Catalase, a therapeutic target in the reversal of estrogen-mediated aging.

Despite increasing interest in the reversal of age-related processes, there is a paucity of data regarding the effects of post-menopausal-associated estrogen loss on cellular function. We studied human adipose-derived mesenchymal stem cells (hASCs) isolated from women younger than 45 years old (pre-menopause, pre-hASC) or older than 55 years old (post-menopause, post-hASC). In this study, we provide proof of concept that the age-related ineffective functionality of ASCs can be reversed to improve their ability in promoting tissue repair. We found reduced estrogen receptor expression, decreased estrogen receptor activation, and reduced sensitivity to 17ß-estradiol in post-hASCs. This correlated with decreased antioxidants (catalase and superoxide dismutase [SOD] expression) and increased oxidative stress compared with pre-hASCs. Increasing catalase expression in post-hASCs restored estrogen receptor (ER) expression and their functional capacity to promote tissue repair as shown in human skin ex vivo wound healing and in vivo mouse model of lung injury. Our results suggest that the consequences of 17ß-estradiol decline on the function of hASCs may be reversible by changing the oxidative stress/antioxidant composition.

Intracellular Staphylococcus aureus triggers pyroptosis and contributes to inhibition of healing due to perforin-2 suppression.

Impaired wound healing associated with recurrent Staphylococcus aureus infection and unresolved inflammation are hallmarks of nonhealing diabetic foot ulcers (DFUs). Perforin-2, an innate immunity molecule against intracellular bacteria, limits cutaneous infection and dissemination of S. aureus in mice. Here, we report the intracellular accumulation of S. aureus in the epidermis of DFUs with no clinical signs of infection due to marked suppression of perforin-2. S. aureus residing within the epidermis of DFUs triggers AIM2 inflammasome activation and pyroptosis. These findings were corroborated in mice lacking perforin-2. The effects of pyroptosis on DFU clinical outcomes were further elucidated in a 4-week longitudinal clinical study in patients with DFUs receiving standard care. Increased AIM2 inflammasome and ASC-pyroptosome coupled with induction of IL-1ß were found in nonhealing DFUs compared with healing DFUs. Our findings revealed that perforin-2 suppression, intracellular S. aureus accumulation, and associated induction of pyroptosis contribute to healing inhibition and prolonged inflammation in patients with DFUs.

Corrigendum: Staphylococcus epidermidis Boosts Innate Immune Response by Activation of Gamma Delta T Cells and Induction of Perforin-2 in Human Skin.

[This corrects the article DOI: 10.3389/fimmu.2020.550946.].

Staphylococcus epidermidis Boosts Innate Immune Response by Activation of Gamma Delta T Cells and Induction of Perforin-2 in Human Skin.

Perforin-2 (P-2) is an antimicrobial protein with unique properties to kill intracellular bacteria. Gamma delta (GD) T cells, as the major T cell population in epithelial tissues, play a central role in protective and pathogenic immune responses in the skin. However, the tissue-specific mechanisms that control the innate immune response and the effector functions of GD T cells, especially the cross-talk with commensal organisms, are not very well understood. We hypothesized that the most prevalent skin commensal microorganism, Staphylococcus epidermidis, may play a role in regulating GD T cell-mediated cutaneous responses. We analyzed antimicrobial protein P-2 expression in human skin at a single cell resolution using an amplified fluorescence in situ hybridization approach to detect P-2 mRNA in combination with immunophenotyping. We show that S. epidermidis activates GD T cells and upregulates P-2 in human skin ex vivo in a cell-specific manner. Furthermore, P-2 upregulation following S. epidermidis stimulation correlates with increased ability of skin cells to kill intracellular Staphylococcus aureus. Our findings are the first to reveal that skin commensal bacteria induce P-2 expression, which may be utilized beneficially to modulate host innate immune responses and protect from skin infections.

A Case Report of a Patient with Turner Syndrome, Multiple Comorbidities, and Pustular Psoriasis: Correlation or Coincidence?

Turner syndrome (TS) is one of the most common chromosomal abnormalities. Patients with TS are at an increased risk for the development of metabolic syndrome, hypertension (HTN), diabetes mellitus type II (DM2), hyperlipidemia (HLD), obesity, and cardiovascular disease. The association between psoriasis and the aforementioned conditions including metabolic syndrome, HTN, HLD, obesity, and cardiovascular disease has also been established. Although the mechanism for heightened risk in TS patients is yet to be elucidated, patients suffering from TS and cardiometabolic diseases are likely to be at an even higher risk for developing psoriasis than patients suffering from TS alone. We present a case of a 53-year-old Hispanic woman with a mosaic TS and multiple comorbidities who presented with pustular psoriasis. For this patient, management can be challenging considering her numerous medical comorbidities and the presence of both TS and psoriasis.

Folliculitis Decalvans and Lichen Planopilaris Phenotypic Spectrum-A Series of 7 New Cases With Focus on Histopathology.

BACKGROUND: Folliculitis decalvans (FD) and lichen planopilaris (LPP) are classified as neutrophilic and lymphocytic cicatricial alopecias according to the North American Hair Research Society. Recently, a clinical phenotype combining concomitant or sequential features for both was described as a FD LPP phenotypic spectrum (FDLPPPS). OBJECTIVES: To review the most common phenotypic presentation of FDLPPPS with a main focus on histopathology. METHODS: We reviewed retrospectively series of 7 patients with a similar phenotypic presentation with special focus on the histologic pattern. All patients presented with concomitant features for FD and LPP and recalcitrant course unresponsive to topical and systemic immunomodulatory/anti-inflammatory agents. RESULTS: The most common clinical phenotype was that of hairless patches on the vertex with lost follicular ostia and perifollicular scale and the following diagnostic findings: (1) polytrichia; (2) positive bacterial culture for Staphylococcus in over 50% of the samples isolated from pustules and hemorrhagic crusts; (3) "mixed" histologic features for primary cicatricial alopecia including multicompound follicular structures of average 2-5 follicles (follicular packs), atrophy of the follicular epithelium, lymphohistiocytic infiltrate with granulomas, and prominent plasma cells, but absence of neutrophilic infiltrate in all cases except scarce neutrophils in one; and (4) clinical improvement with adjuvant systemic antimicrobials. CONCLUSIONS: The FDLPPPS may be underreported and should be considered in all cases of LPP recalcitrant to treatment. Dermatologists and dermatopathologists should recognize this phenotypic spectrum to guide optimal clinical management consisting of immunomodulatory and anti-inflammatory agents along with systemic antimicrobials.

A bioengineered living cell construct activates metallothionein/zinc/MMP8 and inhibits TGFß to stimulate remodeling of fibrotic venous leg ulcers.

Venous leg ulcers (VLU) represent a major clinical unmet need, impairing quality of life for millions worldwide. The bioengineered bilayered living cell construct (BLCC) is the only FDA-approved therapy demonstrating efficacy in healing chronic VLU, yet its in vivo mechanisms of action are not well understood. Previously, we reported a BLCC-mediated acute wounding response at the ulcer edge; in this study we elucidated the BLCC-specific effects on the epidermis-free ulcer bed. We conducted a randomized controlled clinical trial (ClinicalTrials.gov NCT01327937) enrolling 30 subjects with nonhealing VLUs, and performed genotyping, genomic profiling, and functional analysis on wound bed biopsies obtained at baseline and 1 week after treatment with BLCC plus compression or compression therapy (control). The VLU bed transcriptome featured processes of chronic inflammation and was strikingly enriched for fibrotic/fibrogenic pathways and gene networks. BLCC application decreased expression of profibrotic TGFß1 gene targets and increased levels of TGFß inhibitor decorin. Surprisingly, BLCC upregulated metallothioneins and fibroblast-derived MMP8 collagenase, and promoted endogenous release of MMP-activating zinc to stimulate antifibrotic remodeling, a novel mechanism of cutaneous wound healing. By activating a remodeling program in the quiescent VLU bed, BLCC application shifts nonhealing to healing phenotype. As VLU bed fibrosis correlates with poor clinical healing, findings from this study identify the chronic VLU as a fibrotic skin disease and are first to support the development and application of antifibrotic therapies as a successful treatment approach.

Mevastatin promotes healing by targeting caveolin-1 to restore EGFR signaling.

Diabetic foot ulcers (DFUs) are a life-threatening disease that often results in lower limb amputations and a shortened life span. Current treatment options are limited and often not efficacious, raising the need for new therapies. To investigate the therapeutic potential of topical statins to restore healing in patients with DFUs, we performed next-generation sequencing on mevastatin-treated primary human keratinocytes. We found that mevastatin activated and modulated the EGF signaling to trigger an antiproliferative and promigratory phenotype, suggesting that statins may shift DFUs from a hyperproliferative phenotype to a promigratory phenotype in order to stimulate healing. Furthermore, mevastatin induced a migratory phenotype in primary human keratinocytes through EGF-mediated activation of Rac1, resulting in actin cytoskeletal reorganization and lamellipodia formation. Interestingly, the EGF receptor is downregulated in tissue biopsies from patients with DFUs. Mevastatin restored EGF signaling in DFUs through disruption of caveolae to promote keratinocyte migration, which was confirmed by caveolin-1 (Cav1) overexpression studies. We conclude that topical statins may have considerable therapeutic potential as a treatment option for patients with DFUs and offer an effective treatment for chronic wounds that can be rapidly translated to clinical use.

Single cell analyses reveal specific distribution of anti-bacterial molecule Perforin-2 in human skin and its modulation by wounding and Staphylococcus aureus infection.

Perforin-2 (P-2) is a recently described antimicrobial protein with unique properties to kill intracellular bacteria. We investigated P-2 expression pattern and cellular distribution in human skin and its importance in restoration of barrier function during wound healing process and infection with the common wound pathogen Staphylococcus aureus. We describe a novel approach for the measurement of P-2 mRNA within individual skin cells using an amplified fluorescence in situ hybridization (FISH) technique. The unique aspect of this approach is simultaneous detection of P-2 mRNA in combination with immune-phenotyping for cell surface proteins using fluorochrome-conjugated antibodies. We detected P-2 transcript in both hematopoietic (CD45+ ) and non-hematopoietic (CD45- ) cutaneous cell populations, confirming the P-2 expression in both professional and non-professional phagocytes. Furthermore, we found an induction of P-2 during wound healing. P-2 overexpression resulted in a reduction of intracellular S. aureus, while infection of human wounds by this pathogen resulted in P-2 suppression, revealing a novel mechanism by which S. aureus may escape cutaneous immunity to cause persistent wound infections.

Staphylococcus aureus Triggers Induction of miR-15B-5P to Diminish DNA Repair and Deregulate Inflammatory Response in Diabetic Foot Ulcers.

Diabetic foot ulcers (DFUs) are a debilitating complication of diabetes in which bacterial presence, including the frequent colonizer Staphylococcus aureus, contributes to inhibition of healing. MicroRNAs (miRs) play a role in healing and host response to bacterial pathogens. However, the mechanisms by which miR response to cutaneous S. aureus contributes to DFU pathophysiology are unknown. Here, we show that S. aureus inhibits wound closure and induces miR-15b-5p in acute human and porcine wound models and in chronic DFUs. Transcriptome analyses of DFU tissue showed induction of miR-15b-5p to be critical, regulating many cellular processes, including DNA repair and inflammatory response, by suppressing downstream targets IKBKB, WEE1, FGF2, RAD50, MSH2, and KIT. Using a human wound model, we confirmed that S. aureus-triggered miR-15b-5p induction results in suppression of the inflammatory- and DNA repair-related genes IKBKB and WEE1. Inhibition of DNA repair and accumulation of DNA breaks was functionally confirmed by the presence of the pH2AX within colonized DFUs. We conclude that S. aureus induces miR-15b-5p, subsequently repressing DNA repair and inflammatory response, showing a mechanism of inhibition of healing in DFUs previously unreported, to our knowledge. This underscores a previously unknown role of DNA damage repair in the pathophysiology of DFUs colonized with S. aureus.

Topical mevastatin promotes wound healing by inhibiting the transcription factor c-Myc via the glucocorticoid receptor and the long non-coding RNA Gas5.

Diabetic foot ulcers (DFUs), a life-threatening complication of diabetes mellitus, have limited treatment options, often resulting in amputations. HMG-CoA reductase inhibitors such as statins are cholesterol-reducing agents that may provide a new therapeutic option. Statins target the cholesterol pathway and block the synthesis of the wound-healing inhibitors farnesyl pyrophosphate (FPP) and cortisol, ligands for the glucocorticoid receptor (GR). Here we demonstrate that the naturally occurring statin mevastatin reverses FPP's effects and promotes healing by using in vitro wound healing assays, human ex vivo and porcine in vivo wound models, and DFU tissue. Moreover, we measured cortisol levels by ELISA and found that mevastatin inhibited cortisol synthesis in keratinocytes and biopsies from patients with DFU. Of note, topical mevastatin stimulated epithelialization and angiogenesis in vivo Mevastatin also reversed FPP-mediated induction of the GR target, the transcription factor c-Myc (a biomarker of non-healing wounds), in porcine and human wound models. Importantly, mevastatin reversed c-Myc overexpression in DFUs. It induced expression of the long noncoding RNA Gas5 that blocks c-Myc expression, which was confirmed by overexpression studies. We conclude that topical mevastatin accelerates wound closure by promoting epithelialization via multiple mechanisms: modulation of GR ligands and induction of the long noncoding RNA Gas5, leading to c-Myc inhibition. In light of these findings, we propose that repurposing statin drugs for topical treatment of DFUs may offer another option for managing this serious condition.

Wound healing protects against chemotherapy-induced alopecia in young rats via up-regulating interleukin-1ß-mediated signaling.

Wound healing is a complex process regulated by various cell types and a plethora of mediators. While interactions between wounded skin and the hair follicles (HFs) could induce HF neogenesis or promote wound healing, it remains unknown whether the wound healing-associated signaling milieu can be manipulated to protect against alopecia, such as chemotherapy-induced alopecia (CIA). Utilizing a well-established neonatal rat model of CIA, we show here that skin wounding protects from alopecia caused by several clinically relevant chemotherapeutic regimens, and that protection is dependent on the time of wounding and hair cycle stage. Gene expression profiling unveiled a significant increase in interleukin-1 beta (IL-1ß) mediated signaling by skin wounding. Subsequently, we showed that IL-1ß is sufficient and indispensable for mediating the CIA-protective effect. Administration of IL-1ß alone to unwounded rats exhibited local CIA protection while IL-1ß neutralization abrogated CIA protection by wounding. Mechanistically, IL-1ß retarded postnatal HF morphogenesis, making HFs at the wound sites or IL-1ß treated areas damage-resistant while the rats developed total alopecia elsewhere. We conclude that wound healing switches the cutaneous cytokine milieu to an IL-1ß-dominated state thus retarding HF growth progression and rendering the HFs resistant to chemotherapy agents. In the future, manipulation of HF progression through interfering with the IL-1ß signaling milieu may provide therapeutic benefits to a variety of conditions, from prevention of CIA to inhibition of hair growth and treatment of hirsutism.

A bioengineered living cell construct activates an acute wound healing response in venous leg ulcers.

Chronic nonhealing venous leg ulcers (VLUs) are widespread and debilitating, with high morbidity and associated costs; about $15 billion is spent annually on the care of VLUs in the United States. Despite this, there is a paucity of treatments for VLUs because of the lack of pathophysiologic insight into ulcer development as well as the lack of knowledge regarding biologic actions of existing VLU-targeted therapies. The bioengineered bilayered living cellular construct (BLCC) skin substitute is a U.S. Food and Drug Administration-approved biologic treatment for healing VLUs. To elucidate the mechanisms through which the BLCC promotes healing of chronic VLUs, we conducted a clinical trial (NCT01327937) in which patients with nonhealing VLUs were treated with either standard of care (compression therapy) or the BLCC together with standard of care. Tissue was collected from the VLU edge before and 1 week after treatment, and the samples underwent comprehensive microarray mRNA and protein analyses. Ulcers treated with the BLCC skin substitute displayed three distinct transcriptomic patterns, suggesting that BLCC induced a shift from a nonhealing to a healing tissue response, involving modulation of inflammatory and growth factor signaling, keratinocyte activation, and attenuation of Wnt/ß-catenin signaling. In these ways, BLCC application orchestrated a shift from the chronic nonhealing ulcer microenvironment to a distinctive healing milieu resembling that of an acute, healing wound. Our findings provide in vivo evidence in VLU patients of pathways that can be targeted in the design of new therapies to promote healing of chronic VLUs.

Stress Signals, Mediated by Membranous Glucocorticoid Receptor, Activate PLC/PKC/GSK-3ß/ß-catenin Pathway to Inhibit Wound Closure.

Glucocorticoids (GCs), key mediators of stress signals, are also potent wound healing inhibitors. To understand how stress signals inhibit wound healing, we investigated the role of membranous glucocorticoid receptor (mbGR) by using cell-impermeable BSA-conjugated dexamethasone. We found that mbGR inhibits keratinocyte migration and wound closure by activating a Wnt-like phospholipase (PLC)/ protein kinase C (PKC) signaling cascade. Rapid activation of mbGR/PLC/PKC further leads to activation of known biomarkers of nonhealing found in patients, ß-catenin and c-myc. Conversely, a selective inhibitor of PKC, calphostin C, blocks mbGR/PKC pathway, and rescues GC-mediated inhibition of keratinocyte migration in vitro and accelerates wound epithelialization of human wounds ex vivo. This novel signaling mechanism may have a major impact on understanding how stress response via GC signaling regulates homeostasis and its role in development and treatments of skin diseases, including wound healing. To test tissue specificity of this nongenomic signaling mechanism, we tested retinal and bronchial human epithelial cells and fibroblasts. We found that mbGR/PLC/PKC signaling cascade exists in all cell types tested, suggesting a more general role. The discovery of this nongenomic signaling pathway, in which glucocorticoids activate Wnt pathway via mbGR, provides new insights into how stress-mediated signals may activate growth signals in various epithelial and mesenchymal tissues.

MiR-21 and miR-205 are induced in invasive cutaneous squamous cell carcinomas.

Cutaneous squamous cell carcinoma (cSCC) is a malignant proliferation of keratinocytes with an uncertain molecular basis causing significant morbidity. MicroRNAs (miRs) are small RNA molecules that regulate gene expression on post- transcriptional level. MiRs are critical to various biological processes. To determine if miRs play a role in pathogenesis of invasive cSCC, we collected patients' specimens from in situ and invasive cSCC (n = 19) and examined miRs expression levels using qPCR. Specifically, we evaluated miR-21, miR-103a, miR-186, miR-200b, miR-203, and miR-205 expression levels due to their role in skin biology and epithelial to mesenchymal transition. MiR levels were compared between in situ and invasive cSCCs. We found statistically significant (p ≤ 0.05) upregulation of miR-21 and miR-205 in invasive cSCC compared to cSCC in situ. We concluded that miR-21 and miR-205 may have diagnostic value in determining the invasive properties of cSCCs and that each cSCC displays unique miR profile, underscoring the possibility of personalized medicine approach in developing potential novel, less invasive treatments.

Mineralocorticoid Receptor Antagonists-A New Sprinkle of Salt and Youth.

Skin atrophy and impaired cutaneous wound healing are the recognized side effects of topical glucocorticoid (GC) therapy. Although GCs have high affinity for the glucocorticoid receptor, they also bind and activate the mineralocorticoid receptor. In light of this, one can speculate that some of the GC-mediated side effects can be remedied by blocking activation of the mineralocorticoid receptor. Indeed, according to Nguyen et al., local inhibition of the mineralocorticoid receptor via antagonists (spironolactone, canrenoate, and eplerenone) rescues GC-induced delayed epithelialization and accelerates wound closure in diabetic animals by targeting epithelial sodium channels and stimulating keratinocyte proliferation. These findings suggest that the use of mineralocorticoid receptor antagonists coupled with GC therapy may be beneficial in overcoming at least some of the GC-mediated side effects.

Integrative analysis of miRNA and mRNA paired expression profiling of primary fibroblast derived from diabetic foot ulcers reveals multiple impaired cellular functions.

Diabetic foot ulcers (DFUs) are one of the major complications of diabetes. Its molecular pathology remains poorly understood, impeding the development of effective treatments. Although it has been established that multiple cell types, including fibroblasts, keratinocytes, macrophages, and endothelial cells, all contribute to inhibition of healing, less is known regarding contributions of individual cell type. Thus, we generated primary fibroblasts from nonhealing DFUs and evaluated their cellular and molecular properties in comparison to nondiabetic foot fibroblasts (NFFs). Specifically, we analyzed both micro-RNA and mRNA expression profiles of primary DFU fibroblasts. Paired genomic analyses identified a total of 331 reciprocal miRNA-mRNA pairs including 21 miRNAs (FC > 2.0) along with 239 predicted target genes (FC > 1.5) that are significantly and differentially expressed. Of these, we focused on three miRNAs (miR-21-5p, miR-34a-5p, miR-145-5p) that were induced in DFU fibroblasts as most differentially regulated. The involvement of these microRNAs in wound healing was investigated by testing the expression of their downstream targets as well as by quantifying cellular behaviors in prospectively collected and generated cell lines from 15 patients (seven DFUF and eight NFF samples). We found large number of downstream targets of miR-21-5p, miR-34a-5p, miR-145-5p to be coordinately regulated in mRNA profiles, which was confirmed by quantitative real-time PCR. Pathway analysis on paired miRNA-mRNA profiles predicted inhibition of cell movement and cell proliferation, as well as activation of cell differentiation and senescence in DFU fibroblasts, which was confirmed by cellular assays. We concluded that induction of miR-21-5p, miR-34a-5p, miR-145-5p in DFU dermal fibroblasts plays an important role in impairing multiple cellular functions, thus contributing to overall inhibition of healing in DFUs.

Biology and Biomarkers for Wound Healing.

BACKGROUND: As the population grows older, the incidence and prevalence of conditions that lead to a predisposition for poor wound healing also increase. Ultimately, this increase in nonhealing wounds has led to significant morbidity and mortality with subsequent huge economic ramifications. Therefore, understanding specific molecular mechanisms underlying aberrant wound healing is of great importance. It has and will continue to be the leading pathway to the discovery of therapeutic targets, as well as diagnostic molecular biomarkers. Biomarkers may help identify and stratify subsets of nonhealing patients for whom biomarker-guided approaches may aid in healing. METHODS: A series of literature searches were performed using Medline, PubMed, Cochrane Library, and Internet searches. RESULTS: Currently, biomarkers are being identified using biomaterials sourced locally from human wounds and/or systemically using high-throughput "omics" modalities (genomic, proteomic, lipidomic, and metabolomic analysis). In this review, we highlight the current status of clinically applicable biomarkers and propose multiple steps in validation and implementation spectrum, including those measured in tissue specimens, for example, ß-catenin and c-myc, wound fluid, matrix metalloproteinases and interleukins, swabs, wound microbiota, and serum, for example, procalcitonin and matrix metalloproteinases. CONCLUSIONS: Identification of numerous potential biomarkers using different avenues of sample collection and molecular approaches is currently underway. A focus on simplicity and consistent implementation of these biomarkers, as well as an emphasis on efficacious follow-up therapeutics, is necessary for transition of this technology to clinically feasible point-of-care applications.