Rac2 expression and its role in neutrophil functions of zebrafish (Danio rerio)

The neutrophil contributes significantly to the immune response. In particular, their phagocytosis and pathogen-killing functions are vital for defense from invading pathogens. Rac2, a Rho small GTPase, is involved in many key neutrophil functions. Loss of Rac2 activity results in severe bacterial infections and neutrophil function deficits in humans and mice. While the genes rac1, 2, and 3 have been identified in the zebrafish genome, their expression has not been well-characterized. We describe rac1, 2, and 3 expression over the first three days of development, as well as the presence and localization of Rac2 protein in adult zebrafish neutrophils. The mRNA for each Rac isoform was detected in zebrafish embryos as early as 12 h post fertilization. Immunocytochemistry and confocal microscopy of adult zebrafish neutrophils confirmed diffuse Rac2 protein within the cytoplasm. Only rac2 was found in sorted neutrophil samples. Armed with knowledge of its presence and exclusive expression, the role of Rac2 in key antimicrobial zebrafish neutrophil responses was examined by small molecule inhibition of Rac during respiratory burst, NET release, and phagocytosis assays. Inhibition of Rac2 during these assays produced a dose-dependent decrease in each function, as was expected due to previous work in mammals. The expression pattern and role of Rac2 in zebrafish neutrophil function allows for comparative studies of innate immune responses in this animal model.

► rac2 is present by 12 h post fertilization in the developing zebrafish embryo.
► Rac2 shows a cytoplasmic staining pattern in zebrafish neutrophils as in humans.
► rac2 was found in all adult zebrafish neutrophils samples.
► rac1 was present only in macrophage-contaminated samples.
► Rac inhibition decreased neutrophil respiratory burst, NET release, and phagocytosis.