Fig 2: IL-35 cytokine injection delays blood perfusion recovery in the ischemic leg of hindlimb ischemia (HLI) and increases gene expression of IL-12RB2. (A) Experimental schematics. IL-35 group mice were intramuscularly (i.m.) administered with IL-35 [0.3 µg/gastrocnemius (GC) muscle in 20 µl PBS] at the time of HLI procedure and three times a week afterwards until the end of observation. The non-surgical limb was also injected with 20 µl PBS as internal control. (B) Blood perfusion ratio of IL-35- or PBS-injected WT or Il12rb2-/- mice. (C) Bone marrow transplantation experimental schematics. (D) Representative images of the whole GC muscle (left panel) and higher magnification of vascular density (right panel) in vascular area of PBS- or IL-35-injected ischemic muscle at 14 dpi. Tissues were stained with anti-CD31 (red) and DAPI. Lines, where blood vessels were rarely visible. The left two images were taken using tile scan in Leica TCS SP8 system and merged to present the whole tissue section. The avascular area was indicated with red dash at 14 dpi. (E) Statistics of the percentage of the avascular area EGFP+ indicated infiltrated bone marrow-derived cells. (F) Statistics of the percentage of CD31+ cells in the vascular area of the GC muscle at 14 dpi. (G) Statistics of percentage of CD31+ cells in CD45- cell population in IL-35 or PBS-injected groups using flow cytometry. (H) Percentage of CD45+CD31+ ECs in IL-35 or PBS injected ischemic muscle at 3 and 14 dpi using immunofluorescent staining. (I) Normalized gene expression of IL-35 signaling complex in the ischemic muscle at 3 dpi. IL-35 group mice were i.m. injected with IL-35 at the time of HLI surgery. (J) Normalized mRNA expression levels of IL-35 receptor subunits after 6 h of 40 ng/ml of IL-35 treatment in HMVECs under normoxia or hypoxia condition. All animal results were collected from male mice. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001.

Fig 3: Transcriptome analysis of endothelial cells (ECs) at 14 days post-ischemia (dpi) reveals a disturbed extracellular matrix (ECM) reorganization in the IL-35-injected mice. (A) Flow cytometry cell sorting strategy. Hindlimb muscle from control (Ctrl, without surgery) or ischemic mouse (PBS and IL-35 groups) at 14 dpi was dissected and digested for single cell suspension. Cells were stained with the surface markers of CD31 BV605 and CD45 APC-Cy7 and sorted by Aria Cell Sorter (BD Biosciences). 1 × 105 cells of ECs from each sample were collected for RNA extraction and deep-sequencing. (B) Heatmap of the differentially expressed genes (DEGs) in ECs of IL-35- or PBS-injected, and control groups. With the criteria of false discovery rate (FDR) < 0.05, |FC| > 1.5, and max (fragments per kilobase of transcript per million mapped reads, FPKM) > 5, we got 341 DEGs. The green-red color key was scaled based on the z-score of FPKM within the row. (C) Gene set enrichment analysis (GSEA) of the gene expression changes of ECs in IL-35-injected mouse compared to that of PBS group. Four of the most enriched pathways were presented. (D) Representative gene expression changes in different categories of ECM proteins. All results were collected from male mice. n = 3.

Fig 4: Recruited macrophages are distinct in their expression and chromatin profile, relative to resident macrophages at one month post-influenza.RNA-seq and ATAC-seq of AMs FACS-sorted from the lungs of naïve and post-influenza Busulfan chimeras. a, Heat map of genes differentially expressed (FC>1.5, 1-way ANOVA with Benjamini-Hochberg correction, p<0.05) between CD45.2 (Ccr2-/-) resident and CD45.1 recruited AMs. b, Principal component analysis (PCA) plot of RNA-seq data from resident and recruited macrophages from post-influenza and naive lungs of Ccr2-/- Busulfan chimeras. c,d, Gene set enrichment analysis (GSEA) of Flu resident (CD45.2) vs Flu recruited (CD45.1) macrophages using clusters from Lavin et al.30 representing (c) lung macrophages and (d) blood monocytes. e,f, IGV tracks from ATAC-seq data; depicting chromatin in unstimulated resident and recruited macrophages from naïve and post-influenza lungs of Ccr2-/- Busulfan chimeras. All tracks were group-autoscaled to enable comparison. g, PCA plot of ATAC-seq data from unstimulated resident and recruited macrophages from naïve and post-influenza lungs.

Fig 5: Analysis of skin-infiltrating T cells in IMQ-treated K14-I?B?–KO mice.All analyses were performed after 7 days of IMQ treatment. (A) Flow cytometry analysis of T cell subsets in the ears of IMQ-treated Ctrl and K14-KO mice. T cell subsets were detected as CD45+ and CD3+, aßTCR+, or ?dTCR+ cells. Single data points derive from 2 ears. Shown is the mean of 4–12 mice per group ± SEM. (B) Gene expression analysis of Il22 and Il17a in skin tissue of untreated and IMQ-treated control and K14-KO mice, normalized to the reference gene Actin. n = 4–14 ± SEM. (C) Determination of the percentage of IL-17A– and IL-22–producing aß and ?d T cells in IMQ-treated control and K14-KO mice. After fixation and permeabilization, cells were gated as in A, except for an additional gating on either IL-17A+ or IL-22+ cells. n = 3 ± SEM. (D) Gene expression analysis of Il1b and Il23a in untreated and IMQ-treated mice, similar as in B. (E) mRNA levels of Ccl2, Ccl17, and Ccl20 in untreated mice, similar as in B. (F) mRNA levels of Ccr2, Ccr4, and Ccr6 in the skin of untreated control and K14-KO mice, similar as in B. n = 5–14 ± SEM. P values were calculated using 2-tailed Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).