Epithelial cells and DCs initiate and contribute to immune responses; however, the coordination of specific innate immune responses between these two cell types in response to various oral bacteria is not clear. In this report, we show that both epithelial cells and DCs express β-defensins and that products induced in response to oral bacteria by one cell type influence the characteristic responses of the other cell type. Apparently, the crosstalk so well defined between B and T cells  also can occur between GECs and DCs. Bacterially-activated DCs influence and significantly augment multiple innate immune responses of epithelial cells as shown by the expression of hBD2 as well as IL-8, CXCL2/GROβ and CCL-20/MIP3α in epithelial cells. This amplification is primarily mediated via IL-1β from DCs, since induction of these mRNAs in GECs was largely attenuated by IL-1ra. On the other hand, epithelial cells also influence the responses of DCs as shown by selective stimulation of cytokine/chemokine production by DCs in the presence of β-defensins. Furthermore, the responses of both cell types are dependent on the specific bacteria used for stimulation; this is demonstrated by the unique profiles of β-defensin expression as well as the differential cytokine/chemokine responses of both cell types when stimulated by cell wall preparations of various bacteria of different pathogenicity: F. nucleatum, A. naeslundii and P. gingivalis. Our findings highlight the differential and coordinated regulation of innate response in response to oral bacteria. These findings confirm previous studies and provide additional insights into how these two cell types interact in a network that may result in optimal immune regulation in response to various oral bacteria.
The β-defensins are mainly expressed by epithelial cells, although hBD1, but not hBD2, was previously detected in mature DCs, monocytes, and macrophages in response to LPS . Our results show both hBD1 and hBD2 are expressed in DCs with basal level in iDCs approximately 100-fold lower than in epithelial cells. However, in contrast to epithelial cells, in which hBD1 is constitutively expressed and hBD2 and 3 are inducible, both hBD1 and hBD2 were inducible in DCs by bacterial exposure, while hBD3 was only weakly expressed. Furthermore, hBD2 diffusing from the epithelium associated with DCs as shown in an oral full thickness tissue model (Figure 7). Both hBD2 and hBD3 modulated expression of chemokines by DCs including IL-8 and GRO with differential dose-dependent upregulation of IL-6 by hBD2 and MCP-1 by hBD3. Our results agree and extend previous observations that β-defensins influence DC properties. Both hBD1 and hBD3 influence DC maturation with upregulation of costimulatory molecules [36, 37], and hBD1 also stimulates expression of proinflammatory cytokines .
Oral bacteria differ in their effectiveness in inducing hBD1, hBD2 and hBD3 mRNA and proteins in GECs and DCs. The Gram-negative commensal, F. nucleatum, was more effective than a Gram-positive cariogenic bacterium, A. naeslundii, and both are more effective than the periopathogen P. gingivalis in upregulation of hBD1, 2 and 3 in DCs. This dose response pattern is similar to that shown by Chino et al  for chemokine and cytokine secretion by DCs. In addition, in DCs, hBD1 showed a bi-phasic response to the non-pathogens similar to the expression of MCP-1, while hBD2 expression was dose-dependent, similar to the IL-8 response . Maturation of DCs was induced by direct exposure to oral bacteria  and by hBD3  which is highly expressed by epithelial cells in response to F. nucleatum (Figure 2). Thus, low doses of commensal bacteria may play a role in immune surveillance by DCs under non-inflammatory conditions both by direct contact and by cross-talk from epithelial cells via β-defensins. On the other hand, epithelial cells also respond differently to different types of bacteria, as hBD2 and 3 gene expression is greatly induced by F. nucleautm, consistent with the immune-regulatory properties of this bacterium . With greater exposure to commensal bacteria, or under inflammatory conditions, responses would be enhanced by secretion of IL-1β by DCs that stimulates epithelial cell expression of hBD-2 and CCL20, GRO and IL-8. The defensins further stimulate IL-6 and MCP-1 secretion by DCs, which might help to amplify the appropriate immune responses. Thus, epithelial cells and DCs may work together to express antimicrobial peptides and attract monocytes and neutrophils to fight infection in the gingival crevice, a critical factor for the health of the periodontium [27, 38, 39].
IL-1β, secreted by DCs, is the main cytokine responsible for mediating increased hBD2 expression in GECs, as previously demonstrated in epidermal keratinocytes and pulmonary epithelial cells [20, 21]. IL-1ra also attenuated upregulation of other innate immune markers (CCL20, CXCL2) in parallel with that of hBD2. Thus, IL-1β can function in the dialogue or cross-talk between DCs and GECs. However additional factors, such as RANTES, which are differentially secreted by DCs treated with F. nucleatum vs. P. gingivalis, may also contribute to this cross-talk since IL-1ra was only partially effective in blocking F. nucleatum induced responses. Indeed, P. gingivalis (in the absence of its proteases) is a poor stimulant for hBD2 in GECs. However, the GECs can respond to conditioned medium from DCs stimulated with P. gingivalis, suggesting the importance of multiple cell types in the response in situ.
Epithelial secretion of defensins and chemokines that attract neutrophils is much reduced in the presence of the pathogen, P. gingivalis, consistent with its 'stealth-like' properties [40, 41]. However, in response to this pathogen, epithelial cells secreted MDC/CCL22, an inflammatory chemokine that induces Th2 effector responses . These trends could be related to events in gingival inflammation and periodontal disease. Jotwani and coworkers suggested that the prominent response in P. gingivalis-mediated periodontal disease is a Th2 effector response , supported by our findings. However, we did not find secretion of IL-8 or MCP-1 with our P. gingivalis cell wall stimulation in contrast to Kusumoto et al , which used a sonicated extract of P. gingivalis. Neutrophil chemotaxis is critical for periodontal health, and this process is interrupted in periodontal disease commonly associated with P. gingivalis [38, 39]. IL-8 is expressed in F. nucleatum-stimulated GECs, and its expression is strongly enhanced by interaction with DCs stimulated with this commensal bacteria. However, overall levels are low in P. gingivalis-stimulated GECs and in the combination of P. gingivalis-stimulated DCs and GECs. Thus, both DCs and GECs distinguish between these bacteria and have specific responses.
The ability of DCs to stimulate immune responses is related to their activation and maturation status, and activated DCs are significant sources of chemokines that recruit other immune cells, including T cells, natural killer cells, monocytes and additional DCs . The oral bacterial preparations used here induced the maturation of DCs, in agreement with our previous studies , as assessed by up-regulation of surface expression of CD83 (data not shown). Interestingly, both hBD2 and hBD3 induced the maturation of DCs as well, although to a lower extent (10%-30%) than that of bacteria preparations. Defensin treatment produced differential cytokine/chemokine profiles in DCs. Both hBD2 and hBD3 induced IL-8, GRO and MCP-1, in agreement with the findings in peripheral blood mononuclear cells . However, DCs respond differently to hBD2 and hBD3; hBD2 induced IL-6, while hBD3 induced greater levels of MCP-1 than hBD2. The induction of IL-6 and IL-8 may be particularly important in attracting neutrophils and T helper 17 cells . Multiple chemokines, including GRO, have microbicidal effects on both Gram-positive and Gram-negative bacteria . MCP-1 acts as an attractant for monocytes and T-regs . We also observed the increased secretion of TARC, PARC, and TIMP-2 in hBD3-treated DCs, which may attract more immune cells in situ. The evidence that hBD2 and 3 induced selective cytokine expression suggests that these defensins may play unique role in immune responses due to utilizing different receptors to stimulate DCs; TLR4, CCR6 and CD91 have been implicated as receptor for hBD2 [9, 37, 49] and TLR1 and TLR2 as receptors for hBD3 .
An intimate interaction between epithelial cells and DCs has been described in the gut to maintain immune homeostasis in response to various bacteria [50, 51]. Our results for the first time demonstrate that a similar phenomenon of specific bacteria response coordinately may occur in the oral mucosa. Our findings show that DCs amplified the bacterially specific innate immune responses of GECs, while epithelial-derived defensins induced unique chemokine patterns, suggesting the existence of autoregulatory loop between DCs and GECs. GECs and DCs evoke characteristic cytokine patterns upon exposure to different bacterial stimuli and coordinately enhance each other's innate immune responses. Not only do defensins act as chemoattractants to immune cells, but we found that defensins also induce unique cytokine patterns, which could be crucial in amplifying immune responses to oral bacteria. These responses and cross-talk may result in discriminatory signals within oral tissue and gingiva in particular, and yield characteristic and appropriate immune responses in the state of health in the presence of non-pathogenic bacteria, and with inflammation in the presence of pathogens.