Phenotypic profiling of CD8+ T cells during Plasmodium vivax blood-stage infection

Submitted by Repositório Arca ([email protected]) on 2019-04-24T17:38:50Z No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Approved for entry into archive by Janaína Nascimento ([email protected]) on 2019-08-13T14:21:37Z (GMT) No. of bitstreams: 2 ve_ Hojo-Souza_Natália_etal_INI_2015.pdf: 1172050 bytes, checksum: b1948378bfee8669ad90694b3aa2cb60 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-08-13T14:21:37Z (GMT). No. of bitstreams: 2 ve_ Hojo-Souza_Natália_etal_INI_2015.pdf: 1172050 bytes, checksum: b1948378bfee8669ad90694b3aa2cb60 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Centro de Pesquisa em Medicina Tropical. Porto Velho, RO, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Centro de Pesquisa em Medicina Tropical. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Rio de Janeiro. RJ, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Background: For a long time, the role of CD8+ T cells in blood-stage malaria was not considered important because erythrocytes do not express major histocompatibility complex (MHC) class I proteins. While recent evidences suggest that CD8+ T cells may play an important role during the erythrocytic phase of infection by eliminating parasites, CD8+ T cells might also contribute to modulate the host response through production of regulatory cytokines. Thus, the role of CD8+ T cells during blood-stage malaria is unclear. Here, we report the phenotypic profiling of CD8+ T cells subsets from patients with uncomplicated symptomatic P. vivax malaria. Methods: Blood samples were collected from 20 Plasmodium vivax-infected individuals and 12 healthy individuals. Immunophenotyping was conducted by flow cytometry. Plasma levels of IFN-γ, TNF-α and IL-10 were determined by ELISA/CBA. Unpaired t-test or Mann–Whitney test was used depending on the data distribution. Results: P. vivax-infected subjects had lower percentages and absolute numbers of CD8+ CD45RA+ and CD8+ CD45RO+ T cells when compared to uninfected individuals (p ≤ 0.0002). A significantly lower absolute number of circulating CD8+ CD45+ CCR7+ cells (p = 0.002) was observed in P. vivax-infected individuals indicating that infection reduces the number of central memory T cells. Cytokine expression was significantly reduced in the naïve T cells from infected individuals compared with negative controls, as shown by lower numbers of IFN-γ + (p = 0.001), TNF-α+ (p < 0.0001) and IL-10+ (p < 0.0001) CD8+ T cells. Despite the reduction in the number of CD8+ memory T cells producing IFN-γ (p < 0.0001), P. vivax-infected individuals demonstrated a significant increase in memory CD8+ TNF-α+ (p = 0.016) and CD8+ IL-10+ (p = 0.004) cells. Positive correlations were observed between absolute numbers of CD8+ IL-10+ and numbers of CD8+ IFN-γ + (p < 0.001) and CD8+ TNF-α+ T cells (p ≤ 0.0001). Finally, an increase in the plasma levels of TNF-α (p = 0.017) and IL-10 (p = 0.006) and a decrease in the IFN-γ plasma level (p <0.0001) were observed in the P. vivax-infected individuals. Conclusions: P. vivax infection reduces the numbers of different subsets of CD8+ T cells, particularly the memory cells, during blood-stage of infection and enhances the number of CD8+ memory T cells expressing IL-10, which positively correlates with the number of cells expressing TNF-α and IFN-γ

Immunoregulatory mechanisms in Chagas disease: modulation of apoptosis in T-cell mediated immune responses

Submitted by Nuzia Santos ([email protected]) on 2017-07-17T17:53:45Z No. of bitstreams: 1 Chaves_AnaThereza_Immunoregulatory mechanisms_IRR_2016.pdf: 12736177 bytes, checksum: 7182dae7e3675c77254aa3dd4157a0a9 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2017-07-17T18:03:09Z (GMT) No. of bitstreams: 1 Chaves_AnaThereza_Immunoregulatory mechanisms_IRR_2016.pdf: 12736177 bytes, checksum: 7182dae7e3675c77254aa3dd4157a0a9 (MD5)Made available in DSpace on 2017-07-17T18:03:09Z (GMT). No. of bitstreams: 1 Chaves_AnaThereza_Immunoregulatory mechanisms_IRR_2016.pdf: 12736177 bytes, checksum: 7182dae7e3675c77254aa3dd4157a0a9 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, Brazil/Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Morfologia. Laboratório de Biologia das Interações Celulares. Belo Horizonte, MG, Brazil/Universidade Federal de Minas Gerais. Faculdade de Medicina Programa de Pós graduação em Medicina Tropical e Infectologia. Belo Horizonte, MG, Brazil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Biomarcadores de Diagnóstico e Monitoração. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Morfologia. Laboratório de Biologia das Interações Celulares. Belo Horizonte, MG, Brazil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, BrazilUniversidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Laboratório de Imunologia e Genômica de Parasitos. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Fisiologia e Biofísica. Belo Horizonte, MG, Brazil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, BrazilUniversidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Laboratório de Imunologia e Genômica de Parasitos. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Faculdade de Medicina Programa de Pós graduação em Medicina Tropical e Infectologia. Belo Horizonte, MG, Brazil.Laboratório de Imunologia Celular e Molecular, Centro de Pesquisas René Rachou, Fiocruz, Belo Horizonte, Brazil/Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais. Belo Horizonte, MG, Brazil/Universidade Federal de Ouro Preto. Ouro Preto, MG, Brazil.BACKGROUND: Chronic Chagas disease presents different clinical manifestations ranging from asymptomatic (namely indeterminate) to severe cardiac and/or digestive. Previous results have shown that the immune response plays an important role, although no all mechanisms are understood. Immunoregulatory mechanisms such as apoptosis are important for the control of Chagas disease, possibly affecting the morbidity in chronic clinical forms. Apoptosis has been suggested to be an important mechanism of cellular response during T. cruzi infection. We aimed to further understand the putative role of apoptosis in Chagas disease and its relation to the clinical forms of the disease. METHODS: Apoptosis of lymphocytes, under antigenic stimuli (soluble T. cruzi antigens - TcAg) where compared to that of non-stimulated cells. Apoptosis was evaluated using the expression of annexin and caspase 3(+) by T cells and the percentage of cells positive evaluated by flow cytometry. In addition activation and T cell markers were used for the identification of TCD4(+) and TCD8(+) subpopulations. The presence of intracellular and plasma cytokines were also evaluated. Analysis of the activation status of the peripheral blood cells showed that patients with Chagas disease presented higher levels of activation determined by the expression of activation markers, after TcAg stimulation. PCR array were used to evaluate the contribution of this mechanism in specific cell populations from patients with different clinical forms of human Chagas disease. RESULTS: Our results showed a reduced proliferative response associated a high expression of T CD4(+)CD62L(-) cells in CARD patients when compared with IND group and NI individuals. We also observed that both groups of patients presented a significant increase of CD4(+) and CD8(+) T cell subsets in undergoing apoptosis after in vitro stimulation with T. cruzi antigens. In CARD patients, both CD4(+) and CD8(+) T cells expressing TNF-α were highly susceptible to undergo apoptosis after in vitro stimulation. Interestingly, the in vitro TcAg stimulation increased considerably the expression of cell death TNF/TNFR superfamily and Caspase family receptors genes in CARD patients. CONCLUSIONS: Taken together, our results suggest that apoptosis may be an important mechanism for the control of morbidity in T. cruzi infection by modulating the expression of apoptosis genes, the cytokine environment and/or killing of effector cells

IgG Induced by Vaccination With Ascaris suum Extracts Is Protective Against Infection

Human ascariasis has a global and cosmopolitan distribution, and has been characterized as the most prevalent neglected tropical disease worldwide. The development of a preventive vaccine is highly desirable to complement current measures required for this parasitic infection control and to reduce chronic childhood morbidities. In the present study, we describe the mechanism of protection elicited by a preventive vaccine against ascariasis. Vaccine efficacy was evaluated after immunization with three different Ascaris suum antigen extracts formulated with monophosphoryl lipid A (MPLA) as an adjuvant: crude extract of adult worm (ExAD); crude extract of adult worm cuticle (CUT); and crude extract of infective larvae (L3) (ExL3). Immunogenicity elicited by immunization was assessed by measuring antibody responses, cytokine production, and influx of tissue inflammatory cells. Vaccine efficacy was evaluated by measuring the reductions in the numbers of larvae in the lungs of immunized BALB/c mice that were challenged with A. suum eggs. Moreover, lung physiology and functionality were tested by spirometry to determine clinical efficacy. Finally, the role of host antibody mediated protection was determined by passive transfer of serum from immunized mice. Significant reductions in the total number of migrating larvae were observed in mice immunized with ExL3 61% (p &lt; 0.001), CUT 59% (p &lt; 0.001), and ExAD 51% (p &lt; 0.01) antigens in comparison with non-immunized mice. For the Ascaris antigen-specific IgG antibody levels, a significant and progressive increase was observed with each round of immunization, in association with a marked increase of IgG1 and IgG3 subclasses. Moreover, a significant increase in concentration of IL-5 and IL-10 (pre-challenge) in the blood and IL-10 in the lung tissue (post-challenge) was induced by CUT immunization. Finally, ExL3 and CUT-immunized mice showed a marked improvement in lung pathology and tissue fibrosis as well as reduced pulmonary dysfunction induced by Ascaris challenge, when compared to non-immunized mice. Moreover, the passive transfer of specific IgG antibodies from ExL3, CUT, and ExAD elicited a protective response in naïve mice, with significant reductions in parasite burdens in lungs of 65, 64, and 64%, respectively. Taken together, these studies indicated that IgG antibodies contribute to protective immunity

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

The Genome of Anopheles darlingi, the main neotropical malaria vector

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vectorhuman and vectorparasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles- darlingi. © 2013 The Author(s)

Regulatory monocytes in helminth infections: insights from the modulation during human hookworm infection

Abstract Background While the macrophage polarization is well characterized in helminth infections, the natural heterogeneity of monocytes with multiple cell phenotypes might influence the outcome of neglected diseases, such hookworm infection. Here, we report the profile of monocytes in human hookworm infections as a model to study the regulatory subpopulation of monocytes in helminth infections. Methods Blood samples were collected from 19 Necator americanus-infected individuals and 13 healthy individuals. Peripheral blood mononuclear cells (PBMCs) were isolated, and immunophenotyping was conducted by flow cytometry. The expressions of genes encoding human nitric oxide synthase (iNOS), interleukin 4 (IL-4), arginase-1 (Arg-1) and glyceraldehyde 3-phosphate dehydrogenase were quantified by qPCR. Plasma levels of IL-4 were determined by sandwich ELISA. Unpaired t-tests or Mann-Whitney tests were used depending on the data distribution. Results Hookworm infected individuals (HWI) showed a significant increase in the number of monocytes/mm3 (555.2 ± 191.0) compared to that of the non-infected (NI) individuals (120.4 ± 44.7) (p < 0.0001). While the frequencies of CD14+IL-10+ and CD14+IL-12+ cells were significantly reduced in the HWI compared to NI group (p = 0.0289 and p < 0.0001, respectively), the ratio between IL-10/IL-12 producing monocytes was significantly elevated in HWI (p = 0.0004), indicating the potential regulatory activity of these cells. Measurement of IL-4 levels and gene expression of IL-4 and Arg-1 (highly expressed in alternatively activated macrophages) revealed no significant differences between the NI and HWI groups. Interestingly, individuals from the HWI group had higher expression of the iNOS gene (associated with a regulatory profile) (20.27 ± 2.97) compared to the NI group (11.28 ± 1.18, p = 0.0409). Finally, individuals from the HWI group had a significantly higher frequency of CD206+CD23+IL-10+ (7.57 ± 1.96) cells compared to individuals from the NI group (0.35 ± 0.09) (p < 0.001), suggesting that activated monocytes are a potential source of regulatory cytokines during hookworm infection. Conclusions Natural hookworm infection induces a high frequency of circulating monocytes that present a regulatory profile and promote the downmodulation of the proinflammatory response, which may contribute to prolonged survival of the parasite in the host