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Keywords:

  • nitric oxide;
  • nuclear factor κB;
  • chemokines;
  • inflammation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Forkhead transcription factor3 (Foxp3) is critical for generating CD4+CD25+ regulatory T cells. However, its role in microglia has not been identified. Here, we show that Foxp3 is expressed in microglia and is upregulated upon activation. In Foxp3 mutant mice (Foxp3sf), microglia release higher levels of inflammatory cytokines and mediators such as NO, MCP-1, CXCL10, and ROS upon liposaccharide treatment than the wild type, while TNF-α and IL-1β were not significantly different between wild and mutant microglial cells. In addition, Foxp3 silencing enhances inflammatory responses, suggesting that the major role of Foxp3 in microglia is that of a repressor of activation. Similarly, Foxp3 overexpression reduces inflammatory responses in microglia. We also demonstrate that Foxp3 interacts directly with NF-κB and modulates its transcriptional activities. These findings point to the importance of Foxp3 in NF-κB mediated inflammatory responses in microglia. © 2010 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Microglia are the resident immune cells in the central nervous system (CNS) and play a critical role in inflammatory responses in the brain. In the healthy adult brain, microglia are small, highly ramified cells with a flattened appearance. In response to environmental changes, particularly neuronal damage, microglia exhibit marked morphological changes, proliferate, become phagocytic, and upregulate the expression of a large number of molecules including cytokines, adhesion molecules, and transcription factors (Hanisch, 2002; Hanisch and Kettenmann, 2007; Kawanokuchi et al., 2004; Park et al., 2008). For this reason, studies of the role of microglia in promoting or attenuating proinflammatory processes in the brain have a bearing on the mechanisms of many pathophysiologies of the CNS.

The Forkhead transcription factor (Foxp3) was initially identified as defective in a fatal autoimmune disease of mice and humans (Bennett et al., 2001; Wildin et al., 2001). More recently, it was shown to be a key transcription factor in regulatory T cells (Treg) (Hori et al., 2003); it is present at levels comparable to housekeeping genes in CD4+CD25+ Treg (Fontenot et al., 2003) and is found in thymic (Chang et al., 2005) and breast epithelial cells (Zuo et al., 2007b), and in epithelial cells of many lineages (Chen et al., 2008).

The starting point of this study was the serendipitous observation in a microarray analysis that the Foxp3 gene was upregulated in LPS-activated BV-2 microglial cells. This led us to examine Foxp3 expression and its role in microglia. In this study, we used BV-2 cells and then we confirmed the results from BV-2 cells with using primary microglia. The BV-2 is an immortalized microglia cell line made by transfecting primary mouse microglia (C57BL/6 mice) with a v-raf/v-myc-oncogene retrovirus. Although BV-2 cells retain the morphological, phenotypical, and functional properties associated with primary microglia, BV-2 cells are known less responsive to LPS stimulation in comparison to primary microglia (Horvath et al., 2008).

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Materials

N-(1-naphtyl)-ethylenediamine dihydrochloride, LPS (Escherichia coli serotype 0111:B4) and sodium nitrite were from Sigma, St. Louis, MO, and FBS from Hyclone, Logan, UT; 2′,7′-dichlorodihydrofluorescein (H2DCFDA) was purchased from Molecular Probes (Eugene, OR) while 0.2-μm syringe filters, 96- and 24-well tissue culture plates, and 100-mm diameter dishes, were purchased from Corning, NY; we also used DMEM/F12 (Gibco-BRL) and 0.25% trypsin (WelGENE, Korea).

Animals

Scurfy (B.Cg-Foxp3sf/J) and Foxp3EGFP mice (B6.Cg-Foxp3tm2Tch/J) were purchased from the Jackson Laboratories, MA. All mice were maintained in the Kyung Hee University animal facility and cared for in accordance with institutional guidelines.

Cell Cultures

Mouse primary microglial cultures were prepared by mild trypsinization as described previously, with minor modifications (Saura et al., 2003). In brief, cerebral cortices of 0- to 3-day-old scurfy or Foxp3-eGFP transgenic mice were chopped and dissociated by mechanical disruption using a nylon mesh, and the cells were seeded in culture dishes. After in vitro culture for 10–14 days, microglial cells were isolated from these mixed glial cultures by mild trypsinization. The mixed glial cultures were incubated with trypsin solution (0.25% trypsin, 1 mM EDTA in HBS) diluted 1:3 in DMEM/F12 for 30–60 min. This resulted in detachment of an upper layer of astrocytes in one piece, while the microglia remained attached to the bottom of the culture flask. The detached layer of astrocytes was aspirated, and the microglia were used in our experiments. When we analyzed the purity of primary microglia using a pan macrophage marker-F4/80 antibody, the purity of primary microglia was about 90%.

shRNA Transfection

BV-2 cells were grown in 10-cm dishes in DMEM with 10% fetal bovine serum. SureSilencing shRNA Plasmids for mouse FOXP3 were purchased from SABiosciences (MD). The sequences for silencing gene expression were FOXP3, 5′-CCACACCTCTTCTTCCTTGAA-3′; and control, 5′-GGAATCTCATTCGATGCATAC-3′. Transfections were carried out using Lipofectamine 2000 reagent (Invitrogen, MD), in accordance with the manufacturer's instructions. The transfected cells were selected with 20 μg/mL hygromycin B for 3 weeks with medium changes every 3 days. Individual colonies were picked and amplified in the same selection medium.

RNA Extraction

Total RNA was extracted from cultured cells using an EasyBlue RNA extraction kit (iNtRON Biotechnology, Seongnam, Korea). Extracts were assayed to determine the quality and concentration of the RNA using a ND-1000 spectrophotometer (Nanodrop Technologies). Extracts were stored at −20°C.

Real-Time PCR

cDNA was synthesized using a Power cDNA Synthesis Kit (iNtRON Biotechnology) and stored at −20°C. Real-time quantitative PCR was performed using an ABI 7300 Sequence detection system (Applied Biosystems, Foster City, CA) employing SYBR Green I as the dsDNA-specific binding dye for continuous fluorescence monitoring. Amplification was carried out in a total volume of 20 μL containing 375 nM of each gene-specific primer (FOXP3-F, AGC CTG CTC CAT ACC TTG AA; FOXP3-R, GCC CAA GAT GTG CAC TGA TA; GAPDH-F, TTC ACC ACC ATG GAG AAG GC, GAPDH-R, GGC ATG GAC TGT GGT CAT GA), 2× PCR Master Mix (Applied Biosystems) and 2 μL of cDNA. The PCR reactions were then subjected to 40 cycles of denaturation (95°C, 30 s) and annealing and extension at 60°C, with fluorescence measured at the end of each cycle. After the cycles were terminated, the signals at temperatures between 60 and 95°C were also collected to generate a dissociation curve. All reactions were performed in duplicate to confirm reproducibility and included a negative control (without template) to verify that no primer-dimers were being generated. A standard curve for each primer was constructed using serial dilutions of cDNA, and the amount of target mRNA in each sample was normalized using that of the mean GAPDH levels.

Measurement of Nitrite Concentration

Each genotype of microglia (2.5 × 105 cells/well) were treated with LPS (1 μg/mL) and incubated for 24 h. NO synthesis in cell cultures was measured by a microplate assay method, as previously described (Xie et al., 1992).

Cytokine and Chemokine Analysis

Microglia (2.5 × 105 cells/well) were incubated with LPS (1 μg/mL) for 24 h. A Bio-Plex Suspension Array System (Bio-Rad, Hercules, CA) was used to profile the expression of four inflammatory mediators [IL-1β, IL-6, monocyte chemoattractant protein 1 (MCP-1), and Interferon-γ inducible protein 10 (IP10 or CXCL10)]. The results were confirmed using an OptEIA ELISA kit (BD Biosciences Pharmingen, San Diego, CA).

Intracellular Reactive Oxygen Species Assay

Levels of intracellular reactive oxygen species (ROS) were determined from the change in fluorescence resulting from oxidation of the fluorescent probe H2DCFDA. Briefly, 2 × 105 cells/well were exposed to LPS for 24 h. After incubation, the cells were washed once with FBS-free medium and incubated in a 50-μM solution of the fluorescent probe H2DCFDA for 1 h at 37°C. The cells were then washed twice with FBS-free medium, and fluorescence corresponding to intracellular ROS was analyzed by fluorescence microscopy (Axio Observer, Carl Zeiss).

Phagocytosis Assay

Phagocytosis was assayed by means of a commercially available kit (Vybrant phagocytosis assay kit; Molecular Probes, Eugene, OR). Microglia were cultured in 1 μg/mL LPS for 3 h at a cell density of 1 × 104/well in 96-well plates. Then the medium was removed, fluorescein-labeled Escherichia coli were added, and the phagocytosis assay was performed following the manufacturer's instructions. Fluorescence images were taken with a fluorescence microscope.

Immunofluorescence Confocal Microscopy

For these experiments, microglia were cultured on 4-well Lab-Tek™ II Chambered Coverglasses (Nalge Nunc International, Naperville, IL). The cells were fixed in 4% paraformaldehyde/PBS, for 20 min, and washed in PBS with Tween-20 (0.1%). After permeabilization with 0.1% Triton X-100/PBS for 15 min, they were washed with PBST (0.1%) and blocked with blocking buffer (PBS with 1% BSA) at room temperature for 1 h followed by incubation with p65 or Foxp3 antibodies (Santa Cruz Biotechnology, CA) at 4°C overnight. After washing with PBST (0.1%), the cells were incubated with FITC-conjugated secondary antibodies (Sigma) diluted 500-fold in blocking buffer for 1 h at room temperature. They were then washed with PBST (0.1%), mounted in Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA) and analyzed by confocal microscopy (Zeiss LSM Pascal 5).

Flow Cytometric Analysis

Foxp3-eGFP microglia (6 × 105 cells/well) were activated by LPS for 3 or 6 h, harvested and fixed in 70% ethanol at −20°C for 24 h. After washing with cold PBS, the cells were analyzed on a FACSCalibur flow cytometer (BD) using BD CellQuest software.

p-IκBα Western Blotting

Cytoplasmic extracts were prepared as previously described (Chung et al., 2007). Proteins were then separated by Tris-glycine 4–12% (KOMA Biotech, Korea) gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in PBS-Tween-20 for 1 h at room temperature, and then incubated with anti-p-IκB-α or β-actin antibody. After washing three times in PBS-Tween-20, the blot was incubated with secondary antibody for 1 h. Antibody-specific proteins were then visualized with an enhanced chemiluminescence detection system as recommended (Amersham Corp., Newark, NJ). For immunoprecipitation, 500 μg total cell lysate was incubated with the appropriate primary antibody overnight at 4°C using a Catch and Release v2.0 reversible immunoprecipitation system (Millipore, Charlottesville, VA). Immunoprecipitated proteins were resolved in Tris-glycine 4–12% as described above. Normal rabbit IgG was also used in the immunoprecipitation experiments to control for cross-reactivity.

NF-κB Luciferase Assay

BV-2 cells were transfected with an NFκB-dependent reporter construct that contains six copies of the NFκB response element along with mock or Foxp3 plasmid. The Foxp3 expression plasmid and corresponding empty vector (pcDNA 3.1) were kind gifts of Dr. Sin-Hyeog Im (Gwangju Institute of Science and Technology). After 24-h incubation in complete medium, cells were stimulated with LPS for 6 h. Luciferase activity measured with the dual luciferase assay system (Promega) is expressed relative to expression of the cotransfected Renilla luciferase promoter (pRL-SV40), to control for transfection efficiency.

Statistical Analysis

All values are expressed as mean ± SEM. Statistical significance (P < 0.05 for all analyses) was assessed by Student's t-test using GraphPad Prism 4.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Foxp3 mRNA and Protein Are Regulated in Microglia

To assess the expression of Foxp3 mRNA and protein in microglia, we performed real-time PCR and Western blotting in cells of the BV-2 microglial cell line. Real-time PCR analysis showed that Foxp3 mRNA was upregulated upon exposure to LPS with a peak after 3 h (Fig. 1A). Although Foxp3 protein was also expressed in the LPS-activated BV-2 cells, its appearance was delayed compared with the mRNA (Fig. 1B).

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Figure 1. LPS activation induces FoxP3 mRNA and protein production in BV-2 microglial cells. (A) Real-time PCR analysis of Foxp3 expression in BV-2 cells. BV-2 cells were stimulated with LPS (1 μg/ml) for 1, 3, 6, and 9 h. Transcripts of Foxp3 were assayed by real-time PCR and expressed as fold increases relative to normalized Gapdh. Data are representative of at least two independent experiments (means and s.e.m.) (B) Western blotting analysis of Foxp3 expression in BV-2 cells.

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Foxp3 Positive Cell Populations Were Increased upon Microglia Activation

Foxp3EGFP mice were initially described by Haribhai et al. (2007). These mice are transgenic for a bicistronic locus encoding both Foxp3 and EGFP under the control of the Foxp3 promoter (Foxp3EGFP), so that expression of Foxp3 can be followed by measuring the fluorescence signal of EGFP on a real time basis. Using Foxp3EGFP mice, we measured Foxp3 expression in microglial cells. LPS-treated Foxp3EGFP microglia showed a marked increase in EGFP positive microglia (32.1% at 3 h, 44.7% at 6 h) compared with the control (26.6%) (Fig. 2A). When we observed microglia of the Foxp3EGFP mice under a fluorescence microscopy, LPS treatment was found to increase the strength of the EGFP reporter signal (Fig. 2B). In addition, immunocytochemical analysis revealed that endogenous Foxp3 expression also increased in the LPS-activated BV-2 microglia (Fig. 2C).

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Figure 2. Representative analysis of Foxp3-EGFP and Foxp3 protein expression in LPS-activated microglia. (A) EGFP expression in cells concordant with Foxp3 expression. Foxp3-EGFP mouse microglia were isolated and cultured with 1 μg/mL LPS for 3 or 6 h. Histograms show frequency of Foxp3-EGFP-expressing cells. Black line, nontreated cells; green line, LPS-treated for 3 h; blue line, LPS-treated for 6 h (B) Fluorescence images of Foxp3-EGFP mouse microglia with or without 1 μg/mL LPS treatment for 24 h. (C) Immunocytochemical analysis to detect expression of Foxp3. BV-2 cells were stained with anti-Foxp3 polyclonal Ab (green). BV-2 cells were treated with 1 μg/mL of LPS for each time point. Bar graph is expressed as fluorescence intensity in cells. *P < 0.001 compared with nontreated cells.

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The Microglia of Foxp3 Mutant Mice (Foxp3sf) Exhibit Strong Inflammatory Responses

Nitric oxide has been shown to be released by cultured murine microglia and to play a substantial role in mechanisms of neuro-degeneration (Chao et al., 1992; Peterson et al., 1994). To assess whether the Foxp3sf microglia affects NO release, we assayed NO production. Foxp3sf microglia produced substantially more NO in response to LPS than did wild-type microglia (see Fig. 3). To further investigate the immunological functions of Foxp3, we measured cytokine and chemokine production by Foxp3sf and wild-type microglia. LPS stimulation led to significantly more release of CXCL10 and MCP-1 from Foxp3sf microglia than from wild-type microglia. However, LPS treatment did not have a differential effect on the production of proinflammatory cytokines such as IL-1β and IL-6 by Foxp3sf microglia compared with wild-type microglia (see Fig. 3).

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Figure 3. Increased NO, CXCL10, and MCP-1 release from LPS-activated FoxP3sf microglia. Microglia of each genotype were incubated with or without 1 μg/mL of LPS for 24 h. The amounts of CXCL10, MCP-1, IL-1β, and IL-6 secreted were measured by Bio-Plex or ELISA. Graphic representation of the mean ± SEM of six samples. *P < 0.01; **P < 0.001; NS, not significant.

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Since activated microglia have been shown to produce ROS (Keller et al., 1999), we assessed the intracellular concentration of ROS in Foxp3sf microglia and wild-type microglia by H2DCFDA oxidation, and found that the Foxp3sf microglia generated more ROS in response to LPS activation than wild-type microglia (Fig. 4A).

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Figure 4. ROS production and phagocytosis are stimulated by LPS activation in Foxp3sf microglia. (A) Microglia of each genotype were treated with 1 μg/mL of LPS for 24 h. Images were taken with a fluorescence microscope to evaluate ROS production (×50). The bar graph shows the mean fluorescence intensity of the images. (B) Microglia of each genotype were treated with 1 μg/mL of LPS for 3 h. Fluorescein-labeled Escherichia coli were added and fluorescence images were taken with a fluorescence microscopy. The bar graph shows the mean fluorescence intensity of the images.

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As phagocytosis is one of the most important roles of microglia, we studied the phagocytotic activity of LPS-activated Foxp3sf microglia, by measuring the incorporation of fluorescent E. coli. The Foxp3sf microglia displayed greater phagocytotic activity than wild-type microglia (Fig. 4B).

Foxp3 Silencing Increases NO, CXCL10, and MCP-1 Release by BV-2 Microglial Cells

To confirm that the increased inflammatory responses of Foxp3sf microglia are mediated by Foxp3, we also knocked down expression of Foxp3 using a short hairpin RNA (shRNA) plasmid (Fig. 5A). NO, CXCL10, and MCP-1 release were significantly higher in Foxp3 shRNA-transfected BV-2 cells compared with control shRNA-transfected cells (Fig. 5B).

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Figure 5. Increased NO, CXCL10, and MCP-1 release from LPS-activated Foxp3-silenced BV-2 cells. (A) Foxp3-silencing shRNA (FoxP3) or negative control shRNA (Con) transfected BV-2 cells were stimulated with LPS for 9 h, and the expression of Foxp3 protein was analyzed by immunoblot analysis. (B) BV-2 cells were transfected with Foxp3-silencing shRNA (Foxp3 shRNA) or negative control shRNA (Con shRNA) and incubated with or without 1 μg/mL of LPS for 24 h. NO release was measured by the Griess method (to detect nitrite). The amounts of MCP-1 and CXCL10 secreted were measured by Bio-Plex or ELISA. Graphic representation of the mean ± SEM of at least three different experiments. *P < 0.01; **P < 0.001.

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Foxp3 Overexpression Reduces NO, CXCL10, and MCP-1 Production by BV-2 Microglial Cells

We showed above that inflammatory responses are increased in Foxp3-deficient microglia. To determine if Foxp3 represses inflammatory responses, we transfected BV-2 cells with a Foxp3 expression plasmid (Fig. 6A). Overexpression of Foxp3 resulted in lower NO, CXCL10, and MCP-1 production in response to LPS treatment than in mock plasmid-transfected cells (Fig. 6B), confirming that Foxp3 represses microglial inflammatory responses.

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Figure 6. Foxp3 overexpression represses NO, MCP-1, and CXCL10 production in LPS- activated BV-2 cells. (A) BV-2 cells transfected with mock (pcDNA3.1; PC) or Foxp3 plasmids (Foxp3) stimulated with LPS for 6 h, and the expression of Foxp3 protein was analyzed by immunoblot analysis. (B) BV-2 cells transfected with mock (PC) or Foxp3 plasmids (Foxp3) were cultured with or without LPS for 24 h. NO release was measured by the Griess method (to detect nitrite). The amounts of MCP-1 and CXCL10 were measured by ELISA Graphic representation of the mean ± SEM of at least three different experiments. *P < 0.05; **P < 0.01.

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Increased IκB-α Phosphorylation and NF-κB Nuclear Translocation in Foxp3sf Microglia

NF-κB is thought to play a pivotal role in immune and inflammatory responses by regulating genes encoding proinflammatory cytokines, adhesion molecules, chemokines, growth factors, and inducible enzymes. NF-κB is usually kept inactive in the cytoplasm by associating with an endogenous inhibitor protein of the IκB (inhibitor of NF-κB) family. We analyzed the subcellular distribution of p65. Immunocytochemical analysis revealed that treatment of Foxp3sf microglia with LPS resulted in greater nuclear accumulation of p65 than treatment of wild-type microglia (Fig. 7A). A similar result was obtained with microglia transfected with the Foxp3 shRNA plasmid (Fig. 7B), whereas nuclear accumulation of p65 decreased in the LPS-activated Foxp3 overexpressing BV-2 cells (Fig. 7C).

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Figure 7. Nuclear localization of NF-κB induced by LPS. (A) Confocal microscopy images of fluorescence immunocytochemical analysis of p65 (green, left image) in Foxp3sf and wild-type microglia treated with or without 1 μg/mL of LPS for 10 min (×400). Nuclei were counterstained with DAPI (blue, right image). Bar graph is expressed as fluorescence intensity in the DAPI positive nucleus. **P < 0.0001. (B) Immunocytochemical analysis of p65 (green, left image) in the negative control- or Foxp3 shRNA plasmid-transfected BV-2 cells with or without LPS exposure for 10 min. Nuclei were counterstained with DAPI (blue, right image). Bar graph is expressed as fluorescence intensity in the DAPI positive nucleus. **P < 0.01. (C) Immunocytochemical analysis to detect translocation of NF-κB (green, left image) in mock (pcDNA3.1; PC)- or Foxp3 DNA (Foxp3)-transfected cells (×1,000). Nuclei were counterstained with DAPI (blue, right image). Bar graph is expressed as fluorescence intensity in the DAPI positive nucleus. *P < 0.05. (D) Western blot analysis of p-IκB-α and β-actin in cytoplasmic extracts of Foxp3sf or wild-type microglia treated with 1 μg/mL of LPS for 1 h.

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We confirmed these results using cell fractions of Foxp3sf and wild-type microglia: the level of cytoplasmic phosphorylated IκB-α was significantly greater in Foxp3sf microglia than in wild-type microglia (Fig. 7D). Together, these results show that NF-κB translocation to the nucleus is stimulated by an increase of IκB-α phosphorylation upon LPS treatment of Foxp3sf microglia.

Foxp3 Physically Associates with NF-κB and Represses NF-κB Transcriptional Activity

Bettelli et al. showed that Foxp3 physically interacted with NF-κB, and that Foxp3-deficient T cells had increased NF-κB transcriptional activity (Bettelli et al., 2005). Kwon et al. showed further that Foxp3 inhibited nuclear translocation of NF-κB by increasing the stability of the NF-κB inhibitor IκB-α in T cells (Kwon et al., 2008). We tested whether Foxp3 interacted physically with NF-κB in microglia. Co-immunoprecipitation using an anti-Foxp3 antibody, and Western blotting with anti-p65 antibody, revealed that Foxp3 became physically associated with NF-κB when BV-2 cells were activated by LPS treatment (Fig. 8A). To test for transcriptional activity of NF-κB, we performed NF-κB luciferase assay in Foxp3 shRNA-transfected BV-2 cells and Foxp3 overexpressing BV-2 cells. NF-κB transcriptional activity increased in the LPS-activated Foxp3-silenced BV-2 cells, whereas it decreased in the LPS-activated Foxp3 overexpressing BV-2 cells (Fig. 8B).

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Figure 8. Foxp3 physically associates with NF-κB and represses NF-κB transcriptional activity. (A) BV-2 cells lysates were immunoprecipitated with anti-Foxp3 or normal rabbit IgG (negative control) antibody. Proteins were run on a 4–12% Tri-glycine gel and immunoblotted with anti-p65 antibody. M, protein marker (B) Foxp3 inhibits transcriptional activity of NF-κB in BV-2 cells. BV-2 cells were co-transfected with a NF-κB luciferase reporter construct together with mock (pcDNA3.1; PC) or Foxp3 DNA (Foxp3) and luciferase activities were measured in the presence or absence of LPS (top). Negative control (Con shRNA)- or Foxp3 shRNA plasmid (Foxp3 shRNA)-transfected BV-2 cells were transfected with the NF-κB luciferase reporter construct and luciferase activities were measured in the presence or absence of LPS (below). Firefly and renilla luciferase activities were measured using the Dual-Glo Luciferase Assay System. The fold inductions were calculated as ratios of NF-κB firefly luciferase and pRL-SV40 renilla luciferase. Graphic representation of the mean ± SEM of at least three different experiments. *P < 0.05; **P < 0.001.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

We have shown that expression of the Foxp3 gene is upregulated in microglia by LPS activation, and that NO, ROS, MCP-1, CXCL10, and phagocytosis levels are higher in Foxp3sf microglia than in wild-type microglia. The increased inflammatory responses seen in Foxp3sf microglia were also observed in Foxp3-silenced BV-2 cells. Conversely, Foxp3 overexpressing BV-2 cells generated reduced inflammatory responses compared with cells transfected with control plasmid.

Over-production of NO has been correlated with oxidative stress and is regulated by NF-κB, which performs pivotal roles in the immediate early stages of immune, acute phase, and inflammatory responses, as well as in cell survival (Lawrence et al., 2001; Makarov, 2001). NF-κB also plays a critical role in the activation of immune cells by upregulating the expression of many chemokines, including MCP-1 and CXCL10 (Majumder et al., 1998; Piao et al., 2008).

We evaluated whether changes in NF-κB activity were correlated with Foxp3 activation during the increased inflammatory responses, and found that NF-κB physically interacted with Foxp3 upon LPS activation. NF-κB nuclear translocation and cytoplasmic IκB-α phosphorylation were higher in Foxp3sf microglia than in wild-type microglia. Moreover, while NF-κB transcriptional activity increased in Foxp3-silenced BV-2 cells, it decreased in the Foxp3 overexpressing cells.

Recently, many researches have demonstrated that the functions of Foxp3 proteins are not restricted to regulatory T cells. It has been reported that Foxp3 interacts with nuclear factor of activated T cells (NFAT) and NF-κB to repress cytokine gene expression and effector functions of T helper cells (Bettelli et al., 2005). Furthermore, Foxp3 is known to be expressed in epithelial cells such as breast, lung, and prostate (Chen et al., 2008). Zuo et al. reported that Foxp3 is a transcriptional repressor of the breast cancer oncogene SKP2 (Zuo et al., 2007a), and Baratelli et al. showed that prostaglandin E2 induces Foxp3 gene expression and Treg cell function in human CD4+ T cells (Baratelli et al., 2005).

In the present work, we have analyzed the expression and role of Foxp3 in microglia for the first time. Since we found that Foxp3 mutant microglia produced elevated levels of inflammatory factors such as NO, ROS, MCP-1, and CXCL10, we assume that Foxp3 plays a role in repressing inflammatory responses in microglia so as to prevent excessive reactions.

Forkhead transcription factors play critical roles in the maintenance of immune homeostasis. Foxo3 and Foxj1 control the expression of IκBβ and IκBε, and therefore indirectly repress NF-κB activation by preventing its translocation to the nucleus (Lin et al., 2004a, b). Foxp3 is also known to repress NF-κB activation. A previous study showed that overexpression of Foxp3 decreased the expression of IκB-α in activated EL-4 T cells (Kwon et al., 2008). Because phosphorylation of IκB-α, leading to ubiquitination and degradation of IκB-α, is mediated by IKK, our finding of increased IκB-α phosphorylation in Foxp3sf microglia is consistent with the observations in T cells.

Scurfy mice are known to succumb to a CD4+ T cell-mediated, lympho-proliferative disease characterized by wasting and multiorgan lymphocytic infiltration. However, it is not known how Foxp3 functions in the CNS. Our in vitro results suggest that scurfy mice may suffer inflammatory damage to their brains due to over-activated microglia.

In conclusion, this study shows that Foxp3 is upregulated in LPS-activated microglia and that Foxp3 mutant (Foxp3sf) microglia produce excessive amounts of NO CXCL10, MCP-1, and ROS upon LPS activation. We also observed that translocation of NF-κB from the cytoplasm to nucleus was accelerated in Foxp3sf microglia due to increased IκB-α phosphorylation. These results suggest that Foxp3 increases the stability of IκB-α in response to LPS activation and so traps NF-κB in the cytosol. Foxp3 also interacts with NF-κB and interferes with NF-κB transcriptional activity. Together, these results strongly suggest that Foxp3 acts to limit the production of CXCL10 and MCP-1 and the release of ROS and NO in microglial cells by inhibiting the NF-κB pathway.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES