Protective effect of low‐intensity pulsed ultrasound on immune checkpoint inhibitor-related myocarditis via fine-tuning CD4+ T-cell differentiation

Fig. 1. PD-1 inhibitor cause impairment of cardiac function and myocardial inflammatory response. a The schedule of injection. A/J mice were randomly divided into 2 groups: control group (control); ICI-related myocarditis group (PD-1 inhibitor).Mice in ICI-related myocarditis group were immunized with PD-1 inhibitor at day 0, 7 and 14.Mice were sacrificed on day 21. b Serum levels of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) (n = 10). c Representative image of M-mode echocardiography. d Quantifications of ejection fraction (EF) and fraction shortening (FS) (n = 10). e The heart tissue H&E stain represents the image (scale bar, 50 μm). f The H&E images were analyzed based on histological grades (n = 10). g Immunohistochemistry analyze of CD4 + cell infiltration in cardiac sections (scale bars, 50 μm). h The percentages of CD4 + cells were enumerated (n = 10). Error bars show mean ± SD.*p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001

Fig. 2. LIPUS Ameliorated Cardiac Inflammation and Injury Induced by PD-1 inhibitor. a Experimental protocol. b, c Ratio of heart weight to body weight;Ratio of lung weight to body weight (n = 10). d, e Serum levels of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) (n = 10). f Representative image of M-mode echocardiography. g, h Quantifications of ejection fraction (EF) and fraction shortening (FS) (n = 10). i The heart tissue H&E stain represents the image (scale bar, 500 μm). j The H&E images were analyzed based on histological grades (n = 10). k Representative image of Masson staining (scale bar, 500 μm). l The Masson images were analyzed based on fibrotic area (n = 10). m Western Blot was performed to assess relative expression of hypertrophy and fibrosis-related genes. n Representative images and quantitative analysis of immunofluorescence staining of mice heart sections for CD3 and CD4, (scale bar, 200 μm) (n = 10).Error bars show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001

Fig. 3. LIPUS reduced cardiac CD4 + T and Th17 responses. a–b Representative images and quantitative analysis of immunohistochemical analysis of mice heart sections of IL-17A (scale bar, 500 μm) and FOXp3 (scale bar, 100 μm) (n = 10). c Gating strategy for the flow cytometry experiments. d Cardiac infiltrating mononuclear cells were collected from each group for flow cytometry analysis of CD4+T cells. e Cardiac IL-17A+CD4+T cells and Foxp3+CD4+ Tcells for flow cytometry analysis. f Levels of the cytokines IL-17A, and IL-10 were measured by ELISA (n = 5). g Evaluate the expression of RORγt, Il17a, Csf2, Rora and FOXp3 by RT-qPCR; data were normalized to GADPH, (n = 5).Error bars show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001

Fig. 4. The inflammatory relief of LIPUS is mediated by cascade kinase signaling in the HIPPO pathway. a–d Representative images and quantitative analysis of Western blotting analysis of HIPPO pathway proteins (Mst1, TAZ and TEAD-1) in different conditions (n = 3).(Error bars show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001

Fig. 5. Inhibition of the HIPPO signaling pathway partly diminished the therapeutic effect of LIPUS. a Experimental protocol. b Ratio of heart weight to body weight. c Serum levels of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) (n = 10). d Representative image of M-mode echocardiography. e Quantifications of ejection fraction (e, f) and fraction shortening (FS) (n = 10). f The heart tissue H&E stain represents the image. (scale bar, 500 μm). g The H&E images were analyzed based on histological grades (n = 10). h Representative image of Masson staining. (scale bar, 500 μm). i The Masson images were analyzed based on fibrotic area (n = 10). j Representative images and quantitative analysis of immunofluorescence staining of mice heart sections for CD3 and CD4, (scale bar, 200 μm) (n = 10). k Quantifications of immunofluorescence staining. Error bars show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001

Fig. 6. Inhibition of HIPPO signaling pathway partially attenuated the inhibitory effect of LIPUS on CD4+T cell differentiation. a, b epresentative images and quantitative analysis of immunohistochemical analysis of mice heart sections of IL-17A (scale bar, 500 μm) and FOXp3 (scale bar, 500 μm) (n = 10). c Cardiac infiltrating mononuclear cells were collected from each group for flow cytometry analysis of CD4 + T cells (n = 10). d, e Cardiac IL-17A+CD4+T cells and Foxp3+CD4+ T cells for flow cytometry analysis (n = 10). f Levels of the cytokines IL-17A, and IL-10 were measured by ELISA (n = 10). g Evaluate the expression of RORγt, Il17a, Csf2, Rora and FOXp3 by RT-qPCR; data were normalized to GADPH, (n = 5).Error bars show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001