A20 deficiency causes spontaneous neuroinflammation in mice

Journal of Neuroinflammation

Cleide Gonçalves da Silva³  Christiane Ferran³  Averil Ma²  Herwig P Moll³  Eva Csizmadia³  Renata Padilha Guedes¹ 

[1] Current address: Federal University of Health Sciences of Porto Alegre (UFCSPA), Porto Alegre, RS, Brazil;Division of Gastroenterology, University of California, San Francisco, CA, USA;Division of Vascular Surgery, Center for Vascular Biology Research and the Transplant Institute, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

关键词: Oxidative stress;    Inflammatory cytokines;    Reactive gliosis;    Neuroinflammation;    TNFAIP3/A20;   

Others  :  1151344
DOI  :  10.1186/1742-2094-11-122

 received in 2014-02-06, accepted in 2014-06-25,  发布年份 2014

【 摘 要 】

Background

A20 (TNFAIP3) is a pleiotropic NFκB-dependent gene that terminates NFκB activation in response to inflammatory stimuli. The potent anti-inflammatory properties of A20 are well characterized in several organs. However, little is known about its role in the brain. In this study, we investigated the brain phenotype of A20 heterozygous (HT) and knockout (KO) mice.

Methods

The inflammatory status of A20 wild type (WT), HT and KO brain was determined by immunostaining, quantitative PCR, and Western blot analysis. Cytokines secretion was evaluated by ELISA. Quantitative results were statistically analyzed by ANOVA followed by a post-hoc test.

Results

Total loss of A20 caused remarkable reactive microgliosis and astrogliosis, as determined by F4/80 and GFAP immunostaining. Glial activation correlated with significantly higher mRNA and protein levels of the pro-inflammatory molecules TNF, IL-6, and MCP-1 in cerebral cortex and hippocampus of A20 KO, as compared to WT. Basal and TNF/LPS-induced cytokine production was significantly higher in A20 deficient mouse primary astrocytes and in a mouse microglia cell line. Brain endothelium of A20 KO mice demonstrated baseline activation as shown by increased vascular immunostaining for ICAM-1 and VCAM-1, and mRNA levels of E-selectin. In addition, total loss of A20 increased basal brain oxidative/nitrosative stress, as indicated by higher iNOS and NADPH oxidase subunit gp91^phox levels, correlating with increased protein nitration, gauged by nitrotyrosine immunostaining. Notably, we also observed lower neurofilaments immunostaining in A20 KO brains, suggesting higher susceptibility to axonal injury. Importantly, A20 HT brains showed an intermediate phenotype, exhibiting considerable, albeit not statistically significant, increase in markers of basal inflammation when compared to WT.

Conclusions

This is the first characterization of spontaneous neuroinflammation caused by total or partial loss of A20, suggesting its key role in maintenance of nervous tissue homeostasis, particularly control of inflammation. Remarkably, mere partial loss of A20 was sufficient to cause chronic, spontaneous low-grade cerebral inflammation, which could sensitize these animals to neurodegenerative diseases. These findings carry strong clinical relevance in that they question implication of identified A20 SNPs that lower A20 expression/function (phenocopying A20 HT mice) in the pathophysiology of neuroinflammatory diseases.

【 授权许可】

   
2014 Guedes et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

【 参考文献 】

• [1]Unger JW: Glial reaction in aging and Alzheimer’s disease. Microsc Res Tech 1998, 43:24-28.
• [2]Sawada M, Imamura K, Nagatsu T: Role of cytokines in inflammatory process in Parkinson’s disease. J Neural Transm Suppl 2006, 70:373-381.
• [3]Noseworthy JH, Lucchinetti C, Rodriguez M, Weinshenker BG: Multiple sclerosis. N Engl J Med 2000, 343:938-952.
• [4]Tarkowski E, Rosengren L, Blomstrand C, Wikkelso C, Jensen C, Ekholm S, Tarkowski A: Intrathecal release of pro- and anti-inflammatory cytokines during stroke. Clin Exp Immunol 1997, 110:492-499.
• [5]Najjar S, Pearlman DM, Alper K, Najjar A, Devinsky O: Neuroinflammation and psychiatric illness. J Neuroinflammation 2013, 10:43.
• [6]Depino AM: Peripheral and central inflammation in autism spectrum disorders. Mol Cell Neurosci 2013, 53:69-76.
• [7]Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH: Mechanisms underlying inflammation in neurodegeneration. Cell 2010, 140:918-934.
• [8]Brown GC, Neher JJ: Inflammatory neurodegeneration and mechanisms of microglial killing of neurons. Mol Neurobiol 2010, 41:242-247.
• [9]De Cristobal J, Moro MA, Davalos A, Castillo J, Leza JC, Camarero J, Colado MI, Lorenzo P, Lizasoain I: Neuroprotective effect of aspirin by inhibition of glutamate release after permanent focal cerebral ischaemia in rats. J Neurochem 2001, 79:456-459.
• [10]Caso JR, Moro MA, Lorenzo P, Lizasoain I, Leza JC: Involvement of IL-1beta in acute stress-induced worsening of cerebral ischaemia in rats. Eur Neuropsychopharmacol 2007, 17:600-607.
• [11]Koprich JB, Reske-Nielsen C, Mithal P, Isacson O: Neuroinflammation mediated by IL-1beta increases susceptibility of dopamine neurons to degeneration in an animal model of Parkinson’s disease. J Neuroinflammation 2008, 5:8.
• [12]Imielski Y, Schwamborn JC, Luningschror P, Heimann P, Holzberg M, Werner H, Leske O, Puschel AW, Memet S, Heumann R, Israel A, Kaltschmidt C, Kaltschmidt B: Regrowing the adult brain: NF-kappaB controls functional circuit formation and tissue homeostasis in the dentate gyrus. PLoS One 2012, 7:e30838.
• [13]Opipari AW Jr, Boguski MS, Dixit VM: The A20 cDNA induced by tumor necrosis factor alpha encodes a novel type of zinc finger protein. J Biol Chem 1990, 265:14705-14708.
• [14]Pranski EL, Van Sanford CD, Dalal NV, Orr AL, Karmali D, Cooper DS, Costa N, Heilman CJ, Gearing M, Lah JJ, Levey AI, Betarbet RS: Comparative distribution of protein components of the A20 ubiquitin-editing complex in normal human brain. Neurosci Lett 2012, 520:104-109.
• [15]Cooper JT, Stroka DM, Brostjan C, Palmetshofer A, Bach FH, Ferran C: A20 blocks endothelial cell activation through a NF-kappaB-dependent mechanism. J Biol Chem 1996, 271:18068-18073.
• [16]Longo CR, Arvelo MB, Patel VI, Daniel S, Mahiou J, Grey ST, Ferran C: A20 protects from CD40-CD40 ligand-mediated endothelial cell activation and apoptosis. Circulation 2003, 108:1113-1118.
• [17]Boone DL, Turer EE, Lee EG, Ahmad RC, Wheeler MT, Tsui C, Hurley P, Chien M, Chai S, Hitotsumatsu O, McNally E, Pickart C, Ma A: The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses. Nat Immunol 2004, 5:1052-1060.
• [18]Lee EG, Boone DL, Chai S, Libby SL, Chien M, Lodolce JP, Ma A: Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice. Science 2000, 289:2350-2354.
• [19]Daniel S, Arvelo MB, Patel VI, Longo CR, Shrikhande G, Shukri T, Mahiou J, Sun DW, Mottley C, Grey ST, Ferran C: A20 protects endothelial cells from TNF-, Fas-, and NK-mediated cell death by inhibiting caspase 8 activation. Blood 2004, 104:2376-2384.
• [20]Daniel S, Patel VI, Shrikhande GV, Scali ST, Ramsey HE, Csizmadia E, Benhaga N, Fisher MD, Arvelo MB, Ferran C: The universal NF-kappaB inhibitor a20 protects from transplant vasculopathy by differentially affecting apoptosis in endothelial and smooth muscle cells. Transplant Proc 2006, 38:3225-3227.
• [21]Damrauer SM, Studer P, Da Silva CG, Longo CR, Ramsey HE, Csizmadia E, Shrikhande GV, Scali ST, Libermann TA, Bhasin MK, Ferran C: A20 modulates lipid metabolism and energy production to promote liver regeneration. PLoS One 2011, 6:e17715.
• [22]Ramsey HE, Da Silva CG, Longo CR, Csizmadia E, Studer P, Patel VI, Damrauer SM, Siracuse JJ, Daniel S, Ferran C: A20 protects mice from lethal liver ischemia/reperfusion injury by increasing peroxisome proliferator-activated receptor-alpha expression. Liver Transpl 2009, 15:1613-1621.
• [23]Sunaga K, Sugaya E, Kajiwara K, Tsuda T, Sugaya A, Kimura M: Molecular mechanism of preventive effect of peony root extract on neuron damage. J Herb Pharmacother 2004, 4:9-20.
• [24]Yu L, Miao H, Hou Y, Zhang B, Guo L: Neuroprotective effect of A20 on TNF-induced postischemic apoptosis. Neurochem Res 2006, 31:21-32.
• [25]Wang X, Deckert M, Xuan NT, Nishanth G, Just S, Waisman A, Naumann M, Schluter D: Astrocytic A20 ameliorates experimental autoimmune encephalomyelitis by inhibiting NF-kappaB- and STAT1-dependent chemokine production in astrocytes. Acta Neuropathol 2013, 126:711-724.
• [26]Mc Guire C, Rahman M, Schwaninger M, Beyaert R, van Loo G: The ubiquitin editing enzyme A20 (TNFAIP3) is upregulated during permanent middle cerebral artery occlusion but does not influence disease outcome. Cell Death Dis 2013, 4:e531.
• [27]Koumakis E, Giraud M, Dieude P, Cohignac V, Cuomo G, Airo P, Hachulla E, Matucci-Cerinic M, Diot E, Caramaschi P, Mouthon L, Riccieri V, Cracowski JL, Tiev KP, Francès C, Amoura Z, Sibilia J, Cosnes A, Carpentier P, Valentini G, Manetti M, Guiducci S, Meyer O, Kahan A, Boileau C, Chiocchia G, Allanore Y: Brief report: candidate gene study in systemic sclerosis identifies a rare and functional variant of the TNFAIP3 locus as a risk factor for polyautoimmunity. Arthritis Rheum 2012, 64:2746-2752.
• [28]Vereecke L, Beyaert R, van Loo G: Genetic relationships between A20/TNFAIP3, chronic inflammation and autoimmune disease. Biochem Soc Trans 2011, 39:1086-1091.
• [29]Adrianto I, Wen F, Templeton A, Wiley G, King JB, Lessard CJ, Bates JS, Hu Y, Kelly JA, Kaufman KM, Guthridge JM, Alarcón Riquelme ME, Anaya JM, Bae SC, Bang SY, Boackle SA, Brown EE, Petri MA, Gallant C, Ramsey-Goldman R, Reveille JD, Vila LM, Criswell LA, Edberg JC, Freedman BI, Gregersen PK, Gilkeson GS, Jacob CO, James JA, BIOLUPUS and GENLES Networks, et al.: Association of a functional variant downstream of TNFAIP3 with systemic lupus erythematosus. Nat Genet 2011, 43:253-258.
• [30]Bates JS, Lessard CJ, Leon JM, Nguyen T, Battiest LJ, Rodgers J, Kaufman KM, James JA, Gilkeson GS, Kelly JA, Humphrey MB, Harley JB, Gray-McGuire C, Moser KL, Gaffney PM: Meta-analysis and imputation identifies a 109 kb risk haplotype spanning TNFAIP3 associated with lupus nephritis and hematologic manifestations. Genes Immun 2009, 10:470-477.
• [31]McCarthy KD, de Vellis J: Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 1980, 85:890-902.
• [32]Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227:680-685.
• [33]Csizmadia E, Csizmadia V: Detection of apoptosis in tissue sections. Methods Mol Biol 2009, 559:49-63.
• [34]Block ML, Zecca L, Hong JS: Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci 2007, 8:57-69.
• [35]Zhang D, Hu X, Qian L, O’Callaghan JP, Hong JS: Astrogliosis in CNS pathologies: is there a role for microglia? Mol Neurobiol 2010, 41:232-241.
• [36]Moussaud S, Draheim HJ: A new method to isolate microglia from adult mice and culture them for an extended period of time. J Neurosci Methods 2010, 187:243-253.
• [37]He J, Crews FT: Increased MCP-1 and microglia in various regions of the human alcoholic brain. Exp Neurol 2008, 210:349-358.
• [38]Zhang L, Nair A, Krady K, Corpe C, Bonneau RH, Simpson IA, Vannucci SJ: Estrogen stimulates microglia and brain recovery from hypoxia-ischemia in normoglycemic but not diabetic female mice. J Clin Invest 2004, 113:85-95.
• [39]Kumari R, Willing LB, Krady JK, Vannucci SJ, Simpson IA: Impaired wound healing after cerebral hypoxia-ischemia in the diabetic mouse. J Cereb Blood Flow Metab 2007, 27:710-718.
• [40]Eng LF, Ghirnikar RS: GFAP and astrogliosis. Brain Pathol 1994, 4:229-237.
• [41]Farina C, Aloisi F, Meinl E: Astrocytes are active players in cerebral innate immunity. Trends Immunol 2007, 28:138-145.
• [42]Semple BD, Kossmann T, Morganti-Kossmann MC: Role of chemokines in CNS health and pathology: a focus on the CCL2/CCR2 and CXCL8/CXCR2 networks. J Cereb Blood Flow Metab 2010, 30:459-473.
• [43]Harry GJ, Kraft AD: Neuroinflammation and microglia: considerations and approaches for neurotoxicity assessment. Expert Opin Drug Metab Toxicol 2008, 4:1265-1277.
• [44]Dong Y, Benveniste EN: Immune function of astrocytes. Glia 2001, 36:180-190.
• [45]Aloisi F, Care A, Borsellino G, Gallo P, Rosa S, Bassani A, Cabibbo A, Testa U, Levi G, Peschle C: Production of hemolymphopoietic cytokines (IL-6, IL-8, colony-stimulating factors) by normal human astrocytes in response to IL-1 beta and tumor necrosis factor-alpha. J Immunol 1992, 149:2358-2366.
• [46]Zhou L, Zhu DY: Neuronal nitric oxide synthase: structure, subcellular localization, regulation, and clinical implications. Nitric Oxide 2009, 20:223-230.
• [47]Mungrue IN, Bredt DS: nNOS at a glance: implications for brain and brawn. J Cell Sci 2004, 117:2627-2629.
• [48]Endres M, Laufs U, Liao JK, Moskowitz MA: Targeting eNOS for stroke protection. Trends Neurosci 2004, 27:283-289.
• [49]Murphy S: Production of nitric oxide by glial cells: regulation and potential roles in the CNS. Glia 2000, 29:1-13.
• [50]Bolanos JP, Almeida A, Stewart V, Peuchen S, Land JM, Clark JB, Heales SJ: Nitric oxide-mediated mitochondrial damage in the brain: mechanisms and implications for neurodegenerative diseases. J Neurochem 1997, 68:2227-2240.
• [51]Mander P, Brown GC: Activation of microglial NADPH oxidase is synergistic with glial iNOS expression in inducing neuronal death: a dual-key mechanism of inflammatory neurodegeneration. J Neuroinflammation 2005, 2:20.
• [52]Reiter CD, Teng RJ, Beckman JS: Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. J Biol Chem 2000, 275:32460-32466.
• [53]Itoh K, Igarashi K, Hayashi N, Nishizawa M, Yamamoto M: Cloning and characterization of a novel erythroid cell-derived CNC family transcription factor heterodimerizing with the small Maf family proteins. Mol Cell Biol 1995, 15:4184-4193.
• [54]de Vries HE, Witte M, Hondius D, Rozemuller AJ, Drukarch B, Hoozemans J, van Horssen J: Nrf2-induced antioxidant protection: a promising target to counteract ROS-mediated damage in neurodegenerative disease? Free Radic Biol Med 2008, 45:1375-1383.
• [55]Li W, Khor TO, Xu C, Shen G, Jeong WS, Yu S, Kong AN: Activation of Nrf2-antioxidant signaling attenuates NFkappaB-inflammatory response and elicits apoptosis. Biochem Pharmacol 2008, 76:1485-1489.
• [56]Radisavljevic Z, Avraham H, Avraham S: Vascular endothelial growth factor up-regulates ICAM-1 expression via the phosphatidylinositol 3 OH-kinase/AKT/Nitric oxide pathway and modulates migration of brain microvascular endothelial cells. J Biol Chem 2000, 275:20770-20774.
• [57]Berger RP, Adelson PD, Pierce MC, Dulani T, Cassidy LD, Kochanek PM: Serum neuron-specific enolase, S100B, and myelin basic protein concentrations after inflicted and noninflicted traumatic brain injury in children. J Neurosurg 2005, 103:61-68.
• [58]Fuchs E, Cleveland DW: A structural scaffolding of intermediate filaments in health and disease. Science 1998, 279:514-519.
• [59]Kuchel GA, Poon T, Irshad K, Richard C, Julien JP, Cowen T: Decreased neurofilament gene expression is an index of selective axonal hypotrophy in ageing. Neuroreport 1996, 7:1353-1359.
• [60]Somerville MJ, Percy ME, Bergeron C, Yoong LK, Grima EA, McLachlan DR: Localization and quantitation of 68 kDa neurofilament and superoxide dismutase-1 mRNA in Alzheimer brains. Brain Res Mol Brain Res 1991, 9:1-8.
• [61]McKerracher L, Vidal-Sanz M, Essagian C, Aguayo AJ: Selective impairment of slow axonal transport after optic nerve injury in adult rats. J Neurosci 1990, 10:2834-2841.
• [62]O’Callaghan JP, Sriram K: Glial fibrillary acidic protein and related glial proteins as biomarkers of neurotoxicity. Expert Opin Drug Saf 2005, 4:433-442.
• [63]da Silva CG, Studer P, Skroch M, Mahiou J, Minussi DC, Peterson CR, Wilson SW, Patel VI, Ma A, Csizmadia E, Ferran C: A20 promotes liver regeneration by decreasing SOCS3 expression to enhance IL-6/STAT3 proliferative signals. Hepatology 2013, 57:2014-2025.
• [64]Erta M, Quintana A, Hidalgo J: Interleukin-6, a major cytokine in the central nervous system. Int J Biol Sci 2012, 8:1254-1266.
• [65]Matmati M, Jacques P, Maelfait J, Verheugen E, Kool M, Sze M, Geboes L, Louagie E, Mc Guire C, Vereecke L, Chu Y, Boon L, Staelens S, Matthys P, Lambrecht BN, Schimidt-Supprian M, Pasparakis M, Elewaut D, Beyaert R, van Loo G: A20 (TNFAIP3) deficiency in myeloid cells triggers erosive polyarthritis resembling rheumatoid arthritis. Nat Genet 2011, 43:908-912.
• [66]Cheret C, Gervais A, Lelli A, Colin C, Amar L, Ravassard P, Mallet J, Cumano A, Krause KH, Mallat M: Neurotoxic activation of microglia is promoted by a nox1-dependent NADPH oxidase. J Neurosci 2008, 28:12039-12051.
• [67]Dohi K, Ohtaki H, Nakamachi T, Yofu S, Satoh K, Miyamoto K, Song D, Tsunawaki S, Shioda S, Aruga T: Gp91phox (NOX2) in classically activated microglia exacerbates traumatic brain injury. J Neuroinflammation 2010, 7:41.
• [68]Zhu DY, Liu SH, Sun HS, Lu YM: Expression of inducible nitric oxide synthase after focal cerebral ischemia stimulates neurogenesis in the adult rodent dentate gyrus. J Neurosci 2003, 23:223-229.
• [69]Ono K, Suzuki H, Sawada M: Delayed neural damage is induced by iNOS-expressing microglia in a brain injury model. Neurosci Lett 2010, 473:146-150.
• [70]Iadecola C, Zhang F, Xu X: Inhibition of inducible nitric oxide synthase ameliorates cerebral ischemic damage. Am J Physiol 1995, 268:R286-R292.
• [71]Phares TW, Fabis MJ, Brimer CM, Kean RB, Hooper DC: A peroxynitrite-dependent pathway is responsible for blood-brain barrier permeability changes during a central nervous system inflammatory response: TNF-alpha is neither necessary nor sufficient. J Immunol 2007, 178:7334-7343.
• [72]Anrather J, Racchumi G, Iadecola C: NF-kappaB regulates phagocytic NADPH oxidase by inducing the expression of gp91phox. J Biol Chem 2006, 281:5657-5667.
• [73]Saha RN, Pahan K: Regulation of inducible nitric oxide synthase gene in glial cells. Antioxid Redox Signal 2006, 8:929-947.
• [74]Krizanac-Bengez L, Mayberg MR, Janigro D: The cerebral vasculature as a therapeutic target for neurological disorders and the role of shear stress in vascular homeostatis and pathophysiology. Neurol Res 2004, 26:846-853.
• [75]Tei N, Tanaka J, Sugimoto K, Nishihara T, Nishioka R, Takahashi H, Yano H, Matsumoto S, Ohue S, Watanabe H, Kumon Y, Ohnishi T: Expression of MCP-1 and fractalkine on endothelial cells and astrocytes may contribute to the invasion and migration of brain macrophages in ischemic rat brain lesions. J Neurosci Res 2013, 91:681-693.
• [76]Ferran C, Stroka DM, Badrichani AZ, Cooper JT, Wrighton CJ, Soares M, Grey ST, Bach FH: A20 inhibits NF-kappaB activation in endothelial cells without sensitizing to tumor necrosis factor-mediated apoptosis. Blood 1998, 91:2249-2258.
• [77]Turer EE, Tavares RM, Mortier E, Hitotsumatsu O, Advincula R, Lee B, Shifrin N, Malynn BA, Ma A: Homeostatic MyD88-dependent signals cause lethal inflammation in the absence of A20. J Exp Med 2008, 205:451-464.
• [78]Caso JR, Pradillo JM, Hurtado O, Lorenzo P, Moro MA, Lizasoain I: Toll-like receptor 4 is involved in brain damage and inflammation after experimental stroke. Circulation 2007, 115:1599-1608.
• [79]Chakravarty S, Herkenham M: Toll-like receptor 4 on nonhematopoietic cells sustains CNS inflammation during endotoxemia, independent of systemic cytokines. J Neurosci 2005, 25:1788-1796.
• [80]Rivest S: Regulation of innate immune responses in the brain. Nat Rev Immunol 2009, 9:429-439.
• [81]Saijo K, Winner B, Carson CT, Collier JG, Boyer L, Rosenfeld MG, Gage FH, Glass CK: A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death. Cell 2009, 137:47-59.
• [82]Dieguez-Gonzalez R, Calaza M, Perez-Pampin E, Balsa A, Blanco FJ, Canete JD, Caliz R, Carreno L, de la Serna AR, Fernandez-Gutierrez B, Ortiz AM, Herrero-Beaumont G, Pablos JL, Narvaez J, Navarro F, Marenco JL, Gomez_reino JJ, Gonzales A: Analysis of TNFAIP3, a feedback inhibitor of nuclear factor-kappaB and the neighbor intergenic 6q23 region in rheumatoid arthritis susceptibility. Arthritis Res Ther 2009, 11:R42.
• [83]Musone SL, Taylor KE, Lu TT, Nititham J, Ferreira RC, Ortmann W, Shifrin N, Petri MA, Kamboh MI, Manzi S, Seldin MF, Gregersen PK, Behrens TW, Ma A, Kwok PY, Criswell LA: Multiple polymorphisms in the TNFAIP3 region are independently associated with systemic lupus erythematosus. Nat Genet 2008, 40:1062-1064.
• [84]Jurgens HA, Johnson RW: Dysregulated neuronal-microglial cross-talk during aging, stress and inflammation. Exp Neurol 2010, 1:40-48.
• [85]Shrikhande GV, Scali ST, da Silva CG, Damrauer SM, Csizmadia E, Putheti P, Matthey M, Arjoon R, Patel R, Siracuse JJ, Maccariello ER, Andersen ND, Monahan T, Peterson C, Essayagh S, Studer P, Guedes RP, Kocher O, Usheva A, Veves A, Kaczmarek E, Ferran C: O-glycosylation regulates ubiquitination and degradation of the anti-inflammatory protein A20 to accelerate atherosclerosis in diabetic ApoE-null mice. PLoS One 2010, 5:e14240.