Protective Profile Involving CD23/IgE‐mediated NO Release is a Hallmark of Cutaneous Leishmaniasis Patients from the Xakriabá Indigenous Community in Minas Gerais, Brazil - Carvalho‐Gontijo - 2015 - Scandinavian Journal of Immunology - Wiley Online LibraryScandinavian Journal of ImmunologyVolume 81, Issue 6 p. 515-524 Human Immunology Free Access Protective Profile Involving CD23/IgE-mediated NO Release is a Hallmark of Cutaneous Leishmaniasis Patients from the Xakriabá Indigenous Community in Minas Gerais, Brazil R. Carvalho-Gontijo, Laboratory of Biomarkers for Diagnosis and Monitoring, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilSearch for more papers by this authorV. Peruhype-Magalhães, Laboratory of Biomarkers for Diagnosis and Monitoring, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilSearch for more papers by this authorM. F. Costa-Silva, Laboratory of Biomarkers for Diagnosis and Monitoring, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilSearch for more papers by this authorO. A. Martins-Filho, Laboratory of Biomarkers for Diagnosis and Monitoring, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilSearch for more papers by this authorP. F. Quaresma, Laboratory of Biomarkers for Diagnosis and Monitoring, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilSearch for more papers by this authorJ. de Moura Freire, Laboratory of Leishmaniasis, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilSearch for more papers by this authorE. de Castro Moreno, Center of Hematology and Hemotherapy Foundation of Minas Gerais - Hemominas, Belo Horizonte, MG, BrazilSearch for more papers by this authorA. Teixeira-Carvalho, Laboratory of Biomarkers for Diagnosis and Monitoring, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilSearch for more papers by this authorC. M. Ferreira Gontijo, Corresponding Author Laboratory of Leishmaniasis, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilCorrespondence to: C. M. Ferreira Gontijo, Laboratory of Leishmaniasis, René Rachou Research Center – FIOCRUZ/MG, Av. Augusto de Lima 1715, Barro Preto - Belo Horizonte – Minas Gerais – Brazil CEP 30190-002. E-mail: gontijo@cpqrr.fiocruz.brSearch for more papers by this author R. Carvalho-Gontijo, Laboratory of Biomarkers for Diagnosis and Monitoring, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilSearch for more papers by this authorV. Peruhype-Magalhães, Laboratory of Biomarkers for Diagnosis and Monitoring, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilSearch for more papers by this authorM. F. Costa-Silva, Laboratory of Biomarkers for Diagnosis and Monitoring, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilSearch for more papers by this authorO. A. Martins-Filho, Laboratory of Biomarkers for Diagnosis and Monitoring, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilSearch for more papers by this authorP. F. Quaresma, Laboratory of Biomarkers for Diagnosis and Monitoring, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilSearch for more papers by this authorJ. de Moura Freire, Laboratory of Leishmaniasis, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilSearch for more papers by this authorE. de Castro Moreno, Center of Hematology and Hemotherapy Foundation of Minas Gerais - Hemominas, Belo Horizonte, MG, BrazilSearch for more papers by this authorA. Teixeira-Carvalho, Laboratory of Biomarkers for Diagnosis and Monitoring, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilSearch for more papers by this authorC. M. Ferreira Gontijo, Corresponding Author Laboratory of Leishmaniasis, René Rachou Research Center, FIOCRUZ, Belo Horizonte, MG, BrazilCorrespondence to: C. M. Ferreira Gontijo, Laboratory of Leishmaniasis, René Rachou Research Center – FIOCRUZ/MG, Av. Augusto de Lima 1715, Barro Preto - Belo Horizonte – Minas Gerais – Brazil CEP 30190-002. E-mail: gontijo@cpqrr.fiocruz.brSearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract In this study, we described, for the first time, specific aspects of an anti-Leishmania immune response in a Brazilian Xakriabá indigenous community. Induction of an intracellular NO pathway, triggered by the binding of IgE to CD23 receptor in IFN-γ/IL-4 cytokines environment, was evaluated in localized cutaneous leishmaniasis (LCL) carriers and positive Montenegro skin test (MST) individuals without skin lesion (MT+SL−). Our data demonstrated that the higher frequency of CD23+CD14+ monocytes and the increased serum levels of IgE observed in the LCL group were even higher in LCL carriers with late lesions (LCL≥60). Furthermore, patients with LCL presented increased NO production after Leishmania (Viannia) braziliensis stimulation and this NO profile was independent of the time of the lesion (recent LCL 60 or late LCL≥60). We also showed that the increased frequency of IFN-γ+ and IL-4+CD4+ T cells is related to the MT+SL− group. The results of biomarker signature curves demonstrated that in the MT+SL− group, the index signature was characterized by DAF-2T+CD14+/IL-4+CD8+/IFN-γ+CD4+/IL-4+CD4+. On the other hand, the LCL group presented a higher index of DAF-2T+CD14+/CD23+CD14+/IL-4+ CD8+, associated with a lower index of IFN-γ+CD8+. Considering the time of lesion, data analysis demonstrated that the main differences observed were highlighted in LCL 60 patients, with a higher index of CD23+CD14+, which was also present in LCL≥60 patients. In conclusion, our data suggest that the protective immune response involving CD23-IgE-mediated NO release is a hallmark of patients with LCL. However, in MT+SL− individuals, another different leishmanicidal mechanism seems to be involved. Introduction In the New World, leishmaniasis is a zoonosis that involves different species of protozoans of the genus Leishmania and a great variety of mammal hosts and reservoirs. In human leishmaniasis, the clinical manifestations depend mainly on the species of the parasite involved and the immune response of the infected host; however, it is important to emphasize that many factors together function to determine the complex spectrum of clinical manifestations of human leishmaniasis. Tegumentary leishmaniasis (TL) has a great variety of clinical manifestations with the most common being cutaneous leishmaniasis (CL). In CL, innate immunity is preserved, which can be observed through the positive Montenegro skin test (MST) result. This specific cellular response, which is well modulated but with a predominance of type 1 cytokines, reflects the tendency of spontaneous healing and positive response to treatment in patients with CL. Furthermore, about 10% of individuals who live in endemic areas present positive MST results yet do not develop the disease, presenting instead a subclinical form of TL 1. There has been little research on the occurrence of TL outbreaks in indigenous tribes in Brazil. The earliest of these took place in the 1950s and 1960s and studied tribes located in the state of Mato Grosso 2, 3. Aspects of the lifestyle and social condition of indigenous populations, such as poor nutrition, lack of hygiene and sanitation, close interaction with animals in the domestic and peridomestic environments, all favour the transmission of many parasitoses 4, including TL. In a Xakriabá indigenous community, for example, studies revealed that L. (V.) braziliensis, L. (L.) infantum and L. (V.) guyanensis are circulating among domestic and synanthropic mammals, thus favouring leishmaniasis transmission in this area 5. Additionally, a study related to immune response was undertaken in the same community and was able to characterize the immunological biomarkers of the skin lesions of these Brazilian Indians 6. Some previous research has been attracting attention for its importance regarding patterns of immune response in TL. Many studies suggest that the type 1 cellular immune response, characterized by the production of IFN-γ, serves a protective role 7, 8. In murine models, IFN-γ has shown a simultaneous action with another cytokine, the TNF-α, in activating inducible nitric oxide synthase (iNOS or NOS2) to produce nitric oxide (NO) resulting in the killing of intracellular parasites 9, 10. Vouldoukis etal. (1995) demonstrated that the binding of IgE to the low-affinity receptor FcεRII/CD23 in human macrophages is strong and induces NO production after activation with IL-4 and IFN-γ or IFN-γ only, thus restricting the growth of Leishmania major 11. Moreover, it has been described that serum IgE levels and CD23 expression are increased in CL, and human monocytes/macrophages express FcεRII/CD23 after activation with IFN-γ or IL-4 12, 13. In this context, this study evaluated, for the first time, specific aspects of an anti-Leishmania immune response in a Brazilian indigenous community. Induction of the intracellular NO pathway, triggered by the binding of IgE to the CD23 receptor in IFN-γ/IL-4 cytokines environment in CL carriers and positive MST individuals without skin lesions, was evaluated in the Xakriabá indigenous community in the state of Minas Gerais, Brazil. This study provided a rare opportunity to study immunological aspects related to CL in an indigenous population. The Xakriabá Indigenous Reserve is located in the municipality of São João das Missões (14°53′01″ S, 44°05′26″ W), in northern Minas Gerais, Brazil. The reserve covers 78% of the entire municipality and has a population of 7,813 inhabitants. Between 2001 and 2012, 402 cases of cutaneous leishmaniasis (CL) were recorded in the Xakriabá Territory, and since 2008 a large study has been carried out in the reserve to address measures to prevent and control the transmission of CL. All actions have been conducted by the Family Health Program, currently adopted by the Brazilian Unified Health System, and also located in the Xakriabá Territory. The study included indigenous patients with a diagnosis of localized cutaneous leishmaniasis (LCL, n=17) during the 4-year period of 2009–2012 (Table1). The selection criteria for inclusion in the study were patients with skin lesions suggestive of LCL who had a positive Montenegro skin test (MST), presence of Leishmania parasites documented by Giemsa-stained imprints of biopsy fragments and/or parasite isolation in culture medium containing blood agar enriched with LIT (liver infusion tryptose) medium maintained at 26°C. Furthermore, DNA detection of Leishmania by PCR (kDNA was used as DNA target) also made patients eligible for inclusion. Any patients with alcohol dependence, tuberculosis, diabetes, pneumonia, HIV infection or other immunosuppressive disease, use of immunosuppressive agents, pregnancy, patients with severe anemia and patients with previous specific leishmaniasis treatment were excluded. All patients received specific treatment for LCL at the Programa de Saúde da Família (Family Health Program) as recommended by the Brazilian Ministry of Health guidelines. In this study, individuals with positive Montenegro skin test without skin lesions (MT+SL−, n=13) and non-infected individuals who did not present suggestive signals of active LCL or a characteristic scar, plus negative MST (NI, n=10), all residents in Xakriabá indigenous community (Table1), were included. For future comparative analysis, the LCL group was divided in two other groups according to the duration of lesion; patients with early lesions and patients with late lesions. Patients with early lesions were those whose lesion presented a development time of less than 60days (LCL 60, n=7), whereas patients with late lesions were those whose lesions presented development time of greater than or equal to 60days (LCL≥60, n=10). The study was conducted in agreement with the Helsinki Declaration, Resolution #196/1996 at the Comissão Nacional de Ética em Pesquisa – CONEP – (National Commission on Research Ethics) that regulates research involving human subjects in Brazil and Resolution #304/2000 that regulates research involving Indians. The Comitê de Ética em Pesquisa (Research Ethics Committees) at Centro de Pesquisas René Rachou (CEPSH/CPqRR n°: 021/2007) and at the CONEP (protocol n°.14689) previously approved the informed consent forms and procedures. Written informed consent was obtained from all adults, and the children were formally included in the study only if the informed consent form was signed by their parents or legal guardians. This project was conducted with the permission of the Fundação Nacional do Índio – FUNAI – (Indigenous National Foundation, licence no. 149/CGEP/08) to enter into indigenous lands. Promastigotes of Leishmania (V.) braziliensis (MHOM/BR/1986/MSS) were obtained from cultures in complex liquid medium LIT supplemented with 20% fetal bovine sera (FBS) maintained at 26°C in a B.O.D. incubator for 7days 14, 15. The parasites were stained with fluorescein isothiocyanate (fluorescein isothiocyanate, isomer I – C21H11NO5S – FITC; Sigma, MO, USA) at a final concentration of 200μg/ml at 37°C for 30min in a 5% CO2 humidified incubator. After the FITC-labelling procedure, parasites were washed three times with PBS and resuspended in PBS supplemented with 10% FBS. The FITC-labelled parasites suspension was adjusted to 1×108/ml and maintained at 26°C in a B.O.D. incubator until use. Aliquots of FITC-labelled parasites were run in a flow cytometer to evaluate the efficiency of the FITC staining procedure. The ideal FITC-labelled parasite staining would lead to a single peak around 102 and 103 log intervals in FL1/FITC histogram plots. Additionally, the FITC-labelled parasites were monitored by optical microscopy for motility features and by Trypan Blue staining for viability quality control. Phagocytosis assay of L. (V.) braziliensis promastigotes by peripheral blood monocytes Heparinized peripheral blood was centrifuged at 1200g for 10min at room temperature. The plasma was removed, the blood cells were washed with PBS supplemented with 10% FBS, and the final cell suspension was adjusted to 1×107 cells/ml. Short-term in vitro cultures were performed in two distinct platforms referred to as \"ex vivo – unstimulated’ and ‘L. braziliensis – stimulated’ cultures. For the ‘ex vivo – unstimulated’ cultures, aliquots of 1ml of leucocyte suspension were incubated with 1ml of PBS supplemented with 10% FBS. For the ‘L. (V.) braziliensis – stimulated’ cultures, aliquots of 1ml of leucocyte suspension were incubated with 950μl of PBS supplemented with 10% FBS and 50μl of FITC-labelled live L. (V.) braziliensis organisms at 1×108 parasites/ml. Both, unstimulated and stimulated cultures were performed in duplicate. The tubes were incubated under gentle shaking by an orbital shaker for 120min at 37°C in a 5% CO2 humidified incubator. After incubation, each tube received Brefeldin A (BFA) (Sigma, MO, USA) at 10μg/ml and re-incubated for 240min at 37°C in a 5% CO2 humidified incubator. Following the incubation, the cultures were treated with EDTA at 20mm and maintained at room temperature for 15min prior to immunophenotypic staining for cell surface markers and intracytoplasmic cytokine analysis. For cell surface marker analysis, aliquots of 200μl cell cultures obtained from the phagocytosis assay were incubated for 30min at room temperature with PerCP-labelled anti-CD14 mAb and phycoerythrin (PE)-labelled anti-CD23 mAb, all purchased from Becton Dickinson. Following incubation, the red blood cells were lysed with FACS lysing solution, washed with FACS buffer and fixed with FACS FIX solution. A total of 3,000 monocytes were obtained, and analyses of the immunophenotypic and morphometric parameters were determined in a FACScan flow cytometer. For intracytoplasmic cytokine analysis, aliquots of 500μl cell cultures were incubated for 30min at room temperature with anti-CD4PerCP and anti-CD8PerCP mAbs to identify T Lymphocyte subsets. Following incubation, the red blood cells were lysed with FACS lysing solution and the samples were kept with FACS permeabilizing solution. Following incubation, the samples were centrifuged at 600g for 7min at room temperature and the cell pellet washed with FACS buffer. After centrifugation, the cells were resuspended with FACS buffer. Cells were then stained with anticytokine MoAbs, including anti-IFN-γPE and anti-IL-4PE, by incubation for 30min at room temperature in the dark. After incubation, the cells were washed twice and fixed with FACS FIX solution. A total of 20,000 lymphocytes were acquired and analysed. Morphometric, phenotypic and functional parameters were determined in a FACScan flow cytometer. In this study, the intracellular NO assay was performed using plasma-free whole blood samples as previously reported by Schachnik etal. 16 and Gomes etal. 17. Plasma-free whole blood was resuspended to 1×107 leukocytes/ml in PBS 2% BSA. Fifty microlitres of leukocytes was pre-incubated with L. (V.) braziliensis soluble (‘LSA’ culture) antigen preparation at a final concentration of 25μg/ml at 37°C for 120min in a 5% CO2 humidified incubator. Control cultures (‘Ex vivo – Unstimulated’ cultures) were used to determine the basal levels of intracellular nitric oxide. The samples were then incubated in the presence of 4,5-diaminofluorescein-diacetate (DAF-2DA) (Sigma, MO, USA) (final concentrations of 2.0μm) at 37°C for 180min in a 5% CO2 humidified incubator. The samples were labelled and placed in an ice bath for 20min in the presence of PerCP-labelled anti-CD14 mAbs. The cells were lysed with FACS lysing solution and washed with PBS solution, and the cell pellet was gently resuspended with PBS solution for immediate flow cytometer acquisition in a FACScan flow cytometer. A total of 3,000 events/tube were acquired. Positive controls were performed by pre-incubation of leucocyte samples for 60min with a NO inducer (LPS 10μg/ml, Sigma, USA) or 10min pre-incubation with NO inhibitors (Nω-nitro-l-arginine methyl ester 10mm) and aminoguanidine (10mm, Sigma, MO, USA) at 37°C in a 5% CO2 humidified incubator (data not shown)). The data acquired were analysed using flowjo software. Distinct gating strategies were used to analyse CD23 expression, cytokine profile and NO production of specific leucocyte subpopulations. A strategic gate was created for selective analysis of monocytes, gated as SSChighCD14high cells, using FL3/anti-CD14-PerCP versus laser side-scatter (SSC) graph (Fig.1A,D). Following the gating procedure, the percentage of monocytes CD14+ CD23+ and NO producers was determined using double-positive quadrant of FL2/anti-CD23-PE versus FL3/anti-CD14-PerCP and FL1/DAF-2T versus FL3/anti-CD14-PerCP dot plots, respectively (Fig.1B,C and E). Lymphocyte scatter gate was set up using FSC versus SSC dot plots (Fig.1F). Cytokine-expressing T CD4 and T CD8 lymphocyte cells were identified using FL3/anti-CD4 and anti-CD8-PerCP versus FL2/anti-IL-4 and anti-IFN-γ-PE dot plots (Fig.1G). Figure 1Open in figure viewerPowerPoint Analyses of peripheral blood leukocytes of evaluated population by flow cytometry. (A) represents the cell profile of monocytes population, selected on a gate in charts of granulosity density (SSC) versus fluorescence-3 (anti-CD14-PerCP). (B) represents the monocytes profile in the absence of DAF-2DA probe in charts of fluorescence-1 density versus anti-CD14-PerCP. (C) represents the profile of NO-producing monocytes (DAF-2T+) in double-positive quadrant (Q2) in charts of fluorescence-1 density (DAF-2T) versus anti-CD14-PerCP. (D) represents the cell profile of monocytes population, selected on a gate in charts of granulosity density (SSC) versus fluorescence-3 (anti-CD23-PE). (E) represents the profile of CD23-expressing monocytes (anti-CD23-PE) in double-positive quadrant (Q2) in charts of fluorescence-3 density (anti-CD14-PerCP) versus fluorescence-1 anti-CD23-PE. (F) represents the cell profile of T lymphocytes population, selected on a gate in charts of granulosity density (SSC) versus cell size (FSC). (G) represents the profile of IL-4− producers T CD4+ lymphocytes (anti-CD4-PerCP) in double-positive quadrant (Q2) in charts of fluorescence-1 density (anti-CD4-PE) versus fluorescence-3 anti-IL-4-PE. Biomarker signatures were assembled as previously reported by Luiza-Silva etal. 18. The global median value for each biomarker (CD23, NO, IL-4 and IFN-γ) was calculated using all the groups’ values (LCL, LCL 60, LCL≥60, MT+SL− and NI). The global median biomarker index (LEISH-LSA/CC) was used to tag each individual as they display ‘low’ or ‘high’ biomarkers levels. This strategy allowed for a final computation of the percentage of individuals displaying ‘high’ biomarker index. Next, the ‘ascendant biomarker signature’ for the NI group was assembled and taken as the ‘reference curve’ to highlight the changes in biomarker profiles of TM+SL−, LCL, LCL 60 and LCL≥60 groups. Additionally, the ‘ascendant biomarker index signature’ for each clinical group was also assembled and overlaid to identify changes in the overall biomarker profile. The identification of the cell populations of interest, determination of the phagocytic capacity and percentage of phenotypic markers were evaluated though flowjo software (Flow Cytometry Analysis Software, version 7.6.1.). Statistical analyses were performed using graphpad prism software (San Diego, USA, version 5.00). For comparative analyses between two groups, the Mann–Whitney test was used, whereas for comparative analyses among three groups, the Kruskal–Wallis test was used followed by Dunn\'s post-test. In all cases, differences were considered statistically significant when the P value was less than 0.05. A specific CAP-fluorescein-enzyme immunoassay (Cap-FEIA), ImmunoCAP Total IgE kit (Thermo Scientific™), was used to quantify the serum level of total IgE as per the manufacturer\'s instructions. For serum measurements, the samples were stored at −70°C until use. The results were considered elevated when total IgE concentration was higher than 116 KU/L for individuals of 7–10years old and higher than 140 KU/L for individuals 10years old 19. The higher frequency of monocytes expressing CD23 and NO after Leishmania braziliensis stimulation and increased serum levels of IgE are hallmarkers of the LCL group Here we evaluated the impact of L. (V.) braziliensis on CD23 and NO expression by peripheral blood monocytes and serum levels of IgE in MT+SL− individuals and patients with LCL. Our data demonstrated that L. (V.) braziliensis stimulation (LEISH or LSA) induced significant increase in the CD23 (MT+SL− – P=0.0488; LCL – P=0.0008; LCL 60 – P=0.0043; LCL≥60 – P=0.0107) and NO (NI – P=0.0093; MT+SL− – P=0.0001; LCL – P=0.0001; LCL 60 – P=0.0047; LCL≥60 – P=0.0002) expression by monocytes in all groups as compared with the control culture (CC) (Fig.2A, B). Figure 2Open in figure viewerPowerPoint Column chart of functional monocyte profiles and serum IgE concentrations in healthy controls (NI), individuals with positive Montenegro skin test (MT+SL−) and patients with localized cutaneous leishmaniasis (LCL) categorized as recent lesion (LCL 60) and late lesion (LCL≥60). The column in grey shows median values of percentile and interquartile range and highlights the frequency of monocytes CD23+ CD14+ (A) and DAF-2T + CD14 + (B) for the control culture (CC) and the Leishmania braziliensis culture (LEISH or LSA) by flow cytometry and IgE concentration by specific CAP-fluorescein-enzyme immunoassay (Cap-FEIA) (C). Significant differences (P 0.05) among the groups NI, MT+SL−, LCL, LCL 60 and LCL≥60 are represented by the letters a, b and d. Significant differences between the control culture (CC) and the Leishmania braziliensis culture (LEISH or LSA) are represented by asterisks (*). Data analysis demonstrated that the LCL group presented a high frequency of CD23+CD14+ monocytes when compared with NI (P=0.0079) and MT+SL− (P=0.0070) and that this increase was significantly higher in LCL≥60 (P=0.0118; P=0.0121, respectively) (Fig.2A). Aiming to quantify NO production, we performed an analysis of the percentage of DAF-2T+CD14+ monocytes in peripheral blood. The results showed that independent of the time lesion (LCL 60 or LCL≥60), the LCL group presented a high frequency of NO-producing monocytes as compared to the NI (P=0.0155) and MT+SL− (P=0.0273) groups (Fig.2B). Interestingly, we observed elevated IgE serum levels in patients with late LCL lesion (LCL≥60) as compared with the MT+SL− (P=0.0492) and LCL 60 (P=0.0423) groups (Fig.2C). Aiming to evaluate an important NO induction pathway, we characterized the cytokine profile by circulating T lymphocytes in the absence of L. braziliensis/control culture (CC) and in the presence of L. braziliensis (LEISH) stimuli. For this purpose, we analysed the intracytoplasmic expression of IFN-γ and IL-4 by CD4+ and CD8+ T cell subsets from the NI, MT+SL− and LCL groups (Fig.3). Data analysis demonstrated that the increased frequency of IFN-γ+ and IL-4+ CD4+ T cells (Fig.3A, C, respectively) is related to MT+SL− group when compared with NI (IFN-γ → P=0.0007; IL-4 → P=0.0170), LCL (IFN-γ → P=0.0184; IL-4 → P=0.0129), LCL 60 (IFN-γ → P=0.0216; IL-4 → P=0.0075) and LCL≥60 (IFN-γ → P=0.0343; IL-4 → P=0.0327), despite the absence of significant alterations of CD8+ T cells in the evaluated groups (Fig.3B,D). Figure 3Open in figure viewerPowerPoint Column chart of functional T lymphocyte profiles in healthy controls (NI), individuals with a positive Montenegro skin test (MT+SL−) and patients with localized cutaneous leishmaniasis (LCL) categorized as recent lesion (LCL 60) and late lesion (LCL≥60). The columns in grey show median values of percentile and interquartile range and highlight the frequency of T lymphocyte subsets IFN-γ+CD4+/IFN-γ+CD8+ (A and B) and CD4+IL-4+/IL-4+CD8+ (C and D) for the control culture (CC) and the Leishmania braziliensis culture (LEISH). Significant differences (P 0.05) among the groups NI, MT+SL, LCL, LCL 60 and LCL≥60 are represented by the letters a, c, d and e. Significant differences between the control culture (CC) and the Leishmania braziliensis culture (LEISH) are represented by asterisks (*). High producers from MT+SL− individuals display a unique cytokine profile by CD4+ T cells, and L. (V.) braziliensis stimuli induced significant impact in the CD23 and NO expression by monocytes in patients with LCL In a different analysis, the index of the high producers of each group was evaluated for each cellular profile tested. The high producers in the NI group were then aligned to create a reference biomarker index signature that was later on ordered in an ascendant fashion (Fig.4A). The high producers in the other groups were also aligned following the same sequence observed in the NI group for clearer comparisons and overlaid with the reference curve created for the NI group. The results demonstrated that the profile observed in the MT+SL− group is well above the NI reference biomarker index signature, especially for DAF-2T+CD14+, IL-4+CD8+, IFN-γ+CD4+ and IL-4+CD4+ (Fig.4B). On the other hand, when evaluated in the patients with LCL, it was possible to see that the high producers presented a higher index of DAF-2T+CD14+, CD23+CD14+ and IL-4+CD8+, associated with a lower index of IFN-γ+CD8+ high producers (Fig.4C). Considering lesion age, analysis of LCL demonstrated that the main differences observed in the LCL group were that LCL 60 patients had a higher index of CD23+CD14+, which was also present in LCL≥60 patients (Fig.4D, E, respectively). Figure 4Open in figure viewerPowerPoint Signature profiles of biomarkers for individuals with positive Montenegro skin tests (MT+SL−), patients with localized cutaneous leishmaniasis (LCL) and healthy controls (NI). Patients with LCL were categorized as recent lesion (LCL 60) and late lesion (LCL≥60). The graphics were plotted using the global median biomarker index (LEISH-LSA/CC) as the cut-off for identifying each subject as ‘low’ or ‘high’ biomarker producers. (B, C, D) Bar charts showing the frequency of individuals with the ‘high’ biomarker index of DAF-2T +CD14+, CD23+CD14+, IL-4+CD8+, IFN-γ+CD4+, IL-4+CD4+ and IFN-γ+CD8+ in MT+SL−, LCL, LCL 60 and LCL≥60 groups according to the (A) NI ascendant biomarker signature, used as a ‘reference curve’ (). Relevant changes were considered when the frequency of individuals with ‘high’ biomarker levels shifted towards a distinct 50th percentile as compared to the NI reference curve and are highlighted by arrows (↓↑). It has been shown that the different clinical forms of human cutaneous leishmaniasis present distinct immunological features. During the development of the anti-Leishmania immune response, the secretion of soluble factors occurs, such as cytokines and chemokines, associated with the recruitment of circulating monocytes, which differentiate into macrophages in the inflammatory process 20, 21. Macrophages are important cells in leishmaniasis because these cells are targeted by the parasites, thus enabling its development 22, 23. The role played by macrophages depends on the kind of activation and the vulnerability of the parasite against the effector mechanism 24. The intracellular survival of Leishmania is related to its ability to prevent the activation of macrophages 25. Once activated, these cells are able to destroy the parasites, mainly via production of NO and reactive intermediate oxygen species (RIOs) 24. NO is involved in many physiological and pathological processes. In this context, its biological importance is due to the fact that this molecule has the ability to activate or inhibit target molecules in different processes such as regulation of vascular tone, neurotransmission and immune response 26, 27. Guzik etal. 28 demonstrated that during the development of the inflammatory process, the synthesis of large amounts of NO via iNOS occurs (inducible nitric oxide synthase enzyme that catalyses the formation of NO). This NO, produced mainly by activated macrophages, has cytostatic and cytotoxic properties thus promoting the destruction of micro-organisms and tumour cells 28. Diefenbach etal. (1998) showed a dual function of NO. During the innate phase of infection, involving the control of NK cell activity, the early cytokine response and parasite spreading, while during the late phase NOS2 is further upregulated by IFN-γ-producing CD4+ T cells, and its antimicrobial activity is critical for the reduction of parasite burden and clinical resolution of the disease 29. It has been demonstrated that topical treatment of L. major/L. braziliensis ulcers with donor NO was very effective at reducing the size of lesions ant promote healing in mice. These authors suggest that topical application of an NO donor molecule to the lesion may partially compensate for the shortage of NO and promote healing. In addition, the topical therapy of cutaneous leishmaniasis in the mouse model provides the experimental basis for a possible trial of NO in the treatment of LCL 30. Thus, NO is very important as it is associated with the destruction of parasites and the consequent control of infection. In the present study, we evaluated, for the first time, specific aspects of an anti-Leishmania immune response in a Brazilian indigenous community. We investigated aspects of the intracellular NO induction pathway, and its triggering by the binding of IgE to CD23 receptor in a IFN-γ/IL-4 cytokines environment in CL carriers (LCL) and positive MST individuals without skin lesion (MT+SL−) of the Xakriabá indigenous community in Minas Gerais, Brazil. We also examined LCL carriers by the time of lesion (recent LCL 60 and late LCL≥60days) as a strategy to investigate early and late NO-related immunological events that may be triggered by Leishmania infection. Many studies have demonstrated that treatment of human monocytes/macrophages with IFN-γ and IL-4, both secreted during the early phases of the immune response, increases the expression of CD23, the low affinity receptor for IgE 11, 31, 32. The binding of CD23 by its ligand increases mRNA transcription of iNOS (inducible nitric oxide synthase), as well as the production of NO 32. Studies with Leishmania major showed that after treatment of human monocytes/macrophages with IFN and IL-4, the infection had not become established, and when the parasites entered the cell, they were immediately destroyed by NO, suggesting a protective role of this mechanism 11, 33. Our data, corroborated by these studies, suggest that the protective immune response involving CD23-IgE-mediated NO release is a hallmark of patients with LCL, despite the lower percentage of T cell producers IFN-γ and IL-4. We suggest that other leukocytes such as neutrophils and NK cells may be involved in the expression of cytokines needed to produce NO. It is important to mention that Gross etal. 34 affirm that human monocytes do not produce NO under regular cell culture conditions. However, the evaluation of intracellular NO production by monocytes/macrophages has been studied in Leishmaniasis since 2011 35. Besides, another study by our group evaluated intracellular NO production by peripheral blood monocytes in cultures in the presence of Trypanosoma cruzi trypomastigotes 17. In general, in LCL the presence of a strong cellular immune response characterized by a positive Montenegro test has been considered a good prognosis 36. Indeed, it is well known that in areas endemic for Leishmania, there are groups of individuals that, although living in these areas, do not develop the disease but yet have positive Montenegro skin test 37. As mentioned previously, positive MST individuals without skin lesion (MT+SL−) of the Xakriabá indigenous community in Minas Gerais, Brazil, were evaluated in this study. We identified that the increased frequency of IFN-γ+ and IL-4+ CD4+ T cells is related to the MT+SL− group. These results were reinforced by a positive correlation between IFN-γ+ and IL-4+ cytokines (data not shown). In addition, the analysis of high producers demonstrated that in the TM+SL− group, the index signature was characterized by DAF-2T+CD14+/IL-4+CD8+/IFN-γ+ CD4+/IL-4+CD4+. Thus, we suggest that in TM+SL− individuals, another NO-dependent leishmanicidal mechanism is involved. Baratta-Masini etal. (2007) demonstrated that TM+SL− individuals displayed a basal mixed type 0 cytokines profile. However, they observed a low frequency of IL-4+ T cells, a high IFN-γ+/IL-10+ cell ratio, and elevated nitrite and nitrate plasma levels in this group, suggesting that despite basal levels of cytokines, a high proportion of type 1 over type 2 cytokines serves to prevent parasite growth and lesion development 29. It is important to emphasize that there may be immunological differences between indigenous and non-indigenous populations that were not evaluated in the present study. Our hypothesis is that any such differences are likely related to differences in genetic background, behaviour, physiology, life history, stress levels and frequency of contact with Leishmania parasites. Another interesting matter that should be discussed is about the parasite contribution to variable clinical manifestations of cutaneous leishmaniasis. A remarkable genetic variability has previously been shown to the L. (Viannia) subgenus 38. Few reports have shown an association between genetic variability in Leishmania and variable clinical manifestations. The wide variation in the clinical presentation of cutaneous leishmaniasis with respect to the type, number, shape, size, location and immune outspread of the lesions can be explained by individual variations in parasite virulence and host susceptibility 39. Schrieffer etal. 40 studied the genetic variability of L. (V.) braziliensis isolates from patients with cutaneous and disseminated leishmaniasis to test the hypothesis that the polymorphism of L. (V.) braziliensis could explain these different clinical manifestations. They observed an association between distinct L. (V.) braziliensis genotypes and the different clinical forms (cutaneous and disseminated). Results of studies carried out by our collaborative research group showing that L. (V.) braziliensis variants are associated with different lesion types (Quaresma PF, Brito CFA, Freire JM, Moreno EC, Rego FD, Rugani JMN, Carvalho-Gontijo R, Diniz JE, Melo MN, Gontijo CMF; 2015 – personal communication) emphasize the importance of further investigations on the relationship between clinical manifestations and genetic diversity in L. (V.) braziliensis. Future studies will be very important in addressing these questions. Together, the data presented in this paper demonstrate that there are significant differences in the immune response of the individuals evaluated in this study and that these differences are related to the development of resistance or susceptibility to infection. We suggest that the protective immune response involving CD23-IgE-mediated NO release is a hallmark of patients with LCL. However, in TM+SL− individuals, another leishmanicidal mechanism seems to be involved. In addition, this study provided a rare opportunity to study immunological aspects related to CL in an indigenous population. The authors thank the Program for Technological Development in Tools for Health-PDTIS-FIOCRUZ for the use of its facilities. OAMF, ATC and CMFG thank CNPq for fellowships (PQ). This work was supported by FAPEMIG (PPSUS), CNPq, PAHO and European Community. 1Follador I, Araújo C, Bacellar O etal. Epidemiologic and immunologic findings for the subclinical form of Leishmania braziliensis infection. 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