Inflammation is a crucial response to defend the body from infection. However, excess and prolonged inflammation can also be harmful and needs to be tightly regulated (Funes et al., 2018). Macrophages play a leading role in the innate immune system, causing inflammation during infection (Hirayama et al., 2017). Therefore, regulating the condition of macrophages is a major therapeutic strategy for the control of excess inflammation. Various organs including skeletal muscle are known to release anti-inflammatory factors. Skeletal muscle is the largest organ in the human body accounting for 40% of the body weight (Trovato et al., 2019) and responsible for whole-body metabolism, energy homeostasis, and locomotion. Recently, skeletal muscle is attracting attention as a secretory organ of anti-inflammatory factors (Trovato et al., 2019), and extracellular vesicles (EVs) are responsible for transporting various factors from skeletal muscle to target organs or cells. EVs are nano-sized vesicles secreted by most types of cells and facilitate cell-to-cell communication (Trovato et al., 2019; Raposo and Stoorvogel, 2013; EL Andaloussi et al., 2013) through transportation of proteins, mRNAs, and miRNAs (Zomer et al., 2010), leading to regulation of immune response (EL Andaloussi et al., 2013), tissue regeneration (Bittel and Jaiswal, 2019), and cell proliferation/differentiation (Matsuzaka et al., 2016). EVs from skeletal muscle have also been reported to exert therapeutic effects in various dysfunctional organs (Rome et al., 2019; Bei et al., 2017; Madison et al., 2014) and our previous study revealed that skeletal myotube-derived EVs attenuate inflammatory responses of macrophages (Yamaguchi et al., 2023). EVs from mesenchymal stem cells (MSC) also have been reported to suppress inflammatory responses in lipopolysaccharide (LPS)-induced macrophages (Harrell et al., 2019; Xin et al., 2020; Shao et al., 2020) and Kim et al. reported the anti-inflammatory action of MSC EVs depended on the concentration of EVs (Kim et al., 2019). Therefore, an enhancement of EV release may enhance the anti-inflammatory effects of muscle EVs.

Ultrasound (US) irradiation is used as a non-invasive therapy and has physiological effects including cell proliferation, suppression of inflammatory signaling (Ueno et al., 2021), and increase cell membrane permeability (Ma et al., 2022). Our previous study revealed that high-intensity US can promote EV release (Maeshige et al., 2021). An increase in intracellular Ca²⁺ concentration is one of the effects of US irradiation, and elevation of intracellular Ca²⁺ is a key factor for EV secretion (Savina et al., 2003). Previous studies have reported that low-intensity pulsed ultrasound (LIPUS) promotes EV release from cells (Deng et al., 2021; Li et al., 2023). Meanwhile, US increases Ca²⁺ influx into cells by increasing cell membrane permeability through sonoporation (Fan et al., 2010), but its action has been reported to be dependent on US intensity (Zeghimi et al., 2015), so adopting a higher intensity than LIPUS is expected to promote EV release from skeletal muscle cells more efficiently. In addition, stimulus-induced EVs can be altered in their contents and effects compared to EVs released under normal conditions (Kawanishi et al., 2023; Li et al., 2023), thus EVs released from skeletal muscle by US may have different effects. This study aimed to clarify the intensity dependency of EV release enhancement by US and anti-inflammatory effects of US-induced skeletal muscle-derived EVs on macrophages.

This study demonstrated the facilitatory effect of US irradiation on EV release from cultured skeletal myotubes and the enhanced anti-inflammatory effect of EVs from US-irradiated myotubes. These findings contribute to the development of non-invasive complementary therapy using skeletal muscle-derived EVs for various inflammatory diseases.

Skeletal muscle-derived EVs have been reported to exert therapeutic effects in various dysfunctional organs (Rome et al., 2019), including prevention of cardiac damage by ischemia-reperfusion injury (Bei et al., 2017), enhancement of neurite outgrowth in motor neurons (Madison et al., 2014), and enhancement of angiogenesis (Ma et al., 2018). Furthermore, since skeletal muscle EVs mediate cross-talk between skeletal muscle and other tissues (Whitham et al., 2018), methods to enhance the release of skeletal muscle-derived EVs are potentially beneficial in the treatment of various diseases and health management.

Our results showed that the intracellular Ca²⁺ level significantly increased in the US-irradiated group, indicating that US irradiation promoted the uptake of Ca²⁺ to myotubes. This result is supported by previous reports investigating Ca²⁺ uptake by US in various types of cells (Ambattu et al., 2020; Scheffer et al., 2014; Fan et al., 2010; Lentacker et al., 2014; Honda et al., 2004). In the present study, the effect of US on Ca²⁺ uptake was observed in an intensity-dependent manner. US irradiation has been reported to exert a physiological action to promote transient membrane permeabilization, which can promote the Ca²⁺ influx pathways (Fan et al., 2010; Cao et al., 2019; Zhou et al., 2008), and the efficiency of permeabilization depends on the intensity of irradiation (Kumon et al., 2009; Zeghimi et al., 2015). Thus, the change of cell membrane structure is supposed to be the mechanism of the enhancement of Ca²⁺ influx. In this study, EV release was promoted by US of 3.0 W/cm², and this effect was cancelled by inhibition of Ca²⁺. Since intracellular Ca²⁺ level is reported to be a factor that promotes EVs release (Savina et al., 2003; Taylor et al., 2020), US irradiation was shown to enhance EV release from myotubes via promotion of influx of Ca²⁺.

We found that C2C12 myotube conditioned medium was capable of suppressing LPS-induced inflammatory responses in macrophages, and this effect was enhanced by US irradiation to myotubes. In addition, the anti-inflammatory effects were cancelled by eliminating EVs from the culture medium. This indicates that EVs in the culture medium were responsible for this effect, which is consistent with our previous study (Yamaguchi et al., 2023).

IL-1β is essential for an adequate acute inflammatory response to a variety of pathogens and injuries, but its excessive overexpression leads to sepsis, septic shock, or chronic inflammation (Dinarello, 2009; Piccioli and Rubartelli, 2013). IL-6 also contributes to host defense, however, dysregulated continual synthesis of it leads to prolonged chronic inflammation (Tanaka et al., 2014) and can drive tumorigenesis (Chang et al., 2014). Since blockade of these factors is reported to alleviate several inflammatory diseases (Chevalier et al., 2005; Emsley et al., 2005; Choy et al., 2002; Nishimoto et al., 2004; Ito et al., 2004; Yokota et al., 2005), eliciting the anti-inflammatory effects of skeletal muscle-derived EVs by US and suppressing the production of IL-1β and IL-6 suggests a new therapeutic strategy against inflammatory diseases utilizing US.

Meanwhile, it has been reported that the contents of EVs released from cells vary depending on the cellular microenvironment and that certain factors are selectively internalized in response to specific stimuli. The results of our analysis on miRNAs in EVs revealed that the EVs from US-exposed myotubes contain several unique miRNAs. However, only about 0.01% of the total miRNAs were altered by US irradiation, suggesting that the change was very small. Furthermore, no upregulation of the anti-inflammatory effect of myotube EVs by US was observed when the EV concentrations were equalized. Thus, the enhancement of the anti-inflammatory effect of myotube conditioned medium by US irradiation is assumed to be due to changes in the amount of EVs, not to changes in the content of EVs. Consistent with our results, a previous study reported that MSC-EVs regulated inflammatory responses in macrophages concentration dependently (Kim et al., 2019).

Analysis on miRNA profiles in the EVs revealed that the two most abundant miRNAs, miR-206-3p and miR-378a-3p, were common to both control and US EVs, and these two types accounted for more than 60% of the total miRNA contents in the both conditions. The most abundant miR-206 is a member of the skeletal muscle-specific myo-miR family of miRNAs (Carpi et al., 2020). It is reported that miR-206 targets IL-17A and REG3A and suppresses macrophage inflammation (Huang et al., 2020). Furthermore, Lin et al. reported that transfection of miR-mimic-206-3p into macrophages suppressed macrophage inflammation by targeting PPP3CA and transfection of miR-inhibitor-206-3p increased the level of inflammatory factors in macrophages (Lin et al., 2020). miR-378a is highly expressed in skeletal muscle and is involved in metabolism and mitochondrial energy homeostasis (Krist et al., 2015). Rückerl et al. identified miR-378a-3p as a factor contributing to the induction of anti-inflammatory macrophage reprogramming through IL-4-induced gene transcription by targeting Akt (Rückerl et al., 2012). In addition, Kris et al. reported that miR-378a has anti-inflammatory effects on macrophages and its deficiency enhances severity of inflammation (Krist et al., 2020). Based on these previous studies, it is assumed that myotube-derived EVs elicited the anti-inflammatory effects in macrophages by delivering these miRNAs. Additionally, miR-206 has been reported to modulate fat metabolism in diabetes (Wu et al., 2017) and control the tumorigenesis process of cancer (Ding et al., 2017), and miR-378a has been shown to inhibit cardiac hypertrophy (Ganesan et al., 2013) and cancer growth (Zeng et al., 2017) and alleviate spinal cord injury (Zhang et al., 2021), indicating that these miRNAs are expected to have therapeutic effects against various diseases. Therefore, promoting the release of EVs containing these factors by US stimulation to skeletal muscle is potentially effective in the prevention and treatment of various diseases, and further investigations of its effects on other pathological conditions are expected.

On the other hand, while this study attributed the anti-inflammatory effect of US on skeletal muscle-derived EVs to increased EV release, we identified several miRNAs increased in US-induced EVs. Further studies are needed on this point for a more detailed understanding about the effect of US on skeletal muscle.

In summary, this study showed that US irradiation promoted the secretion of myotube-derived EVs which have anti-inflammatory effects and suggested that US irradiation to skeletal muscles is a potent candidate as a novel treatment for various inflammatory disorders.

The RNA-seq data can be found in NCBI database (BioProject ID: PRJNA1044751).

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The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

While the manuscript was reasonably clearly written and the methodology and results sound, it is not clear what the real contribution of the work is. The authors' findings - that ultrasonic stimulation is capable of altering intracellular Ca2+ to effect an increase in EV secretion from cells as long as the irradiation does not affect cell viability-is well established (see, for example, Ambattu et al., Commun Biol 3, 553, 2020; Deng et al., Theranostics, 11, 9 2021; Li et al., Cell Mol Biol Lett 28, 9, 2023). Moreover, the authors' own work (Maeshige et al., Ultrasonics 110, 106243, 2021) using the exact same stimulation (including the same parameters, i.e., intensity and frequency) and cells (C2C12 skeletal myotubes) reported this. Similarly, the authors themselves reported that EV secretion from C2C12 myotubes has the ability to regulate macrophage inflammatory response (Yamaguchi et al., Front Immunol 14, 1099799, 2023). It would then stand to reason that a reasonable and logical deduction from both studies is that the ultrasonic stimulation would lead to the same attenuation of inflammatory response in macrophages through enhanced secretion of EVs from the myotubes.

We appreciate your comments and suggestions. Ambattu et al. in their report stated that the high frequency acoustic stimulation they used has a less effect on cell membranes than the 1 MHz ultrasound that we used in this study. Deng et al. and Li et al. applied low intensity pulsed ultrasound (LIPUS) (about 300 mW/cm2) in their studies. In this study, we assumed that ultrasound induced increase in EV secretion via increased Ca2+ influx into the cell by enhancing cell membrane permeability, and since it has been reported that the effect of ultrasound-induced enhancement in cell membrane permeability increases in an intensity-dependent manner (Zeghimi et al., 2015), we applied intensities of 1-3 W/cm2. While previous studies using LIPUS have used 15 minutes of irradiation, the high intensity employed in this study was capable to promote EV release after 5 minutes of stimulation. We have added the above explanation to the introduction in the revised version of the manuscript. Furthermore, while the previous studies used other types of cells, the main purpose of this study was to determine the optimal ultrasound intensity to promote EV release from skeletal muscle and to determine whether ultrasound-induced EVs are qualitatively altered compared to those released under normal conditions, thereby validating the anti-inflammatory effects of ultrasound-induced muscle EVs. Our previous study (Maeshige et al. 2021) used the same muscle cells but did not investigate an intensity dependence, so this is the first study to show that ultrasound irradiation promotes EV release in an intensity-dependent manner in muscle. In addition, we would like to emphasize that this study also goes beyond our previous study in the method of stimulation. Specifically, the present study a more efficient 5-minute irradiation protocol was used, whereas the previous study have adopted a 9-minute intervention.

We understand that the results of this study are predictable from two of our previous studies, but since stimulus-induced EVs may be qualitatively different compared to EVs released under normal conditions (Kawanishi et al., 2023; Li et al., 2023), it is worthwhile to examine the effects of stimulus-induced EVs. This explanation has been added in the introduction of revised version of the manuscript.

The authors' claim that 'the role of Ca2+ in ultrasound-induced EV release and its intensity-dependency are still unclear', and that the aim of the present work is to clarify the mechanism, is somewhat overstated. That ultrasonic stimulation alters intracellular Ca2+ to lead to EV release, therefore establishing their interdependency and hence demonstrating the mechanism by which EV secretion is enhanced by the ultrasonic stimulation, was detailed in Ambattu et al., Commun Biol 3, 553, 2020. While this was carried out at a slightly higher frequency (10 MHz) and slightly different form of ultrasonic stimulation, the same authors have appeared to since establish that a universal mechanism of transduction across an entire range of frequencies and stimuli (Ambattu, Biophysics Rev 4, 021301, 2023).

In this study, we showed that Ca2+ is involved in ultrasound-induced EV release using Ca2+-depleted culture medium, but since we did not examine the mechanism in more detail than that, we modified the introduction to avoid overstating.

Similarly, the anti-inflammatory effects of EVs on macrophages have also been extensively reported (Li et al., J Nanobiotechnol 20, 38, 2022; Lo Sicco et al., Stem Cells Transl Med 6, 3, 2017; Hu et al., Acta Pharma Sin B 11, 6, 2021), including that by the authors themselves in a recent study on the same C2C12 myotubes (Yamaguchi et al., Front Immunol 14, 1099799, 2023). Moreover, the authors' stated aim for the present work - clarifying the mechanism of the anti-inflammatory effects of ultrasound-induced skeletal muscle-derived EVs on macrophages - appears to be somewhat redundant given that they simply repeated the microRNA profiling study they carried out in Yamaguchi et al., Front Immunol 14, 1099799, 2023. The only difference was that a fraction of the EVs (from identical cells) that they tested were now a consequence of the ultrasound stimulation they imposed.

That the authors have found that their specific type of ultrasonic stimulation maintains this EV content (i.e., microRNA profile) is novel, although this, in itself, appears to be of little consequence to the overall objective of the work which was to show the suppression of macrophage pro-inflammatory response due to enhanced EV secretion under the ultrasonic irradiation since it was the anti-inflammatory effects were attributed to the increase in EV concentration and not their content.

In comparison with the current study, our previous study observed EVs secreted only from muscle in normal condition. However, the purpose of the current study is to answer the question whether ultrasound treatment could enhance the effect of EVs and change the encapsuled miRNAs. Although we identified several miRNAs which are specifically induced by ultrasound, further studies are needed to demonstrate the effect of those miRNAs derived from ultrasound-treated muscles on macrophages. We have mentioned this limitation in the discussion of the revised manuscript.

Reviewer #1 (Recommendations For The Authors):

This reviewer felt that there was a lack of novelty in the manuscript and that the results of the work confirm conclusions that could have been logically deduced from a combination of the authors' preceding work (Maeshige et al., Ultrasonics 110, 106243, 2021 and Yamaguchi et al., Front Immunol 14, 1099799, 2023). The contribution of the work could perhaps be elevated if the authors were to focus more on whether the 0.01% of altered miRNA has any impact on cellular activity.

As mentioned above, the present study is novel compared to our previous studies for examining the effects of ultrasound-induced EVs. In addition, the fact that EV content is maintained after ultrasound stimulation rather indicates that ultrasound can be used as a highly stable and effective method of promoting EV release.

A further, albeit more minor, recommendation is to omit lines 73-80 in the manuscript. The discussion on physical exercise for promoting EV secretion together with the non-invasive nature of ultrasound therapy is very misleading as it creates the impression that the authors' work can be applied as a direct intervention on a patient. This was not shown in the work, which was limited to irradiating cells ex vivo.

We agree and have edited the introduction.

Reviewer #2 (Public Review):

1. The exploration of output parameters for US induction appears limited, with only three different output powers (intensities) tested, thus narrowing the scope of their findings.

We appreciate your comments and suggestions. The intensity of LIPUS is basically in the ~0.3 W/cm2 range, and in clinical practice, ~2.5 W/cm2 is considered to be a safe intensity to irradiate the human body (Draper, 2014). Therefore, 3.0 W/cm2 is also a fairly high intensity for the human body, so 3.0 W/cm2 was set as the maximum intensity in this study.

1. Their claim of elucidating mechanisms seems to be only partially met, with a predominant focus on the correlation between calcium responses and EV release.

The focus of this study was to examine the effects of ultrasound-induced EVs on the inflammatory responses of macrophages and not on the detailed mechanism of calcium involvement. We revised the introduction to make the purpose of this study clearer.

1. While the intracellular calcium response is a dynamic activity, the method used to measure it could risk a loss of kinetic information.

Although we did not examine the kinetic action of calcium, we believe that Ca2+ is at least proven to be involved to the EV-promoting effect of ultrasound on muscle, since the enhancement of EV release by ultrasound was canceled by elimination of calcium from the culture medium. Furthermore, real-time measurement of Ca2+ after ultrasound irradiation has shown that ultrasound irradiation promotes Ca2+ influx into cells immediately after the irradiation. (Fan et al., 2010).

1. The inclusion of miRNA sequencing is commendable; however, the interpretation of this data fails to draw clear conclusions, diminishing the impact of this segment.

Although we identified several miRNAs which are specifically induced by ultrasound, further studies are needed to demonstrate the effect of those miRNAs derived from US-treated muscles on macrophages. We have mentioned this limitation in the discussion of the revised version of manuscript.

While the authors have shown the anti-inflammatory effects of US-induced EVs on macrophages, there are gaps in the comprehensive understanding of the mechanisms underlying US-induced EV release. Certain aspects, like the calcium response and the utility of miRNA sequencing, were not fully explored to their potential. Therefore, while the study establishes some findings, it leaves other aspects only partially substantiated.

As stated above, the main purpose of this study was to examine the effects of ultrasound-induced EVs on the inflammatory responses of macrophages. We set detailed investigation on the mechanism of ultrasound-induced EV release as our next step and have revised the introduction and discussion of the revised manuscript to make the purpose and limitation of this study clearer.

Reviewer #2 (Recommendations For The Authors):

The author's exploration into the role of Ca2+ in the context of US-induced EV release is a timely endeavor, especially given the growing interest in understanding the cellular dynamics associated with external stimulants like ultrasound. Nevertheless, the manuscript does not unambiguously define the mechanism of action and its broader implications.

Ca2+ has long been established as a versatile intracellular messenger, governing a myriad of cellular processes. There is a wealth of methodologies, from specific inhibitors to specialized assays, tailored to dissect the role of Ca2+ in diverse contexts. In the specific case of US-induced Ca2+ activity, the expectation would be for a clear, mechanistic delineation of how this ionic surge drives EV release. Yet, this study stops short of providing those details. It is imperative for the authors to dig deeper, employing a diverse set of tools at their disposal, to fill this knowledge gap.

Recently, it was reported that increased Ca2+ influx causes an increase in EV secretion via the plasma membrane repair protein annexin A6 (Williams et al. 2023). However, the full mechanism of how an increase in intracellular Ca2+, let alone ultrasound-induced Ca2+, promotes EV release has not yet been understood yet, and it is beyond the scope of this study to elucidate this part of the mechanism.

Furthermore, the paper raises another important question: Which specific proteins are pivotal in orchestrating the US-induced Ca2+ entry in myotubes? Addressing this would not only enhance the manuscript's novelty but would also contribute a vital piece to the puzzle of understanding US-cellular interactions.

Ultrasound increases Ca2+ uptake by increasing cell membrane permeability by sonoporation, rather than via protein reactions (Fan et al., 2010). We added this explanation to the introduction in the revised version of manuscript.

Lastly, while the report touches upon the influence of varying US output power on EV concentrations, it piques curiosity about potential effects beyond the 3W/cm2 threshold. It's observed that cell viability isn't compromised at this intensity, suggesting room for further exploration. Would a higher intensity yield a proportionally increased EV release, or is there a saturation point? Conversely, could intensities beyond 3W/cm2 begin to have deleterious effects on the cells? These are crucial considerations that merit investigation to realize the full potential of US as a modulatory tool, both for research and therapeutic applications.

As mentioned above, 3.0 W/cm2 was adopted as the maximum intensity in this study with reference to the intensity used in clinical practice. In addition, since the cytotoxicity and therapeutic effects of ultrasound depend not only on intensity but also on other parameters such as duty cycle, acoustic frequency, pulse repetition frequency and duration, so a comprehensive analysis of the effects of ultrasound on cells at various parameter settings would be valuable as an independent study.

Author details

1. Atomu Yamaguchi

   Department of Rehabilitation Science, Kobe University Graduate School of Health Sciences, Kobe, Japan

   Contribution

   Conceptualization, Validation, Investigation, Writing – original draft

   Competing interests

   No competing interests declared

2. Noriaki Maeshige

   Department of Rehabilitation Science, Kobe University Graduate School of Health Sciences, Kobe, Japan

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Project administration, Writing – review and editing

   For correspondence

   nmaeshige@pearl.kobe-u.ac.jp

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3573-347X

3. Hikari Noguchi

   Department of Rehabilitation Science, Kobe University Graduate School of Health Sciences, Kobe, Japan

   Contribution

   Data curation, Formal analysis, Visualization

   Competing interests

   No competing interests declared

4. Jiawei Yan

   School of Life Sciences and Technology, ShanghaiTech University, Shanghai, China

   Contribution

   Supervision, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

5. Xiaoqi Ma

   Department of Rehabilitation Science, Kobe University Graduate School of Health Sciences, Kobe, Japan

   Contribution

   Data curation, Formal analysis, Writing – review and editing

   Competing interests

   No competing interests declared

6. Mikiko Uemura

   Department of Rehabilitation Science, Kobe University Graduate School of Health Sciences, Kobe, Japan

   Contribution

   Validation, Investigation, Visualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

7. Dongming Su

   Department of Pathology, Nanjing Medical University, Nanjing, China

   Contribution

   Conceptualization, Supervision, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

8. Hiroyo Kondo

   Department of Health and Nutrition , Shubun University, Ichinomiya, Japan

   Contribution

   Conceptualization, Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

9. Kristopher Sarosiek

   John B. Little Center for Radiation Sciences, Harvard University T.H. Chan School of Public Health, Boston, United States

   Contribution

   Supervision, Writing – original draft, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

10. Hidemi Fujino

    Department of Rehabilitation Science, Kobe University Graduate School of Health Sciences, Kobe, Japan

    Contribution

    Conceptualization, Supervision, Funding acquisition, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

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