INTRODUCTION

Section:

ChooseTop of pageABSTRACTINTRODUCTION <<RESULTSDISCUSSIONMETHODSSUPPLEMENTARY MATERIALAmong all immune cells involved in the inflammatory process, macrophages represent the first line of defense against infections. They are located in all body tissues and arise from precursor cells, named monocytes. Tissue-resident macrophages and monocytes recruited from the bloodstream react in response to various inflammatory stimuli of the cellular microenvironment in which they reside or migrate, respectively.11. T. A. Butterfield, T. M. Best, and M. A. Merrick, J. Athletic Train. 41, 457 (2006). When inflammation is active, toll-like receptors (TLRs), especially TLR4, participate in the innate immune response causing the activation of signaling cascades in macrophages, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK),22. Y. C. Lu, W. C. Yeh, and P. S. Ohashi, Cytokine 42, 145–151 (2008). https://doi.org/10.1016/j.cyto.2008.01.006 and the consequent production of high levels of inflammatory cytokines.33. S. Grassin-Delyle, C. Abrial, H. Salvator, M. Brollo, E. Naline, and P. Devillier, J. Innate Immun. 12, 63–73 (2020). https://doi.org/10.1159/000494463Pro-inflammatory (M1-like) macrophages secrete a large variety of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8).4,54. C. D. Mills, K. Kincaid, J. M. Alt, M. J. Heilman, and A. M. Hill, J. Immunol. 164, 6166–6173 (2000). https://doi.org/10.4049/jimmunol.164.12.61665. K. Y. Lee, Med. Biol. Sci. Eng. 2, 1–5 (2019). https://doi.org/10.30579/mbse.2019.2.1.1 Anti-inflammatory (M2-like) macrophages are able to secrete anti-inflammatory cytokines such as interleukin-4 (IL-4) and interleukin-10 (IL-10).66. P. Italiani and D. Boraschi, Immune Networks 15, 167 (2015). https://doi.org/10.4110/in.2015.15.4.167The control of inflammation is a critical problem in the management of several diseases, such as cardiovascular pathologies, cancer, diabetes mellitus, and osteoarthritis.77. D. Furman, J. Campisi, E. Verdin, P. Carrera-Bastos, S. Targ, C. Franceschi, L. Ferrucci, D. W. Gilroy, A. Fasano, G. W. Miller, A. H. Miller, A. Mantovani, C. M. Weyand, N. Barzilai, J. J. Goronzy, T. A. Rando, R. B. Effros, A. Lucia, N. Kleinstreuer, and G. M. Slavich, Nat. Med. 25, 1822–1832 (2019). https://doi.org/10.1038/s41591-019-0675-0 Currently, in the clinical setting, corticosteroids or anti-inflammatory drugs are commonly administered to reduce inflammation; however, all current pharmacological treatments are often far from being satisfactory, and may cause side effects, such as renal impairment, increased cardiovascular risk, and possible secondary infections.88. T. J. Atkinson and J. Fudin, Phys. Med. Rehabil. Clin. North. Am. 31, 219–231 (2020). Low-intensity pulsed ultrasound (LIPUS) is a specific regime of ultrasound (US) stimulation, featured by frequencies > 20 kHz, low intensity (2), and a pulsed waveform to minimize thermal effects. LIPUS is recently attracting more and more attention due to its ability to induce beneficial effects that promote tissue healing and regeneration.99. X. Jiang, O. Savchenko, Y. Li, S. Qi, T. Yang, W. Zhang, and J. Chen, IEEE Trans. Biomed. Eng. 66, 2704 (2018). https://doi.org/10.1109/TBME.2018.2889669So far, the most promising results have been obtained by applying this technology to the field of fracture healing1010. U. Farkash, O. Bain, A. Gam, M. Nyska, and P. Sagiv, J. Orthop. Surg. Res. 10, 1–7 (2015). (LIPUS has been approved by the United States Food and Drug Administration for such an application1111. C. Rubin, M. Bolander, J. P. Ryaby, and M. Hadjiargyrou, J. Bone Jt. Surg., Am. 83, 259 (2001). https://doi.org/10.2106/00004623-200102000-00015) and also for regeneration of soft tissues, such as cartilage, muscles, tendons, and ligaments.1212. A. Khanna, R. T. C. Nelmes, N. Gougoulias, N. Maffulli, and J. Gray, Br. Med. Bull. 89, 169 (2009). https://doi.org/10.1093/bmb/ldn040 The possible mechanisms responsible for the above-mentioned effects on tissues and cells have been widely discussed, but they are still unclear and under continuous investigation.The efficacy of LIPUS in regulating the inflammatory response linked to several diseases has also been recently explored.13–1513. M. Xu, L. Wang, S. Wu, Y. Dong, X. Chen, S. Wang, X. Li, and C. Zou, Quant. Imaging Med. Surg. 11, 443 (2021). https://doi.org/10.21037/qims-20-68014. W. S. Su, C. H. Wu, S. F. Chen, and F. Y. Yang, Sci. Rep. 7, 15524 (2017). https://doi.org/10.1038/s41598-017-15916-215. E. B. Rego, T. Takata, K. Tanne, and E. Tanaka, Open Dent. J. 6, 220 (2013). https://doi.org/10.2174/1874210601206010220 However, only a few groups have investigated the effect of LIPUS on macrophages. The most important results on this line of research are summarized in Table I. Feril et al.1616. L. B. Feril, T. Kondo, Z. G. Cui, Y. Tabuchi, Q. L. Zhao, H. Ando, T. Misaki, H. Yoshikawa, and S. I. Umemura, Cancer Lett. 221, 145 (2005). https://doi.org/10.1016/j.canlet.2004.08.034 applied LIPUS to U937 cells, used as a leukemia model, demonstrating that apoptosis was maximized using a certain intensity, keeping all the other parameters fixed. Tabuchi et al.1717. Y. Tabuchi, I. Takasaki, Q. L. Zhao, S. Wada, T. Hori, L. B. Feril, K. Tachibana, T. Nomura, and T. Kondo, Cancer Lett. 270, 286 (2008). https://doi.org/10.1016/j.canlet.2008.05.018 applied LIPUS on U937 with similar conditions, although with a fixed intensity value, and they observed a downregulation of 193 genes, including estrogen receptor 1 (ESR1), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), and integrin β 1 (ITGB1), as well as an upregulation of 201 genes, including heme oxygenase (decycling) 1 (HMOX1), vimentin (VIM), and chemokine (C–C motif) ligand 3 (CCL3). In the work of Zhang et al.1818. X. Zhang, B. Hu, J. Sun, J. Li, S. Liu, and J. Song, J. Ultrasound Med. 36, 2419 (2017). https://doi.org/10.1002/jum.14239 LIPUS was used with slightly different parameters, varying the intensity between 10 and 90 mW/cm2, and a decrement of the level of pro-inflammatory cytokines such as IL-1β, IL-6, and IL-8 in macrophage-like U937 cells was observed. The authors also observed an increase in cell viability, cell apoptosis inhibition, a suppression of degradation and phosphorylation of kappa-light-chain-enhancer of activated B cells (IκBα), and a translocation of NF-κB p65 subunit into nuclei. Other studies focused on LIPUS-induced bioeffects on the RAW 264.7 cell line, a murine model of macrophages, and on the THP-1 human cell line. In all these studies, the authors showed that LIPUS treatment, although performed with different conditions, inhibited the production of pro-inflammatory cytokines such as interleukin-33 (IL-33), IL-6, IL-8, and IL-1β and suppressed intracellular signaling such as extracellular signal-regulated kinase (ERK) and MAPK.19–2119. X. Zhao, G. Zhao, Z. Shi, C. Zhou, Y. Chen, B. Hu, and S. Yan, Sci. Rep. 7, 45779 (2017). https://doi.org/10.1038/srep4577920. C. Zheng, S. M. Wu, H. Lian, Y. Z. Lin, R. Zhuang, S. Thapa, Q. Z. Chen, Y. F. Chen, and J. F. Lin, J. Cell. Mol. Med. 23, 1963 (2019). https://doi.org/10.1111/jcmm.1409821. B. Zhang, H. Chen, J. Ouyang, Y. Xie, L. Chen, Q. Tan, X. Du, N. Su, Z. Ni, and L. Chen, Autophagy 16, 1262 (2020). https://doi.org/10.1080/15548627.2019.1664705

TABLE I. Relevant in vitro studies focused on LIPUS stimulation of macrophages. Gene and protein symbols are in capital letters. ATP = adenosine triphosphate; CCL3 = chemokine (C-C motif) ligand 3; DC = duty cycle; ERBB2 = v-erb-b2 erythroblastic leukemia viral oncogene homolog 2; ESR1= estrogen receptor 1; ERK = extracellular signal-regulated kinase; F = frequency; HMOX1 = heme oxygenase (decycling) 1; I = intensity; IKBα = nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha; ITGB1 = integrin beta 1; IL= interleukin; LIPUS = low-intensity pulsed ultrasound; LPS = lipopolysaccharide; MAPK = mitogen-activated protein kinase; PKM = pyruvate kinase muscle; PRF = pulse repetition frequency; SQSTM1 = sequestosome 1; t = exposure time; TNF = tumor necrosis factor; VIN = vimentin. N/A = not applicable.

ReferenceStimulation parametersCell lineBio effects1616. L. B. Feril, T. Kondo, Z. G. Cui, Y. Tabuchi, Q. L. Zhao, H. Ando, T. Misaki, H. Yoshikawa, and S. I. Umemura, Cancer Lett. 221, 145 (2005). https://doi.org/10.1016/j.canlet.2004.08.034F = 1 MHz; PRF = 0.1 kHz; I = 100–1000 mW/cm2; DC = 10 %; and t = 1 min.U937Optimal apoptosis with minimal lysis was attained 12 h after sonication at 300 mW/cm2.1717. Y. Tabuchi, I. Takasaki, Q. L. Zhao, S. Wada, T. Hori, L. B. Feril, K. Tachibana, T. Nomura, and T. Kondo, Cancer Lett. 270, 286 (2008). https://doi.org/10.1016/j.canlet.2008.05.018F = 1 MHz; PRF = 0.1 kHz; I = 300 mW/cm2; DC = 10 %; and t = 1 min.U937Six hours after LIPUS treatment, apoptosis without cell lysis was observed. LIPUS downregulated 193 genes and upregulated 201 genes (associated with cellular movement and cell death).1818. X. Zhang, B. Hu, J. Sun, J. Li, S. Liu, and J. Song, J. Ultrasound Med. 36, 2419 (2017). https://doi.org/10.1002/jum.14239F = 1.5 MHz; PRF = 1 kHz; I = 10, 30, 60, and 90 mW/cm2; DC = 20 %; and t = 2 h.U937LIPUS at 60 mW/cm2 was more effective in reducing IL-8 expression. LIPUS reduced the protein expression of IL-6 and IL-8 at both gene and protein levels.LIPUS primarily suppressed the degradation and phosphorylation of IKBα and the translocation of p65 into the nuclei.1919. X. Zhao, G. Zhao, Z. Shi, C. Zhou, Y. Chen, B. Hu, and S. Yan, Sci. Rep. 7, 45779 (2017). https://doi.org/10.1038/srep45779F = 1.5 MHz; PRF = 0.25 kHz; I = 200 mW/cm2; DC = 20 %; and t = 20 min.RAW 264.7LIPUS was found to inhibit inflammation and decrease the levels of IL-1β, IL-33, IL-6, and IL-8.2020. C. Zheng, S. M. Wu, H. Lian, Y. Z. Lin, R. Zhuang, S. Thapa, Q. Z. Chen, Y. F. Chen, and J. F. Lin, J. Cell. Mol. Med. 23, 1963 (2019). https://doi.org/10.1111/jcmm.14098F = 1 MHz; PRF = 0.1 kHz; I = 100 mW/cm2; DC = 20 %; and t = 20 min.RAW 264.7LIPUS treatment on RAW 264.7 inhibited the expression of pro-inflammatory cytokines (TNF-α and IL-6), activated caveolin-1, and suppressed p38 MAPK and ERK signaling.2121. B. Zhang, H. Chen, J. Ouyang, Y. Xie, L. Chen, Q. Tan, X. Du, N. Su, Z. Ni, and L. Chen, Autophagy 16, 1262 (2020). https://doi.org/10.1080/15548627.2019.1664705F = 1.5 MHz; PRF = N/A; I = 30 mW/cm2; DC = 20 %; and t = 20 min.THP-1 and RAW 264.7LIPUS inhibited the production of IL-1β. In addition, LIPUS upregulated the autophagy level and accelerated the formation of an SQSTM1-PKM complex in the LPS-ATP-treated macrophages. In addition, LIPUS downregulated the level of PKM2 in LPS-ATP-treated macrophages.However, in all the above-mentioned studies, a systematic screening of the different possible LIPUS parameters was not performed. Consequently, the optimal parameters producing anti-inflammatory effects are not known. Moreover, the LIPUS setups adopted did not guarantee precise control of the energy dose delivered to the target, thus, producing undesired acoustic artifacts, such as wave attenuations and reflections, which can hamper the repeatability of the experiments and can produce errors in the dose of energy delivered up to 700% with respect to the expected value.22,2322. J. J. Leskinen and K. Hynynen, Ultrasound Med. Biol. 38, 777–794 (2012). https://doi.org/10.1016/j.ultrasmedbio.2012.01.01923. M. Snehota, J. Vachutka, G. Ter Haar, L. Dolezal, and H. Kolarova, Ultrasonics 107, 106167 (2020). https://doi.org/10.1016/j.ultras.2020.106167In this work, we investigated the anti-inflammatory bioeffects induced by LIPUS treatment on a human macrophage-like cell model by using a custom-made in vitro LIPUS stimulation system, with high control of the US dose transmitted to the cells.2424. A. Cafarelli, A. Marino, L. Vannozzi, J. Puigmartí-Luis, S. Pané, G. Ciofani, and L. Ricotti, ACS Nano 15, 11066–11086 (2021). https://doi.org/10.1021/acsnano.1c03087 We assessed the effects of LIPUS on lipopolysaccharide (LPS)-induced U937 cells by exploring different frequencies (F: 38 kHz, 1 MHz, and 5 MHz), intensities (I: 25, 100, 250, and 450 mW/cm2), duty cycles (DC: 10%, 20%, 30%, and 40%) and stimulation times (t: 30, 60, 90, and 120 min) and measuring the release of pro-inflammatory cytokines (IL-1β, IL-8, and TNF-α). At the optimal LIPUS conditions, we also investigated more in-depth metabolic activity and ROS production. Moreover, we assessed the role of Piezo-Type Mechanosensitive Ion Channel Component 1 (PIEZO1), Transient Receptor Potential Cation Channel Subfamily V Member 1 (TRPV1), NF-κB signaling pathway, actin polymerization pathway, and modulation of gene expression pathways downstream of MAPK in the LIPUS-triggered cell response.

RESULTS

Section:

ChooseTop of pageABSTRACTINTRODUCTIONRESULTS <<DISCUSSIONMETHODSSUPPLEMENTARY MATERIAL

Phenotypic differentiation of U937 induced by PMA and selection of LPS concentration

As shown in Fig. S1(a), the exposure to PMA for 48 h led U937 mononuclear monocyte-like cells to differentiate into a M0 phenotype. The cells showed a flat morphology and were attached to the polystyrene (PS) surface. Moreover, as shown in Fig. S1(b), an increase in CD14 gene expression for all PMA-stimulated samples compared to the control sample (U937 without PMA) was observed. M0 cells were further skewed toward an M1-like phenotype by increasing concentrations of LPS for 24 h. The phenotype was confirmed by the increase in CD80 and CD86 markers (both characteristic of M1-like macrophages). As shown in Fig. S1(b), there was a statistical difference in the expression level of TNF-α between control and LPS experimental groups when the LPS concentration was above 1 μg/ml. In addition, no morphological changes were observed in M1-like macrophages induced with 1 μg/ml LPS. Thus, this concentration was used in all the subsequent experiments.

Optimization of stimulation with US

Two custom-designed LIPUS setups,2525. F. Fontana, F. Iberite, A. Cafarelli, A. Aliperta, G. Baldi, E. Gabusi, P. Dolzani, S. Cristino, G. Lisignoli, T. Pratellesi, E. Dumont, and L. Ricotti, Ultrasonics 15, 106495 (2021). https://doi.org/10.1016/j.ultras.2021.106495 dedicated to low and high F, respectively, were adopted in this work to perform highly controlled LIPUS stimulations. The low-F system [Fig. 1(a)] allowed to stimulate biological samples at 38 kHz. The high-F system [Fig. 1(b)] allowed to perform stimulations at 1 MHz and 5 MHz, by also exploring different I, DC, and t values.By means of the above-mentioned systems, different LIPUS conditions were explored, taking into account three experimental groups [Fig. 2(a)]: in M0, cells were not treated with LPS or LIPUS (i.e., negative control); in LPS, cells were treated with LPS for 2 h but they were not treated with LIPUS (i.e., positive control); in LPS+LIPUS, cells were treated with LPS for 2 h and then they were subjected to LIPUS stimulation.At first, three values of F were explored: 38 kHz, 1 MHz, and 5 MHz. In this experiment, I, DC, and t were kept fixed at 250 mW/cm2, 20%, and 120 min, respectively (chosen as quite broadly used values in the literature). The results are shown in Fig. 2(b). It can be observed that LPS treatment increased the level of all the three cytokines (i.e., TNF-α, IL-1β, and IL-8) with respect to the M0 group. With regard to the exploration of different F, 38 kHz considerably decreased the level of all the analyzed pro-inflammatory cytokines, both at protein and gene expression levels, thus, resulting in more effectiveness than other stimulation conditions. Differently, 5 MHz was never able to downregulate IL-1β, IL-8, and TNF-α production both at protein and gene level, compared to the LPS group; a F of 1 MHz, instead, was able to lower only IL-8 production at a protein level. Therefore, 38 kHz was selected and fixed for the following screenings.Then, a similar protocol was used, but exploring four values of I: 25, 100, 250, and 450 mW/cm2 (F was set at the optimal value of 38 kHz found in the previous experiment, whereas DC and t were set at 20% and 120 min, respectively). The results are shown in Fig. 2(c).

Concerning the definition of optimal I, 250 mW/cm2 was found to significantly downregulate cytokines release with respect to other I values. The I of 450 mW/cm2 had no remarkable effect, whereas 25 and 100 mW/cm2 were able to lower only IL-8 production at the protein level. Therefore, the I of 250 mW/cm2 was selected for further analyses.

With F fixed at 38 kHz and I fixed at 250 mW/cm2, the role of DC was explored, considering 10%, 20%, 30%, and 40% values. The results are shown in Fig. 3(a). A DC of 20% resulted in the most effective lowering of the inflammatory level, with respect to the other conditions; 10% was also effective but only on IL-8 and TNF-α protein releases, whereas 30% lowered only the IL-8 protein release. So, together with a F of 38 kHz and an I of 250 mW/cm2, a DC of 20% was chosen among the selected parameters for the following investigation, where t was varied between 30, 60, 90, and 120 min. The results are shown in Fig. 3(b). We found that the treatment efficacy reached a plateau behavior starting from a t of 90 min, so this value was chosen as the minimum optimal t.

Overall, the following combination of parameters was found to be the most effective in decreasing pro-inflammatory cytokine production: F = 38 kHz; I = 250 mW/cm2; DC = 20%, and t = 90 min.

Effects on cell viability, metabolism, and intracellular ROS

Two experimental groups were defined [Fig. 4(a)], named (i) LPS, where M0 cells were treated with LPS for 2 h but were not treated with LIPUS (i.e., positive control), and (ii) LPS+LIPUS (Optimal), where M0 cells were treated with LPS for 2 h and then stimulated at the optimal stimulation conditions for all the US parameters (i.e., F = 38 kHz, I = 250 mW/cm2, DC = 20%, and t = 90 min; PRF =1 kHz).As shown in Fig. 4(b), the maximum temperature increase recorded during LIPUS stimulation was around 1 °C, which is in line with previous reports.26,2726. L. B. Feril and T. Kondo, J. Radiat. Res. 45, 479 (2004). https://doi.org/10.1269/jrr.45.47927. A. R. Salgarella, A. Cafarelli, L. Ricotti, L. Capineri, P. Dario, and A. Menciassi, Ultrasound Med. Biol. 43, 1452–1465 (2017). https://doi.org/10.1016/j.ultrasmedbio.2017.03.003 Therefore, the treatment can be classified as non-thermal.Concerning cell viability, representative images of LPS and “LPS+LIPUS (Optimal)” groups are shown in Fig. 4(c). No significant differences were observed between control and stimulated samples, up to 72 h post-stimulation. These qualitative results were in agreement with dsDNA analyses at the same time point [Fig. 4(d)], in which no statistically significant differences were observed between all the conditions.The results of metabolic activity and ROS production are reported in Figs. 4(e) and 4(f), respectively. The outcome of both these tests was normalized with respect to dsDNA amount. An increase in cell metabolic activity was clearly observed in the LPS+LIPUS (Optimal) group with respect to the LPS one, for all the time points.

Concerning ROS, no statistical difference was found between LPS and LPS+LIPUS (Optimal) groups from 24 to 72 h post-stimulation. However, LIPUS at the optimal stimulation condition was able to considerably reduce ROS production immediately after stimulation.

Temporal evolution of pro-inflammatory cytokines release

Five experimental groups were defined [Fig. 5(a)], named as follows: (i) LPS, where M0 cells were treated with LPS for 2 h but not treated with LIPUS; (ii) LPS+LIPUS (Optimal), where M0 cells were treated with LPS for 2 h and then stimulated with LIPUS at the optimal stimulation conditions found during US parameter screening (i.e., F = 38 kHz, I = 250 mW/cm2, DC = 20%, and t = 90 min); (iii) LPS+Blocker+LIPUS groups, where M0 cells were treated with LPS, then treated with selective ion channel blockers [with (iii) PIEZO1 blocker, (iv) BCTC blocker or (v) both of them] and then stimulated at the optimal stimulation conditions found during US parameter screening (i.e., F = 38 kHz, I = 250 mW/cm2, DC = 20%, t = 90 min, and PRF = 1 kHz).Multiple cytokine production by LPS-induced M0 cells over 72 h post LIPUS treatment at the optimal stimulation conditions were analyzed. The results are shown in Figs. 5 and 6. IL-6 production appeared relatively late, in line with previous studies on human monocytes,2828. J. Abrams, C. G. Figdor, R. De Waal Malefyt, B. Bennett, and J. E. De Vries, J. Exp. Med. 174, 1209–1220 (1991). https://doi.org/10.1084/jem.174.5.1209 while an increase in IL-6-encoding mRNA levels was detected 6 h post-stimulation. For IL-6, both at protein and gene expression levels, the LPS+LIPUS (Optimal) group was statistically lower than the LPS group and the three “LPS+Blocker+LIPUS (Optimal)” groups.

Concerning IL-1β, the behavior was the same as that of IL-6, both at protein and gene expression levels: the LPS+LIPUS (Optimal) group was significantly different from the LPS group for all the time points, and the three LPS+Blocker+LIPUS (Optimal) groups were statistically different from the LPS+LIPUS (Optimal) group for all time points.

As regards IL-8, the LPS+LIPUS (Optimal) group and the “LPS+TRPV1 Blocker+LIPUS (Optimal)” group showed lower values than the other experimental groups, for all considered time points, at protein level. Interestingly, at gene expression level, also the “LPS+PIEZO1 Blocker+LIPUS (Optimal)” group and the “LPS+PIEZO1&TRPV1 Blocker+LIPUS (Optimal)” group were significantly different from the LPS group, up to 6 h post-stimulation. From 12 h post-stimulation, the trend at gene level followed the same kinetics of the protein release.

Regarding TNF-α, all experimental groups were significantly different from the LPS group, at each time point, both at protein and gene levels. For IL-12p35 gene expression, the “LPS+LIPUS” group was significantly different from the LPS group starting from 12 h after stimulation, whereas 24 h post-stimulation, all the LIPUS-stimulated groups were statistically lower with respect to the LPS group. Interestingly, the IL-12p40 subunit exhibited different kinetics, in which the LPS+LIPUS (Optimal) group remained at lower levels than the other groups, up to 12 h post-stimulation. At 24-h time point, only the gene expression of the LPS group was detectable.

Regarding IL-12p70 cytokine production, similar kinetics as IL-12p40 were observed up to 6 h post-stimulation. At the 12-h timepoint, the trend was also identical, with the difference that no protein release was detected in the LPS+LIPUS (Optimal) group. After 24 h, only the LPS group was detected: this latter result was likely due to the additive effect of the anti-inflammatory LIPUS stimulation and the upregulation of IL-4, which, according to the literature, was able to independently suppress IL-12p70 in in vitro monocyte-derived macrophages.2929. C. S. Bonder, J. J. Finlay-Jones, and P. H. Hart, Immunology 96, 529 (1999). https://doi.org/10.1046/j.1365-2567.1999.00711.x Regarding IL-4, a decrease over time was observed in the LPS+LIPUS (Optimal) group at both protein and gene levels; gene expression of IL-10, on the other hand, decreased from 12 h post-stimulation.

Inhibition of NF-κBp65 and modification of actin organization

Two experimental groups were defined [Fig. 7(a)], named (i) LPS, where M0 cells were treated with LPS for 2 h but were not treated with LIPUS (i.e., positive control), and (ii) LPS+LIPUS (Optimal), where M0 cells were treated with LPS for 2 h and then stimulated at the optimal stimulation conditions found during US parameter screening (i.e., F = 38 kHz, I = 250 mW/cm2, DC = 20%, t = 90 min, and PRF = 1 kHz).The LPS treatment induced the nuclear translocation of the p65 subunit. LIPUS treatment significantly reversed this process, as clearly visible in Fig. 7(b); on the left, a purple nuclear halo is visible, resulting from the superposition of blue (chromatin) and red (p65) signals; on the right, the red signal is cytoplasmic. Moreover, the formation of microspikes was evident 30 min after the stimulation [Fig. 7(c)], suggesting that the LIPUS treatment was capable of rearranging the actin cytoskeleton and appeared as a trigger capable of accelerating phagocytosis.

LIPUS induce the overexpression of genes involved in the p38 MAPK Pathway

To determine whether the LIPUS stimulation could affect the MAPK pathway, we performed a transcriptome analysis comparing the LPS group with the LPS+LIPUS(Optimal) group. We used RT2 profiler PCR arrays that analyzed 83 specific genes involved in the MAPK pathway. MAPK cascades are key signaling pathways that regulate a broad variety of cellular processes, including proliferation, differentiation, apoptosis, and stress responses. The MAPK pathway includes three main kinases: MAPK kinase kinase, MAPK kinase, and MAPK, which activate and phosphorylate downstream proteins.3030. Z. Wei and H. T. Liu, Cell Res. 12, 9 (2002). https://doi.org/10.1038/sj.cr.7290105 The MAPKs in mammals include c-Jun NH2-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK). Studies have shown that the JNK and p38 MAPK pathways are mainly related to stress (oxidative, genotoxic, and osmotic stress as well as by proinflammatory cytokines) and apoptosis of cells, while the ERK/MAPK signaling pathway, which is one of the most studied, is closely related to cell proliferation and differentiation.3131. J. M. Kyriakis and J. Avruch, Physiol. Rev. 92, 689 (2012). https://doi.org/10.1152/physrev.00028.2011 We found an upregulation of 17 out of 83 genes, while none was downregulated by the treatment (see Table II and Fig. S4). Therefore, the transcriptomic analysis suggested a general activation of p38 MAPK pathways (genes such as TP53, MAPK9, MAPK14, and MAPK12 were all upregulated).

TABLE II. Transcriptome analysis related to MAPK pathway. Deregulated genes in LPS+LIPUS(Optimal) samples, compared to “LPS” samples with a fold change at least of ±2 and a p-value less than 0.05 are listed. The geometric mean of two housekeeping/reference genes (B2M and RPL0) was used to normalize the raw data. The p-values were calculated based on a Student's t-test of the replicate 2̂ (−ΔCT) values for each gene in the control group (LPS) and treatment group (LPS+LIPUS). The p-value calculation used is based on parametric, unpaired, two-sample equal variance, and two-tailed distribution. ATF2 = Activating transcription factor 2; CCND2 = Cyclin D2; CREBBP = CREB binding protein; ELK1 = ELK1, member of ETS oncogene family; FOS = FBJ murine osteosarcoma viral oncogene homolog; HRAS = V-Ha-ras Harvey rat sarcoma viral oncogene homolog; HSPB1 = heat shock 27 kDa protein 1; LAMTOR3 = late endosomal/lysosomal adaptor, MAPK and MTOR activator 3; MAP2K7 = mitogen-activated protein kinase kinase 7; MAP3K1 = mitogen-activated protein kinase kinase kinase 1; MAPK12 = mitogen-activated protein kinase 12; MAPK13 = mitogen-activated protein kinase 13; MAPK14 = mitogen-activated protein kinase 14; MAPK8IP2= mitogen-activated protein kinase 8 interacting protein; MAPK9 = mitogen-activated protein kinase 9; MAPKAPK2 = mitogen-activated protein kinase-activated protein kinase 2; TP53 = tumor protein p53.

Gene nameFold changep-valueATF22.230.0075CCND22.730.0129CREBBP4.020.0223ELK111.220.0236FOS14.390.0140HRAS3.130.0322HSPB17.320.0365LAMTOR33.190.0107MAP2K73.210.0299MAP3K12.450.0225MAPK123.380.0206MAPK133.810.0416MAPK143.230.0477MAPK8IP23.220.0119MAPK92.570.0469MAPKAPK22.930.0264TP534.650.0097This is an interesting result since the p38 MAPKs pathway plays an important role in the cascades of cellular responses evoked by a wide range of external signals (such as mechanical stress) and responds appropriately by generating a plethora of different biological effects leading to direct activation of transcription factors.32–3832. M. Mayr, C. Li, Y. Zou, U. Huemer, Y. Hu, and Q. Xu, FASEB J. 14, 261 (2000). https://doi.org/10.1096/fasebj.14.2.26133. T. T. Chowdhury, D. M. Salter, D. L. Bader, and D. A. Lee, Inflamm. Res. 57, 306 (2008). https://doi.org/10.1007/s00011-007-7126-y34. L. Yuan, N. Sakamoto, G. Song, and M. Sato, Stem Cells Dev. 21, 2520 (2012). https://doi.org/10.1089/scd.2012.001035. J. Wang, H. Chen, A. Seth, and C. A. McCulloch, Am. J. Physiol.-Hear. Circ. Physiol. 285, H1871 (2003). https://doi.org/10.1152/ajpheart.00387.200336. Y. Kanazawa, J. Nomura, J. Himeta, T. Suzuki, N. Suzuki, S. Yoshimoto, and M. Ichinose, Biol. Sci. 18, 148 (2004).37. I. M. Hdud, A. Mobasheri, and P. T. Loughna, Am. J. Physiol.-Cell Physiol. 306, C1050 (2014). https://doi.org/10.1152/ajpcell.00287.201338. S. H. Hook, H. J. Lee, W. T. Chung, I. H. Hwang, S. A. Lee, B. S. Kim, and J. C. Lee, Mol. Cells 25, 479 (2008).

DISCUSSION

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ChooseTop of pageABSTRACTINTRODUCTIONRESULTSDISCUSSION <<METHODSSUPPLEMENTARY MATERIALMacrophages have a fundamental role in immune response and inflammation.3939. M. A. Elhelu, J. Natl. Med. Assoc. 75, 314–317 (1983). Better control of the inflammatory response remains, at the moment, an open and unsolved line of research, to face several pathologies. Our results showed that M0 macrophages expressed CD14, confirming that PMA activated U937 efficiently, as already shown previously.40,4140. O. P. Barry, D. Praticò, R. C. Savani, and G. A. FitzGerald, J. Clin. Invest. 102, 136–144 (1998). https://doi.org/10.1172/JCI259241. A. Hida, A. Kawakami, T. Nakashima, S. Yamasaki, H. Sakai, S. Urayama, H. Ida, H. Nakamura, K. Migita, Y. Kawabe, and K. Eguchi, Immunology 99, 553–560 (2000). https://doi.org/10.1046/j.1365-2567.2000.00985.x LPS-stimulated macrophages expressed CD80 and CD86 markers and TNF-α starting from 1 μg/mL of the compound, which therefore was chosen as the most appropriate concentration, also in agreement with previous studies.18,4218. X. Zhang, B. Hu, J. Sun, J. Li, S. Liu, and J. Song, J. Ultrasound Med. 36, 2419 (2017). https://doi.org/10.1002/jum.1423942. Y. Shen, S. Yang, Z. Shi, T. Lin, H. Zhu, F. Bi, A. Liu, X. Ying, H. Liu, K. Yu, and S. Yan, Inflammation 38, 736–744 (2015). https://doi.org/10.1007/s10753-014-9984-0Our results clarified what are the optimal LIPUS parameters triggering anti-inflammatory effects on human macrophages (U937 cell line) activated with a strong pro-inflammatory compound such as LPS. No previous studies systematically screened different US parameters (F, I, DC, and t), which may lead to different responses at a cellular level, to identify the optimal protocol for inflammation reduction in vitro. Due to its minimal side effects and non-invasiveness,99. X. Jiang, O. Savchenko, Y. Li, S. Qi, T. Yang, W. Zhang, and J. Chen, IEEE Trans. Biomed. Eng. 66, 2704 (2018). https://doi.org/10.1109/TBME.2018.2889669 LIPUS is used as a treatment for lowering intracellular inflammatory factors. Zhang et al.1818. X. Zhang, B. Hu, J. Sun, J. Li, S. Liu, and J. Song, J. Ultrasound Med. 36, 2419 (2017). https://doi.org/10.1002/jum.14239 observed (at F = 1.5 MHz, I = 60 mW/cm2, DC = 20%, and t = 2 h) a downregulation of apoptotic rate and pro-inflammatory cytokines and increased cell viability, compared to unstimulated control, but their stimulation protocol was not focused on properly controlling the US dose: in fact, all the US parameters were fixed except for the I, for which four different values were tested and their relative bioeffects investigated. However, the poor control of the US dose at the target hampers the reproducibility of the experiments and a full understanding of the interaction between the stimulus and the induced biological effects, thus, slowing down the possible clinical translation.To assess the optimal parameters able to lower the inflammatory level induced by LPS, ELISA analysis and real-time qRT-PCR analysis of three key pro-inflammatory cytokines released by macrophages (TNF-α, IL-1β, and IL-8) were performed. US stimulation at 38 kHz was found to be the best F tested, considerably lowering the inflammatory level of all the three cytokines tested, both at protein and gene levels, compared to 1 and 5 MHz stimulation. This result represented an absolute novelty in the state of the art and, in general, it is the first attempt to explore such low F level, since, as reported by Abrunhosa et al.,4343. V. M. Abrunhosa, C. P. Soares, A. C. Batista Possidonio, A. V. Alvarenga, R. P. B. Costa-Felix, M. L. Costa, and C. Mermelstein, Ultrasound Med. Biol. 40, 504–512 (2014). https://doi.org/10.1016/j.ultrasmedbio.2013.10.013 US is typically used in the 0.5–5 MHz range. The I and DC ranges tested in this study are the typical ones adopted in LIPUS stimulation state of the art, which do not cause significant thermal effects.99. X. Jiang, O. Savchenko, Y. Li, S. Qi, T. Yang, W. Zhang, and J. Chen, IEEE Trans. Biomed. Eng. 66, 2704 (2018). https://doi.org/10.1109/TBME.2018.2889669 Concerning I, although 25 and 100 mW/cm2 triggered positive effects related to cytokines release, a I of 250 mW/cm2 is able to lower the inflammatory level more. When considering DC, 20% guaranteed a greater lowering of the LPS-induced inflammatory level. The exploration of multiple t, as well as multiple DC, was also never explored in the state of the art: in this study, a 90-min stimulation corresponded to the minimal optimal time that guaranteed a significant lowering of proinflammatory cytokines. The results derived from this parameter optimization are of considerable importance because they could be used as a guideline for future pre-clinical and clinical protocols of anti-inflammatory therapies based on LIPUS therapy.Remarkably, additional tests confirmed that this specific stimulation protocol resulted also in an improvement in terms of cell metabolism and intracellular ROS production suppression, without affecting cell viability. Cell proliferation remained at a steady state since PMA induction, which caused cell cycle arrest before the differentiation step into M0 macrophages.4444. Y. S. Baek, S. Haas, H. Hackstein, G. Bein, M. Hernandez-Santana, H. Lehrach, S. Sauer, and H. Seitz, BMC Immunol. 10, 18 (2009). https://doi.org/10.1186/1471-2172-10-18Ion channels are porous membrane proteins, necessary to modify membrane potentials and to tune action potentials and other electrical signals by controlling the passage of ions.4545. L. A. Kadir, M. Stacey, and R. Barrett-Jolley, Front. Physiol. 21, 1161 (2018). https://doi.org/10.3389/fphys.2018.01661 They are involved in the modulation of molecular immune cell environment by acting on inflammatory or anti-inflammatory cascades.4646. S. Feske, H. Wulff, and E. Y. Skolnik, Annu. Rev. Immunol. 33, 291 (2015). https://doi.org/10.1146/annurev-immunol-032414-112212 Early findings have pointed out the key role of ion channels in immune cell behaviors.4747. S. Feske, A. R. Concepcion, and W. A. Coetzee, Sci. Signal. 12, eaaw8014 (2019). PIEZO family of genes were discovered in 2010 as potential channels in the cell line Neuro2A, a glial tumor line.4848. B. Coste, J. Mathur, M. Schmidt, T. J. Earley, S. Ranade, M. J. Petrus, A. E. Dubin, and A. Patapoutian, Science 330, 55 (2010). https://doi.org/10.1126/science.1193270 Among PIEZO channels, PIEZO1 plays a key role in a variety of cell activities, such as c