This study shows for the first time that HIV-1 induces beta-defensin expression in human oral epithelial cells and that beta-defensins block HIV-1 replication via a direct interaction with virions and through modulation of the CXCR4 coreceptor. These properties may be exploited as strategies for mucosal protection against HIV-1 transmission.

Human gingival epithelial cells (HGE) express two antimicrobial peptides of the ␤-defensin family, human ␤-defensin 1 (hBD-1) and hBD-2, as well as cytokines and chemokines that contribute to innate immunity. In the present study, the expression and transcriptional regulation of hBD-2 was examined. HBD-2 mRNA was induced by cell wall extract of Fusobacterium nucleatum, an oral commensal microorganism, but not by that of Porphyromonas gingivalis, a periodontal pathogen. HBD-2 mRNA was also induced by the proinflammatory cytokine tumor necrosis factor alpha (TNF-␣) and phorbol myristate acetate (PMA), an epithelial cell activator. HBD-2 mRNA was also expressed in 14 of 15 noninflamed gingival tissue samples. HBD-2 peptide was detected by immunofluorescence in HGE stimulated with F. nucleatum cell wall, consistent with induction of the mRNA by this stimulant. Kinetic analysis indicates involvement of multiple distinct signaling pathways in the regulation of hBD-2 mRNA; TNF-␣ and F. nucleatum cell wall induced hBD-2 mRNA rapidly (2 to 4 h), while PMA stimulation was slower (ϳ10 h). In contrast, each stimulant induced interleukin 8 (IL-8) within 1 h. The role of TNF-␣ as an intermediary in F. nucleatum signaling was ruled out by addition of anti-TNF-␣ that did not inhibit hBD-2 induction. However, inhibitor studies show that F. nucleatum stimulation of hBD-2 mRNA requires both new gene transcription and new protein synthesis. Bacterial lipopolysaccharides isolated from Escherichia coli and F. nucleatum were poor stimulants of hBD-2, although they up-regulated IL-8 mRNA. Collectively, our findings show inducible expression of hBD-2 mRNA via multiple pathways in HGE in a pattern that is distinct from that of IL-8 expression. We suggest that different aspects of innate immune responses are differentially regulated and that commensal organisms have a role in stimulating mucosal epithelial cells in maintaining the barrier that contributes to homeostasis and host defense.

Antimicrobial peptides (AMPs) are multi-functional peptides whose fundamental biological role in vivo has been proposed to be the elimination of pathogenic microorganisms, including Gram-positive and -negative bacteria, fungi, and viruses. Genes encoding these peptides are expressed in a variety of cells in the host, including circulating phagocytic cells and mucosal epithelial cells, demonstrating a wide range of utility in the innate immune system. Expression of these genes is tightly regulated; they are induced by pathogens and cytokines as part of the host defense response, and they can be suppressed by bacterial virulence factors and environmental factors which can lead to increased susceptibility to infection. New research has also cast light on alternative functionalities, including immunomodulatory activities, which are related to their unique structural characteristics. These peptides represent not only an important component of innate host defense against microbial colonization and a link between innate and adaptive immunity, but also form a foundation for the development of new therapeutic agents.

Porphyromonas gingivalis, a periodontal pathogen, can invade primary cultures of gingival epithelial cells. Optimal invasion occurred at a relatively low multiplicity of infection (i.e., 100) and demonstrated saturation at a higher multiplicity of infection. Following the lag phase, during which bacteria invaded poorly, invasion was independent of growth phase. P. gingivalis was capable of replicating within the epithelial cells. Invasion was an active process requiring both bacterial and epithelial cell energy production. Invasion was sensitive to inhibitors of microfilaments and microtubules, demonstrating that epithelial cell cytoskeletal rearrangements are involved in bacterial entry. P. gingivalis, but not epithelial cell, protein synthesis was necessary for invasion. Invasion within the epithelial cells was not blocked by inhibitors of protein kinase activity. Invasion was inhibited by protease inhibitors, suggesting that P. gingivalis proteases may be involved in the invasion process. Low-passage clinical isolates of P. gingivalis invaded with higher efficiency than the type strain. Serum inhibited invasion of the type strain but had no effect on the invasion of a clinical isolate. Invasion of gingival epithelial cells by P. gingivalis may contribute to the pathology of periodontal diseases.

There is increasing evidence that innate and adaptive immune responses are intimately linked. This linkage is in part mediated through the recognition of conserved microbial products by Tolllike receptors (TLRs). Detection of microbial products by TLRs can result in induction of inflammatory cytokines and activation of professional antigen-presenting cells, thereby enhancing adaptive immune responses. Here, we show that human ␤-defensin-3 (hBD-3), an innate antimicrobial peptide, can induce expression of the costimulatory molecules CD80, CD86, and CD40, on monocytes and myeloid dendritic cells in a TLR-dependent manner. Activation of monocytes by hBD-3 is mediated by interaction with TLRs 1 and 2, resulting in signaling that requires myeloid differentiating factor 88 and results in IL-1 receptor-associated kinase-1 phosphorylation. In studies with HEK cells engineered to express various TLRs, we show that activation of NF-B by hBD-3 depends on the expression of both TLR1 and TLR2. Thus, human TLR signaling is not restricted to recognition of microbial patterns but also can be initiated by host-derived peptides such as hBD-3.defensins ͉ monocytes H uman ␤-defensins (hBDs) are antimicrobial peptides that are produced by epithelial cells (1-3). Some hBDs such as hBD-1 are constitutively expressed, whereas others (hBD-2 and hBD-3) are induced by microbial products, inflammatory cytokines, or epidermal growth factor (1, 2, 4-6). Several different functions have been described for hBDs that relate to host defense including direct killing of microorganisms (3, 5, 7) and chemoattraction of immature dendritic cells and memory T cells through interactions with CCR6 and chemoattraction of monocytes through interactions with an undefined receptor (5,8,9). The chemotactic properties of hBDs suggest a possible role for these peptides in bridging innate and adaptive immunity. Here, we consider another potential mechanism for hBDs to enhance adaptive immune responses by testing the hypothesis that hBD-3 also can induce the differentiation of professional antigen-presenting cells (APCs).Innate host defenses can be activated by exposure to conserved microbial structures that interact with Toll-like receptors (TLRs) (10-12). Signaling through TLRs begins at cytoplasmic TIR (Toll/ IL-1 receptor) domains and proceeds downstream via myeloid differentiating factor 88 (MyD88)-dependent or -independent pathways and subsequently through multiple kinases, including IL-1 receptor-associated kinase-1 (IRAK-1) (13). TLR signaling leads to activation of the transcription factors NF-B and AP-1, resulting in the direct induction of inflammatory cytokine and costimulatory molecule expression by professional APCs.In the present studies, we explore an intersection between the inducible innate host defense molecule, hBD-3, and TLR activation. Only a single previous observation provides indication of this type of interaction, describing the activation of TLR4 by mouse ␤-defensin-2. The lack of homology between mouse ␤-defensin-2 and any known human ...

The stratified epithelia of the oral cavity are continually exposed to bacterial challenge that is initially resisted by innate epithelial factors and by the recruitment of neutrophils. Antimicrobial peptides from phagocytes and epithelia contribute to this antimicrobial barrier. Using antibodies and in situ hybridization, we explored antimicrobial peptide expression in the varied epithelia of the periodontium and in cultured gingival epithelial cells. In gingival tissue, mRNA for the beta-defensins, human beta-defensin 1 (hBD-1) and human beta-defensin 2 (hBD-2) was predominately localized in suprabasal stratified epithelium and the peptides were detected in upper epithelial layers consistent with the formation of the stratified epithelial barrier. In cultured epithelial cells, both hBD-1 and -2 peptides were detected only in differentiating, involucrin-positive epithelial cells, although hBD-2 required stimulation by proinflammatory mediators or bacterial products for expression. Beta-defensins were not detected in junctional epithelium (JE) that serves as the attachment to the tooth surface. In contrast, alpha-defensins and cathelicidin family member LL-37 were detected in polymorphonuclear neutrophils (PMNs) that migrate through the JE, a localization that persists during inflammation, when the JE and surrounding tissue are highly infiltrated with PMNs. Thus, the undifferentiated JE contains exogenously expressed alpha-defensins and LL-37, and the stratified epithelium contains endogenously expressed beta-defensins. These findings show that defensins and other antimicrobial peptides are localized in specific sites in the gingiva, are synthesized in different cell types, and are likely to serve different roles in various regions of the periodontium.

Epithelial tissues provide the first line of defense between an organism and the environment. Disruption of this barrier leads to bacterial invasion and subsequent inflammation. This is precisely the situation existing in the human oral cavity, where tissues are constantly exposed to a variety of microbial challenges that can lead to bacterially induced periodontal diseases, and to infections of the oral mucosa by bacteria, fungi, and viruses. With the recent discoveries of host-derived peptide antibiotics in mammalian mucosal epithelium, a new line of investigation is emerging to test the hypothesis that one class of these peptides, called " -defensins", functions to protect the host against microbial pathogenesis at these critical, confrontational sites. In that light, impairment of f-defensin activity has recently been implicated in chronic bacterial infections in cystic fibrosis patients. The first direct evidence of expression of defensin peptides in the oral mucosa was the identification of a novel epithelial 1-defensin in mammalian tongue. It was shown to be upregulated in inflammation, suggesting that it participates in host defense. It is theorized that epithelial cell-derived antimicrobial peptides function to keep the natural flora of micro-organisms in a steady state in different niches such as the skin, the intestines, the airway, the endocervix, and the mouth.There is now evidence indicating that normal gingival epithelial cells and tissues express two f-defensins, hBD-and the newly described hBD-2. in addition, a cathelin-class antimicrobial peptide, designated and found in human neutrophils, is also expressed in skin and gingiva. It is highly likely that these and/or other epithelial antimicrobial peptides play an important role in determining the outcome of the host-pathogen interaction at the oral mucosal barrier, and that they may have important future applications in antibiotic treatment.

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