The Antimicrobial Peptide Cathelicidin Exerts Immunomodulatory Effects via Scavenger Receptors.

An active form of cathelicidin antimicrobial peptide, LL-37, has immunomodulatory and stimulatory effects, though the specific pathways are not clear. The purpose of this study was to identify the cellular pathways by which LL-37 amplifies the inflammation induced by damage-associated molecular patterns (DAMPs). We performed DNA microarray, reverse transcription polymerase chain reaction, immunoblotting, and proximity ligation assays using cultured keratinocytes treated with LL-37 and/or the DAMP poly(I:C), a synthetic double-stranded RNA. In contrast to the combination of LL-37 and poly(I:C), LL-37 alone induced genes related to biological metabolic processes such as VEGFA and PTGS2 (COX-2). Inhibition of FPR2, a known receptor for cathelicidin, partially suppressed the induction of VEGFA and PTGS2. Importantly, VEGFA and PTGS2 induced by LL-37 alone were diminished by the knockdown of scavenger receptors including SCARB1 (SR-B1), OLR1 (SR-E1), and AGER (SR-J1). Moreover, LL-37 alone, as well as the combination of LL-37 and poly(I:C), showed proximity to the scavenger receptors, indicating that LL-37 acts via scavenger receptors and intermediates between them and poly(I:C). These results showed that the broad function of cathelicidin is generally dependent on scavenger receptors. Therefore, inhibitors of scavenger receptors or non-functional mock cathelicidin peptides may serve as new anti-inflammatory and immunosuppressive agents.

Functional Analysis of the Transcriptional Regulator IκB-ζ in Intestinal Homeostasis.

BACKGROUND: The Toll-like receptor signaling pathway contributes to the regulation of intestinal homeostasis through interactions with commensal bacteria. Although the transcriptional regulator IκB-ζ can be induced by Toll-like receptor signaling, its role in intestinal homeostasis is still unclear. AIMS: To investigate the role of IκB-ζ in gut homeostasis. METHODS: DSS-administration induced colitis in control and IκB-ζ-deficient mice. The level of immunoglobulins in feces was detected by ELISA. The immunological population in lamina propria (LP) was analyzed by FACS. RESULTS: IκB-ζ-deficient mice showed severe inflammatory diseases with DSS administration in the gut. The level of IgM in the feces after DSS administration was less in IκB-ζ-deficient mice compared to control mice. Upon administration of DSS, IκB-ζ-deficient mice showed exaggerated intestinal inflammation (more IFN-g-producing CD4+ T cells in LP), and antibiotic treatment canceled this inflammatory phenotype. CONCLUSION: IκB-ζ plays a crucial role in maintaining homeostasis in the gut.

Psoriasis and Antimicrobial Peptides.

Psoriasis is a systemic inflammatory disease caused by crosstalk between various cells such as T cells, neutrophils, dendritic cells, and keratinocytes. Antimicrobial peptides (AMPs) such as ß-defensin, S100, and cathelicidin are secreted from these cells and activate the innate immune system through various mechanisms to induce inflammation, thus participating in the pathogenesis of psoriasis. In particular, these antimicrobial peptides enhance the binding of damage-associated molecular patterns such as self-DNA and self-RNA to their receptors and promote the secretion of interferon from activated plasmacytoid dendritic cells and keratinocytes to promote inflammation in psoriasis. Neutrophil extracellular traps (NETs), complexes of self-DNA and proteins including LL-37 released from neutrophils in psoriatic skin, induce Th17. Activated myeloid dendritic cells secrete a mass of inflammatory cytokines such as IL-12 and IL-23 in psoriasis, which is indispensable for the proliferation and survival of T cells that produce IL-17. AMPs enhance the production of some of Th17 and Th1 cytokines and modulate receptors and cellular signaling in psoriasis. Inflammation induced by DAMPs, including self-DNA and RNA released due to microinjuries or scratches, and the enhanced recognition of DAMPs by AMPs, may be involved in the mechanism underlying the Köbner phenomenon in psoriasis.

Melanogenesis Connection with Innate Immunity and Toll-Like Receptors.

The epidermis is located in the outermost layer of the living body and is the place where external stimuli such as ultraviolet rays and microorganisms first come into contact. Melanocytes and melanin play a wide range of roles such as adsorption of metals, thermoregulation, and protection from foreign enemies by camouflage. Pigmentary disorders are observed in diseases associated with immunodeficiency such as Griscelli syndrome, indicating molecular sharing between immune systems and the machineries of pigment formation. Melanocytes express functional toll-like receptors (TLRs), and innate immune stimulation via TLRs affects melanin synthesis and melanosome transport to modulate skin pigmentation. TLR2 enhances melanogenetic gene expression to augment melanogenesis. In contrast, TLR3 increases melanosome transport to transfer to keratinocytes through Rab27A, the responsible molecule of Griscelli syndrome. TLR4 and TLR9 enhance tyrosinase expression and melanogenesis through p38 MAPK (mitogen-activated protein kinase) and NFκB signaling pathway, respectively. TLR7 suppresses microphthalmia-associated transcription factor (MITF), and MITF reduction leads to melanocyte apoptosis. Accumulating knowledge of the TLRs function of melanocytes has enlightened the link between melanogenesis and innate immune system.

Artificial Pigmented Human Skin Created by Muse Cells.

The skin composes physiological and chemical barrier and renews skin component cells throughout the human life. Melanocytes locate in the basal layer of the epidermis and produce melanin to protect the skin from ultraviolet. Melanin plays key roles in determining human skin and hair color. Melanocyte dysfunction observed in albinism and vitiligo not only causes cosmetic problems but also increases risk of skin cancer. As rejuvenate therapy, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have been reported to generate melanocytes. Other than ES and iPS cells, human skin tissues maintain pluripotent stem cells, named multilineage-differentiating stress-enduring (Muse) cells. We employ Muse cells isolated from human fibroblasts and adipose tissue to differentiate into melanocytes (Muse-MC). Muse-MC express melanocyte-related molecules, such as tyrosinase and DCT, and show tyrosinase activity. We also succeeded to differentiate Muse cells into fibroblasts and keratinocytes and created three-dimensional (3D) reconstituted skin with Muse cell-derived melanocytes, fibroblasts, and keratinocytes. The 3D reconstituted skin of Muse cell-derived cells coordinately showed epidermis layers and Muse-MC localized in the basal layer of the epidermis. Thus Muse cells in the human skin can be a source of rejuvenation medicine for the skin reconstruction.

Pharmacokinetic Study of Bioactive Flavonoids in the Traditional Japanese Medicine Keigairengyoto Exerting Antibacterial Effects against Staphylococcus aureus.

Recent studies have demonstrated that flavonoid glucuronides can be deconjugated to the active form aglycone by ß-glucuronidase-expressing macrophages. Keigairengyoto (KRT) is a flavonoid-rich traditional Japanese medicine reported to enhance bacterial clearance through immune modulation. Our aims are to examine the pharmacokinetics of KRT flavonoids and to identify active flavonoids contributing to the adjuvant effects of KRT. KRT was evaluated at pharmacokinetic analysis to quantify absorbed flavonoids, and cutaneous infection assay induced in mice by inoculation of Staphylococcus aureus. Preventive or therapeutic KRT administration reduced the number of bacteria in the infection site as well as macroscopic and microscopic lesion scores with efficacies similar to antibiotics. Pharmacokinetic study revealed low plasma levels of flavonoid aglycones after KRT administration; however, plasma concentrations were enhanced markedly by ß-glucuronidase treatment, with baicalein the most abundant (Cmax, 1.32 µg/mL). In random screening assays, flavonoids such as bacalein, genistein, and apigenin enhanced bacteria phagocytosis by macrophages. Glucuronide bacalin was converted to aglycone baicalein by incubation with living macrophages, macrophage lysate, or skin homogenate. Taken together, the adjuvant effect of KRT may be due to some blood-absorbed flavonoids which enhance macrophage functions in host defense. Flavonoid-rich KRT may be a beneficial treatment for infectious skin inflammation.

Inhibition of Human Kallikrein 5 Protease by Triterpenoids from Natural Sources.

Stratum corneum tryptic enzyme kallikrein 5 (KLK5) is a serine protease that is involved in the cell renewal and maintenance of the skin barrier function. The excessive activation of KLK5 causes an exacerbation of dermatoses, such as rosacea and atopic dermatitis. Some triterpenoids are reported to suppress the serine proteases. We aimed to investigate whether bioactive triterpenoids modulate the KLK5 protease. Nineteen triterpenoids occurring in medicinal crude drugs were evaluated using an enzymatic assay to measure the anti-KLK5 activity. The KLK5-dependent cathelicidin peptide LL-37 production in human keratinocytes was examined using immunoprecipitation and Western blotting. Screening assays for evaluating the anti-KLK5 activity revealed that ursolic acid, oleanolic acid, saikosaponin b1, tumulosic acid and pachymic acid suppressed the KLK5 protease activity, although critical molecular moieties contributing to anti-KLK5 activity were unclarified. Ursolic acid and tumulosic acid suppressed the proteolytic processing of LL-37 in keratinocytes at ≤10 µM; no cytotoxicity was observed. Both triterpenoids were detected in the plasma of rats administered orally with triterpenoid-rich crude drug Jumihaidokuto. Our study reveals that triterpenoids, such as ursolic acid and tumulosic acid, modulate the KLK5 protease activity and cathelicidin peptide production. Triterpenoids may affect the skin barrier function via the regulation of proteases.

Minimally-invasive transepidermal potentiometry with microneedle salt bridge.

A commercial painless microneedle was filled with physiological saline agar, and this needle-based salt bridge was inserted into the skin (a piece of porcine skin and a flank skin of a live mouse) to make an electrical contact with its subepidermal region. The transepidermal potential (TEP), the potential difference between the skin surface and the subepidermal region, was measured using this inner electrode and a conventional agar electrode on the surface of the skin. Control of penetration depth of the inner electrode with a spacer and hydrophilic pretreatment with ozone plasma were found to be necessary for stable measurement. The TEP was reduced upon damages on the skin surface by tape stripping and acetone defatting, which indicated the fabricated needle electrode is useful for the minimally-invasive measurement of TEP and evaluation of skin barrier functions. Furthermore, we showed that the device integrating two electrodes into a single compact probe was useful to evaluate the local barrier functions and their mapping on a skin. This device could be a personal diagnostic tool in the fields of medicine and cosmetics in future.

Alarmin function of cathelicidin antimicrobial peptide LL37 through IL-36γ induction in human epidermal keratinocytes.

Several dermatoses, including psoriasis, atopic dermatitis, and rosacea, alter the expression of the innate immune effector human cathelicidin antimicrobial peptide (CAMP). To elucidate the roles of aberrant CAMP in dermatoses, we performed cDNA array analysis in CAMP-stimulated human epidermal keratinocytes, the primary cells responding to innate immune stimuli and a major source of CAMP LL37 in skin. Among LL37-inducible genes, IL-1 cluster genes, particularly IL36G, are of interest because we observed coordinate increases in CAMP and IL-36γ in the lesional skin of psoriasis, whereas virtually no CAMP or IL-36γ was observed in nonlesional skin and normal skin. The production and release of IL-36γ were up to 20-30 ng/ml in differentiated keratinocytes cultured in high-calcium media. G-protein inhibitor pertussis toxin and p38 inhibitor suppressed IL-36γ induction by LL37. As an alarmin, LL37 induces chemokines, including CXCL1, CXCL8/IL8, CXCL10/IP-10, and CCL20/MIP3a, and IL-36 (10-100 ng/ml) augments the production of these chemokines by LL37. Pretreatment with small interfering RNA against IL36γ and IL-36R IL36R/IL1RL2 and IL1RAP suppressed LL37-dependent IL8, CXCL1, CXCL10/IP10, and CCL20 production in keratinocytes, suggesting that the alarmin function of LL37 was partially dependent on IL-36γ and its receptors. Counting on CAMP induction in innate stimuli, such as in infection and wounding, IL-36γ induction by cathelicidin would explain the mechanism of initiation of skin inflammation and occasional exacerbations of psoriasis and skin diseases by general infection.

Milia En Plaque as a Distinct Follicular Hamartoma With Cystic Trichoepitheliomatous Features.

Milia en plaque (MEP) is an uncommon disorder characterized by an erythematous plaque containing numerous milia. The pathogenesis of MEP is not clear. The authors report a man with an erythematous plaque on the right retroauricular area, containing numerous white-yellow cysts varying in size. Histological examination showed that multiple cystic structures at various levels of the dermis that were lined by stratified squamous epithelium and contained keratinous material-these findings were consistent with the diagnosis of multiple milia. In addition to epidermal cysts, however, the lesion consisted of a branched proliferation of pale-staining keratinocytes lined with basal keratinocytes budding from the overlying epidermis. Moreover, some cysts were formed within the branched epithelial proliferation, had thicker cyst walls than the ordinary milium, or had irregular or branched projections toward the surrounding dermis. From these findings, the authors conclude that MEP is a distinct follicular hamartoma with cystic trichoepitheliomatous features.

Abnormal Morphology of Blood Vessels in Erythematous Skin From Atopic Dermatitis Patients.

Previous studies suggest that altered peripheral blood circulation might be associated with erythema or inflammation in atopic dermatitis (AD) patients. However, the overall structure of blood vessels and capillaries in AD skin is poorly understood because most studies have involved light-microscopic observation of thin skin sections. In the present study, we compared the 3-dimensional structures of peripheral blood vessels of healthy subjects and AD patients in detail by means of 2-photon microscopy. In skin from healthy subjects, superficial vascular plexus and capillaries originating from flexous blood vessels were observed. However, skin from AD patients contained thickened, flexuous blood vessels, which might be associated with increased blood flow, in both erythematous and nonlesional areas. However, patients with lichenification did not display these morphological changes. Bifurcation of vessels was not observed in either erythematous or lichenification lesions. These results might be helpful for developing new clinical strategies to treat erythema in AD patients.

Retrospective evaluation of conservative treatment for 140 ingrown toenails with a novel taping procedure.

The aim of this study is retrospectively to review the efficacy of a taping procedure for treating ingrown toe-nails or for supporting other conservative treatments of ingrown toenails. A total of 140 ingrown toenails treated at the Dermatology Clinic in Tohoku University Hospital were retrospectively reviewed for demographic characteristics, association with granulation tissue or infection, treatment modalities and their outcomes, and classified according the treatment modalities. All the ingrown toe-nails were treated with a novel taping procedure, "slit tape-strap procedure" alone or in conjunction with other conservative treatments. The mean?±?SD duration until pain relief and until cure of the ingrown toenail were 4.8?±?4.7 days, range 0-24 and 21.0?±?11.2 weeks, range 4-56, respectively. All of the treatments were all effective, although 18 cases recurred after treatment. The "slit tape-strap procedure" is effective in treating ingrown toenails, either as a monotherapy or as a supportive therapy for other conservative treatments.

Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea.

Acne rosacea is an inflammatory skin disease that affects 3% of the US population over 30 years of age and is characterized by erythema, papulopustules and telangiectasia. The etiology of this disorder is unknown, although symptoms are exacerbated by factors that trigger innate immune responses, such as the release of cathelicidin antimicrobial peptides. Here we show that individuals with rosacea express abnormally high levels of cathelicidin in their facial skin and that the proteolytically processed forms of cathelicidin peptides found in rosacea are different from those present in normal individuals. These cathelicidin peptides are a result of a post-translational processing abnormality associated with an increase in stratum corneum tryptic enzyme (SCTE) in the epidermis. In mice, injection of the cathelicidin peptides found in rosacea, addition of SCTE, and increasing protease activity by targeted deletion of the serine protease inhibitor gene Spink5 each increases inflammation in mouse skin. The role of cathelicidin in enabling SCTE-mediated inflammation is verified in mice with a targeted deletion of Camp, the gene encoding cathelicidin. These findings confirm the role of cathelicidin in skin inflammatory responses and suggest an explanation for the pathogenesis of rosacea by demonstrating that an exacerbated innate immune response can reproduce elements of this disease.

Anti-Melanogenic Activity of Ethanolic Extract from Garcinia atroviridis Fruits Using In Vitro Experiments, Network Pharmacology, Molecular Docking, and Molecular Dynamics Simulation.

Melanin, the pigment responsible for human skin color, increases susceptibility to UV radiation, leading to excessive melanin production and hyperpigmentation disorders. This study investigated the ethanolic extract of Garcinia atroviridis fruits for its phenolic and flavonoid contents, antioxidant activity, and impact on melanogenesis pathways using qRT-PCR and Western blot analysis. Utilizing network pharmacology, molecular docking, and dynamics simulations, researchers explored G. atroviridis fruit extract's active compounds, targets, and pharmacological effects on hyperpigmentation. G. atroviridis fruit extract exhibited antioxidant properties, scavenging DPPH• and ABTS•+ radicals radicals and chelating copper. It inhibited cellular tyrosinase activity and melanin content in stimulated B16F10 cells, downregulating TYR, TRP-1, phosphorylated CREB, CREB, and MITF proteins along with transcription levels of MITF, TYR, and TRP-2. LC-MS analysis identified thirty-three metabolites, with seventeen compounds selected for further investigation. Network pharmacology revealed 41 hyperpigmentation-associated genes and identified significant GO terms and KEGG pathways, including cancer-related pathways. Kaempferol-3-O-α-L-rhamnoside exhibited high binding affinity against MAPK3/ERK1, potentially regulating melanogenesis by inhibiting tyrosinase activity. Stable ligand-protein interactions in molecular dynamics simulations supported these findings. Overall, this study suggests that the ethanolic extract of G. atroviridis fruits possesses significant antioxidant, tyrosinase inhibitory, and anti-melanogenic properties mediated through key molecular targets and pathways.

Diagnostic accuracy of automation and non-automation techniques for identifying Burkholderia pseudomallei: A systematic review and meta-analysis.

BACKGROUND: Burkholderia pseudomallei, a Gram-negative pathogen, causes melioidosis. Although various clinical laboratory identification methods exist, culture-based techniques lack comprehensive evaluation. Thus, this systematic review and meta-analysis aimed to assess the diagnostic accuracy of culture-based automation and non-automation methods. METHODS: Data were collected via PubMed/MEDLINE, EMBASE, and Scopus using specific search strategies. Selected studies underwent bias assessment using QUADAS-2. Sensitivity and specificity were computed, generating pooled estimates. Heterogeneity was assessed using I2 statistics. RESULTS: The review encompassed 20 studies with 2988 B. pseudomallei samples and 753 non-B. pseudomallei samples. Automation-based methods, particularly with updating databases, exhibited high pooled sensitivity (82.79%; 95% CI 64.44-95.85%) and specificity (99.94%; 95% CI 98.93-100.00%). Subgroup analysis highlighted superior sensitivity for updating-database automation (96.42%, 95% CI 90.01-99.87%) compared to non-updating (3.31%, 95% CI 0.00-10.28%), while specificity remained high at 99.94% (95% CI 98.93-100%). Non-automation methods displayed varying sensitivity and specificity. In-house latex agglutination demonstrated the highest sensitivity (100%; 95% CI 98.49-100%), followed by commercial latex agglutination (99.24%; 95% CI 96.64-100%). However, API 20E had the lowest sensitivity (19.42%; 95% CI 12.94-28.10%). Overall, non-automation tools showed sensitivity of 88.34% (95% CI 77.30-96.25%) and specificity of 90.76% (95% CI 78.45-98.57%). CONCLUSION: The study underscores automation's crucial role in accurately identifying B. pseudomallei, supporting evidence-based melioidosis management decisions. Automation technologies, especially those with updating databases, provide reliable and efficient identification.

Cathelicidin, kallikrein 5, and serine protease activity is inhibited during treatment of rosacea with azelaic acid 15% gel.

BACKGROUND: Excess cathelicidin and kallikrein 5 (KLK5) have been hypothesized to play a role in the pathophysiology of rosacea. OBJECTIVE: We sought to evaluate the effects of azelaic acid (AzA) on these elements of the innate immune system. METHODS: Gene expression and protease activity were measured in laboratory models and patients with rosacea during a 16-week multicenter, prospective, open-label study of 15% AzA gel. RESULTS: AzA directly inhibited KLK5 in cultured keratinocytes and gene expression of KLK5, Toll-like receptor-2, and cathelicidin in mouse skin. Patients with rosacea showed reduction in cathelicidin and KLK5 messenger RNA after treatment with AzA gel. Subjects without rosacea had lower serine protease activity (SPA) than patients with rosacea. Distinct subsets of patients with rosacea who had high and low baseline SPA were identified, and patients with high baseline exhibited a statistically significant reduction of SPA with 15% AzA gel treatment. LIMITATIONS: Study size was insufficient to predict clinical efficacy based on the innate immune response to AzA. CONCLUSIONS: These results show that cathelicidin and KLK5 decrease in association with AZA exposure. Our observations suggest a new mechanism of action for AzA and that SPA may be a useful biomarker for disease activity.

A new type of annular erythema with perieccrine inflammation: erythema papulatum centrifugum.

Erythema papulatum centrifugum (EPC) was first described in 1962 by Watanabe [Jpn J Dermatol 1962;72:573] as eczematous lesions extending centrifugally. To date, more than 150 cases have been reported, but only in the Japanese literature. A review of the Japanese literature including our 5 new cases reveals that EPC occurs in middle-aged persons, predominantly in males, as a single lesion on the trunk that is self-limiting but recurs frequently. Although EPC shares several characteristics with the superficial type of erythema annulare centrifugum or asymmetrical periflexural exanthema, EPC differs from erythema annulare centrifugum in clinical manifestation, showing annular rings composed of grouped, tiny papules, and in the histology of perieccrine inflammation, while it differs from asymmetrical periflexural exanthema by male preponderance, onset age, high relapse rate and unique annular configuration. We discuss EPC as compared with other cutaneous disorders showing annular erythema or similar histopathology and suggest that EPC is a distinct clinical entity.