title: A20 deficiency causes spontaneous neuroinflammation in mice.
abstract: 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 gp91phox 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.
authors.forename: Renata Padilha
authors.initials: RP
authors.lastname: Guedes
authors.forename: Eva
authors.initials: E
authors.lastname: Csizmadia
authors.forename: Herwig P
authors.initials: HP
authors.lastname: Moll
authors.forename: Averil
authors.initials: A
authors.lastname: Ma
authors.forename: Christiane
authors.initials: C
authors.lastname: Ferran
authors.affiliation: 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. cdasilva@bidmc.harvard.edu.
authors.forename: Cleide Gonçalves
authors.initials: CG
authors.lastname: da Silva
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000172:deficiency* / Q000235:genetics; D016207:Cytokines / Q000378:metabolism*; D020833:Diffuse Axonal Injury / Q000209:etiology / Q000235:genetics; D004660:Encephalitis / Q000235:genetics* / Q000473:pathology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000172:deficiency* / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D016548:Prosencephalon / Q000166:cytology; D017483:Receptors, Vasopressin / Q000378:metabolism; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: D016207:Cytokines; D005904:Glial Fibrillary Acidic Protein; D015850:Interleukin-6; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D051267:NF-E2-Related Factor 2; D016328:NF-kappa B; C495636:Nfe2l2 protein, mouse; D017483:Receptors, Vasopressin; D014409:Tumor Necrosis Factor-alpha; D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D003546:Cysteine Endopeptidases; C513261:Tnfaip3 protein, mouse