Accepted Manuscript

Title: Canine leishmaniosis. Modulation ofmacrophage/lymphocyte interactions by L. infantum

Authors: Suraya Diaz, Isabel Pereira da Fonseca, ArmandaRodrigues, Catarina Martins, Clara Cartaxeiro, Maria JesusSilva, Teresa Villa de Brito, Graca Alexandre-Pires, GabrielaSantos-Gomes

PII: S0304-4017(12)00256-7DOI: doi:10.1016/j.vetpar.2012.05.004Reference: VETPAR 6375

To appear in: Veterinary Parasitology

Received date: 31-5-2011Revised date: 6-5-2012Accepted date: 7-5-2012

Please cite this article as: Diaz, S., Fonseca, I.P., Rodrigues, A., Martins, C.,Cartaxeiro, C., Silva, M.J., Brito, T.V., Alexandre-Pires, G., Santos-Gomes, G., Canineleishmaniosis. Modulation of macrophage/lymphocyte interactions by L. infantum,Veterinary Parasitology (2010), doi:10.1016/j.vetpar.2012.05.004

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Canine leishmaniosis. Modulation of macrophage/lymphocyte interactions by L. 1

infantum2

3

Suraya Diaz1, Isabel Pereira da Fonseca2, Armanda Rodrigues1, Catarina Martins3, Clara 4

Cartaxeiro2, Maria Jesus Silva2, Teresa Villa de Brito2, Graça Alexandre-Pires2, 5

Gabriela Santos-Gomes16

7

1. Unidade de Ensino e Investigação de Parasitologia Médica, Centro de Malária e 8

Outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova 9

de Lisboa, Rua da Junqueira 100, 1349-008 Lisboa, Portugal10

11

2. Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina 12

Veterinária, Universidade Técnica de Lisboa, Av. Universidade Técnica, 1300-477 13

Lisboa, Portugal14

15

3. Departamento de Imunologia, Faculdade de Ciências Médicas, Universidade Nova de 16

Lisboa, Campo dos Mártires de Pátria, Lisboa, Portugal17

18

Corresponding author: 19

GM Santos-Gomes20

Address: Unidade de Ensino e Investigação de Parasitologia Médica, Centro de Malária 21

e Outras Doenças Tropicais (CMDT), Instituto de Higiene e Medicina Tropical (IHMT), 22

Rua da Junqueira 100, 1349-008 Lisboa - Portugal23

Tel.: +351 21 365 26 00; 24

Fax: +351 21 363 21 05;25

E-mail address: santosgomes@ihmt.unl.pt26

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Abstract 27

Canine leishmaniosis, caused by Leishmania infantum, is a systemic disease with 28

variable clinical signs and a progressive evolution. This disease is characterized by 29

impaired T-cell-mediated immune response, which has been associated with disease 30

chronicity and high mortality. Protective immunity against leishmaniosis is thought to 31

be mediated by T cell and cytokine production. The T cell activation requires a primary 32

signal delivered by the major histocompatibility complex (MHC) molecules present on 33

the surface of antigen presenting cells, and a non-specific signal generated by co-34

stimulatory molecules. To characterize canine immune responses in the presence of L. 35

infantum parasites or their antigens, in vitro cell cultures of canine macrophages and 36

lymphocytes were established, and the macrophages presenting MHC class II molecules37

were evaluated as well as the expression of IL-12 and CD80-86 co-stimulatory 38

molecules and nitric oxide production. The results showed for the first time the up-39

regulation of MHC class II molecules on the surface in canine peripheral blood 40

monocyte-derived macrophages during L. infantum infection in the presence of 41

lymphocytes. In addition, a lack of co-stimulatory expression and a reduced release of 42

nitric oxide were observed, suggesting a loss of T cell function and consequently an 43

inactivation of the macrophage oxidative burst which, in turn, favours the survival of 44

Leishmania. These results constitute a new contribution for the understanding of the 45

interactions between L. infantum and the canine immune system.46

47

Keywords: Dog, Leishmania infantum, macrophage, lymphocyte, co-stimulatory 48

molecules, cytokines, nitric oxide.49

50

51

52

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1. Introduction53

Canine leishmaniosis is a systemic disease with variable clinical signs and degrees of 54

severity, being the domestic dog the main reservoir of Leishmania infantum (syn.55

Leishmania chagasi). This parasite is transmitted to the vertebrate host by the bite of the 56

females of the sand fly of the genera Phlebotomus and Lutzomyia. 57

The canine host when infected with L. infantum mounts innate and acquired immune 58

responses: i) T-cell mediated, conferring protective immunity; and ii) marked non-59

protective humoral immune response with a depressed cell-mediated immunity 60

associated to overt disease (Cabral et al., 1998, Barbieri, 2006, Reis et al., 2006).61

Protective immunity against Leishmania parasites and production of pro-inflammatory 62

cytokines is thought to be mediated by CD4+ type 1 T helper (Th1) cell subset (Strauss-63

Ayali et al., 2007). T cell activation requires the presentation of peptide fragments of an 64

antigen through MHC molecules, and a co-stimulatory signal generated by co-65

stimulatory molecules (Chambers and Allison, 1997). The T-cell receptor (TCR)66

synchronic recognition of MHC–peptide complex and the interaction of B7-1 (CD80) or 67

B7-2 (CD86) with the co-stimulatory molecule CD28 results in T-cell activation, 68

proliferation and differentiation with cytokine production such as interleukin (IL) -12, 69

gamma interferon (IFN-) and tumor necrosis factor (TNF)- (Sperling and Bluestone, 70

1996). In presence of TNF, the IFN-γ-activated macrophages produce nitric oxide (NO) 71

which is considered to be one of the principal macrophage mechanisms for Leishmania 72

parasites elimination (Mauel et al., 1991, Pinelli et al., 2000). In the absence of CD28 73

binding, T cells undergo apoptosis or become anergic. On the other hand, co-binding of74

B7 to cytotoxic T-lymphocyte antigen 4 (CTLA-4) results in cell-cycle arrest and T cell 75

inactivation (Desjeux, 1996, Alegre et al., 2001, Salomon and Bluestone, 2001).76

In canine leishmaniosis, the loss of T cell function late in the infection has been related 77

to a non-coordinated expression of co-stimulatory molecules (Pinelli et al., 1999). 78

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However, the interactions between canine macrophages and lymphocytes during 79

infection with L. infantum promastigotes or in the presence of parasite antigens have 80

never been reported.81

Therefore, this study aims to evaluate the regulation of canine macrophage activity in 82

the presence of L. infantum parasites or antigens by monitoring in vitro; (1) the 83

expression of MHC class II molecules (MHCII) on the surface of macrophages, (2) the 84

interactions between canine macrophages and lymphocytes through the quantification of 85

the expression of co-stimulatory molecules CD80 and CD86 in macrophages, (3) the 86

expression of IL-12, a cytokine that initiates and regulates cellular immune functions,87

and (4) the macrophage oxidative burst by evaluating the production of NO.88

89

2. Materials and Methods90

91

2.1. Animals 92

The present study includes 11 apparently healthy dogs of both sexes (5 females and 6 93

males) aged between 2 and 10 years (median: 5.5 years standard deviation of 2.3 94

years) and with weights ranging between 21 and 54 kg (median: 24 kg standard 95

deviation of 8.1 kg). Six dogs were of a pure breed (Springer Spaniel, German 96

Shepherd, Golden Retriever, Saint Bernard, Rough Collie and Labrador Retriever) and 97

five were of a mixed-breed. The dogs used in this study were from Lisbon, Portugal, 98

considered to be a metropolitan endemic area for visceral leishmaniosis (Abranches et 99

al, 1991) and wore deltametrin collars during the transmission season. 100

All animals were subjected to physical examination and hemoparasites screening, by 101

blood smears, of Leishmania using the specific serodiagnosis indirect 102

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immunofluorescence assay (Kit Leishmania Spot IF, BioMerieux, France) with a cut 103

off of 1:80. 104

Prior to the animals inclusion in this work, the dog owners were informed of the 105

objectives of the study and the nature of the interventions, and their consent was 106

obtained.107

108

2.2. Blood collection109

The peripheral blood collection from all dogs included in this study was performed in 110

accordance with the veterinary norms; using therapeutic shearing under aseptic111

conditions (povidone-iodine and ethanol). The blood collections were divided in two 112

times: i) the first constituted by a collection of 200 mL of blood to a transfusion bag 113

with 250 mL of capacity using CPDA-1 as anticoagulant (Kawasumi, Germany) for 114

isolation and culture of peripheral blood monocyte-derived macrophages (MØ), and ii) 115

5 days after the first bleeding, 20 mL of blood was collected for lymphocytes isolation 116

using heparine as anticoagulant (Biochrom, Germany). 117

118

2.3. Canine peripheral blood monocyte-derived macrophages119

The collected blood (200 mL) was centrifuged at 600g for 10 min at room temperature. 120

The blood cells were re-suspended in phosphate buffered saline (PBS) at a 1:1 ratio and 121

overlaid onto a Ficoll solution (Ficoll-paque Plus, Amersham Biosciences, UK) in 1 122

Ficoll : 2 blood proportion and centrifuged at 400g for 40 min at 18ºC. Isolated 123

peripheral blood mononuclear cells (PBMC) were washed twice in PBS (200g, 10 min 124

and 4 ºC), and re-suspended in 8 ml of Roswell Park Memorial Institute medium 125

(RPMI) -1640 (Gibco, USA) supplemented with 10% fetal calf serum (FCS), L-126

glutamine (200 mM), pyruvate (10 mM), non-essential aminoacids (10 mM), sodium 127

bicarbonate solution (7.5% w/v), penicillin (50 IU/ ml), and streptomycin (50 μl/100ml) 128

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(Bueno et al. 2005). The cell suspension was then transferred to Teflon flasks 129

(NalgeNunc, USA) and cultured at 37ºC with 5% CO2 (Forma Scientific Incubator, 130

USA). Culture medium was replaced after 24h and 72h of incubation by fresh medium 131

to remove non-adherent cells. After 4 days of culture, the Teflon flasks containing 132

canine peripheral blood monocyte-derived macrophages (MØ) were placed onto ice for 133

30 min and harvested by agitation. The macrophages were then centrifuged at 600g for 134

10 min, and resuspended in 2 ml of RPMI-1640. Cell viability and concentration were 135

assessed by trypan blue staining using a Neubauer-counting chamber (Marienfeld, 136

Germany). The purity of peripheral blood monocyte-derived macrophages was 137

evaluated by flow cytometry analysis. Cells were incubated 15 min with 2% 138

paraformaldehyde at room temperature and then incubated 15 min with 0.1% Triton and 139

FITC-labelled mouse anti-human macrophages monoclonal antibody (clone MAC387, 140

AbD Serotec, Germany). This antibody presents a cross reaction with canine141

macrophages. Before acquisition in the flow cytometer, cells were washed with PBS at 142

600g for 10 min. The macrophage purity was approximately 92%.143

Flow cytometric acquisition was performed using a 4-colour flow cytometer (BD 144

FACSCalibur, BD Biosciences, USA) and data was analyzed using Cell Quest3.3TM145

Software (BD Biosciences).146

The duration of monocytes differentiation was established by flow cytometry analysis 147

on the 1st, 5th and 9th day of culture. According to light scattering characteristics 148

macrophages present higher forward and side scatter levels than monocytes. At days 5 149

and 9, cultures presented the same scatter characteristics which differ from day 1th of 150

culture (Fig. 1). Taken into account these results the following in vitro experiments 151

were performed with macrophages obtained after 5 days of culture. 152

153

154

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2.4. Canine peripheral blood lymphocytes155

The 20 mL of peripheral blood collected from each dog were re-suspended in PBS (1:1 156

v/v) and centrifuged at 600g for 10 min. The diluted blood was overlaid onto a 1:2 157

Ficoll solution and then centrifuged at 400g for 30 min at 18ºC. PBMC were separated, 158

washed twice on PBS (600g, 10 min, 4 ºC) and resuspended in 8 mL of RPMI-1640 159

(Gibco, USA) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 10 160

mM pyruvate, 10 mM non-essential amino acids, 7.5% sodium bicarbonate, 20 mM 161

HEPES, penicillin (50 IU/mL) and streptomycin (50 μl/100mL).162

The cell suspension was then transferred to culture flasks (Nunc, Denmark) and cultured 163

at 37 ºC in a humidified atmosphere with 5% of CO2 (Forma Scientific Incubator, 164

USA). The non-adherent cells were collected 24 h later. To confirm that the main 165

component of the suspension were lymphocytes, the cells were microscopically166

examined after methanol (VWR International) fixation and Giemsa (Sigma) staining of 167

cytocentrifuged (StatSpin Cytofuge, IRIS® Sample Processing, USA) slides. Cell 168

viability and concentration were assessed by trypan blue staining in a Neubauer-169

counting chamber. 170

171

2.5. L. infantum parasites culture172

BALB/c mice, aged 6–8 weeks, were purchased from Institute Gulbenkian of Science 173

and housed at the IHMT animal facilities, fulfilling the EU requirements (86/609/CEE), 174

recognised by Portuguese law (DR DL129/92 and Portaria 1005/92). L. infantum MON-175

1 (MHOM/PT/89/IMT151) was maintained by serial passages in BALB/c mice and 176

amastigotes were isolated from infected spleens. After in vitro transformation, virulent 177

promastigotes collected from the stationary phase of a subculture with less than five 178

passages (Santos-Gomes and Abranches, 1996) were used for peripheral blood 179

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monocyte-derived macrophages infection. Parasite concentration was assessed in a180

Neubauer-counting chamber.181

182

2.6. Antigen preparation 183

The antigen (Ag) was obtained from a culture of L. infantum MON-1 184

(MHOM/PT/89/IMT151) in HO-MEM medium supplemented with 20% FCS. When 185

the culture reached a concentration of 108 promastigotes mL-1 parasites were 186

centrifuged at 1830g for 15 min at 4ºC and then washed for three times in PBS/ETDA 187

(Sigma, USA) at 1830g, for 15 min at 4ºC. The pellet was subjected to six cycles of 188

freezing and thawing (-20ºC, -70ºC and room temperature). The protein concentration189

was assessed using a spectrophotometer (Bioteck Gene Quant II, Pharmacia, UK). The 190

antigen was conserved at -20ºC. 191

192

2.7. In vitro assays 193

After four days of culture the canine peripheral blood monocytes-derived macrophages 194

were re-cultured in 96 well plates (Nunc, Denmark) at 3.6x105 cells mL-1 per well in the 195

conditions described in the Table 1.196

After 24h of culture macrophages corresponding to conditions II (MØ infected with L. 197

infantum virulent promastigotes) and IV (MØ infected with L. infantum virulent 198

promastigotes and co-cultured with autologous canine lymphocytes) were infected at a 199

ratio of 7 promastigotes : 1 MØ, and parasite antigen (10µg/mL) was added to 200

conditions III (MØ cultured with L. infantum antigen) and V (MØ cultured with L. 201

infantum antigen and canine lymphocytes). On the 6th day of culture, plates were 202

washed and lymphocytes of the same dog were added to conditions IV, V and VI (MØ 203

co-cultured with canine lymphocytes) at a ratio of 1 MØ : 2 lymphocytes. Plates were 204

then incubated for 72h in 5% CO2 humidified atmosphere, at 37 ºC. 205

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Cells were used for MHCII analysis by flow cytometry and quantification of co-206

stimulatory molecules and cytokine expression by real time PCR. The cells’207

supernatants were used to determine macrophage activation through NO production. 208

209

2.8. MHCII expression at macrophages surface by flow cytometry analysis210

Non-adherent cells were removed and the monolayer of adherent cultured macrophages 211

was harvested with Trypsin-Versene/EDTA at 0.02% for 2 minutes at 37°C and 212

centrifuged at 400g for 5 minutes at 4°C. The cells were then resuspended in culture 213

medium with 10% of FCS. To analyse the MHCII expression, the macrophages were 214

washed with PBS at 600g for 10 min and incubated for 15 min at room temperature 215

with FITC-labelled rat anti-dog MHCII monomorphic (clone YKIX334.2) and APC-216

labelled rat anti-dog CD45 (clone YKIX716.13) monoclonal antibodies (AbD Serotec). 217

After incubation, cells were washed with PBS at 600g for 10 min and analysed in the 218

flow cytometer. 219

220

2.9. Total RNA extraction, reverse transcription and real time PCR221

Total RNA was extracted from parallel cultures using the RNeasy Mini kit (Qiagen, 222

Germany) following the manufacturer´s instructions (Qiagen, Germany). Samples were 223

denatured at 65ºC for 5 min before being reverse transcribed (RT) into cDNA using 200 224

U M-MLV RT (Promega, USA), at 37ºC for 60 min in the presence of 3 mM 5x M-225

MLV RT buffer (250 mM Tris–HCl, pH 8.3, 375 mM KCl and 15 mM MgCl2) 226

(Promega), 10mM BSA (Boehringer, Germany), 0.5 mM dNTPs (Life technologies, 227

Gibco Brl), 40 U rRNAsin ribonuclease inhibitor and 0.1 µg of Oligo (dT)15 (Promega) 228

per 1 µg of RNA. The samples were then heated 5 minutes at 95ºC for RT inactivation, 229

cooled and stored at -20ºC.230

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Following total RNA extraction and cDNA synthesis, Real Time PCR was used to 231

quantify co-stimulation molecules and cytokine expression. 232

Quantitative real-time PCR was performed in the ABI GeneAmp 5700-sequence 233

detection system (Perkin-Elmer/Applied Biosystems, USA). Reaction conditions were 234

processed powered on a Pentium III Dell Opti Plex GX110, linked directly to the 235

sequence detector. PCR amplifications were performed using SYBR® Green as a 236

double-strand DNA-specific binding dye with continuous fluorescence monitoring. The 237

amplifications were carried out in a total volume of 20 µl containing 2 µl of cDNA 238

sample, 10 µl of 2X SYBR® Green I dye PCR Master Mix and primers (300 nM) for -239

actin, IL-12 p(40) (Peters et al., 2005), CD80 and CD86 (Yasunaga et al., 2003). Each 240

PCR reaction was performed in duplicate wells using the following conditions: 10 min 241

at 95°C for AmpliTaq Gold activation followed by a total of 40 cycles (thermal profile 242

for each cycle: 15 s at 95°C, 1 min at 60°C). 243

External cDNA standards were designed as previously described (Rosa et al., 2005) and 244

its concentration was determined by OD measurement at 260 nm. Serial dilutions from 245

the resulting clones were used as standard curves, each containing a known amount of 246

input copy number (Overbergh et al., 1999; Hein et al., 2001; Yasunaga et al., 2003247

Rodrigues et al, 2009). Copy numbers of target genes were normalized to -actin, 248

present at constant levels in all samples, therefore correcting minor variations in RNA 249

isolation and reverse transcription. Final results were expressed as the copy number of 250

each cytokine per 104 cells. Efficiencies of amplification were 96.2% for -actin, 97.5% 251

for IL-12 p(40), and ~100% for CD80 and CD86. 252

253

254

255

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2.10. Quantification of nitric oxide production256

Cell supernatants were collected to assess nitric oxide production (NO) as a means of 257

measuring activation of macrophages. The production of NO was quantified by a classic 258

colorimetric Griess reaction (Sigma-aldrich, USA). Briefly, a mix of each supernatant 259

and Griess’ reagent was incubated for 10 minutes at room temperature and the260

absorbance measured at 550nm in an ELISA reader (Anthos 2010, Austria). The NO 261

concentration was determined using a standard curve generated from serial dilutions of 262

NaNO2 in complete RPMI medium (Gibco-Invitrogen, USA), each one containing a 263

known amount of nitrites. 264

265

2.11. Statistical analysis266

The non-parametric Wilcoxon Signed Ranks Test was used to compare paired groups in 267

relation to each parameter studied. Differences were considered significant with a 5% 268

significance level (p<0.05). The Statistical analysis was performed using the SPSS 19.0 269

for Windows software (SPSS Inc., USA).270

271

3. Results272

The 11 dogs evaluated for this study were negative for hemoparasites and for 273

antileishmania antibodies and therefore were included in the further experiments. The 274

use of molecular techniques to further detect Leishmania infection were disregarded due 275

to the results described above, the relative low prevalence of CanL in the metropolitan 276

region of Lisbon and the fact that the dogs that presented a healthy condition always277

wore deltrametrin collars during the transmission season.278

The number of monocytes present in total blood, and the concentrations of PBMC and 279

macrophages obtained after separation in a Ficoll gradient and 5 days of culture 280

respectively, are indicated in the Table 2. 281

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3.1. Increase of MHCII+ infected-macrophages when in contact with lymphocytes 282

The expression of MHC class II molecules was evaluated by flow cytometry in 283

macrophages cultured in different conditions. The in vitro cultured MØ were able to 284

express MHC class II molecules in all the conditions studied. In general the expression 285

of MHC class II molecules was higher in the presence of lymphocytes suggesting a role 286

for lymphocytes in this process (Fig 2). 287

Statistically significant differences were found between the following conditions: non-288

infected MØ vs infected MØ co-cultured with autologous lymphocytes or MØ cultured 289

with parasite antigens and lymphocytes (p=0.041); infected MØ vs infected MØ co-290

cultured with lymphocytes (p=0.008); MØ cultured with parasite antigens vs infected 291

MØ co-cultured with lymphocytes or MØ cultured with parasite antigens and 292

lymphocytes (p=0.041); MØ co-cultured with lymphocytes vs infected MØ co-cultured 293

with lymphocytes (p=0.004) or MØ cultured with parasite antigens and lymphocytes 294

(p=0.008). 295

296

3.2. Expression of CD80, CD86 and IL-12 by macrophages 297

The expression of CD80, CD86 and IL-12 was evaluated in macrophages cultured in 298

different conditions by real time PCR. No statistically significant differences were 299

observed in the levels of CD80 (Fig 3A), CD86 (Fig 3B) and IL-12 mRNA (Fig 4) 300

expressed in the MØ regardless the different conditions. 301

302

3.3. Quantification of NO production303

The production of NO was quantified in the different MØ culture conditions. Non-304

infected MØ showed higher mean values of NO production when compared to MØ305

infected with virulent L. infantum promastigotes (Fig. 5), pointing to some inhibition or 306

regulatory activity. This reduction in NO is more pronounced when comparing MØ and 307

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infected MØ cultured with lymphocytes or MØ incubated with L. infantum antigen and 308

co-cultured with lymphocytes. 309

Statistical significant differences in the levels of NO production between non-infected 310

MØ vs infected MØ (p=0.008), infected MØ co-cultured with autologous lymphocytes 311

(p=0.015) or MØ cultured with L. infantum antigens and lymphocytes (p=0.004).312

Significant differences were also observed between MØ co-cultured with lymphocytes 313

vs infected MØ co-cultured with lymphocytes (p=0.041) or MØ cultured with parasite 314

antigens and lymphocytes (p=0.021). 315

316

4. Discussion317

Macrophages play an important role against invasive microorganisms including318

Leishmania. They act as antigen presenting cells during infection, processing the 319

foreign antigens into a form in which they can bind MHCII molecules and be 320

recognized by the T cell receptors (TCR) (Kaye et al., 1994). The interactions between 321

MHCII-restricted antigens and the TCR determine whether the T cell becomes tolerant 322

or differentiate into effector cells (Geppert et al., 1990). However, the outcome of 323

Leishmania infection depends on the balance between the host ability to induce 324

leishmanicidal mechanisms and the parasite strategies to suppress or evade the host 325

immune response (Buates and Matlashewski, 2001).326

In the present study, although the sample is comprised by dogs of different sexes,327

breeds and ages, our results show an increase in the expression of MHCII in MØ 328

infected with L. infantum promastigotes when in the presence of autologous canine 329

lymphocytes and in MØ cultured with L. infantum antigens co-cultured with autologous 330

canine lymphocytes. This suggests that the MØ parasite’s antigen presentation in 331

addition with MHCII expression could be potentiated by lymphocytes. The activation of 332

T lymphocytes by MHCII-restricted antigens can induce the production of IFN- a 333

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pleiotropic cytokine with multiple functions in the defense against pathogens. IFN-334

stimulates MHC expression, foreign antigen processing and the presentation of both 335

MHCI and MHCII restricted antigens (St Louis et al., 1993).336

Leishmania spp. are normally characterized by their sophisticated developed 337

mechanisms to evade or interfere with host antigen presentation processes, allowing 338

parasites to modulate T cell mediated immune responses (Pinelli et al., 1999, Panaro et 339

al., 2008). A previous study performed with MØ from beagle dogs has reported 340

unchanged surface MHCI or MHCII expression upon infection with L. infantum (Pinelli 341

et al., 1999). Alternatively, in a different study, despite the up-regulation of MHCII 342

levels observed in in vivo infection with L. donovani a further decrease on APC function 343

was reported (Kaye et al., 1994; Antoine et al., 1999). In both these studies it was 344

proposed that a lack of coordinate expression of co-stimulatory molecules might had 345

lead to subsequent loss of T cell function by a mechanism involving the induction of T 346

cell unresponsiveness (Pinelli et al., 1999).347

The capacity of APCs to activate naive CD4+ T lymphocytes is dependent of co-348

stimulatory molecules like B7-1 (CD80) and B7-2 (CD86). These molecules are 349

involved in the transient binding of T cells to APCs and in signaling cascades leading to 350

T cell activation. In the absence of a co-stimulatory signal delivered by APCs, 351

interacting T cells are inactivated and enter into a state known as anergy, or die by 352

apoptosis (Alegre et al., 2001). In our study we did not find significant differences in 353

the levels of mRNA expression of B7-(CD80) and B7-2 (CD86) between non-infected 354

MØ and those cultured with L. Infantum or with promastigotes antigens either in the 355

presence or absence of lymphocytes. Associated with the lower expression of co-356

stimulatory molecules, the amounts of IL-12mRNA were low and with no statistically 357

significant differences between the different conditions. Taken together these results 358

suggest a regulation of host immune response by L. infantum and respective antigens. 359

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In addition, our results show a statistically significant decrease in the nitric oxide 360

production in MØ infected with virulent L. infantum promastigotes, infected MØ co-361

cultured with lymphocytes, MØ co-cultured with parasite antigen and, MØ co-cultured 362

with parasite antigen and lymphocytes. These results suggest the intervention of 363

lymphocytes, L. infantum parasites and its antigens in the regulation of macrophage 364

activity. The decrease in the production of NO in the presence of lymphocytes could be 365

a consequence of the co-stimulatory signal absence observed in the MØ cultured 366

conditions. Also, the down-regulation of NO could indicate the production of 367

transforming growth factor (TGF)-β and anti-inflammatory cytokines (e.g. IL-4, IL-10, 368

IL-13) by effector lymphocytes co-cultured with MØ infected by L. infantum or 369

cultured with parasite antigen (Panaro et al., 2008). Furthermore, the anti-inflammatory 370

cytokines may activate macrophage through an alternative pathway leading to the 371

production of polyamines, which are essential for the intracellular replication of 372

amastigotes. Alternatively activated macrophages also can present antigenic fractions by 373

MHCII (Rodriguez et al, 2004, Hëlscher et al, 2006).374

The present study suggests a role for L. infantum and parasite antigens in the up-375

regulation of MHCII molecules at MØ surface. Furthermore, both L. infantum and 376

parasite antigens seem to exert a negative regulation on co-stimulatory signals delivered 377

by MØ and NO production. These data also suggests the existence of parasite molecules 378

that are important in a mechanism of survival and proliferation within the host in which 379

parasites do not need to be alive to suppress the host immune responses. According to 380

the methodology used in this study to obtain antigen, it is probable that the parasite 381

molecules involved in this mechanism are not only present in the amastigote form but 382

are also expressed in the promastigote stage. It has been reported that MØ infected with 383

the promastigote stage can transiently present some Leishmania Ags and that only MØ 384

containing parasites that are rapidly killed after internalization are able to present some 385

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Leishmania Ags (McMahon-Pratt et al., 1998, Courret et al., 1999). However, the 386

down-regulation of nitric oxide production observed in MØ infected with L. infantum387

promastigotes in the presence or not of autologous canine lymphocytes and MØ 388

cultured with L. infantum antigens and co-cultured or not with lymphocytes indicates 389

that parasite killing is unlikely to happen. In fact, some Leishmania species have shown 390

typical features of apoptotic death taking place inside the macrophages, which was 391

found to be necessary to regulate cell numbers and to minimize immune reactions. 392

These studies had shown that apoptotic promastigotes were able to inhibit production of 393

nitric oxide by activated macrophages, which is in agreement with our data, which show394

that promastigotes and its antigens down-regulate NO production[0] (Shaha, 2006, van 395

Zandbergen et al., 2006, Wanderley et al., 2009).396

397

In this study, it was described for the first time the expression of MHCII, CD80, CD86398

co-stimulatory molecules, IL-12 and nitric oxide production in MØ from healthy dogs 399

infected in vitro with virulent L. infantum promastigotes or cultured with parasite 400

antigens and co-cultured with lymphocytes. Our results showed that canine MØ infected 401

with L. infantum promastigotes or cultured with the parasite antigens are able to express 402

MHCII molecules being this expression more significant in the presence of 403

lymphocytes. Also, it was shown that both L. infantum parasites and antigens had 404

impaired the MØ co-stimulatory signals and down regulated nitric oxide production. 405

This suggests the implication of promastigote stage specific molecules in the406

suppression of the immune responses allowing the survival, replication and dispersion407

of Leishmania within the host. In this way, the identification of the parasite molecules408

that interfere with the normal activation of the dog immune system seems crucial to409

elucidate Leishmania survival mechanisms in the host and to determine key new targets410

for vaccine and therapy design.411

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Conflict of interest412

The authors declare no conflicts of interest.413

Acknowledgments414

The authors thank Professor Renato Santos for helpful comments and advice, Dr. Joana 415

Gomes, MSc Marisa Ferreira and Dr. Gonçalo Vicente for blood collection, Dr. Lidia 416

Gomes, Dr Gloria Mendes, to all the dogs and owners that allowed the collection of 417

samples and to Antonio Casal for the revision of the manuscript. Funding for this work 418

was provided by a project research grant PTDC/CVT/113121/2009 by the Portuguese 419

Foundation for Science and Technology (FCT) with co-participation of the European 420

Union Fund (FEDER).421

422

References423

Abranches, P., Silva-Pereira, M.C., Conceição-Silva, F.M., Santos-Gomes, G.M., Janz, 424

J.G., 1991, Canine leishmaniasis: pathological and ecological factors influencing 425

transmission of infection. J Parasitol. 77, 557-561.426

Alegre, M.L., Frauwirth, K.A., Thompson, C.B., 2001, T-cell regulation by CD28 and 427

CTLA-4. Nat Rev Immunol 1, 220-228.428

Antoine, J.C., Lang, T., Prina, E., Courret, N., Hellio, R., 1999, H-2M molecules, like 429

MHC class II molecules, are targeted to parasitophorous vacuoles of 430

Leishmania-infected macrophages and internalized by amastigotes of L. 431

amazonensis and L. mexicana. J Cell Sci 112 ( Pt 15), 2559-2570.432

Barbieri, C.L., 2006, Immunology of canine leishmaniasis. Parasite Immunol 28, 329-433

337.434

Page 18 of 30

Accep

ted

Man

uscr

ipt

18

Brodskyn, C., Patricio, J., Oliveira, R., Lobo, L., Arnholdt, A., Mendonca-Previato, L., 435

Barral, A., Barral-Netto, M., 2002, Glycoinositolphospholipids from 436

Trypanosoma cruzi interfere with macrophages and dendritic cell responses. 437

Infect Immun 70, 3736-3743.438

Buates, S., Matlashewski, G., 2001, General suppression of macrophage gene 439

expression during Leishmania donovani infection. J Immunol 166, 3416-3422. 440

Bueno, R., Mello, M.N., Menezes, C.A., Dutra, W.O., Santos, R.L.,: 2005, Phenotypic, 441

functional, and quantitative characterization of canine peripheral blood 442

monocyte-derived macrophages. Mem Inst Oswaldo Cruz, 100m 521-524.. 443

Cabral, M., O'Grady, J.E., Gomes, S., Sousa, J.C., Thompson, H., Alexander, J., 1998, 444

The immunology of canine leishmaniosis: strong evidence for a developing 445

disease spectrum from asymptomatic dogs. Vet Parasitol 76, 173-180.446

Chambers, C.A., Allison, J.P., 1997, Co-stimulation in T cell responses. Curr Opin 447

Immunol 9, 396-404.448

Courret, N., Prina, E., Mougneau, E., Saraiva, E.M., Sacks, D.L., Glaichenhaus, N., 449

Antoine, J.C., 1999, Presentation of the Leishmania antigen LACK by infected 450

macrophages is dependent upon the virulence of the phagocytosed parasites. Eur 451

J Immunol 29, 762-773.452

Desjeux, P., 1996, Leishmaniasis. Public health aspects and control. Clin Dermatol 14, 453

417-423.454

Geppert, T.D., Davis, L.S., Gur, H., Wacholtz, M.C., Lipsky, P.E., 1990, Accessory cell 455

signals involved in T-cell activation. Immunol Rev 117, 5-66.456

Hein, J., Schellenberg, U., Bein, G., Hackstein, H., 2001, Quantification of murine IFN-457

gamma mRNA and protein expression: impact of real-time kinetic RT-PCR 458

using SYBR green I dye. Scand J Immunol 54, 285-291.459

Page 19 of 30

Accep

ted

Man

uscr

ipt

19

Hëlscher, C., Arendse, B., Schwegmann, A., Myburgh, E., Brombacher F., 2006. 460

Impairment of alternative macrophage activation delays cutaneous leishmaniasis 461

in nonhealing BALB/c mice. J Immunol 176, 1115–1121.462

Kaye, P.M., Rogers, N.J., Curry, A.J., Scott, J.C., 1994, Deficient expression of co-463

stimulatory molecules on Leishmania-infected macrophages. Eur J Immunol 24, 464

2850-2854.465

Kuchroo, V.K., Das, M.P., Brown, J.A., Ranger, A.M., Zamvil, S.S., Sobel, R.A., 466

Weiner, H.L., Nabavi, N., Glimcher, L.H., 1995, B7-1 and B7-2 costimulatory 467

molecules activate differentially the Th1/Th2 developmental pathways: 468

application to autoimmune disease therapy. Cell 80, 707-718.469

Lenschow, D.J., Walunas, T.L., Bluestone, J.A., 1996, CD28/B7 system of T cell 470

costimulation. Annu Rev Immunol 14, 233-258.471

Liu, D., Kebaier, C., Pakpour, N., Capul, A.A., Beverley, S.M., Scott, P., Uzonna, J.E., 472

2009, Leishmania major phosphoglycans influence the host early immune 473

response by modulating dendritic cell functions. Infect Immun 77, 3272-3283.474

Mauel, J., Corradin, S.B., Buchmuller Rouiller, Y., 1991, Nitrogen and oxygen 475

metabolites and the killing of Leishmania by activated murine macrophages. Res 476

Immunol 142, 577-580; discussion 593-574.477

McMahon-Pratt, D., Kima, P.E., Soong, L., 1998, Leishmania amastigote target 478

antigens: the challenge of a stealthy intracellular parasite. Parasitol Today 14, 479

31-34.480

Overbergh, L., Valckx, D., Waer, M., Mathieu, C., 1999, Quantification of murine 481

cytokine mRNAs using real time quantitative reverse transcriptase PCR. 482

Cytokine 11, 305-312.483

Panaro, M.A., Brandonisio, O., de Caprariis, D., Cavallo, P., Cianciulli, A., Mitolo, V., 484

Otranto, D., 2008, Canine leishmaniasis in Southern Italy: a role for nitric oxide 485

Page 20 of 30

Accep

ted

Man

uscr

ipt

20

released from activated macrophages in asymptomatic infection? Parasit Vectors486

1, 10.487

Peters, I.R., Helps, C.R., Calvert, E.L., Hall, E.J., Day, M.J., 2005, Cytokine mRNA 488

quantification in histologically normal canine duodenal mucosa by real-time RT-489

PCR. Vet Immunol Immunopathol 103, 101-111.490

Pinelli, E., Gebhard, D., Mommaas, A.M., van Hoeij, M., Langermans, J.A., 491

Ruitenberg, E.J., Rutten, V.P., 2000, Infection of a canine macrophage cell line 492

with Leishmania infantum: determination of nitric oxide production and anti-493

leishmanial activity. Vet Parasitol 92, 181-189.494

Pinelli, E., Rutten, V.P., Bruysters, M., Moore, P.F., Ruitenberg, E.J., 1999, 495

Compensation for decreased expression of B7 molecules on Leishmania 496

infantum-infected canine macrophages results in restoration of parasite-specific 497

T-cell proliferation and gamma interferon production. Infect Immun 67, 237-498

243.499

Reis, A.B., Teixeira-Carvalho, A., Giunchetti, R.C., Guerra, L.L., Carvalho, M.G., 500

Mayrink, W., Genaro, O., Correa-Oliveira, R., Martins-Filho, O.A., 2006, 501

Phenotypic features of circulating leucocytes as immunological markers for 502

clinical status and bone marrow parasite density in dogs naturally infected by 503

Leishmania chagasi. Clin Exp Immunol 146, 303-311. 504

Rodrigues, O., Marques, C., Soares-Clemente, M., Ferronha, M.H., Santos-Gomes, 505

G.M., 2009, Identification of regulatory T cells during experimental Leishmania 506

infantum infection. Immunobiology 214, 101-111.507

Rodriguez, N.E., Chang, H.K., Wilson, M.E., 2004, Novel program of macrophage gene508

expression induced by phagocytosis of Leishmania chagasi. Infect Immun. 272, 509

2111-2122.510

Page 21 of 30

Accep

ted

Man

uscr

ipt

21

Rosa, R., Rodrigues, O.R., Marques, C., Santos-Gomes, G.M., 2005, Leishmania 511

infantum: soluble proteins released by the parasite exert differential effects on 512

host immune response. Exp Parasitol 109, 106-114.513

Salomon, B., Bluestone, J.A., 2001, Complexities of CD28/B7: CTLA-4 costimulatory 514

pathways in autoimmunity and transplantation. Annu Rev Immunol 19, 225-252.515

Santos-Gomes, G.M., Abranches, P., 1996, Comparative study of infectivity caused by 516

promastigotes of Leishmania infantum MON-1, L. infantum MON-24 and L. 517

donovani MON-18. Folia Parasitol (Praha) 43, 7-12.518

Shaha, C., 2006, Apoptosis in Leishmania species & its relevance to disease 519

pathogenesis. Indian J Med Res 123, 233-244.520

Sperling, A.I., Bluestone, J.A., 1996, The complexities of T-cell co-stimulation: CD28 521

and beyond. Immunol Rev 153, 155-182.522

St Louis, J.D., Lederer, J.A., Lichtman, A.H., 1993, Costimulator deficient antigen 523

presentation by an endothelial cell line induces a nonproliferative T cell 524

activation response without anergy. J Exp Med 178, 1597-1605.525

Stempin, C., Giordanengo, L., Gea, S., Cerban, F., 2002, Alternative activation and 526

increase of Trypanosoma cruzi survival in murine macrophages stimulated by 527

cruzipain, a parasite antigen. J Leukoc Biol 72, 727-734.528

Strauss-Ayali, D., Baneth, G., Jaffe, C.L., 2007, Splenic immune responses during 529

canine visceral leishmaniasis. Vet Res 38, 547-564.530

van Zandbergen, G., Bollinger, A., Wenzel, A., Kamhawi, S., Voll, R., Klinger, M., 531

Muller, A., Holscher, C., Herrmann, M., Sacks, D., Solbach, W., Laskay, T., 532

2006, Leishmania disease development depends on the presence of apoptotic 533

promastigotes in the virulent inoculum. Proc Natl Acad Sci U S A 103, 13837-534

13842.535

Page 22 of 30

Accep

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Man

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Wanderley, J.L., Pinto da Silva, L.H., Deolindo, P., Soong, L., Borges, V.M., Prates, 536

D.B., de Souza, A.P., Barral, A., Balanco, J.M., do Nascimento, M.T., Saraiva, 537

E.M., Barcinski, M.A., 2009, Cooperation between apoptotic and viable 538

metacyclics enhances the pathogenesis of Leishmaniasis. PLoS One 4, e5733.539

Yasunaga, S., Masuda, K., Ohno, K., Tsujimoto, H., 2003, Antigen-specific 540

enhancements of CD80 mRNA expression in experimentally sensitized dogs 541

with Japanese cedar pollen. J Vet Med Sci 65, 295-300.542

543

544

545

546

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546

547

Table 1. Conditions used in the in vitro assays. I- non-infected MØ; II- MØ infected 548

with L. infantum virulent promastigotes; III- MØ cultured with L. infantum antigens; 549

IV- MØ infected with L. infantum virulent promastigotes and co-cultured with canine 550

lymphocytes; V- MØ cultured with L. infantum antigens and co-cultured with canine 551

lymphocytes; VI- MØ co-cultured with canine lymphocytes.552

553

Macrophage culture conditions

I MØ

II MØ + Leish

III MØ + Ag

IV MØ + Leish + Lymp MØ + Ag + Lymp

N. MØ /well 1x105 1 x105 1x105 1x105 1

N. L. infantum promastigotes/well - 7x105 - 7x105

L. infantum antigen/well - - 4µg -

N. autologues lymphocytes/well - - - 2x105 2x10

554

555

556

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556

557

558

559

560

561

562

Table 2. Number of isolated monocytes and lymphocyte, monocyte-differentiated 563

macrophage harvested form Teflon flasks after 5 days of culture and, the 564

respective recovery rates.565

566

N. monocytes/200ml total blood

Recovery rate

N. Teflon-adherent cells

Recovery rate

N. lymphocyte/20 ml total blood

Recovery rate

1.5x108 ± 1.7x108 cells

52.4%5.3x106 ± 2.3x106

cell mL-1 3.5% 4.3x107 ± 2.8x107 cells

mL-1 46.3%

567

568

569570

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570

571

572

573

574

575

576

577

578

579

Figure 1. Representative dot plots of Forward Scatter (x axis) vs. Side Scatter (y axis) 580

presenting MØs differentiation along a 9 day culture. A - Day 0; B - Day 5; C – Day 9. 581

The selected gates (R) correspond to: R1 – Monocytes; R2 – MØs.582

583

584

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585

596

597

Figure 2. Expression of MHC class II in MØs. A) Representative histogram of MHC 598

class II expression at surface of MØs cultured alone (black line), MØs cultured with 599

virulent L. infantum promastigotes (bold line) and MØs cultured with virulent L. 600

infantum promastigotes and lymphocytes (grey line). B) Representative dot plots of 601

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Side Scatter (x axis) vs. CD45 (y axis) of MØs cultured alone, cultured with virulent L. 602

infantum promastigotes or with virulent L. infantum promastigotes and lymphocytes.603

The selected gates (R – R1, R3 and R9) correspond to MØs. C) Surface expression of 604

MHC class II in MØs cultured alone (MØ); MØs infected with L. infantum605

promastigotes (MØ+leish), MØs with parasite antigen (MØ+Ag), MØs infected with L. 606

infantum promastigotes and co-cultured with canine lymphocytes (MØ+leish+Lymp), 607

MØs with parasite antigen co-cultured with lymphocytes (MØ+Ag+Lymp), and MØs 608

co-cultured with canine lymphocytes (MØ+Lymp). The interquartile ranges with 609

minimum and maximum values are indicated. * indicates statistically significant 610

differences (p<0.05).611

612

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612

Figure 3. Level of CD80 (A) and CD86 (B) mRNA in non-infected MØ (MØ), MØ 613

infected with L. infantum promastigotes (MØ+leish), MØ with parasite antigen614

(MØ+Ag), MØ infected with L. infantum promastigotes and co-cultured with canine 615

lymphocytes (MØ+leish+Lymp), MØ with parasite antigen and co-cultured with 616

lymphocytes (MØ+Ag+Lymp), and MØ co-cultured with canine lymphocytes 617

(MØ+Lymp). Data are expressed as the number of copies per 104 cells. The interquartile 618

ranges with minimum and maximum values are indicated. 619

620

0

50

100

150

200

250

Conditions

CD

80

mR

NA

co

pie

s (

n-c

op

ies

/100

00 c

ells

)

MO MO+Leish MO+Ag MO+Leish MO+Ag MO+Lymp +Lymp +Lymp

0.0

0.5

1.0

1.5

2.0

Conditions

CD

86 m

RN

A c

op

ies

(n

-co

pie

s/1

0000

ce

lls)

MO MO+Leish MO+Ag MO+Leish MO+Ag MO+Lymp +Lymp +Lymp

A

B

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620

621

622

Figure 4. Level of IL-12 mRNA in MØ cultured alone (MØ), MØ infected with L. 623

infantum promastigotes (MØ+leish), MØ with parasite antigen (MØ+Ag), MØ infected 624

with L. infantum promastigotes and co-cultured with canine lymphocytes 625

(MØ+leish+Lymp), MØ with parasite antigen and co-cultured with lymphocytes 626

(MØ+Ag+Lymp), and MØ co-cultured with canine lymphocytes (MØ+Lymp). Data are 627

expressed as the number of copies per 104 cells. The interquartile ranges with minimum 628

and maximum values are indicated. 629

630631

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Conditions

IL12

mR

NA

co

pie

s (

n-c

op

ies

/100

00 c

ells

)

MO MO+Leish MO+Ag MO+Leish MO+Ag MO+Lymp +Lymp +Lymp

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631

Figure 5. Nitric oxide production by non-infected MØ, MØ co-cultured with infectious 632

L. infantum promastigotas (MØ+leish) or incubated with L. infantum antigen (MØ+Ag), 633

infected MØ co-cultured with lymphocytes (MØ+leish+Lymp), MØ incubated with 634

antigen and co-cultured with lymphocytes (MØ+Ag+Lymp) and MØ co-cultured with 635

lymphocytes (MØ+Lymp). Each point results from independent measurements of the 636

supernatant of three macrophage cultures. The interquartile ranges with minimum and 637

maximum values are indicated. * indicates statistically significant differences, (p638

<0.05).639

640

641642

0

1

2

3

Conditions

µM N

itri

tre

MO MO+Leish MO+Ag MO+Leish MO+Ag MO+Lymp +L ymp +Lymp

*

*

*

*

*