Characterization of mouse allergic skin immune response by single cell RNA transcriptomic analysis

(A) Overview of the experimental setting. Allergic contact dermatitis (ACD)-like skin inflammation was triggered by sequential sensitization and elicitation of ACD by topical application of 1% or 0.2% DNFB on dorsal or ear skin as indicated. Ear skin was collected at 60 hours post-elicitation for analyses.

(B) tSNE plots showing the distribution of various cell clusters marked by a color code.

(C) Bubble plots showing the expression of marker genes for each cell cluster. Abbreviations: dFB, dermal fibroblast; MC, mast cell; MAC, macrophage; BF, basophil; NEU, neutrophil; VSMC, vascular smooth muscle cell; PC, pericytes; KC, keratinocyte; EC, endothelial cell; SC, Schwann cell.

(D) tSNE plots showing how cells were differentially distributed in control and ACD skin samples. Red lines circle key immune cell populations.

(E) Stacked bar graph showing the percentage of each cell cluster in the control and ACD samples.

(F) qRT-PCR analysis showing the expression of indicated genes (n=4∼6/group).

(G) Violin plots showing the expression of indicated genes in the control and ACD samples.

Figure supplement 1. Establishment of the DNFB-elicited ACD mouse model.

T cells are primarily polarized to the IFNγ-producing type-1 inflammatory phenotype in ACD

(A) tSNE plots showing cell distribution of the CD45+CD3+ or THY1+ T cell population after re-clustering.

(B) Bar graphs showing the percentage of each T cell sub-cluster in the control and the ACD samples.

(C) tSNE plots showing cell distribution or the expression of Ifng in the control and the ACD samples.

(D) By sample violin plots showing the expression of indicated genes across various T cell sub-clusters..

(E) WIKI pathway analysis showing the top upregulated (red) or downregulated (blue) pathways in CD4+ and CD8+ T cells in ACD compared to control skin.

(F) Volcano plot showing differentially expressed genes in control and ACD samples within the CD4+ and CD8+ T cells

(G-J) FACS plots and/or quantified bar graphs showing the percentage of CD4+ or CD8+ T cells (upper panel in G), or the percentage of IFNγ+IL4/13-, IFNγ-IL4/13+, or IFNγ-IL17A+ cells in CD8+ (middle panel in G) and CD4+ (lower panel in G) T cells in control, ACD (24 hours) and ACD (60 hours) ear skin samples (n=3/group).

All error bars indicate mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant. Figure supplement 1. Characterization of T cell activation in ACD.

Characterization of myeloid cell activation in ACD

(A) Stacked bar graphs showing the percentage of indicated myeloid cell populations in the control and the ACD samples.

(B) Violin plots showing the expression of indicated genes across major skin resident and immune cells as indicated.

(X) Gene expression plot showing differentially expressed genes in basophils (BF, blue dots) and mast cells (MC, red dots) within the ACD sample.

(D) qRT-PCR analysis of indicated genes in ear skin samples (n=3/group).

(E-F) FACS plots (E) and quantified bar graphs (F) showing the percentage of IL4/13+ mast cells or basophils within all viable cells in ear skin samples.

(G) qRT-PCR analysis of indicated genes in ear skin samples (n=3/group).

(H) Gene expression plot showing differentially expressed genes in control and ACD samples within the macrophage cell cluster.

(I) GO pathway analysis of the upregulated genes (red) or downregulated genes (blue) in macrophage population after ACD elicitation.

(J-L) FACS plots (J) and quantified bar graphs (K-L) showing the percentage of CD11B+Ly6G+ neutrophils (NEU) in all cells (K), or the percentage of Ly6C+ or Ly6C- cells within CD11B+F4/80+ macrophages (L) in ear skin samples. Unstained (UN) plot was shown as negative gating control. All error bars indicate mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant.

Figure supplement 1. Analysis of myeloid cell activation in ACD

Characterization of the immune response of dermal fibroblasts in ACD

(A) tSNE plots showing cell distribution of the Pdgfra+ dermal fibroblasts after re-clustering.

(B) Bubble plots showing the expression of marker genes for each dFB cell cluster. Abbreviations: AP, adipocyte progenitors; Areg, adipogenesis-regulatory cells; pAd, preadipocytes; RET/PAP, reticular and/or papillary dFBs; pF, peri-follicular dFBs.

(C) Violin plots showing the expression of indicated genes across various dFB sub-populations in the control and the ACD samples.

(D) SCENIC analysis showing the top enriched transcriptional factors in various dFB clusters.

(E) GO pathway analysis of the upregulated genes (red) or downregulated genes (blue) in the r5 dFB cluster after ACD elicitation.

(F) Volcano plot showing differentially expressed genes in control and ACD samples within the r5 dFB cluster.

(G) Violin plots showing the expression of indicated genes across various cell populations in the control and the ACD samples.

(H) Frozen sections of control and ACD ear skin samples were subjected to immunostaining analysis using antibodies against CXCL9 (green) and PDGFRA (red). Nuclei were counter stained by DAPI (blue). Dermal CXCL9+ or PDGFRA- cells were highlighted by either red- or green-dotted lines in the zoom-in panel. Scale bar, 200 μm. Zoom-in image is shown on the right-hand side.

(I-J) Quantified results showing the fluorescent intensity (arbitrary unit, AU) of CXCL9, CXCL10 or PDGFRA in the dermal CXCL9/10+ cells or PDGFRA- cells shown in Fig. 4H or Fig. S4G.

All error bars indicate mean ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001, ns, non-significant.

Figure supplement 1. Characterization of the immune response of dermal fibroblasts in ACD.

Interaction between dFBs and T cells via the IFNG-CXCL10-CXCR3 signaling axis in ACD

(A) Circle plot from cell-chat analysis showing the inferred intercellular communication network for IFNγ-IFNGR signaling in the control and the ACD cells.

(B-C) Primary neonatal dFBs were treated with IFNγ, IL4 or IL17A for 48 hours and subjected to RNA-seq analysis. (B) Venn diagram comparing genes up-regulated by IFNγ, IL4. (C) GO pathway analysis of the genes upregulated only by IFNγ.

(D) Primary dFBs were treated with IFNγ for 8h or 48h, and control or IFNγ treated samples were subjected to qRT-PCR analysis of Cxcl9, Cxcl10, Stat1, Apoe, and Col1a1 mRNA expression (n=∼3/group).

(E-H) E: Experiment scheme for collection of IFNγ-primed dFB conditioned medium (dFBIFNγ-CM) or control dFBctrl-CM to stimulate naïve T cells. F: ELISA analysis showing protein levels of CXCL10 in dFBctrl-CM and dFBIFNγ-CM. G-H: Naïve T lymphocytes stimulated without (G) or with CD3/28-Ab (H) were treated with dFBctrl-CM or dFBIFNγ-CM with or w/o CXCR3 neutralizing antibody, and cell supernatants were collected for ELISA analysis of IFNγ protein expression.

All error bars indicate mean ± SEM. *p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001, ns, non-significant. Figure supplement 1. Interaction between dFBs and T cells via the IFNG-CXCL10-CXCR3 signaling axis in ACD.

Targeted deletion of Ifngr in dFBs inhibited the development of type-1 skin inflammation in ACD

(A) Tamoxifen-inducible targeted deletion of Ifngr1 in PDGFRA+ fibroblasts (Ifngr1FB-iKO) was achieved by crossing Ifngr1f/f and Pdgfra-cre/ERT mice, and mice were subjected to DNFB-induced ACD model as indicated.

(B) Multiplex-PCR based genotyping using allele specific primers yields DNA products having sizes specific for the wild-type and Ifngr1-floxed alleles. Lower gel shows gene products for Cre and/or internal product control (IPC) as indicated.

(C) Skin samples were immunostained with IFNGR1 (green) and PDGFRA (blue), and nuclei were counterstained with DAPI (white). Scale, 100 μm.

(D-E) Representative ear skin images (D) for each group at 60 hours after ACD elicitation. Br graphs (E) showing quantified Eczema Area and Severity Index (EASI) scores for each group (n=6∼12 /group).

(F) H&E staining of skin sections for each group at 60 hours after ACD elicitation

(G) qRT-PCR analysis of the mRNA expression levels of indicated genes (ratios to HK gene Tbp were shown, n=3∼8/group).

(H-I) Immunostaining (H) of skin sections with anti-CD8 (red) and anti-PDGFRA (blue) antibodies, and nuclei were counter stained by DAPI (white). Zoom-in images were shown in the lower panel. Quantified results (I) showing the # of CD8+ T cells per 1mm2 of dermal area in ACD skin.

All error bars indicate mean ± SEM. *p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001.

Figure supplement 1. Targeted deletion of Ifngr1 in dFBs inhibited the development of type-1 skin inflammation in ACD.

Activation of dermal T cells and fibroblasts in human ACD skin samples

(A) Skin sections from healthy control (HC) or ACD human skin samples were immunostained with antibodies against CD4 (green), CD8 (blue) and IFNγ (red) (representative of n=3/group). Nuclei were counter stained by DAPI (white). Scale bar, 200 μm. Zoom-in image is shown on the right-hand side.

(B) Upper panel is the zoom-in images from Fig. 6A. Lower panel is the quantified intensity profiles of CD8 (blue) and IFNγ (red) from left to right of the images marked by yellow dashed boxes.

(C) Skin sections from healthy control (HC) or ACD human skin samples were subjected to Masson’s staining, in which collagen bundles were stained in blue.

(D) Skin sections from healthy control (HC) or ACD human skin samples were immunostained with antibodies against CD8 (red) and PDGFRA (blue), and representative images of dermal lobular structures were shown (representative of n=3/group).

(E) Skin sections from healthy control (HC) or ACD human skin samples were immunostained with CXCL10 (green) and PDGFRA (blue). Nuclei were counter stained by DAPI (white). Scale bar, 200 μm. Zoom-in image is shown on the right-hand side

(F) Upper panel is the zoom-in images from Fig. 6E. Lower panel is the quantified intensity profiles of PDGFRA (blue) and CXCL10 (green) from left to right of the images marked by yellow dashed boxes.