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Regulation of neutrophil maturation in zebrafish
expressions of these heme transporter genes were decreased in alas1smu350/smu350 mutants compared with their siblings (Figure 4F), probably due to the feedback regula- tion of aberrant heme contents in alas1smu350/smu350 mutants. Thus, the heme transport deficiency might be one of the reasons that the elevated erythroid heme could not be uti- lized by neutrophils in alas1smu350/smu350 mutants.
Heme was essential for neutrophil maturation
To confirm whether the neutrophil maturation defects were caused by inadequate heme levels, we next treated wild-type zebrafish embryos with SA, an inhibitor of δ-aminolevulinic acid dehydratase, which catalyzes the second step in heme biosynthesis pathway,40 to inhibit the endogenous heme levels. Total heme levels of SA-treated embryos were significantly decreased compared with untreated control embryos (Figure 5A). As reported, the o-Dianisidine staining signal was decreased as hemoglobin synthesis is inhibited without heme (Figure 5B).23,40 When we monitored the neutrophil phenotypes, we found that SA-treated embryos showed loss of SB and DAB staining but intact lyz and mpx expression (Figure 5C-F), which mimics the neutrophil maturation defects in Alas1-defi- cient mutants. These data suggest that the neutrophil defects in alas1smu350/smu350 mutant are indeed caused by inad- equate effective heme in neutrophils.
Neutrophil bactericidal defects in alas1smu350/smu350 mutants
Neutrophils play key roles in various functions, includ- ing action against certain infections, largely depending on granule proteins.1 The neutrophil granule defects suggest that the anti-infection ability of alas1smu350/smu350 mutant neu- trophils may be attenuated. To detect whether alas1 defi- ciency affected neutrophil bactericidal function, alas1smu350/smu350 mutants were challenged with a bacterial infection.26,30 We subcutaneously injected eGFP labeled E. coli over one somite in 3-dpf Tg(lyz:DsRed);alas1smu350/+ inter- crossed embryos, in which DsRed was expressed specifi- cally in neutrophils (Figure 6A). Neutrophil behavior and immune responses were then monitored. We first moni- tored in vivo bacterial growth and detected the kinetic curves of the bacterial burden of infected embryos. In sib- ling embryos, bacteria growth was inhibited effectively in the host, as green fluorescent bacteria decreased rapidly in the infection site (Figure 6B-E). By further plating the homogenized embryos/larvae on LB medium for quantifi- cation, we found that bacterial colonies were gradually decreased from 5 h post injection (hpi) and eventually became almost absent at 24 hpi (Figure 6F), suggesting the inhibition of bacterial growth in the host. In mutant embryos, the eGFP+ bacterial load was similar to siblings within the first 3 hpi (Figure 6B and C), but fluorescent bacteria were still accumulating at 5 hpi and persisted at 24 hpi, when clearance had been completed in the siblings (Figure 6D-E). Quantification data consistently showed that the plated colony numbers from mutants were similar to those of siblings within the first 3 hpi, but the numbers were significantly higher at 5 and 24 hpi than those of the siblings (Figure 6F), suggesting antimicrobial activity was impaired in alas1smu350/smu350 mutants. We further counted the number of neutrophils recruited to the infection site. Within the first 5 hpi, alas1smu350/smu350 embryos showed sim- ilar neutrophil recruitment to sibling embryos (Figure 6B-
D, G). However, neutrophils were still accumulating at the infection site in alas1smu350/smu350 larvae at 24 hpi, when recruited neutrophils had almost completely disappeared in the siblings (Figure 6E and G), confirming the defect in neutrophil-specific antibacterial response in alas1smu350/smu350 mutants.
To gain further insight into the infection-induced inflammatory alterations, we detected the expression of inflammatory factors il1b and cxcl8a,41,42 which induce neu- trophil chemotaxis and promote immune responses. Expression analyses showed that both genes were signifi- cantly up-regulated in infected alas1smu350/smu350 mutants com- pared with siblings. The il1b and cxcl8a expressions peaked in the mutants at 5 hpi, at which point their expressions had already been down-regulated in the sib- lings (Figure 6H and I), suggesting a more dramatic inflam- matory response in alas1smu350/smu350 mutants. Taken together, these data demonstrate that alas1 deficiency causes impaired immune responses to bacterial infection.
Discussion
In this study, we showed a role for the heme biosynthe- sis pathway enzyme Alas1 in regulating neutrophil matu- ration and function. Neutrophils in Alas1-deficient zebrafish had heme deficiency, which led to the loss of heme-related granule protein activities, defective granule formation, and altered immune responses against patho- genic bacteria.
Here, we found that the heme dysregulation caused by the alas1 mutation led to neutrophil defects in zebrafish. Given the important role of neutrophils in immunity, it was expected that Alas1-deficient zebrafish would show impaired bactericidal ability. Inflammatory factors, such as il1b and cxcl8a,41,42 mediate neutrophil recruitment and promote immune responses. The over-elevated expression of inflammatory factors in infected alas1smu350/smu350 mutants might be explained by the ineffectiveness of killing bacte- ria, thereby leading to greater pathogen growth and stronger immune responses in the host. We also noticed that the inflammatory responses eventually subsided while the bacterial burden and recruited neutrophils were still present at 24 hpi in Alas1-deficient zebrafish, so it was likely that the inflammatory factors had been excessively depleted.
As far as we know, the regulatory genes and pathways of heme biosynthesis, degradation and transport are large- ly conserved between mammals and zebrafish in gener- al.20,23,35,36,43 Vertebrates contain two ALAS isozymes encod- ed by 2 distinct genes located on different chromosomes; ALAS2 is expressed in erythrocytes, whereas ALAS1 is ubiquitously expressed.11 By searching the integrated RNA-seq database on BloodSpot,44 we found that, in humans and mice, ALAS1 is highly expressed in myeloid cells, which is consistent with our zebrafish data and part- ly explains the importance of alas1 for neutrophils. In mice, ALAS1 is also highly expressed in the liver, exocrine, and endocrine glands, suggesting specific roles in those tis- sues.24 Accordingly, even though alas1 is ubiquitously expressed, we suspect that alas1 may play specific roles in certain tissues to meet the need of hemoproteins. It is reported that ALAS1-null mice died in utero until E8.5, with a severely retarded morphology, indicating that ALAS1 is essential for the early development of mouse embryos.24
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