Physical energy-based ultrasound shifts M1 macrophage differentiation towards M2 state

Hao-Cheng Qin,Zhi-Wen Luo,Yu-Lian Zhu

Hao-Cheng Qin,Yu-Lian Zhu,Department of Rehabilitation Medicine,Huashan Hospital,Fudan University,Shanghai 200040,China

Zhi-Wen Luo,Department of Sports Medicine,Huashan Hospital,Fudan University,Shanghai 200040,China

Abstract Recently,we read with interest the article entitled “Unveiling the Morphogenetic Code:A New Path at the Intersection of Physical Energies and Chemical Signaling”.In this paper,the investigation into the systematic and comprehensive bio-effects of physical energies prompted us to reflect on our research.We believe that ultrasound,which possesses a special physical energy,also has a certain positive regulatory effect on macrophages,and we have already obtained some preliminary research results that support our hypothesis.

Key Words: Ultrasound;Macrophages;Stem cells;Physical energies;Inflammation

TO THE EDlTOR

Recently,the article named “Unveiling the Morphogenetic Code:A New Path at the Intersection of Physical Energies and Chemical Signaling” contributed by the Editor-in-Chief Carlo Ventura[1]motivated us to reconsider the biological roles of physical energies.In that article,the authors provided a detailed summary of the developmental history of physical energy,especially bioelectricity,as well as its applications and prospects in stem cell research.We strongly support the proposition about the high efficacy of physical energy in tissue repair.An article recently released in “Science Translational Medicine” described how massage,which is also a physical stimulus,regulates muscle repair[2].The systematic and accurate explanation of physical energies by the editor echoes our dedicated research,which is the effect of therapeutic ultrasound on macrophages.

As early as the 1920s,Wood and Loomis began to investigate ultrasound as a therapeutic intervention[3].In recent years,one particular type of ultrasound,low-intensity pulsed ultrasound(LIPUS),has gained much attention.This is due to its non-thermal effects,such as acoustic cavitation and “cellular massage”,which produce a range of biological effects[4].Clinically,LIPUS can be used as a noninvasive adjunctive therapy for a variety of diseases,such as fractures,muscle injury,osteoarthritis,as well as nerve injury[4].Besides,LIPUS has received considerable attention in the discipline of stem cell research.Salgarellaet al[5]proved the bio-effect of ultrasound therapy on the proliferation and differentiation of C2C12 myoblastsin vitro,while the research of Wanget al[6]indicated that LIPUS promotes the production of mesenchymal stem cells(MSC)derived mainly from bone marrow differentiation.Both of these studies verified the positive bio-effects of this therapeutic technique.Tanet al[7]listed recent studies regarding the action of LIPUS on various neural stem cells and concluded that LIPUS can stimulate stem cellsin vitro,promote stem cell proliferation,differentiation,and migration,and maintain stem cell activity.The findings of Wuet al[8]suggested that LIPUS regulates the Notch signalling pathway in the central nervous system,causing neural stem cell proliferation and differentiation.Additionally,LIPUS can also accelerate tissue repair[9]and promote the dissipation of inflammation[10],angiopoiesis[11],etc.

Our research focused on discovering how sound waves,a kind of physical energy,exert potential effects on macrophage polarisation,which is of great significance during the inflammation stage.To ensure accurate muscle regeneration,a balance between M1 and M2 activity(pro- and anti-inflammatory,respectively)shifting over time is required[12].Previous studies have found that ultrasound may regulate macrophages in the spinal fusion model of male Sprague Dawley(SD)rats[13].However,very few studies have explored the direct effect of ultrasound on macrophagesin vitro.We induced macrophages into M1 macrophages using lipopolysaccharide to mimic the inflammatory microenvironmentin vivo.Then,we collected and analysed the secretion composition and gene expression following ultrasound therapy.Details of the experiments are presented in Figure 1.

So far,we have already obtained some preliminary results.As Figure 2 indicates,after LIPUS treatment,the secretion of anti-inflammatory cytokine interleukin(IL)-10 significantly increased,while quantities of pro-inflammatory cytokines tumor necrosis factor-α and IL-6 fell substantially at genetic and secreted proteins levels.Besides,we determined that the phenotypes of the macrophages are polarised into M2 after ultrasound stimulation(Figure 3).According to the above experimental data,we can conclude that ultrasound facilitates the transition of macrophages from the M1 to M2 phenotypein vitro,which is consistent with da Silva Junior's discovery[14].For subsequent research,we will further investigate the underlying mechanism of macrophage polarisation caused by LIPUS,and the potentially affected molecular pathways.Moreover,we will conduct bothin vivo(skeletal muscle contusion model)[15]andin vitroexperiments to verify the mechanism and ascertain how LIPUS exerts a series of downstream bio-effects.

Figure 1 Experimental design for this study in vitro and the method of treating cells.A:At 24 h before lipopolysaccharide(LPS)was added to simulate the inflammatory environment,macrophages at the M1 stage were uniformly subcultured into the six-well plate.Then,The first ultrasound treatment was performed 24 h after the inflammatory environment was maintained.At 48 h,ultrasound was performed on the group requiring a second treatment.The supernatant of culture medium was separated after 24 d of culture(3 d after LPS was added);B:In order to easily adjust the ultrasonic probe to fit the culture holes on the bottom of the six-well plate,we fixed the ultrasonic probe on a sponge pad.Additionally,a box of the same height is used to support the six-hole plate to prevent it from tilting.

Figure 3 Low-intensity pulsed ultrasound shifts M1 macrophages towards M2 state.A:The morphology of macrophages was observed by microscopy.Black,red,and yellow arrows represent M0,M1,and M2 macrophages,respectively;B:Quantitative polymerase chain reaction was used to detect the gene expression of cell phenotypes maker iNOS,CD86,CD206,and ARG1 after being treated by low-intensity pulsed ultrasound.Data are expressed as the mean ±standard error of the mean.aP ＜ 0.05;bP ＜ 0.01;cP ＜ 0.001.Low intensity = 0.25 W/cm2,medium intensity = 0.5 W/cm2,and high intensity = 0.75 W/cm2.

Conclusion and perspective

Various studies have established that physical energies can modulate inflammation and further promote tissue repair.As a conventional form of physical energy,ultrasound has extensive application prospects due to its non-thermal mechanical effect.Accordingly,it warrants further investigation to elucidate its influence on cell signalling.We predict that subsequent research can be extended from the aspect of monotypic cell regulation to the integral tissue,by employing single-cell transcriptomic analysis and spatial transcriptomic analysis[16].Thus,we will gain an increasingly comprehensive insight into the role of ultrasound in tissue repair.

FOOTNOTES

Author contributions:Qin HC and Luo ZW conducted the original search,wrote the first draft of the paper,and contributed to subsequent revisions of the manuscript;Zhu YL generated the original idea of this study and provided suggestions;Qin HC and Luo ZW made equal contributions to the work.

Conflict-of-interest statement:The authors declare no conflict of interest for this article.

Open-Access:This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers.It is distributed in accordance with the Creative Commons Attribution NonCommercial(CC BYNC 4.0)license,which permits others to distribute,remix,adapt,build upon this work non-commercially,and license their derivative works on different terms,provided the original work is properly cited and the use is noncommercial.See:https://creativecommons.org/Licenses/by-nc/4.0/

Country/Territory of origin:China

ORClD number:Hao-Cheng Qin 0000-0003-2723-4299;Zhi-Wen Luo 0000-0002-0524-9951;Yu-Lian Zhu 0000-0001-5530-144X.

S-Editor:Fan JR

L-Editor:Wang TQ

P-Editor:Zhang YL