Interleukin-8 (IL-8) gene polymorphisms have been considered as susceptibility factors in periodontal disease. However, the functional roles of IL-8 gene haplotypes have not been investigated. Here, we demonstrate for the first time the use of the CRISPR/Cas9 system to engineer the IL-8 gene and tested the functionality of different haplotypes. Two sgRNAs vectors targeting the IL-8 gene and the naked homologous repair DNA carrying different haplotypes were used to successfully generate HEK293T cells carrying the AT genotype at the first SNP - rs4073 (alias -251), TT genotype at the second SNP - rs2227307 (alias +396), TC or CC genotypes at the third SNP - rs2227306 (alias +781) at the IL-8 locus. When stimulated with Poly I:C, ATC/TTC haplotype, cells significantly up-regulated the IL-8 at both transcriptional and translational levels. To test whether ATC/TTC haplotype is functional, we used a trans-well assay to measure the transmigration of primary neutrophils incubated with supernatants from the Poly I:C stimulation experiment. ATC/TTC haplotype cells significantly increased transmigration of neutrophils confirming the functional role for this IL-8 haplotype. Taken together, our data provides evidence that carriage of the ATC/TTC haplotype in itself may increase the influx of neutrophils in inflammatory lesions and influence disease susceptibility.

In aggressive periodontitis, the dysbiotic microbial community in the subgingival crevice, which is abundant in Aggregatibacter actinomycetemcomitans, interacts with extra- and intracellular receptors of host cells, leading to exacerbated inflammation and subsequent tissue destruction. Our goal was to understand the innate immune interactions of A. actinomycetemcomitans with macrophages and human gingival epithelial cells (HGECs) on the signaling cascade involved in inflammasome and inflammatory responses. U937 macrophages and HGECs were co-cultured with A. actinomycetemcomitans strain Y4 and key signaling pathways were analyzed using real-time PCR, Western blotting and cytokine production by ELISA. A. actinomycetemcomitans infection upregulated the transcription of TLR2, TLR4, NOD2 and NLRP3 in U937 macrophages, but not in HGECs. Transcription of IL-1β and IL-18 was upregulated in macrophages and HGECs after 1 h interaction with A. actinomycetemcomitans, but positive regulation persisted only in macrophages, resulting in the presence of IL-1β in macrophage supernatant. Immunoblot data revealed that A. actinomycetemcomitans induced the phosphorylation of AKT and ERK1/2, possibly leading to activation of the NF-κB pathway in macrophages. On the other hand, HGEC signaling induced by A. actinomycetemcomitans was distinct, since AKT and 4EBP1 were phosphorylated after stimulation with A. actinomycetemcomitans, whereas ERK1/2 was not. Furthermore, A. actinomycetemcomitans was able to induce the cleavage of caspase-1 in U937 macrophages in an NRLP3-dependent pathway. Differences in host cell responses, such as those seen between HGECs and macrophages, suggested that survival of A. actinomycetemcomitans in periodontal tissues may be favored by its ability to differentially activate host cells.

The role of the adaptor molecule MyD88 is thought to be independent of Toll-like receptor 3 (TLR3) signaling. In this report, we demonstrate a previously unknown role of MyD88 in TLR3 signaling in inducing endogenous ligands of TLR2 to elicit innate immune responses. Of the various TLR ligands examined, the TLR3-specific ligand polyinosinic:polycytidylic acid (poly I:C), significantly induced TNF production and the upregulation of other TLR transcripts, in particular, TLR2. Accordingly, TLR3 stimulation also led to a significant upregulation of endogenous TLR2 ligands mainly, HMGB1 and Hsp60. By contrast, the silencing of TLR3 significantly downregulated MyD88 and TLR2 gene expression and pro-inflammatory IL1β, TNF, and IL8 secretion. The silencing of MyD88 similarly led to the downregulation of TLR2, IL1β, TNF and IL8, thus suggesting MyD88 to somehow act downstream of TLR3. Corroborating in vitro data, Myd88^−/− knockout mice downregulated TNF, CXCL1; and phospho-p65 and phospho-IRF3 nuclear localization, upon poly I:C treatment in a mouse model of skin infection. Taken together, we identified a previously unknown role for MyD88 in the TLR3 signaling pathway, underlying the importance of TLRs and adapter protein interplay in modulating endogenous TLR ligands culminating in pro-inflammatory cytokine regulation.

DNA methylation controls several inflammatory genes affecting bone homeostasis. Hitherto, inhibition of DNA methylation in vivo in the context of periodontitis and osteoclastogenesis has not been attempted. Ligature-induced periodontitis in C57BL/6J mice was induced by placing ligature for five days with Decitabine (5-aza-2′-deoxycytidine) (1 mg/kg/day) or vehicle treatment. We evaluated bone resorption, osteoclast differentiation by tartrate-resistant acid phosphatase (TRAP) and mRNA expression of anti-inflammatory molecules using cluster differentiation 14 positive (CD14⁺) monocytes from human peripheral blood. Our data showed that decitabine inhibited bone loss and osteoclast differentiation experimental periodontitis, and suppressed osteoclast CD14⁺ human monocytes; and conversely, that it increased bone mineralization in osteoblastic cell line MC3T3-E1 in a concentration-dependent manner. In addition to increasing IL10 (interleukin-10), TGFB (transforming growth factor beta-1) in CD14⁺ monocytes, decitabine upregulated KLF2 (Krüppel-like factor-2) expression. Overexpression of KLF2 protein enhanced the transcription of IL10 and TGFB. On the contrary, site-directed mutagenesis of KLF2 binding site in IL10 and TFGB abrogated luciferase activity in HEK293T cells. Decitabine reduces bone loss in a mouse model of periodontitis by inhibiting osteoclastogenesis through the upregulation of anti-inflammatory cytokines via KLF2 dependent mechanisms. DNA methyltransferase inhibitors merit further investigation as a possible novel therapy for periodontitis.