The Function Of Alas1 On Zebrafish Neutrophil Maturation

Neutrophils are the most abundant type of white blood cells in higher animals.They play essential roles in innate immunity and are the first responders to kill foreign micro-organisms by phagocytosis,degranulation,and generation of neutrophil extracellular traps,which means the normal development of neutrophils are required for defending infections in body.The formation,differentiation and maturation of neutrophils are regulated by various genes.Anything wrong will change their quantity,distribution,protein components and activities,leading to specific disorders,such as chronic granulomatous disease,leukocyte adhesion deficiency,specific granule deficiency,and leukopenia.The understanding of neutrophil development network will light up the way in treatment of such diseases.Due to the limitations of traditional animal models and the lack of neutrophil-specific deficient mutant,the regulation mechanism of embryonic neutrophil development remains little understood.In this study,we attempt to reveal a novel regulation mechanism on neutrophil development by the advantage of genetics,embryonic development and live imaging in zebrafish,contributing on understanding of relative diseases.Here we utilized neutrophil-deficient zebrafish to identify a novel role of Alas1,a heme biosynthesis pathway enzyme,in neutrophil development.We previously obtained a neutrophil-deficient mutant zebrafish line named smu350 by large-scale forward genetic screening of the ENU-mutagenized zebrafish.We mapped the mutated gene to alas1.a point mutation of whose exon-intron boundary disrupted mRNA splicing,leading to the disruption of the enzymatic activity domain of the Alas1 protein and the weakness of protein stability.The fact that alas1 mutation caused neutrophil defect was confirmed by another alas1 mutant generated by CRISPR/Cas9,We showed that Alasl-deficient zebrafish exhibited proper neutrophil initiation,but further neutrophil maturation was blocked,with lipid storage and granule formation deficiencies,and loss of heme-dependent granule protein activities.Consequently,Alas1-deficient zebrafish showed impaired bactericidal ability and augmented inflammatory responses when challenged with Escherichia coli.Further research revealed that the functional deficiency of alasl led to heme deficiency in neutrophils,while the heme in erythrocytes catalyzed by Alas2,the isozyme of Alasl,was increased.However,as the heme transport was also impaired by alas1 mutation,the heme deficiency in neutrophils could not be compensated and neutrophils remained defective.These findings demonstrate the important role of Alasl in regulating neutrophil maturation and physiological function through the heme.Our study provides an in vivo model of Alas1 deficiency and may be useful to evaluate the progression of heme-related disorders in order to facilitate the development of drugs and treatment strategies for these diseases.