NON-CLASSICAL MONOCYTE EXHIBIT HEIGHTENED DIFFERENTIATE INTO PRO-INFLAMMATORY VCAN+ SUBSETS IN ACUTE ISCHEMIC STROKE WITH EARLY NEUROLOGICAL DETERIORATION

To investigate the immune mechanisms underlying END after AIS and to develop a functional index based on monocyte subset dynamics for risk stratification.
We performed single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cells (PBMCs) from three END patients and four non-END AIS controls. This study was approved by eijing Tsinghua Changgung Hospital Ethics Review Committee (23019-4-04, ChiCTR2300068780).
Monocytes in AIS patients segregated into two clusters: CD14–FCGR3A+ (non-classical) and VCAN+ subsets (Fig 1A). Pseudotime trajectory analysis revealed a differentiation tendency of non-classical monocytes toward the VCAN+ subset in both groups (Fig 1B-D). Analysis of the top 50 differentially expressed genes (DEGs) identified significantly elevated TCF7L2 expression in non-classical monocytes (p=0.0001; Fig 1F, G), also ranked among the top 10 trajectory-driving genes (Fig 1E), suggesting classical Wnt pathway involvement. Phenotypic analysis indicated the potential of non-classical monocytes to develop into monocyte-derived macrophages (MoMacs) and dendritic cells (MoDCs), supported by high expression of CD83, ETV6, S100A8, and S100A9 in the VCAN+ cluster (p<0.05; Fig 1F, G) and elevated MAFB in the FCGR3A+ subset (p=0.0089; Fig 1F, G). In the END group, S100A9 was significantly upregulated in both subsets (p=0.0183 in FCGR3A+, Fig 1H; trend p=0.0942 in VCAN+, Fig 1I), suggesting enhanced infiltration. An index quantifying non-classical monocyte differentiation propensity—(non-classical monocyte count × PBMC count) / (total monocyte count)²—was significantly elevated in END (p=0.0089, Fig 1J), indicating heightened preparation for MoDC/MoMac differentiation.
In END patients, non-classical monocyte exhibit heightened differentiation into pro-inflammatory VCAN+ subsets.