The onset and progression of MMD are associated with immune dysregulation and inflammatory responses in the body [16, 17]. Studies have indicated an increased risk of MMD with elevated systemic immune and inflammatory markers [18]. Previous investigations have utilized samples from MMD patients, such as cerebrospinal fluid or peripheral blood, to identify specific molecules, including autoantibodies and various RNA molecules [19,20,21,22]. CyTOF, as an advanced cell analysis technology, offers multiple advantages, including high dimensionality, low noise, and multi-parameter analysis, making it suitable for a wide range of biomedical research areas [23, 24]. In a previous study, our team pioneered the use of CyTOF to reveal distinctions in the peripheral immune landscape between healthy individuals and those with unruptured intracranial aneurysms. This work uncovered potential roles for dysfunctions in circulating immune functions in the development of unruptured intracranial aneurysms, indicating a significant link between immune system behavior and the pathophysiology of these aneurysms [25]. However, its application in the field of cerebrovascular diseases, particularly in MMD research, has been limited. In this study, we confirmed that, compared to later-stage MMD, the early-stage MMD group exhibits an increase in non-classical monocytes. As the Suzuki stage level increases, the proportions of plasmacytoid DCs and monocyte-derived DCs decrease. Furthermore, T cells, monocytes, DCs, and PMN-MDSCs in the early-stage MMD group show activation of the canonical NF-κB signaling pathway.

The Suzuki stage system is widely used to assess MMD, serving as a grading system to evaluate the anterior circulation condition of patients. MMD patients undergo a gradual narrowing and occlusion of major intracranial arteries from Suzuki stage I to stage III, accompanied by an increase in collateral vessels. From Suzuki stage IV to stage VI, the collateral vessels decrease, and the supply from the internal carotid arteries diminishes, leading to a gradual dependence on the external carotid or vertebral artery pathways for cerebral circulation [26]. During the disease progression, circulating monocytes may play a role in MMD by leaving the bloodstream, migrating through the endothelium, and differentiating into tissue macrophages. These tissue macrophages can initiate and facilitate further immune responses [9, 17]. Elevated levels of non-classical monocytes, as a distinctive subset, are associated with certain autoimmune diseases and viral infections [27, 28]. In our study, the increased levels of non-classical monocytes in the early-stage MMD group may be linked to a chronic inflammatory state. Additionally, the elevated expression of CX3CR1 in classical monocytes of early-stage MMD patients suggests an enhanced capacity for adhesion and chemotaxis.

DCs are known as highly functional antigen-presenting cells, bridging innate and adaptive immunity through antigen processing and presentation to T and B lymphocytes [29]. In our study, we observed a decrease in the proportion of plasmacytoid DCs and monocyte-derived DCs as Suzuki stages increased. This suggests that as the disease progresses, the extent of inflammation may decrease, leading to a relative reduction in these cell types. The decline in the proportion of DCs may result in a diminished capacity for antigen presentation in the body, and in the later stages of MMD, it could be associated with immune cell depletion. Additionally, in the early-stage MMD, DCs and monocyte-derived DCs exhibited higher expression levels of CD197 (CCR7), possibly indicating an enhanced migratory capacity of these cells to lymphoid tissues. At the same time, we also observed high expression of CD24 and CD1c in DCs of the early-stage MMD group. The increase in CD24 is typically associated with anti-inflammatory characteristics [30], while CD1c is closely related to antigen presentation and the activation of the immune response [31]. Elevated CD1c expression may enhance the antigen presentation capability of DC cells.

Fujimura et al. [32] and Young et al. [33] have reviewed the signaling cascade reactions and histology associated with moyamoya angiopathy, indicating an increase in transforming growth factor (TGF), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) in moyamoya angiopathy patients [34]. These growth factors may be related to angiogenesis and inflammation. In our study, FGFR2, implicated in pathways related to FGF, showed no differences in circulating immune cells. The impact of the FGF-related pathway on disease progression remains unclear and requires further experimental validation.

In our study, increased p-NF-κB/p65 was widely observed in peripheral immune cells, including T cells and T_4 cells, monocytes and classical monocytes, DCs, and PMN-MDSCs. The transcription factor NF-κB is a critical regulator of immune and inflammatory responses, operating through two distinct pathways: (1) the canonical pathway primarily activated by pathogens and inflammatory mediators and (2) the non-canonical pathway mostly activated by developmental cues. The most abundant form of NF-κB activated by pathological stimuli through the canonical pathway is the p65: p50 heterodimer [35]. The disproportionate increase in activated p65 and subsequent transcriptional activation of effector molecules are indispensable mechanisms in the pathogenesis of many chronic diseases, such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, and even neurodegenerative pathologies [36]. The induced activation of p65 in response to various stimuli is typically transient but sufficient to upregulate the transcription of various active target genes, including those involved in cell proliferation, inflammatory cytokines, chemokines, and apoptosis mediators [37, 38]. Research on the susceptibility gene RNF213 in MMD indicates that genetic variations in the RNF213 gene may induce NF-κB related inflammation, leading to vascular smooth muscle cell (VSMC) damage, a characteristic of the pathophysiology of MMD [39]. In our study, there was no statistically significant difference in the mutation rate of the RNF213p.4810 K site between the early-stage and later-stage MMD groups. However, for the early-stage MMD group, there was activation of the NF-κB pathway in circulating peripheral immune cells. This suggests that in MMD, the activation of the NF-κB pathway, unrelated to the mutation, may be involved in the progression of the disease.

Toll-like receptors (TLRs) are transmembrane proteins closely associated with both innate and adaptive immunity, playing a role in the pathogenesis of numerous inflammatory diseases [40]. The TLR family has two main signaling pathways: MyD88-dependent and MyD88-independent, with the MyD88-dependent pathway being common to all TLR signals except TLR3. The TLR family can activate the NF-κB signal pathway through MyD88, leading to increased release of pro-inflammatory cytokines and chemokines [41]. In our study, we observed that in the early-stage MMD group, the expression levels of TLR family members (TLR2 and TLR7) increased in T cells and their subsets (T_3 and T_4 cells). Additionally, there was an increase in p-NF-κB/p65 in T cells, indicating activation of the NF-κB pathway. Furthermore, in DCs, molecules related to the CD14/MyD88/NF-κB pathway showed an increased expression. It can be inferred that in the early stages of MMD, inflammation is closely associated with DCs. Bacterial lipopolysaccharides (high-affinity receptor for CD14) may act as triggers for inflammation, and the resulting activation of the NF-κB pathway is involved in the progression of the disease.

The flow cytometry antibodies we designed can separate neutrophils, PMN-MDSCs, and CD34+ cells from the peripheral CD45+ cell population. Previous studies have indicated an association between neutrophil-related genes and CD34+ cells with the onset of MMD [10, 42]. However, possibly due to our limited sample size, we did not observe significant differences between early and late stages of MMD, except in PMN-MDSCs (pathologically activated neutrophils), where activation of the NF-κB pathway was identified in the early-stage MMD group. Additionally, the increased expression of CX3CR1 in NK cells of the early-stage MMD group suggests enhanced adhesion and chemotaxis capabilities, indicating a more active role of NK cells in inflammation and immune responses in early-stage MMD patients.

While our study is the first to apply CyTOF in examining the differences in the peripheral immune landscape among subgroups of MMD patients, it does have limitations. Firstly, the small sample size necessitates further validation of our findings in a larger cohort to establish their significance. Secondly, the differences in peripheral immune cells and molecular signals found between early and later-stage MMD patients require additional basic research. This will facilitate their clinical application as potential therapeutic targets to delay the progression of MMD. Addressing these issues and expanding upon our research will be the focus of our future efforts.