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) macrophages 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 regulation 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 B-cell differentiation (19), macrophage function (11), and stress IgG IgG Input α -CREB Input α -CREB 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 macrophage 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). 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 γ +LPS/INF γ 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 differences 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. Cell lysates were processed for immunoblotting with antibody ␤⌬Cre macrophage cultures (Fig. 4C), further indicative of their against phospho-CREB (upper panel) followed by stripping and reprobing M1 bias. with antibody against ␣-tubulin (lower panel). (C) IFN␥ primed J774 cells were ␤ pretreated with 5 ␮M Ro 31–8220 or vehicle for 20 min followed by 4 h To evaluate the pro- and anti-inflammatory C/EBP functions in stimulation with IFN␥/LPS. Relative Cebpb mRNA levels in J774 cells were muscle regeneration, a well-established in vivo injury model was measured in triplicate by quantitative real-time PCR, normalized to ubiquitin. used (21). Tibialis anterior (TA) and quadriceps (Q) muscles of one Data are presented as the mean Ϯ SD (ϩ/ϩ n ϭ 3; ␤⌬Cre n ϭ 3). leg were injected with cardiotoxin (CTX), while the contralateral muscles were left uninjured. CTX induces local necrosis (22), followed by an inflammatory response consisting mainly of mac- cance in tissue repair, and provide a model for C/EBP␤ promotion rophage invasion (23), and subsequent tissue reconstitution. Con- of tissue homeostasis. trol and ␤⌬Cre mice developed similar necrotic injuries, as determined by histological analysis at day 2 after CTX injection (Fig. 5 Results A and B). By day 5, the ␤⌬Cre regenerating regions showed To determine if inhibition of CREB activation prevented Cebpb increased inflammation, as indicated by the presence of myofibers induction, we established that LPS stimulation caused the recruit- with eosinophil cytoplasm and accretion of nuclei at the sites of ment of CREB to the Cebpb promoter in the J774 macrophage cell injury (Fig. 5 C and D). At day 10 after CTX injection, where in ϩ/ϩ line (Fig. 1A). In the presence of RO318220, an inhibitor of mice the injury is largely resolved, macroscopic examination of MSK-1/2, CREB phosphorylation in response to LPS/IFN␥ was not ␤⌬Cre muscle showed a highly fibrotic appearance (Fig. S4). induced (Fig. 1B), and Cebpb upregulation was abolished (Fig. 1C), Moreover, at the histological level, multifocal areas of inflamma- consistent with a requirement for recruitment of activated CREB tion and severely calcified myofibers were observed among newly to the Cebpb promoter to achieve transcriptional induction. We formed myofibers in the ␤⌬Cre muscles, whereas ϩ/ϩ muscle was next used homologous recombination in E14.1 ES cells to replace completely regenerated (Fig. 5 E and F). Many of the ␤⌬Cre the Cebpb promoter CREs with a sequence composed of five regenerating myofibers appeared smaller (Fig. 5F) than those in binding sites for the artificial ZFHD transcription factor (16) (Fig. control muscles, as confirmed by a decrease in the cross-sectional S1). This eliminates binding sites for endogenous transcription fiber area (CSA) of ␤⌬Cre myofibers (Fig. 5G) and a 20% factors while maintaining promoter spacing. After germ line trans- reduction in the total regenerated area (Fig. 5H) 10 days after CTX mission, mice were bred to homozygosity for the resulting injection. Together, these results clearly showed that the regener- Cebpb⌬CRE allele (henceforth ␤⌬Cre mice). While CebpbϪ/Ϫ mice ation capacity in the ␤⌬Cre muscles was decreased and confirmed display female infertility (17), ␤⌬Cre females were fertile and increased inflammation as an important element of this impair- lactated. Induction of Cebpb expression by CREB promotes adi- ment. To exclude any contribution to the observed phenotype from

17476 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0908641106 Ruffell et al. Downloaded by guest on October 2, 2021 16.0 A B A 4 untreated 14.0 IFNγ/LPS 12.0 3

10.0 2 * 8.0 (+/+ unst.= 1) 6.0 C 1 Relative mRNA level Relative mRNA 4.0 mg/g of body weight 0 2.0 +/+ DC/DCβ∆Cre 0.0 β∆ +/+ β∆Cre B +/+ Cre –++ – LPS/INFγ D 40 C/EBPβ (4h) 35 +/+ 30 β∆Cre P-CREB (1h) 25 P-ATF1

20 P-CREB (4h) P-ATF1 % BM cells 15

10 Tubulin 5

0 Fig. 3. Cebpb promoter CREs are required for induction by LPS/IFN␥.(A) Mac1+Gr1lo Mac1+Gr1+ Real-time PCR analysis of Cebpb expression in BM-derived primary macrophages from ϩ/ϩ and ␤⌬Cre mice, treated with IFN␥/LPS as indicated. Data are 25 E 16 F presented as the mean Ϯ SD (ϩ/ϩ n ϭ 6; ␤⌬Cre n ϭ 6). Signiﬁcant differences +/+ (P Ͻ 0.05; Student’s t-test) are indicated by asterisk (*). (B) Western blots of 20 ␤ 12 β∆ C/EBP (p33), phospho-CREB (P-CREB), and tubulin (as internal control) from Cre ϩ ϩ ␤⌬ 15 / and Cre primary macrophages, either untreated (-) or treated with LPS 8 for the indicated time after pretreatment with IFN␥ (ϩ).

CFU-M 10

4 % BM cells 5 minor differences in the strain background, these experiments were repeated after backcrossing to C57BL/6 for six generations followed 0 0 +/+ β∆Cre by intercrossing to generate homozygous ␤⌬Cre mice; the pheno- proB preB type remained unaltered (Fig. S5). In addition, no difference in the pre-proB B220+IgM-B220+IgM+ B220++IgM+ regenerative timecourse was observed between C57 and 129 mouse strains (Fig. S6). ␤⌬ G –/– +/+ β∆Cre Since the mutation in Cre mice is ubiquitous, a potential 1.6 muscle-intrinsic role for C/EBP␤ during muscle regeneration could not be excluded. We tested this possibility by conditionally ablating 1.2 Cebpb gene expression exclusively in skeletal muscles (BMKO mice; generated using a Cre-conditional null allele of Cebpb and the 0.8 MCK-Cre transgene (24) (Fig. S7). BMKO mice showed normal

0.4 regeneration after CTX injury (Fig. 5 J and K), consistent with a requirement for C/EBP␤ in cells infiltrating the damage, rather Cebpb mRNA (+/+ = 1) Cebpb mRNA 0 than in resident muscle cells. To further determine if BM-derived Liver Ovary Bone WAT Brain Lung Muscle marrow cells were responsible for the regeneration defect, wild-type mice (CD45.1/2 allotype) were repopulated with ϩ/ϩ or ␤⌬Cre BM Fig. 2. Role of CREB-C/EBP␤ cascade in normal development. (A) Epididymal fat (CD45.2 allotype) (Fig. 5L) and subjected to the injury protocol. IMMUNOLOGY pad weight normalized to total body weight for ␤⌬Cre mice and ϩ/ϩ littermates. The presence of ␤⌬Cre BM cells was sufficient to impair muscle (B and C) Eosin hematoxylin staining on epididymal fat pad histological sections regeneration (Fig. 5 M and N). Conversely, repopulation of ␤⌬Cre ϩ ϩ ϩ from ϩ/ϩ and ␤⌬Cre mice. (D) Frequency of Mac-1 Gr-1lo and Mac-1 Gr-1 cells mice with ϩ/ϩ BM was sufficient to rescue the regeneration defect from ϩ/ϩ and ␤⌬Cre BM determined by flow cytometry phenotyping. Data are ␤⌬ Ϯ ϩ ϩ ϭ ␤⌬ ϭ (Fig. S8). Therefore the presence of the Cre mutation in the presented as the mean SD ( / n 3; Cre n 3). (E) Macrophage colony- hematopoetic system is both necessary and sufficient for the forming activity of BM cells were plated in methylcellulose medium containing M-CSF (10 ng/mL). Macrophage colony-forming units were scored after 8 days regeneration defect. and are presented as average CFU-M/103 BM cells (ϩ/ϩ n ϭ 3; ␤⌬Cre n ϭ 3). To further investigate the nature of the cells infiltrating Data are presented as the mean Ϯ SD. (F) Frequency of prepro-B damaged muscle, we quantified the amount of macrophages (B220ϩCD43ϩAA4.1ϩCD19Ϫ), pro-B (B220ϩCD43ϩAA4.1ϩCD19ϩ), pre-B present in injured ϩ/ϩ and ␤⌬Cre muscle by flow cytometry. As (B220ϩCD43ϪA4.1ϩCD19ϩ), immature B (B220ϩIgMϪ), mature B (B220ϩIgMϩ), expected, injury induced a strong increase in Mac-1ϩF4/80ϩ ϩϩ ϩ and recirculating B cells (B220 IgM ) from ϩ/ϩ and ␤⌬Cre BM determined by macrophage numbers; however, no difference was observed flow cytometry (ϩ/ϩ n ϭ 3; ␤⌬Cre N ϭ 3). Data are presented as the mean Ϯ SD. ␤⌬ ϩ ϩ Ϫ/Ϫ ϩ/ϩ between Cre and / mice (Fig. 6 A and B). We next sorted (G) Cebpb expression levels in tissues extracted from Cebpb , Cebpb and macrophages from injured muscles 6 days after CTX injection to ␤⌬Cre mice measured by real time PCR (n ϭ 3 for each genotype). Although ␤⌬ determine whether Cebpb expression was affected at this stage. Cebpb mRNA levels were somewhat lower in tissues derived from Cre mice ␤⌬ compared to ϩ/ϩ controls, the differences were not significant. The Cebpb gene Cebpb mRNA was reduced 2.5-fold in Cre macrophages is intronless, and CebpbϪ/Ϫ mice were used to control for influence of genomic compared to controls (Fig. 6C), demonstrating the requirement DNA contamination on the analysis. WAT, white adipose tissue. for CREB-dependent Cebpb upregulation in macrophages dur-

Ruffell et al. PNAS ͉ October 13, 2009 ͉ vol. 106 ͉ no. 41 ͉ 17477 Downloaded by guest on October 2, 2021 A control LPS/INFγ Discussion 2 Il12b 2 Tnfa Macrophage polarization has been proposed to play important

1.5 1.5 roles in tissue repair and cancer. In necrotic muscle injury, infiltrating macrophages are essential for regeneration, as depletion of 1 1 peripheral monocytes before injury prevents removal of necrotic 0.5 0.5 tissue (2). Depletion of F4/80ϩ macrophages from regenerating 0 0 muscle led to incomplete repair and reduced muscle fiber size (2) +/+β∆ DC/DCCre +/+β∆ DC/DCCre indicating a direct role for macrophages in promoting fiber forma-

2 Il6 1.5 Il1 tion and growth. Further, muscle fiber regeneration correlated with a transition to an anti-inflammatory macrophage phenotype both 1.5 1 in vivo and in vitro (2). Macrophages isolated from primary tumors 1 have also been found to display an anti-inflammatory M2 pheno- 0.5 0.5 type and are thought to be recruited and/or M2 polarized by the

0 0 tumor cells as a means to prevent their destruction by the immune Relative mRNA level (+/+ LPS/INF γ = 1) Relative mRNA +/+β∆ DC/DCCre +/+β∆ DC/DCCre system (25). This may represent subversive use of a mechanism normally activated to resolve tissue injury by shutting down the control γ B LPS/INF inflammatory response to damage. 1.5 Arg1 1.5 Msr1 The ␤⌬Cre mice described in this report provide a genetic model 1 1 * in which the in vivo role of macrophage polarization may be tested. Our results support a model whereby macrophages recruited to 0.5 * 0.5 muscle injury are M2 polarized by the regenerating environment to stimulate fiber growth; thus a mutation that specifically impairs 0 0 +/+DC/DC+/+DC/DCβ∆Cre β∆Cre M2-specific macrophage gene expression interferes with the later stages of muscle regeneration and fiber replacement, whereas the 2 Il10 1.5 Il13ra1 initial removal of necrotic tissue is maintained. Specifically, the 1.5 ␤⌬ 1 reduction of Arg-1 expression in Cre macrophages is likely to 1 * result in the re-routing of arginine metabolism away from arginase- 0.5 mediated polyamine synthesis (which promotes tissue regenera- 0.5 * tion) toward iNOS-mediated NO production (which promotes 0 0 Relative mRNA level (+/+ LPS/INF γ = 1) Relative mRNA +/+β∆ DC/DCCre +/+β∆ DC/DCCre degradation of transcripts encoding the myocyte differentiation factor MyoD) (26). An essential CREB-dependent pathway for 80 C +/+ inducing Arg-1 and polyamine synthesis during axonal regeneration 70 β∆DC/DCCre has previously been described (27). Our results are consistent with 60 C/EBP␤ acting as an intermediate between CREB and Arg-1 50 expression in this setting as well, implicating the CREB-C/EBP␤- 40 arginase pathway in multiple cell types to promote tissue repair. 30 * Other M2-specific genes (Il13ra1, Msr1, Il10) were not affected in 20 ␤⌬Cre macrophages in vivo. This may be because they are not * Relative NO production 10 significantly induced even in the wild-type macrophages or more

0 likely because signals not present in our in vitro conditions are plain IFN LPS IFN/LPS -10 operating in injured muscle tissue. It is notable that macrophage activation does not involve changes Fig. 4. Defective M2 gene expression in activated ␤⌬Cre macrophages. (A) in the phosphorylation, DNA binding, or translational control of Real-time PCR expression analysis of proinﬂammatory markers in BM-derived C/EBP␤, which appears to be in a fully active state in resting macrophages upon IFN␥/LPS stimulation analyzed as in Fig. 3A (ϩ/ϩ n ϭ 6; ␤⌬ ϭ macrophages (14). These cells appear poised for rapid deployment Cre n 6). (B) Real-time PCR expression analysis of anti-inﬂammatory of an inflammatory response, which requires only a basal C/EBP␤ markers in BM-derived macrophages upon IFN␥/LPS stimulation analyzed as in Fig. 3A (ϩ/ϩ n ϭ 6; ␤⌬Cre n ϭ 6). (C) Relative NO release of thioglycollate- level before activation. Our findings pinpoint CREB-mediated elicited peritoneal macrophages stimulated with IFN␥ and/or LPS, as indicated. Cebpb upregulation as a mechanism whereby activated macro- Data are presented as the mean Ϯ SD (ϩ/ϩ n ϭ 3; ␤⌬Cre n ϭ 3). phages can coordinate M2-specific gene induction, which may serve to temporally organize pro- and anti-inflammatory responses. Interference in this regulatory mechanism results in the normal induction of M1 proinflammatory genes upon macrophage activa- ing muscle regeneration. Expression of M1-specific genes (Il12b, tion, but impaired upregulation of M2-specific genes. The M2 Il6) was unaltered, whereas expression of the M2-specific Arg-1 program thus seems to be specifically sensitive to C/EBP␤ levels. was downregulated, consistent with defective M2 polarization of Importantly, ␤⌬Cre macrophages express lower levels of Cebpb the macrophage population. Deletion of a regulatory element mRNA within the injured muscle, demonstrating that the CREB- from the Cebpb promoter could potentially affect the expression C/EBP␤ cascade that is normally active during tissue repair has of neighboring genes. To address this we performed Affymetrix been perturbed. ϩ ϩ ␤⌬ analysis on / and Cre macrophages isolated from injured Although Cebpb expression is generally induced in response to muscle. None of the neighboring genes were deregulated, physiological stress, including hypoxia and inflammation in several whereas the expected downregulation of Cebpb was observed tissues and cell types, CebpbϪ/Ϫ mice in which C/EBP␤-dependent (Fig. S9A). Finally, Q-PCR analysis of selected genes with gene regulation has been abrogated display a paradoxically im- potential relevance for macrophage activation in in vitro- proved response to both ischemic stroke (28) and inflammatory induced ϩ/ϩ and ␤⌬Cre macrophages also did not reveal any steatohepatitis (29), presumably because both the pro- and anti- significant differences in the expression or regulation (Fig. S9B), inflammatory effects of C/EBP␤ action have been blocked system- consistent with the effect of the ␤⌬Cre deletion being solely on ically. More subtle perturbations, such as the one used in the present the expression of Cebpb. study, will be necessary to tease out various tissue-specific and

17478 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0908641106 Ruffell et al. Downloaded by guest on October 2, 2021 A C E A +/+ β∆Cre uninjured 3.1% 1.4% +/+

B D F β∆ Cre 29.4 27.4% injured Mac-1-FITC

G H F4/80-PE +/+ +/+ ) B 5 2000020 β∆Cre β∆Cre ) x 10 3 2 1500015 % Fibers cells / + CSA ( µ m CSA 10 Mac1 + 5 F4/80 +/+ BMKO thigh muscle (x10 JK 00 UninjWTU d6 WTIInj d6 UninjDCU d6 DCI Inj d6 UninjWTU d10 WTI Inj d10 UninjDCU d10 DCI Inj d10 +/+ β∆Cre +/+ β∆Cre Day 6 Day 10

2 C +/+ β∆Cre L 1.5 B-cell gate Myeloid gate T-cell gate

Myeloid 1 Recipient B-cell ** 0.5

T-cell Donor

WT rec./WT donor 0 mRNA level (control = 1) mRNA C/EBPbCebpb ArginaseArg1 1IL13ra1 !L13raMsr1 Msr1IL10 IL-10 IL12p40.4Il12b Il6 IL-6 CD45.1-PECy7 Mac-1/B220-APC

Fig. 6. Inﬁltrating macrophage phenotype in injured ␤⌬Cre muscle. (A) Phe- notypic analysis from ϩ/ϩ and ␤⌬Cre muscle uninjured and injured 6 days after ϩ ϩ

WT rec./DC donor CTX injection by ﬂow cytometry for the presence of Mac-1 F4/80 cells. Plots are CD4/CD8/B220-PE CD45.2-APCAlexa750 representative of four independent experiments and numbers represent the frequency of cells in the indicated gates. (B) Total mononuclear cells were isolated from injured and control thigh muscle and analyzed for expression of F4/80 and WT rec./DC donor ϩ ϩ M WT rec./WT donor N Mac-1 by FACS. The total number of recovered F4/80 Mac-1 cells is indicated for each condition (n ϭ 2 for uninjured samples; NϾϭ5 for injured samples). Error bars indicate standard deviations. (C) F4/80ϩMac-1ϩ cells were sorted from day 6 injured muscle, and gene expression analyzed by real-time PCR (NϾϭ5/genotype). Data are presented as the mean Ϯ SD normalized to the ϩ/ϩ value (ϭ 1). Asterisks ( ) indicate P Ͻ 0.05 (Student’s t-test).

* IMMUNOLOGY

␤⌬ Fig. 5. Evaluation of regenerating skeletal muscles in Cre mice. (A and B) temporal roles of C/EBP␤, which are likely to be dependent on Trichrome staining of ϩ/ϩ (A) and ␤⌬Cre (B) tibialis anterior muscle shows similar necrosis in both genotypes 2 days after CTX injection. (C and D) Recovery of the relatively small variations in Cebpb expression levels in response to injured muscles and regenerating myofibers (containing centralized nuclei) at different stimuli. The molecular insights obtained here should allow day 5 postinjury in ϩ/ϩ (C) and ␤⌬Cre (D) muscle. Myofibers with eosinophil cytoplasm are indicated with arrowheads in (D). (E and F) At day 10 postinjury, the existence of numerous small fibers (arrowheads) is evident in the ␤⌬Cre injured muscle (F) compared to ϩ/ϩ muscle (E). Note that, in contrast to control regen- muscle. (L) BM cells from ␤⌬Cre mutant or wild-type mice (CD45.1ϪCD45.2ϩ erating muscles, ␤⌬Cre muscles contained many calcified fibers (arrows). (G) allotype) were transferred to lethally irradiated recipient mice (CD45.1ϩCD45.2ϩ Morphometric analysis of muscle regeneration in ϩ/ϩ and ␤⌬Cre mice. The data allotype). Plots show representative FACS analysis of peripheral blood in recipient show the frequency distribution in the tibialis anterior fiber cross-sectional area mice at 4 weeks after transplantation to measure the engraftment of donor (CSA) within the regenerating muscle. (H) Measurement of the total regenerat- (CD45.1ϪCD45.2ϩ) and recipient (CD45.1ϩCD45.2ϩ) cells. (M and N) The mice ing (marked by centralized nuclei) fiber area in mutant injured muscles. Data are transplanted in (L) were subjected to the CTX injury protocol. Trichrome staining presented as the mean Ϯ SD (ϩ/ϩ n ϭ 6; ␤⌬Cre n ϭ 6). Significant differences (P Ͻ of injured TA muscle sections at day 10 postinjury is shown. Note that the 0.05; Student’s t-test) are indicated by asterisks (*). (J and K) Regenerated tibialis observed defect in muscle regeneration in ␤⌬Cre mice was recapitulated in anterior muscle of ϩ/ϩ (J) and BMKO (K) mice 10 days after CTX injection. No wild-type mice transplanted with ␤⌬Cre mutant BM cells (N) while control wild- impairment of regeneration was evident in BMKO muscle compared to ϩ/ϩ type mice transplanted with wild-type BM cells showed normal regeneration (M).

Ruffell et al. PNAS ͉ October 13, 2009 ͉ vol. 106 ͉ no. 41 ͉ 17479 Downloaded by guest on October 2, 2021 for therapeutic manipulation of this regulatory pathway to either ments, peritoneal macrophages were plated in triplicate in a 96-well plate at promote or impair M2 polarization. 0.1 million cells/well. The cells were either left untreated or treated with 10 - U/mL IFN␥ and/or 10 ng/mL LPS for 48 h. NO2 concentration in the medium was - Methods measured with the Griess reagents as described (31). NO2 concentrations were normalized to Thiazolyl blue (MTT; Sigma) vital dye staining to correct for ␤⌬ Mouse Strains. The Cre mouse strain was generated by replacement of the variations in cell number and viability. Macrophage colony-forming unit Cebpb promoter CREs with ZFHD binding sites using ET recombination. The assays were performed as previously described (32). For Western blotting, cells detailed cloning and ES cell targeting strategy is provided in the SI Methods. were washed with cold PBS and lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, ␤ C/EBP null mice have been previously described (17) and were obtained 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 30 mM Na4P205, 1.25 mM NaF, 100 from E. Sterneck, National Cancer Institute (NCI), Bethesda, MD. Conditional ␮MNa3VO4), and lysates were processed as described in the SI Methods. C/EBP␤ knockout mice have been described elsewhere (30). MCK-Cre mice (24) were obtained from R. Kahn, Harvard Medical School, Boston, MA. Mouse Chromatin Immunoprecipitation (ChIP). J774 macrophages were grown to strains were maintained on a mixed C57BL/6–129/Ola background, except subconfluence in 10-cm dishes, and then treated with 1 ␮g/mL LPS for 15 min. where specifically stated otherwise. All animal procedures were performed Details of the ChIP protocol are found in the SI Methods. according to Italian national and European Molecular Biology Laboratory (EMBL) institutional guidelines. Cell Preparation. Injured muscles were placed in warmed DMEM (Gibco), and matrix, fibrotic tissue, and nerves were removed carefully. The muscles were Muscle Injury Induction. For muscle regeneration experiments, animals were chopped into small pieces, and enzymatic disaggregation was performed, first anesthetized using 2.5% Avertin. Tibialis anterior (TA) and quadriceps from 2- using freshly prepared 4 mg/mL collagenase (Sigma) for 30 min (37 °C), and to 3-month-old control, ␤⌬Cre, and BMKO mice were injected with 20 and 40 then using 1 mg/mL collagenase/dispase (Roche) for 25 min (37 °C). Disaggre- ␮L, respectively, of 10 ␮M CTX (Latoxan). The muscles were collected 2, 5, 6, gation was stopped with 5 mL horse serum (heat-inactivated; Gibco), and after and 10 days following the injection of CTX. At least three mice per time point filtration with a 40-␮m cell strainer (BD Bioscience), the cells were mixed with were analyzed. Details of histological analysis is found in the SI Methods. cold PBS plus 1% FCS (Gibco). BM cells were obtained as described (32). For staining of cells for flow cytometry see the SI Methods. Cell Culture. To obtain BM-derived macrophages, femurs and tibias were ϩ ϩ ␤⌬ collected from a mouse, crushed in a mortar in presence of 1% FCS/PBS, and Gene Expression Analysis. RNA isolated from sorted / and Cre macro- filtered. Cells were washed, resuspended, and cultured in differentiation phages 6 days postinjury was subjected to microarray analysis using Affymetrix MOE430.2 arrays. Data were processed as previously described (33). Four biolog- medium consisting of RPMI (Gibco), 20% FCS, 50 ␮M ␤-mercaptoethanol, 100 ical replicates were performed for each genotype, from which average normal- U/mL penicillin, 100 ␮g/mL streptomycin, 2 mM L-glutamine, and 20 ng/mL ized expression values for Cebpb genomic neighbors were calculated. Real-time macrophage colony-stimulating factor (M-CSF; Sigma). The cells were cultured PCR conditions are described in Table S1 and in the SI Methods. in differentiation medium for 6 days, after which M-CSF was depleted. For macrophage activation, cells were treated with 100 U/mL IFN␥ (PeproTech) in Statistical Analysis. Evalation of statistical significance was done using Stu- growth medium, with 5% FCS, for 16 h. Next, the cells were stimulated with dent’s t-test, except for measurement of total regenerated fiber area, which 100 U/mL IFN␥ and 1 ␮g/mL LPS from Escherichia coli (Sigma) for 4 h, after was done by ANOVA . which RNA was extracted. For the preparation of peritoneal macrophages, mice were killed 3 days after an i.p. injection of 1 mL 3% thioglycollate broth ACKNOWLEDGMENTS. We thank Dr. E. Sterneck for providing C/EBP␤ genomic (Sigma). Exudate cells were harvested by washing the peritoneal cavity with 12 clones and Dr. R. Kahn for MCK-Cre mice. This work was supported by the mL PBS and subsequently cultured in DMEM, 5% FCS, 100 U/mL penicillin, 100 Association for International Cancer Research, the European Commission ␮g/mL streptomycin, and 2 mM L-glutamine. For NO production measure- (EuroCSC STREP), and the European Muscle Development Network (MYORES).

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