Etiological treatment of Chagas disease patients with benznidazole lead to a sustained pro-inflammatory profile counterbalanced by modulatory events.

In the present study, we characterized the phagocytic capacity, cytokine profile along with the FCγ-R and TLR expression in leukocytes from Chagas disease patients (indeterminate-IND and cardiac-CARD) before and one-year after Bz-treatment (INDT and CARDT). A down-regulation of IL-17, IFN-γ and IL-10 synthesis by neutrophils was observed in CARDT. The Bz-treatment did not impact on the expression of phagocytosis-related surface molecules or monocyte-derived cytokine profile in INDT. Although CARDT showed unaltered monocyte-phagocytic capacity, up-regulated expression of Fcγ-RI/III and TLR-4 may be related to their ability to produce IL-10 and TGF-ß. Down-regulation of lymphocyte-derived cytokine was observed in INDT whereas up-regulated cytokine profile was observed for lymphocytes in CARDT. Analysis of cytokine network revealed that IND displayed a multifaceted cytokine response characterized by strong connecting axes involving pro-inflammatory/regulatory phagocytes and lymphocytes. On the other hand, CARD presented a modest cytokine network. The Bz-treatment leads to distinct cytokine network: decreasing the links in INDT, with a pivotal role of IL-10(+) monocytes and expanding the connections in CARDT. Our findings highlighted that the Bz-treatment contributes to an overall immunomodulation in INDT and induces a broad change of immunological response in CARDT, eliciting an intricate phenotypic/functional network compatible with beneficial and protective immunological events.

Impaired phagocytic capacity driven by downregulation of major phagocytosis-related cell surface molecules elicits an overall modulatory cytokine profile in neutrophils and monocytes from the indeterminate clinical form of Chagas disease.

The distinct ability of phagocytes to present antigens, produce cytokines and provide co-stimulatory signals may contribute to the severity of the outcome of Chagas disease. In this paper, we evaluate the phenotypic features of phagocytes along with the cytokine signature of circulating T-cells from Chagas disease patients with indeterminate (IND) and cardiac (CARD) clinical forms of the disease. Our data demonstrated that neutrophils from IND patients displayed an impaired ability to produce cytokines. A lower Trypanosoma cruzi phagocytic index and higher nitric oxide levels were characteristics of monocytes from IND. The impaired phagocytic capacity did not reflect on the levels of anti-T. cruzi IgG, but was detectable in the downregulation of Fc-γR, TLR and CR1 molecules. The monocyte-derived cytokine signature demonstrated that a down-regulated synthesis of IL-12 and a modulatory state were evidenced by a positive correlation between IL-12 and IL-10 with a lower synthesis of TNF-α. The down-regulation of MHC-II and CD86 in monocytes supports the occurrence of particularities in the APC-activation-arm in IND, and may be involved in the T-cell pro-inflammatory pattern counterbalanced by a potent IL-10 response. Our findings support the hypothesis that differential phenotypic features of monocytes from IND may be committed to the induction of a distinct immune response related to low morbidity in chronic Chagas disease.

Expression of the chemokine receptor CXCR4 on lymphocytes of leprosy patients

Leprosy is caused by Mycobacterium leprae, which induces chronic granulomatous infection of the skin and peripheral nerves. The disease ranges from the tuberculoid to the lepromatous forms, depending on the cellular immune response of the host. Chemokines are thought to be involved in the immunopathogenesis of leprosy, but few studies have investigated the expression of chemokine receptors on leukocytes of leprosy patients. In the present study, we evaluated 21 leprosy patients (M/F: 16/5) with a new diagnosis from the Dermatology Outpatient Clinic of the University Hospital, Federal University of Minas Gerais. The control group was composed of 20 healthy members (M/F: 15/5) of the community recruited by means of announcements. The expression of CCR2, CCR3, CCR5, and CXCR4 was investigated by flow cytometry on the surface of peripheral blood lymphocytes. There was a decrease in percentage of CD3+CXCR4+ and CD4+CXCR4+ lymphocytes in the peripheral blood of leprosy patients (median [range], 17.6 [2.7-41.9] and 65.3 [3.9-91.9], respectively) compared to the control group (median [range], 43.0 [3.7-61.3] and 77.2 [43.6-93.5], respectively). The percentage of CD4+CXCR4+ was significantly lower in patients with the tuberculoid form (median [range], 45.7 [0.0-83.1]) of the disease, but not in lepromatous patients (median [range], 81.5 [44.9-91.9]). The CXCR4 chemokine receptor may play a role in leprosy immunopathogenesis, probably directing cell migration to tissue lesions in tuberculoid leprosy patients.

Expression of the chemokine receptor CXCR4 on lymphocytes of leprosy patients.

Leprosy is caused by Mycobacterium leprae, which induces chronic granulomatous infection of the skin and peripheral nerves. The disease ranges from the tuberculoid to the lepromatous forms, depending on the cellular immune response of the host. Chemokines are thought to be involved in the immunopathogenesis of leprosy, but few studies have investigated the expression of chemokine receptors on leukocytes of leprosy patients. In the present study, we evaluated 21 leprosy patients (M/F: 16/5) with a new diagnosis from the Dermatology Outpatient Clinic of the University Hospital, Federal University of Minas Gerais. The control group was composed of 20 healthy members (M/F: 15/5) of the community recruited by means of announcements. The expression of CCR2, CCR3, CCR5, and CXCR4 was investigated by flow cytometry on the surface of peripheral blood lymphocytes. There was a decrease in percentage of CD3+CXCR4+ and CD4+CXCR4+ lymphocytes in the peripheral blood of leprosy patients (median [range], 17.6 [2.7-41.9] and 65.3 [3.9-91.9], respectively) compared to the control group (median [range], 43.0 [3.7-61.3] and 77.2 [43.6-93.5], respectively). The percentage of CD4+CXCR4+ was significantly lower in patients with the tuberculoid form (median [range], 45.7 [0.0-83.1]) of the disease, but not in lepromatous patients (median [range], 81.5 [44.9-91.9]). The CXCR4 chemokine receptor may play a role in leprosy immunopathogenesis, probably directing cell migration to tissue lesions in tuberculoid leprosy patients.

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.

Natural killer cell subpopulations in putative resistant individuals and patients with active Mycobacterium tuberculosis infection.

Herein, we intended to perform flow-cytometric analyses of peripheral blood NK-cell subsets in patients with active tuberculosis (TB) and those putative resistant subjects displaying positive tuberculin skin test (TST+) and compared with TST- healthy controls. Our findings demonstrated distinct phenotypic features in TST+ as compared with TB. While lower values of NK-cells with increased frequency of CD3-CD16+ CD56- and CD3-CD16-CD56+ subsets besides lower frequency of CD3-CD16+ CD56+ NK-cells was observed in TST+, unaltered levels of NK-cells with increased levels of CD3-CD16+ CD56- NK-cells with lower frequency of CD3-CD16+ CD56+ NK-cells was found in TB. Additional analysis highlighted a shift towards increased levels of CD3-CD16-/+CD56bright NK-cells as the hallmark of TST+, whereas unaltered frequency was observed in TB. Increased levels of CD3+CD56+ cells were observed in both TST+ and TB. Further focusing on the monocyte/NK-cell network, we have reported that enhanced frequency of CD14+ CD16+ monocytes particularly observed in TST+. Outstanding were the distinct correlation profiles observed between CD3-CD16-CD56+ NK-cells and CD3+ CD56+ cells CD14+ CD16+ monocytes for TST+ and TB. These data suggested that high levels of CD3-CD16-CD56+ NK-cells aside CD14+ CD16+ monocytes as well as non-concurrent increment of CD3+ CD56+ cells, may be involved in protective mechanisms in putative tuberculosis-resistant individuals. On the other hand, the basal levels of macrophage-like monocytes despite its positive correlation with increased levels of CD3+ CD56+ cells may count for the lack of the protective immunity in patients with active tuberculosis. Further studies focusing on the cytokine profiling of peripheral blood innate immunity cells before and after chemotherapeutic treatment are currently under evaluation.

Benznidazole treatment during early-indeterminate Chagas' disease shifted the cytokine expression by innate and adaptive immunity cells toward a type 1-modulated immune profile.

Trypanosoma cruzi-infected children was treated with benznidazole (Bz) during the early-indeterminate disease (E-IND) and the cytokine pattern of innate and adaptive immune compartments were evaluated prior to the treatment and 1 year after it. At first, we observed that the ex vivo cytokine profile of circulating leukocytes from E-IND (n = 6) resembled the one observed for healthy schoolchildren (n = 7). Additionally, in vitro stimulation with T. cruzi antigens drove the E-IND cytokine pattern toward a mixed immune profile with higher levels of IFN-gamma+, TNF-alpha+ and IL-4+ NK cells, increased numbers of IFN-gamma+, TNF-alpha+ and IL-10+ CD4+ T cells in addition to enhanced frequency of TNF-alpha+/IL-4+ CD19+ lymphocytes. Interestingly, upon T. cruzi antigen in vitro stimulation, E-IND CD8+ lymphocytes displayed a selective enhancement of IFN-gamma expression, accounting for a global type 1-modulated cytokine microenvironment. A shift toward a type 1-modulated profile was also the hallmark of Bz-treated children (E-IND(T)). In this context, despite the mixed overall ex vivo cytokine profile observed for NK and CD8+ T cells, increased ability of these leukocytes to produce IFN-gamma in response to T. cruzi antigens was reported. Most noteworthy was the IL-10 production evidenced at T lymphocytes, mainly CD4+ cells, as well as B lymphocytes, both ex vivo and upon antigen stimulation. Together, these findings gave evidence that NK cells and CD8+ T lymphocytes are the major sources of IFN-gamma, a pivotal cytokine for successful therapeutic response in human Chagas' disease. Moreover, our data have also brought additional information, pointing out IL-10 production by CD4+ cells and B lymphocytes, as the putative key element for parasite clearance in the absence of deleterious tissue damage.

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.

Chagasic patients with indeterminate clinical form of the disease have high frequencies of circulating CD3+CD16-CD56+ natural killer T cells and CD4+CD25High regulatory T lymphocytes.

Several studies have demonstrated that different clinical manifestations of human Chagas' disease are associated with distinct and complex host-parasite relationships directly involving the immune system. In this context, it has been proposed that tissue damage might be more severe in the absence of regulatory mechanisms that involve both innate and adaptive immune responses. Herein, we describe a descriptive phenotypic profile focusing on the frequency of major regulatory T cells [CD4+CD25high and natural killer T (NKT) lymphocytes] in different clinical forms of Chagas' disease. Ex vivo immunophenotyping of whole blood demonstrated that the indeterminate clinical form displays a higher frequency of both CD4+CD25high and NKT regulatory cells (CD3+CD16-CD56+), associated with increased levels of circulating cytotoxic NK cells (CD3-CD16+CD56+ and CD3-CD16+CD56dim NK cells). By contrast, the increased percentage of activated CD8+HLA-DR+ T-cell subset was exclusively associated with severe clinical forms of Chagas' disease. We hypothesize that regulatory T cells may be able to control the deleterious cytotoxic activity in the indeterminate clinical form by inhibiting the activation of CD8+HLA-DR+ T cells. The lack of regulated populations in cardiac and digestive clinical forms could account for impaired immune response that culminates in strong cytotoxic activity and tissue damage.

Phenotypic features of peripheral blood leucocytes during early stages of human infection with Trypanosoma cruzi.

We performed a cross-sectional flow cytometric analysis of peripheral blood mononuclear cells to evaluate human immunologic status during early stages of Trypanosoma cruzi infection in children. We identified major immunological features corresponding to three proposed phases of disease: early acute (EA) phase, late acute (LA) phase and recent chronic (RC) phase. EA phase was accompanied by expansion of conventional B cells, up-regulation of CD54 on monocytes and down-regulation of CD54 on T cells and not associated with monocyte-activation phenotypes or changes of natural killer (NK) population. LA phase was characterized by a selective increase in a distinct lineage of NK cells (CD16+CD56-), as well as a persistent expansion of B cells and down-regulation of CD54 on T cells. RC phase showed persistent low levels of CD54 molecule on T cells and an increase of B cells, mainly triggered by expansion of the B1-cell subset, as well as increased expression of human leucocyte antigen (HLA-DR) by monocytes. These findings reinforce the hypothesis that T. cruzi-derived antigens are able to activate NK cells before the development of T-cell-mediated immunity. Moreover, our data support previous findings of increased levels of B1 lymphocytes during human Chagas' disease and show that this event is already present during initial stages of chronic infection.