RESUMO
We have identified an intergenic transcriptional activity that is located between the human HOXA1 and HOXA2 genes, shows myeloid-specific expression, and is up-regulated during granulocytic differentiation. The novel gene, termed HOTAIRM1 (HOX antisense intergenic RNA myeloid 1), is transcribed antisense to the HOXA genes and originates from the same CpG island that embeds the start site of HOXA1. The transcript appears to be a noncoding RNA containing no long open-reading frame; sucrose gradient analysis shows no association with polyribosomal fractions. HOTAIRM1 is the most prominent intergenic transcript expressed and up-regulated during induced granulocytic differentiation of NB4 promyelocytic leukemia and normal human hematopoietic cells; its expression is specific to the myeloid lineage. Its induction during retinoic acid (RA)-driven granulocytic differentiation is through RA receptor and may depend on the expression of myeloid cell development factors targeted by RA signaling. Knockdown of HOTAIRM1 quantitatively blunted RA-induced expression of HOXA1 and HOXA4 during the myeloid differentiation of NB4 cells, and selectively attenuated induction of transcripts for the myeloid differentiation genes CD11b and CD18, but did not noticeably impact the more distal HOXA genes. These findings suggest that HOTAIRM1 plays a role in the myelopoiesis through modulation of gene expression in the HOXA cluster.
Assuntos
DNA Intergênico , Proteínas de Homeodomínio/genética , MicroRNAs/genética , Mielopoese/genética , RNA Mensageiro/genética , Células Cultivadas , DNA Intergênico/genética , Perfilação da Expressão Gênica , Células HL-60 , Células HeLa , Humanos , Células Jurkat , Células K562 , MicroRNAs/análise , Família Multigênica/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Polimerase II/metabolismo , RNA Polimerase II/fisiologia , Sequências Reguladoras de Ácido Ribonucleico/genética , Fatores de Transcrição/genéticaRESUMO
Influenza virus infection of the respiratory tract is characterized by a neutrophil infiltrate accompanied by inflammatory cytokine and chemokine production. We and others have reported that Toll-like receptor (TLR) proteins are present on human neutrophils and that granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment enhances IL-8 (CXCL8) secretion in response to stimulation with TLR ligands. We demonstrate that influenza virus can induce IL-8 and other inflammatory cytokines from GM-CSF-primed human neutrophils. Using heat inactivation of influenza virus, we show that viral entry but not replication is required for cytokine induction. Furthermore, endosomal acidification and viral uncoating are necessary. Finally, using single-cell analysis of intracellular cytokine accumulation in neutrophils from knockout mice, we prove that TLR7 is essential for influenza viral recognition and inflammatory cytokine production by murine neutrophils. These studies demonstrate neutrophil activation by influenza virus and highlight the importance of TLR7 and TLR8 in that response.
Assuntos
Vírus da Influenza A Subtipo H3N2/imunologia , Neutrófilos/imunologia , Neutrófilos/virologia , Receptores Toll-Like/metabolismo , Animais , Linhagem Celular , Citocinas/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Imunidade Inata , Técnicas In Vitro , Vírus da Influenza A Subtipo H3N2/patogenicidade , Vírus da Influenza A Subtipo H3N2/fisiologia , Ligantes , Macrolídeos/farmacologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/imunologia , Neutrófilos/efeitos dos fármacos , RNA Viral/imunologia , RNA Viral/metabolismo , Proteínas Recombinantes , Receptor 7 Toll-Like/deficiência , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Receptores Toll-Like/agonistas , Internalização do Vírus , Replicação ViralRESUMO
This work investigated the functional role of nuclear factor-kappaB (NF-kappaB) in respiratory burst activity and in expression of the human phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase genes CYBB, CYBA, NCF1, and NCF2. U937 cells with a stably transfected repressor of NF-kappaB (IkappaBalpha-S32A/S36A) demonstrated significantly lower superoxide release and lower CYBB and NCF1 gene expression compared with control U937 cells. We further tested Epstein-Barr virus (EBV)-transformed B cells from patients with anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID), an inherited disorder of NF-kappaB function. Superoxide release and CYBB gene expression by EDA-ID cells were significantly decreased compared with healthy cells and similar to cells from patients with X-linked chronic granulomatous disease (X91(0) CGD). NCF1 gene expression in EDA-ID S32I cells was decreased compared with healthy control cells and similar to that in autosomal recessive (A47(0)) CGD cells. Gel shift assays demonstrated loss of recombinant human p50 binding to a NF-kappaB site 5' to the CYBB gene in U937 cells treated with NF-kappaB inhibitors, repressor-transfected U937 cells, and EDA-ID patients' cells. Zymosan phagocytosis was not affected by transfection of U937 cells with the NF-kappaB repressor. These studies show that NF-kappaB is necessary for CYBB and NCF1 gene expression and activation of the phagocyte NADPH oxidase in this model system.