Abstract
Accumulating data suggest that neutrophil extracellular traps (NETs) exert a key function in several diseases. Peptidylarginine deiminase 4 (PAD4) regulates NET formation via citrullination of histones. The aim of this study was to examine the role of miR-155 in controlling PAD4-dependent generation of NETs. Bone marrow neutrophils were stimulated with PMA and MIP-2. Pre-incubation of neutrophils with translational inhibitors (cycloheximide or puromycin) markedly decreased NET formation induced by PMA or MIP-2. Neutrophil transfection with a mimic miR-155 increased PMA-induced PAD4 mRNA expression and NET formation. In contrast, transfection with an antagomiR-155 decreased induction of PAD4 mRNA and NETs in response to PMA challenge. Bioinformatical examination of PAD4 revealed a potential binding site in AU-rich elements at the 3′-UTR region. MiR-155 binding to PAD4 was examined by use of target site blockers and RNA immunoprecipitation, revealing that miR-155 regulation of PAD4 mRNA is mediated via AU-rich elements in the 3′-UTR region. In conclusion, our findings demonstrate that miR-155 positively regulates neutrophil expression of PAD4 and expulsion of extracellular traps. Thus, our novel results indicate that targeting miR-155 might be useful to inhibit exaggerated NET generation in inflammatory diseases.
Highlights
Neutrophils are critical in the host defense against bacterial infections [1] but these cells play a pathological role in a wide range of diseases, including sepsis, acute pancreatitis, inflammatory bowel diseases and ischemia-reperfusion injuries [2,3,4,5]
It was found that 30 min, but not 5 min, of pre-incubation with cycloheximide or puromycin decreased DNA-histone complex formation in neutrophils exposed to MIP-2 (Figure 3A)
This finding is in contrast to three previous studies reporting that there is no requirement of protein translation in the induction of neutrophil extracellular traps (NETs) [34,35,36], which could be related to differences in methodology
Summary
Neutrophils are critical in the host defense against bacterial infections [1] but these cells play a pathological role in a wide range of diseases, including sepsis, acute pancreatitis, inflammatory bowel diseases and ischemia-reperfusion injuries [2,3,4,5]. Hypercitrullination of target histone 2A, 3, and 4 causes chromatin decondensation and constitutes a key process in NET formation [12]. PAD4 has been shown to be a major regulator of NET formation via deimination of histone H3 to citrullinated histone H3 causing chromatin decondensation and release [15, 16] more efforts are needed to understand signaling pathways regulating PAD4 activity and expulsion of DNA from neutrophils
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