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A CREB-C/EBP Cascade Induces M2 Macrophage- Specific Gene

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A CREB-C/EBP Cascade Induces M2 Macrophage- Specific Gene A CREB-C/EBP␤ cascade induces M2 macrophage- specific gene expression and promotes muscle injury repair Daniela Ruffell1, Foteini Mourkioti1, Adriana Gambardella1, Peggy Kirstetter, Rodolphe G. Lopez, Nadia Rosenthal, and Claus Nerlov2 Mouse Biology Unit, European Molecular Biology Laboratory, Via Ramarini 32, 00015 Monterotondo, Italy Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved August 24, 2009 (received for review July 31, 2009) Macrophages play an essential role in the resolution of tissue damage vation of PPAR␥ (7), indicating that in this context PPAR␥ through removal of necrotic cells, thus paving the way for tissue activation suppresses M2 polarization. However, the role of PPAR␥ regeneration. Macrophages also directly support the formation of remains controversial, as PPAR␥ activation has been observed to new tissue to replace the injury, through their acquisition of an promote M2 polarization in adipose tissue (8). anti-inflammatory, or M2, phenotype, characterized by a gene ex- Candidate regulators can be identified by analysis of promoters pression program that includes IL-10, the IL-13 receptor, and arginase of M2-specific genes in macrophages, and among these, the Il10 and 1. We report that deletion of two CREB-binding sites from the Cebpb Arg-1 promoters are regulated by C/EBP␤ (9, 10). C/EBP␤ is a promoter abrogates Cebpb induction upon macrophage activation. member of the C/EBP family of basic region-leucine zipper (bZIP) This blocks the downstream induction of M2-specific Msr1, Il10, II13ra, proteins and is known to be important for the antibacterial activity and Arg-1 genes, whereas the inflammatory (M1) genes Il1, Il6, Tnfa, of macrophages (11). However, macrophage expression of genes and Il12 are not affected. Mice carrying the mutated Cebpb promoter encoding inflammatory molecules, such as Il1b, Inos, Il6, and Tnfa (␤⌬Cre) remove necrotic tissue from injured muscle, but exhibit is also diminished in the absence of C/EBP␤ (12), confounding the severe defects in muscle fiber regeneration. Conditional deletion of role of C/EBP␤ in specifying the M2 gene program. There is the Cebpb gene in muscle cells does not affect regeneration, showing considerable evidence that C/EBP␤ is regulated at the proteomic ␤ that the C/EBP cascade leading to muscle repair is muscle-extrinsic. level through competition between interacting transcriptional reg- ␤⌬ While Cre macrophages efficiently infiltrate injured muscle they ulators (see reference 13 for review). In macrophages, C/EBP␤ fail to upregulate Cebpb, leading to decreased Arg-1 expression. functionally interacts with NF-␬B on inflammatory and synergizes CREB-mediated induction of Cebpb expression is therefore required in with STAT factors on anti-inflammatory promoters, respectively, infiltrating macrophages for upregulation of M2-specific genes and suggesting that its specificity of action may be mediated by com- muscle regeneration, providing a direct genetic link between these petition between cooperating factors, and in this regard the levels two processes. of C/EBP␤ could be critical for determining which transcriptional programs are activated. During macrophage activation, Cebpb is ͉ ͉ macrophage polarization muscle regeneration transcription transcriptionally induced by the CREB transcriptional activator, another bZIP transcription factor (14), which binds two cAMP he resolution of tissue injury involves a complex interaction response elements (CREs) in the proximal Cebpb promoter (15). Tbetween the tissue undergoing repair and the immune system. However, given the presence of a significant basal level of C/EBP␤ Immune cells are critical for the removal of necrotic cells and for protein in resting macrophages, the role of CREB-mediated in- fending off infectious agents. In addition, they may provide support duction is unclear. for stem cells and progenitors as they proliferate and differentiate We report here that in activated primary macrophages, CREB- to repair the inflicted damage. Macrophages may play a key role in mediated induction of Cebpb expression was dispensable for in- this process as the major infiltrating cell population in injured duction of inflammatory (M1) genes (Il1b, Il6, Il12b, Tnfa), but muscle, required for removal of damaged myofibers (1, 2). Mac- required for genes characteristic of anti-inflammatory (M2) mac- rophages also have an important role in the subsequent regrowth rophages (Arg-1, Il10, Il13ra, Msr1). To confirm the role of C/EBP␤ and differentiation of myofibers as depletion of the macrophage in M2 macrophage activation, we generated mice carrying a tar- population after necrotic cell removal leads to a defect in regen- geted deletion of two CREB-binding sites in the Cebpb promoter eration (2). This latter function may require the induction in situ of ␤⌬ ( Cre mice), and analyzed their response to skeletal muscle IMMUNOLOGY an anti-inflammatory or M2 phenotype. Indeed, the M2 phenotype injury. ␤⌬Cre mice were defective in resolution of necrotic damage may be induced in macrophages in vitro through phagocytosis of to skeletal muscle. ␤⌬Cre macrophages infiltrated injured muscle myofiber debris (2). normally in vivo, but failed to upregulate Cebpb and Arg-1, impli- M2 macrophage polarization is induced by anti-inflammatory cating lack of Cebpb induction and defective M2 polarization in cytokines and growth factors, including IL-4, IL-10, and TGF-␤ (3). impaired muscle regeneration. These results define a molecular However, there is limited information about the transcriptional basis for polarized macrophage gene expression, show its signifi- control of M2 genes, and a mechanism for their coordinate regu- lation has yet to be elucidated. Although macrophages lacking the SHIP phosphatase were biased toward an M2 phenotype (4), the Author contributions: D.R., F.M., A.G., N.R., and C.N. designed research; D.R., F.M., A.G., and downstream transcriptional targets involved are not known. Tumor- P.K. performed research; R.G.L. generated conditional Cebpb KO mice; D.R., F.M., A.G., associated macrophages with an M2 phenotype activate signaling P.K., and C.N. analyzed data; and N.R. and C.N. wrote the paper. through IRF3/STAT1 and suppress NF-␬B activation (5). De- The authors declare no conflict of interest. creased NF-␬B signaling is likely to be responsible for the impaired This article is a PNAS Direct Submission. expression of M1 genes, as overexpression of the p50 NF-␬B 1D.R., F.M., and A.G. contributed equally to this work. subunit, which lacks a strong transactivation domain, is sufficient to 2To whom correspondence should be addressed. E-mail: [email protected]. repress M1 gene expression (6). The ability of macrophages to This article contains supporting information online at www.pnas.org/cgi/content/full/ suppress cytotoxic T lymphocyte activity is also impaired by acti- 0908641106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0908641106 PNAS ͉ October 13, 2009 ͉ vol. 106 ͉ no. 41 ͉ 17475–17480 Downloaded by guest on October 2, 2021 A Mock +LPS pogenesis in vitro (18); however, both adipose tissue amounts (Fig. 2A) and histological appearance (Fig. 2 B and C) were normal in ␤⌬Cre mice. In the hematopoietic system, C/EBP␤ is important for -CREB -CREB B-cell differentiation (19), macrophage function (11), and stress IgG IgG Input α Input α granulopoiesis (20). However, the number and distribution of granulocytic cells (Fig. 2D and Fig. S2), as well as the number of Promoter bone marrow (BM) macrophage progenitors (measured as macro- phage colony forming cells; Fig. 2E), B cell progenitors, or mature B cells (Fig. 2F and Fig. S3) were not affected by the ␤⌬Cre 3’UTR mutation. Examination of Cebpb mRNA levels in adult ␤⌬Cre mice B showed no significant downregulation compared to wild-type lit- ␤ +Ro termate controls in any major C/EBP -expressing tissue (Fig. 2G). γ γ From this analysis, we conclude that the Cebpb promoter CREs are dispensable for major physiological functions of C/EBP␤ during normal development. Mock +LPS/INF +LPS/INF To assess the role of the Cebpb promoter CREs in inflammatory P-CREB gene expression, primary macrophages were derived from ␤⌬Cre P-ATF1 and control (ϩ/ϩ) BM in the presence of macrophage-colony stimulating factor (M-CSF). After subsequent activation with LPS/ Tubulin IFN␥, total RNA was isolated, and Cebpb expression analyzed. While significant (3-fold) induction was observed in control mac- C rophage cultures, ␤⌬Cre macrophages failed to induce Cebpb 5 expression (Fig. 3A). C/EBP␤ induction was also impaired at the 4 protein level in ␤⌬Cre macrophages (Fig. 3B), whereas no differ- ences in the levels or kinetics of CREB/ATF1 phosphorylation was 3 observed between ϩ/ϩ and ␤⌬Cre macrophages, indicating that 2 the signal transduction pathways impinging on the Cebpb promoter CREs are functional. Analysis of major proinflammatory C/EBP␤ 1 target genes showed that those encoding the central M1-specific 0 cytokines Tnfa, Il1b, Il6, and Il12b were unaffected by the absence Cebpb mRNA (Mock=1) Cebpb mRNA Mock123 +LPS/ +LPS/ of Cebpb upregulation (Fig. 4A). In contrast, analysis of major M2 γ γ INF INF +Ro specific transcripts showed that Il10, Arg-1, Il13ra, and Msr1 were ␤⌬ Fig. 1. CREB activation induces Cebpb in macrophages. (A) ChIP of CREB on not upregulated in activated Cre macrophages (Fig. 4B). Dele- the Cebpb promoter in J774 macrophages stimulated with LPS. The CRE PCR, tion of the Cebpb promoter CREs thus led to a specific and specific for a 140-bp DNA fragment that spans the CRE elements on the Cebpb coordinated loss of M2-specific gene expression after LPS/IFN␥ promoter, demonstrates the recruitment of CREB onto the CREs of the C/EBP␤ activation. Finally, while high level induction of either ϩ/ϩ and promoter upon LPS treatment. Amplification of a 200-bp fragment in the 3Ј ␤⌬Cre macrophages with LPS/IFN␥ elicited similar production of UTR was used as a control. (B) IFN␥-primed J774 cells were pretreated with 5 nitric oxide (NO), a principal proinflammatory effector molecule, ␮M Ro 31–8220 or vehicle for 20 min followed by 1 h stimulation with treatment with either agent alone generated higher NO levels in IFN␥/LPS.
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