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Bockstiegel J, Engelhardt J, Schuchardt M, Tölle M, Weindl G. The vasoconstrictor adenosine 5'-tetraphosphate is a danger signal that induces IL-1β. Mol Med 2025; 31:72. [PMID: 39984847 PMCID: PMC11844157 DOI: 10.1186/s10020-025-01116-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2024] [Accepted: 02/05/2025] [Indexed: 02/23/2025] Open
Abstract
BACKGROUND The endogenous nucleotide adenosine 5'-tetraphosphate (Ap4) is a potent vasoconstrictor. Despite its structural similarity to the danger signal adenosine 5'-triphosphate (ATP), the immunomodulatory effects of Ap4 remain unclear. METHODS Modulation of interleukin (IL)-1β secretion by Ap4 was studied in both immune cells lines (THP-1, U937) and primary immune cells. Genetic and pharmacological approaches were used to characterize signaling. Cytokine production was measured using ELISA and multiplex assays, while cell viability was determined by MTT and LDH assays. Calcium influx and YO-PRO-1 uptake were assessed via microplate assays and flow cytometry, respectively. RNA sequencing and Western blotting were performed to analyze global gene expression and protein levels. RESULTS We demonstrate that Ap4 stimulates IL-1β release in primed immune cells without affecting the levels of other cytokines, suggesting specificity in its immunomodulatory actions. Mechanistically, Ap4-induced IL-1β release was partially modulated by the P2X7 receptor, a key mediator of inflammation. However, unlike canonical inflammasome activators, this process was independent of potassium efflux, the NLRP3 inflammasome, and caspase-1. Ap4 specifically increased LDH release in macrophages irrespective of priming. Furthermore, Ap4-mediated calcium influx, crucial for immune cell activation, predominantly occurred through P2Y receptors rather than P2X7 receptors. Transcriptomic analysis highlighted Ap4-induced upregulation of metallothioneins, implicating metal ion homeostasis in Ap4-mediated responses. CONCLUSIONS Collectively, our findings suggest Ap4 as a novel pro-inflammatory mediator capable of inducing IL-1β release in innate immune cells through distinct mechanisms from classical NLRP3 inflammasome activators, shedding light on its potential role in inflammatory diseases and vascular disorders.
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Affiliation(s)
- Judith Bockstiegel
- Pharmaceutical Institute, Pharmacology and Toxicology Section, University of Bonn, Gerhard- Domagk-Str. 3, 53121, Bonn, Germany
| | - Jonas Engelhardt
- Pharmaceutical Institute, Pharmacology and Toxicology Section, University of Bonn, Gerhard- Domagk-Str. 3, 53121, Bonn, Germany
| | - Mirjam Schuchardt
- Department of Nephrology and Medical Intensive Care, Charité-Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt Universität zu Berlin, Hindenburgdamm 30, 12203, Berlin, Germany
- Medical School Berlin, Faculty of Medicine, Rüdesheimer Straße 50, 14513, Berlin, Germany
| | - Markus Tölle
- Department of Nephrology and Medical Intensive Care, Charité-Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt Universität zu Berlin, Hindenburgdamm 30, 12203, Berlin, Germany
| | - Günther Weindl
- Pharmaceutical Institute, Pharmacology and Toxicology Section, University of Bonn, Gerhard- Domagk-Str. 3, 53121, Bonn, Germany.
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Du J, Meng X, Ni T, Xiong B, Han Z, Zhu Y, Tu J, Sun H. Mechanism of Innate Immune Response Induced by Albizia julibrissin Saponin Active Fraction Using C2C12 Myoblasts. Vaccines (Basel) 2023; 11:1576. [PMID: 37896979 PMCID: PMC10610972 DOI: 10.3390/vaccines11101576] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2023] [Revised: 10/04/2023] [Accepted: 10/05/2023] [Indexed: 10/29/2023] Open
Abstract
Albizia julibrissin saponin active fraction (AJSAF), is a prospective adjuvant with dual Th1/Th2 and Tc1/Tc2 potentiating activity. Its adjuvant activity has previously been proven to be strictly dependent on its spatial co-localization with antigens, highlighting the role of local innate immunity in its mechanisms. However, its potential targets and pathways remain unclear. Here, its intracellular molecular mechanisms of innate immune response were explored using mouse C2C12 myoblast by integrative analysis of the in vivo and in vitro transcriptome in combination with experimental validations. AJSAF elicited a temporary cytotoxicity and inflammation towards C2C12 cells. Gene set enrichment analysis demonstrated that AJSAF regulated similar cell death- and inflammatory response-related genes in vitro and in vivo through activating second messenger-MAPK-CREB pathways. AJSAF markedly enhanced the Ca2+, cAMP, and reactive oxygen species levels and accelerated MAPK and CREB phosphorylation in C2C12 cells. Furthermore, Ca2+ chelator, CREB inhibitor, and MAPK inhibitors dramatically blocked the up-regulation of IL-6, CXCL1, and COX2 in AJSAF-treated C2C12 cells. Collectively, these results demonstrated that AJSAF induced innate immunity via Ca2+-MAPK-CREB pathways. This study is beneficial for insights into the molecular mechanisms of saponin adjuvants.
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Affiliation(s)
- Jing Du
- College of Animal Sciences, Zhejiang University, Hangzhou 310058, China; (J.D.); (X.M.); (T.N.); (B.X.); (Z.H.); (J.T.)
| | - Xiang Meng
- College of Animal Sciences, Zhejiang University, Hangzhou 310058, China; (J.D.); (X.M.); (T.N.); (B.X.); (Z.H.); (J.T.)
| | - Tiantian Ni
- College of Animal Sciences, Zhejiang University, Hangzhou 310058, China; (J.D.); (X.M.); (T.N.); (B.X.); (Z.H.); (J.T.)
| | - Beibei Xiong
- College of Animal Sciences, Zhejiang University, Hangzhou 310058, China; (J.D.); (X.M.); (T.N.); (B.X.); (Z.H.); (J.T.)
| | - Ziyi Han
- College of Animal Sciences, Zhejiang University, Hangzhou 310058, China; (J.D.); (X.M.); (T.N.); (B.X.); (Z.H.); (J.T.)
| | - Yongliang Zhu
- Laboratory of Gastroenterology Department, Second Affiliated Hospital of School of Medicine, Zhejiang University, Hangzhou 310009, China;
| | - Jue Tu
- College of Animal Sciences, Zhejiang University, Hangzhou 310058, China; (J.D.); (X.M.); (T.N.); (B.X.); (Z.H.); (J.T.)
- Academy of Chinese Medical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
| | - Hongxiang Sun
- College of Animal Sciences, Zhejiang University, Hangzhou 310058, China; (J.D.); (X.M.); (T.N.); (B.X.); (Z.H.); (J.T.)
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Peng X, Zhang Y, Bai X, Li X, Zhao R. Phasic regulation of the ATP/P2X7 receptor signaling pathway affects the function of antigen-presenting cells in experimental autoimmune uveitis. Int Immunopharmacol 2023; 119:110241. [PMID: 37141671 DOI: 10.1016/j.intimp.2023.110241] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2023] [Revised: 04/17/2023] [Accepted: 04/22/2023] [Indexed: 05/06/2023]
Abstract
Purinergic ligand-gated ion channel 7 receptor (P2X7R) is a purine type P2 receptor that is expressed on a variety of immune cells. Recent studies have shown that P2X7R signaling is required to trigger an immune response, and P2X7R antagonist-oxidized ATP (oxATP) effectively blocks P2X7R activation. In this study, we investigated the effect of phasic regulation of the ATP/P2X7R signaling pathway on antigen-presenting cells (APCs) by constructing an experimental autoimmune uveitis (EAU) disease model. Our results demonstrated that APCs isolated from the 1st, 4th, 7th and 11th days of EAU presented antigen function and could stimulate the differentiation of naive T cells. Moreover, after stimulation by ATP and BzATP (a P2X7R agonist), antigen presentation, promoting differentiation and inflammation were enhanced. The regulation of the Th17 cell response was significantly stronger than that of the Th1 cell response. In addition, we verified that oxATP blocked the P2X7R signaling pathway on APCs, attenuated the effect of BzATP, and significantly improved the adoptive transfer EAU induced by antigen-specific T cells cocultured with APCs. Our results demonstrated that at an early stage of EAU, the ATP/P2X7R signaling pathway regulation of APCs was time dependent, and the treatment of EAU could be achieved by intervening in P2X7R function on APCs.
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Affiliation(s)
- Xiaoxiang Peng
- School of Medical Laboratory, Weifang Medical University, Weifang, Shandong, China; Institutional Key Laboratory of Clinical Laboratory Diagnostics, 12th 5-Year Project of Shandong Province, Weifang Medical University, Weifang, Shandong, China
| | - Yunfang Zhang
- School of Medical Laboratory, Weifang Medical University, Weifang, Shandong, China; Institutional Key Laboratory of Clinical Laboratory Diagnostics, 12th 5-Year Project of Shandong Province, Weifang Medical University, Weifang, Shandong, China
| | - Xue Bai
- School of Medical Laboratory, Weifang Medical University, Weifang, Shandong, China; Institutional Key Laboratory of Clinical Laboratory Diagnostics, 12th 5-Year Project of Shandong Province, Weifang Medical University, Weifang, Shandong, China
| | - Xinyu Li
- School of Medical Laboratory, Weifang Medical University, Weifang, Shandong, China; Institutional Key Laboratory of Clinical Laboratory Diagnostics, 12th 5-Year Project of Shandong Province, Weifang Medical University, Weifang, Shandong, China
| | - Ronglan Zhao
- School of Medical Laboratory, Weifang Medical University, Weifang, Shandong, China; Institutional Key Laboratory of Clinical Laboratory Diagnostics, 12th 5-Year Project of Shandong Province, Weifang Medical University, Weifang, Shandong, China.
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Jiang ZF, Wu W, Hu HB, Li ZY, Zhong M, Zhang L. P2X7 receptor as the regulator of T-cell function in intestinal barrier disruption. World J Gastroenterol 2022; 28:5265-5279. [PMID: 36185635 PMCID: PMC9521516 DOI: 10.3748/wjg.v28.i36.5265] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/12/2022] [Revised: 06/20/2022] [Accepted: 09/01/2022] [Indexed: 02/06/2023] Open
Abstract
The intestinal mucosa is a highly compartmentalized structure that forms a direct barrier between the host intestine and the environment, and its dysfunction could result in a serious disease. As T cells, which are important components of the mucosal immune system, interact with gut microbiota and maintain intestinal homeostasis, they may be involved in the process of intestinal barrier dysfunction. P2X7 receptor (P2X7R), a member of the P2X receptors family, mediates the effects of extracellular adenosine triphosphate and is expressed by most innate or adaptive immune cells, including T cells. Current evidence has demonstrated that P2X7R is involved in inflammation and mediates the survival and differentiation of T lymphocytes, indicating its potential role in the regulation of T cell function. In this review, we summarize the available research about the regulatory role and mechanism of P2X7R on the intestinal mucosa-derived T cells in the setting of intestinal barrier dysfunction.
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Affiliation(s)
- Zhi-Feng Jiang
- Center of Emergency & Intensive Care Unit, Jinshan Hospital of Fudan University, Shanghai 201508, China
| | - Wei Wu
- Department of Critical Care Medicine, Zhongshan Hospital of Fudan University, Shanghai 200032, China
| | - Han-Bing Hu
- Center of Emergency & Intensive Care Unit, Jinshan Hospital of Fudan University, Shanghai 201508, China
| | - Zheng-Yang Li
- Department of Gastroenterology, Jinshan Hospital of Fudan University, Shanghai 201508, China
| | - Ming Zhong
- Department of Critical Care Medicine, Zhongshan Hospital of Fudan University, Shanghai 200032, China
| | - Lin Zhang
- Center of Emergency & Intensive Care Unit, Jinshan Hospital of Fudan University, Shanghai 201508, China
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Mukherjee N, Banerjee S, Amin SA, Jha T, Datta S, Das Saha K. Host P2X 7R-p 38MAPK axis mediated intra-macrophage leishmanicidal activity of Spergulin-A. Exp Parasitol 2022; 241:108365. [PMID: 36007587 DOI: 10.1016/j.exppara.2022.108365] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2022] [Revised: 07/14/2022] [Accepted: 08/18/2022] [Indexed: 11/04/2022]
Abstract
Current drugs are inefficient for the treatment of visceral leishmaniasis an immunosuppressive ailment caused by Leishmania donovani. Regrettably, there is no plant-origin antileishmanial drug present. P2X7R is constitutively present on macrophage surfaces and can be a putative therapeutic target in intra-macrophage pathogens with function attributes towards inflammation, host cell apoptosis, altered redox, and phagolysosomal maturation by activating p38MAPK. Here we demonstrated that the initial interaction of Spergulin-A (Sp A), a triterpenoid saponin with RAW 264.7 macrophages was mediated through P2X7R involving the signaling cascade intermediates Ca++, p38MAPK, and NF-κβ. Phospho (P)-p38MAPK involvement is shown to have specific and firm importance in leishmanial killing with increased NF-κβp65. Phago-lysosomal maturation by Sp A also campaigns for another contribution of P2X7R. In vivo evaluation of the anti-leishmanial activity of Sp A was monitored through expression analyses of P2X7R, P-p38MAPK, and NF-κβp65 in murine spleen and bone-marrow macrophages and supported Sp A being a natural compound of leishmanicidal functions which acted through the P2X7R-p38MAPK axis.
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Affiliation(s)
- Niladri Mukherjee
- Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India; Techno India University, EM-4, Sector V, Salt Lake, Kolkata, 700091, West Bengal, India.
| | - Saswati Banerjee
- Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India
| | - Sk Abdul Amin
- Natural Science Laboratory, Department of Pharmaceutical Technology, Division of Medicinal & Pharmaceutical Chemistry, P. O. Box 17020, Jadavpur University, Kolkata, 700032, India
| | - Tarun Jha
- Natural Science Laboratory, Department of Pharmaceutical Technology, Division of Medicinal & Pharmaceutical Chemistry, P. O. Box 17020, Jadavpur University, Kolkata, 700032, India
| | - Sriparna Datta
- Department of Chemical Technology, University of Calcutta, Kolkata, 700009, India
| | - Krishna Das Saha
- Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India.
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Huang CK, Dai D, Xie H, Zhu Z, Hu J, Su M, Liu M, Lu L, Shen W, Ning G, Wang J, Zhang R, Yan X. Lgr4 Governs a Pro-Inflammatory Program in Macrophages to Antagonize Post-Infarction Cardiac Repair. Circ Res 2020; 127:953-973. [PMID: 32600176 DOI: 10.1161/circresaha.119.315807] [Citation(s) in RCA: 83] [Impact Index Per Article: 16.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
RATIONALE Macrophages are critically involved in wound healing following myocardial infarction (MI). Lgr4, a member of LGR (leucine-rich repeat-containing G protein-coupled receptor) family, is emerging as a regulator of macrophage-associated immune responses. However, the contribution of Lgr4 to macrophage phenotype and function in the context of MI remains unclear. OBJECTIVE To determine the role of macrophage Lgr4 in MI and to dissect the underlying mechanisms. METHODS AND RESULTS During early inflammatory phase of MI, infarct macrophages rather than neutrophils expressed high level of Lgr4. Macrophage-specific Lgr4 knockout mice had no baseline cardiovascular defects but manifested improved heart function, modestly reduced infarct size, decreased early mortality due to cardiac rupture, and ameliorated adverse remodeling after MI. Improved outcomes in macrophage-specific Lgr4 knockout mice subjected to MI were associated with mitigated ischemic injury and optimal infarct healing, as determined by reduction of cardiac apoptosis in the peri-infarct zone, attenuation of local myocardial inflammatory response, decrease of matrix metalloproteinase expression in the infarct, enhancement of angiogenesis, myofibroblast proliferation, and collagen I deposition in reparative granulation tissue as well as formation of collagen-rich scar. More importantly, macrophage-specific Lgr4 knockout infarcts had reduced numbers of infiltrating leukocytes and inflammatory macrophages but harbored abundant reparative macrophage subsets. Lgr4-null infarct macrophages exhibited a less inflammatory transcriptional signature. These findings were further supported by transcriptomic profiling data showing repression of multiple pathways and broad-spectrum genes associated with proinflammatory responses in macrophage-specific Lgr4 knockout infarcts. Notably, we discovered that Lgr4-mediated functional phenotype programing in infarct macrophages was at least partly attributed to regulation of AP (activator protein)-1 activity. We further demonstrated that the synergistic effects of Lgr4 on AP-1 activation in inflammatory macrophages occurred via enhancing CREB (cAMP response element-binding protein)-mediated c-Fos, Fosl1, and Fosb transactivation. CONCLUSIONS Together, our data highlight the significance of Lgr4 in governing proinflammatory phenotype of infarct macrophages and postinfarction repair.
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Affiliation(s)
- Chun-Kai Huang
- From the Department of Cardiology (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
- Ruijin Hospital, Institute of Cardiovascular Diseases (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
| | - Daopeng Dai
- From the Department of Cardiology (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
- Ruijin Hospital, Institute of Cardiovascular Diseases (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
| | - Hongyang Xie
- From the Department of Cardiology (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
- Ruijin Hospital, Institute of Cardiovascular Diseases (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
| | - Zhengbin Zhu
- From the Department of Cardiology (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
- Ruijin Hospital, Institute of Cardiovascular Diseases (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
| | - Jian Hu
- From the Department of Cardiology (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
- Ruijin Hospital, Institute of Cardiovascular Diseases (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
| | - Min Su
- Department of Pathology, Institute of Clinical Pathology, Shantou University Medical College, Guangdong, PR China (M.S.)
| | - Mingyao Liu
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, PR China (M.L.)
| | - Lin Lu
- From the Department of Cardiology (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
| | - Weifeng Shen
- From the Department of Cardiology (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
- Ruijin Hospital, Institute of Cardiovascular Diseases (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
| | - Guang Ning
- Department of Endocrinology and Metabolism (G.N., J.W.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
| | - Jiqiu Wang
- Department of Endocrinology and Metabolism (G.N., J.W.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
| | - Ruiyan Zhang
- From the Department of Cardiology (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
- Ruijin Hospital, Institute of Cardiovascular Diseases (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
| | - Xiaoxiang Yan
- From the Department of Cardiology (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
- Ruijin Hospital, Institute of Cardiovascular Diseases (C.-K.H., D.D., H.X., Z.Z., J.H., L.L., W.S., R.Z., X.Y.), Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
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Rivas-Yáñez E, Barrera-Avalos C, Parra-Tello B, Briceño P, Rosemblatt MV, Saavedra-Almarza J, Rosemblatt M, Acuña-Castillo C, Bono MR, Sauma D. P2X7 Receptor at the Crossroads of T Cell Fate. Int J Mol Sci 2020; 21:E4937. [PMID: 32668623 PMCID: PMC7404255 DOI: 10.3390/ijms21144937] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2020] [Revised: 06/26/2020] [Accepted: 07/07/2020] [Indexed: 02/06/2023] Open
Abstract
The P2X7 receptor is a ligand-gated, cation-selective channel whose main physiological ligand is ATP. P2X7 receptor activation may also be triggered by ARTC2.2-dependent ADP ribosylation in the presence of extracellular NAD. Upon activation, this receptor induces several responses, including the influx of calcium and sodium ions, phosphatidylserine externalization, the formation of a non-selective membrane pore, and ultimately cell death. P2X7 receptor activation depends on the availability of extracellular nucleotides, whose concentrations are regulated by the action of extracellular nucleotidases such as CD39 and CD38. The P2X7 receptor has been extensively studied in the context of the immune response, and it has been reported to be involved in inflammasome activation, cytokine production, and the migration of different innate immune cells in response to ATP. In adaptive immune responses, the P2X7 receptor has been linked to T cell activation, differentiation, and apoptosis induction. In this review, we will discuss the evidence of the role of the P2X7 receptor on T cell differentiation and in the control of T cell responses in inflammatory conditions.
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Affiliation(s)
- Elizabeth Rivas-Yáñez
- Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago 7800003, Chile; (E.R.-Y.); (B.P.-T.); (P.B.); (M.V.R.); (J.S.-A.); (M.R.)
| | - Carlos Barrera-Avalos
- Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago 9160000, Chile;
| | - Brian Parra-Tello
- Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago 7800003, Chile; (E.R.-Y.); (B.P.-T.); (P.B.); (M.V.R.); (J.S.-A.); (M.R.)
| | - Pedro Briceño
- Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago 7800003, Chile; (E.R.-Y.); (B.P.-T.); (P.B.); (M.V.R.); (J.S.-A.); (M.R.)
| | - Mariana V. Rosemblatt
- Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago 7800003, Chile; (E.R.-Y.); (B.P.-T.); (P.B.); (M.V.R.); (J.S.-A.); (M.R.)
- Facultad de Medicina y Ciencia, Universidad San Sebastián, Santiago 7510157, Chile
| | - Juan Saavedra-Almarza
- Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago 7800003, Chile; (E.R.-Y.); (B.P.-T.); (P.B.); (M.V.R.); (J.S.-A.); (M.R.)
| | - Mario Rosemblatt
- Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago 7800003, Chile; (E.R.-Y.); (B.P.-T.); (P.B.); (M.V.R.); (J.S.-A.); (M.R.)
- Facultad de Medicina y Ciencia, Universidad San Sebastián, Santiago 7510157, Chile
- Fundación Ciencia & Vida, Santiago 7780272, Chile
| | - Claudio Acuña-Castillo
- Centro de Biotecnología Acuícola, Universidad de Santiago de Chile, Santiago 9160000, Chile
| | - María Rosa Bono
- Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago 7800003, Chile; (E.R.-Y.); (B.P.-T.); (P.B.); (M.V.R.); (J.S.-A.); (M.R.)
| | - Daniela Sauma
- Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago 7800003, Chile; (E.R.-Y.); (B.P.-T.); (P.B.); (M.V.R.); (J.S.-A.); (M.R.)
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Sim JR, Kang SS, Lee D, Yun CH, Han SH. Killed Whole-Cell Oral Cholera Vaccine Induces CCL20 Secretion by Human Intestinal Epithelial Cells in the Presence of the Short-Chain Fatty Acid, Butyrate. Front Immunol 2018; 9:55. [PMID: 29434590 PMCID: PMC5796904 DOI: 10.3389/fimmu.2018.00055] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2017] [Accepted: 01/09/2018] [Indexed: 01/09/2023] Open
Abstract
Short-chain fatty acids (SCFAs), such as acetate, butyrate, and propionate, modulate immune responses in the gut. However, the effect of SCFAs on mucosal vaccine-induced immune cell migration is poorly understood. Here, we investigated whether SCFAs modulate chemokine expression induced by the killed whole-cell oral cholera vaccine, Shanchol™, in human intestinal epithelial cells. Shanchol™ induced expression of CCL2, CCL5, CCL20, and CXCL10 at the mRNA level, but not at the protein level. Interestingly, CCL20 secretion was substantially increased by co-stimulation with Shanchol™ and butyrate, while neither acetate nor propionate showed such effect. Enhanced CCL20 secretion was associated with GPR109A activation, and histone deacetylase (HDAC) inhibition. In addition, co-treatment with Shanchol™ and butyrate synergistically increased the secretion of adenosine triphosphate (ATP). Moreover, CCL20 secretion was decreased by inhibiting the extracellular ATP receptor P2X7. However, neither inflammasomes nor caspases were involved in CCL20 production. The culture supernatant of cells treated with Shanchol™ and butyrate augmented human immature dendritic cell migration. Collectively, these results suggest that butyrate enhances Shanchol™-induced CCL20 production in human intestinal epithelial cells via HDAC inhibition and ATP-P2X7 signaling by activating GPR109A. These effects potentially enhance the mucosal immune responses in the gut induced by this oral cholera vaccine.
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Affiliation(s)
- Ju-Ri Sim
- Department of Oral Microbiology and Immunology, DRI, and BK21 Plus Program, School of Dentistry, Seoul National University, Seoul, South Korea
| | - Seok-Seong Kang
- Department of Food Science and Biotechnology, Dongguk University Seoul, Goyang, South Korea
| | - Daesang Lee
- The 5th R&D Institute, Agency for Defense Development, Daejeon, South Korea
| | - Cheol-Heui Yun
- Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, South Korea
| | - Seung Hyun Han
- Department of Oral Microbiology and Immunology, DRI, and BK21 Plus Program, School of Dentistry, Seoul National University, Seoul, South Korea
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9
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Zhang C, He H, Wang L, Zhang N, Huang H, Xiong Q, Yan Y, Wu N, Ren H, Han H, Liu M, Qian M, Du B. Virus-Triggered ATP Release Limits Viral Replication through Facilitating IFN-β Production in a P2X7-Dependent Manner. THE JOURNAL OF IMMUNOLOGY 2017; 199:1372-1381. [PMID: 28687662 DOI: 10.4049/jimmunol.1700187] [Citation(s) in RCA: 59] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Received: 02/06/2017] [Accepted: 06/10/2017] [Indexed: 12/17/2022]
Abstract
Accumulating evidence shows that innate immune responses are associated with extracellular nucleotides, particularly ATP. In this article, we demonstrate extensive protection of ATP/P2X7 signaling in a host against viral infection. Interestingly, we observed a significant increase in ATP as a danger signal in vesicular stomatitis virus (VSV)-infected cell supernatant and VSV-infected mice in an exocytosis- and pannexin channel-dependent manner. Furthermore, extracellular ATP reduces the replication of VSV, Newcastle disease virus, murine leukemia virus, and HSV in vivo and in vitro through the P2X7 receptor. Meanwhile, ATP significantly increases IFN-β expression in a concentration- and time-dependent manner. Mechanistically, ATP facilitates IFN-β secretion through P38/JNK/ATF-2 signaling pathways, which are crucial in promoting antiviral immunity. Taken together, these results demonstrate the protective role of extracellular ATP and P2X7 in viral infection and suggest a potential therapeutic role for ATP/P2X7 in viral diseases.
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Affiliation(s)
- Chengfei Zhang
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China; and
| | - Hongwang He
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China; and
| | - Li Wang
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China; and
| | - Na Zhang
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China; and
| | - Hongjun Huang
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China; and
| | - Qingqing Xiong
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China; and
| | - Yan Yan
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China; and
| | - Nannan Wu
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China; and
| | - Hua Ren
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China; and
| | - Honghui Han
- Bioray Laboratories Inc., Shanghai 200241, China
| | - Mingyao Liu
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China; and
| | - Min Qian
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China; and
| | - Bing Du
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China; and
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10
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Jacques FJ, Silva TM, da Silva FE, Ornelas IM, Ventura ALM. Nucleotide P2Y13-stimulated phosphorylation of CREB is required for ADP-induced proliferation of late developing retinal glial progenitors in culture. Cell Signal 2017; 35:95-106. [PMID: 28347874 DOI: 10.1016/j.cellsig.2017.03.019] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2017] [Revised: 02/23/2017] [Accepted: 03/24/2017] [Indexed: 12/28/2022]
Abstract
Nucleotides stimulate phosphorylation of CREB to induce cell proliferation and survival in diverse cell types. We report here that ADP induces the phosphorylation of CREB in a time- and concentration-dependent manner in chick embryo retinal progenitors in culture. ADP-induced increase in phospho-CREB is mediated by P2 receptors as it is blocked by PPADS but not by the adenosine antagonists DPCPX or ZM241385. Incubation of the cultures with the CREB inhibitor KG-501 prevents ADP-induced incorporation of [3H]-thymidine, indicating that CREB is involved in retinal cell proliferation. No effect of this compound is observed on the viability of retinal progenitors. While no significant increase in CREB phosphorylation is observed with the P2Y1 receptor agonist MRS2365, ADP-induced phosphorylation of CREB is blocked by the P2Y13 receptor selective antagonist MRS2211, but not by MRS2179 or PSB0739, two antagonists of the P2Y1 and P2Y12 receptors, respectively, suggesting that ADP-induced CREB phosphorylation is mediated by P2Y13 receptors. ADP-induced increase in phospho-CREB is attenuated by the PI3K inhibitor LY294002 and completely prevented by the MEK inhibitor U0126, suggesting that at least ERK is involved in ADP-induced CREB phosphorylation. A pharmacological profile similar to the activation and inhibition of CREB phosphorylation is observed in the phosphorylation of ERK, suggesting that P2Y13 receptors mediate ADP induced ERK/CREB pathway in the cultures. While no increase in [3H]-thymidine incorporation is observed with the P2Y1 receptor agonist MRS2365, both MRS2179 and MRS2211 prevent ADP-mediated increase in [3H]-thymidine incorporation, but not progenitor's survival, suggesting that both P2Y1 and P2Y13 receptor subtypes are involved in ADP-induced cell proliferation. P2Y1 receptor-mediated increase in [Ca2+]i is observed in glial cells only when cultures maintained for 9days are used. In glia from cultures cultivated for only 2days, no increase in [Ca2+]i is detected with MRS2365 and no inhibition of ADP-mediated calcium response is observed with MRS2179. In contrast, MRS2211 attenuates ADP-mediated increase in [Ca2+]i in glial cells from cultures at both stages, suggesting the presence of P2Y13 receptors coupled to calcium mobilization in proliferating retinal glial progenitors in culture.
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Affiliation(s)
- Flavia Jesus Jacques
- Department of Neurobiology, Neuroscience Program, Fluminense Federal University, Outeiro de São João Batista s/n, Centro, Niterói, Rio de Janeiro CEP 24020-141, Brazil
| | - Thayane Martins Silva
- Department of Neurobiology, Neuroscience Program, Fluminense Federal University, Outeiro de São João Batista s/n, Centro, Niterói, Rio de Janeiro CEP 24020-141, Brazil
| | - Flavia Emenegilda da Silva
- Department of Neurobiology, Neuroscience Program, Fluminense Federal University, Outeiro de São João Batista s/n, Centro, Niterói, Rio de Janeiro CEP 24020-141, Brazil
| | - Isis Moraes Ornelas
- Department of Neurobiology, Neuroscience Program, Fluminense Federal University, Outeiro de São João Batista s/n, Centro, Niterói, Rio de Janeiro CEP 24020-141, Brazil
| | - Ana Lucia Marques Ventura
- Department of Neurobiology, Neuroscience Program, Fluminense Federal University, Outeiro de São João Batista s/n, Centro, Niterói, Rio de Janeiro CEP 24020-141, Brazil.
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11
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Nelson S, Kiyono H, Kurashima Y. Epithelial extracellular ATP: an initiator of immunity to parasitic infections. Immunol Cell Biol 2016; 95:117-118. [PMID: 27874014 DOI: 10.1038/icb.2016.106] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Affiliation(s)
- Sean Nelson
- Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Hiroshi Kiyono
- Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.,International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Yosuke Kurashima
- Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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12
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Apostolova P, Zeiser R. The role of danger signals and ectonucleotidases in acute graft-versus-host disease. Hum Immunol 2016; 77:1037-1047. [PMID: 26902992 DOI: 10.1016/j.humimm.2016.02.005] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2015] [Revised: 02/09/2016] [Accepted: 02/18/2016] [Indexed: 12/28/2022]
Abstract
Allogeneic hematopoietic cell transplantation (allo-HCT) represents the only curative treatment approach for many patients with benign or malignant diseases of the hematopoietic system. However, post-transplant morbidity and mortality are significantly increased by the development of acute graft-versus-host disease (GvHD). While alloreactive T cells act as the main cellular mediator of the GvH reaction, recent evidence suggests a critical role of the innate immune system in the early stages of GvHD initiation. Danger-associated molecular patterns released from the intracellular space as well as from the extracellular matrix activate antigen-presenting cells and set pro-inflammatory pathways in motion. This review gives an overview about danger signals representing therapeutic targets with a clinical perspective with a particular focus on extracellular nucleotides and ectonucleotidases.
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Affiliation(s)
- Petya Apostolova
- Department of Hematology, Oncology and Stem Cell Transplantation, Freiburg University Medical Center, Albert-Ludwigs-University, Freiburg, Germany.
| | - Robert Zeiser
- Department of Hematology, Oncology and Stem Cell Transplantation, Freiburg University Medical Center, Albert-Ludwigs-University, Freiburg, Germany.
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13
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Lack of a Functioning P2X7 Receptor Leads to Increased Susceptibility to Toxoplasmic Ileitis. PLoS One 2015; 10:e0129048. [PMID: 26053862 PMCID: PMC4460092 DOI: 10.1371/journal.pone.0129048] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2014] [Accepted: 05/04/2015] [Indexed: 12/23/2022] Open
Abstract
BACKGROUND Oral infection of C57BL/6J mice with the protozoan parasite Toxoplasma gondii leads to a lethal inflammatory ileitis. PRINCIPAL FINDINGS Mice lacking the purinergic receptor P2X7R are acutely susceptible to toxoplasmic ileitis, losing significantly more weight than C57BL/6J mice and exhibiting much greater intestinal inflammatory pathology in response to infection with only 10 cysts of T. gondii. This susceptibility is not dependent on the ability of P2X7R-deficient mice to control the parasite, which they accomplish just as efficiently as C57BL/6J mice. Rather, susceptibility is associated with elevated ileal concentrations of pro-inflammatory cytokines, reactive nitrogen intermediates and altered regulation of elements of NFκB activation in P2X7R-deficient mice. CONCLUSIONS Our data support the thesis that P2X7R, a well-documented activator of pro-inflammatory cytokine production, also plays an important role in the regulation of intestinal inflammation.
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14
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Bartlett R, Stokes L, Sluyter R. The P2X7 receptor channel: recent developments and the use of P2X7 antagonists in models of disease. Pharmacol Rev 2014; 66:638-75. [PMID: 24928329 DOI: 10.1124/pr.113.008003] [Citation(s) in RCA: 328] [Impact Index Per Article: 29.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The P2X7 receptor is a trimeric ATP-gated cation channel found predominantly, but not exclusively, on immune cells. P2X7 activation results in a number of downstream events, including the release of proinflammatory mediators and cell death and proliferation. As such, P2X7 plays important roles in various inflammatory, immune, neurologic and musculoskeletal disorders. This review focuses on the use of P2X7 antagonists in rodent models of neurologic disease and injury, inflammation, and musculoskeletal and other disorders. The cloning and characterization of human, rat, mouse, guinea pig, dog, and Rhesus macaque P2X7, as well as recent observations regarding the gating and permeability of P2X7, are discussed. Furthermore, this review discusses polymorphic and splice variants of P2X7, as well as the generation and use of P2X7 knockout mice. Recent evidence for emerging signaling pathways downstream of P2X7 activation and the growing list of negative and positive modulators of P2X7 activation and expression are also described. In addition, the use of P2X7 antagonists in numerous rodent models of disease is extensively summarized. Finally, the use of P2X7 antagonists in clinical trials in humans and future directions exploring P2X7 as a therapeutic target are described.
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Affiliation(s)
- Rachael Bartlett
- School of Biological Sciences, University of Wollongong, New South Wales, Australia and Illawarra Health and Medical Research Institute, Wollongong, New South Wales, Australia (R.B., R.S.); and Health Innovations Research Institute, School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia (L.S.)
| | - Leanne Stokes
- School of Biological Sciences, University of Wollongong, New South Wales, Australia and Illawarra Health and Medical Research Institute, Wollongong, New South Wales, Australia (R.B., R.S.); and Health Innovations Research Institute, School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia (L.S.)
| | - Ronald Sluyter
- School of Biological Sciences, University of Wollongong, New South Wales, Australia and Illawarra Health and Medical Research Institute, Wollongong, New South Wales, Australia (R.B., R.S.); and Health Innovations Research Institute, School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia (L.S.)
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15
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Gavala ML, Liu YP, Lenertz LY, Zeng L, Blanchette JB, Guadarrama AG, Denlinger LC, Bertics PJ, Smith JA. Nucleotide receptor P2RX7 stimulation enhances LPS-induced interferon-β production in murine macrophages. J Leukoc Biol 2013; 94:759-68. [PMID: 23911869 PMCID: PMC3774844 DOI: 10.1189/jlb.0712351] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2012] [Revised: 05/22/2013] [Accepted: 06/27/2013] [Indexed: 12/22/2022] Open
Abstract
Stimulation of P2RX(7) with extracellular ATP potentiates numerous LPS-induced proinflammatory events, including cytokine induction in macrophages, but the molecular mechanisms underlying this process are not well defined. Although P2RX(7) ligation has been proposed to activate several transcription factors, many of the LPS-induced mediators affected by P2RX(7) activation are not induced by P2RX(7) agonists alone, suggesting a complementary role for P2RX(7) in transcriptional regulation. Type I IFN production, whose expression is tightly controlled by multiple transcription factors that form an enhanceosome, is critical for resistance against LPS-containing bacteria. The effect of purinergic receptor signaling on LPS-dependent type I IFN is unknown and would be of great relevance to a diverse array of inflammatory conditions. The present study demonstrates that stimulation of macrophages with P2RX(7) agonists substantially enhances LPS-induced IFN-β expression, and this enhancement is ablated in macrophages that do not express functional P2RX(7) or when the MAPK MEK1/2 pathways are inhibited. Potentiation of LPS-induced IFN-β expression following P2RX(7) stimulation is likely transcriptionally regulated, as this enhancement is observed at the IFN-β promoter level. Furthermore, P2RX(7) stimulation is able to increase the phosphorylation and subsequent IFN-β promoter occupancy of IRF-3, a transcription factor that is critical for IFN-β transcription by TLR agonists. This newly discovered role for P2RX(7) in IFN regulation may have implications in antimicrobial defense, which has been linked to P2RX(7) activation in other studies.
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Affiliation(s)
- M L Gavala
- 2.University of Wisconsin School of Medicine and Public Health, 600 Highland Ave., CSC H4/472, Madison, WI 53792-9988, USA.
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16
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Norris PC, Dennis EA. A lipidomic perspective on inflammatory macrophage eicosanoid signaling. Adv Biol Regul 2013; 54:99-110. [PMID: 24113376 DOI: 10.1016/j.jbior.2013.09.009] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2013] [Revised: 09/15/2013] [Accepted: 09/17/2013] [Indexed: 12/24/2022]
Abstract
Macrophages are central to essential physiological processes including the regulation of innate and adaptive immunity, but they are also central to a number of inflammatory disease states. These immune cells also possess remarkable plasticity and display various shades of functionalities based on changes in the surrounding molecular environment. Macrophage biology has defined various phenotypes and roles in inflammation based primarily on cytokine and chemokine profiles of cells in different activation states. Importantly, macrophages are elite producers of eicosanoids and other related lipid mediators during inflammation, but specific roles of these molecules have not generally been incorporated into the larger context of macrophage biology. In this review, we discuss the current classification of macrophage types and their roles in inflammation and disease, along with the practical challenges of studying biologically relevant phenotypes ex vivo. Using the latest advances in eicosanoid lipidomics, we highlight several key studies from our laboratory that provide a comprehensive understanding of how eicosanoid metabolism differs between macrophage phenotypes, along with how this metabolism is altered by changes in membrane fatty acid distribution and varied durations of Toll-like receptor (TLR) priming. In conclusion, we summarize several examples of the benefit of macrophage plasticity to develop accurate cellular mechanisms of lipid metabolism, and insights from lipidomic analyses about the differences in eicosanoid pathway enzyme activity in vitro vs. in cells ex vivo. Examples of new techniques to further understand the role of macrophage eicosanoid signaling in vivo are also discussed.
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Affiliation(s)
- Paul C Norris
- Departments of Chemistry/Biochemistry and Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0601, USA
| | - Edward A Dennis
- Departments of Chemistry/Biochemistry and Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0601, USA.
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17
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Sun C, Heid ME, Keyel PA, Salter RD. The second transmembrane domain of P2X7 contributes to dilated pore formation. PLoS One 2013; 8:e61886. [PMID: 23613968 PMCID: PMC3629090 DOI: 10.1371/journal.pone.0061886] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2012] [Accepted: 03/18/2013] [Indexed: 01/07/2023] Open
Abstract
Activation of the purinergic receptor P2X7 leads to the cellular permeability of low molecular weight cations. To determine which domains of P2X7 are necessary for this permeability, we exchanged either the C-terminus or portions of the second transmembrane domain (TM2) with those in P2X1 or P2X4. Replacement of the C-terminus of P2X7 with either P2X1 or P2X4 prevented surface expression of the chimeric receptor. Similarly, chimeric P2X7 containing TM2 from P2X1 or P2X4 had reduced surface expression and no permeability to cationic dyes. Exchanging the N-terminal 10 residues or C-terminal 14 residues of the P2X7 TM2 with the corresponding region of P2X1 TM2 partially restored surface expression and limited pore permeability. To further probe TM2 structure, we replaced single residues in P2X7 TM2 with those in P2X1 or P2X4. We identified multiple substitutions that drastically changed pore permeability without altering surface expression. Three substitutions (Q332P, Y336T, and Y343L) individually reduced pore formation as indicated by decreased dye uptake and also reduced membrane blebbing in response to ATP exposure. Three others substitutions, V335T, S342G, and S342A each enhanced dye uptake, membrane blebbing and cell death. Our results demonstrate a critical role for the TM2 domain of P2X7 in receptor function, and provide a structural basis for differences between purinergic receptors.
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Affiliation(s)
- Chengqun Sun
- Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
| | - Michelle E. Heid
- Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
| | - Peter A. Keyel
- Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
| | - Russell D. Salter
- Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
- * E-mail:
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18
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Massé K, Dale N. Purines as potential morphogens during embryonic development. Purinergic Signal 2012; 8:503-21. [PMID: 22270538 PMCID: PMC3360092 DOI: 10.1007/s11302-012-9290-y] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2011] [Accepted: 01/04/2012] [Indexed: 11/17/2022] Open
Abstract
Components of purinergic signalling are expressed in the early embryo raising the possibility that ATP, ADP and adenosine may contribute to the mechanisms of embryonic development. We summarize the available data from four developmental models—mouse, chick, Xenopus and zebrafish. While there are some notable examples where purinergic signalling is indeed important during development, e.g. development of the eye in the frog, it is puzzling that deletion of single components of purinergic signalling often results in rather minor developmental phenotypes. We suggest that a key step in further analysis is to perform combinatorial alterations of expression of purinergic signalling components to uncover their roles in development. We introduce the concept that purinergic signalling could create novel morphogenetic fields to encode spatial location via the concentration of ATP, ADP and adenosine. We show that using minimal assumptions and the known properties of the ectonucleotidases, complex spatial patterns of ATP and adenosine can be set up. These patterns may provide a new way to assess the potential of purinergic signalling in developmental processes.
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Affiliation(s)
- Karine Massé
- Univ. Bordeaux, CIRID, UMR 5164, F-33000, Bordeaux, France
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19
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Miller CM, Boulter NR, Fuller SJ, Zakrzewski AM, Lees MP, Saunders BM, Wiley JS, Smith NC. The role of the P2X₇ receptor in infectious diseases. PLoS Pathog 2011; 7:e1002212. [PMID: 22102807 PMCID: PMC3213081 DOI: 10.1371/journal.ppat.1002212] [Citation(s) in RCA: 108] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
ATP is an extracellular signal for the immune system, particularly during an inflammatory response. It is sensed by the P2X7 receptor, the expression of which is upregulated by pro-inflammatory cytokines. Activation of the P2X7 receptor opens a cation-specific channel that alters the ionic environment of the cell, activating several pathways, including (i) the inflammasome, leading to production of IL-1β and IL-18; (ii) the stress-activated protein kinase pathway, resulting in apoptosis; (iii) the mitogen-activated protein kinase pathway, leading to generation of reactive oxygen and nitrogen intermediates; and (iv) phospholipase D, stimulating phagosome-lysosome fusion. The P2X7 receptor can initiate host mechanisms to remove pathogens, most particularly those that parasitise macrophages. At the same time, the P2X7 receptor may be subverted by pathogens to modulate host responses. Moreover, recent genetic studies have demonstrated significant associations between susceptibility or resistance to parasites and bacteria, and loss-of-function or gain-of-function polymorphisms in the P2X7 receptor, underscoring its importance in infectious disease.
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Affiliation(s)
- Catherine M. Miller
- Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Broadway, New South Wales, Australia
| | - Nicola R. Boulter
- Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Broadway, New South Wales, Australia
| | - Stephen J. Fuller
- Nepean Clinical School, Nepean Hospital, The University of Sydney, Penrith, New South Wales, Australia
| | - Alana M. Zakrzewski
- Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Broadway, New South Wales, Australia
| | - Michael P. Lees
- Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Broadway, New South Wales, Australia
| | - Bernadette M. Saunders
- Centenary Institute of Cancer Medicine & Cell Biology, The University of Sydney, Camperdown, New South Wales, Australia
| | - James S. Wiley
- Florey Neuroscience Institute, The University of Melbourne, Parkville, Victoria, Australia
| | - Nicholas C. Smith
- Queensland Tropical Health Alliance, Faculty of Medicine, Health and Molecular Sciences, James Cook University, Smithfield, Cairns, Queensland, Australia
- * E-mail:
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20
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Lenertz LY, Gavala ML, Zhu Y, Bertics PJ. Transcriptional control mechanisms associated with the nucleotide receptor P2X7, a critical regulator of immunologic, osteogenic, and neurologic functions. Immunol Res 2011; 50:22-38. [PMID: 21298493 PMCID: PMC3203638 DOI: 10.1007/s12026-011-8203-4] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
The nucleotide receptor P2X(7) is an attractive therapeutic target and potential biomarker for multiple inflammatory and neurologic disorders, and it is expressed in several immune, osteogenic, and neurologic cell types. Aside from its role in the nervous system, it is activated by ATP released at sites of tissue damage, inflammation, and infection. Ligand binding to P2X(7) stimulates many cell responses, including calcium fluxes, MAPK activation, inflammatory mediator release, and apoptosis. Much work has centered on P2X(7) action in cell death and mediator processing (e.g., pro-interleukin-1 cleavage by the inflammasome), but the contribution of P2X(7) to transcriptional regulation is less well defined. This review will focus on the growing evidence for the importance of nucleotide-mediated gene expression, highlight several animal models, human genetic, and clinical studies that support P2X(7) as a therapeutic target, and discuss the latest developments in anti-P2X(7) clinical trials.
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Affiliation(s)
- Lisa Y. Lenertz
- Department of Biomolecular Chemistry, School of Medicine and Public Health, The University of Wisconsin-Madison, Madison, Wisconsin 53706
| | - Monica L. Gavala
- Department of Biomolecular Chemistry, School of Medicine and Public Health, The University of Wisconsin-Madison, Madison, Wisconsin 53706
| | - Yiming Zhu
- Department of Biomolecular Chemistry, School of Medicine and Public Health, The University of Wisconsin-Madison, Madison, Wisconsin 53706
| | - Paul J. Bertics
- Department of Biomolecular Chemistry, School of Medicine and Public Health, The University of Wisconsin-Madison, Madison, Wisconsin 53706
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21
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Wen AY, Sakamoto KM, Miller LS. The role of the transcription factor CREB in immune function. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2010; 185:6413-9. [PMID: 21084670 PMCID: PMC5519339 DOI: 10.4049/jimmunol.1001829] [Citation(s) in RCA: 619] [Impact Index Per Article: 41.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
CREB is a transcription factor that regulates diverse cellular responses, including proliferation, survival, and differentiation. CREB is induced by a variety of growth factors and inflammatory signals and subsequently mediates the transcription of genes containing a cAMP-responsive element. Several immune-related genes possess this cAMP-responsive element, including IL-2, IL-6, IL-10, and TNF-α. In addition, phosphorylated CREB has been proposed to directly inhibit NF-κB activation by blocking the binding of CREB binding protein to the NF-κB complex, thereby limiting proinflammatory responses. CREB also induces an antiapoptotic survival signal in monocytes and macrophages. In T and B cells, CREB activation promotes proliferation and survival and differentially regulates Th1, Th2, and Th17 responses. Finally, CREB activation is required for the generation and maintenance of regulatory T cells. This review summarizes current advances involving CREB in immune function--a role that is continually being defined.
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Affiliation(s)
- Andy Y. Wen
- Division of Pediatric Critical Care, University of California Los Angeles, Los Angeles, CA, 90095
| | - Kathleen M. Sakamoto
- Division of Pediatric Hematology and Oncology, Department of Pediatrics, University of California Los Angeles, Los Angeles, CA, 90095
| | - Lloyd S. Miller
- Division of Dermatology, Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, 90095
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Gavala ML, Hill LM, Lenertz LY, Karta MR, Bertics PJ. Activation of the transcription factor FosB/activating protein-1 (AP-1) is a prominent downstream signal of the extracellular nucleotide receptor P2RX7 in monocytic and osteoblastic cells. J Biol Chem 2010; 285:34288-98. [PMID: 20813842 PMCID: PMC2962527 DOI: 10.1074/jbc.m110.142091] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2010] [Revised: 08/30/2010] [Indexed: 01/25/2023] Open
Abstract
Activation of the ionotropic P2RX7 nucleotide receptor by extracellular ATP has been implicated in modulating inflammatory disease progression. Continuous exposure of P2RX7 to ligand can result in apoptosis in many cell types, including monocytic cells, whereas transient activation of P2RX7 is linked to inflammatory mediator production and the promotion of cell growth. Given the rapid hydrolysis of ATP in the circulation and interstitial space, transient activation of P2RX7 appears critically important for its action, yet its effects on gene expression are unclear. The present study demonstrates that short-term stimulation of human and mouse monocytic cells as well as mouse osteoblasts with P2RX7 agonists substantially induces the expression of several activating protein-1 (AP-1) members, particularly FosB. The potent activation of FosB after P2RX7 stimulation is especially noteworthy considering that little is known concerning the role of FosB in immunological regulation. Interestingly, the magnitude of FosB activation induced by P2RX7 stimulation appears greater than that observed with other known inducers of FosB expression. In addition, we have identified a previously unrecognized role for FosB in osteoblasts with respect to nucleotide-induced expression of cyclooxygenase-2 (COX-2), which is the rate-limiting enzyme in prostaglandin biosynthesis from arachidonic acid and is critical for osteoblastic differentiation and immune behavior. The present studies are the first to link P2RX7 action to FosB/AP-1 regulation in multiple cell types, including a role in nucleotide-induced COX-2 expression, and support a role for FosB in the control of immune and osteogenic function by P2RX7.
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Affiliation(s)
- Monica L. Gavala
- From the Program in Molecular and Cellular Pharmacology and
- the Department of Biomolecular Chemistry, University of Wisconsin School of Medicine, Madison, Wisconsin 53706
| | - Lindsay M. Hill
- From the Program in Molecular and Cellular Pharmacology and
- the Department of Biomolecular Chemistry, University of Wisconsin School of Medicine, Madison, Wisconsin 53706
| | - Lisa Y. Lenertz
- the Department of Biomolecular Chemistry, University of Wisconsin School of Medicine, Madison, Wisconsin 53706
| | - Maya R. Karta
- From the Program in Molecular and Cellular Pharmacology and
- the Department of Biomolecular Chemistry, University of Wisconsin School of Medicine, Madison, Wisconsin 53706
| | - Paul J. Bertics
- From the Program in Molecular and Cellular Pharmacology and
- the Department of Biomolecular Chemistry, University of Wisconsin School of Medicine, Madison, Wisconsin 53706
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Nicotinic receptors on rat alveolar macrophages dampen ATP-induced increase in cytosolic calcium concentration. Respir Res 2010; 11:133. [PMID: 20920278 PMCID: PMC2955664 DOI: 10.1186/1465-9921-11-133] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2010] [Accepted: 09/29/2010] [Indexed: 01/28/2023] Open
Abstract
Background Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine. Methods Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca2+]i) were conducted. Results Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, β1, and β2, with most stable expression being noted for subunits α9, α10, β1, and β2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca2+]i revealed changes in membrane current in response to ACh and in [Ca2+]i in response to nicotine, respectively. However, nicotine (100 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca2+]i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium. Conclusions Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca2+-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential.
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Hill LM, Gavala ML, Lenertz LY, Bertics PJ. Extracellular ATP may contribute to tissue repair by rapidly stimulating purinergic receptor X7-dependent vascular endothelial growth factor release from primary human monocytes. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2010; 185:3028-34. [PMID: 20668222 PMCID: PMC3156583 DOI: 10.4049/jimmunol.1001298] [Citation(s) in RCA: 77] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
Extracellular ATP has been proposed to act as a danger signal to alert the immune system of cell damage. Release of high local concentrations of ATP activates the nucleotide receptor, purinergic receptor X7 (P2RX7), on monocytic cells, which promotes the processing/release of proinflammatory mediators. Although the proinflammatory actions of P2RX7 are well recognized, little is known regarding the potential function of P2RX7 in repair responses. Because the resolution of inflammation is characterized by monocytic cell-dependent production of proangiogenic factors, we evaluated the contribution of P2RX7 to this process. We observed that both short-term and long-term P2RX7 activation promotes the robust release of vascular endothelial growth factor from primary human monocytes. This vascular endothelial growth factor release is calcium dependent and associated with reactive oxygen species production. This previously unrecognized action of P2RX7 suggests that it may not only participate in inflammation and cell death, but that it is also likely to be important in the control of angiogenesis and wound repair.
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Affiliation(s)
- Lindsay M. Hill
- Molecular & Cellular Pharmacology, University of Wisconsin, Madison, WI 53706
| | - Monica L. Gavala
- Dept of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706
| | - Lisa Y. Lenertz
- Dept of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706
| | - Paul J. Bertics
- Dept of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706
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Lenertz LY, Wang Z, Guadarrama A, Hill LM, Gavala ML, Bertics PJ. Mutation of putative N-linked glycosylation sites on the human nucleotide receptor P2X7 reveals a key residue important for receptor function. Biochemistry 2010; 49:4611-9. [PMID: 20450227 PMCID: PMC2895974 DOI: 10.1021/bi902083n] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The nucleotide receptor P2X(7) is an immunomodulatory cation channel and a potential therapeutic target. P2X(7) is expressed in immune cells such as monocytes and macrophages and is activated by extracellular ATP following tissue injury or infection. Ligand binding to P2X(7) can stimulate ERK1/2, the transcription factor CREB, enzymes linked to the production of reactive oxygen species and interleukin-1 isoforms, and the formation of a nonspecific pore. However, little is known about the biochemistry of P2X(7), including whether the receptor is N-linked glycosylated and if this modification affects receptor function. Here we provide evidence that P2X(7) is sensitive to the glycosidases EndoH and PNGase F and that the human receptor appears glycosylated at N187, N202, N213, N241, and N284. Mutation of N187 results in weakened P2X(7) agonist-induced phosphorylation of ERK1/2, CREB, and p90 ribosomal S6 kinase, as well as a decreased level of pore formation. In further support of a role for glycosylation in receptor function, treatment of RAW 264.7 macrophages with the N-linked glycosylation synthesis inhibitor tunicamycin attenuates P2X(7) agonist-induced, but not phorbol ester-induced, ERK1/2 phosphorylation. Interestingly, residue N187 belongs to an N-linked glycosylation consensus sequence found in six of the seven P2X family members, suggesting this site is fundamentally important to P2X receptor function. To address the mechanism whereby N187 mutation attenuates receptor activity, we developed a live cell proteinase K digestion assay that demonstrated altered cell surface expression of P2X(7) N187A. This is the first report to map human P2X(7) glycosylation sites and reveal residue N187 is critical for receptor trafficking and function.
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Affiliation(s)
- Lisa Y. Lenertz
- Department of Biomolecular Chemistry, The University of Wisconsin-Madison, Madison, Wisconsin 53706
| | - Ziyi Wang
- Department of Biomolecular Chemistry, The University of Wisconsin-Madison, Madison, Wisconsin 53706
| | - Arturo Guadarrama
- Department of Biomolecular Chemistry, The University of Wisconsin-Madison, Madison, Wisconsin 53706
| | - Lindsay M. Hill
- Department of Biomolecular Chemistry, The University of Wisconsin-Madison, Madison, Wisconsin 53706
| | - Monica L. Gavala
- Department of Biomolecular Chemistry, The University of Wisconsin-Madison, Madison, Wisconsin 53706
| | - Paul J. Bertics
- Department of Biomolecular Chemistry, The University of Wisconsin-Madison, Madison, Wisconsin 53706
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Hecker A, Mikulski Z, Lips KS, Pfeil U, Zakrzewicz A, Wilker S, Hartmann P, Padberg W, Wessler I, Kummer W, Grau V. Pivotal Advance: Up-regulation of acetylcholine synthesis and paracrine cholinergic signaling in intravascular transplant leukocytes during rejection of rat renal allografts. J Leukoc Biol 2009; 86:13-22. [PMID: 19201987 DOI: 10.1189/jlb.1107722] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
During acute rejection, large numbers of leukocytes accumulate in the blood vessels of experimental renal allografts. About 70% of them are activated, cytotoxic monocytes that appear to be involved in allograft destruction. ACh exerts anti-inflammatory effects upon monocytes/macrophages and has been proposed to be a key player in neuroimmunological interactions. Its short half-life, however, makes it unlikely that neuronal ACh affects blood leukocytes. Renal transplantation was performed in the allogeneic DA to LEW and in the isogeneic LEW to LEW rat strain combination. Intravascular leukocytes were harvested after 4 days, and the expression of CHT1, cChAT, pChAT, and nAChR subunits was investigated by RT-PCR, immunoblotting, and immunohistochemistry. Monocytes were identified by double-labeling with ED1-antibody, directed to a CD68-like antigen. ACh content was measured by HPLC. [Ca(2+)](i) was monitored by Fura-2. Intravascular graft leukocytes express CHT1 and cChAT mRNA and protein and pChAT protein. Their expression is strongly up-regulated in vivo during acute allograft rejection. Immunohistochemistry revealed CHT1, cChAT, and pChAT protein in ED1-positive monocytes. The ACh content of allograft intravascular leukocytes was sixfold higher than that of isografts. Intravascular leukocytes express nAChR subunits, and an ATP-induced increase in [Ca(2+)](i) was augmented in vitro by a nAChR inhibitor in allograft but not isograft leukocytes. Intravascular graft leukocytes, among them monocytes, up-regulate non-neuronal ACh synthesis and develop auto-/paracrine cholinergic attenuation of ATP signaling during acute allograft rejection.
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Affiliation(s)
- Andreas Hecker
- Department of General and Thoracic Surgery, Justus-Liebig-University Giessen, Giessen, Germany
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