The leukotriene B4 receptor, BLT1, is required for the induction of experimental autoimmune encephalomyelitis

Leukotriene B₄ (LTB₄) is a potent chemoattractant and activator of neutrophils, macrophages and T cells. These cells are a key component of inflammation and all express BLT1, a high affinity G-protein-coupled receptor for LTB₄. However, little is known about the neuroimmune functions of BLT1. In this study, we describe a distinct role for BLT1 in the pathology of experimental autoimmune encephalomyelitis (EAE) and T_H1/T_H17 immune responses. BLT1 mRNA was highly upregulated in the spinal cord of EAE mice, especially during the induction phase. BLT1^−/− mice had delayed onset and less severe symptoms of EAE than BLT1^+/+ mice. Additionally, inflammatory cells were recruited to the spinal cord of asymptomatic BLT1^+/+, but not BLT1^−/− mice before the onset of disease. Ex vivo studies showed that both the proliferation and the production of IFN-γ, TNF-α, IL-17 and IL-6 were impaired in BLT1^−/− cells, as compared with BLT1^+/+ cells. Thus, we suggest that BLT1 exacerbates EAE by regulating the migration of inflammatory cells and T_H1/T_H17 immune responses. Our findings provide a novel therapeutic option for the treatment of multiple sclerosis and other T_H17-mediated diseases.     

Leukotriene B4 receptors contribute to house dust mite-induced eosinophilic airway inflammation via TH2 cytokine production

Leukotriene B₄ (LTB₄) is a lipid mediator of inflammation that is generated from arachidonic acid via the 5-lipoxygenase pathway. Previous studies have reported that the receptors of LTB₄, BLT1, and BLT2 play mediatory roles in the allergic airway inflammation induced by ovalbumin (OVA). However, considering that house dust mites (HDMs) are the most prevalent allergen and well-known risk factor for asthmatic allergies, we are interested in elucidating the contributory roles of BLT1/2 in HDM-induced allergic airway inflammation. Our aim in this study was to investigate whether BLT1/2 play any roles in HDM-induced allergic airway inflammation. In this study, we observed that the levels of ligands for BLT1/2 [LTB₄ and 12(S)-HETE (12(S)-hydroxyeicosatetraenoic acid)] were significantly increased in bronchoalveolar lavage fluid (BALF) after HDM challenge. Block-ade of BLT1 or BLT2 as well as of 5-lipoxygenase (5-LO) or 12-lipoxygenase (12-LO) markedly suppressed the production of T_H2 cytokines (IL-4, IL-5, and IL-13) and alleviated lung inflammation and mucus secretion in an HDM-induced eosinophilic airway-inflammation mouse model. Together, these results indicate that the 5-/12-LO-BLT1/2 cascade plays a role in HDM-induced airway inflammation by mediating the production of T_H2 cytokines. Our findings suggest that BLT1/2 may be a potential therapeutic target for patients with HDM-induced allergic asthma.     

KDM4A regulates myogenesis by demethylating H3K9me3 of myogenic regulatory factors

Histone lysine demethylase 4A (KDM4A) plays a crucial role in regulating cell proliferation, cell differentiation, development and tumorigenesis. However, little is known about the function of KDM4A in muscle development and regeneration. Here, we found that the conditional ablation of KDM4A in skeletal muscle caused impairment of embryonic and postnatal muscle formation. The loss of KDM4A in satellite cells led to defective muscle regeneration and blocked the proliferation and differentiation of satellite cells. Myogenic differentiation and myotube formation in KDM4A-deficient myoblasts were inhibited. Chromatin immunoprecipitation assay revealed that KDM4A promoted myogenesis by removing the histone methylation mark H3K9me3 at MyoD, MyoG and Myf5 locus. Furthermore, inactivation of KDM4A in myoblasts suppressed myoblast differentiation and accelerated H3K9me3 level. Knockdown of KDM4A in vitro reduced myoblast proliferation through enhancing the expression of the cyclin-dependent kinase inhibitor P21 and decreasing the expression of cell cycle regulator Cyclin D1. Together, our findings identify KDM4A as an important regulator for skeletal muscle development and regeneration, orchestrating myogenic cell proliferation and differentiation.Subject terms: Differentiation, Muscle stem cells, Epigenetics     

Contributory Role of BLT2 in the Production of Proinflammatory Cytokines in Cecal Ligation and Puncture-Induced Sepsis

BLT2 is a low-affinity receptor for leukotriene B₄, a potent lipid mediator of inflammation generated from arachidonic acid via the 5-lipoxygenase pathway. The aim of this study was to investigate whether BLT2 plays any role in sepsis, a systemic inflammatory response syndrome caused by infection. A murine model of cecal ligation and puncture (CLP)-induced sepsis was used to evaluate the role of BLT2 in septic inflammation. In the present study, we observed that the levels of ligands for BLT2 (LTB₄ [leukotriene B₄] and 12(S)-HETE [12(S)-hydroxyeicosatetraenoic acid]) were significantly increased in the peritoneal lavage fluid and serum from mice with CLP-induced sepsis. We also observed that the levels of BLT2 as well as 5-lipoxygenase (5-LO) and 12-LO, which are synthesizing enzymes for LTB₄ and 12(S)-HETE, were significantly increased in lung and liver tissues in the CLP mouse model. Blockade of BLT2 markedly suppressed the production of sepsis-associated cytokines (IL-6 [interleukin-6], TNF-α[[tumor necrosis factor alpha], and IL-1β [interleukin-1β] as well as IL-17 [interleukin-17]) and alleviated lung inflammation in the CLP group. Taken together, our results suggest that BLT2 cascade contributes to lung inflammation in CLP-induced sepsis by mediating the production of inflammatory cytokines. These findings suggest that BLT2 may be a potential therapeutic target for sepsis patients.     

Leukotriene B4 receptors as therapeutic targets for ophthalmic diseases

Leukotriene B₄ (LTB₄) is an inflammatory lipid mediator produced from arachidonic acid by multiple reactions catalyzed by two enzymes 5-lipoxygenase (5-LOX) and LTA₄ hydrolase (LTA₄H). The two receptors for LTB₄ have been identified: a high-affinity receptor, BLT1, and a low-affinity receptor, BLT2. Our group identified 12(S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid (12-HHT) as a high-affinity BLT2 ligand. Numerous studies have revealed critical roles for LTB₄ and its receptors in various systemic diseases. Recently, we also reported the roles of LTB₄, BLT1 and BLT2 in the murine ophthalmic disease models of mice including cornea wound, allergic conjunctivitis, and age-related macular degeneration. Moreover, other groups revealed the evidence of the ocular function of LTB₄. In the present review, we introduce the roles of LTB₄ and its receptors both in ophthalmic diseases and systemic inflammatory diseases. LTB₄ and its receptors are putative novel therapeutic targets for systemic and ophthalmic diseases.     

EETs Attenuate Ox-LDL-Induced LTB4 Production and Activity by Inhibiting p38 MAPK Phosphorylation and 5-LO/BLT1 Receptor Expression in Rat Pulmonary Arterial Endothelial Cells

Cytochrome P-450 epoxygenase (EPOX)-derived epoxyeicosatrienoic acids (EETs), 5-lipoxygenase (5-LO), and leukotriene B₄ (LTB₄), the product of 5-LO, all play a pivotal role in the vascular inflammatory process. We have previously shown that EETs can alleviate oxidized low-density lipoprotein (ox-LDL)-induced endothelial inflammation in primary rat pulmonary artery endothelial cells (RPAECs). Here, we investigated whether ox-LDL can promote LTB₄ production through the 5-LO pathway. We further explored how exogenous EETs influence ox-LDL-induced LTB₄ production and activity. We found that treatment with ox-LDL increased the production of LTB₄ and further led to the expression and release of both monocyte chemoattractant protein-1 (MCP-1/CCL2) and intercellular adhesion molecule-1 (ICAM-1). All of the above ox-LDL-induced changes were attenuated by the presence of 11,12-EET and 14,15-EET, as these molecules inhibited the 5-LO pathway. Furthermore, the LTB₄ receptor 1 (BLT1 receptor) antagonist U75302 attenuated ox-LDL-induced ICAM-1 and MCP-1/CCL2 expression and production, whereas LY255283, a LTB₄ receptor 2 (BLT2 receptor) antagonist, produced no such effects. Moreover, in RPAECs, we demonstrated that the increased expression of 5-LO and BLT1 following ox-LDL treatment resulted from the activation of nuclear factor-κB (NF-κB) via the p38 mitogen-activated protein kinase (MAPK) pathway. Our results indicated that EETs suppress ox-LDL-induced LTB₄ production and subsequent inflammatory responses by downregulating the 5-LO/BLT1 receptor pathway, in which p38 MAPK phosphorylation activates NF-κB. These results suggest that the metabolism of arachidonic acid via the 5-LO and EPOX pathways may present a mutual constraint on the physiological regulation of vascular endothelial cells.     

Six family genes control the proliferation and differentiation of muscle satellite cells

Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4^+/−Six5^−/− mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications.     

BLT1-mediated O-GlcNAcylation is required for NOX2-dependent migration,exocytotic degranulation and IL-8 release of human mast cell induced by Trichomonas vaginalis-secreted LTB4

Trichomonas vaginalis is a sexually-transmitted protozoan parasite that causes vaginitis and cervicitis. Although mast cell activation is important for provoking tissue inflammation during infection with parasites, information regarding the signaling mechanisms in mast cell activation and T. vaginalis infection is limited. O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of serine and threonine residues that functions as a critical regulator of intracellular signaling, regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). We investigated if O-GlcNAcylation was associated with mast cell activation induced by T. vaginalis-derived secretory products (TvSP). Modified TvSP collected from live trichomonads treated with the 5-lipooxygenase inhibitor AA861 inhibited migration of mast cells. This result suggested that mast cell migration was caused by stimulation of T. vaginalis-secreted leukotrienes. Using the BLT1 antagonist U75302 or BLT1 siRNA, we found that migration of mast cells was evoked via LTB₄ receptor (BLT1). Furthermore, TvSP induced protein O-GlcNAcylation and OGT expression in HMC-1 cells, which was prevented by transfection with BLT1 siRNA. TvSP-induced migration, ROS generation, CD63 expression and IL-8 release were significantly suppressed by pretreatment with OGT inhibitor ST045849 or OGT siRNA. These results suggested that BLT1-mediated OGlcNAcylation was important for mast cell activation during trichomoniasis.     

Myostatin Suppression of Akirin1 Mediates Glucocorticoid-Induced Satellite Cell Dysfunction

Glucocorticoids production is increased in many pathological conditions that are associated with muscle loss, but their role in causing muscle wasting is not fully understood. We have demonstrated a new mechanism of glucocorticoid-induced muscle atrophy: Dexamethasone (Dex) suppresses satellite cell function contributing to the development of muscle atrophy. Specifically, we found that Dex decreases satellite cell proliferation and differentiation in vitro and in vivo. The mechanism involved Dex-induced upregulation of myostatin and suppression of Akirin1, a promyogenic gene. When myostatin was inhibited in Dex-treated mice, Akirin1 expression increased as did satellite cell activity, muscle regeneration and muscle growth. In addition, silencing myostatin in myoblasts or satellite cells prevented Dex from suppressing Akirin1 expression and cellular proliferation and differentiation. Finally, overexpression of Akirin1 in myoblasts increased their expression of MyoD and myogenin and improved cellular proliferation and differentiation, theses improvements were no longer suppressed by Dex. We conclude that glucocorticoids stimulate myostatin which inhibits Akirin1 expression and the reparative functions of satellite cells. These responses attribute to muscle atrophy. Thus, inhibition of myostatin or increasing Akirin1 expression could lead to therapeutic strategies for improving satellite cell activation and enhancing muscle growth in diseases associated with increased glucocorticoid production.     

TAM kinase signaling is indispensable for proper skeletal muscle regeneration in mice

Skeletal muscle regeneration following injury results from the proliferation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibers. Infiltrating macrophages play an essential role in the process partly by clearing the necrotic cell debris, partly by producing cytokines that guide myogenesis. Infiltrating macrophages are at the beginning pro-inflammatory, but phagocytosis of dead cells induces a phenotypic change to become healing macrophages that regulate inflammation, myoblast fusion and growth, fibrosis, vascularization and return to homeostasis. The TAM receptor kinases Mer and Axl are known efferocytosis receptors in macrophages functioning in tolerogenic or inflammatory conditions, respectively. Here we investigated their involvement in the muscle regeneration process by studying the muscle repair following cardiotoxin-induced injury in Mer^−/− mice. We found that Axl was the only TAM kinase receptor expressed on the protein level by skeletal muscle and C2C12 myoblast cells, while Mer was the dominant TAM kinase receptor in the CD45⁺ cells, and its expression significantly increased during repair. Mer ablation did not affect the skeletal muscle weight or structure, but following injury it resulted in a delay in the clearance of necrotic muscle cell debris, in the healing phenotype conversion of macrophages and consequently in a significant delay in the full muscle regeneration. Administration of the TAM kinase inhibitor BMS-777607 to wild type mice mimicked the effect of Mer ablation on the muscle regeneration process, but in addition, it resulted in a long-persisting necrotic area. Finally, in vitro inhibition of TAM kinase signaling in C2C12 myoblasts resulted in decreased viability and in impaired myotube growth. Our work identifies Axl as a survival and growth receptor in the mouse myoblasts, and reveals the contribution of TAM kinase-mediated signaling to the skeletal muscle regeneration both in macrophages and in myoblasts.Subject terms: Mechanisms of disease, Immunological disorders     

白三烯B4受体对类风湿关节炎的免疫调节作用

目的：白三烯B4（LTB4）受体包括BLT1和BLT2两个亚型,利用各自的特异性拮抗剂U-75302、LY255283,通过体外实验分析LTB4受体在炎症免疫反应中介导的作用。方法：①以U-75302和LY255283分别阻断BLT1和BLT2,用OH—TdR掺入法检测大鼠滑膜细胞的增殖情况。②流式细胞术检测U-75302或LY255283对大鼠脾CD4^＋T细胞中IFN-γ和IL-4表迭的影响,分析Th1、Th2细胞的百分率。结果：①U-75302仅在浓度为10μmol／L时方可检测到对滑膜细胞增殖的明显抑制作用（P〈0．05）,而LY255283在10nmol／L～10μmol／L范围内对滑膜细胞均有明显的抑制作用（P〈0．05）。②流式细胞术分析结果表明,当U-75302或LY255283达到10μmol／L时,可明显升高Th2细胞的百分率,而对Th1细胞的百分率影响不明显。结论：BLT1和（或）BLT2介入了大鼠滑膜细胞增殖、Th1／Th2淋巴细胞百分率变化,对于类风湿关节炎（rheumatoid arthritis,RA）等炎症疾病的防治而言,BLT1、BLT2可能具有重要的药物靶点意义。     

Mediatory role of BLT2 in the proliferation of KRAS mutant colorectal cancer cells

Inflammatory lipid mediators play various roles in colorectal cancer progression through complex pathways. However, the mechanism by which lipoxygenase-derived inflammatory lipid mediators contribute to colorectal cancer progression remains elusive. In this study, we found that BLT2, a cell surface GPCR for leukotriene B₄ and 12‑hydroxyeicosatetraenoic acid, is highly upregulated in KRAS mutant LOVO and SW480 colorectal cancer cells and plays critical roles in mediating proliferation through activation of phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (Akt) and subsequent upregulation of cyclin D1. Exposure to BLT2 siRNA or LY255283, a specific BLT2 inhibitor, clearly suppressed the proliferation of KRAS mutant colorectal cancer cells and markedly increased cell cycle arrest by downregulating the PI3K/Akt-cyclin D1 cascade. Xenograft tumor formation by LOVO and SW480 cells in athymic mice was also substantially reduced by treatment with the BLT2 inhibitor in vivo. Together, our study demonstrates that BLT2 is necessary for the proliferation of LOVO and SW480 cells and thus may be a potential therapeutic target for the treatment of KRAS mutant colorectal cancer.     

Bioactive lipoxygenase metabolites stimulation of NADPH oxidases and reactive oxygen species

In mammalian cells, reactive oxygen species (ROS) are produced via a variety of cellular oxidative processes, including the activity of NADPH oxidases (NOX), the activity of xanthine oxidases, the metabolism of arachidonic acid (AA) by lipoxygenases (LOX) and cyclooxygenases (COX), and the mitochondrial respiratory chain. Although NOX-generated ROS are the best characterized examples of ROS in mammalian cells, ROS are also generated by the oxidative metabolism (e.g., via LOX and COX) of AA that is released from the membrane phospholipids via the activity of cytosolic phospholipase A₂ (cPLA₂). Recently, growing evidence suggests that LOX- and COX-generated AA metabolites can induce ROS generation by stimulating NOX and that a potential signaling connection exits between the LOX/COX metabolites and NOX. In this review, we discuss the results of recent studies that report the generation of ROS by LOX metabolites, especially 5-LOX metabolites, via NOX stimulation. In particular, we have focused on the contribution of leukotriene B₄ (LTB₄), a potent bioactive eicosanoid that is derived from 5-LOX, and its receptors, BLT1 and BLT2, to NOX stimulation through a signaling mechanism that leads to ROS generation.     

A new method to evaluate anti-allergic effect of food component by measuring leukotriene B<Subscript>4</Subscript> from a mouse mast cell line

Leukotrienes (LTs), chemical mediators produced by mast cells, play an important role in allergic symptoms such as food allergies and hay fever. We tried to construct an evaluation method for the anti-LTB₄ activity of chemical substances using a mast cell line, PB-3c. PB-3c pre-cultured with or without arachidonic acid (AA) was stimulated by calcium ionophore (A23187) for 20 min, and LTB₄ production by the cells was determined by HPLC with UV detection. LTB₄ was not detected when PB-3c was pre-cultured without AA. On the other hand, LTB₄ production by PB-3c pre-cultured with AA was detectable by HPLC, and the optimal conditions of PB-3c for LTB₄ detection were to utilize the cells pre-cultured with 50 µM AA for 48 h. MK-886 (5-lipoxygenase inhibitor) completely inhibited LTB₄ production, but AACOCF₃ (phospholipase A₂ inhibitor) slightly increased LTB₄ production, suggesting that LTB₄ was generated from exogenous free AA through 5-lipoxygenase pathway. We applied this technique to the evaluation of the anti-LTB₄ activity of food components. PB-3c pre-cultured with 50 µM AA for 48 h was stimulated with A23187 in the presence of 50 µM soybean isoflavones (daidzin, genistin, daidzein, and genistein), equol, quercetin, or kaempferol. Genistein, equol, quercetin, and kaempferol strongly inhibited LTB₄ production without cytotoxicity. These results suggest that a new assay system using PB-3c is convenient to evaluate LTB₄ inhibition activity by food components. This method could be utilized for elucidation of the mechanisms of LTB₄ release suppression by food components such as flavonoids and the structure–activity relationship.     

Isolation,Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.     

Brain-derived Neurotrophic Factor Regulates Satellite Cell Differentiation and Skeltal Muscle Regeneration

In adult skeletal muscle, brain-derived neurotrophic factor (BDNF) is expressed in myogenic progenitors known as satellite cells. To functionally address the role of BDNF in muscle satellite cells and regeneration in vivo, we generated a mouse in which BDNF is specifically depleted from skeletal muscle cells. For comparative purposes, and to determine the specific role of muscle-derived BDNF, we also examined muscles of the complete BDNF^−/− mouse. In both models, expression of the satellite cell marker Pax7 was significantly decreased. Furthermore, proliferation and differentiation of primary myoblasts was abnormal, exhibiting delayed induction of several markers of differentiation as well as decreased myotube size. Treatment with exogenous BDNF protein was sufficient to rescue normal gene expression and myotube size. Because satellite cells are responsible for postnatal growth and repair of skeletal muscle, we next examined whether regenerative capacity was compromised. After injury, BDNF-depleted muscle showed delayed expression of several molecular markers of regeneration, as well as delayed appearance of newly regenerated fibers. Recovery of wild-type BDNF levels was sufficient to restore normal regeneration. Together, these findings suggest that BDNF plays an important role in regulating satellite cell function and regeneration in vivo, particularly during early stages.     

Exosomes derived from inflammatory myoblasts promote M1 polarization and break the balance of myoblast proliferation/differentiation

BACKGROUNDAcute muscle injuries are one of the most common injuries in sports. Severely injured muscles are prone to re-injury due to fibrotic scar formation caused by prolonged inflammation. How to regulate inflammation and suppress fibrosis is the focus of promoting muscle healing. Recent studies have found that myoblasts and macrophages play important roles in the inflammatory phase following muscle injury; however, the crosstalk between these two types of cells in the inflammatory environment, particularly the exosome-related mechanisms, had not been well studied. AIMTo evaluate the effects of exosomes from inflammatory C2C12 myoblasts (IF-C2C12-Exos) on macrophage polarization and myoblast proliferation/differentiation.METHODSA model of inflammation was established in vitro by lipopolysaccharide stimulation of myoblasts. C2C12-Exos were isolated and purified from the supernatant of myoblasts by gradient centrifugation. Multiple methods were used to identify the exosomes. Gradient concentrations of IF-C2C12-Exos were added to normal macrophages and myoblasts. PKH67 fluorescence tracing was used to identify the interaction between exosomes and cells. Microscopic morphology, Giemsa stain, and immunofluorescence were carried out for histological analysis. Additionally, ELISA assays, flow cytometry, and western blot were conducted to analyze molecular changes. Moreover, myogenic proliferation was assessed by the BrdU test, scratch assay, and CCK-8 assay.RESULTSWe found that the PKH-67-marked C2C12-Exos can be endocytosed by both macrophages and myoblasts. IF-C2C12-Exos induced M1 macrophage polarization and suppressed the M2 phenotype in vitro. In addition, these exosomes also stimulated the inflammatory reactions of macrophages. Furthermore, we demonstrated that IF-C2C12-Exos disrupted the balance of myoblast proliferation/differentiation, leading to enhanced proliferation and suppressed fibrogenic/myogenic differentiation.CONCLUSIONIF-C2C12-Exos can induce M1 polarization, resulting in a sustained and aggravated inflammatory environment that impairs myoblast differentiation, and leads to enhanced myogenic proliferation. These results demonstrate why prolonged inflammation occurs after acute muscle injury and provide a new target for the regulation of muscle regeneration.