In our study, inflammation and endothelial dysfunction in KD were improved by the transplantation of Huc-MSCs. Moreover, Huc-MSCs promoted the proliferation, migration, invasion, and tube formation of HUVECs that were stimulated with KD serum and THP-1 cells. In addition, NLRP3 inflammasome activation and the expression of ASC and downstream inflammatory cytokine release were reduced after transplantation. It was shown that the reduction in NLRP3 inflammasome activation played a significant role in stem cell therapy for KD vasculitis. Thus, we hypothesized that the protective effects of Huc-MSCs on KD were mediated by inhibiting NLRP3 inflammatory activation.
Many studies have shown that KD is an acute systemic vasculitis; however, the pathogenesis is still unclear, and immune disorders[4, 39, 40] and endothelial dysfunction[9, 41] are important mechanisms. Currently, there is a lack of reliable and effective treatments for children with severe cardiovascular lesions and endothelial dysfunction. Inflammatory and autoimmune responses lead to endothelial injury, which is one of the vital causes of the development of KD[14]. Therefore, targeted suppression of excessive inflammatory activation is a focus of therapy for KD.
In recent years, many studies have shown that pyroptosis is a pathophysiological event in KD[12, 13, 15, 16, 29]. Its activation could increase the expression of RAGE and cathepsin B activity, resulting in NLRP3 inflammasome-dependent pyroptotic death in ECs. Moreover, NLRP3 inflammasome activation and IL-1β production were obviously present in KD cardiovascular lesions, as determined by scRNA-seq and ST analysis, which was verified in the LCWE-induced murine model of KD. Our research also reveals that endothelial injury is related to pyroptosis in vitro and in vivo, suggesting that the NLRP3 inflammasome plays an important role in KD vasculitis. Thus, targeted suppression of excessive NLRP3 inflammasome activation is a promising therapeutic strategy for KD. Among them, in Fig. 1A, we found that IL-18 in convalescent period seems to be significantly upregulated compared with the acute stage, which was speculated that it may be involved in the repair process of KD inflammation damage, but the specific mechanism process has not been further explored in this research, and our group may further study it later.
MSCs can not only differentiate into various types of cells but also have unique immunomodulatory properties through paracrine signaling or the release of extracellular vesicles, which provides new opportunities for the treatment of autoimmune diseases, such as RA, SLE and inflammatory bowel disease (IBD)[32–36, 42]. However, autoimmune diseases do not have a precise pathogenesis, and several studies have reported that the NLRP3 inflammasome is associated with susceptibility, disease severity and treatment effects[43–45]. Preliminary experiments demonstrated that Huc-MSCs could suppress endothelial inflammation by regulating the expression of CD54 and CD105 in KD models vivo and in vitro, suggesting that stem cell therapy may be a new direction[30]. In addition, Ryoichi Uchimura et al. found that by transplanting fat-derived MSCs into mice, CAWS-induced heart inflammation, which is another in vivo model of KD, was inhibited, and the survival rate was dramatically improved[21]. This also validates our hypothesis.
An increasing number of studies have considered the significant role of the NLRP3 inflammasome in a variety of autoimmune diseases[46]. We examined the anti-inflammatory effects of Huc-MSCs on cardiovascular inflammation in KD induced by LCWE and found that macrophage infiltration around the coronary artery and CALs could be reduced by the transplantation of Huc-MSCs via the tail vein. Moreover, we found that the expression of molecules related to pyroptosis was reduced on week after transplantation, as shown by slice staining and quantitative analysis, which was consistent with our earlier conclusions. However, in our single-cell sequencing analysis, we also found no significant differences between the endothelial cells in Fig. 2c, which we analyzed may be related to the fact that the cells used for sequencing were not abdominal aorta rather than endothelial cells alone. However, combined with the relevant literature and research basis we reviewed in the early stage, we still chose endothelial cells as the research object for this time.
In addition, we used KD serum and THP-1 cells to stimulate HUVECs in vitro to imitate the in vivo inflammatory environment, and the pyroptosis pathway was activated in ECs, which was consistent with the conclusion of Chang Jia et al[15, 47]. Furthermore, the biological function of ECs in the model was also dysfunctional. Therefore, we separately added the NLRP3 inhibitor MCC950 and agonist nigericin to an in vitro KD model to observe changes in ECs, including the level of inflammation and the function of ECs, compared with the effect of Huc-MSCs. We found that Huc-MSCs not only reduced activation of the NLRP3 inflammasome in damaged HUVECs but also improved proliferation, migration, invasion, tube formation and apoptosis, ameliorating longterm damage to ECs in KD.
The present results suggested that Huc-MSCs could treat KD coronary artery damage and vasculitis by suppressing activation of the NLRP3 inflammasome. Moreover, we also found that Huc-MSCs were more effective than MCC950 in improving endothelial dysfunction and cell death. The mechanism might involve the paracrine effect of Huc-MSCs and should be further studied in follow-up experiments. In vitro, although the NLRP3 inhibitor MCC950 could inhibit inflammasome activation, our study showed that compared to MCC950, Huc-MSCs could significantly improve the biological function of the endothelium while inhibiting inflammasome activation. Due to the inconvenience of frequent injections and the potential infection risk associated with NLRP3 inhibitors, these agents may be difficult to use in the clinic[48]. However, Huc-MSCs have unique advantages, including strong anti-inflammatory, angiogenic, antioxidant and immunomodulatory potential. These cells are also easy to isolate, have self-renewal properties and lack of immunogenicity, and clinical applications have been carried out in some diseases, which may be a new direction of future research[49]. In addition, a clear safety profile for Huc-MSCs as well as further pre-clinical and clinical studies in KD are necessary prior to any treatment.
The mechanism by which Huc-MSCs regulate NLRP3 remain unclear. In vivo, we used GFP-labeled Huc-MSCs and examined their localization using noninvasive imaging. However, no obvious fluorescence signal was observed at the coronary artery damage site, and we did not have time to examine it further. Thus, we hypothesized that Huc-MSC-mediated therapeutic effects on the KD vasculitis model involve paracrine activity rather than direct cell-to-cell contact, and we will further study this hypothesis in the future.