Therapeutic strategies of EAM that can modulate CD4+ Th17/Treg immune responses, inhibit cytokine-producing from macrophages, and restrain migration of activated macrophages into the myocardium have been reported to show benefits in ameliorating cardiac inflammation and reducing myocardial injury [
30]. Considering the long-term legacy of inflammatory damage to myocarditis caused by immune imbalance and the lack of a cure that patients have a poor quality of life, the noninvasive portable treatment is an available option. In this study, LIPUS was used to stimulate the spleen nerve and alleviate autoimmune myocarditis. We evaluated the dependence of the spleen and cholinergic anti-inflammatory pathway in treating autoimmune myocarditis with LIPUS in mice. It revealed that splenic ultrasound could alleviate the immune response, regulate the proportion and function of CD4+ Treg and macrophages by activating CAP, and finally reduce heart inflammatory injury and improve cardiac remodeling. While investigating the therapeutic efficacy and optimizing the therapeutic parameters for EAM with LIPUS, the targeted organ and enough acoustic pressure have played an important role in EAM treatment.
(1) The spleen is suitable for ultrasound stimulation
Firstly, we determined the center of the spleen by following the research published in
Nature Communication by Zachs et al., which shows that the midpoint of the connecting line from the shoulder and hip joints of mice is considered the spleen’s location [
16]. And we measured the thickness and depth of the spleen of normal BALB/c mice (width * thickness: 4.84 mm*2.02 mm) and EAM mice (width * thickness: 5.77 mm * 2.48 mm) used in this study (Additional file
2). The ultrasound transducer’s sound field was measured using a needle hydrophone (ONDA HNA-0400) to clarify the transducer’s sound pressure distribution. As shown in Additional file
4, from the tip of the coupling cone to the other end of the ultrasound focus, the long axis of the ultrasound focus is about 4 mm (red line). Therefore, the ultrasound focal depth is comparable to the depth of the spleen from the skin, which can be sonicated properly. In summary, we use an external transducer to emit focused ultrasound waves that penetrate the skin to a certain depth to work on the spleen. This method does not guarantee that it only works on the spleen and avoids all other organs. However, according to the above explanation and the ultrasound foci size and depth, the ultrasound wave cannot deliver enough sound energy to stimulate the surrounding tissues, so it should have little impact on the surrounding intestinal tract and celiac plexus. Moreover, the LIPUS-Contra group, shown in Fig.
4, is ultrasound stimulation of the contralateral intestinal tract of the spleen, which has no significant therapeutic effect on EAM. To some extent, it is proved that the LIPUS stimulus caused therapeutic effects through the spleen rather than the surrounding intestinal tissues and celiac nerves.
(2) The therapeutic effect of ultrasound stimulation depends on acoustic pressure
In the study of Cotero et al., splenic NE levels averaged 140 nmol/L in rodents (wild-type C57black/6 mice or Sprague-Dawley rats), whereas the LPS group dropped NE levels to near zero, demonstrating suppression of CAP signaling during LPS-induced inflammation [
15]. With the gradual increase of acoustic pressure, the concentration of NE from spleen increases first and then decreases in ultrasound-stimulated mice. At 0.25 MPa, the average NE concentration reached its peak. In our Additional file
1, under the treatment of different stimulated pressure, the concentration of NE in spleen indeed increased in varying degrees. It suggested that the response of NE to the ultrasound is dependent on stimulated pressure. However, under the effect of 0.3 MPa and 0.473 MPa, there was no significant difference in NE level. Similarly, there was no significant difference in the therapeutic effect between the 0.35 MPa and 0.473 MPa acoustic pressure as shown in Figs.
1,
2. There may be an acoustic pressure threshold for neuronal activation to release neurotransmitters and produce neuromodulation effects. In addition, the LIPUS stimulation duration of each treatment was another determinant, which was even more important than the total treatment length across the LIPUS experiment. Spleen stimulation for 12 min at 0.35 MPa was more effective at reducing myocarditis than 6 min, and the optimized parameters were consistently effective in subsequent experiments. Combining our results with previous studies, we speculated that the production of neurotransmitters is an accumulation process that requires adequate stimulation time.
(3) The selection of vagus nerve segment for ultrasound stimulation
The vagus nerve is the main nerve of the parasympathetic division of the autonomic nervous system. Its efferent arm is distributed in major body organs, including the heart, liver, digestive system and spleen [
31]. The efferent vagal nerve-mediated cholinergic signal controls the immune function and proinflammatory response through inflammatory reflex. The intervention of it can produce an anti-inflammatory reaction and have a therapeutic effect on sepsis, renal ischemia, colitis, arthritis, etc. [
32‐
35] Compared with the systemic regulation by oral drugs, physical stimulation of a certain vagus nerve segment can avoid dysfunction of other important tissues and organs caused by activation of the vagus nerve in the whole body. In previous studies, most of the physical stimulation sites of vagus nerve were selected to be controlled by cervical vagus nerve [
36‐
38], apical splenic nerve [
39] or celiac ganglion [
40‐
42]. Electrical stimulation is the most common form of physical therapy. Implantation of electrodes and generation of different voltages to stimulate nerves are the main intervention methods, but there are some side effects, such as dyspnea, pain and cough. Stimulation of cervical vagus nerve causes the excitation of branches of cardiac afferent nerve, leading to a long-term decrease in heart rate and increased heart rate variability. In addition, since electrodes need to be implanted surgically, many postoperative complications may occur, such as bleeding, infection, permanent vocal cord paralysis, coma, etc. [
43,
44]. Therefore, it is of great significance to find a noninvasive means of vagus nerve regulation, which is also the purpose of this study. We choose the spleen, one of the vagus nerve branch, as the stimulated organ. The spleen is an important immune organ of the human body, and is an indispensable part for the activation, development, differentiation and maturation of a variety of immune cells. Previous studies have shown that the spleen can be used as the cross-talk bridge between nerve and immunity, and that the cholinergic anti-inflammatory pathway of the splenic nerve can be used to regulate the differentiation and response of T cells and macrophages [
45,
46]. This intervention approach will not affect the normal operation of other organs, especially to avoid the risk of inhibiting heart rate and arrhythmia, which is very important for cardiovascular disease treatment. The denervation of splenic nerve surgery was performed in EAM mice in Fig.
5. LIPUS treatment of mice with denervation of splenic nerve did not show statistically significant differences compared with the EAM group alone. The therapeutic effect of LIPUS on mice with denervation of splenic nerve was not as good as that on mice with intact splenic nerve. Since the splenic nerve denervation operation only inactivated most of the splenic nerve fibers, the spleen vessels were not harmed and the blood supply in the spleen was normal, and the immune cells were still alive but not regulated by the splenic nerve. Therefore, the comparison between LIPUS + EAM group and LIPUS + EAM + denervation of splenic nerve group at least partially explains the splenic nerve dependence of the ultrasound treatment in this study.
In the progress of EAM, the proliferative or imbalance of CD4+ Th17/Treg and activated macrophages play a prominent role, contributing to the severity of EAM inflammation [
22,
47,
48]. Therefore, the dynamic change of different immune cell subsets is one of our research's focuses, contributing to the further understanding of myocarditis pathogenesis. We found that the modulatory effect of LIPUS stimulation was more evident on Treg than Th17 cells. The reduction of inflammatory CCR2+ macrophages with antigen-presenting function and the increase of immune-tolerant Treg cells work synergistically in LIPUS immune process control. Among the CCR2+ macrophage, the proportion of high MHC II expression in F480+/CD11b+ CCR2+ positive macrophages decreased significantly after LIPUS treatment. As MHC II represents the antigen presentation ability to T cells, we speculated that the CCR2+ macrophages with MHC II high expression showed the function of antigen-presenting cells to regulate the Treg immune response. Since the classification of M1/M2 macrophages has been replaced gradually due to many overlapping functions and markers [
48], the blood-derived CCR2+ macrophages, which are involved in cardiac dysfunction and fibrosis, inhibited oxidative stress, myocardial apoptosis and cardiac inflammation, have been used to distinguish between proinflammatory and cardiac resident macrophage [
49,
50]. Leuschner reported that mice silenced CCR2 exhibit a reduced inflammation in autoimmune myocarditis [
51]. In addition, transcriptome sequencing results showed that DEGs were associated with T cell differentiation, metabolism and antigen presentation and involved in oxidative stress response. Taken together, both adaptive and innate immunity is involved in the biological mechanism underlying LIPUS treatment.
Noninvasive ultrasound stimulation is demonstrated to be effective in treating EAM at both the organ level and the cellular and molecular levels. We also determined that ultrasound treatment in EAM mice activates the splenic nerve. As an essential signaling pathway for neuro-immune modulation, the CAP of the spleen provides a crucial bridge for LIPUS to exert cardiac protective effects. Stimulating other body areas such as the heart or the contralateral abdomen opposite the spleen did not effectively improve myocarditis. Hence, the positive results of LIPUS therapy appeared to be not mediated by a direct effect on the heart but immunomodulation via CAP that is dependent upon the spleen. Moreover, ultrasound stimulation showed similar therapeutic efficacy as the α7nAChR agonists, GTS-21, which decreased heart inflammation in a murine virus and autoimmune myocarditis model [
53,
54]. Moreover, right cervical vagotomy inhibited the CAP, aggravated myocardial lesions, and upregulated the expression of TNF-α, IL-1β, and IL-6. It worsened the impaired left ventricular function in murine viral myocarditis, and these changes were reversed by co-treatment with nicotine by activating CAP [
55]. Therefore, the ultrasound stimulation can function via CAP like vagal stimulation, which has been demonstrated to improve the survival rate and prevent the progression of cardiac dysfunction and remodeling [
56]. Considering the insidious onset and rapid progress of myocarditis, LIPUS may not participate in the clinical treatment in the acute phase timely, but applying it in the convalescence stage is encouraging. Given that LIPUS is used in the clinic, this adds a valuable treatment option for patients with EAM. With the advantage of safety, availability and portability of LIPUS, it could be worn by patients over their abdominal area, significantly improving their quality of life and demonstrating a perspective of clinical transformation. There is an emerging need for noninvasive neuromodulation techniques to minimize adverse events and morbidity during improving patient outcomes. The intensity of ultrasound used in the LIPUS therapy is below the upper limit of acoustic output standards for diagnostic ultrasound devices, making it suitable for home care during recovery. For the first time, LIPUS has become an adjuvant therapy for EAM.