Cytotoxic Natural Killer Cells Disrupt Nerve Fibres Through Granzyme H in Atheriosclerotic Cerebral Small Vessel Disease

Background: Circulating natural killer cells (NK cells) are enriched in the central nervous system in atheriosclerotic cerebral small vessel disease (aCSVD) rat models, but their resulting effects and underlying mechanism remain to be investigated.Methods: A total of 32 patients with aCSVD and 28 healthy control patients were recruited for this study. According to white matter hyperintensity (WMH) burden, which is closely related to the severity of aCSVD, 20 participants were divided into two groups: burden of 0-1 (n=10) and burden of 2-3 (n=10). All participants participated in proteomics analysis of their cytotoxic NK cells and serum, and 3 participated in proteomics analysis of their cerebrospinal fluid (CSF). In vitro BBB models and a co-culture system with primary human neurons were utilized to verify the pathogenic behaviours of cytotoxic NK cells.Results: In aCSVD patients with a high WMH burden, integrin β2 (ITGB2), cathepsin D (CTSD) and granzyme H (GZMH) were highly expressed in cytotoxic NK cells. ITGB2 interacted with intercellular adhesion molecule 1 (ICAM1) in vascular endothelial cells and promoted the adhesion of cytotoxic NK cells in vitro. Moreover, inhibition of CTSD reduced the destruction of type IV collagen (COL4A) in the extracellular matrix of the BBB and the leakiness of the BBB in vitro and in vivo, indicating that synthetic CTSD in cytotoxic NK cells participates in BBB damage. After passing through the leaky BBB, GZMH disruption on demyelinated nerve fibres was reversed by cotreatment with the inhibitor 3,4-DCIC, suggesting that cytotoxic NK cell-released GZMH is crucial for the disruption of demyelinated nerve fibres during WMH in aCSVD.Conclusions: Cytotoxic NK cells contribute to the CTSD-induced damage to the BBB and GZMH-induced disruption of demyelinated nerve fibres during WMH in aCSVD. Our work highlights the important role of cytotoxic NK cells in the disruption of nerve fibres in patients with aCSVD with a high WMH burden for the first time.