A CREB-C/EBP� cascade induces M2 macrophage-specific gene expression and promotes muscleinjury repairDaniela 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 damagethrough removal of necrotic cells, thus paving the way for tissueregeneration. Macrophages also directly support the formation ofnew tissue to replace the injury, through their acquisition of ananti-inflammatory, or M2, phenotype, characterized by a gene ex-pression program that includes IL-10, the IL-13 receptor, and arginase1. We report that deletion of two CREB-binding sites from the Cebpbpromoter abrogates Cebpb induction upon macrophage activation.This blocks the downstream induction of M2-specific Msr1, Il10, II13ra,and Arg-1 genes, whereas the inflammatory (M1) genes Il1, Il6, Tnfa,and Il12 are not affected. Mice carrying the mutated Cebpb promoter(��Cre) remove necrotic tissue from injured muscle, but exhibitsevere defects in muscle fiber regeneration. Conditional deletion ofthe Cebpb gene in muscle cells does not affect regeneration, showingthat the C/EBP� cascade leading to muscle repair is muscle-extrinsic.While ��Cre macrophages efficiently infiltrate injured muscle theyfail to upregulate Cebpb, leading to decreased Arg-1 expression.CREB-mediated induction of Cebpb expression is therefore required ininfiltrating macrophages for upregulation of M2-specific genes andmuscle regeneration, providing a direct genetic link between thesetwo processes.

macrophage polarization � muscle regeneration � transcription

The resolution of tissue injury involves a complex interactionbetween the tissue undergoing repair and the immune system.

Immune cells are critical for the removal of necrotic cells and forfending off infectious agents. In addition, they may provide supportfor stem cells and progenitors as they proliferate and differentiateto repair the inflicted damage. Macrophages may play a key role inthis process as the major infiltrating cell population in injuredmuscle, required for removal of damaged myofibers (1, 2). Mac-rophages also have an important role in the subsequent regrowthand differentiation of myofibers as depletion of the macrophagepopulation after necrotic cell removal leads to a defect in regen-eration (2). This latter function may require the induction in situ ofan anti-inflammatory or M2 phenotype. Indeed, the M2 phenotypemay be induced in macrophages in vitro through phagocytosis ofmyofiber debris (2).

M2 macrophage polarization is induced by anti-inflammatorycytokines and growth factors, including IL-4, IL-10, and TGF-� (3).However, there is limited information about the transcriptionalcontrol of M2 genes, and a mechanism for their coordinate regu-lation has yet to be elucidated. Although macrophages lacking theSHIP phosphatase were biased toward an M2 phenotype (4), thedownstream transcriptional targets involved are not known. Tumor-associated macrophages with an M2 phenotype activate signalingthrough IRF3/STAT1 and suppress NF-�B activation (5). De-creased NF-�B signaling is likely to be responsible for the impairedexpression of M1 genes, as overexpression of the p50 NF-�Bsubunit, which lacks a strong transactivation domain, is sufficient torepress M1 gene expression (6). The ability of macrophages tosuppress cytotoxic T lymphocyte activity is also impaired by acti-

vation of PPAR� (7), indicating that in this context PPAR�activation suppresses M2 polarization. However, the role of PPAR�remains controversial, as PPAR� activation has been observed topromote M2 polarization in adipose tissue (8).

Candidate regulators can be identified by analysis of promotersof M2-specific genes in macrophages, and among these, the Il10 andArg-1 promoters are regulated by C/EBP� (9, 10). C/EBP� is amember of the C/EBP family of basic region-leucine zipper (bZIP)proteins and is known to be important for the antibacterial activityof macrophages (11). However, macrophage expression of genesencoding inflammatory molecules, such as Il1b, Inos, Il6, and Tnfais also diminished in the absence of C/EBP� (12), confounding therole of C/EBP� in specifying the M2 gene program. There isconsiderable evidence that C/EBP� is regulated at the proteomiclevel through competition between interacting transcriptional reg-ulators (see reference 13 for review). In macrophages, C/EBP�functionally interacts with NF-�B on inflammatory and synergizeswith STAT factors on anti-inflammatory promoters, respectively,suggesting that its specificity of action may be mediated by com-petition between cooperating factors, and in this regard the levelsof C/EBP� could be critical for determining which transcriptionalprograms are activated. During macrophage activation, Cebpb istranscriptionally induced by the CREB transcriptional activator,another bZIP transcription factor (14), which binds two cAMPresponse elements (CREs) in the proximal Cebpb promoter (15).However, given the presence of a significant basal level of C/EBP�protein in resting macrophages, the role of CREB-mediated in-duction is unclear.

We report here that in activated primary macrophages, CREB-mediated induction of Cebpb expression was dispensable for in-duction of inflammatory (M1) genes (Il1b, Il6, Il12b, Tnfa), butrequired for genes characteristic of anti-inflammatory (M2) mac-rophages (Arg-1, Il10, Il13ra, Msr1). To confirm the role of C/EBP�in M2 macrophage activation, we generated mice carrying a tar-geted deletion of two CREB-binding sites in the Cebpb promoter(��Cre mice), and analyzed their response to skeletal muscleinjury. ��Cre mice were defective in resolution of necrotic damageto skeletal muscle. ��Cre macrophages infiltrated injured musclenormally in vivo, but failed to upregulate Cebpb and Arg-1, impli-cating lack of Cebpb induction and defective M2 polarization inimpaired muscle regeneration. These results define a molecularbasis for polarized macrophage gene expression, show its signifi-

Author contributions: D.R., F.M., A.G., N.R., and C.N. designed research; D.R., F.M., A.G., andP.K. performed research; R.G.L. generated conditional Cebpb KO mice; D.R., F.M., A.G.,P.K., and C.N. analyzed data; and N.R. and C.N. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1D.R., F.M., and A.G. contributed equally to this work.

2To whom correspondence should be addressed. E-mail: nerlov@embl.it.

This article contains supporting information online at www.pnas.org/cgi/content/full/0908641106/DCSupplemental.

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cance in tissue repair, and provide a model for C/EBP� promotionof tissue homeostasis.

ResultsTo determine if inhibition of CREB activation prevented Cebpbinduction, we established that LPS stimulation caused the recruit-ment of CREB to the Cebpb promoter in the J774 macrophage cellline (Fig. 1A). In the presence of RO318220, an inhibitor ofMSK-1/2, CREB phosphorylation in response to LPS/IFN� was notinduced (Fig. 1B), and Cebpb upregulation was abolished (Fig. 1C),consistent with a requirement for recruitment of activated CREBto the Cebpb promoter to achieve transcriptional induction. Wenext used homologous recombination in E14.1 ES cells to replacethe Cebpb promoter CREs with a sequence composed of fivebinding sites for the artificial ZFHD transcription factor (16) (Fig.S1). This eliminates binding sites for endogenous transcriptionfactors while maintaining promoter spacing. After germ line trans-mission, mice were bred to homozygosity for the resultingCebpb�CRE allele (henceforth ��Cre mice). While Cebpb�/� micedisplay female infertility (17), ��Cre females were fertile andlactated. Induction of Cebpb expression by CREB promotes adi-

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 forB-cell differentiation (19), macrophage function (11), and stressgranulopoiesis (20). However, the number and distribution ofgranulocytic cells (Fig. 2D and Fig. S2), as well as the number ofbone marrow (BM) macrophage progenitors (measured as macro-phage colony forming cells; Fig. 2E), B cell progenitors, or matureB cells (Fig. 2F and Fig. S3) were not affected by the ��Cremutation. Examination of Cebpb mRNA levels in adult ��Cre miceshowed no significant downregulation compared to wild-type lit-termate controls in any major C/EBP�-expressing tissue (Fig. 2G).From this analysis, we conclude that the Cebpb promoter CREs aredispensable for major physiological functions of C/EBP� duringnormal development.

To assess the role of the Cebpb promoter CREs in inflammatorygene expression, primary macrophages were derived from ��Creand control (�/�) BM in the presence of macrophage-colonystimulating factor (M-CSF). After subsequent activation with LPS/IFN�, total RNA was isolated, and Cebpb expression analyzed.While significant (3-fold) induction was observed in control mac-rophage cultures, ��Cre macrophages failed to induce Cebpbexpression (Fig. 3A). C/EBP� induction was also impaired at theprotein level in ��Cre macrophages (Fig. 3B), whereas no differ-ences in the levels or kinetics of CREB/ATF1 phosphorylation wasobserved between �/� and ��Cre macrophages, indicating thatthe signal transduction pathways impinging on the Cebpb promoterCREs are functional. Analysis of major proinflammatory C/EBP�target genes showed that those encoding the central M1-specificcytokines Tnfa, Il1b, Il6, and Il12b were unaffected by the absenceof Cebpb upregulation (Fig. 4A). In contrast, analysis of major M2specific transcripts showed that Il10, Arg-1, Il13ra, and Msr1 werenot upregulated in activated ��Cre macrophages (Fig. 4B). Dele-tion of the Cebpb promoter CREs thus led to a specific andcoordinated loss of M2-specific gene expression after LPS/IFN�activation. Finally, while high level induction of either �/� and��Cre macrophages with LPS/IFN� elicited similar production ofnitric oxide (NO), a principal proinflammatory effector molecule,treatment with either agent alone generated higher NO levels in��Cre macrophage cultures (Fig. 4C), further indicative of theirM1 bias.

To evaluate the pro- and anti-inflammatory C/EBP� functions inmuscle regeneration, a well-established in vivo injury model wasused (21). Tibialis anterior (TA) and quadriceps (Q) muscles of oneleg were injected with cardiotoxin (CTX), while the contralateralmuscles were left uninjured. CTX induces local necrosis (22),followed by an inflammatory response consisting mainly of mac-rophage invasion (23), and subsequent tissue reconstitution. Con-trol and ��Cre mice developed similar necrotic injuries, as deter-mined by histological analysis at day 2 after CTX injection (Fig. 5A and B). By day 5, the ��Cre regenerating regions showedincreased inflammation, as indicated by the presence of myofiberswith eosinophil cytoplasm and accretion of nuclei at the sites ofinjury (Fig. 5 C and D). At day 10 after CTX injection, where in �/�mice the injury is largely resolved, macroscopic examination of��Cre muscle showed a highly fibrotic appearance (Fig. S4).Moreover, at the histological level, multifocal areas of inflamma-tion and severely calcified myofibers were observed among newlyformed myofibers in the ��Cre muscles, whereas �/� muscle wascompletely regenerated (Fig. 5 E and F). Many of the ��Creregenerating myofibers appeared smaller (Fig. 5F) than those incontrol muscles, as confirmed by a decrease in the cross-sectionalfiber area (CSA) of ��Cre myofibers (Fig. 5G) and a 20%reduction in the total regenerated area (Fig. 5H) 10 days after CTXinjection. Together, these results clearly showed that the regener-ation capacity in the ��Cre muscles was decreased and confirmedincreased inflammation as an important element of this impair-ment. To exclude any contribution to the observed phenotype from

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Fig. 1. CREB activation induces Cebpb in macrophages. (A) ChIP of CREB onthe Cebpb promoter in J774 macrophages stimulated with LPS. The CRE PCR,specific for a 140-bp DNA fragment that spans the CRE elements on the Cebpbpromoter, demonstrates the recruitment of CREB onto the CREs of the C/EBP�

promoter upon LPS treatment. Amplification of a 200-bp fragment in the 3�UTR was used as a control. (B) IFN�-primed J774 cells were pretreated with 5�M Ro 31–8220 or vehicle for 20 min followed by 1 h stimulation withIFN�/LPS. Cell lysates were processed for immunoblotting with antibodyagainst phospho-CREB (upper panel) followed by stripping and reprobingwith antibody against �-tubulin (lower panel). (C) IFN� primed J774 cells werepretreated with 5 �M Ro 31–8220 or vehicle for 20 min followed by 4 hstimulation with IFN�/LPS. Relative Cebpb mRNA levels in J774 cells weremeasured in triplicate by quantitative real-time PCR, normalized to ubiquitin.Data are presented as the mean � SD (�/� n � 3; ��Cre n � 3).

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minor differences in the strain background, these experiments wererepeated after backcrossing to C57BL/6 for six generations followedby intercrossing to generate homozygous ��Cre mice; the pheno-type remained unaltered (Fig. S5). In addition, no difference in theregenerative timecourse was observed between C57 and 129 mousestrains (Fig. S6).

Since the mutation in ��Cre mice is ubiquitous, a potentialmuscle-intrinsic role for C/EBP� during muscle regeneration couldnot be excluded. We tested this possibility by conditionally ablatingCebpb gene expression exclusively in skeletal muscles (BMKOmice; generated using a Cre-conditional null allele of Cebpb and theMCK-Cre transgene (24) (Fig. S7). BMKO mice showed normalregeneration after CTX injury (Fig. 5 J and K), consistent with arequirement for C/EBP� in cells infiltrating the damage, ratherthan in resident muscle cells. To further determine if BM-derivedcells were responsible for the regeneration defect, wild-type mice(CD45.1/2 allotype) were repopulated with �/� or ��Cre BM(CD45.2 allotype) (Fig. 5L) and subjected to the injury protocol.The presence of ��Cre BM cells was sufficient to impair muscleregeneration (Fig. 5 M and N). Conversely, repopulation of ��Cremice with �/� BM was sufficient to rescue the regeneration defect(Fig. S8). Therefore the presence of the ��Cre mutation in thehematopoetic system is both necessary and sufficient for theregeneration defect.

To further investigate the nature of the cells infiltratingdamaged muscle, we quantified the amount of macrophagespresent in injured �/� and ��Cre muscle by flow cytometry. Asexpected, injury induced a strong increase in Mac-1�F4/80�

macrophage numbers; however, no difference was observedbetween ��Cre and �/� mice (Fig. 6 A and B). We next sortedmacrophages from injured muscles 6 days after CTX injection todetermine whether Cebpb expression was affected at this stage.Cebpb mRNA was reduced 2.5-fold in ��Cre macrophagescompared to controls (Fig. 6C), demonstrating the requirementfor CREB-dependent Cebpb upregulation in macrophages dur-

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Fig. 2. Role of CREB-C/EBP� cascade in normal development. (A) Epididymal fatpad weight normalized to total body weight for ��Cre mice and �/� littermates.(B and C) Eosin hematoxylin staining on epididymal fat pad histological sectionsfrom �/� and ��Cre mice. (D) Frequency of Mac-1�Gr-1lo and Mac-1�Gr-1� cellsfrom �/� and ��Cre BM determined by flow cytometry phenotyping. Data arepresented as the mean � SD (�/� n � 3; ��Cre n � 3). (E) Macrophage colony-forming activity of BM cells were plated in methylcellulose medium containingM-CSF (10 ng/mL). Macrophage colony-forming units were scored after 8 daysand are presented as average CFU-M/103 BM cells (�/� n � 3; ��Cre n � 3).Data are presented as the mean � SD. (F) Frequency of prepro-B(B220�CD43�AA4.1�CD19�), pro-B (B220�CD43�AA4.1�CD19�), pre-B(B220�CD43�A4.1�CD19�), immature B (B220�IgM�), mature B (B220�IgM�),and recirculating B cells (B220��IgM�) from �/� and ��Cre BM determined byflow cytometry (�/� n � 3; ��Cre N � 3). Data are presented as the mean � SD.(G) Cebpb expression levels in tissues extracted from Cebpb�/�, Cebpb�/� and��Cre mice measured by real time PCR (n � 3 for each genotype). AlthoughCebpb mRNA levels were somewhat lower in tissues derived from ��Cre micecompared to �/� controls, the differences were not significant. The Cebpb geneis intronless, and Cebpb�/� mice were used to control for influence of genomicDNA contamination on the analysis. WAT, white adipose tissue.

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ing muscle regeneration. Expression of M1-specific genes (Il12b,Il6) was unaltered, whereas expression of the M2-specific Arg-1was downregulated, consistent with defective M2 polarization ofthe macrophage population. Deletion of a regulatory elementfrom the Cebpb promoter could potentially affect the expressionof neighboring genes. To address this we performed Affymetrixanalysis on �/� and ��Cre macrophages isolated from injuredmuscle. None of the neighboring genes were deregulated,whereas the expected downregulation of Cebpb was observed(Fig. S9A). Finally, Q-PCR analysis of selected genes withpotential relevance for macrophage activation in in vitro-induced �/� and ��Cre macrophages also did not reveal anysignificant differences in the expression or regulation (Fig. S9B),consistent with the effect of the ��Cre deletion being solely onthe expression of Cebpb.

DiscussionMacrophage polarization has been proposed to play importantroles in tissue repair and cancer. In necrotic muscle injury, infil-trating macrophages are essential for regeneration, as depletion ofperipheral monocytes before injury prevents removal of necrotictissue (2). Depletion of F4/80� macrophages from regeneratingmuscle led to incomplete repair and reduced muscle fiber size (2)indicating a direct role for macrophages in promoting fiber forma-tion and growth. Further, muscle fiber regeneration correlated witha transition to an anti-inflammatory macrophage phenotype bothin vivo and in vitro (2). Macrophages isolated from primary tumorshave also been found to display an anti-inflammatory M2 pheno-type and are thought to be recruited and/or M2 polarized by thetumor cells as a means to prevent their destruction by the immunesystem (25). This may represent subversive use of a mechanismnormally activated to resolve tissue injury by shutting down theinflammatory response to damage.

The ��Cre mice described in this report provide a genetic modelin which the in vivo role of macrophage polarization may be tested.Our results support a model whereby macrophages recruited tomuscle injury are M2 polarized by the regenerating environment tostimulate fiber growth; thus a mutation that specifically impairsM2-specific macrophage gene expression interferes with the laterstages of muscle regeneration and fiber replacement, whereas theinitial removal of necrotic tissue is maintained. Specifically, thereduction of Arg-1 expression in ��Cre macrophages is likely toresult in the re-routing of arginine metabolism away from arginase-mediated polyamine synthesis (which promotes tissue regenera-tion) toward iNOS-mediated NO production (which promotesdegradation of transcripts encoding the myocyte differentiationfactor MyoD) (26). An essential CREB-dependent pathway forinducing Arg-1 and polyamine synthesis during axonal regenerationhas previously been described (27). Our results are consistent withC/EBP� acting as an intermediate between CREB and Arg-1expression in this setting as well, implicating the CREB-C/EBP�-arginase pathway in multiple cell types to promote tissue repair.Other M2-specific genes (Il13ra1, Msr1, Il10) were not affected in��Cre macrophages in vivo. This may be because they are notsignificantly induced even in the wild-type macrophages or morelikely because signals not present in our in vitro conditions areoperating in injured muscle tissue.

It is notable that macrophage activation does not involve changesin the phosphorylation, DNA binding, or translational control ofC/EBP�, which appears to be in a fully active state in restingmacrophages (14). These cells appear poised for rapid deploymentof an inflammatory response, which requires only a basal C/EBP�level before activation. Our findings pinpoint CREB-mediatedCebpb upregulation as a mechanism whereby activated macro-phages can coordinate M2-specific gene induction, which may serveto temporally organize pro- and anti-inflammatory responses.Interference in this regulatory mechanism results in the normalinduction of M1 proinflammatory genes upon macrophage activa-tion, but impaired upregulation of M2-specific genes. The M2program thus seems to be specifically sensitive to C/EBP� levels.Importantly, ��Cre macrophages express lower levels of CebpbmRNA within the injured muscle, demonstrating that the CREB-C/EBP� cascade that is normally active during tissue repair hasbeen perturbed.

Although Cebpb expression is generally induced in response tophysiological stress, including hypoxia and inflammation in severaltissues and cell types, Cebpb�/� mice in which C/EBP�-dependentgene regulation has been abrogated display a paradoxically im-proved response to both ischemic stroke (28) and inflammatorysteatohepatitis (29), presumably because both the pro- and anti-inflammatory effects of C/EBP� action have been blocked system-ically. More subtle perturbations, such as the one used in the presentstudy, will be necessary to tease out various tissue-specific and

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temporal roles of C/EBP�, which are likely to be dependent onrelatively small variations in Cebpb expression levels in response todifferent stimuli. The molecular insights obtained here should allow

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Fig. 5. Evaluation of regenerating skeletal muscles in ��Cre mice. (A and B)Trichrome staining of �/� (A) and ��Cre (B) tibialis anterior muscle shows similarnecrosis in both genotypes 2 days after CTX injection. (C and D) Recovery of theinjured muscles and regenerating myofibers (containing centralized nuclei) atday 5 postinjury in �/� (C) and ��Cre (D) muscle. Myofibers with eosinophilcytoplasmare indicatedwitharrowheads in (D). (EandF)Atday10postinjury, theexistence of numerous small fibers (arrowheads) is evident in the ��Cre injuredmuscle (F) compared to �/� muscle (E). Note that, in contrast to control regen-erating muscles, ��Cre muscles contained many calcified fibers (arrows). (G)Morphometric analysis of muscle regeneration in �/� and ��Cre mice. The datashow the frequency distribution in the tibialis anterior fiber cross-sectional area(CSA) within the regenerating muscle. (H) Measurement of the total regenerat-ing (marked by centralized nuclei) fiber area in mutant injured muscles. Data arepresented as the mean � SD (�/� n � 6; ��Cre n � 6). Significant differences (P �0.05; Student’s t-test) are indicated by asterisks (*). (J and K) Regenerated tibialisanterior muscle of �/� (J) and BMKO (K) mice 10 days after CTX injection. Noimpairment of regeneration was evident in BMKO muscle compared to �/�

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Fig. 6. Infiltrating macrophage phenotype in injured ��Cre muscle. (A) Phe-notypic analysis from �/� and ��Cre muscle uninjured and injured 6 days afterCTX injection by flow cytometry for the presence of Mac-1�F4/80� cells. Plots arerepresentative of four independent experiments and numbers represent thefrequencyofcells in the indicatedgates. (B)Totalmononuclearcellswere isolatedfrom injured and control thigh muscle and analyzed for expression of F4/80 andMac-1 by FACS. The total number of recovered F4/80�Mac-1� cells is indicated foreach condition (n � 2 for uninjured samples; N� � 5 for injured samples). Errorbars indicate standard deviations. (C) F4/80�Mac-1� cells were sorted from day 6injured muscle, and gene expression analyzed by real-time PCR (N� � 5/geno-type). Data are presented as the mean � SD normalized to the �/� value (� 1).Asterisks (*) indicate P � 0.05 (Student’s t-test).

muscle. (L) BM cells from ��Cre mutant or wild-type mice (CD45.1�CD45.2�

allotype) were transferred to lethally irradiated recipient mice (CD45.1�CD45.2�

allotype).Plots showrepresentativeFACSanalysisofperipheralblood inrecipientmice at 4 weeks after transplantation to measure the engraftment of donor(CD45.1�CD45.2�) and recipient (CD45.1�CD45.2�) cells. (M and N) The micetransplanted in (L) were subjected to the CTX injury protocol. Trichrome stainingof injured TA muscle sections at day 10 postinjury is shown. Note that theobserved defect in muscle regeneration in ��Cre mice was recapitulated inwild-type mice transplanted with ��Cre mutant BM cells (N) while control wild-typemice transplantedwithwild-typeBMcells showednormal regeneration (M).

Ruffell et al. PNAS � October 13, 2009 � vol. 106 � no. 41 � 17479

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for therapeutic manipulation of this regulatory pathway to eitherpromote or impair M2 polarization.

MethodsMouse Strains. The ��Cre mouse strain was generated by replacement of theCebpb promoter CREs with ZFHD binding sites using ET recombination. Thedetailed cloning and ES cell targeting strategy is provided in the SI Methods.

C/EBP� null mice have been previously described (17) and were obtainedfrom E. Sterneck, National Cancer Institute (NCI), Bethesda, MD. ConditionalC/EBP� knockout mice have been described elsewhere (30). MCK-Cre mice (24)were obtained from R. Kahn, Harvard Medical School, Boston, MA. Mousestrains were maintained on a mixed C57BL/6–129/Ola background, exceptwhere specifically stated otherwise. All animal procedures were performedaccording to Italian national and European Molecular Biology Laboratory(EMBL) institutional guidelines.

Muscle Injury Induction. For muscle regeneration experiments, animals wereanesthetized using 2.5% Avertin. Tibialis anterior (TA) and quadriceps from 2-to 3-month-old control, ��Cre, and BMKO mice were injected with 20 and 40�L, respectively, of 10 �M CTX (Latoxan). The muscles were collected 2, 5, 6,and 10 days following the injection of CTX. At least three mice per time pointwere analyzed. Details of histological analysis is found in the SI Methods.

Cell Culture. To obtain BM-derived macrophages, femurs and tibias werecollected from a mouse, crushed in a mortar in presence of 1% FCS/PBS, andfiltered. Cells were washed, resuspended, and cultured in differentiationmedium consisting of RPMI (Gibco), 20% FCS, 50 �M �-mercaptoethanol, 100U/mL penicillin, 100 �g/mL streptomycin, 2 mM L-glutamine, and 20 ng/mLmacrophage colony-stimulating factor (M-CSF; Sigma). The cells were culturedin differentiation medium for 6 days, after which M-CSF was depleted. Formacrophage activation, cells were treated with 100 U/mL IFN� (PeproTech) ingrowth medium, with 5% FCS, for 16 h. Next, the cells were stimulated with100 U/mL IFN� and 1 �g/mL LPS from Escherichia coli (Sigma) for 4 h, afterwhich 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(Sigma). Exudate cells were harvested by washing the peritoneal cavity with 12mL PBS and subsequently cultured in DMEM, 5% FCS, 100 U/mL penicillin, 100�g/mL streptomycin, and 2 mM L-glutamine. For NO production measure-

ments, peritoneal macrophages were plated in triplicate in a 96-well plate at0.1 million cells/well. The cells were either left untreated or treated with 10U/mL IFN� and/or 10 ng/mL LPS for 48 h. NO2

- concentration in the medium wasmeasured with the Griess reagents as described (31). NO2

- concentrations werenormalized to Thiazolyl blue (MTT; Sigma) vital dye staining to correct forvariations in cell number and viability. Macrophage colony-forming unitassays were performed as previously described (32). For Western blotting, cellswere washed with cold PBS and lysed in lysis buffer (50 mM Tris-HCl, pH 7.5,150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 30 mM Na4P205, 1.25 mM NaF, 100�M Na3VO4), and lysates were processed as described in the SI Methods.

Chromatin Immunoprecipitation (ChIP). J774 macrophages were grown tosubconfluence in 10-cm dishes, and then treated with 1 �g/mL LPS for 15 min.Details of the ChIP protocol are found in the SI Methods.

Cell Preparation. Injured muscles were placed in warmed DMEM (Gibco), andmatrix, fibrotic tissue, and nerves were removed carefully. The muscles werechopped into small pieces, and enzymatic disaggregation was performed, firstusing freshly prepared 4 mg/mL collagenase (Sigma) for 30 min (37 °C), andthen using 1 mg/mL collagenase/dispase (Roche) for 25 min (37 °C). Disaggre-gation was stopped with 5 mL horse serum (heat-inactivated; Gibco), and afterfiltration with a 40-�m cell strainer (BD Bioscience), the cells were mixed withcold PBS plus 1% FCS (Gibco). BM cells were obtained as described (32). Forstaining of cells for flow cytometry see the SI Methods.

Gene Expression Analysis. RNA isolated from sorted �/� and ��Cre macro-phages 6 days postinjury was subjected to microarray analysis using AffymetrixMOE430.2 arrays. Data were processed as previously described (33). Four biolog-ical replicates were performed for each genotype, from which average normal-ized expression values for Cebpb genomic neighbors were calculated. Real-timePCR conditions are described in Table S1 and in the SI Methods.

Statistical Analysis. Evalation of statistical significance was done using Stu-dent’s t-test, except for measurement of total regenerated fiber area, whichwas done by ANOVA .

ACKNOWLEDGMENTS. We thank Dr. E. Sterneck for providing C/EBP� genomicclones and Dr. R. Kahn for MCK-Cre mice. This work was supported by theAssociation for International Cancer Research, the European Commission(EuroCSC STREP), and the European Muscle Development Network (MYORES).

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