The innate immune response is the first line of host defense. Recognition of different classes of microorganisms involves signaling through specific receptors, one major group of which is TLRs . TLRs recognize various PAMPS conserved in microorganisms, including triacylated lipoproteins (TLR1/2 agonist), diacylated lipoproteins (TLR2/6 agonist), double-stranded RNA (TLR3 agonist), LPS (TLR4 agonist), flagellin (TLR5 agonist), single-stranded RNA (TLR7, TLR8 agonist)  and CpG motifs in DNA (TLR9 agonist) . Following pathogen detection, these TLRs mediate activation of innate and adaptive immune responses through modulation of gene expression by immune cells . Recently, the connective tissue and epithelial cell layers of both healthy and infected gingival tissues were shown to express various TLRs , implying that oral keratinocytes and fibroblasts play an essential role in orchestrating immune responses against pathogens via TLRs.
Interleukin-8, a potent neutrophil chemoattractant and activator, has been associated with the pathogenesis of various forms of periodontitis, accumulation and degranulation of neutrophils with subsequent destruction of normal tissue being a common feature of these diseases . On the other hand, CXCL10, which is highly expressed in aggressive periodontitis and lichen planus, plays an important role in T-cell-mediated oral inflammation through its T-cell chemotactic and adhesion-promoting activities [6, 7]. TLR agonists cause secretion of proinflammatory chemokines and double-stranded RNA (TLR3) was found to dramatically increase IL-8 and CXCL10 in airway epithelial cells [8, 9]. Also, stimulation with LTA (TLR2) and LPS (TLR4) increases IL-8 production by intestinal myofibroblasts . On the other hand, TNF-α is a major inflammatory cytokine in inflammatory diseases related to periodontitis . Oral keratinocytes and fibroblasts produce IL-8 and CXCL10 in response to TNF-α production of these chemokines by oral keratinocytes is thought to lead to oral inflammation [12, 13]. However, it remains unknown whether direct recognition of PAMPS, rather than immune cells, regulates IL-8 and CXCL10 or augments TNF-α-mediated inflammatory responses by oral keratinocytes and fibroblasts in oral inflammatory diseases.
We postulated that oral keratinocytes, as well as oral fibroblasts, recognize PAMPS from bacterial pathogens via TLRs and cooperatively participate in oral inflammatory diseases. In the present study, we examined IL-8 and CXCL10 production by immortalized human oral keratinocytes (RT7) and gingival fibroblasts (GT1) in response to various TLR agonists. Subsequently, we investigated the effects of TLR agonists combined with TNF-α on production of IL-8 and CXCL10.
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- MATERIALS AND METHODS
Toll-like receptors have been found in many types of cells and are known to play a central role in pathogen recognition by the innate immune system . Some investigators have reported that mRNAs of various TLRs are expressed in oral keratinocytes and fibroblasts [17, 18]. The present results demonstrate that both oral keratinocytes and gingival fibroblasts constitutively express mRNA of TLRs 1–10 (Fig. 1). An immunohistochemical study has shown that TLRs 1–9 (i.e. excluding TLR10) are present in epithelial and fibroblastic cells in gingival tissue . Thus, TLR10 mRNA may not have the ability to produce protein in oral keratinocytes and fibroblasts.
It has also been reported that various TLR agonists (e.g., TLR1/2, 4, 9 for gingival epithelial cells, TLR3 and 5 for gingival fibroblasts) induce IL-8 in primary cultures [19, 20]. In agreement with these findings, we found that various TLR agonists induce IL-8 production by RT7 and GT1 (Figs. 3 and 5). The marked IL-8 production by RT7 in response to MALP-2 (TLR2/6 agonist) is noteworthy (Fig. 3) because it implies that oral keratinocytes are specifically involved in host defense responses against mycoplasma through TLR2/6, which has not been previously reported.
CXCL10 binds to the chemokine receptor CXCR3, which is mainly expressed by memory/activated T cells associated with Th1-type responses . In a more recent study, expressions of CXCL10 and CXCR3 were more prevalent and stronger in gingival tissues from patients with aggressive periodontitis than in those from healthy subjects . Poly I:C (TLR3 agonist) has been shown to increase CXCL10 in cultures of human dendritic cells, macrophages, endothelial cells and synovial fibroblasts isolated from rheumatoid arthritis joint tissue . The present findings demonstrate that poly I:C directly induces production of CXCL10 in oral keratinocytes and fibroblasts (Fig. 5), suggesting that oral keratinocytes and fibroblasts infected by viruses may attract T cells toward the site of infection via CXCL10 secretion, leading to the development of T-cell-mediated oral inflammation.
A previous study found that TNF-α induces increases in expression of various chemokines, such as IL-8 and CXCL10, in oral keratinocytes and fibroblasts . Also, the amounts of TNF-α in tissue biopsy specimens correlate strongly with the severity of periodontitis . TLR agonists are known to enhance TNF-α-mediated inflammatory responses in ASMCs; a recent study reported that Pam3CSK4 increases TNF-α-induced IL-8 production via TLR1/2 . Poly I:C (TLR3 agonist) treatment also results in marked enhancement of TNF-α-mediated CXCL10 production in ASMCs . In the present study, we found that almost all TLR agonists, except for imiquimod (TLR7 agonist), enhanced TNF-α-dependent production of IL-8 by RT7 and GT1, whereas marked enhancement of TNF-α-dependent CXCL10 was induced only by poly I:C and Pam3CSK4 in these cells (Figs. 4 and 6). Therefore, TNF-α-dependent acute and chronic inflammation may be affected by gingival tissues through direct recognition of microorganisms by TLRs.
Some TLR agonists act not only as immune stimulatory agents but also induce strong immune suppression in inflammatory conditions . For example, LPS (TLR4 agonist) abolishes IFN-γ-induced CXCL10 in peripheral blood mononuclear cells , whereas other investigations have demonstrated suppressive effects of CpG ODNs (TLR9 agonist) on skin and intestinal inflammation in mouse models [28, 29]. In our study, addition of LPS and CpG-ODN suppressed TNF-α dependent CXCL10 production by GT1, but not by RT7 (Figs. 4 and 6). These results indicate that microorganisms with specific PAMPS, such as CpG-ODN, and LPS, may take advantage of the anti-inflammatory properties of oral fibroblasts to escape from TNF-α dependent inflammatory protection.
Most TLR agonists activate NF-κB and MAPK via a MyD88-dependent pathway for induction of inflammatory cytokines . Although the same TLR agonists, such as poly I:C, flagellin and Pam3CSK4 in GT1, tended to dramatically increase both IL-8 and CXCL10, the extent of induction of IL-8 and CXCL10 by the same TLR agonist differed in RT7 (Figs. 4 and 6). This differential induction of IL-8 and CXCL10 may be related to activation of diverse signaling pathways in RT7 by the TLR agonists. On the other hand, TNF-α activates transcription factors such as NF-κB, which are involved in transcription of IL-8 through MAPK in human airway epithelial cells . Nearly all of the tested TLR agonists enhanced TNF-α-induced IL-8 production regardless of cell type (Figs. 4 and 6), which might be explained by TNF-α and TLR agonists (e.g., NF-kB and MAPK) sharing some signaling pathways for inducing IL-8. However, some TLR agonists differently modulate induction of TNF-α-induced CXCL10 production in different cell types. For example, in our study both flagellin and poly I:C alone increased CXCL10 production, whereas flagellin failed to enhance TNF-α-induced CXCL10 production (Figs. 4 and 6). Moreover, LPS and CpG-ODN decreased TNF-α-induced CXCL10 in GT1. Since specific TLRs (e.g., TLR3, TLR4, and TLR9) are able to activate the IRF signaling pathway , CXCL10 induction may be regulated through complex modification of or interference with the signal transduction pathway between TNF-α and TLR agonists in different cell types. Thus, differential modulation between keratinocytes and fibroblasts of IL-8 and CXCL10 by specific PAMPS and TNF-α may shape various leukocyte migration patterns in epithelium and connective tissue in oral inflammation disease.
In conclusion, our study demonstrates that: (i) oral keratinocytes and fibroblasts express mRNA of TLRs 1–10 mRNA; (ii) IL-8 and CXCL10 production by oral keratinocytes and fibroblasts is induced by treatment with various TLR agonists; and (iii) TLR agonists differentially regulate TNF-α-induced IL-8 and CXCL10 production by the tested cell types. Therefore, direct recognition of microorganism pathogens by those cell types may have an important role in promoting hyper/hypo reactions associated with oral inflammatory diseases.