Receptor Tyrosine Kinase EPHA2 Drives Epidermal Differentiation through Regulation of EGFR Signaling.

Intricate signaling systems are required to maintain homeostasis and promote differentiation in the epidermis. Receptor tyrosine kinases are central in orchestrating these systems in epidermal keratinocytes. In particular, EPHA2 and EGFR transduce distinct signals to dictate keratinocyte fate, yet how these cell communication networks are integrated has not been investigated. Our work shows that loss of EPHA2 impairs keratinocyte stratification, differentiation, and barrier function. To determine the mechanism of this dysfunction, we drew from our proteomics data of potential EPHA2 interacting proteins. We identified EGFR as a high-ranking EPHA2 interactor and subsequently validated this interaction. We found that when EPHA2 is reduced, EGFR activation and downstream signaling are intensified and sustained. Evidence indicates that prolonged SRC association contributes to the increase in EGFR signaling. We show that hyperactive EGFR signaling underlies the differentiation defect caused by EPHA2 knockdown because EGFR inhibition restores differentiation in EPHA2-deficient 3-dimensional skin organoids. Our data implicate a mechanism whereby EPHA2 restrains EGFR signaling, allowing for fine tuning in the processes of terminal differentiation and barrier formation. Taken together, we purport that crosstalk between receptor tyrosine kinases EPHA2 and EGFR is critical for epidermal differentiation.

Substance use among pregnant women in NSW prisons.

INTRODUCTION AND AIMS: Little is published about pregnant women in custody. Existing data on the prevalence of substance use among incarcerated pregnant women or their needs are scant. This study sought to determine the prevalence and characteristics of women with substance use histories who present to prison pregnant in New South Wales (NSW), Australia. DESIGN AND METHODS: A retrospective review of prison medical health records was completed for pregnant women entering New South Wales custodial settings between January 2020 and June 2021. RESULTS: We identified 158 prison receptions among 141 pregnant women (median age 28 years [IQR 25-33 years]), 42 % identified as Aboriginal or Torres Strait Islander. Eighty four percent of the women (n = 119) reported recent use of one or more than one substance and 36 % had injected drugs. The most commonly used substances were (meth)amphetamine (60 %), cannabis (40 %) and opioids (28 %). We found discrepancies between drug problems self-reported at reception screening on entry to prison and self-reported drug use collected during the subsequent drug and alcohol specialist assessment while incarcerated. Most (88 %) women described their current pregnancy as unplanned and half (52 %) were unaware they were pregnant before incarceration. CONCLUSIONS: These findings highlight the high prevalence of substance use in incarcerated pregnant women and that many women are unaware of their pregnancy prior to incarceration. Findings emphasize the importance of timely and appropriate drug and alcohol assessment and treatment to minimize harm for both the mother and foetus and also underscore the urgent need for enhanced access to contraception for these women.

GWAS meta-analysis of psoriasis identifies new susceptibility alleles impacting disease mechanisms and therapeutic targets.

Psoriasis is a common, debilitating immune-mediated skin disease. Genetic studies have identified biological mechanisms of psoriasis risk, including those targeted by effective therapies. However, the genetic liability to psoriasis is not fully explained by variation at robustly identified risk loci. To move towards a saturation map of psoriasis susceptibility we meta-analysed 18 GWAS comprising 36,466 cases and 458,078 controls and identified 109 distinct psoriasis susceptibility loci, including 45 that have not been previously reported. These include susceptibility variants at loci in which the therapeutic targets IL17RA and AHR are encoded, and deleterious coding variants supporting potential new drug targets (including in STAP2, CPVL and POU2F3). We conducted a transcriptome-wide association study to identify regulatory effects of psoriasis susceptibility variants and cross-referenced these against single cell expression profiles in psoriasis-affected skin, highlighting roles for the transcriptional regulation of haematopoietic cell development and epigenetic modulation of interferon signalling in psoriasis pathobiology.

Skin Infiltrate Composition as a Telling Measure of Responses to Checkpoint Inhibitors.

Checkpoint inhibitors treat a variety of tumor types with significant benefits. Unfortunately, these therapies come with diverse adverse events. Skin rash is observed early into treatment and might serve as an indicator of downstream responses to therapy. We studied the cellular composition of cutaneous eruptions and whether their contribution varies with the treatment applied. Skin samples from 18 patients with cancer and 11 controls were evaluated by mono- and multiplex imaging, quantification, and statistical analysis. T cells were the prime contributors to skin rash, with T cells and macrophages interacting and proliferating on site. Among T cell subsets examined, type 1 and 17 T cells were relatively increased among inflammatory skin infiltrates. A combination of increased cytotoxic T cell content and decreased macrophage abundance was associated with dual checkpoint inhibition over PD1 inhibition alone. Importantly, responders significantly separated from nonresponders by greater CD68+ macrophage and either CD11c+ antigen-presenting cell or CD4+ T cell abundance in skin rash. The microenvironment promoted epidermal proliferation and thickening as well. The combination of checkpoint inhibitors used affects the development and composition of skin infiltrates, whereas the combined abundance of two cell types in cutaneous eruptions aligns with responses to checkpoint inhibitor therapy.

Health and correctional staff acceptability of depot buprenorphine in NSW prisons.

BACKGROUND: Provision of opioid agonist treatment (OAT) in custodial settings is resource-intensive and may be associated with diversion, non-medical use, and violence. A clinical trial of a new OAT, depot buprenorphine (the UNLOC-T study), provided the opportunity to obtain health and correctional staff perspectives regarding this treatment prior to widespread roll-out. METHODS: Sixteen focus groups with 52 participants were conducted, including 44 health staff (nurses, nurse practitioners, doctors, and operational staff) and eight correctional staff. RESULTS: Key challenges to providing OAT identified as potentially being addressed by depot buprenorphine included 1) patient access, 2) OAT program capacity, 3) treatment administration procedures, 4) medication diversion and other safety issues and, 5) impact on other service delivery. CONCLUSIONS: The introduction of depot buprenorphine into correctional settings was considered to have the potential to increase safety for patients, improve staff / patient relations and advance patient health outcomes via expanded treatment coverage and efficiencies gained through enhanced health service delivery. Support was almost universal from both correctional and health staff participating in this study. These findings build on emerging research regarding the positive impact of more flexible OAT programs and could be used to engage support for the implementation of depot buprenorphine from staff in other secure settings.

Keratinocytes sense and eliminate CRISPR DNA through STING/IFN-κ activation and APOBEC3G induction.

CRISPR/Cas9 has been proposed as a treatment for genetically inherited skin disorders. Here we report that CRISPR transfection activates STING-dependent antiviral responses in keratinocytes, resulting in heightened endogenous interferon (IFN) responses through induction of IFN-κ, leading to decreased plasmid stability secondary to induction of the cytidine deaminase gene APOBEC3G. Notably, CRISPR-generated KO keratinocytes had permanent suppression of IFN-κ and IFN-stimulated gene (ISG) expression, secondary to hypermethylation of the IFNK promoter region by the DNA methyltransferase DNMT3B. JAK inhibition via baricitinib prior to CRISPR transfection increased transfection efficiency, prevented IFNK promoter hypermethylation, and restored normal IFN-κ activity and ISG responses. This work shows that CRISPR-mediated gene correction alters antiviral responses in keratinocytes, has implications for future gene therapies for inherited skin diseases using CRISPR technology, and suggests pharmacologic JAK inhibition as a tool for facilitating and attenuating inadvertent selection effects in CRISPR/Cas9 therapeutic approaches.

Nucleoli and the nucleoli-centromere association are dynamic during normal development and in cancer.

Centromeres are known to cluster around nucleoli in Drosophila and mammalian cells, but the significance of the nucleoli-centromere interaction remains underexplored. To determine whether the interaction is dynamic under different physiological and pathological conditions, we examined nucleolar structure and centromeres at various differentiation stages using cell culture models and the results showed dynamic changes in nucleolar characteristics and nucleoli-centromere interactions through differentiation and in cancer cells. Embryonic stem cells usually have a single large nucleolus, which is clustered with a high percentage of centromeres. As cells differentiate into intermediate states, the nucleolar number increases and the centromere association decreases. In terminally differentiated cells, including myotubes, neurons, and keratinocytes, the number of nucleoli and their association with centromeres are at the lowest. Cancer cells demonstrate the pattern of nucleoli number and nucleoli-centromere association that is akin to proliferative cell types, suggesting that nucleolar reorganization and changes in nucleoli-centromere interactions may play a role in facilitating malignant transformation. This idea is supported in a case of pediatric rhabdomyosarcoma, in which induced differentiation reduces the nucleolar number and centromere association. These findings suggest active roles of nucleolar structure in centromere function and genome organization critical for cellular function in both normal development and cancer.

Reduced SPAG17 Expression in Systemic Sclerosis Triggers Myofibroblast Transition and Drives Fibrosis.

Systemic sclerosis (SSc) is a clinically heterogeneous fibrotic disease with no effective treatment. Myofibroblasts are responsible for unresolving synchronous skin and internal organ fibrosis in SSc, but the drivers of sustained myofibroblast activation remain poorly understood. Using unbiased transcriptome analysis of skin biopsies, we identified the downregulation of SPAG17 in multiple independent cohorts of patients with SSc, and by orthogonal approaches, we observed a significant negative correlation between SPAG17 and fibrotic gene expression. Fibroblasts and endothelial cells explanted from SSc skin biopsies showed reduced chromatin accessibility at the SPAG17 locus. Remarkably, mice lacking Spag17 showed spontaneous skin fibrosis with increased dermal thickness, collagen deposition and stiffness, and altered collagen fiber alignment. Knockdown of SPAG17 in human and mouse fibroblasts and microvascular endothelial cells was accompanied by spontaneous myofibroblast transformation and markedly heightened sensitivity to profibrotic stimuli. These responses were accompanied by constitutive TGF-ß pathway activation. Thus, we discovered impaired expression of SPAG17 in SSc and identified, to our knowledge, a previously unreported cell-intrinsic role for SPAG17 in the negative regulation of fibrotic responses. These findings shed fresh light on the pathogenesis of SSc and may inform the search for innovative therapies for SSc and other fibrotic conditions through SPAG17 signaling.

The Desmosome-Keratin Scaffold Integrates ErbB Family and Mechanical Signaling to Polarize Epidermal Structure and Function.

While classic cadherin-actin connections in adherens junctions (AJs) have ancient origins, intermediate filament (IF) linkages with desmosomal cadherins arose in vertebrate organisms. In this mini-review, we discuss how overlaying the IF-desmosome network onto the existing cadherin-actin network provided new opportunities to coordinate tissue mechanics with the positioning and function of chemical signaling mediators in the ErbB family of receptor tyrosine kinases. We focus in particular on the complex multi-layered outer covering of the skin, the epidermis, which serves essential barrier and stress sensing/responding functions in terrestrial vertebrates. We will review emerging data showing that desmosome-IF connections, AJ-actin interactions, ErbB family members, and membrane tension are all polarized across the multiple layers of the regenerating epidermis. Importantly, their integration generates differentiation-specific roles in each layer of the epidermis that dictate the form and function of the tissue. In the basal layer, the onset of the differentiation-specific desmosomal cadherin desmoglein 1 (Dsg1) dials down EGFR signaling while working with classic cadherins to remodel cortical actin cytoskeleton and decrease membrane tension to promote cell delamination. In the upper layers, Dsg1 and E-cadherin cooperate to maintain high tension and tune EGFR and ErbB2 activity to create the essential tight junction barrier. Our final outlook discusses the emerging appreciation that the desmosome-IF scaffold not only creates the architecture required for skin's physical barrier but also creates an immune barrier that keeps inflammation in check.

Cytokine responses in nonlesional psoriatic skin as clinical predictor to anti-TNF agents.

BACKGROUND: A major issue with the current management of psoriasis is our inability to predict treatment response. OBJECTIVE: Our aim was to evaluate the ability to use baseline molecular expression profiling to assess treatment outcome for patients with psoriasis. METHODS: We conducted a longitudinal study of 46 patients with chronic plaque psoriasis treated with anti-TNF agent etanercept, and molecular profiles were assessed in more than 200 RNA-seq samples. RESULTS: We demonstrated correlation between clinical response and molecular changes during the course of the treatment, particularly for genes responding to IL-17A/TNF in keratinocytes. Intriguingly, baseline gene expressions in nonlesional, but not lesional, skin were the best marker of treatment response at week 12. We identified USP18, a known regulator of IFN responses, as positively correlated with Psoriasis Area and Severity Index (PASI) improvement (P = 9.8 × 10-4) and demonstrate its role in regulating IFN/TNF responses in keratinocytes. Consistently, cytokine gene signatures enriched in baseline nonlesional skin expression profiles had strong correlations with PASI improvement. Using this information, we developed a statistical model for predicting PASI75 (ie, 75% of PASI improvement) at week 12, achieving area under the receiver-operating characteristic curve value of 0.75 and up to 80% accurate PASI75 prediction among the top predicted responders. CONCLUSIONS: Our results illustrate feasibility of assessing drug response in psoriasis using nonlesional skin and implicate involvement of IFN regulators in anti-TNF responses.

Social isolation among older veterans: findings from the National Health and Aging Trends Study.

OBJECTIVES: Social isolation is a critical public health issue that affects multiple domains of well-being among older adults, but little is known about social isolation among older military veterans. As such, the purpose of this study was to estimate the prevalence of social isolation among older veterans and to examine risk factors for social isolation among older veterans. METHOD: Data were derived from Round 1 of the National Health and Aging Trends Study, which is an annual, longitudinal panel survey of Medicare beneficiaries aged 65 and older. The sample included 1,683 veterans, who were primarily White and male. Weighted logistic regression models were used to predict severe social isolation (having no social participation) and social isolation (having only one source of social participation) among older veterans, while controlling for age, sex, race, marital status, education, income, and metropolitan residency. RESULTS: After accounting for other predictors, results show that veterans who are 85 and older, male, White, unmarried or unpartnered, with lower educational attainment and lower income are greatly at risk of both severe social isolation and social isolation. CONCLUSION: The results of this study support past research showing that veterans with limited social and economic capital are at great risk of experiencing adverse outcomes in older adulthood, including social isolation. Interventions should therefore aim to improve social connectedness among this population and should address the risk-factors that contribute to social isolation among older veterans.

Inhibition of dipeptidyl-peptidase 4 induces upregulation of the late cornified envelope cluster in keratinocytes.

Dipeptidyl-peptidase 4 (DPP4) is a multifunctional type II transmembrane glycoprotein that is expressed on various cell surfaces. While DPP4 inhibitors have a therapeutic role in the treatment of diabetes mellitus, they are an independent risk factor in the development of bullous pemphigoid. Contrarily, there are reports of improvement in psoriasis with DPP4 inhibition. We investigated the effect of DPP4 inhibition on primary human keratinocytes to determine whether DPP4 modulates keratinocyte inflammatory signaling and keratinocyte homeostasis. We performed RNA sequencing of primary adult human keratinocytes treated with DPP4 inhibitor, identifying 424 differentially expressed genes. Gene ontology analysis revealed significant enrichment of epidermal differentiation and cornified envelope genes. Using three-dimensional organotypic cultures and a pan-late cornified envelope 2 (LCE2) antibody, we demonstrate a dose dependent relationship between DPP4 inhibition and increased expression of LCE2 during epidermal development. The late cornified envelope gene clusters are expressed at the late stages of epithelial development, responding to stimuli such as calcium and ultraviolet light. While its biologic function is not fully understood, mutations in LCE3B/LCE3C confer a 40% increased risk in the development of plaque psoriasis. While we did not identify significant modulation of keratinocyte inflammatory markers, DPP4 inhibition increased expression of the late cornified envelope may offer a potential alternative therapeutic mechanism in psoriasis.

Treatment of opioid dependence with depot buprenorphine (CAM2038) in custodial settings.

BACKGROUND AND AIMS: Opioid agonist treatment is effective but resource intensive to administer safely in custodial settings, leading to significant under-treatment of opioid dependence in these settings world-wide. This study assessed the safety of subcutaneous slow-release depot buprenorphine in custody. DESIGN: Open-label, non-randomized trial. SETTING: Correctional centres in New South Wales, Australia. PARTICIPANTS: Sixty-seven men and women, aged ≥ 18 years of various security classifications with a diagnosis of moderate to severe DSM-5 opioid use disorder currently serving a custodial sentence of ≥ 6 months were recruited between November 2018 and July 2019. Patients not in opioid agonist treatment at recruitment commenced depot buprenorphine; patients already stable on oral methadone treatment were recruited to the comparison arm. INTERVENTION AND COMPARATOR: Depot buprenorphine (CAM2038 weekly for 4 weeks then monthly) and daily oral methadone. MEASUREMENTS: Safety was assessed by adverse event (AE) monitoring and physical examinations at every visit. Participants were administered a survey assessing self-reported diversion and substance use at baseline and weeks 4 and 16. FINDINGS: Retention in depot buprenorphine treatment was 92.3%. Ninety-four per cent of patients reported at least one adverse event, typically mild and transient. No diversion was identified. The prevalence of self-reported non-prescribed opioid use among depot buprenorphine patients decreased significantly between baseline (97%) and week 16 (12%, odds ratio = 0.0035, 95% confidence interval = 0.0007-0.018, P < 0.0001). CONCLUSIONS: This first study of depot buprenorphine in custodial settings showed treatment retention and outcomes comparable to those observed in community settings and for other opioid agonist treatment used in custodial settings, without increased risk of diversion.

Gene expression profiling of laminin α3-blocked keratinocytes reveals an immune-independent mechanism of blistering.

Laminin-332 pemphigoid is a rare and chronic autoimmune blistering disease which results in subepidermal blisters and erosive lesions predominantly localized to mucous membranes. As histologic inflammation is variable and non-complement-fixing IgG antibodies against laminin-332 are the predominant class of autoantibodies deposited at the epidermal basement membrane zone, we hypothesized that complement-independent pro-inflammatory and blistering pathways existed similarly to that previously shown in bullous pemphigoid. As autoantibodies to laminin α3 are most prevalent, we studied the major cellular response to blockade of laminin α3 using a well-characterized monoclonal antibody (P3H9-2). RNA-seq revealed upregulation of numerous desmosomal genes (DSG1, DSG3, DSC1, DSC3 and DSP) as well as KRT1 and KRT10. Additionally, P3H9-2-treated cells demonstrated downregulation of most hemidesmosomal genes. A pro-inflammatory response was not appreciated. Using pharmacological inhibitors, we identified both protein kinase C and NOTCH as key regulators of P3H9-2 induced differentiation. We lastly utilized 3D human skin equivalents to determine whether blockade of laminin α3 would lead to delayed blistering, consistent with keratinocyte differentiation. Significant blistering was noted after 72 h of treatment, with only minimal separation at 24 h. In summary, blockade of laminin α3 alters keratinocyte differentiation, representing a potential complement-independent mechanism of blistering.

Transcriptome Analysis Reveals Intrinsic Proinflammatory Signaling in Healthy African American Skin.

Differences in the morphology and physiology of darkly pigmented skin compared with those of lightly pigmented skin are well-recognized. There are also disparities in the prevalence and clinical features for many inflammatory skin diseases, including atopic dermatitis and psoriasis; however, the underlying mechanisms are largely unknown. We compared the baseline gene expression in full-thickness skin biopsies from healthy individuals self-reporting as African American (AA) or as White non-Hispanic (WNH). Extensively validated RNA-sequencing analysis identified 570 differentially expressed genes in AA skin, including Igs and their receptors such as FCER1G; proinflammatory genes such as TNFα and IL32; and epidermal differentiation cluster and keratin genes. Differentially expressed genes were functionally enriched for inflammatory responses, keratinization, and cornified envelope formation. RNA-sequencing analysis of three-dimensional human skin equivalents made from AA and WNH primary keratinocytes revealed 360 differentially expressed genes (some shared with skin) that were enriched by similar functions. AA human skin equivalents appeared more responsive to TNF-α proinflammatory effects. Finally, AA-specific differentially expressed genes in the skin and human skin equivalents significantly overlapped with molecular signatures of skin in patients with atopic dermatitis and psoriasis. Overall, these findings suggest the existence of intrinsic proinflammatory circuits in AA keratinocytes/skin that may account for disease disparities and will help to build a foundation for the development of targeted skin disease prevention.

Ciliogenesis and autophagy are coordinately regulated by EphA2 in the cornea to maintain proper epithelial architecture.

PURPOSE: To understand the relationship between ciliogenesis and autophagy in the corneal epithelium. METHODS: siRNAs for EphA2 or PLD1 were used to inhibit protein expression in vitro. Morpholino-anti-EphA2 was used to knockdown EphA2 in Xenopus skin. An EphA2 knockout mouse was used to conduct loss of function studies. Autophagic vacuoles were visualized by contrast light microscopy. Autophagy flux, was measured by LC3 turnover and p62 protein levels. Immunostaining and confocal microscopy were conducted to visualize cilia in cultured cells and in vivo. RESULTS: Loss of EphA2 (i) increased corneal epithelial thickness by elevating proliferative potential in wing cells, (ii) reduced the number of ciliated cells, (iii) increased large hollow vacuoles, that could be rescued by BafA1; (iv) inhibited autophagy flux and (v) increased GFP-LC3 puncta in the mouse corneal epithelium. This indicated a role for EphA2 in stratified epithelial assembly via regulation of proliferation as well as a positive role in both ciliogenesis and end-stage autophagy. Inhibition of PLD1, an EphA2 interacting protein that is a critical regulator of end-stage autophagy, reversed the accumulation of vacuoles, and the reduction in the number of ciliated cells due to EphA2 depletion, suggesting EphA2 regulation of both end-stage autophagy and ciliogenesis via PLD1. PLD1 mediated rescue of ciliogenesis by EphA2 depletion was blocked by BafA1, placing autophagy between EphA2 signaling and regulation of ciliogenesis. CONCLUSION: Our findings demonstrate a novel role for EphA2 in regulating both autophagy and ciliogenesis, processes that are essential for proper corneal epithelial homeostasis.

Developing a model of care for substance use in pregnancy and parenting services, Sydney, Australia: Service provider perspectives.

INTRODUCTION: The absence of a clear model of care for services supporting pregnant women and mothers with substance use disorders has impeded opportunities to build an evidence base for the effectiveness of these services. Previous research has typically focused on the needs of pregnant women or mothers, as two distinct groups. This paper explores service providers' perceptions of key components of a model of care, extending from perinatal care to community-based support for up to 17 years post-delivery. A model of care is outlined and feasibility factors affecting implementation are highlighted. METHODS: Qualitative methods were adopted as a first step to informing development of the model of care. Semi-structured in-depth interviews were conducted with thirty-eight staff providing a range of substance use in pregnancy and parenting services (SUPPS) in hospital and community-based agencies, in a metropolitan health district in Sydney, Australia. Interview data was entered into NVivo and analysed using constant comparative methods. RESULTS: Guiding principles for the model of care included integrated care, harm reduction and person-centredness. Practice approaches integral to the model of care were promoting engagement with women, flexible service provision, trauma-informed care, and continuity of care. Feasibility factors influencing implementation of the model of care included fragmentation or siloing of the service network and workforce sustainability. CONCLUSIONS: A harm reduction approach was crucial for promoting engagement of women with SUPPS, particularly child protection services. A greater focus on providing ongoing community-based support for mothers also has the potential to achieve sustainable positive outcomes for women and children. Implementation of the SUPPS model of care could be undermined by threats to integration of service delivery and continuity of care. Further research is needed to explore consumer perspectives and inform the model of care as a framework for evaluation.

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.