Leishmania infantum induces high phagocytic capacity and intracellular nitric oxide production by human proinflammatory monocyte

BACKGROUND The mechanism of resistance to SbIII in Leishmania is complex, multifactorial and involves not only biochemical mechanisms, but also other elements, such as the immune system of the host. OBJECTIVES In this study, putative changes in the immunological profile of human monocytes infected with wild-type (WT) and antimony (SbIII)-resistant Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum lines were evaluated. METHODS Susceptibility assays WT and SbIII-resistant L. braziliensis and L. infantum were performed using lines THP-1 human monocytic lineage. Phagocytic capacity, cytokine profile, intracellular nitric oxide (NO) production and surface carbohydrate residues profile were performed in peripheral blood monocytes by flow cytometry. FINDINGS The phagocytic capacity and intracellular NO production by classical (CD14++CD16-) and proinflammatory (CD14++CD16+) monocytes were higher in the presence of L. infantum lines compared to L. braziliensis lines. The results also highlight proinflammatory monocytes as the cellular subpopulation of major relevance in a phagocytosis event and NO expression. It is important to note that L. infantum induced a proinflammatory cytokine profile characterised by higher levels of TNF-α in culture supernatant than L. braziliensis. Conversely, both Leishmania lines induce high levels of IL-6 in culture supernatant. Analysis of the expression profile of surface carbohydrates showed that L. braziliensis presents 4.3-fold higher expression of galactose(β1,4)N-acetylglucosamine than L. infantum line. Interestingly, the expression level of α-N-acetylgalactosamine residues was 2-fold lower in the SbIII-resistant L. braziliensis line than its counterpart WT line, indicating differences in surface glycoconjugates between these lines. MAIN CONCLUSIONS Our results showed that L. braziliensis and L. infantum induce different innate immune responses and a highly inflammatory profile, which is characteristic of infection by L. infantum, the species associated with visceral disease.

drug for the treatment of human leishmaniasis in many countries. (3,4) Among the mechanisms of drug resistance in Leishmania spp., the main one is the decrease in drug concentration in parasite. (5) In addition, changes in the surface glycoconjugates expression profile of parasites have also been related to resistance and for this evaluation, lectins, which are glycoproteins of non-immunological origin, that interact in a reversible and specific way to carbohydrates or glycoconjugates are used. Studies involving an analysis of the surface glycoconjugates expression profile using specific lectins revealed that antimony-resistant L. donovani clinical isolates from patients in Bihar overexpress terminal glycoconjugates of N-acetyl-D-galactosamine residues. (6,7) The mechanism of resistance to Sb III in Leishmania is complex, multifactorial and involves not only biochemical mechanisms, but also other elements, such as the immune system of the host. A study using an isolate of L. infantum, with in vitro induced resistance to Sb III , reported resistance to NO and the cytotoxic effects of activated macrophages. (8) Another study demonstrated that L. braziliensis promastigotes isolated from refractory patients are generally less susceptible to nitric oxide (NO). (9) Moura et al. (10) observed that Sb III -resistant isolates of L. infantum showed a higher rate of infectivity in macrophages and higher resistance to Sb III and NO in vitro compared to wild-type (WT) samples. However, the involvement of the host immune system in the mechanisms of antimonyresistance in Leishmania requests to be better elucidated.
Clinical aspects and cure of leishmaniasis are related to the development of an effective and balanced immune response in the host. The intracellular parasite must be contained, whereas the immune response must be strictly controlled to avoid further tissue damage. (11,12) The characteristics of the immunological responses developed during Leishmania sp. infections are quite variable among the affected individuals, resulting in several clinical aspects. These natural differences arise from factors such as the host's immune status, the Leishmania species, parasite exposure, co-infections, among others. (11,12) Immunity against Leishmania is mediated through a complex immunological parameters network, including the innate and adaptive immune response. The immune response plays an important role in the clinical cure of the disease or its progression. (11) Thus, the presence of effector cells such as monocytes/macrophages, is critical for the control or development of leishmaniasis. (12) Monocytes/macrophages are antigen presenting cells that represent one of the first steps in the innate immune response aimed at killing the Leishmania parasite. These cells use important mechanisms that modulate inflammatory response activation and kill the parasite by producing cytokines as TNF-α, and NO, reactive oxygen intermediates (ROI). (13,14) Interestingly, Leishmania spp. modulates monocytes/ macrophage functions by preventing oxidative burst and the effector functions that lead to its elimination and by improvement a bursting microenvironment of modulatory IL-4, IL-10, and TGF-β cytokines. (14,15,16,17,18) A better understanding of the immune response against Leishmania spp. is very important to establishing a rational approach to chemotherapy and to comprehend mechanism of drug resistance. In this context, the objective of this study was to analyse changes in the immunological profile of monocytes after WT and Sb IIIresistant L. braziliensis and L. infantum lines infection and evaluate the agglutination pattern these different parasite species by applying tests that assess the lectins' binding specificity.

SUBJECTS AND METHODS
Human subjects -The study population consisted of 10 non-infected volunteers. The individuals were five men and five women, ranging from 20 to 31 years of age. They did not report previous infection by Leishmania spp. All individuals live in Belo Horizonte, Minas Gerais, Brazil, and voluntarily agreed to donate blood samples for the research.
Genotyping of Leishmania species using Hsp70 and ITS1 markers -The WT and Sb III -resistant L. braziliensis and L. infantum lines were submitted to polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for genotyping analysis using specific primers for HSP70 and ITS1 genes, as previously described. (20) Briefly, genomic DNA of these lines was obtained using the QIAquick DNA Extraction Kit (Qiagen), according to the manufacturer's recommendations. After amplification, the PCR products were digested with the restriction endonuclease enzyme HaeIII to analyse restriction fragments length polymorphisms (RFLP). Restriction profiles were analysed on 4% agarose GelRed (Biotium) stained and compared to the standard reference Leishmania species.
Susceptibility assays WT and Sb III -resistant L. braziliensis and L. infantum lines -THP-1 human monocytic lineage (ATCC TIB 202) were cultured in a complete RPMI-1640 medium [supplemented 10% foetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin] and differentiated into macrophages by adding 20 ng/mL phorbol myristate acetate (PMA) in culture. Macrophages (4 x 10 5 cells/well) were seeded in 24 well cell culture plates containing 13 mm round coverslips and incubated for 72 h at 37ºC in a humid atmosphere containing 5% CO 2 . Then, the adhered macrophages were exposed to promastigote forms of WT and Sb III -resistant L. braziliensis and L. infantum lines at the stationary phase (4 x 10 6 parasites/well) (10:1 parasites/ macrophage). After 5 h of infection, the free parasites were removed and a RPMI-1640 medium was added in the absence or presence of Sb III at a concentration ranging from 12.5 to 200 µM. After 72 h of incubation, the adhered macrophages were stained by panoptic staining. The infected cells and the number of intracellular amastigotes were determined using ImageJ software (1.50i version, Wayne Rasband National Institute of Health). EC 50 values were obtained from three independent measurements in triplicate, using the linear interpolation method.
L. braziliensis and L. infantum Alexa Fluor 647-labelling procedures -Promastigote forms of WT and Sb III -resistant L. braziliensis and L. infantum lines were obtained from cultures in a M199 medium supplemented with 10% FBS, maintained at 26ºC in BOD incubator for five and seven days, respectively. The parasites were stained with Alexa Fluor 647 at a final concentration of 3.2 µg/mL at 37ºC for 45 min in a 5% CO 2 incubator. After the Alexa Fluor 647-labelling procedure, parasites were washed once with phosphate buffered saline (PBS) pH 7.4 and resuspended in PBS supplemented with 10% FBS. The Alexa Fluor 647-labelled parasite suspension was adjusted to 1 x 10 8 /mL and maintained at 26ºC in a BOD incubator until use. Aliquots of Alexa Fluor 647-labelled parasites were run in LSR Fortessa (BD) to evaluate the efficiency of the Alexa Fluor 647-labelling procedure. The ideal Alexa Fluor 647-labelled parasite staining would lead to a single peak around 10 3 and 10 4 log intervals in histogram plots.
In vitro L. braziliensis and L. infantum promastigote peripheral blood monocyte infection -Human heparinised whole blood was centrifuged at 800 x g for 10 min at 18ºC. The plasma was removed, the whole blood cells (erythrocytes and leukocytes) washed two times with RPMI 1640 medium, and the final cell suspension adjusted to 1 x 10 7 cells/mL. For the Alexa Fluor 647-labelled promastigotes platform, 1 x 10 7 leukocytes were incubated with 1 mL of RPMI 1640 medium and 50 µL of Alexa Fluor 647-labelled WT or Sb III -resistant L. infantum or L. braziliensis promastigotes at 5 x 10 6 parasites/mL. Short-term in vitro cultures were performed in five distinct platforms for promastigote forms referred to as "uninfected culture", "infected WT promastigote of L. infantum culture", "infected Sb III -resistant promastigote of L. infantum culture", "infected WT promastigote of L. braziliensis culture", and "infected Sb III -resistant promastigote of L. braziliensis culture" by 5 h of incubation under gentle shaking in an orbital shaker, at 37ºC in a 5% CO 2 incubator. After incubation, each tube was centrifuged at 600 x g for 7 min at 18ºC. The supernatants were maintained for 1 month at -70ºC for cytokine analysis.
Intracellular NO expression in peripheral blood monocytes after WT and Sb III -resistant L. braziliensis and L. infantum in vitro infection -The NO expression was performed using peripheral blood monocytes as previously reported. (14,21) Membrane-permeable fluorescent NO indicator, DAF-2DA is deacetylated by intracellular esterases to DAF-2 that reacts with NO to yield the highly fluorescent, a membrane-impermeable compost, the triazolofluorescein (DAF-2T), allow to distinguish between NO generated inside the monocytes and NO-derived from an exogenous source. The culture was performed using 1 x 10 7 leukocytes incubated with 5 µL of WT or Sb III -resistant L. braziliensis or L. infantum Alexa Fluor 647-labelled promastigotes for 1 h at 37ºC in a 5% CO 2 incubator. A control culture ("uninfected culture") was used to determine the basal levels of intracellular NO. The samples were then incubated in the presence of 4,5-diaminofluorescein diacetate (DAF-2DA) (10 µM, Sigma, MO, USA) for 3 h at 37ºC in a 5% CO 2 incubator. The samples were labelled for 20 min at room temperature in the presence of PerCP-labelled anti-CD16 mAb, V450-labelled anti-CD14 mAb and PE-labelled anti-HLA-DR mAb. Following incubation, the red blood cells were lysed with FACS lysing solution (Becton Dickinson, CA, USA) and the cell pellet was washed and centrifuged at 600 x g for 7 min at room temperature and resuspended with PBS solution for immediate acquisition in the LSR Fortessa cytometer. A total of 3,000 events/tube on CD14 + monocyte gate was acquired. In order, to evaluate the enzymatic origin of the monocytes NO detected, the selective iNOS inducer [lipopolysaccharide (LPS) 10 µg/mL, Sigma, USA] and inhibitor (Aminoguanidine-AG 10mM, Sigma, MO, USA) were used. The LPS and AG cultures were used as functional specificity controls associated with monocytes NO production by iNOS pathway in all experiments. The data were analysed using Flow Jo 10.1 software.
Detection of supernatant cytokine levels by cytometric bead array -The cytometric bead array (CBA) is a method in which the simultaneous measurement of multiple cytokines is performed in a single sample. In this study, culture supernatant aliquots were used to quantify the secreted cytokine levels using CBA Human Inflammatory kit (Becton Dickinson, San Diego, CA, USA) for evaluation of IL-8, IL-1b, IL-6, IL-10, TNF-a, IL-12 p70 (limit of detection: 3.6 pg/mL, 7.2 pg/mL, 2.5 pg/mL, 3.3 pg/mL, 3.7 pg/mL and 1.9 pg/mL, respectively). Briefly, 25 µL of supernatants or standards were added to 17 µL of mixture beads and incubated for 3 h at room temperature in the dark. After incubation, the samples and standards were washed with 500 µL of wash buffer and centrifuged at 600 x g for 7 min at room temperature. Subsequently, 17 µL of detection cocktail consisting of mix PE-conjugated mAbs were added to each tube and the mixture reincubated for 180 min at room temperature in the dark. Following incubation, the samples and standards were washed again with 500 µL of wash buffer and centrifuged at 600 x g for 7 min at room temperature. After washing, 250 µL of wash buffer were added to each tube prior to data acquisition (300 events/tube) using FACSVerse flow cytometer (Becton Dickinson, San Diego, CA, USA). Data analysis was performed using FCAP Array 3.1 software (Becton Dickinson, San Diego, CA, USA). Secreted cytokines concentrations were expressed as pg/mL and the results plotted in bar graphs presenting mean + standard error of the mean (SEM) in log scale.

Expression profile of surface carbohydrates using Erythrina cristagalli, Dolichos biflorus, and Concanavalin A lectins in WT and Sb III -resistant L. braziliensis and L. infantum lines -The
Erythrina cristagalli (ECA), Dolichos biflorus (DBA), and Concanavalin-A (Con-A) lectins were used to compare the expression profile of the surface carbohydrates in WT and antimony-resistant L. braziliensis and L. infantum lines, as they have different carbohydrate binding specificities. ECA binds to residues of galactose (β1,4) N-acetylglucosamine, DBA recognises α-N-acetylgalactosamine residues, and Con-A binds to terminal portions of α-D-mannose and α-D-glucose residues. Promastigote forms were incubated separately with each of these lectins conjugated to fluorescein isothiocyanate (FITC). Briefly, promastigote forms of the WT and Sb III -resistant L. braziliensis and L. infantum lines in the stationary growth phase (2 x 10 6 parasites/mL) were washed with PBS and incubated with each lectin conjugated to FITC: ECA and DBA (EY Laboratories, INC, San Mateo, CA, USA) and Con A (Vector Laboratories, Burlingame, CA, USA) at a final concentration of 10 mg/mL for 30 min at 37ºC in a 5% CO 2 incubator. The total of 30.000 parasites/tube were then acquired to flow cytometer (LSR Fortessa) and the data were analysed using Flow Jo v.10 software. The geometric mean fluorescence intensity (gMFI) and the Con-A labelled-Leishmania percentage (%) for each L. braziliensis and L. infantum sample was determined.
Statistical analysis -Statistical analyses were performed using GraphPad Prism software (San Diego, USA, version 5.00). First, the Kolmogorov-Smirnov test was performed as normality test to evaluate the Gaussian distribution of data. The unpaired t test was used for comparative analyses between two groups, whereas, the one-way analysis of variance followed by Tukey's posttest were used for comparative analyses among three or more groups. For susceptibility assays, bidirectional analysis of variance (ANOVA two-way) followed by the Bonferroni post-test was used. In all cases, the differences were considered statistically significant when the p value was less than 0.05.
Ethics -This work complied with resolution number 466/2012 from the National Health Council for research involving human subjects and was approved by the Ethical Committee at Instituto René Rachou (CEPSH/ IRR/FIOCRUZ protocol:1.368.058), Belo Horizonte, Minas Gerais, Brazil.

RESULTS
Genotyping of Leishmania lines using Hsp70 and ITS1 markers -The ITS1 and Hsp70 molecular markers for genotyping were used to confirm the Leishmania species used in this study, of WT and Sb III -resistant L. braziliensis and L. infantum lines. Both pairs of Hsp70 and ITS1 primers amplified fragments of 1300 and 300-350 bp, respectively, in all Leishmania species analysed. The restriction profiles of the PCR products were compared to the standard reference Leishmania species: Susceptibility assays of WT and Sb III -resistant L. braziliensis and L. infantum lines using THP-1 macrophages -To analyse whether the Sb III -resistant phenotype persists in parasite intracellular forms, amastigote forms of WT and Sb III -resistant L. braziliensis and L. infantum lines were submitted to susceptibility assays with Sb III . The data showed that the numbers of infected macrophages were higher in the Sb III -resistant lines of both Leishmania species than in their WT counterparts (Fig. 1A). The number of amastigotes/100 infected macrophages was also higher in the Sb III -resistant than in the WT L. infantum line. The amastigote forms from both Sb III -resistant L. braziliensis and L. infantum lines were about 2.05 and 2.65-fold more resistant to Sb III (EC 50 34.8 and 133.3 µM for LbSbR and LiSbR, respectively) than to their respective counterpart WT lines (EC 50 17 and 50.2 µM for LbWT and LiWT) (Fig. 1B).
Phagocytic capacity of proinflammatory and classical monocytes -In order to evaluate the phagocytic ca- Classical (CD14 ++ CD16 -) and proinflammatory (CD14 ++ CD16 + ) monocyte subpopulations were plotted in anti-CD14-V450 versus anti-CD16-PerCP graphs, and the evaluation of the phagocytic capacity was obtained in anti-Leishmania AF647 + versus anti-CD14-V450 graphs to classical (CD14 ++ CD16 -) and proinflammatory (CD14 ++ CD16 + ) monocyte subpopulations. An illustrative graph of phagocytic capacity of proinflammatory (CD14 ++ CD16 + ) monocyte was presented in the last image. (B) Analysis of the phagocytic capacity of CD14 ++ CD16and CD14 ++ CD16 + monocytes of healthy volunteers were shown in bar graphs presenting mean + SEM, after incubation with wild-type (WT) and Sb III -resistant L. braziliensis and L. infantum lines. The results were presented by percentage (%) and geometric mean fluorescence intensity (gMFI); statistically significant differences (*P < 0.05, **P < 0.01) between WT and Sb III -resistant L. braziliensis and L. infantum lines were shown by connector lines. (C) Analysis of the phagocytic capacity of CD14 ++ CD16versus CD14 ++ CD16 + monocytes of healthy volunteers were shown in bar graphs presenting mean + SEM, after incubation with WT and Sb IIIresistant L. braziliensis and L. infantum lines. Statistically significant differences (P < 0.05) between the promastigote of WT and Sb III -resistant Leishmania lines in different monocyte subpopulations were shown by asterisks (*). pacity (percentage of monocytes Leish + ) of peripheral blood proinflammatory/ (CD14 ++ CD16 + ) and classical (CD14 ++ CD16 -) monocytes, they were infected with WT and Sb III -resistant L. braziliensis and L. infantum lines and evaluated by flow cytometry using phenotypicspecific selection strategy (anti-HLA-DR PE, anti-CD14 V450, and anti-CD16 PerCP antibodies) (Fig. 2A). The results showed that the phagocytic capacity of proinflammatory and classical monocytes was higher for WT and Sb III -resistant L. infantum lines as compared to L. braziliensis lines (Fig. 2B). Moreover, our data high-light proinflammatory monocytes as the cellular subpopulation of major relevance in a phagocytosis event (Fig. 2C). A second approach was used as complementary analysis with the purpose to evaluate the infection density of the monocytes (gMFI of infected cells). Our data demonstrated that despite the high percentage of L. braziliensis + proinflammatory monocytes (above 50% of infected cells), the number of parasites internalised or present in the cell membrane (interaction) is small as compared to L. infantum challenge (Fig. 2B -top  graphs). On the other hand, no differences were found Classical (CD14 ++ CD16 -) and proinflammatory (CD14 ++ CD16 + ) monocyte subpopulations were plotted in anti-CD14-V450 versus anti-CD16-PerCP graphs, and the intracellular NO expression was obtained in a DAF-2T FITC versus count histogram. (B) Analysis of the intracellular NO expression by CD14 ++ CD16and CD14 ++ CD16 + monocytes of healthy volunteers were shown in bar graphs presenting mean + SEM, after incubation with promastigotes of wild-type (WT) and Sb III -resistant Leishmania braziliensis and L. infantum lines. The control culture (basal NO expression) was shown by dashed rectangle (----) presenting the 95% confidence interval (95%CI). Statistically significant differences (p < 0.05) between promastigote of WT and Sb III -resistant Leishmania lines as compared to control culture were shown by asterisks (*). (C) Analysis of the intracellular NO expression by CD14 ++ CD16versus CD14 ++ CD16 + monocytes of healthy volunteers were shown in bar graphs presenting mean + SEM, after incubation with promastigotes of WT and SbIII-resistant L. braziliensis and L. infantum lines. The results were presented by percentage (%) and geometric mean fluorescence intensity (gMFI). Statistically significant differences (p < 0.05) between promastigotes of WT and Sb III -resistant Leishmania lines in different monocyte subpopulations were identified by asterisks (*). (D) Secreted cytokine profile was performed in culture supernatant of whole blood after promastigotes of WT and Sb III -resistant L. braziliensis and L. infantum lines infection using flow cytometric CBA immunoassay. Secreted IL-6, TNF-a, IL-4, and IL-10 concentrations were expressed as pg/mL and the results plotted in bar graphs presenting mean + SEM in log scale. The control culture (basal secreted cytokines) was shown by dashed rectangle (----) presenting the 95%CI. Statistically significant differences (p < 0.05) between promastigotes of WT and Sb III -resistant Leishmania lines cultures and control culture were shown by asterisks (*).
between Sb III -resistant parasites as compared to WT of the same Leishmania spp.

Functional biomarkers evaluation after WT and Sb III -resistant promastigote L. braziliensis and L. infantum infection
Intracellular NO expression by proinflammatory and classical monocytes -The reagent 4,5-diaminofluorescein diacetate (DAF-2DA) was used to evaluate intracellular NO expression. The monocytes were evaluated by flow cytometry in anti-CD14 V450 versus anti-CD16 PerCP plots, and NO expression was evaluated in histogram graphs highlighting the displacement of DAF-2T FITC in X axis (Fig. 3A). NO inducer (LPS) and NO inhibitor (AG) stimuli were used as functional and specificity controls, respectively. The control culture and WT and Sb III -resistant L. braziliensis and L. infantum promastigote cultures were used to evaluate the basal and Leishmania-specific NO production by proinflammatory and classical monocytes. The LPS data presented mean + SEM equal to 62.29 ± 8.45 (% -proinflammatory monocytes) and 24.23 ± 8.21 (% -classical monocytes). Already AG analyses showed mean + SEM equal to 6.69 ± 3.37 (% -proinflammatory monocytes) and 3.22 ± 0.35 (% -classical monocytes), indicative of perfect protocol controls. Data analyses were performed in comparison with control culture (basal cytokine profile -dashed rectangle presenting the 95%CI ; Fig. 3B). The results Fig. 4: flow cytometric analysis of the differential expression of surface carbohydrates in wild-type (WT) and Sb III -resistant Leishmania lines using Erythrina cristagalli (ECA), Dolichos biflorus (DBA), and Concanavalin-A (Con-A) lectins conjugated to fluorescein isothiocyanate (FITC). ECA binds to galactose (β 1,4) N-acetylglucosamine residues, DCA recognises α-N-acetylgalactosamine residues, and Con-A binds to terminal portions of α-D-mannose and α-D-glucose residues. (A) The Leishmania spp. population was selected in SSC-A versus FSC-A graph, and ECA, DBA and Con-A lectin profiles were performed in lectin-FITC versus count histograms. (B) Comparison of the ECA, DBA, and Con-A lectins binding profiles were presented in bar graphs showing the mean + SEM for WT and Sb III -resistant L. braziliensis and L. infantum lines. The results were presented by percentage (%) and geometric mean fluorescence intensity (gMFI), and statistically significant differences (*p < 0.05; **p < 0.01) between Sb III -resistant L. braziliensis and L. infantum lines were shown by connector lines. (C) Evaluation of the ECA, DBA, and Con-A lectins binding profiles for each Leishmania spp. line was presented in bar graphs showing the mean + SEM. Statistically significant differences (***p < 0.001) between Con-A expression and the other lectins in WT and Sb III -resistant L. braziliensis and L. infantum lines were shown by connector lines.
showed that Sb III -resistant L. braziliensis and both WT and Sb III -resistant L. infantum lines induced a statistically significant increase in fluorescence intensity and percentage of DAF-2T + proinflammatory monocytes (p < 0.05; Fig. 3B -Top Graphs). Interestingly, our data demonstrated a significant increase in fluorescence intensity and percentage of classical DAF-2T + monocytes subpopulation after L. infantum stimulation (Fig. 3B -Bottom Graphs). It also highlighted proinflammatory monocytes as the principal source of NO as compared to classical monocytes (Fig. 3C). No differences were found between Sb III -resistant parasites as compared to WT of the same Leishmania spp.
Proinflammatory and modulatory cytokine profile in culture supernatant -The Cytometric Bead Array (CBA) system was used to quantify cytokine supernatant levels. Promastigote forms of all Leishmania lines analysed induced high levels of proinflammatory IL-6 cytokine as observed in the supernatants of the different culture conditions. Data analyses were performed in comparison with control culture [basal cytokine profile -dashed rectangle presenting the 95% confidence interval (95%CI); Fig.3D]. Interestingly, both WT and Sb III -resistant L. infantum lines also induced higher levels of TNF-α (p < 0.05; Fig. 3D). The data did not show differentiated expression of modulatory cytokines, IL-4 and IL-10, in the culture conditions (Fig. 3D).
Evaluation of surface carbohydrate residues in L. braziliensis and L. infantum lines -We also investigated the expression profile of the surface carbohydrates galactose (β 1,4) N-acetylglucosamine (ECA), N-acetylgalactosamine (DBA), and Concanavalin-A (Con-A) in the WT and Sb III -resistant lines of each species. The Leishmania parasites were selected in SSC-A versus FSC-A graphs and the surface carbohydrates profile was performed by lectin-FITC expression (Fig. 4A). The results showed that the WT L. braziliensis line (LbWT) expresses 4.3-fold more galactose (β 1,4) N-acetylglucosamine residue compared to the WT L. infantum line (LiWT) (ECA-Lectin + ). In addition, a 2-fold lower expression of N-acetylgalactosamine residue (DBA-Lectin + ) was detected in the Sb III -resistant L. braziliensis as compared to its WT counterpart (Fig. 4B). A similar parasite percentage Con-A-labelled for each Leishmania sample was observed, indicating the homogeneity of the parasites regarding the expression of molecules containing mannose or glucose residues (Fig. 4C). It is important to emphasize that regardless of antimony-resistance phenotype, α-D-mannose and α-D-glucose are the most prevalent carbohydrates in the membrane of parasites compared to other evaluated.

DISCUSSION
Parasites of the genus Leishmania appear to be related to escape mechanisms that repress the normal functions of the macrophage, an immune system effector cell. (11,12) Monocytes/macrophages are antigen-presenting cells that act as parasite hosts and are responsible for eliminating them. Phagocytosed parasites are an important mechanism modulating the activation of the inflammatory response and controlling their growth. (11,12) According to the phenotypic-functional properties, the monocytes population is segregated into classical (CD14 ++ CD16 -), proinflammatory (CD14 ++ CD16 + ) and patrolling (CD14 dim CD16 + ) monocytes. (22) The classical monocyte subset shows a high expression of genes associated with tissue repair, anti-apoptotic properties, and acts as the first line of defense of the innate immune response against intracellular pathogens due to its phagocytic capacity. Proinflammatory monocytes express cytoskeletal rearrangement genes at high levels, presenting high phagocytosis capacity, anti-proliferative and pro-apoptotic properties, and produce higher levels of proinflammatory cytokines. (23) Previous studies have shown that a good Type 1 immune response (proinflammatory profile) is required, aiding in parasite protection. If the response is Type 1, cytokines such as IL-2, IFN-g, TNF-a, and IL-12 will be produced, promoting the activation of macrophages, NO synthesis, and parasite destruction. Conversely, if the response is Type 2, IL-4 and IL-10 will be produced, which cause a low macrophage activation and, consequently, clinical forms will appear. (11,12,24) Several Leishmania species have the ability to suppress the response to infection through inhibition of NO synthesis and proinflammatory cytokine production. (11,12,24) However, recent literature has showed that L. braziliensis-induced infection has been characterised by an inflammatory response. Cytokines such as IL-6, IL-1β, IL-17 and TNF-a have been associated with exacerbation of the infection, with TNF-a, an important NO inducer, being particularly significant in mucosal leishmaniasis evolution. (24) In VL, the increased production of inflammatory cytokines without parallel infection control has also contributed to pathogenesis. Several human VL studies have demonstrated elevated serum levels of IL-4, IL-6, IL-10, IL-12, IL-13, IFN-g, and TNF-a in active disease compared to asymptomatic infection. (15,16,25) In this sense, the progression/worsening of the clinical status would be promoting a lack of control of the inflammatory response and this "cytokine storm" without modulation favors the systemic inflammatory syndrome in VL patients and disfiguring mucosal lesions. (25,26) On the other hand, as in other infectious and parasitic diseases, individuals who development efficient immunological response to the infection and do not presented the apparent disease allows to understand the immunological mechanisms of protection against infection. These individuals present satisfactory balance between modulatory and inflammatory biomarkers. In Tegumentary Leishmaniasis the subclinical infection is characterised by a lower IFN-g and TNF-a production, suggesting that this modulated immune response has the ability to control infection without causing pathology. (27) Similarly, asymptomatic VL individuals presented basal profile cytokine. (15,16,25) In this study, the main objective was to analyse changes in the immunological profile of human peripheral blood monocytes after infection with WT and Sb IIIresistant L. braziliensis and L. infantum lines. Our data demonstrated that L. infantum induced higher levels of TNF-a compared to L. braziliensis. However, both Leish-mania species induce high levels of IL-6 in monocyte culture supernatants. Our results showed that L. infantum induced a higher phagocytic capacity, associated with NO and TNF-a production by human monocytes as compared to L. braziliensis. However, our group, in cutaneous leishmaniasis, showed protective immune response associate with CD23-IgE-mediated NO release, iNOS specific and increase of intracellular NO after treatment. (14,18) Thus, our data are in concordance with literature studies.
Regarding cytokine profile, although we not determine the source, we believed that the monocytes were the main cell population associated with this cytokine profile because they are physiologically the peripheral blood cells that most contribute with TNF-a and IL-6 production. Our data of proinflammatory monocytes reinforce this hypothesis since proinflammatory monocytes are highlighted as the cellular subpopulation of greater relevance in TNF-a and IL-6 production. In our study, we observed increase of Leishmania phagocytosis, specially L. infantum and NO expression and suggest that the monocytes are the main responsible by the TNF-a and IL-6 production.
This interspecific difference showing that L. infantum induced greater phagocytic capacity and production of NO and TNF-a by human monocytes compared to L. braziliensis. In the literature, studies point to the existence of polymorphisms in the structure of LPG intra and interspecies of Leishmania. (28,29) Polymorphisms of the glycoconjugates (LPG, GIPLs and gp63), are important in the differential regulation of the initial events of the immune response, as well as in the establishment of the infection. The variation in LPG structure results in a differential activation of macrophages, resulting in distinct production of cytokines and/or NO levels. (29) The presence of genetic diversity among lines allows a distinct ability to infect macrophages and thus the parasite surface molecules can modulate the interaction between Leishmania and host cells, influencing the clinical course of the disease (30) and on the appearance of different immunopathology in leishmaniasis. (28) As mentioned, discrepancies in the structure and composition of these molecules can influence the innate immune response and the differential establishment of the infection, generating a variety of clinical manifestations, such as LV and LT. (28) Interesting, no difference was observed in the phagocytic capacity and NO/cytokine production by human monocytes between WT and Sb III -resistant L. braziliensis and L. infantum lines. The resistant lines studied here have a low antimony-resistance index (2.0 and 2.65-fold) in THP-1 macrophages. We believe that if this resistance index had been higher, it would possibly alter the phagocytic capacity and other complementary parameters evaluated in the study. The Sb III -resistance mechanism in Leishmania is complex, multifactorial, and involves not only biochemical mechanisms but also other elements, such as the host immune system. Literature data using other antimony-resistant models have revealed some differences in Leishmania lines. Holzmuller et al. (8) reported that an isolate of L. infantum with resistance induced in vitro to Sb III was resistant to NO donors and the cytotoxic effects of activated macrophages. It has also been shown that L. braziliensis promastigotes isolated from refractory patients are generally less susceptible to NO. (9) Moura et al. (10) observed that antimony-resistant L. infantum isolates had a higher rate of infectivity in macrophages and a greater resistance to Sb III and NO in vitro as compared to WT samples. The authors further suggested that clinical isolates of L. infantum resistant to Sb III may induce the production of inflammatory cytokines and inhibit the mechanisms associated with macrophage death.
We also investigated the expression profile of the surface carbohydrates galactose (β 1,4) N-acetylglucosamine, using Erythrina cristagalli lectin (ECA), and Nacetylgalactosamine, using Dolichos biflorus (DBA) in the WT and Sb III -resistant L. braziliensis and L. infantum lines. The data showed a higher expression of galactose (β1,4) N-acetylglucosamine in WT L. braziliensis than in L. infantum line, indicating interspecific differences of the surface carbohydrates. The DBA expression profile showed that the Sb III -resistant L. braziliensis line expresses lower α-N-acetylgalactosamine residues than its counterpart WT line. In contrast, Mukhopadhyay et al., (7) using the same ECA and DBA lectins, showed that antimony-resistant L. donovani isolates overexpress surface glycoconjugates containing N-acetylgalactosaminyl residues. This difference may be due to different Leishmania species analysed. Drug resistance mechanism is complex, multifactorial and varies among the different Leishmania species analysed. The role of these surface glycoconjugates in the antimony-resistance phenotype should be further investigated. Concanavalin-A was used as a control and our data also showed the homogeneity of parasite populations in both species regarding the expression of molecules containing mannose and glucose residues, possibly LPG, since approximately 90% of the parasites were labelled with Con-A.
In this study, we used promastigote forms of only one strain of each Leishmania species: L. braziliensis (2904) and L. infantum (PP75), then further studies analysing a greater number of strains are need to support better the results.
In conclusion, our results showed that the phagocytic capacity and intracellular NO and TNF-a cytokine production by human monocytes were higher in the presence of L. infantum lines as compared to L. braziliensis lines. In addition, the data also highlighted proinflammatory monocytes as the cellular subpopulation of major relevance in phagocytosis events and NO expressions.