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Veterinary Immunology and Immunopathology 163 (2015) 67–76

Contents lists available at ScienceDirect

Veterinary Immunology and Immunopathology

j ourna l h omepa ge: www.elsev ier .com/ locate /vet imm

esearch Paper

ranscription of innate immunity genes and cytokineecretion by canine macrophages resistant or susceptible tontracellular survival of Leishmania infantum

ndréia Pereira Turchetti a, Luciana Fachini da Costac,verton de Lima Romãoa, Ricardo Toshio Fujiwarab, Tatiane Alves da Paixãoc,enato Lima Santosa,∗

Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, MG,razilDepartamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, BrazilDepartamento de Patologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

r t i c l e i n f o

rticle history:eceived 12 July 2014eceived in revised form0 September 2014ccepted 13 November 2014

eywords:eishmania infantumacrophageog

nnate immunityytokines

a b s t r a c t

In this study we assessed the basal transcription of genes associated with innate immu-nity (i.e. Nramp1, NOD1, NOD2, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR9) incanine monocyte-derived macrophages from Leishmania-free dogs. Additionally, secretionof cytokines (IL-10, IL-12, TNF-� and IFN-�) and nitric oxide in culture supernatants ofmacrophages with higher or lower resistance to intracellular survival of Leishmania infan-tum was also measured. Constitutive transcription of TLR9 and NOD2 were negligible;NOD1, TLR1, and TLR7 had low levels of transcription, whereas Nramp1 and TLR2, 3, 4, 5, and6 had higher levels of constitutive transcription in canine monocyte-derived macrophages.There were no significant differences in transcription between macrophages with higheror lower resistance to intracellular survival of L. infantum. Secretion of TNF-� was higherin more resistant macrophages (designated as resistant) at 24 h after infection when com-pared to less resistant macrophages (designated as susceptible), as well as the secretion ofIFN-� at 72 h post infection. Secretion of IL-10 was lower in resistant macrophages at 24 hafter infection. No detectable production of nitric oxide was observed. Interestingly, therewas a negative correlation between NOD2 transcript levels and intracellular survival of L.

infantum in resistant macrophages. This study demonstrated that decreased intracellularsurvival of L. infantum in canine macrophages was associated with increased production ofTNF-� and IFN-� and decreased production of IL-10; and that constitutive transcription ofNramp1, TLR and NLR does not interfere in intracellular survival of L. infantum.

© 2014 Elsevier B.V. All rights reserved.

∗ Corresponding author at: Departamento de Clínica e Cirurgia Vet-rinária, Escola de Veterinária, Universidade Federal de Minas Gerais,v. Presidente Antônio Carlos 6627, CEP 30161-970, Belo Horizonte, MG,razil. Tel.: +55 31 3409 2239; fax: +55 31 3409 2230.

E-mail address: rsantos@vet.ufmg.br (R.L. Santos).

http://dx.doi.org/10.1016/j.vetimm.2014.11.010165-2427/© 2014 Elsevier B.V. All rights reserved.

1. Introduction

The innate immune system is the first line of defense,

programmed to quickly respond in a non-specific mannerto structures common to groups of similar organisms, alsoknown as microbe-associated molecular patterns (MAMP).Two of the receptor families responsible for recognizing

ology an

68 A.P. Turchetti et al. / Veterinary Immun

MAMP are the Toll and the NOD like receptors (TLR and NLR,respectively) (Lee et al., 2012). Innate immune response ofdogs against Leishmania sp. is poorly understood (Reis et al.,2009).

In the Americas, visceral leishmaniasis (VL) is caused byLeishmania infantum (synonym Leishmania chagasi) proto-zoa and transmitted by sand flies (Grimaldi and Tesh, 1993).In dogs, VL may have variable clinical manifestations,which vary from asymptomatic infections to a chronic,systemic, debilitating disease (Blavier et al., 2001). Charac-terization of the innate immune response of dogs naturallyresistant to leishmaniasis is an important step towards thecontrol of the disease. A few genes from the innate immunesystem had their roles investigated in canine visceral leish-maniasis, such as Nramp1 and MHC (Bueno et al., 2009).TLR had their role demonstrated in human VL as well as inanimal models leishmaniasis, but the role of TLR has notbeen investigated in dogs (Cezário et al., 2011). This studyaimed to evaluate the profile of constitutive transcriptionof genes associated with innate immunity as well as quan-tify the secretion of selected cytokines and nitric oxide (NO)secretion by primary canine macrophages in response to L.infantum infection.

2. Materials and methods

2.1. Selection of dogs

The experimental protocol for this study was approvedby the Ethics Committee in Animal Experimentation atthe Universidade Federal de Minas Gerais (CEUA-UFMGprotocol 140/2009). Blood samples from male and femalemongrel dogs, one to eight years old, were collected toobtain serum and buffy coat. The absence of infection byLeishmania sp. was evaluated by ELISA using the rK39antigen. Briefly, 96 well plates were coated with rK39in carbonate buffer before being washed four times withPBS-Tween 20. Unspecific reactions were minimized byincubating the plates with 2% PBS-casein. Serum samples(1:80) were added and incubated for 12 h. The plates werewashed and secondary antibody IgG anti-dog (1:5000)incubated for 40 min. The plates were washed one moretime and incubated with o-phenylenediamine (OPD) inH2O2 for 10 min. Optical density was measured at 490 nmwave length. Samples were considered negative when ODwas smaller than the cut off (medium of the OD of thenegative controls plus two times the standard deviationof the negative controls). To confirm the absence of Leish-mania sp., DNA was extracted from blood samples fromdogs that were negative by ELISA, DNA was extracted fromthe buffy coat according to Pitcher et al. (1989) and PCRperformed according to Diniz et al. (2008) using the fol-lowing primers: 5′-CTTTTCTGGTCCCGCGGGTAGG-3′ and5′-CCACCTGGCCTATTTTACACCA-3′.

Macrophages from six dogs that presented the lowestpercentage of intracellular survival of L. infantum, and were

considered the most resistant (designated as resistant), andsix that had the highest percentage, considered the lessresistant (designated as susceptible), were selected for fur-ther analyses as detailed below.

d Immunopathology 163 (2015) 67–76

2.2. Canine macrophage isolation and culture

Canine macrophages derived from peripheral bloodmonocytes were obtained according to Bueno et al. (2005).Briefly, Peripheral blood was collected into heparanizedtubes. Blood (60 mL) was centrifuged at 1600 × g for 10 minat room temperature, plasma was separated, blood cellswere ressuspended (1:1 proportion) in phosphate bufferedsaline (PBS), overlaid onto a Ficoll solution (Ficoll-paquePlus, Amershan Biosciences, Little Chalfont, UK) (at 1 Ficoll:2 blood proportion), and then centrifuged at 1200 × g for40 min at 18 ◦C. Peripheral blood mononuclear cells (PBMC)were separated, washed twice on PBS, and ressuspendedin 8 mL of RPMI-1640 (Gibco, Carlsbad, USA) supplementedwith 10% foetal bovine serum, l-glutamine (200 mM), pyru-vate (10 mM), non essential aminoacids (10 mM), sodiumbicarbonate solution (7.5%, w/v) (Gibco, Carlsbad, USA),penicillin (50 IU/mL), and streptomicin (50 �L/100 mL). Thecell suspension was then transferred to Teflon flasks (Nal-geNunc, Rochester, USA), and cultured at 37 ◦C with 5%CO2. The medium was changed to remove non-adherentcells 24 h later, and the culture was kept under the sameconditions for 10 days, changing the medium every 3days. After 10 days in culture, the majority of the cellsare phenotypically characterized as macrophages based onCD14 expression as measured by flow cytometry as well asphagocytic activity, as previously described (Bueno et al.,2005).

2.3. Leishmania infantum culture

L. infantum World Health Organization strainMHOM/BR/1970/BH46 promastigotes were cultivateduntil reaching infective metacyclic stage in Schneider’smedium supplemented with 5% foetal calf serum, penicillin(100 U/mL) and streptomycin (50 �g/mL) at 25 ◦C.

2.4. Canine macrophage in vitro infection withLeishmania infantum

Infection with L. infantum was performed as previouslydescribed by Bueno et al. (2009). Briefly, macrophageswere harvested and distributed in four chamber slides(4 × 104 macrophages/well), after adherence to the slide,macrophages were inoculated with L. infantum BH46promastigotes with a multiplicity of infection (MOI) of10. The remaining macrophages that were cultured inteflon flasks as described above, were used for extractionof total RNA by adding 1 mL of Trizol followed by storageat −80 ◦C, until RNA extraction. After 24 h, supernatantwas harvested and macrophages infected with L. infantumBH46 at a 1:10 MOI. At 24 or 72 h post infection, super-natants were harvested and the chamber slides stainedwith May-Grunwald/Giemsa. In order to determine thepercentage of intracellular survival, the numbers of intra-cellular amastigotes at 24 and 72 h were determined by

direct counting of 200 randomly chosen macrophages.The survival percentage was obtained dividing the meannumber of intracellular amastigotes at 72 h post infectionby the mean number of intracellular amastigotes at 24 h

A.P. Turchetti et al. / Veterinary Immunology and Immunopathology 163 (2015) 67–76 69

Fig. 1. Observed frequency for 5% class intervals of intracellular survival percentage of Leishmania infantum in canine macrophages. The survival percentagewas obtained dividing the mean number of intracellular amastigotes at 72 h post infection by the mean number of intracellular amastigotes at 24 h posti

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relation (GraphPad Prism 5). Differences were consideredsignificant when p < 0.05.

Table 1Primers used in this study.

Gene Primers

TLR1 5′-TGCCATCCTACCGTGAACCTCA-3′

5′-ACTGTGTGGCACTCAACCCCAGA-3′

TLR2 5′-TCCGACACAAGAATGCAAAG-3′

5′-GGCAAAATCAGGGAAAATGA-3′

TLR3 5′-ACAGCTCCCCTTCACCTTTT-3′

5′-CCCTCCAACAGCTCATCATT-3′

TLR4 5′-GCCATTGCTTCTCCAACTTC-3′

5′-TGGTTTAGGCCCTGATATGC-3′

TLR5 5′-CCAGGACCAGACGTTCAGAT-3′

5′-GCCCAGGAAGATGGTGTCTA-3′

TLR6 5′-GGGCAAGAAACACACCCTTA-3′

5′-CTTCACCCAGGCAGAATCAT-3′

TLR7 5′-GGAAGACCCAAGGGAGAAAC-3′

5′-GCTGTATGCTCTGGGAAAGG-3′

TLR9 5′-CGCAGACTCAACCTGTCCTT-3′

5′-GCCATGTATGTCCAGCTCCT-3′

NOD1 5′-AGAGCAAGGGTGAAGAAGCA-3′

5′-GAGAAGCCAATCTCCGACAG-3′

NOD2 5′-ACTGCTGTTGGCCTGACTTT-3′

5′-TGAACTCATCAAAGCCATCG-3′

Nramp1 5′-CAGGCCTTCTACCAGCAAAC-3′

5′-ATGTCCACCTCCACCGTAAG-3′

′ ′

nfection.

ost infection, and frequencies of intracellular survivalere distributed at 5% class intervals (Fig. 1).

.5. Quantification of innate immunity related genexpression by qRT-PCR

Macrophages had their RNA extracted using Trizoleagent (Life Technologies, Carlsbad, CA, USA), cDNA wasynthesized using TaqMan Reverse Transcription ReagentsLife Technologies, Carlsbad, CA, USA) and qPCR performedo determine the expression of innate immune genes.

Transcription of TLR1, 2, 3, 4, 5, 6, 7 and 9; NOD1 and 2;nd Nramp1; as well as �-actin and GAPDH (house-keepingenes) was measured by qRT-PCR using the Step One PlusLife Technologies, Carlsbad, CA, USA) and primers listed inable 1. Data were analyzed by the comparative CT methodLivak and Schmittgen, 2001).

.6. Quantification of cytokines by ELISA

Detection of canine IL-10, IL-12/IL-23 p40, IFN-�, andNF-� supernatants of primary macrophages obtained at 0,4 and 72 h post L. infantum infection was performed usinguoSet ELISA Development Systems kits (R&D Systems,inneapolis, MN, USA), according to the manufacturer’s

nstruction.

.7. Nitric oxide assay

Nitric oxide quantification was indirectly determinedsing the Griess reagent (Promega, USA) according to theanufacturer’s recommendations. Briefly, macrophagesere seeded on a 96-well plate, inoculated with L. infan-

um as described above, and supernatants were harvestedt 0 (prior to inoculation), 24 and 72 h after inoculation.upernatants were mixed to the Griess reagent, and ODas measured at 540 nm. Standard curve was made using

erial dilutions of sodium nitrite.

.8. Statistical analysis

Cytokines and NO assays were compared by two-wayNOVA (GraphPad Prism 5). Differences were considered

significant when p < 0.05. Gene expression data were nor-malized based on the �-actin expression. After logarithmictransformation, means of resistant and susceptible ani-mals were compared by Student’s T test (GraphPad Prism5). Differences were considered significant when p < 0.05.Correlation between L. infantum intracellular survival andgene expression was evaluated by using the Pearson’s cor-

GAPDH 5 -AAGGCTGAGAACGGGAAACT-35′-TACTCAGCACCAGCATCACC-3′

�-actin 5′-GGCATCCTGACCCTGAAGTA-3′

5′-CGCAGCTCGTTGTAGAAGGT-3′

70 A.P. Turchetti et al. / Veterinary Immunology and Immunopathology 163 (2015) 67–76

Table 2Mean number of amastigotes per macrophage at 24 and 72 h post experimental infection with Leishmania infantum and individual intracellular survivalpercentage. Macrophages considered resistant or susceptible to intracellular survival of L. infantum are highlighted in bold.

Animal number Mean number of amastigotesper macrophage at 24 h postinfection

Mean number of amastigotesper macrophage at 72 h postinfection

Intracellularsurvival percentage

1 6.78 0.63 9.222 7.18 0.75 10.383 5.81 0.62 10.584 11.97 1.44 11.995 5.82 0.71 12.206 7.28 0.92 12.577 6.36 0.96 15.058 8.85 1.40 15.799 12.12 2.12 17.46

10 9.18 1.62 17.6511 3.95 0.72 18.1112 6.10 1.12 18.3813 9.56 1.86 19.4114 4.37 0.86 19.6115 9.13 1.93 21.1516 8.35 1.77 21.2217 8.52 1.81 21.3018 8.37 1.80 21.5119 8.28 1.84 22.2220 7.09 1.61 22.7221 9.49 2.21 23.3022 12.30 2.92 23.7323 7.93 1.89 23.8224 6.85 1.63 23.8525 7.24 1.73 23.9426 7.45 1.83 24.5027 8.59 2.47 28.7028 4.34 1.29 29.7429 10.21 3.07 30.0330 9.21 2.81 30.4731 9.64 2.97 30.7632 12.64 4.28 33.8633 4.52 1.57 34.7234 11.19 3.94 35.1735 11.27 4.12 36.5336 9.04 3.32 36.6937 8.19 3.32 40.5638 5.71 2.32 40.6639 11.98 4.96 41.4040 14.37 6.22 43.2741 10.31 5.27 51.1442 11.84 6.33 53.4243 11.09 6.11 55.1244 8.84 4.88 55.1545 16.05 10.22 63.6446 10.61 7.14 67.2847 10.17 7.22 71.03

48 12.24

3. Results

3.1. Serological and PCR screening

Initially, dogs were screened for inclusion in this studybased on negative test results for Leishmania sp. infection.A total of 234 dogs were subjected to blood and serumsampling, and 96 out of the 234 (41.03%) had antibod-ies anti-Leishmania, as detected by rK39 ELISA. Out of the

138 serologically negative dogs, 12 had Leishmania sp. DNAamplified (8.7%) from blood samples. Therefore, 126 out of234 dogs (53.85%) were considered negative to Leishmaniasp. infection.

9.74 79.58

3.2. Intracellular survival of Leishamania infantum inprimary canine macrophages

Forty eight clinically healthy dogs, negative to visceralleishmaniasis, by rK39 ELISA and buffy coat PCR, wereselected and had their peripheral blood monocytes iso-lated, cultured and differentiated into macrophages, whichwere in vitro infected with L. infantum. The number ofintracellular amastigotes of L. infantum in macrophages

was determined and percentage of intracellular survivalcalculated for each dog (Table 2). Fig. 1 shows the distribu-tion of the intracellular survival percentage of L. infantumfor 5% class interval. Representative images of infected

A.P. Turchetti et al. / Veterinary Immunology and Immunopathology 163 (2015) 67–76 71

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ig. 2. Representative images of macrophages that are resistant (A and B)nd C) and 72 (B and D) hours post infection. May-Grunwald/Giemsa, 40×

acrophages at 24 and 72 h post infection from resistantr susceptible macrophages are shown in Fig. 2.

.3. Quantification innate immunity-related transcripts

qRT-PCR results are shown in Figs. 3 and 4. Constitutivexpression of TLR9 and NOD2 were negligible, NOD1, LR1nd TLR7 had low levels of transcription, and Nramp1 andLR2, 3, 4, 5, and 6 were higher in canine monocyte-derived

ig. 3. Transcription of Nramp1, NOD1, NOD2, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6epresents the arithmetic mean of macrophages from 10 dogs in duplicate with s

eptible (C and D) to intracellular survival of Leishmania infantum at 24 (A

macrophages. There were no significant differences in tran-scription between macrophages that were resistant orsusceptible to intracellular survival of L. infantum (Fig. 4).

3.4. Cytokine secretion by primary canine macrophages

during Leishmania infantum infection

IL-10, IL-12/IL-23 p40, TNF-�, and IFN-� secretion byprimary canine macrophages infected with L. infantum are

, TLR7, and TLR9 in canine monocyte-derived macrophages. Each columntandard error bar.

72 A.P. Turchetti et al. / Veterinary Immunology and Immunopathology 163 (2015) 67–76

Fig. 4. Transcription of innate immunity genes in macrophages resistant or susceptible to intracellular survival of Leishmania infantum. (A) Nramp1, (B)NOD1, (C) NOD2, (D) TLR1, (E) TLR2, (F) TLR3, (G) TLR4, (H) TLR5, (I) TLR6, (J) TLR7 and (K) TLR9. The dots represent the mean of duplicate from each of fivedogs analyzed. Horizontal lines represent the average.

A.P. Turchetti et al. / Veterinary Immunology and Immunopathology 163 (2015) 67–76 73

Fig. 5. Quantification of cytokines by ELISA in macrophage supernatants. Macrophages (4 × 104/well) resistant (n = 6) or susceptible (n = 6) to intracellularsurvival of Leishmania infantum strain BH 46. MOI 1:10 in duplicate, and supernatants harvested 0, 24 and 72 h post infection. Positive control consists ofmacrophages from four dogs treated with 1 �g/mL of LPS. (A) IL-10, (B) IL-12, (C) TNF-� and (D) IFN-�. Each column represents the arithmetic mean withstandard error bar. One asterisk (*) indicates that the difference between resistant and susceptible animals in the same post infection period is statisticallysignificant (p < 0.05).

74 A.P. Turchetti et al. / Veterinary Immunology and Immunopathology 163 (2015) 67–76

Fig. 6. Indirect nitric oxide (NO) quantification by the Griess method in macrophage supernatants. Macrophages (4 × 104/well) resistant (n = 6) or susceptible(n = 6) to intracellular survival of Leishmania infantum strain BH 46 MOI 1:10 in duplicate, and supernatants harvested 0, 24 and 72 h post infection. Positive

PS. Each

control consists of macrophages from four dogs treated with 1 �g/mL of L

shown in Fig. 5. Secretion of TNF-� was higher in culturesof resistant macrophages at 24 h after infection when com-pared to susceptible macrophages, as well as the secretionof IFN-� at 72 h post infection. Secretion of IL-10 was lowerin resistant macrophages at 24 h after infection when com-pared to susceptible macrophages. There was no significantdifference in secretion of IL-12 between resistant or sus-ceptible macrophages, and levels IL-12 were very low. Asexpected, LPS induced secretion of IL-10, IL-12 and TNF-�.

3.5. Nitric oxide (NO) generation during infection

Generation of NO is demonstrated in Fig. 6. As expected,LPS induced generation of NO. However, there wasno detectable production of NO in monocyte-derivedmacrophages infected with L. infantum.

3.6. Correlation between gene expression andLeishmania infantum intracellular survival

Pearson’s correlation demonstrated a significant nega-tive correlation between NOD2 gene expression and intra-cellular survival of L. infantum in resistant macrophages(Fig. 7).

Fig. 7. Pearson’s correlation between NOD2 gene expression and intracel-lular Leishmania infantum survival in canine macrophages. P and r valuesindicate negative correlation between NOD 2 expression and intracellularsurvival in macrophages resistant to intracellular survival of L. infantum.

column represents the arithmetic mean with standard error bar.

4. Discussion

This study described for the profile of constitutivetranscription of genes associated with innate immunity,including NLR and TLR families, in canine peripheral bloodmonocyte-derived macrophages. Although transcription ofthese innate immunity genes have been previously studiedin other species (Fukui et al., 2001; Chang et al., 2009), andrecent studies begin to focus on the expression of thesegenes in the dog (Figueiredo et al., 2013), this study con-tributes to a better understanding of the role of TLR andNLR in canine innate immunity. Our data demonstratedhigh levels of constitutive expression of Nramp1 and TLR2, 3, 4, 5 and 6, receptors of Gram positive and nega-tive bacteria, fungi and viruses PAMP, may suggest theirroles in the recognition and control of pathogens by caninemacrophages. Conversely, NOD2 and TLR9 likely have sec-ondary roles in macrophage-mediated innate immunity ofdogs, due to their low expression in macrophages. In thisstudy, there were no significant differences in constitu-tive expression of these selected innate immunity genesbetween macrophages that were resistant or susceptibleto intracellular survival of L. infantum. However, these find-ings do not exclude the possibility of involvement of thesegenes in resistance against VL. Importantly, TLR4 polymor-phisms have been linked to increased susceptibility andseverity of Leishmania major infection in human patients(Ajdary et al., 2011).

Supernatants from macrophages that were resistant tointracellular survival of L. infantum had increased amountsof TNF-� and IFN-� and smaller amounts of IL-10. Thesefindings are consistent with previous studies. Cezário et al.(2011) evaluating the infection by L. infantum in Balb/cmice, which are considered susceptible to Leishmania infec-tion, demonstrated that in susceptible mice IL-10 secretionremains increased throughout the course of infection, andthat production of IFN-� and IL-12 is significantlydecreased. Furthermore, Muraille et al. (2003) demon-strated that C57BL/6 MyD88 knockout mice, which lack

natural resistance to L. major, have lower levels of IFN-� andIL-12. Interestingly, dogs vaccinated against visceral leish-maniasis have higher levels of IFN-� (Araújo et al., 2009).In another study, a live attenuated vaccine of Leishmania

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onovani led to increased production of IFN-�, TNF-�,nd IL-12 (Fiuza et al., 2013). Importantly, although weave previously demonstrated that the cells that remainsdhered to the teflon flasks as performed in this study havehenotypic features of macrophages (Bueno et al., 2005),he possibility of contamination of these primary culturesf peripheral blood monocyte-derived macrophages withymphocytes cannot be completely excluded. Therefore,FN-� production under these experimental conditions

ay be partially derived from lymphocytes.This study demonstrated that control of intracellular

urvival of L. infantum in canine macrophages is associ-ted with increased production of TNF-� and IFN-� andecreased production of IL-10. Clearance of L. donovanirom macrophages in vitro and in vivo is associated withnduction of IL-12 mediated by TLR4, and inhibition ofL-10 mediated by TLR2 (Paul et al., 2012). Furthermore,uman patients with low production of TNF-� are not ableo spontaneously heal lesions caused by L. major (Safaiyant al., 2011), confirming the important role of TNF-� inisease resistance. Additionally, monocytes from asymp-omatic human carriers of L. infantum produced highermounts of TNF-� when challenged in vitro when com-ared to monocytes from uninfected patients (Viana et al.,008). Both IL-12 and NO quantification were very low inhis study, probably due to the low detection limit of theechniques used, and the small number of macrophages inulture (4 × 104/well), which are intrinsic limitations of thisxperimental design.

Despite the low expression of NOD2, there was annteresting negative correlation between gene expres-ion and intracellular survival of L. infantum in resistantacrophages, i.e. the higher the expression of NOD2,

he greater the chance the macrophage is resistant tontracellular survival of L. infantum. The role of NOD2 ineishmaniasis has not been fully investigated (Clay et al.,014). Therefore, it may be a relevant target for future

nvestigations.

. Conclusion

This study demonstrated that decreased intracellu-ar survival of L. infantum in canine peripheral blood

onocyte-derived macrophages seems to be associatedith increased production of TNF-� and IFN-� andecreased production of IL-10. Furthermore, constitutiveranscription of Nramp1, TLR and NLR does not interferen intracellular survival of L. infantum in primary canine

acrophages. Importantly, this is the first description ofasal levels of transcription of innate immunity genes (i.e.ramp1, TLR, and NLR) in canine macrophages.

onflict of interest

The authors confirm that they have no conflicts of inter-st in relation to this work.

cknowledgements

We thank Daniel Menezes Souza and Michele Silva deatos for technical assistance. Work in RLS lab is supported

d Immunopathology 163 (2015) 67–76 75

by CNPq (Conselho Nacional de Desenvolvimento Cien-tífico e Tecnológico - grant # 480506/2012-4), FAPEMIG(Fundac ão de Amparo a Pesquisa do Estado de Minas Gerais- grant # CVZ-PPM-00464-13), and CAPES (Coordenac ãode Aperfeic oamento de Pessoal de Nível Superior - grant #PNPD 561/2012).

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