Mem Inst Oswaldo Cruz, Rio de Janeiro, VOLUME 115 | FEBRUARY 2020
Original Article

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

Christiana Vargas Ribeiro1, Bruna Fonte Boa Rocha1, Edward Oliveira1, Andrea Teixeira-Carvalho2, Olindo Assis Martins-Filho2, Silvane Maria Fonseca Murta1/+, Vanessa Peruhype-Magalhães2

1Fundação Oswaldo Cruz-Fiocruz, Instituto René Rachou, Grupo de Genômica Funcional e Proteômica de Leishmania spp. e Trypanosoma cruzi, Belo Horizonte, MG, Brasil
2Fundação Oswaldo Cruz-Fiocruz, Instituto René Rachou, Grupo Integrado de Pesquisas em Biomarcadores, Belo Horizonte, MG, Brasil

DOI: 10.1590/0074-02760190408
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ABSTRACT

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.

 Leishmaniasis is caused by over 21 different species of unicellular protozoan parasites belonging to the ge­nus Leishmania. It is classified as a neglected tropical disease, according to the World Health Organization (WHO), and is a public health issue in many develop­ing countries.(1) Leishmaniasis is characterised by a spectrum of clinical manifestations ranging from self-healing cutaneous (CL) and mucocutaneous (MCL) skin lesions to a visceral (VL) form, which is lethal if un­treated. In the New World, L. (L.) infantum (syn. L. (L.) chagasi) is the causative agent of VL, whereas L. (V.) braziliensis causes CL and MCL.(2)

Many studies have been carried out to identify genes that may be involved in the N-methylglucamine antimo­niate (Glucantime®)-resistance phenotype, first-choice 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 SbIII 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 SbIII, 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 SbIII-resistant isolates of L. infan­tum showed a higher rate of infectivity in macrophages and higher resistance to SbIII and NO in vitro compared to wild-type (WT) samples. However, the involvement of the host immune system in the mechanisms of antimony-resistance 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 SbIII-resistant 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.

Leishmania samples and culture conditions - In this study, we used promastigote forms of Leishmania (Vi­annia) braziliensis (MHOM/BR/1975/2904) and Leish­mania (Leishmania) infantum (MHOM/BR/1974/PP75) lines WT and resistant to potassium antimony tartrate (SbIII). The SbIII-resistant lines were previously selected in vitro by step-wise increase of SbIII drug pressure.(19) The parasites were grown at 26ºC in M199 medium sup­plemented with 2 mM L-glutamine, 5 μg/mL hemin, 50 μg/mL streptomycin, 2 μg/mL biopterin, 1 μg/mL bio­tin, 40 mM HEPES pH 7.4, 50 U/mL penicillin, and 10% v/v heat-inactivated fetal calf serum. All experiments were carried out with parasites in the stationary phase.

Genotyping of Leishmania species using Hsp70 and ITS1 markers - The WT and SbIII-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 SbIII-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 105 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% CO2. Then, the adhered mac­rophages were exposed to promastigote forms of WT and SbIII-resistant L. braziliensis and L. infantum lines at the stationary phase (4 x 106 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 SbIII 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). EC50 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 SbIII-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% CO2 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 108/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 103 and 104 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 107 cells/mL. For the Alexa Fluor 647-labelled promastigotes platform, 1 x 107 leukocytes were incubated with 1 mL of RPMI 1640 medium and 50 μL of Alexa Fluor 647-la­belled WT or SbIII-resistant L. infantum or L. braziliensis promastigotes at 5 x 106 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 SbIII-resistant promastigote of L. infantum culture”, “infected WT promastigote of L. braziliensis culture”, and “infected SbIII-resistant promastigote of L. brazilien­sis culture” by 5 h of incubation under gentle shaking in an orbital shaker, at 37ºC in a 5% CO2 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 SbIII-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 107 leukocytes incubated with 5 μL of WT or SbIII-resistant L. braziliensis or L. infantum Alexa Fluor 647-labelled promastigotes for 1 h at 37ºC in a 5% CO2 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% CO2 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 re-incubated 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 SbIII-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 SbIII-resistant L. braziliensis and L. infantum lines in the stationary growth phase (2 x 106 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% CO2 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 post-test 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 SbIII-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: L. amazonensis (IFLA/BR/67/PH8), L. braziliensis (MHOM/BR/75/M2903), L. infantum (MHOM/BR/74/ PP75), and L. guyanensis (MHOM/BR/75/M4147). The results confirmed that the Leishmania species used in this work correspond to L. braziliensis and L. infantum [Supplementary data (Figure)].

Susceptibility assays of WT and SbIII-resistant L. bra­ziliensis and L. infantum lines using THP-1 macrophages - To analyse whether the SbIII-resistant phenotype persists in parasite intracellular forms, amastigote forms of WT and SbIII-resistant L. braziliensis and L. infantum lines were sub­mitted to susceptibility assays with SbIII. The data showed that the numbers of infected macrophages were higher in the SbIII-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 SbIII-resistant than in the WT L. infantum line. The amastigote forms from both SbIII-resistant L. braziliensis and L. infantum lines were about 2.05 and 2.65-fold more resistant to SbIII (EC50 34.8 and 133.3 μM for LbSbR and LiSbR, respectively) than to their respective counterpart WT lines (EC50 17 and 50.2 μM for LbWT and LiWT) (Fig. 1B).

Phagocytic capacity of proinflammatory and classi­cal monocytes - In order to evaluate the phagocytic ca­pacity (percentage of monocytes Leish<sup+>) of peripheral blood proinflammatory/ (CD14++CD16+) and classical (CD14++CD16-) monocytes, they were infected with WT and SbIII-resistant L. braziliensis and L. infantum lines and evaluated by flow cytometry using phenotypic-specific 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 SbIII-resistant L. infantum lines as compared to L. braziliensis lines (Fig. 2B). Moreover, our data high­light 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 between SbIII-resistant parasites as compared to WT of the same Leishmania spp.

Functional biomarkers evaluation after WT and SbIII-resistant promastigote L. braziliensis and L. infantum infection

 

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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 SbIII-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 showed that SbIII-resistant L. braziliensis and both WT and SbIII-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 SbIII-resistant parasites as compared to WT of the same Leishmania spp.

 

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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 SbIII-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 SbIII-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 SbIII-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.

 

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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 (CD14dimCD16+) 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 SbIII-resistant L. braziliensis and L. infantum lines. Our data demonstrated that L. infantum induced higher levels of TNF-a compared to L. braziliensis. However, both Leish­mania 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 SbIII-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 SbIII-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 SbIII 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 SbIII and NO in vitro as compared to WT samples. The authors further suggested that clini­cal isolates of L. infantum resistant to SbIII 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 N-acetylgalactosamine, using Dolichos biflorus (DBA) in the WT and SbIII-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 SbIII-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.

ACKNOWLEDGEMENTS

To the Program for Technological Development in Tools for Health-PDTIS-FIOCRUZ for use of its facilities.

AUTHORS’ CONTRIBUTION

CVR, BFBR, SMFM and VPM designed, performed the experiments and analysed the data; CVR, BFBR, EO, ATC, OAMF, SMFM and VPM wrote and reviewed the full manu­script. All authors read and approved the final manuscript. The authors declare that there is no conflict of interests.

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Financial support: CNPq, FAPEMIG (CBB-PPM00610/15), CAPES.EO, ATC, SMFM and BFBR are research fellows supported by CNPq; VPMP by FAPEMIG; CVR by CAPES.
+ Corresponding author: silvane@cpqrr.fiocruz.br
 http://orcid.org/0000-0002-8523-2155
Received 31 October 2019
Accepted 14 February 2020

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