Fig 2: STING knock-out and activation in THP1-derived macrophages promote pro-inflammatory subtype differentiation. (A) Left panel, immunoblot analysis of STING expression in human THP1-derived macrophages treated as indicated. a-Tubulin was used as a loading control; right panel, the ratio of STING/a-Tubulin was quantified; ***, p < 0.001; *, p < 0.05. (B) Luciferase activity driven by the IFN-ß promoter in THP1-derived macrophages treated as indicated. ***, p < 0.001. (C) Flow cytometric analysis of surface marker expression for pro-inflammatory macrophages (CD11b+CD80+) in human THP1-derived macrophages treated as indicated. *, p < 0.05. (D) RT-PCR analysis of the macrophage-subtype markers in stable THP1-derived macrophages treated as indicated. ***, p < 0.01; p < 0.01; *, p < 0.05. Data are presented as the mean±SD (n=3). Control stands for the average of undistinguishable controls of scrambled sequence (SC; for shSTING), and PBS treatment (for 2'3'-c-GAMP treatment), empty vector (EV) was used as a control of STING overexpression.

Fig 3: Identification of NP-derived G-MDSCs. A) Uniform manifold approximation and projection (UMAP) visualization showing immune cells inside NP tissues. B) Heatmap showing the typically expressed genes in each cell type. C) Dot plot showing scaled expression of selected signature genes for GMP, G-MDSCs, and neutrophils, by average expression of each gene in each cluster scaled across all clusters. Dot size represents the percentage of cells in each cluster with more than one read of the corresponding gene. D) Monocle method reconstructed pseudospace trajectory for GMP, G-MDSC, and neutrophils. E) FACS isolation for CD45+ CD11b+OLR+ cells from mild/severe degenerative NP tisues. F) Safranin O/Fast Green staining of the intervertebral discs sham and experimental rats. Scale bar, 1 mm in left and 100 µm in right. G) Merged immunofluorescence staining of DAPI, CD11b, OLR1 in the intervertebral discs of sham and experimental rat. Scale bar, 1 mm in merge images and 100 µm in others.

Fig 4: Characterization of PMA-treated THP-1 cells. (A) Morphological changes in THP-1 cells caused by PMA treatment. THP-1 cells were treated with 100 ng/ml PMA for 24 h for differentiation into resting macrophages. Magnification, ×100. (B) Flow cytometry characterization of macrophages derived from THP-1 cells stimulated with PMA; CD11b and CD14 are specific markers of macrophages. PMA, phorbol 12-myristate 13-acetate.

Fig 5: STING knocking-down and activation regulate macrophage differentiation through JAK-IL24 pathway. (A) Representative pictures of IL6R in a cytokine array analysis of supernatants from PBMC-DM cultures treated as indicated, and quantified in (B); ***, p<0.001; **, p< 0.01. (C) Flow cytometric analysis of surface marker expression for pro-inflammatory macrophages (CD11b+/HLA-DR+) and anti-inflammatory macrophages (CD11b+/CD163+) in human PBMC-DMs treated as indicated. ***, p < 0.001; **, p < 0.01; *, p < 0.05. (D) RT-PCR analysis of the macrophage subtype-markers in human PBMC-DMs treated as indicated. ***, p < 0.001; **, p < 0.01; *, p < 0.05. (E) Left panel, representative flow cytometric plots of apoptosis markers (FITC-Annexin V+) in human PBMC-DMs treated as indicated; right panel, quantification of percentage of Annexin V+ apoptotic cells; **, p < 0.01; *, p < 0.05. (F) Immunoblot analysis of phospho-STAT1, total STAT1, phospho-STAT3, total STAT3, IL24 expression in PBMC-DMs from two healthy donors treated as indicated; ß-Actin was used as a loading control; quantification of the immunoblot analysis of PBMC-DMs from each healthy donor was below the bands; ***, p < 0.001; **, p < 0.01; *, p < 0.05. (G) Immunofluorescent staining of IL24 (green) in human PBMC-DMs. DAPI (blue) stained for nuclei. Scale bar represents 50 µm. (H) IL24 expression measured by ELISA in supernatants of human PBMC-DMs (left) or mouse BM-DMs (right) cultures treated as indicated. ***, p < 0.001; **, p < 0.01; *, p < 0.05. (I) Left panel, representative flow cytometric plots of apoptosis markers (FITC-Annexin V+) in human PBMC-DMs treated as indicated; right panel, quantification of percentage of Annexin V+ apoptotic cells; **, p < 0.01; *, p < 0.05. Data in B,C, D, E and F are presented as the mean±SD (n=3). (A-E, H) Control stands for a representative sample of undistinguishable controls of scrambled sequence (for shSTING), empty vector (for STING overexpression), and PBS treatment (for 2'3'-c-GAMP treatment). (F, G, I) Control stands for a representative sample of undistinguishable controls of scrambled sequence (for shSTING and/or shIL24) and PBS treatment (for 2'3'-c-GAMP treatment).