Spatial Distribution and Functional Impact of Human Scalp Hair Follicle Microbiota.

Human hair follicles (HFs) constitute a unique microbiota habitat that differs substantially from the skin surface. Traditional HF sampling methods fail to eliminate skin microbiota contaminants or assess the HF microbiota incompletely, and microbiota functions in human HF physiology remain ill explored. Therefore, we used laser-capture microdissection, metagenomic shotgun sequencing, and FISH to characterize the human scalp HF microbiota in defined anatomical compartments. This revealed significant compartment-, tissue lineage-, and donor age-dependent variations in microbiota composition. Greatest abundance variations between HF compartments were observed for viruses, archaea, Staphylococcus epidermidis, Cutibacterium acnes, and Malassezia restricta, with the latter 2 being the most abundant viable HF colonizers (as tested by propidium monoazide assay) and, surprisingly, most abundant in the HF mesenchyme. Transfection of organ-cultured human scalp HFs with S. epidermidis-specific lytic bacteriophages ex vivo downregulated transcription of genes known to regulate HF growth and development, metabolism, and melanogenesis, suggesting that selected microbial products may modulate HF functions. Indeed, HF treatment with butyrate, a metabolite of S. epidermidis and other HF microbiota, delayed catagen and promoted autophagy, mitochondrial activity, and gp100 and dermcidin expression ex vivo. Thus, human HF microbiota show spatial variations in abundance and modulate the physiology of their host, which invites therapeutic targeting.

Calreticulin: a multifunctional protein with potential therapeutic applications for chronic wounds.

Calreticulin is recognized as a multifunctional protein that serves an essential role in diverse biological processes that include wound healing, modification and folding of proteins, regulation of the secretory pathway, cell motility, cellular metabolism, protein synthesis, regulation of gene expression, cell cycle regulation and apoptosis. Although the role of calreticulin as an endoplasmic reticulum-chaperone protein has been well described, several studies have demonstrated calreticulin to be a highly versatile protein with an essential role during wound healing. These features make it an ideal molecule for treating a complex, multifactorial diseases that require fine tuning, such as chronic wounds. Indeed, topical application of recombinant calreticulin to wounds in multiple models of wound healing has demonstrated remarkable pro-healing effects. Among them include enhanced keratinocyte and fibroblast migration and proliferation, induction of extracellular matrix proteins, recruitment of macrophages along with increased granulation tissue formation, all of which are important functions in promoting wound healing that are deregulated in chronic wounds. Given the high degree of diverse functions and pro-healing effects, application of exogenous calreticulin warrants further investigation as a potential novel therapeutic option for chronic wound patients. Here, we review and highlight the significant effects of topical application of calreticulin on enhancing wound healing and its potential as a novel therapeutic option to shift chronic wounds into healing, acute-like wounds.

Cutaneous changes in diabetic patients: Primed for aberrant healing?

Cutaneous manifestations affect most patients with diabetes mellitus, clinically presenting with numerous dermatologic diseases from xerosis to diabetic foot ulcers (DFUs). Skin conditions not only impose a significantly impaired quality of life on individuals with diabetes but also predispose patients to further complications. Knowledge of cutaneous biology and the wound healing process under diabetic conditions is largely limited to animal models, and studies focusing on biology of the human condition of DFUs remain limited. In this review, we discuss the critical molecular, cellular, and structural changes to the skin in the hyperglycaemic and insulin-resistant environment of diabetes with a focus specifically on human-derived data. Elucidating the breadth of the cutaneous manifestations coupled with effective diabetes management is important for improving patient quality of life and averting future complications including wound healing disorders.

Chronic wound microenvironment mediates selection of biofilm-forming multi drug resistant Staphylococcus epidermidis with capability to impair healing.

Venous leg ulcers (VLU) are the most common chronic wounds characterized by bacterial biofilms and perturbed microbiome. Staphylococcus epidermidis is primarily known as skin commensal beneficial for the host, however, some strains can form biofilms and cause infections. By employing shotgun metagenomic sequencing we show that genetic signatures of antimicrobial resistance, adhesion and biofilm formation in VLU isolates correlate with in vitro bacterial traits. We demonstrate that the capability of chronic wound isolates to form biofilms and elicit IL-8 and IL-1ß expression in human ex vivo wounds, correlates with the non-healing outcomes in patients with VLU. In contrast, commensal strains were incapable of surviving in the human ex vivo wounds. We show that major fitness traits of S. epidermis from VLU involve genes for resistance to methicillin and mupirocin, while the biofilm formation relied on the minimal number of genetic elements responsible for bacterial binding to fibronectin and fibrinogen. This underscores the importance of the emergence of treatment resistant virulent lineages in patients with non-healing wounds.

Molecular Pathophysiology of Chronic Wounds: Current State and Future Directions.

Venous leg ulcers, diabetic foot ulcers, and pressure ulcers are complex chronic wounds with multifactorial etiologies that are associated with high patient morbidity and mortality. Despite considerable progress in deciphering the pathologies of chronic wounds using "omics" approaches, considerable gaps in knowledge remain, and current therapies are often not efficacious. We provide a comprehensive overview of current understanding of the molecular mechanisms that impair healing and current knowledge on cell-specific dysregulation including keratinocytes, fibroblasts, immune cells, endothelial cells and their contributions to impaired reepithelialization, inflammation, angiogenesis, and tissue remodeling that characterize chronic wounds. We also provide a rationale for further elucidation of ulcer-specific pathologic processes that can be therapeutically targeted to shift chronic nonhealing to acute healing wounds.

Urine-derived exosomes from individuals with IPF carry pro-fibrotic cargo.

Background: MicroRNAs (miRNA) and other components contained in extracellular vesicles may reflect the presence of a disease. Lung tissue, sputum, and sera of individuals with idiopathic pulmonary fibrosis (IPF) show alterations in miRNA expression. We designed this study to test whether urine and/or tissue derived exosomal miRNAs from individuals with IPF carry cargo that can promote fibrosis. Methods: Exosomes were isolated from urine (U-IPFexo), lung tissue myofibroblasts (MF-IPFexo), serum from individuals with IPF (n=16) and age/sex-matched controls without lung disease (n=10). We analyzed microRNA expression of isolated exosomes and their in vivo bio-distribution. We investigated the effect on ex vivo skin wound healing and in in vivo mouse lung models. Results: U-IPFexo or MF-IPFexo expressed miR-let-7d, miR-29a-5p, miR-181b-3p and miR-199a-3p consistent with previous reports of miRNA expression obtained from lung tissue/sera from patients with IPF. In vivo bio-distribution experiments detected bioluminescent exosomes in the lung of normal C57Bl6 mice within 5 min after intravenous infusion, followed by distribution to other organs irrespective of exosome source. Exosomes labeled with gold nanoparticles and imaged by transmission electron microscopy were visualized in alveolar epithelial type I and type II cells. Treatment of human and mouse lung punches obtained from control, non-fibrotic lungs with either U-IPFexo or MF-IPFexo produced a fibrotic phenotype. A fibrotic phenotype was also induced in a human ex vivo skin model and in in vivo lung models. Conclusions: Our results provide evidence of a systemic feature of IPF whereby exosomes contain pro-fibrotic miRNAs when obtained from a fibrotic source and interfere with response to tissue injury as measured in skin and lung models. Funding: This work was supported in part by Lester and Sue Smith Foundation and The Samrick Family Foundation and NIH grants R21 AG060338 (SE and MKG), U01 DK119085 (IP, RS, MTC).

Innate immunity and microbial dysbiosis in hidradenitis suppurativa - vicious cycle of chronic inflammation.

Hidradenitis Suppurativa (HS) is a chronic multifactorial inflammatory skin disease with incompletely understood mechanisms of disease pathology. HS is characterized by aberrant activation of the innate immune system, resulting in activation of pathways that aim to protect against pathogenic microorganisms, and also contribute to failure to resolve inflammation. Imbalance in innate immunity is evident in deregulation of host antimicrobial peptides (AMPs) and the complement system associated with the microbiome dysbiosis. The pathology is further complicated by ability of pathogens associated with HS to overcome host immune response. Potential roles of major AMPs, cathelicidin, defensins, dermcidin, S100 proteins, RNAse 7 and complement proteins are discussed. Dysregulated expression pattern of innate immunity components in conjunction with bacterial component of the disease warrants consideration of novel treatment approaches targeting both host immunity and pathogenic microbiome in HS.

Integrative small and long RNA omics analysis of human healing and nonhealing wounds discovers cooperating microRNAs as therapeutic targets.

MicroRNAs (miR), as important epigenetic control factors, reportedly regulate wound repair. However, our insufficient knowledge of clinically relevant miRs hinders their potential therapeutic use. For this, we performed paired small and long RNA-sequencing and integrative omics analysis in human tissue samples, including matched skin and acute wounds collected at each healing stage and chronic nonhealing venous ulcers (VUs). On the basis of the findings, we developed a compendium (https://www.xulandenlab.com/humanwounds-mirna-mrna), which will be an open, comprehensive resource to broadly aid wound healing research. With this first clinical, wound-centric resource of miRs and mRNAs, we identified 17 pathologically relevant miRs that exhibited abnormal VU expression and displayed their targets enriched explicitly in the VU gene signature. Intermeshing regulatory networks controlled by these miRs revealed their high cooperativity in contributing to chronic wound pathology characterized by persistent inflammation and proliferative phase initiation failure. Furthermore, we demonstrated that miR-34a, miR-424, and miR-516, upregulated in VU, cooperatively suppressed keratinocyte migration and growth while promoting inflammatory response. By combining miR expression patterns with their specific target gene expression context, we identified miRs highly relevant to VU pathology. Our study opens the possibility of developing innovative wound treatment that targets pathologically relevant cooperating miRs to attain higher therapeutic efficacy and specificity.

FOXM1 network in association with TREM1 suppression regulates NET formation in diabetic foot ulcers.

Diabetic foot ulcers (DFU) are a serious complication of diabetes mellitus and associated with reduced quality of life and high mortality rate. DFUs are characterized by a deregulated immune response with decreased neutrophils due to loss of the transcription factor, FOXM1. Diabetes primes neutrophils to form neutrophil extracellular traps (NETs), contributing to tissue damage and impaired healing. However, the role of FOXM1 in priming diabetic neutrophils to undergo NET formation remains unknown. Here, we found that FOXM1 regulates reactive oxygen species (ROS) levels in neutrophils and inhibition of FOXM1 results in increased ROS leading to NET formation. Next generation sequencing revealed that TREM1 promoted the recruitment of FOXM1+ neutrophils and reversed effects of diabetes and promoted wound healing in vivo. Moreover, we found that TREM1 expression correlated with clinical healing outcomes of DFUs, indicating TREM1 may serve as a useful biomarker or a potential therapeutic target. Our findings highlight the clinical relevance of TREM1, and indicates FOXM1 pathway as a novel regulator of NET formation during diabetic wound healing, revealing new therapeutic strategies to promote healing in DFUs.

Dichotomous role of miR193b-3p in diabetic foot ulcers maintains inhibition of healing and suppression of tumor formation.

Despite the hyperproliferative environment marked by activation of ß-catenin and overexpression of c-myc, the epidermis surrounding chronic diabetic foot ulcers (DFUs) is clinically hypertrophic and nonmigratory yet does not undergo malignant transformation. We identified miR193b-3p as a master regulator that contributes to this unique cellular phenotype. We determined that induction of tumor suppressor miR193b-3p is a unique feature of DFUs that is not found in venous leg ulcers, acute wounds, or cutaneous squamous cell carcinoma (SCC). Genomic analyses of DFUs identified suppression of the miR193b-3p target gene network that orchestrates cell motility. Inhibition of migration and wound closure was further confirmed by overexpression of miR193b-3p in human organotypic and murine in vivo wound models, whereas miR193b-3p knockdown accelerated wound reepithelialization in human ex vivo and diabetic murine wounds in vivo. The dominant negative effect of miR193b-3p on keratinocyte migration was maintained in the presence of promigratory miR31-5p and miR15b-5p, which were also overexpressed in DFUs. miR193b-3p mediated antimigratory activity by disrupting stress fiber formation and by decreasing activity of GTPase RhoA. Conversely, miR193b-3p targets that typically participate in malignant transformation were found to be differentially regulated between DFUs and SCC, including the proto-oncogenes KRAS (Kirsten rat sarcoma viral proto-oncogene) and KIT (KIT proto-oncogene). Although miR193b-3p acts as a tumor suppressor contributing to low tumor incidence in DFUs, it also acts as a master inhibitor of cellular migration and epithelialization in DFUs. Thus, miR193b-3p may represent a target for wound healing induction, cancer therapeutics, and diagnostics.

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.

Angiogenin Released from ABCB5+ Stromal Precursors Improves Healing of Diabetic Wounds by Promoting Angiogenesis.

Severe angiopathy is a major driver for diabetes-associated secondary complications. Knowledge on the underlying mechanisms essential for advanced therapies to attenuate these pathologies is limited. Injection of ABCB5+ stromal precursors at the edge of nonhealing diabetic wounds in a murine db/db model, closely mirroring human type 2 diabetes, profoundly accelerates wound closure. Strikingly, enhanced angiogenesis was substantially enforced by the release of the ribonuclease angiogenin from ABCB5+ stromal precursors. This compensates for the profoundly reduced angiogenin expression in nontreated murine chronic diabetic wounds. Silencing of angiogenin in ABCB5+ stromal precursors before injection significantly reduced angiogenesis and delayed wound closure in diabetic db/db mice, implying an unprecedented key role for angiogenin in tissue regeneration in diabetes. These data hold significant promise for further refining stromal precursors-based therapies of nonhealing diabetic foot ulcers and other pathologies with impaired angiogenesis.

Novel Diagnostic Technologies and Therapeutic Approaches Targeting Chronic Wound Biofilms and Microbiota.

Purpose of Review: To provide an up-to-date overview of recent developments in diagnostic methods and therapeutic approaches for chronic wound biofilms and pathogenic microbiota. Recent Findings: Biofilm infections are one of the major contributors to impaired wound healing in chronic wounds, including diabetic foot ulcers, venous leg ulcers, pressure ulcers, and nonhealing surgical wounds. As an organized microenvironment commonly including multiple microbial species, biofilms develop and persist through methods that allow evasion from host immune response and antimicrobial treatments. Suppression and reduction of biofilm infection have been demonstrated to improve wound healing outcomes. However, chronic wound biofilms are a challenge to treat due to limited methods for accurate, accessible clinical identification and the biofilm's protective properties against therapeutic agents. Here we review recent approaches towards visual markers for less invasive, enhanced biofilm detection in the clinical setting. We outline progress in wound care treatments including investigation of their antibiofilm effects, such as with hydrosurgical and ultrasound debridement, negative pressure wound therapy with instillation, antimicrobial peptides, nanoparticles and nanocarriers, electroceutical dressings, and phage therapy. Summary: Current evidence for biofilm-targeted treatments has been primarily conducted in preclinical studies, with limited clinical investigation for many therapies. Improved identification, monitoring, and treatment of biofilms require expansion of point-of-care visualization methods and increased evaluation of antibiofilm therapies in robust clinical trials.

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.].

Novel Cyclic Lipopeptides Fusaricidin Analogs for Treating Wound Infections.

Both acute and chronic cutaneous wounds are often difficult to treat due to the high-risk for bacterial contamination. Once hospitalized, open wounds are at a high-risk for developing hospital-associated infections caused by multi drug-resistant bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. Treating these infections is challenging, not only because of antibiotic resistance, but also due to the production of biofilms. New treatment strategies are needed that will help in both stimulating the wound healing process, as well as preventing and eliminating bacterial wound infections. Fusaricidins are naturally occurring cyclic lipopeptides with antimicrobial properties that have shown to be effective against a variety of fungi and Gram-positive bacteria, with low toxicity. Continuing with our efforts toward the identification of novel cyclic lipopeptides Fusaricidin analogs, herein we report the synthesis and evaluation of the antimicrobial activity for two novel cyclic lipopeptides (CLP), CLP 2605-4 and CLP 2612-8.1 against methicillin resistant S. aureus and P. aeruginosa, respectively, in in vivo porcine full thickness wound model. Both CLPs were able to reduce bacterial counts by approximately 3 log CFU/g by the last assessment day. Peptide 2612-8.1 slightly enhanced the wound healing, however, wounds treated with peptide 2605-4, have shown higher levels of inflammation and impaired wound healing process. This study highlights the importance of identifying new antimicrobials that can combat bacterial infection while not impeding tissue repair.

Glucocorticoid-mediated induction of caveolin-1 disrupts cytoskeletal organization, inhibits cell migration and re-epithelialization of non-healing wounds.

Although impaired keratinocyte migration is a recognized hallmark of chronic wounds, the molecular mechanisms underpinning impaired cell movement are poorly understood. Here, we demonstrate that both diabetic foot ulcers (DFUs) and venous leg ulcers (VLUs) exhibit global deregulation of cytoskeletal organization in genomic comparison to normal skin and acute wounds. Interestingly, we found that DFUs and VLUs exhibited downregulation of ArhGAP35, which serves both as an inactivator of RhoA and as a glucocorticoid repressor. Since chronic wounds exhibit elevated levels of cortisol and caveolin-1 (Cav1), we posited that observed elevation of Cav1 expression may contribute to impaired actin-cytoskeletal signaling, manifesting in aberrant keratinocyte migration. We showed that Cav1 indeed antagonizes ArhGAP35, resulting in increased activation of RhoA and diminished activation of Cdc42, which can be rescued by Cav1 disruption. Furthermore, we demonstrate that both inducible keratinocyte specific Cav1 knockout mice, and MßCD treated diabetic mice, exhibit accelerated wound closure. Taken together, our findings provide a previously unreported mechanism by which Cav1-mediated cytoskeletal organization prevents wound closure in patients with chronic wounds.

Cellular reprogramming of diabetic foot ulcer fibroblasts triggers pro-healing miRNA-mediated epigenetic signature.

Diabetic foot ulcers (DFUs), a prevalent complication of diabetes, constitute a major medical challenge with a critical need for development of cell-based therapies. We previously generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts derived from the DFU patients, location-matched skin of diabetic patients and normal healthy donors and re-differentiated them into fibroblasts. To assess the epigenetic microRNA (miR) regulated changes triggered by cellular reprogramming, we performed miRs expression profiling. We found let-7c, miR-26b-5p, -29c-3p, -148a-3p, -196a-5p, -199b-5p and -374a-5p suppressed in iPSC-derived fibroblasts in vitro and in 3D dermis-like self-assembly tissue, whereas their corresponding targets involved in cellular migration were upregulated. Moreover, targets involved in organization of extracellular matrix were induced after fibroblast reprogramming. PLAT gene, the crucial fibrinolysis factor, was upregulated in iPSC-derived fibroblasts and was confirmed as a direct target of miR-196a-5p. miR-197-3p and miR-331-3p were found upregulated specifically in iPSC-derived diabetic fibroblasts, while their targets CAV1 and CDKN3 were suppressed. CAV1, an important negative regulator of wound healing, was confirmed as a direct miR-197-3p target. Together, our findings demonstrate that iPSC reprogramming is an effective approach for erasing the diabetic non-healing miR-mediated epigenetic signature and promoting a pro-healing cellular phenotype.

Wounds and Skin and Soft Tissue Infections in People Who Inject Drugs and the Utility of Syringe Service Programs in Their Management.

Significance: Opioid use disorder and transition to injection drug use (IDU) are an urgent, nationwide public health crisis. Wounds and skin and soft tissue infections (SSTIs) are common complications of IDU that disproportionately affect people who inject drugs (PWID) and are a major source of morbidity and mortality for this population. Critical Issues: Injections in a nonsterile environment and reusing or sharing needles facilitates bacterial inoculation, with subsequent risk of serious complications such as sepsis, gangrene, amputation, and death. PWID are susceptible to infections with a wide spectrum of organisms beyond common culprits of SSTI, including Clostridium and Bacillus spp., as well as Candida. Recent Advances: Syringe services programs (SSPs) are cost-effective and successful in reducing harms associated with IDU. SSPs provide new equipment to PWID and aid in discarding used equipment. SSPs aim to reduce the risks of unhygienic injecting practices, which are associated with transmission of infections and blood-borne pathogens. Future Directions: Concurrently run SSPs and wound care clinics are uniquely positioned to facilitate care to PWID. Providing new, sterile equipment as well as early wound care intervention can reduce morbidity and mortality as well as health care expenditures by reducing the number of SSTI and injection-related wounds that require hospital admission. Establishment of wound care clinics as part of an SSP represents an untapped potential to reduce harm.