Hypoxia modulates expression of the 70-kD heat shock protein and reduces Leishmania infection in macrophages

Hypoxia, a microenvironmental factor present in diseased tissues, has been recognized as a specific metabolic stimulus or a signal of cellular response. Experimental hypoxia has been reported to induce adaptation in macrophages such as differential migration, elevation of proinflammatory cytokines and glycolytic enzyme activities, and decreased phagocytosis of inert particles. In this study we demonstrate that although exposure to hypoxia (5% O-2, 5% CO2, and balanced N-2) did not change macrophage viability, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cleavage and proliferation, it significantly reduced expression of the 70-kD heat shock protein (HSP70), which was restored to prehypoxia levels after reoxygenation. The influence of low oxygen tension on macrophage functional activity was also studied, i.e. the ability of these cells to maintain or resist infection by a microorganism. We demonstrate that macrophages from two different sources (a murine cell line and primary cells) exposed to hypoxia were efficiently infected with Leishmania amazonensis, but after 24 h showed a reduction in the percentage of infected cells and of the number of intracellular parasites per macrophage, indicating that hypoxia induced macrophages to kill the intracellular parasites. These results support the notion that hypoxia, a microenvironmental factor, can modulate macrophage protein expression and functional activity. Copyright (C) 2004 National Science Council, ROC and S. Karger AG, Basel.11684785

Hypoxia promotes liver stage malaria infection in primary human hepatocytes in vitro

Homeostasis of mammalian cell function strictly depends on balancing oxygen exposure to maintain energy metabolism without producing excessive reactive oxygen species. In vivo, cells in different tissues are exposed to a wide range of oxygen concentrations, and yet in vitro models almost exclusively expose cultured cells to higher, atmospheric oxygen levels. Existing models of liver stage malaria that utilize primary human hepatocytes typically exhibit low in vitro infection efficiencies, possibly due to missing microenvironmental support signals. One cue that may influence the infection capacity of cultured human hepatocytes is the dissolved oxygen concentration. We developed a microscale human liver platform comprised of precisely patterned primary human hepatocytes and nonparenchymal cells (MPCC) to model liver stage malaria, but the oxygen concentrations are typically higher in the in vitro liver platform than anywhere along the hepatic sinusoid. Indeed, we observed that liver stage Plasmodium parasite development in vivo correlates with hepatic sinusoidal oxygen gradients. Therefore, we hypothesized that in vitro liver stage malaria infection efficiencies may improve under hypoxia. Using the infection of MPCCs with P. berghei or P. yoelii as a model, we observed that ambient hypoxia resulted in increased survival of exo-erythrocytic forms (EEFs) in hepatocytes, and improved parasite development in a subset of surviving EEFs, based on EEF size. Further, the effective cell surface oxygen tensions (pO2) experienced by the hepatocytes, as predicted by a mathematical model, were systematically perturbed by varying culture parameters like hepatocyte density and media height, uncovering an optimal cell surface pO2 to maximize the number of mature EEFs. Initial mechanistic experiments reveal that treatment of primary human hepatocytes with the hypoxia mimetic, cobalt (II) chloride, as well as a HIF-1α activator, dimethyloxalylglycine, also enhance P. berghei infection, suggesting that the effect of hypoxia on infection is mediated in part by host-dependent HIF-1α mechanisms.Bill & Melinda Gates Foundation (Award 51066)Howard Hughes Medical Institut

Studies of funcional alterations of macrophages submetted to hypoxia in ana in vitro model of leishmaniasis

Orientador: Selma GiorgioTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Diversas patologias provocam mudanças na pressão parcial de oxigênio, tornando o microambiente tecidual hipóxico. O interesse em analisar as alterações fenotípicas de células em hipóxia deve-se a necessidade de entender os mecanismos patológicos e a resistência aos tratamentos e ao desenvolvimento de terapias celulares. Células como os macrófagos adaptam-se a hipóxia modificando o metabolismo e a produção de citocinas. As lesões causadas pelo parasita intracelular Leishmania amazonensis são hipóxicas e o cultivo de macrófagos (células hospedeiras da Leishmania) em hipóxia induz redução da infecção com o parasita e modula a expressão de proteínas do choque térmico, indicando alterações funcionais e estruturais em ambiente hipóxico. Neste trabalho avaliamos os mecanismos responsáveis pela resistência destas células ao parasita em hipóxia e as modificações dos macrófagos causadas por este microambiente. Macrófagos cultivados em hipóxia não apresentam alterações na produção de óxido nítrico (NO) e na expressão da sua enzima produtora, óxido nítrico sintase (iNOS). Além disso, macrófagos knockout para enzima iNOS, que não produzem NO, são capazes de reduzir a infecção por L. amazonensis semelhante a macrófagos selvagens, o que sugere que o efeito leishmanicida da hipóxia não se deve ao NO. A liberação das citocinas TNF-a, IL-6, IL-12 e IL- 10 pelos macrófagos é alterada quando estes são cultivados em microambiente hipóxico. A produção destas moléculas pelos macrófagos infectados com L. amazonensis é semelhante em hipóxia e normóxia, indicando que estas citocinas não participam do efeito leishmanicida. O metabolismo energético dos macrófagos infectados, avaliado pela produção de ATP, não é modificado pela hipóxia, indicando que este fator não está envolvido na morte do parasita em macrófagos cultivados em hipóxia. Embora macrófagos fagocitem menos partículas inertes em hipóxia do que macrófagos em normóxia, a fagocitose do parasita vivo não é alterada pela hipóxia, sugerindo que o processo fagocítico também não está relacionado à diminuição da infecção pela hipóxia. Macrófagos cultivados em hipóxia aumentam a produção de ROS em relação a normóxia, mas a produção de ROS por macrófagos infectados com L. amazonensis em hipóxia não é alterada. Mas a inibição do efeito leishmanicida pelos antioxidantes N-acetilcisteína e Ebselen sugere que ROS tem papel na resistência dos macrófagos a L. amazonensis em hipóxia. A expressão de duas isoformas do fator transcricional hypoxia-inducible factor (HIF) (HIF-1alfa e 2alfa) é induzida em macrófagos cultivados em ambiente hipóxico e, interessantemente, em macrófagos infectados com L. amazonensis, mesmo quando estes são cultivados em normóxia. A inibição do HIF-1alfa pelo cloreto de cobalto impediu a sobrevivência do parasita dentro do macrófago, indicando que este fator é importante na manutenção do macrófago como célula hospedeira da L. amazonensis. Nossos resultados também demonstraram que a hipóxia alterou a capacidade proliferativa dos linfócitos T e o processamento e apresentação de antígeno de L. amazonensis pelos macrófagos. Assim, concluímos que a hipóxia induz alterações funcionais e estruturais nos macrófagos e que ROS são importantes para o efeito leishmanicida da hipóxia. Palavras-chave: Leishmania, macrófagos, hipóxiaAbstract: Regions of low oxygen tension (hypoxia) are common features of inflamed/infected tissues. The analyses of cell phenotypic alterations by hypoxia are helpful for understanding of the pathological mechanisms and treatment resistance and for the development of cellular therapies. Macrophages, cells involved in the clearance of microorganisms from infected tissues, are influenced by oxygen tension changing metabolism and cytokines production. Lesions caused by intracellular protozoan Leishmania amazonensis are hypoxic and macrophages (host cells for Leishmania) cultured in hypoxia are resistant to the infection and change heat shock proteins expression, suggesting functional and structural alterations of these cells in hypoxic microenvironment. In the present work we evaluated mechanisms involved in the macrophage resistance to the parasite as well as macrophages phenotypic alterations in hypoxia. Macrophages cultured in hypoxia did not show alterations in nitric oxide (NO) production and oxide nitric synthase (iNOS) enzyme expression. Furthermore, iNOS knockout macrophages lacking NO production are also able to reduce L. amazonensis infection as wild type macrophages, what suggests the leishmanicidal effect of hypoxia is not related to NO. The cytokines TNF-a, IL-6, IL-12 e IL-10 release is altered when macrophages are cultured in hypoxia. However, the production of these cytokines by L. amazonensis-infected macrophages in hypoxia is similar to normoxia, indicating these cytokines do not participate of the leishmanicidal effect of hypoxia. The energetic metabolism of infected macrophages, evaluated through ATP production, is not modified by hypoxia, suggesting this factor is not responsible for parasite death by macrophages cultured in hypoxia. Although the uptake of inert particles by macrophages in hypoxia is lower than in normoxia, living L. amazonensis phagocytosis by macrophages is not altered by hypoxia. This result suggests that phagocytic process is also not related to low infection in hypoxia. Cultured macrophages in hypoxia have shown higher ROS production than in normoxia, but the ROS production by L. amazonensis-infected macrophages is not altered by hypoxia. Indeed, the inhibition of leishmanicidal effect of hypoxia by antioxidants N-acetylcystein and Ebselen suggests ROS play a role in the macrophage resistance to L. amazonensis in hypoxia. The expression of two isoforms of the transcriptional factor hypoxia-inducible factor (HIF) (HIF- 1alpha e 2alpha) is induced in macrophages cultured in hypoxia and, interestingly, in L. amazonensis-infected macrophages in normoxia and hypoxia. The inhibition of HIF-1alpha by cadmium chloride impaired survival of intracellular parasite, suggesting this factor is important for macrophages as host cell for L. amazonensis. Our results also demonstrated hypoxia altered the proliferative capacity of T lymphocytes and the L. amazonensis antigen presentation by macrophages. We conclude hypoxia induces macrophages functional and structural alterations and ROS are important for the leishmanicidal effect of hypoxia. Keywords: Leishmania, macrophages, hypoxiaDoutoradoImunologiaDoutor em Genetica e Biologia Molecula

Functional alterations in macrophages after hypoxia selection

Regions of low oxygen tension are common features of inflamed and infected tissues and provide physiologic selective pressure for the expansion of cells with enhanced hypoxia tolerance. The aim of this study was to investigate whether macrophages resistant to death induced by hypoxia were accompanied by functional alterations. A mouse macrophage cell line (J774 cells) was used to obtain subpopulations of death-resistant macrophages induced by long-term exposure to severe hypoxia (< 1% 02). The results indicated that exposing J774 macrophages to periods of severe hypoxia results in the selection of cells with phenotypes associated with the modulation of heat-shock protein 70 kDa (HSP70) expression, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) production and reduced susceptibility to parasite Leishmania infection. Thus, we suggest that hypoxia-selected macrophages may influence the outcome of inflammation and infection.2321889

Functional Alterations In Macrophages After Hypoxia Selection.

Regions of low oxygen tension are common features of inflamed and infected tissues and provide physiologic selective pressure for the expansion of cells with enhanced hypoxia tolerance. The aim of this study was to investigate whether macrophages resistant to death induced by hypoxia were accompanied by functional alterations. A mouse macrophage cell line (J774 cells) was used to obtain subpopulations of death-resistant macrophages induced by long-term exposure to severe hypoxia (<1% O(2)). The results indicated that exposing J774 macrophages to periods of severe hypoxia results in the selection of cells with phenotypes associated with the modulation of heat-shock protein 70 kDa (HSP70) expression, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) production and reduced susceptibility to parasite Leishmania infection. Thus, we suggest that hypoxia-selected macrophages may influence the outcome of inflammation and infection.23288-9

Expression of hypoxia-inducible factor 1 alpha in mononuclear phagocytes infected with Leishmania amazonensis

Increasing evidence indicates that hypoxia-inducible factor 1 alpha (HIF-1 alpha) can be upregulated indifferent cell types by nonhypoxic stimuli such as growth factors, cytokines, nitric oxide, lipopolysaccharides and a range of infectious microorganisms. In this study, the ability of the following mononuclear phagocytes to express HIF-1 alpha is reported: mouse macrophages (mM Phi)), human macrophages (W(D) and human dendritic cells (DC), parasitized in vitro with Leishmania amazonensis; as assessed by immunofluorescence microscopy. A logical explanation for HIF-1 alpha expression might be that the mononuclear phagocytes became hypoxic after L. amazonensis infection. Using the hypoxia marker pimonidazole, observation revealed that L. amazonensis-infected cells were not hypoxic. In addition, experiments using a HIF-1 alpha inhibitor, CdCl2, to treat L. amazonensis-infected macrophage cultures showed reduced parasite survival. These studies indicated that HIF-1 alpha could play a role in adaptative and immune responses of mononuclear phagocytes presenting infection by the parasite L. amazonensis. (c) 2007 Elsevier B.V. All rights reserved.114211912

Expression Of Hypoxia-inducible Factor 1α In Mononuclear Phagocytes Infected With Leishmania Amazonensis

Increasing evidence indicates that hypoxia-inducible factor 1α (HIF-1α) can be upregulated in different cell types by nonhypoxic stimuli such as growth factors, cytokines, nitric oxide, lipopolysaccharides and a range of infectious microorganisms. In this study, the ability of the following mononuclear phagocytes to express HIF-1α is reported: mouse macrophages (mMΦ), human macrophages (hMΦ) and human dendritic cells (DC), parasitized in vitro with Leishmania amazonensis; as assessed by immunofluorescence microscopy. A logical explanation for HIF-1α expression might be that the mononuclear phagocytes became hypoxic after L. amazonensis infection. Using the hypoxia marker pimonidazole, observation revealed that L. amazonensis-infected cells were not hypoxic. In addition, experiments using a HIF-1α inhibitor, CdCl2, to treat L. amazonensis-infected macrophage cultures showed reduced parasite survival. 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The Influence of Low Oxygen on Macrophage Response to Leishmania Infection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Hypoxia (low oxygen tension) is a common feature of inflamed and infected tissues. The influence of hypoxia on macrophage responses to micro-organisms has only recently been studied. This study demonstrates that hypoxia induced macrophages to control Leishmania amazonensis, an intracellular parasite that causes cutaneous and cutaneous metastatic lesions. The mechanisms that contribute to the control of macrophages against L. amazonensis infection under a hypoxic microenvironment are not known. Nitric oxide, TNF-alpha, IL-10 or IL-12 is not responsible for the decrease in parasitism under hypoxia. Live L. amazonensis entry or exocytosis of internalized particles as well as energetic metabolism was not impaired in infected macrophages; no apoptosis-like death was detected in intracellular parasites. Reactive oxygen species (ROS) is likely to be involved, because treatment with antioxidants N-acetylcysteine (NAC) and ebselen inhibits the leishmanicidal effect of macrophages under hypoxia. Leishmania amazonensis infection induces macrophages to express hypoxia-inducible factor-1 (HIF-1 alpha) and -2 (HIF-2 alpha). Data indicate that hypoxia affects the microbial activities and protein expression of macrophages leading to a different phenotype from that of the normoxic counterpart and that it plays a role in modulating Leishmania infection.742165175Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Fluorescence spectroscopy of roGFP2-based redox probes responding to various physiologically relevant oxidant species in vitro

This article contains representative fluorescence excitation spectra of roGFP2-based probes used for ratiometric analysis of redox changes as presented in the article "Systematic in vitro assessment of responses of roGFP2-based probes to physiologically relevant oxidant species" [1]. The recombinant probes roGFP2, roGFP2-Orp1, and Grx1-roGFP2 were exposed to various oxidative and nitrosative species, including hydrogen peroxide (H2O2), aldrithiol-2 (AT-2), glutathione disulfide (GSSG), hypochlorous acid (HOCl), S-nitrosoglutathione (GSNO), peroxynitrite (ONOO−), potassium polysulfide (K2Sx), spermine NONOate (SperNO), and diethyl amino NONOate (DeaNO) at different molar ratios. Fluorescence excitation spectra of the probes were recorded in the excitation wavelength range between 350 and 500 nm and for a total of 60 min. Analysis and interpretation of the data is presented in an associated article [1]