Innate immune responses of lumpfish (Cyclopterus lumpus) : Transcriptome analysis and characterization of pro-inflammatory cytokines

Abstract

In recent years, lumpfish and different species of wrasse are used as cleaner fishes for removal of sea lice from farmed Atlantic salmon in Europe and Canada. The production is successful, but there are challenges with high mortality due to bacterial infections. In-depth knowledge of the immune system in lumpfish, particularly immune responses upon bacterial infections and identification of immune genes, will make an important basis for development of immune-prophylactic measures. Prior to this study, there were no available sequences of immune genes in lumpfish in public databases. To obtain sequence information from lumpfish, RNA sequencing of head kidney leukocytes (HKLs) exposed to Vibrio anguillarum O1, followed by de novo transcriptome assembly, was performed. The transcriptome encompassed 221659 trinity genes, 9033 differentially expressed genes (DEG) at 6 hours post exposure (hpe) and 15225 DEG at 24 hpe. The DEG analysis of the lumpfish transcriptome clearly showed that the alternative pathway of the complement cascade is one of the chief mechanisms in the Vibrio anguillarum response of lumpfish HKLs. Furthermore, the DEG analysis also clearly showed that the TLR signaling through the canonical NFκB pathway was the major pathway in the adaptation of the innate immune response. Further, a wider MAPK pathway regulation was observed, involving all of its sub pathways, however, at much less potent regulation levels compared with NFκB. Globally, the DEG analysis displayed a picture of a pro-inflammatory reaction, initiated by toll-like receptor TLR5S, and the alternative complement pathway, and resulting in high up-regulation of the cytokines interleukin (IL)-1β, IL-6, CXCL8 (also known as IL-8) and tumor necrosis factor (TNF)-α. Further characterization of the lumpfish IL-1 family, both ligands and receptors (IL-1R), were performed. Downstream signaling pathways were also characterized. Full-length sequences of the ligands IL-1β, IL-18, and the fish-specific IL-1 family members nIL-1F and IL-1Fm2, the receptors IL-1R1, IL-1R2, IL18-R1, suppression of tumorgenicity (ST2), and partial sequences of double immunoglobulin IL-1R related molecule (DIGIRR) and IL- receptor accessory protein (IL-Ra) were identified. In vitro stimulation of lumpfish leukocytes with a selection of PAMPs, showed that lumpfish IL-1β and nIL-1F were upregulated, most potently by flagellin. The phylogenetic analysis of the IL-1 family ligands showed that IL-1β, nIL-1F1 and IL1Fm2 are more similar to each other than to IL-18. Furthermore, it placed sequences from lobe-finned fish and shark within the nIL-1F clade, suggesting that nIL-1F, together with IL-1β are ancestral genes. This is the first report describing the occurrence of nIL-1F in non-teleost species. Characterization of lumpfish TNF-α revealed that it contained the hallmark properties of TNF-family at nucleotide and peptide levels. Characterization of lumpfish IL-6 revealed a novel gene arrangement, with one less exon, than other described IL-6 teleost sequences. Furthermore, the predicted peptide sequence of lumpfish IL-6 contained a signal peptide first revealed after proteolytic cleavage. Caspase 1 was predicted to cleave in this region of lumpfish IL-6. It is therefore hypothesized that lumpfish IL-6 may be dependent on caspase 1 processing. In the normal tissues and unstimulated leukocytes, the levels of TNF-α transcripts were higher than IL-6, except for eye and brain where the transcript levels of IL-6 were unexpectedly high. Both genes displayed a similar induction pattern to PAMP stimulation in vitro and both were most potently stimulated by flagellin. IL-6 was more potently stimulated that TNF-α. In conclusion, the lumpfish innate immune responses are potent and consist of most of the molecules for a modern teleost. The alternative complement pathway and TLR signaling pathway are fundamental to the in vitro response to Vibrio anguillarum O1 by lumpfish leukocytes. The identification and characterization of the major pro-inflammatory cytokines and design of qPCR assays in lumpfish provides a valuable tool to measure innate immune responses in lumpfish e.g. upon immune modulation, such as vaccination, microbial disease or physiological trials.