Advertisement
Journal of Investigative Dermatology Home

Single-Cell Analysis Reveals Major Histocompatibility Complex II‒Expressing Keratinocytes in Pressure Ulcers with Worse Healing Outcomes

  • Author Footnotes
    8 These authors contributed equally to this work.
    Dongqing Li
    Footnotes
    8 These authors contributed equally to this work.
    Affiliations
    Dermatology and Venereology Division, Department of Medicine Solna, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
    Search for articles by this author
  • Author Footnotes
    8 These authors contributed equally to this work.
    Shangli Cheng
    Footnotes
    8 These authors contributed equally to this work.
    Affiliations
    Department of Physiology and Pharmacology, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden

    Ming Wai Lau Centre for Reparative Medicine, Karolinska Institute, Stockholm, Sweden
    Search for articles by this author
  • Author Footnotes
    9 These authors contributed equally to this work.
    Yu Pei
    Footnotes
    9 These authors contributed equally to this work.
    Affiliations
    Department of Physiology and Pharmacology, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
    Search for articles by this author
  • Author Footnotes
    9 These authors contributed equally to this work.
    Pehr Sommar
    Footnotes
    9 These authors contributed equally to this work.
    Affiliations
    Department of Reconstructive Plastic Surgery, Karolinska University Hospital, Stockholm, Sweden
    Search for articles by this author
  • Author Footnotes
    9 These authors contributed equally to this work.
    Jaanika Kärner
    Footnotes
    9 These authors contributed equally to this work.
    Affiliations
    Division of Rheumatology, Department of Medicine Solna, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
    Search for articles by this author
  • Eva K. Herter
    Affiliations
    Dermatology and Venereology Division, Department of Medicine Solna, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
    Search for articles by this author
  • Maria A. Toma
    Affiliations
    Dermatology and Venereology Division, Department of Medicine Solna, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
    Search for articles by this author
  • Letian Zhang
    Affiliations
    Dermatology and Venereology Division, Department of Medicine Solna, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
    Search for articles by this author
  • Kim Pham
    Affiliations
    Dermatology and Venereology Division, Department of Medicine Solna, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
    Search for articles by this author
  • Yuen Ting Cheung
    Affiliations
    Dermatology and Venereology Division, Department of Medicine Solna, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
    Search for articles by this author
  • Zhuang Liu
    Affiliations
    Dermatology and Venereology Division, Department of Medicine Solna, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
    Search for articles by this author
  • Xingqi Chen
    Affiliations
    Department of Immunology, Genetics and Pathology, Faculty of Medicine, Uppsala University, Uppsala, Sweden
    Search for articles by this author
  • Liv Eidsmo
    Affiliations
    Division of Rheumatology, Department of Medicine Solna, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden

    Leo Foundation Skin Immunology Center, Department of Immunology and Microbiology, Copenhagen University, Copenhagen, Denmark
    Search for articles by this author
  • Author Footnotes
    10 These authors contributed equally to this work.
    Qiaolin Deng
    Footnotes
    10 These authors contributed equally to this work.
    Affiliations
    Department of Physiology and Pharmacology, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
    Search for articles by this author
  • Author Footnotes
    10 These authors contributed equally to this work.
    Ning Xu Landén
    Correspondence
    Correspondence: Ning Xu Landén, Dermatology and Venereology Division, Department of Medicine Solna, Center for Molecular Medicine L8:02, Karolinska Institutet, 17176 Stockholm, Sweden.
    Footnotes
    10 These authors contributed equally to this work.
    Affiliations
    Dermatology and Venereology Division, Department of Medicine Solna, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden

    Ming Wai Lau Centre for Reparative Medicine, Karolinska Institute, Stockholm, Sweden
    Search for articles by this author
  • Author Footnotes
    8 These authors contributed equally to this work.
    9 These authors contributed equally to this work.
    10 These authors contributed equally to this work.
Open AccessPublished:September 15, 2021DOI:https://doi.org/10.1016/j.jid.2021.07.176
      Pressure ulcer (PU) is a chronic wound often seen in patients with spinal cord injury and other bed-bound individuals, particularly in the elderly population. Despite its association with high mortality, the pathophysiology of PU remains poorly understood. In this study, we compared single-cell transcriptomic profiles of human epidermal cells from PU wound edges with those from uninjured skin and acute wounds in healthy donors. We identified significant shifts in the cell composition and gene expression patterns in PU. In particular, we found that major histocompatibility complex class II‒expressing keratinocytes were enriched in patients with worse healing outcomes. Furthermore, we showed that the IFN-γ in PU-derived wound fluid could induce major histocompatibility complex II expression in keratinocytes and that these wound fluid‒treated keratinocytes inhibited autologous T-cell activation. In line with this observation, we found that T cells from PUs enriched with major histocompatibility complex II+ keratinocytes produced fewer inflammatory cytokines. Overall, our study provides a high-resolution molecular map of human PU compared with that of acute wounds and intact skin, providing insights into PU pathology and the future development of tailored wound therapy.

      Abbreviations:

      AW (acute wound), GO (gene ontology), IF (immunofluorescence), K (keratin), KC (keratinocyte), MHC (major histocompatibility complex), PU (pressure ulcer), scRNA-seq (single-cell RNA sequencing)

      Introduction

      A pressure ulcer (PU) is a chronic nonhealing wound that is caused by the continuous pressure of the bodyweight on the skin. PUs are often seen in patients with spinal cord injury and among bed-bound individuals, especially in the elderly population (
      • Hajhosseini B.
      • Longaker M.T.
      • Gurtner G.C.
      Pressure injury.
      ). Most patients with PU receive conservative treatment consisting of pressure relief and dressing changes, which can last for months to years (
      • Raetz J.G.
      • Wick K.H.
      Common questions about pressure ulcers.
      ). A minority of patients with PU receive reconstructive surgery with resection of the wound and flap coverage of the defect after the failure of conservative therapy. This is an extensive procedure and is unsuitable for elderly individuals and critically ill patients with multiple comorbidities (
      • Hajhosseini B.
      • Longaker M.T.
      • Gurtner G.C.
      Pressure injury.
      ). Therefore, a deeper understanding of PU pathophysiology and the identification of therapeutic targets are pressing needs.
      The advancement of single-cell RNA sequencing (scRNA-seq) technology has enabled the generation of molecular maps of human tissues at an unprecedented resolution, and this technology has recently been used to characterize human skin (
      • Cheng J.B.
      • Sedgewick A.J.
      • Finnegan A.I.
      • Harirchian P.
      • Lee J.
      • Kwon S.
      • et al.
      Transcriptional programming of normal and inflamed human epidermis at single-cell resolution.
      ;
      • Philippeos C.
      • Telerman S.B.
      • Oulès B.
      • Pisco A.O.
      • Shaw T.J.
      • Elgueta R.
      • et al.
      Spatial and single-cell transcriptional profiling identifies functionally distinct human dermal fibroblast subpopulations.
      ;
      • Tabib T.
      • Morse C.
      • Wang T.
      • Chen W.
      • Lafyatis R.
      SFRP2/DPP4 and FMO1/LSP1 define major fibroblast populations in human skin [published correction appears in J Invest Dermatol 2018;138:2086].
      ;
      • Wang S.
      • Drummond M.L.
      • Guerrero-Juarez C.F.
      • Tarapore E.
      • MacLean A.L.
      • Stabell A.R.
      • et al.
      Single cell transcriptomics of human epidermis identifies basal stem cell transition states.
      ) and related diseases, such as psoriasis (
      • Cheng J.B.
      • Sedgewick A.J.
      • Finnegan A.I.
      • Harirchian P.
      • Lee J.
      • Kwon S.
      • et al.
      Transcriptional programming of normal and inflamed human epidermis at single-cell resolution.
      ), atopic dermatitis (
      • He H.
      • Suryawanshi H.
      • Morozov P.
      • Gay-Mimbrera J.
      • Del Duca E.
      • Kim H.J.
      • et al.
      Single-cell transcriptome analysis of human skin identifies novel fibroblast subpopulation and enrichment of immune subsets in atopic dermatitis.
      ), and melanoma (
      • Tirosh I.
      • Izar B.
      • Prakadan S.M.
      • Wadsworth 2nd, M.H.
      • Treacy D.
      • Trombetta J.J.
      • et al.
      Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq.
      ), as well as fibroblast heterogeneity (
      • Guerrero-Juarez C.F.
      • Dedhia P.H.
      • Jin S.
      • Ruiz-Vega R.
      • Ma D.
      • Liu Y.
      • et al.
      Single-cell analysis reveals fibroblast heterogeneity and myeloid-derived adipocyte progenitors in murine skin wounds.
      ) and epidermal stem cells (
      • Haensel D.
      • Jin S.
      • Sun P.
      • Cinco R.
      • Dragan M.
      • Nguyen Q.
      • et al.
      Defining epidermal basal cell states during skin homeostasis and wound healing using single-cell transcriptomics.
      ;
      • Joost S.
      • Jacob T.
      • Sun X.
      • Annusver K.
      • La Manno G.
      • Sur I.
      • et al.
      Single-cell transcriptomics of traced epidermal and hair follicle stem cells reveals rapid adaptations during wound healing.
      ) in murine wound models. Although rodent wound models exhibit a wound-healing procedure similar to that of humans, they cannot fully reflect the complexity of disordered wound healing in humans (
      • Darwin E.
      • Tomic-Canic M.
      Healing chronic wounds: current challenges and potential solutions.
      ;
      • Volk S.W.
      • Bohling M.W.
      Comparative wound healing--are the small animal veterinarian's clinical patients an improved translational model for human wound healing research?.
      ). An in-depth understanding of wound healing mechanisms in humans has the potential to impact wound therapy and diagnosis directly.
      In this study, we performed a full-length single-cell transcriptomic analysis of human PUs in comparison with that of normal acute wounds (AWs) and skin from matched healthy donors. This dataset can be freely explored at a browsable web portal (https://www.xulandenlab.com/data), serving as a useful resource to enhance the understanding of wound healing biology and chronic wound pathogenesis. Among the many previously unknown or poorly characterized cellular and molecular events uncovered by this scRNA-seq analysis, we focused on a subset of keratinocytes (KCs) expressing major histocompatibility complex (MHC) class II because they were specifically enriched in PUs with worse healing outcomes after reconstructive surgery. We identified IFN-γ in PU wound fluid as a major inducer of MHC II expression in KCs. These MHC II+ KCs inhibit T-cell activation and may be one of the crucial contributors to the dysregulated immune response of PU.

      Results

       Characterization of epidermal cell composition of human skin and wounds

      To construct a gene expression map of the human wound-edge epidermis, we collected samples of chronic nonhealing wounds and nearby intact skin from five patients with spinal cord injury with grade IV PU. We also collected uninjured skin and day-7 AW from four healthy donors who were matched to these patients with PU in terms of age, sex, ethnicity, and body location (Figure 1a and Supplementary Figure S1 and Supplementary Table S1). We isolated viable epidermal cells and performed Smart-seq2 scRNA-seq, a highly sensitive method for full-length mRNA sequencing at the single-cell level (
      • Picelli S.
      • Björklund A.K.
      • Reinius B.
      • Sagasser S.
      • Winberg G.
      • Sandberg R.
      Tn5 transposase and tagmentation procedures for massively scaled sequencing projects.
      ). After stringent quality control, we retained data from 1,170 epidermal cells with an average of 5,223 genes detected in each cell (Supplementary Figure S2a). The average expression level in the scRNA-seq data was highly correlated with the bulk cell RNA sequencing data of the same sample (Spearman correlation coefficient > 0.95), confirming that the single cells we analyzed provided an unbiased representation of all the cell populations in the samples (Supplementary Figure S2b). We also evaluated these cells for their fraction of mitochondrial RNA, which was 9.5% on average, indicating good cell integrity (
      • Wang X.
      • He Y.
      • Zhang Q.
      • Ren X.
      • Zhang Z.
      Direct comparative analyses of 10X Genomics chromium and Smart-seq2 [e-pub ahead of print].
      ). There was no significant difference in mitochondrial RNA ratio between samples or clusters (Supplementary Figure S2c and d).
      Figure thumbnail gr1
      Figure 1Epidermal cell composition of human skin and wounds. (a) Demographics of the subjects analyzed by scRNA-seq. (b) t-SNE projection of cells sorted by cell clusters (left), disease conditions (middle), or donors (right) (n = 1,170 cells). (c) Cell type annotation with SingleR. (d) Selected marker gene expression in all the cells. (e) Immunofluorescence costaining of K14 and K10 or IFITM1 or S100A7; in situ hybridization of IGFBP3 and K14 in the skin (n = 3), AW (n = 3), and PU (n = 5). Red arrows indicate wound edge, and white arrows indicate a few positively stained cells. Nuclei were stained with DAPI. Bar = 50 μm or 10 μm (enlarged regions). (f–i), Positively stained KCs were counted and normalized with the area of each field. ∗P < 0.05, ∗∗∗P < 0.001; Student's t-test for f‒i. AW, acute wound; K, keratin; KC, keratinocyte; NS, not significant; PU, pressure ulcer; RPKM, Reads Per Kilobase of transcript per Million mapped reads; scRNA-seq, single-cell RNA sequencing; t-SNE, t-distributed stochastic neighbor embedding.
      These 1,170 epidermal cells were segregated into six clusters by Seurat2 clustering (after canonical correlation analysis to minimize batch effects from individual sample variability), which results were further confirmed by Seurat3 clustering (
      • Butler A.
      • Hoffman P.
      • Smibert P.
      • Papalexi E.
      • Satija R.
      Integrating single-cell transcriptomic data across different conditions, technologies, and species.
      ) (Figure 1b and Supplementary Figure S2e–h). Each of these clusters contained cells from at least eight of a total of nine donors and from all the three sample types, that is, skin, AW, and PU (Figure 1b). According to the expression of established markers, we annotated one melanocyte cluster (TYRP1+, n = 149), one immune cell cluster (CD74high, n = 43), and four KC clusters (KC_1–4) (Figure 1b and Supplementary Figure S3a and b and Supplementary Dataset S1). Furthermore, we characterized these cell clusters with the automated annotation tool SingleR (
      • Aran D.
      • Looney A.P.
      • Liu L.
      • Wu E.
      • Fong V.
      • Hsu A.
      • et al.
      Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage.
      ). A comparison of our data with a recently published human epidermis scRNA-seq dataset revealed that KC_1 (n = 367) and KC_3 (n = 189) represented spinous and granular KCs, respectively, whereas both KC_2 (n = 269) and KC_4 (n = 153) were basal layer KCs (Figure 1c) (
      • Cheng J.B.
      • Sedgewick A.J.
      • Finnegan A.I.
      • Harirchian P.
      • Lee J.
      • Kwon S.
      • et al.
      Transcriptional programming of normal and inflamed human epidermis at single-cell resolution.
      ). Next, we selected the top differentially expressed genes of each cell cluster as marker genes, that is, keratin (K) gene K10 for KC_1, IFITM1 for KC_2, S100A7 for KC_3, and IGFBP3 for KC_4 (Figure 1d and Supplementary Dataset S1 and Supplementary Table S2). We confirmed the spatial localization of each KC cluster in human skin and wounds by detecting these markers at the protein level (by immunofluorescence [IF] staining) or RNA level (by FISH) (Figure 1e–i and Supplementary Figure S3c).

       Molecular features of epidermal KCs

      We aligned the KCs on the basis of the expression patterns of five established differentiation markers (i.e., K5, K14, K1, K10, and CALML5), which divided all the KCs into five differentiation states (I–V) (Figure 2a ) (
      • Méhul B.
      • Bernard D.
      • Schmidt R.
      Calmodulin-like skin protein: a new marker of keratinocyte differentiation.
      ). Monocle3 pseudotime analysis set the root of differentiation origin to state I and the cells progress along transcriptional trajectories to state V (Figure 2b). Most KC_2 and KC_4 cells were assigned to the undifferentiated (K5/14+K1/10CALML5) or early differentiation states (K5/14+K1/10+CALML5), whereas KC_1 and KC_3 cells were enriched with KCs at a more differentiated state (K5/14+K1/10+CALML5+) (Figure 2c). In addition, analysis of cell cycle‒related gene expression revealed more proliferative cells in the KC_2/4 clusters than in the KC_1/3 clusters (Figure 2d–g) (
      • Macosko E.Z.
      • Basu A.
      • Satija R.
      • Nemesh J.
      • Shekhar K.
      • Goldman M.
      • et al.
      Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets.
      ). Functional predictions of the marker genes with gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes, and Wiki pathway analysis suggested different roles for each KC cluster, for example, lipid metabolism, Ras signal transduction, and Notch signaling pathway for spinous KCs (KC_1) and immune response and epidermis/hair follicle development for granular KCs (KC_3). Both basal layer KC clusters (KC_2 and KC_4) played a role in focal adhesion and extracellular matrix organization, whereas KC_2 KCs were also involved in the response to external stimuli and stem cell development (Figure 2h and Supplementary Figure S4a and b and Supplementary Dataset S2). In particular, both KC_2 and KC_4 express the known basal KC signature, for example, K14, ITGA6, and ITGB1 (
      • Jones P.H.
      • Harper S.
      • Watt F.M.
      Stem cell patterning and fate in human epidermis.
      ;
      • Li A.
      • Simmons P.J.
      • Kaur P.
      Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype.
      ) (Figure 2i). However, KC_2 differs from KC_4 in its higher expression of immunomodulatory genes, for example, IFITM1, IFITM3, CCL2, IL1R2, and TIMP1. Meanwhile, KC_2 has a lower level of IGFBP3, which is expressed exclusively in the basal KCs of the suprapapillary epidermis and inhibits cell proliferation (
      • Edmondson S.R.
      • Thumiger S.P.
      • Kaur P.
      • Loh B.
      • Koelmeyer R.
      • Li A.
      • et al.
      Insulin-like growth factor binding protein-3 (IGFBP-3) localizes to and modulates proliferative epidermal keratinocytes in vivo.
      ;
      • Wraight C.J.
      • Edmondson S.R.
      • Fortune D.W.
      • Varigos G.
      • Werther G.A.
      Expression of insulin-like growth factor binding protein-3 (IGFBP-3) in the psoriatic lesion.
      ) (Figure 2i and Supplementary Dataset S3).
      Figure thumbnail gr2
      Figure 2Molecular features of KC clusters. (a) Hierarchical cluster of differentiation marker expression in KCs. (b) Monocle3 pseudotime analysis of KCs. (c) Cell densities along PC1 for cells in each differentiation stage or cluster. (d) Cell cycle scores were calculated for each KC. (e) MKI67 and (f) PCNA expression shown in the cell cycle plots. (g) Proliferating cell (cells in the quadrants I, II, and IV of the cell cycle plot) ratio in each cluster. (h) GO analysis for the top 100 upregulated genes of each cluster compared with that in the rest KC clusters. The donut charts show the percentage of the genes associated with the GO terms with the lowest P-values. (i) Comparison of gene expression between KC_2 and KC_4. AMP, antimicrobial peptide; AW, acute wound; GO, gene ontology; K, keratin; KC, keratinocyte; PC, principal component; PCNA, proliferating cell nuclear antigen; PU, pressure ulcer; RPKM, Reads Per Kilobase of transcript per Million mapped reads; UMAP, uniform manifold approximation and projection.

       Epidermal cell heterogeneity in AWs and PUs

      The integrated view of cells from healthy skin, AW, and PU allowed us to compare the cellular composition and the gene expression of the same cell type under healthy and disease conditions. We found fewer melanocytes at the wound edges of AW and PU than in healthy skin (Figure 3a and b ), in line with previous observations in burn injuries (
      • Vachiramon V.
      • Thadanipon K.
      Postinflammatory hypopigmentation.
      ). In contrast, more epidermal immune cells were detected in the PU than in the skin or AW (Figure 3a and b). By comparing our results with a published scRNA-seq dataset of 236 epidermal immune cells (
      • Cheng J.B.
      • Sedgewick A.J.
      • Finnegan A.I.
      • Harirchian P.
      • Lee J.
      • Kwon S.
      • et al.
      Transcriptional programming of normal and inflamed human epidermis at single-cell resolution.
      ), we identified three hematopoietic subsets in our samples, that is, CD207+CD1A+ Langerhans cells, CD1C+CD301A+ myeloid dendritic cells, and CD3+ T cells, among which epidermal dendritic cells and T cells were mainly detected in PU (Figure 3c–e and Supplementary Figure S4c). Comparing AW and skin KCs, we observed increased frequencies of spinous (KC_1) and granular (KC_3) KCs during wound repair. Moreover, we detected fewer spinous (KC_1) and basal layer (KC_4) KCs and more granular (KC_3) KCs in the PU than in healthy skin or AW (Figures 1e–i and 3a, b and f).
      Figure thumbnail gr3
      Figure 3Altered epidermal cell heterogeneity in AWs and PUs. (a) Frequency distribution and (b) t-SNE projections of the six epidermal cell clusters in the skin (n = 391 cells), AW (n = 398 cells), and PU (n = 381 cells). (c–e) The number of immune cells identified by scRNA-seq, including CD207+CD1A+ LCs, CD1C+CD301A+ myeloid DCs, and CD3+ αβ T cells, in each sample of the skin, AW, and PU. (f) tSNE overlay of marker gene expression in the skin, AW, and PU scRNA-seq datasets. AW, acute wound; DC, dendritic cell; K, keratin; LC, Langerhans cell; PU, pressure ulcer; scRNA-seq, single-cell RNA sequencing; t-SNE, t-distributed stochastic neighbor embedding.

       Differential gene expression in epidermal cells of AWs and PUs

      Unlike melanocytes, which share similar gene expression profiles between AW and PU (Figure 4a and Supplementary Figure S5a and Supplementary Dataset S4), KCs exhibited contrasting molecular signatures in PU and AW (Figure 4b and Supplementary Figure S5b and Supplementary Dataset S4). GO analysis revealed that genes involved in neutrophil-mediated immunity (e.g., FABP5, S100A7, S100A8, and S100A9 [
      • Federici M.
      • Giustizieri M.L.
      • Scarponi C.
      • Girolomoni G.
      • Albanesi C.
      Impaired IFN-gamma-dependent inflammatory responses in human keratinocytes overexpressing the suppressor of cytokine signaling 1.
      ;
      • Ogawa E.
      • Owada Y.
      • Ikawa S.
      • Adachi Y.
      • Egawa T.
      • Nemoto K.
      • et al.
      Epidermal FABP (FABP5) regulates keratinocyte differentiation by 13(S)-HODE-mediated activation of the NF-κB signaling pathway.
      ]) were strongly upregulated in PU KCs compared with those in KCs from AW or uninjured skin. However, during normal wound healing (AW vs. skin), the expression of these genes was only slightly enhanced (Figure 4c and Supplementary Figure S5b and Supplementary Dataset S5). In addition, the expression levels of genes essential for cellular homeostasis of transition metals, in particular, zinc ions (e.g., MT2A, MT1E, FTH1, and FTL [
      • Emri E.
      • Miko E.
      • Bai P.
      • Boros G.
      • Nagy G.
      • Rózsa D.
      • et al.
      Effects of non-toxic zinc exposure on human epidermal keratinocytes.
      ;
      • Theil E.C.
      Ferritin: the protein nanocage and iron biomineral in health and in disease.
      ]), were higher in PU KCs than in AW KCs (Figure 4c and Supplementary Figure S5b and Supplementary Dataset S5). GO, Kyoto Encyclopedia of Genes and Genomes, and Wiki pathway analysis revealed that PU KCs expressed higher levels of apoptosis-related genes (e.g., DUSP1 and RHOB [
      • Canguilhem B.
      • Pradines A.
      • Baudouin C.
      • Boby C.
      • Lajoie-Mazenc I.
      • Charveron M.
      • et al.
      RhoB protects human keratinocytes from UVB-induced apoptosis through epidermal growth factor receptor signaling.
      ;
      • Yang J.
      • Sun L.
      • Han J.
      • Zheng W.
      • Peng W.
      DUSP1/MKP-1 regulates proliferation and apoptosis in keratinocytes through the ERK/Elk-1/Egr-1 signaling pathway.
      ]) than KCs from AW and uninjured skin, which may be related to the massive activation of the unfolded protein response (e.g., HSPH1 and DNAJB1 [
      • Fribley A.
      • Zhang K.
      • Kaufman R.J.
      Regulation of apoptosis by the unfolded protein response.
      ]) (Figure 4c and Supplementary Figures S5b and S6 and Supplementary Dataset S5). The enhanced apoptosis in PU was confirmed by immunostaining, which showed increased cleaved caspase-3‒positive cells in the PU samples compared with those in the skin and AW samples (Figure 4d).
      Figure thumbnail gr4
      Figure 4Differential gene expression in epidermal cells of AWs and PUs. Heatmap illustrates the genes differentially expressed in (a) melanocytes and (b) keratinocytes from the skin, AW, and PU. (c) GO analysis for the top 150 upregulated genes in comparisons of AW with PU, skin with PU, and skin with AW. The five biological processes with the lowest P-values are shown. (d) Immunofluorescence staining of cleaved Caspase 3 in the skin (n = 2), AW (n = 2), and PU (n = 5). Nuclei: DAPI. Bar = 50 μm. (e) Cell densities along PC1 for cells in each differentiation stage (upper) or sample type (lower). (f) Cell cycle scores of the skin, AW, and PU keratinocytes. (g) The percentage of proliferative keratinocytes with low or high K1/10 expression. AW, acute wound; GO, gene ontology; K, keratin; ND, none detected; PC, principal component; PU, pressure ulcer.
      We further compared KC differentiation and proliferation in the skin, AW, and PU. Along the differentiation trajectory reconstructed with pseudotime analysis (Figure 2b), we found that the proportion of differentiated KCs was higher in AW than in the skin (Figure 4e). In contrast to the skin and AW KCs that were distributed along the differentiation axis, PU KCs clustered in differentiation state I (for donors PU1 and PU2) or IV (for donors PU3, PU4, and PU5), suggesting a dysregulation of the differentiation program (Figure 4e). Furthermore, we identified proliferating KCs on the basis of their expression of cell cycle‒related genes (Figure 4f and Supplementary Dataset S6) (
      • Macosko E.Z.
      • Basu A.
      • Satija R.
      • Nemesh J.
      • Shekhar K.
      • Goldman M.
      • et al.
      Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets.
      ). We found that a significant proportion of these proliferating cells expressed the early differentiation markers K1 and K10, confirming previous findings showing the proliferative and tissue-regenerative capacity of human early differentiating KCs (
      • Li A.
      • Pouliot N.
      • Redvers R.
      • Kaur P.
      Extensive tissue-regenerative capacity of neonatal human keratinocyte stem cells and their progeny.
      ). We detected more proliferating KCs in AW than in the skin (Figure 4g). In addition, in PU3, PU4, and PU5 but not PU1 and PU2, the proportion of proliferating cells was lower than that in the AWs (Figure 4g). This finding not only revealed dysregulated KC differentiation and proliferation in PU but also suggested that PU exhibited a higher degree of interindividual variability than uninjured skin or AW.

       Stratification of PU into two subtypes with distinct molecular features correlating with clinical outcomes

      To stratify the patients with PU, we performed principal component analysis using the 4,000 most variable genes on all PU KCs, which separated PU3, PU4, and PU5 (named PU group 1, i.e., PU_G1 in the remaining part of this paper) from PU1 and PU2 (PU group 2, i.e., PU_G2) (Figure 5a ). Interestingly, functional predictions of the differentially expressed genes with GO, Kyoto Encyclopedia of Genes and Genomes, and Wiki pathway analysis revealed that the PU_G1 KCs expressed genes involved in MHC II‒mediated antigen presentation (Figure 5b and Supplementary Figure S7a and b and Supplementary Dataset S7). After this observation, we discovered a subset of KCs expressing the canonical components of the MHC II machinery (e.g., CD74 and HLA-DRB) (Figure 5c). The presence of MHC II on KCs was unlikely to be due to doublets of KCs and antigen-presenting cells because the MHC II+ and MHC II KCs had similar mean gene expression, and the External RNA Controls Consortium spike-in RNA was used as control (Supplementary Figure S7c and d). Next, we costained K14 and HLA-DR mRNAs using FISH and proteins using IF staining in additional clinical samples. We confirmed that MHC II+ KCs were overrepresented in PU_G1 compared with those in PU_G2, AWs, or the skin, as shown by scRNA-seq (Figure 5d–g and Supplementary Figure S7e and Supplementary Movie S1). On the other hand, the PU_G2 KCs expressed higher levels of proproliferation and proangiogenesis genes than the PU_G1 KCs (Figure 5b and Supplementary Dataset S8). We confirmed the GO analysis results by IF costaining of K14 and the proliferation marker Ki-67 (Figure 5e and h). We detected more proliferative KCs in PU_G2 than in PU_G1, which was in line with the cell cycle analysis results (Figure 4g). Moreover, the scRNA-seq analysis showed that there were more epidermal immune cells in the PU_G1 than in the PU_G2 samples (Supplementary Figure S7f–i). In addition, IF staining revealed a higher density of T cells in PU-G1 ulcers than in PU-G2 ulcers, five-fold in the epidermis and three-fold in the dermis (Supplementary Figure S7j–l).
      Figure thumbnail gr5
      Figure 5Stratification of PU into subtypes. (a) PC analysis of PU KCs. (b) GO analysis of the top 100 upregulated genes in KC from two PU subtypes. MHC II gene, MHC II, expression in (c) CD74HLA-DRB KC, CD74highHLA-DRBhigh KC, and immune cells and in (d) KC from the skin, AW, and PUs. (e) HLADR+ or KI-67+ KC (K14+) identified by IF and FISH were counted in the (f‒h) skin (n = 3), AW (n = 3), PU G1 (n = 7), and G2 (n = 9). Nuclei: DAPI. Arrows: HLA-DR+ KC. Bar = 50 μm or 10 μm (enlarged regions). (i) HLA-DR+ and KI-67+ KCs were counted in 16 PUs analyzed by IF. (j) t-SNE projection of the PUs based on CIITA, CD74, CIITA, and VEGFA expression. ∗P < 0.05, ∗∗P < 0.01; Student's t-test for fh. AW, acute wound; GO, gene ontology; IF, immunofluorescence; K, keratin; KC, keratinocyte; MHC, major histocompatibility complex; ND, none detected; PC, principal component; PU, pressure ulcer; RPKM, Reads Per Kilobase of transcript per Million mapped reads; t-SNE, t-distributed stochastic neighbor embedding.
      To validate these findings, we performed IF staining of MHC II and Ki-67 in wound-edge biopsies from 16 patients with PU treated with reconstructive surgery. We found seven PUs enriched with MHC II+ KCs but lacking proliferating KCs (PU_G1), whereas nine PUs had few MHC II+ KCs but active proliferating KCs (PU_G2) (Figure 5i and Supplementary Figure S8). The healing outcomes in these 16 patients were evaluated 1 month after the operation independently by two surgeons who had not seen the experimental data associated with these patients. Wounds with closed edges and without signs of infection, exudate, or necrotic tissue were diagnosed as healed wounds. Interestingly, all the patients in PU_G2 were identified to have healed wounds; however, none of the patients in PU_G1 fulfilled these criteria of healing (Figure 5i and Supplementary Tables S1 and S3). Moreover, on the basis of the quantitative real-time reverse transcriptase–PCR analysis of wound biopsies for the expression of CD74, CIITA, HLA-DRA, and VEGFA, which are among the top differentially expressed genes identified in scRNA-seq between PU-G1 and PU-G2, the healing and nonhealing PUs could also be separated in the t-distributed stochastic neighbor embedding plot (Figure 5j and Supplementary Figure S9a–d). We also checked clinical parameters, including patient age, wound size and duration, circulating CRP and leukocytes, and bacterial colonization and infection, and found that none of them could be used to distinguish the two groups of PUs with varied healing outcomes (Supplementary Table S1 and Supplementary Figure S9e–i). Despite the small patient cohort, our study provides proof of the principle that PU can be stratified into subtypes with distinct molecular features correlating with clinical outcomes.

       The formation and function of MHC II+ KCs in PUs

      To understand the mechanism triggering MHC II expression in KCs, we performed a gene set enrichment analysis for the differentially expressed genes between the MHC II+ and MHC II KCs, which revealed an upregulation of IFN-γ signaling in MHC II+ KCs (Figure 6a ). In line with this, we found that stimulation of human primary KCs with IFN-γ strongly induced the expression of MHC II and its transactivator CIITA at the mRNA level (Figure 6b). In addition, we observed increased HLA-DR protein expression in IFNγ-treated KCs (Figure 6c). Furthermore, we showed that the cell-free wound fluid from patients in PU_G1 but not those in PU_G2 induced KC expression of CD74 and HLA-DRB (Figure 6d and e and Supplementary Figure S10a). Importantly, this effect was blocked by neutralizing IFN-γ in the wound fluids, suggesting that IFN-γ may account for MHC II expression in PU KCs (Figure 6d and e). Of note, we observed that the application of the IFN-γ antibody 24 hours after wound fluid treatment could not reverse CD74 and HLA levels in the cultured KCs (Supplementary Figure S10b and c). Moreover, gene set enrichment analysis of scRNA-seq data revealed higher expression of IFN-γ response‒related genes in PU_G1 KCs than in PU_G2 KCs (Figure 6f). Quantitative real-time reverse transcriptase–PCR analysis showed that IFNγ expression was increased in PU_G1 compared with that in PU_G2 biopsies. The levels of IFN-γ and MHC II were positively correlated in human skin and wounds in vivo (Figure 6g and h and Supplementary Figure S10d and e). Together, our data suggest IFN-γ as a major inducer of KC-intrinsic MHC II expression in PU.
      Figure thumbnail gr6
      Figure 6MHC II+ keratinocytes in PUs. GSEA of IFN-γ–response genes among the DEG between (a) CD74highHLA-DRBhigh and CD74HLA-DRB keratinocytes or (f) between PU_G1 and PU_G2 keratinocytes. (b) QRT-PCR of HLADRB, CD74, and CIITA and (c) IF of K14 and HLA-DR in IFN-γ–treated keratinocytes. (d, e) QRT-PCR of HLADRB and CD74 in keratinocytes treated with PU WF and IFN-γ antibody or isotype Ctrl. (g) QRT-PCR of IFNG and (h) Spearman’s correlation between IFNG and CD74 in the skin (n = 10), AW (n = 10), PU_G1 (n = 7), and PU_G2 (n = 10). (i) IF of HLA-DR, CD3, K14, and DAPI. Bar = 50 μm. (j) Flow cytometry analysis of cytokines in T cells from two PU subtypes. (k) T-cell proliferation 5 days after coculture with WF-treated keratinocytes (n = 3 donors). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; Student’s t-test. Ab, antibody; AW, acute wound; Ctrl, control; DEG, differentially expressed gene; ES, enrichment score; GSEA, gene set enrichment analysis; IF, immunofluorescence; K, keratin; MHC, major histocompatibility complex; ND, none detected; NS, not significant; PU, pressure ulcer; QRT-PCR, quantitative real-time reverse transcriptase–PCR; WF, wound fluid.
      Next, we explored the potential role of MHC II+ KCs in PU pathology. Gene set enrichment analysis of scRNA-seq data revealed that MHC II+ KCs expressed more antigen presentation‒ and processing‒related genes than MHC II KCs (Supplementary Figure S10f). In line with this, we found that MHC II+ KCs were close to T cells in PU wound edges by confocal imaging (Figure 6i). Surprisingly, flow cytometry analysis showed that αβ T cells in the PUs with MHC II+ KC overrepresentation (PU_G1) indeed produced fewer inflammatory cytokines, for example, TNF-α, IL-17a, and IFN-γ, than the PUs lacking MHC II+ KCs (PU_G2) (Figure 6j and Supplementary Figure S11). Moreover, we detected increased epidermal γδ T cells in the PU compared with that in the skin by flow cytometry (Supplementary Figure S12a). Similar to αβ T cells (Figure 6j), γδ T cells from PU_G1 also produced fewer IFN-γ than those from PU_G2 (Supplementary Figure S12b).
      To directly study whether MHC II+ KCs may impact T-cell activation, we cocultured KCs and autologous T cells isolated from healthy donors' skin and blood, respectively. We found that pretreatment of KCs with PU_G1‒derived wound fluids reduced the concentration of several cytokines important for T-cell recruitment and activation, for example, IFN-γ, IL-9, IL-21, GM-CSF, CXCL1, CXCL8, CCL4, and CXCL12, in cell culture supernatants (Supplementary Figure S12c and d). Moreover, we found that TCR-induced T-cell proliferation decreased from 7.6% to 1.2% (P = 0.03) by coculturing with KCs pretreated with PU_G1‒derived wound fluid (Figure 6k and Supplementary Figure S11e). These results suggested that KCs might function as atypical antigen-presenting cells and exert an inhibitory effect on T-cell activation in PU wound edges.

      Discussion

      Despite the high mortality of patients with PU, the molecular pathology of PU remains mostly elusive, which hampers the development of more effective treatment. For this, we generated a cellular landscape of human wound-edge epidermis at the single-cell level, charting the differences in cell composition and molecular state between nonhealing PU, healing AW, and uninjured skin. Comparing PU with AW, we found that most pathological changes occurred in KCs and immune cells but not in melanocytes because melanocytes in the healing and nonhealing wounds exhibited similar cellular proportions and transcriptomes. At the PU wound edge, suprabasal KC_3 and basal KC_2 KCs predominated, whereas suprabasal KC_1 and basal KC_4 KCs were lacking. Interestingly, both the KC_3 and KC_2 clusters had gene signatures associated with immune functions. This cellular composition shift was also reflected in the gene expression profile of PU KCs, revealing their intense inflammatory response. Although a low number of immune cells were captured in this study, a sharp increase of epidermal T cells, myeloid dendritic cells, and Langerhans cells was detected in PU but not in AW, which further advanced our view of the different immune microenvironments between healing and nonhealing wounds.
      The lack of scRNA-seq analysis of wound dermal cells is a major limitation of this study. The choice of Smart-seq2 for this study was made before the availability of the droplet-based commercial platform, that is, 10X Genomics Chromium (Pleasanton, CA); therefore, much fewer cells were sequenced owing to relatively high cost and labor intensity. However, Smart-seq2 is still a much more sensitive method with full-length coverage (
      • Wang X.
      • He Y.
      • Zhang Q.
      • Ren X.
      • Zhang Z.
      Direct comparative analyses of 10X Genomics chromium and Smart-seq2 [e-pub ahead of print].
      ). Owing to the crucial immune and structural functions of KCs in wound healing, we performed scRNA-seq in the wound-edge epidermis as a first step. Further analysis of full-thickness skin and wound specimens with single-cell technology is needed to complete the picture regarding the complex immune environments in human wounds.
      In this study, we discovered an MHC II+ KC population that is overrepresented in PUs with worse healing outcomes. KCs are known to play an active role in skin immunity and inflammation by producing many cytokines and chemokines (
      • Landén N.X.
      • Li D.
      • Ståhle M.
      Transition from inflammation to proliferation: a critical step during wound healing.
      ). Our findings suggest that they may also directly interact with T cells through antigen presentation and reduce T-cell activation. We postulated that MHC II+ KCs might interfere with T-cell function in PU, which was endorsed by previous studies showing that KC–T cell interactions could lead to T-cell anergy or tolerance (
      • Bal V.
      • McIndoe A.
      • Denton G.
      • Hudson D.
      • Lombardi G.
      • Lamb J.
      • et al.
      Antigen presentation by keratinocytes induces tolerance in human T cells.
      ;
      • Gaspari A.A.
      • Katz S.I.
      Induction of in vivo hyporesponsiveness to contact allergens by hapten-modified Ia+ keratinocytes.
      ). In addition, epidermal T cells isolated from human chronic wounds have been shown to be less responsive to stimulation than T cells from AWs (
      • Toulon A.
      • Breton L.
      • Taylor K.R.
      • Tenenhaus M.
      • Bhavsar D.
      • Lanigan C.
      • et al.
      A role for human skin-resident T cells in wound healing.
      ). Together, our findings support the recently raised hypothesis that chronic wound inflammation is persistent but ineffective in combatting infection and healing wounds (
      • MacLeod A.S.
      • Mansbridge J.N.
      The innate immune system in acute and chronic wounds.
      ;
      • Toulon A.
      • Breton L.
      • Taylor K.R.
      • Tenenhaus M.
      • Bhavsar D.
      • Lanigan C.
      • et al.
      A role for human skin-resident T cells in wound healing.
      ). Interestingly, a small fraction of MHC II+ KCs was also found in humans (
      • Carr M.M.
      • McVittie E.
      • Guy K.
      • Gawkrodger D.J.
      • Hunter J.A.
      MHC class II antigen expression in normal human epidermis.
      ) and in the mouse epidermis under homeostasis (
      • Tamoutounour S.
      • Han S.J.
      • Deckers J.
      • Constantinides M.G.
      • Hurabielle C.
      • Harrison O.J.
      • et al.
      Keratinocyte-intrinsic MHCII expression controls microbiota-induced Th1 cell responses.
      ). In mouse skin, MHC II+ KCs were shown to control homeostatic type one responses to the microbiota (
      • Tamoutounour S.
      • Han S.J.
      • Deckers J.
      • Constantinides M.G.
      • Hurabielle C.
      • Harrison O.J.
      • et al.
      Keratinocyte-intrinsic MHCII expression controls microbiota-induced Th1 cell responses.
      ). Thus, MHC II expression by KCs occurs in both physiological and pathological conditions but may have different roles in these different contexts.
      Our study suggests the potential significance of IFN-γ in human PU pathology by identifying it as a major inducer of MHC II+ KCs and associating its upregulation in PUs with delayed healing. IFN-γ has been shown to induce MHC class II expression in KCs (
      • Albanesi C.
      • Cavani A.
      • Girolomoni G.
      Interferon-gamma-stimulated human keratinocytes express the genes necessary for the production of peptide-loaded MHC class II molecules.
      ;
      • Gaspari A.A.
      • Jenkins M.K.
      • Katz S.I.
      Class II MHC-bearing keratinocytes induce antigen-specific unresponsiveness in hapten-specific Th1 clones.
      ;
      • Skov L.
      • Baadsgaard O.
      MHC class II+ keratinocytes from IFN gamma-treated human skin activate T cells in the presence of staphylococcal superantigen despite UVB irradiation.
      ;
      • Takagi A.
      • Nishiyama C.
      • Kanada S.
      • Niwa Y.
      • Fukuyama K.
      • Ikeda S.
      • et al.
      Prolonged MHC class II expression and CIITA transcription in human keratinocytes.
      ;
      • Tamoutounour S.
      • Han S.J.
      • Deckers J.
      • Constantinides M.G.
      • Hurabielle C.
      • Harrison O.J.
      • et al.
      Keratinocyte-intrinsic MHCII expression controls microbiota-induced Th1 cell responses.
      ) and to inhibit KC proliferation (
      • Hancock G.E.
      • Kaplan G.
      • Cohn Z.A.
      Keratinocyte growth regulation by the products of immune cells.
      ). In rodent models, IFN-γ has also been found to inhibit angiogenesis and collagen deposition, thus hampering wound repair (
      • Ishida Y.
      • Kondo T.
      • Takayasu T.
      • Iwakura Y.
      • Mukaida N.
      The essential involvement of cross-talk between IFN-gamma and TGF-beta in the skin wound-healing process.
      ;
      • Laato M.
      • Heino J.
      • Gerdin B.
      • Kähäri V.M.
      • Niinikoski J.
      Interferon-gamma-induced inhibition of wound healing in vivo and in vitro.
      ). On the basis of these pieces of evidence, it would be tempting to test whether the local blockage of IFN-γ signaling may improve wound healing in patients with PU. Under this paradigm, humanized anti–IFN-γ antibody, which is under development to treat Crohn’s disease, might be repurposed for wound therapy (
      • Reinisch W.
      • de Villiers W.
      • Bene L.
      • Simon L.
      • Rácz I.
      • Katz S.
      • et al.
      Fontolizumab in moderate to severe Crohn's disease: a phase 2, randomized, double-blind, placebo-controlled, multiple-dose study.
      ).
      Recently, scRNA-seq technology has also been leveraged to study another type of chronic wound, diabetic foot ulcers (
      • Januszyk M.
      • Chen K.
      • Henn D.
      • Foster D.S.
      • Borrelli M.R.
      • Bonham C.A.
      • et al.
      Characterization of diabetic and non-diabetic foot ulcers using single-cell RNA-sequencing.
      ;
      • Theocharidis G.
      • Baltzis D.
      • Roustit M.
      • Tellechea A.
      • Dangwal S.
      • Khetani R.S.
      • et al.
      Integrated skin transcriptomics and serum multiplex assays reveal novel mechanisms of wound healing in diabetic foot ulcers.
      ). These studies started with full-thickness skin specimens, but the majority of the cells captured were dermal cells, for example, fibroblasts, smooth muscle cells, vascular or lymphatic endothelial cells, and immune cells (
      • Januszyk M.
      • Chen K.
      • Henn D.
      • Foster D.S.
      • Borrelli M.R.
      • Bonham C.A.
      • et al.
      Characterization of diabetic and non-diabetic foot ulcers using single-cell RNA-sequencing.
      ;
      • Theocharidis G.
      • Baltzis D.
      • Roustit M.
      • Tellechea A.
      • Dangwal S.
      • Khetani R.S.
      • et al.
      Integrated skin transcriptomics and serum multiplex assays reveal novel mechanisms of wound healing in diabetic foot ulcers.
      ). Although these findings in the dermis could not be directly compared with our dataset of epidermal cells, we found that some biological processes dysregulated in PU also surfaced in diabetic foot ulcers, including elevated inflammatory state and cell apoptosis but reduced cell mitogenic activity.
      The high patient-to-patient variability in PU underscores the need for personalized wound treatments. However, biomarkers to stratify subsets of nonhealing patients and to guide therapy are lacking (
      • Lindley L.E.
      • Stojadinovic O.
      • Pastar I.
      • Tomic-Canic M.
      Biology and biomarkers for wound healing.
      ). Our study showed that even within a group of patients with similar clinical manifestations, the gene expression profiles of PU wound-edge KCs could distinguish between different subtypes. More interestingly, those patients with MHC II+ KC overrepresentation accompanied by Ki-67+ KC reduction at their wound edges had worse healing outcomes after operation. Despite the limited number of patients included, our study provides a proof of principle that PU can be stratified into subtypes with distinct molecular hallmarks highly correlated with clinical outcomes, underscoring the importance of molecular wound diagnosis. Further validation of our findings in larger patient cohorts may lead to the identification of biomarkers for early recognition of patients with PU who may require attentive therapeutic interference. Thus, our study provides insights for future precision medicine in wound care.

      Materials and Methods

       Human wound samples

      We enrolled 25 healthy donors and 18 patients with PU at the Karolinska University Hospital (Stockholm, Sweden) (Supplementary Table S1). All the clinical materials were taken after written informed patient consent. The study was approved by the Stockholm Regional Ethics Committee and was conducted according to the Declaration of Helsinki’s principles.

       Statistics

      All data were expressed as mean  ± SD or mean ± SEM and plotted using GraphPad Prism, version 6 (GraphPad Software, San Diego, CA). Statistical significance was determined by a two-tailed Student’s t-test or Mann‒Whitney U Test. The correlation between the expressions of different genes in the same sample set was made using Spearman’s correlation test on log-transformed data. For all statistical tests, P < 0.05 was considered to be statistically significant.
      The detailed methods used for human sample collection and processing, scRNA-seq and data analysis, IF staining, FISH, image analysis, cell culture and treatments, RNA extraction and quantitative real-time reverse transcriptase–PCR, analysis of cytokine production of T cells, flow cytometry and autologous KC–T cell coculture are provided in Supplementary Experimental Procedures. Patients’ information is listed in Supplementary Table S1. Antibodies, primers, and chemicals used in this study are listed in Supplementary Table S4.

       Data availability statement

      The raw data and the processed matrices have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus database (GSE137897). The codes are available at https://github.com/shanglicheng/HumanKeratinocyte. A web resource for browsing the single-cell RNA-sequencing data can be accessed at our resource webpage https://www.xulandenlab.com/data.

      ORCIDs

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We express our gratitude to all the patients and healthy donors who took part in this study. We thank Maria Kasper (Karolinska Institutet, Stockholm, Sweden) and Stanley Sing Hoi Cheuk (Göteborgs University, Gothenburg, Sweden) for discussion and advice. We thank Borislav Ignatov (Karolinska Institutet), Hua Zhang, and Yonglong Dang (Uppsala University, Uppsala, Sweden) for technical support. We thank Madeleine Stenius (Rehab Station Stockholm Academy) for clinical sample collection. This work was supported by Swedish Research Council (Vetenskapsradet, 2016-02051 , 2018-02557 , and 2020-01400 ), Ragnar Söderbergs Foundation (M31/15), Cancerfonden ( 200930Pj ), Hedlunds Foundation, Welander and Finsens Foundation (Hudfonden), Åke Wibergs Foundation, Jeanssons Foundation, Swedish Psoriasis Foundation, Ming Wai Lau Centre for Reparative Medicine, Tore Nilson's Foundation, Lars Hiertas Foundation, and Karolinska Institutet.

      Author Contributions

      Conceptualization: NXL, QD; Formal Analysis: SC, DL, YTC, ZL, XC, LE; Investigation: DL, YP, PS, KP, JK, EKH, MAT, LZ; Resources: PS, KP; Software: SC, DL, YTC, ZL, XC, LE; Supervision: NXL, QD; Visualization: DL, SC; Writing - Original Draft Preparation: NXL; Writing - Review and Editing: DL, SC, YP, PS, JK, EKH, MAT, LZ, KP, YTC, ZL, XC, LE, QD, NXL

      Supplementary Material

      References

        • Albanesi C.
        • Cavani A.
        • Girolomoni G.
        Interferon-gamma-stimulated human keratinocytes express the genes necessary for the production of peptide-loaded MHC class II molecules.
        J Invest Dermatol. 1998; 110: 138-142
        • Aran D.
        • Looney A.P.
        • Liu L.
        • Wu E.
        • Fong V.
        • Hsu A.
        • et al.
        Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage.
        Nat Immunol. 2019; 20: 163-172
        • Bal V.
        • McIndoe A.
        • Denton G.
        • Hudson D.
        • Lombardi G.
        • Lamb J.
        • et al.
        Antigen presentation by keratinocytes induces tolerance in human T cells.
        Eur J Immunol. 1990; 20 (1893–7)
        • Butler A.
        • Hoffman P.
        • Smibert P.
        • Papalexi E.
        • Satija R.
        Integrating single-cell transcriptomic data across different conditions, technologies, and species.
        Nat Biotechnol. 2018; 36: 411-420
        • Canguilhem B.
        • Pradines A.
        • Baudouin C.
        • Boby C.
        • Lajoie-Mazenc I.
        • Charveron M.
        • et al.
        RhoB protects human keratinocytes from UVB-induced apoptosis through epidermal growth factor receptor signaling.
        J Biol Chem. 2005; 280: 43257-43263
        • Carr M.M.
        • McVittie E.
        • Guy K.
        • Gawkrodger D.J.
        • Hunter J.A.
        MHC class II antigen expression in normal human epidermis.
        Immunology. 1986; 59: 223-227
        • Cheng J.B.
        • Sedgewick A.J.
        • Finnegan A.I.
        • Harirchian P.
        • Lee J.
        • Kwon S.
        • et al.
        Transcriptional programming of normal and inflamed human epidermis at single-cell resolution.
        Cell Rep. 2018; 25: 871-883
        • Darwin E.
        • Tomic-Canic M.
        Healing chronic wounds: current challenges and potential solutions.
        Curr Dermatol Rep. 2018; 7: 296-302
        • Edmondson S.R.
        • Thumiger S.P.
        • Kaur P.
        • Loh B.
        • Koelmeyer R.
        • Li A.
        • et al.
        Insulin-like growth factor binding protein-3 (IGFBP-3) localizes to and modulates proliferative epidermal keratinocytes in vivo.
        Br J Dermatol. 2005; 152: 225-230
        • Emri E.
        • Miko E.
        • Bai P.
        • Boros G.
        • Nagy G.
        • Rózsa D.
        • et al.
        Effects of non-toxic zinc exposure on human epidermal keratinocytes.
        Metallomics. 2015; 7: 499-507
        • Federici M.
        • Giustizieri M.L.
        • Scarponi C.
        • Girolomoni G.
        • Albanesi C.
        Impaired IFN-gamma-dependent inflammatory responses in human keratinocytes overexpressing the suppressor of cytokine signaling 1.
        J Immunol. 2002; 169: 434-442
        • Fribley A.
        • Zhang K.
        • Kaufman R.J.
        Regulation of apoptosis by the unfolded protein response.
        Methods Mol Biol. 2009; 559: 191-204
        • Gaspari A.A.
        • Jenkins M.K.
        • Katz S.I.
        Class II MHC-bearing keratinocytes induce antigen-specific unresponsiveness in hapten-specific Th1 clones.
        J Immunol. 1988; 141: 2216-2220
        • Gaspari A.A.
        • Katz S.I.
        Induction of in vivo hyporesponsiveness to contact allergens by hapten-modified Ia+ keratinocytes.
        J Immunol. 1991; 147: 4155-4161
        • Guerrero-Juarez C.F.
        • Dedhia P.H.
        • Jin S.
        • Ruiz-Vega R.
        • Ma D.
        • Liu Y.
        • et al.
        Single-cell analysis reveals fibroblast heterogeneity and myeloid-derived adipocyte progenitors in murine skin wounds.
        Nat Commun. 2019; 10: 650
        • Haensel D.
        • Jin S.
        • Sun P.
        • Cinco R.
        • Dragan M.
        • Nguyen Q.
        • et al.
        Defining epidermal basal cell states during skin homeostasis and wound healing using single-cell transcriptomics.
        Cell Rep. 2020; 30 (3932–47.e6)
        • Hajhosseini B.
        • Longaker M.T.
        • Gurtner G.C.
        Pressure injury.
        Ann Surg. 2020; 271: 671-679
        • Hancock G.E.
        • Kaplan G.
        • Cohn Z.A.
        Keratinocyte growth regulation by the products of immune cells.
        J Exp Med. 1988; 168: 1395-1402
        • He H.
        • Suryawanshi H.
        • Morozov P.
        • Gay-Mimbrera J.
        • Del Duca E.
        • Kim H.J.
        • et al.
        Single-cell transcriptome analysis of human skin identifies novel fibroblast subpopulation and enrichment of immune subsets in atopic dermatitis.
        J Allergy Clin Immunol. 2020; 145: 1615-1628
        • Ishida Y.
        • Kondo T.
        • Takayasu T.
        • Iwakura Y.
        • Mukaida N.
        The essential involvement of cross-talk between IFN-gamma and TGF-beta in the skin wound-healing process.
        J Immunol. 2004; 172: 1848-1855
        • Januszyk M.
        • Chen K.
        • Henn D.
        • Foster D.S.
        • Borrelli M.R.
        • Bonham C.A.
        • et al.
        Characterization of diabetic and non-diabetic foot ulcers using single-cell RNA-sequencing.
        Micromachines (Basel). 2020; 11: 815
        • Jones P.H.
        • Harper S.
        • Watt F.M.
        Stem cell patterning and fate in human epidermis.
        Cell. 1995; 80: 83-93
        • Joost S.
        • Jacob T.
        • Sun X.
        • Annusver K.
        • La Manno G.
        • Sur I.
        • et al.
        Single-cell transcriptomics of traced epidermal and hair follicle stem cells reveals rapid adaptations during wound healing.
        Cell Rep. 2018; 25: 585-597.e7
        • Laato M.
        • Heino J.
        • Gerdin B.
        • Kähäri V.M.
        • Niinikoski J.
        Interferon-gamma-induced inhibition of wound healing in vivo and in vitro.
        Ann Chir Gynaecol. 2001; 90: 19-23
        • Landén N.X.
        • Li D.
        • Ståhle M.
        Transition from inflammation to proliferation: a critical step during wound healing.
        Cell Mol Life Sci. 2016; 73: 3861-3885
        • Li A.
        • Pouliot N.
        • Redvers R.
        • Kaur P.
        Extensive tissue-regenerative capacity of neonatal human keratinocyte stem cells and their progeny.
        J Clin Invest. 2004; 113: 390-400
        • Li A.
        • Simmons P.J.
        • Kaur P.
        Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype.
        Proc Natl Acad Sci USA. 1998; 95: 3902-3907
        • Lindley L.E.
        • Stojadinovic O.
        • Pastar I.
        • Tomic-Canic M.
        Biology and biomarkers for wound healing.
        Plast Reconstr Surg. 2016; 138: 18S-28S
        • MacLeod A.S.
        • Mansbridge J.N.
        The innate immune system in acute and chronic wounds.
        Adv Wound Care (New Rochelle). 2016; 5: 65-78
        • Macosko E.Z.
        • Basu A.
        • Satija R.
        • Nemesh J.
        • Shekhar K.
        • Goldman M.
        • et al.
        Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets.
        Cell. 2015; 161: 1202-1214
        • Méhul B.
        • Bernard D.
        • Schmidt R.
        Calmodulin-like skin protein: a new marker of keratinocyte differentiation.
        J Invest Dermatol. 2001; 116: 905-909
        • Ogawa E.
        • Owada Y.
        • Ikawa S.
        • Adachi Y.
        • Egawa T.
        • Nemoto K.
        • et al.
        Epidermal FABP (FABP5) regulates keratinocyte differentiation by 13(S)-HODE-mediated activation of the NF-κB signaling pathway.
        J Invest Dermatol. 2011; 131: 604-612
        • Philippeos C.
        • Telerman S.B.
        • Oulès B.
        • Pisco A.O.
        • Shaw T.J.
        • Elgueta R.
        • et al.
        Spatial and single-cell transcriptional profiling identifies functionally distinct human dermal fibroblast subpopulations.
        J Invest Dermatol. 2018; 138: 811-825
        • Picelli S.
        • Björklund A.K.
        • Reinius B.
        • Sagasser S.
        • Winberg G.
        • Sandberg R.
        Tn5 transposase and tagmentation procedures for massively scaled sequencing projects.
        Genome Res. 2014; 24: 2033-2040
        • Raetz J.G.
        • Wick K.H.
        Common questions about pressure ulcers.
        Am Fam Physician. 2015; 92: 888-894
        • Reinisch W.
        • de Villiers W.
        • Bene L.
        • Simon L.
        • Rácz I.
        • Katz S.
        • et al.
        Fontolizumab in moderate to severe Crohn's disease: a phase 2, randomized, double-blind, placebo-controlled, multiple-dose study.
        Inflamm Bowel Dis. 2010; 16: 233-242
        • Skov L.
        • Baadsgaard O.
        MHC class II+ keratinocytes from IFN gamma-treated human skin activate T cells in the presence of staphylococcal superantigen despite UVB irradiation.
        Arch Dermatol Res. 1996; 288: 255-257
        • Tabib T.
        • Morse C.
        • Wang T.
        • Chen W.
        • Lafyatis R.
        SFRP2/DPP4 and FMO1/LSP1 define major fibroblast populations in human skin [published correction appears in J Invest Dermatol 2018;138:2086].
        J Invest Dermatol. 2018; 138 (802–10)
        • Takagi A.
        • Nishiyama C.
        • Kanada S.
        • Niwa Y.
        • Fukuyama K.
        • Ikeda S.
        • et al.
        Prolonged MHC class II expression and CIITA transcription in human keratinocytes.
        Biochem Biophys Res Commun. 2006; 347: 388-393
        • Tamoutounour S.
        • Han S.J.
        • Deckers J.
        • Constantinides M.G.
        • Hurabielle C.
        • Harrison O.J.
        • et al.
        Keratinocyte-intrinsic MHCII expression controls microbiota-induced Th1 cell responses.
        Proc Natl Acad Sci USA. 2019; 116: 23643-23652
        • Theil E.C.
        Ferritin: the protein nanocage and iron biomineral in health and in disease.
        Inorg Chem. 2013; 52: 12223-12233
        • Theocharidis G.
        • Baltzis D.
        • Roustit M.
        • Tellechea A.
        • Dangwal S.
        • Khetani R.S.
        • et al.
        Integrated skin transcriptomics and serum multiplex assays reveal novel mechanisms of wound healing in diabetic foot ulcers.
        Diabetes. 2020; 69: 2157-2169
        • Tirosh I.
        • Izar B.
        • Prakadan S.M.
        • Wadsworth 2nd, M.H.
        • Treacy D.
        • Trombetta J.J.
        • et al.
        Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq.
        Science. 2016; 352: 189-196
        • Toulon A.
        • Breton L.
        • Taylor K.R.
        • Tenenhaus M.
        • Bhavsar D.
        • Lanigan C.
        • et al.
        A role for human skin-resident T cells in wound healing.
        J Exp Med. 2009; 206: 743-750
        • Vachiramon V.
        • Thadanipon K.
        Postinflammatory hypopigmentation.
        Clin Exp Dermatol. 2011; 36: 708-714
        • Volk S.W.
        • Bohling M.W.
        Comparative wound healing--are the small animal veterinarian's clinical patients an improved translational model for human wound healing research?.
        Wound Repair Regen. 2013; 21: 372-381
        • Wang S.
        • Drummond M.L.
        • Guerrero-Juarez C.F.
        • Tarapore E.
        • MacLean A.L.
        • Stabell A.R.
        • et al.
        Single cell transcriptomics of human epidermis identifies basal stem cell transition states.
        Nat Commun. 2020; 11: 4239
        • Wang X.
        • He Y.
        • Zhang Q.
        • Ren X.
        • Zhang Z.
        Direct comparative analyses of 10X Genomics chromium and Smart-seq2 [e-pub ahead of print].
        Genomics Proteomics Bioinformatics. 2021; (accessed June 11, 2021)https://doi.org/10.1016/j.gpb.2020.02.005
        • Wraight C.J.
        • Edmondson S.R.
        • Fortune D.W.
        • Varigos G.
        • Werther G.A.
        Expression of insulin-like growth factor binding protein-3 (IGFBP-3) in the psoriatic lesion.
        J Invest Dermatol. 1997; 108: 452-456
        • Yang J.
        • Sun L.
        • Han J.
        • Zheng W.
        • Peng W.
        DUSP1/MKP-1 regulates proliferation and apoptosis in keratinocytes through the ERK/Elk-1/Egr-1 signaling pathway.
        Life Sci. 2019; 223: 47-53