Identification and characterization of ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) involved in regulating extracellular ATP-mediated innate immune responses in Japanese flounder (Paralichthys olivaceus)

doi:10.1016/j.molimm.2019.04.024

Molecular Immunology

Volume 112, August 2019, Pages 10-21

https://doi.org/10.1016/j.molimm.2019.04.024 Get rights and content

Highlights

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CD39 gene (poCD39) was identified from Japanese flounder Paralichthys olivaceus.
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poCD39 is dominantly expressed in Japanese flounder head kidney macrophages.
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poCD39 expression was significantly upregulated by extracellular ATP stimulation and immune challenges.
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poCD39 protein is a functional membrane glycoprotein for hydrolysis of extracellular ATP.
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poCD39 is involved in regulating extracellular ATP-mediated innate immune responses in fish.

Abstract

Extracellular adenosine triphosphate (eATP), released following inflammatory stimulation or infection, is a potent signaling molecule in activating innate immune responses in fish. However, the regulation of eATP-mediated innate immunity in fish remains unknown. Ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) is a critical molecular switch for controlling the ATP levels in the extracellular space. CD39 plays a key role in regulating eATP-activated innate immune responses through the phosphohydrolysis of pro-inflammatory eATP to inactive AMP. Here, we identified and characterized a CD39 homolog (namely, poCD39) in the Japanese flounder Paralichthys olivaceus and analyzed its regulatory role in eATP-mediated innate immunity. Real-time quantitative PCR analysis revealed that poCD39 is ubiquitously present in all tested normal tissues with dominant expression in enriched Japanese flounder head kidney macrophages (HKMs). Immune challenge experiments demonstrated that poCD39 expression was upregulated by inflammatory stimulation and Edwardsiella tarda infection. Biochemical and immunofluorescence analysis revealed that poCD39 is a functional glycosylated membrane protein for the hydrolysis of eATP. Inhibition of poCD939 activity with the ecto-NTPDase inhibitor ARL 67156 resulted in increased IL-1beta gene expression and ROS production in Japanese flounder HKMs. In contrast, overexpression of poCD39 in Japanese flounder FG-9307 cells reduced eATP-induced pro-inflammatory cytokine IL-1beta gene expression. Finally, poCD39 expression was significantly induced by eATP stimulation in the HKMs, suggesting that eATP may provide a feedback mechanism for transcriptional regulation of fish CD39. Taken together, we identified and characterized a functional fish CD39 protein involved in regulating eATP-mediated innate immune responses in fish.

Introduction

Extracellular ATP (eATP), released from host cells under conditions of stress, inflammatory stimulation or infection, is an important endogenous signaling molecule with pro-inflammatory properties through action on ATP-gated P2 receptors. Our previous studies revealed that inflammatory challenge or pathogen infections can elicit significant ATP release into the extracellular space through pannexin 1 and/or connexin 43 channels in fish (Li et al., 2018d, a; Li et al., 2016c). In addition, several ATP-gated purinergic receptor subtypes, including P2X2, P2X4, P2X7 and P2Y₂, were detected in various Japanese flounder (Paralichthys olivaceus) tissues and immune cells (Li et al., 2016b, 2015b, 2018b). Furthermore, we also showed that eATP plays critical roles in regulating pro-inflammatory cytokine gene expression (Li et al., 2014b), NOD-like receptor family CARD domain containing 3 (NLRC3) inflammasome immune signaling (Li et al., 2016a), and the production of inflammatory mediators including ROS and NO in Japanese flounder head kidney macrophages (Li et al., 2018a). Moreover, we recently found that eATP broadly modulated the expression and enzyme activities of several caspases including caspase 1, 2, 3, 6 and 8 in Japanese flounder immune cells (Li et al., 2019, 2017), suggesting the involvement of eATP in fish apoptosis. These findings indicate that eATP is a potent signaling molecule that plays important roles in P. olivaceus innate immunity.

Despite the positive roles of eATP in triggering host innate immune responses, the contents of eATP must be tightly controlled as excessive accumulation of eATP may lead to excessive inflammatory responses and result in detrimental effects or damage to the immune system. To restore homeostasis and avoid excessive inflammation-induced tissue damage, timely attenuation or termination of eATP-induced inflammatory responses is thus critically required. The metabolism of eATP is one of the critical mechanisms that attenuate eATP-stimulated pro-inflammatory responses in innate immunity.

Ecto-nucleoside triphosphate diphosphohydrolase, namely, NTPDase, is an enzyme family that catalyzes the hydrolysis of γ- and β-phosphate residues of nucleotides, albeit to different degrees of effectiveness (Zimmermann, 2001). NTPDase 1 (EC 3.6.1.5), also known as CD39 or ecto-apyrase, was initially described as an activation marker of lymphoid cells (Maliszewski et al., 1994). A growing number of studies, however, have demonstrated that CD39 is a dominant NTPDase in immune tissues/cells and plays an important role in regulating eATP-mediated innate immunity by reducing the levels of pro-inflammatory ATP in the extracellular space (Mizumoto et al., 2002). CD39 inhibits inflammation by phosphohydrolyzing ATP to AMP, and the latter is further metabolized by another ecto-5′-nucleotidase (CD73) to anti-inflammatory adenosine (Colgan et al., 2006). CD39 is thus tightly linked with purinergic signaling in inflammatory responses (Dwyer et al., 2007; Mizumoto et al., 2002). For instance, through the hydrolysis of eATP, CD39 can terminate P2 receptor activation and plays a key role in the control of P2X7R-dependent macrophage responses (Levesque et al., 2010) and P2X7R-mediated inflammatory cell death in mast cells (Kuhny et al., 2014). In addition, it has been found that CD39 controls IL-8 production via regulation of P2Y₂ receptor activation in human neutrophils (Kukulski et al., 2011). Furthermore, CD39 can protect against excessive organ inflammation and damage, enhancing the survival of septic mice (Csoka et al., 2015). In contrast, suppression of constitutive CD39 expression may result in elevated concentrations of ATP and ADP at the vascular interface that could lead to a predisposition to thrombosis and inflammation (Imai et al., 1999). Finally, CD39 has been suggested to play a role in dampening the general immune activation in T cells (Schulze zur Wiesch et al., 2011).

Although intensive investigations in mammals have been conducted, the properties and immune relevance of CD39 in fish have not been investigated. In this report, we identified and characterized a CD39 gene, namely, poCD39, in the Japanese flounder (Paralichthys olivaceus) and analyzed its regulatory role in eATP-mediated innate immune responses. We demonstrated for the first time that poCD39 is a functional membrane glycoprotein involved in regulating eATP-mediated innate immune responses in fish.

Section snippets

Experimental animals

Japanese flounder P. olivaceus obtained from a local fish farm in Tianjin, China, were cultured in an aerated recirculating seawater system at 21 °C in laboratory for two weeks before experimentation commenced. Fish were handled and clinically examined as described in our previous study (Li et al., 2014a); only healthy animals without any pathological signs were used in experiments. All experiments were carried out in accordance with the NIH guidelines for the care and use of experimental

Cloning and sequence analysis of CD39 from Japanese flounder P. olivaceus

Using a combined approach of RT-PCR and RACE technology, the CD39 gene (namely, poCD39) was successfully cloned from Japanese flounder liver tissue. The complete Japanese flounder poCD39 cDNA sequence comprised a 97 bp 5′-untranslated region, a 1523 bp open reading frame and a 48 bp 3′-untranslated region with a poly(A) tail (Supplementary Fig. 1). This cDNA sequence was deposited into the GenBank database with accession number KP347442. The deduced Japanese flounder poCD39 protein is composed

Discussion

Our previous studies demonstrated that ATP released from fish cells under conditions of inflammatory stimulation or infection, is a critical extracellular signaling molecule that plays important roles in the activation of fish innate immune responses through activation of purinergic signaling cascades (Li et al., 2018a, 2014b). Although proper inflammation is essential for the repair and resolution of infection and damage, excessive inflammation is deleterious and may cause serious diseases or

Declarations of interest

None.

Acknowledgements

This research was funded by the Natural Science Foundation of Tianjin (NO. 18JCZDJC33600), the National Natural Science Foundation of China (No. 31572645), the National Key R&D Program of China (No. 2018YFD0901301), the University Yong Talent Cultivation Program provided by Tianjin Municipal Education Commission, the Program for Innovative Research Team in Universities of Tianjin (TD13-5076), the Tianjin Development Program for Innovation and Entrepreneurship, and the Innovation Team of Tianjin

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Identification and characterization of two novel functional CD39 splice variants that negatively modulate the proinflammatory response in the Japanese flounder Paralichthys olivaceus
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Citation Excerpt :
In addition, it has been reported that C-terminal splicing of rat NTPDase2 (CD39L1) exhibits distinctive catalytic properties, cellular distribution and enzyme regulation (Carol et al., 2005). We have previously demonstrated that CD39 is a functional cell membrane glycoprotein that is involved in regulating eATP-mediated innate immune responses in P. olivaceus (Li et al., 2019a), an important mariculture teleost fish species in East Asia. However, even though the presence of multiple CD39 splice variants has been reported in mammals, their expression, transcriptional regulation and functions in lower vertebrates, such as teleost fish, remain unknown.
Ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) is a key regulator of inflammatory responses by scavenging proinflammatory extracellular ATP (eATP) and promoting the generation of immunosuppressive adenosine. In addition to the previously identified CD39 homolog (designated CD39a), here we identified two novel functional CD39 splice variants (termed CD39b and CD39c) from teleost fish Japanese flounder (Paralichthys olivaceus). Transcriptional expression analysis revealed that all three CD39 splicing variants were coexpressed in Japanese flounder head kidney macrophages (HKMs) and a variety of normal tissues but with differential tissue/immune cell expression preferences. Immune challenge experiments showed that the expression of CD39 splice variants was modulated by both in vitro inflammatory stimulation and in vivo bacterium Edwardsiella piscicida infections. However, even though all CD39 splice variants were functional cell surface-expressed proteins and shared generally similar domain structures, their varied cytoplasmic N-terminal sequences resulted in a varied capability for hydrolysis of eATP and attenuation of eATP/P2XR-mediated proinflammatory cytokine expression. Pharmacological inhibition of endogenous CD39 activity in Japanese flounder HKMs or overexpression of CD39 splice variants in Japanese flounder FG-9307 cells further revealed the role of CD39 variants in down-regulating LPS-induced proinflammatory cytokine production. In addition, luciferase reporter analysis demonstrated that the CD39 splice variant promoters had varied transcriptional activities and that Sp1 was a potential transcription factor for CD39c transcription regulation. Taken together, we report for the first time that functional CD39 splice variants provide additional means to downregulate eATP- and LPS-induced proinflammatory response(s) in fish.
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Extracellular ATP-mediated purinergic immune signaling in teleost fish: A review
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It was found that extracellular ATP-induced IL-1beta gene expression and ROS production were increased in Japanese flounder HKMs by pharmacological inhibition of CD39 activity with the ecto-NTPDase inhibitor ARL 67156 (Li et al., 2019a). In contrast, overexpression of ENTPDase1 in Japanese flounder FG-9307 cells reduced extracellular ATP-induced proinflammatory cytokine IL-1beta gene expression (Li et al., 2019a; Li et al., 2020). Given the high expression level of ENTPDase1 in Japanese flounder HKMs, it was proposed that ENTPDase1 may serve as a “brake” of the proinflammatory actions of extracellular ATP in fish by controlling the available extracellular ATP levels (Li et al., 2019a) .
Extracellular nucleotides, including adenosine triphosphate (ATP), adenosine 5′-diphosphate (ADP), uridine 5′-triphosphate (UTP) and uridine 5′-diphosphate (UDP), released under inflammation or infection conditions, are important danger-associated signaling molecules that regulate host immune responses and inflammation by binding to and activating specific surface-localized ionotropic P2X and metabotropic P2Y purinergic receptors on immune cells in an autocrine and/or paracrine manner. In this review, we summarize recent studies regarding the molecular mechanisms of immune challenge-induced ATP release in fish, the role of extracellular ATP signaling in the activation of fish innate immune responses, the purinergic receptors involved in regulating fish innate immune responses, the crosstalk between nucleotide oligomerization domain-like receptor (NLR) inflammasome signaling and extracellular ATP-mediated immune signaling, and the role of ectonucleoside triphosphate diphosphohydrolases in regulating extracellular ATP-induced innate immune responses in fish. In addition, we also discuss the immunomodulatory role of other extracellular nucleotides, including UTP, UDP and ADP, in fish.
Characterization of three connexin32 genes and their role in inflammation-induced ATP release in the Japanese flounder Paralichthys olivaceus
2020, Fish and Shellfish Immunology
Citation Excerpt :
We also demonstrated that extracellular ATP is a potent signaling molecule in the activation of pro-inflammatory responses in fish [16]. In addition, crosstalk between extracellular ATP-mediated immune signaling and NOD-like receptor family CARD domain containing 3 (NLRC3) inflammasome immune signaling was also found in Japanese flounder (Paralichthys olivaceus) [17]. Furthermore, our recent studies revealed a apoptotic role of extracellular ATP in fish [18,19].
Extracellular ATP (eATP) is a potent singling molecule in activation of fish innate immunity while the molecular determinants for eATP release in fish were not completely understood. Connexin32 (Cx32) is a member of gap junction protein family that plays important immunological functions in mammals. However, the immune relevance of Cx32 and its role in ATP release in fish has not been investigated. Here, we identified, characterized three Cx32 isoform genes (Cx32.2, Cx32.2x and Cx32.7) from the Japanese flounder Paralichthys olivaceus, and investigated their role in inflammation-induced ATP release in fish. Expression analysis revealed that even though all the three Cx32 genes are constitutively expressed in all examined Japanese flounder tissues, Cx32.2 and Cx32.2x are dominantly expressed in liver, and Cx32.7 is highly expressed in intestine and head kidney macrophages. In addition, we showed that gene expression of all the three Cx32 isoforms was modulated by cAMP stimulation and inflammatory challenges. Furthermore, we revealed that Cx32 expression was upregulated in TNF-alpha overexpressed Japanese flounder FG-9307 cells. Moreover, overexpression of the three Cx32 isoforms significantly reduced the gene expression level of LPS-induced pro-inflammatory cytokine IL-8 and TNF-alpha, indicating that Cx32 is involved in modulating inflammatory response in fish. Finally, we showed that inflammation-induced ATP release was significantly increased in Cx32-overexpressed Japanese flounder FG-9307 cells, and this increased ATP release could be attenuated by pre-incubation with gap junction protein blocker carbenoxolone. Taken together, we for the first time reported the involvement of Cx32 in fish immunity. Our findings suggested that in addition to Cx43 and pannexin1 channels, Cx32 also plays a role in inflammation-induced ATP release in fish.
Purinergic signaling and gene expression of purinoceptors in the head kidney of the silver catfish Rhamdia quelen experimentally infected by Flavobacterium columnare
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The head kidney is a lymphoid immune organ that plays a key role in the immune and inflammatory responses of teleost fish. It is associated with immunoglobulin G production and differentiation of B cells. The presence of a multi-enzymatic complex found anchored in the plasma membrane makes the head kidney an important purinergic-dependent tissue. Purinergic signaling has been associated with these responses under pathological conditions via regulation of extracellular adenosine triphosphate (ATP), the main damage molecular associated pattern agent released during bacterial infections. The aim of this study was to determine whether purinergic signaling is a pathway associated with impairment of immune responses in silver catfish (Rhamdia quelen) experimentally infected by Flavobacterium columnare, as well as to evaluate the role of P2 purine receptors in this response. Triphosphate diphosphohydrolase (NTPDase) activity in the head kidney was significantly lower in silver catfish experimentally-infected F. columnare 72 h post-infection (hpi) than in the control group, while no significant difference was observed with respect NTPDase activity on adenosine diphosphate, as well as on 5′-nucleotidase and adenosine deaminase activities. Extracellular ATP levels were significantly higher in the head kidney of experimentally-infected fish than in the control group at 72 hpi. Finally, p2ry11 and p2rx3 purine receptor levels were significantly higher in experimentally-infected fish than in the control group at 72 hpi. We conclude that purinergic signaling in the head kidney of silver catfish infected by F. columnare creates a pro-inflammatory profile that may contribute to impairment of immune and inflammatory responses via reduction of ATP hydrolysis and its accumulation in the extracellular milieu, accompanied by upregulation of p2ry11 and p2rx3 purine receptors, leading to pro-inflammatory status.
Functional characterization of two ecto-nucleoside triphosphate diphosphohydrolase 2 genes in Japanese flounder (Paralichthys olivaceus) head kidney macrophages
2020, Fish and Shellfish Immunology
Citation Excerpt :
The expression of recombinant ENTPDase1, ENTPDase2 and ENTPDase2a proteins was confirmed by western blotting analysis using anti-Flag antibody (Fig. 9B). By blocking ecto-NTPDases activities with the ecto-NTPDase inhibitor ARL 67156, we showed previously that ENTPDase1 plays important roles in regulating eATP-mediated innate immune responses in P. olivaceus [23]. However, in addition to ENTPDase1, ARL 67156 can also inhibit the activities of other ecto-NTPDases [27].
Ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases) are pivotal regulators of extracellular ATP-mediated purinergic immune signaling. ENTPDase2 is a member of the cell surface-bound ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) protein family that hydrolyzes extracellular nucleoside 5′-triphosphates and nucleoside 5′-diphosphates. However, the immune relevance of ENTPDase2 in fish has not been elucidated. In the present study, from a comparative immunological perspective, we functionally characterized two ENTPDase2 transcript variants (namely ENTPDase2 and ENTPDase2a) from Japanese flounder (Paralichthys olivaceus). Sequence analysis indicates that the deduced Japanese flounder ENTPDase2 and ENTPDase2a proteins possess two conserved transmembrane domains and five apyrase conserved regions that are present in ENTPDase family proteins. However, these proteins only share 54% amino acid sequence identity. Tissue expression analysis revealed that both ENTPDase2 and ENTPDase2a mRNA transcripts are ubiquitously expressed in all examined Japanese flounder tissues, whereas ENTPDase2 is dominantly expressed in blood and ENTPDase2a is abundantly expressed in muscle. Immune challenge experiments showed that ENTPDase2 and ENTPDase2a were significantly upregulated by both inflammatory stimulation and Edwardsiella tarda infection. In addition, the expression of ENTPDase2 and ENTPDase2a was modulated by extracellular ATP (eATP) stimulation in a dose-dependent manner. Furthermore, immunolocalization and functional studies demonstrated that both ENTPDase2 and ENTPDase2a are functional glycosylated plasma membrane proteins. However, ENTPDase2a exhibits greater activity in the hydrolysis of eATP than ENTPDase2 and ENTPDase1 proteins. Finally, knockdown of the ENTPDase2 gene by small interfering RNA significantly upregulated the expression of eATP-induced proinflammatory cytokines IL-1beta, TNF-alpha and G-CSF in Japanese flounder head kidney macrophages, while knockdown of ENTPDase2a only upregulated eATP-induced IL-1beta expression. Taken together, our findings suggest that the two functional Japanese flounder ENTPDase2 isoforms play an essential role in the downregulation of eATP-induced proinflammatory cytokine expression in fish by degrading the available ATP levels in the extracellular milieu.

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Identification and characterization of ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) involved in regulating extracellular ATP-mediated innate immune responses in Japanese flounder (Paralichthys olivaceus)

Highlights

Abstract

Introduction

Section snippets

Experimental animals

Cloning and sequence analysis of CD39 from Japanese flounder P. olivaceus

Discussion

Declarations of interest

Acknowledgements

Trends Mol. Med.

J. Invest. Dermatol.

Cell

Fish Shellfish Immunol.

Fish Shellfish Immunol.

Fish Shellfish Immunol.

Fish Shellfish Immunol.

Fish Shellfish Immunol.

Fish Shellfish Immunol.

Fish Shellfish Immunol.

Fish Shellfish Immunol.

Fish Shellfish Immunol.

BMC Vet. Res.

Biochim. Biophys. Acta (BBA) - Protein Structure and Molecular Enzymology

Vascular smooth muscle cell expression of ectonucleotidase CD39 (ENTPD1) is required for neointimal formation in mice

Purinergic Signal.

Extracellular ATP selectively upregulates ecto-nucleoside triphosphate diphosphohydrolase 2 and ecto-5’-nucleotidase by rat cortical astrocytes in vitro

J. Mol. Neurosci.

Physiological roles for ecto-5’-nucleotidase (CD73)

Purinergic Signal.

CD39 improves survival in microbial sepsis by attenuating systemic inflammation

FASEB J.

CD39 and control of cellular immune responses

Purinergic Signal.

The C-terminal cysteine-rich region dictates specific catalytic properties in chimeras of the ectonucleotidases NTPDase1 and NTPDase2

Eur. J. Biochem.