In vivo β-defensin gene expression in rat gingival epithelium in response to Actinobacillus actinomycetemcomitans infection

The innate immune response provides the mucosal epithelium with its first line of defense against invading pathogens. The cells that comprise the innate immune response are primarily phagocytes (including neutrophils and macrophages) and the cells lining the epithelial mucosa. As part of this defense, these cells express the genes that encode antimicrobial peptides. These small peptides are characterized by a cationic charge and a broad spectrum of antimicrobial activity against gram-positive and -negative bacteria, fungi and enveloped viruses (1). Among these peptides are the defensins – cationic peptides 20–40 amino acids in length, containing six cysteines which form three intramolecular disulfide bonds. The two major families of defensins (the α- and β-defensins) differ based upon the position of their disulfide bonds (2). The most well studied human β-defensins (HBDs) are HBD-1, HBD-2 and HBD-3, which have been isolated from the kidney, genitourinary tract, small intestine, lung, gingival epithelium and skin (3). While HBD-1 is constitutively expressed in the epithelium, HBD-2 mRNA and protein levels can be induced in airway epithelium by live gram-negative bacteria through a Toll-like receptor (TLR)-4-mediated pathway (4), and by lipopeptide through a TLR-2 pathway (5). Furthermore, both HBD-2 and -3 can be induced by lipopolysaccharide, interleukin-1β and tumor necrosis factor-α (TNF-α) in vitro (6), and by bacterial infection and inflammation in vivo (7). In the oral cavity, the magnitude of microbial organisms necessitates a protective mechanism to prevent the onset of disease, but which has no detrimental effect on commensal organisms. In human gingival tissue, HBD-1 and HBD-2 are both expressed in normal, uninflamed tissue, at the highest levels at the gingival margin near the site of plaque formation, and within the sulcular epithelium during states of inflammation (8). In vitro studies have demonstrated that Porphyromonas gingivalis induces HBD-1 expression in cultured human gingival epithelial cells, while Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans stimulate the production of HBD-2 and -3 (9,10). In neither case, however, is the induction mediated by lipopolysaccharide, suggesting that regulation of the induction of these mediators of innate immunity in the oral mucosa is controlled by more complex mechanisms of innate immunity. To elucidate the in vivo response of β-defensin genes to oral pathogens in the gingival epithelium we used a rat model of A. actinomycetemcomitans-induced periodontitis (11). A. actinomycetemcomitans is a gram-negative, capnophilic coccobacillus, which is associated both with chronic periodontitis as well as localized aggressive periodontitis (12). This affects mainly prepubescent adolescents of African–American descent (13), and is defined by destruction of the periodontal ligament and alveolar bone of the first molar and central incisors (14). Feeding A. actinomycetemcomitans to rats in their food results in stable colonization by A. actinomycetemcomitans (15) and consequently leads to gingival detachment and alveolar bone loss (11). Studies in the rat initially demonstrated the expression of homologues to HBD-1 and -2 in kidney and lung (16), respectively, and later in several other tissues (17). Subsequently, numerous new rat β-defensin (RBD) genes were identified, with the expression of at least three new molecules (Defb 3, 5 and 36) in the intestinal epithelium (18). Here, we describe the identification of β-defensins in the rat gingival epithelium and examine the response of these genes in A. actinomycetemcomitans-induced periodontal disease.