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. 2014 Jul 16:11:122.
doi: 10.1186/1742-2094-11-122.

A20 deficiency causes spontaneous neuroinflammation in mice

Renata Padilha Guedes, Eva Csizmadia, Herwig P Moll, Averil Ma, Christiane Ferran, Cleide Gonçalves da Silva  1
Affiliations

A20 deficiency causes spontaneous neuroinflammation in mice

Renata Padilha Guedes et al. J Neuroinflammation. .

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.

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Figures

Figure 1
Figure 1
Baseline A20 mRNA expression in mouse cerebral cortex and hippocampus. (A) A20 and (C) IκBα mRNA levels in cerebral cortex (CX) and hippocampus (HC) of wild type (WT), A20 heterozygous (HT) and A20 knockout (KO) mice, measured by qPCR. Graph shows relative mRNA levels after normalization with mRNA levels of housekeeping gene βactin. Results are expressed as mean ± SEM of three to four animals per genotype. **P < 0.01. ND: not detectable. (B) Western blot (WB) analysis of IκBα expression in CX and HC of A20 WT, HT and KO mice. Immunoblotting for the housekeeping protein βactin was used to control for loading. Results are representative of three animals per genotype.
Figure 2
Figure 2
Loss of A20 leads to spontaneous microglia and astrocyte activation. Representative (A) F4/80 and (C) GFAP immunohistochemistry (brown) in the cerebral cortex (CX) and hippocampus (HC) of A20 wild type (WT), heterozygous (HT) and knockout (KO) mice. Yellow arrows indicate hypertrophied activated microglia, noted by their stout, dense appearance with shorter and thicker branched projections. Blue arrows indicate reactive astrocytes displaying thick cell bodies and processes, evident in the outer layers of the CX and throughout the HC. Photomicrographs are representative of three animals per genotype. Bar = 20 μm, magnification = 400x. (B) A1 and (D) GFAP mRNA levels measured by qPCR. Graph shows relative mRNA levels after normalization with mRNA levels of housekeeping gene βactin. Results are expressed as mean ± SEM of five to seven animals per genotype. *P < 0.05, **P < 0.01 and ***P < 0.001.
Figure 3
Figure 3
Levels of pro-inflammatory mediators are increased in cerebral cortex and hippocampus of A20 deficient mice. (A) TNF, IL-6, IL-1β and MCP-1 mRNA levels in cerebral cortex (CX) and hippocampus (HC) of wild type (WT), A20 heterozygous (HT) and A20 knockout (KO), measured by qPCR. Graph shows relative mRNA levels after normalization with mRNA levels of housekeeping gene βactin. Results are expressed as mean ± SEM of four to seven animals per genotype. *P < 0.05, **P < 0.01 and ***P < 0.001. (B) Representative images of TNF, IL-6 and MCP-1 immunohistochemistry (brown) in HC (TNF and IL-6) and CX (MCP-1) of A20 WT, HT and KO mice. Photomicrographs are representative of three to four animals per genotype. Top images: Bar = 50 μm, magnification = 200x. Bottom images are close-up images of the area delineated by the black box in top images.
Figure 4
Figure 4
Astrocytes and microglia contribute to higher IL-6 levels in A20 deficient brains. Representative images of double IL-6 (red) and GFAP (green), or IL-6 (red) and F4/80 (green) positive cells in the hippocampus (HC) of A20 wild type (WT), heterozygous (HT) and knockout (KO) mice, as determined by immunofluorescence staining. White arrows show IL-6 co-localization with glial fibrillary acidic protein (GFAP) (astrocytes) or F4/80 (microglia), as evidenced by the yellow overlay. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Photomicrographs are representative of four animals per genotype. Top images: Bar = 20 μm, magnification = 400x. Bottom images are close-up images of the area delineated by the white box in top images.
Figure 5
Figure 5
Cytokine production in response to inflammatory stimuli is enhanced in A20 deficient primary astrocytes. (A) TNF and (B) IL-6 levels, measured by ELISA, in cell culture supernatant from A20 wild type (WT), heterozygous (HT) and knockout (KO) mouse primary astrocytes following 24 hour stimulation with LPS (10 μg/mL). (C) IL-6 levels measured by ELISA, in cell culture supernatant from WT, HT and KO mouse primary astrocytes following 24 hours stimulation with TNF (100 UI/mL). NS: non-stimulated cells. Data represent mean ± SEM of primary astrocytes isolated from littermate pups (WT n = 2; HT n = 8 to 10, KO n = 3 to 4). *P < 0.05, **P < 0.01.
Figure 6
Figure 6
A20 knockdown enhances lipopolysaccharide (LPS)-mediated cytokine production in mouse primary astrocytes and microglia cell line. (A) TNF and (B) IL-6 mRNA levels in mouse primary astrocytes 6 hours after LPS (1 μg/mL) stimulation, as measured by qPCR. (D) TNF and (E) IL-6 mRNA levels in the microglia cell line N13, 1 hour and 6 hour respectively, after LPS (1 μg/mL) stimulation, as measured by qPCR. Graph shows relative mRNA levels after normalization with mRNA levels of housekeeping gene βactin. IL-6 protein levels, measured by ELISA, in cell culture supernatant of (C) mouse primary astrocytes and (F) microglia cell line N13 6 hours after LPS (1 μg/mL) stimulation. Results are expressed as mean ± SEM of three to five independent experiments. NS: non-stimulated cells. Ctrl: non-transfected control cells. A20 siRNA: cells transfected with A20 silencing RNA. C siRNA: cells transfected with All Star control silencing RNA. *P < 0.05, **P < 0.01 and ***P < 0.001.
Figure 7
Figure 7
Loss of A20 increases oxidative/nitrosative stress in the brain. (A) iNOS, eNOS and nNOS, (C) NADPH oxidase gp91phox subunit and (E) Nrf2 mRNA levels in cerebral cortex (CX) and hippocampus (HC) of wild type (WT), A20 heterozygous (HT) and A20 knockout (KO) mice, measured by qPCR. Graphs show the statistical analysis of relative mRNA levels after normalization with βactin. Results are expressed as mean ± SEM of five to seven animals per genotype. (B) iNOS and nitrotyrosine (NTY) immunostaining (brown) in CX of A20 WT, HT and KO mice. Top images: Bar = 50 μm, magnification = 200x. Bottom images are close-up images of the area delineated by the black box in top images. (D) NADPH oxidase gp91phox subunit expression in CX and HC protein lysates of A20 WT, HT and KO brains evaluated by Western blot (WB). Housekeeping protein GAPDH was used as loading control for semi-quantitative densitometry as shown in the graph. Graph shows semi-quantitative densitometry data using GAPDH as loading control. Results are expressed as mean ± SEM for four animals per genotype. (F) Representative images of Nrf-2 (red) and 4′,6-diamidino-2-phenylindole (DAPI, nuclear staining, blue) immunofluorescence staining in HC of WT, HT and KO mice. Photomicrographs are representative of three animals per genotype. Top images: Bar = 50 μm, magnification = 200x. Bottom images are close-up images of the area delineated by the white box in top images.*P < 0.05 and **P < 0.01.
Figure 8
Figure 8
Expression of adhesion molecules is increased in A20 deficient brain vasculature. (A) ICAM-1 and VCAM-1 immunohistochemistry (brown) in cerebral cortex (CX) of A20 wild type (WT), heterozygous (HT) and knockout (KO) mice. Black arrows indicate blood vessels. Photomicrographs are representative of three animals per genotype. Bar = 20 μm, magnification = 400x. (B) E-selectin mRNA levels in CX and hippocampus (HC) of A20 WT, HT and KO mice, measured by qPCR. Graph shows the statistical analysis of relative mRNA levels after normalization with βactin. Results are expressed as mean ± SEM for four to six animals per genotype. *P < 0.05, **P < 0.01 and ***P < 0.001.
Figure 9
Figure 9
Loss of A20 decreases the expression of neurofilaments in mice cerebral cortex and hippocampus. Immunostaining for neurofilaments (NF) (brown) in cerebral cortex (CX) and hippocampus (HC) of wild type (WT), A20 heterozygous (HT) and A20 knockout (KO) mice. Photomicrographs are representative of three animals per genotype. Top images: Bar = 50 μm, magnification = 200x. Bottom images are close-up images of the area delineated by the black box in top images.
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