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1.
Front Mol Biosci ; 6: 106, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31681794

RESUMO

Restimulation-induced cell death (RICD) is an apoptotic pathway triggered in activated effector T cells after T cell receptor (TCR) re-engagement. RICD operates at the peak of the immune response to ensure T cell expansion remains in check to maintain immune homeostasis. Understanding the biochemical regulation of RICD sensitivity may provide strategies for tuning the magnitude of an effector T cell response. Metabolic reprogramming in activated T cells is not only critical for T cell differentiation and effector functions, but also influences apoptosis sensitivity. We previously demonstrated that aerobic glycolysis correlates with optimum RICD sensitivity in human effector CD8 T cells. However, metabolic programming in CD4 T cells has not been investigated in this context. We employed a pharmacological approach to explore the effects of fatty acid and glycolytic metabolism on RICD sensitivity in primary human CD4 T cells. Blockade of fatty acid synthase (FASN) with the compound C75 significantly protected CD4 effector T cells from RICD, suggesting that fatty acid biosynthesis contributes to RICD sensitivity. Interestingly, sphingolipid synthesis and fatty acid oxidation (FAO) were dispensable for RICD. Disruption of glycolysis did not protect CD4 T cells from RICD unless glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymatic activity was targeted specifically, highlighting important differences in the metabolic control of RICD in effector CD4 vs. CD8 T cell populations. Moreover, C75 treatment protected effector CD4 T cells derived from naïve, effector memory, and central memory T cell subsets. Decreased RICD in C75-treated CD4 T cells correlated with markedly reduced FAS ligand (FASL) induction and a Th2-skewed phenotype, consistent with RICD-resistant CD4 T cells. These findings highlight FASN as a critical metabolic potentiator of RICD in human effector CD4 T cells.

2.
Cell Mol Immunol ; 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31659245

RESUMO

The adaptive immune response relies on specific apoptotic programs to maintain homeostasis. Conventional effector T cell (Tcon) expansion is constrained by both forkhead box P3 (FOXP3)+-regulatory T cells (Tregs) and restimulation-induced cell death (RICD), a propriocidal apoptosis pathway triggered by repeated stimulation through the T-cell receptor (TCR). Constitutive FOXP3 expression protects Tregs from RICD by suppressing SLAM-associated protein (SAP), a key adaptor protein that amplifies TCR signaling strength. The role of transient FOXP3 induction in activated human CD4 and CD8 Tcons remains unresolved, but its expression is inversely correlated with acquired RICD sensitivity. Here, we describe a novel role for FOXP3 in protecting human Tcons from premature RICD during expansion. Unlike FOXP3-mediated protection from RICD in Tregs, FOXP3 protects Tcons through a distinct mechanism requiring de novo transcription that does not require SAP suppression. Transcriptome profiling and functional analyses of expanding Tcons revealed that FOXP3 enhances expression of the SLAM family receptor CD48, which in turn sustains basal autophagy and suppresses pro-apoptotic p53 signaling. Both CD48 and FOXP3 expression reduced p53 accumulation upon TCR restimulation. Furthermore, silencing FOXP3 expression or blocking CD48 decreased the mitochondrial membrane potential in expanding Tcons with a concomitant reduction in basal autophagy. Our findings suggest that FOXP3 governs a distinct transcriptional program in early-stage effector Tcons that maintains RICD resistance via CD48-dependent protective autophagy and p53 suppression.

3.
Cell Immunol ; 327: 54-61, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29454648

RESUMO

Restimulation-induced cell death (RICD) is an apoptotic program that regulates effector T cell expansion, triggered by repeated stimulation through the T cell receptor (TCR) in the presence of interleukin-2 (IL-2). Although CD4+ regulatory T cells (Tregs) consume IL-2 and experience frequent TCR stimulation, they are highly resistant to RICD. Resistance in Tregs is dependent on the forkhead box P3 (FOXP3) transcription factor, although the mechanism remains unclear. T cells from patients with X-linked lymphoproliferative disease (XLP-1), that lack the adaptor molecule SLAM-associated protein (SAP), are also resistant to RICD. Here we demonstrate that normal Tregs express very low levels of SAP compared to conventional T cells. FOXP3 reduces SAP expression by directly binding to and repressing the SH2D1A (SAP) promoter. Indeed, ectopic SAP expression restores RICD sensitivity in human FOXP3+ Tregs. Our findings illuminate the mechanism behind FOXP3-mediated RICD resistance in Tregs, providing new insight into their long-term persistence.


Assuntos
Fatores de Transcrição Forkhead/fisiologia , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/metabolismo , Linfócitos T Reguladores/metabolismo , Adulto , Apoptose/imunologia , Morte Celular/imunologia , Fatores de Transcrição Forkhead/metabolismo , Voluntários Saudáveis , Humanos , Ativação Linfocitária/fisiologia , Regiões Promotoras Genéticas , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/genética
5.
Cancer Lett ; 408: 190-196, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28866092

RESUMO

An effective adaptive immune response hinges on the rapid clonal expansion of T cells in response to antigen. The sensitivity of these T cells to programmed cell death (i.e. apoptosis) is carefully calibrated at various stages to ensure a robust yet measured reaction that resolves without inflicting unintended damage to host tissues. To meet bioenergetic demands associated with vigorous proliferation, acquisition of effector functions, and memory formation, T cells also undergo dynamic changes in their metabolism at every stage of this response. In this review, we focus on relatively recent studies that illuminate intimate links between metabolic programs and apoptosis sensitivity in T cells. We then examine how these connections ultimately influence T cell survival and function within the metabolically taxing environs of the tumor microenvironment.


Assuntos
Apoptose , Reprogramação Celular , Metabolismo Energético , Memória Imunológica , Linfócitos T/imunologia , Animais , Diferenciação Celular , Humanos , Transdução de Sinais , Linfócitos T/metabolismo
6.
Nat Genet ; 49(8): 1192-1201, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28628108

RESUMO

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Through next-generation sequencing on a cohort of patients with severe atopic dermatitis with and without comorbid infections, we found eight individuals, from four families, with novel heterozygous mutations in CARD11, which encodes a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant CARD11 expression constructs into T cell lines demonstrated both loss-of-function and dominant-interfering activity upon antigen receptor-induced activation of nuclear factor-κB and mammalian target of rapamycin complex 1 (mTORC1). Patient T cells had similar defects, as well as low production of the cytokine interferon-γ (IFN-γ). The mTORC1 and IFN-γ production defects were partially rescued by supplementation with glutamine, which requires CARD11 for import into T cells. Our findings indicate that a single hypomorphic mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Dermatite Atópica/genética , Mutação em Linhagem Germinativa , Guanilato Ciclase/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Estudos de Coortes , Análise Mutacional de DNA , Dermatite Atópica/imunologia , Feminino , Genes Dominantes , Glutamina/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Antígenos de Histocompatibilidade Menor/metabolismo , Complexos Multiproteicos/metabolismo , NF-kappa B/metabolismo , Linhagem , Linfócitos T/imunologia , Linfócitos T/metabolismo , Serina-Treonina Quinases TOR/metabolismo
7.
Cell Death Discov ; 3: 17031, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28580175

RESUMO

CD8+ central memory (CM) and effector memory (EM) T-cell subsets exhibit well-established differences in proliferative and protective capacity after infectious challenge. However, their relative sensitivity to apoptosis has been largely overlooked, despite the importance of programmed cell death in regulating effector T-cell homeostasis. Here we demonstrate that primary human effector T cells derived from the CD8+ EM subset exhibit significantly higher sensitivity to cytokine withdrawal-induced cell death (CWID), a critical intrinsic apoptosis program responsible for culling cells once an infection is cleared and interleukin-2 (IL-2) levels diminish. Interestingly, we found no differences in the expression of IL-2 or IL-2 receptor components in cells originating from either subset. Relative to CM-derived effectors, however, EM-derived T cells displayed more mitochondrial instability and greater caspase activity. Indeed, we found that heightened CWID sensitivity in EM-derived effectors coincided with higher expression of the pro-apoptotic Bcl-2 family protein BIM, both at steady state and with de novo induction following withdrawal of exogenous IL-2. These data point to 'imprinted' differences in BIM protein regulation, preserved by CD8+ CM and EM progeny, which govern their relative sensitivity to CWID. In addition, we detected a burst of autophagy after IL-2 withdrawal, which was better maintained in CM-derived T cells. Both subsets showed increased, equivalent CWID sensitivity upon treatment with autophagy inhibitors, suggesting sustained autophagy could preferentially protect CM-derived T cells from apoptosis. These findings offer new insight into how CM CD8+ T cells display superior effector cell expansion and more persistent memory responses in vivo relative to EM-derived T cells, based in part on decreased CWID sensitivity.

8.
PLoS One ; 10(4): e0124792, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25927990

RESUMO

Many viruses have been implicated in utilizing or modulating the Ubiquitin Proteasome System (UPS) to enhance viral multiplication and/or to sustain a persistent infection. The mosquito-borne Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is an important biodefense pathogen and select agent. There are currently no approved vaccines or therapies for VEEV infections; therefore, it is imperative to identify novel targets for therapeutic development. We hypothesized that a functional UPS is required for efficient VEEV multiplication. We have shown that at non-toxic concentrations Bortezomib, a FDA-approved inhibitor of the proteasome, proved to be a potent inhibitor of VEEV multiplication in the human astrocytoma cell line U87MG. Bortezomib inhibited the virulent Trinidad donkey (TrD) strain and the attenuated TC-83 strain of VEEV. Additional studies with virulent strains of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) demonstrated that Bortezomib is a broad spectrum inhibitor of the New World alphaviruses. Time-of-addition assays showed that Bortezomib was an effective inhibitor of viral multiplication even when the drug was introduced many hours post exposure to the virus. Mass spectrometry analyses indicated that the VEEV capsid protein is ubiquitinated in infected cells, which was validated by confocal microscopy and immunoprecipitation assays. Subsequent studies revealed that capsid is ubiquitinated on K48 during early stages of infection which was affected by Bortezomib treatment. This study will aid future investigations in identifying host proteins as potential broad spectrum therapeutic targets for treating alphavirus infections.


Assuntos
Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Western Blotting , Bortezomib/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Cobaias , Imunoprecipitação , Hibridização in Situ Fluorescente , Espectrometria de Massas em Tandem
9.
Virology ; 468-470: 490-503, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25261871

RESUMO

New World alphaviruses belonging to the family Togaviridae are classified as emerging infectious agents and Category B select agents. Our study is focused on the role of the host extracellular signal-regulated kinase (ERK) in the infectious process of New World alphaviruses. Infection of human cells by Venezuelan equine encephalitis virus (VEEV) results in the activation of the ERK-signaling cascade. Inhibition of ERK1/2 by the small molecule inhibitor Ag-126 results in inhibition of viral multiplication. Ag-126-mediated inhibition of VEEV was due to potential effects on early and late stages of the infectious process. While expression of viral proteins was down-regulated in Ag-126 treated cells, we did not observe any influence of Ag-126 on the nuclear distribution of capsid. Finally, Ag-126 exerted a broad-spectrum inhibitory effect on New World alphavirus multiplication, thus indicating that the host kinase, ERK, is a broad-spectrum candidate for development of novel therapeutics against New World alphaviruses.


Assuntos
Vírus da Encefalite Equina Venezuelana/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transdução de Sinais/fisiologia , Replicação Viral/fisiologia , Animais , Linhagem Celular , Sobrevivência Celular , Vírus da Encefalite Equina do Leste/fisiologia , Vírus da Encefalite Equina do Oeste/fisiologia , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Regulação Viral da Expressão Gênica , Fosforilação , Tirfostinas/farmacologia , Replicação Viral/efeitos dos fármacos
10.
PLoS One ; 9(2): e86745, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24586253

RESUMO

Venezuelan equine encephalitis virus (VEEV) belongs to the genus Alphavirus, family Togaviridae. VEEV infection is characterized by extensive inflammation and studies from other laboratories implicated an involvement of the NF-κB cascade in the in vivo pathology. Initial studies indicated that at early time points of VEEV infection, the NF-κB complex was activated in cells infected with the TC-83 strain of VEEV. One upstream kinase that contributes to the phosphorylation of p65 is the IKKß component of the IKK complex. Our previous studies with Rift valley fever virus, which exhibited early activation of the NF-κB cascade in infected cells, had indicated that the IKKß component underwent macromolecular reorganization to form a novel low molecular weight form unique to infected cells. This prompted us to investigate if the IKK complex undergoes a comparable macromolecular reorganization in VEEV infection. Size-fractionated VEEV infected cell extracts indicated a macromolecular reorganization of IKKß in VEEV infected cells that resulted in formation of lower molecular weight complexes. Well-documented inhibitors of IKKß function, BAY-11-7082, BAY-11-7085 and IKK2 compound IV, were employed to determine whether IKKß function was required for the production of infectious progeny virus. A decrease in infectious viral particles and viral RNA copies was observed with inhibitor treatment in the attenuated and virulent strains of VEEV infection. In order to further validate the requirement of IKKß for VEEV replication, we over-expressed IKKß in cells and observed an increase in viral titers. In contrast, studies carried out using IKKß(-/-) cells demonstrated a decrease in VEEV replication. In vivo studies demonstrated that inhibitor treatment of TC-83 infected mice increased their survival. Finally, proteomics studies have revealed that IKKß may interact with the viral protein nsP3. In conclusion, our studies have revealed that the host IKKß protein may be critically involved in VEEV replication.


Assuntos
Vírus da Encefalite Equina Venezuelana/fisiologia , Encefalomielite Equina Venezuelana/metabolismo , Quinase I-kappa B/metabolismo , Animais , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Encefalomielite Equina Venezuelana/patologia , Cobaias , Interações Hospedeiro-Patógeno , Humanos , Camundongos , NF-kappa B/metabolismo , Neurônios/virologia , Nitrilos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Carga Viral/efeitos dos fármacos , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
11.
Pathog Dis ; 71(2): 177-89, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24488789

RESUMO

Viruses have developed numerous and elegant strategies to manipulate the host cell machinery to establish a productive infectious cycle. The interaction of viral proteins with host proteins plays an important role in infection and pathogenesis, often bypassing traditional host defenses such as the interferon response and apoptosis. Host-viral protein interactions can be studied using a variety of proteomic approaches ranging from genetic and biochemical to large-scale high-throughput technologies. Protein interactions between host and viral proteins are greatly influenced by host signal transduction pathways. In this review, we will focus on comparing proteomic information obtained through differing technologies and how their integration can be used to determine the functional aspect of the host response to infection. We will briefly review and evaluate techniques employed to elucidate viral-host interactions with a primary focus on Protein Microarrays (PMA) and Mass Spectrometry (MS) as potential tools in the discovery of novel therapeutic targets. As many potential molecular markers and targets are proteins, proteomic profiling is expected to yield both clearer and more direct answers to functional and pharmacologic questions.


Assuntos
Testes Diagnósticos de Rotina/métodos , Descoberta de Drogas/métodos , Proteômica/métodos , Viroses/diagnóstico , Viroses/tratamento farmacológico , Biomarcadores/análise , Interações Hospedeiro-Patógeno , Humanos , Espectrometria de Massas/métodos , Análise Serial de Proteínas/métodos
12.
Virology ; 449: 270-86, 2014 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-24418562

RESUMO

Rift Valley fever virus (RVFV) infection is often associated with pronounced liver damage. Previously, our studies revealed altered host phospho-signaling responses (NFκB, MAPK and DNA damage responses) in RVFV infected epithelial cells that correlated with a cellular stress response. Here, we report that RVFV infection of liver cells leads to an increase in reactive oxygen species (ROS). Our data suggests the presence of the viral protein NSs in the mitochondria of infected cells, hence contributing to early increase in ROS. Increased ROS levels correlated with activation of NFκB (p65) and p53 responses, which in conjunction with infection, was also reflected as macromolecular rearrangements observed using size fractionation of protein lysates. Additionally, we documented an increase in cytokine expression and pro-apoptotic gene expression with infection, which was reversed with antioxidant treatment. Collectively, we identified ROS and oxidative stress as critical contributors to apoptosis of liver cells during RVFV infection.


Assuntos
Apoptose , Fígado/citologia , Espécies Reativas de Oxigênio/metabolismo , Febre do Vale de Rift/metabolismo , Vírus da Febre do Vale do Rift/fisiologia , Fator de Transcrição RelA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Humanos , Fígado/metabolismo , Fígado/virologia , Estresse Oxidativo , Febre do Vale de Rift/fisiopatologia , Febre do Vale de Rift/virologia , Fator de Transcrição RelA/genética , Proteína Supressora de Tumor p53/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
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