Diabetes downregulates the antimicrobial peptide psoriasin and increases E. coli burden in the urinary bladder.

Diabetes is known to increase susceptibility to infections, partly due to impaired granulocyte function and changes in the innate immunity. Here, we investigate the effect of diabetes, and high glucose on the expression of the antimicrobial peptide, psoriasin and the putative consequences for E. coli urinary tract infection. Blood, urine, and urine exfoliated cells from patients are studied. The influence of glucose and insulin is examined during hyperglycemic clamps in individuals with prediabetes and in euglycemic hyperinsulinemic clamped patients with type 1 diabetes. Important findings are confirmed in vivo in type 2 diabetic mice and verified in human uroepithelial cell lines. High glucose concentrations induce lower psoriasin levels and impair epithelial barrier function together with altering cell membrane proteins and cytoskeletal elements, resulting in increasing bacterial burden. Estradiol treatment restores the cellular function with increasing psoriasin and bacterial killing in uroepithelial cells, confirming its importance during urinary tract infection in hyperglycemia. In conclusion, our findings present the effects and underlying mechanisms of high glucose compromising innate immunity.

Influence of FLG loss-of-function mutations in host-microbe interactions during atopic skin inflammation.

BACKGROUND: Loss-of-function mutations in the filaggrin (FLG) gene directly alter skin barrier function and critically influence atopic inflammation. While skin barrier dysfunction, Th2-associated inflammation and bacterial dysbiosis are well-known characteristics of atopic dermatitis (AD), the mechanisms interconnecting genotype, transcriptome and microbiome remain largely elusive. OBJECTIVE: In-depth analysis of FLG genotype-associated skin gene expression alterations and host-microbe interactions in AD. METHODS: Multi-omics characterization of a cohort of AD patients carrying heterozygous loss-of-function mutations in the FLG gene (ADMut) (n = 15), along with matched wild-type (ADWt) patients and healthy controls. Detailed clinical characterization, microarray gene expression and 16 S rRNA-based microbial marker gene data were generated and analyzed. RESULTS: In the context of filaggrin dysfunction, the transcriptome was characterized by dysregulation of barrier function and water homeostasis, while the lesional skin of ADWt demonstrated the specific upregulation of pro-inflammatory cytokines and T-cell proliferation. S. aureus dominated the microbiome in both patient groups, however, shifting microbial communities could be observed when comparing healthy with non-lesional ADWt or ADMut skin, offering the opportunity to identify microbe-associated transcriptomic signatures. Moreover, an AD core signature with 28 genes, including CCL13, CCL18, BTC, SCIN, RAB31 and PCLO was identified. CONCLUSIONS: Our integrative approach provides molecular insights for the concept that FLG loss-of-function mutations are a genetic shortcut to atopic inflammation and unravels the complex interplay between genotype, transcriptome and microbiome in the human holobiont.

Microbial and transcriptional differences elucidate atopic dermatitis heterogeneity across skin sites.

It is well established that different sites in healthy human skin are colonized by distinct microbial communities due to different physiological conditions. However, few studies have explored microbial heterogeneity between skin sites in diseased skin, such as atopic dermatitis (AD) lesions. To address this issue, we carried out deep analysis of the microbiome and transcriptome in the skin of a large cohort of AD patients and healthy volunteers, comparing two physiologically different sites: upper back and posterior thigh. Microbiome samples and biopsies were obtained from both lesional and nonlesional skin to identify changes related to the disease process. Transcriptome analysis revealed distinct disease-related gene expression profiles depending on anatomical location, with keratinization dominating the transcriptomic signatures in posterior thigh, and lipid metabolism in the upper back. Moreover, we show that relative abundance of Staphylococcus aureus is associated with disease severity in the posterior thigh, but not in the upper back. Our results suggest that AD may select for similar microbes in different anatomical locations-an "AD-like microbiome," but distinct microbial dynamics can still be observed when comparing posterior thigh to upper back. This study highlights the importance of considering the variability across skin sites when studying the development of skin inflammation.

Microbe-host interplay in atopic dermatitis and psoriasis.

Despite recent advances in understanding microbial diversity in skin homeostasis, the relevance of microbial dysbiosis in inflammatory disease is poorly understood. Here we perform a comparative analysis of skin microbial communities coupled to global patterns of cutaneous gene expression in patients with atopic dermatitis or psoriasis. The skin microbiota is analysed by 16S amplicon or whole genome sequencing and the skin transcriptome by microarrays, followed by integration of the data layers. We find that atopic dermatitis and psoriasis can be classified by distinct microbes, which differ from healthy volunteers microbiome composition. Atopic dermatitis is dominated by a single microbe (Staphylococcus aureus), and associated with a disease relevant host transcriptomic signature enriched for skin barrier function, tryptophan metabolism and immune activation. In contrast, psoriasis is characterized by co-occurring communities of microbes with weak associations with disease related gene expression. Our work provides a basis for biomarker discovery and targeted therapies in skin dysbiosis.

Seeing Is Believing: Vitamin A Promotes Skin Health through a Host-Derived Antibiotic.

In this issue of Cell Host & Microbe, Harris et al. (2019) show that members of the resistin-like molecule (RELM) family are bactericidal, antimicrobial peptides that promote skin defenses in a vitamin-A-dependent manner. Administration of vitamin A analogs triggers RELM, which improves host resistance against skin pathogens in mice.

Psoriasin, a novel anti-Candida albicans adhesin.

Candida albicans belongs to the normal microbial flora on epithelial surfaces of humans. However, under certain, still not fully understood conditions, it can become pathogenic and cause a spectrum of diseases, from local infections to life-threatening septicemia. We investigated a panel of antimicrobial proteins and peptides (AMPs), potentially involved in mucosal immunity against this pathogen. Out of six studied AMPs, psoriasin was most up-regulated during a mucosal infection, an acute episode of recurrent Candida vulvovaginitis, although candidacidal activity has not been demonstrated. We here show that psoriasin binds to ß-glucan, a basic component of the C. albicans cell wall, and thereby inhibits adhesion of the pathogen to surfaces and increases IL-8 production by mucosal epithelial cells. In conclusion, we show a novel mechanism of action of psoriasin. By inhibiting C. albicans adhesion and by enhancing cytokine production, psoriasin contributes to the immune response against C. albicans. KEY MESSAGES: The antimicrobial peptide psoriasin is highly up-regulated during a local mucosal infection, Candida albicans vulvovaginitis. Psoriasin binds to ß-glucan in the Candida albicans cell wall and thereby inhibits adhesion of the pathogen. Binding of psoriasin to Candida albicans induces an immune response by mucosal epithelial cells.

Focal targeting by human ß-defensin 2 disrupts localized virulence factor assembly sites in Enterococcus faecalis.

Virulence factor secretion and assembly occurs at spatially restricted foci in some Gram-positive bacteria. Given the essentiality of the general secretion pathway in bacteria and the contribution of virulence factors to disease progression, the foci that coordinate these processes are attractive antimicrobial targets. In this study, we show in Enterococcus faecalis that SecA and Sortase A, required for the attachment of virulence factors to the cell wall, localize to discrete domains near the septum or nascent septal site as the bacteria proceed through the cell cycle. We also demonstrate that cationic human ß-defensins interact with E. faecalis at discrete septal foci, and this exposure disrupts sites of localized secretion and sorting. Modification of anionic lipids by multiple peptide resistance factor, a protein that confers antimicrobial peptide resistance by electrostatic repulsion, renders E. faecalis more resistant to killing by defensins and less susceptible to focal targeting by the cationic antimicrobial peptides. These data suggest a paradigm in which focal targeting by antimicrobial peptides is linked to their killing efficiency and to disruption of virulence factor assembly.

Epidermal EGFR controls cutaneous host defense and prevents inflammation.

The epidermal growth factor receptor (EGFR) plays an important role in tissue homeostasis and tumor progression. However, cancer patients treated with EGFR inhibitors (EGFRIs) frequently develop acneiform skin toxicities, which are a strong predictor of a patient's treatment response. We show that the early inflammatory infiltrate of the skin rash induced by EGFRI is dominated by dendritic cells, macrophages, granulocytes, mast cells, and T cells. EGFRIs induce the expression of chemokines (CCL2, CCL5, CCL27, and CXCL14) in epidermal keratinocytes and impair the production of antimicrobial peptides and skin barrier proteins. Correspondingly, EGFRI-treated keratinocytes facilitate lymphocyte recruitment but show a considerably reduced cytotoxic activity against Staphylococcus aureus. Mice lacking epidermal EGFR (EGFR(Δep)) show a similar phenotype, which is accompanied by chemokine-driven skin inflammation, hair follicle degeneration, decreased host defense, and deficient skin barrier function, as well as early lethality. Skin toxicities were not ameliorated in a Rag2-, MyD88-, and CCL2-deficient background or in mice lacking epidermal Langerhans cells. The skin phenotype was also not rescued in a hairless (hr/hr) background, demonstrating that skin inflammation is not induced by hair follicle degeneration. Treatment with mast cell inhibitors reduced the immigration of T cells, suggesting that mast cells play a role in the EGFRI-mediated skin pathology. Our findings demonstrate that EGFR signaling in keratinocytes regulates key factors involved in skin inflammation, barrier function, and innate host defense, providing insights into the mechanisms underlying EGFRI-induced skin pathologies.

Fungal protein MGL_1304 in sweat is an allergen for atopic dermatitis patients.

BACKGROUND: Sweat is a major aggravating factor of atopic dermatitis (AD) and approximately 80% of patients with AD show type I hypersensitivity against sweat. OBJECTIVE: To identify and characterize an antigen in sweat that induces histamine release from basophils of patients with AD. METHODS: Basophil histamine-releasing activity in sweat was purified by a combination of chromatographies, and proteins were analyzed with mass spectrometry. Recombinant proteins of the sweat antigen were generated, and their biological characteristics were studied by immunoblots, histamine release tests, and neutralization assays. RESULTS: We identified a fungal protein, MGL_1304, derived from Malassezia globosa (M globosa) in the purified sweat antigen. Recombinant MGL_1304 induced histamine release from basophils of most of the patients with AD, in accordance with the semi-purified sweat antigen. Moreover, recombinant MGL_1304 abolished the binding of serum IgE of patients with AD to the semi-purified sweat antigen, or vice versa in immunoblot analysis, and attenuated the sensitization of RBL-48 mast cells expressing human FcɛRI by serum IgE. Studies of truncated mutants of MGL_1304 indicated that IgE of patients with AD recognized the conformational structure of MGL_1304 rather than short peptide sequences. Western blot analysis of the whole lysate, the culture supernatant of M globosa, and the semi-purified sweat antigen showed that MGL_1304 was produced as a minor immunological antigen of M globosa with posttranslational modification, cleaved, and secreted as a 17-kDa major histamine-releasing sweat antigen. CONCLUSION: MGL_1304 is a major allergen in human sweat and could cause type I allergy in patients with AD.

Prenatal human skin expresses the antimicrobial peptide RNase 7.

Antimicrobial peptides and proteins (AMPs) play important roles in skin immune defense due to their capacity to inhibit growth of microbes. During intrauterine life, the skin immune system has to acquire the prerequisites to protect the newborn from infection in the hostile environment after birth, which includes the production of skin AMPs. The aim of this study was to analyze the expression of RNase 7, HBD-2/3 and psoriasin during human skin development, thus, providing a deeper insight about the maturity of a fundamental component of the innate immune system. We found low RNase 7 expression levels in the periderm but no expression of HBD-2/3 and psoriasin in first trimester human skin using immunohistochemistry. At the end of the second trimester, RNase 7 is expressed weakly in all epidermal layers with a marked signal in the stratum corneum. HBD-3 and psoriasin are focally expressed while HBD-2 is not detectable. Analysis of supernatants from cultured prenatal skin cells showed that in contrast to adult control, RNase 7 and psoriasin are not found in prenatal skin, suggesting that AMPs are detectable but are not secreted. This study shows the differential expression of AMPs in developing, non-perturbed human prenatal skin. It is conceivable that the combined expression of RNase 7, HBD-3 and psoriasin in fetal skin constitutes a developmental program to exert a broad spectrum of antimicrobial activity to maintain sterility in the amniotic cavity.

Antimicrobial peptides in healthy skin and atopic dermatitis.

Antimicrobial Peptides and Proteins (AMPs) represent effector molecules of the innate defense system in all organisms. AMPs are either constitutively or inducibly produced mainly by various epithelial cells, including keratinocytes. This report reviews our current knowledge about the major yet known keratinocyte-derived AMPs, its role in healthy skin and atopic dermatitis.

Purification of antimicrobial peptides from human skin.

Human skin is a rich source of human antimicrobial peptides. Its cellular source is the keratinocyte, which terminally differentiates in the uppermost parts of the skin, eventually forming the stratum corneum, the horny layer. The easy availability of human stratum corneum makes it possible to identify and characterize human antimicrobial peptides with a biochemical approach. Moreover, the availability of lesional scales of patients with psoriasis, an inflammatory skin disease, allows the identification of human-inducible peptide antibiotics, which are absent in healthy skin. With this strategy, the beta-defensins hBD-2 and hBD-3, RNase-7 as well as psoriasin/S100A7 have been discovered as human antimicrobial peptides and proteins. A detailed description of the strategies and methods is presented, which allowed a successful identification and characterization of human antimicrobial peptides. We used various HPLC techniques, combined with antimicrobial testing as read-out system. In parallel, SDS-PAGE analyses as well as electrospray ionization mass spectrometry were used for further biochemical characterization as well as purity assessment.

UV-B radiation induces the expression of antimicrobial peptides in human keratinocytes in vitro and in vivo.

BACKGROUND: Suppression of the adaptive immune system by UV radiation plays an important role in photocarcinogenesis. Exacerbation of skin infections has been proposed as a further consequence of UV-induced immunosuppression. Clinically bacterial infections are not a problem. For defense against bacteria, the innate immune response including the release of antimicrobial peptides is much more relevant than the adaptive immune response. Keratinocytes have the capacity to release antimicrobial peptides. OBJECTIVE: We asked whether UV radiation induces antimicrobial peptides in vitro and in vivo. METHODS: Antimicrobial peptide expression by normal human keratinocytes was measured by real-time PCR and fluorescence-activated cell sorting analysis. Biopsies taken from human volunteers and skin explants were studied with immunohistochemistry. RESULTS: Real-time PCR of normal human keratinocytes revealed a dose-dependent increase of human beta-defensin-2, -3, ribonuclease 7, and psoriasin (S100A7) after UV radiation. This was confirmed at the protein level by intracellular fluorescence-activated cell sorting and in vitro immunofluorescence analysis. Immunohistochemistry of biopsies taken from healthy volunteers exposed to different UV radiation doses revealed enhanced epidermal expression of antimicrobial peptides after UV exposure. This was also confirmed by exposing human skin explants to UV radiation. CONCLUSION: UV radiation exerts diverse effects on the immune system, suppressing the adaptive but inducing the innate immune response. This may explain why T-cell-mediated immune reactions are suppressed on UV exposure but not host defense reactions against bacterial attacks.

The angiogenesis inhibitor thrombospondin-1 inhibits acute cutaneous hypersensitivity reactions.

There is increasing evidence that vascular remodeling and endothelial cell activation promote acute and chronic inflammation. Thrombospondin 1 (TSP-1) is a potent endogenous angiogenesis inhibitor thought to play an important role in maintaining cutaneous vascular quiescence. We first investigated TSP-1 expression in human and contact hypersensitivity (CHS) reactions and found that TSP-1 was upregulated in the inflamed skin of patients and in mice. To elucidate the function of TSP-1 in cutaneous inflammation, we induced CHS reactions in the skin of mice with targeted epidermal TSP-1 overexpression in TSP-1-deficient mice and in wild-type mice. We found decreased edema formation, angiogenesis, and inflammatory infiltrate in the inflamed skin of TSP-1 transgenic mice. Conversely, TSP-1-deficient mice exhibited an enhanced and prolonged inflammation, characterized by increased edema formation, enhanced vascular remodeling, and increased neutrophilic infiltrate, when compared with wild-type mice. Moreover, we found strong upregulation of the proinflammatory cytokines IL-1beta, macrophage inflammatory protein 2, and tumor necrosis factor-alpha in the inflamed skin of TSP-1-deficient mice. Our results indicate that TSP-1 downregulates cutaneous delayed-type hypersensitivity reactions by acting on several distinct pathways mediating skin inflammation.

Uncovering the evolutionary history of innate immunity: the simple metazoan Hydra uses epithelial cells for host defence.

Although many properties of the innate immune system are shared among multicellular animals, the evolutionary origin remains poorly understood. Here we characterize the innate immune system in Hydra, one of the simplest multicellular animals known. In the complete absence of both protective mechanical barriers and mobile phagocytes, Hydra's epithelium is remarkably well equipped with potent antimicrobial peptides to prevent pathogen infection. Induction of antimicrobial peptide production is mediated by the interaction of a leucine-rich repeats (LRRs) domain containing protein with a TIR-domain containing protein lacking LRRs. Conventional Toll-like receptors (TLRs) are absent in the Hydra genome. Our findings support the hypothesis that the epithelium represents the ancient system of host defence.

Mouse beta-defensin-14, an antimicrobial ortholog of human beta-defensin-3.

Searching the database for mouse homologs of the antimicrobial peptide human beta-defensin-3 (hBD-3) revealed highest identity (69%) to mouse beta-defensin-14 (mBD-14). Recombinant mBD-14 exhibited broad-spectrum, nanomolar microbicidal activity. Treatment of keratinocytes with gamma interferon or transforming growth factor alpha increased mBD-14 gene expression. These data suggest that mBD-14 is the functional ortholog of hBD-3.

Co-regulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers.

Human epidermis elaborates two small cationic, highly hydrophobic antimicrobial peptides (AMP), beta-defensin 2 (hBD2), and the carboxypeptide cleavage product of human cathelicidin (hCAP18), LL-37, which are co-packaged along with lipids within epidermal lamellar bodies (LBs) before their secretion. Because of their colocalization, we hypothesized that AMP and barrier lipid production could be coregulated by altered permeability barrier requirements. mRNA and immunostainable protein levels for mBD3 and cathelin-related antimicrobial peptide (CRAMP) (murine homologues of hBD2 and LL-37, respectively) increase 1-8 hours after acute permeability barrier disruption and normalize by 24 hours, kinetics that mirror the lipid metabolic response to permeability barrier disruption. Artificial permeability barrier restoration, which inhibits the lipid-synthetic response leading to barrier recovery, blocks the increase in AMP mRNA/protein expression, further evidence that AMP expression is linked to permeability barrier function. Conversely, LB-derived AMPs are also important for permeability barrier homeostasis. Despite an apparent increase in mBD3 protein, CRAMP-/- mice delayed permeability barrier recovery, attributable to defective LB contents and abnormalities in the structure of the lamellar membranes that regulate permeability barrier function. These studies demonstrate that (1) the permeability and antimicrobial barriers are coordinately regulated by permeability barrier requirements and (2) CRAMP is required for permeability barrier homeostasis.

Novel anti-inflammatory properties of the angiogenesis inhibitor vasostatin.

Endothelial cells are critically involved in the pathogenesis of inflammation, which is characterized by vasopermeability, plasma leakage, leukocyte recruitment, and neovascularization. Therefore, inhibitors of endothelial cell function could reduce inflammation. In this study, we evaluated the effects of the angiogenesis inhibitor vasostatin on inflammations induced by contact hypersensitivity reactions in mouse ears. Vasostatin-treated mice revealed significantly reduced edema formation, resulting from lower plasma leakage and inhibition of inflammation-associated vascular remodeling. Intravital microscopy studies of inflamed ears showed a decrease in the fraction of rolling leukocytes in vasostatin-treated mice, and Lycopersicon esculentum lectin-perfused ears revealed fewer leukocytes adherent to the vessel wall. The inflammatory infiltrate from vasostatin-treated mice was characterized by fewer CD8+ T cells, neutrophils, and macrophages compared to the saline-treated animals. In a modified Miles assay, vasostatin inhibited vascular endothelial growth factor-A-induced permeability, and inflamed ear tissues from vasostatin-treated mice expressed significantly reduced levels of the vascular destabilizer angiopoietin-2. These results reveal a previously unrecognized anti-inflammatory property of the angiogenesis inhibitor vasostatin, and suggest that vasostatin is a potential candidate drug for the treatment of inflammation.

Identification of RNase 8 as a novel human antimicrobial protein.

The antimicrobial activity of the human RNase A superfamily member RNase 8 was evaluated. Recombinant RNase 8 exhibited broad-spectrum microbicidal activity against potential pathogenic microorganisms (including multidrug-resistant strains) at micro- to nanomolar concentrations. Thus, RNase 8 was identified as a novel antimicrobial protein and may contribute to host defense.