A homologue of interleukin 18 has been identified from rainbow trout, Oncorhynchus mykiss. The trout IL‐18 gene spans 3.7 kb and consists of six exons and five introns, sharing the same gene organization with its human counterpart. The putative translated protein is 199 amino acids in length with no predicted signal peptide. Analysis of the multiple sequence alignment reveals a conserved ICE cut site, resulting in a mature peptide of 162 amino acids. The trout IL‐18 shares 41–45% similarity with known IL‐18 molecules and contains an IL‐1 family signature motif. It is constitutively expressed in a wide range of tissues including brain, gill, gut, heart, kidney, liver, muscle, skin and spleen. Transcription is not modulated by lipopolysaccharide, poly(I:C) or trout recombinant IL‐1β in primary head kidney leucocyte cultures and RTS‐11 cells, a macrophage cell line. However, expression is downregulated by lipopolysaccharide and rIL‐1β in RTG‐2 cells, a fibroblast‐like cell line. An alternatively spliced form of IL‐18 mRNA has also been found and translates into a 182 amino acid protein with a 17 amino acid deletion in the precursor region of the authentic form. This alternatively spliced form is also widely expressed although much lower than the authentic form. Interestingly, its expression is upregulated by lipopolysaccharide and poly(I:C), but is not affected by rIL‐1β in RTG‐2 cells. The present study suggests that alternative splicing may play an important role in regulating IL‐18 activities in rainbow trout.

In mammals, type I IFNs, which commonly contain one or two disulfide bonds, activate the JAK-STAT signaling pathway through binding to the common cell surface receptor formed by IFN-α/β receptor (IFNAR)1 and IFNAR2 subunits. Although type I IFNs are also known to be essential for antiviral defense in teleost fish, very little is known about mechanisms underlying the recognition of fish type I IFNs by associated receptors. In this study, we demonstrate that a type I IFN of large yellow croaker Larimichthys crocea (LcIFNi), belonging to a new subgroup of fish type I IFNs, triggers antiviral response via the conserved JAK-STAT pathway through stable binding with a heterodimeric receptor comprising subunits LcCRFB5 and LcCRFB2. LcIFNi binds to LcCRFB5 with a much higher affinity than to LcCRFB2. Furthermore, we determined the crystal structure of LcIFNi at a 1.39 Å resolution. The high-resolution structure is, to our knowledge, the first reported structure of a type I IFN with three disulfide bonds, all of which were found to be indispensable for folding and stability of LcIFNi. Using structural analysis, mutagenesis, and biochemical assays, we identified key LcIFNi residues involved in receptor interaction and proposed a structural model of LcIFNi bound to the LcCRFB2–LcCRFB5 receptor. The results show that LcIFNi–LcCRFB2 exhibits a similar binding pattern to human IFN-ω–IFNAR2, whereas the binding pattern of LcIFNi–LcCRFB5 is quite different from that of IFN-ω–IFNAR1. Altogether, our findings reveal the structural basis for receptor interaction and signaling of a type I IFN with three disulfide bonds and provide new insights into the mechanisms underlying type I IFN recognition in teleosts.

Interleukin (IL)-22 plays an important role in regulating inflammation and clearance of infectious pathogens. IL-22 homologs have been discovered in fish, but the functions and sources of IL-22 have not been fully characterized. In this study, an IL-22 homolog was identified in grass carp and its bioactivities were investigated. The grass carp IL-22 was constitutively expressed in tissues, with the highest expression detected in the gills and hindgut. It was upregulated in the spleen after infection with Flavobacterium columnare and grass carp reovirus and in the primary head kidney and spleen leukocytes stimulated with LPS and IL-34. Conversely, it was downregulated by Th2 cytokines such as IL-4/13B and IL-10. The recombinant IL-22 produced in bacteria showed a stimulatory effect on the expression of inflammatory cytokines and STAT3 in the primary head kidney leukocytes and CIK cells. Moreover, the IL-22-positive cells were found to be induced in the hindgut and head kidney 24 h after infection by F. columnare. Our data suggest that IL-22 plays an important role in regulating mucosal and systemic immunity against bacterial and viral infection.