RESUMO
Japanese encephalitis virus (JEV) is a plus strand RNA virus, which infects brain. MicroRNAs are regulatory non-coding RNAs which regulate the expression of various genes in cells. Viruses modulate the expression of various microRNAs to suppress anti-viral signaling and evade the immune response. SOCS (Suppressor of cytokine signalling) family of proteins are negative regulators of anti-viral Jak-STAT pathway. In this study, we demonstrated the regulatory role of SOCS5 in Jak-STAT signaling and its exploitation by JEV through a microRNA mediated mechanism. JEV infection in human brain microglial cells (CHME3) downregulated the expression of miR-432, and upregulated SOCS5 levels. SOCS5 was validated as a target of miR-432 by using 3'UTR clone of SOCS5 in luciferase vector along with miR-432 mimic. The overexpression of miR-432 prior to JEV infection enhanced the phosphorylation of STAT1 resulting into increased ISRE activity and cellular inflammatory response resulting into diminished viral replication. The knockdown of SOCS5 resulted into increased STAT1 phosphorylation and suppressed viral replication. JEV infection mediated downregulation of miR-432 leads to SOCS5 upregulation, which helps the virus to evade cellular anti-viral response. This study demonstrated that JEV utilizes this microRNA mediated strategy to manipulate cellular immune response promoting JEV pathogenesis.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/fisiologia , Encefalite Japonesa/metabolismo , MicroRNAs/genética , Proteínas Supressoras da Sinalização de Citocina/genética , Regiões 3' não Traduzidas , Animais , Linhagem Celular , Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Encefalite Japonesa/virologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/metabolismo , Microglia/metabolismo , Microglia/virologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Suínos , Replicação ViralRESUMO
Microglia are the resident macrophages of the CNS, which secrete several pro- and anti-inflammatory cyto-chemokines including interleukin-1ß (IL-1ß), in response to pathogenic stimuli. Once secreted, IL-1ß binds to IL-1 receptor present on microglia and initiates the production of inflammatory cytokines in microglia. However, the detailed information regarding the molecular mechanisms of IL-1ß triggered inflammatory pathways in microglia is lacking. Our studies focused on the role of Krüppel-like factor 4 (Klf4) in mediating the regulation of pro-inflammatory gene expression upon IL-1ß stimulation in microglia. Our studies show that stimulation of microglia with IL-1ß robustly induces Klf4 via PI3K/Akt pathway which positively regulates the production of endogenous IL-1ß as well as other pro-inflammatory markers, cyclooxygenase-2, monocyte chemoattractant protein-1 and interleukin-6 (IL-6). In addition, we report that Klf4 negatively regulates the expression of inducible nitric oxide synthase, thereby playing a key role in regulating the immunomodulatory activities of microglia.