Abstract
The interleukin (IL)-1 family play a fundamental role as immune system modulators. Our previous transcriptome-analyses of leukocytes from lumpfish (Cyclopterus lumpus L.) showed that IL-1β was among the most highly upregulated genes following bacterial exposure. In the present study, we characterized IL-1 signaling pathways, identified and characterized four ligands of the IL-1 family in lumpfish; IL-1β type I and type II, IL-18, and the novel IL-1 family members (nIL-1F), both at mRNA and gene levels. The two IL-1β in lumpfish is termed IL-1β1 (type II) and IL-1β2 (type I). Furthermore, a comprehensive phylogenetic analysis of 277 IL-1 ligands showed that nIL-1F, in common with IL-1β, likely represents an ancestral gene, as representatives for nIL-1F were found in cartilaginous and lobe-finned fish, in addition to teleosts. This shows that nIL-1F is not exclusively present in teleosts as previously suggested. Our analyses of exon-intron structures, intron phases, phylogeny and synteny clearly show the separation of IL-1β into groups; type I and type II, which likely is a result of the third whole genome duplication (3R WGD). The phylogenetic analysis shows that most teleosts have both type I and type II. Furthermore, we have determined transcription levels of the IL-1 ligands in leukocytes and 16 different tissues, and their responses upon in vitro stimulation with seven different ligands. In addition, we have identified the IL-1 receptors IL-1R1, IL-1R2, IL-1R4 (ST2/IL-33 receptor/IL-1RL), IL-1R5 (IL-18R1), and partial sequences of DIGIRR and IL-1R3 (IL-RAcP). Identification of immune molecules and description of innate responses in lumpfish is interesting for comparative and evolutionary studies and our study constitutes a solid basis for further functional analyses of IL-1 ligands and receptors in lumpfish. Furthermore, since lumpfish are now farmed in large numbers to be used as cleaner fish for removal of sea lice on farmed salmon, in-depth knowledge of key immune molecules, signaling pathways and innate immune responses is needed, as the basis for design of efficient immune prophylactic measures such as vaccination.
Highlights
Cytokines belonging to the IL-1 family are key mediators of the body’s response to microbial invasion, inflammation, immunological reactions, and tissue injury
The differential gene expression (DEG) analysis upon bacterial exposure showed that the transcript level of genes belonging to the canonical NF-kappa B pathway (e.g., IL-1β1, IL-8, TNFα, and COX2) were most highly upregulated compared with the atypical and non-canonical pathway (Figure 1A and Supplemental Table 2), and the level of expression was higher at 24 hpe than 6 hpe (Figure 1A)
In a previous transcriptome-wide study of lumpfish leukocytes, we identified IL-1β1 and a partial sequence of a new interleukin1 family member, novel IL-1 family members (nIL-1F)
Summary
Cytokines belonging to the IL-1 family are key mediators of the body’s response to microbial invasion, inflammation, immunological reactions, and tissue injury. Multiple paralogs of many cytokines [2] and multiple IL-1β have been identified in several fish species, including channel catfish [3], salmon, trout [4, 5] and carp [3, 6]. The genes encoding IL1β in teleost fish are divided into two groups (type I and II) based on the number of exon/intron and synteny analyses [4, 7]. Type I has been identified in species belonging to Neoteleostei and Protacanthopterygii, e.g., carp, cod, salmon, and in the most evolutionary advanced fishes, such as gilthead seabream (Sparus aurata), European seabass, three-spined stickleback, Nile tilapia, southern platyfish, Japanese rice fish and Japanese flounder [8]. Gene expression studies of IL-1β have shown that it is significantly upregulated in immune tissues, in primary cultures and cell lines in response to immunostimulants, immune response modifiers and/or pathogens [reviewed in [2]]
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