Dual Roles for Rac2 in Neutrophil Motility and Active Retention in Zebrafish Hematopoietic Tissue

Abstract

Summary Neutrophil homeostasis is essential for host defense. Here we identify dual roles for Rac2 during neutrophil homeostasis using a zebrafish model of primary immune deficiency induced by the human inhibitory Rac2D57N mutation in neutrophils. Non-invasive live imaging of Rac2 morphants or Rac2D57N zebrafish larvae demonstrates an essential role for Rac2 in regulating 3D motility and the polarization of F-actin dynamics and PI(3)K signaling in vivo. Tracking of photolabeled Rac2-deficient neutrophils from hematopoietic tissue also shows increased mobilization into the circulation, indicating that neutrophil mobilization does not require traditionally defined cell motility. Moreover, excessive neutrophil retention in hematopoietic tissue resulting from a constitutively-active CXCR4 mutation in zebrafish WHIM syndrome is partially rescued by the inhibitory Rac2 mutation. These findings reveal that Rac2 signaling is necessary for both neutrophil 3D motility and CXCR4-mediated neutrophil retention in hematopoietic tissue, thereby limiting neutrophil mobilization, a critical first step in the innate immune response.

Control of Mucosal Candidiasis in the Zebrafish Swim Bladder Depends on Neutrophils That Block Filament Invasion and Drive Extracellular-Trap Production

“…Rac2, PI3K, and CXCR2 are all important in neutrophil activity in both zebrafish and mammals (34,38,(47)(48)(49)(50). Further, the unchanged neutrophil recruitment upon CXCR2 inhibition suggests that neutrophil activation may be an important function of CXCR2.…”

“…We used a fish line developed in the laboratory of Anna Huttenlocher in which the dominant negative Rac2-D57N mutant protein is expressed only in neutrophils, inhibiting their recruitment and rendering this fish line more susceptible to several infections (38)(39)(40). Using this genetic strategy for inhibition of neutrophil recruitment, we found that neutrophil recruitment itself is required for resistance to mucosal candidiasis in the swim bladder (Fig.…”

Migration of cardiomyocytes is essential for heart regeneration in zebrafish

“…7). Recent studies also showed neutrophil mobilization in zebrafish larvae (Deng et al, 2011) and developmental timing assays during zebrafish heart development (de Pater et al, 2009) with similar approaches. Thus, localized photoconversion in combination with the use of cell-type-specific promoters/enhancers would be a valuable approach for cell migration and lineage analysis in a variety of biological processes.…”

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“…The potential of advanced microfluidic platforms to study chemotaxis mechanisms in immune cells is strengthened by recent development of molecular probes and imaging technol ogies (Wang et al, 2008;Deng et al, 2011). Immunofluores cence staining against specific cell surface and intracellular markers is a well established technique (Fritschy and Härtig, 2001;Robinson et al, 2010).…”