Fig 2: Schematic illustration of the development of the hydrogel components for post-discectomy care of IVDs.a Engineering process of NPPC-derived exosomes and their incorporation into the injectable hydrogel based on ECM analogs. b A sketch showing treatment of IDD rats by HACS@CPP-miR-Exo. c The HACS@CPP-miR-Exo therapy potently inhibits the ferroptosis programs in NPCs through leveraging IRF8-STAT1 pathway, leading to enhanced post-discectomy IVD recovery. OCS oxidized chondroitin sulfate, HA-ADH adipic acid dihydrazide-modified hyaluronic acid, CPP cell-penetrating peptide, CPP-miR-Exo miR-221-3p overexpressed NPPC-derived exosomes with CPP modification, IRF8 interferon-regulatory factor 8, STAT1 signal transducer and activator of transcription 1. Schematic illustrations were generated using BioRender (Wang, V., 2025; accessible at: https://BioRender.com/i62o243). Source data are provided as a Source Data file.

Fig 3: Aff3ir-ORF2 mitigates disturbed shear stress-induced inflammation by interacting with Irf5 and retaining it within the cytosol.(A–D) Mouse embryonic fibroblasts (MEFs) were isolated from wild-type (WT) and Aff3ir-ORF2-/- mice. (A) Principal component analysis (PCA) analysis of RNA-seq data to visualize sample-to-sample variation. (B) Volcano map showing mRNA profiles of WT and Aff3ir-ORF2-/- MEFs (n=3). (C) Gene Ontology enrichment pathway analysis of the differentially expressed genes. (D) Venn diagrams of the top 20 transcription factors from the ChEA3 and DisGENET analysis related to atherosclerosis. (E) Immunoprecipitation performed using antibodies against Aff3ir-ORF2, Irf5, and Irf8. n=3 independent experiments. (F–H), WT and Aff3ir-ORF2-/- MEFs were subjected to silence of Control (siNC), Irf5 (siIrf5), or Irf8 (siIrf8) with siRNAs for 24 hr, followed by exposure to static (ST) or oscillatory shear stress (OSS, 0.5±4 dyn/cm2, 1 Hz) for another 6 hr. (F) RT-PCR analysis of the mRNA levels of Vcam1, Icam1, Il6, and Il1b. The relative expression values were compared to WT MEFs transfected with siNC and treated with ST. Data are mean ± SEM (n=6 independent experiments). *p<0.05, two-way ANOVA with Tukey post-test. (G–H), Representative western blots of Irf5 and Icam1 expression. Data are mean ± SEM (n=5 independent experiments). *p<0.05, two-way ANOVA with Tukey post-test. (I–J), WT, and Aff3ir-ORF2-/- MEFs were exposed to ST or OSS for 6 hr. Nuclear and cytoplasmic proteins were extracted from the cells. Representative western blots of the indicated proteins and quantification of Irf5 expression in nucleus are shown. The expression of these proteins was relative to the level of nuclear Irf5 in ST-treated WT MEFs. Data are mean ± SEM (n=6 independent experiments). *p<0.05, one-way ANOVA with Tukey post-test. (K) HEK293 cells were transfected with the firefly luciferase reporter plasmid containing the Irf5-responsive ZNF217 promoter along with a β-galactosidase reporter plasmid for 24 hr. Cells were infected with the indicated adenoviruses (Ad-Scramble or Ad-Aff3ir-ORF2) for 24 h. Promoter activity was measured using luciferase, which was normalized to β-gal. Data are mean ± SEM (n=6 independent experiments). *p<0.05, two-way ANOVA with Tukey post-test. Figure 4—source data 1.The table summarizes the data in the statistical graph for Figure 4F, H, J and K. Figure 4—source data 2.The original file of the full raw uncropped, unedited polyacrylamide gels for Figure 4E. Figure 4—source data 3.Figures with the uncropped polyacrylamide gels with the relevant bands clearly labeled for Figure 4E. Figure 4—source data 4.The original file of the full raw uncropped, unedited polyacrylamide gels for Figure 4G. Figure 4—source data 5.Figures with the uncropped polyacrylamide gels with the relevant bands clearly labeled for Figure 4G. Figure 4—source data 6.The original file of the full raw uncropped, unedited polyacrylamide gels for Figure 4I. Figure 4—source data 7.Figures with the uncropped polyacrylamide gels with the relevant bands clearly labeled for Figure 4I.

Fig 4: An essential role of TGFβ1 autocrine signal in microglial response to pathological axonal degeneration.A–C 8-week-old Cx3cr1CreER/+; Tgfbr1 fl/fl and control Cx3cr1CreER/+; Tgfbr1+/+ female mice were subjected to the 4-hydroxytamoxifen-induced Cre-recombinase activity and then utilized for the model of optic nerve injury. Microglial phospho-SMAD2 (p-SMAD2) in the optic nerves at 3 days post-injury was assessed by co-immunostaining with CD11b. A Representative images are shown. Microglia densities (B) and p-SMAD2+ microglia (C) were quantified. n = 3 mice per condition, mean ± SEM, two-way ANOVA test. D 8-week-old Cx3cr1CreER/+; Tgfb1 fl/fl, Cx3cr1CreER/+; Tgfbr1 fl/fl, and control Cx3cr1CreER/+ female mice were subjected to the 4-hydroxytamoxifen-induced Cre-recombinase activity and then utilized for the model of optic nerve injury. Microglia were FACS-sorted from the injured optic nerves and analyzed by RNA-seq. Pooled RNA-seq data from 4 mice per condition are shown for GO enrichment analysis of microglia from the injured optic nerves of control Cx3cr1CreER/+ versus Cx3cr1CreER/+; Tgfb1 fl/fl mice and control Cx3cr1CreER/+ versus Cx3cr1CreER/+; Tgfbr1 fl/fl mice. E–G 8-week-old Cx3cr1CreER/+; Irf8 fl/fl, Cx3cr1CreER/+; Tgfb1 fl/fl, Cx3cr1CreER/+; Tgfbr1 fl/fl, and control Cx3cr1CreER/+ female mice were subjected to the 4-hydroxytamoxifen-induced Cre-recombinase activity and then utilized for the model of optic nerve injury. Microglia were FACS-sorted from the optic nerves and analyzed by RNA-seq. Pooled RNA-seq data from 4 mice per condition are shown. Expression levels of homeostatic markers (E), signature genes for microglial response to neurodegenerative insults (F), and proinflammatory cytokines and chemokines (G) in microglia of the indicated conditions.

Fig 5: Microglial TGFβ1 expression in the prefrontal cortices of Alzheimer’s disease patients.A–C The published snRNA-seq datasets of total cells in the prefrontal cortices of Alzheimer’s disease patients (syn21670836, information in Supplementary Table 2) were analyzed. A UMAP plot of defined neuronal and non-neuronal cell types. B Violin plots of the expression of signature genes in defined cell types. OPCs, oligodendrocyte progenitor cells. C The expression of MERTK, PU.1, IRF8, or TGFB1 was projected onto the UMAP plot of defined cell types. D–F The published snRNA-seq datasets of total cells in the prefrontal cortices of Alzheimer’s disease patients (syn18681734, information in Supplementary Table 2) were analyzed. A UMAP plot of defined neuronal and non-neuronal cell types. B Violin plots of the expression of signature genes in defined cell types. OPCs, oligodendrocyte progenitor cells. C The expression of MERTK, PU.1, IRF8, or TGFB1 was projected onto the UMAP plot of defined cell types.