Transcriptional landscape of epithelial and immune cell populations revealed through FACS-seq of healthy human skin.

Human skin consists of multiple cell types, including epithelial, immune, and stromal cells. Transcriptomic analyses have previously been performed from bulk skin samples or from epithelial and immune cells expanded in cell culture. However, transcriptomic analysis of bulk skin tends to drown out expression signals from relatively rare cells while cell culture methods may significantly alter cellular phenotypes and gene expression profiles. To identify distinct transcriptomic profiles of multiple cell populations without substantially altering cell phenotypes, we employed a fluorescence activated cell sorting method to isolate keratinocytes, dendritic cells, CD4+ T effector cells, and CD8+ T effector cells from healthy skin samples, followed by RNA-seq of each cell population. Principal components analysis revealed distinct clustering of cell types across samples, while differential expression and coexpression network analyses revealed transcriptional profiles of individual cell populations distinct from bulk skin, most strikingly in the least abundant CD8+ T effector population. Our work provides a high resolution view of cutaneous cellular gene expression and suggests that transcriptomic profiling of bulk skin may inadequately capture the contribution of less abundant cell types

The distribution of cutaneous metastases correlates with local immunologic milieu.

BackgroundMetastases to the skin are found with increased frequency at certain sites, such as the scalp, but the biological factors that influence this distribution are not understood.ObjectiveWe aimed to compare the proportional frequency of metastases at various cutaneous locations with the immunologic microenvironments at those sites.MethodsWe retrospectively identified all biopsy specimens of cutaneous metastases diagnosed at our institution from 1991 to 2014 (n = 1984) and mapped their anatomic distribution while controlling for regional surface area. Using a separate, mapped cohort of normal-appearing skin samples (n = 140), we measured the density of regulatory T cells, CD4(+) effector T cells, and CD8(+) T cells by flow cytometry.ResultsPer unit surface area, cutaneous metastases arise most commonly on the head and neck, followed by the trunk, upper extremities, and lower extremities, respectively. Sites with more frequent metastases tend to contain a greater density of regulatory T cells and a lower proportion of CD8(+) T cells (P < .05).LimitationsImmunologic factors were only assessed in control tissue and were not measured from patients with metastatic disease in this correlative single-center study.ConclusionThe distribution of cutaneous metastases follows the distribution of regulatory and effector T cells in skin. Further studies are required to prove a mechanistic association between local immunologic factors and the development of cutaneous metastases

Skin-infiltrating, interleukin-22–producing T cells differentiate pediatric psoriasis from adult psoriasis

BackgroundEvidence from adult psoriasis studies implicates an imbalance between regulatory and effector T cells, particularly TH-17-producing T cells, in the pathogenesis of psoriasis. Little is known about the immunopathology of psoriasis in children.ObjectiveWe sought to functionally characterize the inflammatory cell profiles of psoriatic plaques from pediatric patients and compare them with healthy, age-matched controls and adult psoriasis patients.MethodsSkin samples from pediatric psoriasis patients and healthy controls were analyzed by multiparameter flow cytometry to determine the dominant immune cell subsets present and cytokines produced.ResultsLesional tissue from pediatric psoriasis patients had significantly increased interleukin (IL) 22 derived from CD4+ and CD8+ cells compared with the tissues from healthy pediatric controls and adult psoriasis patients. Tissue from pediatric psoriasis patients had significantly less elevation of IL-17 derived from CD4+ and CD8+ cells compared with the tissue from adult psoriasis patients. In contrast with the lesions from adult patients, lesional skin in pediatric patients with psoriasis did not have increases in regulatory T cells.LimitationsThis is a pilot study, thus the sample size is small.ConclusionSignificant differences in IL-17 and IL-22 expression were observed in the pediatric psoriasis patients compared with pediatric healthy controls and adult psoriasis patients. IL-22 might be relevant in the pathogenesis of pediatric psoriasis and represents a potential treatment target unique to pediatric psoriasis

Transcriptional landscape of epithelial and immune cell populations revealed through FACS-seq of healthy human skin.

Human skin consists of multiple cell types, including epithelial, immune, and stromal cells. Transcriptomic analyses have previously been performed from bulk skin samples or from epithelial and immune cells expanded in cell culture. However, transcriptomic analysis of bulk skin tends to drown out expression signals from relatively rare cells while cell culture methods may significantly alter cellular phenotypes and gene expression profiles. To identify distinct transcriptomic profiles of multiple cell populations without substantially altering cell phenotypes, we employed a fluorescence activated cell sorting method to isolate keratinocytes, dendritic cells, CD4+ T effector cells, and CD8+ T effector cells from healthy skin samples, followed by RNA-seq of each cell population. Principal components analysis revealed distinct clustering of cell types across samples, while differential expression and coexpression network analyses revealed transcriptional profiles of individual cell populations distinct from bulk skin, most strikingly in the least abundant CD8+ T effector population. Our work provides a high resolution view of cutaneous cellular gene expression and suggests that transcriptomic profiling of bulk skin may inadequately capture the contribution of less abundant cell types

Fas ligand promotes an inducible TLR-dependent model of cutaneous lupus-like inflammation

Toll-like receptors TLR7 and TLR9 are both implicated in the activation of autoreactive B cells and other cell types associated with systemic lupus erythematosus (SLE) pathogenesis. However, Tlr9-/- autoimmune-prone strains paradoxically develop more severe disease. We have now leveraged the negative regulatory role of TLR9 to develop an inducible rapid-onset murine model of systemic autoimmunity that depends on T cell detection of a membrane-bound OVA fusion protein expressed by MHC class II+ cells, expression of TLR7, expression of the type I IFN receptor, and loss of expression of TLR9. These mice are distinguished by a high frequency of OVA-specific Tbet+, IFN-gamma+, and FasL-expressing Th1 cells as well as autoantibody-producing B cells. Unexpectedly, contrary to what occurs in most models of SLE, they also developed skin lesions that are very similar to those of human cutaneous lupus erythematosus (CLE) as far as clinical appearance, histological changes, and gene expression. FasL was a key effector mechanism in the skin, as the transfer of FasL-deficient DO11gld T cells completely failed to elicit overt skin lesions. FasL was also upregulated in human CLE biopsies. Overall, our model provides a relevant system for exploring the pathophysiology of CLE as well as the negative regulatory role of TLR9

RNA-seq and flow-cytometry of conventional, scalp, and palmoplantar psoriasis reveal shared and distinct molecular pathways.

It has long been recognized that anatomic location is an important feature for defining distinct subtypes of plaque psoriasis. However, little is known about the molecular differences between scalp, palmoplantar, and conventional plaque psoriasis. To investigate the molecular heterogeneity of these psoriasis subtypes, we performed RNA-seq and flow cytometry on skin samples from individuals with scalp, palmoplantar, and conventional plaque psoriasis, along with samples from healthy control patients. We performed differential expression analysis and network analysis using weighted gene coexpression network analysis (WGCNA). Our analysis revealed a core set of 763 differentially expressed genes common to all sub-types of psoriasis. In contrast, we identified 605, 632, and 262 genes uniquely differentially expressed in conventional, scalp, and palmoplantar psoriasis, respectively. WGCNA and pathway analysis revealed biological processes for the core genes as well as subtype-specific genes. Flow cytometry analysis revealed a shared increase in the percentage of CD4+ T regulatory cells in all psoriasis subtypes relative to controls, whereas distinct psoriasis subtypes displayed differences in IL-17A, IFN-gamma, and IL-22 production. This work reveals the molecular heterogeneity of plaque psoriasis and identifies subtype-specific signaling pathways that will aid in the development of therapy that is appropriate for each subtype of plaque psoriasis

Developing Human Skin Contains Lymphocytes Demonstrating a Memory Signature.

Lymphocytes in barrier tissues play critical roles in host defense and homeostasis. These cells take up residence in tissues during defined developmental windows, when they may demonstrate distinct phenotypes and functions. Here, we utilized mass and flow cytometry to elucidate early features of human skin immunity. Although most conventional αβ T (Tconv) cells in fetal skin have a naive, proliferative phenotype, a subset of CD4+ Tconv and CD8+ cells demonstrate memory-like features and a propensity for interferon (IFN)γ production. Skin regulatory T cells dynamically accumulate over the second trimester in temporal and regional association with hair follicle development. These fetal skin regulatory T cells (Tregs) demonstrate an effector memory phenotype while differing from their adult counterparts in expression of key effector molecules. Thus, we identify features of prenatal skin lymphocytes that may have key implications for understanding antigen and allergen encounters in utero and in infancy

Adoptive Treg Cell Therapy in a Patient With Systemic Lupus Erythematosus.

ObjectiveAdoptive Treg cell therapy has great potential to treat autoimmune disease. Currently, very little is known about how these cells impact inflamed tissues. This study was undertaken to elucidate how autologous Treg cell therapy influences tissue inflammation in human autoimmune disease.MethodsWe describe a systemic lupus erythematosus (SLE) patient with active skin disease who received adoptive Treg therapy. We comprehensively quantified Treg cells and immune activation in peripheral blood and skin, with data obtained at multiple time points posttreatment.ResultsDeuterium tracking of infused Treg cells revealed the transient presence of cells in peripheral blood, accompanied by increased percentages of highly activated Treg cells in diseased skin. Flow cytometric analysis and whole transcriptome RNA sequencing revealed that Treg cell accumulation in skin was associated with a marked attenuation of the interferon-γ pathway and a reciprocal augmentation of the interleukin-17 (IL-17) pathway. This phenomenon was more pronounced in skin relative to peripheral blood. To validate these findings, we investigated Treg cell adoptive transfer of skin inflammation in a murine model and found that it also resulted in a pronounced skewing away from Th1 immunity and toward IL-17 production.ConclusionWe report the first case of a patient with SLE treated with autologous adoptive Treg cell therapy. Taken together, our results suggest that this treatment leads to increased activated Treg cells in inflamed skin, with a dynamic shift from Th1 to Th17 responses

Developing Human Skin Contains Lymphocytes Demonstrating a Memory Signature.

Lymphocytes in barrier tissues play critical roles in host defense and homeostasis. These cells take up residence in tissues during defined developmental windows, when they may demonstrate distinct phenotypes and functions. Here, we utilized mass and flow cytometry to elucidate early features of human skin immunity. Although most conventional αβ T (Tconv) cells in fetal skin have a naive, proliferative phenotype, a subset of CD4+ Tconv and CD8+ cells demonstrate memory-like features and a propensity for interferon (IFN)γ production. Skin regulatory T cells dynamically accumulate over the second trimester in temporal and regional association with hair follicle development. These fetal skin regulatory T cells (Tregs) demonstrate an effector memory phenotype while differing from their adult counterparts in expression of key effector molecules. Thus, we identify features of prenatal skin lymphocytes that may have key implications for understanding antigen and allergen encounters in utero and in infancy

The distribution of cutaneous metastases correlates with local immunologic milieu

BACKGROUND: Metastases to the skin are found with increased frequency at certain sites, such as the scalp, but the biological factors that influence this distribution are not understood. OBJECTIVE: We aimed to compare the proportional frequency of metastases at various cutaneous locations with the immunologic microenvironments at those sites. METHODS: We retrospectively identified all biopsies of cutaneous metastases diagnosed at our institution from 1991 to 2014 (n = 1984) and mapped their anatomic distribution while controlling for regional surface area. Using a separate, mapped cohort of normal skin samples (n = 140), we measured the density of regulatory T cells (Tregs), CD4+ effector T cells, and CD8+ T cells by flow cytometry. RESULTS: Per unit surface area, cutaneous metastases arise most commonly on the head and neck, followed by the trunk, upper extremities, and lower extremities, respectively. Sites with more frequent metastases tend to contain a greater density of Tregs and a lower proportion of CD8+ T cells (p < 0.05). LIMITATIONS: Immunologic factors were only assessed in control tissue and were not measured from patients with metastatic disease in this correlative single-center study. CONCLUSION: The distribution of cutaneous metastases follows the distribution of regulatory and effector T cells in skin. Further studies are required to prove a mechanistic association between local immunologic factors and the development of cutaneous metastases