Andréa Teixeira-Carvalho

Systemic and compartmentalized immune response in canine visceral leishmaniasis

Human visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL) are the most important emerging diseases with high prevalence in Latin American countries and are mainly caused by Leishmania (L.) chagasi (Syn=L. infantum). CVL has a great impact on Brazilian public health because domestic dogs are the most important VL peri-domicile reservoirs in both urban and peri-urban areas. Our findings highlight the complexity of cellular immunological events related to the natural infection from dogs by L. chagasi, additionally correlating major peripheral blood phenotypic markers with clinical status and tissues parasite density. Our main results demonstrated that lower frequency of circulating B cells and monocytes are important markers of severe CVL, whereas increased levels of CD8+ lymphocytes appear to be the major phenotypic feature of asymptomatic disease. Determination of the isotypes patterns during CVL demonstrated that asymptomatic dogs and those with low parasitism are associated with an increase of IgG1, while the symptomatic dogs and those with high parasitism are associated with an increase of IgG, IgG2, IgM, IgA and IgE immunoglobulins. Pioneer findings obtained by our group showed a correlation between clinical status of CVL with degree of tissue parasite density. This data demonstrated that asymptomatic dogs presented low parasitism while symptomatic dogs are associated with high parasite load in various tissues such as skin, bone marrow and spleen. We have also investigated the association between tissue parasitism and CVL clinical forms. Regardless of clinical status, skin and spleen are the major sites of high parasite density during ongoing CVL. Furthermore, we demonstrated that bone marrow and spleen parasite density are the most reliable parasitological markers to decode the clinical status of CVL. In this article, we have reviewed some aspects of the histopathological and immunological events occurring in natural and experimental L. chagasi/L. infantum infection, pointing out the main L. chagasi-parasitized tissue. We have discussed the importance of the association between parasite density, immunological/histopathological aspects and clinical status of the CVL, their current applications, challenges for the future and potential opportunities in CVL research.

Mixed inflammatory/regulatory cytokine profile marked by simultaneous raise of interferon-gamma and interleukin-10 and low frequency of tumour necrosis factor-alpha(+) monocytes are hallmarks of active human visceral Leishmaniasis due to Leishmania chagasi infection.

Considering the complexity of the immunological events triggered during active visceral Leishmaniasis (VL), the relevance of the segregation of the immune response during human VL into type 1 and type 2 still remains unclear. For this purpose, in individuals living in risk areas for VL, we have evaluated especially asymptomatic individuals and patients with active VL, the plasmatic levels of cytokines and reactive nitrogen species under ex vivo conditions. In addition, we have also performed an analysis of intracellular cytokine patterns of circulating leucocytes after short‐term culture, particularly in the absence of antigenic‐specific stimulation, in order to reflect dynamic events of immune response in vivo during Leishmania chagasi infection. Although asymptomatic individuals and non‐infected subjects presented a similar immunological profile, an outstanding inflammatory/regulatory profile, based on higher plasmatic levels of cytokines such as interleukin (IL)‐8, interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α, IL‐6 and IL‐10, was associated with clinical status observed in active VL. In this context, we hypothesize that IL‐10, through its ability to inhibit anti‐leishmanial macrophage activation, associated with the lower frequency of TNF‐α+ monocytes and ordinary levels of nitrite and nitrate are the major mechanisms associated with disease onset.

Phenotypic features of circulating leucocytes as immunological markers for clinical status and bone marrow parasite density in dogs naturally infected by Leishmania chagasi

Canine visceral leishmaniasis (CVL) manifests itself as a broad clinical spectrum ranging from asymptomatic infection to patent severe disease. Despite relevant findings suggesting changes on lymphocytes subsets regarding the CVL clinical forms, it still remains to be elucidated whether a distinct phenotypic profile would be correlated with degree of tissue parasite density. Herein, we have assessed the correlation between the clinical status as well as the impact of bone marrow parasite density on the phenotypic profile of peripheral blood leucocytes in 40 Brazilian dogs naturally infected by Leishmania chagasi. Our major findings describe the lower frequency of B cells and monocytes as the most important markers of severe CVL. Our main statistically significant findings reveal that the CD8+ T cell subset reflects most accurately both the clinical status and the overall bone marrow parasite density, as increased levels of CD8+ lymphocytes appeared as the major phenotypic feature of asymptomatic disease and dogs bearing a low parasite load. Moreover, enhanced major histocompatibility complex (MHC)‐II density as well as a higher CD45RB/CD45RA expression index seems to represent a key element to control disease morbidity. The association between clinical status, bone marrow parasitism and CD8+ T cells re‐emphasizes the role of the T cell‐mediated immune response in the resistance mechanisms during ongoing CVL. Higher levels of circulating T lymphocytes (both CD4+ and CD8+ T cells) and lower MHC‐II expression by peripheral blood lymphocytes seem to be the key for the effective immunological response, a hallmark of asymptomatic CVL.

Augmented plasma microparticles during acute Plasmodium vivax infection

BackgroundIn the last few years, the study of microparticles (MPs) - submicron vesicles released from cells upon activation or apoptosis - has gained growing interest in the field of inflammation and in infectious diseases. Their role in the human malaria parasite Plasmodium vivax remains unexplored. Because acute vivax malaria has been related to pro-inflammatory responses, the main hypothesis investigated in this study was that Plasmodium vivax infection is associated with elevated levels of circulating MPs, which may play a role during acute disease in non-immune patients.MethodsPlasma MPs were analysed among thirty-seven uncomplicated P. vivax infections from an area of unstable malaria transmission in the Brazilian Amazon. The MP phenotype was analysed by flow cytometry using the classical MP marker, annexin, and fluorochrome-labeled monoclonal antibodies against specific cell surface markers. The frequencies of plasma MPs in P. vivax patients (n = 37) were further compared to malaria-unexposed controls (n = 15) and ovarian carcinoma patients (n = 12), a known MPs-inducing disease non-related to malaria.ResultsThe frequencies of plasma circulating MPs were markedly increased in P. vivax patients, as compared to healthy age-matched malaria-unexposed controls. Although platelets, erythrocytes and leukocytes were the main cellular sources of MPs during vivax malaria, platelet derived-MPs (PMPs) increased in a linear fashion with the presence of fever at the time of blood collection (β = 0.06, p < 0.0001) and length of acute symptoms (β = 0.36, p < 0.0001). Finally, the results suggest that plasma levels of PMPs diminish as patient experience more episodes of clinical malaria (β = 0.07, p < 0.003).ConclusionsAbundant circulating MPs are present during acute P. vivax infection, and platelet derived-MPs may play a role on the acute inflammatory symptoms of malaria vivax.

Are increased frequency of macrophage-like and natural killer (NK) cells, together with high levels of NKT and CD4+CD25high T cells balancing activated CD8+ T cells, the key to control Chagas’ disease morbidity?

The immunological response during early human Trypanosoma cruzi infection is not completely understood, despite its role in driving the development of distinct clinical manifestations of chronic infection. Herein we report the results of a descriptive flow cytometric immunophenotyping investigation of major and minor peripheral blood leucocyte subpopulations in T. cruzi‐infected children, characterizing the early stages of the indeterminate clinical form of Chagas’ disease. Our results indicated significant alterations by comparison with uninfected children, including increased values of pre‐natural killer (NK)‐cells (CD3– CD16+ CD56–), and higher values of proinflammatory monocytes (CD14+ CD16+ HLA‐DR++). The higher values of activated B lymphocytes (CD19+ CD23+) contrasted with impaired T cell activation, indicated by lower values of CD4+ CD38+ and CD4+ HLA‐DR+ lymphocytes, a lower frequency of CD8+ CD38+ and CD8+ HLA‐DR+ cells; a decreased frequency of CD4+ CD25HIGH regulatory T cells was also observed. These findings reinforce the hypothesis that simultaneous activation of innate and adaptive immunity mechanisms in addition to suppression of adaptive cellular immune response occur during early events of Chagas’ disease. Comparative cross‐sectional analysis of these immunophenotypes with those exhibited by patients with late chronic indeterminate and cardiac forms of disease suggested that a shift toward high values of macrophage‐like cells extended to basal levels of proinflammatory monocytes as well as high values of mature NK cells, NKT and regulatory T cells, may account for limited tissue damage during chronic infection favouring the establishment/maintenance of a lifelong indeterminate clinical form of the disease. On the other hand, development of an adaptive cell‐mediated inflammatory immunoprofile characterized by high levels of activated CD8+ cells and basal levels of mature NK cells, NKT and CD4+ CD25HIGH cells might lead to late chronic pathologies associated with chagasic heart disease.

Immunogenicity of a killed Leishmania vaccine with saponin adjuvant in dogs

Abstract Cellular and humoral immune responses of dogs to a candidate vaccine, composed of Leishmania braziliensis promastigote protein plus saponin as adjuvant, have been investigated as a pre-requisite to understanding the mechanisms of immunogenicity against canine visceral leishmaniasis (CVL). The candidate vaccine elicited strong antigenicity related to the increases of anti-Leishmania IgG isotypes, together with higher levels of lymphocytes, particularly of circulating CD8+ T-lymphocytes and Leishmania chagasi antigen-specific CD8+ T-lymphocytes. As indicated by the intense cell proliferation and increased nitric oxide production during in vitro stimulation by L. chagasi soluble antigens, the candidate vaccine elicited an immune activation status potentially compatible with effective control of the etiological agent of CVL.

Cytokine Signatures of Innate and Adaptive Immunity in 17DD Yellow Fever Vaccinated Children and Its Association With the Level of Neutralizing Antibody

BACKGROUND The live attenuated yellow fever (YF) vaccines have been available for decades and are considered highly effective and one of the safest vaccines worldwide. METHODS The impact of YF-17DD-antigens recall on cytokine profiles of YF-17DD-vaccinated children were characterized using short-term cultures of whole blood samples and single-cell flow cytometry. This study enrolled seroconverters and nonseroconverters after primovaccination (PV-PRNT⁺ and PV-PRNT⁻), seroconverters after revaccination (RV-PRNT⁺), and unvaccinated volunteers (UV-PRNT⁻). RESULTS The analysis demonstrated in the PV-PRNT⁺ group a balanced involvement of pro-inflammatory/regulatory adaptive immunity with a prominent participation of innate immunity pro-inflammatory events (IL-12⁺ and TNF-α⁺ NEU and MON). Using the PV-PRNT⁺ cytokine signature as a reference profile, PV-PRNT⁻ presented a striking lack of innate immunity proinflammatory response along with an increased adaptive regulatory profile (IL-4⁺CD4⁺ T cells and IL-10⁺ and IL-5⁺CD8⁺ T cells). Conversely, the RV-PRNT⁺ shifted the overall cytokine signatures toward an innate immunity pro-inflammatory profile and restored the adaptive regulatory response. CONCLUSIONS The data demonstrated that the overall cytokine signature was associated with the levels of PRNT antibodies with a balanced innate/adaptive immunity with proinflammatory/regulatory profile as the hallmark of PV-PRNT(MEDIUM⁺), whereas a polarized regulatory response was observed in PV-PRNT⁻ and a prominent proinflammatory signature was the characteristic of PV-PRNT(HIGH⁺).

Strategy to Assess the Overall Cytokine Profile of Circulating Leukocytes and its Association with Distinct Clinical Forms of Human Chagas Disease

Herein we have employed an alternative strategy to assess the cytokine patterns of circulating leukocytes and correlate dominant cytokine profiles with indeterminate‐IND and cardiac‐CARD clinical forms of Chagas disease. We have first calculated median percentages of cytokine‐positive leukocytes of our study sample to establish, for each cytokine‐positive cell population, the cut‐off edge that would segregate ‘low’ and ‘high’ cytokine producers to build colour diagrams and draw a panoramic cytokine chart. Using this approach we demonstrated that most IND individuals presented a dominant regulatory cytokine profile, whereas CARD individuals displayed a dominant inflammatory cytokine pattern. In addition, radar chart analysis confirmed the dichotomic cytokine balance between IND and CARD groups and further allowed the identification of the relative contribution of each cell population for the global cytokine pattern. Data analysis demonstrated that CD4+ T cells were the major cell population defining the regulatory profile in IND, whereas monocytes and CD4+ T cells determined the inflammatory cytokine pattern in CARD individuals. Interestingly, in vitro stimulation with trypomastigote Trypanosoma cruzi antigen was able to invert the cytokine balances in IND and CARD groups. Upon antigenic stimulation, changes in the frequencies of IL‐10‐producing CD4+ T cells and monocytes drove IND individuals towards an inflammatory pattern and CARD towards a regulatory cytokine profile. A similar inversion could be found after in vivo treatment of IND and CARD individuals with benzonidazole. Altogether, these findings shed some light into the complex cytokine network underlying the immunopathogenesis of Chagas disease and provide putative immunological biomarkers of disease severity and therapeutic response.

Immune Response in Human Visceral Leishmaniasis: Analysis of the Correlation Between Innate Immunity Cytokine Profile and Disease Outcome

We investigated the cytokine profile of cells of the innate immune response and its association with active (ACT), asymptomatic (AS) and cured (CUR) human visceral leishmaniasis (VL), as well as noninfected (NI) subjects. The frequency of cytokine‐producing cells was determined after short‐term in vitro incubation of whole peripheral blood samples with soluble Leishmania antigen (SLA). Our data demonstrated a predominant type 2 cytokine profile in NI and ACT. In NI, we observed an increase of IL‐4+ neutrophils, IL‐10+ eosinophils besides a decrease of tumour necrosis factor (TNF)‐α+ eosinophils/monocytes. Yet in ACT, we observed an increase of IL‐4+ neutrophils and natural killer (NK) cells and IL‐10+ monocytes, a reduced frequency of IL‐12+ and IFN‐γ+ eosinophils and lower levels of TNF‐α+ and IL‐12+ monocytes. AS presented a mixed profile, characterized by an increase of IFN‐γ+ neutrophils/eosinophils and NK cells, of IL‐12+ eosinophils/monocytes, as well as increase of IL‐4+ neutrophils and NK cells and IL‐10+ eosinophils/monocytes. In contrast, CUR was characterized by a type 1 response with an increase of IFN‐γ+ neutrophils/eosinophils and NK cells, associated with an increase in IL‐12+ monocytes. In conclusion, we show a correlation between innate immune cytokine patterns and clinical status of VL, suggesting that these cells, in addition to other factors, may contribute to the cytokine microenvironment in which Leishmania‐specific T cells are primed and to disease outcome.

Foxp3+CD25high CD4+ regulatory T cells from indeterminate patients with Chagas disease can suppress the effector cells and cytokines and reveal altered correlations with disease severity

Immunoregulatory mechanisms are important to control the intense immune activity induced in Chagas disease. We evaluated the phenotypic profile and the mechanisms by which Treg cells function in patients with the indeterminate (IND) and cardiac (CARD) clinical forms of Chagas disease. The frequency of Foxp3(+)CD25(high) CD4(+)-T cells is augmented and correlated with the maintenance of a better cardiac function in IND. Treg cells from IND present suppressive activity, although the mechanism is not IL-10 or CTLA-4 dependent and are able to produce augmented levels of IL-17, IL-10 and granzyme B being its frequency correlated with percentage of Annexin V(+) CD4(+)-cells. In contrast, CARD presents higher frequency of IL-6(+), IFN-gamma(+), TNF-alpha(+) and CTLA-4(+) Treg-cells than IND. Thus, our data suggest that Treg cells have an important role in controlling the exacerbated immune response and morbidity in Trypanosoma cruzi infection, probably modulating the cytokine environment and/or killing effector cells.