Abstract
Mammalian granulocyte colony-stimulating factor (G-CSF; CSF3) is a primary cytokine that promotes the development, mobilization, and activation of neutrophils and their precursors. Teleosts have been reported to possess two paralogs as a likely result of the teleost-wide whole genome duplication (WGD) event, but functional divergence of G-CSF paralogs remains poorly understood. Common carp are an allotetraploid species owing to an additional WGD event in the carp lineage and here, we report on genomic synteny, sequence similarity, and phylogeny of four common carp G-CSF paralogs (g-csfa1 and g-csfa2; g-csfb1 and g-csfb2). G-csfa1 and g-csfa2 show differential and relatively high gene expression levels, while g-csfb1 and g-csfb2 show low basal gene expression levels in most tissues. All paralogs are expressed higher in macrophages than in other leukocyte sub-types and are highly up-regulated by treatment of macrophages with mitogens. Recombinant G-CSFa1 and G-CSFb1 both promoted the proliferation of kidney hematopoietic cells, while only G-CSFb1 induced the differentiation of kidney cells along the neutrophil-lineage. Colony-forming unit assays revealed that G-CSFb1 alone stimulates the formation of CFU-G colonies from head- and trunk-kidney whereas the combination of G-CSFa1 and G-CSFb1 stimulates the formation of both CFU-G and CFU-GM colonies. Recombinant G-CSFa1 and G-CSFb1 also exhibit chemotactic activity against kidney neutrophils and up-regulation of cxcr1 mRNA expression was highest in neutrophils after G-CSFb1 stimulation. Furthermore, G-CSFb1 more than G-CSFa1 induced priming of kidney neutrophils through up-regulation of a NADPH-oxidase component p47phox. In vivo administration of G-CSF paralogs increased the number of circulating blood neutrophils of carp. Our findings demonstrate that gene duplications in teleosts can lead to functional divergence between paralogs and shed light on the sub-functionalization of G-CSF paralogs in cyprinid fish.
Highlights
Granulocyte colony-stimulating factor (G-CSF), called colony-stimulating factor 3 (CSF3), is a primary cytokine that promotes the proliferation, differentiation and survival of neutrophil progenitors and enhances trafficking and immunological functions of mature neutrophils in mammals [1]
In order to determine induction of the different granulocyte colony-stimulating factor (G-CSF) paralogs upon antigenic stimulation, we investigated expression levels in freshly isolated kidney leukocytes and head kidney-derived macrophages following the stimulation with LPS, concanavaline A (ConA), phorbol 12-myristate 13-acetate (PMA) and poly I:C
To characterize the roles of G-CSFa1 and G-CSFb1, we examined the gene expressions of transcription factors (TFs) and cell surface markers involved in the development of various cell lineages in carp kidney leukocytes treated with G-CSFa1, G-CSFb1 and a combination of G-CSFa1 and G-CSFb1
Summary
Granulocyte colony-stimulating factor (G-CSF), called colony-stimulating factor 3 (CSF3), is a primary cytokine that promotes the proliferation, differentiation and survival of neutrophil progenitors and enhances trafficking and immunological functions of mature neutrophils in mammals [1]. Mice lacking G-CSF/G-CSFR signaling (G-csf -deficient or G-csfr-deficient mice) exhibit a reduction in myeloid progenitors and impaired neutrophil mobilization into the circulation, resulting in chronic neutropenia [9, 10]. This suggests that G-CSF is a major regulator of neutrophil development and contributes to the regulation of multipotent hematopoietic progenitors. G-CSF influences the phenotype and function of mature neutrophils and does so by modulating expression of for example chemokine receptors, up-regulating phagocytosis and production of reactive oxygen species (ROS) and enhancing bactericidal activity of neutrophils [11]
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