Illustrating the functional heterogeneity of M-MDSCs to predict sepsis outcomes

• Three subsets of M-MDSCs, including IL1R2_M-MDSC, THBS1_M-MDSC, and S100A_M-MDSC, account for the majority of the population in sepsis. • The typical characteristics of M-MDSCs in sepsis during the early, middle, and late stages are pro-inflammation, concurrent pro-inflammation and immunosuppression, and robust immunosuppression, respectively. • VSIG4 is a functional marker of immunosuppressive M-MDSCs and is specifically expressed in THBS1_M-MDSC. • The combined detection of surface VSIG4 and IL1R2 on HLA-DR low CD14 + monocytes shows potential for accurately predicting sepsis outcomes. Sepsis remains one of the leading causes of death worldwide. Monocytes play a pivotal role in sepsis due to their dual role in both pro-inflammation and immunosuppression. However, the phenotypic markers and developmental characteristics of immunosuppressive monocytic myeloid-derived suppressor cells (M-MDSCs) in sepsis remain largely unknown. This study aimed to investigate the functional heterogeneity of M-MDSCs in sepsis. The frequency of M-MDSCs was assessed for the prognosis of sepsis. Single-cell RNA sequencing was conducted on purified HLA-DR high CD14 + and HLA-DR low CD14 + monocytes, respectively, from patients with sepsis to study their heterogeneity. We find that the frequency of M-MDSCs, as defined by classical markers, has limited value in the prognosis of sepsis due to their broad heterogeneity. Based on scRNA-seq analysis, M-MDSCs in sepsis are established as HLA-DR low CD14 + monocytes, which display high expression of RETN and low expression of HLA-DPB1. We further segregate M-MDSCs into five subsets: IL1R2_M-MDSC, THBS1_M-MDSC, S100A_M-MDSC, IFN-sti_M-MDSC, and PPBP_M-MDSC, with the first three subsets accounting for the majority of the population. Pro-inflammatory S100A_M-MDSC dominates early-stage population and is characterized by high expression of S100A. Middle-stage IL1R2_M-MDSC exhibits cytokine production, while THBS1_M-MDSC is associated with TGF-β signaling, revealing concurrent pro-inflammatory and immunosuppressive functions. In the late stage, both THBS1_M-MDSC and IL1R2_M-MDSC display immunosuppressive functions. Furthermore, our data suggest a metabolic shift from oxidative phosphorylation to fatty acid and amino acid biosynthesis as pro-inflammatory monocytes transition into immunosuppressive M-MDSCs. VSIG4, a novel functional marker of immunosuppressive M-MDSCs, is specifically expressed in THBS1_M-MDSC. Subsequently, our preliminary results suggest that the combined detection of surface VSIG4 and IL1R2 on HLA-DR low CD14 + monocytes shows potential for predicting sepsis outcomes. This study illuminates the characteristics of M-MDSCs in sepsis, providing a new direction for disease prognosis.