Abstract
Immune-modulatory effects of β-glucans are generally considered beneficial to fish health. Despite the frequent application of β-glucans in aquaculture practice, the exact receptors and downstream signalling remains to be described for fish. In mammals, Dectin-1 is a member of the C-type lectin receptor (CLR) family and the best-described receptor for β-glucans. In fish genomes, no clear homologue of Dectin-1 could be identified so far. Yet, in previous studies we could activate carp macrophages with curdlan, considered a Dectin-1-specific β-(1,3)-glucan ligand in mammals. It was therefore proposed that immune-modulatory effects of β-glucan in carp macrophages could be triggered by a member of the CLR family activating the classical CLR signalling pathway, different from Dectin-1. In the current study, we used primary macrophages of common carp to examine immune modulation by β-glucans using transcriptome analysis of RNA isolated 6 h after stimulation with two different β-glucan preparations. Pathway analysis of differentially expressed genes (DEGs) showed that both β-glucans regulate a comparable signalling pathway typical of CLR activation. Carp genome analysis identified 239 genes encoding for proteins with at least one C-type Lectin Domains (CTLD). Narrowing the search for candidate β-glucan receptors, based on the presence of a conserved glucan-binding motif, identified 13 genes encoding a WxH sugar-binding motif in their CTLD. These genes, however, were not expressed in macrophages. Instead, among the β-glucan-stimulated DEGs, a total of six CTLD-encoding genes were significantly regulated, all of which were down-regulated in carp macrophages. Several candidates had a protein architecture similar to Dectin-1, therefore potential conservation of synteny of the mammalian Dectin-1 region was investigated by mining the zebrafish genome. Partial conservation of synteny with a region on the zebrafish chromosome 16 highlighted two genes as candidate β-glucan receptor. Altogether, the regulation of a gene expression profile typical of a signalling pathway associated with CLR activation and, the identification of several candidate β-glucan receptors, suggest that immune-modulatory effects of β-glucan in carp macrophages could be a result of signalling mediated by a member of the CLR family.
Highlights
Immunomodulation by β-glucans has been widely studied in teleost fish
Subsets of candidate receptors were selected from the restricted number of proteins based on the following three criteria: [1] presence of a conserved WxH motif in the C-type Lectin Domains (CTLD); [2] corresponding expression of ≥50 reads per kilobase million (RPKM) in unstimulated macrophages (n = 5); [3] differential regulation of expression in carp macrophages stimulated with β-glucans
Primary macrophages of common carp had previously been shown to respond to prototypical Dectin-1 ligands, which led to the hypothesis that the C-type lectin receptor (CLR) pathway must play an important role in the recognition of β-glucans in carp macrophages
Summary
Immunomodulation by β-glucans has been widely studied in teleost fish. Regardless of the administration route or fish species, β-glucan administration often has an immune stimulatory effect and can result in increased resistance to both viral and bacterial infections [reviewed by [1,2,3]]. Β-glucan was shown to regulate the expression of several pattern recognition receptors including tlr in primary macrophages of European eel (Anguilla anguilla) [26], regulation of tlr and cxc receptors in common carp [27, 28], the purinergic receptor p2x4 in Japanese flounder (Paralichthys olivaceus) [29] and nod and tlr in zebrafish (Danio rerio) [30]. Subsequent investigation of the common carp genome for candidate β-glucan receptors identified a number of genes based on their architecture or expression profile, all encoding proteins with at least one C-type Lectin Domain (CTLD). Our study identifies several teleost CLRs of interest for future functional studies aimed at further specifying the modulatory effects of β-glucans on the fish immune system
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