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.

Defects of filaggrin-like proteins in both lesional and nonlesional atopic skin.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by a disturbed epidermal barrier. In a subset of patients, this is explained by nonsense mutations in the gene encoding filaggrin (FLG). OBJECTIVES: We sought to evaluate the respective role of FLG mutations and proinflammatory cytokines and to assess the expression of FLG, hornerin (HRNR), and FLG2, 2 FLG-like proteins, which are involved in epidermal barrier functions, in normal skin and both lesional and nonlesional skin of patients with AD. METHODS: An FLG-genotyped cohort of 73 adults with AD and 73 aged-matched control subjects was analyzed by using immunohistochemistry and immunoblotting. Normal primary human keratinocytes were differentiated in either the absence or presence of IL-4, IL-13, and IL-25. RESULTS: Compared with control subjects, FLG, HRNR, and FLG2 were detected at significantly lower levels in the skin of patients with AD, irrespective of their FLG genotype. The reduction was greater in lesional compared with nonlesional skin. In addition, the proFLG/FLG ratio was found to be higher in the skin of wild-type patients than in control subjects. Cytokine treatment of keratinocytes induced a dramatic reduction in FLG, FLG2, and HRNR expression both at the mRNA and protein levels. CONCLUSION: The stratum corneum of lesional but also clinically unaffected skin of adults with AD is abnormal, with reduced expression of FLG and FLG-like proteins. In addition to nonsense mutations, proinflammatory cytokines and some defects in the proFLG processing can contribute to the FLG downregulation. Our study suggests that skin inflammation reduces the expression of FLG-like proteins, contributing to the AD-related epidermal barrier dysfunction.

Deimination of human filaggrin-2 promotes its proteolysis by calpain 1.

Filaggrin-2 (FLG2), a member of the S100-fused type protein family, shares numerous features with filaggrin (FLG), a key protein implicated in the epidermal barrier functions. Both display a related structural organization, an identical pattern of expression and localization in human epidermis, and proteolytic processing of a large precursor. Here, we tested whether FLG2 was a substrate of calpain 1, a calcium-dependent protease directly involved in FLG catabolism. In addition, deimination being critical for FLG degradation, we analyzed whether FLG2 deimination interfered with its proteolytic processing. With this aim, we first produced a recombinant form of FLG2 corresponding to subunits B7 to B10 fused to a COOH-terminal His tag. Incubation with calpain 1 in the presence of calcium induced a rapid degradation of the recombinant protein and the production of several peptides, as shown by Coomassie Blue-stained gels and Western blotting with anti-FLG2 or anti-His antibodies. MALDI-TOF mass spectrometry confirmed this result and further evidenced the production of non-immunoreactive smaller peptides. The degradation was not observed when a calpain 1-specific inhibitor was added. The calpain cleavage sites identified by Edman degradation were regularly present in the B-type repeats of FLG2. Moreover, immunohistochemical analysis of normal human skin revealed colocalization of FLG2 and calpain 1 in the upper epidermis. Finally, the FLG2 deiminated by human peptidylarginine deiminases was shown to be more susceptible to calpain 1 than the unmodified protein. Altogether, these data demonstrate that calpain 1 is essential for the proteolytic processing of FLG2 and that deimination accelerates this process.

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.

Isolation of SPINK6 in human skin: selective inhibitor of kallikrein-related peptidases.

Kallikrein-related peptidases (KLKs) play a central role in skin desquamation. They are tightly controlled by specific inhibitors, including the lymphoepithelial Kazal-type inhibitor (LEKTI) encoded by SPINK5 and LEKTI-2 encoded by SPINK9. Herein, we identify SPINK6 as a selective inhibitor of KLKs in the skin. Unlike LEKTI but similar to LEKTI-2, SPINK6 possesses only one typical Kazal domain. Its mRNA was detected to be expressed at low levels in several tissues and was induced during keratinocyte differentiation. Natural SPINK6 was purified from human plantar stratum corneum extracts. Immunohistochemical analyses revealed SPINK6 expression in the stratum granulosum of human skin at various anatomical localizations and in the skin appendages, including sebaceous glands and sweat glands. SPINK6 expression was decreased in lesions of atopic dermatitis. Using KLK5, KLK7, KLK8, KLK14, thrombin, trypsin, plasmin, matriptase, prostasin, mast cell chymase, cathepsin G, neutrophil elastase, and chymotrypsin, inhibition with recombinant SPINK6 was detected only for KLK5, KLK7, and KLK14, with apparent K(i) values of 1.33, 1070, and 0.5 nm, respectively. SPINK6 inhibited desquamation of human plantar callus in an ex vivo model. Our findings suggest that SPINK6 plays a role in modulating the activity of KLKs in human skin. A selective inhibition of KLKs by SPINK6 might have therapeutic potential when KLK activity is elevated.

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.

Molecular identification and expression analysis of filaggrin-2, a member of the S100 fused-type protein family.

Genes of the S100 fused-type protein (SFTP) family are clustered within the epidermal differentiation complex and encode essential components that maintain epithelial homeostasis and barrier functions. Recent genetic studies have shown that mutations within the gene encoding the SFTP filaggrin cause ichthyosis vulgaris and are major predisposing factors for atopic dermatitis. As a vital component of healthy skin, filaggrin is also a precursor of natural moisturizing factors. Here we present the discovery of a member of this family, designated as filaggrin-2 (FLG2) that is expressed in human skin. The FLG2 gene encodes a histidine- and glutamine-rich protein of approximately 248 kDa, which shares common structural features with other SFTP members, in particular filaggrin. We found that FLG2 transcripts are present in skin, thymus, tonsils, stomach, testis and placenta. In cultured primary keratinocytes, FLG2 mRNA expression displayed almost the same kinetics as that of filaggrin following Ca(2+) stimulation, suggesting an important role in molecular regulation of epidermal terminal differentiation. We provide evidences that like filaggrin, FLG2 is initially expressed by upper granular cells, proteolytically processed and deposited in the stratum granulosum and stratum corneum (SC) layers of normal epidermis. Thus, FLG2 and filaggrin may have overlapping and perhaps synergistic roles in the formation of the epidermal barrier, protecting the skin from environmental insults and the escape of moisture by offering precursors of natural moisturizing factors.

Highly complex peptide aggregates of the S100 fused-type protein hornerin are present in human skin.

Human hornerin (HRNR) is a 245 kDa S100 fused-type protein which contains 95% tandem quasi-repeating glycine- and serine-rich domains. Previously HRNR was not thought to be expressed in healthy skin; however, we purified an HRNR peptide fragment from stratum corneum. Moreover, we found that HRNR mRNA is expressed in skin biopsies from different sites as head, trunk, legs, hands, and feet. In cultured human epidermal keratinocytes, HRNR mRNA expression was transiently induced during Ca(2+)-dependent differentiation. Immunostaining using distinct antibodies generated against four putative HRNR domains revealed strong HRNR immunoreactivity in healthy epidermis as well as in the entire outer root sheath of normal human scalp hair follicles. In lesions from psoriasis and atopic dermatitis patients, HRNR immunoreactivity was reduced compared with uninvolved skin of these patients. Electrospray ionization mass spectrometry and Western blot analyses revealed that HRNR is a highly degradable protein that forms complex high molecular weight peptide aggregates. Our findings suggest that HRNR is expressed in healthy skin and give insight into the complex biology of this protein. HRNR and its degradation products might contribute to the barrier function of healthy human skin.