Fig 2: Microglia and macrophages display distinct transcriptional responses and HDAC3 influences in SCI.(A) Volcano plots showing microglial DEGs of the indicated comparisons. n = 187 microglia in CTRL, 512 in SCI, and 510 in SCI_HDAC3i. (B) Venn diagram showing overlap of microglial injury response DEGs and HDAC3-dependent DEGs. Bottom: Pathway enrichment analyses of 55 overlapping genes and 168 HDAC3-dependent genes. (C) Volcano plots showing macrophage DEGs of the indicated comparisons. n = 4 macrophages in CTRL, 337 in SCI, and 659 in SCI_HDAC3i. (D) Top: Venn diagram and Enrichr pathway enrichment analysis of HDAC3-dependent genes shared by activated microglia or macrophages in SCI. Bottom: Comparative IPA showing enriched canonical pathways or diseases/functions for HDAC3-regulated genes unique to microglia or macrophages. (E) Comparative IPA of different sets of DEGs revealing distinct pathway enrichment for activated microglia and macrophages in SCI, both heavily dependent on HDAC3. (F) Unbiased subclustering analysis identified four microglia (MG1 to MG4) and four macrophage subclusters (Mac1 to Mac4), shown as UMAP plots. n = 1209 microglia and 1000 macrophage cells from all conditions combined. (G) Violin plots showing expression of marker genes for myeloid subclusters. (H) UMAP plots showing relative representation of myeloid subclusters in different conditions. Right: Frequency heatmaps showing myeloid subclusters relative to all cells in the entire sample in each condition.

Fig 3: Diversified transcriptional responses and functional specialization of macrophages in SCI.(A) Left: UMAP plot of macrophage subclusters (Mac1 to Mac4). Middle: Graphs showing relative abundance of Mac1 to Mac4 as compared to all cells in the entire sample in each condition. Right: Pie charts showing relative proportions of Mac1 to Mac4 among macrophages in each condition. n = 4 macrophages in CTRL, 337 in SCI, and 659 in SCI_HDAC3i. (B) Enriched GO terms associated with the top 30 up-regulated marker genes of each macrophage subcluster. Bottom: Violin plots show expression of selected marker genes related to the pathway ontologies indicated above. (C and D) Enrichment of IAM core genes (C) or DAM signature genes (D), separated as up- and down-regulated groups relative to control microglia, plotted as signature scores on the UMAPs of myeloid cells under different conditions. Notably, 9 of the 131 IAM core genes identified by INTACT RNA-seq were not detectable in scRNA-seq. Results showed that the IAM and DAM signature genes were predominantly enriched in subclusters MG2 and Mac1. Right: Reference UMAP plot of myeloid subclusters.

Fig 4: Identification of IL-6R as a potentially novel receptor of eCIRP in macrophages.(A) RAW264.7 cells (1 × 106/mL) were stimulated with rmCIRP (1 µg/mL), and surface expression of IL-6R was assessed at 24 and 48 hours following rmCIRP treatment by flow cytometry. Data are expressed as mean ± SEM (n = 4 samples/group). Experiments were repeated, and the repeated experimental data are shown in Supplemental Figure 9. The groups were compared by 1-way ANOVA and SNK method (*P < 0.05 vs. PBS). (B) SPR (Biacore assay) was performed between rhCIRP and rhIL-6R. rhIL-6R as a ligand was immobilized on the chip. rhCIRP was injected as an analyte in concentrations of 0–1000 nM. The association and dissociation of analyte with ligand at the indicated concentration were recorded, and binding kinetics of rhCIRP and rhIL-6R was calculated. (C) Peritoneal macrophages (4 × 105/mL) were treated with rmCIPR (5 µg/mL) at 4°C for 10 minutes, immediately fixed with paraformaldehyde, and stained with rabbit anti-mouse CIRP Ab, goat anti–mouse IL-6R Ab, and goat anti–mouse CD11b Ab followed by Cy3-conjugated donkey anti–rabbit IgG and Cy5-conjugated donkey anti–goat IgG. The images were obtained by using a Zeiss confocal microscope under 63× objective. The colocalization of rmCIRP and IL-6R is indicated by the merged images (shown in yellow color). Scale bar: 20 µm. (D) After the staining protocol described in (C), cell-associated fluorescence was measured on a Biotek Synergy Neo2 at 579 nm upon excitation at 540 nm (E1), at 681 nm after excitation at 640 nm (E2), and at 681 nm after excitation at 540 nm (E3) for FRET unit calculation. The transfer of fluorescence was calculated as FRET units. FRET unit = (E3both - E3none) - ([E3Cy5 - E3none] × [E2both/E2Cy5]) - ([E3Cy3 - E3none] × [E1both/E1Cy3]). Data are expressed as mean ± SEM (n = 5–6 wells/group). Experiments were repeated 3 times. Groups compared by 2-tailed Student’s t test (*P < 0.01 vs. CD11b).