Fish and Shellfish Immunology 93 (2019) 200–207 Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Full length article Functional characterization of purinergic receptor P2Y14 in the Japanese flounder (Paralichthys olivaceus) head kidney macrophages T Shuo Lia,1, Nan Wanga, Yu Fenga, Jiafang Lia, Xuyun Gengb, Jinsheng Suna,* a Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin, 300387, China b Tianjin Fisheries Research Institute, 442 South Jiefang Road, Hexi District, Tianjin, 300221, China ARTICLE INFO ABSTRACT Keywords: Extracellular nucleotides and nucleotide sugars are important danger-associated signaling molecules that play P2Y receptor critical roles in regulation of immune responses in mammals through activation of purinergic receptors located Inflammation regulation on the cell surface. However, the immunological role of extracellular UDP-glucose-activated P2Y14 receptor ERK1/2 signaling (P2Y14R) in fish still remains unknown. In this study, we identified and characterized a P2Y14R paralog in the Paralichthys olivaceus Japanese flounder (Paralichthys olivaceus). The mRNA transcripts of P2Y14R are detected in all examined Japanese flounder tissues. Compared with the UDP-activated P2Y6 receptor, however, P2Y14R gene is highly expressed in Japanese flounder head kidney macrophages (HKMs). In addition, P2Y14R is significantly upre- gulated following inflammatory stimulation with LPS and poly (I:C) in the HKMs, suggesting a role of P2Y14Rin response to inflammation in fish. Furthermore, activation of P2Y14 receptor with its potent and selective agonist MRS 2905 resulted in a decreased expression of LPS-induced pro-inflammatory cytokine IL-1beta gene in the HKMs. In contrast, inhibition of P2Y14 receptor activity or down-regulation of the endogenous expression of P2Y14R by small interfering RNA significantly upregulates the LPS-induced pro-inflammatory cytokine IL-1beta gene expression in the HKMs, demonstrating that P2Y14R is involved in inflammation regulation in fish. Moreover, stimulation of the Japanese flounder HKMs with UDP-glucose evoked a rapid increase of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in a dose- and time-dependent manner, indicating the involvement of P2Y14R in activation of ERK1/2 signaling in fish immune cells. Taken together, we demonstrated that the inducible P2Y14R plays an important role in regulation of fish innate immunity. 1. Introduction UDP and UDP-sugars [7], is highly expressed in immune and in- flammatory cells [7,8]. Activation of P2Y14R regulates a range of im- Extracellular nucleotides such as ATP, ADP, UTP, and UDP and mune responses including maturation of dendritic cells [9], inhibition nucleotide sugars such as uridine 5′-diphosphoglucose (UDP-glucose) of T lymphocyte proliferation [10], chemotaxis of bone-marrow he- are important danger signaling molecules that play an important role in matopoietic stem cells [11] and neutrophils [12], and secretion of the the regulation of immune responses through binding to and activating potent neutrophil chemoattractant CXCL8/IL-8 in airway epithelial of different members of plasma membrane P2 receptors, which are cells [13]. In addition, it has been demonstrated that UDP-glucose and widely expressed on immune cells [1–3]. P2Y receptors belonging to its receptor P2Y14R are key front line players able to trigger innate the G-protein-coupled receptors (GPCRs) superfamily, are a family of mucosal immune responses in mouse female reproductive tract [14]. the metabotropic purinergic receptors for adenine and uridine nucleo- We showed previously that extracellular UDP-activated P2Y6 re- tides and nucleotide sugars. Eight P2Y receptor subtypes (P2Y1, P2Y2, ceptors functions importantly in the Japanese flounder Paralichthys P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14) with differences in both olivaceus peripheral blood leukocytes [15]. Because extracellular UDP pharmacology and downstream signaling pathways have been identi- can also activate P2Y14 receptors [8], we presumed the hypothesis that fied in mammals [4–6]. P2Y14 receptors could play a role in Japanese flounder innate im- In particular, P2Y14R that is potently and selectively activated by munity. For this purpose, we cloned P2Y14 receptor gene and * Corresponding author. College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin, 300387, China. E-mail addresses: [email protected] (S. Li), [email protected] (J. Sun). 1 S. Li is a senior author. https://doi.org/10.1016/j.fsi.2019.07.053 Received 22 April 2019; Received in revised form 15 July 2019; Accepted 18 July 2019 Available online 18 July 2019 1050-4648/ © 2019 Elsevier Ltd. All rights reserved. S. Li, et al. Fish and Shellfish Immunology 93 (2019) 200–207 characterized its expression under normal and inflammatory conditions Table 1 in Japanese flounder head kidney macrophages (HKMs). By pharma- Sequence of primers used in this study. cological activation or inhibition of P2Y14R activities, and down-reg- Primers Sequence (5'→3′) ulation of the P2Y14R endogenous expression in the HKMs, we explored the potential role of the P2Y14 receptor in Japanese flounder innate F ATGGATCCCTTCAACGTCAG R TCCTGTTGTCGGTTTTCTCA immunity. In addition, we investigated the consequence of P2Y14R fi qP2Y14R-f GGGTCGCTGTTC TGGGTGA activation on ERK signaling in the HKMs. Taken our ndings together, qP2Y R-r ATCCAACCGCAGCATCTTC fi 14 we for the rst time demonstrated that the inducible P2Y14R plays an qP2Y6R-f AGTGCGGAGATGCGGGAC important role in regulation of fish innate immune response and in qP2Y6R-r GGTATCGTGGCTGTGTGAAGTA activation of ERK signaling pathway in fish immune cells. qP2Y2R-f TTCCTCCCAGCGAAGACG qP2Y2R-r CAGACGGACGTAGCAACCAG qP2Y12R-f ACTGGCCTGCGTGTGTTTG 2. Material and methods qP2Y12R-r AGATGGAGCCCGAGAGAAGC qβ-actin-f AGGTTCCGTTGTCCCG 2.1. Experimental animals qβ-actin-r TGGTTCCTCCAGATAGCAC qIL-1β-f CCTGTCGTTCTGGGCATCAA qIL-1β-r CACCCCGCTGTCCTGCTT Japanese flounder P. olivaceus were obtained from a local fish farm in Tianjin, China. Fish were cultured in an aerated recirculating sea- q: quantitative real-time PCR; f: forward; r: reverse. water system at 21 °C in the laboratory for two weeks before use in experiments. Fish were handled and clinically examined as described in 2.5. Expression analysis of P2Y14R mRNA transcripts in Japanese flounder our previous study [16]. Only animals with healthy appearance and tissues and head kidney macrophages normal activity were used in experiments. qRT-PCR analysis (see section 2.8) was performed to determine the 2.2. Chemicals and antibodies relative basal gene expression level of P2Y14R in various Japanese flounder tissues including blood, gill, head kidney, trunk kidney, heart, UDP-glucose, LPS and poly (I:C) were obtained from Sigma-Aldrich. liver, skin, muscle, intestine and spleen with a protocol described in the ′ α The potent and selective P2Y14 receptor agonist 2-Thiouridine-5 -O-( , previous study [21]. In addition, qRT-PCR analysis was also applied to β-methylene)diphosphate trisodium salt (MRS 2905) and high affinity compare the relative gene expression level of P2Y2, P2Y6, P2Y12 and and selective P2Y receptor antagonist 4-[4-(4-Piperidinyl)-phenyl]-7- 14 P2Y14 receptors in the Japanese flounder HKMs. To this aim, total RNA ® [4-(trifluoromethyl)-phenyl]-2-naphthalenecarboxylic acid (PPTN) from the HKM cells and tissues was purified with a PureLink RNA Mini were purchased from Tocris Bioscience. Rabbit monoclonal antibodies Kit and TRIzol reagent (Invitrogen), respectively, according to the against phospho-p44/p42 MAPK (p-Erk1/2) and against total p44/p42 manufacturer's recommendations. RNA was quantified by a NanoDrop MAPK (Erk1/2) were purchased from Cell Signaling Technology. spectrophotometer and treated with RNase Free DNase I (Invitrogen) to ® Lipofectamine RNAiMAX transfection reagent, HRP-labeled goat anti- digest possible contaminating genomic DNA following the manufac- mouse and goat anti-rabbit secondary antibodies and mouse mono- turer's protocol. The treated RNA was then transcribed into cDNAs clonal antibody against beta-actin were obtained from ThermoFisher using a SuperScript III reverse transcriptase kit (Invitrogen) according Scientific. to the manufacturer's directions. The relative gene expression levels for the individual P2Y receptor genes were determined by qRT-PCR (see 2.3. Preparation and cell culture of Japanese flounder head kidney section 2.8) with beta-actin serving as an internal reference gene. macrophages 2.6. In vitro immune challenge experiments in the Japanese flounder HKMs To collect head kidney tissues, Japanese flounder were anesthetized using 0.25 g/L tricaine methanesulfonate (Sigma-Aldrich) and the head To study the involvement of P2Y R in Japanese innate immune kidney was rapidly dissected under sterilized conditions. The dissected 14 responses, Japanese flounder HKMs were challenged with pathogen- head kidney tissues were used to prepare head kidney primary cells associated molecular patterns (PAMPs) LPS and poly (I:C) as described with the procedure described previously [17]. The head kidney mac- in a previous study [22]. Briefly, overnight-cultured Japanese flounder rophages (HKMs) were further purified from the obtained primary head HKMs (5 × 106 cells/well) were stimulated with 20 μg/ml LPS or poly kidney cell suspensions by discontinuous Percoll (GE Biosciences)