Heritability of Immunity Traits and Resistance of Atlantic Salmon against the Sea Louse Caligus rogercresseyi.

The immune response of Atlantic salmon to sea lice has been extensively studied, but we still do not know the mechanisms by which some fish become resistant and others do not. In this study, we estimated the heritabilities of three key proteins associated with the innate immunity and resistance of Salmo salar against the sea louse Caligus rogercresseyi. In particular, we quantified the abundance of 2 pro-inflammatory cytokines, Tnfα and Il-8, and an antioxidant enzyme, Nkef, in Atlantic salmon skin and gill tissue from 21 families and 268 individuals by indirect ELISA. This covers a wide parasite load range from low or resistant (mean sea lice ± SE = 8.7 ± 0.9) to high or susceptible (mean sea lice ± SE = 43.3 ± 2.0). Our results showed that susceptible fish had higher levels of Nkef and Tnfα than resistant fish in their gills and skin, although gill Il-8 was higher in resistant fish, while no significant differences were found in the skin. Furthermore, moderate to very high heritable genetic variation was estimated for Nkef (h2 skin: 0.96 ± 0.14 and gills: 0.97 ± 0.11) and Tnfα (h2 skin: 0.53 ± 0.17 and gills: 0.32 ± 0.14), but not for Il-8 (h2 skin: 0.22 ± 0.12 ns and gills: 0.09 ± 0.08 ns). This work provides evidence that Nkef and Tnfα protein expressions are highly heritable and related to resistance against sea lice in Atlantic salmon.

Antiviral Function of NKEF against VHSV in Rainbow Trout.

Natural killer enhancing factor (NKEF) belongs to the peroxiredoxin family of proteins, a group of antioxidants that has been extensively studied in mammals. Recently, we identified NKEF in the immunoprecipitated proteome of rainbow trout red blood cells (RBCs) exposed to viral hemorrhagic septicemia virus (VHSV). In the present study, we evaluated the role of NKEF in the antiviral response of rainbow trout against VHSV by examining the expression profile of NKEF in VHSV-exposed RBCs and rainbow trout gonad-2 (RTG-2) cell line. We found an in vitro correlation between decreased VHSV replication and increased NKEF expression after RBCs were exposed to VHSV, however this was not found in RTG-2 cells where the infection highly increased and nkef transcripts remained almost unchanged. In addition, siRNA silencing of the nkef gene in rainbow trout RBCs and RTG-2 cells resulted in increased VHSV replication. We also found a correlation between nkef gene silencing and a decrease in the expression of genes related to type 1 interferon (IFN1) pathway. These findings indicated that NKEF is involved in the antiviral mechanisms of rainbow trout RBCs against VHSV and thus support its antiviral role and implication in the modulation of their immune response. Finally, overexpression of NKEF in an EPC cell line significantly reduced VHSV infectivity and was coupled to an increment in IFN1-related genes. In conclusion, NKEF may be a potential target for new therapeutic strategies against viral infections.

Fish natural killer enhancing factor-A (NKEF-A) enhance cytotoxicity of nonspecific cytotoxic cells against bacterial infection.

Natural killer enhancing factor (NKEF)-A/B is a member of Peroxiredoxin (Prxs) family, which is named for the function of enhancing NK cells activity. NKEF also plays essential roles in multiple physiology/pathology processes including inflammation regulation, cancer development and redox reactions. However, the regulatory effects of fish NKEF on immune cells remain largely unknown. In this study, the full-length cDNA of NKEF-A (Accession No. MK584553, designated as On-NKEF-A) was identified from Nile tilapia (Oreochromis niloticus). On-NKEF-A encoded a 198 amino acid peptide with molecular mass of 22.085 kDa. On-NKEF-A protein contained a typical 2-Cys family domain, two active sites (51aa and 172aa) that were conserved in mammals, birds, amphibians and fish. Phylogenetic analysis showed that On-NKEF-A had the closest relationship with Zebra mbuna (Maylandia zebra) NKEF. The On-NKEF-A transcription was present in all examined tissues or organs. And the relative high expression levels of On-NKEF-A was found in head kidney leucocytes and nonspecific cytotoxic cells (NCC). After Streptococcus agalactiae stimulation, On-NKEF-A was significantly up-regulated in head kidney, spleen, gill and skin. Also, On-NKEF-A was markedly induced post S. agalactiae, LPS and poly I:C stimulation in head kidney-derived NCC. Moreover, On-NKEF-A was mainly distributed in cytoplasm of fathead minnow cells (FHM cells). The further in vitro analysis found that the recombinant protein of On-NKEF-A (rOn-NKEF-A) could induce the expression of various molecular markers of B cells, macrophages and NCC, enhanced the cytotoxicity of NCC via increasing the effectors expression. The present data collectively indicate that On-NKEF-A participates in anti-bacterial immune response via regulating NCC activity, which will provide new ideas to further explore the defense mechanism of fish against bacteria.

Cloning and functional characterisation of natural killer enhancing factor-B (NKEF-B) gene of Labeo rohita: Anti-oxidant and antimicrobial activities of its recombinant protein.

Natural killer enhancing factor (NKEF) of peroxiredoxin family is an important innate immune molecule with having anti-oxidant activity. Although this gene has already been studied in a few fish species, it is yet to be identified and functionally characterised in Indian major carps. In the present study, the complete NKEF-B cDNA of rohu, Labeo rohita was cloned that encoded a putative protein of 197 amino acids. The phylogenetic study showed that L. rohita NKEF-B (LrNKEF-B) is closely related to NKEF-B of Cyprinus carpio and Danio rerio species. Tissue-specific expression of LrNKEF-B gene revealed the highest transcript level in the liver tissue. In the ontogeny study, the highest level of the expression was observed in milt and at 18 h post-development. The expression pattern of this gene was also studied in various pathogen models viz., Gram-negative bacteria (Aeromonas hydrophila), ectoparasite (Argulus siamensis) and a dsRNA viral analogue (poly I:C) in the liver and anterior kidney tissues of L. rohita juveniles. During A. hydrophila infection, the increase in expression of transcripts was observed at 3 h post-infection in both liver (15-fold) and anterior kidney (8-fold). In A. siamensis infection, the expression gradually increased up to 3 d post-infection in the anterior kidney, whereas in liver 3-fold up-regulation was noticed at 12 h post-infection. Similarly, during poly I:C stimulation, up-regulation of NKEF-B transcript was observed in anterior kidney from 1 h to 24 h post-stimulation and down-regulated afterwards whereas, the transcript level increased gradually from 6 h to 15 d post-stimulation in liver tissue. In vitro exposure to concanavalin, A and formalin-killed A. hydrophila upregulated NKEF-B gene expression in anterior kidney and peripheral blood leukocytes of L. rohita, however, down-regulated the same in the splenic leukocytes. A recombinant protein of LrNKEF-B (rLrNKEF-B) of 22 kDa was produced and it showed anti-oxidant activity by protecting supercoiled DNA and reducing insulin disulfide bonds. The minimum bactericidal concentration of this recombinant protein was found to be 4.54 µM against A. hydrophila and Staphylococcus aureus. Interestingly, rLrNKEF-B showed relative percent survival of 72.6 % in A. hydrophila challenged L. rohita, and the survival was found to be associated with a high level of expression of different cytokines, anti-oxidant genes and perforin in the rLrNKEF-B treated L. rohita. An indirect ELISA assay for estimation of NKEF was developed in L. rohita, and the concentrations of NKEF-B increased with time periods post A. hydrophila challenge viz., 0 h (42.56 ng/mL), 12 h (174 ng/mL) and 48 h (370 ng/mL) in rohu serum. Our results suggest a crucial role of LrNKEF-B in innate immunity against biotic stress and oxidative damage and also having antibacterial activity.

Insights into the multifunctional role of natural killer enhancing factor-A (NKEF-A/Prx1) in big-belly seahorse (Hippocampus abdominalis): DNA protection, insulin reduction, H2O2 scavenging, and immune modulation activity.

Natural killer enhancing factor A (NKEF-A), also known as peroxiredoxin 1 (Prx1), is a well-known antioxidant involved in innate immunity. Although NKEF-A/Prx1 has been studied in different fish species, the present study broadens the knowledge of NKEF-A gene in terms of molecular structure, function, and immune responses in fish species. Hippocampus abdominalis NKEF-A (HaNKEF-A) cDNA encoded a putative protein of 198 amino acids containing a thioredoxin_2 domain, VCP motifs, and three conserved cysteine residues including peroxidatic and resolving cysteines. Amino acid sequence comparison and phylogenetic breakdown showed the higher sequence identity and closer evolutionary position of HaNKEF-A to those of other fish counterparts. A recombinant protein of HaNKEF-A was shown to i) protect supercoiled DNA against mixed catalyzed oxidation, ii) reduce insulin disulfide bonds, and iii) scavenge extracellular H2O2. Results of in vitro assays demonstrated the concentration dependent antioxidant function of recombinant HaNKEF-A. In addition, qPCR assessments revealed that the HaNKEF-A transcripts were constitutively expressed in fourteen tissues with the highest expression in liver. As an innate immune response, HaNKEF-A transcripts were up-regulated in liver post injection of LPS, Edwardsiella tarda, Streptococcus iniae, and polyinosinic-polycytidylic acid. Thus, HaNKEF-A can safeguards big-belly seahorse from oxidative damage and pathogenic infections. This study provides insight into the functions of NKEF-A/Prx1 in fish species.

Molecular identification and expression analysis of a natural killer cell enhancing factor (NKEF) from rock bream Oplegnathus fasciatus and the biological activity of its recombinant protein.

Natural killer cell enhancing factor (NKEF) belongs to the defined peroxiredoxin (Prx) family. Rock bream NKEF cDNA was identified by expressed sequence tag (EST) analysis of rock bream liver that was stimulated with the LPS. The full-length RbNKEF cDNA (1062 bp) contained an open reading frame (ORF) of 594 bp encoding 198 amino acids. RbNKEF was significantly expressed in the gill, liver, and intestine. mRNA expression of NKEF in the head kidney was examined under viral and bacterial challenge via real-time RT-PCR. Experimental challenge of rock bream with Edwardsiella tarda, Streptococcus iniae, and RSIV resulted in significant increases in RbNKEF mRNA in the head kidney. To obtain a recombinant NKEF, the RbNKEF ORF was expressed in Escherichia coli BL21 (DE3), and the purified soluble protein exhibited a single band corresponding to the predicted molecular mass. When kidney leucocytes were treated with a high concentration of rRbNKEF (10 µg/mL), they exhibited significantly enhanced cell proliferation and viability under oxidative stress.