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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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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.
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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.

Mentions: IL-6, mostly produced by astrocytes, achieves higher concentration than other soluble pro-inflammatory mediators in the brain, and hence has been designated as a key contributor to neuroinflammation [44,45]. To check whether enhanced IL-6 levels in the brain of A20 KO mice resulted from heightened activators, that is higher TNF levels, or also related to heightened production by A20 deficient astrocytes in response to a similar level of activator, we isolated and cultured primary astrocytes from A20 KO, HT and WT mice, exposed them for 24 hours to similar concentrations of exogenous LPS (10 μg/mL) and measured, by ELISA, TNF and IL-6 levels in cell culture supernatant. We noted a trend towards higher (albeit not significant) TNF and IL-6 levels in 24 hours culture supernatants of A20 KO as compared to WT astrocytes (2.25- and 1.5-fold, respectively), in the absence of any inflammatory stimuli. Following LPS treatment, TNF and IL-6 levels increased in response to LPS in all groups, albeit these levels were significantly higher in KO, as compared to WT astrocytes (Figure 5A and B). HT astrocytes showed an intermediate response, that is LPS treatment increased IL-6 production by two to three-fold, as compared to a ten-fold upregulation in KO astrocytes (Figure 5B). Since higher LPS-induced TNF levels is the master inducer of IL-6 in astrocytes, and hence could account for higher IL-6 levels in LPS treated A20 deficient astrocytes, we independently evaluated TNF-induced production (100 U/ml) of IL-6 in these cells. Here again, TNF-induced upregulation of IL-6 production was significantly higher in A20 KO as compared to WT and HT astrocytes (Figure 5C). We confirmed these findings in mouse primary astrocytes that had undergone siRNA mediated A20 knockdown. Transfection of astrocytes with A20 siRNA reduced by 50% LPS-induced A20 upregulation, as evidenced by mRNA levels measured 1 hour after LPS (1 μg/mL) stimulation (Additional file 2: Figure S2). Inadequate A20 upregulation following LPS (mimicking A20 knockdown in A20 HT mice) correlated with significantly higher TNF and IL-6 mRNA levels six hours after LPS, as compared to levels measured in non-transfected and All Star siRNA (C siRNA) control cells (Figure 6A and B). This was paralleled by significantly higher IL-6 protein levels in the cell culture supernatant of A20siRNA versus control cells (Figure 6C). As in astrocytes, siRNA mediated A20 knockdown in microglia cells (N13) decreased by 50% LPS induced upregulation of A20 (Additional file 2: Figure S2). Here again, this correlated with significantly higher LPS-induced upregulation of TNF and IL-6 mRNA (Figure 6D and E), and of IL-6 protein (Figure 6F) levels in these cells as compared to non-transfected or C siRNA transfected cells.


A20 deficiency causes spontaneous neuroinflammation in mice.

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

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

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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.
Mentions: IL-6, mostly produced by astrocytes, achieves higher concentration than other soluble pro-inflammatory mediators in the brain, and hence has been designated as a key contributor to neuroinflammation [44,45]. To check whether enhanced IL-6 levels in the brain of A20 KO mice resulted from heightened activators, that is higher TNF levels, or also related to heightened production by A20 deficient astrocytes in response to a similar level of activator, we isolated and cultured primary astrocytes from A20 KO, HT and WT mice, exposed them for 24 hours to similar concentrations of exogenous LPS (10 μg/mL) and measured, by ELISA, TNF and IL-6 levels in cell culture supernatant. We noted a trend towards higher (albeit not significant) TNF and IL-6 levels in 24 hours culture supernatants of A20 KO as compared to WT astrocytes (2.25- and 1.5-fold, respectively), in the absence of any inflammatory stimuli. Following LPS treatment, TNF and IL-6 levels increased in response to LPS in all groups, albeit these levels were significantly higher in KO, as compared to WT astrocytes (Figure 5A and B). HT astrocytes showed an intermediate response, that is LPS treatment increased IL-6 production by two to three-fold, as compared to a ten-fold upregulation in KO astrocytes (Figure 5B). Since higher LPS-induced TNF levels is the master inducer of IL-6 in astrocytes, and hence could account for higher IL-6 levels in LPS treated A20 deficient astrocytes, we independently evaluated TNF-induced production (100 U/ml) of IL-6 in these cells. Here again, TNF-induced upregulation of IL-6 production was significantly higher in A20 KO as compared to WT and HT astrocytes (Figure 5C). We confirmed these findings in mouse primary astrocytes that had undergone siRNA mediated A20 knockdown. Transfection of astrocytes with A20 siRNA reduced by 50% LPS-induced A20 upregulation, as evidenced by mRNA levels measured 1 hour after LPS (1 μg/mL) stimulation (Additional file 2: Figure S2). Inadequate A20 upregulation following LPS (mimicking A20 knockdown in A20 HT mice) correlated with significantly higher TNF and IL-6 mRNA levels six hours after LPS, as compared to levels measured in non-transfected and All Star siRNA (C siRNA) control cells (Figure 6A and B). This was paralleled by significantly higher IL-6 protein levels in the cell culture supernatant of A20siRNA versus control cells (Figure 6C). As in astrocytes, siRNA mediated A20 knockdown in microglia cells (N13) decreased by 50% LPS induced upregulation of A20 (Additional file 2: Figure S2). Here again, this correlated with significantly higher LPS-induced upregulation of TNF and IL-6 mRNA (Figure 6D and E), and of IL-6 protein (Figure 6F) levels in these cells as compared to non-transfected or C siRNA transfected cells.

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