Neutrophil-specific knockout demonstrates a role for mitochondria in regulating neutrophil motility in zebrafish

Abstract

Neutrophils are fast-moving cells essential for host immune functions. Although they primarily rely on glycolysis for ATP, isolated primary human neutrophils depend on mitochondrial membrane potential for chemotaxis. However, it is not known whether mitochondria regulate neutrophil motility in vivo, and the underlying molecular mechanisms remain obscure. Here, we visualized mitochondria in an interconnected network that localizes to the front and rear of migrating neutrophils using a novel transgenic zebrafish line. To disrupt mitochondrial function genetically, we established a gateway system harboring the CRISPR/Cas9 elements for tissue-specific knockout. In a transgenic line, neutrophil-specific disruption of mitochondrial DNA polymerase, polg, significantly reduced the velocity of neutrophil interstitial migration. In addition, inhibiting the mitochondrial electron transport chain or the enzymes that reduce mitochondrial reactive oxygen species also inhibited neutrophil motility. The reduced cell motility that resulted from neutrophil-specific knockout of sod1 was rescued with sod1 mRNA overexpression, or by treating with scavengers of reactive oxygen species. Together, our work has provided the first in vivo evidence that mitochondria regulate neutrophil motility, as well as tools for the functional characterization of mitochondria-related genes in neutrophils and insights into immune deficiency seen in patients with primary mitochondrial disorders.This article has an associated First Person interview with the first author of the paper.

Mitofusin 2 regulates neutrophil adhesive migration and the actin cytoskeleton

“…In our previous work, we have confirmed the requirement of mitochondrial membrane potential and electron transport chain in the migration of zebrafish neutrophils (Zhou et al, 2018a). In addition, we have visualized a highly fused tubular network of mitochondria in zebrafish neutrophils, which is consistent with a previous report investigating primary human neutrophils (Maianski et al, 2002).…”

“…In vascular endothelial cells, mitochondria also serve as signaling rather than energy-producing moieties (Lugus et al, 2011). In our study, in addition to the altered Ca 2+ level, mitochondrial membrane potential and ROS, both critical for neutrophil chemotaxis and migration (Fossati et al, 2003;Zhou et al, 2018a), were reduced in stimulated MFN2-deficient dHL-60 cells. It remains to be determined whether the modest decreases in mitochondria membrane potential or ROS contribute to the defect of neutrophil migration upon MFN2 depletion.…”

“…Human neutrophils are terminally differentiated and undergo apoptosis within 24 hours in culture and thus are not genetically tractable. We have overcome this hurdle by developing a neutrophil-specific knockout platform in zebrafish (Zhou et al, 2018a). The zebrafish is a suitable model for neutrophil research because of its highly conserved innate immune system.…”

“…To generate transgenic zebrafish lines, plasmids with the tol2 backbone were coinjected with Tol2 transposase mRNA into embryos of the AB strain at one-cell stage as described (Zhou et al, 2018a). Constructs for neutrophil-specific knockout in zebrafish were generated as described (Zhou et al, 2018a) All mice used in this study were purchased from Jackson Laboratories. Conditional Mfn2 knockout mice (B6.129(Cg)-Mfn2 tm3Dcc /J) were crossed to S100A8-Cre (B6.Cg-Tg(S100A8-cre, -EGFP)1Ilw/J) transgenic mice to obtain a homozygous floxed Mfn2 alleles with or without the Cre.…”

Hypoxia-inducible factor-1α regulation of myeloid cells

“…To combat this, studies have focused on neutrophil-centric models of inflammation, but to tease apart the importance of neutrophil HIF-1α activity broadly, more sophistication is required. One opportunity lies in the use of clustered regularly interspaced short palindromic repeats (CRISPER)/Cas9 to target neutrophils in zebrafish [94,95]. Additionally, mice with Cre recombinase expressed at the Ly6G promoter (neutrophil specific) could generate mice with neutrophil-specific HIF-1α deletion for infection studies [96].…”