Avaliação in vitro da ação de enzimas oriundas das NETs na replicação viral do hRSV
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Data
2021-03-05
Autores
Lopes, Bruno Rafael Pereira
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Universidade Estadual Paulista (Unesp)
Resumo
As infecções do trato respiratório são extremamente comuns sendo, o Vírus Sincicial Respiratório Humano (hRSV) o principal agente etiológico das infecções virais agudas do trato respiratório inferior, resultando mundialmente em mais de 3 milhões de casos muito graves de bronquiolite por ano. A infecção promove o influxo de células imunes populando o tecido predominantemente com neutrófilos. Estimulado pelo epitélio infectado e por proteínas virais, os neutrófilos secretam seu material genético imiscuído com enzimas granulares que formam as armadilhas extracelulares neutrofílicas (NETs). Essas estruturas possuem ampla atividade microbicida, porém apresentam grande potencial citotóxico, sendo comumente associadas a grande parte do dano tecidual, com consequente agravamento do quadro clínico. Pacientes infectados com hRSV apresentam grande quantidade de neutrófilos e elevado nível de enzimas neutrofílicas nas secreções do trato respiratório, entretanto pouco se sabe sobre a capacidade anti-hRSV dessas proteínas. Intuindo contribuir na elucidação do papel das NETs na infecção por hRSV, propôs-se avaliar o efeito de algumas enzimas sobre a replicação do hRSV in vitro. Para tanto, o efeito da replicação do hRSV, isoladamente ou na presença de diversas concentrações de enzimas das NETs (Mieloperoxidase, Proteínase 3, Catepsina G e Elastase) foi caraterizado e quantificado através de análises morfológicas, ensaios de viabilidade utilizando células HEp-2, perfil eletroforético e ensaios de redução de placas de lise. A presença de Proteinase 3, Catepsina G e Elastase reduziu a morte celular e a formação de placas de lise induzidas pela infecção, bem como a formação de sincícios, uma característica da replicação in vitro. O perfil eletroforético sugere que a proteína F viral, envolvida nos processos de adesão e fusão, interage com estas enzimas gerando pesados complexos ou mesmo clivagem da proteína viral. O conjunto dos resultados, associado ao descrito na literatura, indica que as enzimas das NETs possuem relevante efeito anti-hRSV, provavelmente via interação com as proteínas de superfície do vírion, como a proteína de fusão F.
Respiratory infections are extremely common, and the Human Respiratory Syncytial Virus (hRSV) is the main etiologic agent of acute viral infections of the lower respiratory tract, resulting in more than 3 million very serious cases of bronchiolitis worldwide each year. The infection promotes the influx of immune cells populating the tissue predominantly with neutrophils. Stimulated by the infected epithelium and viral proteins, neutrophils secrete their genetic material intermingled with granular enzymes that form the neutrophil extracellular traps (NETs). These structures have huge microbicidal activity, but have cytotoxic potential, being commonly associated with a large part of tissue damage, with consequent worsening of the clinical condition. Patients infected with hRSV have a large amount of neutrophils and a high level of neutrophilic enzymes in respiratory tract secretions, however little is known about the anti-hRSV capacity of these proteins. Intending to contribute to the elucidation of the role of NETs in hRSV infection, it was proposed to evaluate the effect of some enzymes of NETs upon hRSV replication in vitro. Therefore, the effect of hRSV replication, alone or in the presence of different concentrations of NETs enzymes (Myeloperoxidase, Proteinase 3, Cathepsin G and Elastase) was characterized and quantified through morphological analyzes, cell viability tests, electrophoretic profile and reduction of lysis plates. The presence of Proteinase 3, Cathepsin G and Elastase reduced cell death and the formation of lysis plaques induced by infection, as well as the formation of syncytia, a characteristic of in vitro replication. The electrophoretic profile suggests that the viral F protein, involved in the adhesion and fusion processes, interacts with those enzymes generating heavy complexes or even cleavage of the viral protein. Our results, in association with literature, indicates that NETs enzymes have a relevant anti-hRSV effect, probably via interaction with virion surface proteins, such as the fusion protein F.
Respiratory infections are extremely common, and the Human Respiratory Syncytial Virus (hRSV) is the main etiologic agent of acute viral infections of the lower respiratory tract, resulting in more than 3 million very serious cases of bronchiolitis worldwide each year. The infection promotes the influx of immune cells populating the tissue predominantly with neutrophils. Stimulated by the infected epithelium and viral proteins, neutrophils secrete their genetic material intermingled with granular enzymes that form the neutrophil extracellular traps (NETs). These structures have huge microbicidal activity, but have cytotoxic potential, being commonly associated with a large part of tissue damage, with consequent worsening of the clinical condition. Patients infected with hRSV have a large amount of neutrophils and a high level of neutrophilic enzymes in respiratory tract secretions, however little is known about the anti-hRSV capacity of these proteins. Intending to contribute to the elucidation of the role of NETs in hRSV infection, it was proposed to evaluate the effect of some enzymes of NETs upon hRSV replication in vitro. Therefore, the effect of hRSV replication, alone or in the presence of different concentrations of NETs enzymes (Myeloperoxidase, Proteinase 3, Cathepsin G and Elastase) was characterized and quantified through morphological analyzes, cell viability tests, electrophoretic profile and reduction of lysis plates. The presence of Proteinase 3, Cathepsin G and Elastase reduced cell death and the formation of lysis plaques induced by infection, as well as the formation of syncytia, a characteristic of in vitro replication. The electrophoretic profile suggests that the viral F protein, involved in the adhesion and fusion processes, interacts with those enzymes generating heavy complexes or even cleavage of the viral protein. Our results, in association with literature, indicates that NETs enzymes have a relevant anti-hRSV effect, probably via interaction with virion surface proteins, such as the fusion protein F.
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Palavras-chave
hRSV, Armadilhas extracelulares neutrofílicas, Antiviral, Neutrophil extracellular traps, Antiviral