RESUMO
Microglia proliferate during brain development and brain lesions, but how this is coordinated at the transcriptional level is not well understood. Here, we investigated fundamental aspects of the transcriptional process associated with proliferation of mouse microglia during postnatal development and in adults in a model of induced microglial depletion-repopulation. While each proliferative subset displayed globally a distinct signature of gene expression, they also co-expressed a subgroup of 1370 genes at higher levels than quiescent microglia. Expression of these may be coordinated by one of two mechanisms of regulation with distinct properties. A first mechanism augments expression of genes already expressed in quiescent microglia and is subject to regulation by Klf/Sp, Nfy, and Ets transcription factors. Alternatively, a second mechanism enables de novo transcription of cell cycle genes and requires additional regulatory input from Lin54 and E2f transcription factors. Of note, transcriptional upregulation of E2f1 and E2f2 family members may represent a critical regulatory checkpoint to enable microglia to achieve efficient cell cycling. Furthermore, analysis of the activity profile of the repertoire of promoter-distal genomic regulatory elements suggests a relatively restricted role for these elements in coordinating cell cycle gene expression in microglia. Overall, proliferating microglia integrates regulation of cell cycle gene expression with their broader, context-dependent, transcriptional landscape.
Assuntos
Regulação da Expressão Gênica , Microglia , Animais , Proliferação de Células/genética , Camundongos , Microglia/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Pattern-recognition receptors such as Toll-like receptors (TLRs) are essential sensors implicated in the early and efficient innate immune response against pathogens. We have previously demonstrated that leukotriene B(4)(LTB(4)) has the capacity to enhance leukocyte responses to TLR9 ligands and to control viral infection. In this report, we provide evidence that LTB(4) treatment of human neutrophils leads to a potentiation in proinflammatory cytokine secretion induced by various myeloid differentiation factor 88-dependent TLR agonists. LTB(4) failed to enhance TLR mRNA levels as well as expression of TLR2 and TLR4 receptors, suggesting that LTB(4) acts through intracellular mechanism(s) to potentiate neutrophil responses to TLR ligands. We found that while IRAK can be activated by LTB(4), this process is dispensable to LTB(4) to potentiate neutrophil responses to TLR ligands since pretreatment of neutrophils with IRAK1/4 inhibitor did not affect its potentiating effects. However, our data clearly show that LTB(4) treatment of neutrophils led to the phosphorylation of downstream signaling molecules, TAK1 and p38, a process found essential to observe an increased secretion of cytokines by neutrophils activated with TLR ligands. Pretreatment of neutrophils with TAK1 or p38 kinase inhibitors strongly repressed the effect of LTB(4) on cytokine synthesis by neutrophils stimulated with LTA, LPS or CpG. The same pattern was observed in agonist-treated human embryonic kidney 293 cells transfected with TAK1-targeting siRNA where secretion of IL-8 was significantly reduced to basal levels. These results indicate that TAK1 and p38 kinases appear to be central in the 'priming effect' of LTB(4) on neutrophils to enhance response to TLR ligands.
Assuntos
Leucotrieno B4/imunologia , MAP Quinase Quinase Quinases/imunologia , Neutrófilos/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Citometria de Fluxo , Células HEK293 , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/imunologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-8/imunologia , Interleucina-8/metabolismo , Leucotrieno B4/farmacologia , Ligantes , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Oligodesoxirribonucleotídeos/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Inibidores de Proteínas Quinases/imunologia , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácidos Teicoicos/imunologia , Ácidos Teicoicos/farmacologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/agonistas , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Bacterial peptidoglycan-derived muramyl dipeptide (MDP) and derivatives have long-recognized antiviral properties but their mechanism of action remains unclear. In recent years, the pattern-recognition receptor NOD2 has been shown to mediate innate responses to MDP. Here, we show that MDP treatment of mice infected with Influenza A virus (IAV) significantly reduces mortality, viral load and pulmonary inflammation in a NOD2-dependent manner. Importantly, the induction of type I interferon (IFN) and CCL2 chemokine was markedly increased in the lungs following MDP treatment and correlated with a NOD2-dependent enhancement in circulating monocytes. Mechanistically, the protective effect of MDP could be explained by the NOD2-dependent transient increase in recruitment of Ly6C(high) "inflammatory" monocytes and, to a lesser extent, neutrophils to the lungs. Indeed, impairment in both Ly6C(high) monocyte recruitment and survival observed in infected Nod2-/- mice treated with MDP was recapitulated in mice deficient for the chemokine receptor CCR2 required for CCL2-mediated Ly6C(high) monocyte migration from the bone marrow into the lungs. MDP-induced pulmonary monocyte recruitment occurred normally in IAV-infected and MDP-treated Ips-1-/- mice. However, IPS-1 was required for improved survival upon MDP treatment. Finally, mycobacterial N-glycolyl MDP was more potent than N-acetyl MDP expressed by most bacteria at reducing viral burden while both forms of MDP restored pulmonary function following IAV challenge. Overall, our work sheds light on the antiviral mechanism of a clinically relevant bacterial-derived compound and identifies the NOD2 pathway as a potential therapeutic target against IAV.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/farmacologia , Adjuvantes Imunológicos/farmacologia , Antígenos Ly , Vírus da Influenza A/metabolismo , Pulmão/metabolismo , Monócitos/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Pulmão/patologia , Camundongos , Camundongos Knockout , Monócitos/patologia , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/genética , Neutrófilos/metabolismo , Neutrófilos/patologia , Proteína Adaptadora de Sinalização NOD2/genética , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologiaRESUMO
TLR9 plays an important role in innate defense against viruses by the detection of CpG motifs of foreign DNA within intracellular compartments. In this study, we evaluated the ability of EBV to promote monocyte and plasmacytoid dendritic cell (pDC) activation and cytokine release through TLR9 activation. We demonstrated that treatment of primary monocytes with EBV and with purified EBV DNA induced the release of IL-8 through TLR9. Activation of TLR9 by viral DNA requires endosomal maturation because pretreatment of monocytes with chloroquine strongly reduced IL-8 secretion. However, pretreatment of monocytes with siRNA directed against TLR2, with inhibitory ODN (iODN) or with a combination of both inhibitors strongly reduced the secretion of IL-8, providing evidence of a dual action of TLR2 and TLR9 in EBV recognition by monocytes. In contrast, production of MCP-1 and IL-10 in EBV-treated monocytes was mainly regulated through TLR2. Although EBV does not establish infection in pDCs, challenge with either live EBV particles or isolated EBV DNA was found to induce the release of IFN-alpha through TLR9, as supported by blockage of TLR9 activity with iODN or chloroquine. The role of TLR9 in the recognition of EBV by pDCs appears to be dominant, as confirmed by the marked inhibitory effect of iODN observed on the synthesis of IFN-alpha, IL-6, and IL-8 by pDCs. These results demonstrate that recognition of EBV by TLR9 is differently orchestrated in primary monocytes and pDCs to optimize viral recognition and antiviral response.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/virologia , Herpesvirus Humano 4/imunologia , Monócitos/imunologia , Monócitos/virologia , Receptor Toll-Like 9/fisiologia , Comunicação Celular/genética , Comunicação Celular/imunologia , Células Cultivadas , Citocinas/biossíntese , Células Dendríticas/metabolismo , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/patologia , Regulação da Expressão Gênica/imunologia , Herpesvirus Humano 4/patogenicidade , Humanos , Mediadores da Inflamação/fisiologia , Monócitos/metabolismo , Transporte Proteico/genética , Transporte Proteico/imunologia , Receptor Toll-Like 9/biossíntese , Receptor Toll-Like 9/genética , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Epstein-Barr virus (EBV) is a gammaherpesvirus infecting the majority of the human adult population in the world. TLR2, a member of the Toll-like receptor (TLR) family, has been implicated in the immune responses to different viruses including members of the herpesvirus family, such as human cytomegalovirus, herpes simplex virus type 1, and varicella-zoster virus. In this report, we demonstrate that infectious and UV-inactivated EBV virions lead to the activation of NF-kappaB through TLR2 using HEK293 cells cotransfected with TLR2-expressing vector along with NF-kappaB-Luc reporter plasmid. NF-kappaB activation in HEK293-TLR2 cells (HEK293 cells transfected with TLR2) by EBV was not enhanced by the presence of CD14. The effect of EBV was abrogated by pretreating HEK293-TLR2 cells with blocking anti-TLR2 antibodies or by preincubating viral particles with neutralizing anti-EBV antibodies 72A1. In addition, EBV infection of primary human monocytes induced the release of MCP-1 (monocyte chemotactic protein 1), and the use of small interfering RNA targeting TLR2 significantly reduced such a chemokine response to EBV. Taken together, these results indicate that TLR2 may be an important pattern recognition receptor in the immune response directed against EBV infection.