No abstract

In mammals, cytosolic sensors retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) activate multiple signaling cascades initiating IFN-α/β expression. IFN regulatory factor 3 (IRF3) is required for the activation of IFN-β, which, in turn, primes the expression of most IFN-α genes by IFN-induced IRF7 through the STAT1 pathway. In fish, RIG-I overexpression inhibits virus infection by induction of IFN response; however, the subtle signaling cascade mechanism remains to be identified. In this study, we clone an ortholog of MITA, a recently identified adaptor responsible for RLR pathway, from crucian carp (Carassius auratus L.), and demonstrate its ability to suppress viral replication through IRF3/7-dependent IFN response. The pivotal signaling molecules of RLR pathway, including RIG-I, melanoma differentiation-associated gene 5, laboratory of genetics and physiology 2, and TANK-binding kinase 1, are also cloned and characterized, confirming that the RLR-mediated IFN activation is conserved from fish to mammals. Further characterization of distinct IFN gene activation reveals that zebrafish IFN1 and IFN3 are induced by the MITA pathway but are dependent on distinct transcription factors. Whereas fish IFN genes cannot be classified into IFN-α or IFN-β, zebrafish IFN1 is primarily regulated by IRF3, thereby resembling that of IFN-β, and zebrafish IFN3 is regulated by IRF7, thereby resembling of those of IFN-αs. In contrast with mammalian IFN-α/β, zebrafish IFN1 and IFN3 are induced by the basally expressed IRF3 or IRF7, both of which are upregulated by IFN and virus infection. Collectively, these data suggest that IFN genes in fish and mammals have evolved independently to acquire a similar mechanism triggering their expression.

Dectin-1 is a C-type lectin-like receptor on leukocytes that mediates phagocytosis and inflammatory mediator production in innate immunity to fungal pathogens. Dectin-1 lacks residues involved in calcium ligation that mediates carbohydrate-binding by classical C-type lectins; nevertheless, it binds zymosan, a particulate ␤-glucan-rich extract of Saccharomyces cerevisiae, and binding is inhibited by polysaccharides rich in ␤1,3-or both ␤1,3-and ␤1,6-linked glucose. The oligosaccharide ligands on glucans recognized by Dectin-1 have not yet been delineated precisely. It is also not known whether Dectin-1 can interact with other types of carbohydrates. We have investigated this, since Dectin-1 shows glucan-independent binding to a subset of T-lymphocytes and is involved in triggering their proliferation. Here we assign oligosaccharide ligands for Dectin-1 using the neoglycolipid-based oligosaccharide microarray technology, a unique approach for constructing microarrays of lipid-linked oligosaccharide probes from desired sources. We generate "designer" microarrays from three glucan polysaccharides, a neutral soluble glucan isolated from S. cerevisiae and two bacterial glucans, curdlan from Alcaligenes faecalis and pustulan from Umbilicaria papullosa, and use these in conjunction with 187 diverse, sequence-defined, predominantly mammalian-type, oligosaccharide probes. Among these, Dectin-1 binding is detected exclusively to 1,3-linked glucose oligomers, the minimum length required for detectable binding being a 10-or 11-mer. Thus, the ligands assigned so far are exogenous rather than endogenous. We further show that Dectin-1 ligands, 11-13 gluco-oligomers, in clustered form (displayed on liposomes), mimic the macromolecular ␤-glucans and compete with zymosan binding and triggering of tumor necrosis factor-␣ secretion by a Dectin-1-expressing macrophage cell line.Dectin-1 is the main receptor on leukocytes that mediates innate immunity to fungal pathogens (1, 2). It is a germ line-encoded membrane-associated cell surface glycoprotein, first identified on a mouse dendritic cell line and cultured Langerhans cells (3) and on a murine macrophage cell line (4). It is now known to be also expressed at the surface of monocytes and neutrophils and at low levels on a subpopulation of T-cells (5). Studies of the tissue distribution of the murine Dectin-1 by immunohistochemistry have shown it to be expressed in the spleen, thymus, liver, lung, and gut and at low levels in the skin, but it is not detectable in the brain, heart, kidney, or eye (6). The human homologue of Dectin-1 has also been characterized (4, 7-9) and resembles the murine protein but differs in that the transcript is alternatively spliced, resulting in two major and several minor isoforms. The major isoforms appear to serve the same immune function as the murine receptor (10).The deduced amino acid sequence of Dectin-1 is that of a type II transmembrane protein, with an extracellular lectin-like domain at the C terminus followed by a stalk region and a short cyto...