Role of Exosomal miR‐223 in Chronic Skeletal Muscle Inflammation

As skeletal muscle is one of the largest organs in the body, its damage can directly reflect a decline in somatic function, thus, further affecting daily life and health. Inflammation is a prerequisite for the repair of injured skeletal muscles. Chronic inflammation induced by inadequate repair in skeletal muscle aggravates tissue injury. Exosomes regulate inflammatory responses to facilitate the repair of skeletal muscle injury. Moreover, exosomal miR‐223 with high specificity is the most abundant miRNA in peripheral blood and regarded as biomarkers for inflammation post skeletal muscle injury, which warrants further investigation. Available studies have demonstrated that exosomal miR‐223 negatively correlates with TNF‐α levels in serum and regulates the canonical inflammatory NF‐κB signaling pathway. miR‐223 is a negative feedback regulator with great potential for adjusting inflammatory imbalance and promoting skeletal muscle repair. The research on the regulation of negative feedback factors in the inflammatory signaling pathway is essential in biology and medicine. Therefore, this review mainly elaborates the formation, heterogeneity and markers of exosomes and points out exosomal miR‐223 as a beneficial role in chronic skeletal muscle inflammation and can be expected to be a potential therapeutic target for skeletal muscle damage.


Introduction
A s one of the largest organs in the human body 1,2 , skeletal muscle plays a key role in exercise 3 , breathing 4 , and metabolism 5 , participating in many vitalphysiological functions of the living body 6,7 . Generally, 50% of the cases of acute injury are transformed into chronic due to the limitation of diagnosis and treatment and long-term negligence of the harmfulness of skeletal muscle inflammation. For instance, chronic inflammation after lumbar multifidus muscle injury could result in recurrent low back pain, which is refractory for a long time 8,9 . Therefore, it is one of the clinical problems that chronic inflammation after skeletal muscle injury demands urgent attention. Current studies have unraveled exosomes correlate with inflammatory diseases 10,11 and have potential advantages in the targeted therapy of chronic inflammation after muscle injury 12 to emerge as a state-of-the-art therapeutic strategy for treating chronic inflammation of skeletal muscle 13,14 .
Most cells secrete exosomes, and the functions of exosomes depend on the type of cell from which they originate 15 . Exosomes contain specific microRNAs (miRNAs), proteins, and other biologically active substances, which have great potential to be applied as non-invasive markers of diseases 16,17 . Patients with different diseases release exosomes containing specific RNAs and proteins into the circulation [18][19][20] . An existing study has demonstrated that miR-223 is the most abundant miRNA in the microvesicles isolated from peripheral blood of healthy donors 21 . These microvesicles may be originated from peripheral blood mononuclear cells which are the main source of circulating exosomes 22 . Furthermore, several studies have substantiated the implications of exosomal miR-223 in the inflammatory response to skeletal muscle injury 17,23,24 . It has been reported that miR-223 can down-regulate TNF-α and other pro-inflammatory factors [25][26][27] , suppresses inflammatory infiltration, and reduce the area of necrotic muscle tissues 28 . Therefore, miR-223 may be a potential biomarker and therapeutic target in chronic inflammation related to insufficient regeneration and repair following muscle injury, although which warrants further verification.

Formation of Exosomes
Since 2011, the International Society for Extracellular Vesicles (ISEV: www.isev.org/) has paid attention to unifying the name and isolation methods of extracellular vesicles (EVs) 29 . EVs are classified in the latest research into two categories, namely, ectosomes and exosomes 30 . Ectosomes are vesicles (50 nm-1 μm in diameter) directly derived from the plasma membrane that include microvesicles, microparticles, and large vesicles; whereas, exosomes are nanosized extracellular membrane vesicles of endosomal origin secreted by most cell types with a diameter of 40-160 nm (average diameter of 100 nm) and a density of 1.13-1.19 g/ml 31 . They are typically cup-shaped and shuttle-shaped vesicles wrapped by a double-layer lipid membrane with an average thickness less than 5 nm 32,33 .
The production of exosomes is a special formation process. In brief, cytoskeleton proteins (such as actin and tubulin) interact with clathrin to form vesicles covered with clathrin through endocytosis and in vagination of the cell membrane. These vesicles after clathrin uncoating are known as early sorting endosomes (ESEs) 34 . Small molecules including proteins, mRNAs, and miRNAs derived from organelles such as endoplasmic reticulum, Golgi bodies, and mitochondria could be selectively transferred into early endosomes by two pathways, one of which is endosomal sorting complex required for transport (ESCRT)-dependent and another one is ESCRT-independent. The ESCRT-independent pathway is mediated bytetraspanin membrane proteins (CD63, CD81, CD82, and CD9)and neutral sphingomyelinases 2 (nSMase2). Subsequently, reverse budding of early endosomes leads to the formation of intracellular vesicles enveloping small molecules, namely late sorting endosomes (LSEs) 35 . LSEs are gradually matured and transformed into multivesicular bodies (MVBs) containing exosomes. MVBs could not only be formed into autophagosomes or degraded by lysosomes but also be transported to the plasma membrane through the cytoskeleton and microtubule network. Depending on the Rab GTPase family, MVBs can release exosomes outside through exocytosis after fusion with the cell membrane 36 . During the formation process of exosomes, the specific endosomal proteins and some cellular contents are selectively sorted before the plasma membrane is sealed, forming the final contents of exosomes ( Figure 1A-G). The ongoing advancement of new technologies will also improve their classification. The classification of exosomes is particularly meaningful in biology as their production involves a unique intracellular regulatory process. Once secreted into the extracellular space, their composition and function are determined 37 .

Heterogeneity and Markers of Exosomes
Exosomes reflecting the source of cells are highly heterogeneous. The heterogeneity of exosomes is manifested with their size, content, functional effect on the recipient cells. Different combinations of these characteristics lead to the complex heterogeneity of exosomes. Proteomic analysis of EVs potentially reveals the heterogeneity of exosomes and reflects the specific proteins derived from parental cells 38 . Previous studies have shown that proteomics has been widely used for investigations on the role of exosomes in inflammatory diseases to identify the proteins that are differentially expressed in disease and normal conditions. Meanwhile, specific proteins rich in exosomes are usually used as marker proteins, including tetraspanin membrane proteins (CD9, CD63, CD81, CD82, CD151, and Tspan8), specific stress proteins such as the heat shock protein (HSP) family, TSG101, endosome sorting complex required for the transport-III (ESCRT-3)-binding protein ALIX, Rab GTPase family, cytoplasmic proteins, etc 39 . CD9, CD63, and CD81 are especially abundant in the exosomal membrane, and therefore frequently used for the identification of exosomes 40 .

Exosomal miRNAs
The protein, mRNA, and miRNA composition of exosomes are dependent on the parental cell origin. Exosomes can transfer these cargoes to regulate the function of the recipient cell. These biologically active molecules perform a fundamental role in cellular communication and are also a targeted marker to assess the progression and treatment efficacy of disease 41 . Since exosomes are rich in resident mRNA and miRNA 42 , they treat diseases by carrying specific RNA molecules at the gene level. With the development of molecular biology techniques, researchers have gradually discovered the critical role of exosomal miRNAs in the progression of many diseases 43 .

Exosomal miRNAs and Chronic Inflammation
Inflammation is defined as a preventive response to stimuli (such as pathogens or damages), the purpose of which is to eliminate stimuli, and to remove dead cells, inducing tissue repair. If controlled, beneficial outcomes are realized, otherwise, the consequences will be devastating if the stimuli persist or the inflammation has not been effectively resolved and become chronic. Typically, inflammation is classified as acute or chronic according to its intensity or duration. Exosome derived therapy emerges as one of the most advanced therapeutic strategies to modulate an overactive immune system. Immune cells actively secrete exosomes after noxious stimulation (such as pathogens or injuries). It has been reported that exosomes could act as either a promoter or inhibitor of inflammation, participating in immune regulation 44,45 . The exosome-based targeted therapy is a cell-free treatment that is long-term stability, and low or no immune response less toxic and produces fewer immune reactions 46 . There isincreasing concern for its role in the excessive inflammatory immunemediated response. Previous studies have evaluated the role of exosomal miRNAs in chronic inflammatory bowel disease (IBD) 47 , sepsis 48 , arthritis, 49 , diabetes 50 , atherosclerosis 51 , and neurodegenerative disease 52 , which suggests exosomes may potentially participate in the development of chronic inflammatory diseases.

Exosomal miRNAs and Chronic Inflammation in Skeletal Muscle
Following skeletal muscle injury, macrophages will be activated to polarize and a variety of cytokines are released to result in the inflammatory response. Regarding the process of inflammation after skeletal muscle injury, acute inflammatory damage has long been concerned while chronic inflammatory damage is ignored. In fact, injury 53 , dystrophy 54 , and aging 55 of skeletal muscle are accompanied by a state of lowlevel chronic inflammation, pro-inflammatory M1 macrophages were recruited into pathological muscle tissues, which shows the hidden dangers. Chronic inflammation is also a crucial cause of many diseases and complications, such as inflammation-cancer transformation 56,57 , type 2 diabetes 58 , dementia 59 and sarcopenia 60 . Currently, it is beneficial to use anti-inflammatory agents in the treatment of type 2 diabetes 61 . Long-term sustained chronic inflammation can induce degeneration of skeletal muscle and related organs, showing weakened muscle energy and muscular function in mild cases, and connective tissue excessive accumulation in severe cases which is manifested with severe pathological reactions. As chronic inflammation further impairs the normal regeneration and repair process after skeletal muscle injury, the skeletal muscle cannot be fully repaired 62,63 .
Current research on the inflammation after skeletal muscle injury mainly focuses on examining the implication of a single cytokine in skeletal muscle damage. Different respective contents of exosomes can be optimized for treatment at different times of skeletal muscle injury. Although it is the first important step in the investigation of mechanisms underlying inflammation in skeletal muscle, the comprehensive roles of selected cytokines (such as pro-inflammatory factors) may not be predictable in a more complex in vivo environment, because dozens of regulatory proteins and their receptors fluctuate rapidly in vivo. Therefore, more critical information about inflammatory cells and their specific inflammatory factors in the process of skeletal muscle regeneration and repair is needed. Exosomes have gradually emerged as messengers of this key information on muscle damage [64][65][66] . They could mediate intercellular communication to produce various biological effects and high specificity of targeting. M1-macrophages and M2-macrophages release different inflammatory factors correspondingly. In general, NF-κB binds to its inhibitory protein (IκBs) in the cytoplasm and remains in an inactive state. When IKK is activated, IκB-α can be phosphorylated to remove its inhibition on NF-κB, and allow free NF-κB transferring into the nucleus to play the role of the transcription factor to produce TNF-α. The long-term presence of pro-inflammatory TNF-α continuously activates the NF-κB signaling pathway, which forms a vicious circle, eventually leading to chronic inflammation. MiR-223-mediated inhibition of IKK-α (a target of miR-223) in macrophages, inhibits the activation of the NF-κB inflammatory pathway and reduces the production of proinflammatory cytokines such as TNF-α, thus breaking the vicious circle of chronic inflammation.

Exosomal miR-223
Exosomes are cell-to-cell communication vesicles that transfer abundant miRNAs across long distances and ubiquitously exist in the circulation 67,68 . Then miR-223 is the most abundant miRNA in human peripheral blood microvesicles (exosomes, etc.). These microvesiclesmay be derived from peripheral blood mononuclear cells, the primary source of circulating exosomes 21,22 . MiR-223 was first bioinformatically identified, then specifically expressed in the hematopoietic system. It can limit inflammation and prevent indirect damage during infection 26,69 . Since persistently excessive expression of inflammatory factors may lead to chronic inflammation, miR-223 is a negative feedback inhibitor that has the potential to adjust the inflammatory imbalance and accelerate the resolution of inflammatory processes 70,71 . It can be seen that the research on the regulation of negative feedback factors in the inflammatory signaling pathway is essential in biology and medicine.

Negative Feedback of miR-223 in Chronic Inflammation
It is reported that miR-223 can regulate neutrophil activity and enhance macrophage IL-6 and IL-1β production. Notably, miR-223 plays a vital role in inhibiting the development of pro-inflammatory cells 69 . Certified targets for miR-223 that affect inflammation and infection include Pknox1 72 , granzyme B 73,74 , IKKα 75 , Roquin 76 , STAT3 74 . Specifically, miR-223 directly suppresses Pknox1 expression, which induces macrophage phenotype switch towards M2, as well as it attenuates NF-κB-mediated inflammation by targeting IKK-α expression. miR-223 can target Roquin (a negative regulator of IL-17 production in lymphocytes) early in the myeloid lineage. Additionally, the up-regulation of inducible miR-223 reduces TLR-triggered IL-6 and IL-1β production in macrophages by targeting STAT3 at the transcriptional level. For other immune cells, miR-223 was down-modulated, thereby up-regulating its target gene, granzyme B, a significant component of cytotoxic T lymphocytes (CTLs) and NK cells granules. It is revealed that multiple functions of miR-223 are associated with inhibition of many different target genes with specificity not merely used as a biomarker and addresses the indispensable role in negatively regulating the inflammatory process 77 .
A large body of evidence supports that miR-223 is abnormally expressed in patient plasma in influenza 78 , chronic hepatitis B 79 , inflammatory bowel disease 80 , type 2 diabetes 81 , leukemic 82 , and lymphoma 83 .

Negative Feedback Mechanism of Exosomal miR-223 in Chronic Inflammation of Skeletal Muscle
Skeletal muscle macrophages participate in repair and regeneration following injury. Previous studies have reported that macrophages polarize into different phenotypes. It depends on which pathological mechanism of skeletal muscle injury is dominant. Exosomal MiR-223 involved in the chronic inflammation of skeletal muscle injury 12,84 . It could negatively regulate pro-inflammatory factors such as TNF-α 25-27 , inhibit inflammatory infiltration 28 , and eventually drive the ongoing inflammation to resolve.
An existing study has substantiated that the myeloid cells that initially invade into damaged muscle tissues are mainly neutrophils and pro-inflammatory M1-macrophages expressing iNOSat the early stages of chronic skeletal muscle inflammation, which clear away the damaged tissue 85 . As with acute muscle inflammatory injury, neutrophils and M1-macrophages aggravate muscle injury through iNOSmediated arginine metabolism. However, differing from acute inflammatory injury, the influx of neutrophils and M1-macrophages may be accompanied by the infiltration of M2-macrophages which exhibit an M2a-like phenotype during chronic inflammatory injury 85 . M2-macrophages are characterized by elevation of IL-4, IL-10, CD206, and CD163 as well as the expression of arginases 86 . M2a-like macrophages correlate with wound healing in other injured tissues and they can accelerate tissue repair and reduce inflammation. However, a pro-inflammatory/anti-inflammatory cytokine imbalance is found in chronic inflammatory injury, which may contribute to chronic condition.
An in-depth investigation has revealed the presence of a series of key pro-inflammatory factors, such as TNF-α, in the regeneration and repair process after skeletal muscle injury. TNF-α, a type of cytokine highly expressed by M1-macrophages, exacerbates muscle damage. After an acute injury, the expression of TNF-α in the muscle reaches its peak about 24 h after injury, which coincides with the time point when neutrophils and M1-macrophages invade into the muscle tissues and induce damag. 87 . The pro-inflammatory effect of TNF-α is mainly attributed to activation of NF-κB. In general, NF-κB binds to its inhibitory protein (IκBs) in the cytoplasm and remains as an inactive state. When IKK is activated by TNF-α stimulation, IκB-α can be phosphorylated to remove its inhibition on NF-κB, and allow free NF-κB transferring into the nucleus to play the role of the transcription factor to produce TNF-α. The long-term presence of proinflammatory TNF-α continuously activates NF-κB signaling pathway, which forms a vicious circle, eventually leading to chronic inflammation. It has been shown that exosome-derived miR-223 induces remission of chronic progressive inflammation. and maintains cellular homeostass. 26,72,[88][89][90] . miR-223-mediated inhibition of IKK-α (a target of miR-223) in macrophages, inhibits the activation of the NF-κB inflammatory pathway and reduces the production of pro-inflammatory cytokines such as TNF-α, thus breaking the vicious circle of chronic inflammation 65,66 (Fig. 2).
Cell experiments have also confirmed that exosomal miR-223 could regulate the classical NF-κB signaling pathway. NF-κB remained in the cytoplasm in an inactive form after binding to the endogenous inhibitor of exosomal miR-223. It is unraveled that miR-223 can down-regulate TNF-α in the exosomes induced by inflammation, thereby significantly repressing the inflammatory response and reducing the area of muscle necrosis 31,32 . miR-223 has been demonstrated to negatively regulate NF-κB activation and down-regulate the expression of pro-inflammatory factors such as NF-κB, IL-1β and IL-6 in macrophages, potentially reducing the development of chronic inflammatory responses to skeletal muscle injury 12,91,92 . It follows that overexpression of miR-223 decreases the level of cytokines by targeting corresponding genes to curb the excessive inflammation, making it possible to a positive transition from out-ofcontrol to in-control.
Meanwhile, animal experiments confirmed this relationship between exosomal miR-223 and inflammatory damage as well 93 . For instance, miR-223 can enhance the wound healing of mice after infection with Staphylococcus aureus. Additionally, restoration of miR-223 in miR-223-deficient (miR-223-/Y) neutrophils at the wound potentially improves wound healing, indicating that miR-223 may guide wound healing in a cell-autonomous and non-cell-autonomous manner, and miR-223 may inhibit the NF-κB inflammatory signaling pathway in mouse epithelial cells 94 .

Conclusions
E xosomes harbor nucleic acids, proteins, lipids and metabolites, making them not only important in cell communication, but also a replacement therapy to treating inflammation. Meanwhile, exosomes are regarded as "the third wagon (secondary to Circulation Tumor Cell (CTC) and Circulating Tumor DNA (ctDNA))" in the field of liquid biopsy secondary to ctDNA and CTC, which could be used as biomarkers of disease progression and treatment efficacy. MiR-223 is the most abundant miRNA in human peripheral blood microvesicles (exosomes, etc.). As an antiinflammatory miRNA of chronic inflammation in skeletal muscle, it is negatively correlated with pro-inflammatory factors such as TNF-α in serum. Exosomal miR-223 also plays an important role in the dysregulated inflammation of skeletal muscle and is expected to be a therapeutic target for chronic inflammation in skeletal muscle.
Additionally, miR-223 is a negative feedback regulator with potentials to address complex chronic condition and is therapeutically viable. Therefore, Further research on more convincing experimental proof on the biological function of miR-223 to improve clinical efficacy is urgently required. With the progress of molecular biotechnology in the future, it is also still a challenge to accurately regulate the effect of exosomal miR-223 on target cells.