Short communicationNovel functions of grass carp three p40 isoforms as modulators of Th17 signature cytokine expression in head kidney leukocytes
Introduction
In mammals, interleukin (IL)-12p40 subunit (also named as p40), can pair with p35 or p19 to form IL-12 or IL-23, respectively [1,2]. IL-12 and IL-23 are proinflammatory or prostimulatory cytokines that execute important regulatory functions [3] and both of them are produced by inflammatory myeloid cells and influence the development of Th1 cell and Th17 cell responses, respectively [2,4]. Unlike p35 and p19, the p40 subunit is also secreted as monomer or as homodimer (p40)2, and large amounts of p40 more than that of the IL-12 and IL-23 can be detected in some cell models [1,[5], [6], [7]]. For functionality, the most notable function of free p40 is as an antagonist for IL-12 and IL-23 because the p40 homodimer or monomer competitively binds to the IL-12/IL-23 common receptor in vitro and in vivo [[8], [9], [10], [11]]. In contrast to this antagonistic effect, several datasets have also suggested an independent agonist effect for p40 [9]. For example, it acts as a chemoattractant for macrophages and promotes the migration of the activated dendritic cells from the lung to the draining lymph node following a challenge by mycobacteria [12,13]. One of the earliest established activities of recombinant p40 homodimer is up-regulating IFN-γ production by CD8+ T cells and stimulating CD8+ T helper (Th) 1 development in vitro [14]. In addition, p40 can induce the production of immune-related molecules, such as tumor necrosis factor (TNF-α) and nitric oxide (NO) via p38 and ERK signaling pathway in mouse microglia and macrophages [6,15].
Unlike a single gene for p40 in mammals, three distinct p40 genes (p40a, p40b and p40c) were found in some fish species, such as common carp (Cyprinus carpio) [16], amberjack (Seriola dumerili) [17] and grass carp (Ctenopharyngodon idellus) [18]. Using Co-Immunoprecipitation assay, we reveal that all three p40 paralogues can combine with p19 subunit to form three soluble grass carp Il-23 isoforms [18]. Moreover, excessive secretion of grass carp p40 isoforms as monomer triggered by various immune stimuli is observed in grass carp head kidney leukocytes (HKLs), and their recombinant proteins (rgcp40a, rgcp40b and rgcp40c) display the activities to stimulate the secretion of proinflammatory cytokines Il-1β and tumor necrosis factor-α (Tnf-α) in the same cell model [19]. In accordance with the regulatory roles of three rgcp40 proteins, administration of recombinant p40 protein increases the transcript levels of immune gene expression in the kidney of rock bream [20]. Recently studies indicated that recombinant p40 and (p40)2 proteins of Chinese sea bass possess high potential antimicrobial activity against eight bacterial-tested strains [21]. These observations suggest the regulatory role of p40 in an independent manner in fish immunity.
In the present study, we unexpectedly found the bioactivity of three rgcp40 isoforms in mediating Th17-like response in HKLs. The novel regulatory effects of three gcp40 isoforms were reinforced by the signaling mechanisms governing rgcp40 isoforms-modulated gcil-17a/f1 expression. Therefore, our findings for the first time uncovered the role of free p40 paralogues involved in Th17-like responses in teleost.
Section snippets
Fish
Grass carp with an average body weight of 1.0 kg were obtained from Chengdu Tongwei Aquatic Science and Technology Company (Chengdu, China) and were acclimatized in laboratory tanks at room temperature for 2 weeks before experimental processing. The HKLs were isolated from freshly sacrificed fish. All animal experiments were conducted according to the Regulation of Animal Use in Sichuan province, China and were approved by the ethics committee of the University of Electronic Science and
Effects of rgcp40s on the expression of Th17 related cytokines in grass carp HKLs
Grass carp HKLs were incubated with rgcp40a, rgcp40b or rgcp40c for different times and RT-qPCR assay showed that all the recombinant proteins induced a time-dependent increase in gcil-17a/f1 gene expression from 4 to 8 h for rgcp40a (Fig. 1A), from 12 to 24 h for rgcp40b (Fig. 1B) and from 2 to 24 h for rgcp40c (Fig. 1C), separately. In parallel experiments, increasing concentrations of rgcp40a (100–1000 ng/ml), rgcp40b (300–1000 ng/ml) and rgcp40c (300–1000 ng/ml) were able to up-regulate
Acknowledgments
This work was supported by the grants from the National Natural Science Foundation of China (31572650) and the Applied Basic Research Program of Sichuan Province, China (19YYJC0036).
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