RESUMO
Reactivation of human cytomegalovirus (HCMV) from latency is a major health consideration for recipients of stem-cell and solid organ transplantations. With over 200,000 transplants taking place globally per annum, virus reactivation can occur in more than 50% of cases leading to loss of grafts as well as serious morbidity and even mortality. Here, we present the most extensive screening to date of epigenetic inhibitors on HCMV latently infected cells and find that histone deacetylase inhibitors (HDACis) and bromodomain inhibitors are broadly effective at inducing virus immediate early gene expression. However, while HDACis, such as myeloid-selective CHR-4487, lead to production of infectious virions, inhibitors of bromodomain (BRD) and extraterminal proteins (I-BETs), including GSK726, restrict full reactivation. Mechanistically, we show that BET proteins (BRDs) are pivotally connected to regulation of HCMV latency and reactivation. Through BRD4 interaction, the transcriptional activator complex P-TEFb (CDK9/CycT1) is sequestered by repressive complexes during HCMV latency. Consequently, I-BETs allow release of P-TEFb and subsequent recruitment to promoters via the superelongation complex (SEC), inducing transcription of HCMV lytic genes encoding immunogenic antigens from otherwise latently infected cells. Surprisingly, this occurs without inducing many viral immunoevasins and, importantly, while also restricting viral DNA replication and full HCMV reactivation. Therefore, this pattern of HCMV transcriptional dysregulation allows effective cytotoxic immune targeting and killing of latently infected cells, thus reducing the latent virus genome load. This approach could be safely used to pre-emptively purge the virus latent reservoir prior to transplantation, thereby reducing HCMV reactivation-related morbidity and mortality.
Assuntos
Proteínas de Ciclo Celular/genética , Citomegalovirus/imunologia , DNA Viral/genética , Epigênese Genética , Histona Desacetilases/genética , Fator B de Elongação Transcricional Positiva/genética , Fatores de Transcrição/genética , Azepinas/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzodiazepinas/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/imunologia , Ciclina T/genética , Ciclina T/imunologia , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/imunologia , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Replicação do DNA/efeitos dos fármacos , DNA Viral/antagonistas & inibidores , DNA Viral/imunologia , Genes Precoces , Genes Reporter , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/imunologia , Interações Hospedeiro-Patógeno , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Fator B de Elongação Transcricional Positiva/imunologia , Cultura Primária de Células , Regiões Promotoras Genéticas , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Células THP-1 , Talidomida/análogos & derivados , Talidomida/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/imunologia , Transcrição Gênica , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacosRESUMO
Osteoarthritis (OA) is generally considered to be characterized by progressive articular cartilage destruction. Increasing evidence demonstrates that CDK9, which is a member of cyclin-dependent kinase family, plays a significant role in the regulation of acute and chronic inflammatory diseases. IL-1ß, a major proinflammatory cytokine, was used to establish a model of OA in vitro after stimulating chondrocytes. We found that CDK9 was highly expressed in in vitro and in vivo models of inflammation. The role of LDC000067 (abbreviated as LDC067), a specific inhibitor of CDK9, in protecting articular cartilage from immune response has not been fully clarified. Intriguingly, in this study, we demonstrated that LDC067 prevented IL-1ß-induced production of metalloproteinases (MMPs) and inflammatory cytokines, including MMP3, MMP9, MMP13, IL-6, IL-8 and TNF-É. Furthermore, we revealed that LDC067 inhibited IL-1ß-induced NF-κB signaling pathway activation in chondrocytes. The inhibition of CDK9 could also delay cartilage degeneration in an anterior cruciate ligament transection (ACLT) mouse model in vivo. Taken together, these results highlighted the significance of this CDK9 inhibitor in preventing cartilage destruction and indicated that LDC067 might serve as a potential therapeutic agent for OA.