In a previous study was showed that when microglia are activated, the glucocorticoid receptor is downregulated. In this study it was examined if DNA methylation plays an important role in this downregulation of the GR. Here, we used qPCR to measure the state of downregulation of the GR and the state of microglia activation after LPS stimulation in vitro. As expected, microglia were activated after 6 and 24 h of LPS stimulation and the GR was downregulated after 6 and 24 h of LPS stimulation. DNA methylation occurs in or near the beginning of the transcription start site. Two regions around the transcription start site of the Nr3C1 (GR gene) were used to measure the methylation pattern of the GR promoter region after LPS stimulation. gDNA was isolated, and a bisulfide treatment was performed to make the DNA ready for sequence comparison. After ligation and transformation of the two regions, DNA was sequenced to compare methylation pattern of the Gr promoter region of LPS stimulated and unstimulated cells. Probably, DNA methylation does not play a role in the downregulation of the GR. How the GR is downregulated if DNA methylation is not involved must be examined in further studies. Other Epigenetic Mechanisms, such as miRNA and histon modification may play an important role in the downregulation of the GR.
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Microglia are the resident macrophages of the central nervous system (Perry and Gordon, 1991). In chronic neurodegenerative diseases, such as Alzheimer, Huntington's and Parkinson disease, microglia activation is an early sign that often precedes neuronal death. Increasing evidence indicates that in these chronic pathologies activated microglia sustain a local inflammatory response. (Streit, 2002). Microglia are a major component of the innate immune response in the brain, as they regulate the beginning of the inflammatory process after injury or inflammation. Microglia also clean up cell debris and pathogens (Bohn et al., 1991). Another major function is the ability to generate an adaptive immune response (Hwang et al., 2006). As a consequence of brain injury or disease, resident microglia change phenotype from their downregulated state to an activated phenotype, which is characterized by hypertrophy of the cell body. The ability to identify activated microglia has provoked interest in their role as indicators of pathology. Therefore it is interesting to look at the possible role of activated microglia in disease pathogenesis (Perry et al., 2010).
When microglia cells are at rest they express steroid hormone receptors, thereby inhibiting any source of immune activity. Glucocorticoids are known to be one of those steroid hormones. These hormones binds to the glucocorticoid receptor (GR). Steroid hormone receptors such as GR are expressed by all the cell types involved in the inflammatory process, including macrophages (Cutolo et al., 1996), dendritic cells (Moa et al., 2005), T lymphocytes (Cohan et al., 1983) and endothelian cells (Dietrich, 2004). Glucocorticoids act as anti-inflammatory hormones and immunoregulatory agents. They have numerous effects on cells of the macrophage/microglia lineage. An example of this effect is the downregulation of the MHC complex (Snyder and Unanue, 1982). Recent studies showed that after lipopolysaccharides (LPS) stimulation the GR is downregulated as a mechanism to relieve microglia from the control of steroid hormones (Siarra et al, 2008). The contrary is described for macrophages (Salkowski and Vogel, 1992). The downregulation of the GR might be controlled by an epigenetic mechanism called DNA methylation. Methylation takes place in promoter regions in so called CpG islands.
CpG islands are regions in the genome that contain a high frequency of CpG sites. The definition of a CpG island is a region with at least 200 base pairs and a CG percentage of 50% or more, and a CpG region greater than 60%. The p refers to the phosphodiester between the C and the G (Bird, 2002). DNA methylation occurs mainly at the cytosine residue of a CpG dinucleotide where a methyl group is bound to the 5-carbon position of the cytosine pyrimidine ring (Zhang et al., 2005). This process is catalyzed by DNA methyltranferase. In mammalian genomes, CpG islands are 300-3000 base pairs in length. They are found in and near 40% of promoters of genes, especially in housekeeping genes (Razin, 1998). These CpG islands normally occur at or near the transcription start site of genes. DNA methylation of a CpG island in a promoter gene may inhibit the expression of a gene, and prevents transcription factors to access DNA (Down et al., 2002)
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In order to better understand the mechanism by which GR expression in microglia is regulated, we have assessed whether DNA methylation in the promoter region has an effect on the downregulation of the GR. First we examined if the expression in the GR was downregulated when cell lines BV2 and N9 were stimulated with LPS. We also examined if the promoter region of the GR was methylated when it was stimulated with LPS. Comparing methylation patterns from promoter region of the GR receptor in microglia that are stimulated and non-stimulated we can examine if the methylation pattern of the promoter region also changes after stimulation
Material & Method
The murine microglia cell lines, BV2 and N9 were obtained from the Department 'Medical Physiology' from the UMCG. Cells were stored under liquid nitrogen storage. Cells were maintained in supplemented 15 ml DMEM/10%/FCS (Salkowski et al., 1992), until there were 6x10^6 cells/ml, measured with haemocytometer. Cells were divided into 4 groups in each of the two experiments (Methylation and Expression): (1) BV2 + LPS, (2) BV2 control, (3) N9 + LPS and (4) N9 control.
LPS and IL-4 stimulation
Cells where seeded in a 24well plate at a density of 2.5x10^5/ cells a well in 500 ul DMEM/10%FCS. After 2-3 h. in which they could attach, the cells were treated with LPS (200ng/ml) or IL-4 (10ng/ml). Cells were stimulated both for LPS and IL-4 for 6 and 24 h after the stimulation cells were ready for RNA isolation.
RNA isolation and QPCR
RNA from stimulated cells was isolated adding 500 Î¼l RNA phenol and 100 Î¼l chloroform isoamylalcohol (CI:IAA) (49:1). After that the samples were centrifuged for 20 min at 13200 rpm. Then the upper phase was transferred and into this upper phase 1/5 of total volume of 2M NaAc PH 4.7 and 1 volume of total volume isopropanol was added. After that 1.5Î¼l of glycogen was added and samples were stored for at least one h at -25Â°C. Centrifuge the sample for 35 minutes at 13200 rpm at 4Â°C. After this the supernatant was discarded and the pellet was resuspended in 50Î¼l DNAse buffer and 1Î¼l of DNAse1 was added. Then the sample was incubated for 25 min. at 37Â°C, and 350 Î¼l of pure water was added. 400 Î¼l of RNA phenol and 80 Î¼l of CI:IAA was added and the samples were centrifuged for 20 min. 13200 rpm at 4Â°C. Then again the upper phase was transferred and into this upper phase 1/5 of total volume of 2M NaAc PH 4.7 and 1 volume of total volume isopropanol was added. After that again 1.5Î¼l of glycogen was added and samples were stored for at least one h. at -25Â°C. Centrifuge the sample for 35 minutes at 13200 rpm at 4Â°C. After that the supernatant was discarded and the cell pellet was washed with 500 Î¼l of 70% EtOH. The sample was centrifuged for 5 min at 13200 rpm at 4Â°C. Again, the supernatant was discarded and the cell pellet was washed with 500 Î¼l of 70% EtOH, and centrifuged 5 min. 13200 rpm at 4 Â°C. The pellet air dried for 10 min. and then the pellet was dissolved in 11 Î¼l pure water. With the isolated RNA, a QPCR was performed. Expression was measured of 4 genes: 2 housekeeping genes as control, Nr3C1 (GR gene), and IL1ß. IL1ß is produced by microglia when they are in the activated state. QPCR measures the threshold cycle value (Ct). The Ct represents the cycle at which the fluorescence reaches the threshold intensity, thus, the amount of the target RNA. Each sample was run in triplicate and the average Ct was used to calculate the relative amount of the product. Expression rates were calculated with the formula: 2^(-Î”Î”Ct).
LPS Stimulation and gDNA isolation
Cells where stimulated with 2 ml LPS from E.coli (200ng/ml), obtained from Sigma-Alderich (Germany), and incubated for 24 h. at 37Â°C.
The isolation of the gDNA was performed as in the in protocol 'Genomic DNA purification KIT' from Fermentas. After measuring with a spectrophotometer (nanodrop) whether the gDNA concentration was sufficient (concentration > 100ng/Î¼l), the bisulfide treatment (EZ DNA Methylationâ„¢Kit) was performed. The bisulfide treatment converts unmethylated cytosines into uracil and methylated cytosines remained unchanged during the treatment.
Designing primers and visualize CpG islands
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The gene sequence of the GR promotor gene, Nr3C1, was obtained from the genbank database. First CpG islands in the GR promotor region were visualized and divided into 4 regions. The primers used were 17-28 bases in length with a GC level between 20-60%, and had a melting temperature (TM) around 64Â°C. Specific backward and forward primers were made for every region, and nested primers were used for more specificity (see table 1). In this study only 2 regions were used, region 2 and 3. (Primers were obtained from Biolegio BV, Nijmegen)
PCR and Electrophoresis
A PCR with the designed BSP primers (2.5Î¼l PCR buffer,1.5 Î¼l 25 mM mgcl2, 0.5 Î¼l primermix region 2 and 3 , 0.1 Î¼l taq polymerase, 0.5 Î¼l 10 mM dNTP, 10 Î¼l sterile demi, 10 Î¼l template gDNA) of region 2 and 3 (35 cycles of 95Â°C; 3 min; 94Â°C; 1 min; 56Â°C; 2 min; 72Â°C; 1 min, 72Â°C; 10 min) was carried out. To confirm whether the PCR had been succeeded the samples were loaded into a 1% agarose gel to perform electrophoresis (90V, 40 min, Tris-acetate-EDTA (TAE) buffer, 12 Î¼l ethylium bromide, 6X loading buffer, 1000bp marker). Another PCR (2.5 Î¼l PCR buffer, 1.5 Î¼l 25 mM MgCl2, 0.5 Î¼l primermix region 2 and 3, 0.1 Î¼l taq polymerase, 0.5 Î¼l 10 mM dNTP, 19 Î¼l sterile demi, 1 Î¼l template gDNA from first PCR) ((35 cycles of 95Â°C; 3 min, 94Â°C; 1 min, 56Â°C; 1.5 min, 72Â°C; 1 min, 72Â°C; 10 min) with the designed BSP nested primers (region 2 and 3) was carried out in order to obtain the right PCR product. Here, a 1.5% agarose gel was used for electrophoresis. In order to verify our experiment, a negative and positive control was used. The negative control was sterile demi and the positive control Oct4. The gDNA of the Oct4, a fibroblast gene, is always methylated (Yamanaka and Takahashi, 2006).
Ligation and transformation
Directly after the nested PCR the bands of the electrophoresis (gDNA) was extracted and purified from the agarose gel (MinElute Gel Extraction Kit Protocol; QIAGEN). The PCR product was cloned in a PCRII plasmid (invitrogen) from 3900 base pairs (3 Î¼l insert gDNA, 2 Î¼l vector, 1.5 Î¼l ligase buffer, 0.4 Î¼l T4 DNA ligase, 8.1 Î¼l sterile demi). The samples were incubated for 1 night at 14 Â°C. The transformation was performed as in protocol transformation (Invitrogen). Competent top 10 E.coli were used for the transformation and were chemically transferred in the presence of X-galactosidase (X-gal) and isopropyl-B-D- thiogalactopyranoside (IPTG). When the transformation was complete, the white colony's were picked out and brought into 3 ml LB medium, Then the sample was incubated in the shaker (37Â°C, 200 rpm). From these colonies the plasmids were isolated with the genejet plasmid miniprep KIT (Fermentas) and concentration of plasmids were measured with spectrophotometer (nanodrop). Then the samples were send away to ServiceXS for sequencing. The sequences were aligned with megaline to detect if CpG residues were methylated or unmethylated. To test if our PCR product was cloned into the plasmids a digestion control was performed using 5 Î¼l of miniprep sample, 1 Î¼l EcoR1 buffer, 0.5 Î¼l EcoR1 and 3.5 Î¼l sterile demi.
LPS activates microglia
Status of activity of microglia was measured by expression of the IL1ß gene. IL1ß upregulation marks activated microglia. The BV2 and N9 cells were stimulated with LPS and IL-4 for 6 and 24 h After BV2 and N9 cells were stimulated with LPS and IL-4, RNA was isolated and analyzed for expression of the IL1ß gene with QPCR. After stimulation with IL-4, no upregulation of the microglia was measured. In BV2 cells, upregulation was detected in the expression of the IL1ß gene after LPS stimulation in comparison to the control housekeeping genes HPRT1 and HMBS. By 6 h, IL1ß expression was upregulated to 7212% and 836% after 24 h, compared to control gene HPRT1. Nearly the same results were found in comparison to control to HMBS; 7306% after 6 h, 1036% after 24 h (Fig. 1A). IL1ß was even more upregulated in N9 cells, 45306% after 6 h, 10751% after 24 h in comparison to HMBS. In comparison to HPRT1; 45884% after 6h, 19985% after 24h (Fig. 1B).
LPS downregulates the expression of the GR in microglia
The gene that encodes the GR is NR3C1. The BV2 and N9 cells were stimulated with LPS and IL-4 for 6 and 24 h, IL-4 stimulation did not activate the microglia, therefore the data of IL-4 stimulation on the NR3C1 gene is not written down. In BV2 cells we detected a downregulation in the expression of the NR3C1 gene after LPS stimulation in comparison for both the control housekeeping genes; 80% after 6 h, 66% after 24 h in comparison with HMBS. In comparison to HPRT1; 79% after 6 h, 91% after 24 h (Fig. 2A) NR3C1 was even further downregulated in N9 cells; 25% after 6 h and 15% after 24 h (HMBS), 26% after 6 h and 28 % after 24 h (HPRT1) (Fig. 2B).
BV2 region 3 gDNA and Oct3/4 capable for ligation into plasmid vector
A PCR with primers specific for the region of interest, was performed on the gDNA isolated from the cells. No PCR product was formed for the N9 cells stimulated with LPS. Because no specific PCR product was formed with the gDNA from the N9 cells, the experiment was continued with only the BV2 cells. With the gDNA from the BV2 cells a PCR was performed with a nested primer to obtain a more specific product (Fig. 3). As can be seen in figure 3, no specific product was formed for region 2. For this reason the experiment was continued with region 3 from BV2 cells only. Figure 3 also the positive control used in the experiment, Oct3/4. Oct3/4 is always methylated
BV2 reg3 gDNA present in plasmid
To control if the ligation product was ligated into the plasmid vector, a digestion control was performed (Fig. 4). As shown in figure 6 all the BV2 control gDNA was ligated into the plasmids. For BV2 LPS, only 1 plasmid took up the gDNA (lane 11).
Methylation not involved in the downregulation of the GR after LPS stimulation
After the CT conversion the unmethylated cytosines were converted into thymine. The samples with the promoter region 3 Nr3C1 gene were analyzed by serviceXS and obtained in an electropherogram. Megaline was used to perform an alignment with the results of the electropherogram. With this alignment methylated and unmethylated cytosine's were compared (Fig. 5) One cytosine in the control was methylated in the promotor region3 of the Nr3C1 gene.
Based on the results from our study, our conclusion is that DNA methylation is not involved in the downregulation of the GR. Not one CpG residue was methylated in the LPS stimulated cells. In this pilot study, only a single test was performed to compare the methylation pattern from the promoter region of the GR in microglia, with or without stimulation of cells with LPS. IL1ß gene expression was upregulated after of LPS and IL-4 stimulation. This data confirms the data suggested by Sierra et al. (2008) that after LPS stimulation microglia are activated. After IL-4 stimulation the GR was downregulated, but the microglia were not activated. This confirms the data by Shet er al. (1994) that IL-4 downregulates the GR Because IL-4 downregulates the GR, we thought that this effect would cause activation of microglia, but this was not the case. The fact that the microglia were downregulated, but not activated needs further investigation. The GR play an essential role in protecting the brain against inflammatory challenge. When the GR is downregulated microglia can reach their activated state. Sierra et al (2008) suggested that after stimulation cells with LPS the GR receptor was downregulated. In this study, the same results were found. In the expression study only RNA was measured. To precisely measure the upregulation of IL1ß a western blot should have been performed to measure the real protein transcription. Why the PCR of N9 with LPS did not gave any product, is not known. MgCl2 concentration was changed, and also annealing time and temperature were changed but still the PCR did not give any result. More effective results of downregulation of the GR were founded in N9 cells stimulated with LPS then in BV2 cells stimulated with LPS. Unfortunately only BV2 cells were appropriate for PCR. Exactly the same problem was found in the BV2 region 2. Here, also MgCl2 concentration, annealing time and temperature were changed but the PCR did not gave any product. For next studies, only N9 cells or primary microglia must be used for this experiment. This study was only performed in vitro, a study in vivo will be more reliable for what happens in organisms. Furthermore, not the same cells were used for the experiments in the expression and the methylation study. Cells were stimulated at different times and only for 1 experiment. To be sure that the GR is downregulated in both the experiments the cells must be stimulated and after that the cells must be divided into two groups. This experiment was only a pilot study to control if the GR is really downregulated and microglia are activated after LPS stimulation. Only a single test was performed for the expression study, so significance could not be measured. In summary, microglia are activated after stimulation with LPS. After LPS stimulation, microglia are activated by the mechanism of GR downregulation. DNA methylation is not involved in the downregulation of the GR. To be sure that DNA methylation is not involved, this study must be repeated in primary microglia, or for N9 cells region 2 and 3. How the GR is downregulated if DNA methylation is not involved must be examined in further studies. Other epigenetic mechanisms, such as miRNA and histon modification may play an important role in the downregulation of the GR and activation if microglia. If the mechanism of microglia activation and GR downregulation is clarified, more research can be done about these mechanisms.