome is engaged in the response against L ama-zonensis restricting parasite replication Additionally IL-1120573seems to be important for host resistance to infection byinducible (i) NOS-mediated production of NO a major hostdefense mechanism against Leishmania spp [26]

Taking into account the above-mentioned evidenceKA was evaluated for its in vitro effect on susceptiblemacrophages from BALBc mice infected with L ama-zonensis promastigote forms Accordingly we performedin vitro assays to investigate the direct effect of KA onparasites as well as its modulatory action on Leishmania-infected macrophages We further investigated a putativemechanism of action of this compound as a modulator ofproinflammatory molecules such as oxygen reactive speciesNO cytokines and inflammasome

2 Materials and Methods

21 Parasite L amazonensis (MHOMBR1989166MJO)was used in promastigote forms kept in culture medium199 (Invitrogen-GIBCO) supplemented with 10 fetal bovineserum (Invitrogen-GIBCO) 1M Hepes 01 human urine01 L-glutamine 10UmL penicillin and 10 120583gmL strep-tomycin (Invitrogen-GIBCO) and 10 sodium bicarbonate(complete medium for promastigotesmdashCMP) Cell cultureswere incubated at 25∘C in 25 cm2 flasks

22 Animals Female BALBc mice weighing approximately25ndash30 g and aged 6ndash8 weeks were obtained from FundacaoOsvaldo Cruz FIOCRUZ Curitiba Brazil Mice were keptunder pathogen-free conditions and used according to proto-cols approved by the Ethics Committee of the State University

of Londrina (protocol number 33064201242) Every effortwas made to minimize the number of animals used and theirsuffering

23 Plant Material KA used in this paper was obtainedfrom Sphagneticola trilobata The crude extract was obtainedfrom dried roots which were pulverized and extracted withdichloromethane and partitioned with n-hexane and ethylacetate all solvents were dried under reduced pressure Thehexane fraction was subjected to vacuum liquid chromatog-raphy (VLC) by increasing gradient polarity The secondfraction produced an amorphous compound (200mg) whichwas washed with cold methanol and analyzed by high per-formance liquid chromatography (HPLC) methods yielding96 purity The identification was performed by 1H and13 C nuclear magnetic resonance (NMR) electron impactmass spectrometry (EIMS) and comparison with literaturedata [27] The stock solution of KA was dissolved in 2dimethyl sulfoxide (DMSO) (Invitrogen-Gibco) HoweverDMSO concentration did not exceed 02 in all experiments

24 Viability of Promastigotes Theviability of L amazonensispromastigote forms treated with KA was evaluated using the3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bro-mide (MTT) assay as previously described [28] Promastigoteforms (106100 120583L) were incubated with different concentra-tions of KA (10 30 50 70 and 90 120583M) or with KA solvent(02 DMSO) and maintained in culture for 24 48 and 72 hat 25∘C Thereafter 10 120583L of MTT (5mgmL) were addedfollowed by incubation for an additional 4 h at 24∘C TheMTT formazan product was diluted with 300120583L of DMSOtransferring to 96-well plates and measured in a spectro-photometer with absorbance determined at 550 nm Theresults were expressed as percentage MTT reduction relativeto the control group calculated as the following formula(viable promastigotes) = (OD of drug-treated sampleODof untreated sample) times 100

25 Cell Proliferation Kinetics Promastigote forms (106mL)incubated in CMP were treated with different concentrationsof KA (10 30 50 70 and 90 120583M) or with KA solvent (02DMSO) and cultured for 5 days at 25∘C Promastigotes werecounted in a Neubauer chamber after 24 48 72 and 120 h

26 Phagocytic Assay Macrophages (5 times 105mL) wereobtained from the peritoneal cavity by the injection of 2mLof RPMI 1640 culture medium (Invitrogen-GIBCO) supple-mented with 10 fetal bovine serum (Invitrogen-GIBCO)and cultured on 24-well plates containing 13mm diameterglass coverslips Cells were preincubated with 200120583L ofRPMI medium for 2 h for adherence and incubated withpromastigote forms (5 1) for 2 h KA (50 70 or 90120583M) ormedium was added followed by 24 h incubation at 37∘C and5 CO

2 The cells were stained with Giemsa to establish

the phagocytic index of infection (by percentage) and theparasitesmacrophage (mean) The supernatant was utilizedto measure the levels of malondialdehyde (MDA) total

Mediators of Inflammation 3

antioxidant capacity of plasma (TRAP) nitric oxide (NO)and cytokines

27 Measurement of the Total Antioxidant Capacity of Samples(Trapping Antioxidant Parameter (TRAP)) Samples (50 120583Lof supernatant with cells) obtained from the phagocyticassay were analyzed as previously described by Repetto etal [29] by using the chemiluminescence-based methodSoluble vitamin E (Trolox) was employed as a standardantioxidant The chemiluminescence curves were obtainedusing the Glomax luminometer (Promega) and the resultswere expressed in nM Trolox

28 Measurement of Malondialdehyde Levels (MDA) by HighPerformance Chromatography MDA levels were determinedas resultant of oxidative stress occurrence which causes lipidperoxidation and the production of this metabolite Accord-ingly we used HPLC as previously described by Victorinoet al [30] with slight modifications The analyses were con-ducted with an Alliance e2695 HPLC (Waters Milford MAUSA) equipped with a SecurityGuard ODS-C18 (4 times 30mmPhenomenex) C18 reverse phase column (Eclipse XDB-C18 46 times 250mm 5 120583m Agilent) and a photodiode arraydetector (Photodiode Array Detector (PDA) 2998) Analyseswere conducted using Empower 2 software (Waters MilfordMA USA) MDA standards were prepared using 1133-tetraethoxypropane (TEP) Aliquots containing 250 120583L ofcells + supernatants were deproteinized by adding 20trichloroacetic acid and reacted with 1mL of thiobarbituricacid The mobile phase was 70 10mM KH

2PO4buffer pH

70 and 40HPLC-grade methanol Readings were obtainedat 532 nm following an 8min isocratic flow at the rate of1mLmin The results were expressed in nMMDA

29 Determination of Nitrite Levels as Estimate of NOProduction The determination of nitrite in supernatantscollected from the phagocytic tests was used to measurethe concentration of nitric oxide (NO) according to Paniset al [31] with some modifications Briefly the supernatantaliquotswere deproteinized by adding 50120583Lof 75mMZnSO

4

and 70 120583L of NaOH and shaking and centrifuging for 5minat 10000 rpm and 25∘C The supernatant was recovered anddiluted in glycine buffer (45 gL pH 97) Cadmium granuleswere rinsed with distilled sterile water and added to a 5mMCuSO

4in glycine-NaOH buffer (15 gL pH 97) followed by

5min incubation and the copper-coated cadmium granuleswere used within 10min Activated granules were added toglycine buffer-diluted supernatant and the suspension stirredfor 10min Aliquots of 200120583L were recovered in appropriatetubes for nitrite determination and the same volume ofGriess reagent was added After 10min incubation at roomtemperature tubes were centrifuged at 10000 rpm for 2minat 25∘C and added to 96-well microplates in triplicate Acalibration curvewas prepared using dilutions ofNaNO

2 and

the absorbance was determined at 550 nm in a microplatereader

210 Cytokine Determination The supernatants obtainedfrom the phagocytic assay were used to determine the levelsof IL-1120573 IL-12 TNF-120572 IFN-120574 TGF-120573 and IL-10 usingeBioscience commercial kits capture enzyme-linked immunesorbent assay (ELISA) (San Diego CA USA) Accordingto the manufacturerrsquos instructions absorbance was readat 450 nm using a spectrophotometer and the results areexpressed in pgmL based on a standard curveThe sensitivityof the test was 8 pgmL for IL-1120573 TNF-120572 and TGF-12057315 pgmL for IL-12 and IFN-120574 and 32 pgmL for IL-10

211 Immunocytochemical Labeling for NLRP12 and iNOSImmunocytochemistry of NLRP12 and inducible nitric oxidesynthase (iNOS) was performed on coverslip-adherent cells(cells prepared according to the protocol described inthe phagocytic assay) using the labeled streptavidin biotinmethod with the LSAB kit (DAKO Japan Kyoto Japan)without microwave treatmentThe coverslips were incubatedwith 10 Triton X-100 for 1 h washed 3 times with PBSand treated for 40min at room temperature with 10 BSAIn addition coverslips were incubated overnight at 4∘Cwith the primary antibody (anti-NLRP12 rabbit polyclonalantibody diluted 1 300 (Abcam catalog number ab93113)and anti-iNOS rabbit monoclonal antibody diluted 1 200(BD Biosciences catalog number 610599)) After secondaryantibody treatment (2 h room temperature) horseradishperoxidase activity was visualized by treatment with H

2O2

and 331015840-diaminobenzidine (DAB) for 5min In the laststep the sections were weakly counterstained with Harryrsquoshematoxylin (Merck) For each case negative controls wereperformed by omitting the primary antibody Intensity andlocalization of immunoreactivity against primary antibodyused were examined in all coverslips using a photomicro-scope (Olympus BX41 Olympus Optical Co Ltd TokyoJapan) Color photomicrographs of representative areas (times40objective lens) were digitally acquired for image analysisFor determining a semiquantitative scoring images wereevaluated by using the color deconvolution tool from Image Jsoftware (NIH USA) Pixels were categorized as previouslydescribed by Chatterjee et al [32] as strong positive (3+)positive (2+) weak positive (1+) and negative (0)

212 cNOS Inhibition Assay Peritoneal macrophages werechallenged with L amazonensis and treated with KA asdescribed in the phagocytic assay method Before the treat-ment with KA the cells were incubated with 20120583M NG-nitro-L-arginine methyl ester (L-NAME) for 2 h at 36∘C and5 CO

2[33] aiming to cause pharmacological blockage of

constitutive NOS The supernatants were utilized to measureNO levels (as previously described)

213 Statistical Analysis Three independent experimentswere performed each with triplicate datasets Data wereexpressed as mean plusmn standard error of the mean Datawere analyzed using the GraphPad Prism statistical software(GraphPad Software Inc USA 500288) Significant differ-ences between the treatments were determined by one-way


Viab

le p

rom

astig

ote f

orm

s (

) 100

80

60

40

20

0

24 48 72

H2O2

DMSO10120583M30120583M

50120583M70120583M90120583M

(h)

(a)

lowast

lowast

lowast

lowast

lowastlowast

lowastlowastlowast

lowast

Prom

astig

otes

(106)

8

6

4

2

0

24 48 72 96 120

ControlDMSO10120583M30120583M

50120583M70120583M90120583M

(h)

(b)

Infe

cted

mac

roph

ages

()

60

40

20

0

lowast lowast lowast

Infected control 50 70 90

Kaurenoic acid (120583M)

(c)

Am

astig

otes

mac

roph

ages

25

20

15

10

05

00

lowast lowast



(d)

Figure 1 Kaurenoic acid has a leishmanicidal effect against promastigote and amastigote forms of L amazonensis MTT assay in promastigoteforms of L amazonensis treated with kaurenoic acid (10 30 50 70 and 90 120583M) or 02 DMSO for 24 48 and 72 h (Panel (a)) Proliferationkinetics of L amazonensis promastigote forms after pretreatment for 24 h with kaurenoic acid (10 30 50 70 and 90 120583M) or 02 DMSOfor 24 48 72 and 120 h (Panel (b)) Percentage of infected macrophages (Panel (c)) and number of amastigotes per macrophage after24 h of incubation with kaurenoic acid (50 70 and 90 120583M) (Panel (d)) Data represent mean plusmn SEM of three independent experiments(promastigotes) and six independent experiments (amastigotes) (lowastSignificantly different from control (119875 lt 005 compared with controlgroup promastigotes in culture medium) (one-way ANOVA followed by Tukeyrsquos test))

ANOVA followed by Tukeyrsquos test for multiple comparisons119875 lt 005 was considered statistically significant

3 Results

31 Kaurenoic Acid Exerts Leishmanicidal Effect against Pro-mastigote and Amastigote Forms of L Amazonensis In thefirst set of experiments the antileishmanial effect of KAwas investigated against the promastigote forms of L ama-zonensis We observed that KA at concentrations of 5070 and 90 120583M reduced promastigote viability according toan MTT assay of 24 h by 30 31 and 34 respectively(Figure 1(a)) the reduction was maintained for 72 h We alsoobserved 282 458 and 515 decrease in the proliferation

of the promastigote forms at concentrations of 50 70 and90 120583M respectively after 120 h with 24 h of pretreatment(Figure 1(b)) Therefore we chose testing all concentrationsof KA for 24 h treatment of macrophages

In attempt to verify if KA could enhance the leish-manicidal capacity of macrophages we initially challengedthese cells with promastigote forms of L amazonensis for2 h for phagocytosis Afterwards the cells were treated withKA (at concentrations ranging from 50 to 90 120583M) for 24 hMacrophages and amastigotes were counted to establish thephagocytic index indicating the extent of infection as thenumber of parasites per macrophage

Regarding the percentage of infected macrophages 5070 and 90 120583M KA respectively caused a decrease of 266256 and 284 of infected macrophages when compared to


10

8

6

4

2

0

Trol

ox (n

M)

Control 50 70 90

lowast

lowastlowast


Infectedcontrol

L amazonensis

(a)

MD

A (n

M)

300

200

100

0

Control 50 70 90

lowast


Infectedcontrol

L amazonensis

(b)

Figure 2 The effect of kaurenoic acid on macrophages is not associated with oxidative stress generation Total antioxidant capacity (TRAP)(Panel (a)) and MDA levels (Panel (b)) were evaluated as markers of the oxidative status in supernatant or macrophages infected with Lamazonensis and treated with kaurenoic acid (50 70 and 90120583M) for 24 h Data represent the mean plusmn SEM of three independent experiments(lowastSignificantly different from infected cells (119875 lt 005)) (one-way ANOVA followed by Tukeyrsquos test)

the untreated infected macrophages (Figure 1(c)) Moreoverthe mean number of amastigotes per macrophage was signif-icantly decreased at the concentrations of 70 and 90120583M by215 and 203 respectively (Figure 1(d))

32 Effect of Kaurenoic Acid onMacrophages Is Not Associatedwith ROS Production In order to assess the involvementof KA in modulating the respiratory burst of macrophagesduring the Leishmania challenge we measured the oxidativestress status of these cells by quantifying its total antioxidantcapacity (TRAP) and MDA formation

There was an increase in TRAP of macrophages infectedwith L amazonensis treated with 50 and 70 120583MKA whileTRAP was reduced by 90120583MKA compared to the infectedcontrol (Figure 2(a)) The MDA level was reduced at allKA concentrations with significant difference at 50120583MKA(Figure 2(b))

33 Kaurenoic Acid Upregulates NO Levels in a cNOS-Dependent Mechanism Concerning NO levels our resultsshowed that untreated macrophages infected with L ama-zonensis displayed decreased levels of NO Interestingly thetreatment with KA reestablished baseline NO levels at allconcentrations tested (Figure 3(a)) Thus we next investi-gated the enzymatic pathway involved in KA-induced NOproduction

The expression of iNOS was assessed by immunocyto-chemistry Our data showed that KA did not alter iNOSexpression (Figure 3(b))

Aiming to investigate the involvement of cNOS wepretreated macrophages with the preferential cNOS inhibitorL-NAME After the Leishmania challenge macrophages werepretreated with L-NAME and then incubated with KAresulting in a substantial reduction in NO production only

at 90120583M indicating that the augmented NO previouslyobserved at 90 120583M KA was probably dependent on cNOSactivity (Figure 3(c))

34 Kaurenoic Acid Promotes the Production of Active IL-1120573 in Macrophages Infected with L amazonensis In order todetermine the immunomodulatory action ofKAon cytokinesin infected macrophages we measured the levels of IL-12TNF-120572 IFN-120574 TGF-120573 IL-10 and IL-1120573We observed that theproduction of IL-12 TNF-120572 IFN-120574 TGF-120573 and IL-10was notsignificantly different between the KA-treated groups and thecontrol group (Figure 4) On the other hand 70 and 90 120583MKA augmented the levels of IL-1120573 (Figure 4(f))

35 Kaurenoic Acid Upregulates NLRP12 Expression inMacrophages Infected with L amazonensis In this presentstudy the activation and participation of the inflammasomeduring the immune response to infection by intracellularpathogen were investigated The augmented levels of IL-1120573 induced during KA treatment of infected macrophagescombined with the lack of information about this complex inACL led us to investigate the role of NLRP12 one memberof the subfamily of NLRP innate receptors As shown inFigure 5 KA at 90 120583Mwas able to upregulate NLRP12 expres-sion in macrophages infected with L amazonensis Theseresults indicate that the stimulation of macrophages with KAtriggered the overexpression of NLRP12 with consequentactivation of IL-1120573

4 Discussion

The success of chemotherapy in ACL is mainly dependenton two factors the microbicidal activity of the drug and the


150

100

50

0

NO2minus

(120583M

)

Control

Infectedcontrol

50 70 90

lowast

lowast


L amazonensis

(a)

iNO

S sc

orin

g

20

15

10

05

00


Control Infectedcontrol

50 70 90

L amazonensis

(b)

lowast

NO2minus

(120583M

)

150

100

50

0

50 70 90

L-NAMEKaurenoic acid (120583M)

+ + + +

+ + +

minus

minus minus

L amazonensis

(c)

Figure 3 Kaurenoic acid exerted its effect by upregulating NO levels in a cNOS-dependent mechanism NO levels (Panel (a)) immunocy-tochemistry scoring for inducible nitric oxide synthase (iNOS) Peritoneal macrophages infected with L amazonensis and treated withkaurenoic acid (50 70 and 90120583M) for 24 h (Panel (b)) Determination of NO in peritoneal macrophages infected with L amazonensis andblocked with 20 120583M L-NAME and treated with kaurenoic acid (50 70 and 90 120583M) for 24 h (Panel (c)) Data represent the mean plusmn SEM ofthree independent experiments (lowastSignificantly different from infected cells (119875 lt 005) Significantly different from control cells (119875 lt 005)(one-way ANOVA followed by Tukeyrsquos test))

protective immune response triggered in the host during thetreatment

In the present study we evaluated the therapeutic poten-tial of KA treatment which directly inhibited the viabilityand proliferation of L amazonensis promastigote forms(Figure 1(a)) Previous in vitro studies demonstrated thatKA has direct antileishmanial activity against promastigoteand amastigote forms of L amazonensis [19 34 35] andthe main proposed mechanism was related to mitochondrialmembrane depolarization in the protozoan However theimmunomodulatory activity of KA in themacrophage exper-imental leishmaniasis model still remained to be determined

Our results indicated that infected macrophages treatedwith KA were more effective during the leishmanicidalresponse against the intracellular forms of L amazonensis(Figures 1(b) and 1(c)) The solvent (02 DMSO) did notaffect the viability and proliferation of L amazonensis pro-mastigotes (Figures 1(a) and 1(b)) These findings suggested

that this diterpene was able to reverse the downregulation ofthe killer machinery of macrophages caused by Leishmaniainfection [20 36]

In order to elucidate the microbicidal effects of KA weinvestigated the main leishmanicidal molecules produced bymacrophages The results showed that KA treatment was notable to enhance the oxidative burst of infected macrophagesOn the other hand even though L amazonensis is capable ofdepleting NO levels [20] the results showed that treatmentwith KA was able to restore the levels of this microbicidalmolecule (Figure 3(a)) but no alteration in the expression ofiNOS was found

In fact NO is the main antileishmanial molecule pro-duced in the early macrophage response against intracellularparasites Besides iNOS cNOS is also an important route forNO production [37 38] Some studies have demonstratedthat KA induces cNOS-dependent activity in the disease


IL-12

(pg

mL)

15

10

5

0


50 70 90


L amazonensis

(a)


50 70 90


TNF-120572

(pg

mL)

3000

2000

1000

0

L amazonensis

(b)


50 70 90

IFN

-120574(p

gm

L)

150

100

50

0


L amazonensis

(c)


50 70 90

TGF-120573

(pg

mL)

25

20

15

10

5

0


L amazonensis

(d)


50 70 90

IL-10

(pg

mL)

800

600

400

200

0


L amazonensis

(e)


50 70 90


IL-1120573

(pg

mL)

150

100

50

0

lowast lowast

L amazonensis

(f)

Figure 4 Kaurenoic acid promotes the production of active IL-1120573 in macrophages infected with L amazonensis Mapping the cytokineprofiling produced in vitro by macrophages infected with L amazonensis and treated with kaurenoic acid (50 70 and 90 120583M) for 24 hdetermined by ELISA IL-12 production (Panel (a)) TNF-120572 production (Panel (b)) IFN-120574 production (Panel (c)) TGF-120573 production (Panel(d)) IL-10 production (Panel (e)) and IL-1120573 production (Panel (f)) Data represent the mean plusmn SEM of three independent experiments(lowastSignificantly different from infected cells (119875 lt 005) (one-way ANOVA followed by Tukeyrsquos test))

context [13 14] but the involvement of a cNOS mechanismfor KA in parasitic infections has not been well elucidated

Studies have demonstrated the importance of innateimmune response-triggered cytokine production during theearly stages of experimental leishmaniasis and have shown

that some cytokines may drive the clinical manifestation ofACL by modulating resistance or susceptibility to infection[39] IL-1120573 IL-12 TNF-120572 and IFN-120574 are essential cytokinesfor the development of an effective immune response againstLeishmania spp leading to the activation of macrophages

8 Mediators of InflammationN

LRP-12

scor

ing

4

3

2

1

0


50 70 90


lowast

L amazonensis

Figure 5 Kaurenoic acid upregulated the NLRP12 expression inmacrophages infected with L amazonensis Immunocytochemistryscoring for NLRP12 in macrophages infected with L amazonensisand treated with kaurenoic acid (50 70 and 90 120583M) for 24 hData represent the mean plusmn SEM of three independent experiments(lowastSignificantly different from infected cells (119875 lt 005) (One-wayANOVA followed by Tukeyrsquos test))

and promoting the microbicidal effects against this parasite[23 26 40 41]

In the present experimental conditions KA was able toincrease the production of IL-1120573 despite of showing no effecton other cytokines evaluated suggesting a selective effect ofKA on IL-1120573 productionmaturation system IL-1120573 is a proin-flammatory cytokine that becomes active after its cleavageby the inflammasome complex [42] and when active thiscytokine helps the activation of macrophages by enhancingthe response against pathogens In fact a recent study showedthat IL-1120573 was associated with resistance to L amazonensisL braziliensis and L major infections IL-1120573 maturationis dependent on the inflammasome NLRP3ASCcaspase-1 complex [26] In addition to NLRP3 which is endoge-nously expressed during Leishmania infection NLRP12 isan important NLR involved in the inflammatory responseagainst parasites such as Trypanosoma cruzi [43] In thissense the present study also addressed whether the expres-sion of NLRP12 increased during Leishmania infection ofmacrophages to determine if KA induces IL-1120573 produc-tionmaturation by a previously unrecognized mechanism

Our findings showed that there was no induction ofNLRP12 in macrophages during Leishmania infection Onthe other hand KA at 90120583M upregulated the expressionof NLRP12 in infected macrophages thus explaining theincreased production of IL-1120573 induced by KAThese data arealso in line with the increased cNOS-dependent productionof NO since it has been shown that during Leishmaniainfection a NLRP3ASCcaspase-1IL-1120573cNOSNO pathwayis triggered to kill this parasite In the present study the datasuggest that KA triggers a NLRP12IL-1120573cNOSNO killingmechanism during Leishmania infection of macrophagesfrom BALBc mice Importantly KA-induced the expressionof a NLR (NLRP12) that is not endogenously activated to

kill Leishmania thereby upregulating unused endogenousmechanisms valuable to protect the host against Leishmaniainfection with additive effects for other NLRs such as NLRP3as observed in T cruzi infection [43] Therefore KA seemsto unequivocally provide additional protective mechanismsagainst Leishmania infection It is also possible that KAtriggers similarmechanisms in other parasitic diseases whichremains to be determined

In conclusion the present study demonstrated that kau-renoic acid has therapeutic potential as a pharmacologicalapproach against Leishmania infection The mechanism ofaction of kaurenoic acid depends at least in part on trig-gering the NLRP12IL-1120573cNOSNO leishmanicidal pathwayTherefore KA merits further preclinical and clinical studiesas a possible therapy for Leishmania infection

Conflict of Interests

The authors have no conflict of interests to declare

Authorsrsquo Contribution

Milena Menegazzo Miranda Carolina Panis Suelen Santosda Silva and Juliana Aparecida Macri equally contributed tothis study

Acknowledgments

The authors gratefully acknowledge Juliano Bordignon forsupplying the animals used in this paper This study wassupported by Conselho Nacional de Pesquisa (CNPq Brazil)Coordenadoria de Aperfeicoamento Pessoal de Nıvel Supe-rior (CAPES Brazil) and Fundacao Araucaria by Governodo Estado do Parana (Brazil) Dr A Leyva helped with theEnglish editing of the paper

References

[1] P Kaye and P Scott ldquoLeishmaniasis complexity at the host-pathogen interfacerdquo Nature Reviews Microbiology vol 9 no 8pp 604ndash615 2011

[2] Ministerio da Saude and Secretaria de Vigilancia em SaudeManual for Surveillance of American Tegumentary Leishmani-asis Serie A Normas e Manuais Tecnicos Ministerio da SaudeSecretaria de Vigilancia em Saude Editora do Ministerio daSaude Brasılia Brazil 2nd edition 2010

[3] H Goto and J A L Lindoso ldquoCurrent diagnosis and treatmentof cutaneous andmucocutaneous leishmaniasisrdquo Expert Reviewof Anti-Infective Therapy vol 8 no 4 pp 419ndash433 2010

[4] R E Silva-Lopez ldquoProteases de Leishmania novos alvos para odesenvolvimento racional de farmacosrdquo Quımica Nova vol 33no 7 pp 1541ndash1548 2010

[5] L F Oliveira A O Schubach M M Martins et al ldquoSystematicreview of the adverse effects of cutaneous leishmaniasis treat-ment in the NewWorldrdquo Acta Tropica vol 118 no 2 pp 87ndash962011

[6] T S Tiuman A O Santos T Ueda-Nakamura B P DFilho and C V Nakamura ldquoRecent advances in leishmaniasis


treatmentrdquo International Journal of Infectious Diseases vol 15no 8 pp e525ndashe532 2011

[7] S S da Silva G D S Thome A H D Cataneo et al ldquoBrazil-ian propolis antileishmanial and immunomodulatory effectsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 673058 7 pages 2013

[8] T Baccarin A I Czepula R A Ferreira and R M Lucinda-Silva ldquoAnalise morfoanatomica das partes aereas de Wedeliapaludosa DC (Acmela brasiliensis Sphagneticola trilobata)Asteraceaerdquo Revista Brasileira de Farmacognosia vol 19 pp612ndash616 2009

[9] R Batista G C Brandao F C Braga and A B Oliveira ldquoCyto-toxicity of Wedelia paludosa DC extracts and constituentsrdquoBrazilian Journal of Pharmacognosy vol 19 no 1 pp 36ndash402009

[10] R Batista E Chiari and A B de Oliveira ldquoTrypanosomicidalkaurane diterpenes fromWedelia paludosardquo Planta Medica vol65 no 3 pp 283ndash284 1999

[11] R Batista J L Humberto E Chiari and A B de OliveiraldquoSynthesis and trypanocidal activity of ent-kaurane glycosidesrdquoBioorganic and Medicinal Chemistry vol 15 no 1 pp 381ndash3912007

[12] M Wilkens C Alarcon A Urzua and L Mendoza ldquoCharac-terization of the bactericidal activity of the natural diterpenekaurenoic acidrdquo PlantaMedica vol 68 no 5 pp 452ndash454 2002

[13] S SMizokami N S Arakawa S R Ambrosio et al ldquoKaurenoicacid from Sphagneticola trilobata inhibits inflammatory paineffect on cytokine production and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channelsignaling pathwayrdquo Journal of Natural Products vol 75 no 5pp 896ndash904 2012

[14] C R Tirapelli S R Ambrosio F B da Costa S T Coutinho DC R de Oliveira and A M de Oliveira ldquoAnalysis of the mech-anisms underlying the vasorelaxant action of kaurenoic acid inthe isolated rat aortardquo European Journal of Pharmacology vol492 no 2-3 pp 233ndash241 2004

[15] C R Tirapelli S R Ambrosio A M de Oliveira and R CTostes ldquoHypotensive action of naturally occurring diterpenesa therapeutic promise for the treatment of hypertensionrdquoFitoterapia vol 81 no 7 pp 690ndash702 2010

[16] H Lim H A Jung J S Choi Y S Kim S S Kang and HP Kim ldquoAnti-inflammatory activity of the constituents of theroots of Aralia continentalisrdquo Archives of Pharmacal Researchvol 32 no 9 pp 1237ndash1243 2009

[17] R J Choi E M Shin H A Jung J S Choi and Y SKim ldquoInhibitory effects of kaurenoic acid from Aralia conti-nentalis on LPS-induced inflammatory response in RAW2647macrophagesrdquo Phytomedicine vol 18 no 8-9 pp 677ndash682 2011

[18] E Izumi T Ueda-Nakamura V F Veiga A C Pinto andC V Nakamura ldquoTerpenes from copaifera demonstrated invitro antiparasitic and synergic activityrdquo Journal of MedicinalChemistry vol 55 no 7 pp 2994ndash3001 2012

[19] A O dos Santos E Izumi T Ueda-Nakamura B P Dias-FilhoV F da Veiga-Junior and C V Nakamura ldquoAntileishmanialactivity of diterpene acids in copaiba oilrdquoMemorias do InstitutoOswaldo Cruz vol 108 no 1 pp 59ndash64 2013

[20] T Van Assche M Deschacht R A I Da Luz L Maes andP Cos ldquoLeishmania-macrophage interactions insights into theredox biologyrdquo Free Radical Biology and Medicine vol 51 no 2pp 337ndash351 2011

[21] M S Faria F C G Reis and A P C A Lima ldquoToll-likereceptors in Leishmania infections guardians or promotersrdquo

Journal of Parasitology Research vol 2012 Article ID 930257 12pages 2012

[22] E Handman and D V Bullen ldquoInteraction of Leishmania withthe host macrophagerdquo Trends in Parasitology vol 18 no 8 pp332ndash334 2002

[23] D Sacks and N Noben-Trauth ldquoThe immunology of suscep-tibility and resistance to Leishmania major in micerdquo NatureReviews Immunology vol 2 no 11 pp 845ndash858 2002

[24] J K Krishnaswamy T Chu and S C Eisenbarth ldquoBeyondpattern recognition NOD-like receptors in dendritic cellsrdquoTrends in Immunology vol 34 no 5 pp 224ndash233 2013

[25] M Takahashi ldquoNLRP3 inflammasome as a novel player inmyocardial infarctionrdquo International Heart Journal vol 55 no2 pp 101ndash105 2014

[26] D S Lima-Junior D L Costa V Carregaro et al ldquoInflamma-some-derived IL-1120573 production induces nitric oxide-mediatedresistance to Leishmaniardquo Nature Medicine vol 19 no 7 pp909ndash915 2013

[27] F B da Costa S Albuquerque andW Vichnewski ldquoDiterpenesand synthetic derivatives from Viguiera aspillioides with trypa-nomicidal activityrdquo Planta Medica vol 62 no 6 pp 557ndash5591996

[28] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[29] M Repetto C Reides M L Gomez Carretero M Costa GGriemberg and S Llesuy ldquoOxidative stress in blood of HIVinfected patientsrdquo Clinica Chimica Acta vol 255 no 2 pp 107ndash117 1996

[30] V J Victorino C Panis F C Campos et al ldquoDecreasedoxidant profile and increased antioxidant capacity in naturallypostmenopausal womenrdquoAge vol 35 no 4 pp 1411ndash1421 2013

[31] C Panis A C S A Herrera V J Victorino et al ldquoOxidativestress and hematological profiles of advanced breast cancerpatients subjected to paclitaxel or doxorubicin chemotherapyrdquoBreast Cancer Research and Treatment vol 133 no 1 pp 89ndash972012

[32] S Chatterjee R Malhotra F Varghese et al ldquoQuantitativeimmunohistochemical analysis reveals association betweensodium iodide symporter and estrogen receptor expression inbreast cancerrdquo PLoS ONE vol 8 no 1 Article ID e54055 2013

[33] K-S Lee D-K Lee D Jeoung et al ldquoDifferential effects ofsubstrate-analogue inhibitors on nitric oxide synthase dimer-izationrdquoBiochemical and Biophysical Research Communicationsvol 418 no 1 pp 49ndash55 2012

[34] AO Santos T Ueda-Nakamura B P D Filho V F V Junior AC Pinto and C V Nakamura ldquoEffect of Brazilian copaiba oilson Leishmania amazonensisrdquo Journal of Ethnopharmacologyvol 120 no 2 pp 204ndash208 2008

[35] A O dos Santos T Ueda-Nakamura B P D Filho V F DV Junior and C V Nakamura ldquoCopaiba oil an alternativeto development of new drugs against leishmaniasisrdquo Evidence-Based Complementary and Alternative Medicine vol 2012Article ID 898419 7 pages 2012

[36] F M Balestieri A R Queiroz C Scavone V M CostaM Barral-Netto and A Abrahamsohn Ide ldquoLeishmania (L)amazonensis-induced inhibition of nitric oxide synthesis in hostmacrophagesrdquo Microbes and Infection vol 4 no 1 pp 23ndash292002


[37] Z Huang F W Hoffmann J D Fay et al ldquoStimulation ofunprimed macrophages with immune complexes triggers a lowoutput of nitric oxide by calcium-dependent neuronal nitric-oxide synthaserdquoThe Journal of Biological Chemistry vol 287 no7 pp 4492ndash4502 2012

[38] L Connelly A T Jacobs M Palacios-Callender S MoncadaandA J Hobbs ldquoMacrophage endothelial nitric-oxide synthaseautoregulates cellular activation and pro-inflammatory proteinexpressionrdquoThe Journal of Biological Chemistry vol 278 no 29pp 26480ndash26487 2003

[39] S Nylen and S Gautam ldquoImmunological perspectives ofleishmaniasisrdquo Journal of Global Infectious Diseases vol 2 pp135ndash146 2010

[40] B M Babior ldquoPhagocytes and oxidative stressrdquo AmericanJournal of Medicine vol 109 no 1 pp 33ndash44 2000

[41] A C Cunningham ldquoParasitic adaptivemechanisms in infectionby Leishmaniardquo Experimental and Molecular Pathology vol 72no 2 pp 132ndash141 2002

[42] F L van de Veerdonk M G Netea C A Dinarello and LA B Joosten ldquoInflammasome activation and IL-1120573 and IL-18processing during infectionrdquo Trends in Immunology vol 32 no3 pp 110ndash116 2011

[43] M A Ataide W A Andrade D S Zamboni et al ldquoMalaria-inducedNLRP12NLRP3-dependent caspase-1 activationmedi-ates inflammation and hypersensitivity to bacterial superinfec-tionrdquo PLoS Pathogens vol 10 no 1 Article ID e1003885 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


a diterpene obtained from various Brazilian plants [8 9]Thismolecule has been reported as showing a wide variety of bio-logical activities such as antiprotozoal [10 11] antimicrobial[12] antinociceptive [13] vasorelaxant hypotensive [14 15]and anti-inflammatory [13 16 17] activities Moreover someimmunomodulatory properties of KA have been reported inthese models

The antiprotozoal activity of this diterpene involves itsdirect action in altering cell membrane integrity and mito-chondrial membrane depolarization in promastigote andamastigote forms of L amazonensis [18] and epimastigoteforms of Trypanosoma cruzi [19] respectively This directactivity of KA against the protozoa is not enough to reflectits overall potential as a therapeutic leishmanicidal drugsince Leishmania spp parasites replicate intracellularly inmacrophages and have several escape mechanisms againstmicrobicidal molecules that are not dependent on the directaction of antileishmanial drugs [20ndash23]

Members of the Nod-like receptor (NLR) family ofproteins have emerged as important innate immune sen-sors of pathogen-associated molecular patterns (PAMPS)and damage-associated molecular patterns (DAMPS) [24]NLRs are the key components of the inflammasome thatregulate the maturation of the potent inflammatory cytokineinterleukin- (IL-) 1120573 [25]

Accordingly Lima-Junior et al found that the NLRP-3inflammasome is engaged in the response against L ama-zonensis restricting parasite replication Additionally IL-1120573seems to be important for host resistance to infection byinducible (i) NOS-mediated production of NO a major hostdefense mechanism against Leishmania spp [26]

Taking into account the above-mentioned evidenceKA was evaluated for its in vitro effect on susceptiblemacrophages from BALBc mice infected with L ama-zonensis promastigote forms Accordingly we performedin vitro assays to investigate the direct effect of KA onparasites as well as its modulatory action on Leishmania-infected macrophages We further investigated a putativemechanism of action of this compound as a modulator ofproinflammatory molecules such as oxygen reactive speciesNO cytokines and inflammasome

2 Materials and Methods

21 Parasite L amazonensis (MHOMBR1989166MJO)was used in promastigote forms kept in culture medium199 (Invitrogen-GIBCO) supplemented with 10 fetal bovineserum (Invitrogen-GIBCO) 1M Hepes 01 human urine01 L-glutamine 10UmL penicillin and 10 120583gmL strep-tomycin (Invitrogen-GIBCO) and 10 sodium bicarbonate(complete medium for promastigotesmdashCMP) Cell cultureswere incubated at 25∘C in 25 cm2 flasks

22 Animals Female BALBc mice weighing approximately25ndash30 g and aged 6ndash8 weeks were obtained from FundacaoOsvaldo Cruz FIOCRUZ Curitiba Brazil Mice were keptunder pathogen-free conditions and used according to proto-cols approved by the Ethics Committee of the State University

of Londrina (protocol number 33064201242) Every effortwas made to minimize the number of animals used and theirsuffering

23 Plant Material KA used in this paper was obtainedfrom Sphagneticola trilobata The crude extract was obtainedfrom dried roots which were pulverized and extracted withdichloromethane and partitioned with n-hexane and ethylacetate all solvents were dried under reduced pressure Thehexane fraction was subjected to vacuum liquid chromatog-raphy (VLC) by increasing gradient polarity The secondfraction produced an amorphous compound (200mg) whichwas washed with cold methanol and analyzed by high per-formance liquid chromatography (HPLC) methods yielding96 purity The identification was performed by 1H and13 C nuclear magnetic resonance (NMR) electron impactmass spectrometry (EIMS) and comparison with literaturedata [27] The stock solution of KA was dissolved in 2dimethyl sulfoxide (DMSO) (Invitrogen-Gibco) HoweverDMSO concentration did not exceed 02 in all experiments

24 Viability of Promastigotes Theviability of L amazonensispromastigote forms treated with KA was evaluated using the3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bro-mide (MTT) assay as previously described [28] Promastigoteforms (106100 120583L) were incubated with different concentra-tions of KA (10 30 50 70 and 90 120583M) or with KA solvent(02 DMSO) and maintained in culture for 24 48 and 72 hat 25∘C Thereafter 10 120583L of MTT (5mgmL) were addedfollowed by incubation for an additional 4 h at 24∘C TheMTT formazan product was diluted with 300120583L of DMSOtransferring to 96-well plates and measured in a spectro-photometer with absorbance determined at 550 nm Theresults were expressed as percentage MTT reduction relativeto the control group calculated as the following formula(viable promastigotes) = (OD of drug-treated sampleODof untreated sample) times 100

25 Cell Proliferation Kinetics Promastigote forms (106mL)incubated in CMP were treated with different concentrationsof KA (10 30 50 70 and 90 120583M) or with KA solvent (02DMSO) and cultured for 5 days at 25∘C Promastigotes werecounted in a Neubauer chamber after 24 48 72 and 120 h

26 Phagocytic Assay Macrophages (5 times 105mL) wereobtained from the peritoneal cavity by the injection of 2mLof RPMI 1640 culture medium (Invitrogen-GIBCO) supple-mented with 10 fetal bovine serum (Invitrogen-GIBCO)and cultured on 24-well plates containing 13mm diameterglass coverslips Cells were preincubated with 200120583L ofRPMI medium for 2 h for adherence and incubated withpromastigote forms (5 1) for 2 h KA (50 70 or 90120583M) ormedium was added followed by 24 h incubation at 37∘C and5 CO

2 The cells were stained with Giemsa to establish

the phagocytic index of infection (by percentage) and theparasitesmacrophage (mean) The supernatant was utilizedto measure the levels of malondialdehyde (MDA) total





2PO4buffer pH



4




2 and




2O2







Viab

le p

rom

astig

ote f

orm

s (

) 100

80

60

40

20

0

24 48 72

H2O2

DMSO10120583M30120583M

50120583M70120583M90120583M

(h)

(a)

lowast

lowast

lowast

lowast

lowastlowast

lowastlowastlowast

lowast

Prom

astig

otes

(106)

8

6

4

2

0

24 48 72 96 120


50120583M70120583M90120583M

(h)

(b)

Infe

cted

mac

roph

ages

()

60

40

20

0




(c)

Am

astig

otes

mac

roph

ages

25

20

15

10

05

00

lowast lowast



(d)



3 Results






10

8

6

4

2

0

Trol

ox (n

M)

Control 50 70 90

lowast

lowastlowast


Infectedcontrol

L amazonensis

(a)

MD

A (n

M)

300

200

100

0

Control 50 70 90

lowast


Infectedcontrol

L amazonensis

(b)











4 Discussion



150

100

50

0

NO2minus

(120583M

)

Control

Infectedcontrol

50 70 90

lowast

lowast


L amazonensis

(a)

iNO

S sc

orin

g

20

15

10

05

00



50 70 90

L amazonensis

(b)

lowast

NO2minus

(120583M

)

150

100

50

0

50 70 90


+ + + +

+ + +

minus

minus minus

L amazonensis

(c)









IL-12

(pg

mL)

15

10

5

0


50 70 90


L amazonensis

(a)


50 70 90


TNF-120572

(pg

mL)

3000

2000

1000

0

L amazonensis

(b)


50 70 90

IFN

-120574(p

gm

L)

150

100

50

0


L amazonensis

(c)


50 70 90

TGF-120573

(pg

mL)

25

20

15

10

5

0


L amazonensis

(d)


50 70 90

IL-10

(pg

mL)

800

600

400

200

0


L amazonensis

(e)


50 70 90


IL-1120573

(pg

mL)

150

100

50

0

lowast lowast

L amazonensis

(f)






LRP-12

scor

ing

4

3

2

1

0


50 70 90


lowast

L amazonensis











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2PO4buffer pH



4




2 and




2O2







Viab

le p

rom

astig

ote f

orm

s (

) 100

80

60

40

20

0

24 48 72

H2O2

DMSO10120583M30120583M

50120583M70120583M90120583M

(h)

(a)

lowast

lowast

lowast

lowast

lowastlowast

lowastlowastlowast

lowast

Prom

astig

otes

(106)

8

6

4

2

0

24 48 72 96 120


50120583M70120583M90120583M

(h)

(b)

Infe

cted

mac

roph

ages

()

60

40

20

0




(c)

Am

astig

otes

mac

roph

ages

25

20

15

10

05

00

lowast lowast



(d)



3 Results






10

8

6

4

2

0

Trol

ox (n

M)

Control 50 70 90

lowast

lowastlowast


Infectedcontrol

L amazonensis

(a)

MD

A (n

M)

300

200

100

0

Control 50 70 90

lowast


Infectedcontrol

L amazonensis

(b)











4 Discussion



150

100

50

0

NO2minus

(120583M

)

Control

Infectedcontrol

50 70 90

lowast

lowast


L amazonensis

(a)

iNO

S sc

orin

g

20

15

10

05

00



50 70 90

L amazonensis

(b)

lowast

NO2minus

(120583M

)

150

100

50

0

50 70 90


+ + + +

+ + +

minus

minus minus

L amazonensis

(c)









IL-12

(pg

mL)

15

10

5

0


50 70 90


L amazonensis

(a)


50 70 90


TNF-120572

(pg

mL)

3000

2000

1000

0

L amazonensis

(b)


50 70 90

IFN

-120574(p

gm

L)

150

100

50

0


L amazonensis

(c)


50 70 90

TGF-120573

(pg

mL)

25

20

15

10

5

0


L amazonensis

(d)


50 70 90

IL-10

(pg

mL)

800

600

400

200

0


L amazonensis

(e)


50 70 90


IL-1120573

(pg

mL)

150

100

50

0

lowast lowast

L amazonensis

(f)






LRP-12

scor

ing

4

3

2

1

0


50 70 90


lowast

L amazonensis











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Viab

le p

rom

astig

ote f

orm

s (

) 100

80

60

40

20

0

24 48 72

H2O2

DMSO10120583M30120583M

50120583M70120583M90120583M

(h)

(a)

lowast

lowast

lowast

lowast

lowastlowast

lowastlowastlowast

lowast

Prom

astig

otes

(106)

8

6

4

2

0

24 48 72 96 120


50120583M70120583M90120583M

(h)

(b)

Infe

cted

mac

roph

ages

()

60

40

20

0




(c)

Am

astig

otes

mac

roph

ages

25

20

15

10

05

00

lowast lowast



(d)



3 Results






10

8

6

4

2

0

Trol

ox (n

M)

Control 50 70 90

lowast

lowastlowast


Infectedcontrol

L amazonensis

(a)

MD

A (n

M)

300

200

100

0

Control 50 70 90

lowast


Infectedcontrol

L amazonensis

(b)











4 Discussion



150

100

50

0

NO2minus

(120583M

)

Control

Infectedcontrol

50 70 90

lowast

lowast


L amazonensis

(a)

iNO

S sc

orin

g

20

15

10

05

00



50 70 90

L amazonensis

(b)

lowast

NO2minus

(120583M

)

150

100

50

0

50 70 90


+ + + +

+ + +

minus

minus minus

L amazonensis

(c)









IL-12

(pg

mL)

15

10

5

0


50 70 90


L amazonensis

(a)


50 70 90


TNF-120572

(pg

mL)

3000

2000

1000

0

L amazonensis

(b)


50 70 90

IFN

-120574(p

gm

L)

150

100

50

0


L amazonensis

(c)


50 70 90

TGF-120573

(pg

mL)

25

20

15

10

5

0


L amazonensis

(d)


50 70 90

IL-10

(pg

mL)

800

600

400

200

0


L amazonensis

(e)


50 70 90


IL-1120573

(pg

mL)

150

100

50

0

lowast lowast

L amazonensis

(f)






LRP-12

scor

ing

4

3

2

1

0


50 70 90


lowast

L amazonensis











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















10

8

6

4

2

0

Trol

ox (n

M)

Control 50 70 90

lowast

lowastlowast


Infectedcontrol

L amazonensis

(a)

MD

A (n

M)

300

200

100

0

Control 50 70 90

lowast


Infectedcontrol

L amazonensis

(b)











4 Discussion



150

100

50

0

NO2minus

(120583M

)

Control

Infectedcontrol

50 70 90

lowast

lowast


L amazonensis

(a)

iNO

S sc

orin

g

20

15

10

05

00



50 70 90

L amazonensis

(b)

lowast

NO2minus

(120583M

)

150

100

50

0

50 70 90


+ + + +

+ + +

minus

minus minus

L amazonensis

(c)









IL-12

(pg

mL)

15

10

5

0


50 70 90


L amazonensis

(a)


50 70 90


TNF-120572

(pg

mL)

3000

2000

1000

0

L amazonensis

(b)


50 70 90

IFN

-120574(p

gm

L)

150

100

50

0


L amazonensis

(c)


50 70 90

TGF-120573

(pg

mL)

25

20

15

10

5

0


L amazonensis

(d)


50 70 90

IL-10

(pg

mL)

800

600

400

200

0


L amazonensis

(e)


50 70 90


IL-1120573

(pg

mL)

150

100

50

0

lowast lowast

L amazonensis

(f)






LRP-12

scor

ing

4

3

2

1

0


50 70 90


lowast

L amazonensis











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















150

100

50

0

NO2minus

(120583M

)

Control

Infectedcontrol

50 70 90

lowast

lowast


L amazonensis

(a)

iNO

S sc

orin

g

20

15

10

05

00



50 70 90

L amazonensis

(b)

lowast

NO2minus

(120583M

)

150

100

50

0

50 70 90


+ + + +

+ + +

minus

minus minus

L amazonensis

(c)









IL-12

(pg

mL)

15

10

5

0


50 70 90


L amazonensis

(a)


50 70 90


TNF-120572

(pg

mL)

3000

2000

1000

0

L amazonensis

(b)


50 70 90

IFN

-120574(p

gm

L)

150

100

50

0


L amazonensis

(c)


50 70 90

TGF-120573

(pg

mL)

25

20

15

10

5

0


L amazonensis

(d)


50 70 90

IL-10

(pg

mL)

800

600

400

200

0


L amazonensis

(e)


50 70 90


IL-1120573

(pg

mL)

150

100

50

0

lowast lowast

L amazonensis

(f)






LRP-12

scor

ing

4

3

2

1

0


50 70 90


lowast

L amazonensis











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IL-12

(pg

mL)

15

10

5

0


50 70 90


L amazonensis

(a)


50 70 90


TNF-120572

(pg

mL)

3000

2000

1000

0

L amazonensis

(b)


50 70 90

IFN

-120574(p

gm

L)

150

100

50

0


L amazonensis

(c)


50 70 90

TGF-120573

(pg

mL)

25

20

15

10

5

0


L amazonensis

(d)


50 70 90

IL-10

(pg

mL)

800

600

400

200

0


L amazonensis

(e)


50 70 90


IL-1120573

(pg

mL)

150

100

50

0

lowast lowast

L amazonensis

(f)






LRP-12

scor

ing

4

3

2

1

0


50 70 90


lowast

L amazonensis











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















LRP-12

scor

ing

4

3

2

1

0


50 70 90


lowast

L amazonensis











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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