Significance of stress keratin expression in normal and diseased epithelia.

A group of keratin intermediate filament genes, the type II KRT6A-C and type I KRT16 and KRT17, are deemed stress responsive as they are induced in keratinocytes of surface epithelia in response to environmental stressors, in skin disorders (e.g., psoriasis) and in carcinomas. Monitoring stress keratins is widely used to identify keratinocytes in an activated state. Here, we analyze single-cell transcriptomic data from healthy and diseased human skin to explore the properties of stress keratins. Relative to keratins occurring in healthy skin, stress-induced keratins are expressed at lower levels and show lesser type I-type II pairwise regulation. Stress keratins do not "replace" the keratins expressed during normal differentiation nor reflect cellular proliferation. Instead, stress keratins are consistently co-regulated with genes with roles in differentiation, inflammation, and/or activation of innate immunity at the single-cell level. These findings provide a roadmap toward explaining the broad diversity and contextual regulation of keratins.

Retrospective pharmacogenetic study of psoriasis highlights the role of KLK7 in tumour necrosis factor signalling.

BACKGROUND: Multiple treatment options are available for the management of psoriasis, but clinical response varies among individual patients and no biomarkers are available to facilitate treatment selection for improved patient outcomes. OBJECTIVES: To utilize retrospective data to conduct a pharmacogenetic study to explore the potential genetic pathways associated with drug response in the treatment of psoriasis. METHODS: We conducted a retrospective pharmacogenetic study using self-evaluated treatment response from 1942 genotyped patients with psoriasis. We examined 6 502 658 genetic markers to model their associations with response to six treatment options using linear regression, adjusting for cohort variables and demographic features. We further utilized an integrative approach incorporating epigenomics, transcriptomics and a longitudinal clinical cohort to provide biological implications for the topmost signals associated with drug response. RESULTS: Two novel markers were revealed to be associated with treatment response: rs1991820 (P = 1.30 × 10-6) for anti-tumour necrosis factor (TNF) biologics; and rs62264137 (P = 2.94 × 10-6) for methotrexate, which was also associated with cutaneous mRNA expression levels of two known psoriasis-related genes KLK7 (P = 1.0 × 10-12) and CD200 (P = 5.4 × 10-6). We demonstrated that KLK7 expression was increased in the psoriatic epidermis, as shown by immunohistochemistry, as well as single-cell RNA sequencing, and its responsiveness to anti-TNF treatment was highlighted. By inhibiting the expression of KLK7, we further illustrated that keratinocytes have decreased proinflammatory responses to TNF. CONCLUSIONS: Our study implicates the genetic regulation of cytokine responses in predicting clinical drug response and supports the association between pharmacogenetic loci and anti-TNF response, as shown here for KLK7.

Regulation of Photosensitivity by the Hippo Pathway in Lupus Skin.

OBJECTIVE: Photosensitivity is one of the most common manifestations of systemic lupus erythematosus (SLE), yet its pathogenesis is not well understood. The normal-appearing epidermis of patients with SLE exhibits increased ultraviolet B (UVB)-driven cell death that persists in cell culture. Here, we investigated the role of epigenetic modification and Hippo signaling in enhanced UVB-induced apoptosis seen in SLE keratinocytes. METHODS: We analyzed DNA methylation in cultured keratinocytes from SLE patients compared to keratinocytes from healthy controls (n = 6/group). Protein expression was validated in cultured keratinocytes using immunoblotting and immunofluorescence. An immortalized keratinocyte line overexpressing WWC1 was generated via lentiviral vector. WWC1-driven changes were inhibited using a large tumor suppressor kinase 1/2 (LATS1/2) inhibitor (TRULI) and small interfering RNA (siRNA). The interaction between the Yes-associated protein (YAP) and the transcriptional enhancer associate domain (TEAD) was inhibited by overexpression of an N/TERT cell line expressing a tetracycline-inducible green fluorescent protein-tagged protein that inhibits YAP-TEAD binding (TEADi). Apoptosis was assessed using cleaved caspase 3/7 and TUNEL staining. RESULTS: Hippo signaling was the top differentially methylated pathway in SLE versus control keratinocytes. SLE keratinocytes (n = 6) showed significant hypomethylation (Δß = -0.153) and thus overexpression of the Hippo regulator WWC1 (P = 0.002). WWC1 overexpression increased LATS1/2 kinase activation, leading to YAP cytoplasmic retention and altered proapoptotic transcription in SLE keratinocytes. Accordingly, UVB-mediated apoptosis in keratinocytes could be enhanced by WWC1 overexpression or YAP-TEAD inhibition, mimicking SLE keratinocytes. Importantly, inhibition of LATS1/2 with either the chemical inhibitor TRULI or siRNA effectively eliminated enhanced UVB-apoptosis in SLE keratinocytes. CONCLUSION: Our work unravels a novel driver of photosensitivity in SLE: overactive Hippo signaling in SLE keratinocytes restricts YAP transcriptional activity, leading to shifts that promote UVB apoptosis.

Single-cell Transcriptomics Reveals Dynamic Role of Smooth Muscle Cells and Enrichment of Immune Cell Subsets in Human Abdominal Aortic Aneurysms.

OBJECTIVE: To determine cell-specific gene expression profiles that contribute to development of abdominal aortic aneurysms (AAAs). BACKGROUND: AAAs represent the most common pathological aortic dilation leading to the fatal consequence of aortic rupture. Both immune and structural cells contribute to aortic degeneration, however, gene specific alterations in these cellular subsets are poorly understood. METHODS: We performed single-cell RNA sequencing (scRNA-seq) analysis of AAAs and control tissues. AAA-related changes were examined by comparing gene expression profiles as well as detailed receptor-ligand interactions. An integrative analysis of scRNA-seq data with large genome-wide association study data was conducted to identify genes critical for AAA development. RESULTS: Using scRNA-seq we provide the first comprehensive characterization of the cellular landscape in human AAA tissues. Unbiased clustering analysis of transcriptional profiles identified seventeen clusters representing 8 cell lineages. For immune cells, clustering analysis identified 4 T-cell and 5 monocyte/macrophage subpopulations, with distinct transcriptional profiles in AAAs compared to controls. Gene enrichment analysis on immune subsets identified multiple pathways only expressed in AAA tissue, including those involved in mitochondrial dysfunction, proliferation, and cytokine secretion. Moreover, receptor-ligand analysis defined robust interactions between vascular smooth muscle cells and myeloid populations in AAA tissues. Lastly, integrated analysis of scRNA-seq data with genome-wide association study studies determined that vascular smooth muscle cell expression of SORT1 is critical for maintaining normal aortic wall function. CONCLUSIONS: Here we provide the first comprehensive evaluation of single-cell composition of the abdominal aortic wall and reveal how the gene expression landscape is altered in human AAAs.

IFN-κ is critical for normal wound repair and is decreased in diabetic wounds.

Wound repair following acute injury requires a coordinated inflammatory response. Type I IFN signaling is important for regulating the inflammatory response after skin injury. IFN-κ, a type I IFN, has recently been found to drive skin inflammation in lupus and psoriasis; however, the role of IFN-κ in the context of normal or dysregulated wound healing is unclear. Here, we show that Ifnk expression is upregulated in keratinocytes early after injury and is essential for normal tissue repair. Under diabetic conditions, IFN-κ was decreased in wound keratinocytes, and early inflammation was impaired. Furthermore, we found that the histone methyltransferase mixed-lineage leukemia 1 (MLL1) is upregulated early following injury and regulates Ifnk expression in diabetic wound keratinocytes via an H3K4me3-mediated mechanism. Using a series of in vivo studies with a geneticall y engineered mouse model (Mll1fl/fl K14cre-) and human wound tissues from patients with T2D, we demonstrate that MLL1 controls wound keratinocyte-mediated Ifnk expression and that Mll1 expression is decreased in T2D keratinocytes. Importantly, we found the administration of IFN-κ early following injury improves diabetic tissue repair through increasing early inflammation, collagen deposition, and reepithelialization. These findings have significant implications for understanding the complex role type I IFNs play in keratinocytes in normal and diabetic wound healing. Additionally, they suggest that IFN may be a viable therapeutic target to improve diabetic wound repair.

DDX58(RIG-I)-related disease is associated with tissue-specific interferon pathway activation.

BACKGROUND: Singleton-Merten syndrome (SGMRT) is a rare immunogenetic disorder that variably features juvenile open-angle glaucoma (JOAG), psoriasiform skin rash, aortic calcifications and skeletal and dental dysplasia. Few families have been described and the genotypic and phenotypic spectrum is poorly defined, with variants in DDX58 (DExD/H-box helicase 58) being one of two identified causes, classified as SGMRT2. METHODS: Families underwent deep systemic phenotyping and exome sequencing. Functional characterisation with in vitro luciferase assays and in vivo interferon signature using bulk and single cell RNA sequencing was performed. RESULTS: We have identified a novel DDX58 variant c.1529A>T p.(Glu510Val) that segregates with disease in two families with SGMRT2. Patients in these families have widely variable phenotypic features and different ethnic background, with some being severely affected by systemic features and others solely with glaucoma. JOAG was present in all individuals affected with the syndrome. Furthermore, detailed evaluation of skin rash in one patient revealed sparse inflammatory infiltrates in a unique distribution. Functional analysis showed that the DDX58 variant is a dominant gain-of-function activator of interferon pathways in the absence of exogenous RNA ligands. Single cell RNA sequencing of patient lesional skin revealed a cellular activation of interferon-stimulated gene expression in keratinocytes and fibroblasts but not in neighbouring healthy skin. CONCLUSIONS: These results expand the genotypic spectrum of DDX58-associated disease, provide the first detailed description of ocular and dermatological phenotypes, expand our understanding of the molecular pathogenesis of this condition and provide a platform for testing response to therapy.

Noninvasive Tape-Stripping with High-Resolution RNA Profiling Effectively Captures a Preinflammatory State in Nonlesional Psoriatic Skin.

Tape stripping is a minimally invasive, nonscarring method that can be utilized to assess gene expression in the skin but is infrequently used given technical constraints. By comparing different tape stripping technologies and full-thickness skin biopsy results of lesional and nonlesional psoriatic skin from the same patients, we demonstrate that tape stripping with optimized high-resolution transcriptomic profiling can be used to effectively assess and characterize inflammatory responses in the skin. Upon comparison with single-cell RNA-sequencing data from psoriatic full-thickness skin biopsies, we illustrate that tape-stripping efficiently captures the transcriptome of the upper layers of the epidermis with sufficient resolution to assess the molecular components of the feed-forward immune amplification pathway in psoriasis. Notably, nonlesional psoriatic skin sampled by tape stripping demonstrates activated, proinflammatory changes when compared to healthy control skin, suggesting a prepsoriatic state, which is not captured on full-thickness skin biopsy transcriptome profiling. This work illustrates an approach to assess inflammatory response in the epidermis by combining noninvasive sampling with high throughput RNA-sequencing, providing a foundation for biomarker discoveries and mechanism of action studies for inflammatory skin conditions.

IRAK2 Has a Critical Role in Promoting Feed-Forward Amplification of Epidermal Inflammatory Responses.

Many inflammatory skin diseases are characterized by altered epidermal differentiation. Whether this altered differentiation promotes inflammatory responses has been unknown. Here, we show that IRAK2, a member of the signaling complex downstream of IL-1 and IL-36, correlates positively with disease severity in both atopic dermatitis and psoriasis. Inhibition of epidermal IRAK2 normalizes differentiation and inflammation in two mouse models of psoriasis- and atopic dermatitis-like inflammation. Specifically, we demonstrate that IRAK2 ties together proinflammatory and differentiation-dependent responses and show that this function of IRAK2 is specific to keratinocytes and acts through the differentiation-associated transcription factor ZNF750. Taken together, our findings suggest that IRAK2 has a critical role in promoting feed-forward amplification of inflammatory responses in skin through modulation of differentiation pathways and inflammatory responses.

Inhibition of macrophage histone demethylase JMJD3 protects against abdominal aortic aneurysms.

Abdominal aortic aneurysms (AAAs) are a life-threatening disease for which there is a lack of effective therapy preventing aortic rupture. During AAA formation, pathological vascular remodeling is driven by macrophage infiltration, and the mechanisms regulating macrophage-mediated inflammation remain undefined. Recent evidence suggests that an epigenetic enzyme, JMJD3, plays a critical role in establishing macrophage phenotype. Using single-cell RNA sequencing of human AAA tissues, we identified increased JMJD3 in aortic monocyte/macrophages resulting in up-regulation of an inflammatory immune response. Mechanistically, we report that interferon-ß regulates Jmjd3 expression via JAK/STAT and that JMJD3 induces NF-κB-mediated inflammatory gene transcription in infiltrating aortic macrophages. In vivo targeted inhibition of JMJD3 with myeloid-specific genetic depletion (JMJD3f/fLyz2Cre+) or pharmacological inhibition in the elastase or angiotensin II-induced AAA model preserved the repressive H3K27me3 on inflammatory gene promoters and markedly reduced AAA expansion and attenuated macrophage-mediated inflammation. Together, our findings suggest that cell-specific pharmacologic therapy targeting JMJD3 may be an effective intervention for AAA expansion.

Epigenetic regulation of the PGE2 pathway modulates macrophage phenotype in normal and pathologic wound repair.

Macrophages are a primary immune cell involved in inflammation, and their cell plasticity allows for transition from an inflammatory to a reparative phenotype and is critical for normal tissue repair following injury. Evidence suggests that epigenetic alterations play a critical role in establishing macrophage phenotype and function during normal and pathologic wound repair. Here, we find in human and murine wound macrophages that cyclooxygenase 2/prostaglandin E2 (COX-2/PGE2) is elevated in diabetes and regulates downstream macrophage-mediated inflammation and host defense. Using single-cell RNA sequencing of human wound tissue, we identify increased NF-κB-mediated inflammation in diabetic wounds and show increased COX-2/PGE2 in diabetic macrophages. Further, we identify that COX-2/PGE2 production in wound macrophages requires epigenetic regulation of 2 key enzymes in the cytosolic phospholipase A2/COX-2/PGE2 (cPLA2/COX-2/PGE2) pathway. We demonstrate that TGF-ß-induced miRNA29b increases COX-2/PGE2 production via inhibition of DNA methyltransferase 3b-mediated hypermethylation of the Cox-2 promoter. Further, we find mixed-lineage leukemia 1 (MLL1) upregulates cPLA2 expression and drives COX-2/PGE2. Inhibition of the COX-2/PGE2 pathway genetically (Cox2fl/fl Lyz2Cre+) or with a macrophage-specific nanotherapy targeting COX-2 in tissue macrophages reverses the inflammatory macrophage phenotype and improves diabetic tissue repair. Our results indicate the epigenetically regulated PGE2 pathway controls wound macrophage function, and cell-targeted manipulation of this pathway is feasible to improve diabetic wound repair.

IL18-containing 5-gene signature distinguishes histologically identical dermatomyositis and lupus erythematosus skin lesions.

Skin lesions in dermatomyositis (DM) are common, are frequently refractory, and have prognostic significance. Histologically, DM lesions appear similar to cutaneous lupus erythematosus (CLE) lesions and frequently cannot be differentiated. We thus compared the transcriptional profile of DM biopsies with CLE lesions to identify unique features. Type I IFN signaling, including IFN-κ upregulation, was a common pathway in both DM and CLE; however, CLE also exhibited other inflammatory pathways. Notably, DM lesions could be distinguished from CLE by a 5-gene biomarker panel that included IL18 upregulation. Using single-cell RNA-sequencing, we further identified keratinocytes as the main source of increased IL-18 in DM skin. This study identifies a potentially novel molecular signature, with significant clinical implications for differentiating DM from CLE lesions, and highlights the potential role for IL-18 in the pathophysiology of DM skin disease.