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.

Standardized Protocols for Clinical and Histopathological Characterization of Hidradenitis Suppurativa Tissue Specimens.

Methods for describing and reporting the clinical and histological characteristics of cutaneous tissue samples from patients with hidradenitis suppurativa (HS) are not currently standardized, limiting clinicians' and scientists' ability to uniformly record, report, and communicate about the characteristics of tissue used in translational experiments. A recently published consensus statement outlined morphological definitions of typical HS lesions, but no consensus has been reached regarding clinical characterization and examination of HS tissue samples. Here we aimed to establish a protocol for reporting histopathologic and clinical characteristics of HS tissue specimens. This study was conducted from May 2023 to August 2023. Experts in clinical care, dermatopathology, and translational research were recruited, and a modified Delphi technique was used to develop a protocol for histologic reporting and clinical characterization of submitted tissue specimens from HS patients. A total of 27 experts participated (14 dermatologists, 3 fellowship-trained dermatopathologists, 3 plastic surgeons, 3 general surgeons, and 4 research scientists) in creating and reviewing protocols for the clinical and histopathological examination of HS tissue specimens. The protocols were formatted as a synoptic report and will help consistently classify specimens in biobanks based on histological features and more accurately report and select samples used in translational research projects.

International Consensus Definition and Diagnostic Criteria for Generalized Pustular Psoriasis From the International Psoriasis Council.

Importance: Generalized pustular psoriasis (GPP) lacks internationally accepted definitions and diagnostic criteria, impeding timely diagnosis and treatment and hindering cross-regional clinical and epidemiological study comparisons. Objective: To develop an international consensus definition and diagnostic criteria for GPP using the modified Delphi method. Evidence Review: The rarity of GPP presents a challenge in acquiring comprehensive published clinical data necessary for developing standardized definition and criteria. Instead of relying on a literature search, 43 statements that comprehensively addressed the fundamental aspects of the definitions and diagnostic criteria for GPP were formulated based on expert reviews of 64 challenging GPP cases. These statements were presented to a panel of 33 global GPP experts for voting, discussion, and refinements in 2 virtual consensus meetings. Consensus during voting was defined as at least 80% agreement; the definition and diagnostic criteria were accepted by all panelists after voting and in-depth discussion. Findings: In the first and second modified Delphi round, 30 (91%) and 25 (76%) experts participated. In the initial Delphi round, consensus was achieved for 53% of the statements, leading to the approval of 23 statements that were utilized to develop the proposed definitions and diagnostic criteria for GPP. During the second Delphi round, the final definition established was, "Generalized Pustular Psoriasis is a systemic inflammatory disease characterized by cutaneous erythema and macroscopically visible sterile pustules." It can occur with or without systemic symptoms, other psoriasis types, and laboratory abnormalities. GPP may manifest as an acute form with widespread pustules or a subacute variant with an annular phenotype. The identified essential criterion was, "Macroscopically visible sterile pustules on erythematous base and not restricted to the acral region or within psoriatic plaques." Conclusions and Relevance: The achievement of international consensus on the definition and diagnostic criteria for GPP underscores the importance of collaboration, innovative methodology, and expert engagement to address rare diseases. Although further validation is needed, these criteria can serve as a reference point for clinicians, researchers, and patients, which may contribute to more accurate diagnosis and improved management of GPP.

ERK hyperactivation in epidermal keratinocytes impairs intercellular adhesion and drives Grover disease pathology.

Grover disease is an acquired dermatologic disorder characterized by pruritic vesicular and eroded skin lesions. While its pathologic features are well-defined, including impaired cohesion of epidermal keratinocytes, the etiology of Grover disease remains unclear and it lacks any FDA-approved therapy. Interestingly, drug-induced Grover disease occurs in patients treated with B-RAF inhibitors that can paradoxically activate C-RAF and the downstream kinase MEK. We recently identified hyperactivation of MEK and ERK as key drivers of Darier disease, which is histologically identical to Grover disease, supporting our hypothesis that they share a pathogenic mechanism. To model drug-induced Grover disease, we treated human keratinocytes with clinically utilized B-RAF inhibitors dabrafenib or vemurafenib and leveraged a fluorescent biosensor to confirm they activated ERK, which disrupted intercellular junctions and compromised keratinocyte sheet integrity. Consistent with clinical data showing concomitant MEK blockade prevents Grover disease in patients receiving B-RAF inhibitors, we found that MEK inhibition suppressed excess ERK activity to rescue cohesion of B-RAF-inhibited keratinocytes. Validating these results, we demonstrated ERK hyperactivation in skin biopsies of vemurafenib-induced Grover disease, but also in spontaneous Grover disease. In sum, our data define a pathogenic role for ERK hyperactivation in Grover disease and support MEK inhibition as a therapeutic strategy.

A Critical Role for IFN-ß Signaling for IFN-κ Induction in Keratinocytes.

Background/Purpose: Cutaneous lupus erythematosus (CLE) affects up to 70% of patients with systemic lupus erythematosus (SLE), and type I interferons (IFNs) are important promoters of SLE and CLE. Our previous work identified IFN-kappa (IFN-κ), a keratinocyte-produced type I IFN, as upregulated in non-lesional and lesional lupus skin and as a critical regulator for enhanced UVB-mediated cell death in SLE keratinocytes. Importantly, the molecular mechanisms governing regulation of IFN-κ expression have been relatively unexplored. Thus, this study sought to identify critical regulators of IFN-κ and identified a novel role for IFN-beta (IFN-ß). Methods: Human N/TERT keratinocytes were treated with the RNA mimic poly (I:C) or 50 mJ/cm2 ultraviolet B (UVB), followed by mRNA expression quantification by RT-qPCR in the presence or absence neutralizing antibody to the type I IFN receptor (IFNAR). IFNB and STAT1 knockout (KO) keratinocytes were generated using CRISPR/Cas9. Results: Time courses of poly(I:C) and UVB treatment revealed a differential expression of IFNB, which was upregulated between 3-6 hours and IFNK, which was upregulated 24 hours after stimulation. Intriguingly, only IFNK expression was substantially abrogated by neutralizing antibodies to IFNAR, suggesting that IFNK upregulation required type I IFN signaling for induction. Indeed, deletion of IFNB abrogated IFNK expression. Further exploration confirmed a role for type I IFN-triggered STAT1 activation. Conclusion: Collectively, our work describes a novel mechanistic paradigm in keratinocytes in which initial IFN-κ induction in response to poly(I:C) and UVB is IFNß1-dependent, thus describing IFNK as both an IFN gene and an interferon-stimulated gene.

Metabolic coordination between skin epithelium and type 17 immunity sustains chronic skin inflammation.

Inflammatory epithelial diseases are spurred by the concomitant dysregulation of immune and epithelial cells. How these two dysregulated cellular compartments simultaneously sustain their heightened metabolic demands is unclear. Single-cell and spatial transcriptomics (ST), along with immunofluorescence, revealed that hypoxia-inducible factor 1α (HIF1α), downstream of IL-17 signaling, drove psoriatic epithelial remodeling. Blocking HIF1α in human psoriatic lesions ex vivo impaired glycolysis and phenocopied anti-IL-17 therapy. In a murine model of skin inflammation, epidermal-specific loss of HIF1α or its target gene, glucose transporter 1, ameliorated epidermal, immune, vascular, and neuronal pathology. Mechanistically, glycolysis autonomously fueled epithelial pathology and enhanced lactate production, which augmented the γδ T17 cell response. RORγt-driven genetic deletion or pharmacological inhibition of either lactate-producing enzymes or lactate transporters attenuated epithelial pathology and IL-17A expression in vivo. Our findings identify a metabolic hierarchy between epithelial and immune compartments and the consequent coordination of metabolic processes that sustain inflammatory disease.

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.

Interferon alpha promotes caspase-8 dependent ultraviolet light-mediated keratinocyte apoptosis via interferon regulatory factor 1.

Introduction: Ultraviolet (UV) light is a known trigger of both cutaneous and systemic disease manifestations in lupus patients. Lupus skin has elevated expression of type I interferons (IFNs) that promote increased keratinocyte (KC) death after UV exposure. The mechanisms by which KC cell death is increased by type I IFNs are unknown. Methods: Here, we examine the specific cell death pathways that are activated in KCs by type I IFN priming and UVB exposure using a variety of pharmacological and genetic approaches. Mice that overexpress Ifnk in the epidermis were exposed to UVB light and cell death was measured. RNA-sequencing from IFN-treated KCs was analyzed to identify candidate genes for further analysis that could drive enhanced cell death responses after UVB exposure. Results: We identify enhanced activation of caspase-8 dependent apoptosis, but not other cell death pathways, in type I IFN and UVB-exposed KCs. In vivo, overexpression of epidermal Ifnk resulted in increased apoptosis in murine skin after UVB treatment. This increase in KC apoptosis was not dependent on known death ligands but rather dependent on type I IFN-upregulation of interferon regulatory factor 1 (IRF1). Discussion: These data suggest that enhanced sensitivity to UV light exhibited by lupus patients results from type I IFN priming of KCs that drives IRF1 expression resulting in caspase-8 activation and increased apoptosis after minimal exposures to UVB.

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.

Vaccination recommendations for adults receiving biologics and oral therapies for psoriasis and psoriatic arthritis: Delphi consensus from the medical board of the National Psoriasis Foundation.

BACKGROUND: For psoriatic patients who need to receive nonlive or live vaccines, evidence-based recommendations are needed regarding whether to pause or continue systemic therapies for psoriasis and/or psoriatic arthritis. OBJECTIVE: To evaluate literature regarding vaccine efficacy and safety and to generate consensus-based recommendations for adults receiving systemic therapies for psoriasis and/or psoriatic arthritis receiving nonlive or live vaccines. METHODS: Using a modified Delphi process, 22 consensus statements were developed by the National Psoriasis Foundation Medical Board and COVID-19 Task Force, and infectious disease experts. RESULTS: Key recommendations include continuing most oral and biologic therapies without modification for patients receiving nonlive vaccines; consider interruption of methotrexate for nonlive vaccines. For patients receiving live vaccines, discontinue most oral and biologic medications before and after administration of live vaccine. Specific recommendations include discontinuing most biologic therapies, except for abatacept, for 2-3 half-lives before live vaccine administration and deferring next dose 2-4 weeks after live vaccination. LIMITATIONS: Studies regarding infection rates after vaccination are lacking. CONCLUSION: Interruption of antipsoriatic oral and biologic therapies is generally not necessary for patients receiving nonlive vaccines. Temporary interruption of oral and biologic therapies before and after administration of live vaccines is recommended in most cases.

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.

Epidermal ZBP1 stabilizes mitochondrial Z-DNA to drive UV-induced IFN signaling in autoimmune photosensitivity.

Photosensitivity is observed in numerous autoimmune diseases and drives poor quality of life and disease flares. Elevated epidermal type I interferon (IFN) production primes for photosensitivity and enhanced inflammation, but the substrates that sustain and amplify this cycle remain undefined. Here, we show that IFN-induced Z-DNA binding protein 1 (ZBP1) stabilizes ultraviolet (UV)B-induced cytosolic Z-DNA derived from oxidized mitochondrial DNA. ZBP1 is significantly upregulated in the epidermis of adult and pediatric patients with autoimmune photosensitivity. Strikingly, lupus keratinocytes accumulate extensive cytosolic Z-DNA after UVB, and transfection of keratinocytes with Z-DNA results in stronger IFN production through cGAS-STING activation compared to B-DNA. ZBP1 knockdown abrogates UV-induced IFN responses, whereas overexpression results in a lupus-like phenotype with spontaneous Z-DNA accumulation and IFN production. Our results highlight Z-DNA and ZBP1 as critical mediators for UVB-induced inflammation and uncover how type I IFNs prime for cutaneous inflammation in photosensitivity.

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.

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.