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Rock bream (Oplegnathus fasciatus) IL-12p40: Identification, expression, and effect on bacterial infection

https://doi.org/10.1016/j.fsi.2014.05.026Get rights and content

Highlights

  • An IL-12p40 homologue, OfIL-12p40, was identified in rock bream.

  • OfIL-12p40 possesses a conserved IL-12p40 domain and a WSxWS signature motif.

  • Bacterial infection enhanced OfIL-12p40 expression in kidney and spleen.

  • rOfIL-12p40 stimulated the respiratory burst activity of peripheral blood leukocytes.

  • rOfIL-12p40 enhanced the resistance of rock bream against bacterial infection.

Abstract

IL-12p40, also called IL-12β, is a subunit of the proinflammatory cytokines interleukin (IL)-12 and IL-23. In teleost, IL-12p40 homologues have been identified in several species, however, the biological function of fish IL-12p40 is essentially unknown. In this work, we reported the identification and analysis of an IL-12p40, OfIL-12p40, from rock bream (Oplegnathus fasciatus). OfIL-12p40 is composed of 361 amino acids and possesses a conserved IL-12p40 domain and a WSxWS signature motif characteristic of known IL-12p40. Constitutive expression of OfIL-12p40 occurred in multiple tissues and was highest in kidney. Experimental infection with bacterial pathogen upregulated the expression of OfIL-12p40 in kidney and spleen in a time-dependent manner. Purified recombinant OfIL-12p40 (rOfIL-12p40) stimulated the respiratory burst activity of peripheral blood leukocytes in a dose-dependent manner. rOfIL-12p40 also enhanced the resistance of rock bream against bacterial infection and upregulated the expression of innate immune genes in kidney. Taken together, these results indicate that OfIL-12p40 possesses cytokine-like property and plays a role in immune defense against bacterial infection.

Introduction

The interleukin 12 (IL-12) family, which includes IL-12, IL-23, IL-27, and IL-35, is unique in containing the only heterodimeric cytokines [1]. IL-12 and IL-23 are proinflammatory or prostimulatory cytokines that execute positive regulatory functions, whereas IL-27 and IL-35 are inhibitory cytokines [1]. IL-12 is a heterodimeric cytokine consisting of two disulphide-linked subunits, α-chain (p35) and β-chain (p40) [1]. The p35 subunit has a four-helix bundle structure characteristic of the IL-6 superfamily, while the p40 subunit resembles the extracellular domain of hematopoietic cytokine receptor family members, such as the IL-6 receptor α-chain (IL-6Rα) and the ciliary neurotrophic factor receptor (CNTFR) [2], [3]. The p40 subunit can pair with p35 or p19 to form IL-12 or IL-23 respectively. p40 and p35 subunits have to be co-expressed in the same cell to yield functional IL-12 [4]. In mammals, p40 expression is restricted to cells that produce bioactive IL-12 while p35 is more ubiquitously expressed [5], [6].

IL-12 is a key proinflammatory cytokine that bridges innate and adaptive immunity. It is produced early in the immune response by macrophages, dendritic cells, and B cells in response to microbial pathogens, and can induce or promote generation of proinflammatory T helper type 1 (Th1) [1], [7], [8]. IL-12 stimulates the production of IFNγ, an important activator of macrophage, mostly in natural killer (NK) cells and T cells. IL-12 can also enhance the proliferation and cytolytic activities of NK cells and T cells and participate in the regulation of the cell-mediated immune response [9]. IL-12 binds to a receptor complex, IL-12Rβ1 and IL-12Rβ2, and the ensued signal transduction is mediated via members of the Jak-STAT family [1], [2], [5], [10].

In teleost fish, the p40 of IL-12 (IL-12p40) was first found in fugu (Fugu rubripes) [11], and then identified in other teleost species, including sea bass (Dicentrarchus labrax L.), Atlantic halibut (Hippoglossus hippoglossus L.), common carp (Cyprinus carpio), zebrafish (Danio rerio) and grouper (Epinephelus coioides) [3], [9], [12], [13], [14]. Unlike higher vertebrates, fish express three distinct p40-like isoforms, p40a, p40b, and p40c, which suggest an expansion of the vertebrate heterodimeric cytokine family [15]. In fish, IL-12p40 induces immune responses similar to its mammalian orthologues [9], [16]. It has been reported that the expression of fish IL12p40 is upregulated by bacterial and viral infection [3], [12], [17], [18], stimulation with LPS [12], [19], ConA and PMA [16], and parasite challenge [14]. Grouper IL-12p40 is known to increase the proliferation of Con A-stimulated PBL, enhance the expression of TNFα, and induce the chemotactic effect of PBL [14]. In addition, IL-12p40 together with p35 can drive Th-17 response in fish [16]. In this paper, we identified and characterized an IL-12p40 homologue (named OfIL-12p40) from rock bream (Oplegnathus fasciatus), an economic fish species farmed in China. We examined the expression pattern and immune effect of OfIL-12p40. Our results indicate that OfIL-12p40 expression was modulated by bacterial pathogen, and that recombinant OfIL-12p40 reduced bacterial infection in vivo.

Section snippets

Fish

Four hundred clinically healthy rock bream (average 28.5 g, age 5-month old) were purchased from a fish farm in Shandong Province, China, and maintained at 18 °C in aerated seawater. Fish were acclimatized in the laboratory for two weeks before experimental manipulation. Before experiment, fish were randomly sampled for the examination of potential existence of bacteria in blood, liver, kidney, and spleen as reported previously [20]. No bacteria were detected from sampled fish. Before tissue

cDNA and amino acid sequence analysis of OfIL-12p40

The cDNA of OfIL-12p40 contains a 5′-UTR of 81 bp, an ORF of 1086 bp, and a 3′-UTR of 642 bp (Fig. 1). The 3′-UTR has three ATTTA motifs that are typically found in cytokine mRNA [31], and a polyadenylation signal (ATTAAA) at 23 bp upstream of the poly(A) tail. The ORF of OfIL-12p40 encodes a protein of 361 amino acid residues with an isoelectric point of 4.64. OfIL-12p40 possesses a 21-aa signal peptide and is predicted to be an extracellular protein. The mature protein of OfIL-12p40 is

Discussion

In this study, we analyzed the expression and biological effect of an IL-12p40 homologue, OfIL-12p40, from rock bream. OfIL-12p40 possesses an IL-12p40 domain and contains multiple conserved cysteine residues that are known to form disulfide bonds essential to IL-12 activity [3]. Like mammalian IL-12p40, OfIL-12p40 has a C-terminal WSxWS motif, which, however, differs slightly from the mammalian counterpart in sequence, and this difference is preserved in a number of fish species. These data

Acknowledgments

This work was supported by the grants of the 863 High Technology Project (2011AA10A216) and the Taishan Scholar Program of Shandong Province.

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