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1.
Nat Immunol ; 21(2): 158-167, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932809

RESUMO

STING (stimulator of interferon genes) is an important innate immune protein, but its homeostatic regulation at the resting state is unknown. Here, we identified TOLLIP as a stabilizer of STING through direct interaction to prevent its degradation. Tollip deficiency results in reduced STING protein in nonhematopoietic cells and tissues, and renders STING protein unstable in immune cells, leading to severely dampened STING signaling capacity. The competing degradation mechanism of resting-state STING requires IRE1α and lysosomes. TOLLIP mediates clearance of Huntington's disease-linked polyQ protein aggregates. Ectopically expressed polyQ proteins in vitro or endogenous polyQ proteins in Huntington's disease mouse striatum sequester TOLLIP away from STING, leading to reduced STING protein and dampened immune signaling. Tollip-/- also ameliorates STING-mediated autoimmune disease in Trex1-/- mice. Together, our findings reveal that resting-state STING protein level is strictly regulated by a constant tug-of-war between 'stabilizer' TOLLIP and 'degrader' IRE1α-lysosome that together maintain tissue immune homeostasis.


Assuntos
Homeostase/imunologia , Imunidade Inata/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais/imunologia , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Exodesoxirribonucleases/deficiência , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas de Membrana/imunologia , Camundongos Knockout , Fosfoproteínas/deficiência
2.
Immunity ; 56(2): 444-458.e5, 2023 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-36720220

RESUMO

Crohn's disease (CD) is a chronic gastrointestinal disease that is increasing in prevalence worldwide. CD is multifactorial, involving the complex interplay of genetic, immune, and environmental factors, necessitating a system-level understanding of its etiology. To characterize cell-type-specific transcriptional heterogeneity in active CD, we profiled 720,633 cells from the terminal ileum and colon of 71 donors with varying inflammation status. Our integrated datasets revealed organ- and compartment-specific responses to acute and chronic inflammation; most immune changes were in cell composition, whereas transcriptional changes dominated among epithelial and stromal cells. These changes correlated with endoscopic inflammation, but small and large intestines exhibited distinct responses, which were particularly apparent when focusing on IBD risk genes. Finally, we mapped markers of disease-associated myofibroblast activation and identified CHMP1A, TBX3, and RNF168 as regulators of fibrotic complications. Altogether, our results provide a roadmap for understanding cell-type- and organ-specific differences in CD and potential directions for therapeutic development.


Assuntos
Doença de Crohn , Humanos , Transcriptoma , Colo , Íleo , Inflamação/genética , Ubiquitina-Proteína Ligases/genética
3.
Nat Immunol ; 17(5): 495-504, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27019227

RESUMO

Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response.


Assuntos
DNA Polimerase I/metabolismo , DNA/biossíntese , Interferon Tipo I/metabolismo , RNA/biossíntese , Sequência de Bases , Células Cultivadas , Citosol/metabolismo , DNA/genética , DNA Polimerase I/genética , Saúde da Família , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Masculino , Microscopia Confocal , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Linhagem , Transtornos da Pigmentação/genética , Transtornos da Pigmentação/metabolismo , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Res Sq ; 2022 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-35313592

RESUMO

SARS-CoV-2 infection leads to a broad range of outcomes and immune responses, with the development of neutralizing antibodies generally correlated with protection against reinfection. Here, we have characterized both neutralizing activity and T cell responses in a cluster of subjects with mild disease linked to a single spreading event. Surprisingly, we observed sex-specific associations between spike- and particularly nucleoprotein-specific T cell responses and neutralization, with pro-inflammatory cytokines being linked to higher titers only in males. Using single cell immunoprofiling, which provided matched transcriptome and T-cell receptor (TCR) profiles in restimulated CD4 + and CD8 + cells from these subjects, we identified differences in type I IFN signaling that may underlie this difference in antibody generation. Finally, we also identified several TCRs associated with cytokine producing T cells. Altogether, our work maps the breadth of immunological outcomes of SARS-CoV2 infections and highlight the potential role of sex-specific feedback loops during the generation of neutralizing antibodies.

6.
Methods Mol Biol ; 1656: 167-174, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28808969

RESUMO

The signaling adapter protein STING is crucial for the host immune response to cytosolic DNA and cyclic dinucleotides. Under basal conditions, STING resides on the endoplasmic reticulum (ER ) , but upon activation, it traffics through secretory pathway to cytoplasmic vesicles, where STING activates downstream immune signaling. Classical STING activation and trafficking are triggered by binding of cyclic dinucleotide ligands. STING signaling can also be activated by gain-of-function mutations that lead to constitutive trafficking of STING. These gain-of-function mutations are associated with several human diseases such as STING-associated vasculopathy with onset in infancy (SAVI), systemic lupus erythematosus (SLE), or familial chilblain lupus (FCL). This dynamic activation pathway presents a challenge to study. We describe methods here for measuring ligand-dependent and ligand-independent activation of STING signaling in HEK293T cells. We also describe a retroviral-based reconstitution assay to study STING protein trafficking and activation in immune competent cells such as mouse embryonic fibroblasts (MEF), which avoids the use of plasmid DNA. These methods will expedite research regarding STING trafficking and signaling dynamics in the settings of infection and autoimmune diseases.


Assuntos
Bioensaio/métodos , Proteínas de Membrana/imunologia , Transdução de Sinais/imunologia , Animais , Pérnio/genética , Pérnio/imunologia , Células HEK293 , Humanos , Lúpus Eritematoso Cutâneo/genética , Lúpus Eritematoso Cutâneo/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Proteínas de Membrana/genética , Camundongos , Mutação , Transporte Proteico/genética , Transporte Proteico/imunologia , Transdução de Sinais/genética
7.
Cell Rep ; 21(11): 3234-3242, 2017 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-29241549

RESUMO

STING is an endoplasmic reticulum (ER)-associated transmembrane protein that turns on and quickly turns off downstream signaling as it translocates from the ER to vesicles. How STING signaling is attenuated during trafficking remains poorly understood. Here, we show that trafficking-mediated STING degradation requires ER exit and function of vacuolar ATPase complex. Late-stage STING vesicles are sorted to Rab7-positive endolysosomes for degradation. Based on analysis of existing structures, we also identified the helix amino acid 281 (aa281)-297 as a motif required for trafficking-mediated STING degradation. Immuno-electron microscopy (EM) reveals the size and clustering of STING vesicles and topology of STING on the vesicle. Importantly, blockade of trafficking-mediated STING degradation using bafilomycin A1 specifically enhanced cyclic guanosine monophosphate (GMP)-AMP (cGAMP)-mediated immune response and anti-tumor effect in mice. Together, our findings provide biochemical and imaging evidence for STING degradation by the lysosome and pinpoint trafficking-mediated STING degradation as a previously unanticipated therapeutic target for enhancing STING signaling in cancer therapy.


Assuntos
Lisossomos/efeitos dos fármacos , Macrolídeos/farmacologia , Melanoma Experimental/tratamento farmacológico , Proteínas de Membrana/genética , Proteólise/efeitos dos fármacos , Animais , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Expressão Gênica , Humanos , Lipopolissacarídeos/farmacologia , Lisossomos/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Nucleotídeos Cíclicos/farmacologia , Poli I-C/farmacologia , Estrutura Secundária de Proteína , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/imunologia , proteínas de unión al GTP Rab7
8.
J Exp Med ; 213(3): 329-36, 2016 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-26880576

RESUMO

The neuroinflammatory autoimmune disease Aicardi-Goutières syndrome (AGS) develops from mutations in genes encoding several nucleotide-processing proteins, including RNase H2. Defective RNase H2 may induce accumulation of self-nucleic acid species that trigger chronic type I interferon and inflammatory responses, leading to AGS pathology. We created a knock-in mouse model with an RNase H2 AGS mutation in a highly conserved residue of the catalytic subunit, Rnaseh2a(G37S/G37S) (G37S), to understand disease pathology. G37S homozygotes are perinatal lethal, in contrast to the early embryonic lethality previously reported for Rnaseh2b- or Rnaseh2c-null mice. Importantly, we found that the G37S mutation led to increased expression of interferon-stimulated genes dependent on the cGAS-STING signaling pathway. Ablation of STING in the G37S mice results in partial rescue of the perinatal lethality, with viable mice exhibiting white spotting on their ventral surface. We believe that the G37S knock-in mouse provides an excellent animal model for studying RNASEH2-associated autoimmune diseases.


Assuntos
Doenças Autoimunes do Sistema Nervoso/imunologia , Imunidade Inata , Proteínas de Membrana/metabolismo , Mutação/genética , Malformações do Sistema Nervoso/imunologia , Nucleotidiltransferases/metabolismo , Ribonuclease H/química , Ribonuclease H/metabolismo , Animais , Doenças Autoimunes do Sistema Nervoso/genética , Domínio Catalítico , Células Cultivadas , Cruzamentos Genéticos , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Homozigoto , Humanos , Interferons/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , Masculino , Camundongos , Malformações do Sistema Nervoso/genética , Fenótipo , Transdução de Sinais
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