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1.
Sci Rep ; 11(1): 15715, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344959

RESUMO

Key elements for viral pathogenesis include viral strains, viral load, co-infection, and host responses. Several studies analyzing these factors in the function of disease severity of have been published; however, no studies have shown how all of these factors interplay within a defined cohort. To address this important question, we sought to understand how these four key components interplay in a cohort of COVID-19 patients. We determined the viral loads and gene expression using high throughput sequencing and various virological methods. We found that viral loads in the upper respiratory tract in COVID-19 patients at an early phase of infection vary widely. While the majority of nasopharyngeal (NP) samples have a viral load lower than the limit of detection of infectious viruses, there are samples with an extraordinary amount of SARS-CoV-2 RNA and a high viral titer. No specific viral factors were identified that are associated with high viral loads. Host gene expression analysis showed that viral loads were strongly correlated with cellular antiviral responses. Interestingly, however, COVID-19 patients who experience mild symptoms have a higher viral load than those with severe complications, indicating that naso-pharyngeal viral load may not be a key factor of the clinical outcomes of COVID-19. The metagenomics analysis revealed that the microflora in the upper respiratory tract of COVID-19 patients with high viral loads were dominated by SARS-CoV-2, with a high degree of dysbiosis. Finally, we found a strong inverse correlation between upregulation of interferon responses and disease severity. Overall our study suggests that a high viral load in the upper respiratory tract may not be a critical factor for severe symptoms; rather, dampened antiviral responses may be a critical factor for a severe outcome from the infection.


Assuntos
COVID-19/patologia , Interferons/metabolismo , SARS-CoV-2/genética , Adulto , Idoso , COVID-19/virologia , Disbiose/etiologia , Feminino , Humanos , Masculino , Metagenômica , Microbiota/genética , Pessoa de Meia-Idade , Nasofaringe/virologia , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real , Sistema Respiratório/microbiologia , Sistema Respiratório/virologia , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença , Transcriptoma , Regulação para Cima , Carga Viral
2.
Int J Mol Sci ; 22(14)2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34299101

RESUMO

Children with the new coronavirus disease 2019 (COVID-19) have milder symptoms and a better prognosis than adult patients. Several investigations assessed type I, II, and III interferon (IFN) signatures in SARS-CoV-2 infected adults, however no data are available for pediatric patients. TRIM28 and SETDB1 regulate the transcription of multiple genes involved in the immune response as well as of human endogenous retroviruses (HERVs). Exogenous viral infections can trigger the activation of HERVs, which in turn can induce inflammatory and immune reactions. Despite the potential cross-talks between SARS-CoV-2 infection and TRIM28, SETDB1, and HERVs, information on their expressions in COVID-19 patients is lacking. We assessed, through a PCR real time Taqman amplification assay, the transcription levels of six IFN-I stimulated genes, IFN-II and three of its sensitive genes, three IFN-lIIs, as well as of TRIM28, SETDB1, pol genes of HERV-H, -K, and -W families, and of env genes of Syncytin (SYN)1, SYN2, and multiple sclerosis-associated retrovirus (MRSV) in peripheral blood from COVID-19 children and in control uninfected subjects. Higher expression levels of IFN-I and IFN-II inducible genes were observed in 36 COVID-19 children with mild or moderate disease as compared to uninfected controls, whereas their concentrations decreased in 17 children with severe disease and in 11 with multisystem inflammatory syndrome (MIS-C). Similar findings were found for the expression of TRIM-28, SETDB1, and every HERV gene. Positive correlations emerged between the transcriptional levels of type I and II IFNs, TRIM28, SETDB1, and HERVs in COVID-19 patients. IFN-III expressions were comparable in each group of subjects. This preserved induction of IFN-λs could contribute to the better control of the infection in children as compared to adults, in whom IFN-III deficiency has been reported. The upregulation of IFN-I, IFN-II, TRIM28, SETDB1, and HERVs in children with mild symptoms, their declines in severe cases or with MIS-C, and the positive correlations of their transcription in SARS-CoV-2-infected children suggest that they may play important roles in conditioning the evolution of the infection.


Assuntos
COVID-19/epidemiologia , COVID-19/metabolismo , Retrovirus Endógenos/metabolismo , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença , COVID-19/patologia , COVID-19/virologia , Estudos de Casos e Controles , Criança , Retrovirus Endógenos/genética , Feminino , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Interferons/genética , Interferons/metabolismo , Itália/epidemiologia , Masculino , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo
3.
Nat Commun ; 12(1): 4303, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262037

RESUMO

Lipid droplets (LDs) are increasingly recognized as critical organelles in signalling events, transient protein sequestration and inter-organelle interactions. However, the role LDs play in antiviral innate immune pathways remains unknown. Here we demonstrate that induction of LDs occurs as early as 2 h post-viral infection, is transient and returns to basal levels by 72 h. This phenomenon occurs following viral infections, both in vitro and in vivo. Virally driven in vitro LD induction is type-I interferon (IFN) independent, and dependent on Epidermal Growth Factor Receptor (EGFR) engagement, offering an alternate mechanism of LD induction in comparison to our traditional understanding of their biogenesis. Additionally, LD induction corresponds with enhanced cellular type-I and -III IFN production in infected cells, with enhanced LD accumulation decreasing viral replication of both Herpes Simplex virus 1 (HSV-1) and Zika virus (ZIKV). Here, we demonstrate, that LDs play vital roles in facilitating the magnitude of the early antiviral immune response specifically through the enhanced modulation of IFN following viral infection, and control of viral replication. By identifying LDs as a critical signalling organelle, this data represents a paradigm shift in our understanding of the molecular mechanisms which coordinate an effective antiviral response.


Assuntos
Interferons/imunologia , Gotículas Lipídicas/imunologia , Viroses/imunologia , Animais , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Herpesvirus Humano 1/fisiologia , Humanos , Imunidade Inata , Interferons/genética , Interferons/metabolismo , Gotículas Lipídicas/metabolismo , Camundongos , Ácidos Nucleicos/metabolismo , Replicação Viral/efeitos dos fármacos , Zika virus/fisiologia
4.
Int J Mol Sci ; 22(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205295

RESUMO

Mesenchymal stem cells (MSCs) maintain the musculoskeletal system by differentiating into multiple lineages, including osteoblasts and adipocytes. Mechanical signals, including strain and low-intensity vibration (LIV), are important regulators of MSC differentiation via control exerted through the cell structure. Lamin A/C is a protein vital to the nuclear architecture that supports chromatin organization and differentiation and contributes to the mechanical integrity of the nucleus. We investigated whether lamin A/C and mechanoresponsiveness are functionally coupled during adipogenesis in MSCs. siRNA depletion of lamin A/C increased the nuclear area, height, and volume and decreased the circularity and stiffness. Lamin A/C depletion significantly decreased markers of adipogenesis (adiponectin, cellular lipid content) as did LIV treatment despite depletion of lamin A/C. Phosphorylation of focal adhesions in response to mechanical challenge was also preserved during loss of lamin A/C. RNA-seq showed no major adipogenic transcriptome changes resulting from LIV treatment, suggesting that LIV regulation of adipogenesis may not occur at the transcriptional level. We observed that during both lamin A/C depletion and LIV, interferon signaling was downregulated, suggesting potentially shared regulatory mechanism elements that could regulate protein translation. We conclude that the mechanoregulation of adipogenesis and the mechanical activation of focal adhesions function independently from those of lamin A/C.


Assuntos
Adipogenia , Adesões Focais/fisiologia , Lamina Tipo A/fisiologia , Células-Tronco Mesenquimais/fisiologia , Animais , Módulo de Elasticidade , Interferons/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Transdução de Sinais , Proteínas de Ligação a Telômeros/metabolismo , Vibração
5.
Sci Rep ; 11(1): 14536, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267262

RESUMO

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) hospitalizations and deaths disportionally affect males and older ages. Here we investigated the impact of male sex and age comparing sex-matched or age-matched ferrets infected with SARS-CoV-2. Differences in temperature regulation was identified for male ferrets which was accompanied by prolonged viral replication in the upper respiratory tract after infection. Gene expression analysis of the nasal turbinates indicated that 1-year-old female ferrets had significant increases in interferon response genes post infection which were delayed in males. These results provide insight into COVID-19 and suggests that older males may play a role in viral transmission due to decreased antiviral responses.


Assuntos
COVID-19/virologia , Furões/virologia , Interferons/metabolismo , Fatores Etários , Animais , Anticorpos Antivirais , COVID-19/metabolismo , Modelos Animais de Doenças , Feminino , Furões/metabolismo , Interações entre Hospedeiro e Microrganismos , Interferons/genética , Masculino , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia , Fatores Sexuais , Carga Viral , Replicação Viral
6.
Viruses ; 13(6)2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207750

RESUMO

An inefficient immune response against the hepatitis C virus (HCV), combined with viral evasion mechanisms, is responsible for the chronicity of infection. The need to evaluate the innate mechanisms of the immune response, such as TLR3 and IFN-λ3, and their relationship with the virus-host interaction is important for understanding the pathogenesis of chronic hepatitis C. The present study aimed to investigate the gene expressions of TRL3 and IFNL3 in liver tissue, seeking to evaluate whether these could be potential biomarkers of HCV infection. A total of 23 liver biopsy samples were collected from patients with chronic HCV, and 8 biopsies were collected from healthy control patients. RNA extraction, reverse transcription and qPCR were performed to quantify the relative gene expressions of TLR3 and IFNL3. Data on the viral load; AST, ALT, GGT and AFP levels; and the viral genotype were collected from the patients' medical records. The intrahepatic expression of TLR3 (p = 0.0326) was higher in chronic HCV carriers than in the control group, and the expression of IFNL3 (p = 0.0037) was lower in chronic HCV carriers than in the healthy control group. The expression levels of TLR3 (p = 0.0030) and IFNL3 (p = 0.0036) were higher in the early stages of fibrosis and of necroinflammatory activity in the liver; in contrast, TLR3 and IFNL3 expressions were lower in the more advanced stages of fibrosis and inflammation. There was no correlation between the gene expression and the serum viral load. Regarding the initial METAVIR scale scores, liver transaminase levels were lower in patients with advanced fibrosis when correlated with TLR3 and IFNL3 gene expressions. The results suggest that in the early stages of the development of hepatic fibrosis, TLR3 and IFN-λ3 play important roles in the antiviral response and in the modulation of the tolerogenic liver environment because there is a decrease in the intrahepatic expressions of TLR3 and IFNL3 in the advanced stages of fibrosis, probably due to viral evasion mechanisms.


Assuntos
Hepacivirus , Hepatite C Crônica/complicações , Hepatite C Crônica/genética , Interferons/genética , Cirrose Hepática/diagnóstico , Cirrose Hepática/etiologia , Receptor 3 Toll-Like/genética , Biomarcadores , Biópsia , Estudos Transversais , Suscetibilidade a Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Genoma Viral , Genótipo , Hepacivirus/genética , Hepatite C Crônica/virologia , Humanos , Interferons/metabolismo , Masculino , Índice de Gravidade de Doença , Receptor 3 Toll-Like/metabolismo , Carga Viral
7.
PLoS Pathog ; 17(7): e1009753, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34260666

RESUMO

To understand the diversity of immune responses to SARS-CoV-2 and distinguish features that predispose individuals to severe COVID-19, we developed a mechanistic, within-host mathematical model and virtual patient cohort. Our results suggest that virtual patients with low production rates of infected cell derived IFN subsequently experienced highly inflammatory disease phenotypes, compared to those with early and robust IFN responses. In these in silico patients, the maximum concentration of IL-6 was also a major predictor of CD8+ T cell depletion. Our analyses predicted that individuals with severe COVID-19 also have accelerated monocyte-to-macrophage differentiation mediated by increased IL-6 and reduced type I IFN signalling. Together, these findings suggest biomarkers driving the development of severe COVID-19 and support early interventions aimed at reducing inflammation.


Assuntos
COVID-19/imunologia , Modelos Imunológicos , SARS-CoV-2 , Biomarcadores/metabolismo , Linfócitos T CD8-Positivos/imunologia , COVID-19/virologia , Estudos de Coortes , Biologia Computacional , Simulação por Computador , Suscetibilidade a Doenças/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Imunidade Inata , Imunossupressão , Interferons/metabolismo , Interleucina-6/metabolismo , Macrófagos/imunologia , Pandemias , SARS-CoV-2/imunologia , Índice de Gravidade de Doença , Interface Usuário-Computador
8.
Nat Commun ; 12(1): 4558, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315904

RESUMO

Patients with advanced stage cancers frequently suffer from severe pain as a result of bone metastasis and bone destruction, for which there is no efficacious treatment. Here, using multiple mouse models of bone cancer, we report that agonists of the immune regulator STING (stimulator of interferon genes) confer remarkable protection against cancer pain, bone destruction, and local tumor burden. Repeated systemic administration of STING agonists robustly attenuates bone cancer-induced pain and improves locomotor function. Interestingly, STING agonists produce acute pain relief through direct neuronal modulation. Additionally, STING agonists protect against local bone destruction and reduce local tumor burden through modulation of osteoclast and immune cell function in the tumor microenvironment, providing long-term cancer pain relief. Finally, these in vivo effects are dependent on host-intrinsic STING and IFN-I signaling. Overall, STING activation provides unique advantages in controlling bone cancer pain through distinct and synergistic actions on nociceptors, immune cells, and osteoclasts.


Assuntos
Neoplasias Ósseas/complicações , Dor do Câncer/etiologia , Dor do Câncer/imunologia , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Analgésicos/farmacologia , Animais , Neoplasias Ósseas/sangue , Dor do Câncer/sangue , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Fêmur/diagnóstico por imagem , Fêmur/efeitos dos fármacos , Fêmur/patologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Proteínas de Homeodomínio/metabolismo , Hiperalgesia/complicações , Interferons/sangue , Interferons/metabolismo , Masculino , Neoplasias Mamárias Animais/complicações , Proteínas de Membrana/agonistas , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Neurônios/efeitos dos fármacos , Nociceptividade/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Osteogênese/efeitos dos fármacos , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Xantonas/farmacologia
9.
Biomed Pharmacother ; 141: 111794, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34153851

RESUMO

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) is the causative agent of Corona Virus Disease 2019 (COVID-19). Lower production of type I and III interferons and higher levels of inflammatory mediators upon SARS-CoV2 infection contribute to COVID-19 pathogenesis. Optimal interferon production and controlled inflammation are essential to limit COVID-19 pathogenesis. However, the aggravated inflammatory response observed in COVID-19 patients causes severe damage to the host and frequently advances to acute respiratory distress syndrome (ARDS). Toll-like receptor 7 and 8 (TLR7/8) signaling pathways play a central role in regulating induction of interferons (IFNs) and inflammatory mediators in dendritic cells. Controlled inflammation is possible through regulation of TLR mediated response without influencing interferon production to reduce COVID-19 pathogenesis. This review focuses on inflammatory mediators that contribute to pathogenic effects and the role of TLR pathways in the induction of interferon and inflammatory mediators and their contribution to COVID-19 pathogenesis. We conclude that potential TLR7/8 agonists inducing antiviral interferon response and controlling inflammation are important therapeutic options to effectively eliminate SARS-CoV2 induced pathogenesis. Ongoing and future studies may provide additional evidence on their safety and efficacy to treat COVID-19 pathogenesis.


Assuntos
COVID-19/metabolismo , Mediadores da Inflamação/metabolismo , Interferons/metabolismo , Transdução de Sinais/fisiologia , Receptor 7 Toll-Like/fisiologia , Receptor 8 Toll-Like/fisiologia , Anti-Inflamatórios/administração & dosagem , COVID-19/imunologia , COVID-19/terapia , Humanos , Mediadores da Inflamação/imunologia , Interferons/imunologia , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas
10.
J Exp Med ; 218(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34128960

RESUMO

Initial replication of SARS-CoV-2 in the upper respiratory tract is required to establish infection, and the replication level correlates with the likelihood of viral transmission. Here, we examined the role of host innate immune defenses in restricting early SARS-CoV-2 infection using transcriptomics and biomarker-based tracking in serial patient nasopharyngeal samples and experiments with airway epithelial organoids. SARS-CoV-2 initially replicated exponentially, with a doubling time of ∼6 h, and induced interferon-stimulated genes (ISGs) in the upper respiratory tract, which rose with viral replication and peaked just as viral load began to decline. Rhinovirus infection before SARS-CoV-2 exposure accelerated ISG responses and prevented SARS-CoV-2 replication. Conversely, blocking ISG induction during SARS-CoV-2 infection enhanced viral replication from a low infectious dose. These results show that the activity of ISG-mediated defenses at the time of SARS-CoV-2 exposure impacts infection progression and that the heterologous antiviral response induced by a different virus can protect against SARS-CoV-2.


Assuntos
COVID-19/imunologia , COVID-19/virologia , Imunidade Inata/fisiologia , Nasofaringe/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Enzima de Conversão de Angiotensina 2/genética , Estudos de Casos e Controles , Quimiocina CXCL10/metabolismo , Suscetibilidade a Doenças/imunologia , Feminino , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Interferons/genética , Interferons/imunologia , Interferons/metabolismo , Masculino , Pessoa de Meia-Idade , Infecções por Picornaviridae/imunologia , Infecções por Picornaviridae/virologia , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Carga Viral , Replicação Viral
11.
Viruses ; 13(5)2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064444

RESUMO

Bats are reservoirs of many pathogenic viruses, including the lyssaviruses rabies virus (RABV) and Australian bat lyssavirus (ABLV). Lyssavirus strains are closely associated with particular host reservoir species, with evidence of specific adaptation. Associated phenotypic changes remain poorly understood but are likely to involve phosphoprotein (P protein), a key mediator of the intracellular virus-host interface. Here, we examine the phenotype of P protein of ABLV, which circulates as two defined lineages associated with frugivorous and insectivorous bats, providing the opportunity to compare proteins of viruses adapted to divergent bat species. We report that key functions of P protein in the antagonism of interferon/signal transducers and activators of transcription 1 (STAT1) signaling and the capacity of P protein to undergo nuclear trafficking differ between lineages. Molecular mapping indicates that these differences are functionally distinct and appear to involve modulatory effects on regulatory regions or structural impact rather than changes to defined interaction sequences. This results in partial but significant phenotypic divergence, consistent with "fine-tuning" to host biology, and with potentially distinct properties in the virus-host interface between bat families that represent key zoonotic reservoirs.


Assuntos
Biodiversidade , Quirópteros/virologia , Lyssavirus/fisiologia , Fenótipo , Sequência de Aminoácidos , Animais , Reservatórios de Doenças , Interações Hospedeiro-Patógeno , Interferons/metabolismo , Lyssavirus/classificação , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
13.
J Phys Chem Lett ; 12(23): 5608-5615, 2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34110168

RESUMO

Papain-like protease (PLpro) from SARS-CoV-2 plays essential roles in the replication cycle of the virus. In particular, it preferentially interacts with and cleaves human interferon-stimulated gene 15 (hISG15) to suppress the innate immune response of the host. We used small-angle X-ray and neutron scattering combined with computational techniques to study the mechanism of interaction of SARS-CoV-2 PLpro with hISG15. We showed that hISG15 undergoes a transition from an extended to a compact state after binding to PLpro, a conformation that has not been previously observed in complexes of SARS-CoV-2 PLpro with ISG15 from other species. Furthermore, computational analysis showed significant conformational flexibility in the ISG15 N-terminal domain, suggesting that it is weakly bound to PLpro and supports a binding mechanism that is dominated by the C-terminal ISG15 domain. This study fundamentally improves our understanding of the SARS-CoV-2 deISGylation complex that will help guide development of COVID-19 therapeutics targeting this complex.


Assuntos
Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Citocinas/química , Citocinas/metabolismo , Interferons/metabolismo , SARS-CoV-2/metabolismo , Ubiquitinas/química , Ubiquitinas/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/genética , Citocinas/genética , Humanos , Difração de Nêutrons , Conformação Proteica , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , Espalhamento a Baixo Ângulo , Ubiquitinas/genética , Difração de Raios X
14.
Nat Commun ; 12(1): 2624, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976143

RESUMO

The etiology of ulcerative colitis is poorly understood and is likely to involve perturbation of the complex interactions between the mucosal immune system and the commensal bacteria of the gut, with cytokines acting as important cross-regulators. Here we use IFN receptor-deficient mice in a dextran sulfate sodium (DSS) model of acute intestinal injury to study the contributions of type I and III interferons (IFN) to the initiation, progression and resolution of acute colitis. We find that mice lacking both types of IFN receptors exhibit enhanced barrier destruction, extensive loss of goblet cells and diminished proliferation of epithelial cells in the colon following DSS-induced damage. Impaired mucosal healing in double IFN receptor-deficient mice is driven by decreased amphiregulin expression, which IFN signaling can up-regulate in either the epithelial or hematopoietic compartment. Together, these data underscore the pleiotropic functions of IFNs and demonstrate that these critical antiviral cytokines also support epithelial regeneration following acute colonic injury.


Assuntos
Colite Ulcerativa/imunologia , Interferons/metabolismo , Mucosa Intestinal/patologia , Reepitelização/imunologia , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/patologia , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Células Epiteliais , Feminino , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Masculino , Camundongos , Camundongos Knockout , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Organismos Livres de Patógenos Específicos
15.
J Biol Chem ; 297(1): 100811, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34023381

RESUMO

A20 is a potent anti-inflammatory protein that mediates both inflammation and ubiquitination in mammals, but the related mechanisms are not clear. In this study, we performed mass spectrometry (MS) screening, gene ontology (GO) analysis, and coimmunoprecipitation (co-IP) in a lipopolysaccharide (LPS)-induced inflammatory cell model to identify novel A20-interacting proteins. We confirmed that the E3 ubiquitin ligase Nrdp1, also known as ring finger protein 41 (RNF41), interacted with A20 in LPS-stimulated cells. Further co-IP analysis demonstrated that when A20 was knocked out, degradation-inducing K48-linked ubiquitination of inflammatory effector MyD88 was decreased, but protein interaction-mediating K63-linked ubiquitination of another inflammatory effector TBK1 was increased. Moreover, western blot experiments showed that A20 inhibition induced an increase in levels of MyD88 and phosphorylation of downstream effector proteins as well as of TBK1 and a downstream effector, while Nrdp1 inhibition induced an increase in MyD88 but a decrease in TBK1 levels. When A20 and Nrdp1 were coinhibited, no further change in MyD88 was observed, but TBK1 levels were significantly decreased compared with those upon A20 inhibition alone. Gain- and loss-of-function analyses revealed that the ZnF4 domain of A20 is required for Nrdp1 polyubiquitination. Upon LPS stimulation, the inhibition of Nrdp1 alone increased the secretion of IL-6 and TNF-α but decreased IFN-ß secretion, as observed in other studies, suggesting that Nrdp1 preferentially promotes the production of IFN-ß. Taken together, these results demonstrated that A20/Nrdp1 interaction is important for A20 anti-inflammation, thus revealing a novel mechanism for the anti-inflammatory effects of A20.


Assuntos
Inflamação/metabolismo , Lisina/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Poliubiquitina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Ativação Enzimática , Inflamação/patologia , Interferons/metabolismo , Macrófagos/metabolismo , Camundongos , Modelos Biológicos , Ligação Proteica , Domínios Proteicos , Proteólise , Células RAW 264.7 , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/química
16.
Mol Cell Biochem ; 476(10): 3671-3679, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34050900

RESUMO

The trend of consuming food high in calories, fat, and sugar with little nutritional value and reduced physical exercise has resulted in an alarming ratio of overweight and obese subjects worldwide. Low-grade chronic inflammation is the key feature of obesity that causes an increase in pro-inflammatory cytokines and decrease in anti-inflammatory cytokines in circulation. The current study was aimed to investigate the effect of high-fat diet on the architecture of spleen, liver, and skeletal muscle and changes in the expression of hepatic cytokines. Two groups of experimental rats were established, against control that were given different percentage of fats in their diet. After a period of sixteen weeks, rats were dissected and their organs were excised out and processed accordingly. Spleen sections of experimental groups, revealed increased recruitment of lymphocytes, sinusoidal dilatations, necrotic lymphocytes, increased ratio of white-to-red pulp, and hemosiderin and iron deposits in red pulp indicating immune system activation. Hepatic sections showed enlarged sinusoidal spaces, disruptive hepatocytes, necrosis and dilation of portal veins. Sections of skeletal muscle showed degenerating fibers, increased fat accumulation, and recruitment of macrophages. Elevated expression of IFN-γ and decreased expression of IFN-α and IFN-ß cytokines verified the adverse effect of high-fat diet on immune system as well. Fats tend to accumulate in organs due to increased intake of fat-rich diet disturbing their normal function and histology. In addition, gene expression analysis of cytokines confirmed the effect of high-fat diet as an inflammatory agent.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Interferons/metabolismo , Fígado/metabolismo , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Baço/metabolismo , Animais , Fígado/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Músculo Esquelético/patologia , Obesidade/induzido quimicamente , Obesidade/patologia , Ratos , Ratos Wistar , Baço/patologia
17.
J Leukoc Biol ; 110(3): 409-424, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33988272

RESUMO

Deficiency of adenosine deaminase 2 (DADA2) is a rare autosomal recessive disease caused by loss-of-function variants in the ADA2 gene. DADA2 typically presents in childhood and is characterized by vasculopathy, stroke, inflammation, immunodeficiency, as well as hematologic manifestations. ADA2 protein is predominantly present in stimulated monocytes, dendritic cells, and macrophages. To elucidate molecular mechanisms in DADA2, CD14+ monocytes from 14 patients and 6 healthy donors were analyzed using single-cell RNA sequencing (scRNA-seq). Monocytes were purified by positive selection based on CD14 expression. Subpopulations were imputed from their transcriptomes. Based on scRNA-seq, monocytes could be classified as classical, intermediate, and nonclassical. Further, we used gene pathway analytics to interpret patterns of up- and down-regulated gene transcription. In DADA2, the frequency of nonclassical monocytes was higher compared with that of healthy donors, and M1 macrophage markers were up-regulated in patients. By comparing gene expression of each monocyte subtype between patients and healthy donors, we identified upregulated immune response pathways, including IFNα/ß and IFNγ signaling, in all monocyte subtypes. Distinctively, the TNFR2 noncanonical NF-κB pathway was up-regulated only in nonclassical monocytes. Patients' plasma showed increased IFNγ and TNFα levels. Our results suggest that elevated IFNγ activates cell signaling, leading to differentiation into M1 macrophages from monocytes and release of TNFα. Immune responses and more general response to stimuli pathways were up-regulated in DADA2 monocytes, and protein synthesis pathways were down-regulated, perhaps as stress responses. Our identification of novel aberrant immune pathways has implications for therapeutic approaches in DADA2 (registered at clinicaltrials.gov NCT00071045).


Assuntos
Agamaglobulinemia/genética , Agamaglobulinemia/patologia , Monócitos/patologia , Análise de Sequência de RNA , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/patologia , Análise de Célula Única , Adenosina Desaminase/genética , Adolescente , Adulto , Agamaglobulinemia/sangue , Agamaglobulinemia/enzimologia , Criança , Pré-Escolar , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Interferons/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação/genética , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Imunodeficiência Combinada Severa/sangue , Imunodeficiência Combinada Severa/enzimologia , Transdução de Sinais , Doadores de Tecidos , Adulto Jovem
18.
Cell Host Microbe ; 29(7): 1052-1062, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34022154

RESUMO

COVID-19 can result in severe disease characterized by significant immunopathology that is spurred by an exuberant, yet dysregulated, innate immune response with a poor adaptive response. A limited and delayed interferon I (IFN-I) and IFN-III response results in exacerbated proinflammatory cytokine production and in extensive cellular infiltrates in the respiratory tract, resulting in lung pathology. The development of effective therapeutics for patients with severe COVID-19 depends on our understanding of the pathological elements of this unbalanced innate immune response. Here, we review the mechanisms by which SARS-CoV-2 both activates and antagonizes the IFN and inflammatory response following infection, how a dysregulated cytokine and cellular response contributes to immune-mediated pathology in COVID-19, and therapeutic strategies that target elements of the innate response.


Assuntos
COVID-19/imunologia , Imunidade Inata/imunologia , Interferons/uso terapêutico , SARS-CoV-2/imunologia , Animais , Anti-Inflamatórios/uso terapêutico , COVID-19/tratamento farmacológico , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Evasão da Resposta Imune , Interferon Tipo I/metabolismo , Interferons/metabolismo , Cinética
19.
Biochem J ; 478(10): 1853-1859, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34003254

RESUMO

The current SARS-CoV-2 pandemic has spurred new interest in interferon signaling in response to viral pathogens. Much of what we know about the signaling molecules and associated signal transduction induced during the host cellular response to viral pathogens has been gained from research conducted from the 1990's to the present day, but certain intricacies of the mechanisms involved, still remain unclear. In a recent study by Vaughn et al. the authors examine one of the main mechanisms regulating interferon induction following viral infection, the RIG-I/MAVS/IRF3 pathway, and find that similar to PKR both DICER interacting proteins, PACT and TRBP, regulate RIG-I signaling in an opposing manner. More specifically, the reported findings demonstrate, like others, that PACT stimulates RIG-I-mediated signaling in a manner independent of PACT dsRNA-binding ability or phosphorylation at sites known to be important for PACT-dependent PKR activation. In contrast, they show for the first time that TRBP inhibits RIG-I-mediated signaling. RIG-I inhibition by TRBP did not require phosphorylation of sites shown to be important for inhibiting PKR, nor did it involve PACT or PKR, but it did require the dsRNA-binding ability of TRBP. These findings open the door to a complex co-regulation of RIG-I, PKR, MDA5, miRNA processing, and interferon induction.


Assuntos
COVID-19/imunologia , Interferons/metabolismo , SARS-CoV-2/imunologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , COVID-19/virologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Regulação da Expressão Gênica/imunologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferons/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Coativadores de Receptor Nuclear/genética , Coativadores de Receptor Nuclear/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo
20.
PLoS Pathog ; 17(5): e1009229, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34029358

RESUMO

While MERS-CoV (Middle East respiratory syndrome Coronavirus) provokes a lethal disease in humans, camelids, the main virus reservoir, are asymptomatic carriers, suggesting a crucial role for innate immune responses in controlling the infection. Experimentally infected camelids clear infectious virus within one week and mount an effective adaptive immune response. Here, transcription of immune response genes was monitored in the respiratory tract of MERS-CoV infected alpacas. Concomitant to the peak of infection, occurring at 2 days post inoculation (dpi), type I and III interferons (IFNs) were maximally transcribed only in the nasal mucosa of alpacas, while interferon stimulated genes (ISGs) were induced along the whole respiratory tract. Simultaneous to mild focal infiltration of leukocytes in nasal mucosa and submucosa, upregulation of the anti-inflammatory cytokine IL10 and dampened transcription of pro-inflammatory genes under NF-κB control were observed. In the lung, early (1 dpi) transcription of chemokines (CCL2 and CCL3) correlated with a transient accumulation of mainly mononuclear leukocytes. A tight regulation of IFNs in lungs with expression of ISGs and controlled inflammatory responses, might contribute to virus clearance without causing tissue damage. Thus, the nasal mucosa, the main target of MERS-CoV in camelids, seems central in driving an efficient innate immune response based on triggering ISGs as well as the dual anti-inflammatory effects of type III IFNs and IL10.


Assuntos
Camelídeos Americanos , Infecções por Coronavirus/imunologia , Interferon Tipo I/metabolismo , Interferons/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Camelídeos Americanos/imunologia , Camelídeos Americanos/metabolismo , Camelídeos Americanos/virologia , Chlorocebus aethiops , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/veterinária , Reservatórios de Doenças/veterinária , Resistência à Doença/efeitos dos fármacos , Resistência à Doença/genética , Resistência à Doença/imunologia , Regulação da Expressão Gênica , Imunidade Inata/fisiologia , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/veterinária , Inflamação/virologia , Interferon Tipo I/genética , Interferon Tipo I/farmacologia , Interferons/genética , Interferons/farmacologia , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/imunologia , Mucosa Nasal/metabolismo , Mucosa Nasal/virologia , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/imunologia , Sistema Respiratório/metabolismo , Sistema Respiratório/virologia , Células Vero , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
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