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1 extra view Cell Cycle 11:5, ; March 1, 2012; 2012 Landes Bioscience extra view MKP-1 coordinates ordered macrophage-phenotype transitions essential for stem cell-dependent tissue repair Eusebio Perdiguero, 1, * Yacine Kharraz, 1 Antonio L. Serrano 1, * and Pura Muñoz-Cánoves 1,2, * 1 Cell Biology Group; Department of Experimental and Health Sciences; Pompeu Fabra University (UPF); CIBER on Neurodegenerative diseases (CIBERNED); Barcelona, Spain; 2 Institució Catalana de Recerca i Estudis Avançats (ICREA); Barcelona, Spain Key words: MKP-1, p38 MAP kinase, regeneration, inflammation, satellite cells Submitted: 01/12/12 Accepted: 01/15/12 *Correspondence to: Eusebio Perdiguero, Antonio Serrano and Pura Muñoz-Cánoves; eusebio. perdiguero@upf.edu, antonio.serrano@upf.edu and pura.munoz@upf.edu Re-establishing tissue homeostasis in response to injury requires infiltration of inflammatory cells and activation of resident stem cells. However, full tissue recovery also requires that the inflammation is resolved. While it is known that disturbing the interactions between inflammatory cells and tissue resident cells prevents successful healing, the molecular mechanisms underlying the paracrine interactions between these cell types are practically unknown. Here, and in a recent study, we provide mechanistic evidence that macrophages control stem cell-dependent tissue repair. In particular, we found that the temporal spacing of the pro- to anti-inflammatory macrophage polarization switch is controlled by the balance of p38 MAPK (termed here p38) and the MAPK phosphatase MKP-1 during the muscle healing process. Moreover, we demonstrate a new function for MKP-1-regulated p38 signaling in deactivating macrophages during inflammation resolution after injury. Specifically, at advanced stages of regeneration, MKP-1 loss caused an unscheduled exhaustion-like state in muscle macrophages, in which neither pro- nor anti-inflammatory cytokines are expressed despite persistent tissue damage, leading to dysregulated reparation by the tissue stem cells. Mechanistically, we demonstrate that p38 and MKP-1 control the AKT pathway through a mir-21-dependent PTEN regulation. Importantly, both genetic and pharmacological interference with the individual components of this pathway restored inflammation-dependent tissue homeostasis in MKP-1-deficient mice and delayed inflammation resolution and tissue repair dysregulation in wild-type mice. Because the process of tolerance to bacterial infection involves a progressive attenuation of pro-inflammatory gene expression, we discuss here the potential similarities between the mechanisms underlying inflammation resolution during tissue repair and those controlling endotoxin tolerance. The Inflammatory Response to Tissue Damage Tissue damage elicits a highly coordinated response of several cell types in response to local and systemic signals that begin the repair process. Acute tissue injury leads to the orchestrated activities of the infiltrating inflammatory cells and resident stem cells that restore tissue homeostasis. In this situation, the resident macrophage populations are likely to be insufficient to mediate phagocytosis of the necrotic debris and its subsequent substitution by healthy tissue. To help with this task, monocytes enter the damaged tissue and differentiate into a spectrum of mononuclear phagocytes. Since these are pro-inflammatory, an inflammatory state exists in damaged tissues and a balance must be struck between producing the pro-inflammatory mediators and the anti-inflammatory factors that protect tissue integrity. 1-4 In response to prolonged bacterial infection or endotoxin stimulation, the progressive attenuation of pro-inflammatory gene expression, known as endotoxin tolerance, exerts a protective role to the host from excessive Cell Cycle 877

2 inflammation. 5-7 Whether such tolerance in response to prolonged tissue damage also occurs in vivo remains unknown. It is thought that macrophages represent a spectrum of activated phenotypes rather than discrete, permanent subpopulations. Indeed, numerous studies have shown that macrophages can switch from a functional pro-inflammatory phenotype to an anti-inflammatory one in response to the variable microenvironmental signals of the local tissue milieu, suggesting a high amount of flexibility in their programming. Thus, we can consider that, in response to tissue injury, there is an ordered process of inflammation aimed at preserving tissue homeostasis. By analogy with the host response to bacterial infection, we propose that this orderly inflammation encompasses two major concepts during tissue repair after injury: tolerance and polarization. Here, we discuss our data regarding how changes in inflammatory conditions influence the activity of adult muscle stem cells and muscle tissue repair and the mechanisms by which these occur. We propose that dysregulated macrophage transitions, with a precocious pro- to anti-inflammatory polarization, lead to the immediate cessation of cytokine expression and impair both tissue damage resolution and repair. Mechanistically, we provide novel evidence that the macrophage-specific MKP-1 regulates the evolution and the resolution of inflammation during muscle stem cell-dependent tissue repair, and that it does this by controlling the p38-mediated AKT activation. We therefore conclude that the p38/mkp-1 axis plays a role not only in sepsis prevention but also as an important regulator of inflammation-mediated tissue repair. We discuss these findings in the context of the emerging similarities between signaling pathways that control the attenuation of inflammation during endotoxin tolerance and tissue repair. Evolution of the Macrophage Response to Infection: Polarization and Tolerance Following bacterial infection, the endotoxins engage a Toll-like receptor (such as TLR4) to stimulate the first-responder macrophages, which usually exhibit an inflammatory phenotype and secrete pro-inflammatory mediators, such as the tumor necrosis factor α (TNFα) and interleukin (IL)-1. After this first inflammatory burst, there is a period known as endotoxin tolerance, in which the genes involved in the antimicrobial response as well as some anti-inflammatory genes (such as IL-10) are not attenuated, but rather induced and can reach expression levels similar to or higher than those achieved during the primary challenge. Endotoxin tolerance can be therefore viewed as a gene-specific response that decreases the risks of excessive inflammation while maintaining active antimicrobial systems (reviewed in ref. 1, 2 and 5 11). In the context of non-infectious tissue injury, infiltrating inflammatory monocytes have the potential to increase the damage, and the blood-derived monocytes quickly differentiate into macrophages at the damage site. These macrophages also produce reactive oxygen and nitrogen intermediates, including NO and superoxide, which can be highly damaging to tissues and, if the exposure is prolonged, can lead to aberrant inflam- mation (reviewed in ref. 3, 4 and 12). The pro-inflammatory (M1) macrophage response must be carefully controlled, as it can lead to extensive tissue damage and has been implicated in different chronic inflammatory and autoimmune diseases. This is achieved in part through the antagonistic activities of the M2 macrophages, which have strong anti-inflammatory activities and are active in wound healing. M2 macrophages can be classified into the three major subtypes of M2a, M2b and M2c, each of which responds to distinct stimuli and is endowed with different functions. The M2a or alternatively activated macrophages are polarized by the cytokines IL-4 and IL-13 and thereby mirror the Th2 polarization of T cells. These macrophages produce high levels of the so-called M2 markers, such as non-opsonic receptors (e.g., the mannose receptor MCR1/CD206), Ym1, Ym2, arginase 1 (Arg1) and selected chemokines. 13 They are involved in resistance to parasites and allergy and have been associated to fibrosis development in organs and tissues. 3,14,15 M2b or regulatory macrophages require two different signals to be polarized, the first one from immune complexes, prostaglandins, adenosine or apoptotic cells, followed by the ligation of a Toll-like receptor (such as the TLR4) by either damage-associated molecular patterns (DAMP) or pathogen-associated molecular patterns (PAMP). These macrophages exert immunoregulatory functions and drive type II responses. Finally, M2c or anti-inflammatory macrophages (also called deactivated macrophages) are induced by glucorticoid and antiinflammatory cytokines, such IL-10 or transforming growth factor β (TGFβ); in response, these macrophages then produce high levels of these two cytokines. M2c macrophages are involved in immune response suppression and tissue remodeling and in some ways, resemble endotoxin-tolerant macrophages. Hence, it can be speculated that macrophage polarization after injury serves a function similar to that of endotoxin tolerance over time. The mechanistic details of the macrophage polarization during tissue repair have still to be worked out. In contrast, the mechanisms of endotoxin tolerance have been studied in further detail. Endotoxin tolerance has been linked with particular regulatory events, such as upregulation of the negative regulators IRAK-M 16 or SOCS1, 17 or to the presence of the nuclear receptor PPARγ. 18 Interestingly, some of the proteins involved in endotoxin tolerance are also required for proper macrophage polarization within tissues/organs. For example, IRAK-M is necessary for the differentiation of pro-inflammatory monocytes into M2 tumor-associated macrophages (TAMs). 19 Systems biology and bioinformatics approaches have recently reinforced the concept that similarities exist between M2 polarization and endotoxin tolerance in macrophages. 20 Along these lines, endotoxin tolerance conditions were shown to enhance the wound-healing properties of epithelial cells. Interestingly, TLR signaling, which is classically associated with bacterial infection, was shown to regulate the resolution of inflammation during tissue remodeling. Indeed, macrophages deficient in the histone demethylase Jmjd3, which is induced by TLR stimulation, fail to polarize to an M2 phenotype in response to helminth infection; 878 Cell Cycle volume 11 Issue 5

3 Figure 1. Dysregulation of the p38/mkp-1 balance impairs skeletal muscle repair. (A) Cryosections from cardiotoxin (Ctx)-injured limb muscle from wild-type (wt) and MKP-1 -/- mice were stained with H/E and with an anti-emhc antibody. Ten days post-injury (P.I.) sections are shown. Bar = 50 μm. (B) Cryosections from WT and MKP-1 -/- mice muscles were obtained 21 d after a laceration-induced muscle injury and analyzed as in (A). Bar = 50 μm. (C) Schematic representation of macrophages (F4/80 + cells) in WT and MKP-1 -/- muscles after Ctx-injury at the indicated time points. (D) Cryosections from 10 d Ctx-injured muscles from WT and MKP-1 -/- mice, previously transplanted with bone marrow (BM) from WT and/or MKP-1 -/- mice, were stained with H/E. The origin of the BM donors is indicated in brackets. Bar = 50 μm. moreover, M2 but not M1 markers were affected in response to LPS, suggesting that TLR-induced Jmjd3 provides a feedback mechanism during inflammatory damage reparation. 21 Several other transcription-coupled signal transduction molecules are known to activate endotoxin tolerance by regulating the pro-inflammatory gene silencing in response to LPS and/or by inducing the expression of anti-inflammatory cytokines; these include ATF3, an ATF/CREB family member and p Activating p38 in macrophages in response to bacterial infection, in turn, activates downstream kinases, resulting in the nuclear translocation of NFκB and stabilization of proinflammatory cytokine mrna, 23 thus supporting its pro-inflammatory action. However, by inducing tolerance, 24 p38 has also been shown to exert an anti-inflammatory role through its capacity to induce the expression of SOCS3, IRAK-M, IL-10 and its inactivating phosphatase MKP-1, 25 thereby negatively regulating pathogeninduced inflammation. 26 Activation of the PI3K/AKT pathway has also been shown to regulate the process of tolerization by blocking LPS-induced MAPK and NFκB signaling cascades in monocytes. 27 Akt1-deficient macrophages demonstrate enhanced responsiveness to endotoxins, and Akt1-deficient mice do not exhibit LPS tolerance in vivo. 28 Thus, a potential axis has been proposed for bacterial tolerance occurring through the action of SOCS and modulated via the PI3K/AKT/ p38 pathway. 29 The anti-inflammatory capacity of p38 is further demonstrated in mouse models of pneumococcal pneumonia and tuberculosis, in which its inhibition results in impaired bacterial clearance and increased TNFα production both in vitro and in vivo. 11,30 However, a global understanding of the evolution of the macrophage gene expression program during sustained bacterial stimulation is still lacking; further, the question remains unanswered as to whether similar tolerance processes and functional evolution of macrophages, involving re-programming (e.g., activating and silencing) of distinct cytokine gene programs, also occur in vivo in response to tissue injury. The p38/mkp-1 Balance in Muscle Tissue Repair: Inflammatory vs. Muscle Intrinsic Function? The activation of MAPKs through tissue insult, both exogenous (i.e., infection) and endogenous (i.e., tissue injury), plays a central role in regulating the resulting inflammatory responses. 31,32 For instance, JNK, p38 and ERK MAPKs are rapidly activated in response to bacterial endotoxins to induce pro-inflammatory mediators by inflammatory cells It is critical that the MAPK activation state is tightly controlled, as it helps to determine both the magnitude and the duration of the inflammation and unchecked inflammation underlies numerous chronic diseases. 36 MKP-1, an archetypal member of the MKP/DUSP family, controls the threshold and magnitude of MAPK activation by reversing MAPK phosphorylation (predominantly that of p38). 37,38 Of note, there are four p38 MAPKs, p38α, p38β, p38γ and p38δ, displaying distinct and/or functions Interestingly, the MAPKs are involved in a negative feedback loop, Cell Cycle 879

4 Figure 2. MKP-1 is dispensable for muscle growth after overloading. (A) Plantaris muscles from WT and MKP-1-/- mice were obtained before surgery and 3 d after induction of compensatory hypertrophy by tenotomy of the gastrocnemius muscle. Muscle cryosections were immunostained with antibodies against Pax7, MyoD, Myogenin and emhc, and the number of positive cells was quantified. (B) As in (A), Pax7, MyoD, Myogenin, emhc mrna expression levels were analyzed by qrt-pcr. (C) Cryosections from muscles obtained at different time points after overloading (OVL) were stained with H/E. Bar = 50 μm. (D) Frequency histograms of myofiber size distribution in WT (left) and MKP-1-/- (right) plantaris muscles before (control) and after 14 d of OVL. Values are mean ± SEM. Data are representative of at least three independent experiments. n.s., not significantly different; p > as they induce the transcription of the MKP-1 gene during the anti-inflammatory response to bacterial infection44 and in the bacterial tolerance state.45 Similarly, the expression of TNFα, IL-6 and IL-10 that is induced by p38 activation, the concomitantly enhanced inos/no expression and reduced arginase synthesis expression, have been proposed to underlie the exacerbated response to endotoxin challenge in mice genetically deficient in MKP In contrast to the well-defined role of MPK-1 in the inflammatory response to 880 bacterial infection, little is known about its role in regulating the macrophage transitions during tissue healing, that is, from the initial inflammatory response to the injury resolution. Interestingly, it has been demonstrated that infiltrating macrophages changed from an initial pro-inflammatory activation state (M1) to an anti-inflammatory one (M2) during the course of muscle regeneration and the depletion of intramuscular macrophages at late stages of regeneration resulted in a impaired growth of the regenerating myofibers.49 To address the question of the role of MPK-1 during tissue healing, we undertook a combination of genetic and pharmacological approaches using MKP1-deficient mice and specific p38α/β inhibitors in models of tissue repair induced by experimental injury. The process of muscle regeneration, which strictly relies on the activation, proliferation and fusion of muscle stem cells (satellite cells), was severely affected by MKP-1 loss after cardiotoxin- or laceration-induced injuries, thus indicating that the formation and Cell Cycle Volume 11 Issue 5

5 growth of new myofibers in the mutant mice were delayed and/or impaired in a satellite cell-dependent manner (Fig. 1A and B). Considering the important role of MKP-1 macrophage functions after endotoxin challenge, we postulated that MKP-1-dependent alterations may occur during the macrophage inflammatory response that could influence the course of muscle regeneration. We found that while the number of infiltrating macrophages was similar in wild-type and MKP-1- deficient muscle one day after injury, this number was significantly higher in MKP- 1-deficient than in wild-type muscle three days after injury. Important differences were still observed at late stages of the repair process (10 d after injury), at which point the macrophage number decreased sharply in wild-type muscle but persisted at significant levels in the MKP-1-deficient muscle (Fig. 1C). Thus, MKP-1 may regulate muscle repair progression by affecting the properties of satellite cells and/or macrophages. To address these possibilities, we restored MKP-1 expression in the bone marrow of MKP-1-deficient mice by transplanting wild-type bone marrow, and found that this could revert both the exacerbated inflammatory response after injury and the defective muscle regeneration phenotype 50 (Fig. 1C). Hence, MKP-1-mediated control of inflammation appears to be critical to achieve optimal satellite cell-mediated myofiber growth during skeletal muscle regeneration. Since various studies had anticipated a relevant function for MKP-1 in myogenesis, 47,51-53 we undertook further studies to test the potential additional contribution of MKP-1 to satellite cell-intrinsic functions during muscle tissue repair. First, we analyzed satellite cell behavior in wild-type and MKP-1 -/- mice during overloading-induced compensatory muscle growth, a process that requires satellite cells to be incorporated into the preexisting fibers. 54 Myofiber overloading is similar to injury-induced myofiber repair, but, unlike this process, it involves limited inflammation, making it an ideal model to evaluate satellite cell-intrinsic functions in vivo. After the overloading stimulus, the numbers of activated and proliferating satellite cells (which express MyoD) and of differentiated satellite cells (which express Figure 3. Representation of macrophage phenotype transitions in WT and MKP-1 -/- muscles during early/medium stages of muscle repair. The shown macrophage phenotypes are based on the cytokine expression profiles of WT and MKP-1 -/- macrophages at early-medium stages after Ctx-injury: PRO, pro-inflammatory (i.e., high TNFα, IL-1β); ANti, anti-inflammatory (i.e., high TGFβ, IL-10); PRO/ANti, expression of both pro-and anti-inflammatory cytokines. Figure 4. Anticipated deactivation of MKP-1 -/- macrophages during tissue repair. Macrophages undergo cytokine expression silencing (i.e., deactivated) at late stages of muscle repair in WT mice. In the absence of MKP-1, macrophage deactivation occurs at earlier stages. The shown macrophage phenotypes are based on the cytokine expression profiles of WT and MKP-1 -/- macrophages at medium-late stages after Ctx-injury: PRO, pro-inflammatory (i.e., high TNFα, IL-1β); ANti, anti-inflammatory (i.e., high TGFβ, IL-10); PRO/ANti, expression of both pro-and anti-inflammatory cytokines; Deactivated, absence of pro- and anti-inflammatory cytokine expression. myogenin and emhc) increased similarly in both wild-type and MKP-1 -/- muscles, consistent with the observation that comparable levels of Pax7 + quiescent satellite cells were present in resting wild-type and MKP-1 -/- muscles (Fig. 2A). These results were confirmed by qrt-pcr expression analysis of overloaded muscle tissue (Fig. 2B) and strongly suggest that MKP-1 is dispensable for satellite cell-mediated adult myofiber growth. Indeed, the final CSA of overloaded myofibers was not significantly different in either mouse genotypes (Fig. 2C and D). Finally, an ex vivo analysis of the behavior of satellite cells isolated from wild-type and MKP-1 -/- muscle confirmed that muscle-intrinsic MKP-1 is not critically involved in myogenesis. 50 Together, these results unveil a central function for macrophage-intrinsic MKP-1 in controlling the extent of inflammation in damaged muscle tissue, which, in turn, impacts satellite cell-mediated repair via a paracrine pathway. Previous studies have suggested that MKP-1 has a function in myogenesis; 47,51-53 however, distinctions between our results and the previously reported findings could be due to the different experimental models used in the referred studies, thereby understriking the need for a future evaluation of MKP-1 function using muscle-specific deletion approaches. Ordered Transition of Macrophage Phenotypic States during Tissue Repair: A Tolerance-like State after Tissue Injury? FACS analyses of skeletal muscle have shown that, in response to injury, Ly-6C high monocytes/macrophages with an M1 pro-inflammatory cytokine activation profile are initially recruited, whereas predominantly Ly-6C low macrophages with an M2 anti-inflammatory profile Cell Cycle 881

6 Figure 5. p38/mkp-1 and PI3K/AKT pathways crosstalk in macrophages during tissue repair. (A) Schematic representation of the p38 and AKT activities and PteN expression in WT and MKP-1 -/- macrophages during muscle regeneration at the indicated time points after Ctx injury. (B) Proposed model of the crosstalk between the p38/mkp-1 and PI3K/AKT pathways during muscle regeneration, involving mir-21-mediated downregulation of PteN. Alternative pathways of AKT activation by p38 cannot be discarded. Figure 6. The p38/mkp-1 and AKT pathways as regulators of pro- to anti-inflammatory cytokine expression transition in macrophages during endotoxin tolerance and tissue repair. (A) Diagram illustrating the major common pathways regulating cytokine gene expression in macrophages in endotoxin tolerance and tissue repair (as discussed in the text). are present at later repair stages. 49 We have found that loss of MKP-1 results in a higher number of muscle macrophages with activated p38 at three days post injury 50 (see Fig. 5A, left). Moreover, in contrast to wild-type macrophages, MKP-1 -/- muscle macrophages expressed high levels of anti-inflammatory cytokines in addition to pro-inflammatory cytokines three days after injury, thus resulting in a temporal overlap of pro- and anti-inflammatory cytokine expression 50 (Fig. 3); of note, the anticipated anti-inflammatory gene expression program was p38-dependent, since it could be neutralized with the p38α/β-specific inhibitor SB The Ly-6C low macrophage population present in 6-d-damaged wild-type muscle 882 Cell Cycle volume 11 Issue 5

7 expressed high levels of anti-inflammatory cytokines, 50 suggesting that, as reparative myofiber growth commences, the antiinflammatory cytokine program is required to shut down the initial pro-inflammatory macrophage response. This resembles the period of endotoxin tolerance in which the anti-inflammatory genes are induced to levels comparable to those genes activated immediately after a bacterial challenge. Importantly, we also found that macrophages were still present in muscle tissue at very advanced regeneration stages (8 and 10 d after injury) (Fig. 1C). Interestingly, at these late stages, we have previously found that muscle macrophages ceased to express either type of cytokines, 50 implying that as satellite cell functions are turned down and tissue approaches full recovery, macrophages turn to a deactivated state (Fig. 4). Unexpectedly, in 6-d-injured muscle, Ly-6C low macrophages expressed almost undetectable levels of anti-inflammatory (and pro-inflammatory) cytokines in the absence of MKP-1 (Fig. 4), indicating that premature muscle macrophage deactivation occurred during the repair process, and that this could be prevented by inhibiting p38 activity. 50 Thus, as tissue repair advances, the balance struck between p38 and MKP-1 controls, first, the polarization of pro- to anti-inflammatory macrophages and, later, the transition to the deactivated state. Macrophage Activation- Deactivation Transitions during Tissue Repair are Mediated by the p38/akt-regulated AKT/PTEN Axis The regulatory mechanisms controlling cytokine gene silencing and macrophage deactivation remain largely undeciphered. As indicated above, not only the p38/ MKP-1 balance but also the PI3K/AKT pathway have been implicated in bacterial tolerance; however, the function of the PI3K/AKT pathway remains controversial, as it exerts both positive and negative actions on cytokine expression in endotoxin-activated macrophages. 55,56 We investigated the AKT activation status of wild-type and MKP-1 -/- macrophages during the deactivating period of the muscle repair process. The activity of AKT was higher in MKP-1 -/- than in wild-type macrophages at day 6 after muscle injury (Fig. 5A), correlating with a loss of proand anti-inflammatory cytokine gene expression in the absence of MKP-1, and this effect was reverted by pharmacologically inhibiting p38 activity. 50 Treating wild-type mice with the PI3K/AKT inhibitor wortmannin prolonged the activated status in macrophages by preventing their final deactivation. 50 Taken together, these results strongly support a role for MKP-1 in neutralizing p38 and thereby controlling sequential macrophage activationdeactivation transitions during satellite cell-controlled tissue repair by restraining AKT activation. This newly identified macrophage state is characterized by cytokine gene silencing and may serve a function similar during tissue repair similar to that of endotoxin tolerance during bacterial infection. The final transition during tissue repair is regulated by a p38- AKT pathway. Whether an inverse AKTp38 signaling loop (as occurs in endotoxin tolerance) is also operative during macrophage deactivation in tissue repair remains to be investigated. Our recent results also unveiled a potential link between the p38/mkp-1 axis and the activation of AKT 50 (Fig. 5). Consistent with an enhanced AKT activation, PTEN levels were lower in MKP-1 -/- as compared with wild-type macrophages at day 6 after injury (Fig. 5A). PTEN is a bona fide target of mir-21, 57 an mir classically associated with cancer and fibrosis, and was reported to be expressed in RAW macrophages, thus suggesting that the mir-21/pten axis is operative in macrophages. Since mir-21 expression increased in deactivated macrophages in a p38-dependent manner, 50 we speculate that the loss of MKP-1, through regulating the mir-21/pten/akt pathway, extends macrophage cell persistence while provoking their premature deactivation during the tissue repair process. Recent studies have shown that inhibiting mir-21 may increase NFκB activation and the proinflammatory cytokine IL-6 and decrease the levels of the anti-inflammatory cytokine IL ,59 Thus, mir-21 may act as an anti-inflammatory agent within a negative regulatory loop in response to bacterial infection. This stresses that context-specific differences may underlie the diverse pathways utilized by mir-21 to regulate inflammation: in LPS-stimulated macrophages, mir-21 leads to NFκB activation and thus promotes both inflammation and anti-inflammatory gene expression. In injured muscle tissue, mir-21 acts to downregulate PTEN in macrophages, thereby activating AKT and leading to resolution of inflammation 50 (Fig. 5B). Therefore, p38/mkp-1 provides a signaling pathway that regulates the initiation of inflammation and the subsequent endotoxin tolerance. Additionally, it also functions through mir-21/akt as part of the positive feedback loop that underlies the switch linking the initial pro-inflammatory response after injury to inflammation resolution associated with tissue repair (Figs. 5B and 6). The p38/mkp-1-creb Pathway as a Common Mechanism for Macrophage Polarization in Response to Bacterial Infection and Tissue Injury Interestingly, some studies have shown that the camp response element-binding protein (CREB) has an important antiinflammatory role in macrophages in response to LPS that is mediated by the mitogen- and stress-activated kinases 1 and 2 (MSK1 and MSK2), which are, in turn, activated by p38. 60,61 In particular, CREB-induced expression of IL-10 and MKP-1 was shown to inhibit the expression of pro-inflammatory genes associated with M1 macrophage activation in a model of toxic contact eczema induced by phorbol-12-myristate-13-acetate, thereby supporting the link between p38/mapk-1 and CREB in macrophage polarization transitions. In the context of tissue repair, an important regulatory function for CREB in macrophage polarization has been revealed by the finding that M2, but not M1, gene expression was impaired by deleting two CREB-binding sites from the C/EBPβ gene promoter, leading to abnormal muscle regeneration. 62 Indeed, macrophages from the C/EBPβ promoter mutant mice had a reduced expression of the M2 arginase gene after LPS stimulation; it was hypothesized that this leads to a switch in arginine metabolism from Cell Cycle 883

8 arginase-mediated polyamine synthesis to inos-mediated NO production. 62 In line with this, it was observed that shifts in macrophage polarization and macrophage competition for arginine metabolism influenced the severity of the muscle pathology in mdx dystrophic mice. 63 These studies strongly support the idea that CREB might be a pivotal transcription factor in macrophage polarization that functions by promoting M2-associated genes while repressing M1 activation, with CREB transcriptional activity regulated by the p38/msk1/2-mkp-1 balance (Fig. 6). Further investigating the mechanistic basis of these complex macrophage phenotype dynamics in vivo will be necessary but appears extremely challenging. Concluding Remarks Unsuccessful repair after damage leads to the derangement of tissue structure and loss of its normal function. Our recent findings clearly demonstrate that dysregulated macrophage transitions, with precocious pro- to anti-inflammatory polarization followed by immediate cessation of cytokine expression, impair tissue damage resolution and efficient repair. In particular, we have uncovered a new function of macrophage-specific MKP-1 in regulating the evolution as well as the resolution of inflammation during muscle stem cell-dependent tissue repair by controlling p38-mediated AKT activation. Generating and characterizing mouse models with cell type-specific inactivation of MKP-1 should provide powerful biological tools to address the role of this phosphatase in the regulation of inflammation and stem cell responses during tissue repair in physiological and pathological conditions. For instance, dysregulated p38 activity has been associated with the aging process, 64,65 which, considering the prominent role of inflammation in the control of regeneration, might be linked to the decline of muscle regenerative capacity during aging. 3 Furthermore, we propose that the pro- to anti-inflammatory polarization switch of macrophages during the process of tissue repair after injury shares a similar function to that occurring during endotoxin tolerance. Notably, the p38/mkp-1 balance also appears to be critically involved in the process of bacterial tolerization. More interestingly, crosstalk between the p38 and AKT pathways appear to occur in both processes. Nevertheless, mechanistic insight into the inflammatory responses to distinct types of insults remains to be elucidated. Identifying the relative role of the distinct p38 (and MKP) family members and their upstream signaling mechanisms, extracellular activating cues and downstream effectors will help us to understand how balancing the activities of p38 MAPKs and MKPs can regulate both the initiation and the resolution of inflammation in distinct stress contexts. Acknowledgements We are indebted to Drs. C. Caelles, M. Jardí, V. Ruiz-Bonilla and P. Souza- Victor for their contributions to this study and Dr. J. Martín-Caballero for assistance in animal care. The authors acknowledge funding from the Ministerio de Ciencia e Innovación (PLE , SAF , FIS-PS09/01267 and Centro de Investigación Biomédica en Red, Enfermedades Neurodegenerativas), AFM, Fundació La Marató de TV3, Muscular Dystrophy Association and MYOAGE, OptiStem and EndoStem (EU-FP7). We thank V.A. Raker and M. Raya for help in preparing the manuscript. References 1. Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. 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