A CREB-C/EBPbeta cascade induces M2 macrophage-specific gene expression and promotes muscle injury repair

Abstract

Macrophages play an essential role in the resolution of tissue damage through removal of necrotic cells, thus paving the way for tissue regeneration. Macrophages also directly support the formation of new tissue to replace the injury, through their acquisition of an anti-inflammatory, or M2, phenotype, characterized by a gene expression program that includes IL-10, the IL-13 receptor, and arginase 1. We report that deletion of two CREB-binding sites from the Cebpb promoter 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 (betaDeltaCre) remove necrotic tissue from injured muscle, but exhibit severe defects in muscle fiber regeneration. Conditional deletion of the Cebpb gene in muscle cells does not affect regeneration, showing that the C/EBPbeta cascade leading to muscle repair is muscle-extrinsic. While betaDeltaCre macrophages efficiently infiltrate injured muscle they fail to upregulate Cebpb, leading to decreased Arg-1 expression. CREB-mediated induction of Cebpb expression is therefore required in infiltrating macrophages for upregulation of M2-specific genes and muscle regeneration, providing a direct genetic link between these two processes.

Fig. 1.

CREB activation induces Cebpb in macrophages. (A) ChIP of CREB on the 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 Cebpb promoter, demonstrates the recruitment of CREB onto the CREs of the C/EBPβ promoter upon LPS treatment. Amplification of a 200-bp fragment in the 3′ UTR was used as a control. (B) IFNγ-primed J774 cells were pretreated with 5 μM Ro 31–8220 or vehicle for 20 min followed by 1 h stimulation with IFNγ/LPS. Cell lysates were processed for immunoblotting with antibody against phospho-CREB (upper panel) followed by stripping and reprobing with antibody against α-tubulin (lower panel). (C) IFNγ primed J774 cells were pretreated with 5 μM Ro 31–8220 or vehicle for 20 min followed by 4 h stimulation with IFNγ/LPS. Relative Cebpb mRNA levels in J774 cells were measured in triplicate by quantitative real-time PCR, normalized to ubiquitin. Data are presented as the mean ± SD (+/+ n = 3; βΔCre n = 3).

Fig. 2.

Role of CREB-C/EBPβ cascade in normal development. (A) Epididymal fat pad weight normalized to total body weight for βΔCre mice and +/+ littermates. (B and C) Eosin hematoxylin staining on epididymal fat pad histological sections from +/+ and βΔCre mice. (D) Frequency of Mac-1⁺Gr-1^lo and Mac-1⁺Gr-1⁺ cells from +/+ and βΔCre BM determined by flow cytometry phenotyping. Data are presented as the mean ± SD (+/+ n = 3; βΔCre n = 3). (E) Macrophage colony-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 and are presented as average CFU-M/10³ BM cells (+/+ n = 3; βΔCre 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 βΔCre BM determined by flow cytometry (+/+ n = 3; βΔCre N = 3). Data are presented as the mean ± SD. (G) Cebpb expression levels in tissues extracted from Cebpb^−/−, Cebpb^+/+ and βΔCre mice measured by real time PCR (n = 3 for each genotype). Although Cebpb mRNA levels were somewhat lower in tissues derived from βΔCre mice compared to +/+ controls, the differences were not significant. The Cebpb gene is intronless, and Cebpb^−/− mice were used to control for influence of genomic DNA contamination on the analysis. WAT, white adipose tissue.

Fig. 3.

Cebpb promoter CREs are required for induction by LPS/IFNγ. (A) Real-time PCR analysis of Cebpb expression in BM-derived primary macrophages from +/+ and βΔCre mice, treated with IFNγ/LPS as indicated. Data are presented as the mean ± SD (+/+ n = 6; βΔCre n = 6). Significant differences (P < 0.05; Student's t-test) are indicated by asterisk (*). (B) Western blots of C/EBPβ (p33), phospho-CREB (P-CREB), and tubulin (as internal control) from +/+ and βΔCre primary macrophages, either untreated (-) or treated with LPS for the indicated time after pretreatment with IFNγ (+).

Fig. 4.

Defective M2 gene expression in activated βΔCre macrophages. (A) Real-time PCR expression analysis of proinflammatory markers in BM-derived macrophages upon IFNγ/LPS stimulation analyzed as in Fig. 3A (+/+ n = 6; βΔCre n = 6). (B) Real-time PCR expression analysis of anti-inflammatory markers in BM-derived macrophages upon IFNγ/LPS stimulation analyzed as in Fig. 3A (+/+ n = 6; βΔCre n = 6). (C) Relative NO release of thioglycollate-elicited peritoneal macrophages stimulated with IFNγ and/or LPS, as indicated. Data are presented as the mean ± SD (+/+ n = 3; βΔCre n = 3).

Fig. 5.

Evaluation of regenerating skeletal muscles in βΔCre mice. (A and B) Trichrome staining of +/+ (A) and βΔCre (B) tibialis anterior muscle shows similar necrosis in both genotypes 2 days after CTX injection. (C and D) Recovery of the injured muscles and regenerating myofibers (containing centralized nuclei) at day 5 postinjury in +/+ (C) and βΔCre (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 βΔCre injured muscle (F) compared to +/+ muscle (E). Note that, in contrast to control regenerating muscles, βΔCre muscles contained many calcified fibers (arrows). (G) Morphometric analysis of muscle regeneration in +/+ and βΔCre mice. The data show the frequency distribution in the tibialis anterior fiber cross-sectional area (CSA) within the regenerating muscle. (H) Measurement of the total regenerating (marked by centralized nuclei) fiber area in mutant injured muscles. Data are presented as the mean ± SD (+/+ n = 6; βΔCre n = 6). Significant differences (P < 0.05; Student's t-test) are indicated by asterisks (*). (J and K) Regenerated tibialis anterior muscle of +/+ (J) and BMKO (K) mice 10 days after CTX injection. No impairment of regeneration was evident in BMKO muscle compared to +/+ muscle. (L) BM cells from βΔCre mutant or wild-type mice (CD45.1⁻CD45.2⁺ allotype) were transferred to lethally irradiated recipient mice (CD45.1⁺CD45.2⁺ allotype). Plots show representative FACS analysis of peripheral blood in recipient mice 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 mice transplanted in (L) were subjected to the CTX injury protocol. Trichrome staining of injured TA muscle sections at day 10 postinjury is shown. Note that the observed defect in muscle regeneration in βΔCre mice was recapitulated in wild-type mice transplanted with βΔCre mutant BM cells (N) while control wild-type mice transplanted with wild-type BM cells showed normal regeneration (M).

Fig. 6.

Infiltrating macrophage phenotype in injured βΔCre muscle. (A) Phenotypic analysis from +/+ and βΔCre muscle uninjured and injured 6 days after CTX injection by flow cytometry for the presence of Mac-1⁺F4/80⁺ cells. Plots are 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 Mac-1 by FACS. The total number of recovered F4/80⁺Mac-1⁺ cells is indicated for each condition (n = 2 for uninjured samples; N> = 5 for injured samples). Error bars indicate standard deviations. (C) F4/80⁺Mac-1⁺ cells were sorted from day 6 injured muscle, and gene expression analyzed by real-time PCR (N> = 5/genotype). Data are presented as the mean ± SD normalized to the +/+ value (= 1). Asterisks (*) indicate P < 0.05 (Student's t-test).