Identification and establishment of type IV interferon and the characterization of interferon-υ including its class II cytokine receptors IFN-υR1 and IL-10R2.

Interferons (IFNs) are critical soluble factors in the immune system and are composed of three types, (I, II and III) that utilize different receptor complexes IFN-αR1/IFN-αR2, IFN-γR1/IFN-γR2, and IFN-λR1/IL-10R2, respectively. Here we identify IFN-υ from the genomic sequences of vertebrates. The members of class II cytokine receptors, IFN-υR1 and IL-10R2, are identified as the receptor complex of IFN-υ, and are associated with IFN-υ stimulated gene expression and antiviral activity in zebrafish (Danio rerio) and African clawed frog (Xenopus laevis). IFN-υ and IFN-υR1 are separately located at unique and highly conserved loci, being distinct from all other three-type IFNs. IFN-υ and IFN-υR1 are phylogenetically clustered with class II cytokines and class II cytokine receptors, respectively. Therefore, the finding of this IFN ligand-receptor system may be considered as a type IV IFN, in addition to the currently recognized three types of IFNs in vertebrates.

Cloning and functional characterization of IRAK1 from rainbow trout (Oncorhynchus mykiss).

As a key molecule in innate immune signalling pathway, interleukin (IL)-1 receptor-associated kinase 1 (IRAK1) mediates downstream signalling cascades in immune response. In the present study, an IRAK1 orthologue was characterized from rainbow trout (Oncorhynchus mykiss), with a 2115 bp open reading frame (ORF), encoding a protein of 704 amino acids (aa). Multiple alignments showed that IRAK1 contains highly conserved features among different species, with a conservative N-terminal death domain (DD) and a C-terminal conserved serine/threonine protein kinase (STKc) domain. Expression analysis indicated that IRAK1 was widely expressed in examined organs/tissues, with the highest level observed in muscle and lowest in stomach. In RTG-2 cell line, the induced expression of IRAK1 was observed following the stimulation by the fish bacterial pathogen Flavobacterium columnare. Luciferase activity assays revealed that IRAK1 induced significantly the activity of NF-κB in Human embryonic kidney 293T (HEK293T) cell line; but after co-transfected with rainbow trout IL-1 receptor-associated kinase 4 (IRAK4), the induction was significantly down-regulated when compared with the expression of IRAK1 alone. Co-immunoprecipitation (Co-IP) assays indicated that IRAK1 was associated with rainbow trout myeloid differentiation factor 88 (MyD88), IRAK4 and TNF receptor associated factor 6 (TRAF6) in transfected HEK293T cells, and may form a complex with MyD88, IRAK4 and TRAF6 during the signalling pathway.

In Primitive Zebrafish, MHC Class II Expression Is Regulated by IFN-γ, IRF1, and Two Forms of CIITA.

Mammalian CIITA isoforms are tightly regulated by independent promoters. These promotors are induced by IFN-γ through JAK-STAT signaling pathway. The induction of CIITA controls the expression of MHC class II (MHCII) and Ag presentation to the adaptive immune system. In the current study, to our knowledge, we first identified two independent promoters, p1 and p2, in the zebrafish (Danio rerio) that control the expression of the two variants of CIITA, CIITA variant 1 (CIITAv1), and CIITA variant 2 (CIITAv2), respectively. Moreover, although IRF1 in an IFN-γ signaling pathway induced CIITAv2, which has two ISRE motifs in its promoter, CIITAv1 expression was not induced by this signal. Further, the transcription of MHCII DAB was controlled by IRF1 via two distinct mechanisms: 1) the transcription of MHCII DAB was controlled by IRF1 indirectly through the two ISREs in p2; and 2) directly via the ISRE in MHCII DAB promoter. We also found that IRF1 associated with CIITAv1 and CIITAv2 via protein-protein interactions to synergistically drive the transcription of MHCII DAB. The IFN-γ-IRF1-CIITA-MHCII signaling cascade was functional in early life stages of CIITA-/- and IRF1-/- zebrafish. Our findings imply that the immune system develops early in fishes and that the IFN-γ signaling cascade-induced CIITA and MHCII DAB is conserved in teleost fishes and mammals.

Unique Composition of Intronless and Intron-Containing Type I IFNs in the Tibetan Frog Nanorana parkeri Provides New Evidence To Support Independent Retroposition Hypothesis for Type I IFN Genes in Amphibians.

In vertebrates, intron-containing and intronless type I IFN genes have recently been reported in amphibian model species Xenopus tropicalis and X. laevis. However, whether intronless type I IFNs in amphibians are the ancestral genes of type I IFNs in amniotes or just represent the independent divergence in amphibians is unknown or even uninvestigated. In this study, both intron-containing and intronless type I IFN genes, as well as their receptor genes, were identified in the Tibetan frog Nanorana parkeri The evidence obtained from homology, synteny, phylogeny, and divergence time showed that intronless type I IFN genes in N. parkeri and in Xenopus might have arisen from two independent retroposition events occurred in these two lineages, and the retrotransposition causing the generation of intronless type I IFN genes in amniotes is another independent event beyond the two in amphibians. It can then be proposed that intronless type I IFNs in N. parkeri and Xenopus may not be the ancestral genes of intronless type I IFNs in amniotes but may just represent two independent bifurcations in the amphibian lineage. Furthermore, both intronless and intron-containing type I IFNs in N. parkeri showed strong ability in inducing the expression of IFN-stimulated genes and the strong antiviral activity against frog virus 3. The present study thus provides the evolutionary evidence to support the independent retroposition hypothesis for the occurrence of intronless type I IFN genes in amphibians and contributes to a functional understanding of type I IFNs in this group of vertebrates.