CD73 alleviates GSDMD-mediated microglia pyroptosis in spinal cord injury through PI3K/AKT/Foxo1 signaling
Shun Xu
Department of Orthopedics, Shanghai Fifth People's Hospital, Fudan University, Shanghai, China
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
These three authors contributed equally to this work and are the first coauthors.Search for more papers by this authorJin Wang
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
These three authors contributed equally to this work and are the first coauthors.Search for more papers by this authorJunjie Zhong
Department of Neurosurgery, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China
Neurosurgical Institute of Fudan University, Fudan University, Shanghai, China
Shanghai Clinical Medical Center of Neurosurgery, Shanghai, China
Shanghai Key Laboratory of Brain Function and Restoration and Neural Regeneration, Shanghai, China
These three authors contributed equally to this work and are the first coauthors.Search for more papers by this authorMinghao Shao
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorJianyuan Jiang
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorJian Song
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorWei Zhu
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorFan Zhang
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorHaocheng Xu
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorGuangyu Xu
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorYuxuan Zhang
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorCorresponding Author
Xiaosheng Ma
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Correspondence
Feizhou Lyu, Department of Orthopedics, Shanghai Fifth People's Hospital, Fudan University, Shanghai 200240, China.
Email: [email protected]
Xiaosheng Ma, Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai 200040, China.
Email: [email protected]
Search for more papers by this authorCorresponding Author
Feizhou Lyu
Department of Orthopedics, Shanghai Fifth People's Hospital, Fudan University, Shanghai, China
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Correspondence
Feizhou Lyu, Department of Orthopedics, Shanghai Fifth People's Hospital, Fudan University, Shanghai 200240, China.
Email: [email protected]
Xiaosheng Ma, Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai 200040, China.
Email: [email protected]
Search for more papers by this authorShun Xu
Department of Orthopedics, Shanghai Fifth People's Hospital, Fudan University, Shanghai, China
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
These three authors contributed equally to this work and are the first coauthors.Search for more papers by this authorJin Wang
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
These three authors contributed equally to this work and are the first coauthors.Search for more papers by this authorJunjie Zhong
Department of Neurosurgery, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China
Neurosurgical Institute of Fudan University, Fudan University, Shanghai, China
Shanghai Clinical Medical Center of Neurosurgery, Shanghai, China
Shanghai Key Laboratory of Brain Function and Restoration and Neural Regeneration, Shanghai, China
These three authors contributed equally to this work and are the first coauthors.Search for more papers by this authorMinghao Shao
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorJianyuan Jiang
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorJian Song
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorWei Zhu
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorFan Zhang
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorHaocheng Xu
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorGuangyu Xu
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorYuxuan Zhang
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Search for more papers by this authorCorresponding Author
Xiaosheng Ma
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Correspondence
Feizhou Lyu, Department of Orthopedics, Shanghai Fifth People's Hospital, Fudan University, Shanghai 200240, China.
Email: [email protected]
Xiaosheng Ma, Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai 200040, China.
Email: [email protected]
Search for more papers by this authorCorresponding Author
Feizhou Lyu
Department of Orthopedics, Shanghai Fifth People's Hospital, Fudan University, Shanghai, China
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China
Correspondence
Feizhou Lyu, Department of Orthopedics, Shanghai Fifth People's Hospital, Fudan University, Shanghai 200240, China.
Email: [email protected]
Xiaosheng Ma, Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai 200040, China.
Email: [email protected]
Search for more papers by this authorAbstract
Background
Neuroinflammation-induced secondary injury is an important cause of sustained progression of spinal cord injury. Inflammatory programmed cell death pyroptosis executed by the pore-forming protein gasdermin D (GSDMD) is an essential step of neuroinflammation. However, it is unclear whether CD73, a widely accepted immunosuppressive molecule, can inhibit pyroptosis via mediating GSDMD.
Methods
C57BL/6J CD73 deficient mice and wild-type mice, Lipopolysaccharide (LPS)-induced primary microglia and BV2 cells were respectively used to illustrate the effect of CD73 on microglia pyroptosis in vivo and in vitro. A combination of molecular and histological methods was performed to assess pyroptosis and explore the mechanism both in vivo and in vitro.
Results
We have shown molecular evidence for CD73 suppresses the activation of NLRP3 inflammasome complexes to reduce the maturation of GSDMD, leading to decreased pyroptosis in microglia. Further analysis reveals that adenosine-A2B adenosine receptor-PI3K-AKT-Foxo1 cascade is a possible mechanism of CD73 regulation. Importantly, we determine that CD73 inhibits the expression of GSDMD at the transcriptional level through Foxo1. What's more, we confirm the accumulation of HIF-1α promotes the overexpression of CD73 after spinal cord injury (SCI), and the increased CD73 in turn upregulates the expression of HIF-1α, eventually forming a positive feedback regulatory loop.
Conclusion
Our data reveal a novel function of CD73 on microglia pyroptosis, suggesting a unique therapeutic opportunity for mitigating the disease process in SCI.
CONFLICT OF INTEREST
We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
Open Research
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Supporting Information
| Filename | Description |
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| ctm2269-sup-0001-FigureS1.tif1.4 MB | Supporting Information |
| ctm2269-sup-0002-SuppMat.xlsx67 KB | Supporting Information |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
REFERENCES
- 1Singh A, Tetreault L, Kalsi-Ryan S, Nouri A, Fehlings MG. Global prevalence and incidence of traumatic spinal cord injury. Clin Epidemiol. 2014; 6: 309-331.
- 2Putatunda R, Bethea JR, Hu W-H. Potential immunotherapies for traumatic brain and spinal cord injury. Chinese J Traumatol. 2018; 21(3): 125-136.
- 3Mortazavi MM, Verma K, Harmon OA, et al. The microanatomy of spinal cord injury: a review. Clin Anat. 2015; 28(1): 27-36.
- 4Beck KD, Nguyen HX, Galvan MD, Salazar DL, Woodruff TM, Anderson AJ. Quantitative analysis of cellular inflammation after traumatic spinal cord injury: evidence for a multiphasic inflammatory response in the acute to chronic environment. Brain. 2010; 133(Pt 2): 433-447.
- 5Hayden MS, Ghosh S. NF-κB in immunobiology. Cell Res. 2011; 21(2): 223-244.
- 6Wang Y-T, Lu X-M, Chen K-T, Shu Y-H, Qiu C-H. Immunotherapy strategies for spinal cord injury. Curr Pharm Biotechnol. 2015; 16(6): 492-505.
- 7Mortezaee K, Khanlarkhani N, Beyer C, Zendedel A. Inflammasome: its role in traumatic brain and spinal cord injury. J Cell Physiol. 2018; 233(7): 5160-5169.
- 8McKee CA, Lukens JR. Emerging roles for the immune system in traumatic brain injury. Front Immunol. 2016; 7: 556.
- 9Zendedel A, Johann S, Mehrabi S, et al. Activation and regulation of NLRP3 inflammasome by intrathecal application of SDF-1a in a spinal cord injury model. Mol Neurobiol. 2016; 53(5): 3063-3075.
- 10Vande Walle L, Lamkanfi M. Pyroptosis. Curr Biol. 2016; 26(13): R568-R572.
- 11Broz P, Dixit VM. Inflammasomes: mechanism of assembly, regulation and signalling. Nat Rev Immunol. 2016; 16(7): 407-420.
- 12Aglietti RA, Estevez A, Gupta A, et al. GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes. Proc Natl Acad Sci. 2016; 113(28): 7858-7863.
- 13Shi J, Zhao Y, Wang K, et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature. 2015; 526(7575): 660-665.
- 14Liu X, Zhang Z, Ruan J, et al. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature. 2016; 535(7610): 153-158.
- 15Aglietti RA, Dueber EC. Recent insights into the molecular mechanisms underlying pyroptosis and gasdermin family functions. Trends Immunol. 2017; 38(4): 261-271.
- 16Zhang Y, Chen X, Gueydan C, Han J. Plasma membrane changes during programmed cell deaths. Cell Res. 2018; 28(1): 9-21.
- 17David S, Greenhalgh AD, Kroner A. Macrophage and microglial plasticity in the injured spinal cord. Neuroscience. 2015; 307: 311-318.
- 18Walsh JG, Muruve DA, Power C. Inflammasomes in the CNS. Nat Rev Neurosci. 2014; 15(2): 84-97.
- 19Jiang W, Li M, He F, Zhou S, Zhu L. Targeting the NLRP3 inflammasome to attenuate spinal cord injury in mice. J Neuroinflammation. 2017; 14(1): 207.
- 20Liu W, Chen Y, Meng J, et al. Ablation of caspase-1 protects against TBI-induced pyroptosis in vitro and in vivo. J Neuroinflammation. 2018; 15(1): 48.
- 21Antonioli L, Pacher P, Vizi ES, Haskó G. CD39 and CD73 in immunity and inflammation. Trends Mol Med. 2013; 19(6): 355-367.
- 22Yu J, Wang X, Lu Q, et al. Extracellular 5′-nucleotidase (CD73) promotes human breast cancer cells growth through AKT/GSK-3β/β-catenin/cyclinD1 signaling pathway. Int J Cancer. 2018; 142(5): 959-967.
- 23Bynoe MS, Waickman AT, Mahamed DA, Mueller C, Mills JH, Czopik A. CD73 is critical for the resolution of murine colonic inflammation. J Biomed Biotechnol. 2012; 2012: 1-13.
10.1155/2012/260983 Google Scholar
- 24Xu S, Zhu W, Shao M, et al. Ecto-5′-nucleotidase (CD73) attenuates inflammation after spinal cord injury by promoting macrophages/microglia M2 polarization in mice. J Neuroinflammation. 2018; 15(1): 155.
- 25Silva-Vilches C, Ring S, Mahnke K. ATP and its metabolite adenosine as regulators of. dendritic cell activity. Front Immunol. 2018; 9: 2581.
- 26Vuerich M, Robson SC, Longhi MS. Ectonucleotidases in intestinal and hepatic inflammation. Front Immunol. 2019; 10: 507.
- 27Synnestvedt K, Furuta GT, Comerford KM, et al. Ecto-5′-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. J Clin Invest. 2002; 110(7): 993-1002.
- 28Merighi S, Borea PA, Stefanelli A, et al. A 2a and a 2b adenosine receptors affect HIF-1α signaling in activated primary microglial cells. Glia. 2015; 63(11): 1933-1952.
10.1002/glia.22861 Google Scholar
- 29Sekhon LHS, Fehlings MG. Epidemiology, demographics, and pathophysiology of acute spinal cord injury. Spine (Phila Pa 1976). 2001; 26(24 Suppl): S2-S12.
- 30Lin W-P, Xiong G-P, Lin Q, et al. Heme oxygenase-1 promotes neuron survival through down-regulation of neuronal NLRP1 expression after spinal cord injury. J Neuroinflammation. 2016; 13(1): 52.
- 31Lozano D, Gonzales-Portillo GS, Acosta S, et al. Neuroinflammatory responses to traumatic brain injury: etiology, clinical consequences, and therapeutic opportunities. Neuropsychiatr Dis Treat. 2015; 11: 97-106.
- 32Rathinam VAK, Fitzgerald KA. Inflammasome complexes: emerging mechanisms and effector functions. Cell. 2016; 165(4): 792-800.
- 33Wallisch JS, Simon DW, Bayır H, Bell MJ, Kochanek PM, Clark RSB. Cerebrospinal fluid NLRP3 is increased after severe traumatic brain injury in infants and children. Neurocrit Care. 2017; 27(1): 44-50.
- 34Liu H-D, Li W, Chen Z-R, et al. Expression of the NLRP3 inflammasome in cerebral cortex after traumatic brain injury in a rat model. Neurochem Res. 2013; 38(10): 2072-2083.
- 35Adamczak SE, de Rivero Vaccari JP, Dale G, et al. Pyroptotic neuronal cell death mediated by the AIM2 inflammasome. J Cereb Blood Flow Metab. 2014; 34(4): 621-629.
- 36David S, Kroner A. Repertoire of microglial and macrophage responses after spinal cord injury. Nat Rev Neurosci. 2011; 12(7): 388-399.
- 37Loane DJ, Byrnes KR. Role of microglia in neurotrauma. Neurotherapeutics. 2010; 7(4): 366-377.
- 38Resta R, Yamashita Y, Thompson LF. Ecto-enzyme and signaling functions of lymphocyte CD73. Immunol Rev. 1998; 161: 95-109.
- 39Kulesskaya N, Võikar V, Peltola M, et al. CD73 is a major regulator of adenosinergic signalling in mouse brain. PLoS One. 2013; 8(6):e66896.
10.1371/journal.pone.0066896 Google Scholar
- 40Csóka B, Haskó G. Adenosine, inflammation pathways and therapeutic challenges. Jt Bone Spine. 2011; 78(1): 4-6.
10.1016/j.jbspin.2010.08.010 Google Scholar
- 41Phillis JW, Goshgarian HG. Adenosine and neurotrauma: therapeutic perspectives. Neurol Res. 2001; 23(2-3): 183-189.
10.1179/016164101101198316 Google Scholar
- 42Fredholm BB, IJzerman AP, Jacobson KA, Klotz KN, Linden J. International union of pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev. 2001; 53(4): 527-552.
- 43Haskó G, Linden J, Cronstein B, Pacher P. Adenosine receptors: therapeutic aspects for inflammatory and immune diseases. Nat Rev Drug Discov. 2008; 7(9): 759-770.
- 44Ohta A, Sitkovsky M. Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage. Nature. 2001; 414(6866): 916-920.
- 45Boros D, Thompson J, Larson D. Adenosine regulation of the immune response initiated by ischemia reperfusion injury. Perfusion. 2016; 31(2): 103-110.
- 46Eckle T, Grenz A, Laucher S, Eltzschig HK. A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice. J Clin Invest. 2008; 118(10): 3301-3315.
- 47Eckle T, Krahn T, Grenz A, et al. Cardioprotection by ecto-5′-nucleotidase (CD73) and A 2B adenosine receptors. Circulation. 2007; 115(12): 1581-1590.
- 48Fruman DA, Chiu H, Hopkins BD, Bagrodia S, Cantley LC, Abraham RT. The PI3K pathway in human disease. Cell. 2017; 170(4): 605-635.
- 49Yin H, Zhou H, Kang Y, et al. Syk negatively regulates TLR4-mediated IFNβ and IL-10 production and promotes inflammatory responses in dendritic cells. Biochim Biophys Acta. 2016; 1860(3): 588-598.
10.1016/j.bbagen.2015.12.012 Google Scholar
- 50Wang L, Lu Y, Zhang X, et al. Mindin is a critical mediator of ischemic brain injury in an experimental stroke model. Exp Neurol. 2013; 247: 506-516.
- 51Kayagaki N, Lee BL, Stowe IB, et al. IRF2 transcriptionally induces GSDMD expression for pyroptosis. Sci Signal. 2019; 12(582):eaax4917.
- 52Link W. Introduction to FOXO biology. Methods Mol Biol. 2019; 1890: 1-9.
- 53Brown J, Wang H, Suttles J, Graves DT, Martin M. Mammalian target of rapamycin complex 2 (mTORC2) negatively regulates toll-like receptor 4-mediated inflammatory response via FoxO1. J Biol Chem. 2011; 286(52): 44295-44305.
- 54Wang Y, Zhou Y, Graves DT. FOXO transcription factors: their clinical significance and regulation. Biomed Res Int. 2014; 2014: 1-13.
- 55de Lemos ML, de la Torre AV, Petrov D, et al. Evaluation of hypoxia inducible factor expression in inflammatory and neurodegenerative brain models. Int J Biochem Cell Biol. 2013; 45(7): 1377-1388.
- 56Semenza GL. Expression of hypoxia-inducible factor 1: mechanisms and consequences. Biochem Pharmacol. 2000; 59(1): 47-53.
- 57Siddiq A, Aminova LR, Troy CM, et al. Selective inhibition of hypoxia-inducible factor (HIF) prolyl-hydroxylase 1 mediates neuroprotection against normoxic oxidative death via HIF- and CREB-independent pathways. J Neurosci. 2009; 29(27): 8828-8838.
- 58Nordal RA. Hypoxia and hypoxia-inducible factor-1 target genes in central nervous system radiation injury: a role for vascular endothelial growth factor. Clin Cancer Res. 2004; 10(10): 3342-3353.
- 59Karhausen J, Furuta GT, Tomaszewski JE, Johnson RS, Colgan SP, Haase VH. Epithelial hypoxia-inducible factor-1 is protective in murine experimental colitis. J Clin Invest. 2004; 114(8): 1098-1106.
10.1172/JCI200421086 Google Scholar
- 60Henn A, Lund S, Hedtjärn M, Schrattenholz A, Pörzgen P, Leist M. The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation. ALTEX. 2009; 26(2): 83-94.
- 61Butovsky O, Jedrychowski MP, Moore CS, et al. Identification of a unique TGF-β–dependent molecular and functional signature in microglia. Nat Neurosci. 2014; 17(1): 131-143.
- 62Kayagaki N, Warming S, Lamkanfi M, et al. Non-canonical inflammasome activation targets caspase-11. Nature. 2011; 479(7371): 117-121.
- 63Rathinam VAK, Zhao Y, Shao F. Innate immunity to intracellular LPS. Nat Immunol. 2019; 20(5): 527-533.
