Single‐cell transcriptomics reveals a senescence‐associated IL ‐6/ CCR6 axis driving radiodermatitis

Article4 July 2022Open Access Transparent process Single-cell transcriptomics reveals a senescence-associated IL-6/CCR6 axis driving radiodermatitis Mor Paldor Mor Paldor orcid.org/0000-0001-7210-1351 The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: Conceptualization, ​Investigation, Writing - original draft Search for more papers by this author Orr Levkovitch-Siany Orr Levkovitch-Siany orcid.org/0000-0002-3798-2576 The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: ​Investigation, Methodology Search for more papers by this author Dana Eidelshtein Dana Eidelshtein The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: ​Investigation Search for more papers by this author Revital Adar Revital Adar The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: ​Investigation Search for more papers by this author Claes D Enk Claes D Enk Department of Dermatology, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: Conceptualization, Writing - review & editing Search for more papers by this author Yitzhak Marmary Yitzhak Marmary The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: Conceptualization, Writing - review & editing Search for more papers by this author Sharona Elgavish Sharona Elgavish Info-CORE, Bioinformatics Unit of the I-CORE, Hebrew University of Jerusalem, Jerusalem, Israel Contribution: Formal analysis, Writing - review & editing Search for more papers by this author Yuval Nevo Yuval Nevo Info-CORE, Bioinformatics Unit of the I-CORE, Hebrew University of Jerusalem, Jerusalem, Israel Contribution: Data curation, Formal analysis Search for more papers by this author Hadar Benyamini Hadar Benyamini Info-CORE, Bioinformatics Unit of the I-CORE, Hebrew University of Jerusalem, Jerusalem, Israel Contribution: Formal analysis Search for more papers by this author Inbar Plaschkes Inbar Plaschkes Info-CORE, Bioinformatics Unit of the I-CORE, Hebrew University of Jerusalem, Jerusalem, Israel Contribution: Formal analysis Search for more papers by this author Shiri Klein Shiri Klein The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: Conceptualization Search for more papers by this author Alex Mali Alex Mali Department of Pathology, Hadassah Hebrew University Hospital, Jerusalem, Israel Search for more papers by this author Stefan Rose-John Stefan Rose-John orcid.org/0000-0002-7519-3279 Institut für Biochemie, Christian-Albrechts-Universität zu Kiel, Kiel, Germany Contribution: Conceptualization, Resources, Writing - review & editing Search for more papers by this author Amnon Peled Amnon Peled The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: Conceptualization, Resources, Writing - review & editing Search for more papers by this author Eithan Galun Corresponding Author Eithan Galun [email protected] orcid.org/0000-0002-6243-6702 The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: Conceptualization, Supervision, Funding acquisition, Writing - review & editing Search for more papers by this author Jonathan H Axelrod Corresponding Author Jonathan H Axelrod [email protected] orcid.org/0000-0002-8996-3916 The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, ​Investigation, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Mor Paldor Mor Paldor orcid.org/0000-0001-7210-1351 The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: Conceptualization, ​Investigation, Writing - original draft Search for more papers by this author Orr Levkovitch-Siany Orr Levkovitch-Siany orcid.org/0000-0002-3798-2576 The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: ​Investigation, Methodology Search for more papers by this author Dana Eidelshtein Dana Eidelshtein The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: ​Investigation Search for more papers by this author Revital Adar Revital Adar The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: ​Investigation Search for more papers by this author Claes D Enk Claes D Enk Department of Dermatology, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: Conceptualization, Writing - review & editing Search for more papers by this author Yitzhak Marmary Yitzhak Marmary The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: Conceptualization, Writing - review & editing Search for more papers by this author Sharona Elgavish Sharona Elgavish Info-CORE, Bioinformatics Unit of the I-CORE, Hebrew University of Jerusalem, Jerusalem, Israel Contribution: Formal analysis, Writing - review & editing Search for more papers by this author Yuval Nevo Yuval Nevo Info-CORE, Bioinformatics Unit of the I-CORE, Hebrew University of Jerusalem, Jerusalem, Israel Contribution: Data curation, Formal analysis Search for more papers by this author Hadar Benyamini Hadar Benyamini Info-CORE, Bioinformatics Unit of the I-CORE, Hebrew University of Jerusalem, Jerusalem, Israel Contribution: Formal analysis Search for more papers by this author Inbar Plaschkes Inbar Plaschkes Info-CORE, Bioinformatics Unit of the I-CORE, Hebrew University of Jerusalem, Jerusalem, Israel Contribution: Formal analysis Search for more papers by this author Shiri Klein Shiri Klein The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: Conceptualization Search for more papers by this author Alex Mali Alex Mali Department of Pathology, Hadassah Hebrew University Hospital, Jerusalem, Israel Search for more papers by this author Stefan Rose-John Stefan Rose-John orcid.org/0000-0002-7519-3279 Institut für Biochemie, Christian-Albrechts-Universität zu Kiel, Kiel, Germany Contribution: Conceptualization, Resources, Writing - review & editing Search for more papers by this author Amnon Peled Amnon Peled The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: Conceptualization, Resources, Writing - review & editing Search for more papers by this author Eithan Galun Corresponding Author Eithan Galun [email protected] orcid.org/0000-0002-6243-6702 The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: Conceptualization, Supervision, Funding acquisition, Writing - review & editing Search for more papers by this author Jonathan H Axelrod Corresponding Author Jonathan H Axelrod [email protected] orcid.org/0000-0002-8996-3916 The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, ​Investigation, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Mor Paldor1, Orr Levkovitch-Siany1, Dana Eidelshtein1, Revital Adar1, Claes D Enk2, Yitzhak Marmary1, Sharona Elgavish3, Yuval Nevo3, Hadar Benyamini3, Inbar Plaschkes3, Shiri Klein1, Alex Mali4, Stefan Rose-John5, Amnon Peled1, Eithan Galun *,1,† and Jonathan H Axelrod *,1,† 1The Goldyne-Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel 2Department of Dermatology, Hadassah Hebrew University Hospital, Jerusalem, Israel 3Info-CORE, Bioinformatics Unit of the I-CORE, Hebrew University of Jerusalem, Jerusalem, Israel 4Department of Pathology, Hadassah Hebrew University Hospital, Jerusalem, Israel 5Institut für Biochemie, Christian-Albrechts-Universität zu Kiel, Kiel, Germany † These authors contributed equally to this work as senior authors *Corresponding author. Tel: +972 50 7874395; E-mail: [email protected] author. Tel: +972 52 4369425; E-mail: [email protected] EMBO Mol Med (2022)14:e15653https://doi.org/10.15252/emmm.202115653 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Irradiation-induced alopecia and dermatitis (IRIAD) are two of the most visually recognized complications of radiotherapy, of which the molecular and cellular basis remains largely unclear. By combining scRNA-seq analysis of whole skin-derived irradiated cells with genetic ablation and molecular inhibition studies, we show that senescence-associated IL-6 and IL-1 signaling, together with IL-17 upregulation and CCR6+-mediated immune cell migration, are crucial drivers of IRIAD. Bioinformatics analysis colocalized irradiation-induced IL-6 signaling with senescence pathway upregulation largely within epidermal hair follicles, basal keratinocytes, and dermal fibroblasts. Loss of cytokine signaling by genetic ablation in IL-6−/− or IL-1R−/− mice, or by molecular blockade, strongly ameliorated IRIAD, as did deficiency of CCL20/CCR6-mediated immune cell migration in CCR6−/− mice. Moreover, IL-6 deficiency strongly reduced IL-17, IL-22, CCL20, and CCR6 upregulation, whereas CCR6 deficiency reciprocally diminished IL-6, IL-17, CCL3, and MHC upregulation, suggesting that proximity-dependent cellular cross talk promotes IRIAD. Therapeutically, topical application of Janus kinase blockers or inhibition of T-cell activation by cyclosporine effectively reduced IRIAD, suggesting the potential of targeted approaches for the treatment of dermal side effects in radiotherapy patients. Synopsis Irradiation-induced alopecia and dermatitis (IRIAD) represent two of the most prominent side effects of radiotherapy in the skin. This study utilized scRNA-seq analysis to identify driving factors of IRIAD in order to facilitate development of targeted therapeutic approaches for its prevention. IRIAD in mice is strongly associated with upregulation of senescence pathways involving IL-6 and IL-1 signaling, together with IL-17 signaling. Loss of IL-6 and IL-1 signaling, or of CCL20/CCR6-mediated immune cell migration strongly ameliorated IRIAD, while simultaneously reducing irradiation-induced cellular senescence. IL-6-deficiency strongly reduced IL-17, CCL20, and CCR6 upregulation, while loss of CCR6-mediated immune cell migration reciprocally diminished IL-6 and IL-17 upregulation, and loss of immune privilege. IRIAD is effectively ameliorated by treatment with Janus kinases blockers or cyclosporine A. The paper explained Problem Radiotherapy-treated patients with cancer frequently suffer from side effects including irradiation-induced alopecia and dermatitis (IRIAD). In addition to psychological stress, these side effects can also generate severe chronic radiation injury, including the delayed formation of ulcers, fibrosis, telangiectasia, and opiate-resistant chronic pain. Moreover, the serious complications in wound healing of the skin following radiotherapy constitute a major impediment to reconstructive surgery. The mechanism(s) underlying IRIAD remain poorly understood, thus limiting the development of effective rationally targeted therapies. Results scRNA-seq analysis of an IRIAD model in mice reveals pivotal cellular and molecular cross-interactions involving inflammatory and senescence-associated cytokine signaling pathways, including IL-6, IL-1, and type 17-mediated cytokines (IL-17, IL-22). Genetic or pharmacological blockade of these pathways strongly reduced major effects of irradiation, including acanthosis, alopecia, and senescence. Moreover, IL-6 signaling blockade prevented IL-17 IL-22, CCL20, and CCR6 upregulation, and the infiltration of CD3+ T cells to degenerating hair follicles, and, vis-a-versa; IR-induced IL-6 upregulation was strongly dependent upon IL-17 upregulation, CCR6-dependent immune cell infiltration upon loss of HF immune privilege. Moreover, inhibition of T-cell activation by treatment with cyclosporine A, or inhibition of STAT3-mediated signaling by topical application of pharmacological inhibitors of Janus kinases also effectively prevented IRIAD. Impact The findings of this study point to the clinical potential of cytokine, chemokine, and T cell-targeted therapeutics for the prevention of IRIAD in radiotherapy patients, in particular via topical application of low-molecular-weight pharmaceuticals. Introduction Irradiation-induced alopecia (hair loss) and radiodermatitis (IRIAD) are two of the most common and psychologically stressful side effects in radiotherapy patients (Lawenda et al, 2004; Hymes et al, 2006; Ryan, 2012). Radiodermatitis occurs in about 95% of radiotherapy patients and ranges in severity from mild erythema to moist desquamation and ulceration (Hymes et al, 2006; Ryan, 2012). Late or chronic irradiation (IR)-induced injury, including the delayed formation of ulcers, fibrosis, telangiectasias, and opiate-resistant chronic pain, can also spontaneously appear months and even years after IR exposure (Hymes et al, 2006; Brown & Rzucidlo, 2011; Ryan, 2012). Moreover, problematic wound healing in irradiated skin constitutes a major surgical impediment and remains a substantial therapeutic challenge (Gieringer et al, 2011; Haubner et al, 2012). Despite recent improvements in skin-sparing radiation technology, such as intensity-modulated radiation therapy, alopecia, and injury to the skin remain markedly problematic (Salvo et al, 2010; McQuestion, 2011). Surprisingly, although the effects of irradiation on the skin and hair follicles have been known for more than a century, their underlying molecular and cellular mechanism(s) remain poorly understood, thus limiting the development of effective, rationally targeted therapies (Malkinson & Keane, 1981; Hymes et al, 2006; Ryan, 2012). The cellular events encompassing IRIAD have been described in considerable depth (Ryan, 2012). Immediate damage to hair follicle stem cells and basal keratinocytes that follows the burst of IR-released free radicals leading to persistent double-stranded DNA breaks is largely thought to initiate the inflammation-mediated process of IR-induced skin injury (Ryan, 2012). Subsequent infiltration of immune cells, including neutrophils, macrophages, and T cells to irradiated skin, and hyperkeratosis constitute commonly recognized hallmarks of acute IR-induced skin injury (Muller & Meineke, 2007; Holler et al, 2009; Ryan, 2012). Nevertheless, much of the cellular and molecular interactions leading to IRIAD remain unclear. In consideration of the given complexity of the skin, involving numerous cell types, we have applied quantitative single-cell RNA sequence (scRNA-seq) analysis to an established IRIAD model in mice (Vegesna et al, 1988; Burdelya et al, 2012; Marmary et al, 2016) in order to elucidate molecular and cellular events that drive IRIAD. Results A single-cell transcriptome atlas of irradiated skin Irradiation (15 Gy) of mice (C57BL/6) to the head and neck generated initial external signs of skin injury with the appearance of serous exudates on the chin and throat beginning at about 8 days post-IR that progressed in severity with time and spread upward to the cheeks and downward to include the chest. Radiodermatitis reached maximal levels about 2 weeks post-IR and subsided thereafter (Fig 1A). The appearance of alopecia followed that of radiodermatitis, reaching maximal levels about 3–4 weeks post-IR, and hair growth returned to roughly normal levels by weeks 7–8, but with striking loss of hair color (depigmentation), as reported previously (Inomata et al, 2009; Fig 1A and Appendix Fig S1A). A similar outcome resulted following exposure of mice to 30 Gy irradiation administered in fractionated doses over 5 consecutive days (5 × 6 Gy; Appendix Fig S1B). Importantly, different areas of skin displayed varying degrees of irradiation sensitivity, with the ventral neck, the chest, and the muzzle exhibiting the highest sensitivity levels, thus appearing to closely recapitulate the presentation in human radiotherapy patients (Lawenda et al, 2004; Hymes et al, 2006; Brown & Rzucidlo, 2011). Histological analysis of H&E stained skin thin sections from mice 2–3 weeks post-IR revealed striking morphological changes in both the epidermal and dermal layers, which included keratinocyte hyperplasia, and acanthosis involving a fourfold thickening of the epidermis, capillary stasis, edema, and infiltration of immune cells (Fig 1A and Appendix Fig S1C). Figure 1. scRNA-seq analysis reveals heterogeneity in radiodermatitis A. Schematic of scRNA-seq experimental workflow. B. UMAP projection of 8,805 cells from irradiated and naïve mouse skin integrated into 21 clusters. Cells are colored by the assigned cluster (middle). (Right) UMAP projection showing cluster composition according to cell origin in naïve or irradiated skin. C. Naïve expression of the hair follicle (Ptn), IFE Basal (Krt14), IFE Differentiated (Krt10), fibroblast (Col3a1), and immune T cell (Cd3d) transcripts visualized by UMAP across scRNA-seq datasets from integrated naïve and irradiated skin. D. Dot plot depicting integrated naïve-irradiated clusters according to skin cell-type-specific marker expression in the naïve cells. Data information: See also Fig EV1 and Appendix Figs S1–S6. Download figure Download PowerPoint To dissect the cellular and molecular events in IRIAD, we performed scRNA-seq analysis on unsorted living cells isolated separately from the whole skin of either naïve or irradiated (15 Gy) mice taken 14 days post-IR (Fig 1A). Dissected skin samples were separated into epidermal and dermal layers prior to enzymatic dissociation and treated with a cocktail of protease inhibitors upon termination to enhance cell viability. Viable dissociated cell populations were then pooled and characterized using the 10× Genomics® platform for random capture and 3′ sequencing analysis of cDNAs. In total, 8,805 sequenced skin cells (n = 4 biological replicates per experimental group) met our quality control and inclusion criterion. Using the 10× Genomics platform, we obtained about 5,135 and 3,670 transcriptomes for naïve and irradiated skin cells, respectively, totaling about 25,000 detected genes. Cells with less than 200 expressed genes or more than 5% mitochondrial gene expression were excluded. Unsupervised cluster analysis using the Seurat software package (Stuart et al, 2019) segregated the naïve and irradiated skin cells into 21 distinct clusters which were overlaid and visualized in two-dimensional space by Uniform Manifold Approximation and Projection (UMAP; Becht et al, 2018; Fig 1B and C). Cell clusters were classified according to cell type and lineage within naïve cell populations by comparison with previously reported and validated scRNA-seq datasets for epidermal (Joost et al, 2016) and dermal (Guerrero-Juarez et al, 2019) cell populations using GSEA analysis. Immune and stromal cell populations were further classified by comparison with community-based immune-related gene sets of the Immunological Genome Project (Heng et al, 2008). In this way, we assigned clusters to epidermal or dermal/stromal layers and identified specific cell types, including immune cells. Epidermal-derived clusters, identified by gene expression patterns and GSEA, included hair follicle (HF) inner bulge cells clustered together with outer bulge (OB/IB, Cl-11) cells expressing Lgr5/Lgr6/Postn/Ptn; upper hair follicle (uHF) I/III and infundibulum B (INFU B) cells showing a likeness to OB cells (uHF I/INFU, Cl-8) and expressing Postn/Sostdc1/Aqp3; uHF II with uHF V (uHF II/V; Cl-9) cells expressing Krt79/Defb6/Krt17/Cst6; and sebaceous gland (SG, Cl-6) cells expressing marker genes Cers4/Cidea/Soat1/Zfp655 (Joost et al, 2016; Fig 1C and D, and Appendix Fig S2). Cluster analysis by GSEA also identified epidermal cells divided into three distinct interfollicular epidermis basal (IFE-B) cell clusters (IFE-B, Cl-2; IFE-BII, Cl-0; and IFE-BI, Cl-3) characterized by Krt5/Krt14/Krt15/Cxcl14/Mt2/Bhlhe40 and distinguished between themselves by Ptn/Pde4b/Krt16 expression (Joost et al, 2016); and three distinct clusters of differentiated or keratinized IFE cells (IFE-DI, Cl-1; IFE-DI/KII, Cl-14; and IFE-KII, Cl-4) expressing Krt1/krt10/Krt77/Ptsg1/Mt4. Two distinct clusters representing stromal fibroblast (FIB) populations were also identified: FIB-III/I (Cl-5) expressing Col1a1/Col3a1/CD34/Dcn, with strong similarities to FIB types 1, 2, and 3 as defined previously (Guerrero-Juarez et al, 2019); and FIB (Cl-10) expressing Spon1/Serpine2, with remote resemblances to FIB types 1, 2, and 3 and also sharing similarities with skin lymph node reticular fibroblasts (Fig 1D, and Appendix Figs S2 and S3). GSEA and ImmGen database comparisons identified four cutaneous stromal endothelial clusters expressing Pecam1/Cdh5/Cav1/Cald1 that included blood endothelial cells (BEC, Cl-17), lymphatic endothelial cells (LEC, Cl-16), and two small, endothelial-related clusters with similarities to stromal double-negative cells (DN, Cl-13, and EC, Cl-19; Link et al, 2007; Fig 1C and D, and Appendix Fig S3). Four distinct immune cell populations, including dendritic cells (Cl-12; Lyz2/Ccl9/Ctss/Cd74), Langerhans cells (Cl-18; Cd74, Ctss, Cd86, Cd207), resident αβT cells that appeared to cluster together with innate lymphoid cells (αβT/ILC, Cl-7; Cd3d, Ctla4), and γδT cells (Cl-15; Cd3d, Cd3e, Trdc, Ctla2a) were identified by comparisons using GSEA or ImmGen databases (Fig 1C and D, and Appendix Fig S3). Further analysis of naïve αβT/ILC (CL-7) cells for individual marker genes indicated that most cells in the cluster expressed at least one Cd3 allele (d,e,g), Cd28, and/or Trac, suggesting an adaptive T-cell lineage. However, about 10% (5/46) of the cells in the cluster expressed ILC-associated genes (Klrk1, Runx, or Fuca1; Koues et al, 2016) and were negative for T-cell markers, thus suggesting that these cells may represent ILCs (ILC1, ILC3, or NK cells; Appendix Fig S4). Additionally, 10% (4/46) of naive αβT/ILC cells were identified as Foxp3+ Tregs (Appendix Fig S4). Dendritic cells of Cl-12 appeared largely of cDC2 (Itgam, Sirpa) lineage that cluster together with monocyte-derived macrophages (Adgre1, C1qc, Mafb; Guilliams et al, 2014; Saba et al, 2022; Appendix Fig S4). A comparison of the effect of irradiation on cluster populations revealed substantial IR-induced changes in cell numbers within specific clusters. These included striking relative increases (> 4-fold) in populations of IFE-B and SG cells, and in two immune clusters, αβT/ILC cells and dendritic cells (Appendix Table S1). Relative increased population sizes in the epidermal clusters aligned with the upregulation of cell cycle markers, Ki67 (Mki67), and geminin (Gmnn), indicating a strong mitotic response within these HF clusters drives the keratinocyte hyperplasia observed in H&E thin sections of irradiated skin (Appendix Fig S5). In contrast, relatively few αβT/ILC cells showed upregulation of cell cycle markers, suggesting that infiltration may largely contribute to their increased number. Additionally, following irradiation almost all αβT/ILC cells express one or more of the T-cell marker genes, substantially outnumbering the ILC population (Appendix Fig S4). FoxP3+ Treg cell numbers remained relatively stable but were reduced to about 1% (5/365) of the cluster against the accumulating T-helper population (Appendix Fig S4). The composition of cluster-12 (dendritic cells) also appeared to shift following irradiation showing higher numbers of Adgre1, C1qc, Mafb, and Ly6c2 expressing cells that overlap with higher numbers of cells expressing Itgam and Sirpa, perhaps suggesting an influx of monocyte-derived macrophages and dendritic cells in addition to resident cDC2 cells (Appendix Fig S4). In addition, two novel minor cell clusters representing innate NK/ILCs immune cells (Cl-20) and granulocytes (Cl-21) appeared exclusively following irradiation (Appendix Fig S3). At the same time, notable reductions (> 2-fold) were evident in epidermal OB/IB, IFE-BII, and IFE-DI cells population sizes, as well as in both stromal fibroblast clusters, whereas other cluster populations remained proportionally unchanged. Bioinformatics comparison of irradiated versus naïve cell clusters using QIAGEN Ingenuity Pathway Analysis (IPA) revealed multiple significant physiological and molecular pathways that characterize the cellular responses to IR. Among the most prominently ranked pathways (i.e. pathways with the highest significance: −log(B-H P-value) > 1.3, and z-scores > 2 or < −2) appearing across both epidermal and dermal cell clusters were pathways associated with aging-related functions, including Mitochondrial Dysfunction, Senescence Pathways upregulation, and senescence-related Sirtuin Signaling downregulation (Lee et al, 2019; Table EV1). Together with the senescence-associated pathways, IPA also revealed strong upregulation of multiple cytokine signaling pathways, including both IL-6 and IL-1 signaling, as reflected in their positive IPA z-score values. Notably, upregulation of IL-6 and IL-1 signaling pathways, which are essential cytokines contributing to the senescence-associated secretory phenotype (SASP; He & Sharpless, 2017), conspicuously overlapped within irradiated epidermal and dermal clusters that also displayed highly significant upregulation in Senescence Pathways (Table EV1). Curiously, we also noted that, although not conventionally thought to be associated with senescence, IPA reported significant upregulation of IL-17 Signaling, reminiscent of IL-17A Signaling in Airway Cells, Th17 Activation, and the Role of IL-17A in Psoriasis (Table EV1) that appeared to parallel those of IL-6 signaling and senescence pathways in many clusters. Interrogation of the scRNA-seq datasets by CellPhoneDB analysis (Efremova et al, 2020) concurred with the IPA findings showing significantly increased potential ligand–receptor interactions in irradiated skin (Appendix Fig S6). Thus, irradiation increased the levels of multiple ligand–receptor interactions that spanned across multiple cell clusters, particularly within FIB-III/I, BEC, IFE-B, and uHF-I/INFU-B cells, and, as described below in detail, included those involving IL-6, IL-1, IL-17, and their respective cognate receptors (Appendix Fig S6). To localize senescence pathway upregulation within the skin scRNA-seq cell clusters, we employed an arbitrary "Senescence Score" based on the combined relative expression of select IPA designated senescence-related genes (Kras, Mtor, Smad1, Smad3, Il6, Il1a, Plaur, Serpine1, Igfbp2, Tgfb1, Tgfbr1, Ccl2, Tmem173, Mb21d1) to stain the clusters (Fig EV1A and B). This depiction suggested that cellular senescence, which in naive skin is associated with roughly half of the FIB-III/I and FIB dermal fibroblasts, expands following irradiation to include nearly all FIB-III/I fibroblasts, and many HF cells, including some OB/IB, uHF/INFU-B, and SG cells and most basal keratinocytes (IFE-B, -BI, -BII), largely in agreement with the IPA analysis (Table EV1). Click here to expand this figure. Figure EV1. (Related to Fig 1). Relative mutually exclusive senescence and cell cycle scoring in naïve and irradiated epidermal cells A. Relative expression and co-localization of Senescence Score (red) and Cell Cycle Score (Mki67, Gmnn, Ccna1; green) in integrated skin cells by blended UMAPs. Blowup: Regions of IEF-2 and uHF-I/INFU-B showing exclusive mitotic activity (outlined in blue). B. Dot plot depicting the relative expression of selected IPA-defined senescence-related markers and cell cycle markers (framed in red) (x-axis) in naïve (blue dots) and irradiated (red dots) scRNA-seq skin cell clusters. C. UMAP plots depicting relative expression and localization of urokinase-type plasminogen activator receptor (Plaur) mRNA in irradiated and naïve skin-derived clusters. D. Immunostaining and histochemical staining (brown) of urokinase-type plasminogen activator receptor (uPAR), p16INK4a, and lipofuscin (SenTraGor®) in paraffin-embedded skin thin sections from ventral neck and upper chest region of naïve and irradiated (15 Gy) wild type mice taken at 21 days post-IR. p16INK4a immunostaining appears as nuclear and/or cytoplasmic staining (Arrows) in IFE keratinocytes, in cells comprising discrete hair follicle layers, and in distinct cells in the dermis. Scale bars, 20 μm. Download figure Download PowerPoint Expression of Plaur mRNA, which encodes the urokinase-type plasminogen activator receptor (uPAR), a protein that is broadly induced on the surface of senescent cells (Amor et al, 2020), mirrored the increased senescence score, particularly within irradiated HF and basal keratinocytes (uHF/INFU-B, IFE-B) and SG-derived cells (Fig EV1C). Anti-uPAR immunostaining confirmed the substantially increased IR-induced uPAR protein expression in keratinocytes particularly within basal keratinocytes and degenerating hair follicles, but notably less so in