Polycomb group protein Bmi1 negatively regulates IL ...

Immunology and Cell Biology (2011), 1–5 & 2011 Australasian Society for Immunology Inc. All rights reserved 0818-9641/11 www.nature.com/icb

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Polycomb group protein Bmi1 negatively regulates IL-10 expression in activated macrophages Arnold R Sienerth, Christian Scheuermann, Antoine Galmiche, Ulf R Rapp and Matthias Becker Macrophages exert a wide variety of functions, which necessitate a high level of plasticity on the chromatin level. In the work presented here, we analyzed the role of the polycomb group protein Bmi1 during the acute response of bone marrow derived macrophages (BMDM) to lipopolysaccharide (LPS). Unexpectedly, we observed that Bmi1 was rapidly induced at the protein level and transiently phosphorylated upon LPS treatment. The induction of Bmi1 was dependent on MAP-kinase signaling. LPS treatment of BMDM in the absence of Bmi1 resulted in a pronounced increase in expression of the anti-inflammatory cytokine interleukin-10 (IL-10). Our results identify Bmi1 as a repressor of IL-10 expression during macrophage activation. Immunology and Cell Biology advance online publication, 11 January 2011; doi:10.1038/icb.2010.160 Keywords: macrophage; LPS; polycomb; Bmi1; TLR

Macrophages participate in various physiologically important processes, such as pathogen recognition and elimination as well as wound healing.1 Associated with these functions is the ability to mount either pro- or anti-inflammatory responses characterized by the execution of differential gene expression programs.2 Membrane bound receptors such as Toll-like receptors (TLRs) are crucially involved in the activation of specific gene expression programs.3 Specifically mitogen-activated protein kinase (MAPK) signaling has been found to be essential for the execution of pro- and anti-inflammatory gene expression programs.4 In this connection recently published data implicates extracellular signal-regulated kinase (ERK) activity in mediating specific chromatin modulations such as histone H3 S10 phosphorylation at the interleukin-10 (il-10) locus in response to TLR4 signaling.5 Polycomb group (PcG) proteins constitute a large group of evolutionary conserved factors, which mediate the establishment and maintenance of differentiation programs.6 Recent genome wide chromatin association studies implicate PcG proteins in the regulation of a multitude of genes.7–9 PcG proteins are also required for the establishment and maintenance of differentiation specific expression programs of various hematopoietic lineages, among them the B-lymphoid and the myeloid lineages.10 Recent studies suggest that IL-4 and IL-13, two markers of T helper type 1 cell differentiation, are regulated by PcG proteins.11 At least two distinct polycomb repressive complexes (PRC1 and PRC2) exist that are characterized by specific histone modification activities. The PRC2 histone methyl transferase Ezh2 tri-methylates histone H3 at lysine 27 (H3 K27me3). This methylation mark is recognized by PRC1 through affinity binding of chromodomain

containing proteins.12 The PRC1 protein Bmi1 has been shown to be essential for PRC1-specific histone H2A ubiquitin E3 ligase activity.13 Bmi1 is involved in the maintenance of hematopoietic stem cell self-renewal through repression of the ink4a/arf tumor suppressor locus.14 Moreover, Bmi1 has recently been described to affect cell survival through regulation of mitochondrial genes.15 Previously, Bmi1 has been shown to be phosphorylated in a cell cycle dependent manner by a so far undescribed kinase. Phosphorylation of the protein peaked at the S/G2 phase.16 A second report showed that 3pK, a downstream effector kinase of three MAPK pathways: RAS-RAF-MEK-ERK, p38 and JNK,17 is targeting Bmi1 as a consequence of mitogenic and stress kinase signaling. In this instance phosphorylation of Bmi1 resulted in derepression of the ink4a/arf locus.18 Here, we investigated the role of Bmi1 as a potential downstream target of the TLR4-induced MAPK signaling in bone marrow derived macrophages (BMDM). We found an unexpectedly rapid upregulation of Bmi1 on the protein level in response to treatment with the TLR4 ligand lipopolysaccharide (LPS) in terminally differentiated BMDM. Gene abrogation studies identified Bmi1 as a suppressor of IL-10 expression after LPS treatment. RESULTS AND DISCUSSION Bmi1 is a downstream target of LPS-induced MAPK signaling in macrophages Western blot analysis of Bmi1 protein levels in the absence or presence of LPS (Figure 1a) showed that while Bmi1 protein levels were close to the detection limit in untreated cells, a strong increase was observed

Institut fu¨r Medizinische Strahlenkunde und Zellforschung, Wu¨rzburg, Germany Correspondence: Dr M Becker, Institut fu¨r Medizinische Strahlenkunde und Zellforschung (MSZ), Zentrum fu¨r Experimentelle Molekulare Medizin (ZEMM), Zinklesweg 10, D-97078 Wu¨rzburg, Germany. E-mail: [email protected] Received 4 January 2010; revised 22 November 2010; accepted 23 November 2010

Bmi1 function in macrophages AR Sienerth et al 2

Figure 1 Rapid upregulation of Bmi1 and transient phosphorylation in macrophages after LPS treatment. (a, b) BMDM from WT mice were treated with 100 ng ml 1 LPS for the indicated time periods. Whole cell lysates (WCLs) of BMDM were immunoblotted with antibodies against Bmi1 and GAPDH as loading control. (b) Total RNA was isolated from BMDM and subjected to quantitative real-time PCR (RT-qPCR) for the analysis of Bmi1 mRNA expression. Bmi1 mRNA levels were quantified relative to HPRT mRNA levels. (c) BMDM were pretreated for 30 min with either 3 mg ml 1 lethal factor (LF), 3 mg ml 1 protective antigen (PA) alone or 3 mg ml 1 LF and 3 mg ml 1 PA in combination followed by 100 ng ml 1 LPS treatment at indicated time points. WCLs were immunoblotted with indicated antibodies. (d) J774 cells were treated with 100 ng ml 1 LPS for indicated time periods and subsequently subjected to biochemical fractionation. Equal protein ratios of the fractions were blotted and probed with specific antibodies for Bmi1. The quality of fractionation was assessed by probing with C-RAF and H4 specific antibodies.

10 min after LPS treatment. The protein migrated as three distinct bands. Previous work has shown that the individual bands correspond to differentially phosphorylated versions of Bmi1 with the slowest migrating band representing the hyperphosphorylated form of the protein.16 Concomitant with the increase in protein levels, the fastest migrating band disappeared and reappeared 3 h after LPS treatment indicating a change in the phosphorylation status of the protein. Bmi1 levels varied to some extend, but stayed above the levels observed in the uninduced state over a period of 24 h. The rapid induction of Bmi1 on the protein level was not paralleled by a rapid upregulation on the RNA level as mildly (twofold) increased Bmi1 RNA levels could only be detected at 12–24 h after LPS treatment (Figure 1b). Thus, Bmi1 is not regulated on the transcriptional level in the rapid response to LPS treatment. The rapid nature of Bmi1 upregulation after LPS treatment, however, suggests regulation at the translational/post-translational level. Immunology and Cell Biology

We next addressed which signaling pathway(s) downstream of LPS/ TLR4 was/were targeting Bmi1. Previous results placed Bmi1 downstream of the mitogenic and stress MAPK cascades.18 Anthrax lethal toxin consists of the two components protective antigen and lethal factor, and is a highly specific blocker of the RAS/RAF/MEK/ERK, p38 and JNK MAPK pathways.19 As the combination of anthrax lethal toxin and TLR4 activation has previously been shown to induce apoptosis in macrophages,20 we chose experimental conditions that allowed the abrogation of MAPK signaling, but did not affect viability of BMDM (Supplementary Figures S1b and c). Figure 1c shows that the rapid induction of the Bmi1 protein was abrogated in the presence of anthrax lethal toxin while the individual components of the toxin had no effect. Therefore, either one pathway alone or the pathways in combination are involved in the regulation of Bmi1. As the use of individual inhibitors for p38, JNK and MEK did not yield significant


effects on Bmi1 protein levels (Supplementary Figure S1a), we conclude that Bmi1 is a target of coordinate MAPK signaling pathways activation downstream of TLR4. Voncken et al.18 have previously shown that arsenite-induced phosphorylation of Bmi1 in the human osteosarcoma cell line U2OS leads to the dissociation of the protein from chromatin. We, therefore, analyzed chromatin association of Bmi1 after LPS treatment. To this end, we used the murine macrophage cell line J774A.1 (further on referred to as J774). Shrimp alkaline phosphatase treatment of J774 protein extracts showed that Bmi1 was phosphorylated in response to LPS treatment (Supplementary Figure S2a). Bmi1 phosphorylation followed the same kinetics as observed in BMDM, however, in contrast to WT BMDM, the Bmi1 protein level did not increase after LPS treatment in J774 cells (Supplementary Figure S2b). Differential extraction of non-chromatin bound and chromatin bound fractions showed no changes in Bmi1 chromatin association in response to LPS treatment. Irrespective of phosphorylation status, Bmi1 was exclusively found in the chromatin bound fractions (Figure 1d). Therefore, LPS-mediated phosphorylation does not interfere with chromatin binding of Bmi1 in J774 macrophages. This finding is in contrast to what has previously been reported for the chromatin association of phosphorylated Bmi1 in human cell lines in response to MAPK activation18 and the association of phosphorylated Bmi1, during the S/G2 phase of the cell cycle.16 Thus, our observations argue for a cell cycle regulation independent function of Bmi1 in response to LPS and suggest a cell type/stimulus-specific regulation of Bmi1 chromatin association. Bmi1 abrogation leads to increased LPS-induced IL-10 expression in macrophages We next addressed the consequences of Bmi1 abrogation on LPSactivated BMDM. To this end, we generated BMDM from WT mice and from mice with a constitutive Bmi1 knockout (Bmi1 / ).21 Bmi1 / BMDM are comparable to WT BMDM with respect to surface marker expression, bacterial uptake and clearance, as well as activation kinetics of TLR4 downstream signaling pathways (Supplementary Figures S3a–c). Analysis of cytokine secretion of Bmi1 / and WT BMDM after LPS treatment (Supplementary Figure S4) showed similar concentrations of IL-1b, interferon gamma, transforming growth factor-b and IL-12(p70) whereas IL-12(p40), IL-6 and tumor necrosis factor-a levels were slightly reduced in supernatants of Bmi1 / BMDM over a period of 12 h. In contrast, IL-10 was found in significantly higher concentrations in the supernatants of Bmi1 / BMDM. As LPS is not a potent stimulator of IL-10 gene transcription,22 we next asked whether the increased IL-10 secretion of Bmi1 / BMDM correlated with higher IL-10 mRNA expression. Figure 2a shows that IL-10 mRNA levels were three- to fivefold higher in Bmi1 / BMDM in response to LPS over a period of 20 h. Note that the basal level in Bmi1 / BMDM was only slightly elevated in the uninduced state. Though consistently higher, IL-10 mRNA expression in Bmi1 / BMDM followed the same kinetic as in WT BMDM with peak expression at 8 h. In summary, these data show that the loss of Bmi1 in BMDM leads to elevated LPS-induced IL-10 expression. Previous reports have shown an effect of aging/senescence on the cytokine expression profile of macrophages.23,24 As we observed senescent cells in the Bmi1 / BMDM cultures judged by morphology, p16ink4a expression and SA b-GAL staining (Supplementary Figures S5a–c) senescence could not be excluded as a possible cause for the increase in IL-10 expression. To avoid senescence mediated effects, we performed small interfering RNA (siRNA)mediated knockdown of Bmi1 expression in J774 cells, which led to a significant

decrease in Bmi1 protein levels whereas Ezh2 levels were unaffected (Figure 2b). Bmi1 knockdown did not result in a senescence phenotype as evidenced by an unchanged cell cycle profile (Supplementary Figure S6a), the lack of p16ink4a expression (Supplementary Figure S6b) and unchanged cellular morphology (Supplementary Figure S6c). This observation indicates that the residual Bmi1 protein levels were sufficient to maintain repression of negative cell cycle regulators after Bmi1 knockdown. LPS treatment of J774 cells after siRNA-mediated Bmi1 knockdown yielded a two- to threefold increase of IL-10 expression (Figure 2c). In conclusion, these data are consistent with a senescence/cell cycle regulation-independent acute role of Bmi1 in regulation of IL-10 expression during LPS-mediated activation. In line with previously published data,25 we found lower LPSdependent expression of IL-10 in J774 cells in comparison with WT BMDM (Figure 2d). As our data so far indicated that the presence of LPS-induced Bmi1 protein levels negatively influenced IL-10 expression, we asked how Bmi1 protein levels compare in J774 cells and BMDMs. The results presented in Figures 2e and f show that Bmi1 protein levels were considerably higher in J774 cells irrespective of LPS treatment (Figures 2e–f). Therefore, the low IL-10 expression in J774 cells correlated with higher Bmi1 protein levels. In conclusion, these data in combination with the above described Bmi1 abrogation studies, are consistent with an inverse correlation of Bmi1 protein levels with il-10 gene expression. So far, it remains elusive whether Bmi1 directly regulates IL-10. As PcG proteins have been shown to regulate a multitude of genes,7–9 an indirect effect of Bmi1 on the expression of IL-10 seems likely. Indeed not only IL-10, but also other cytokines were found to be differentially regulated in our initial cytokine profile analysis (Supplementary Figure S3) albeit the effect on IL-10 was most pronounced. To address whether PcG proteins directly target the IL-10 locus, we reanalyzed previously published ChIP-Seq data sets addressing the influence of combined interferon gamma and LPS treatment on genome wide H3 K27me3 in BMDM.26 The H3 K27me3 modification is a result of PRC2 action, and serves as a platform for PRC1 binding.12 As the H3 K27me3 modification has been found in close proximity to transcriptional start sites as well as in broad local enrichments spanning genomic regions of up to several 100 bp,27 we analyzed B160 kbp 5¢ and 80 kbp 3¢ of the IL-10 transcriptional start site for the distribution of the H3 K27me3 mark. This analysis revealed no significant enrichment of H3 K27me3 at the il-10 promoter and surrounding regions irrespective of treatment conditions (Supplementary Figure S7). Thus, the H3 K27me3 modification is most likely not directly involved in the regulation of il-10 gene transcription. Therefore, at this stage, our data is consistent with two models: (1) H3 K27me3-independent binding of Bmi1 at the il-10 locus similar to what has previously been described for the binding and function of other PRC1 members at various genomic locations;28,29 (2) indirect Bmi1-dependent regulation of IL-10 expression by so far unidentified mediators. In summary, the data presented here show a previously undescribed rapid regulation of the Bmi1 protein level in response to the bacterial compound LPS in macrophages and implicates this protein in the regulation of IL-10 expression, a key component of the anti-inflammatory immune response.

METHODS Animals Bmi1+/ mice were obtained from Maarten van Lohuizen (The Netherlands Cancer Institute, Amsterdam, The Netherlands) and maintained at in house facilities. Immunology and Cell Biology


Figure 2 Bmi1 levels are inversely correlated with LPS-induced IL-10 expression. (a) RT-qPCR analysis of IL-10 mRNA levels in WT and Bmi1 / BMDM. Cells were treated with 100 ng ml 1 LPS at indicated time points. Total RNA was isolated and RT-qPCR was carried out with primers specific for IL-10 and AcRP0. IL-10 mRNA levels are presented relative to AcRP0 mRNA levels. (b–c) J774 macrophages were transfected with scrambled small interfering RNA (siRNA) and siRNA directed against Bmi1 respectively and treated with LPS for the indicated time periods. (b) The obtained WCLs were immunoblotted with antibodies against Bmi1, Ezh2 and GAPDH as loading control. (c) Total RNA was isolated and RT-qPCR analysis was carried out with primers specific for IL-10 and AcRP0. IL-10 expression levels are presented relative to AcRP0 mRNA levels (*Po0.05; **Po0.01 paired Student’s t-test). (d) IL-10 mRNA levels in WT BMDM and in J774 macrophages were analysed by RT-qPCR. The cells were treated with 100 ng ml 1 LPS at indicated time points. Total RNA was isolated and RT-qPCR analysis was carried out with primers specific for IL-10 and AcRP0. IL-10 mRNA levels are presented relative to AcRP0 mRNA levels. (e, f) WCLs were prepared from equal cell numbers of WT BMDM and J774 cells followed by western blot analysis using antibodies specific for Bmi1 and TLR4. (e) Analysis of untreated cells. (f) Analysis of cells that were treated with 100 ng ml 1 LPS for 30 min.

Cell culture Cells were kept at 37 1C under 7% CO2 atmosphere. The mouse macrophage cell line J774A.1 was cultured in Dulbecco’s modified Eagle’s medium medium supplemented with 10% fetal bovine serum, 100 U ml 1 penicillin, 100 mg ml 1 streptomycin and 2 mM glutamine. Primary BMDM were isolated from mouse femurs of wild type and Bmi1 / mice genotyped by PCR. Bone marrow derived cells were prepared as previously described and propagated in RPMI 1640 supplemented with 10% fetal bovine serum, 100 U ml 1 penicillin, 100 mg ml 1 streptomycin and 2 mM glutamine in the presence of 10 ng ml 1 recombinant M-CSF (Calbiochem, La Jolla, CA, USA) and 1 ng ml 1 recombinant IL-3 (Calbiochem) for 8 days before experiments were performed. J774A.1 cells and BMDM were treated with LPS (100 ng ml 1) purified from Escherichia coli 0111:B4 (Sigma, St Loius, MO, USA). Immunology and Cell Biology

Recombinant protective antigen and lethal factor were a gift from Prof. Emmanuel Lemichez (University of Nice). BMDM were pre-treated for 30 min with endotoxin-free lethal factor and protective antigen at a final concentration of 3 mg ml 1.

Western blot analysis Whole-cell lysates were prepared in lysis buffer (50 mM Tris-HCL, pH 6.8, 5% 2-mercaptoethanol, 0.005% (w/v) Bromphenol Blue, 4% SDS and 20% glycerol). Whole-cell lysates extracts were sheared by syringing through a 22G needle and subsequently heated at 95 1C for 5 min. Whole-cell lysates were separated on 14% acrylamide gels and transferred to PROTRAN nitrocellulose transfer membrane (Whatman, Springfield Mill, Kent, UK). The membrane was blocked for 1 h in blocking buffer (Phosphate-buffered saline, 0.05% Tween 20, 5%

Bmi1 function in macrophages AR Sienerth et al 5 non-fat dry milk). Primary antibodies were diluted between 1:500 and 1:10 000 in blocking buffer. Corresponding secondary antibodies (PERBIO SCIENCE, Helsingborg, Sweden) were used at a dilution of 1:1000 and 1:5000. Western blots were developed using Supersignal West Pico or Femto ECL KIT (PERBIO SCIENCE). The following antibodies were used: mouse monoclonal anti-Bmi1 (clone F6, Millipore, Billerica, MA, USA), mouse monoclonal anti-GAPDH (clone 6C5, Millipore), rabbit polyclonal anti-TLR4 (clone H-80, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-C-RAF (clone C20, Santa Cruz Biotechnology), rabbit monoclonal anti-H4, pan (clone 62-14113, Millipore), rabbit polyclonal anti-MKK3 (clone H-70, Santa Cruz Biotechnology), rabbit polyclonal anti-phospho-JNK (Thr183/Tyr185, Cell Signaling, Danvers, MA, USA), mouse monoclonal anti-phospho-ERK (clone 12D4, Santa Cruz Biotechnology), rabbit monoclonal anti-phospho-p38 (anti-ACTIVE, Promega, Madison, WI, USA), rabbit polyclonal anti-JNK (Cell Signaling), polyclonal rabbit anti-Ezh2 (Cell Signaling) and monoclonal mouse anti-Ezh2 (clone AC22, Cell Signaling).

Quantitative real-time PCR Total RNA was isolated using peqGOLD TriFast (PEQLAB, Erlangen, Germany) according to the manufacturer instructions. Isolated RNA was subjected to DNAseI treatment before preparation of complementary DNAs using random hexamer primers provided by the First Strand cDNA Synthesis Kit (Fermentas, St Leon-Rot, Germany). Real-time PCR was performed monitoring SYBR green incorporation using the DyNAmo HS SYBR Green qPCR Kit (Finnzymes, Espoo, Finland) in a Rotogene 2000 thermocycler (Qiagen, Hilden, Germany). RTprimer sequences: AcRP0for 5¢-TTATCAGCTGCACATCACTCAG-3¢; AcRP0rev 5¢-CGAGAA GACCTCCTTCTTCCA-3¢;30 IL-10for 5¢-CAGGGATCTTAGCTAACGGAAA-3¢; IL-10rev 5¢-GCTCAGTGAATAAATAGAATGGGAAC-3¢; Bmi1for 5¢-CTGAT GCTGCCAATGGCTCC-3¢; Bmi1rev 5¢-AGTCATTGCTGCTGGGCATC-3¢; HPRTfor 5¢-CACAGGACTAGAACACCT-3¢; HPRTrev 5¢-GCTGGTGAAAAG GACCTCT-3¢; p16for 5¢-CGTGAGGGCACTGCTGGAAG-3¢; p16rev 5¢-ACCA GCGTGTCCAGGAAGCC-3¢.

siRNA interference J774A.1 cells were transfected with siRNA directed against Bmi1 (Santa Cruz) and non-silencing/scrambled siRNAs (Santa Cruz) as controls. siRNAs were transfected twice at intervals of 24 h using the HiPerFect Transfection Reagent (Qiagen) and analysed 12 h later.

Chromatin fractionation assay 4107 J774A.1 cells were collected for preparation of subcellular fractions as previously described.31

Statistical analysis The data were analysed by the paired or unpaired t-test using STATGRAPHICS Centurion XV (STATPOINT TECHNOLOGIES, Warrenton, VA, USA). A value of Po0.05 was accepted as statistically significant.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS The authors would like to express their gratitude to Antje Becker, Tamara Potapenko and Katharina Galmbacher for excellent technical assistance, Nadine Obier and Tom Misteli for critical reading of the manuscript, Gioacchino Natoli and Albrecht M Mu¨ller for helpful discussions, Emmanuel Lemichez for reagents and Simon Andrews for bioinformatic analysis of ChIP-Seq data.

ARS and CS were supported by the GRK1141/1. MB was supported by DFG TR17. Authors contribution: ARS, CS, MB and AG performed experiments. ARS and MB analyzed results and made the figures. MB, URR and ARS designed the research. MB wrote the manuscript.

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Supplementary Information accompanies the paper on Immunology and Cell Biology website (http://www.nature.com/icb) Immunology and Cell Biology