Anti-IL-17 Inhibits PINK1/Parkin Autophagy and M1 Macrophage Polarization in Rheumatic Heart Disease.

Rheumatic heart disease (RHD) is an important and preventable cause of cardiovascular death and disability, but the lack of clarity about its exact mechanisms makes it more difficult to find alternative methods or prevention and treatment. We previously demonstrated that increased IL-17 expression plays a crucial role in the development of RHD-related valvular inflammatory injury. Macrophage autophagy/polarization may be a pro-survival strategy in the initiation and resolution of the inflammatory process. This study investigated the mechanism by which IL-17 regulates autophagy/polarization activation in macrophages. A RHD rat model was generated, and the effects of anti-IL-17 and 3-methyladenine (3-MA) were analyzed. The molecular mechanisms underlying IL-17-induced macrophage autophagy/polarization were investigated via in vitro experiments. In our established RHD rat model, the activation of the macrophage PINK1/Parkin autophagic pathway in valve tissue was accompanied by M1 macrophage infiltration, and anti-IL-17 treatment inhibited autophagy and reversed macrophage inflammatory infiltration, thereby attenuating endothelial-mesenchymal transition (EndMT) in the valve tissue. The efficacy of 3-MA treatment was similar to that of anti-IL-17 treatment. Furthermore, in THP-1 cells, the pharmacological promotion of autophagy by IL-17 induced M1-type polarization, whereas the inhibition of autophagy by 3-MA reversed this process. Mechanistically, silencing PINK1 in THP-1 blocked autophagic flux. Moreover, IL-17-induced M1-polarized macrophages promoted EndMT in HUVECs. This study revealed that IL-17 plays an important role in EndMT in RHD via the PINK1/Parkin autophagic pathway and macrophage polarization, providing a potential therapeutic target.

Potential Involvement of M1 Macrophage and VLA4/VCAM-1 Pathway in the Valvular Damage Due to Rheumatic Heart Disease.

BACKGROUND: Rheumatic heart disease (RHD) is caused by inflammatory cells mistakenly attacking the heart valve due to Group A Streptococcus (GAS) infection, but it is still unclear which cells or genes are involved in the process of inflammatory cells infiltrating the valve. Inflammatory infiltration into the target tissue requires an increase in the expression of phosphorylated vascular endothelial-cadherin (p-VE-cad), p-VE-cad can increase the endothelial permeability and promote the migration of inflammatory cells across the endothelium. P-VE-cad is potentially regulated by RAS-related C3 botulinum substrate 1 (RAC1), together with phosphorylated proline-rich tyrosine kinase 2 (p-PYK2). While RAC1/p-PYK2/p-VE-cad is triggered by the activation of vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 is related to M1 macrophages adhering to the endothelium via very late antigen 4 (VLA4). Inflammatory infiltration into the valve is extremely important in the early pathogenesis of RHD. However, there is no relevant research on whether M1/VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad is involved in RHD; therefore, what we explored in this study was whether M1/VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad is involved. METHODS: We established a rat model of RHD and a cell model of M1 macrophage and endothelial cell cocultivation. Subsequently, we measured the degree of inflammatory cell infiltration, the levels of IL-6/IL-17, the degree of fibrosis (COL3/1), and the expression levels of fibrosis markers (FSP1, COL1A1 and COL3A1) in the heart valves of RHD rats. Additionally, we detected the expression of M1/M2 macrophage biomarkers in rat model and cell model, as well as the expression of M1/VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad. We also tested the changes in endothelial permeability after coculturing M1 macrophages and endothelial cells. RESULTS: Compared to those in the control group, the levels of inflammatory cell infiltration and fibrotic factors in the heart valves of RHD rats were significantly higher; the expression of M1 macrophage biomarkers (iNOS, CD86 and TNF-α) in RHD rats was significantly higher; and significantly higher than the expression of M2 macrophage biomarkers (Arg1 and TGF-ß). And the expression levels of VLA4/VCAM-1 and RAC1/p-PYK2/p-VE-cad in the hearts of RHD rats were significantly higher. At the cellular level, after coculturing M1 macrophages with endothelial cells, the expression levels of VLA4/VCAM-1 and RAC1/p-PYK2/p-VE-cad were significantly higher, and the permeability of the endothelium was significantly greater due to cocultivation with M1 macrophages. CONCLUSIONS: All the results suggested that M1 macrophages and the VLA4/VCAM-1 pathway are potentially involved in the process of inflammatory infiltration in RHD.

Interference with the expression of S1PR1 or STAT3 attenuates valvular damage due to rheumatic heart disease.

Rheumatic heart disease (RHD) affects numerous individuals annually; however, its pathogenesis remains unclear. The sphingosine 1­phosphate receptor 1 (S1PR1) and signal transducer and activator of transcription 3 (STAT3) have recently been shown to be involved in valvular damage via the promotion of the differentiation of T helper 17 (Th17) cells during the development of RHD­induced valvular damage. The present study investigated whether altering the expression of S1PR1 or STAT3 attenuates valvular damage due to RHD. Inactivated group A streptococcus (GAS) was used to establish a rat model of RHD. Recombinant adeno­associated viral vectors carrying an S1PR1 overexpression sequence were used to overexpress S1PR1. STAT3 small interfering RNA (STAT3­siRNA) was used to inhibit STAT3 expression. Reverse transcription­quantitative PCR (RT­qPCR) was performed to detect the mRNA expression of S1PR1, STAT3, collagen type III α1 chain (Col3a1) and fibroblast­specific protein 1. Western blotting (WB) and immunohistochemistry were used to detect the levels of S1PR1, STAT3, phosphorylated (p­) STAT3, and retinoic acid­related orphan receptor Î³T (RORγt) proteins. Enzyme­linked immunosorbent assays (ELISAs) and immunohistochemistry were used to detect the levels of interleukin (IL)­6 and IL­17. Hematoxylin and eosin (H&E) staining and Sirius Red staining were performed to evaluate the degree of inflammation and fibrosis in the valvular tissues. S1PR1 expression was decreased in the valvular tissues of the rats with RHD. The levels of IL­6, IL­17 and p­STAT3 in the rats with RHD were increased. The degree of valvular inflammation and fibrosis in the rats with RHD was also increased. The overexpression of S1PR1 and the inhibition of STAT3 reduced the total p­STAT3 level, resulting in decreased levels of IL­6, IL­17 and RORγt, and a reduced degree of valvular inflammation and fibrosis. These results suggest that the expression of S1PR1 and STAT3 may be involved in valvular tissue damage due to RHD. Thus, strategies designed to interfere with the expression of S1PR1 or STAT3 may affect the expression of Th17 cell­related cytokines and may thus attenuate valvular damage due to RHD.

Signalling pathways implicated in the pathogenesis of rheumatic heart disease (Review).

Rheumatic heart disease (RHD) is frequently encountered in underdeveloped areas and primarily affects patients under the age of 40 years old. The pathogenesis of RHD has yet to be fully elucidated and surgical treatment remains the only option, which is expensive and technically demanding for patients in less developed areas. Signalling pathways are crucial for the occurrence and development of several diseases, and researchers worldwide have made progress in elucidating the signalling pathways associated with the pathogenesis of RHD. The aim of the present review was to discuss 6 signalling pathways implicated in the pathogenesis of RHD, summarize the methods and progress of these studies and propose future research directions. Important information on the pathogenesis of RHD according to the current progress of signalling pathway studies was also summarized, in the hope that this review may serve as a reference for future research on the signalling pathways involved in the pathogenesis of RHD.

Activation of activin/Smad2 and 3 signaling pathway and the potential involvement of endothelial­mesenchymal transition in the valvular damage due to rheumatic heart disease.

Rheumatic heart disease (RHD) is an autoimmune disease caused by rheumatic fever following group A hemolytic streptococcal infection and primarily affects the mitral valve. RHD is currently a major global health problem. However, the exact pathological mechanisms associated with RHD­induced cardiac valve damage remain to be elucidated. The endothelial­mesenchymal transition (EndMT) serves a key role in a number of diseases with an important role in cardiac fibrosis and the activin/Smad2 and 3 signaling pathway is involved in regulating the EndMT. Nevertheless, there are no studies to date, to the best of the authors' knowledge, investigating the association between RHD and EndMT. Thus, the aim of the current study was to investigate the potential role of EndMT in cardiac valve damage and assess whether activin/Smad2 and 3 signaling was activated during RHD­induced valvular injury in a rat model of RHD induced by inactivated Group A streptococci and complete Freund's adjuvant. Inflammation and fibrosis were assessed by hematoxylin and eosin and Sirius red staining. Serum cytokine and rheumatoid factor levels were measured using ELISA kits. Expression levels of activin/Smad2 and 3 signaling pathway­related factors [activin A, Smad2, Smad3, phosphorylated (p­)Smad2 and p­Smad3], EndMT­related factors [lymphoid enhancer factor­1 (LEF­1), Snail1, TWIST, zinc finger E­box­binding homeobox (ZEB)1, ZEB2, α smooth muscle actin (α­SMA) and type I collagen α 1 (COL1A1)], apoptosis­related markers (BAX and cleaved caspase­3) and valvular inflammation markers (NF­κB and p­NF­κB) were detected using reverse transcription­quantitative PCR and western blot analyses. Compared with the control group, the degree of valvular inflammation and fibrosis, serum levels of IL­6, IL­17, TNF­α and expression of apoptosis­related markers (BAX and cleaved caspase­3) and valvular inflammation marker (p­NF­κB), activin/Smad2 and 3 signaling pathway­related factors (activin A, p­Smad2 and p­Smad3), EndMT­related factors (LEF­1, Snail1, TWIST, ZEB 1, ZEB2, α­SMA and COL1A1) were significantly increased in the RHD group. These results suggested that the activin/Smad2 and 3 signaling pathway was activated during the development of valvular damage caused by RHD and that the EndMT is involved in RHD­induced cardiac valve damage.

Inhibition of miR­155­5p attenuates the valvular damage induced by rheumatic heart disease.

Autoimmunity is involved in the valvular damage caused by rheumatic heart disease (RHD). Increased evidence has linked microRNAs (miRNAs/miRs) to autoimmune disease. Signal transducer and activator of transcription 3 (STAT3) and sphingosine­1­phosphate receptor 1 (S1PR1) and suppressor of cytokine signaling 1 (SOCS1) have been widely studied for their roles in autoimmunity and inflammation. Thus, the current study aims to investigate the role played by miR­155­5p in RHD­induced valvular damage via the S1PR1, SOCS1/STAT3 and interleukin (IL)­6/STAT3 signaling pathways. An RHD rat model was induced by inactivated Group A streptococci and complete Freund's adjuvant. A recombinant adeno­associated virus (AAV­miR155­inhibitor) was used to inhibit the expression of miR­155­5p in the heart. Inflammation and fibrosis were assessed by hematoxylin and eosin staining and Sirius red staining. The expression of miR­155­5p in valvular tissues and serum exosomes was detected by reverse transcription­quantitative PCR. S1PR1, SOCS1, STAT3, phosphorylated STAT3, IL­6 and IL­17 protein expression was detected by western blotting and immunohistochemistry. The relationships between miR­155­5p and S1PR1 and SOCS1 were detected by dual luciferase assays. Cytokine concentrations were measured by ELISA. The expression of miR­155­5p in valve tissues and serum exosomes was increased along with decreased S1PR1 and activated SOCS1/STAT3 signaling in the RHD model. The expression of IL­6 and IL­17 was increased in the valves and the serum. Dual luciferase assays showed that miR­155­5p directly targeted S1PR1 and SOCS1. Inhibition of valvular miR­155­5p through AAV pretreatment increased S1PR1 expression and inhibited activation of the SOCS1/STAT3 signal pathway as a result of attenuated valvular inflammation and fibrosis as well as a decrease in IL­6 and IL­17 in the valves and serum. These results suggest that inhibition of miR­155­5p can reduce RHD­induced valvular damage via the S1PR1, SOCS1/STAT3 and IL­6/STAT3 signaling pathways.