Increased cytotoxic NK cells was related to sparse nerve fibres in patients with aCSVD with a high WMH burden.
This prospective randomized controlled study included 28 controls and 32 patients with aCSVD. There were 28 (100.00 %) controls with low WMH burden (0-1 Fazekas scale) by T2 FLAIR imaging and 22 (68.70 %) patients with aCSVD with a high WMH burden (2-3 Fazekas scale; Table S1) . WMH burden was strongly correlated with the severity of aCSVD (r=0.712, p=0.000), including WMH around the ventricle and in the corona compared with the LI, PVS and CMB burdens (Fig. 1, B).
We regrouped the 60 participants according to their WMH burden. As shown in Table S1, 38 participants were in the WMH burden 0-1 group, and the other 21 patients with aCSVD were in the WMH burden 2-3 group. The patients with a high WMH burden had hypoperfusion as observed by arterial spin labelling (ASL) imaging and sparse white matter fibres (WMFs) by diffusion tensor imaging (DTI) (both p<0.05, Fig. 1, A; Table S1). We measured balance and gait impairment and found that high WMH burden patients had a prolonged sitting time (p=0.011, Fig. 1, C) and a higher number of NK cells in their blood (p=0.011, Fig. 1, D). Furthermore, a high WMH burden was moderately correlated with hypoperfusion (r=0.573, p=0.000), sparse WMFs (r=0.639, p=0.000), sitting time (r=0.476, p=0.025) and number of NK cells (r=0.509, p=0.000) (Fig. 1, E). NK cells also had a medium correlation with hypoperfusion (r=0.342, p=0.026) and sparse WMFs (r=0.511, p=0.002) (Fig. 1, E). These findings suggested that the causal relationship between NK cells and sparse WMFs deserves further investigation. Next, we analysed the five subtypes of NK cells by FC (Fig. 1, F). Compared with WMH burden 0-1 participants, the proportion of cytotoxic NK cells (CD56dimCD16bright) increased most significantly in the patients with WMH burdens of 2-3 (p=0.000, Fig. 1, G). These results indicate that an increase in the number of cytotoxic NK cells was closely related to sparse nerve fibres during WMH of aCSVD.
Cytotoxic NK cells in aCSVD migrated through ITGB2 and negatively regulated neural projection development by FLNA.
To explore the relationship between cytotoxic NK cells and sparse nerve fibres in aCSVD, we isolated cytotoxic NK cells (CD56dimCD16bright) from the WMH burden 0-1 and WMH burden 2-3 groups by FC (n=10). Based on proteomics analysis of the cytotoxic NK cells, 976 proteins were upregulated in the cytotoxic NK cells from the high WMH burden group, which indicated that they were in a highly activated state (all p<0.05, Fig. S1). We showed the cluster of the first 300 differential proteins in the heat map (all p<0.01, Fig. 2, A). Gene ontology (GO) enrichment of cytotoxic NK cells found that the cellular component (CC) mainly included focal adhesions and extracellular exosomes (including cathepsin D; CTSD) (both p=0.000, Fig. 1, B), molecular function (MF) integrin binding (p=0.028), actin filament binding (p=0.000), and peptidase activity (CTSD, p=0.024, Fig. 2, E). The main biological processes (BPs), including NK cell activation (p=0.047), cell migration (p=0.028), regulation of cell shape (p=0.001), cell-cell adhesion (p=0.005) and cellular extravasation (p=0.008) were closely related to the key protein integrin β2 (ITGB2) (Fig. 2, C). GZMH was responsible for the BP of cytolysis (p=0.001, Fig. 2, C). Filamin-A (FLNA) was involved in cell junction assembly (p=0.000), positive regulation of actin filament bundle assembly (p=0.011), negative regulation of neuron projection development (p=0.021) and negative regulation of apoptotic processes (p=0.000, Fig. 2, C). In addition, FLNA participated in cell-extracellular matrix interactions through reactome pathway analysis (p=0.020, Fig. 2, D). The levels of ITGB2, GZMH, CTSD and FLNA were 2-fold higher in cytotoxic NK cells from patients with aCSVD with a 2-3 WMH burden than in NK cells from patients with aCSVD with a 0-1 WMH burden (all p<0.01, Fig. 2, F and G). In the interaction map, ITGB2 in cytotoxic NK cells interacted with intercellular adhesion molecule 1 (ICAM1) expressed in endothelial cells (interaction score: 0.989, Fig. 3, H) and promoted the adhesion of cytotoxic NK cells and endothelial cells. The above results suggest that the cytotoxic NK cells crossed the damaged BBB through ITGB2 and negatively regulated neural projection development through FLNA.
B2M mediated the lymphocyte immunoreaction in serum from patients with aCSVD.
To explore whether the serum components are involved in the inflammatory response of cytotoxic NK cells, we collected serum from the above mentioned patients for proteomic tests. The results showed that 35 proteins were upregulated and 16 proteins were downregulated (all p<0.05, Fig. 3, A). Among these upregulated proteins, differential β2-microglobulin (B2M) not only was related to extracellular exosomes in CC (p=0.000, Fig. 3, B), positive regulation of receptor binding (p=0.017) and negative regulation of neurogenesis (p=0.049) in BP (p=0.017, Fig. 3, E) but also participated in the immunoregulatory interactions between lymphoid and non-lymphoid cells from the reactome pathway analysis (p=0.000, Fig. 3, D). An interaction map clarified that B2M in serum interacted with ICAM1 (interaction score: 0.944, Fig. 3, H) and then promoted the adhesion of NK cells and endothelial cells in the vasculature [29, 30]. B2M in serum also interacted with CTSD (interaction score: 0.920, Fig. 3, H), which promoted the immune response of cytotoxic NK cells . Correspondingly, downregulated sphingosine 1-phosphate receptor 1 (S1PR1) was involved in S1PR receptor activity in MF (p=0.017, Fig. 3, C), blood vessel maturation (p=0.019) and the S1PR signalling pathway in BP (p=0.019, Fig. S2, A). Both B2M and S1PR1 were changed by more than 2-fold (both p<0.05, Fig. 3, F and G). These results strongly suggest that upregulated B2M in serum might promote cytotoxic NK cell binding and interaction with other cells, such as endothelial cells, through ICAM1. In addition, downregulation of S1PR1 might be harmful to blood vessel maturation.
Axon, dendritic, and neurofilament fragments were observed in the CSF from patients with aCSVD.
To further identify the disruption of nerve fibres in patients with aCSVD, we examined 3 CSF samples from the two groups by proteomics. There were 20 upregulated proteins and 44 downregulated proteins in the CSF of patients with aCSVD with a high WMH burden (all p<0.05, Fig. 4, A). The CC for CSF contained extracellular exosomes, axonal growth cones, focal adhesions, dendritic spine membranes and neurofilaments (all p<0.05, Fig. 4, B). Neurofilament medium polypeptide (NEFM) took part in the structural constituent of the cytoskeleton in MF (p=0.039, Fig. 4, C) and neurofilament bundle assembly in BP (p=0.010, Fig. 4, D). On the other hand, the downregulation of neuronal pentraxin-2 (NPTX2) was mainly responsible for the regulation of postsynaptic neurotransmitter receptor activity in BP (p=0.001, Fig. 4, D). Both NEFM and NPTX2 changed by more than 2-fold (both p<0.01, Fig. 4, E and F), indicating that a large number of nerve fibres were disrupted and the transmission of synaptic activity was reduced in patients with aCSVD with a high WMH burden. The 29 common BPs in the CSF and cytotoxic NK cells included the regulation of lymphocyte migration, negative regulation of neuron projection development and negative regulation of neuron apoptotic processes (all p<0.05, Fig. S2, B), demonstrating that cytotoxic NK cells migrated and disrupted nerve fibres in the CNS of patients with aCSVD.
The volcano plot displayed a two-fold change in cadherin-5 (CDH5, p=0.006, Fig. 4, F and G) in the CSF located on the external side of the plasma membrane in CC (p=0.007, Fig. 4, B), showing functions in signalling receptor binding (p=0.039, Fig. 4, C) and participating in cell-cell adhesion via plasma membrane adhesion molecules (p=0.008, Fig. 4, D). Even though CDH5 interacted with FLNA in cytotoxic NK cells (interaction score: 0.227, Fig. 3, H), we still thought that CDH5 was not the target protein for cytotoxic NK cell-targeting neurons because CDH5 is often expressed in endothelial cells and not neurons . Instead, differential CDH5 in CSF proved the leakage of the damaged BBB in patients with aCSVD with a high WMH burden.
Cytotoxic NK cells exacerbated the BBB damage by CTSD to cross the damaged BBB.
To understand whether cytotoxic NK cells pass through the BBB in the WMH area of patients with aCSVD, we used an OGD cell model to simulate hypoperfusion in WMH [23, 32]. We observed the inner sides and outer sides of Transwell membranes covered with HBMECs and HA, respectively, by examining the ECM and found that the connections between these cells were very tight, similar to the normal BBB (Fig. 5, A). Intercellular connections of HBMECs consist of intercellular adhesion molecule 1 (ICAM1), collagen (COL4A), laminin-R (LR), and so on [33-35]. ICAM1, COL4A and LR were highly expressed by HBMECs in vitro on the fourth day (all p<0.05, Fig. 5, B and C; Fig. S3, A and B). High expression of ITGB2, a receptor for ICAM1 in endothelial cells, was found in the cytotoxic NK cells, which promotes NK cell binding to ICAM1 in the endothelial cells of the BBB in vitro (Fig. 5, D) and in vivo (p<0.05, Fig. S4, C and E). HBMECs that fused to 100 % were co-cultured with cytotoxic NK cells from patients with aCSVD with WMH burdens of 2-3 and participants with WMH burdens of 0-1. Four hours later, the COL4A between the endothelial cells was degraded (p<0.05, Fig. 5, E and F). Considering that CTSD can degrade collagen in the extracellular matrix [36, 37], we found that the CTSD secreted by cytotoxic NK cells was closely related to the decrease in COL4A in the aCSVD group, and the degradation of COL4A was significantly inhibited by 10 μM 3,4-DCIC in vitro (all p<0.05, Fig. 5, G and H; Fig. S3, C and D) and in vivo (all p<0.05, Fig. S4, D and F-I). SEM directly showed that the number of cytotoxic NK cell immune synapses (NKISs) increased, and the gaps between the HBMECs of the BBB model expanded to allow the crossing of cytotoxic NK cells in aCSVD with high WMH (both p<0.05, Fig. 5, I and J). With the decrease in NKIS, the damage between endothelial cells also decreased significantly under after intervention with 3,4-DCIC (Fig. 5, I and J). Increased B2M and decreased S1PR1 in vitro (both p<0.05, Fig. 5, K and L) and in vivo (both p<0.05, Fig. S4, A and B) were consistent with the results from the serum of the participants, which synergistically promoted cytotoxic NK cell migration, adhesion and damage to the BBB [38, 39]. We also observed that cytotoxic NK cells from patients with aCSVD with a high WMH burden crossed the damaged BBB model in the lower layer (Fig. 5, M; Fig. S3, E). These results suggested that cytotoxic NK cells adhered to endothelial cells with ITGB2, exacerbated BBB damage through CTSD and then crossed the leaky BBB in patients with aCSVD.
Cytotoxic NK cells disrupted nerve fibres through GZMH.
Due to the decrease in nerve fibres as observed by MRI and the leaky BBB, we explored the effects of cytotoxic NK cells on neurons from patients with aCSVD with a high WMH burden. In the rat model of aCSVD, the thicknesses of both the subcortical white matter (SWM) and corpus callosum (CC) decreased (both p<0.05, Fig. S5, A and B). Additionally, in this area, myelin was vacuolated (p<0.05, Fig. S5, A and C). Combined with the high WMH signal in patients with aCSVD, we speculated that most nerve fibres were demyelinated [1, 23]. Moreover, the intensities of the nerve fibres also decreased in this area of the aCSVD model (p<0.05, Fig. S5, A and D), which was consistent with the sparse WMF observed from DTI in patients with aCSVD (Fig. 1, A). To explore the relationship between increased cytotoxic NK cells and demyelinated nerve fibres in white matter, we co-cultured cytotoxic NK cells isolated from participants with HAs for 4 h in vitro (Fig. 6, A). Interestingly, cytotoxic NK cells from patients with aCSVD with a high WMH burden were enriched around neural axon hillocks and disrupted the nerve fibres without myelin sheaths, which decreased the number and length of neural axons and dendrites (all p<0.05, Fig. 6, A, D and E). In the co-cultured supernatants and CSF from the rat model, the increase in NEFM and decrease in NPTX2 verified nerve fibre injury (all p<0.05, Fig. 6, B and C; Fig. S5, E and F). Cytotoxic NK cells from patients with aCSVD adhered to neural axon hillocks by highly expressing FLNA in vitro (Fig. 6, F) and in vivo (p<0.05, Fig. S5, G and H). In vivo, cytotoxic NK cells from the aCSVD model synthesized a large number of GZMHs (p<0.05, Fig. S5, I). The damaged neurons were also rich in GZMH in the SWM of the aCSVD model (Fig. S5, K and J). To further explore the mechanism by which cytotoxic NK cells disrupt nerve fibres, an inhibitor of GZMH was injected into each rat’s tail vein and co-cultured with cytotoxic NK cells from patients with aCSVD, which reduced the disruption of nerve fibres (all p<0.05, Fig. 6, G and H; Fig. S5, A, D and E). Additionally, phosphorylation of the death receptor (p-DR), which is involved in apoptosis, did not change in vitro or in vivo, indicating that the apoptotic pathway was not activated (Fig. 6, H and G; Fig. S5, K and J). These results demonstrated that GZMH released by cytotoxic NK cells was a key determinant of the disruption of demyelinated nerve fibres during WMH in patients with aCSVD.