Specific function and modulation of teleost monocytes/macrophages: polarization and phagocytosis

Macrophages exist in most tissues and play a variety of functions in vertebrates.Teleost fish species are found in most aquatic environments throughout the world and are quite diverse for a group of vertebrate animals.Due to whole genome duplication and en vironme ntal adaptati on,teleost monocytes/macrophages possess a variety of different functions and modulations compared with those of mammals.A deeper understanding of teleost monocytes/macrophages in the immune system will not only help develop teleost-specific methods of disease prevention but will also help improve our understanding of the various immune mechanisms in mammals.In this review,we summarize the differences in polarizati on and phagocytosis of teleost and mammalian macrophages to improve our understanding of the various immune mechanisms in vertebrates.

Molecular characterization and functional analysis of a piscidin gene in large yellow croaker (Larimichthys crocea)

The piscidin family, which includes potent antimicrobial peptides with broad-spectrum activity, plays an important role in the innate immune system of fish. In this study, we cloned piscidin-5-1ike type 3 （Lcpis51t3） in large yellow croaker （Larimichthys crocea）. Multiple alignments with other known piscidins revealed amino acid conservation throughout the fish, especially at the signal peptide （22 amino acids）. The phylogenetic tree confirmed that Lcpis51t3 and large yellow croaker piscidin-5-1ike proteins were grouped together to form a branch. Quantitative real-time PCR revealed that Lcpis51t3 was expressed in a wide range of tissues, including the brain, muscle, gill, head kidney, intestine, kidney, liver, and spleen. The highest mRNA expression level of Lcpis51t3 was found in the spleen. After Vibrio alginolyticus infection, mRNA expression was rapidly upregulated in the liver, head kidney, gill, kidney, and intestine at 4, 8, 12, and 24 h post infection （hpi）, whereas there were no significant changes in the spleen. The antimicrobial spectrum showed that the synthetic mature peptide of Lcpis51t3 exhibited different activity in vitro against various bacteria, such as Aeromonas hydrophila, V. anguillarum, V. alginolyticus, V. parahaemolyticus, Staphylococcus aureus, and Listeria monocytogenes In addition, survival rates from the in vivo assay indicated that the synthetic peptide of Lcpis51t3 increased the survival rate of large yellow croaker after V. alginolyticus challenge, resulting in a decline in bacterial burden and mRNA expression levels of interleukin-ll3, interleukin-10, and tumor necrosis factor-（]. These data suggest that Lcpis51t3 plays an important role in innate immunity in large yellow croaker and might represent a potential therapeutic agent against pathogen invasion.

Molecular cloning, pathologically-correlated expression and functional characterization of the colony- stimulating factor 1 receptor （CSF-1R） gene from a teleost, Plecoglossus altivelis

Colony-stimulating factor 1 receptor （CSF-1R） is an important regulator of monocytes/macrophages （MO/MФ）. Although several CSF-1R genes have been identified in teleosts, the precise role of CSF- 1R in ayu （Plecoglossus altivelis） remains unclear. In this study, we characterized the CSF-1R homologue from P. altivelis, and named it PaCSF-1R. Multiple sequence alignment and phylogenetic tree analysis showed that PaCSF-1R was most closely related to that of Japanese ricefish （Oryzias latipes）. Tissue distribution and expression analysis showed that the PaCSF-1R transcript was mainly expressed in the head kidney-derived MO/MФ, spleen, and head kidney, and its expression was significantly altered in various tissues upon Vibrio anguillarum infection. After PaCSF-1R neutralization for 48 h, the phagocytic activity of MO/MФ was significantly decreased, suggesting that PaCSF-1R plays a role in regulating the phagocytic function of ayu MO/M（P.

Comparative transcriptome analysis on the alteration of gene expression in ayu （Plecoglossus altivelis）larvae associated with salinity change

Ayu （Plecoglossus altivelis） fish, which are an amphidromous species distributed in East Asia, live in brackish water （BW） during their larval stage and in fresh water （FW） during their adult stage. In this study, we found that FW-acclimated ayu larvae exhibited a slower growth ratio compared with that of BW-acclimated larvae. However, the mechanism underlying FW acclimation on growth suppression is poorly known. We employed transcriptome analysis to investigate the differential gene expression of FW acclimation by RNA sequencing. We identified 158 upregulated and 139 downregulated transcripts in FW-acclimated ayu larvae compared with that in BW-acclimated larvae. As determined by Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway mapping, functional annotation of the genes covered diverse biological functions and processes, and included neuroendo- crinology, osmotic regulation, energy metabolism, and the cytoskeleton. Transcriptional expression of several differentially expressed genes in response to FW acclimation was further confirmed by real-time quantitative PCR. In accordance with transcriptome analysis, iodothyronine deiodinase （ID）, pro-opiom- elanocortin （POMC）, betaine-homocysteine S-meth- yltransferase 1 （BHMT）, fructose-bisphosphate aldolase B （aldolase B）, tyrosine aminotransferase （TAT）, and Na＋-K＋ ATPase （NKA） were upregulated after FW acclimation. Furthermore, the mRNA expressions of b-type natriurefic peptide （BNP） and transgelin were downregulated after FW acclimation. Our data indicate that FW acclimation reduced the growth rate of ayu larvae, which might result from the expression alteration of genes related to endocrine hormones, energy metabolism, and direct osmoregulation.

Characterization and expression of sweetfish （Plecoglossus altivelis） cathepsin D

Cathepsin D(CTSD) is a lysosomal acidic endoproteinase that plays an important role in immune response. In this study, we obtained sweetfish(Plecoglossus altivelis) CTSD(PaCTSD) via de-novo transcriptome sequencing of sweetfish macrophages. The full length cDNA sequence of PaCTSD was 1 955 bp encoding a propeptide of 397 amino acids. The deduced protein had a calculated molecular weight of 43.17×103. Multiple alignment with other known CTSD amino acid sequences revealed amino acid conservation through the teleosts. Phylogenetic tree analysis showed that PaCTSD grouped tightly with other fish CTSD, and was close to that of Atlantic salmon and rainbow trout. Subsequently, PaCTSD was prokaryotically expressed and refolded by the urea gradient method on a nickel-nitrilotriacetic acid column. Enzyme activity analysis showed that PaCTSD exhibited pH-dependent proteolytic activity. Quantitative real-time PCR showed that PaCTSD mRNA was expressed in all detected tissues in healthy sweetfish. The highest expression was observed in the spleen and white blood cells, followed by liver, head-kidney, kidney, intestine, gill, and muscle. After Listonella anguillarum infection, PaCTSD transcripts were up-regulated significantly in liver, spleen, white blood cells, and head-kidney of sweetfish. In summary, PaCTSD has proteolytic activity and is closely involved in the immune response of sweetfish.