In the current study, we describe a novel mechanism of platelet activation and thrombosis induced by TSLP in KD. Using clinical samples, we found significantly upregulated expression of plasma TSLP in KD patients relative to healthy controls, which was exacerbated in patients complicated with thrombosis. Furthermore, TSLP receptor (TSLPR and IL-7R) expression was significantly enhanced on platelets of KD patients complicated with thrombosis. Interestingly, we found increased platelet mitophagy and apoptosis in KD patients complicated with thrombosis, which TSLP induced in vitro. Lastly, TSLPR bound to mitophagy regulators Parkin and VDAC1 respectively following TSLP treatment, suggesting a novel TSLP-mediated mitophagy pathway in platelets. Taken together, our findings uncover a novel mechanism of platelet activation and thrombosis in KD and suggest TSLP as a novel anti-thrombotic target.
Our work identifies that upregulated TSLP expression at least partially underlies platelet activation and thrombosis in KD. TSLP-induced platelet mitophagy and activation likely promotes thrombosis by promoting platelets to directly bind its ligands mediating platelet aggregation46–48, and to harbor phosphatidylserine (PS) on its surface, which promotes a hypercoagulable state through cell-based thrombin generation34,49. Current anti-thrombotic therapies may not effectively inhibit TSLP-induced thrombosis in KD given its unique mechanism, which may explain the persistent platelet activation and thrombosis in treated patients. A previous report identified that TSLP activates platelets through the PI3K/AKT pathway. However, since we found that the mitophagy inhibitor Mdivi-1 significantly attenuated TSLP-mediated in vitro thrombosis, we propose that its effect on PI3K/AKT signaling may be downstream of its induction of mitophagy23,50.
Mitophagy is mainly regulated by the PINK1/Parkin signaling pathway51. Recombinant Voltage Dependent Anion Channel Protein 1(VDAC1) is a critical substrate of Parkin responsible for the regulation of mitophagy and apoptosis52. Interestingly, we found that TSLPR bound to Parkin and VDAC1 and TSLP treatment enhanced the independent binding of TSLPR/Parkin/VDAC1. TSLP binding TSLPR is reported to cause receptor internalization44, which is consistent with our findings of TSLPR co-localization with the mitochondria in platelets following TSLP treatment. This TSLPR/Parkin/VDAC1 protein complex may be an important driver of TSLP-mediated platelet mitophagy in KD.
Several reports suggest platelet activation may be a major driver of inflammation in KD8,53. Platelets contain and release several proinflammatory cytokines upon activation such as TNF-α, IFN-β, and IL-6 that may propagate an inflammatory state40,54. Activated platelets also express P-selectin, which promotes the formation of platelet-leukocyte aggregates, an important contributor in the progression of KD55,56. Inflammatory responses, platelet activation and thrombosis are inextricably linked. We propose that in addition to its role in thrombosis, TSLP may also contribute to systemic inflammation through its activation of platelets and release of pro-inflammatory factors. Recent studies have shown that TSLP upregulates inflammatory responses by inducing autophagy in T cells, which partially validating our hypothesis57.
To our knowledge, we are the first to report evidence of increased platelet apoptosis in KD. It is interesting to note that the therapeutic mechanism of IVIG in KD is unclear, yet has previously been reported to inhibit platelet apoptosis in immune thrombocytopenia (ITP)58. Thus, it is conceivable that IVIG partially exerts its therapeutic effect in KD through its inhibition of platelet apoptosis. The cause of platelet apoptosis in KD remains unclear, however, considering the intimate link of platelet apoptosis and mitophagy, TSLP likely has a faciliatory role.
The origin of TSLP in KD remains unclear. A variety of stimuli and cytokines (IL-4, IL-13, IL-5, NF-κB, and TNF-α etc.) can activate TSLP production59. Interestingly, a recent report found that TSLP production in human dermal microvascular endothelial cells is also triggered by activated platelets in an IL-1β dependent manner60. Based on this report, TSLP may activate platelets, which triggers endothelial cells to produce TSLP creating a positive feedback cycle that drives TSLP production and platelet activation. Thus, TSLP neutralization may effectively normalize its plasma concentration and reduce platelets activation.
Many experts suggest targeting TSLP-mediated signaling as a novel therapeutic strategy against allergic diseases to neutralize its inflammatory function61. Interestingly, KD disease is marked by a persistent inflammatory state over many months that share several features to allergic disease inflammation including abnormal type 2 inflammation, Th17/Treg imbalance, and other immunopathogenesis62. In addition, anti-TSLP monoclonal antibodies are already used for the treatment of severe asthma, such as Tezepelumab and CSJ11763. Thus, TSLP may serve as a novel therapeutic target that effectively treats the inflammatory and thrombotic risks associated with KD.
A limitation of our research is that we did not use the TSLPR knockout KD mouse model to verify our results. However, our study utilized a large number of rare human clinical specimens, including convalesce stage KD patients with thrombosis, to identify the pivotal role of TSLP in KD.
Our results demonstrate a close relationship between TSLP and thrombosis in vivo and in vitro. TSLP induced platelet mitophagy and activation via the TSLPR/Parkin/VDAC1 signaling pathway to promote thrombosis in KD. Our findings highlight TSLP as an important contributor and novel therauptic target for KD-associated thrombosis.