Fig 1: Distinct chemotactic SMCs express high levels of MΦ chemoattractants, ameliorating atheroprogression(A–C) Reanalyzed single-cell RNA-seq data from human coronary arteries from Wirka et al., GEO: GSE131780. (A) Violin plots (calculated on all cells expressing detectable baseline levels of the respective gene) of highly expressed cytokines and chemokines in chemotactic SMCs. Dots represent single cells, only cells exhibiting detectable expression of the particular gene are included (B) interactome depicting cell-cell interactions between MΦ and SMC subsets, prominent SMC → MΦ interactions are depicted in red. Intensity of red color depicts the respective portion of the CCL2-CCR2 axis for the concrete interaction (the darker the red color, the more the CCL2-CCR2 axis accounts for the respective inter-cluster interplay among all detected chemokine-receptor interactions). (C) Heatmap further unraveling SMC → MΦ chemokine:chemokine-receptor interactions. Blue box depicts interactions of chemotactic SMC subset, red box depicts CCL2-mediated interactions between SMC and MΦ subsets.(D) Ccl2 and Mif expression in Ng2+ SMCs FACS-sorted from western-diet fed atherosclerotic MCRFP-rep mice compared to chow-diet fed non-atherosclerotic control mice. n = 3–4 mice per group.(E) Representative images of BCA sections from Ccl2SMC +/+ and Ccl2SMCΔ/Δ littermates after 14 weeks of western diet stained for ACTA2 (green), LGALS3 (red), and Hoechst (blue). Scale bars, 100 μm.(F and G) Morphometric analysis of plaque size (F) and vascular remodeling (G) from BCA sections at three consecutive locations from Ccl2SMC +/+ (n = 11) and Ccl2SMCΔ/Δ (n = 10) littermates.(H and I) Quantification of ACTA2+ smooth muscle cell content as ACTA2+ area in percentage of total plaque area and percentage of 30 μm plaque surface area in valves (H) and in the BCA at three consecutive locations (I). (H and I) n = 10–11 mice per group.(J and K) Analysis of intimal LGALS3+ area as percentage of plaque size in BCA sections at three consecutive locations (J) and in plaques from aortic valves (K) (n = 10–11 each).(L) Schematic illustration of media and intima processing from aortae of Ccl2SMC+/+ and Ccl2SMCΔ/Δ littermates after 14 weeks of western diet (left). Heatmap displaying expression of differentially regulated genes in bulk RNA-seq of Ccl2SMC+/+ mice (n = 3) and Ccl2SMCΔ/Δ mice (n = 4). Rows represent individual replicates, differentially expressed genes are illustrated in columns (right).(M) Volcano plots of intima/media RNA-seq showing differentially expressed genes in Ccl2SMC+/+ mice (n = 3) and Ccl2SMCΔ/Δ mice (n = 4), x-axis depicts Log2FC, y-axis depicts -Log10(adj. p-value). Data are shown as mean and SEM. (H and K), Student’s t test was used. (F, G, I, and J) Repeated measures two-way ANOVA or mixed-effects model was used. ∗p < 0.05; NS, not significant. Bar graphs show mean with SEM. Violin plots with matching boxplot and mean expression.
Fig 2: IL-33 signaling is necessary for recruiting angiogenic LY6C+ monocytes(A) Top: UMAP of leukocytes from day 2 wounds in control vs. ΔIL33Col1α2 mice. Bottom: log2fold-ratio change in cell numbers from each cluster comparing control and ΔIL33Col1α2 mice.(B) GO analysis for the monocyte cluster in control groups truncated to seven biological processes.(C) Violin plots of Ly6c2 and Vegfa expression in each immune cluster.(D) Mif expression levels in Treg cells stimulated with saline, recombinant IL-33, or IL-33 plus soluble ST2. Left: mRNA levels by qPCR. Right: protein levels by ELISA.(E) Cytometry plots (left) and quantification (right) of migrated LY6C+ monocyte numbers in Transwell experiments, where Treg cells were primed with saline, IL-33, or IL-33+ST2.(F) Migrated LY6C+ monocyte numbers where IL-33-primed Treg cells were treated with isotype immunoglobulin G (IgG) or an anti-MIF (αMIF) antibody.(G) Migrated LY6C+ monocytes after monocytes were treated with isotype IgG or an anti-CD74 (αCD74) antibody.(H) Left: flow cytometry plot of cells pre-gated for live CD45+ cells, expressing CD74 and LY6C from day 2 wounds of control and ΔIL33Col1α2 mice. Right: CD74+LY6C+ cell percentage normalized by total CD45+ cells.(I) Left: immunofluorescence images of 2-day oral wounds stained with antibodies against LY6C (green) and VEGFA (red). Arrows, LY6C+VEGFA+ cells; arrowheads, LY6C+VEGFA− cells. Scale bar, 50 μm. Right: LY6C+ VEGFA+ cells normalized by wound area.(J) Immunofluorescence images of oral wounds stained with CD31 (white) antibody in control and ΔIL33Col1α2 mice. Dashed lines encircle connective tissues. Scale bar, 500 μm.(K) Blood vessel numbers in oral wounds across healing time points from control and ΔIL33Col1α2 mice, raw count per wound (left), and normalized by stromal tissue area (right).Data represent mean ± SEM. n = 3 for (D)–(G), in which each N is an independent experiment from individual control mouse; n = 6–10 mice each for (H)–(K). Student’s t test (F–K) or one-way ANOVA and post hoc test (D and E); *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 3: Treg cell ablation in oral mucosa impairs angiogenesis, inflammation, and wound healing(A) H&E-stained images (left) and epithelial gap quantification (right) of day 2 oral wounds from Foxp3-DTR mice that received saline or diphtheria toxin (DT). Scale bar, 500 μm.(B) Left: COL3A1 staining images from day 4 oral wounds. Right: COL3A1+ area in control and Treg cell ablation mice. Scale bar, 500 μm.(C) LY6C+VEGFA+ monocyte numbers normalized by stromal area in day 2 or 4 oral wounds.(D) F4/80+CD206+ macrophage numbers normalized by stromal area in day 2 or 4 oral wounds.(E) MIF and TGF-β1 levels by ELISA from day 2 wound lysates, normalized by tissue weight.(F) CD31+ blood vessel numbers normalized by stromal area in day 2 or 4 oral wounds.(G and H) Flow cytometry plots (G) and quantification (H) of CD74+LY6C+ monocytes and F4/80+CD206+ macrophages in day 4 oral wounds of Treg cell ablation mice that received vehicle (+saline) or recombinant MIF and TGF-β1 (+rMIF/TGF-β1, 5 ng/1 ng per wound).(I and J) CD31+ blood vessels (I) and COL3A1+ stromal area (J) in the wounds of Treg cell ablation mice that received saline or rMIF/TGF-β1.Data represent mean ± SEM. n = 5–10 mice each. Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 4: scRNA-seq analysis of oral wounds in ΔIL33Col1α2 mice reveals changes in Treg cell activation(A) Representative flow cytometry plot and gating strategy for determining ST2+ cell identity in day 2 oral wounds.(B) EPCAM+ (epithelial), PDGFRA+ (fibroblast), CD31+ (endothelial), and CD45+ (leukocyte) cells that express ST2, normalized by total ST2+ cell count per time point.(C) scRNA-seq analysis and UMAP plot of CD45+ sorted cells from day 2 wounds of control Il33f/f mice (Ctrl) and experimental ΔIL33Col1α2 mice. 8–10 wounds from 4–5 mice were pooled per condition.(D) Left: UMAP of subset clusters that express Il1rl1+ (encoding ST2). Right: feature plot of Il1rl1 expression in three clusters.(E) Dot plot for genes that are enriched in Treg cells, including Foxp3.(F) Il1rl1+ cell numbers in each cluster normalized by total number of Il1rl1+ cells. Treg, regulatory T cell; MC, mast cell; ILC2, type 2 innate lymphoid cell.(G) Pseudobulk analysis of DEGs, comparing Treg cells from control versus ΔIL33Col1α2 groups.(H) GO analysis for the upregulated gene list in control Treg cells. Biological process terms associated with immune responses are shown.(I) CellChat analysis of ligand-receptor interaction pairs. Treg cells are set as the ligand source and other leukocytes as the recipients. Communication probability from the control and ΔIL33Col1α2 (conditional knockout [cKO]) groups are shown.(J) Violin plots of Mif and Tgfb1 expression in Treg cell cluster from control and ΔIL33Col1α2 groups.(K) Flow cytometry plots of MIF and TGF-β1 expression in FOXP3+ Treg cells from day 2 wounds of control or ΔIL33Col1α2 mice.(L) Mean fluorescent intensity (MFI) for MIF or TGF-β1 expression in FOXP3+ Treg cells in day 2 wounds.Data represent mean ± SEM; n = 3–5 mice per group. Student’s t test, *p < 0.05.
Fig 5: Evidence of IL-33+ fibroblast-Treg cell signaling in the deeper lamina propria in humans(A) H&E staining and immunofluorescence images of normal human gingiva and skin. Dashed lines demarcate the boundaries of reticular connective tissue. Scale bar, 500 μm. Inset: respective areas (i–iv) in gingiva and skin, showing immunopositive signals for IL-33, PI16, and CD31. Scale bar, 50 μm.(B) PI16+IL33+ cell counts per area in the deeper connective tissue of skin dermis and oral lamina propria.(C) Illustrative diagram of inadvertent implant abutment removal and tissue resection for histological assessment.(D) H&E staining and immunofluorescence images of human gingiva that “healed” for 4 days after abutment removal; n = 3 individual patients (56M, 56F, and 54M). Scale bar, 500 μm. Inset: deeper lamina propria showing immunopositive signals for KI67, KRT14, and CD45 antigen. Scale bar, 100 μm.(E) RNAscope in situ hybridization images with specific probes against FOXP3, MIF, and TGFB1. The upper and lower aspect of healing gingival connective tissue is shown. Scale bar, 100 μm. Inset: representative Treg cells in the upper (i) and lower (ii) lamina propria (LP). Scale bar, 5 μm.(F) FOXP3+MIF+ (left) and FOXP3+TGFB1+ Treg cell count per area (right) from the upper versus lower lamina propria in healing gingiva.(G) MIF and TGFB1 puncta in FOXP3+ cells from the lower lamina propria.Data represent mean ± SEM; n = 5–7 individuals per group (B), 3 specimens (F), and 30 fields of view (FOV) (G) from three patients. Student’s t test, *p < 0.05, **p < 0.01.
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