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1.
Nat Immunol ; 25(4): 607-621, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38589621

RESUMO

One in ten severe acute respiratory syndrome coronavirus 2 infections result in prolonged symptoms termed long coronavirus disease (COVID), yet disease phenotypes and mechanisms are poorly understood1. Here we profiled 368 plasma proteins in 657 participants ≥3 months following hospitalization. Of these, 426 had at least one long COVID symptom and 233 had fully recovered. Elevated markers of myeloid inflammation and complement activation were associated with long COVID. IL-1R2, MATN2 and COLEC12 were associated with cardiorespiratory symptoms, fatigue and anxiety/depression; MATN2, CSF3 and C1QA were elevated in gastrointestinal symptoms and C1QA was elevated in cognitive impairment. Additional markers of alterations in nerve tissue repair (SPON-1 and NFASC) were elevated in those with cognitive impairment and SCG3, suggestive of brain-gut axis disturbance, was elevated in gastrointestinal symptoms. Severe acute respiratory syndrome coronavirus 2-specific immunoglobulin G (IgG) was persistently elevated in some individuals with long COVID, but virus was not detected in sputum. Analysis of inflammatory markers in nasal fluids showed no association with symptoms. Our study aimed to understand inflammatory processes that underlie long COVID and was not designed for biomarker discovery. Our findings suggest that specific inflammatory pathways related to tissue damage are implicated in subtypes of long COVID, which might be targeted in future therapeutic trials.


Assuntos
Pesquisa Biomédica , COVID-19 , Humanos , Síndrome de COVID-19 Pós-Aguda , Hospitalização , Imunoglobulina G
2.
Clin Exp Immunol ; 215(1): 1-14, 2024 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-37556759

RESUMO

Early life is a time of increased susceptibility to infectious diseases and development of allergy. Innate lymphocytes are crucial components of the initiation and regulation of immune responses at mucosal surfaces, but functional differences in innate lymphocytes early in life are not fully described. We aimed to characterize the abundance and function of different innate lymphocyte cell populations in cord blood in comparison to that of adults. Blood was collected from adult donors and umbilical vessels at birth. Multicolor flow cytometry panels were used to identify and characterize lymphocyte populations and their capacity to produce hallmark cytokines. Lymphocytes were more abundant in cord blood compared to adults, however, mucosal-associated invariant T cells and natural killer T (NKT)-like cells, were far less abundant. The capacity of NKT-like cells to produce cytokines and their expression of the cytotoxic granule protein granzyme B and the marker of terminal differentiation CD57 were much lower in cord blood than in adults. In contrast, natural killer (NK) cells were as abundant in cord blood as in adults, they could produce IFNγ, and their expression of granzyme B was not significantly different from that of adult NK cells, although CD57 expression was lower. All innate lymphoid cell (ILC) subsets were more abundant in cord blood, and ILC1 and ILC2 were capable of production of IFNγ and IL-13, respectively. In conclusion, different innate lymphoid cells differ in both abundance and function in peripheral blood at birth and with important implications for immunity in early life.


Assuntos
Imunidade Inata , Células Matadoras Naturais , Humanos , Adulto , Recém-Nascido , Citocinas/metabolismo , Subpopulações de Linfócitos , Expressão Gênica
3.
J Lipid Res ; 59(9): 1671-1684, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29946055

RESUMO

In patients with asthma or chronic obstructive pulmonary disease, rhinovirus (RV) infections can provoke acute worsening of disease, and limited treatment options exist. Viral replication in the host cell induces significant remodeling of intracellular membranes, but few studies have explored this mechanistically or as a therapeutic opportunity. We performed unbiased lipidomic analysis on human bronchial epithelial cells infected over a 6 h period with the RV-A1b strain of RV to determine changes in 493 distinct lipid species. Through pathway and network analysis, we identified temporal changes in the apparent activities of a number of lipid metabolizing and signaling enzymes. In particular, analysis highlighted FA synthesis and ceramide metabolism as potential anti-rhinoviral targets. To validate the importance of these enzymes in viral replication, we explored the effects of commercially available enzyme inhibitors upon RV-A1b infection and replication. Ceranib-1, D609, and C75 were the most potent inhibitors, which confirmed that FAS and ceramidase are potential inhibitory targets in rhinoviral infections. More broadly, this study demonstrates the potential of lipidomics and pathway analysis to identify novel targets to treat human disorders.


Assuntos
Brônquios/citologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Metabolismo dos Lipídeos , Terapia de Alvo Molecular , Rhinovirus/fisiologia , Replicação Viral , Antivirais/farmacologia , Células HeLa , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Rhinovirus/efeitos dos fármacos
4.
J Virol ; 91(9)2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28228588

RESUMO

Picornavirus replication is known to cause extensive remodeling of Golgi and endoplasmic reticulum membranes, and a number of the host proteins involved in the viral replication complex have been identified, including oxysterol binding protein (OSBP) and phosphatidylinositol 4-kinase III beta (PI4KB). Since both OSBP and PI4KB are substrates for protein kinase D (PKD) and PKD is known to be involved in the control of Golgi membrane vesicular and lipid transport, we hypothesized that PKD played a role in viral replication. We present multiple lines of evidence in support of this hypothesis. First, infection of HeLa cells with human rhinovirus (HRV) induced the phosphorylation of PKD. Second, PKD inhibitors reduced HRV genome replication, protein expression, and titers in a concentration-dependent fashion and also blocked the replication of poliovirus (PV) and foot-and-mouth disease virus (FMDV) in a variety of cells. Third, HRV replication was significantly reduced in HeLa cells overexpressing wild-type and mutant forms of PKD1. Fourth, HRV genome replication was reduced in HAP1 cells in which the PKD1 gene was knocked out by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Although we have not identified the molecular mechanism through which PKD regulates viral replication, our data suggest that this is not due to enhanced interferon signaling or an inhibition of clathrin-mediated endocytosis, and PKD inhibitors do not need to be present during viral uptake. Our data show for the first time that targeting PKD with small molecules can inhibit the replication of HRV, PV, and FMDV, and therefore, PKD may represent a novel antiviral target for drug discovery.IMPORTANCE Picornaviruses remain an important family of human and animal pathogens for which we have a very limited arsenal of antiviral agents. HRV is the causative agent of the common cold, which in itself is a relatively trivial infection; however, in asthma and chronic obstructive pulmonary disease (COPD) patients, this virus is a major cause of exacerbations resulting in an increased use of medication, worsening symptoms, and, frequently, hospital admission. Thus, HRV represents a substantial health care and economic burden for which there are no approved therapies. We sought to identify a novel host target as a potential anti-HRV therapy. HRV infection induces the phosphorylation of PKD, and inhibitors of this kinase effectively block HRV replication at an early stage of the viral life cycle. Moreover, PKD inhibitors also block PV and FMDV replication. This is the first description that PKD may represent a target for antiviral drug discovery.


Assuntos
Replicação do DNA/genética , Vírus da Febre Aftosa/crescimento & desenvolvimento , Poliovirus/crescimento & desenvolvimento , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Rhinovirus/crescimento & desenvolvimento , Rhinovirus/genética , Replicação Viral/genética , Animais , Linhagem Celular Tumoral , Cricetinae , DNA Viral/biossíntese , Vírus da Febre Aftosa/genética , Técnicas de Inativação de Genes , Células HeLa , Humanos , Interferon Tipo I/metabolismo , Fosforilação , Poliovirus/genética , Proteína Quinase C/metabolismo , Pirimidinas/farmacologia
5.
J Virol ; 88(20): 11671-85, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25100828

RESUMO

The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion. Importance: The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be differences between different members of the family and inconsistent results when comparing infection with live virus to expression of individual nonstructural proteins. We demonstrate that individual nonstructural HRV16 proteins, when expressed in HeLa cells, can both fragment the Golgi apparatus and block secretion, whereas viral infection fragments the Golgi apparatus without blocking secretion. This has major implications for how we interpret mechanistic evidence derived from the expression of single viral proteins.


Assuntos
Complexo de Golgi/fisiologia , Rhinovirus/fisiologia , Proteínas Virais/metabolismo , Sequência de Bases , Primers do DNA , Células HeLa , Humanos , Microscopia de Fluorescência , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/fisiologia , Replicação Viral
7.
EBioMedicine ; 87: 104402, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36543718

RESUMO

BACKGROUND: Most studies of immunity to SARS-CoV-2 focus on circulating antibody, giving limited insights into mucosal defences that prevent viral replication and onward transmission. We studied nasal and plasma antibody responses one year after hospitalisation for COVID-19, including a period when SARS-CoV-2 vaccination was introduced. METHODS: In this follow up study, plasma and nasosorption samples were prospectively collected from 446 adults hospitalised for COVID-19 between February 2020 and March 2021 via the ISARIC4C and PHOSP-COVID consortia. IgA and IgG responses to NP and S of ancestral SARS-CoV-2, Delta and Omicron (BA.1) variants were measured by electrochemiluminescence and compared with plasma neutralisation data. FINDINGS: Strong and consistent nasal anti-NP and anti-S IgA responses were demonstrated, which remained elevated for nine months (p < 0.0001). Nasal and plasma anti-S IgG remained elevated for at least 12 months (p < 0.0001) with plasma neutralising titres that were raised against all variants compared to controls (p < 0.0001). Of 323 with complete data, 307 were vaccinated between 6 and 12 months; coinciding with rises in nasal and plasma IgA and IgG anti-S titres for all SARS-CoV-2 variants, although the change in nasal IgA was minimal (1.46-fold change after 10 months, p = 0.011) and the median remained below the positive threshold determined by pre-pandemic controls. Samples 12 months after admission showed no association between nasal IgA and plasma IgG anti-S responses (R = 0.05, p = 0.18), indicating that nasal IgA responses are distinct from those in plasma and minimally boosted by vaccination. INTERPRETATION: The decline in nasal IgA responses 9 months after infection and minimal impact of subsequent vaccination may explain the lack of long-lasting nasal defence against reinfection and the limited effects of vaccination on transmission. These findings highlight the need to develop vaccines that enhance nasal immunity. FUNDING: This study has been supported by ISARIC4C and PHOSP-COVID consortia. ISARIC4C is supported by grants from the National Institute for Health and Care Research and the Medical Research Council. Liverpool Experimental Cancer Medicine Centre provided infrastructure support for this research. The PHOSP-COVD study is jointly funded by UK Research and Innovation and National Institute of Health and Care Research. The funders were not involved in the study design, interpretation of data or the writing of this manuscript.


Assuntos
COVID-19 , SARS-CoV-2 , Adulto , Humanos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Seguimentos , Vacinação , Hospitalização , Imunoglobulina A , Imunoglobulina G , Anticorpos Antivirais , Anticorpos Neutralizantes
8.
Eur J Pharmacol ; 893: 173839, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33359650

RESUMO

Inhaled corticosteroids (ICS) are recommended treatments for all degrees of asthma severity and in combination with bronchodilators are indicated for COPD patients with a history of frequent exacerbations. However, the long-term side effects of glucocorticoids (GCs) may include increased risk of respiratory infections, including viral triggered exacerbations. Rhinovirus (RV) infection is the main trigger of asthma and COPD exacerbations. Thus, we sought to explore the influence of GCs on viral replication. We demonstrate the ICS fluticasone propionate (FP) and two selective non-steroidal (GRT7) and steroidal (GRT10) glucocorticoid receptor (GR) agonists significantly suppress pro-inflammatory (IL-6 and IL-8) and antiviral (IFN-λ1) cytokine production and the expression of the interferon-stimulated genes (ISGs) OAS and viperin in RV-infected bronchial epithelial cells, with a consequent increase of viral replication. We also show that FP, GRT7 and GRT10 inhibit STAT1 Y701 and/or STAT2 Y690 phosphorylation and ISG mRNA induction following cell stimulation with recombinant IFN-ß. In addition, we investigated the effects of the ICS budesonide (BD) and the long-acting ß2 agonist (LABA) formoterol, alone or as an ICS/LABA combination, on RV-induced ISG expression and viral replication. Combination of BD/formoterol increases the suppression of OAS and viperin mRNA observed with both BD and formoterol alone, but an increase in viral RNA was only observed with BD treatment and not with formoterol. Overall, we provide evidence of an impairment of the innate antiviral immune response by GC therapy and the potential for GCs to enhance viral replication. These findings could have important clinical implications.


Assuntos
Brônquios/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Glucocorticoides/toxicidade , Mediadores da Inflamação/metabolismo , Interferon Tipo I/metabolismo , Rhinovirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/toxicidade , Brônquios/imunologia , Brônquios/metabolismo , Brônquios/virologia , Quimioterapia Combinada , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Fumarato de Formoterol/toxicidade , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Proteínas/genética , Proteínas/metabolismo , Rhinovirus/crescimento & desenvolvimento , Rhinovirus/imunologia , Transdução de Sinais
9.
Nat Chem ; 10(6): 599-606, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29760414

RESUMO

Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections.


Assuntos
Aciltransferases/antagonistas & inibidores , Antivirais/farmacologia , Capsídeo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Rhinovirus/efeitos dos fármacos , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/química , Inibidores Enzimáticos/química , Células HeLa , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Rhinovirus/enzimologia , Rhinovirus/fisiologia
11.
Nat Med ; 23(6): 681-691, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28459437

RESUMO

Respiratory viral infections represent the most common cause of allergic asthma exacerbations. Amplification of the type-2 immune response is strongly implicated in asthma exacerbation, but how virus infection boosts type-2 responses is poorly understood. We report a significant correlation between the release of host double-stranded DNA (dsDNA) following rhinovirus infection and the exacerbation of type-2 allergic inflammation in humans. In a mouse model of allergic airway hypersensitivity, we show that rhinovirus infection triggers dsDNA release associated with the formation of neutrophil extracellular traps (NETs), known as NETosis. We further demonstrate that inhibiting NETosis by blocking neutrophil elastase or by degrading NETs with DNase protects mice from type-2 immunopathology. Furthermore, the injection of mouse genomic DNA alone is sufficient to recapitulate many features of rhinovirus-induced type-2 immune responses and asthma pathology. Thus, NETosis and its associated extracellular dsDNA contribute to the pathogenesis and may represent potential therapeutic targets of rhinovirus-induced asthma exacerbations.


Assuntos
Asma/imunologia , Citocinas/imunologia , DNA/imunologia , Armadilhas Extracelulares/imunologia , Infecções por Picornaviridae/imunologia , Hipersensibilidade Respiratória/imunologia , Infecções Respiratórias/imunologia , Células Th2/imunologia , Adulto , Animais , Estudos de Casos e Controles , Dermatophagoides farinae/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Interferon gama/imunologia , Interleucina-13/imunologia , Interleucina-4/imunologia , Interleucina-5/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Rhinovirus , Adulto Jovem
12.
Eur J Pharmacol ; 729: 75-85, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24534492

RESUMO

The chemokine receptor CCR4 has at least two natural agonist ligands, MDC (CCL22) and TARC (CCL17) which bind to the same orthosteric site with a similar affinity. Both ligands are known to evoke chemotaxis of CCR4-bearing T cells and also elicit CCR4 receptor internalization. A series of small molecule allosteric antagonists have been described which displace the agonist ligand, and inhibit chemotaxis. The aim of this study was to determine which cellular coupling pathways are involved in internalization, and if antagonists binding to the CCR4 receptor could themselves evoke receptor internalization. CCL22 binding coupled CCR4 efficiently to ß-arrestin and stimulated GTPγS binding however CCL17 did not couple to ß-arrestin and only partially stimulated GTPγS binding. CCL22 potently induced internalization of almost all cell surface CCR4, while CCL17 showed only weak effects. We describe four small molecule antagonists that were demonstrated to bind to two distinct allosteric sites on the CCR4 receptor, and while both classes inhibited agonist ligand binding and chemotaxis, one of the allosteric sites also evoked receptor internalization. Furthermore, we also characterize an N-terminally truncated version of CCL22 which acts as a competitive antagonist at the orthosteric site, and surprisingly also evokes receptor internalization without demonstrating any agonist activity. Collectively this study demonstrates that orthosteric and allosteric antagonists of the CCR4 receptor are capable of evoking receptor internalization, providing a novel strategy for drug discovery against this class of target.


Assuntos
Quimiotaxia/fisiologia , Endocitose/fisiologia , Receptores CCR4/antagonistas & inibidores , Receptores CCR4/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Basófilos/efeitos dos fármacos , Basófilos/metabolismo , Células CHO , Células Cultivadas , Quimiocina CCL17/farmacologia , Quimiocina CCL22/farmacologia , Quimiotaxia/efeitos dos fármacos , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Humanos
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