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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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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.
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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.

Mentions: Activated microglia and reactive astrocytes are key defense mechanisms of the CNS to injury, in part through their ability to modulate immune and inflammatory responses by secreting pro-inflammatory cytokines and chemokines such as TNF, IL-6, IL-1β, and MCP-1 [41,42]. As in all inflammatory responses, this defense system needs to be tightly modulated in order to avoid unfettered inflammation that would counterproductively cause neurotoxicity [43]. Accordingly, we probed by qPCR for mRNA levels of TNF, IL-6, IL-1β and MCP-1 in CX and HC of A20 WT, HT and KO mice. Our results show significantly increased mRNA levels of all these pro-inflammatory mediators in the brain of KO, as compared to WT mice, further confirming glial activation (Figure 3A). HT mouse brains also showed a tendency (albeit not significant) for higher mRNA levels of all these molecules when compared to WT brains. This tendency was more prominent in the CX than in the HC. We confirmed by IHC that higher TNF, IL-6, and MCP-1 mRNA levels in the brain of A20 deficient mice correlated with higher protein levels (Figure 3B). Double immunofluorescence staining using antibodies against microglia surface marker F4/80 or astrocyte marker GFAP in combination with anti-IL-6 demonstrate that both cell types produce IL-6 and contribute to its increased levels in the brains of A20 KO mice (Figure 4). Altogether, these results indicate a heightened basal level of inflammation in the brain of A20 deficient mice, especially when A20 expression is totally knocked-out.


A20 deficiency causes spontaneous neuroinflammation in mice.

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

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.
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Related In: Results  -  Collection

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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.
Mentions: Activated microglia and reactive astrocytes are key defense mechanisms of the CNS to injury, in part through their ability to modulate immune and inflammatory responses by secreting pro-inflammatory cytokines and chemokines such as TNF, IL-6, IL-1β, and MCP-1 [41,42]. As in all inflammatory responses, this defense system needs to be tightly modulated in order to avoid unfettered inflammation that would counterproductively cause neurotoxicity [43]. Accordingly, we probed by qPCR for mRNA levels of TNF, IL-6, IL-1β and MCP-1 in CX and HC of A20 WT, HT and KO mice. Our results show significantly increased mRNA levels of all these pro-inflammatory mediators in the brain of KO, as compared to WT mice, further confirming glial activation (Figure 3A). HT mouse brains also showed a tendency (albeit not significant) for higher mRNA levels of all these molecules when compared to WT brains. This tendency was more prominent in the CX than in the HC. We confirmed by IHC that higher TNF, IL-6, and MCP-1 mRNA levels in the brain of A20 deficient mice correlated with higher protein levels (Figure 3B). Double immunofluorescence staining using antibodies against microglia surface marker F4/80 or astrocyte marker GFAP in combination with anti-IL-6 demonstrate that both cell types produce IL-6 and contribute to its increased levels in the brains of A20 KO mice (Figure 4). Altogether, these results indicate a heightened basal level of inflammation in the brain of A20 deficient mice, especially when A20 expression is totally knocked-out.

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