RESUMO
Children typically experience more mild symptoms of Coronavirus Disease 2019 (COVID-19) when compared to adults. There is a strong body of evidence that children are also less susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remain to be determined. Here, we use primary nasal epithelial cells (NECs) from children and adults, differentiated at an air-liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the NECs of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the NECs of children. Importantly, the Delta variant also replicated to significantly lower titers in the NECs of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves.
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COVID-19 , SARS-CoV-2 , Adulto , Criança , Células Epiteliais , Humanos , SARS-CoV-2/genéticaRESUMO
Understanding the airborne survival of viruses is important for public health and epidemiological modeling and potentially to develop mitigation strategies to minimize the transmission of airborne pathogens. Laboratory experiments typically involve investigating the effects of environmental parameters on the viability or infectivity of a target airborne virus. However, conflicting results among studies are common. Herein, the results of 34 aerovirology studies were compared to identify links between environmental and compositional effects on the viability of airborne viruses. While the specific experimental apparatus was not a factor in variability between reported results, it was determined that the experimental procedure was a major factor that contributed to discrepancies in results. The most significant contributor to variability between studies was poorly defined initial viable virus concentration in the aerosol phase, causing many studies to not measure the rapid inactivation, which occurs quickly after particle generation, leading to conflicting results. Consistently, studies that measured their reference airborne viability minutes after aerosolization reported higher viability at subsequent times, which indicates that there is an initial loss of viability which is not captured in these studies. The composition of the particles which carry the viruses was also found to be important in the viability of airborne viruses; however, the mechanisms for this effect are unknown. Temperature was found to be important for aerosol-phase viability, but there is a lack of experiments that directly compare the effects of temperature in the aerosol phase and the bulk phase. There is a need for repeated measurements between different research groups under identical conditions both to assess the degree of variability between studies and also to attempt to better understand already published data. Lack of experimental standardization has hindered the ability to quantify the differences between studies, for which we provide recommendations for future studies. These recommendations are as follows: measuring the reference airborne viability using the "direct method"; use equipment which maximizes time resolution; quantify all losses appropriately; perform, at least, a 5- and 10-min sample, if possible; report clearly the composition of the virus suspension; measure the composition of the gas throughout the experiment. Implementing these recommendations will address the most significant oversights in the existing literature and produce data which can more easily be quantitatively compared.
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Vírus , AerossóisRESUMO
AIMS: To investigate the effect of Lactobacillus rhamnosus on viral replication and cellular response to human rhinovirus (HRV) infection, including the secretion of antiviral and inflammatory mediators from well-differentiated nasal epithelial cells (WD-NECs). METHODS AND RESULTS: The WD-NECs from healthy adult donors (N = 6) were cultured in vitro, exposed to different strains of L. rhamnosus (D3189, D3160, or LB21), and infected with HRV (RV-A16) after 24 h. Survival and adherence capacity of L. rhamnosus in a NEC environment were confirmed using CFSE-labelled isolates, immunofluorescent staining, and confocal microscopy. Shed virus and viral replication were quantified using TCID50 assays and RT-qPCR, respectively. Cytotoxicity was measured by lactate dehydrogenase (LDH) activity. Pro-inflammatory mediators were measured by multiplex immunoassay, and interferon (IFN)-λ1/3 was measured using a standard ELISA kit. Lactobacillus rhamnosus was able to adhere to and colonize WD-NECs prior to the RV-A16 infection. Lactobacillus rhamnosus did not affect shed RV-A16, viral replication, RV-A16-induced IFN-λ1/3 production, or LDH release. Pre-exposure to L. rhamnosus, particularly D3189, reduced the secretion of RV-A16-induced pro-inflammatory mediators by WD-NECs. CONCLUSIONS: These findings demonstrate that L. rhamnosus differentially modulates RV-A16-induced innate inflammatory immune responses in primary NECs from healthy adults.
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Infecções por Enterovirus , Lacticaseibacillus rhamnosus , Adulto , Humanos , Citocinas , Rhinovirus/fisiologia , Células Cultivadas , Células Epiteliais , Inflamação , Quimiocinas/farmacologia , Mediadores da Inflamação/farmacologiaRESUMO
Lung transplant recipients frequently encounter immune-related complications, including chronic lung allograft dysfunction (CLAD). Monitoring immune cells within the lung microenvironment is pivotal for optimizing post-transplant outcomes. This study examined the proportion of T cell subsets in paired bronchoalveolar lavage (BAL) and peripheral PBMC comparing healthy (n = 4) and lung transplantation patients (n = 6, no CLAD and n = 14 CLAD) using 14-color flow cytometry. CD4+ T cell proportions were reduced in CD3 cells in both PBMC and BAL, and positive correlations were discerned between T cell populations in peripheral PBMC and BAL, suggesting the prospect of employing less invasive PBMC sampling as a means of monitoring lung T cells. Furthermore, regulatory T cells (Tregs) were enriched in BAL when compared to peripheral PBMC for transplant recipients. A parallel positive correlation emerged between Treg proportions in BAL and peripheral PBMC, underscoring potential avenues for monitoring lung Tregs. Finally, the most promising biomarker was the Teff (CD8+Granzyme B+)-Treg ratio, which was higher in both the PBMC and BAL of transplant recipients compared to healthy individuals, and increased in the patients with CLAD compared to no CLAD and healthy patients. Conclusions: Distinct T cell profiles in BAL and peripheral PBMC underscore the significance of localized immune monitoring in lung transplantation. The Teff (CD8+granzyme B+)-Treg ratio, particularly within the context of CLAD, emerges as a promising blood and BAL biomarker reflective of inflammation and transplant-related complications. These findings emphasize the imperative need for personalized immune monitoring strategies that tailored to address the unique immunological milieu in post-transplant lungs.
Assuntos
Leucócitos Mononucleares , Transplante de Pulmão , Humanos , Granzimas , Líquido da Lavagem Broncoalveolar , Lavagem Broncoalveolar , BiomarcadoresRESUMO
The factors governing the viability of airborne viruses embedded within respiratory particles are not well understood. This study aimed to investigate the relative humidity (RH)-dependent viability of airborne respiratory syncytial virus (RSV) in simulated respiratory particles suspended in various indoor air conditions. We tested airborne RSV viability in three static indoor air conditions, including sub-hysteresis (RH < 39%), hysteresis (39% < RH < 65%), and super-hysteresis (RH > 65%) air as well as in three dynamic indoor air conditions, including the transitions between the static conditions. The dynamic conditions were hysteresis â super-hysteresis â hysteresis, sub-hysteresis â hysteresis, and super-hysteresis â hysteresis. We found that after 45 min of particle aging in static conditions, the viability of RSV in sub-hysteresis, hysteresis, and super-hysteresis air was 0.72% ± 0.06%, 0.03% ± 0.006%, and 0.27% ± 0.008%, respectively. After 45 min of aging in dynamic conditions, the RSV viability decreased for particles that remained in a liquid (deliquesced) state during aging when compared with particles in a solid (effloresced) state. The decreased viability of airborne RSV for deliquesced particles is consistent with prolonged exposure to elevated aqueous solutes. These results represent the first measurements of the survival of airborne RSV over particle aging time, with equal viability in low, intermediate, and high RHs at 5 and 15 min and a V-shaped curve after 45 min.
Assuntos
Poluição do Ar em Ambientes Fechados , Vírus Sinciciais Respiratórios , Poluição do Ar em Ambientes Fechados/análise , Umidade , Monitoramento Ambiental/métodosRESUMO
Rationale: The alarmins IL-33 and HMGB1 (high mobility group box 1) contribute to type 2 inflammation and asthma pathogenesis. Objectives: To determine whether P2Y13-R (P2Y13 receptor), a purinergic GPCR (G protein-coupled receptor) and risk allele for asthma, regulates the release of IL-33 and HMGB1. Methods: Bronchial biopsy specimens were obtained from healthy subjects and subjects with asthma. Primary human airway epithelial cells (AECs), primary mouse AECs, or C57Bl/6 mice were inoculated with various aeroallergens or respiratory viruses, and the nuclear-to-cytoplasmic translocation and release of alarmins was measured by using immunohistochemistry and an ELISA. The role of P2Y13-R in AEC function and in the onset, progression, and exacerbation of experimental asthma was assessed by using pharmacological antagonists and mice with P2Y13-R gene deletion. Measurements and Main Results: Aeroallergen exposure induced the extracellular release of ADP and ATP, nucleotides that activate P2Y13-R. ATP, ADP, and aeroallergen (house dust mite, cockroach, or Alternaria antigen) or virus exposure induced the nuclear-to-cytoplasmic translocation and subsequent release of IL-33 and HMGB1, and this response was ablated by genetic deletion or pharmacological antagonism of P2Y13. In mice, prophylactic or therapeutic P2Y13-R blockade attenuated asthma onset and, critically, ablated the severity of a rhinovirus-associated exacerbation in a high-fidelity experimental model of chronic asthma. Moreover, P2Y13-R antagonism derepressed antiviral immunity, increasing IFN-λ production and decreasing viral copies in the lung. Conclusions: We identify P2Y13-R as a novel gatekeeper of the nuclear alarmins IL-33 and HMGB1 and demonstrate that the targeting of this GPCR via genetic deletion or treatment with a small-molecule antagonist protects against the onset and exacerbations of experimental asthma.
Assuntos
Asma/imunologia , Proteína HMGB1/metabolismo , Interleucina-33/metabolismo , Receptores Purinérgicos P2/metabolismo , Animais , Asma/metabolismo , Asma/fisiopatologia , Biomarcadores/metabolismo , Estudos de Casos e Controles , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The bactericidal effects of inhalable ciprofloxacin (CIP) loaded-poly(2-ethyl-2-oxazoline) (PEtOx) nanoparticles (NPs) with traces of zinc oxide (ZnO) were investigated against clinical strains of the respiratory pathogens Staphylococcus aureus and Pseudomonas aeruginosa. CIP-loaded PEtOx NPs retained their bactericidal activity within the formulations compared to free CIP drugs against these two pathogens, and bactericidal effects were enhanced with the inclusion of ZnO. PEtOx polymer and ZnO NPs did not show bactericidal activity alone or in combination against these pathogens. The formulations were tested to determine the cytotoxic and proinflammatory effects on airway epithelial cells derived from healthy donors (NHBE), donors with chronic obstructive pulmonary disease (COPD, DHBE), and a cell line derived from adults with cystic fibrosis (CFBE41o-) and macrophages from healthy adult controls (HCs), and those with either COPD or CF. NHBE cells demonstrated maximum cell viability (66%) against CIP-loaded PEtOx NPs with the half maximal inhibitory concentration (IC50) value of 50.7 mg/mL. CIP-loaded PEtOx NPs were more toxic to epithelial cells from donors with respiratory diseases than NHBEs, with respective IC50 values of 0.103 mg/mL for DHBEs and 0.514 mg/mL for CFBE41o- cells. However, high concentrations of CIP-loaded PEtOx NPs were toxic to macrophages, with respective IC50 values of 0.002 mg/mL for HC macrophages and 0.021 mg/mL for CF-like macrophages. PEtOx NPs, ZnO NPs, and ZnO-PEtOx NPs with no drug were not cytotoxic to any cells investigated. The in vitro digestibility of PEtOx and its NPs was investigated in simulated lung fluid (SLF) (pH 7.4). The analysed samples were characterized using Fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), and UV-Vis spectroscopy. Digestion of PEtOx NPs commenced one week following incubation and was completely digested after four weeks; however, the original PEtOx was not digested after six weeks of incubation. The outcome of this study revealed that PEtOx polymer could be considered an efficient drug delivery carrier in respiratory linings, and CIP-loaded PEtOx NPs with traces of ZnO could be a promising addition to inhalable treatments against resistant bacteria with reduced toxicity.
Assuntos
Nanopartículas Metálicas , Nanopartículas , Doença Pulmonar Obstrutiva Crônica , Óxido de Zinco , Humanos , Ciprofloxacina/farmacologia , Óxido de Zinco/química , Antibacterianos/farmacologia , Nanopartículas/química , Espectroscopia de Infravermelho com Transformada de Fourier , Nanopartículas Metálicas/química , Testes de Sensibilidade MicrobianaRESUMO
This study investigated immune cell characteristics in chronic hypersensitivity pneumonitis (HP), focusing on CD39-expressing cells' impact on inflammation and tissue remodelling. Lung tissue from an HP patient was analysed using single-cell transcriptomics, flow cytometry, and gene expression profiling. The tissue revealed diverse cell types like macrophages, T cells, fibroblasts, and regulatory T cells (Tregs). CD39-expressing Tregs exhibited heightened ATP hydrolysis capacity and regulatory gene expression. CD39hi cells displayed markers of both Tregs and proinflammatory Th17 cells, suggesting transitional properties. Communication networks involving molecules like SPP1, collagen, CSF1, and IL-1ß were identified, hinting at interactions between cell types in HP pathogenesis. This research provides insights into the immune response and cell interactions in chronic HP. CD39-expressing cells dual nature as Tregs and Th17 cells suggests a role in modulating lung inflammation, potentially affecting disease progression. These findings lay the groundwork for further research, underscoring CD39-expressing cells as potential therapeutic targets in HP.
Assuntos
Alveolite Alérgica Extrínseca , Antígenos CD , Humanos , Adenosina Trifosfatases/metabolismo , Alveolite Alérgica Extrínseca/patologia , Antígenos CD/metabolismo , Pulmão/metabolismo , Fenótipo , Linfócitos T Reguladores , Análise de Célula ÚnicaRESUMO
Type-2 immunity elicits tissue repair and homeostasis, however dysregulated type-2 responses cause aberrant tissue remodelling, as observed in asthma. Severe respiratory viral infections in infancy predispose to later asthma, however, the processes that mediate tissue damage-induced type-2 inflammation and the origins of airway remodelling remain ill-defined. Here, using a preclinical mouse model of viral bronchiolitis, we find that increased epithelial and mesenchymal high-mobility group box 1 (HMGB1) expression is associated with increased numbers of IL-13-producing type-2 innate lymphoid cell (ILC2s) and the expansion of the airway smooth muscle (ASM) layer. Anti-HMGB1 ablated lung ILC2 numbers and ASM growth in vivo, and inhibited ILC2-mediated ASM cell proliferation in a co-culture model. Furthermore, we identified that HMGB1/RAGE (receptor for advanced glycation endproducts) signalling mediates an ILC2-intrinsic IL-13 auto-amplification loop. In summary, therapeutic targeting of the HMGB1/RAGE signalling axis may act as a novel asthma preventative by dampening ILC2-mediated type-2 inflammation and associated ASM remodelling.
Assuntos
Remodelação das Vias Aéreas/imunologia , Proteína HMGB1/imunologia , Inflamação/imunologia , Linfócitos/imunologia , Músculo Liso/imunologia , Animais , Camundongos , Músculo Liso/patologia , Receptor para Produtos Finais de Glicação Avançada/imunologiaRESUMO
Laboratory-generated bioaerosols are widely used in aerobiology studies of viruses; however, few comparisons of alternative nebulizers exist. We compared aerosol production and virus survival for a Collison nebulizer, vibrating mesh nebulizer (VMN), and hydraulic spray atomizer (HSA). We also measured the dry size distribution of the aerosols produced and calculated the droplet sizes before evaporation and the dry size distribution from normal saline solution. Dry count median diameters of 0.11, 0.22, and 0.30 µm were found for normal saline from the Collison nebulizer, VMN, and HSA, respectively. The volume median diameters were 0.323, 1.70, and 1.30 µm, respectively. The effect of nebulization on the viability of two influenza A viruses (IAVs) (H1N1 and H3N2) and human rhinovirus 16 (HRV-16) was assessed by nebulization into an SKC BioSampler. The HSA had the least impact on surviving fractions (SFs) of H1N1 and H3N2 (89% ± 3% and 94% ± 2%, respectively), followed by the Collison nebulizer (83% ± 1% and 82% ± 2%, respectively). The VMN yielded SFs of 78% ± 2% and 76% ± 2%, respectively. Conversely, for HRV-16, the VMN produced higher SFs (87% ± 8%). Our findings indicate that there were no statistical differences between SFs of the viruses nebulized by these nebulizers. However, VMN produced higher aerosol concentrations within the airborne size range, making it more suitable where high aerosol mass production is required. IMPORTANCE Viral respiratory tract infections cause millions of lost days of work and physician visits globally, accounting for significant morbidity and mortality. Respiratory droplets and droplet nuclei from infected hosts are the potential carriers of such viruses within indoor environments. Laboratory-generated bioaerosols are applied in understanding the transmission and infection of viruses, modeling the physiological aspects of bioaerosol generation in a controlled environment. However, little comparative characterization exists for nebulizers used in infectious disease aerobiology, including Collison nebulizer, vibrating mesh nebulizer, and hydraulic spray atomizer. This study characterized the physical features of aerosols generated by laboratory nebulizers and their performance in producing aerosols at a size relevant to airborne transmission used in infectious disease aerobiology. We also determined the impact of nebulization mechanisms of these nebulizers on the viability of human respiratory viruses, including IAV H1N1, IAV H3N2, and HRV-16.
Assuntos
Aerossóis/análise , Microbiologia do Ar , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Nebulizadores e Vaporizadores/virologia , Rhinovirus/fisiologia , Humanos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Tamanho da Partícula , Rhinovirus/crescimento & desenvolvimentoRESUMO
The viability of airborne respiratory viruses varies with ambient relative humidity (RH). Numerous contrasting reports spanning several viruses have failed to identify the mechanism underlying this dependence. We hypothesized that an "efflorescence/deliquescence divergent infectivity" (EDDI) model accurately predicts the RH-dependent survival of airborne human rhinovirus-16 (HRV-16). We measured the efflorescence and deliquescence RH (RHE and RHD, respectively) of aerosols nebulized from a protein-enriched saline carrier fluid simulating the human respiratory fluid and found the RH range of the aerosols' hygroscopic hysteresis zone (RHE-D) to be 38-68%, which encompasses the preferred RH for indoor air (40-60%). The carrier fluid containing HRV-16 was nebulized into the sub-hysteresis zone (RH
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Rhinovirus , Aerossóis , Humanos , Umidade , MolhabilidadeRESUMO
Rationale: Respiratory syncytial virus (RSV) bronchiolitis causes significant infant mortality. Bronchiolitis is characterized by airway epithelial cell (AEC) death; however, the mode of death remains unknown.Objectives: To determine whether necroptosis contributes to RSV bronchiolitis pathogenesis via HMGB1 (high mobility group box 1) release.Methods: Nasopharyngeal samples were collected from children presenting to the hospital with acute respiratory infection. Primary human AECs and neonatal mice were inoculated with RSV and murine Pneumovirus, respectively. Necroptosis was determined via viability assays and immunohistochemistry for RIPK1 (receptor-interacting protein kinase-1), MLKL (mixed lineage kinase domain-like pseudokinase) protein, and caspase-3. Necroptosis was blocked using pharmacological inhibitors and RIPK1 kinase-dead knockin mice.Measurements and Main Results: HMGB1 levels were elevated in nasopharyngeal samples of children with acute RSV infection. RSV-induced epithelial cell death was associated with increased phosphorylated RIPK1 and phosphorylated MLKL but not active caspase-3 expression. Inhibition of RIPK1 or MLKL attenuated RSV-induced HMGB1 translocation and release, and lowered viral load. MLKL inhibition increased active caspase-3 expression in a caspase-8/9-dependent manner. In susceptible mice, Pneumovirus infection upregulated RIPK1 and MLKL expression in the airway epithelium at 8 to 10 days after infection, coinciding with AEC sloughing, HMGB1 release, and neutrophilic inflammation. Genetic or pharmacological inhibition of RIPK1 or MLKL attenuated these pathologies, lowered viral load, and prevented type 2 inflammation and airway remodeling. Necroptosis inhibition in early life ameliorated asthma progression induced by viral or allergen challenge in later life.Conclusions: Pneumovirus infection induces AEC necroptosis. Inhibition of necroptosis may be a viable strategy to limit the severity of viral bronchiolitis and break its nexus with asthma.
Assuntos
Bronquiolite/virologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proteína HMGB1/metabolismo , Necroptose , Mucosa Respiratória/citologia , Infecções por Vírus Respiratório Sincicial/metabolismo , Animais , Pré-Escolar , Humanos , Lactente , Camundongos , Estudos ProspectivosRESUMO
The current COVID-19 pandemic is stretching both the global supply for face masks and personal protective equipment (PPE). Production capacity is severely limited in many countries. This is a call for the R&D community, particularly to those in the polymer degradation and stability field. We have not only an opportunity but an obligation to engage and collaborate with virology and bio-medical experts. We require comparative R&D for extended, reuse and recyclability options. There is urgent need for large scale institutional approaches and methods that can be quickly applied locally by non-experts with limited resources.
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BACKGROUND: Rhinovirus infection triggers acute asthma exacerbations. IL-33 is an instructive cytokine of type 2 inflammation whose expression is associated with viral load during experimental rhinovirus infection of asthmatic patients. OBJECTIVE: We sought to determine whether anti-IL-33 therapy is effective during disease progression, established disease, or viral exacerbation using a preclinical model of chronic asthma and in vitro human primary airway epithelial cells (AECs). METHODS: Mice were exposed to pneumonia virus of mice and cockroach extract in early and later life and then challenged with rhinovirus to model disease onset, progression, and chronicity. Interventions included anti-IL-33 or dexamethasone at various stages of disease. AECs were obtained from asthmatic patients and healthy subjects and treated with anti-IL-33 after rhinovirus infection. RESULTS: Anti-IL-33 decreased type 2 inflammation in all phases of disease; however, the ability to prevent airway smooth muscle growth was lost after the progression phase. After the chronic phase, IL-33 levels were persistently high, and rhinovirus challenge exacerbated the type 2 inflammatory response. Treatment with anti-IL-33 or dexamethasone diminished exacerbation severity, and anti-IL-33, but not dexamethasone, promoted antiviral interferon expression and decreased viral load. Rhinovirus replication was higher and IFN-λ levels were lower in AECs from asthmatic patients compared with those from healthy subjects. Anti-IL-33 decreased rhinovirus replication and increased IFN-λ levels at the gene and protein levels. CONCLUSION: Anti-IL-33 or dexamethasone suppressed the magnitude of type 2 inflammation during a rhinovirus-induced acute exacerbation; however, only anti-IL-33 boosted antiviral immunity and decreased viral replication. The latter phenotype was replicated in rhinovirus-infected human AECs, suggesting that anti-IL-33 therapy has the additional benefit of enhancing host defense.
Assuntos
Antivirais/farmacologia , Asma/tratamento farmacológico , Asma/imunologia , Inflamação/imunologia , Interleucina-33/imunologia , Vírus da Pneumonia Murina/efeitos dos fármacos , Vírus da Pneumonia Murina/imunologia , Animais , Antivirais/imunologia , Asma/virologia , Suscetibilidade a Doenças/imunologia , Suscetibilidade a Doenças/virologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/virologia , Inflamação/tratamento farmacológico , Inflamação/virologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Pneumovirus/tratamento farmacológico , Infecções por Pneumovirus/imunologia , Infecções por Pneumovirus/virologia , Carga Viral/efeitos dos fármacos , Carga Viral/imunologiaRESUMO
Cellular protein eukaryotic translation elongation factor 1A (eEF1A) is an actin binding protein that plays a role in the formation of filamentous actin (F-actin) bundles. F-Actin regulates multiple stages of respiratory syncytial virus (RSV) replication including assembly and budding. Our previous study demonstrated that eEF1A knock-down significantly reduced RSV replication. Here we investigated if the eEF1A function in actin bundle formation was important for RSV replication and release. To investigate this, eEF1A function was impaired in HEp-2 cells by either knock-down of eEF1A with siRNA, or treatment with an eEF1A inhibitor, didemnin B (Did B). Cell staining and confocal microscopy analysis showed that both eEF1A knock-down and treatment with Did B resulted in disruption of cellular stress fiber formation and elevated accumulation of F-actin near the plasma membrane. When treated cells were then infected with RSV, there was also reduced formation of virus-induced cellular filopodia. Did B treatment, similarly to eEF1A knock-down, reduced the release of infectious RSV, but unlike eEF1A knock-down, did not significantly affect RSV genome replication. The lower infectious virus production in Did B treated cells also reduced RSV-induced cell death. In conclusion, the cellular factor eEF1A plays an important role in the regulation of F-actin stress fiber formation required for RSV assembly and release.
Assuntos
Actinas/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Vírus Sincicial Respiratório Humano/fisiologia , Fibras de Estresse/fisiologia , Replicação Viral , Actinas/genética , Linhagem Celular Tumoral , Depsipeptídeos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Técnicas de Silenciamento de Genes , Humanos , Pseudópodes/fisiologia , Pseudópodes/virologia , Vírus Sincicial Respiratório Humano/genéticaRESUMO
Background: Human metapneumovirus (hMPV) infection is implicated in exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Research into the pathogenesis of infection is restricted to animal models, and information about hMPV replication and inflammatory and immune responses in human disease is limited. Methods: Human primary bronchial epithelial cells (PBECs) from healthy and asthmatic subjects and those with COPD were infected with hMPV, with or without glucocorticosteroid (GCS) exposure. Viral replication, inflammatory and immune responses, and apoptosis were analyzed. We also determined whether adjuvant interferon (IFN) can blunt hMPV infection in vitro and in a murine model. Results: hMPV infected human PBECs and viral replication was enhanced in cells from patients with COPD. The virus induced gene expression of IFN-stimulated gene 56 (ISG56) and IFN-ß, as well as IFN-γ-inducible protein 10 (IP-10) and regulated on activation, normal T cell expressed and secreted (RANTES), and more so in cells from patients with COPD. GCS exposure enhanced hMPV replication despite increased IFN expression. Augmented virus replication associated with GCS was mediated by reduced apoptosis via induction of antiapoptotic genes. Adjuvant IFN treatment suppressed hMPV replication in PBECs and reduced hMPV viral titers and inflammation in vivo. Conclusions: hMPV infects human PBECs, eliciting innate and inflammatory responses. Replication is enhanced by GCS and adjuvant IFN is an effective treatment, restricting virus replication and proinflammatory consequences of hMPV infections.
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Glucocorticoides/farmacologia , Interferon gama/farmacologia , Metapneumovirus , Infecções por Paramyxoviridae/virologia , Doença Pulmonar Obstrutiva Crônica/virologia , Animais , Apoptose/efeitos dos fármacos , Asma/virologia , Brônquios/citologia , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Mucosa Respiratória/citologia , Replicação Viral/efeitos dos fármacosRESUMO
UNLABELLED: It is well established that glycosaminoglycans (GAGs) function as attachment factors for human metapneumovirus (HMPV), concentrating virions at the cell surface to promote interaction with other receptors for virus entry and infection. There is increasing evidence to suggest that multiple receptors may exhibit the capacity to promote infectious entry of HMPV into host cells; however, definitive identification of specific transmembrane receptors for HMPV attachment and entry is complicated by the widespread expression of cell surface GAGs. pgsA745 Chinese hamster ovary (CHO) cells are deficient in the expression of cell surface GAGs and resistant to HMPV infection. Here, we demonstrate that the expression of the Ca(2+)-dependent C-type lectin receptor (CLR) DC-SIGN (CD209L) or L-SIGN (CD209L) rendered pgsA745 cells permissive to HMPV infection. Unlike infection of parental CHO cells, HMPV infection of pgsA745 cells expressing DC-SIGN or L-SIGN was dynamin dependent and inhibited by mannan but not by pretreatment with bacterial heparinase. Parental CHO cells expressing DC-SIGN/L-SIGN also showed enhanced susceptibility to dynamin-dependent HMPV infection, confirming that CLRs can promote HMPV infection in the presence or absence of GAGs. Comparison of pgsA745 cells expressing wild-type and endocytosis-defective mutants of DC-SIGN/L-SIGN indicated that the endocytic function of CLRs was not essential but could contribute to HMPV infection of GAG-deficient cells. Together, these studies confirm a role for CLRs as attachment factors and entry receptors for HMPV infection. Moreover, they define an experimental system that can be exploited to identify transmembrane receptors and entry pathways where permissivity to HMPV infection can be rescued following the expression of a single cell surface receptor. IMPORTANCE: On the surface of CHO cells, glycosaminoglycans (GAGs) function as the major attachment factor for human metapneumoviruses (HMPV), promoting dynamin-independent infection. Consistent with this, GAG-deficient pgaA745 CHO cells are resistant to HMPV. However, expression of DC-SIGN or L-SIGN rendered pgsA745 cells permissive to dynamin-dependent infection by HMPV, although the endocytic function of DC-SIGN/L-SIGN was not essential for, but could contribute to, enhanced infection. These studies provide direct evidence implicating DC-SIGN/L-SIGN as an alternate attachment factor for HMPV attachment, promoting dynamin-dependent infection via other unknown receptors in the absence of GAGs. Moreover, we describe a unique experimental system for the assessment of putative attachment and entry receptors for HMPV.
Assuntos
Moléculas de Adesão Celular/metabolismo , Glicosaminoglicanos/metabolismo , Lectinas Tipo C/metabolismo , Metapneumovirus/fisiologia , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo , Ligação Viral , Animais , Células CHO , Cricetinae , Cricetulus , HumanosRESUMO
Reverse transcription is the central defining feature of HIV-1 replication. We previously reported that the cellular eukaryotic elongation factor 1 (eEF1) complex associates with the HIV-1 reverse transcription complex (RTC) and the association is important for late steps of reverse transcription. Here we show that association between the eEF1 and RTC complexes occurs by a strong and direct interaction between the subunit eEF1A and reverse transcriptase (RT). Using biolayer interferometry and co-immunoprecipitation (co-IP) assays, we show that association between the eEF1 and RTC complexes occurs by a strong (KD ~3-4 nM) and direct interaction between eEF1A and reverse transcriptase (RT). Biolayer interferometry analysis of cell lysates with titrated levels of eEF1A indicates it is a predominant cellular RT binding protein. Both the RT thumb and connection domains are required for interaction with eEF1A. A single amino acid mutation, W252A, within the thumb domain impaired co-IP between eEF1A and RT, and also significantly reduced the efficiency of late reverse transcription and virus replication when incorporated into infectious HIV-1. Molecular modeling analysis indicated that interaction between W252 and L303 are important for RT structure, and their mutation to alanine did not impair heterodimerisation, but negatively impacted interaction with eEF1A. Didemnin B, which specifically binds eEF1A, potently inhibited HIV-1 reverse transcription by greater than 2 logs at subnanomolar concentrations, especially affecting reverse transcription late DNA synthesis. Analysis showed reduced levels of RTCs from HIV-1-infected HEK293T treated with didemnin B compared to untreated cells. Interestingly, HIV-1 with a W252A RT mutation was resistant to didemnin B negative effects showing that didemnin B affects HIV-1 by targeting the RT-eEF1A interaction. The combined evidence indicates a direct interaction between eEF1A and RT is crucial for HIV reverse transcription and replication, and the RT-eEF1A interaction is a potential drug target.
Assuntos
Infecções por HIV/metabolismo , Transcriptase Reversa do HIV/metabolismo , HIV-1/fisiologia , Fator 1 de Elongação de Peptídeos/metabolismo , Transcrição Reversa/fisiologia , Replicação Viral/fisiologia , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , ImunoprecipitaçãoRESUMO
BACKGROUND: Frequent viral lower respiratory infections in early life are an independent risk factor for asthma onset. This risk and the development of persistent asthma are significantly greater in children who later become sensitized. OBJECTIVE: We sought to elucidate the pathogenic processes that underlie the synergistic interplay between allergen exposures and viral infections. METHODS: Mice were inoculated with a murine-specific Pneumovirus species (pneumonia virus of mice [PVM]) and exposed to low-dose cockroach extract (CRE) in early and later life, and airway inflammation, remodeling, and hyperreactivity assessed. Mice were treated with anti-IL-33 or apyrase to neutralize or block IL-33 release. RESULTS: PVM infection or CRE exposure alone did not induce disease, whereas PVM/CRE coexposure acted synergistically to induce the hallmark features of asthma. CRE exposure during viral infection in early life induced a biphasic IL-33 response and impaired IFN-α and IFN-λ production, which in turn increased epithelial viral burden, airway smooth muscle growth, and type 2 inflammation. These features were ameliorated when CRE-induced IL-33 release was blocked or neutralized, whereas substitution of CRE with exogenous IL-33 recapitulated the phenotype observed in PVM/CRE-coexposed mice. Mechanistically, IL-33 downregulated viperin and interferon regulatory factor 7 gene expression and rapidly degraded IL-1 receptor-associated kinase 1 expression in plasmacytoid dendritic cells both in vivo and in vitro, leading to Toll-like receptor 7 hyporesponsiveness and impaired IFN-α production. CONCLUSION: We identified a hitherto unrecognized function of IL-33 as a potent suppressor of innate antiviral immunity and demonstrate that IL-33 contributes significantly to the synergistic interplay between respiratory virus and allergen exposures in the onset and progression of asthma.