The molecular, functional and phylogenetic characterization of PGE₂ receptors reveals their different roles in the immune response of the teleost fish gilthead seabream (Sparus aurata L.)

Highlights

• PGE₂ receptors ptger1-4 genes have been identified in gilthead seabream.

• Ptger2 has 2 paralogs in gilthead seabream.

• Macrophages highly express ptger2 and ptger3.

• PGE₂ and PAMP stimulation robustly induces ptger1-4 in granulocytes.

Abstract

Prostaglandin E₂ (PGE₂) plays an important role in immune activities in teleost fish, including seabream. However, receptors involved in PGE₂ signaling, as well as the pathways activated downstream, are largely unknown. In this study, one ortholog of mammalian PTGER1, PTGER3 and PTGER4, and two of PTGER2 (Ptger2a and Ptger2b) were identified and characterized in gilthead seabream. In silico analysis showed that all these receptors possessed the organization domain of G protein-coupled receptors, with the exception of Ptger2b. The corresponding in vivo studies revealed that they were expressed in all the tissues examined, the highest mRNA levels of ptger1 and ptger3 being observed in the spleen and of ptger2a and ptger4 in the blood. Bacterial infection induced higher mRNA levels of ptger2a, ptger3 and ptger4 in peritoneal exudate (the site of bacterial injection). In addition, head kidney acidophilic granulocytes and macrophages displayed different ptger1, ptger2a, ptger3 and ptger4 expression profiles. Furthermore, in macrophages the expression of the receptors was weakly affected by stimulation with bacterial DNA or with PGE₂, while in acidophilic granulocytes stimulation resulted in the upregulation of ptger2a and ptger4. Taken together, these results suggest different roles for seabream PGE₂ receptors in the regulation of the immune responses.

Introduction

Prostanoids including PGE₂ are bioactive lipid-derived autacoids (Ricciotti and FitzGerald, 2011) involved in a broad array of physiological and pathological processes (Gamil et al., 2015; Kobayashi and Narumiya, 2002). They are derived from the arachidonic acid released from cell membranes (Calder and Grimble, 2002) in response to a variety of stimuli (Harris et al., 2002; MacKenzie et al., 2010; Ricciotti and FitzGerald, 2011; Sargent et al., 1999), which is subsequently converted into to PGH₂ through the action of two rate-limiting enzymes: constitutive (COX-1) and inducible (COX-2) cyclooxygenases (Wang and Dubois, 2010). PGH₂, is converted into several prostanoids by the action of the respective synthases (Narumiya et al., 1999).

The divergent effects of PGE₂ have been involved in a large spectrum of biological processes (Hao and Breyer, 2008; Ning et al., 2014; Miller, 2006), including both pro- and anti-inflammatory mechanisms, ranging from lower vertebrates (fishes) (MacKenzie et al., 2010; Gómez-Abellán and Sepulcre, 2016; Tyrkalska et al., 2016) to human (Calder and Grimble, 2002; Hsu et al., 2014; Fattahi and Mirshafiey, 2014; Miller, 2006).

In mammals, the pleiotropic effects of PGE₂ are mediated by binding to one or to a combination of four receptors subtypes PTGER1-PTGER4 (also named EP1-EP4) (Harris et al., 2002; Qu et al., 2015), belonging to the family of rhodopsin-like seven transmembrane-spanning, G-protein-coupled receptors (Guo et al., 2015). These receptors can be grouped into three groups of prostanoid receptors according to the corresponding associated G-protein subunit, and hence to the intracellular second messengers and the effect triggered by them (Yokoyama et al., 2013). The group of relaxant receptors include EP2 and EP4, whose activation results in increased cAMP/PKA through Gα_s and adenyl cyclase (Fujino et al., 2002), leading to smooth muscle relaxation. EP1 is coupled to Gα_q, which increases Ca²⁺ levels following the activation of phospholipase C (Fujino and Regan, 2005) and belongs to the group of contractile receptors. EP3 is generally coupled to Gi, leading to a decrease in cAMP via Gα_i and resulting in the inhibition of muscle contraction (Narumiya et al., 1999), although different isoforms resulting from alternative splicing in the EP3 gene are coupled to different types of G protein, such as Gαs, Gαi and Gα12/13 (Tang et al., 2017).

Mammalian EP1-EP4 receptors exhibit distinctive levels of ligand-binding affinity, different selective expression, tissue distribution and subcellular localization (Sugimoto and Narumiya, 2007; Yokoyama et al., 2013). These receptors have been characterized at genomic (Locker and Buzard, 1990; Smock et al., 1999; Suganami et al., 2016; Yokoyama et al., 2013), proteomic (Margan et al., 2012; Stillman et al., 1999; Sugimoto and Narumiya, 2007; Yokoyama et al., 2013) and expression levels, both in vitro and in vivo, in response to a variety of stimuli (Birrell et al., 2015; Sakamoto et al., 2004; St-Jacques and Ma, 2011; Sheppe et al., 2018). Mouse EP3 and EP4 are the most widely PGE2 receptor distributed (Yokoyama et al., 2013) and both have a much higher affinity than EP1 and EP2 for PGE₂ (Fujino et al., 2002).

In teleost fish, the production of PGE₂ by immune cells has been described in a broad range of species, including black seabream (Spondyliosoma cantharus), black rockfish (Sebastes melanops), red seabream (Pagrus major), Atlantic cod (Gadus morhua), yellow croaker (Larimichthys polyactis), rainbow trout (Oncorhynchus mykiss), goldfish (Carassius auratus) and gilthead seabream (Sparus aurata L.) (Gómez-Abellán and Sepulcre, 2016). Furthermore, there is evidence concerning the immunoregulatory activity of PGE₂ in fish: for example, the modulation of cytokine expression, ROS production and phagocytic responses (Gómez-Abellán and Sepulcre, 2016). More specifically, we have shown that PGE₂ leads to M2 polarization of macrophages in gilthead seabream (Montero et al., 2016), in a similar way to that seen in mammals (Harris et al., 2002). PGE₂ deactivates acidophilic granulocytes (AGs, the functionally equivalent cell to mammalian neutrophils) through the modulation of the cytokine expression profile (Montero et al., 2016). However, studies concerning the molecular characterization of PGE₂ receptors and their role in the immune response in teleost fish are scant. Mammalian homologs of EP1-4 have been identified and characterized in several fish species: zebrafish (Danio rerio H.) (Kwok et al., 2012; Tsuge et al., 2013), Atlantic salmon (Salmo salar L.) (Guo et al., 2015), Bostrichthys sinensis (Lai et al., 2014) and ayu (Plecoglossus altivelis) (Rong et al., 2016), EP4 being the most characterized (Fujimori et al., 2011; Gamil et al., 2015; Guo et al., 2015; Tsuge et al., 2013). They are involved in several functions such as reproduction (Fujimori et al., 2011, 2012), metabolic regulation (Busby et al., 2002) and the immune response (Guo et al., 2015; Rong et al., 2016). Ptger4 has also been considered as an early marker of T cells precursor in zebrafish development (Villablanca et al., 2007). However, our knowledge of the role of PGE₂ receptors in the teleost immune response is still limited, which makes a comparative analysis with their higher vertebrate counterparts quite difficult.

In previous studies, we showed that the cell-permeable analog of cAMP (dbcAMP) mimicked the effect of PGE₂ in the regulation of cytokine mRNA levels in stimulated macrophages but not in AGs. The variety of effects that PGE₂ elicits in both phagocytic cell types could reflect the presence of specific receptors in those cells and suggest that PGE₂ signals through EP2 and/or EP4 in seabream macrophages but not in AGs. Taking into consideration all the above, the aim of this study was the identification and in silico characterization of gilthead seabream PGE₂ receptors (Ptger1-4) and to gain further insight into their role in the resolution of inflammation in vivo an in vitro. In particular, their distribution in vivo and modulation after bacterial challenge and their contribution to phagocytic cell functions were analyzed. These data pave the way for analysis of the role of PGE2 receptors during the immune response of teleost fish.