Induction of CD4+CD25+FOXP3+ Regulatory T Cells during Human Hookworm Infection Modulates Antigen-Mediated Lymphocyte Proliferation

Hookworm infection is considered one of the most important poverty-promoting neglected tropical diseases, infecting 576 to 740 million people worldwide, especially in the tropics and subtropics. These blood-feeding nematodes have a remarkable ability to downmodulate the host immune response, protecting themselves from elimination and minimizing severe host pathology. While several mechanisms may be involved in the immunomodulation by parasitic infection, experimental evidences have pointed toward the possible involvement of regulatory T cells (Tregs) in downregulating effector T-cell responses upon chronic infection. However, the role of Tregs cells in human hookworm infection is still poorly understood and has not been addressed yet. In the current study we observed an augmentation of circulating CD4+CD25+FOXP3+ regulatory T cells in hookworm-infected individuals compared with healthy non-infected donors. We have also demonstrated that infected individuals present higher levels of circulating Treg cells expressing CTLA-4, GITR, IL-10, TGF-β and IL-17. Moreover, we showed that hookworm crude antigen stimulation reduces the number of CD4+CD25+FOXP3+ T regulatory cells co-expressing IL-17 in infected individuals. Finally, PBMCs from infected individuals pulsed with excreted/secreted products or hookworm crude antigens presented an impaired cellular proliferation, which was partially augmented by the depletion of Treg cells. Our results suggest that Treg cells may play an important role in hookworm-induced immunosuppression, contributing to the longevity of hookworm survival in infected people

Membrane Cholesterol Regulates Lysosome-Plasma Membrane Fusion Events and Modulates Trypanosoma cruzi Invasion of Host Cells

Trypanosoma cruzi, is the etiological agent of a neglected tropical malady known as Chagas' disease, which affects about 8 million people in Latin America. 30–40% of affected individuals develop a symptomatic chronic infection, with cardiomyopathy being the most prevalent condition. T. cruzi utilizes an interesting strategy for entering cells: T. cruzi enhances intracellular calcium levels, which in turn trigger the exocytosis of lysosomal contents. Lysosomes then donate their membrane for the formation of the parasitophorous vacuole. Membrane rafts, cholesterol-enriched microdomains in the host cell plasma membrane, have also been implicated in T. cruzi invasion process. Since both plasma membrane and lysosomes collaborate in parasite invasion, we decided to study the importance of these membrane domains for lysosomal recruitment and fusion during T. cruzi invasion into host cells. Our results show that drug dependent depletion of plasma membrane cholesterol changes raft organization and induces excessive lysosome exocytosis in the earlier stages of treatment, leading to a depletion of lysosomes near the cell cortex, which in turn compromises T. cruzi invasion. Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events of pre-docked lysosomes, reducing lysosome availability at the cell cortex and consequently compromising T. cruzi infection

Urogenital schistosomiasis in Cabo Delgado, northern Mozambique: baseline findings from the SCORE study

Abstract Background The results presented here are part of a five-year cluster-randomised intervention trial that was implemented to understand how best to gain and sustain control of schistosomiasis through different preventive chemotherapy strategies. This paper presents baseline data that were collected in ten districts of Cabo Delgado province, northern Mozambique, before treatment. Methods A cross-sectional study of 19,039 individuals was sampled from 144 villages from May to September 2011. In each village prevalence and intensity of S. haematobium were investigated in 100 children first-year students (aged 5–8 years), 100 school children aged 9–12 years (from classes 2 to 7) and 50 adults (20–55 years). Prevalence and intensity of S. haematobium infection were evaluated microscopically by two filtrations, each of 10 ml, from a single urine specimen. Given that individual and community perceptions of schistosomiasis influence control efforts, community knowledge and environmental risk factors were collected using a face-to-face interview. Data were entered onto mobile phones using EpiCollect. Data summary was made using descriptive statistics. Chi-square and logistic regression were used to determine the association between dependent and independent variables. Results The overall prevalence of urogenital schistosomiasis was 60.4% with an arithmetic mean intensity of infection of 55.8 eggs/10 ml of urine. Heavy infections were detected in 17.7%, of which 235 individuals (6.97%) had an egg count of 1000 eggs/10 ml or more. There was a significantly higher likelihood of males being infected than females across all ages (62% vs 58%; P < 0.0005). Adolescents aged 9–12 years had a higher prevalence (66.6%) and mean infection intensity (71.9 eggs/10 ml) than first-year students (63.1%; 58.2 eggs/10 ml). This is the first study in Mozambique looking at infection rates among adults. Although children had higher levels of infection, it was found here that adults had a high average prevalence and intensity of infection (44.5%; 23.9 eggs/10 ml). Awareness of schistosomiasis was relatively high (68.6%); however, correct knowledge of how schistosomiasis is acquired was low (23.2%) among those who had heard of the disease. Schistosomiasis risk behaviour such as washing (91.3%) and bathing (86.7%) in open water sources likely to be infested with host snails was high. Conclusions Urogenital schistosomiasis is widespread in Cabo Delgado. In addition, poor community knowledge about the causes of schistosomiasis and how to prevent it increases the significant public health challenge for the national control program. This was the first study in Mozambique that examined infection levels among adults, where results showed that S. haematobium infection was also extremely high. Given that this controlled trial aims to understand the impact of different combinations of schistosomiasis control through treatment of communities, schools, and treatment holidays over a five-year period, these findings highlight the importance of examining the impact of different treatment approaches also in adults. Trial registration The trials have been registered with the International Standard Randomised Controlled Trial registry under ISRCT 14117624 Mozambique (14 December 2015)

CD4+ T cells apoptosis in Plasmodium vivax infection is mediated by activation of both intrinsic and extrinsic pathways

Submitted by Repositório Arca ([email protected]) on 2019-04-24T17:44:00Z No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Approved for entry into archive by Janaína Nascimento ([email protected]) on 2019-08-22T12:31:14Z (GMT) No. of bitstreams: 2 ve_Hojo-Souza_Natália_etal_INI_2015.pdf: 673291 bytes, checksum: 5de19dfdb1440f05f52dfb3676dbc81a (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-08-22T12:31:14Z (GMT). No. of bitstreams: 2 ve_Hojo-Souza_Natália_etal_INI_2015.pdf: 673291 bytes, checksum: 5de19dfdb1440f05f52dfb3676dbc81a (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Research Centre in Tropical Medicine. Porto Velho, RO, Brazil.Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Research Centre in Tropical Medicine. Porto Velho, RO, Brazil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Rio de Janeiro, RJ, Brasil.Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Background: Reduction in the number of circulating blood lymphocytes (lymphocytopaenia) has been reported during clinical episodes of malaria and is normalized after treatment with anti-malaria drugs. While this phenomenon is well established in malaria infection, the underlying mechanisms are still not fully elucidated. In the present study, the occurrence of apoptosis and its pathways in CD4+ T cells was investigated in naturally Plasmodium vivax-infected individuals from a Brazilian endemic area (Porto Velho – RO). Methods: Blood samples were collected from P. vivax-infected individuals and healthy donors. The apoptosis was characterized by cell staining with Annexin V/FITC and propidium iodide and the apoptosis-associated gene expression profile was carried out using RT2 Profiler PCR Array–Human Apoptosis. The plasma TNF level was determined by ELISA. The unpaired t-test or Mann–Whitney test was applied according to the data distribution. Results: Plasmodium vivax-infected individuals present low number of leukocytes and lymphocytes with a higher percentage of CD4+ T cells in early and/or late apoptosis. Increased gene expression was observed for TNFRSF1B and Bid, associated with a reduction of Bcl-2, in individuals with P. vivax malaria. Furthermore, these individuals showed increased plasma levels of TNF compared to malaria-naive donors. Conclusions: The results of the present study suggest that P. vivax infection induces apoptosis of CD4+ T cells mediated by two types of signaling: by activation of the TNFR1 death receptor (extrinsic pathway), which is amplified by Bid, and by decreased expression of the anti-apoptotic protein Bcl-2 (intrinsic pathway). The T lymphocytes apoptosis could reflect a strategy of immune evasion triggered by the parasite, enabling their persistence but also limiting the occurrence of immunopathology

Treatment with MβCD leads to changes in membrane raft organization of cardiomyocytes.

<p>Confocal images of control (A) and cardiomyocytes pre-treated with 10 mM MβCD (B) or HγCD (C). Cells were washed, fixed and then labeled with CTXb-Alexa 488, which recognizes GM1, a raft marker. In comparison to control cells, which show a homogenous strong labeling for GM1, cholesterol-depleted cardiomyocytes reveals a more discrete labeling. Cells treated with HγCD show GM1 labeling similar to control cells whereas cholesterol-replenished cells (D) exhibit both patterns of cholesterol-depleted (arrows) as well as control (asterisks) GM1 labeling. Scale bar: 0.9 µm.</p

<i>T. cruzi</i> invasion of cells and association with LAMP-1 in cardiomyocytes decreases after cholesterol depletion.

<p>Cardiomyocytes pre-treated or not with different cyclodextrins were washed and challenged with <i>T. cruzi</i> trypomastigotes at a M.O.I of 50, for 40 minutes at 37°C, then fixed and processed for immunofluorescence detection of total intracellular parasites, as well as intracellular parasites associated with LAMP-1 (a lysosomal marker). Both <i>T. cruzi</i> internalization (A) and association with host LAMP-1 (B) diminishes after incubation with 10 and 15 mM of MβCD but not after treatment with 10 and 15 mM of HγCD. Cholesterol replenishment after treatment with 15 mM MβCD reverts the effect of the drug on parasite cell invasion (A), and at least partially on LAMP-1 association (B). The average number of cardiomyocytes ±SD per 10 counted fields in each coverslip is shown above the bars (A). Data are shown as mean of triplicates ±SD. Asteriks indicate statistically significant differences (p < 0.05, Student's t test) between control and treated cells. (C) Representative panels of <i>T. cruzi</i> invasion and association with host cell lysosomes, revealed by immunocytochemistry. Total cell and parasite nuclei, as well as parasite kinteoplast DNA were labeled with DAPI; lysosomes were labeled with anti-LAMP 1 antibody followed by secondary IgG labeled with Alexa Fluor 488; extracellular parasites in the field were labeled with anti-<i>T.cruzi</i> antibody followed by secondary IgG labeled with Alexa Fluor 546. From top to bottom: control cells, 15 mM MβCD treated cells, 15 mM HγCD treated cells and 15 mM MβCD treated cells followed by incubation with 0.05 mM of WSC. Blue arrows show total <i>T. cruzi</i> trypomastigotes in the field, yellow ellipsoids show lysosomal associated trypomastigotes, red triangles points out extracellular trypomastigotes and the last column shows the merge of the three previous. Scale bar: 10 µm.</p

Cholesterol depletion leads to changes in lysosomal distribution within cells.

<p>Representative panels of lysosomal distribution in control cardiomyocyte (A) and cardiomyocytes pre-treated either with 10 mM (B) or 15 mM MβCD (C), 10 mM or (D) 15 mM HγCD (E), or 15 mM MβCD followed by 0.05 mM WSC (F). MβCD treated cardiomyocytes show significant changes in lysosomal dispersion in cell cytoplasm. After drug treatment, cells were washed and incubated with <i>T. cruzi</i> TCTs, M.O.I. of 50 parasites per cell for 40 minutes at 37°C. After cell invasion, cells were washed, fixed and immunostained for LAMP-1 (green) DAPI (blue) and analyzed under fluorescence microscope. In comparison to untreated cardiomyocytes (A), which exhibit homogenous and well distributed LAMP-1 labeling, MβCD treated cardiomyocytes, (B) and (C), show a heterogeneous LAMP-1 labeling with lysosomes localized predominantly near cell nuclei. On the other hand, HγCD treated cardiomyocytes and cholesterol-replenished cells, (E), (F) and (D), present lysosomal distribution similar to control cells. Scale bar: 10 µm.</p

MβCD but not HγCD cell incubation leads to lysosomal exocytosis in cardiomyocytes.

<p>Cardiomyocytes were exposed to either 10 mM MβCD or HγCD for 10, 20 or 40 minutes at 37°C, in the absence (white bars) or presence (black bars) of calcium. Both extracellular media and lysates were collected and exposed to 4-methylumbelliferyl-N-acetyl-B-D-glucosaminide, the fluorescent substrate of beta-hexosaminidase, an enzyme resident within lysosomes. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001583#s3" target="_blank">Results</a> are shown as ratio between β-hexosaminidase activity in extracellular media and total β-hexosaminidase activity (extracellular media over extracellular media plus β- hexosaminidase cell lysate hexosaminidase activity). Cells treated with 10 µM Ionomycin (Calbiochem) for 10 minutes were used as lysosomal exocytosis positive control. Data are shown as mean of triplicates ±SD. Asteriks indicate statistically significant differences (p < 0.05,Student's t test) between control and treated cells.</p

Lysosomal exocytosis events after cholesterol depletion are not due to cell death.

<p>After treatment with MβCD or HγCD, in the absence (white bars) or presence (black bars) of calcium, cardiomyocytes were trypsinized, collected and incubated with HFS solution, containing propidium iodide (PI). Cells that became inviable after drug treatment acquired PI labeling in their nuclei due to membrane permeability, and were counted as dead cells by flow cytometer. Data are shown as mean of triplicates ±SD.</p