Microglia and blood-borne macrophages in injured or diseased brains are difficult to distinguish because they share many common characteristics. However, the identification of microglia-specific markers and the use of flow cytometry have recently made it easy to discriminate these types of cells. In this study, we analyzed the features of blood-borne macrophages, and activated and resting microglia in a rat traumatic brain injury (TBI) model. Oxidative injury was indicated in macrophages and neurons in TBI lesions by the presence of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Generation of mitochondrial reactive oxygen species (ROS) was markedly observed in granulocytes and macrophages, but not in activated or resting microglia. Dihydroethidium staining supported microglia not being the major source of ROS in TBI lesions. Furthermore, macrophages expressed NADPH oxidase 2, interleukin-1β (IL-1β), and CD68 at higher levels than microglia. In contrast, microglia expressed transforming growth factor β1 (TGFβ1), interleukin-6 (IL-6), and tumor necrosis factor α at higher levels than macrophages. A hypnotic, bromovalerylurea (BU), which has anti-inflammatory effects, reduced both glycolysis and mitochondrial oxygen consumption. BU administration inhibited chemokine CCL2 expression, accumulation of monocytes/macrophages, 8-OHdG generation, mitochondrial ROS generation, and proinflammatory cytokine expression, and markedly ameliorated the outcome of the TBI model. Yet, BU did not inhibit microglial activation or expression of TGFβ1 and insulin-like growth factor 1 (IGF-1). These results indicate that macrophages are the major aggravating cell type in TBI lesions, in particular during the acute phase. Activated microglia may even play favorable roles. Reduction of cellular energy metabolism in macrophages and suppression of CCL2 expression in injured tissue may lead to amelioration of TBI.

An old sedative and hypnotic bromovalerylurea (BU) has anti-inflammatory effects. BU suppressed nitric oxide (NO) release and proinflammatory cytokine expression by lipopolysaccharide (LPS)-treated BV2 cells, a murine microglial cell line. However, BU did not inhibit LPS-induced nuclear translocation of nuclear factor-κB and subsequent transcription. BU suppressed LPS-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) and expression of interferon regulatory factor 1 (IRF1). The Janus kinase 1 (JAK1) inhibitor filgotinib suppressed the NO release much more weakly than that of BU, although filgotinib almost completely prevented LPS-induced STAT1 phosphorylation. Knockdown of JAK1, STAT1, or IRF1 did not affect the suppressive effects of BU on LPS-induced NO release by BV2 cells. A combination of BU and filgotinib synergistically suppressed the NO release. The mitochondrial complex I inhibitor rotenone, which did not prevent STAT1 phosphorylation or IRF1 expression, suppressed proinflammatory mediator expression less significantly than BU. BU and rotenone reduced intracellular ATP (iATP) levels to a similar extent. A combination of rotenone and filgotinib suppressed NO release by LPS-treated BV2 cells as strongly as BU. These results suggest that anti-inflammatory actions of BU may be attributable to the synergism of inhibition of JAK1/STAT1-dependent pathways and reduction in iATP level.

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Presently, there are no approved drugs to treat sepsis, despite clinical trials of many agents that have distinct targets. Therefore a novel effective treatment should be developed. Objectives: We recently observed marked immunosuppressive effects of an old hypnotic drug, bromvalerylurea (BU), on lipopolysaccharide (LPS)-activated macrophages. The objective was to elucidate molecular mechanisms underlying the anti-inflammatory effects of BU and to determine whether BU ameliorated cecum ligation and puncture (CLP)-induced sepsis. Interventions: LPS-activated macrophages were incubated in vitro with BU. After CLP, some rats were subjected to subcutaneous injection with BU. Methods: Immunoblotting, quantitative real-time RT-PCR, immunohistochemical staining and, protein-knock down with siRNA. Results: BU suppressed a variety of pro- and anti-inflammatory mediators. LPS induced a two-step program of pro-inflammatory activation of macrophages; LPS first induced nuclear translocation of NF-κB, and then caused activation of Janus kinase 1/Signal transducer and activator 1 (JAK1/STAT1) that lead to interferon regulatory factor 1 expression as a consequence of NF-κB translocation. BU suppressed JAK1 activity. BU prevented the onset of MOF and increased survival in the septic rats. Conclusions: The JAK1/STAT1-dependent pro-inflammatory pathway could represent a promising novel drug target for treating sepsis. BU is worth reevaluating as a novel agent for sepsis and related disorders.