Real-time functional analysis of Hv1 channel in neutrophils: a new approach from zebrafish model

• Published in: American journal of physiology. Regulatory, integrative and comparative physiology Vol. 316; no. 6; pp. R819 - R831
• Main Authors: Ratanayotha, Adisorn, Kawai, Takafumi, Okamura, Yasushi
• Format: Journal Article
• Language: English
• Published: United States American Physiological Society 01.06.2019
• Subjects: Animals; Animals, Genetically Modified; Bacterial infections; Calcium; Calcium Signaling; Danio rerio; Embryos; Functional analysis; Immune response; Immune system; Immunity, Innate; In vivo methods and tests; Infections; Innate immunity; Ion Channel Gating; Ion Channels - deficiency; Ion Channels - genetics; Ion Channels - metabolism; Larvae; Leukocytes (neutrophilic); Mammals; Membrane Potentials; Neutrophils; Neutrophils - immunology; Neutrophils - metabolism; Phagocytosis; Phagosomes; Phagosomes - immunology; Phagosomes - metabolism; Physiology; Protons; Reactive oxygen species; Reactive Oxygen Species - metabolism; Real time; Survival analysis; Time Factors; Vertebrates; Zebrafish; Zebrafish - embryology; Zebrafish - genetics; Zebrafish - immunology; Zebrafish - metabolism; Zebrafish Proteins - deficiency; Zebrafish Proteins - genetics; Zebrafish Proteins - metabolism; proton channel; phagocytosis; in vivo model; zebrafish; reactive oxygen species
• ISSN: 0363-6119; 1522-1490
• DOI: 10.1152/ajpregu.00326.2018

Voltage-gated proton channel (Hv1) has been studied in various immune cells, including neutrophils. However, most studies have taken an in vitro approach using isolated cells or primary cultured cells of mammals; therefore, limited evidence is available on the function of Hv1 in a physiological context. In this study, we have developed the in vivo system that enables real-time functional analysis of Hv1 using zebrafish embryos ( ). -deficiency ( ) in zebrafish completely abolished voltage-gated proton current, which is typically observed in wild-type neutrophils. Importantly, -deficiency significantly reduced reactive oxygen species production and calcium response of zebrafish neutrophils, comparable to the results observed in mammalian models. These findings verify zebrafish Hv1 (DrHv1) as the primary contributor for native Hv1-derived proton current in neutrophils and suggest the conserved function of Hv1 in the immune cells across vertebrate animals. Taking advantage of Hv1 zebrafish model, we compared real-time behaviors of neutrophils between wild-type and zebrafish in response to tissue injury and acute bacterial infection. Notably, we observed a significant increase in the number of phagosomes in neutrophils, raising a possible link between Hv1 and phagosomal maturation. Furthermore, survival analysis of zebrafish larvae potentially supports a protective role of Hv1 in the innate immune response against systemic bacterial infection. This study represents the influence of Hv1 on neutrophil behaviors and highlights the benefits of in vivo approach toward the understanding of Hv1 in a physiological context.