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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 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.
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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.

Mentions: NO, when produced in physiologic levels by the low-throughput and constitutively expressed nNOS and eNOS NO synthases (NOS), mostly serves a homeostatic function through regulation of synaptic signaling and plasticity [46,47], as well as vasoprotection, through combined anti-apoptotic, anti-inflammatory, and reactive oxygen radicals’ scavenging properties [48]. However, overproduction of NO in pathophysiological conditions is implicated in oxidative-dependent neuronal death and dysfunction. High throughput NFκB-dependent iNOS, mainly produced by activated glia, is the primary NOS involved in inflammatory neurodegenerative disorders [49]. Accordingly, we evaluated iNOS mRNA and protein levels in CX and HC of A20-competent and A20 deficient mice. Our results show that iNOS mRNA levels significantly increase in the CX of KO mice as compared to WT, with HT demonstrating an intermediate level (Figure 7A). We confirmed this by IHC that depicted increased iNOS immunostaining in the CX of A20 deficient mice (Figure 5B). Levels of iNOS mRNA in HC were similar in all groups. Besides high NO production by iNOS, drastic increase of nNOS expression in certain pathophysiologic conditions could promote excitotoxicity causing neuronal death [50]. Interestingly, nNOS mRNA levels were significantly decreased in CX and HC of A20 KO, as compared to WT mice, with HT showing an intermediate phenotype (Figure 7A), while brain eNOS mRNA levels were comparable in all three genotypes (Figure 7A). Increased expression of iNOS in inflammatory conditions is often associated with increased levels of NADPH oxidase, the major enzymatic complex involved in the production of superoxide anion (O2-) [51]. Increased NO levels, in the setting of oxidative stress, favors formation of highly reactive peroxynitrite (NO/O2) species, enhancing formation of the protein adduct, nitrotyrosine [52]. Accordingly, we evaluated, by qPCR and Western blot, the expression level of the transmembrane catalytic subunit of NADPH oxidase, gp91phox. Our results show that gp91phox mRNA and protein levels were significantly higher in CX and HC of A20 KO as compared to WT and HT mice, with HT mice showing slightly higher levels then their WT littermates (Figure 7C and D). Combined increase of gp91phox and iNOS (hence likely NO) production in brains of A20 KO mice correlated with increased immunostaining for nitrotyrosine, indicating heightened levels of protein nitration, implying nitrosative stress (Figure 7B).


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 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.
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Related In: Results  -  Collection

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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.
Mentions: NO, when produced in physiologic levels by the low-throughput and constitutively expressed nNOS and eNOS NO synthases (NOS), mostly serves a homeostatic function through regulation of synaptic signaling and plasticity [46,47], as well as vasoprotection, through combined anti-apoptotic, anti-inflammatory, and reactive oxygen radicals’ scavenging properties [48]. However, overproduction of NO in pathophysiological conditions is implicated in oxidative-dependent neuronal death and dysfunction. High throughput NFκB-dependent iNOS, mainly produced by activated glia, is the primary NOS involved in inflammatory neurodegenerative disorders [49]. Accordingly, we evaluated iNOS mRNA and protein levels in CX and HC of A20-competent and A20 deficient mice. Our results show that iNOS mRNA levels significantly increase in the CX of KO mice as compared to WT, with HT demonstrating an intermediate level (Figure 7A). We confirmed this by IHC that depicted increased iNOS immunostaining in the CX of A20 deficient mice (Figure 5B). Levels of iNOS mRNA in HC were similar in all groups. Besides high NO production by iNOS, drastic increase of nNOS expression in certain pathophysiologic conditions could promote excitotoxicity causing neuronal death [50]. Interestingly, nNOS mRNA levels were significantly decreased in CX and HC of A20 KO, as compared to WT mice, with HT showing an intermediate phenotype (Figure 7A), while brain eNOS mRNA levels were comparable in all three genotypes (Figure 7A). Increased expression of iNOS in inflammatory conditions is often associated with increased levels of NADPH oxidase, the major enzymatic complex involved in the production of superoxide anion (O2-) [51]. Increased NO levels, in the setting of oxidative stress, favors formation of highly reactive peroxynitrite (NO/O2) species, enhancing formation of the protein adduct, nitrotyrosine [52]. Accordingly, we evaluated, by qPCR and Western blot, the expression level of the transmembrane catalytic subunit of NADPH oxidase, gp91phox. Our results show that gp91phox mRNA and protein levels were significantly higher in CX and HC of A20 KO as compared to WT and HT mice, with HT mice showing slightly higher levels then their WT littermates (Figure 7C and D). Combined increase of gp91phox and iNOS (hence likely NO) production in brains of A20 KO mice correlated with increased immunostaining for nitrotyrosine, indicating heightened levels of protein nitration, implying nitrosative stress (Figure 7B).

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