Fusobacterium nucleatum and Human Beta-Defensins Modulate the Release of Antimicrobial Chemokine CCL20/Macrophage Inflammatory Protein 3α

Abstract

Cells of the innate immune system regulate immune responses through the production of antimicrobial peptides, chemokines, and cytokines, including human beta-defensins (hBDs) and CCL20. In this study, we examined the kinetics of primary human oral epithelial cell (HOEC) production of CCL20 and hBDs in response to Fusobacterium nucleatum, a commensal bacterium of the oral cavity, which we previously showed promotes HOEC induction of hBDs. HOECs secrete maximal levels of CCL20 at 6 h, following stimulation by F. nucleatum cell wall (FnCW). The kinetics of CCL20 release is distinct from that of hBD-2 and -3, which peaks after 24 h and 48 h of FnCW stimulation, respectively. FnCW-induced release of CCL20 by HOECs requires both transcriptional and translational activation. Release of CCL20 by HOECs is inhibited by brefeldin A, suggesting that it is secreted through a vesicle transport pathway. Other epithelium-derived agents that FnCW induces, such as hBD-2, hBD-3, tumor necrosis factor alpha (TNF-␣) and interleukin-1␤ (IL-1␤), are also able to release CCL20. By focusing on mitogen-activated protein kinases, we show that both extracellular signalregulated kinase 1/2 and p38, but not JNK, are required for hBD-, TNF-␣-, and IL-1␤-induced secretion of CCL20 by HOECs. The ability of FnCW and its induced hBDs to produce proinflammatory cytokines and CCL20 suggests the broad role of F. nucleatum and human antimicrobial peptides in primary immune responses elicited by oral epithelium.

Tumor necrosis factor–like weak inducer of apoptosis increases CC chemokine ligand 20 production in interleukin 1β–stimulated human gingival fibroblasts

“…However, the inhibitor of JNK did not modulate CCL20 production. Ghosh et al reported that a p38 MAPK inhibitor and an ERK inhibitor decreased CCL20 production from IL-1␤-or TNF-␣-stimulated human oral epithelial cells, although a JNK inhibitor did not change CCL20 production [17] Kao et al reported that inhibitors of p38 MAPK, ERK, and NF-B decreased CCL20 production in IL-17A-treated human trancheobronchial epithelial cells, although inhibitors of JNK and Akt did not inhibit CCL20 production [18], similar to our results. However, we observed that the activation of JNK in HGFs was enhanced by TWEAK and IL-1␤ stimulation.…”

Diverse Toll-Like Receptors Mediate Cytokine Production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in Macrophages

“…Cellular signaling elicited by F. nucleatum and A. actinomycetemcomitans seems to be differently regulated, depending on target molecules or host cell types. Ghosh et al showed that all MAPK (p38, ERK, and JNK) inhibitors showed little or no effect on F. nucleatum cell wall-induced CCL20 secretion in human oral epithelial cells (26). F. nucleatum induces collagenase 3 gene expression in human keratinocytes (HaCaT cells) via a p38-but not ERK-dependent pathway (27).…”

FAD-I, a Fusobacterium nucleatum Cell Wall-Associated Diacylated Lipoprotein That Mediates Human Beta Defensin 2 Induction through Toll-Like Receptor-1/2 (TLR-1/2) and TLR-2/6

“…Primers were designed using Primer3 primer design software (29) and were analyzed using Oligo Analysis (Integrated DNA Technologies [IDT]) in order to avoid secondary structures. The medium supernatants from HOECs were analyzed for hBD-2 protein levels following our previously described protocol (9,10).…”

“…By virtue of their antimicrobial and immunoregulatory properties, these epithelial-cell-derived innate response peptides contribute to the homeostasis between the bacterium and the host (4). Human beta defensin 2 (hBD-2) and hBD-3 are the two inducible members of the hBD peptide family that we and others have described in the oral cavity (1,(5)(6)(7)(8)(9)(10). Interestingly, while hBD-3 is associated with the highly proliferating, nondifferentiated stratum basale of the oral mucosa, hBD-2 is compartmentalized in the more superficial stratum spinosum and stratum granulosum; i.e., nonproliferating yet differentiating regions of the oral mucosa (11,12).…”

“…In addition to its role in oral community architecture, we and others have shown that the cell wall of F. nucleatum induces hBD-2 expression in normal primary human oral epithelial cells (HOECs) (1,5,10,23). The presence of F. nucleatum in oral biofilms colonizing oral surfaces may be a reason why the generally inducible hBD-2 is constitutively expressed in the upper strata of the oral mucosa, a trait apparently unique to this site compared to other mucosal body sites (5,11,17).…”