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Production of inducible antimicrobial peptides offers a first and rapid defense response of epithelial cells against invading microbes. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide induced in various epithelia upon extracellular as well as intracellular bacterial challenge. Nucleotide-binding oligomerization domain protein 2 (NOD2/CARD15) is a cytosolic protein involved in intracellular recognition of microbes by sensing peptidoglycan fragments (e.g. muramyl dipeptide). We used luciferase as a reporter gene for a 2.3-kb hBD-2 promoter to test the hypothesis that NOD2 mediates the induction of hBD-2. Activation of NOD2 in NOD2-overexpressing human embryonic kidney 293 cells through its ligand muramyl dipeptide (MDP) induced hBD-2 expression. In contrast, overexpression of NOD2 containing the 3020insC frameshift mutation, the most frequent NOD2 variant associated with Crohn disease, resulted in defective induction of hBD-2 through MDP. Luciferase gene reporter analyses and site-directed mutagenesis experiments demonstrated that functional binding sites for NF-B and AP-1 in the hBD-2 promoter are required for NOD2-mediated induction of hBD-2 through MDP. Moreover, the NF-B inhibitor Helenalin as well as a super-repressor form of the NF-B inhibitor IB strongly inhibited NOD2-mediated hBD-2 promoter activation. Expression of NOD2 was detected in primary keratinocytes, and stimulation of these cells with MDP induced hBD-2 peptide release. In contrast, small interference RNA-mediated downregulation of NOD2 expression in primary keratinocytes resulted in a defective induction of hBD-2 upon MDP treatment. Together, these data suggest that NOD2 serves as an intracellular pattern recognition receptor to enhance host defense by inducing the production of antimicrobial peptides such as hBD-2.Human epithelia are in permanent contact with various potential pathogenic microorganisms. To overcome these microbial threats, epithelia have developed a chemical defense system based on the production of various antimicrobial proteins (1-4). Human beta-defensin-2 (hBD-2) 2 was the first inducible human antimicrobial protein discovered and was originally isolated from lesional psoriatic skin (5). hBD-2 belongs to the beta-defensin family, a group of small (4 -5 kDa), cationic antibiotic peptides first discovered in cattle (6). hBD-2 exhibits a broad spectrum of antimicrobial activity, and its capacity to kill bacteria in vivo has been demonstrated in a mouse gene therapy study with hBD-2-transfected tumor cells. Following a bacterial infection, mice with hBD-2-bearing tumors bore fewer viable bacteria than controls (7). In addition to its capacity to serve as an antibiotic peptide, hBD-2 may promote adaptive immune responses by recruiting dendritic and T cells to the site of microbial invasion through interaction with CCR6 (8). Furthermore hBD-2 was found to be a specific chemoattractant for tumor necrosis factor-␣ (TNF-␣)-treated human neutrophils (9). hBD-2 is expressed in many epithelia (e.g. skin, respiratory tract, dig...

Human skin is able to mount a fast response against invading harmful bacteria through the rapid production of inducible peptide antibiotics such as the human beta-defensins (hBD). To gain more insight into the role and regulation of inducible beta-defensins in the innate immunity of human skin, we investigated whether gene induction of the human beta-defensins hBD-1, -2, -3, and -4 in keratinocytes is regulated in a similar manner. Therefore, we performed a comparative study of gene expression of these four hBD in primary cultured keratinocytes using real-time PCR. A basal mRNA expression was observed for all four hBD in primary keratinocytes, which strongly increased for hBD-2, -3, and -4 during Ca(2+)-induced differentiation of the keratinocytes. This effect was completely abolished when the keratinocytes were pre-treated with all-trans-retinoic acid (RA). Furthermore, the differential induction of hBD-2, -3, and -4 gene expression in keratinocytes by proinflammatory cytokines, phorbol-myristate-acetate (PMA), and bacteria was inhibited by more than 90% when the keratinocytes were pre-incubated with RA. Inhibition of IL-1beta-mediated hBD-2 induction through RA was further confirmed by gene reporter assays and western-blot analysis. We conclude that RA is a potent inhibitor of beta-defensin induction in keratinocytes and might downregulate the inducible innate chemical defense system of human skin.

Human beta-defensin-2 (hBD-2) is an inducible epithelial peptide antibiotic involved in cutaneous defense. Expression of hBD-2 in keratinocytes is strongly induced by IL-1beta and culture supernatants of Pseudomonas aeruginosa (PA). The use of an IL-1 receptor antagonist revealed that PA-mediated induction of hBD-2 is not dependent on IL-1. Luciferase gene reporter experiments, demonstrated that a 2,338 bp promoter fragment of hBD-2 containing three putative NF-kappaB as well as one activator protein-1 (AP-1) binding site was strongly activated by IL-1beta and PA. Mutation of all NF-kappaB binding sites together with mutation of the AP-1 binding site completely abolished hBD-2 promoter activation by IL-1beta and PA. Treatment with the NF-kappaB inhibitor Helenalin as well as with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the p38 mitogen-activated protein kinase inhibitor SB 202190 blocked hBD-2 induction by IL-1beta and PA. PD 98059, a selective inhibitor of extracellular signal-regulated kinase 1/2 demonstrated no significant influence. Transcription factor ELISAs indicated that the NF-kappaB heterodimer p50-p65 binds to all three NF-kappaB sites in the hBD-2 promoter upon stimulation of primary keratinocytes with IL-1beta and PA. We conclude that the activation of NF-kappaB (p50-p65) and AP-1 are crucial events for induction of hBD-2 in keratinocytes upon IL-1beta and PA stimulation.

IntroductionShared decision-making (SDM) is not yet widely used when making decisions in German hospitals. Making SDM a reality is a complex task. It involves training healthcare professionals in SDM communication and enabling patients to actively participate in communication, in addition to providing sound, easy to understand information on treatment alternatives in the form of evidence-based patient decision aids (EbPDAs). This project funded by the German Innovation Fund aims at designing, implementing and evaluating a multicomponent, large-scale and integrative SDM programme—called SHARE TO CARE (S2C)—at all clinical departments of a University Hospital Campus in Northern Germany within a 4-year time period.Methods and analysisS2C tackles the aforementioned components of SDM: (1) training physicians in SDM communication, (2) activating and empowering patients, (3) developing EbPDAs in the most common/relevant diseases and (4) training other healthcare professionals in SDM coaching. S2C is designed together with patients and providers. The physicians’ training programme entails an online and an in situ training module. The decision coach training is based on a similar but less comprehensive approach. The development of online EbPDAs follows the International Patient Decision Aid Standards and includes written, graphical and video-based information. Validated outcomes of SDM implementation are measured in a preintervention and postintervention evaluation design. Process evaluation accompanies programme implementation. Health economic impact of the intervention is investigated using a propensity-score-matched approach based on potentially preference-sensitive hospital decisions.Ethics and disseminationEthics committee review approval has been obtained from Medical Ethics Committee of the Medical Faculty of the Christian-Albrechts-University Kiel. Project information and results will be disseminated at conferences, on project-hosted websites at University Hospital Medical Center Schleswig Holstein and by S2C as well as in peer-reviewed and professional journals.