First in-depth analysis of the novel Th2-type cytokines in salmonid fish reveals distinct patterns of expression and modulation but overlapping bioactivities.

IL-4 and IL-13 are closely related canonical type-2 cytokines in mammals and have overlapping bioactivities via shared receptors. They are frequently activated together as part of the same immune response and are the signature cytokines produced by T-helper (Th)2 cells and type-2 innate lymphoid cells (ILC2), mediating immunity against extracellular pathogens. Little is known about the origin of type-2 responses, and whether they were an essential component of the early adaptive immune system that gave a fitness advantage by limiting collateral damage caused by metazoan parasites. Two evolutionary related type-2 cytokines, IL-4/13A and IL-4/13B, have been identified recently in several teleost fish that likely arose by duplication of an ancestral IL-4/13 gene as a consequence of a whole genome duplication event that occurred at the base of this lineage. However, studies of their comparative expression levels are largely missing and bioactivity analysis has been limited to IL-4/13A in zebrafish. Through interrogation of the recently released salmonid genomes, species in which an additional whole genome duplication event has occurred, four genomic IL-4/13 loci have been identified leading to the cloning of three active genes, IL-4/13A, IL-4/13B1 and IL-4/13B2, in both rainbow trout and Atlantic salmon. Comparative expression analysis by real-time PCR in rainbow trout revealed that the IL-4/13A expression is broad and high constitutively but less responsive to pathogen-associated molecular patterns (PAMPs) and pathogen challenge. In contrast, the expression of IL-4/13B1 and IL-4/13B2 is low constitutively but is highly induced by viral haemorrhagic septicaemia virus (VHSH) infection and during proliferative kidney disease (PKD) in vivo, and by formalin-killed bacteria, PAMPs, the T cell mitogen PHA, and the T-cell cytokines IL-2 and IL-21 in vitro. Moreover, bioactive recombinant cytokines of both IL-4/13A and B were produced and found to have shared but also distinct bioactivities. Both cytokines rapidly induce the gene expression of antimicrobial peptides and acute phase proteins, providing an effector mechanism of fish type-2 cytokines in immunity. They are anti-inflammatory via up-regulation of IL-10 and down-regulation of IL-1β and IFN-γ. They modulate the expression of cellular markers of T cells, macrophages and B cells, the receptors of IFN-γ, the IL-6 cytokine family and their own potential receptors, suggesting multiple target cells and important roles of fish type-2 cytokines in the piscine cytokine network. Furthermore both cytokines increased the number of IgM secreting B cells but had no effects on the proliferation of IgM+ B cells in vitro. Taken as a whole, fish IL-4/13A may provide a basal level of type-2 immunity whilst IL-4/13B, when activated, provides an enhanced type-2 immunity, which may have an important role in specific cell-mediated immunity. To our knowledge this is the first in-depth analysis of the expression, modulation and bioactivities of type-2 cytokines in the same fish species, and in any early vertebrate. It contributes to a broader understanding of the evolution of type-2 immunity in vertebrates, and establishes a framework for further studies and manipulation of type-2 cytokines in fish.