Unconventional role of voltage-gated proton channels (VSOP/Hv1) in regulation of microglial ROS production.

It has been established that voltage-gated proton channels (VSOP/Hv1), encoded by Hvcn1, support reactive oxygen species (ROS) production in phagocytic activities of neutrophils (El Chemaly et al. ) and antibody production in B lymphocytes (Capasso et al. ). VSOP/Hv1 is a potential therapeutic target for brain ischemia, since Hvcn1 deficiency reduces microglial ROS production and protects brain from neuronal damage (Wu et al. ). In the present study, we report that VSOP/Hv1 has paradoxical suppressive role in ROS production in microglia. Extracellular ROS production was lower in neutrophils of Hvcn1-/- mice than WT mice as reported. In contrast, it was drastically enhanced in isolated Hvcn1-/- microglia as compared with cells from WT mice. Actin dynamics was altered in Hvcn1-/- microglia and intracellular distribution of cytosolic NADPH oxidase subunit, p67, was changed. When expression levels of oxidative stress responsive antioxidant genes were compared between WT and Hvcn1-/- in cerebral cortex at different ages of animals, they were slightly decreased in Hvcn1-/- mice at younger stage (1 day, 5 days, 3 weeks old), but drastically increased at aged stage (6 months old), suggesting that the regulation of microglial ROS production by VSOP/Hv1 is age-dependent. We also performed brain ischemic stroke experiments and found that the neuroprotective effect of VSOP/Hv1deficiency on infarct volume depended on the age of animals. Taken together, regulation of ROS production by VSOP/Hv1 is more complex than previously thought and significance of VSOP/Hv1 in microglial ROS production depends on age.

Kawai T ，Okochi Y ，Ozaki T ，Imura Y ，Koizumi S ，Yamazaki M ，Abe M ，Sakimura K ，Yamashita T ，Okamura Y ... -  《-》

Mechanistic insight into the suppression of microglial ROS production by voltage-gated proton channels (VSOP/Hv1).

Kawai T ，Tatsumi S ，Kihara S ，Sakimura K ，Okamura Y ... -  《-》

Deficiency in the voltage-gated proton channel Hv1 increases M2 polarization of microglia and attenuates brain damage from photothrombotic ischemic stroke.

Microglia become activated during cerebral ischemia and exert pro-inflammatory or anti-inflammatory role dependent of microglial polarization. NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) production in microglia plays an important role in neuronal damage after ischemic stroke. Recently, NOX and ROS are consistently reported to participate in the microglial activation and polarization; NOX2 inhibition or suppression of ROS production are shown to shift the microglial polarization from M1 toward M2 state after stroke. The voltage-gated proton channel, Hv1, is selectively expressed in microglia and is required for NOX-dependent ROS generation in the brain. However, the effect of Hv1 proton channel on microglial M1/M2 polarization state after cerebral ischemia remains unknown. In this study, we investigated the role of microglial Hv1 proton channel in modulating microglial M1/M2 polarization during the pathogenesis of ischemic cerebral injury using a mouse model of photothrombosis. Following photothrombotic ischemic stroke, wild-type mice presented obvious brain infarct, neuronal damage, and impaired motor coordination. However, mice lacking Hv1 (Hv1(-/-)) were partially protected from brain damage and motor deficits compared to wild-type mice. These rescued phenotypes in Hv1(-/-) mice in ischemic stroke is accompanied by reduced ROS production, shifted the microglial polarization from M1 to M2 state. Hv1 deficiency was also found to shift the M1/M2 polarization in primary cultured microglia. Our study suggests that the microglial Hv1 proton channel is a unique target for modulation of microglial M1/M2 polarization in the pathogenesis of ischemic stroke. The voltage-gated proton channel, Hv1, is selectively expressed in microglia and is required for NOX-dependent generation of reactive oxygen species (ROS) in the brain. ROS participate in microglial activation and polarization. However, the effect of Hv1 on microglial M1/M2 polarization state after cerebral ischemia remains unknown. Hv1 deficiency was found to shift the microglial polarization from M1 to M2 state in ischemic stroke accompanied by reduced ROS production. Our study suggests that the microglial Hv1 proton channel is a unique target for modulation of microglial M1/M2 polarization in the pathogenesis of ischemic stroke.

Tian DS ，Li CY ，Qin C ，Murugan M ，Wu LJ ，Liu JL ... -  《-》

Regulation of hepatic oxidative stress by voltage-gated proton channels (Hv1/VSOP) in Kupffer cells and its potential relationship with glucose metabolism.

Voltage-gated proton channels (Hv1/VSOP), encoded by Hvcn1, are important regulator of reactive oxygen species (ROS) production in many types of immune cells. While in vitro studies indicate that Hv1/VSOP regulates ROS production by maintaining pH homeostasis, there are few studies investigating the functional importance of Hv1/VSOP in vivo. In the present study, we first show that Hv1/VSOP is functionally expressed in liver resident macrophage, Kupffer cells, regulating the hepatic oxidative stress in vivo. Our immunocytochemistry and electrophysiology data showed that Hvcn1 is specifically expressed in Kupffer cells, but not in hepatocytes. Furthermore, Hvcn1-deficiency drastically altered the hepatic oxidative stress. The Hvcn1-deficient mice showed high blood glucose and serum insulin but normal insulin sensitivity, indicating that these phenotypes were not linked to insulin resistance. Transcriptome analysis indicated that the gene expression of glycogen phosphorylase (Pygl) and Glucose-6-phosphatase, catalytic subunit (G6pc) were upregulated in Hvcn1-deficient liver tissues, and quantitative PCR confirmed the result for Pygl. Furthermore, we observed higher amount of glucose-6-phosphate, a key sugar intermediate for glucose in Hvcn1-deficient liver than WT, suggesting that glucose production in liver is accelerated in Hvcn1-deficient mice. The present study sheds light on the functional importance of Kupffer cells in hepatic oxidative stress and its potential relationship with glucose metabolism.

Kawai T ，Kayama K ，Tatsumi S ，Akter S ，Miyawaki N ，Okochi Y ，Abe M ，Sakimura K ，Yamamoto H ，Kihara S ，Okamura Y ... -  《-》

Hv1/VSOP regulates neutrophil directional migration and ERK activity by tuning ROS production.

High-level reactive oxygen species (ROS) production in neutrophils is tightly regulated, as it can damage host cells. Neutrophils also undergo low-level ROS production when stimulated by cytokines or chemoattractants, but its biologic significance remains largely unknown. Voltage-gated proton channels (Hv1/VSOP) activity reportedly supports ROS production in neutrophils; however, we show here that Hv1/VSOP balances ROS production to suppress neutrophil directional migration in the presence of low concentrations of N-formyl-Met-Leu-Phe (fMLF). Neutrophils derived from Hvcn1 gene knockout mice produced more ROS than neutrophils from wild-type mice in the stimulation with fMLF at concentration of 1 µM and nonstimulus condition. They also exhibited stronger chemotactic responses to low concentrations of fMLF than did wild-type neutrophils. Receptor sensitivity to fMLF and evoked Ca2+ responses did not differ between Hv1/VSOP-deficient and wild-type neutrophils. Activation of ERK, but not p38, was enhanced and prolonged during the increased ROS production seen after fMLF stimulation in Hv1/VSOP-deficient neutrophils. Inhibiting ROS production suppressed the enhanced ERK activation in Hv1/VSOP-deficient neutrophils and their directional migration. These results indicate that Hv1/VSOP balances ROS production to reduce ERK signaling and suppress excessive neutrophil migration in response to fMLF. Our findings thus reveal a novel role for ROS in the directional migration of neutrophils.

Okochi Y ，Umemoto E ，Okamura Y 《-》