Discovery of natural bispecific antibodies: Is psoriasis induced by a toxigenic Corynebacterium simulans and maintained by CIDAMPs as autoantigens?

The high abundance of Corynebacterium simulans in psoriasis skin suggests a contribution to the psoriasis aetiology. This hypothesis was tested in an exploratory study, where western blot (WB) analyses with extracts of heat-treated C. simulans and psoriasis serum-derived IgG exhibited a single 16 kDa-WB-band. Proteomic analyses revealed ribosomal proteins as candidate C. s.-antigens. A peptidomic analysis unexpectedly showed that psoriasis serum-derived IgG already contained 31 immunopeptides of Corynebacteria ssp., suggesting the presence of natural bispecific antibodies (BsAbs). Moreover, peptidomic analyses gave 372 DECOY-peptides with similarity to virus- and phage proteins, including Corynebacterium diphtheriae phage, and similarity to diphtheria toxin. Strikingly, a peptidomic analysis for human peptides revealed 64 epitopes of major psoriasis autoantigens such as the spacer region of filaggrin, hornerin repeats and others. Most identified immunopeptides represent potential cationic intrinsically disordered antimicrobial peptides (CIDAMPs), which are generated within the epidermis. These may form complexes with bacterial disordered protein regions, representing chimeric antigens containing discontinuous epitopes. In addition, among 128 low-abundance immunopeptides, 48 are putatively psoriasis-relevant such as epitope peptides of PGE2-, vitamin D3- and IL-10-receptors. Further, 47 immunopeptides originated from tumour antigens, and the endogenous retrovirus HERV-K. I propose that persistent infection with a toxigenic C. simulans initiates psoriasis, which is exacerbated as an autoimmune disease by CIDAMPs as autoantigens. The discovery of natural BsAbs allows the identification of antigen epitopes from microbes, viruses, autoantigens and tumour-antigens, and may help to develop epitope-specific peptide-vaccines and therapeutic approaches with antigen-specific regulatory T cells to improve immune tolerance in an autoimmune disease-specific-manner.

Evolution of innate defense in human skin.

More often as compared to other barrier systems (gastrointestinal, urogenital, and respiratory linings) human skin over millions of years has been subject to fundamental changes in structure and function. When life on land started, the first changes consisted in the formation of a coherent impermeable stratum corneum. Two-legged locomotion was followed by loss of body hair and formation of sweat glands. Major changes took place after the agricultural revolution, investigating settlements with domestication of animals and plants. Living together after giving up nomadic life, hairless skin became a battlefield for pathogens, members of the skin microbiome, and arthropod visits. Human skin became exceptional in showing a boosted, highly developed immune system which is much more complex as compared to the "skins" of other species. A recently found skin disinfection system ("Cationic Intrinsically Disordered Antimicrobial Peptides, CIDAMPs") dates back to the origins of life and still is active in present-day integuments. As a skin-restricted and effective principle, keratinocyte- myeloid synergy (KMS) is recognized. As a consequence of such highly developed immune defense, the basic contributions of KMS - cells (keratinocytes, neutrophils, macrophages) in regulating innate immunity is emphasized. Antimicrobial peptides and chemokines became major keratinocyte products. The formation of impermeable str. corneum membrane has enabled KMS - cells to accumulate within upper skin levels and cause a special group of human skin diseases, pustular dermatoses.

Cationic Intrinsically Disordered Antimicrobial Peptides (CIDAMPs) Represent a New Paradigm of Innate Defense with a Potential for Novel Anti-Infectives.

In the search for potential mechanisms underlying the remarkable resistance of healthy skin against infection by soil bacteria like Pseudomonas (P.) aeruginosa we identified fragments of the intrinsically disordered protein hornerin as potent microbicidal agents in the stratum corneum. We found that, independent of the amino acid (AA)-sequence, any tested linear cationic peptide containing a high percentage of disorder-promoting AA and a low percentage of order-promoting AA is a potent microbicidal antimicrobial. We further show that the antimicrobial activity of these cationic intrinsically disordered antimicrobial peptides (CIDAMPs) depends on the peptide chain length, its net charge, lipidation and environmental conditions. The ubiquitous presence of latent CIDAMP sources in nature suggests a common and yet overlooked adapted innate disinfection system of body surfaces. The simple structure and virtually any imaginable sequence or composition of disorder-promoting AA allow the generation of a plethora of CIDAMPs. These are potential novel microbicidal anti-infectives for various bacterial pathogens, including P. aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA) and fungal pathogens like Candida albicans and Cryptococcus neoformans.

Skin-Derived SPINK9 Kills Escherichia coli.

Antimicrobial peptides play a critical role in the barrier function of human skin. They offer a fast response to invading microorganisms and protect from external microbial infection. Here we show the isolation of the kallikrein-related peptidase inhibitor SPINK9 as a major antibacterial factor from healthy stratum corneum. In total, six N-terminal SPINK9 variants were identified in the stratum corneum. Whereas all variants exhibited similar inhibition activities against kallikrein-related peptidase, only three variants with either lysine or glutamine as their first N-terminal residues were able to kill various Escherichia coli strains, but not other bacteria or fungi. The killing activity also depended on the sequence essential for kallikrein-related peptidase inhibition. Ultrastructural electron microscopy analyses suggested that SPINK9 entered the cell and killed growing bacteria. A bacterial chaperone, SKP, was identified as the major SPINK9 interacting partner in E. coli cells. The Skp-deleted mutant was more sensitive to SPINK9 than the wild-type control, suggesting that the bactericidal activity of SPINK9 should first overcome the resistance from the bacterial chaperone SKP. Thus, SPINK9 is a member of epidermal antimicrobial peptides for selective killing of E. coli, which might contribute to the innate barrier function of human skin.

Hornerin contains a Linked Series of Ribosome-Targeting Peptide Antibiotics.

Cationic intrinsically disordered antimicrobial peptides (CIDAMPs) belong to a novel class of epithelial peptide antibiotics with microbicidal activity against various pathogens, including Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Here we show that treatment of distinct bacteria with different hornerin (HRNR)-derived CIDAMPs cause formation of unique cytoplasmic protein aggregates, suggesting a common intracellular mode of action. We further found that, unlike most amphipathic antimicrobial peptides, HRNR traverses bacterial membranes energy-dependently and accumulates within the cytoplasm. Strikingly, certain structurally different, HRNR-based CIDAMPs were found to bind to an identical panel of distinct bacterial ribosomal proteins, thereby manifesting features of several known classes of antibiotics. This may cause the formation of aberrant proteins and toxic protein aggregates in HRNR-treated pathogens which eventually may induce its death. Our study reveals evidence that structurally distinct CIDAMPs of an abundant body surface protein simultaneously target multiple sites of the bacterial protein synthesis machinery.

Expression and Regulation of S100 Fused-Type Protein Hornerin at the Ocular Surface and Lacrimal Apparatus.

Purpose: The S100 fused-type proteins hornerin (HRNR) and filaggrin-2 (FLG2) are members of the epidermal differentiation complex, which is involved in terminal differentiation of keratinocytes via cornification as well as maintenance of the epidermal antimicrobial barrier. We investigated the expression and possible regulation of HRNR and FLG2 at the ocular surface and in the lacrimal apparatus. Methods: Tissues of the lacrimal apparatus and ocular surface were analyzed systematically by means of RT-PCR, immunohistochemistry, and immuntransmission electron microscopy (iTEM) for their ability to express and produce HRNR and FLG2. In addition, inducibility and regulation of HRNR were studied in cultivated human corneal (HCE), conjunctival (HCjE), as well as meibomian gland (HMGEC) epithelial cell line by real-time RT-PCR. Results: RT-PCR, immunohistochemistry, and iTEM revealed constitutive expression of HRNR in the epithelium of cornea, conjunctiva, nasolacrimal ducts, and acinus cells of lacrimal and meibomian glands. HRNR also was detected in tears of healthy volunteers. No expression of FLG2 could be detected in tissue samples of the ocular surface and lacrimal apparatus. Real-time RT-PCR revealed a decreased HRNR gene expression after challenge with proinflammatory cytokines and supernatants of Escherichia coli and Pseudomonas aeroginosa in HCE cells, whereas HCjE cells revealed no changes. In HMGECs serum-induced differentiation and application of all-trans retinoic acid significantly increased HRNR gene expression. Conclusions: The data suggest that HRNR, but not FLG2, is a component of the ocular surface and lacrimal apparatus, including meibomian glands. HRNR seems to contribute to the maintenance of the epithelial barrier at the ocular surface and, thus, also may be involved in ocular surface diseases.

Molecular Basis of the Receptor Interactions of Polysialic Acid (polySia), polySia Mimetics, and Sulfated Polysaccharides.

Polysialic acid (polySia) and polySia glycomimetic molecules support nerve cell regeneration, differentiation, and neuronal plasticity. With a combination of biophysical and biochemical methods, as well as data mining and molecular modeling techniques, it is possible to correlate specific ligand-receptor interactions with biochemical processes and in vivo studies that focus on the potential therapeutic impact of polySia, polySia glycomimetics, and sulfated polysaccharides in neuronal diseases. With this strategy, the receptor interactions of polySia and polySia mimetics can be understood on a submolecular level. As the HNK-1 glycan also enhances neuronal functions, we tested whether similar sulfated oligo- and polysaccharides from seaweed could be suitable, in addition to polySia, for finding potential new routes into patient care focusing on an improved cure for various neuronal diseases. The knowledge obtained here on the structural interplay between polySia or sulfated polysaccharides and their receptors can be exploited to develop new drugs and application routes for the treatment of neurological diseases and dysfunctions.

Skin-Derived C-Terminal Filaggrin-2 Fragments Are Pseudomonas aeruginosa-Directed Antimicrobials Targeting Bacterial Replication.

Soil- and waterborne bacteria such as Pseudomonas aeruginosa are constantly challenging body surfaces. Since infections of healthy skin are unexpectedly rare, we hypothesized that the outermost epidermis, the stratum corneum, and sweat glands directly control the growth of P. aeruginosa by surface-provided antimicrobials. Due to its high abundance in the upper epidermis and eccrine sweat glands, filaggrin-2 (FLG2), a water-insoluble 248 kDa S100 fused-type protein, might possess these innate effector functions. Indeed, recombinant FLG2 C-terminal protein fragments display potent antimicrobial activity against P. aeruginosa and other Pseudomonads. Moreover, upon cultivation on stratum corneum, P. aeruginosa release FLG2 C-terminus-containing FLG2 fragments from insoluble material, indicating liberation of antimicrobially active FLG2 fragments by the bacteria themselves. Analyses of the underlying antimicrobial mechanism reveal that FLG2 C-terminal fragments do not induce pore formation, as known for many other antimicrobial peptides, but membrane blebbing, suggesting an alternative mode of action. The association of the FLG2 fragment with the inner membrane of treated bacteria and its DNA-binding implicated an interference with the bacterial replication that was confirmed by in vitro and in vivo replication assays. Probably through in situ-activation by soil- and waterborne bacteria such as Pseudomonads, FLG2 interferes with the bacterial replication, terminates their growth on skin surface and thus may contributes to the skin's antimicrobial defense shield. The apparent absence of FLG2 at certain body surfaces, as in the lung or of burned skin, would explain their higher susceptibility towards Pseudomonas infections and make FLG2 C-terminal fragments and their derivatives candidates for new Pseudomonas-targeting antimicrobials.

Revealing the Achilles heel of bacterial toxins.

In addition to having antimicrobial properties, defensins inactivate various structurally unrelated bacterial toxins by a yet unknown manner. In this issue of Immunity, Kudryashova et al. (2014b) provide insights into mechanisms by which human ?-defensins destabilize and inactivate bacterial toxins.

The skin microbiome of caspase-14-deficient mice shows mild dysbiosis.

Caspase-14, an important proteinase involved in filaggrin catabolism, is mainly active in terminally differentiating keratinocytes, where it is required for the generation of skin natural moisturizing factors (NMFs). Consequently, caspase-14 deficient epidermis is characterized by reduced levels of NMFs such as urocanic acid and 2-pyrrolidone-5-carboxylic acid. Patients suffering from filaggrin deficiency are prone to develop atopic dermatitis, which is accompanied with increased microbial burden. Among several reasons, this effect could be due to a decrease in filaggrin breakdown products. In this study, we found that caspase-14(-/-) mice show enhanced antibacterial response compared to wild-type mice when challenged with bacteria. Therefore, we compared the microbial communities between wild-type and caspase-14(-/-) mice by sequencing of bacterial 16S ribosomal RNA genes. We observed that caspase-14 ablation leads to an increase in bacterial richness and diversity during steady-state conditions. Although both wild-type and caspase-14(-/-) skin were dominated by the Firmicutes phylum, the Staphylococcaceae family was reduced in caspase-14(-/-) mice. Altogether, our data demonstrated that caspase-14 deficiency causes the imbalance of the skin-resident bacterial communities.

Characterization of Spink6 in mouse skin: the conserved inhibitor of kallikrein-related peptidases is reduced by barrier injury.

The proteolytic regulation of the desquamation process by kallikrein-related peptidases (KLKs) is crucial for epidermal barrier function, and elevated KLK levels have been reported in atopic dermatitis. KLKs are controlled by specific inhibitors of the serine protease inhibitor of Kazal-type (Spink) family. Recently, SPINK6 was shown to be present in human stratum corneum. In order to investigate its role in epidermal barrier function, we studied mouse Spink6. Sequence alignment revealed that the Kazal domain of Spink6 is highly conserved in animals. Recombinant Spink6 efficiently inhibited mouse Klk5 and human KLK2, KLK4, KLK5, KLK6, KLK7, KLK12, KLK13, and KLK14, whereas human KLK1 and KLK8 were not inhibited. Spink6 was expressed in mouse epidermis mainly in the stratum granulosum, and the inner root sheath of hair follicles. Stimulation with flagellin, EGF, and IL-1ß did not alter Spink6 expression, whereas stimulation with tumor necrosis factor-α (TNFα)/IFNγ and all-trans retinoic acid resulted in a significant downregulation of Spink6 expression in cultured primary mouse keratinocytes. Mechanically and metabolically induced skin barrier dysfunction resulted both in a downregulation of Spink6 expression. Our study indicates that Spink6 is a potent inhibitor of KLKs and involved in skin barrier function.

Coal tar induces AHR-dependent skin barrier repair in atopic dermatitis.

Topical application of coal tar is one of the oldest therapies for atopic dermatitis (AD), a T helper 2 (Th2) lymphocyte-mediated skin disease associated with loss-of-function mutations in the skin barrier gene, filaggrin (FLG). Despite its longstanding clinical use and efficacy, the molecular mechanism of coal tar therapy is unknown. Using organotypic skin models with primary keratinocytes from AD patients and controls, we found that coal tar activated the aryl hydrocarbon receptor (AHR), resulting in induction of epidermal differentiation. AHR knockdown by siRNA completely abrogated this effect. Coal tar restored filaggrin expression in FLG-haploinsufficient keratinocytes to wild-type levels, and counteracted Th2 cytokine-mediated downregulation of skin barrier proteins. In AD patients, coal tar completely restored expression of major skin barrier proteins, including filaggrin. Using organotypic skin models stimulated with Th2 cytokines IL-4 and IL-13, we found coal tar to diminish spongiosis, apoptosis, and CCL26 expression, all AD hallmarks. Coal tar interfered with Th2 cytokine signaling via dephosphorylation of STAT6, most likely due to AHR-regulated activation of the NRF2 antioxidative stress pathway. The therapeutic effect of AHR activation herein described opens a new avenue to reconsider AHR as a pharmacological target and could lead to the development of mechanism-based drugs for AD.

The skin surface as antimicrobial barrier: present concepts and future outlooks.

Human skin protects the body as an outer barrier from various environmental threats and has to deal with an immense quantity of microbes comprising members of the own microbiota as well as potential pathogenic microbes. In this viewpoint, we highlight important cutaneous first-line defense barriers from the perspective of a microbe that tries to colonize and invade human skin. We place special emphasis on the role of antimicrobial peptides and the microbiota. In addition, we discuss and try to provide answers to some emerging issues concerning the various defense strategies of the epidermis.

Pseudomonas aeruginosa-derived rhamnolipids subvert the host innate immune response through manipulation of the human beta-defensin-2 expression.

Pseudomonas aeruginosa is a well-known cause of infections especially in compromised patients. To neutralize this pathogen, the expression of antimicrobial factors in epithelial cells is crucial. In particular the human beta-defensin hBD-2 is especially active against P. aeruginosa. In this study, we identified rhamnolipids in P. aeruginosa culture supernatants that are able to prevent the pathogen-induced hBD-2 response in keratinocytes. The presence of rhamnolipids within the host cells and inhibition assays suggest that calcium-regulated pathways and protein kinase C activation are impaired by rhamnolipids. In consequence, the induction of hBD-2 in keratinocytes by P. aeruginosa-derived flagellin as well as the host's own hBD-2 mediator interleukin IL-1ß is inhibited. Strikingly, rhamnolipids did not affect the release of the proinflammatory mediator interleukin IL-8 by flagellin. Thus, in addition to their function in establishment and persistence of P. aeruginosa infections, rhamnolipids can be engaged by P. aeruginosa for a targeted attenuation of the innate immunity to manage its survival and colonization on compromised epithelia.

Murine filaggrin-2 is involved in epithelial barrier function and down-regulated in metabolically induced skin barrier dysfunction.

The S100 fused-type proteins (SFTPs) are thought to be involved in the barrier formation and function of the skin. Mutations in the profilaggrin gene, one of the best investigated members of this family, are known to be the major risk factors for ichthyosis vulgaris and atopic dermatitis. Recently, we identified human filaggrin-2 as a new member of the SFTP family. To achieve further insight into its function, here the murine filaggrin-2 was analysed as a possible orthologue. The 5' and 3' ends of the mouse filaggrin-2 cDNA of the BALB/c strain were sequenced and confirmed an organization typical for SFTPs. Murine filaggrin-2 showed an expression pattern mainly in keratinizing epithelia in the upper cell layers on both mRNA and protein levels. The expression in cultured mouse keratinocytes was increased upon elevated Ca(2+) levels. Immunoblotting experiments indicated an intraepidermal processing of the 250-kDa full-length protein. In metabolically (essential fatty acid-deficient diet) induced skin barrier dysfunction, filaggrin-2 expression was significantly reduced, whereas filaggrin expression was up-regulated. In contrast, mechanical barrier disruption with acetone treatment did not affect filaggrin-2 mRNA expression. These results suggest that filaggrin-2 may contribute to epidermal barrier function and its regulation differs, at least in parts, from that of filaggrin.

Antimicrobial RNases in cutaneous defense.

Antimicrobial proteins (AMP) are small endogenous proteins which are capable of rapidly inactivating microorganisms at low micro- and nanomolar concentrations. Their significance in host defense is reflected by their wide distribution in nature. Several AMP have been isolated from human skin, and there is increasing evidence that AMP may play an important role in cutaneous defense. One important human AMP class comprises several antimicrobial members of the RNase A superfamily. Of these, two members, RNase 7 and RNase 5, have been implicated in cutaneous defense. This review gives an overview about our current knowledge on the potential role of RNase 7 and RNase 5 in protecting human skin from infection.

Epidermal proteases in the pathogenesis of rosacea.

A number of different proteases and their inhibitors have a role in skin physiology and in the pathophysiology of inflammatory skin diseases. Proteases are important in the desquamation process and orderly regulation of the skin's barrier function. On the basis of the catalytic domain, proteases are classified into aspartate-, cysteine-, glutamate-, metallo-, serine-, and threonine proteases. Particularly, serine proteases (SPs) contribute to epidermal permeability barrier homeostasis, as acute barrier disruption increases SP activity in skin and inhibition by topical SP inhibitors accelerated recovery of barrier function after acute abrogation. In rosacea, increased levels of the vasoactive and inflammatory host-defense peptide cathelicidin LL-37 and its proteolytic peptide fragments were found, which were explained by an abnormal production of tryptic activity originating from kallikrein-related peptidase (KLK) 5. It is therefore possible that also other proteases, even from microbial or parasite origin, have a role in rosacea by forming alternate angiogenic and proinflammatory cathelicidin peptides. Further, the regulation of protease activity, in particular KLK-5 activity, might have a role in rosacea. This review briefly summarizes our current knowledge about keratinocyte-derived proteases and protease inhibitors, which might have a role in the pathophysiology of rosacea.

Aggregation of Kanamycin A: dimer formation with physiological cations.

Global cluster geometry optimization has focused so far on clusters of atoms or of compact molecules. We are demonstrating here that present-day techniques also allow to globally optimize clusters of extended, flexible molecules, and that such studies have immediate relevance to experiment. For example, recent experimental findings point to production of larger clusters of an aminoglycoside closely related to Kanamycin A (KA), together with certain preferred physiological cations, by Pseudomonas aeruginosa. The present study provides first theoretical support for KA clustering, with a close examination of the monomer, the bare dimer, and dimers with sodium and potassium cations, employing global cluster structure optimization, in conjunction with force fields, semiempirical methods, DFT and ab-initio approaches. Interestingly, already at this stage the theoretical findings support the experimental observation that sodium cations are preferred over potassium cations in KA clusters, due to fundamentally different cationic embedding. Theoretically predicted NMR and IR spectra for these species indicate that it should be possible to experimentally detect the aggregation state and even the cationic embedding mode in such clusters.

Inhibition of kallikrein-related peptidases by the serine protease inhibitor of Kazal-type 6.

Kallikrein-related peptidases (KLKs) are a group of serine proteases, expressed in several tissues. Their activity is regulated by inhibitors including members of the serine protease of Kazal-type (SPINK) family. Recently, we discovered that SPINK6 is expressed in human skin and inhibits KLK5, KLK7, KLK14 but not KLK8. In this study we tested whether SPINK6 inhibits other members of the KLK family and caspase-14. Using chromogenic substrates, SPINK6 exhibited inhibitory activity against KLK12 and KLK13 with K(i) around 1nM, KLK4 with K(i)=27.3nM, KLK6 with K(i)=140nM, caspase-14 with a K(i) approximating 1µM and no activity against KLK1, KLK3 and KLK11. Taken together, SPINK6 is a potent inhibitor of distinct KLKs members.