NOD2/RICK-dependent β-defensin 2 regulation is protective for nontypeable Haemophilus influenzae-induced middle ear infection.

Middle ear infection, otitis media (OM), is clinically important due to the high incidence in children and its impact on the development of language and motor coordination. Previously, we have demonstrated that the human middle ear epithelial cells up-regulate β-defensin 2, a model innate immune molecule, in response to nontypeable Haemophilus influenzae (NTHi), the most common OM pathogen, via TLR2 signaling. NTHi does internalize into the epithelial cells, but its intracellular trafficking and host responses to the internalized NTHi are poorly understood. Here we aimed to determine a role of cytoplasmic pathogen recognition receptors in NTHi-induced β-defensin 2 regulation and NTHi clearance from the middle ear. Notably, we observed that the internalized NTHi is able to exist freely in the cytoplasm of the human epithelial cells after rupturing the surrounding membrane. The human middle ear epithelial cells inhibited NTHi-induced β-defensin 2 production by NOD2 silencing but augmented it by NOD2 over-expression. NTHi-induced β-defensin 2 up-regulation was attenuated by cytochalasin D, an inhibitor of actin polymerization and was enhanced by α-hemolysin, a pore-forming toxin. NOD2 silencing was found to block α-hemolysin-mediated enhancement of NTHi-induced β-defensin 2 up-regulation. NOD2 deficiency appeared to reduce inflammatory reactions in response to intratympanic inoculation of NTHi and inhibit NTHi clearance from the middle ear. Taken together, our findings suggest that a cytoplasmic release of internalized NTHi is involved in the pathogenesis of NTHi infections, and NOD2-mediated β-defensin 2 regulation contributes to the protection against NTHi-induced otitis media

Human beta defensin 2 selectively inhibits HIV-1 in highly permissive CCR6+CD4+ T cells

Chemokine receptor type 6 (CCR6)+CD4+ T cells are preferentially infected and depleted during HIV disease progression, but are preserved in non-progressors. CCR6 is expressed on a heterogeneous population of memory CD4+ T cells that are critical to mucosal immunity. Preferential infection of these cells is associated, in part, with high surface expression of CCR5, CXCR4, and α4β7. In addition, CCR6+CD4+ T cells harbor elevated levels of integrated viral DNA and high levels of proliferation markers. We have previously shown that the CCR6 ligands MIP-3α and human beta defensins inhibit HIV replication. The inhibition required CCR6 and the induction of APOBEC3G. Here, we further characterize the induction of apolipoprotein B mRNA editing enzyme (APOBEC3G) by human beta defensin 2. Human beta defensin 2 rapidly induces transcriptional induction of APOBEC3G that involves extracellular signal-regulated kinases 1/2 (ERK1/2) activation and the transcription factors NFATc2, NFATc1, and IRF4. We demonstrate that human beta defensin 2 selectively protects primary CCR6+CD4+ T cells infected with HIV-1. The selective protection of CCR6+CD4+ T cell subsets may be critical in maintaining mucosal immune function and preventing disease progression

Current Status of Defensins and Their Role in Innate and Adaptive Immunity

Naturally occurring antimicrobial cationic polypeptides play a major role in innate and adaptive immunity. These polypeptides are found to be either linear and unstructured or structured through disulfide bonds. Among the structured antimicrobial polypeptides, defensins comprise a family of cysteine-rich cationic polypeptides that contribute significantly to host defense against the invasion of microorganisms in animals, humans, insects and plants. Their wide-spread occurrence in various tissues of these diverse organisms, and their importance in innate and adaptive immunity have led to their identification, isolation and characterization. A large volume of literature is available on defensins’ occurrence, structural characterization, gene expression and regulation under normal and pathological conditions. Much has also been published regarding their antimicrobial, antiviral and chemoattractive properties, and their molecular and cellular interactions. In this review, we describe the current status of our knowledge of defensins with respect to their molecular, cellular and structural biology, their role in host defense, future research paradigms and the possibility of their utilization as a new class of non-toxic antimicrobial agents and immuno-modulators

Antimicrobial peptides: agents of border protection for companion animals.

Over the past 20 years, there have been significant inroads into understanding the roles of antimicrobial peptides in homeostatic functions and their involvement in disease pathogenesis. In addition to direct antimicrobial activity, these peptides participate in many cellular functions, including chemotaxis, wound healing and even determination of canine coat colour. Various biological and genetic approaches have helped to elucidate the role of antimicrobial peptides with respect to innate immunity and host defense. Associations of antimicrobial peptides with various skin diseases, including psoriasis, rosacea and atopic dermatitis, have been documented in humans. In the longer term, therapeutic modulation of antimicrobial peptide expression may provide effective new treatments for disease. This review highlights current knowledge about antimicrobial peptides of the skin and circulating leukocytes, with particular focus on relevance to physiology and disease in companion animals

Synergistic effect of interleukin 1 alpha on nontypeable Haemophilus influenzae-induced up-regulation of human beta-defensin 2 in middle ear epithelial cells

BACKGROUND: We recently showed that beta-defensins have antimicrobial activity against nontypeable Haemophilus influenzae (NTHi) and that interleukin 1 alpha (IL-1 alpha) up-regulates the transcription of beta-defensin 2 (DEFB4 according to new nomenclature of the Human Genome Organization) in human middle ear epithelial cells via a Src-dependent Raf-MEK1/2-ERK signaling pathway. Based on these observations, we investigated if human middle ear epithelial cells could release IL-1 alpha upon exposure to a lysate of NTHi and if this cytokine could have a synergistic effect on beta-defensin 2 up-regulation by the bacterial components. METHODS: The studies described herein were carried out using epithelial cell lines as well as a murine model of acute otitis media (OM). Human cytokine macroarray analysis was performed to detect the released cytokines in response to NTHi exposure. Real time quantitative PCR was done to compare the induction of IL-1 alpha or beta-defensin 2 mRNAs and to identify the signaling pathways involved. Direct activation of the beta-defensin 2 promoter was monitored using a beta-defensin 2 promoter-Luciferase construct. An IL-1 alpha blocking antibody was used to demonstrate the direct involvement of this cytokine on DEFB4 induction. RESULTS: Middle ear epithelial cells released IL-1 alpha when stimulated by NTHi components and this cytokine acted in an autocrine/paracrine synergistic manner with NTHi to up-regulate beta-defensin 2. This synergistic effect of IL-1 alpha on NTHi-induced beta-defensin 2 up-regulation appeared to be mediated by the p38 MAP kinase pathway. CONCLUSION: We demonstrate that IL-1 alpha is secreted by middle ear epithelial cells upon exposure to NTHi components and that it can synergistically act with certain of these molecules to up-regulate beta-defensin 2 via the p38 MAP kinase pathway

A Bacterial Mutant Library as a Tool to Study the Attack of a Defensin Peptide

Defensin attack! Here we report the screening of human defensin 5 against the Keio Collection of E. coli strains. The results of this screen further our understanding of how this important hostdefense peptide kills bacteria and how bacteria protect themselves against the attack from the human host.United States. National Institutes of Health (DP2OD007045

Large-scale Synthesis and Functional Elements for the Antimicrobial Activity of Defensins

Human neutrophil defensins, and their analogues incorporating anionic, hydrophobic or cationic residues at the N- and C-termini, were synthesized by solid-phase procedures. The synthetic defensins were examined for their microbicidal activity against Candida albicans, two Gram-negative bacteria (Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis) and two Gram-positive bacteria (Streptococcus gordonii and Streptococcus mutans). The human neutrophil peptide 1 (HNP1) and HNP2 were found to be potent candidacidal agents. HNP3, which differs by one amino acid at the N-terminus of its sequence, was totally inactive. The Gram-negative bacteria A. actinomycetemcomitans and P. gingivalis and the Gram-positive bacteria S. gordonii and S. mutans were insensitive to human defensins. However, the insertion of two basic residues, such as arginine, at both the N-terminus and the C-terminus of HNP2 significantly enhanced antifungal and antibacterial activity. The addition of anionic residues, such as aspartic acid, at the N- and C-termini rendered the molecule totally inactive. The presence of two hydrophobic amino acids, such as valine, at the N-terminus of HNP2 and of two basic arginine residues at its C-terminus resulted in molecules that were optimally active against these oral pathogens. The results suggest that the N- and C-terminal residues in defensin peptides are the crucial functional elements that determine their microbicidal potency. The three-dimensional structure of all defensins constitutes the same amphiphilic beta-sheet structure, with the polar face formed by the N- and C-terminal residues playing an important role in defining microbicidal potency and the antimicrobial spectrum. The enhanced microbicidal activity observed for defensin peptides with two basic residues at both the N- and C-termini could be due to optimization of the amphiphilicity of the structure, which could facilitate specific interactions with the microbial membranes