SummaryRecent evidence suggests that probiotic bacteria may stabilize gut barrier function via induction of anti-microbial peptides such as defensins. This study aimed to elucidate the induction mechanism of the human beta defensin-2 (hBD-2) gene by different probiotic lactobacillus strains. The expression of hBD-2 mRNA peaked at 6 h of incubation upon treatment of Caco-2 cells and increased with higher dosage of various probiotic bacteria. Deletion of nuclear factor (NF)-kB and activator protein-1 (AP-1) binding sites on the hBD-2 promoter resulted in a complete abrogation of promoter activation by probiotics. As revealed by the use of specific mitogen-activated protein kinase (MAPK) inhibitors the hBD-2 induction was dependent on the MAPK extracellular regulated kinase (ERK 1/2), p38 and c-Jun N-terminal kinase (JNK), although to varying degrees. Several Lactobacillus strains and VSL#3, a probiotic cocktail of four lactobacilli, three bifidum and one streptococcus species, induced the secretion of the hBD-2 peptide into the culture media as shown by enzyme-linked immunosorbent assay (ELISA). Thus, the present study suggests that lactobacilli and the VSL#3 bacterial mixture strengthen intestinal barrier functions through the up-regulation of hBD-2 via induction of proinflammatory pathways including NF-kB and AP-1 as well as MAPKs.
BackgroundHuman skin is able to mount a fast response against invading microorganisms by the release of antimicrobial proteins such as the ribonuclease RNase 7. Because RNase 7 exhibits high activity against Enterococcus faecium the aim of this study was to further explore the role of RNase 7 in the cutaneous innate defense system against E. faecium.Methodology/Principal FindingsAbsolute quantification using real-time PCR and ELISA revealed that primary keratinocytes expressed high levels of RNase 7. Immunohistochemistry showed RNase 7 expression in all epidermal layers of the skin with an intensification in the upper more differentiated layers. Furthermore, RNase 7 was secreted by keratinocytes in vitro and in vivo in a site-dependent way. RNase 7 was still active against E. faecium at low pH (5.5) or high NaCl (150 mM) concentration and the bactericidal activity of RNase 7 against E. faecium required no ribonuclease activity as shown by recombinant RNase 7 lacking enzymatic activity. To further explore the role of RNase 7 in cutaneous defense against E. faecium, we investigated whether RNase 7 contributes to the E. faecium killing activity of skin extracts derived from stratum corneum. Treatment of the skin extract with an RNase 7 specific antibody, which neutralizes the antimicrobial activity of RNase 7, diminished its E. faecium killing activity.Conclusions/SignificanceOur data indicate that RNase 7 contributes to the E. faecium-killing activity of skin extracts and suggest an important role for RNase 7 in the protection of human skin against E. faecium colonization.
Acute eczema is an inflammatory skin disease characterized by the formation of small intraepidermal blisters, reduction of the adhesion molecule E-cadherin from the keratinocyte surface, and impaired keratinocyte cohesion. Here, we reveal that the disintegrin and metalloprotease ADAM10 is critically involved in regulating E-cadherin cell-surface expression in cultured primary human keratinocytes and in diseased human skin. Proinflammatory cytokines, transforming growth factor-beta, and lipopolysaccharide led to increased release of soluble E-cadherin by activating mitogen-activated protein kinase signaling in cultured keratinocytes. Moreover, these stimuli decreased the amount of pro-ADAM10 and increased the level of the active protease, leading to loss of E-cadherin from the cell surface and decreased keratinocyte cohesion. In situ examination and immunoblot analyses of E-cadherin and ADAM10 expression in lesional skin of eczema revealed that the reduction of E-cadherin expression in areas of blister formation closely correlated with increased level of ADAM10 expression and elevated E-cadherin shedding. Our data suggest that ADAM10-mediated E-cadherin proteolysis leads to the impaired cohesion of keratinocytes observed in eczematous dermatitis and provide previously unreported insights into the understanding of the molecular mechanisms involved in inflammatory diseases with loss in epithelial integrity.
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.
We propose here a feedback model in which misfolded mutant K14 triggers enhanced expression of K14, which in turn amplifies the JNK-/AP1-mediated MAPK stress response. The exact mechanism cannot be deduced from these preliminary data. However, based on the observation that extrachromosomal overexpression of wild-type K14 in non-mutant cells also leads to the same effects, we suggest that an imbalance between K14 incorporated into the IF network and K14 existing as cytoplasmic keratin monomers triggers elevated MAPK signalling, potentially altering IF dynamics by phosphorylation. In this context, the MAPK family member p38 has previously been shown to modulate IF organization through phosphorylation of keratins (14).These data should help broaden the still narrow field of therapeutic approaches to EBS (17) and raise awareness to the possibility that as a secondary effect of the K14 mutation, imbalance of IF components and the associated amplification of MAPK signalling could play an important role in the pathophysiology of the disease. AcknowledgementsMW performed the research, analysed the data and wrote the manuscript, KO designed the research study, AT contributed essential tools and expertise in laser microscopy, and JWB and HH critically reviewed the manuscript. We gratefully thank Prof. Birgit Lane for kindly supplying the wild-type NEB1 and the patient KEB7 keratinocyte cell lines. This work was supported by the Dystrophic Epidermolysis Bullosa Research Association (DEBRA), Austria and by the Paracelsus Medical University, Salzburg, Austria (project number E-11/13/067-OEN). Conflict of interestsThe authors have declared no conflict of interest. Supporting InformationAdditional Supporting Information may be found in the online version of this article: Kiel, Schittenhelmstr. 7, 24105 Kiel, Germany, Tel.: 49-431-5971598, Fax: 49-431-5975243, e-mail: jharder@dermatology.uni-kiel.de Abstract: The Gram-positive bacterium Staphylococcus aureus is a frequent skin colonizer that often causes severe skin infections. It has been reported that neutralizing the negatively charged bacterial surface through the incorporation of D-alanine in its teichoic acids confers reduced susceptibility of S. aureus towards cationic antimicrobial peptides (AMPs). Using a S. aureus strain deficient in D-alanylated teichoic acids (dltA mutant), we demonstrate that D-alanylation of its surface reduces the susceptibility of S. aureus to skin-derived AMPs such as RNase 7 and human beta-defensins. This is accompanied by a higher killing activity of skin extracts towards the S. aureus dltA mutant as well as towards clinical isolates expressing lower levels of dltA. We conclude that modulation of cell envelope D-alanylation may help S. aureus to persist on human skin through evasion of cutaneous innate defense provided by cationic skin-derived AMPs.
SummaryPsoriasin (S100 A7) was discovered two decades ago as a protein abundantly expressed in psoriatic keratinocytes. Even though much scientific research has been carried out on the characterization of psoriasin, only recent studies point to an important role of psoriasin as an antimicrobial and immunomodulatory protein in skin and other epithelia. In this review, we provide an overview of the major findings in psoriasin research and discuss novel studies highlighting the role of psoriasin as an important effector molecule of the cutaneous barrier.
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