Defensins and cathelicidins constitute the two major groups of antimicrobial peptides in most mammalian species. The most abundant group of antimicrobial peptides is comprised by the α-, β- and θ-defensins.[alpha, beta, theta (circle w/horizontal line)] Mammalian defensins are endogenous cysteine-rich peptide antibiotics classically produced either by epithelial cells of the respiratory, urogenital and digestive tracts, or by circulating cells including granulocytes and macrophages. More recently, however, β-defensins have also been identified in the heart of different species.
β-defensins are small (3.5-4.5 kDa) highly basic cationic peptides. These peptides are ancient and universal molecules of innate immunity with functions extending far beyond simple antibiotics, including anti-tumor and mitogenic activity, as well as immunomodulation and signal transduction characteristics. The overall effectiveness of an innate immunity based host defense is shown by the clearly successful survival of plants and invertebrates, organisms which completely lack adaptive immunity. β-defensins are structurally defined by the conserved cysteine-rich motif forming three disulphide bonds, which stabilize a β-sheet formation. The amphipathicity of these molecules determines their folding pattern and is believed to govern the antimicrobial effect. The mechanism of action appears to rely on permeabilization of the microbial membrane and lysis of invading organisms, as explained by the Shai-Matsuzaki-Huang model. Epithelial β-defensins constitute a rapidly mobilized local defense against microbial intruders at the epithelial and mucosal surfaces, and different studies have reported induction of epithelial β-defensins at sites of inflammation, injury, infection as well as other disease processes.
TOLL-LIKE RECEPTORS AND THE NUCLEAR FACTOR KAPPA-B PATHWAY
The innate immune system utilizes a group of pattern recognition receptors (PRRs) that are capable of recognizing specific pathogen-associated molecular patterns (PAMPs), such as LPS (lipopolysaccharide) of Gram-negative bacteria. LPS initially binds to the LBP (LPS-binding protein) and transfer it via the CD14-enhanced mechanism to a receptor complex including TLR-4 and MD-2. This recognition results in different intracellular pathways being activated, involving the adaptor molecules AP-1 and MyD88 and the transcription factor nuclear factor kappa B (NF-κB). The NF-κB transcription factor is a central mediator of the innate immune response, regulating infectious as well as non-infectious stress responses. Upon recognition of microbial components by TLRs the innate as well as the adaptive immune response is activated. NF-κB and AP-1 are chronically activated in cardiomyocytes in human heart failure. Inflammatory cytokine activation and up-regulation of TLR expression have been reported in experimental as well as human heart failure regardless of etiology. In vertebrates, the best characterized of the PRRs are the Toll-like receptors (TLRs), of which a prototype is the interleukin-1 receptor. The mammalian TLR family includes a minimum of ten members (TLR1-TLR10), each apparently recognizing distinct microbial structures. Recently an eleventh TLR was discovered by two independent groups and named TLR11 by one and TLR12 by the other. The TLRs are pivotal in sensing and defeating infection at its initial stages, and in controlling the cellular responses to infection. The TLR family is subdivided into three groups, which recognize specific components conserved among microorganisms and pathogens in particular. TLR4 is linked to the recognition of LPS and hence Gram-negative bacteria, whereas TLR2 is critical to the recognition of lipoproteins and peptidoglycan from Gram-positive bacteria. TLR6 functionally cooperates with TLR-2 to recognize microbial lipoproteins. The activation of the TLRs and the NF-κB pathway is responsible for the induction of the expression of antimicrobial peptide genes, including beta-defensins. To our knowledge, this is the only pathway which has been described as a route for β-defensin production.
INNATE IMMUNITY OF THE HEART
The heart contains a functionally intact local innate immune system that can be activated in response to different types of injury. Both beta-defensins and their signaling molecules, Toll-Like Receptors, have been identified in the heart of various species. β-defensin-1 mRNA expression has been identified in the heart of mice (murine beta-defensin-1, mBD1), pig (porcine beta-defensin-1, pBD1), and horse (equine beta-defensin-1, eBD1). Human beta-defensin-3 (HBD-3) has been detected in the adult heart, and its expression may be regulated by inflammatory stimuli. Toll-like receptor-4 (TLR4) expression has been identified in normal mouse, rat and human myocardium. Furthermore, TLR2 and TLR6 expression have been identified in the human heart. Since antimicrobial peptides are ubiquitous in different tissue types and organs of living organisms, it is very likely that the heart may express different isoforms of β-defensins as well as other types of antimicrobial peptides, and that these peptides might become up-regulated in various pathologic processes. More elaborative studies on the expression patterns of different isoforms and the actual significance of beta-defensins' presence in the heart has, however, not yet been performed.
Our research is currently focusing on identifying the gene expression pattern of antimicrobial peptides, more specifically beta-defensins, in the heart. We are currently investigating the mRNA expression of beta-defensins in the canine heart. Our studies include assessment of beta-defensin gene-expression in canine whole heart homogenate as well as in canine cardiomyocytes isolated using the pressure assisted laser microdissection technique (P.A.L.M.). It is likely that the expression of these important peptides changes in the light of cardiovascular pathology, and part of our study aims at investigating and comparing the expression of beta-defensins in myocardial samples from normal dogs and dogs with heart disease. We have furthermore recently identified beta-defensin 1 gene-expression in cardiomyocytes from the rat heart. We have cloned the full cDNA of rat beta-defensin 1 (rBD1) from whole heart homogenate, and in a separate study assessed the gene-expression of beta-defensin 1 in neonatal cardiomyocytes and adult rat cardiomyocytes excised via pressure-assisted laser microdissection.
To our knowledge, these studies are the first which demonstrate the expression of a natural antibiotic peptide in cardiomyocytes from the rat and dog heart. Our findings support that the heart possesses a complex innate immune defense system, but more research is warranted in this field. Still, the knowledge we possess thus far from other types of tissue suggest that the role of these peptides goes far beyond that of 'simple antibiotics'.
References are available upon request.