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1.
Nat Immunol ; 24(9): 1473-1486, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37580603

RESUMO

Omnivorous animals, including mice and humans, tend to prefer energy-dense nutrients rich in fat over plant-based diets, especially for short periods of time, but the health consequences of this short-term consumption of energy-dense nutrients are unclear. Here, we show that short-term reiterative switching to 'feast diets', mimicking our social eating behavior, breaches the potential buffering effect of the intestinal microbiota and reorganizes the immunological architecture of mucosa-associated lymphoid tissues. The first dietary switch was sufficient to induce transient mucosal immune depression and suppress systemic immunity, leading to higher susceptibility to Salmonella enterica serovar Typhimurium and Listeria monocytogenes infections. The ability to respond to antigenic challenges with a model antigen was also impaired. These observations could be explained by a reduction of CD4+ T cell metabolic fitness and cytokine production due to impaired mTOR activity in response to reduced microbial provision of fiber metabolites. Reintroducing dietary fiber rewired T cell metabolism and restored mucosal and systemic CD4+ T cell functions and immunity. Finally, dietary intervention with human volunteers confirmed the effect of short-term dietary switches on human CD4+ T cell functionality. Therefore, short-term nutritional changes cause a transient depression of mucosal and systemic immunity, creating a window of opportunity for pathogenic infection.


Assuntos
Mucosa , Salmonella typhimurium , Humanos , Camundongos , Animais , Linfócitos T , Imunidade nas Mucosas
2.
Immunity ; 44(6): 1392-405, 2016 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-27287411

RESUMO

Although numerous polymorphisms have been associated with inflammatory bowel disease (IBD), identifying the function of these genetic factors has proved challenging. Here we identified a role for nine genes in IBD susceptibility loci in antibacterial autophagy and characterized a role for one of these genes, GPR65, in maintaining lysosome function. Mice lacking Gpr65, a proton-sensing G protein-coupled receptor, showed increased susceptibly to bacteria-induced colitis. Epithelial cells and macrophages lacking GPR65 exhibited impaired clearance of intracellular bacteria and accumulation of aberrant lysosomes. Similarly, IBD patient cells and epithelial cells expressing an IBD-associated missense variant, GPR65 I231L, displayed aberrant lysosomal pH resulting in lysosomal dysfunction, impaired bacterial restriction, and altered lipid droplet formation. The GPR65 I231L polymorphism was sufficient to confer decreased GPR65 signaling. Collectively, these data establish a role for GPR65 in IBD susceptibility and identify lysosomal dysfunction as a potentially causative element in IBD pathogenesis with effects on cellular homeostasis and defense.


Assuntos
Colite/imunologia , Células Epiteliais/imunologia , Doenças Inflamatórias Intestinais/genética , Lisossomos/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Infecções por Salmonella/imunologia , Salmonella enterica/imunologia , Salmonella typhimurium/imunologia , Animais , Predisposição Genética para Doença , Células HeLa , Humanos , Doenças Inflamatórias Intestinais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagossomos/fisiologia , Polimorfismo Genético , Receptores Acoplados a Proteínas G/genética , Risco
3.
Cell ; 143(5): 789-801, 2010 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-21111238

RESUMO

The mechanism by which CD4 T cells are depleted in HIV-infected hosts remains poorly understood. In ex vivo cultures of human tonsil tissue, CD4 T cells undergo a pronounced cytopathic response following HIV infection. Strikingly, >95% of these dying cells are not productively infected but instead correspond to bystander cells. We now show that the death of these "bystander" cells involves abortive HIV infection. Inhibitors blocking HIV entry or early steps of reverse transcription prevent CD4 T cell death while inhibition of later events in the viral life cycle does not. We demonstrate that the nonpermissive state exhibited by the majority of resting CD4 tonsil T cells leads to accumulation of incomplete reverse transcripts. These cytoplasmic nucleic acids activate a host defense program that elicits a coordinated proapoptotic and proinflammatory response involving caspase-3 and caspase-1 activation. While this response likely evolved to protect the host, it centrally contributes to the immunopathogenic effects of HIV.


Assuntos
Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , HIV-1/imunologia , Tecido Linfoide/virologia , Apoptose , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Replicação do DNA , DNA Viral/metabolismo , Transcriptase Reversa do HIV/metabolismo , Humanos , Tecido Linfoide/citologia , Tecido Linfoide/imunologia
4.
PLoS Genet ; 15(5): e1008084, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31059511

RESUMO

The transition from mitotic to meiotic cell cycles is essential for haploid gamete formation and fertility. Stimulated by retinoic acid gene 8 (Stra8) is an essential gatekeeper of meiotic initiation in vertebrates; yet, the molecular role of STRA8 remains principally unknown. Here we demonstrate that STRA8 functions as a suppressor of autophagy during spermatogenesis in mice. Stra8-deficient germ cells fail to enter meiosis and present aberrant upregulation of autophagy-lysosome genes, commensurate with autophagy activation. Biochemical assays show that ectopic expression of STRA8 alone is sufficient to inhibit both autophagy induction and maturation. Studies also revealed that, Nr1d1, a nuclear hormone receptor gene, is upregulated in Stra8-deficient testes and that STRA8 binds to the Nr1d1 promoter, indicating that Nr1d1 is a direct target of STRA8 transcriptional repression. In addition, it was found that NR1D1 binds to the promoter of Ulk1, a gene essential for autophagy initiation, and that Nr1d1 is required for the upregulated Ulk1 expression in Stra8-deficient testes. Furthermore, both genetic deletion of Nr1d1 and pharmacologic inhibition of NR1D1 by its synthetic antagonist SR8278 exhibit rescuing effects on the meiotic initiation defects observed in Stra8-deficient male germ cells. Together, the data suggest a novel link between STRA8-mediated autophagy suppression and meiotic initiation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Fertilidade/genética , Meiose , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Espermatogênese/genética , Espermatozoides/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Sequência de Bases , Regulação da Expressão Gênica no Desenvolvimento , Isoquinolinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Espermatozoides/citologia , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Tiofenos/farmacologia
5.
J Immunol ; 203(7): 1820-1829, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31451676

RESUMO

The clear role of autophagy in human inflammatory diseases such as Crohn disease was first identified by genome-wide association studies and subsequently dissected in multiple mechanistic studies. ATG16L1 has been particularly well studied in knockout and hypomorph settings as well as models recapitulating the Crohn disease-associated T300A polymorphism. Interestingly, ATG16L1 has a single homolog, ATG16L2, which is independently implicated in diseases, including Crohn disease and systemic lupus erythematosus. However, the contribution of ATG16L2 to canonical autophagy pathways and other cellular functions is poorly understood. To better understand its role, we generated and analyzed the first, to our knowledge, ATG16L2 knockout mouse. Our results show that ATG16L1 and ATG16L2 contribute very distinctly to autophagy and cellular ontogeny in myeloid, lymphoid, and epithelial lineages. Dysregulation of any of these lineages could contribute to complex diseases like Crohn disease and systemic lupus erythematosus, highlighting the value of examining cell-specific effects. We also identify a novel genetic interaction between ATG16L2 and epithelial ATG16L1. These findings are discussed in the context of how these genes may contribute distinctly to human disease.


Assuntos
Morte Celular Autofágica , Proteínas Relacionadas à Autofagia , Proteínas de Transporte , Doença de Crohn , Lúpus Eritematoso Sistêmico , Animais , Morte Celular Autofágica/genética , Morte Celular Autofágica/imunologia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Doença de Crohn/genética , Doença de Crohn/imunologia , Modelos Animais de Doenças , Humanos , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Knockout , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia
6.
J Immunol ; 202(11): 3256-3266, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31010852

RESUMO

Tetraspanins are a family of proteins possessing four transmembrane domains that help in lateral organization of plasma membrane proteins. These proteins interact with each other as well as other receptors and signaling proteins, resulting in functional complexes called "tetraspanin microdomains." Tetraspanins, including CD82, play an essential role in the pathogenesis of fungal infections. Dectin-1, a receptor for the fungal cell wall carbohydrate ß-1,3-glucan, is vital to host defense against fungal infections. The current study identifies a novel association between tetraspanin CD82 and Dectin-1 on the plasma membrane of Candida albicans-containing phagosomes independent of phagocytic ability. Deletion of CD82 in mice resulted in diminished fungicidal activity, increased C. albicans viability within macrophages, and decreased cytokine production (TNF-α, IL-1ß) at both mRNA and protein level in macrophages. Additionally, CD82 organized Dectin-1 clustering in the phagocytic cup. Deletion of CD82 modulates Dectin-1 signaling, resulting in a reduction of Src and Syk phosphorylation and reactive oxygen species production. CD82 knockout mice were more susceptible to C. albicans as compared with wild-type mice. Furthermore, patient C. albicans-induced cytokine production was influenced by two human CD82 single nucleotide polymorphisms, whereas an additional CD82 single nucleotide polymorphism increased the risk for candidemia independent of cytokine production. Together, these data demonstrate that CD82 organizes the proper assembly of Dectin-1 signaling machinery in response to C. albicans.


Assuntos
Candida albicans/fisiologia , Candidíase/metabolismo , Membrana Celular/metabolismo , Proteína Kangai-1/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Fagossomos/metabolismo , Animais , Candidíase/imunologia , Linhagem Celular , Predisposição Genética para Doença , Humanos , Imunidade Celular , Interleucina-1beta/metabolismo , Proteína Kangai-1/genética , Lectinas Tipo C/genética , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polimorfismo de Nucleotídeo Único , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
7.
Proc Natl Acad Sci U S A ; 112(31): E4281-7, 2015 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-26195741

RESUMO

Studies of human genetics and pathophysiology have implicated the regulation of autophagy in inflammation, neurodegeneration, infection, and autoimmunity. These findings have motivated the use of small-molecule probes to study how modulation of autophagy affects disease-associated phenotypes. Here, we describe the discovery of the small-molecule probe BRD5631 that is derived from diversity-oriented synthesis and enhances autophagy through an mTOR-independent pathway. We demonstrate that BRD5631 affects several cellular disease phenotypes previously linked to autophagy, including protein aggregation, cell survival, bacterial replication, and inflammatory cytokine production. BRD5631 can serve as a valuable tool for studying the role of autophagy in the context of cellular homeostasis and disease.


Assuntos
Autofagia/efeitos dos fármacos , Genética Médica , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Bibliotecas de Moléculas Pequenas/farmacologia , Bactérias/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Agregação Celular/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/metabolismo , Peptídeos/metabolismo , Fenótipo , Bibliotecas de Moléculas Pequenas/química
8.
Proc Natl Acad Sci U S A ; 111(21): 7741-6, 2014 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-24821797

RESUMO

A coding polymorphism (Thr300Ala) in the essential autophagy gene, autophagy related 16-like 1 (ATG16L1), confers increased risk for the development of Crohn disease, although the mechanisms by which single disease-associated polymorphisms contribute to pathogenesis have been difficult to dissect given that environmental factors likely influence disease initiation in these patients. Here we introduce a knock-in mouse model expressing the Atg16L1 T300A variant. Consistent with the human polymorphism, T300A knock-in mice do not develop spontaneous intestinal inflammation, but exhibit morphological defects in Paneth and goblet cells. Selective autophagy is reduced in multiple cell types from T300A knock-in mice compared with WT mice. The T300A polymorphism significantly increases caspase 3- and caspase 7-mediated cleavage of Atg16L1, resulting in lower levels of full-length Atg16Ll T300A protein. Moreover, Atg16L1 T300A is associated with decreased antibacterial autophagy and increased IL-1ß production in primary cells and in vivo. Quantitative proteomics for protein interactors of ATG16L1 identified previously unknown nonoverlapping sets of proteins involved in ATG16L1-dependent antibacterial autophagy or IL-1ß production. These findings demonstrate how the T300A polymorphism leads to cell type- and pathway-specific disruptions of selective autophagy and suggest a mechanism by which this polymorphism contributes to disease.


Assuntos
Proteínas de Transporte/genética , Doença de Crohn/imunologia , Celulas de Paneth/patologia , Polimorfismo de Nucleotídeo Único/genética , Infecções por Salmonella/imunologia , Animais , Autofagia/genética , Proteínas Relacionadas à Autofagia , Western Blotting , Cromatografia Líquida , Doença de Crohn/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Técnicas de Introdução de Genes , Células Caliciformes/patologia , Camundongos , Proteômica , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas em Tandem
9.
Gut ; 62(10): 1505-10, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24037875

RESUMO

Studies of the genetics underlying inflammatory bowel diseases have increased our understanding of the pathways involved in both ulcerative colitis and Crohn's disease and focused attention on the role of the microbiome in these diseases. Full understanding of pathogenesis will require a comprehensive grasp of the delicate homeostasis between gut bacteria and the human host. In this review, we present current evidence of microbiome-gene interactions in the context of other known risk factors and mechanisms, and describe the next steps necessary to pair genetic variant and microbiome sequencing data from patient cohorts. We discuss the concept of dysbiosis, proposing that the functional composition of the gut microbiome may provide a more consistent definition of dysbiosis and may more readily provide evidence of genome-microbiome interactions in future exploratory studies.


Assuntos
Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/microbiologia , Microbiota , Bactérias/imunologia , Predisposição Genética para Doença , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Intestinos/microbiologia , Fenótipo , Transdução de Sinais/genética
10.
PLoS Pathog ; 7(8): e1002184, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21876670

RESUMO

The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear.We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51) mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50) mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb). We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1) as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells.Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through demethylation of K51 in Tat. Small molecule inhibitors of LSD1/KDM1 show therapeutic promise by enforcing HIV latency in infected T cells.


Assuntos
Histona Desmetilases/metabolismo , Transcrição Gênica/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Acetilação , Animais , Epigênese Genética/fisiologia , Genes Virais/efeitos dos fármacos , Histona Desmetilases/antagonistas & inibidores , Metilação , Fenelzina/farmacologia , Fator B de Elongação Transcricional Positiva/metabolismo , Coelhos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/imunologia
11.
bioRxiv ; 2023 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-37425769

RESUMO

Extraintestinal autoimmune diseases are multifactorial with translocating gut pathobionts implicated as instigators and perpetuators in mice. However, the microbial contributions to autoimmunity in humans remain largely unclear, including whether specific pathological human adaptive immune responses are triggered by such pathobionts. We show here that the translocating pathobiont Enterococcus gallinarum induces human IFNγ + Th17 differentiation and IgG3 subclass switch of anti- E. gallinarum RNA and correlating anti-human RNA autoantibody responses in patients with systemic lupus erythematosus and autoimmune hepatitis. Human Th17 induction by E. gallinarum is cell-contact dependent and involves TLR8-mediated human monocyte activation. In murine gnotobiotic lupus models, E. gallinarum translocation triggers IgG3 anti-RNA autoantibody titers that correlate with renal autoimmune pathophysiology and with disease activity in patients. Overall, we define cellular mechanisms of how a translocating pathobiont induces human T- and B-cell-dependent autoimmune responses, providing a framework for developing host- and microbiota-derived biomarkers and targeted therapies in extraintestinal autoimmune diseases. One Sentence Summary: Translocating pathobiont Enterococcus gallinarum promotes human Th17 and IgG3 autoantibody responses linked to disease activity in autoimmune patients.

12.
Med ; 3(7): 481-518.e14, 2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35649411

RESUMO

BACKGROUND: Pro-inflammatory fibroblasts are critical for pathogenesis in rheumatoid arthritis, inflammatory bowel disease, interstitial lung disease, and Sjögren's syndrome and represent a novel therapeutic target for chronic inflammatory disease. However, the heterogeneity of fibroblast phenotypes, exacerbated by the lack of a common cross-tissue taxonomy, has limited our understanding of which pathways are shared by multiple diseases. METHODS: We profiled fibroblasts derived from inflamed and non-inflamed synovium, intestine, lungs, and salivary glands from affected individuals with single-cell RNA sequencing. We integrated all fibroblasts into a multi-tissue atlas to characterize shared and tissue-specific phenotypes. FINDINGS: Two shared clusters, CXCL10+CCL19+ immune-interacting and SPARC+COL3A1+ vascular-interacting fibroblasts, were expanded in all inflamed tissues and mapped to dermal analogs in a public atopic dermatitis atlas. We confirmed these human pro-inflammatory fibroblasts in animal models of lung, joint, and intestinal inflammation. CONCLUSIONS: This work represents a thorough investigation into fibroblasts across organ systems, individual donors, and disease states that reveals shared pathogenic activation states across four chronic inflammatory diseases. FUNDING: Grant from F. Hoffmann-La Roche (Roche) AG.


Assuntos
Artrite Reumatoide , Membrana Sinovial , Animais , Artrite Reumatoide/genética , Fibroblastos/metabolismo , Fenótipo , Células Estromais/metabolismo
13.
J Biol Chem ; 285(38): 29326-35, 2010 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-20624919

RESUMO

Approximately half of all human genes undergo alternative mRNA splicing. This process often yields homologous gene products exhibiting diverse functions. Alternative splicing of APOBEC3G (A3G) and APOBEC3F (A3F), the major host resistance factors targeted by the HIV-1 protein Vif, has not been explored. We investigated the effects of alternative splicing on A3G/A3F gene expression and antiviral activity. Three alternatively spliced A3G mRNAs and two alternatively spliced A3F mRNAs were detected in peripheral blood mononuclear cells in each of 10 uninfected, healthy donors. Expression of these splice variants was altered in different cell subsets and in response to cellular stimulation. Alternatively spliced A3G variants were insensitive to degradation by Vif but displayed no antiviral activity against HIV-1. Conversely, alternative splicing of A3F produced a 37-kDa variant lacking exon 2 (A3FΔ2) that was prominently expressed in macrophages and monocytes and was resistant to Vif-mediated degradation. Alternative splicing also produced a 24-kDa variant of A3F lacking exons 2-4 (A3FΔ2-4) that was highly sensitive to Vif. Both A3FΔ2 and A3FΔ2-4 displayed reduced cytidine deaminase activity and moderate antiviral activity. These alternatively spliced A3F gene products, particularly A3FΔ2, were incorporated into HIV virions, albeit at levels less than wild-type A3F. Thus, alternative splicing of A3F mRNA generates truncated antiviral proteins that differ sharply in their sensitivity to Vif.


Assuntos
Citosina Desaminase/metabolismo , HIV-1/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Western Blotting , Linhagem Celular , Citosina Desaminase/genética , Humanos , Ligação Proteica/genética , Ligação Proteica/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genética
14.
PLoS Pathog ; 5(4): e1000377, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19360131

RESUMO

Elite suppressors (ES) are a rare subset of HIV-1-infected individuals who are able to maintain HIV-1 viral loads below the limit of detection by ultra-sensitive clinical assays in the absence of antiretroviral therapy. Mechanism(s) responsible for this elite control are poorly understood but likely involve both host and viral factors. This study assesses ES plasma-derived envelope glycoprotein (env) fitness as a function of entry efficiency as a possible contributor to viral suppression. Fitness of virus entry was first evaluated using a novel inducible cell line with controlled surface expression levels of CD4 (receptor) and CCR5 (co-receptor). In the context of physiologic CCR5 and CD4 surface densities, ES envs exhibited significantly decreased entry efficiency relative to chronically infected viremic progressors. ES envs also demonstrated slow entry kinetics indicating the presence of virus with reduced entry fitness. Overall, ES env clones were less efficient at mediating entry than chronic progressor envs. Interestingly, acute infection envs exhibited an intermediate phenotypic pattern not distinctly different from ES or chronic progressor envs. These results imply that lower env fitness may be established early and may directly contribute to viral suppression in ES individuals.


Assuntos
Produtos do Gene env/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/patogenicidade , Internalização do Vírus , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Citometria de Fluxo , Humanos , Receptores CCR5/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carga Viral
15.
J Virol ; 83(21): 11016-26, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19692480

RESUMO

The affinity of human immunodeficiency virus (HIV) envelope for CD4 and CCR5 appears to be associated with aspects of R5 virus (virus using the CCR5 coreceptor) pathogenicity. However, entry efficiency results from complex interactions between the viral envelope glycoprotein and both CD4 and CCR5, which limits attempts to correlate viral pathogenicity with surrogate measures of envelope CD4 and CCR5 affinities. Here, we present a system that provides a quantitative and comprehensive characterization of viral entry efficiency as a direct interdependent function of both CD4 and CCR5 levels. This receptor affinity profiling system also revealed heretofore unappreciated complexities underlying CD4/CCR5 usage. We first developed a dually inducible cell line in which CD4 and CCR5 could be simultaneously and independently regulated within a physiologic range of surface expression. Infection by multiple HIV type 1 (HIV-1) and simian immunodeficiency virus isolates could be examined simultaneously for up to 48 different combinations of CD4/CCR5 expression levels, resulting in a distinct usage pattern for each virus. Thus, each virus generated a unique three-dimensional surface plot in which viral infectivity varied as a function of both CD4 and CCR5 expression. From this functional form, we obtained a sensitivity vector along with corresponding metrics that quantified an isolate's overall efficiency of CD4/CCR5 usage. When applied to viral isolates with well-characterized sensitivities to entry/fusion inhibitors, the vector metrics were able to encapsulate their known biological phenotypes. The application of the vector metrics also indicated that envelopes derived from elite suppressors had overall-reduced entry efficiencies compared to those of envelopes derived from chronically infected viremic progressors. Our affinity-profiling system may help to refine studies of R5 virus tropism and pathogenesis.


Assuntos
Antígenos CD4/fisiologia , HIV-1/fisiologia , Receptores CCR5/fisiologia , Vírus da Imunodeficiência Símia/fisiologia , Internalização do Vírus , Marcadores de Afinidade , Animais , Antígenos CD4/genética , Linhagem Celular , Ecdisterona/análogos & derivados , Ecdisterona/metabolismo , Humanos , Conceitos Matemáticos , Minociclina/metabolismo , Receptores CCR5/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
16.
Sci Adv ; 6(21): eaaz6717, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32671214

RESUMO

The functional relevance and mechanistic basis of the effects of the pyroptosis executioner Gasdermin D (GSDMD) on colitis remain unclear. In this study, we observed that GSDMD protein was activated during intestinal inflammation in a model of chemically induced colitis. GSDMD deficiency exacerbated experimental colitis independent of changes in the microbiota and without affecting the production of antimicrobial peptides. GSDMD deficiency in macrophages, but not epithelial cells, was sufficient to drive this exacerbated experimental colitis. We further demonstrate that GSDMD functions in macrophages as a negative regulator to control cyclic GMP-AMP synthase (cGAS)-dependent inflammation, thereby protecting against colitis. Moreover, the administration of cGAS inhibitor can rescue the colitogenic phenotype in GSDMD-deficient mice. Collectively, these findings provide the first demonstration of GSDMD's role in controlling colitis and a detailed delineation of the underlying mechanism.

17.
Cell Rep ; 33(6): 108371, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33176151

RESUMO

Transcription factor EB (TFEB) activates lysosomal biogenesis genes in response to environmental cues. Given implications of impaired TFEB signaling and lysosomal dysfunction in metabolic, neurological, and infectious diseases, we aim to systematically identify TFEB-directed circuits by examining transcriptional responses to TFEB subcellular localization and stimulation. We reveal that steady-state nuclear TFEB is sufficient to activate transcription of lysosomal, autophagy, and innate immunity genes, whereas other targets require higher thresholds of stimulation. Furthermore, we identify shared and distinct transcriptional signatures between mTOR inhibition and bacterial autophagy. Using a genome-wide CRISPR library, we find TFEB targets that protect cells from or sensitize cells to lysosomal cell death. BHLHE40 and BHLHE41, genes responsive to high, sustained levels of nuclear TFEB, act in opposition to TFEB upon lysosomal cell death induction. Further investigation identifies genes counter-regulated by TFEB and BHLHE40/41, adding this negative feedback to the current understanding of TFEB regulatory mechanisms.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Técnicas de Inativação de Genes , Células HeLa , Proteínas de Homeodomínio/genética , Humanos , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Transcrição Gênica
18.
J Virol ; 82(24): 12291-303, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18829756

RESUMO

The molecular mechanisms utilized by human immunodeficiency virus (HIV) to enter latency are poorly understood. Following the infection of Jurkat T cells with lentiviral vectors that express Tat in cis, gene expression is progressively silenced. Silencing is greatly enhanced when the lentiviral vectors carry an attenuated Tat gene with the H13L mutation. Individual clones of lentivirus-infected cells showed a wide range of shutdown rates, with the majority showing a 50% silencing frequency between 30 to 80 days. The silenced clones characteristically contained a small fraction (0 to 15%) of activated cells that continued to express d2EGFP. When d2EGFP(+) and d2EGFP(-) cell populations were isolated from the shutdown clones, they quickly reverted to the original distribution of inactive and active cells, suggesting that the d2EGFP(+) cells arise from stochastic fluctuations in gene expression. The detailed analysis of transcription initiation and elongation using chromatin immunoprecipitation (ChIP) assays confirms that Tat levels are restricted in the latently infected cells but gradually rise during proviral reactivation. ChIP assays using clones of latently infected cells demonstrate that the latent proviruses carry high levels of deacetylated histones and trimethylated histones. In contrast, the cellular genes IkappaB alpha and GAPDH had high levels of acetylated histones and no trimethylated histones. The levels of trimethylated histone H3 and HP1-alpha associated with HIV proviruses fell rapidly after tumor necrosis factor alpha activation. The progressive shutdown of HIV transcription following infection suggests that epigenetic mechanisms targeting chromatin structures selectively restrict HIV transcription initiation. This decreases Tat production below the levels that are required to sustain HIV gene expression.


Assuntos
Cromatina/genética , Inativação Gênica , HIV/genética , HIV/metabolismo , Sequências Repetidas Terminais/genética , Transcrição Gênica/genética , Latência Viral , Linhagem Celular , Proliferação de Células , Regulação Viral da Expressão Gênica , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Vetores Genéticos/genética , Humanos , Cinética , NF-kappa B/metabolismo
19.
Elife ; 82019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30666959

RESUMO

Inflammatory bowel disease (IBD) is driven by dysfunction between host genetics, the microbiota, and immune system. Knowledge gaps remain regarding how IBD genetic risk loci drive gut microbiota changes. The Crohn's disease risk allele ATG16L1 T300A results in abnormal Paneth cells due to decreased selective autophagy, increased cytokine release, and decreased intracellular bacterial clearance. To unravel the effects of ATG16L1 T300A on the microbiota and immune system, we employed a gnotobiotic model using human fecal transfers into ATG16L1 T300A knock-in mice. We observed increases in Bacteroides ovatus and Th1 and Th17 cells in ATG16L1 T300A mice. Association of altered Schaedler flora mice with B. ovatus specifically increased Th17 cells selectively in ATG16L1 T300A knock-in mice. Changes occur before disease onset, suggesting that ATG16L1 T300A contributes to dysbiosis and immune infiltration prior to disease symptoms. Our work provides insight for future studies on IBD subtypes, IBD patient treatment and diagnostics.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Doença de Crohn/genética , Doença de Crohn/microbiologia , Microbioma Gastrointestinal , Células Th1/citologia , Células Th17/citologia , Alelos , Animais , Bacteroides , Disbiose/genética , Disbiose/microbiologia , Transplante de Microbiota Fecal , Fezes/microbiologia , Técnicas de Introdução de Genes , Genótipo , Humanos , Sistema Imunitário , Camundongos , Polimorfismo Genético , Risco , Células Th1/microbiologia , Células Th17/microbiologia
20.
PLoS Pathog ; 2(7): e68, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16839202

RESUMO

HIV-1 latency in resting CD4+ T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART). We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4+ T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS) HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4+ T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4+ T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB) was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4+ T cells. Overexpression of PTB in resting CD4+ T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4+ T cells. Virus culture experiments showed that overexpression of PTB in resting CD4+ T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications.


Assuntos
Linfócitos T CD4-Positivos/química , Núcleo Celular/química , Regulação Viral da Expressão Gênica , HIV-1/genética , RNA Viral/análise , Síndrome da Imunodeficiência Adquirida/genética , Síndrome da Imunodeficiência Adquirida/fisiopatologia , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/virologia , Núcleo Celular/fisiologia , Núcleo Celular/virologia , Produtos do Gene rev/análise , Produtos do Gene rev/genética , Produtos do Gene rev/fisiologia , Produtos do Gene tat/análise , Produtos do Gene tat/genética , Produtos do Gene tat/fisiologia , HIV-1/patogenicidade , HIV-1/fisiologia , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/fisiologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/análise , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/fisiologia , Splicing de RNA , RNA Viral/genética , Latência Viral/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologia , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência Humana
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