NOD2/CARD15 Mediates Induction of the Antimicrobial Peptide Human Beta-defensin-2

Abstract

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...

Characteristic changes in microbial community composition and expression of innate immune genes in acute appendicitis

“…46 One of its crucial functions is the defense against intracellular bacteria and invasion. For the regulation of hBD-2 a role of NOD2 has been reported, 47 but for other AMPs only few data exist. 48 The observed regulation of the mRNA of AMPs, cytokines and PRR suggested the involvement of bacteria in the pathogenesis of appendicitis.…”

NOD2 polymorphisms predict severe acute graft-versus-host and treatment-related mortality in T-cell-depleted haematopoietic stem cell transplantation

“…Their production has been shown decreased when there are NOD2 polymorphisms. 9,10 The increased incidence of CoNS bacteraemia found in patients with an NOD2 polymorphism suggests that lower number or activity of the peptides influences infectious complications in HSCT. This might explain how the gut may be an important origin of staphylococcal and streptococcal bacteraemias.…”

“…NOD2 is important in the antibacterial defences especially by regulating the production of defensins at the epithelial barriers of gut and lung. 9,28,29 Defensins possess anti-staphylococcal and streptococcal activity 30 and, in the gut, are elaborated predominantly by Paneth cells. Their production has been shown decreased when there are NOD2 polymorphisms.…”

Cloning of two rainbow trout nucleotide-binding oligomerization domain containing 2 (NOD2) splice variants and functional characterization of the NOD2 effector domains

“…The best characteristic of these is the activation of NF-kB and MAPKs through interactions with the CARD domain of receptor-interacting protein 2 (RIP2) [11]. NOD2 signaling by activating NF-kB and MAPKs leads to the induction of proinflammatory cytokines and chemokines [12], and anti-microbial peptides [13], which mediate the antimicrobial response. Besides the function in antibacterial defence, new studies show that NOD2 also has an important role in recognizing viruses and inducing type I IFN production through association with the mitochondrial antiviral signaling protein (MAVS) [14].…”