Mehmet A. Eskan

The leukocyte integrin antagonist Del-1 inhibits IL-17-mediated inflammatory bone loss

Aging is linked to greater susceptibility to chronic inflammatory diseases, several of which, including periodontitis, involve neutrophil-mediated tissue injury. Here we found that aging-associated periodontitis was accompanied by lower expression of Del-1, an endogenous inhibitor of neutrophil adhesion dependent on the integrin LFA-1, and by reciprocal higher expression of interleukin 17 (IL-17). Consistent with that, IL-17 inhibited gingival endothelial cell expression of Del-1, thereby promoting LFA-1-dependent recruitment of neutrophils. Young Del-1-deficient mice developed spontaneous periodontitis that featured excessive neutrophil infiltration and IL-17 expression; disease was prevented in mice doubly deficient in Del-1 and LFA-1 or in Del-1 and the IL-17 receptor. Locally administered Del-1 inhibited IL-17 production, neutrophil accumulation and bone loss. Therefore, Del-1 suppressed LFA-1-dependent recruitment of neutrophils and IL-17-triggered inflammatory pathology and may thus be a promising therapeutic agent for inflammatory diseases.

Modulation of TLR2 Protein Expression by miR-105 in Human Oral Keratinocytes

Mammalian biological processes such as inflammation, involve regulation of hundreds of genes controlling onset and termination. MicroRNAs (miRNAs) can translationally repress target mRNAs and regulate innate immune responses. Our model system comprised primary human keratinocytes, which exhibited robust differences in inflammatory cytokine production (interleukin-6 and tumor necrosis factor-α) following specific Toll-like receptor 2 and 4 (TLR-2/TLR-4) agonist challenge. We challenged these primary cells with Porphyromonas gingivalis (a Gram-negative bacterium that triggers TLR-2 and TLR-4) and performed miRNA expression profiling. We identified miRNA (miR)-105 as a modulator of TLR-2 protein translation in human gingival keratinocytes. There was a strong inverse correlation between cells that had high cytokine responses following TLR-2 agonist challenge and miR-105 levels. Knock-in and knock-down of miR-105 confirmed this inverse relationship. In silico analysis predicted that miR-105 had complementarity for TLR-2 mRNA, and the luciferase reporter assay verified this. Further understanding of the role of miRNA in host responses may elucidate disease susceptibility and suggest new anti-inflammatory therapeutics.

Defective Neutrophil Recruitment in Leukocyte Adhesion Deficiency Type I Disease Causes Local IL-17–Driven Inflammatory Bone Loss

Inflammatory bone loss from periodontal infections in leukocyte adhesion deficiency disease can be reversed by treatment with antibodies to IL-17 or IL-23. Lessening Bone Loss and Bacterial Burden Nothing dazzles like a beautiful smile, but it’s hard to flash a red carpet–worthy grin when you have leukocyte adhesion deficiency type I (LAD-I). These patients suffer inflammatory degeneration of the bone that cradles the pearly whites (periodontium) as well as other oral pathologies. The periodontal bone loss was thought to result from a lack of neutrophil surveillance of LAD-associated frequent periodontal infections. Now, Moutsopoulos et al. show that cytokine IL-17 secreted by immune T lymphocytes drives periodontal bone loss, thus pinpointing a therapeutic target for LAD-I. Neutrophils are white blood cells that form the first line of defense against microbial infections. These cells carry on their surfaces the LFA-1 β2 integrin (CD11a/CD18), a leukocyte adhesion molecule that is essential for neutrophil movement from the blood to peripheral tissues (extravasation) in response to infection. LAD-I is caused by mutations in the CD18 subunit of β2 integrins and is associated with frequent oral microbial infections and severe periodontal bone loss. The authors showed that defective neutrophil recruitment to the periodontal space in LAD-I patients or in LFA-1–deficient mice (which exhibit the LAD-I periodontal phenotype) was associated with excessive production of the inflammatory cytokine IL-17, mostly from T cells. Local treatment with antibodies to IL-17 in LFA-1–deficient mice blocked inflammatory periodontal bone loss and reduced the total bacterial burden. These findings suggest that IL-17–targeted therapy for periodontitis might be effective in LAD-I patients, which would clearly be a reason to smile. Leukocyte adhesion deficiency type I (LAD-I), a disease syndrome associated with frequent microbial infections, is caused by mutations on the CD18 subunit of β2 integrins. LAD-I is invariably associated with severe periodontal bone loss, which historically has been attributed to the lack of neutrophil surveillance of the periodontal infection. We provide an alternative mechanism by showing that the cytokine interleukin-17 (IL-17) plays a major role in the oral pathology of LAD-I. Defective neutrophil recruitment in LAD-I patients or in LFA-1 (CD11a/CD18)–deficient mice—which exhibit the LAD-I periodontal phenotype—was associated with excessive production of predominantly T cell–derived IL-17 in the periodontal tissue, although innate lymphoid cells also contributed to pathological IL-17 elevation in the LFA-1–deficient mice. Local treatment with antibodies to IL-17 or IL-23 in LFA-1–deficient mice not only blocked inflammatory periodontal bone loss but also caused a reduction in the total bacterial burden, suggesting that the IL-17–driven pathogenesis of LAD-I periodontitis leads to dysbiosis. Therefore, our findings support an IL-17–targeted therapy for periodontitis in LAD-I patients.

Gingival epithelial cells heterozygous for Toll-like receptor 4 polymorphisms Asp299Gly and Thr399Ile are hypo-responsive to Porphyromonas gingivalis

The Toll-like receptor (TLR)4 is the major sensor for bacterial lipopolysaccharide and its two common co-segregating polymorphisms, Asp299Gly and Thr399Ile, which occur at a frequency of between 6 and 10%, have been associated with infectious diseases, LPS hypo-responsiveness and cardiovascular disease. Porphyromonas gingivalis is a Gram-negative bacterium implicated in chronic periodontitis and is a known TLR4 and TLR2 agonist. We obtained two gingival epithelial cell primary cultures from subjects heterozygous for the TLR4 polymorphism Asp299Gly and compared response characteristics with similar cells from patients (four) with the wild-type TLR4 genes. Cytokine responses and transcriptome profiles of gingival epithelial cell primary culture cells to TNFα challenge were similar for all primary epithelial cell cultures. P. gingivalis challenge, however, gave markedly different responses for Asp299Gly heterozygous and wild-type epithelial cell cultures. The epithelial cells heterozygous for the TLR4 polymorphism Asp299Gly were functionally hypo-responsive, evidenced by differences in BD-2 mRNA expression, mRNA response profile by microarray analysis and by pro-inflammatory and chemokine cytokines at the protein and mRNA level. These findings emphasize variance in human epithelial cell TLRs, linked with Asp299Gly carriage, which results in a hypo-responsive epithelial cell phenotype less susceptible to Gram-negative diseases and associated systemic conditions.

Interleukin-1β Modulates Proinflammatory Cytokine Production in Human Epithelial Cells

ABSTRACT Periodontitis is a chronic human inflammatory disease initiated and sustained by dental plaque microorganisms. A major contributing pathogen is Porphyromonas gingivalis, a gram-negative bacterium recognized by Toll-like receptor 2 (TLR2) and TLR4, which are expressed by human gingival epithelial cells (HGECs). However, it is still unclear how these cells respond to P. gingivalis and initiate inflammatory and immune responses. We have reported previously that HGECs produce a wide range of proinflammatory cytokines, including interleukin-6 (IL-6), IL-8, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha (TNF-α), and IL-1β. In this study, we show that IL-1β has a special role in the modulation of other inflammatory cytokines in HGECs challenged with P. gingivalis. Our results show that the increased production of IL-1β correlates with the cell surface expression of TLR4, and more specifically, TLR4-normal HGECs produce fourfold more IL-1β than do TLR4-deficient HGECs after challenge. Moreover, blocking the IL-1β receptor greatly reduces the production of “secondary” proinflammatory cytokines such as IL-8 or IL-6. Our data indicate that the induction of IL-1β plays an important role in mediating the release of other proinflammatory cytokines from primary human epithelial cells following challenge with P. gingivalis, and this process may be an inflammatory enhancement mechanism adopted by epithelial cells.

Differential Activation of Human Gingival Epithelial Cells and Monocytes by Porphyromonas gingivalis Fimbriae

ABSTRACT Humans develop periodontitis in response to challenge by microbial dental plaque. Inflammation begins after perturbation of gingival epithelial cells by subgingival bacteria interacting through pattern-recognition receptors, including the Toll-like receptors (TLR). Porphyromonas gingivalis is a major periodontopathogen that interacts with epithelial cells through its cell surface fimbriae (FimA), leading to colonization and/or invasion. Previous work by our group has established membrane CD14 as an essential coreceptor for TLR2-mediated activation of transfected cell lines by P. gingivalis FimA. We have shown that gingival epithelial cells express TLR2 but not CD14 on their cell surfaces. We thus speculated that P. gingivalis FimA does not readily activate epithelial innate immune responses but rather functions to promote P. gingivalis colonization in the absence of a vigorous FimA-induced response. This hypothesis was verified by the findings that primary human gingival epithelial cells responded poorly to FimA in terms of interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha responses, in stark contrast to the marked response to other TLR2 agonists (Pam3Cys, FSL-1) that are not strictly dependent on CD14. On the other hand, CD14-expressing human primary monocytes responded with high levels of the same cytokines to both FimA and the control TLR2 agonists. The gingival epithelial cells failed to respond to FimA even in the presence of exogenously added soluble CD14. These data indicate that the gingival epithelial cell hyporesponsiveness to FimA is attributable to the lack of membrane-expressed but not soluble CD14. In conclusion, P. gingivalis FimA differentially activates human monocytes and epithelial cells, perhaps reflecting different tactics used by P. gingivalis when interacting with different host cell types or a host strategy to limit inflammation.

TLR4 and S1P receptors cooperate to enhance inflammatory cytokine production in human gingival epithelial cells

Toll‐like receptors (TLR) are pattern recognition receptors for highly conserved microbial molecular patterns. Activation of TLR is a pivotal step in the initiation of innate, inflammatory, and immune defense mechanisms. Recent findings indicate that G protein‐coupled receptors (GPCR) may modulate TLR signaling, but it is unclear which GPCR are involved in this process. One such cooperation between GPCR and TLR can be attributed to the sphingosine 1‐phosphate (S1P) receptor family. The S1P receptors (S1P1–5) are a family of GPCR with a high affinity for S1P, a serum‐borne bioactive lipid associated with diverse biological activities such as inflammation and healing. In this study, we show that pro‐inflammatory cytokine production, including IL‐6 and IL‐8, was increased with LPS and concomitant S1P stimulation. Furthermore, elevated cytokine production following LPS and S1P challenge in human gingival epithelial cells (HGEC) was significantly reduced when TLR4, S1P1 or S1P3 signaling was blocked. Our study also shows that S1P1 and S1P3 expression was induced by LPS in HGEC, and this elevated expression enhanced the influence of S1P in its cooperation with TLR4 to increase cytokine production. This cooperation between TLR4 and S1P1 or S1P3 demonstrates that TLR4 and GPCR can interact to enhance cytokine production in epithelial cells.

Sphingosine 1-Phosphate 1 and TLR4 Mediate IFN-β Expression in Human Gingival Epithelial Cells

IFN-β production is a critical step in human innate immune responses and is primarily controlled at the transcription level by highly ordered mechanisms. IFN-β can be induced by pattern-recognition receptors such as the TLR4. S1P1 is a G protein-coupled receptor, which has a high affinity for sphingosine 1-phosphate (S1P). Although many of the receptors and signaling pathways leading to the expression of IFN-β have been identified and characterized, it is still unclear how IFN-β is regulated in primary human gingival epithelial cells (HGECs). In this study, we demonstrate that S1P1 and TLR4, acting in unison, play an important role in IFN-β expression at the protein and mRNA level in HGECs. We demonstrate that the expression of both IFN-β and IFN-inducible protein-10 (CXCL-10) is significantly up-regulated by LPS and S1P or LPS and a specific S1P1 agonist. This enhanced innate immune response is attenuated in HGECs by small interfering RNA knockdown of either TLR4 or S1P1. Moreover, we show that triggering of TLR4 results in the increased expression of S1P1 receptors. Furthermore, we found that IFN-regulatory factor 3 activation was maximized by LPS and S1P through PI3K. Our data show that triggering TLR4 increases S1P1, such that both TLR4 and S1P1 acting through PI3K enhancement of IFN-regulatory factor 3 activation increase IFN-β expression in epithelial cells. The functional association between TLR4 and the S1P1 receptor demonstrates a novel mechanism in the regulation of IFN-β and CXCL-10 in human primary gingival epithelial cells.

Platelet-Rich Plasma–Assisted Guided Bone Regeneration for Ridge Augmentation: A Randomized, Controlled Clinical Trial

BACKGROUND Platelet-rich plasma (PRP) contains a number of biologically active growth factors, and previous studies have reported conflicting ridge augmentation results. The primary aim of this randomized, controlled, masked, clinical trial was to determine if PRP combined with a rapidly resorbing cancellous allograft would enhance the regenerative result compared with an allograft without PRP. METHODS Thirty-two patients with an edentulous ridge defect were sequentially entered into the study; four were excluded from data analysis. Fourteen patients received a cancellous allograft (CAN group) and the other 14 received a cancellous allograft mixed with PRP (PRP group). All 28 grafted sites were covered with a resorbable polylactide membrane. After elevation of a full-thickness flap, horizontal ridge dimensions were measured with a digital caliper at the crest and 5 mm apical to the crest. Vertical ridge dimensions were measured from a tooth-supported stent. All sites were reentered at 4 months, and a trephine core was obtained for histologic analysis before implant placement. RESULTS The crestal ridge width for the CAN group had a mean gain of 2.0 ± 1.2 mm, whereas the PRP group gained 2.9 ± 1.0, and the difference was statistically significant between groups (P <0.05). The percent vital bone was 36% ± 14% for the CAN group compared with 51% ± 15% for the PRP group and was statistically significant between groups (P <0.05). Loss of augmented ridge width was 34% ± 17% for the CAN group and 28% ± 17% for the PRP group (P >0.05). CONCLUSION These clinical and histologic findings suggest that PRP enhanced bone regeneration and resulted in increased horizontal bone gain and percentage vital bone.