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Abstract Regulatory T cells (Treg) are CD4+ T cells with immune-suppressive function, which is defined by Foxp3 expression. However, the molecular determinants defining the suppressive population of T cells have yet to be discovered. Here we report that the cell surface protein Lrig1 is enriched in suppressive T cells and controls their suppressive behaviors. Within CD4+ T cells, Treg cells express the highest levels of Lrig1, and the expression level is further increasing with activation. The Lrig1+ subpopulation from T helper (Th) 17 cells showed higher suppressive activity than the Lrig1- subpopulation. Lrig1-deficiency impairs the suppressive function of Treg cells, while Lrig1-deficient naïve T cells normally differentiate into other T cell subsets. Adoptive transfer of CD4+Lrig1+ T cells alleviates autoimmune symptoms in colitis and lupus nephritis mouse models. A monoclonal anti-Lrig1 antibody significantly improves the symptoms of experimental autoimmune encephalomyelitis. In conclusion, Lrig1 is an important regulator of suppressive T cell function and an exploitable target for treating autoimmune conditions.

Bardet-Biedl syndrome (BBS) is a ciliopathy with pleiotropic effects on multiple tissues, including the kidney. Here we have compared renal differentiation of iPS cells from healthy and BBS donors. High content image analysis of WT1-expressing kidney progenitors showed that cell proliferation, differentiation and cell shape were similar in healthy, BBS1, BBS2, and BBS10 mutant lines. We then examined three patient lines with BBS10 mutations in a 3D kidney organoid system. The line with the most deleterious mutation, with low BBS10 expression, expressed kidney marker genes but failed to generate 3D organoids. The other two patient lines expressed near normal levels of BBS10 mRNA and generated multiple kidney lineages within organoids when examined at day 20 of organoid differentiation. However, on prolonged culture (day 27) the proximal tubule compartment degenerated. Introducing wild type BBS10 into the most severely affected patient line restored organoid formation, whereas CRISPR-mediated generation of a truncating BBS10 mutation in a healthy line resulted in failure to generate organoids. Our findings provide a basis for further mechanistic studies of the role of BBS10 in the kidney.

Keratinocytes of the mammalian skin provide not only mechanical protection for the tissues, but also transmit mechanical, chemical, and thermal stimuli from the external environment to the sensory nerve terminals. Sensory nerve fibers penetrate the epidermal basement membrane and function in the tight intercellular space among keratinocytes. Here we show that epidermal keratinocytes produce hydrogen peroxide upon the activation of the NADPH oxidase dual oxidase 1 (DUOX1). This enzyme can be activated by increasing cytosolic calcium levels. Using DUOX1 knockout animals as a model system we found an increased sensitivity towards certain noxious stimuli in DUOX1-deficient animals, which is not due to structural changes in the skin as evidenced by detailed immunohistochemical and electron-microscopic analysis of epidermal tissue. We show that DUOX1 is expressed in keratinocytes but not in the neural sensory pathway. The release of hydrogen peroxide by activated DUOX1 alters both the activity of neuronal TRPA1 and redox-sensitive potassium channels expressed in dorsal root ganglia primary sensory neurons. We describe hydrogen peroxide, produced by DUOX1 as a paracrine mediator of nociceptive signal transmission.Our results indicate that a novel, hitherto unknown redox mechanism modulates noxious sensory signals.

Abstract To establish whether 4-nitroquinoline N-oxide-induced carcinogenesis mirrors the heterogeneity of human oral squamous cell carcinoma (OSCC), we have performed genomic analysis of mouse tongue lesions. The mutational signatures of human and mouse OSCC overlap extensively. Mutational burden is higher in moderate dysplasias and invasive SCCs than in hyperplasias and mild dysplasias, although mutations in p53, Notch1 and Fat1 occur in early lesions. Laminin-α3 mutations are associated with tumour invasiveness and Notch1 mutant tumours have an increased immune infiltrate. Computational modelling of clonal dynamics indicates that high genetic heterogeneity may be a feature of those mild dysplasias that are likely to progress to more aggressive tumours. These studies provide a foundation for exploring OSCC evolution, heterogeneity and progression.

Although acne vulgaris is the most common human inflammatory skin disease, its pathogenic mechanisms remain incompletely understood. Here the authors show that GATA6 is involved in maintaining homeostasis of the upper pilosebaceous unit of human skin and may contribute to acne pathogenesis.

Mammalian epidermis is a striking example of the role of lipids in tissue biology. In this stratified epithelium, highly specialized structures are formed that leverage the hydrophobic properties of lipids to form an impermeable barrier and protect the humid internal environment of the body from the dry outside. This is achieved through tightly regulated lipid synthesis that generates the molecular species unique to the tissue. Beyond their fundamental structural role, lipids are involved in the active protection of the body from external insults. Lipid species present on the surface of the body possess antimicrobial activity and directly contribute to shaping the commensal microbiota. Lipids belonging to a variety of classes are also involved in the signaling events that modulate the immune responses to environmental stress as well as differentiation of the epidermal keratinocytes themselves. Recently, high-resolution methods are beginning to provide evidence for the involvement of newly identified specific lipid molecules in the regulation of epidermal homeostasis. In this review we give an overview of the wide range of biological functions of mammalian epidermal lipids.

Cellular quiescence is a dormant but reversible cellular state in which cell-cycle entry and proliferation are prevented. Recent studies both in vivo and in vitro demonstrate that quiescence is actively maintained through synergistic interactions between intrinsic and extrinsic signals. Subtypes of adult mammalian stem cells can be maintained in this poised, quiescent state, and subsequently reactivated upon tissue injury to restore homeostasis. However, quiescence can become deregulated in pathological settings. In this review, we discuss the recent advances uncovering intracellular signaling pathways, transcriptional changes, and extracellular cues within the stem cell niche that control induction and exit from quiescence in tissue stem cells. We discuss the implications of quiescence as well as the pharmacological and genetic approaches that are being explored to either induce or prevent quiescence as a therapeutic strategy. : Stem cell quiescence is a dormant but reversible cellular state in which cell-cycle entry and proliferation are prevented. Intrinsic regulators include the cell-cycle machinery, autophagy, chromatin modifiers, and microRNAs. Extrinsic regulators include cell-adhesion molecules and secreted factors. Therapeutic modulation of stem cell quiescence could restore tissue homeostasis. Keywords: stem cells, cancer, quiescence, cell cycle, autophagy, niche, cell therapy

Frontal fibrosing alopecia (FFA) features lichenoid cutaneous inflammation and scarring hair loss. Here, Tziotzios et al. identify four genetic loci associated with FFA by GWAS followed by Bayesian fine-mapping, co-localisation and HLA imputation which highlights HLA-B*07:02 as a risk factor.

We have examined the developmental origins of Ng2+ perivascular cell populations that adhere to the basement membrane of blood vessels, and their contribution to wound healing. Neural/glial antigen 2 (Ng2) labeled most perivascular cells (70–80%) in developing and adult mouse back skin, a higher proportion than expressed by other pericyte markers Tbx18, Nestin and Pdgfrβ. In adult mouse back skin Ng2+ perivascular cells could be categorized into 4 populations based on whether they expressed Pdgfrα and Pdgfrβ individually or in combination or were Pdgfr-negative. Lineage tracing demonstrated that although Ng2+ cells in embryonic and neonatal back skin contributed to multiple cell types they did not give rise to interfollicular fibroblasts within the dermis. Lineage tracing of distinct fibroblast populations during skin development showed that papillary fibroblasts (Lrig1+) gave rise to Ng2+ perivascular cells in the upper dermis, whilst Ng2+ perivascular cells in the lower dermis were primarily derived from reticular Dlk1+ fibroblasts. Following wounding of adult skin, Ng2+ dermal cells only give rise to Ng2+ blood vessel associated cells and did not contribute to other fibroblast lineages. The relative abundance of Ng2+ Pdgfrβ+ perivascular populations was comparable in wounded and non-wounded skin, indicating that perivascular heterogeneity was maintained during full thickness skin repair. In the wound bed Ng2+ perivascular populations were primarily derived from Lrig1+ papillary or Dlk1+ reticular fibroblast lineages, according to the location of the regenerating blood vessels. We conclude that Ng2+ perivascular cells represent a heterogeneous lineage restricted population that is primarily recruited from the papillary or reticular fibroblast lineages during tissue regeneration.

Although human dermis contains distinct fibroblast subpopulations, the functional heterogeneity of fibroblast lines from different donors is under-appreciated. We identified one commercially sourced fibroblast line (c64a) that failed to express α-smooth muscle actin (α-SMA), a marker linked to fibroblast contractility, even when treated with transforming growth factor-β1 (TGF-β1). Gene expression profiling identified insulin-like growth factor 1 (IGF1) as being expressed more highly, and Asporin (ASPN) and Wnt family member 4 (WNT4) expressed at lower levels, in c64a fibroblasts compared to three fibroblast lines that had been generated in-house, independent of TGF-β1 treatment. TGF-β1 increased expression of C-X-C motif chemokine ligand 1 (CXCL1) in c64a cells to a greater extent than in the other lines. The c64a gene expression profile did not correspond to any dermal fibroblast subpopulation identified by single-cell RNAseq of freshly isolated human skin cells. In skin reconstitution assays, c64a fibroblasts did not support epidermal stratification as effectively as other lines tested. In fibroblast lines generated in-house, shRNA-mediated knockdown of IGF1 increased α-SMA expression without affecting epidermal stratification. Conversely, WNT4 knockdown had no consistent effect on α-SMA expression, but increased the ability of fibroblasts to support epidermal stratification. Thus, by comparing the properties of different lines of cultured dermal fibroblasts, we have identified IGF1 and WNT4 as candidate mediators of two distinct dermal functions: myofibroblast formation and epidermal maintenance.

Keratin 76 (Krt76) is an epithelial differentiation marker that is downregulated in oral squamous cell carcinomas, correlating with poor prognosis. Here the authors show that genetic ablation of Krt76 in a mouse model results in increased susceptibility to carcinogenesis via enhanced accumulation of Tregs.

Abstract Background Canonical Wnt/beta-catenin signalling regulates self-renewal and lineage selection within the mammalian epidermis. Although the transcriptional response of keratinocytes that receive a Wnt signal is well characterized, little is known about the mechanism by which keratinocytes in proximity to the Wnt-receiving cell are co-opted to undergo a change in cell fate. Results To address this, we perform single-cell RNA-sequencing on mouse keratinocytes co-cultured with and without beta-catenin-activated neighbouring cells. We identify five distinct cell states in cultures that had not been exposed to the beta-catenin stimulus and show that the stimulus redistributes wild-type subpopulation proportions. Using temporal single-cell analysis, we reconstruct the cell fate change induced by Wnt activation from neighbouring cells. Gene expression heterogeneity is reduced in neighbouring cells and this effect is most dramatic for protein synthesis-associated genes. Changes in gene expression are accompanied by a shift to a more proliferative stem cell state. By integrating imaging and reconstructed sequential gene expression changes during the state transition we identify transcription factors, including Smad4 and Bcl3, that are responsible for effecting the transition in a contact-dependent manner. Conclusions Our data indicate that non-cell autonomous Wnt/beta-catenin signalling decreases transcriptional heterogeneity. This furthers our understanding of how epidermal Wnt signalling orchestrates regeneration and self-renewal.

Regenerative medicine aims to replace damaged tissues by stimulating endogenous tissue repair or by transplanting autologous or allogeneic cells. Due to their capacity to produce unlimited numbers of cells of a given cell type, pluripotent stem cells, whether of embryonic origin or induced via the reprogramming of somatic cells, are of considerable therapeutic interest in the regenerative medicine field. However, regardless of the cell type, host immune responses present a barrier to success. The aim of this study was to investigate in vitro the immunological properties of human pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs). These cells expressed MHC class I molecules while they lacked MHC class II and co-stimulatory molecules, such as CD80 and CD86. Following stimulation with IFN-γ, HLCs upregulated CD40, PD-L1 and MHC class I molecules. When co-cultured with allogeneic T cells, HLCs did not induce T cell proliferation; furthermore, when T cells were stimulated via αCD3/CD28 beads, HLCs inhibited their proliferation via IDO1 and tryptophan deprivation. These results demonstrate that PSC-derived HLCs possess immunoregulatory functions, at least in vitro.

Abstract The regenerative capacity of adult human tissues and organs is limited, but recent developments have seen the advent of promising new technologies for regenerative therapy. The human heart is of particular interest for regenerative medicine, as cardiac tissue damage is repaired by the formation of rigid scar tissue, which causes inevitable structural changes and progressive functional decline leading to heart failure. Cardiac regenerative medicine aims to prevent scar formation or replace existing scars to halt or reverse adverse remodeling and therapeutic approaches include the use of biomaterials, gene therapies, delivery of growth factors, and (stem) cell therapies. Regenerative therapies, however, face significant obstacles in a hostile microenvironment. While the early immune response to a myocardial infarct is essential to ensure tissue integrity and to avoid fatal cardiac rupture, excessive activation of endogenous repair mechanisms may lead to ongoing inflammation, fibrosis, and sustained autoimmune-mediated tissue damage. Anti-cardiac autoreactivity of the adaptive immune system has been suggested to be involved in structural remodeling, functional decline, and the development of heart failure. It is, therefore, crucial to first understand the endogenous response to cardiac tissue damage and how to restore immune tolerance to cardiac tissue, before additional regenerative therapies can achieve their full potential.

Individual human epidermal cells differ in their self-renewal ability. Here the authors perform genome-wide pooled RNAi screens to uncover the molecular basis for this heterogeneity, and identify genes conferring a clonal growth advantage on normal and neoplastic human epidermal cells.

Summary: Large cohorts of human induced pluripotent stem cells (iPSCs) from healthy donors are a potentially powerful tool for investigating the relationship between genetic variants and cellular behavior. Here, we integrate high content imaging of cell shape, proliferation, and other phenotypes with gene expression and DNA sequence datasets from over 100 human iPSC lines. By applying a dimensionality reduction approach, Probabilistic Estimation of Expression Residuals (PEER), we extracted factors that captured the effects of intrinsic (genetic concordance between different cell lines from the same donor) and extrinsic (cell responses to different fibronectin concentrations) conditions. We identify genes that correlate in expression with intrinsic and extrinsic PEER factors and associate outlier cell behavior with genes containing rare deleterious non-synonymous SNVs. Our study, thus, establishes a strategy for examining the genetic basis of inter-individual variability in cell behavior. : Cell behavior reflects both the intrinsic state of the cell and extrinsic signals it receives from its microenvironment. By integrating genomic, gene expression, and cell biology datasets from a large number of human iPSCs from healthy donors, Vigilante et al. show how genetic variation contributes to phenotypic variation. Keywords: iPSC, SNV, genetic variation, high content imaging, stem cells, dimensionality reduction, fibronectin, stem cell niche, cell adhesion

The molecular mechanisms regulating skin dermal changes are unclear. Here, the authors show that deletion of Hedgehog (Hh) in the upper dermis alters the response to epidermal Wnt signalling, which, together with changes in extracellular matrix production, influences distinct fibroblast lineages differently.

Wnt/β-catenin activation in adult epidermis can induce new hair follicle formation and tumor development. We used lineage tracing to uncover the relative contribution of different stem cell populations. LGR6+ and LRIG1+ stem cells contributed to ectopic hair follicles formed in the sebaceous gland upon β-catenin activation, whereas LGR5+ cells did not. Lgr6, but not Lrig1 or Lgr5, was expressed in a subpopulation of interfollicular epidermal cells that were competent to form new hair follicles. Oncogenic β-catenin expression in LGR5+ cells led to formation of pilomatricomas, while LRIG1+ cells formed trichoadenomas and LGR6+ cells formed dermatofibromas. Tumor formation was always accompanied by a local increase in dermal fibroblast density and transient extracellular matrix remodeling. However, each tumor had a distinct stromal signature in terms of immune cell infiltrate and expression of CD26 and CD44. We conclude that compartmentalization of epidermal stem cells underlies different responses to β-catenin and skin tumor heterogeneity.

Summary: Metastatic colonization of distant organs underpins the majority of human-cancer-related deaths, including deaths from head and neck squamous cell carcinoma (HNSCC). We report that miR-203, a miRNA that triggers differentiation in multilayered epithelia, inhibits multiple postextravasation events during HNSCC lung metastasis. Inducible reactivation of miR-203 in already established lung metastases reduces the overall metastatic burden. Using an integrated approach, we reveal that miR-203 inhibits metastasis independently of its effects on differentiation. In vivo genetic reconstitution experiments show that miR-203 inhibits lung metastasis by suppressing the prometastatic activities of three factors involved in cytoskeletal dynamics (LASP1), extracellular matrix remodeling (SPARC), and cell metabolism (NUAK1). Expression of miR-203 and its downstream effectors correlates with HNSCC overall survival outcomes, indicating the therapeutic potential of targeting this signaling axis. : Benaich et al. have identified miR-203, a microRNA that triggers differentiation in multilayered epithelia, as an inhibitor of lung metastasis in head and neck squamous cell carcinoma (HNSCC) cells. They show that miR-203 inhibits metastasis independently of its effects on differentiation. Rather, miR-203 suppresses the prometastatic activities of three factors involved in cytoskeletal dynamics (LASP1), extracellular matrix remodeling (SPARC), and cell metabolism (NUAK1). Expression of miR-203 and its downstream effectors correlates with survival in HNSCC patients.

B-lymphocyte-induced nuclear maturation protein 1 (BLIMP1) was previously reported to define a sebaceous gland (SG) progenitor population in the epidermis. However, the recent identification of multiple stem cell populations in the hair follicle junctional zone has led us to re-evaluate its function. We show, in agreement with previous studies, that BLIMP1 is expressed by postmitotic, terminally differentiated epidermal cells within the SG, interfollicular epidermis, and hair follicle. Epidermal overexpression of c-Myc results in loss of BLIMP1+ cells, an effect modulated by androgen signaling. Epidermal-specific deletion of Blimp1 causes multiple differentiation defects in the epidermis in addition to SG enlargement. In culture, BLIMP1+ sebocytes have no greater clonogenic potential than BLIMP1− sebocytes. Finally, lineage-tracing experiments reveal that, under steady-state conditions, BLIMP1-expressing cells do not divide. Thus, rather than defining a sebocyte progenitor population, BLIMP1 functions in terminally differentiated cells to maintain homeostasis in multiple epidermal compartments.

Although the sebaceous gland (SG) plays an important role in skin function, the mechanisms regulating SG differentiation and carcinoma formation are poorly understood. We previously reported that c-MYC overexpression stimulates SG differentiation. We now demonstrate roles for the androgen receptor (AR) and p53. MYC-induced SG differentiation was reduced in mice lacking a functional AR. High levels of MYC triggered a p53-dependent DNA damage response, leading to accumulation of proliferative SG progenitors and inhibition of AR signaling. Conversely, testosterone treatment or p53 deletion activated AR signaling and restored MYC-induced differentiation. Poorly differentiated human sebaceous carcinomas exhibited high p53 and low AR expression. Thus, the consequences of overactivating MYC in the SG depend on whether AR or p53 is activated, as they form a regulatory axis controlling proliferation and differentiation.

Solar ultraviolet radiation (UVR) is a major source of skin damage, resulting in inflammation, premature ageing, and cancer. While several UVR-induced changes, including extracellular matrix reorganisation and epidermal DNA damage, have been documented, the role of different fibroblast lineages and their communication with immune cells has not been explored. We show that acute and chronic UVR exposure led to selective loss of fibroblasts from the upper dermis in human and mouse skin. Lineage tracing and in vivo live imaging revealed that repair following acute UVR is predominantly mediated by papillary fibroblast proliferation and fibroblast reorganisation occurs with minimal migration. In contrast, chronic UVR exposure led to a permanent loss of papillary fibroblasts, with expansion of fibroblast membrane protrusions partially compensating for the reduction in cell number. Although UVR strongly activated Wnt signalling in skin, stimulation of fibroblast proliferation by epidermal β-catenin stabilisation did not enhance papillary dermis repair. Acute UVR triggered an infiltrate of neutrophils and T cell subpopulations and increased pro-inflammatory prostaglandin signalling in skin. Depletion of CD4- and CD8-positive cells resulted in increased papillary fibroblast depletion, which correlated with an increase in DNA damage, pro-inflammatory prostaglandins, and reduction in fibroblast proliferation. Conversely, topical COX-2 inhibition prevented fibroblast depletion and neutrophil infiltration after UVR. We conclude that loss of papillary fibroblasts is primarily induced by a deregulated inflammatory response, with infiltrating T cells supporting fibroblast survival upon UVR-induced environmental stress.

The heterogeneity and compartmentalization of stem cells is a common principle in many epithelia, and is known to function in epithelial maintenance, but its other physiological roles remain elusive. Here we show transcriptional and anatomical contributions of compartmentalized epidermal stem cells in tactile sensory unit formation in the mouse hair follicle. Epidermal stem cells in the follicle upper-bulge, where mechanosensory lanceolate complexes innervate, express a unique set of extracellular matrix (ECM) and neurogenesis-related genes. These epidermal stem cells deposit an ECM protein called EGFL6 into the collar matrix, a novel ECM that tightly ensheathes lanceolate complexes. EGFL6 is required for the proper patterning, touch responses, and αv integrin-enrichment of lanceolate complexes. By maintaining a quiescent original epidermal stem cell niche, the old bulge, epidermal stem cells provide anatomically stable follicle–lanceolate complex interfaces, irrespective of the stage of follicle regeneration cycle. Thus, compartmentalized epidermal stem cells provide a niche linking the hair follicle and the nervous system throughout the hair cycle.

Epidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation. The transition between the two cell states, termed commitment, is poorly understood. Here, we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension to induce differentiation. Cell detachment induces several protein phosphatases, five of which - DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA – promote differentiation by negatively regulating ERK MAPK and positively regulating AP1 transcription factors. Conversely, DUSP10 expression antagonises commitment. The phosphatases form a dynamic network of transient positive and negative interactions that change over time, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem and differentiated) via an unstable (committed) state. Phosphatase expression is also spatially regulated in vivo and in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.

Type XVII collagen (COL17) is a transmembrane protein located at the epidermal basement membrane zone. COL17 deficiency results in premature hair aging phenotypes and in junctional epidermolysis bullosa. Here, we show that COL17 plays a central role in regulating interfollicular epidermis (IFE) proliferation. Loss of COL17 leads to transient IFE hypertrophy in neonatal mice owing to aberrant Wnt signaling. The replenishment of COL17 in the neonatal epidermis of COL17-null mice reverses the proliferative IFE phenotype and the altered Wnt signaling. Physical aging abolishes membranous COL17 in IFE basal cells because of inactive atypical protein kinase C signaling and also induces epidermal hyperproliferation. The overexpression of human COL17 in aged mouse epidermis suppresses IFE hypertrophy. These findings demonstrate that COL17 governs IFE proliferation of neonatal and aged skin in distinct ways. Our study indicates that COL17 could be an important target of anti-aging strategies in the skin.