Calcitriol modulates epidermal tight junction barrier function in human keratinocytes.

BACKGROUND: The aberrant expression of tight junction (TJ) proteins play an important role in several diseases with impaired skin barriers, including atopic dermatitis, psoriasis, and chronic wounds. The evidence provided thus far suggests an important role of calcitriol in skin homeostasis. However, it is not known whether calcitriol improves the impaired skin barrier. OBJECTIVE: To investigate the effect of calcitriol on TJ barrier function in human primary keratinocytes. METHODS: Normal human primary keratinocytes were stimulated with calcitriol, and the expression of TJ-related proteins was measured by real-time PCR and Western blotting. Immunofluorescence was used to examine the intercellular distribution of TJ-related proteins. TJ barrier function was assessed by the transepithelial electrical resistance (TER) assay. RESULTS: We demonstrated that calcitriol increased the expression levels of TJ-related proteins, including claudin-4, claudin-7, occludin, and zonula occludens (ZO)- 1. Calcitriol enhanced the distribution of TJ-related proteins at cellcell borders and induced the phosphorylation of pathways involved in the regulation of TJ barrier function, such as atypical protein kinase C (aPKC), Ras-related C3 botulinum toxin substrate 1 (Rac1), phosphoinositide 3-kinase (PI3K), and protein kinase B (Akt), as evidenced by the effects of specific inhibitors on the above pathways. Indeed, we confirmed that calcitriol enhanced TER in keratinocyte monolayers. CONCLUSION: These findings showed that calcitriol could modify the expression of keratinocyte TJ proteins, contributing to the maintenance of homeostatic barrier function.

A Dual Pharmacological Strategy against COVID-19: The Therapeutic Potential of Metformin and Atorvastatin.

Metformin (MET) and atorvastatin (ATO) are promising treatments for COVID-19. This review explores the potential of MET and ATO, commonly prescribed for diabetes and dyslipidemia, respectively, as versatile medicines against SARS-CoV-2. Due to their immunomodulatory and antiviral capabilities, as well as their cost-effectiveness and ubiquitous availability, they are highly suitable options for treating the virus. MET's effect extends beyond managing blood sugar, impacting pathways that can potentially decrease the severity and fatality rates linked with COVID-19. It can partially block mitochondrial complex I and stimulate AMPK, which indicates that it can be used more widely in managing viral infections. ATO, however, impacts cholesterol metabolism, a crucial element of the viral replicative cycle, and demonstrates anti-inflammatory characteristics that could modulate intense immune reactions in individuals with COVID-19. Retrospective investigations and clinical trials show decreased hospitalizations, severity, and mortality rates in patients receiving these medications. Nevertheless, the journey from observing something to applying it in a therapeutic setting is intricate, and the inherent diversity of the data necessitates carefully executed, forward-looking clinical trials. This review highlights the requirement for efficacious, easily obtainable, and secure COVID-19 therapeutics and identifies MET and ATO as promising treatments in this worldwide health emergency.

Calcitriol, an Active Form of Vitamin D3, Mitigates Skin Barrier Dysfunction in Atopic Dermatitis NC/Nga Mice.

Atopic dermatitis and psoriasis are prevalent chronic inflammatory skin diseases that are characterized by dysfunctional skin barriers and substantially impact patients' quality of life. Vitamin D3 regulates immune responses and keratinocyte differentiation and improves psoriasis symptoms; however, its effects on atopic dermatitis remain unclear. Here, we investigated the effects of calcitriol, an active form of vitamin D3, on an NC/Nga mouse model of atopic dermatitis. We observed that the topical application of calcitriol decreased the dermatitis scores and epidermal thickness of NC/Nga mice with atopic dermatitis compared to untreated mice. In addition, both stratum corneum barrier function as assessed by the measurement of transepidermal water loss and tight junction barrier function as evaluated by biotin tracer permeability assay were improved following calcitriol treatment. Moreover, calcitriol treatment reversed the decrease in the expression of skin barrier-related proteins and decreased the expression of inflammatory cytokines such as interleukin (IL)-13 and IL-33 in mice with atopic dermatitis. These findings suggest that the topical application of calcitriol might improve the symptoms of atopic dermatitis by repairing the dysfunctional epidermal and tight junction barriers. Our results suggest that calcitriol might be a viable therapeutic agent for the treatment of atopic dermatitis in addition to psoriasis.

The Antimicrobial Peptide AMP-IBP5 Suppresses Dermatitis-like Lesions in a Mouse Model of Atopic Dermatitis through the Low-Density Lipoprotein Receptor-Related Protein-1 Receptor.

The antimicrobial peptide derived from insulin-like growth factor-binding protein 5 (AMP-IBP5) exhibits antimicrobial activities and immunomodulatory functions in keratinocytes and fibroblasts. However, its role in regulating skin barrier function remains unclear. Here, we investigated the effects of AMP-IBP5 on the skin barrier and its role in the pathogenesis of atopic dermatitis (AD). 2,4-Dinitrochlorobenzene was used to induce AD-like skin inflammation. Transepithelial electrical resistance and permeability assays were used to investigate tight junction (TJ) barrier function in normal human epidermal keratinocytes and mice. AMP-IBP5 increased the expression of TJ-related proteins and their distribution along the intercellular borders. AMP-IBP5 also improved TJ barrier function through activation of the atypical protein kinase C and Rac1 pathways. In AD mice, AMP-IBP5 ameliorated dermatitis-like symptoms restored the expression of TJ-related proteins, suppressed the expression of inflammatory and pruritic cytokines, and improved skin barrier function. Interestingly, the ability of AMP-IBP5 to alleviate inflammation and improve skin barrier function in AD mice was abolished in mice treated with an antagonist of the low-density lipoprotein receptor-related protein-1 (LRP1) receptor. Collectively, these findings indicate that AMP-IBP5 may ameliorate AD-like inflammation and enhance skin barrier function through LRP1, suggesting a possible role for AMP-IBP5 in the treatment of AD.

Cathelicidin LL-37 Activates Human Keratinocyte Autophagy through the P2X7, Mechanistic Target of Rapamycin, and MAPK Pathways.

Human cathelicidin LL-37 is a multifunctional antimicrobial peptide that exhibits antimicrobial and immunomodulatory activities. LL-37 regulates skin barrier function and was recently reported to activate autophagy in macrophages. Because autophagy deficiency is associated with skin diseases characterized by a dysfunctional epidermal barrier, we hypothesized that LL-37 might regulate the skin barrier through autophagy modulation. We showed that LL-37 activated autophagy in human keratinocytes and three-dimensional skin equivalent models as indicated by increases in LC3 puncta formation, decreases in p62, and autophagosome and autolysosome formation. LL-37‒induced autophagy was suppressed by P2X7 receptor, adenosine monophosphate‒activated protein kinase, and unc-51-like kinase 1 inhibitors, suggesting that the P2X7, adenosine monophosphate‒activated protein kinase, and unc-51-like kinase 1 pathways are involved. Moreover, LL-37 enhanced the phosphorylation of adenosine monophosphate‒activated protein kinase and unc-51-like kinase 1. In addition, LL-37‒mediated autophagy involves the mechanistic target of rapamycin and MAPK pathways. Interestingly, the LL-37‒induced distribution of tight junction proteins and improvement in the tight junction barrier were inhibited in autophagy-deficient keratinocytes and keratinocytes and skin models treated with autophagy inhibitors, indicating that the LL-37‒mediated tight junction barrier is associated with autophagy activation. Collectively, these findings suggest that LL-37 is a potential therapeutic target for skin diseases characterized by dysfunctional autophagy and skin barriers.

The Antimicrobial Peptides Human ß-Defensins Induce the Secretion of Angiogenin in Human Dermal Fibroblasts.

The skin produces a plethora of antimicrobial peptides that not only show antimicrobial activities against pathogens but also exhibit various immunomodulatory functions. Human ß-defensins (hBDs) are the most well-characterized skin-derived antimicrobial peptides and contribute to diverse biological processes, including cytokine production and the migration, proliferation, and differentiation of host cells. Additionally, hBD-3 was recently reported to promote wound healing and angiogenesis, by inducing the expression of various angiogenic factors and the migration and proliferation of fibroblasts. Angiogenin is one of the most potent angiogenic factors; however, the effects of hBDs on angiogenin production in fibroblasts remain unclear. Here, we investigated the effects of hBDs on the secretion of angiogenin by human dermal fibroblasts. Both in vitro and ex vivo studies demonstrated that hBD-1, hBD-2, hBD-3, and hBD-4 dose-dependently increased angiogenin production by fibroblasts. hBD-mediated angiogenin secretion involved the epidermal growth factor receptor (EGFR), Src family kinase, c-Jun N-terminal kinase (JNK), p38, and nuclear factor-kappa B (NF-κB) pathways, as evidenced by the inhibitory effects of specific inhibitors for these pathways. Indeed, we confirmed that hBDs induced the activation of the EGFR, Src, JNK, p38, and NF-κB pathways. This study identified a novel role of hBDs in angiogenesis, through the production of angiogenin, in addition to their antimicrobial activities and other immunomodulatory properties.

Antimicrobial peptide derived from insulin-like growth factor-binding protein 5 improves diabetic wound healing.

Impaired keratinocyte functions are major factors that are responsible for delayed diabetic wound healing. In addition to its antimicrobial activity, the antimicrobial peptide derived from insulin-like growth factor-binding protein 5 (AMP-IBP5) activates mast cells and promotes keratinocyte and fibroblast proliferation and migration. However, its effects on diabetic wound healing remain unclear. Human keratinocytes were cultured in normal or high glucose milieus. The production of angiogenic growth factor and cell proliferation and migration were evaluated. Wounds in normal and streptozotocin-induced diabetic mice were monitored and histologically examined. We found that AMP-IBP5 rescued the high glucose-induced attenuation of proliferation and migration as well as the production of angiogenin and vascular endothelial growth factors in keratinocytes. The AMP-IBP5-induced activity was mediated by the epidermal growth factor receptor, signal transducer and activator of transcription 1 and 3, and mitogen-activated protein kinase pathways, as indicated by the inhibitory effects of pathway-specific inhibitors. In vivo, AMP-IBP5 markedly accelerated wound healing, increased the expression of angiogenic factors and promoted vessel formation in both normal and diabetic mice. Overall, the finding that AMP-IBP5 accelerated diabetic wound healing by protecting against glucotoxicity and promoting angiogenesis suggests that AMP-IBP5 might be a potential therapeutic target for treating chronic diabetic wounds.

The Antimicrobial Peptide Human ß-Defensin-3 Accelerates Wound Healing by Promoting Angiogenesis, Cell Migration, and Proliferation Through the FGFR/JAK2/STAT3 Signaling Pathway.

In addition to its antimicrobial activity, the skin-derived antimicrobial peptide human ß-defensin-3 (hBD-3) promotes keratinocyte proliferation and migration to initiate the wound healing process; however, its effects on fibroblasts, which are the major cell type responsible for wound healing, remain unclear. We investigated the role of hBD-3 in cell migration, proliferation and production of angiogenic growth factors in human fibroblasts and evaluated the in vivo effect of hBD-3 on promoting wound healing and angiogenesis. Following hBD-3 treatment, the mouse wounds healed faster and showed accumulation of neutrophils and macrophages in the early phase of wound healing and reduction of these phagocytes 4 days later. hBD-3-treated wounds also displayed an increased number of fibroblasts and newly formed vessels compared to those of the control mice. Furthermore, the expression of various angiogenic growth factors was increased in the hBD-3-treated wounds. Additionally, in vitro studies demonstrated that hBD-3 enhanced the secretion of angiogenic growth factors such as fibroblast growth factor, platelet-derived growth factor and vascular endothelial growth factor and induced the migration and proliferation of human fibroblasts. The hBD-3-mediated activation of fibroblasts involves the fibroblast growth factor receptor 1 (FGFR1)/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathways, as evidenced by the inhibitory effects of pathway-specific inhibitors. We indeed confirmed that hBD-3 enhanced the phosphorylation of FGFR1, JAK2 and STAT3. Collectively, the current study provides novel evidence that hBD-3 might be a potential candidate for the treatment of wounds through its ability to promote wound healing, angiogenesis and fibroblast activation.

Intractable Itch in Atopic Dermatitis: Causes and Treatments.

Itch or pruritus is the hallmark of atopic dermatitis and is defined as an unpleasant sensation that evokes the desire to scratch. It is also believed that itch is a signal of danger from various environmental factors or physiological abnormalities. Because histamine is a well-known substance inducing itch, H1-antihistamines are the most frequently used drugs to treat pruritus. However, H1-antihistamines are not fully effective against intractable itch in patients with atopic dermatitis. Given that intractable itch is a clinical problem that markedly decreases quality of life, its treatment in atopic dermatitis is of high importance. Histamine-independent itch may be elicited by various pruritogens, including proteases, cytokines, neuropeptides, lipids, and opioids, and their cognate receptors, such as protease-activated receptors, cytokine receptors, Mas-related G protein-coupled receptors, opioid receptors, and transient receptor potential channels. In addition, cutaneous hyperinnervation is partly involved in itch sensitization in the periphery. It is believed that dry skin is a key feature of intractable itch in atopic dermatitis. Treatment of the underlying conditions that cause itch is necessary to improve the quality of life of patients with atopic dermatitis. This review describes current insights into the pathophysiology of itch and its treatment in atopic dermatitis.

Exogenous factors in the pathogenesis of atopic dermatitis: Irritants and cutaneous infections.

Atopic dermatitis (AD) is a chronic relapsing inflammatory cutaneous disease that is often associated with other atopic symptoms, such as food allergy, allergic rhinitis and asthma, leading to significant morbidity and healthcare costs. The pathogenesis of AD is complicated and multifactorial. Although the aetiology of AD remains incompletely understood, recent studies have provided further insight into AD pathophysiology, demonstrating that the interaction among genetic predisposition, immune dysfunction and environmental provocation factors contributes to its development. However, the increasing prevalence of AD suggests that environmental factors such as irritation and cutaneous infection play a crucial role in triggering and/or aggravating the disease. Of note, AD skin is susceptible to bacterial, fungal and viral infections, and microorganisms may colonize the skin and aggravate AD symptoms. Overall, understanding the mechanisms by which these risk factors affect the cutaneous immunity of patients with AD is of great importance for developing a precision medicine approach for treatment. This review summarizes recent developments in exogenous factors involved in the pathogenesis of AD, with special emphasis on irritants and microbial infections.

Role of Antimicrobial Peptides in Skin Barrier Repair in Individuals with Atopic Dermatitis.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease that exhibits a complex interplay of skin barrier disruption and immune dysregulation. Patients with AD are susceptible to cutaneous infections that may progress to complications, including staphylococcal septicemia. Although most studies have focused on filaggrin mutations, the physical barrier and antimicrobial barrier also play critical roles in the pathogenesis of AD. Within the physical barrier, the stratum corneum and tight junctions play the most important roles. The tight junction barrier is involved in the pathogenesis of AD, as structural and functional defects in tight junctions not only disrupt the physical barrier but also contribute to immunological impairments. Furthermore, antimicrobial peptides, such as LL-37, human b-defensins, and S100A7, improve tight junction barrier function. Recent studies elucidating the pathogenesis of AD have led to the development of barrier repair therapy for skin barrier defects in patients with this disease. This review analyzes the association between skin barrier disruption in patients with AD and antimicrobial peptides to determine the effect of these peptides on skin barrier repair and to consider employing antimicrobial peptides in barrier repair strategies as an additional approach for AD management.