Novel functions of grass carp three p40 isoforms as modulators of Th17 signature cytokine expression in head kidney leukocytes

Highlights

• Three grass carp p40 isoforms induce the transcription of Th17 signature cytokines.

• Three p40 isoforms significantly stimulate Il-17a/f1 protein release.

• Three p40 isoforms can activate Erk, Jnk and Stat3 pathways.

• Erk, Jnk and Stat3 pathways mediate three isoforms-induced il-17a/f1 transcription.

Abstract

Interleukin (IL)-12p40, a component of IL-12 and IL-23, can be secreted as monomer and homodimer in mammals. Our previous study has proved the existence of natural three p40 isoforms and their proinflammatory properties in grass carp. In the present study, we unexpectedly found that recombinant grass carp p40a/b/c (rgcp40a, rgcp40b and rgcp40c) were able to enhance the mRNA levels of grass carp il-17a/f1 (gcil-17a/f1) in a dose- and time-dependent manner in head kidney leukocytes (HKLs). In agreement with these findings, the enzyme-linked immunosorbent assay (ELISA) showed that rgcp40a, rgcp40b and rgcp40c markedly stimulated gcIl-17a/f1 secretion from the HKLs. Together with their stimulatory effects on grass carp gcil-22 and gcil-26 expression, our data suggested their potential to mediate Th17-like response in grass carp. To support this notion, we investigated the underlying mechanisms for the regulation of rgcp40 isoforms on gcil-17a/f1 expression, and found that three rgcp40 isoforms significantly induced the activation of Erk, Jnk and Stat3 pathways in a time-dependent oscillation in the same cell model. Moreover, three rgcp40 isoforms-induced gcil-17a/f1 mRNA expression was suppressed by the inhibition on Erk, Jnk and Stat3 pathways, suggesting the signaling pathways in the p40 isoforms-mediating il-17a/f1 transcription. These studies for the first time proved the involvement of three gcp40 isoforms in mediating Th17 signature cytokine expression in fish immune cells, therefore providing new insights into the roles of p40 in teleost immunity.

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.