Tumors evade immunity through the overexpression of immune inhibitory molecules in the tumor microenvironment such as PD-L1/B7-H1. An immune inhibitory molecule named PD-1 homolog (also known as V-domain Ig-containing suppressor of T cell activation [VISTA]) functions to control both T cells and myeloid cells. Current clinical trials using anti-VISTA-blocking agents for treatment of cancer are ongoing. We sought to determine the extent of VISTA expression in primary cutaneous melanomas (n = 190), identify the critical cell types expressing VISTA, and correlate its expression with PD-L1 expression using multiplexed quantitative immunofluorescence. Within the tumor subcompartments, VISTA is most highly expressed on CD11b myeloid cells, and PD-L1 is most highly expressed on CD68 myeloid cells in our melanoma cohort. There is little correlation between VISTA and PD-L1 expression intensity, suggesting that individual tumors have distinct immunosuppressive tumor microenvironments. High levels of VISTA expression on CD11b myeloid cells but not PD-L1 expression were associated with greater melanoma recurrence and greater all-cause mortality. Our findings suggest that cell-specific VISTA expression may be a negative prognostic biomarker for melanoma and a future potential therapeutic target.

Absorption of dietary iron is largely regulated by the liver hormone hepcidin, which is released under conditions of iron overload and inflammation. Although hepcidin-dependent regulation of iron uptake and circulation is well-characterized, recent studies have suggested that the skin may play an important role in iron homeostasis, including transferrin receptor-mediated epidermal iron uptake and direct hepcidin production by keratinocytes. In this study, we characterized direct keratinocyte responses to conditions of high and low iron. We observed potent iron storage capacity by keratinocytes in vitro and in vivo and the effects of iron on epidermal differentiation and gene expression associated with inflammation and barrier function. In mice, systemic iron was observed to be coupled to epidermal iron content. Furthermore, topical inflammation, as opposed to systemic inflammation, resulted in a primary iron-deficiency phenotype associated with low liver hepcidin. These studies suggest a role for keratinocytes and epidermal iron storage as regulators of iron homeostasis with direct contribution by the cutaneous inflammatory state.

Mycosis fungoides with large-cell transformation (MF-LCT) occurs in a minor proportion of aggressive lesions, which express CD30 similar to primary cutaneous anaplastic large-cell lymphoma (cALCL). We investigated differences in spatially resolved transcriptome profiles of MF-LCT and cALCL using CD30 morphology markers and 28 and 24 regions of interest (ROIs) in MF-LCT and cALCL, respectively. Differentially expressed genes (DEGs), pathway analysis, and immune-cell deconvolution by selective analysis of CD30-positive tumor-cell and CD30-negative extra-tumoral areas were undertaken. In CD30-postive ROIs of MF-LCT, 190 DEGs were upregulated (29 were directly or indirectly associated with extracellular matrix [ECM] remodeling), whereas 255 DEGs were downregulated compared to those of cALCL. Except for cornified envelope formation and keratinization, all six pathways enriched in CD30-positive ROIs of MF-LCT were associated with ECM remodeling. In CD30-positive ROIs in MF-LCT compared to cALCL, immune-cell deconvolution revealed significantly increased fibroblasts and M2 macrophages (p=0.012 and p=0.023, respectively) but decreased M1 macrophages (p=0.031). In CD30-negative ROIs in MF-LCT compared to cALCL, memory B (p=0.021), plasma (p=0.023), and CD8 memory T (p=0.001) cells significantly decreased whereas regulatory T cells (p=0.024) increased. Predomination of ECM remodeling pathways and immunosuppressive microenvironment in MF-LCT indicates pathophysiological differences from MF-LCT and cALCL.

Innate lymphoid cells (ILCs) are essential players in the skin-associated immune system, nevertheless little is known about their proteomes and proteomic diversity. Here, we describe over 6,600 proteins constitutively expressed by ILC2s and ILC3s from healthy human skin and blood using state-of-the-art proteomics. Although the vast majority of proteins was expressed by both ILC subsets and in both compartments, the skin ILC2s and ILC3s were more distinct than their counterparts in blood. Only skin ILC3s expressed uniquely detected proteins. Our in-depth proteomic dataset allowed us to define the cluster of differentiation marker profiles of the ILC subsets, explore distribution and abundance of proteins known to have immunological functions, as well as identify subset-specific proteins that have not previously been implicated in ILC biology. Taken together, our analyses substantially expand understanding of the protein expression signatures of ILC subsets. Going forward, these proteomic datasets will serve as valuable resources for future studies of ILC biology.

The p63 transcription factor is critical for epidermis formation in embryonic development, but its role in the adult epidermis is poorly understood. Here we show that acute genetic ablation of ΔNp63, the main p63 isoform, in adult epidermis disrupts keratinocyte proliferation and self-maintenance and, unexpectedly, triggers an inflammatory psoriasis-like condition. Mechanistically, single-cell RNA sequencing revealed down-regulation of cell cycle genes, up-regulation of differentiation markers, and induction of several pro-inflammatory pathways in ΔNp63-ablated keratinocytes. Intriguingly, ΔNp63-ablated cells disappear by 3 weeks post-ablation, at the expense of the remaining non-ablated cells. This is not associated with active cell death and is likely due to reduced self-maintenance and enhanced differentiation. Indeed, in vivo wound healing, a physiological readout of the epidermal stem cell function, is severely impaired upon ΔNp63 ablation. We found that the Wnt signaling pathway (Wnt10a, Fzd6, Fzd10) and the AP1 factors (JunB, Fos, FosB) are the likely ΔNp63 effectors responsible for keratinocyte proliferation/stemness and suppression of differentiation, respectively, while interleukins IL-1a, IL-18, IL-24, and IL-36γ are the likely negative effectors responsible for suppression of inflammation. These data establish ΔNp63 as a critical node that coordinates epidermal homeostasis, stemness, and suppression of inflammation, upstream of known regulatory pathways.

Wound healing is a complex process involving phases of hemostasis, inflammation, proliferation, and remodeling. The regenerative process in the skin requires coordination between many regulators, including signaling molecules, transcription factors, and the epigenetic machinery. In this study, we show that chromatin regulators HDAC1 and LSD1, key components of the CoREST repressor complex, are upregulated in the regenerating epidermis during wound repair. We also show that corin, a synthetic dual inhibitor of the CoREST complex and HDAC1/LSD1 activities, significantly accelerates wound closure through enhanced re-epithelialization in a mouse tail wound model. Acetylated H3K9 (methylation of histone H3 at lysine 9) expression, a histone modification targeted by HDAC1, is increased in keratinocytes after topical treatment with 100 nM and 1 μM of corin. In vitro experiments demonstrate that corin promotes migration and inhibits the proliferation of human keratinocytes. Furthermore, expression levels of genes promoting keratinocyte migration, such as AREG, CD24, EPHB2, ITGAX, PTGS, SCT1, SERPINB2, SERPINE1, SLPI, SNAI2, and TWIST, increased in keratinocytes treated with corin. These data demonstrate that dual inhibition of class I histone deacetylases and LSD1 by corin may serve as a new approach for promoting wound re-epithelialization and provide a platform for further applications of corin for the treatment of chronic wounds.

Excessive oxidative stress is thought to play pathologic roles in cellular senescence and autoimmune disorders by inducing inflammation and breaking down immune tolerance. In this study, we sought to identify the factors linking oxidative stress to autoimmunity and cellular senescence in vitiligo, where elevated oxidative stress plays an important role. RNA sequencing analysis of H2O2-treated melanocytes revealed upregulation of interferon-stimulated gene 15 (ISG15). The upregulation of ISG15 was observed in vitiligo skin tissues as well as the blood of vitiligo patients, while ubiquitin specific peptidase 18 (USP18) downregulation was observed in vitiligo melanocytes and vitiligo skin tissues. Oxidative stress induced hypermethylation of the USP18 promoter region in keratinocytes and melanocytes and USP18 promoter hypermethylation was also confirmed in vitiligo skin tissues. Our results indicate that USP18 promoter hypermethylation caused by oxidative stress increases ISG15 expression in keratinocytes and melanocytes along with senescence changes, leading CD8+ T cells to produce IFN-γ, the main pathogenic cytokine in vitiligo. Therefore, the ISG15-USP18 network may be important in oxidative stress-induced autoimmunity and cellular senescence in vitiligo pathogenesis.

Diabetic chronic ulcers are plagued with persistent non-resolving inflammation. However, diabetic wound environment early after injury suffers from inadequate inflammatory responses due to reductions the proinflammatory cytokines levels. Diabetic neutrophils have known impairments in bactericidal functions. We hypothesized that reduced bacterial killing by diabetic neutrophils, due to their bactericidal functional impairments, results in reduced bioactive bacterial products, known as pathogen associated molecular patterns (PAMPs), which in turn contribute to reduced signaling through TLRs, leading to inadequate production of proinflammatory cytokines in infected diabetic wound early after injury. We tested our hypothesis in db/db type 2 obese diabetic mouse wound infection model with Pseudomonas aeruginosa. Our data indicate that despite substantially higher levels of infection, TLR4-mediated signaling is reduced in diabetic wound early after injury, due to reduced bioactive levels of lipopolysaccharide (LPS). We further demonstrate that topical treatment with LPS enhances TLR4 signaling, increases proinflammatory cytokine production, restores leukocytes trafficking, reduces infection burden, and stimulates healing in diabetic wound. We posit that LPS may be a viable therapeutic option for the treatment of diabetic foot ulcers if it is applied topically after the surgical debridement process which is intended to reset chronic ulcers into acute fresh wounds.

Efficient clearance of dead cells is critical for tissue regeneration after injuries. How dead cells are removed from the skin after radiotherapy and chemotherapy is unclear. In this study, we found that radiotherapeutic and chemotherapeutic damage induced extensive apoptosis of highly proliferative transit-amplifying cells in hair follicles. These apoptotic cells disappeared rapidly in the early stage of regenerative attempts, and the lost structures were regenerated with transient and low-level inflammation. Without the recruitment of macrophages as scavengers, the dying cells were engulfed directly by adjacent surviving transit-amplifying cells, which produced mature phagosomes through fusion with lysosomes in a manner similar to professional phagocytosis and remained active in proliferation. Autophagy did not contribute significantly to the clearance of engulfed cell debris. Perturbing phagocytosis in the transit-amplifying cells hindered apoptotic cell removal, delayed structural recovery, and aggravated hair loss. Therefore, transit-amplifying cells are capacitated with both proliferative and efferocytic functions that facilitate tissue regeneration after tissue injury.

Tissue transcriptomics is used to uncover molecular dysregulations underlying diseases. However, the majority of transcriptomics studies focus on single diseases with limited relevance for understanding the molecular relationship between diseases or for identifying disease-specific markers. Here, we used a normalization approach to compare gene expression across nine inflammatory skin diseases. The normalized datasets were found to retain differential expression signals that allowed unsupervised disease clustering and identification of disease-specific gene signatures. Using the NS-Forest algorithm, we identified a minimal set of biomarkers and validated their use as diagnostic disease classifier. Among them, PTEN was identified as being a specific marker for cutaneous lupus erythematosus (CLE) and found to be strongly expressed by lesional keratinocytes in association with pathogenic type I interferons (IFNs). In fact, PTEN facilitated expression of IFN-β and IFN-κ in keratinocytes by promoting activation and nuclear translocation of IRF3. Thus, cross-comparison of tissue transcriptomics is a valid strategy to establish a molecular disease classification and identify pathogenic disease biomarkers.

Vitiligo is the most common depigmenting skin disorder. Given the ongoing development of new targeted therapies, it has become important to evaluate adequately the surface area involved. Assessment of vitiligo scores can be time-consuming with variation between investigators. Therefore, the aim of this study was to build an artificial intelligence (AI) system capable of assessing facial vitiligo severity. One hundred pictures of vitiligo patients' faces were used to train and validate the AI model. Sixty-nine additional pictures of facial vitiligo were then used as a final data set. Three expert physicians scored the facial vitiligo on the same 69 pictures. Inter- and intra-rater performances were evaluated by comparing the scores between raters and AI. Algorithm assessment achieved an accuracy of 93%. Overall, the scores reached good agreement between vitiligo raters and the AI model. Results demonstrate the potential of the model. It provides an objective evaluation of facial vitiligo and could become a complementary/alternative tool to human assessment in clinical practice and/or clinical research.

In SARS-CoV-2 infected patients, vasculopathic changes of the skin are associated with a severe prognosis. However, the pathogenesis of this vasculopathy is not conclusively clarified. In this study, 25 prospectively collected skin samples from patients with COVID-19 related skin lesions were examined for vasculopathic changes and in case of vasculitis further analyzed with electron microscopy and immunohistochemistry. Vasculopathy was observed in 76 % of all COVID-19 related inflammatory skin lesions. Visual endothelial changes without manifest leukocytoclastic vasculitis were found in 60 % of the COVID-19 related skin lesions, whereas leukocytoclastic vasculitis was diagnosed in 16 %. In the cases of vasculitis, there were extensive spike protein depositions in microvascular endothelial cells that colocalized with the autophagosome proteins LC3B and LC3C. The autophagy protein complex LC3-associated endocytosis in microvascular endothelial cells seems to be an important pathogenic factor for SARS-CoV-2 related vasculitis in the skin. On ultrastructural morphology, the vasculitic process was dominated by intracellular vesicle formation and endothelial cell disruption. Direct presence of SARS-CoV-2 particles in the skin was not observed. Therefore, our results suggest that instead of direct viral infection, dermal vasculitic lesions in COVID-19 are caused by SARS-CoV-2 spike protein deposition followed by endothelial damage with the activation of autophagy.