Molecular characterization and expression analysis of IL-1β and two types of IL-1 receptor in barbel steed (Hemibarbus labeo)

Highlights

• We cloned the cDNA sequence of il1b, il1r1, and il1r2 genes from barbel steed.

• il1b and its two receptors gene expression rapidly responded to LPS stimulation.

• il1r1 and il1r2 genes were induced after recombinant IL-1β stimulation in monocytes/macrophages.

Abstract

Interleukin-1β (IL-1β) is a pivotal proinflammatory cytokine that plays important roles in regulating immune responses and in inducing a series of inflammatory reactions in response to infection. Recently, increasing attention has focused on the regulatory mechanisms of IL-1β activity in teleosts. In this regard, IL-1 receptor type 1 plays a crucial role in immune responses, whereas IL-1 receptor type 2 is a decoy receptor that functions as an IL-1β signaling inhibitor. However, the interactions of these three proteins with respect to fish immunity have rarely been studied. In the present study, cDNAs of the il1b, il1r1, and il1r2 genes of the barbel steed (Hemibarbus labeo) were cloned and sequenced. Amino acid sequence analysis revealed that the IL-1β protein and its two receptors identified in barbel steed are conserved in most teleosts, whereas phylogenetic tree analysis indicated that these three proteins are closely related to those of cyprinids. In response to lipopolysaccharide treatment, expression of the genes encoding IL-1β and its two receptors was significantly upregulated in the immune-related tissues of barbel steed. Furthermore, expression of the il1r1 and il1r2 genes was induced in monocytes/macrophages in response to stimulation with recombinant IL-1β.

Introduction

The barbel steed (Hemibarbus labeo) is a bottom-dwelling freshwater cyprinid that inhabits streams where it feeds on aquatic insects. It is distributed in the major drainages of east mainland China, Japan, and Korea. The development of barbel steed as an aquacultural species has been hampered to a certain extent by bacterial fish diseases, which adversely affect fish production. Although proinflammatory cytokines have been proven to be useful for evaluating the inflammatory responses of fish following pathogen infection (Xiao et al., 2019), to date, no cytokine sequences have been reported for barbel steed. Gaining a better understanding of the involvement of cytokines in barbel steed immune regulation would predictably enable the modulation of its immune responses.

The cytokine interleukin-1β (IL-1β) exerts a plethora of systemic and localized biological effects and plays a central role in the initiation and regulation of immune and inflammatory responses in animals (Hong et al., 2003). IL-1β sequences have previously been studied in many economically important teleosts (Bo et al., 2015; Fujiki et al., 2000; Herath et al., 2016; Ho et al., 2016; Lepen Pleić et al., 2014; Liao et al., 2018; Lu et al., 2013a; Scapigliati et al., 2001; Yang et al., 2017; Zou et al., 1999), and some studies have shown that IL-1β is closely associated with the defense-related reactions of teleosts that are activated in response to pathogen infection. For example, upon infection with Vibrio anguillarum or V. harveyi, the expression level of the il1b gene was found to be upregulated in the spleen of the miiuy croaker (Yang et al., 2017). Some studies have also shown that the biological function of IL-1β in teleosts is similar to that observed in mammals (Buonocore et al., 2005; Hong et al., 2003; Lu et al., 2013a; Wu et al., 2015). Furthermore, a recombinant IL-1β has been found to enhance the resistance of the large yellow croaker (Larimichthys crocea) to Vibrio alginolyticus and to increase the migration and phagocytic activities of head kidney-derived macrophages in vitro (Wu et al., 2015).

In mammals, there are two distinct receptors for IL-1, namely, IL-1 receptor type 1 (IL-1R1) and IL-1 receptor type 2 (IL-1R2). IL-1 signaling, mediated by IL-1α/IL-1β, occurs exclusively via IL-1R1 (Sims et al., 1993), whereas IL-1R2, which lacks the Toll/IL-1 receptor (TIR) domain, non-productively binds IL-1β, thereby preventing signal transduction (Colotta et al., 1993). Unlike IL-1β, IL-1R1 and IL-1R2 have been less well studied in teleosts, having to date been reported in the rainbow trout (Oncorhynchus mykiss) (Sangrador-Vegas et al., 2000), Atlantic salmon (Salmo salar) (Morrison et al., 2012; Subramaniam et al., 2002), common carp (Cyprinus carpio) (Metz et al., 2006), gilthead seabream (Sparus aurata) (Gloria et al., 2007), Japanese flounder (Paralichthys olivaceus) (Fan et al., 2010), grass carp (Ctenopharyngodon idella) (Yang et al., 2013), orange-spotted grouper (Epinephelus coioides) (Lu et al., 2013b), miiuy croaker (Miichthys miiuy) (Yang et al., 2017), and yellow catfish (Pelteobagrus fulvidraco) (Liao et al., 2018). On the basis previous observations, the activity of these two receptors appear to be closely related to inflammatory responses (Gloria et al., 2007; Liao et al., 2018; Subramaniam et al., 2002; Yang et al., 2017; Yang et al., 2013). Thus, the expression of il1r1 and il1r2 was found to be significantly increased in immune-related tissues of the miiuy croaker after stimulation with V. anguillarum, lipopolysaccharide (LPS) or polyinosinic–polycytidylic acid [poly(I:C)] (Yang et al., 2017). However, the interactions of IL-1β with its two receptors have rarely been studied in relation to fish immunity.

Therefore, in the present study, we evaluated the transcript levels of barbel steed il1b, il1r1, and il1r2 in healthy and LPS-treated fish. Moreover, we investigated the regulatory effects of IL-1β on the expression of the IL-1R1- and IL-1R2-encoding genes in the monocytes/macrophages (MO/MΦ) of this species.