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1.
Annu Rev Immunol ; 30: 115-48, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22224780

RESUMO

Only a handful of the more than 100,000 fungal species on our planet cause disease in humans, yet the number of life-threatening fungal infections in patients has recently skyrocketed as a result of advances in medical care that often suppress immunity intensely. This emerging crisis has created pressing needs to clarify immune defense mechanisms against fungi, with the ultimate goal of therapeutic applications. Herein, we describe recent insights in understanding the mammalian immune defenses deployed against pathogenic fungi. The review focuses on adaptive immune responses to the major medically important fungi and emphasizes how dendritic cells and subsets in various anatomic compartments respond to fungi, recognize their molecular patterns, and signal responses that nurture and shape the differentiation of T cell subsets and B cells. Also emphasized is how the latter deploy effector and regulatory mechanisms that eliminate these nasty invaders while also constraining collateral damage to vital tissue.


Assuntos
Imunidade Adaptativa , Fungos/imunologia , Micoses/imunologia , Animais , Diferenciação Celular/imunologia , Quimiocinas/imunologia , Citocinas/imunologia , Células Dendríticas/imunologia , Humanos , Imunidade Inata , Imunoglobulinas/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo
2.
Clin Infect Dis ; 76(3): 531-534, 2023 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35767272

RESUMO

In the combination antiretroviral era, there are limited data regarding the pathogenesis of histoplasmosis immune reconstitution inflammatory syndrome (IRIS) in people with human immunodeficiency virus (HIV). We immunologically characterized 10 cases of histoplasmosis, 4 of whom developed histoplasmosis IRIS. CD4+ T cells in histoplasmosis IRIS demonstrated a significant polyfunctional cytokine response to histoplasma antigen.


Assuntos
Síndrome da Imunodeficiência Adquirida , Infecções por HIV , Histoplasmose , Síndrome Inflamatória da Reconstituição Imune , Humanos , Linfócitos T CD4-Positivos , Síndrome da Imunodeficiência Adquirida/complicações , HIV , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico
3.
J Immunol ; 206(3): 524-530, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33328214

RESUMO

Although fibrotic disorders are frequently assumed to be linked to TH2 cells, quantitative tissue interrogation studies have rarely been performed to establish this link and certainly many fibrotic diseases do not fall within the type 2/allergic disease spectrum. We have previously linked two human autoimmune fibrotic diseases, IgG4-related disease and systemic sclerosis, to the clonal expansion and lesional accumulation of CD4+CTLs. In both these diseases TH2 cell accumulation was found to be sparse. Fibrosing mediastinitis linked to Histoplasma capsulatum infection histologically resembles IgG4-related disease in terms of the inflammatory infiltrate and fibrosis, and it provides an example of a fibrotic disease of infectious origin in which the potentially profibrotic T cells may be induced and reactivated by fungal Ags. We show in this study that, in this human disease, CD4+CTLs accumulate in the blood, are clonally expanded, infiltrate into disease lesions, and can be reactivated in vitro by H. capsulatum Ags. TH2 cells are relatively sparse at lesional sites. These studies support a general role for CD4+CTLs in inflammatory fibrosis and suggest that fibrosing mediastinitis is an Ag-driven disease that may provide important mechanistic insights into the pathogenesis of idiopathic fibrotic diseases.


Assuntos
Histoplasma/fisiologia , Histoplasmose/imunologia , Doença Relacionada a Imunoglobulina G4/imunologia , Mediastinite/imunologia , Esclerose/imunologia , Linfócitos T Citotóxicos/imunologia , Células Th2/imunologia , Adulto , Antígenos CD4/metabolismo , Células Cultivadas , Estudos de Coortes , Feminino , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade
4.
Immunity ; 39(4): 697-710, 2013 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-24138881

RESUMO

Macrophages possess numerous mechanisms to combat microbial invasion, including sequestration of essential nutrients, like zinc (Zn). The pleiotropic cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) enhances antimicrobial defenses against intracellular pathogens such as Histoplasma capsulatum, but its mode of action remains elusive. We have found that GM-CSF-activated infected macrophages sequestered labile Zn by inducing binding to metallothioneins (MTs) in a STAT3 and STAT5 transcription-factor-dependent manner. GM-CSF upregulated expression of Zn exporters, Slc30a4 and Slc30a7; the metal was shuttled away from phagosomes and into the Golgi apparatus. This distinctive Zn sequestration strategy elevated phagosomal H⁺ channel function and triggered reactive oxygen species generation by NADPH oxidase. Consequently, H. capsulatum was selectively deprived of Zn, thereby halting replication and fostering fungal clearance. GM-CSF mediated Zn sequestration via MTs in vitro and in vivo in mice and in human macrophages. These findings illuminate a GM-CSF-induced Zn-sequestration network that drives phagocyte antimicrobial effector function.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Histoplasma/imunologia , Histoplasmose/metabolismo , Macrófagos Peritoneais/imunologia , Superóxidos/metabolismo , Zinco/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/imunologia , Regulação da Expressão Gênica , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/imunologia , Complexo de Golgi/microbiologia , Histoplasma/efeitos dos fármacos , Histoplasmose/imunologia , Histoplasmose/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Ativação de Macrófagos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/microbiologia , Metalotioneína/genética , Metalotioneína/imunologia , Camundongos , Camundongos Transgênicos , NADPH Oxidases/genética , NADPH Oxidases/imunologia , Fagossomos/efeitos dos fármacos , Fagossomos/imunologia , Fagossomos/microbiologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Transdução de Sinais , Superóxidos/imunologia , Zinco/imunologia
6.
J Immunol ; 201(11): 3352-3361, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30348735

RESUMO

Allergens such as house dust mites (HDM) and papain induce strong Th2 responses, including elevated IL-4, IL-5, and IL-13 and marked eosinophilia in the airways. Histoplasma capsulatum is a dimorphic fungal pathogen that induces a strong Th1 response marked by IFN-γ and TNF-α production, leading to rapid clearance in nonimmunocompromised hosts. Th1 responses are generally dominant and overwhelm the Th2 response when stimuli for both are present, although there are instances when Th2 stimuli downregulate a Th1 response. We determined if the Th2 response to allergens prevents the host from mounting a Th1 response to H. capsulatum in vivo. C57BL/6 mice exposed to HDM or papain and infected with H. capsulatum exhibited a dominant Th2 response early, characterized by enhanced eosinophilia and elevated Th2 cytokines in lungs. These mice manifested exacerbated fungal burdens, suggesting that animals skewed toward a Th2 response by an allergen are less able to clear the H. capsulatum infection despite an intact Th1 response. In contrast, secondary infection is not exacerbated by allergen exposure, indicating that the memory response may suppress the Th2 response to HDM and quickly clear the infection. In conclusion, an in vivo skewing toward Th2 by allergens exacerbates fungal infection, even though there is a concurrent and unimpaired Th1 response to H. capsulatum.


Assuntos
Eosinófilos/imunologia , Histoplasma/fisiologia , Histoplasmose/imunologia , Hipersensibilidade/imunologia , Pulmão/imunologia , Células Th1/imunologia , Células Th2/imunologia , Poluição do Ar em Ambientes Fechados , Animais , Antígenos de Dermatophagoides/imunologia , Citocinas/metabolismo , Memória Imunológica , Interleucina-33/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pyroglyphidae/imunologia , Equilíbrio Th1-Th2
7.
Int J Mol Sci ; 21(20)2020 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-33050608

RESUMO

Allergic asthma is a chronical pulmonary disease with high prevalence. It manifests as a maladaptive immune response to common airborne allergens and is characterized by airway hyperresponsiveness, eosinophilia, type 2 cytokine-associated inflammation, and mucus overproduction. Alveolar macrophages (AMs), although contributing to lung homeostasis and tolerance to allergens at steady state, have attracted less attention compared to professional antigen-presenting and adaptive immune cells in their contributions. Using an acute model of house dust mite-driven allergic asthma in mice, we showed that a fraction of resident tissue-associated AMs, while polarizing to the alternatively activated M2 phenotype, exhibited signs of polynucleation and polyploidy. Mechanistically, in vitro assays showed that only Granulocyte-Macrophage Colony Stimulating Factor and interleukins IL-13 and IL-33, but not IL-4 or IL-5, participate in the establishment of this phenotype, which resulted from division defects and not cell-cell fusion as shown by microscopy. Intriguingly, mRNA analysis of AMs isolated from allergic asthmatic lungs failed to show changes in the expression of genes involved in DNA damage control except for MafB. Altogether, our data support the idea that upon allergic inflammation, AMs undergo DNA damage-induced stresses, which may provide new unconventional therapeutical approaches to treat allergic asthma.


Assuntos
Asma/etiologia , Asma/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interleucina-33/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Poliploidia , Animais , Asma/patologia , Biomarcadores , Modelos Animais de Doenças , Suscetibilidade a Doenças , Imunofluorescência , Expressão Gênica , Células Gigantes/efeitos dos fármacos , Células Gigantes/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Ativação de Macrófagos , Macrófagos Alveolares/citologia , Camundongos
8.
Infect Immun ; 87(7)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31036602

RESUMO

The fungal pathogen Histoplasma capsulatum causes a spectrum of disease, ranging from local pulmonary infection to disseminated disease. The organism seeks residence in macrophages, which are permissive for its survival. Hypoxia-inducible factor 1α (HIF-1α), a principal regulator of innate immunity to pathogens, is necessary for macrophage-mediated immunity to H. capsulatum in mice. In the present study, we analyzed the effect of HIF-1α in human macrophages infected with this fungus. HIF-1α stabilization was detected in peripheral blood monocyte-derived macrophages at 2 to 24 h after infection with viable yeast cells. Further, host mitochondrial respiration and glycolysis were enhanced. In contrast, heat-killed yeasts induced early, but not later, stabilization of HIF-1α. Since the absence of HIF-1α is detrimental to host control of infection, we asked if large amounts of HIF-1α protein, exceeding those induced by H. capsulatum, altered macrophage responses to this pathogen. Exposure of infected macrophages to an HIF-1α stabilizer significantly reduced recovery of H. capsulatum from macrophages and produced a decrement in mitochondrial respiration and glycolysis compared to those of controls. We observed recruitment of the autophagy-related protein LC3-II to the phagosome, whereas enhancing HIF-1α reduced phagosomal decoration. This finding suggested that H. capsulatum exploited an autophagic process to survive. In support of this assertion, inhibition of autophagy activated macrophages to limit intracellular growth of H. capsulatum Thus, enhancement of HIF-1α creates a hostile environment for yeast cells in human macrophages by interrupting the ability of the pathogen to provoke host cell autophagy.


Assuntos
Histoplasma/imunologia , Histoplasmose/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Macrófagos/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Animais , Autofagia , Histoplasmose/genética , Histoplasmose/microbiologia , Histoplasmose/fisiopatologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Macrófagos/microbiologia , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Fagossomos/imunologia , Fagossomos/microbiologia
9.
PLoS Pathog ; 13(5): e1006356, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28542595

RESUMO

Our understanding of persistence and plasticity of IL-17A+ memory T cells is clouded by conflicting results in models analyzing T helper 17 cells. We studied memory IL-17A+ CD8+ T-cell (Tc17) homeostasis, persistence and plasticity during fungal vaccine immunity. We report that vaccine-induced memory Tc17 cells persist with high fidelity to the type 17 phenotype. Tc17 cells persisted durably for a year as functional IL-17A+ memory cells without converting to IFNγ+ (Tc1) cells, although they produced multiple type I cytokines in the absence of residual vaccine antigen. Memory Tc17 cells were canonical CD8+ T cells with phenotypic features distinct from Tc1 cells, and were Ror(γ)thi, TCF-1hi, T-betlo and EOMESlo. In investigating the bases of Tc17 persistence, we observed that memory Tc17 cells had much higher levels of basal homeostatic proliferation than did Tc1 cells. Conversely, memory Tc17 cells displayed lower levels of anti-apoptotic molecules Bcl-2 and Bcl-xL than Tc1 cells, yet were resistant to apoptosis. Tc1 cells required Bcl-2 for their survival, but Bcl-2 was dispensable for the maintenance of Tc17 cells. Tc17 and Tc1 cells displayed different requirements for HIF-1α during effector differentiation and sustenance and memory persistence. Thus, antifungal vaccination induces durable and stable memory Tc17 cells with distinct requirements for long-term persistence that distinguish them from memory Tc1 cells.


Assuntos
Blastomyces/imunologia , Blastomicose/imunologia , Vacinas Fúngicas/imunologia , Memória Imunológica , Interferon gama/imunologia , Células Th17/imunologia , Animais , Blastomicose/microbiologia , Blastomicose/fisiopatologia , Blastomicose/prevenção & controle , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Humanos , Interleucina-17/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Células Th17/citologia
10.
Infect Immun ; 86(6)2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29581197

RESUMO

Tumor necrosis factor (TNF) antagonists are popular therapies for inflammatory diseases. These agents enhance the numbers and function of regulatory T cells (Tregs), which are important in controlling inflammatory diseases. However, elevated Treg levels increase susceptibility to infections, including histoplasmosis. We determined the mechanism by which Tregs expand in TNF-neutralized mice infected with Histoplasma capsulatum Lung CD11c+ CD11b+ dendritic cells (DCs), but not alveolar macrophages, from H. capsulatum-infected mice treated with anti-TNF induced a higher percentage of Tregs than control DCs in vitro CD11b+ CD103+ DCs, understood to be unique to the intestines, were augmented in lungs with anti-TNF treatment. In the absence of this subset, DCs from anti-TNF-treated mice failed to amplify Tregs in vitro CD11b+ CD103+ DCs from TNF-neutralized mice displayed higher retinaldehyde dehydrogenase 2 (RALDH2) gene expression, and CD11b+ CD103+ RALDH+ DCs exhibited greater enzyme activity. To determine if CD11b+ CD103+ DCs migrated from gut to lung, fluorescent beads were delivered to the gut via oral gavage, and the lungs were assessed for bead-containing DCs. Anti-TNF induced migration of CD11b+ CD103+ DCs from the gut to the lung that enhanced the generation of Tregs in H. capsulatum-infected mice. Therefore, TNF neutralization promotes susceptibility to pulmonary H. capsulatum infection by promoting a gut/lung migration of DCs that enhances Tregs.


Assuntos
Histoplasmose/imunologia , Pneumopatias Fúngicas/imunologia , Linfócitos T Reguladores/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anticorpos Neutralizantes , Histoplasma , Histoplasmose/metabolismo , Pneumopatias Fúngicas/microbiologia , Macrófagos Alveolares/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator de Necrose Tumoral alfa/metabolismo
11.
J Immunol ; 197(2): 565-79, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27271565

RESUMO

Hypoxia-inducible factor (HIF)-1α is a transcription factor that regulates metabolic and immune response genes in the setting of low oxygen tension and inflammation. We investigated the function of HIF-1α in the host response to Histoplasma capsulatum because granulomas induced by this pathogenic fungus develop hypoxic microenvironments during the early adaptive immune response. In this study, we demonstrated that myeloid HIF-1α-deficient mice exhibited elevated fungal burden during the innate immune response (prior to 7 d postinfection) as well as decreased survival in response to a sublethal inoculum of H. capsulatum The absence of myeloid HIF-1α did not alter immune cell recruitment to the lungs of infected animals but was associated with an elevation of the anti-inflammatory cytokine IL-10. Treatment with mAb to IL-10 restored protective immunity to the mutant mice. Macrophages (Mϕs) constituted most IL-10-producing cells. Deletion of HIF-1α in neutrophils or dendritic cells did not alter fungal burden, thus implicating Mϕs as the pivotal cell in host resistance. HIF-1α was stabilized in Mϕs following infection. Increased activity of the transcription factor CREB in HIF-1α-deficient Mϕs drove IL-10 production in response to H. capsulatum IL-10 inhibited Mϕ control of fungal growth in response to the activating cytokine IFN-γ. Thus, we identified a critical function for Mϕ HIF-1α in tempering IL-10 production following infection. We established that transcriptional regulation of IL-10 by HIF-1α and CREB is critical for activation of Mϕs by IFN-γ and effective handling of H. capsulatum.


Assuntos
Histoplasmose/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Interleucina-10/imunologia , Macrófagos/imunologia , Animais , Western Blotting , Proteína de Ligação a CREB/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Histoplasma/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Reação em Cadeia da Polimerase em Tempo Real
12.
J Immunol ; 197(5): 1864-76, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27465530

RESUMO

Zinc (Zn) is an essential metal for development and maintenance of both the innate and adaptive compartments of the immune system. Zn homeostasis impacts maturation of dendritic cells (DCs) that are important in shaping T cell responses. The mechanisms by which Zn regulates the tolerogenic phenotype of DCs remain largely unknown. In this study, we investigated the effect of Zn on DC phenotype and the generation of Foxp3(+) regulatory T cells (Tregs) using a model of Histoplasma capsulatum fungal infection. Exposure of bone marrow-derived DCs to Zn in vitro induced a tolerogenic phenotype by diminishing surface MHC class II (MHCII) and promoting the tolerogenic markers, programmed death-ligand (PD-L)1, PD-L2, and the tryptophan degrading enzyme, IDO. Zn triggered tryptophan degradation by IDO and kynurenine production by DCs and strongly suppressed the proinflammatory response to stimulation by TLR ligands. In vivo, Zn supplementation and subsequent H. capsulatum infection supressed MHCII on DCs, enhanced PD-L1 and PD-L2 expression on MHCII(lo) DCs, and skewed the Treg-Th17 balance in favor of Foxp3(+) Tregs while decreasing Th17 cells. Thus, Zn shapes the tolerogenic potential of DCs in vitro and in vivo and promotes Tregs during fungal infection.


Assuntos
Células Dendríticas/efeitos dos fármacos , Histoplasmose/imunologia , Tolerância Imunológica , Linfócitos T Reguladores/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Zinco/farmacologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células Dendríticas/imunologia , Genes MHC da Classe II/imunologia , Histoplasma/imunologia , Histoplasma/fisiologia , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Cinurenina/metabolismo , Ativação Linfocitária , Camundongos , Fenótipo , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Triptofano/metabolismo , Zinco/fisiologia
13.
Chembiochem ; 18(20): 2069-2078, 2017 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-28783875

RESUMO

Ferrostatin-1 (Fer-1) is a lipophilic antioxidant that effectively blocks ferroptosis, a distinct non-apoptotic form of cell death caused by lipid peroxidation. During many infections, both pathogens and host cells are subjected to oxidative stress, but the occurrence of ferroptosis had not been investigated. We examined ferroptosis in macrophages infected with the pathogenic yeast Histoplasma capsulatum. Unexpectedly, Fer-1 not only reduced the death of macrophages infected in vitro, but inhibited the growth of H. capsulatum and related species Paracoccidioides lutzii and Blastomyces dermatitidis at concentrations under 10 µm. Other antioxidant ferroptosis inhibitors, including liproxstatin-1, did not prevent fungal growth or reduce macrophage death. Structural analysis revealed a potential similarity of Fer-1 to inhibitors of fungal sterol synthesis, and ergosterol content of H. capsulatum decreased more than twofold after incubation with Fer-1. Strikingly, additional Fer-1 analogues with slight differences from Fer-1 had limited impact on fungal growth. In conclusion, the ferroptosis inhibitor Fer-1 has unexpected antifungal potency distinct from its antiferroptotic activity.


Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Cicloexilaminas/química , Cicloexilaminas/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Fenilenodiaminas/química , Fenilenodiaminas/farmacologia , Histoplasma/efeitos dos fármacos , Relação Estrutura-Atividade
14.
Anal Bioanal Chem ; 409(26): 6163-6172, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28801743

RESUMO

This work sought to assess optimal extraction conditions in the study of the metalloproteome of the dimorphic fungus Histoplasma capsulatum. One of the body's responses to H. capsulatum infection is sequestration of zinc within host macrophage (MØ), as reported by Vignesh et al. (Immunity 39:697-710, 2013) and Vignesh et al. (PLOS Pathog 9:E1003815, 2013). Thus, metalloproteins containing zinc were of greatest interest as it plays a critical role in survival of the fungus. One challenge in metalloproteomics is the preservation of the native structure of proteins to retain non-covalently bound metals. Many of the conventional cell lysis, separation, and identification techniques in proteomics are carried out under conditions that could lead to protein denaturation. Various cell lysis techniques were investigated in an effort to both maintain the metalloproteins during lysis and subsequent analysis while, at the same time, serving to be strong enough to break the cell wall, allowing access to cytosolic metalloproteins. The addition of 1% Triton x-100, a non-ionic detergent, to the lysis buffer was also studied. Seven lysis methods were considered and these included: Glass Homogenizer (H), Bead Beater (BB), Sonication Probe (SP), Vortex with 1% Triton x-100 (V, T), Vortex with no Triton x-100 (V, NT), Sonication Bath, Vortex, and 1% Triton x-100 (SB, V, T) and Sonication Bath, Vortex, and no Triton x-100 (SB, V, NT). A Qubit® Assay was used to compare total protein concentration and inductively coupled plasma-mass spectrometry (ICP-MS) was utilized for total metal analysis of cell lysates. Size exclusion chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS) was used for separation of the metalloproteins in the cell lysate and the concentration of Zn over a wide molecular weight range was examined. Additional factors such as potential contamination sources were also considered. A cell lysis method involving vortexing H. capsulatum yeast cells with 500 µm glass beads in a 1% Triton x-100 lysis buffer (V, T) was found to be most advantageous to extract intact zinc metalloproteins as demonstrated by the highest Zn to protein ratio, 1.030 ng Zn/µg protein, and Zn distribution among high, mid, and low molecular weights suggesting the least amount of protein denaturation. Graphical abstract In this work, several cell lysis techniques and two lysis buffers were investigated to evaluate the preservation of the zinc metalloproteome of H. capsulatum while maintaining compatibility with the analytical techniques employed.


Assuntos
Proteínas Fúngicas/isolamento & purificação , Histoplasma/química , Metaloproteínas/isolamento & purificação , Zinco/isolamento & purificação , Detergentes/química , Proteínas Fúngicas/química , Histoplasma/citologia , Histoplasmose/microbiologia , Humanos , Metaloproteínas/química , Octoxinol/química , Proteômica/métodos , Sonicação/métodos , Zinco/química
15.
Int J Mol Sci ; 18(10)2017 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-29065550

RESUMO

Metallothioneins (MTs) are a family of metal-binding proteins virtually expressed in all organisms including prokaryotes, lower eukaryotes, invertebrates and mammals. These proteins regulate homeostasis of zinc (Zn) and copper (Cu), mitigate heavy metal poisoning, and alleviate superoxide stress. In recent years, MTs have emerged as an important, yet largely underappreciated, component of the immune system. Innate and adaptive immune cells regulate MTs in response to stress stimuli, cytokine signals and microbial challenge. Modulation of MTs in these cells in turn regulates metal ion release, transport and distribution, cellular redox status, enzyme function and cell signaling. While it is well established that the host strictly regulates availability of metal ions during microbial pathogenesis, we are only recently beginning to unravel the interplay between metal-regulatory pathways and immunological defenses. In this perspective, investigation of mechanisms that leverage the potential of MTs to orchestrate inflammatory responses and antimicrobial defenses has gained momentum. The purpose of this review, therefore, is to illumine the role of MTs in immune regulation. We discuss the mechanisms of MT induction and signaling in immune cells and explore the therapeutic potential of the MT-Zn axis in bolstering immune defenses against pathogens.


Assuntos
Sistema Imunitário/metabolismo , Infecções/metabolismo , Metalotioneína/imunologia , Animais , Citocinas , Humanos , Metalotioneína/metabolismo , Metais/metabolismo , Transdução de Sinais
16.
Arch Biochem Biophys ; 611: 66-78, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26921502

RESUMO

The importance of Zn ions (Zn) in regulating development and functions of the immune system is well established. However, recent years have witnessed a surge in our knowledge of how immune cells choreograph Zn regulatory mechanisms to combat the persistence of pathogenic microbes. Myeloid and lymphoid populations manipulate intracellular and extracellular Zn metabolism via Zn binding proteins and transporters in response to immunological signals and infection. Rapid as well as delayed changes in readily exchangeable Zn, also known as free Zn and the Zn proteome are crucial in determining activation of immune cells, cytokine responses, signaling and nutritional immunity. Recent studies have unearthed distinctive Zn modulatory mechanisms employed by specialized immune cells and necessitate an understanding of the Zn handling behavior in immune responses to infection. The focus of this review, therefore, stems from novel revelations of Zn intoxication, sequestration and signaling roles deployed by different immune cells, with an emphasis on innate immunity, to challenge microbial parasitization and cope with pathogen insult.


Assuntos
Interações Hospedeiro-Patógeno , Sistema Imunitário , Imunidade Inata , Zinco/imunologia , Animais , Anti-Infecciosos/química , Citocinas/metabolismo , Homeostase , Humanos , Inflamação , Complexo Antígeno L1 Leucocitário/química , Macrófagos/metabolismo , Manganês/química , Neutrófilos/metabolismo , Estresse Oxidativo , Transdução de Sinais
17.
Med Mycol ; 54(8): 865-75, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27335059

RESUMO

Histoplasma capsulatum (Hc) exists in the soil and is capable of adapting to the shift in environment during infection to ensure survival. Yeast encounter a restrictive host environment low in nutrients such as zinc. In this study we functionally analyzed a putative zinc regulated transporter, HcZrt2, in zinc limiting conditions by complementation of HcZrt2 and gene knockdown through RNA interference (RNAi). Complementation analysis demonstrated HcZrt2's ability to functionally replace the characterized Saccharomyces cerevisiae zinc plasma membrane transporters Zrt1 and Zrt2 in zinc deficient medium. Gene silencing revealed that HcZrt2 is essential for growth in zinc deficient medium and plays a role in zinc accumulation. Fungal burden was reduced in mice infected with HcZrt2 silenced strains compared to a control strain. Sixty-seven percent of mice infected with a lethal dose of HcZrt2-RNAi#1 survived, and 100% of mice infected with HcZrt2-RNAi#2 withstood lethal infection. Our data suggest that HcZrt2 is a vital part of zinc homeostasis and essential for the pathogenesis of histoplasmosis.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Histoplasma/fisiologia , Histoplasmose/microbiologia , Fatores de Virulência/metabolismo , Zinco/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Sobrevivência Celular , Contagem de Colônia Microbiana , Meios de Cultura/química , Modelos Animais de Doenças , Deleção de Genes , Técnicas de Silenciamento de Genes , Teste de Complementação Genética , Histoplasma/genética , Histoplasma/crescimento & desenvolvimento , Masculino , Camundongos Endogâmicos C57BL , Interferência de RNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Análise de Sobrevida , Fatores de Virulência/genética
19.
J Immunol ; 188(1): 334-44, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22102723

RESUMO

Apoptosis of leukocytes is known to strongly influence the immunopathogenesis of infection. In this study, we dissected the death pathways of murine macrophages (MΦs) infected with the intracellular pathogen Histoplasma capsulatum. Yeast cells caused apoptosis of MΦs at a wide range of multiplicity of infection, but smaller inocula resulted in delayed detection of apoptosis. Upon infection, caspases 3 and 1 were activated, and both contributed to cell death; however, only the former was involved in apoptosis. The principal driving force for apoptosis involved the extrinsic pathway via engagement of TNFR1 by TNF-α. Infected MΦs produced IL-10 that dampened apoptosis. The chronology of TNF-α and IL-10 release differed in vitro. The former was detected by 2 h postinfection, and the latter was not detected until 8 h postinfection. In vivo, the lungs of TNFR1(-/-) mice infected for 1 d contained fewer apoptotic MΦs than wild-type mice, whereas the lungs of IL-10(-/-) mice exhibited more. Blockade of apoptosis by a pan-caspase inhibitor or by simvastatin sharply reduced the release of TNF-α but enhanced IL-10. However, these treatments did not modify the fungal burden in vitro over 72 h. Thus, suppressing cell death modulated cytokine release but did not alter the fungal burden. These findings provide a framework for the early pathogenesis of histoplasmosis in which yeast cell invasion of lung MΦs engenders apoptosis, triggered in part in an autocrine TNF-α-dependent manner, followed by release of IL-10 that likely prevents apoptosis of newly infected neighboring phagocytes.


Assuntos
Apoptose/imunologia , Histoplasma/imunologia , Histoplasmose/imunologia , Macrófagos Alveolares/imunologia , Animais , Anticolesterolemiantes/farmacologia , Apoptose/genética , Caspase 3/genética , Caspase 3/imunologia , Inibidores de Caspase , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Histoplasmose/genética , Interleucina-10/genética , Interleucina-10/imunologia , Macrófagos Alveolares/microbiologia , Camundongos , Camundongos Knockout , Inibidores de Proteases/farmacologia , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Sinvastatina/farmacologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
20.
mSphere ; 9(2): e0070423, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38259064

RESUMO

Histoplasma capsulatum is a dimorphic fungal pathogen acquired via inhalation of soil-resident spores. Upon exposure to mammalian body temperatures, these fungal elements transform into yeasts that reside primarily within phagocytes. Macrophages (MΦ) provide a permissive environment for fungal replication until T cell-dependent immunity is engaged. MΦ activated by granulocyte macrophage colony stimulating factor (GM-CSF) induces metallothioneins (MTs) that bind zinc (Zn) and deprive yeast cells of labile Zn, thereby disabling fungal growth. Prior work demonstrated that the zinc transporter, ZRT2, was important for fungal survival in vivo. Hence, we constructed a yeast cell reporter strain that expresses green fluorescent protein (GFP) under control of the ZRT2 zinc-regulated promoter. This reporter accurately responds to a medium devoid of Zn. ZRT2 expression increased in GM-CSF, but not interferon-γ, stimulated MΦ. To examine the in vivo response, we infected mice with a reporter yeast strain and assessed ZRT2 expression at 0, 3, 7, and 14 days post-infection (dpi). ZRT2 expression minimally increased at 3 dpi and peaked at 7 dpi, corresponding with the onset of adaptive immunity. We discovered that the major MΦ populations that restrict Zn from the fungus are interstitial MΦ and exudate MΦ. Neutralizing GM-CSF blunted the control of infection but unexpectedly increased ZRT2 expression. This increase was dependent on another cytokine that activates MΦ to control H. capsulatum replication, M-CSF. These findings illustrate the reporter's ability to sense Zn in vitro and in vivo and correlate ZRT2 expression with GM-CSF and M-CSF activation of MΦ.IMPORTANCEPhagocytes use an arsenal of defenses to control the replication of Histoplasma yeasts, one of which is the limitation of trace metals. On the other hand, H. capsulatum combats metal restriction by upregulating metal importers such as the Zn importer ZRT2. This transporter contributes to H. capsulatum pathogenesis upon activation of adaptive immunity. We constructed a fluorescent ZRT2 transcriptional reporter to probe H. capsulatum Zn sensing during infection and exposed the role for M-CSF activation of macrophages when GM-CSF is absent. These data highlight the ways in which fungal pathogens sense metal deprivation in vivo and reveal the potential of metal-sensing reporters. The work adds a new dimension to study how intracellular pathogens sense and respond to the changing environments of the host.


Assuntos
Histoplasma , Histoplasmose , Camundongos , Animais , Histoplasma/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Histoplasmose/microbiologia , Zinco/metabolismo , Saccharomyces cerevisiae , Mamíferos
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