Differential expression, modulation and bioactivity of distinct fish IL‐12 isoforms: Implication towards the evolution of Th1‐like immune responses

Abstract

IL-12 is a heterodimeric cytokine composed of an α-chain (p35) and a β-chain (p40). Primarily produced by APCs, IL-12 induces IFN-γ production in T, B and NK cells. IL-12 drivesTh1-cell differentiation and IFN-γ secretion to promote cell-mediated immunity, which is essential in the defence against intracellular pathogens. The importance of IL-12 in Th1 responses is echoed by its targeted suppression by intracellular pathogens evading cell-mediated immunity. IL-12 subunits have been identified recently in fish, although reported bioactivities are limited to higher vertebrates. Here, we report the cloning of a p35 gene and two divergent p40 genes (p40b and p40c), capable of producing two functional IL-12 isoforms (p35/p40b and p35/p40c) in rainbow trout. Trout IL-12 isoforms possess distinct bioactivities with respect to the induction of IFN-γ, IL-10 and p40c expression. Trout IL-12 isoforms are differentially expressed and modulated in vivo, exhibiting specific gene expression profiles in bacterial, viral and parasitic infection models, and in vitro in stimulated macrophage and leucocyte cultures. These data imply that alternative or additional pathogen-specific Th-like cell populations may exist in fish. This study will facilitate a broader understanding of the evolutionary processes driving host-pathogen interactions and Th1-like immune responses in lower vertebrates.Keywords: Bioactivity r IL-12 r Infection r Rainbow trout r Th1 immune responses Additional supporting information may be found in the online version of this article at the publisher's web-site Introduction IL-12 is a pleiotropic cytokine belonging to the IL-12 family of composite heterodimeric cytokines, with each member consisting of one of three α-chains (p35, p28 and p19) and one of two β-chains (p40 and Ebi3). Structurally related to the IL-6 ligand and receptor families, each chain in mammals is encoded by a single gene [1][2][3][4][5]. Currently, the IL-12 family consists of four cytokine members each with a unique α/β subunit pairing, namely, IL-12 (p35/p40), Correspondence: Dr. Tiehui Wang e-mail: t.h.wang@abdn.ac.uk (p19/p40), IL-27 (p28/Ebi3) and IL-35 (p35/Ebi3), all of which are known to have key roles in the regulation of T-cell responses [2,3,6].Also known as NK-cell stimulatory factor [7] and cytotoxic lymphocyte maturation factor [8], IL-12 is produced primarily by APCs, such as macrophages and DCs, and has been shown to have direct proliferative and cytolytic activities towards pre-activated T cells and NK cells [7][8][9]. IL-12 is a major player in the activation of innate immunity and in the induction of IFN-γ, the cytokine that defines Th1-type immune responses in higher vertebrates. In doing so, IL-12 acts as a crucial molecular switch between innate and adaptive immunity driving Th1 cell differentiation and expansion, whilst inhibiting IL-4 and antagonising Th2 responses [2,3,6,10]. Eur. J. Immunol. 2014Immunol. . 44: 1541Immunol. -1551 Hence, IL-12 is an important prerequisite for the effective clearance of mostly intrace...

Identification and expression analysis of an atypical chemokine receptor-2 (ACKR2)/CC chemokine binding protein-2 (CCBP2) in rainbow trout (Oncorhynchus mykiss)

“…Four day old primary HK macrophages were prepared as described previously [40] and stimulated with a variety of PAMPS and cytokines, including polyinosinic: polycytidylic acid (poly I:C, 50 mg/ml, Sigma), peptidoglycan (PGN, 5 mg/ml, Invivogen), rIL-6 (100 ng/ml) [40], rIFN-g (20 ng/ml) [41], rTNF-a (10 ng/ml) [42], rIL-1b (20 ng/ml) [43], and two rIL-12 isoforms that differ in the p40 chain [44] for 4 h, 8 h and 24 h. As above, the concentration chosen for each stimulant was deemed optimal for immune gene expression experiments based on our previous studies [40e44]. Real-time PCR quantification of expression was as described above and the results were expressed as a fold change relative to the time-matched unstimulated controls.…”

Key cytokines of adaptive immunity are differentially induced in rainbow trout kidney by a group of structurally related geranyl aromatic derivatives

“…It has been reported that the recombinant IL-12 induces IFNg expression in head kidney (HK) cells of the rainbow trout [27], indicating that the cytokine have functions similar to those described in mammals. In this regard, although Filifolinol induced IL-12 expression, increase of IFN-g transcripts was not detected in the treated fish at the same time point of analysis.…”

The analysis of correlation between IL-12 gene expression and hepatitis B virus in the affected patients

“…Signals from interleukins are essential for the development of these different types of T cells and for their effector functions [1]. Interleukin-12 (IL-12) is the main cytokine that regulates cell-mediated immunity, Th1 differentiation, Th1 maintenance and IFN-c production in T and NK cells [3,10,11,13,24,27,28,30]. IFN-c, tumor necrosis factoralpha (TNF-a) and IL-2 as inflammatory cytokines are critical molecules for modulating the intensity and duration of the host immune responses against HBV infections.…”

“…As a matter of fact, they are able to inhibit HBV gene expression and replication and play pivotal roles in the clearance of viruses from the rest of infected cells by non-cytolytic mechanisms [3,32]. IL-12 is an important pro-inflammatory cytokine that consists of p35 and p40 subunits [3,20,27,30]. The genes encoding human p35 and p40 are located on chromosomes 3 and 5, respectively [28].…”

“…The genes encoding human p35 and p40 are located on chromosomes 3 and 5, respectively [28]. IL-12 is mainly secreted by antigen-presenting cells (APC): macrophages (MQ) and dendritic cells (DCs) in response to infection caused by intracellular pathogens and host immune stimuli [3,4,25,27,28,30]. The cellular effects of IL-12 are mediated by binding to its receptor (IL-12R) which includes b1-and b2-plasma membrane-traversing proteins [10,13,22].…”