Disease heterogeneity and molecular classification of inflammatory palmoplantar diseases.

BACKGROUND: Palmoplantar pustulosis (PPP) is an inflammatory disease characterized by relapsing eruptions of neutrophil-filled, sterile pustules on the palms and soles that can be clinically difficult to differentiate from non-pustular palmoplantar psoriasis (palmPP) and dyshidrotic palmoplantar eczema (DPE). OBJECTIVE: To identify overlapping and unique PPP, palmPP, and DPE drivers to provide molecular insight into their pathogenesis. METHODS: We performed bulk RNA sequencing of lesional PPP (n=33), palmPP (n=5), and DPE (n=28) samples, as well as 5 healthy non-acral and 10 healthy acral skin samples. RESULTS: Acral skin shows a unique immune environment, likely contributing to a unique niche for palmoplantar inflammatory diseases. Compared with healthy acral skin, PPP, palmPP, and DPE displayed a broad overlapping transcriptomic signature characterized by shared upregulation of pro-inflammatory cytokines (TNF, IL36), chemokines, and T cell-associated genes, along with unique disease features of each disease state, including enriched neutrophil processes in PPP and to a lesser extent in palmPP, and lipid antigen processing in DPE. Strikingly, unsupervised clustering and trajectory analyses demonstrated divergent inflammatory profiles within the three disease states. These identified putative key upstream immunological switches, including eicosanoids, interferon responses, and neutrophil degranulation, contributing to disease heterogeneity. CONCLUSION: We demonstrate the molecular overlap between different inflammatory palmoplantar diseases that supersedes clinical and histologic assessment, yet highlighting the heterogeneity within each condition, suggesting limitations of current disease classification and the need to move toward a molecular classification of inflammatory acral diseases.

Multi-ancestry Genome-Wide Association Meta-Analysis Identifies Novel Loci in Atopic Dermatitis.

Atopic dermatitis (AD) is a highly heritable and common inflammatory skin condition affecting children and adults worldwide. Multi-ancestry approaches to AD genetic association studies are poised to boost power to detect genetic signal and identify ancestry-specific loci contributing to AD risk. Here, we present a multi-ancestry GWAS meta-analysis of twelve AD cohorts from five ancestral populations totaling 56,146 cases and 602,280 controls. We report 101 genomic loci associated with AD, including 15 loci that have not been previously associated with AD or eczema. Fine-mapping, QTL colocalization, and cell-type enrichment analyses identified genes and cell types implicated in AD pathophysiology. Functional analyses in keratinocytes provide evidence for genes that could play a role in AD through epidermal barrier function. Our study provides new insights into the etiology of AD by harnessing multiple genetic and functional approaches to unveil the mechanisms by which AD-associated variants impact genes and cell types.

Epigenetic Alteration of Smooth Muscle Cells Regulates Endothelin-Dependent Blood Pressure and Hypertensive Arterial Remodeling.

Long-standing hypertension (HTN) affects multiple organ systems and leads to pathologic arterial remodeling, which is driven largely by smooth muscle cell (SMC) plasticity. Although genome wide association studies (GWAS) have identified numerous variants associated with changes in blood pressure in humans, only a small percentage of these variants actually cause HTN. In order to identify relevant genes important in SMC function in HTN, we screened three separate human GWAS and Mendelian randomization studies to identify SNPs located within non-coding gene regions, focusing on genes encoding epigenetic enzymes, as these have been recently identified to control SMC fate in cardiovascular disease. We identified SNPs rs62059712 and rs74480102 in the promoter of the human JMJD3 gene and show that the minor C allele increases JMJD3 transcription in SMCs via increased SP1 binding to the JMJD3 promoter. Using our novel SMC-specific Jmjd3-deficient murine model ( Jmjd3 flox/flox Myh11 CreERT ), we show that loss of Jmjd3 in SMCs results in HTN, mechanistically, due to decreased EDNRB expression and a compensatory increase in EDNRA expression. As a translational corollary, through single cell RNA-sequencing (scRNA-seq) of human arteries, we found strong correlation between JMJD3 and EDNRB expression in SMCs. Further, we identified that JMJD3 is required for SMC-specific gene expression, and loss of JMJD3 in SMCs in the setting of HTN results in increased arterial remodeling by promoting the SMC synthetic phenotype. Our findings link a HTN-associated human DNA variant with regulation of SMC plasticity, revealing therapeutic targets that may be used in the screening and/or personalized treatment of HTN.

Targeting IL-1 controls refractory pityriasis rubra pilaris.

Pityriasis rubra pilaris (PRP) is a rare inflammatory skin disease with a poorly understood pathogenesis. Through a molecularly driven precision medicine approach and an extensive mechanistic pathway analysis in PRP skin samples, compared to psoriasis, atopic dermatitis, healed PRP, and healthy controls, we identified IL-1ß as a key mediator, orchestrating an NF-κB-mediated IL-1ß-CCL20 axis, including activation of CARD14 and NOD2. Treatment of three patients with the IL-1 antagonists anakinra and canakinumab resulted in rapid clinical improvement and reversal of the PRP-associated molecular signature with a 50% improvement in skin lesions after 2 to 3 weeks. This transcriptional signature was consistent with in vitro stimulation of keratinocytes with IL-1ß. With the central role of IL-1ß underscoring its potential as a therapeutic target, our findings propose a redefinition of PRP as an autoinflammatory keratinization disorder. Further clinical trials are needed to validate the efficacy of IL-1ß antagonists in PRP.

Positionally distinct interferon stimulated dermal immune acting fibroblasts promote neutrophil recruitment in Sweet's syndrome.

Sweet's syndrome is a poorly understood inflammatory skin disease characterized by neutrophil infiltration to the dermis. Single-nucleus and bulk transcriptomics of archival clinical samples of Sweet's syndrome revealed a prominent interferon signature in Sweet's syndrome skin that was reduced in tissue from other neutrophilic dermatoses. This signature was observed in different subsets of cells, including fibroblasts that expressed interferon-induced genes. Functionally, this response was supported by analysis of cultured primary human dermal fibroblasts that were observed to highly express neutrophil chemokines in response to activation by type I interferon. Furthermore, single-molecule resolution spatial transcriptomics of skin in Sweet's syndrome identified positionally distinct immune acting fibroblasts that included a CXCL1+ subset proximal to neutrophils and a CXCL12+ subset distal to the neutrophilic infiltrate. This study defines the cellular landscape of neutrophilic dermatoses and suggests dermal immune acting fibroblasts play a role in the pathogenesis of Sweet's syndrome through recognition of type I interferons.

Histone demethylase JARID1C/KDM5C regulates Th17 cells by increasing IL-6 expression in diabetic plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are first responders to tissue injury, where they prime naive T cells. The role of pDCs in physiologic wound repair has been examined, but little is known about pDCs in diabetic wound tissue and their interactions with naive CD4+ T cells. Diabetic wounds are characterized by increased levels of inflammatory IL-17A cytokine, partly due to increased Th17 CD4+ cells. This increased IL-17A cytokine, in excess, impairs tissue repair. Here, using human tissue and murine wound healing models, we found that diabetic wound pDCs produced excess IL-6 and TGF-ß and that these cytokines skewed naive CD4+ T cells toward a Th17 inflammatory phenotype following cutaneous injury. Further, we identified that increased IL-6 cytokine production by diabetic wound pDCs is regulated by a histone demethylase, Jumonji AT-rich interactive domain 1C histone demethylase (JARID1C). Decreased JARID1C increased IL-6 transcription in diabetic pDCs, and this process was regulated upstream by an IFN-I/TYK2/JAK1,3 signaling pathway. When inhibited in nondiabetic wound pDCs, JARID1C skewed naive CD4+ T cells toward a Th17 phenotype and increased IL-17A production. Together, this suggests that diabetic wound pDCs are epigenetically altered to increase IL-6 expression that then affects T cell phenotype. These findings identify a therapeutically manipulable pathway in diabetic wounds.

Diabetic wound keratinocytes induce macrophage JMJD3-mediated Nlrp3 expression via IL-1R signaling.

Macrophage (Mφ) plasticity is critical for normal wound repair; however, in type 2 diabetic wounds, Mφs persist in a low-grade inflammatory state that prevents the resolution of wound inflammation. Increased NLRP3 inflammasome activity has been shown in diabetic wound Mφs; however, the molecular mechanisms regulating NLRP3 expression and activity are unclear. Here, we identified that diabetic wound keratinocytes induce Nlrp3 gene expression in wound Mφs through IL-1 receptor-mediated signaling, resulting in enhanced inflammasome activation in the presence of PAMPs and DAMPs. We found that IL-1 alpha is increased in human and murine wound diabetic keratinocytes compared to non-diabetic controls and directly induces Mφ Nlrp3 expression through IL-1 receptor signaling. Mechanistically, we report that the histone demethylase, JMJD3, is increased in wound Mφs late post-injury and is induced by IL-1 alpha from diabetic wound keratinocytes, resulting in Nlrp3 transcriptional activation through an H3K27me3-mediated mechanism. Using genetically engineered mice deficient in JMJD3 in myeloid cells (Jmjd3fl/fllyz2cre+), we demonstrate that JMJD3 controls Mφ-mediated Nlrp3 expression during diabetic wound healing. Thus, our data suggest a role for keratinocyte-mediated IL-1 alpha/IL-1R signaling in driving enhanced NLRP3 inflammasome activity in wound Mφs. These data also highlight the importance of cell crosstalk in wound tissues and identify JMJD3 and the ILR signaling cascade as important upstream therapeutic targets for Mφ NLRP3 inflammasome hyperactivity in nonhealing diabetic wounds.

tauFisher predicts circadian time from a single sample of bulk and single-cell pseudobulk transcriptomic data.

As the circadian clock regulates fundamental biological processes, disrupted clocks are often observed in patients and diseased tissues. Determining the circadian time of the patient or the tissue of focus is essential in circadian medicine and research. Here we present tauFisher, a computational pipeline that accurately predicts circadian time from a single transcriptomic sample by finding correlations between rhythmic genes within the sample. We demonstrate tauFisher's performance in adding timestamps to both bulk and single-cell transcriptomic samples collected from multiple tissue types and experimental settings. Application of tauFisher at a cell-type level in a single-cell RNAseq dataset collected from mouse dermal skin implies that greater circadian phase heterogeneity may explain the dampened rhythm of collective core clock gene expression in dermal immune cells compared to dermal fibroblasts. Given its robustness and generalizability across assay platforms, experimental setups, and tissue types, as well as its potential application in single-cell RNAseq data analysis, tauFisher is a promising tool that facilitates circadian medicine and research.

Suppression of TCF4 promotes a ZC3H12A-mediated self-sustaining inflammatory feedback cycle involving IL-17RA/IL-17RE epidermal signaling.

IL-17C is an epithelial cell-derived proinflammatory cytokine whose transcriptional regulation remains unclear. Analysis of the IL17C promoter region identified TCF4 as putative regulator, and siRNA knockdown of TCF4 in human keratinocytes (KCs) increased IL17C. IL-17C stimulation of KCs (along with IL-17A and TNF-α stimulation) decreased TCF4 and increased NFKBIZ and ZC3H12A expression in an IL-17RA/RE-dependent manner, thus creating a feedback loop. ZC3H12A (MCPIP1/Regnase-1), a transcriptional immune-response regulator, also increased following TCF4 siRNA knockdown, and siRNA knockdown of ZC3H12A decreased NFKBIZ, IL1B, IL36G, CCL20, and CXCL1, revealing a proinflammatory role for ZC3H12A. Examination of lesional skin from the KC-Tie2 inflammatory dermatitis mouse model identified decreases in TCF4 protein concomitant with increases in IL-17C and Zc3h12a that reversed following the genetic elimination of Il17c, Il17ra, and Il17re and improvement in the skin phenotype. Conversely, interference with Tcf4 in KC-Tie2 mouse skin increased Il17c and exacerbated the inflammatory skin phenotype. Together, these findings identify a role for TCF4 in the negative regulation of IL-17C, which, alone and with TNF-α and IL-17A, feed back to decrease TCF4 in an IL-17RA/RE-dependent manner. This loop is further amplified by IL-17C-TCF4 autocrine regulation of ZC3H12A and IL-17C regulation of NFKBIZ to promote self-sustaining skin inflammation.

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.

HERC6 regulates STING activity in a sex-biased manner through modulation of LATS2/VGLL3 Hippo signaling.

Interferon (IFN) activity exhibits a gender bias in human skin, skewed toward females. We show that HERC6, an IFN-induced E3 ubiquitin ligase, is induced in human keratinocytes through the epidermal type I IFN; IFN-κ. HERC6 knockdown in human keratinocytes results in enhanced induction of interferon-stimulated genes (ISGs) upon treatment with a double-stranded (ds) DNA STING activator cGAMP but not in response to the RNA-sensing TLR3 agonist. Keratinocytes lacking HERC6 exhibit sustained STING-TBK1 signaling following cGAMP stimulation through modulation of LATS2 and TBK1 activity, unmasking more robust ISG responses in female keratinocytes. This enhanced female-biased immune response with loss of HERC6 depends on VGLL3, a regulator of type I IFN signature. These data identify HERC6 as a previously unrecognized negative regulator of ISG expression specific to dsDNA sensing and establish it as a regulator of female-biased immune responses through modulation of STING signaling.

Topical Ruxolitinib in the Treatment of Necrobiosis Lipoidica: A Prospective, Open-Label Study.

Necrobiosis lipoidica (NL) is a rare granulomatous disease. There are few effective treatments for NL. We sought to investigate the efficacy and safety of the Jak1/2 inhibitor, ruxolitnib, in the treatment of NL and identify the biomarkers associated with the disease and treatment response. We conducted an open-label, phase 2 study of ruxolitinib in 12 patients with NL. We performed transcriptomic analysis of tissue samples before and after treatment. At week 12, the mean NL lesion score decreased by 58.2% (SD = 28.7%, P = .003). Transcriptomic analysis demonstrated enrichment of type I and type II IFN pathways in baseline disease. Weighted gene coexpression network analysis demonstrated post-treatment changes in IFN pathways with key hub genes IFNG and signal transducer and activator of transcription 1 gene STAT1. Limitations include small sample size and a study group limited to patients with <10% body surface area. In conclusion, ruxolitinib is an effective treatment for NL and targets the key pathogenic mediators of the disease.

Lupus dermal fibroblasts are proinflammatory and exhibit a profibrotic phenotype in scarring skin disease.

Fibroblasts are stromal cells known to regulate local immune responses important for wound healing and scar formation; however, the cellular mechanisms driving damage and scarring in patients with cutaneous lupus erythematosus (CLE) remain poorly understood. Dermal fibroblasts in patients with systemic lupus erythematosus (SLE) experience increased cytokine signaling in vivo, but the effect of inflammatory mediators on fibroblast responses in nonscarring versus scarring CLE subtypes is unclear. Here, we examined responses to cytokines in dermal fibroblasts from nonlesional skin of 22 patients with SLE and CLE and 34 individuals acting as healthy controls. Notably, inflammatory cytokine responses were exaggerated in SLE fibroblasts compared with those from individuals acting as healthy controls. In lesional CLE biopsies, these same inflammatory profiles were reflected in single-cell RNA-Seq of SFRP2+ and inflammatory fibroblast subsets, and TGF-ß was identified as a critical upstream regulator for inflammatory fibroblasts in scarring discoid lupus lesions. In vitro cytokine stimulation of nonlesional fibroblasts from patients who scar from CLE identified an upregulation of collagens, particularly in response to TGF-ß, whereas inflammatory pathways were more prominent in nonscarring patients. Our study revealed that SLE fibroblasts are poised to hyperrespond to inflammation, with differential responses among patients with scarring versus nonscarring disease, providing a potential skin-specific target for mitigating damage.

Comparative analyses of Netherton syndrome patients and Spink5 conditional knock-out mice uncover disease-relevant pathways.

Netherton syndrome (NS) is a rare skin disease caused by loss-of-function mutations in the serine peptidase inhibitor Kazal type 5 (SPINK5) gene. Disease severity and the lack of efficacious treatments call for a better understanding of NS mechanisms. Here we describe a novel and viable, Spink5 conditional knock-out (cKO) mouse model, allowing to study NS progression. By combining transcriptomics and proteomics, we determine a disease molecular profile common to mouse models and NS patients. Spink5 cKO mice and NS patients share skin barrier and inflammation signatures defined by up-regulation and increased activity of proteases, IL-17, IL-36, and IL-20 family cytokine signaling. Systemic inflammation in Spink5 cKO mice correlates with disease severity and is associated with thymic atrophy and enlargement of lymph nodes and spleen. This systemic inflammation phenotype is marked by neutrophils and IL-17/IL-22 signaling, does not involve primary T cell immunodeficiency and is independent of bacterial infection. By comparing skin transcriptomes and proteomes, we uncover several putative substrates of tissue kallikrein-related proteases (KLKs), demonstrating that KLKs can proteolytically regulate IL-36 pro-inflammatory cytokines. Our study thus provides a conserved molecular framework for NS and reveals a KLK/IL-36 signaling axis, adding new insights into the disease mechanisms and therapeutic targets.

Systems-based identification of the Hippo pathway for promoting fibrotic mesenchymal differentiation in systemic sclerosis.

Systemic sclerosis (SSc) is a devastating autoimmune disease characterized by excessive production and accumulation of extracellular matrix, leading to fibrosis of skin and other internal organs. However, the main cellular participants in SSc skin fibrosis remain incompletely understood. Here using differentiation trajectories at a single cell level, we demonstrate a dual source of extracellular matrix deposition in SSc skin from both myofibroblasts and endothelial-to-mesenchymal-transitioning cells (EndoMT). We further define a central role of Hippo pathway effectors in differentiation and homeostasis of myofibroblast and EndoMT, respectively, and show that myofibroblasts and EndoMTs function as central communication hubs that drive key pro-fibrotic signaling pathways in SSc. Together, our data help characterize myofibroblast differentiation and EndoMT phenotypes in SSc skin, and hint that modulation of the Hippo pathway may contribute in reversing the pro-fibrotic phenotypes in myofibroblasts and EndoMTs.

GZMK+CD8+ T cells Target A Specific Acinar Cell Type in Sjögren's Disease.

Sjögren's Disease (SjD) is a systemic autoimmune disease without a clear etiology or effective therapy. Utilizing unbiased single-cell and spatial transcriptomics to analyze human minor salivary glands in health and disease we developed a comprehensive understanding of the cellular landscape of healthy salivary glands and how that landscape changes in SjD patients. We identified novel seromucous acinar cell types and identified a population of PRR4+CST3+WFDC2- seromucous acinar cells that are particularly targeted in SjD. Notably, GZMK+CD8 T cells, enriched in SjD, exhibited a cytotoxic phenotype and were physically associated with immune-engaged epithelial cells in disease. These findings shed light on the immune response's impact on transitioning acinar cells with high levels of secretion and explain the loss of this specific cell population in SjD. This study explores the complex interplay of varied cell types in the salivary glands and their role in the pathology of Sjögren's Disease.

Oral Baricitinib in the Treatment of Cutaneous Lichen Planus.

Background: Cutaneous lichen planus (LP) is a recalcitrant, difficult-to-treat, inflammatory skin disease characterized by pruritic, flat-topped, violaceous papules on the skin. Baricitinib is an oral Janus kinase (JAK) 1/2 inhibitor that interrupts the signaling pathway of interferon (IFN)-γ, a cytokine implicated in the pathogenesis of LP. Methods: In this phase II trial, twelve patients with cutaneous LP received baricitinib 2 mg daily for 16 weeks, accompanied by in-depth spatial, single-cell, and bulk transcriptomic profiling of pre-and post-treatment samples. Results: An early and sustained clinical response was seen with 83.3% of patients responsive at week 16. Our molecular data identified a unique, oligoclonal IFN-γ, CD8+, CXCL13+ cytotoxic T-cell population in LP skin and demonstrate a rapid decrease in interferon signature within 2 weeks of treatment, most prominent in the basal layer of the epidermis. Conclusion: This study demonstrates the efficacy and molecular mechanisms of JAK inhibition in LP. Trial Registration Number : NCT05188521.

Single-cell sequencing reveals Hippo signaling as a driver of fibrosis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory disease characterized by abscesses, nodules, dissecting/draining tunnels, and extensive fibrosis. Here, we integrate single-cell RNA sequencing, spatial transcriptomics, and immunostaining to provide an unprecedented view of the pathogenesis of chronic HS, characterizing the main cellular players and defining their interactions. We found a striking layering of the chronic HS infiltrate and identified the contribution of 2 fibroblast subtypes (SFRP4+ and CXCL13+) in orchestrating this compartmentalized immune response. We further demonstrated the central role of the Hippo pathway in promoting extensive fibrosis in HS and provided preclinical evidence that the profibrotic fibroblast response in HS can be modulated through inhibition of this pathway. These data provide insights into key aspects of HS pathogenesis with broad therapeutic implications.

Monogenic etiologies of persistent human papillomavirus infections: A comprehensive systematic review.

PURPOSE: Persistent human papillomavirus infection (PHPVI) causes cutaneous, anogenital, and mucosal warts. Cutaneous warts include common warts, Treeman syndrome, and epidermodysplasia verruciformis, among others. Although more reports of monogenic predisposition to PHPVI have been published with the development of genomic technologies, genetic testing is rarely incorporated into clinical assessments. To encourage broader molecular testing, we compiled a list of the various monogenic etiologies of PHPVI. METHODS: We conducted a systematic literature review to determine the genetic, immunological, and clinical characteristics of patients with PHPVI. RESULTS: The inclusion criteria were met by 261 of 40,687 articles. In 842 patients, 83 PHPVI-associated genes were identified, including 42, 6, and 35 genes with strong, moderate, and weak evidence for causality, respectively. Autosomal recessive inheritance predominated (69%). PHPVI onset age was 10.8 ± 8.6 years, with an interquartile range of 5 to 14 years. GATA2,IL2RG,DOCK8, CXCR4, TMC6, TMC8, and CIB1 are the most frequently reported PHPVI-associated genes with strong causality. Most genes (74 out of 83) belong to a catalog of 485 inborn errors of immunity-related genes, and 40 genes (54%) are represented in the nonsyndromic and syndromic combined immunodeficiency categories. CONCLUSION: PHPVI has at least 83 monogenic etiologies and a genetic diagnosis is essential for effective management.