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A20 deficiency causes spontaneous neuroinflammation in mice.

Guedes RP, Csizmadia E, Moll HP, Ma A, Ferran C, da Silva CG - J Neuroinflammation (2014)

Bottom Line: Quantitative results were statistically analyzed by ANOVA followed by a post-hoc test.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.Importantly, A20 HT brains showed an intermediate phenotype, exhibiting considerable, albeit not statistically significant, increase in markers of basal inflammation when compared to WT.

View Article: PubMed Central - HTML - PubMed

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.

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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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.
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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.

Mentions: Immunohistochemistry analysis of mouse brain sections using a macrophage/microglia cell surface marker F4/80 [36] revealed increased number of activated microglia throughout the A20 KO brain, as evidenced by their typical hypertrophied phenotype, that is enlarged cells with shorter and thicker branched processes [37] (Figure 2A). This picture totally contrasted with WT brains that showed resting/quiescent microglia harboring a ramified morphology with slender sensing arms. We confirmed the activation status of microglia by probing for mRNA levels of the microglial activation marker A1 [38,39]. A1 mRNA levels were significantly higher in CX and HC (approximately seven-fold) of A20 KO as compared to WT mice (Figure 2B). Astrocyte activation in the brain of A20 KO mice was also evident, as demonstrated by enhanced GFAP immunoreactivity [40]. Astrocytes displaying thick cell bodies and processes characteristic of astrocyte reactivity were especially marked in the outer layers of the CX and throughout the HC (Figure 2C). Astrocyte activation was confirmed at the mRNA levels by qPCR. GFAP mRNA levels were significantly (approximately 1.8-fold) higher in the CX and HC of A20 KO mice as compared to WT (Figure 2D). Brains from HT mice showed an intermediate phenotype with a consistent trend for greater microglia and astrocyte activation when compared to WT mice, and for significantly lower microglia and astrocyte activation when compared to KO mice, as showed by IHC and qPCR (Figure 2).


A20 deficiency causes spontaneous neuroinflammation in mice.

Guedes RP, Csizmadia E, Moll HP, Ma A, Ferran C, da Silva CG - J Neuroinflammation (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4128606&req=5

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.
Mentions: Immunohistochemistry analysis of mouse brain sections using a macrophage/microglia cell surface marker F4/80 [36] revealed increased number of activated microglia throughout the A20 KO brain, as evidenced by their typical hypertrophied phenotype, that is enlarged cells with shorter and thicker branched processes [37] (Figure 2A). This picture totally contrasted with WT brains that showed resting/quiescent microglia harboring a ramified morphology with slender sensing arms. We confirmed the activation status of microglia by probing for mRNA levels of the microglial activation marker A1 [38,39]. A1 mRNA levels were significantly higher in CX and HC (approximately seven-fold) of A20 KO as compared to WT mice (Figure 2B). Astrocyte activation in the brain of A20 KO mice was also evident, as demonstrated by enhanced GFAP immunoreactivity [40]. Astrocytes displaying thick cell bodies and processes characteristic of astrocyte reactivity were especially marked in the outer layers of the CX and throughout the HC (Figure 2C). Astrocyte activation was confirmed at the mRNA levels by qPCR. GFAP mRNA levels were significantly (approximately 1.8-fold) higher in the CX and HC of A20 KO mice as compared to WT (Figure 2D). Brains from HT mice showed an intermediate phenotype with a consistent trend for greater microglia and astrocyte activation when compared to WT mice, and for significantly lower microglia and astrocyte activation when compared to KO mice, as showed by IHC and qPCR (Figure 2).

Bottom Line: Quantitative results were statistically analyzed by ANOVA followed by a post-hoc test.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.Importantly, A20 HT brains showed an intermediate phenotype, exhibiting considerable, albeit not statistically significant, increase in markers of basal inflammation when compared to WT.

View Article: PubMed Central - HTML - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus