RESUMO
Toll-like receptor (TLR)-mediated signaling are critical for host defense against pathogen invasion. However, excessive responses would cause harmful damages to the host. Here we show that deficiency of the E3 ubiquitin ligase TRIM32 increases poly(I:C)- and LPS-induced transcription of downstream genes such as type I interferons (IFNs) and proinflammatory cytokines in both primary mouse immune cells and in mice. Trim32-/- mice produced higher levels of serum inflammatory cytokines and were more sensitive to loss of body weight and inflammatory death upon Salmonella typhimurium infection. TRIM32 interacts with and mediates the degradation of TRIF, a critical adaptor protein for TLR3/4, in an E3 activity-independent manner. TRIM32-mediated as well as poly(I:C)- and LPS-induced degradation of TRIF is inhibited by deficiency of TAX1BP1, a receptor for selective autophagy. Furthermore, TRIM32 links TRIF and TAX1BP1 through distinct domains. These findings suggest that TRIM32 negatively regulates TLR3/4-mediated immune responses by targeting TRIF to TAX1BP1-mediated selective autophagic degradation.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Autofagia , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citocinas/metabolismo , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Proteínas de Neoplasias/genética , Receptor 3 Toll-Like/genéticaRESUMO
Toll-like receptors (TLRs) are pattern recognition receptors that sense a variety of pathogens, initiate innate immune responses, and direct adaptive immunity. All TLRs except TLR3 recruit the adaptor MyD88 to ultimately elicit inflammatory gene expression, whereas TLR3 and internalized TLR4 use TIR-domain-containing adaptor TRIF for the induction of type I interferon and inflammatory cytokines. Here, we identify the WD repeat and FYVE-domain-containing protein WDFY1 as a crucial adaptor protein in the TLR3/4 signaling pathway. Overexpression of WDFY1 potentiates TLR3- and TLR4-mediated activation of NF-κB, interferon regulatory factor 3 (IRF3), and production of type I interferons and inflammatory cytokines. WDFY1 depletion has the opposite effect. WDFY1 interacts with TLR3 and TLR4 and mediates the recruitment of TRIF to these receptors. Our findings suggest a crucial role for WDFY1 in bridging the TLR-TRIF interaction, which is necessary for TLR signaling.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Nucleares/genética , Receptor 3 Toll-Like/genética , Receptor 4 Toll-Like/genética , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Motivos de Aminoácidos , Citocinas/genética , Citocinas/imunologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Indutores de Interferon/farmacologia , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Dados de Sequência Molecular , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/imunologia , Plasmídeos/química , Plasmídeos/imunologia , Poli I-C/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Receptor 3 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/imunologia , TransfecçãoRESUMO
Viral infection triggers induction of type I interferons (IFNs), which are critical mediators of innate antiviral immune response. Mediator of IRF3 activation (MITA, also called STING) is an adapter essential for virus-triggered IFN induction pathways. How post-translational modifications regulate the activity of MITA is not fully elucidated. In expression screens, we identified RING finger protein 26 (RNF26), an E3 ubiquitin ligase, could mediate polyubiquitination of MITA. Interestingly, RNF26 promoted K11-linked polyubiquitination of MITA at lysine 150, a residue also targeted by RNF5 for K48-linked polyubiquitination. Further experiments indicated that RNF26 protected MITA from RNF5-mediated K48-linked polyubiquitination and degradation that was required for quick and efficient type I IFN and proinflammatory cytokine induction after viral infection. On the other hand, RNF26 was required to limit excessive type I IFN response but not proinflammatory cytokine induction by promoting autophagic degradation of IRF3. Consistently, knockdown of RNF26 inhibited the expression of IFNB1 gene in various cells at the early phase and promoted it at the late phase of viral infection, respectively. Furthermore, knockdown of RNF26 inhibited viral replication, indicating that RNF26 antagonizes cellular antiviral response. Our findings thus suggest that RNF26 temporally regulates innate antiviral response by two distinct mechanisms.
Assuntos
Antivirais/metabolismo , Imunidade Inata/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Vírus/imunologia , Western Blotting , Células Cultivadas , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Fator Regulador 3 de Interferon/genética , Interferon Tipo I/genética , Proteínas de Membrana/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Ativação Viral , Viroses/imunologia , Viroses/virologia , Replicação ViralRESUMO
The transcription factor NF-κB plays a pivotal role in a broad range of physiological and pathological processes, including development, inflammation, and immunity. How NF-κB integrates activating signals to expression of specific sets of target genes is of great interest. Here, we identified Krüppel-like factor 6 (KLF6) as a co-activator of NF-κB after TNFα and IL-1ß stimulation. Overexpression of KLF6 enhanced TNFα- and IL-1ß-induced activation of NF-κB and transcription of a subset of downstream genes, whereas knockdown of KLF6 had opposite effects. KLF6 interacted with p65 in the nucleus and bound to the promoters of target genes. Upon IL-1ß stimulation, KLF6 was recruited to promoters of a subset of NF-κB target genes in a p65-dependent manner, which was in turn required for the optimal binding of p65 to the target gene promoters. Our findings thus identified KLF6 as a previously unknown but essential co-activator of NF-κB and provided new insight into the molecular regulation of p65-dependent gene expression.
Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição RelA/metabolismo , Transcrição Gênica , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Núcleo Celular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HEK293 , Humanos , Interleucina-1beta/farmacologia , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , NF-kappa B/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição RelA/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Ag receptor engagement triggers lymphocyte activation and proliferation by activating several transcription factors including NF-κB. Caspase recruitment domain (CARD) containing membrane-associated guanylate kinase (MAGUK) protein 1 (CARMA1) is an essential adaptor protein that links Ag receptors to NF-κB activation. Here, we identify stress-induced-phosphoprotein 1 homology and U-box containing protein 1 (STUB1) as a CARMA1-associated protein. STUB1 constitutively interacted with CARMA1, and the interaction was intensified by TCR stimulation. Downregulation of STUB1 expression by RNAi markedly diminished TCR-induced canonical NF-κB activation and IL-2 production. Furthermore, overexpression of STUB1 enhanced the ubiquitination of CARMA1, whereas knockdown of STUB1 abolished the endogenous ubiquitination of CARMA1 induced by TCR stimulation. Subsequently, the ubiquitination of CARMA1 catalyzed by STUB1 was identified as Lys-27 linked, which is important for CARMA1-mediated NF-κB activation. These data provide the first evidence that ubiquitination of CARMA1 by STUB1 promotes TCR-induced NF-κB signaling.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Guanilato Ciclase/metabolismo , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Catálise , Linhagem Celular , Ativação Enzimática , Humanos , Interleucina-2/biossíntese , NF-kappa B/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
T-cell receptor (TCR)-induced T-cell activation is a critical event in adaptive immune responses. The engagement of TCR complex by antigen along with the activation of the costimulatory receptors trigger a cascade of intracellular signaling, in which caspase recruitment domain-containing membrane-associated guanylate kinase 1 (CARMA1) is a crucial scaffold protein. Upon stimulation, CARMA1 recruits downstream molecules including B-cell CLL/lymphoma 10 (Bcl10), mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1), and TRAF6 to assemble a specific TCR-induced signalosome that triggers NF-κB and JNK activation. In this report, we identified protein kinase Cδ (PKCδ) as a CARMA1-associated protein by a biochemical affinity purification approach. PKCδ interacted with CARMA1 in TCR stimulation-dependent manner in Jurkat T cells. Overexpression of PKCδ inhibited CARMA1-mediated NF-κB activation, whereas knockdown of PKCδ potentiated TCR-triggered NF-κB activation and IL-2 secretion in Jurkat T cells. Reconstitution experiments with PKCδ kinase-dead mutant indicated that the kinase activity of PKCδ was dispensable for its ability to inhibit TCR-triggered NF-κB activation. Furthermore, we found that PKCδ inhibited the interaction between MALT1 and TRAF6, but not the association of CARMA1 with PKCθ, Bcl10, or MALT1. These observations suggest that PKCδ is a negative regulator in T cell activation through inhibiting the assembly of CARMA1 signalosome.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Guanilato Ciclase/metabolismo , Ativação Linfocitária/fisiologia , NF-kappa B/metabolismo , Proteína Quinase C-delta/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/fisiologia , Linfócitos T/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína 10 de Linfoma CCL de Células B , Proteínas Adaptadoras de Sinalização CARD/genética , Caspases/genética , Caspases/metabolismo , Guanilato Ciclase/genética , Células HEK293 , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Células Jurkat , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , NF-kappa B/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína Quinase C-delta/genética , Receptores de Antígenos de Linfócitos T/genética , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
Objective: Coronavirus disease 2019 (COVID-19) and tuberculosis (TB) are major public health and social issues worldwide. The long-term follow-up of COVID-19 with pulmonary TB (PTB) survivors after discharge is unclear. This study aimed to comprehensively describe clinical outcomes, including sequela and recurrence at 3, 12, and 24 months after discharge, among COVID-19 with PTB survivors. Methods: From January 22, 2020 to May 6, 2022, with a follow-up by August 26, 2022, a prospective, multicenter follow-up study was conducted on COVID-19 with PTB survivors after discharge in 13 hospitals from four provinces in China. Clinical outcomes, including sequela, recurrence of COVID-19, and PTB survivors, were collected via telephone and face-to-face interviews at 3, 12, and 24 months after discharge. Results: Thirty-two COVID-19 with PTB survivors were included. The median age was 52 (45, 59) years, and 23 (71.9%) were men. Among them, nearly two-thirds (62.5%) of the survivors were moderate, three (9.4%) were severe, and more than half (59.4%) had at least one comorbidity (PTB excluded). The proportion of COVID-19 survivors with at least one sequela symptom decreased from 40.6% at 3 months to 15.8% at 24 months, with anxiety having a higher proportion over a follow-up. Cough and amnesia recovered at the 12-month follow-up, while anxiety, fatigue, and trouble sleeping remained after 24 months. Additionally, one (3.1%) case presented two recurrences of PTB and no re-positive COVID-19 during the follow-up period. Conclusion: The proportion of long symptoms in COVID-19 with PTB survivors decreased over time, while nearly one in six still experience persistent symptoms with a higher proportion of anxiety. The recurrence of PTB and the psychological support of COVID-19 with PTB after discharge require more attention.
Assuntos
COVID-19 , Tuberculose Pulmonar , Masculino , Humanos , Pessoa de Meia-Idade , Feminino , COVID-19/complicações , Seguimentos , Estudos Prospectivos , Tuberculose Pulmonar/complicações , Tuberculose Pulmonar/epidemiologia , Tuberculose Pulmonar/diagnóstico , SobreviventesRESUMO
Toll-like receptor (TLR) signaling pathways need to be tightly controlled to avoid excessive inflammation and unwanted damage to the host. Myeloid differentiation primary response gene 88 (MyD88) is a critical adaptor of TLR signaling. Here, we identified the speckle-type POZ protein (SPOP) as a MyD88-associated protein. SPOP was recruited to MyD88 following TLR4 activation. TLR4 activation also caused the translocation of SPOP from the nucleus to the cytoplasm. SPOP depletion promoted the aggregation of MyD88 and recruitment of the downstream signaling kinases IRAK4, IRAK1 and IRAK2. Consistently, overexpression of SPOP inhibited the TLR4-mediated activation of NF-κB and production of inflammatory cytokines, whereas SPOP depletion had the opposite effects. Furthermore, knockdown of SPOP increased MyD88 aggregation and inflammatory cytokine production upon TLR2, TLR7 and TLR9 activation. Our findings reveal a mechanism by which MyD88 is regulated and highlight a role for SPOP in limiting inflammatory responses.
Assuntos
Fator 88 de Diferenciação Mieloide , Receptores Toll-Like , Humanos , Inflamação/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras , Receptores Toll-Like/metabolismoRESUMO
OBJECTIVE: Previous studies mainly reported the clinical characteristics of novel coronavirus 2019 (COVID-19) infections, but the research on clinical characteristics and treatment outcomes of COVID-19 patients with stroke is still rare. METHODS: A multi-center retrospective study was conducted at 11 hospitals in 4 provinces of China, and COVID-19 patients with stroke were enrolled from February 24 to May 4, 2020. We analyzed epidemiological, demographic, and clinical characteristics of cases as well as the laboratory test results, treatment regimens and outcomes, and the clinical characteristics and therapeutic outcomes were compared between severe and nonsevere patients, and by age group, respectively. RESULTS: A total of 27 patients [mean age: 66.41 (SD 12.1) years] were enrolled. Among them, 9 (33.3%) were severe patients and 18 (66.7%) were nonsevere patients; 17 (63.0%) were female; 19 (70.4%) were aged 60 years and above. The most common symptoms were fever [19 (70.4%)], fatigue [12 (44.4%)] and cough [11 (40.7%)], respectively. Abnormal laboratory findings of COVID-19 patients with stroke included high levels of C-reactive protein [19 (73.1%)], D-dimer [14 (58.3%)], blood glucose [14 (53.8%)], fibrinogen [13 (50.0%)], and decreased lymphocytes [12 (44.4%)]. Comparing to nonsevere cases with stroke, severe patients with stroke were likely to be older, susceptible to receiving oxygen inhalation, and had more complications (p < 0.05). In addition, there were significant differences in lymphocytes, neutrophils, lactate dehydrogenase, C-reactive protein, creatine kinase between the severe cases and nonsevere cases (p < 0.05). The older patients had a decreased platelet count and elevated fibrinogen, compared with the younger (p < 0.05). All patients (100%) received antiviral treatment, 12 (44.4%) received antibiotics treatment, 26 (96.3%) received Traditional Chinese Medicine (Lung cleansing & detoxifying decoction), and oxygen inhalation was in 18 (66.7%). The median duration of hospitalization was 16 days. By May 4, 2020, a total of 26 (96.3%) patients were cured and discharged, and 1 (3.7%) patients died. CONCLUSION: COVID-19 patients with stroke had poor indicators of coagulation system, and severe and older patients might have a higher risk of complications and unfavorable coagulation system. However, the overall treatment outcome is favorable.
Assuntos
COVID-19/complicações , COVID-19/terapia , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/terapia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Antivirais/uso terapêutico , Transtornos da Coagulação Sanguínea/complicações , Transtornos da Coagulação Sanguínea/terapia , COVID-19/epidemiologia , China/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oxigenoterapia , Estudos Retrospectivos , Acidente Vascular Cerebral/epidemiologia , Resultado do TratamentoRESUMO
OBJECTIVE: Several COVID-19 patients have overlapping comorbidities. The independent role of each component contributing to the risk of COVID-19 is unknown, and how some non-cardiometabolic comorbidities affect the risk of COVID-19 remains unclear. METHODS: A retrospective follow-up design was adopted. A total of 1,160 laboratory-confirmed patients were enrolled from nine provinces in China. Data on comorbidities were obtained from the patients' medical records. Multivariable logistic regression models were used to estimate the odds ratio ( OR) and 95% confidence interval (95% CI) of the associations between comorbidities (cardiometabolic or non-cardiometabolic diseases), clinical severity, and treatment outcomes of COVID-19. RESULTS: Overall, 158 (13.6%) patients were diagnosed with severe illness and 32 (2.7%) had unfavorable outcomes. Hypertension (2.87, 1.30-6.32), type 2 diabetes (T2DM) (3.57, 2.32-5.49), cardiovascular disease (CVD) (3.78, 1.81-7.89), fatty liver disease (7.53, 1.96-28.96), hyperlipidemia (2.15, 1.26-3.67), other lung diseases (6.00, 3.01-11.96), and electrolyte imbalance (10.40, 3.00-26.10) were independently linked to increased odds of being severely ill. T2DM (6.07, 2.89-12.75), CVD (8.47, 6.03-11.89), and electrolyte imbalance (19.44, 11.47-32.96) were also strong predictors of unfavorable outcomes. Women with comorbidities were more likely to have severe disease on admission (5.46, 3.25-9.19), while men with comorbidities were more likely to have unfavorable treatment outcomes (6.58, 1.46-29.64) within two weeks. CONCLUSION: Besides hypertension, diabetes, and CVD, fatty liver disease, hyperlipidemia, other lung diseases, and electrolyte imbalance were independent risk factors for COVID-19 severity and poor treatment outcome. Women with comorbidities were more likely to have severe disease, while men with comorbidities were more likely to have unfavorable treatment outcomes.
Assuntos
COVID-19/complicações , Adulto , Idoso , COVID-19/epidemiologia , COVID-19/terapia , COVID-19/virologia , China/epidemiologia , Comorbidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
Interferon-induced transmembrane protein 3 (IFITM3) is a restriction factor that can be induced by viral infection and interferons (IFNs). It inhibits the entry and replication of many viruses, which are independent of receptor usage but dependent on processes that occur in endosomes. In this study, we demonstrate that IFITM3 plays important roles in regulating the RNA-virus-triggered production of IFN-ß in a negative-feedback manner. Overexpression of IFITM3 inhibited Sendai virus-triggered induction of IFN-ß, whereas knockdown of IFITM3 had the opposite effect. We also showed that IFITM3 was constitutively associated with IRF3 and regulated the homeostasis of IRF3 by mediating the autophagic degradation of IRF3. These findings suggest a novel inhibitory function of IFITM3 on the RNA-virus-triggered production of type I IFNs and cellular antiviral responses.