Chronic exposure of gingival fibroblasts to TLR2 or TLR4 agonist inhibits osteoclastogenesis but does not affect osteogenesis

Chronic exposure to periodontopathogenic bacteria such as Porphyromonas gingivalis and the products of these bacteria that interact with the cells of the tooth surrounding tissues can ultimately result in periodontitis. This is a disease that is characterized by inflammation-related alveolar bone degradation by the bone-resorbing cells, the osteoclasts. Interactions of bacterial products with Toll-like receptors (TLRs), in particular TLR2 and TLR4, play a significant role in this chronic inflammatory reaction, which possibly affects osteoclastic activity and osteogenic capacity. Little is known about how chronic exposure to specific TLR activators affects these two antagonistic activities. Here, we studied the effect of TLR activation on gingival fibroblasts (GF), cells that are anatomically close to infiltrating bacterial products in the mouth. These were co-cultured with naive osteoclast precursor cells (i.e., monocytes), as part of the peripheral blood mononuclear cells (PBMCs). Activation of GF co-cultures (GF + PBMCs) with TLR2 or TLR4 agonists resulted in a weak reduction of the osteoclastogenic potential of these cultures, predominantly due to TLR2. Interestingly, chronic exposure, especially to TLR2 agonist, resulted in increased release of TNF-α at early time points. This effect, was reversed at later time points, thus suggesting an adaptation to chronic exposure. Monocyte cultures primed with M-CSF + RANKL, led to the formation of bone-resorbing osteoclasts, irrespective of being activated with TLR agonists. Late activation of these co-cultures with TLR2 and with TLR4 agonists led to a slight decrease in bone resorption. Activation of GF with TLR2 and TLR4 agonists did not affect the osteogenic capacity of the GF cells. In conclusion, chronic exposure leads to diverse reactions; inhibitory with naive osteoclast precursors, not effecting already formed (pre-)osteoclasts. We suggest that early encounter of naive monocytes with TLR agonists may result in differentiation toward the macrophage lineage, desirable for clearing bacterial products. Once (pre-)osteoclasts are formed, these cells may be relatively insensitive for direct TLR stimulation. Possibly, TLR activation of periodontal cells indirectly stimulates osteoclasts, by secreting osteoclastogenesis stimulating inflammatory cytokines

Mapping of DNA methylationsensitive cellular processes in gingival and periodontal ligament fibroblasts in the context of periodontal tissue homeostasis

Interactions between gingival fibroblasts (GFs) and oral pathogens contribute to the chronicity of inflammation in periodontitis. Epigenetic changes in DNA methylation are involved in periodontitis pathogenesis, and recent studies indicate that DNA methyltransferase (DNMT) inhibitors may protect against epithelial barrier disruption and bone resorption. To assess the impact of DNMT inhibition on GFs, cells were cultured with decitabine (5-aza-2’-deoxycytidine, DAC) for 12 days to induce DNA hypomethylation. We observed several potentially detrimental effects of DAC on GF biological functions. First, extended treatment with DAC reduced GF proliferation and induced necrotic cell death. Second, DAC amplified Porphyromonas gingivalis- and cytokine-induced expression and secretion of the chemokine CCL20 and several matrix metalloproteinases (MMPs), including MMP1, MMP9, and MMP13. Similar pro-inflammatory effects of DAC were observed in periodontal ligament fibroblasts. Third, DAC upregulated intercellular adhesion molecule-1 (ICAM-1), which was associated with increased P. gingivalis adherence to GFs and may contribute to bacterial dissemination. Finally, analysis of DAC-induced genes identified by RNA sequencing revealed increased expression of CCL20, CCL5, CCL8, CCL13, TNF, IL1A, IL18, IL33, and CSF3, and showed that the most affected processes were related to immune and inflammatory responses. In contrast, the genes downregulated by DAC were associated with extracellular matrix and collagen fibril organization. Our observations demonstrate that studies of DNMT inhibitors provide important insights into the role of DNA methylation in cells involved in periodontitis pathogenesis. However, the therapeutic potential of hypomethylating agents in periodontal disease may be limited due to their cytotoxic effects on fibroblast populations and stimulation of pro-inflammatory pathways.</p

Regulation of histone deacetylase expression and activity by Porphyromonas gingivalis and proinflammatory cytokines in human gingival fibroblasts

Zapalenie przyzębia, nazywane potocznie paradontozą, jest chorobą związaną z chronicznym stanem zapalnym, która wywoływana jest przez zaburzenia w równowa-dze mikrobiologicznej. Patogenem odrywającym kluczową rolę w rozwoju tego scho-rzenia jest Porphyromonas gingivalis. Pomimo licznych badań dotyczących paradontozy, nadal niewiele wiadomo na temat epigenetycznej regulacji w tej chorobie. Podejrzewa się, że proces acetylacji histonów może być patologicznie regulowany przez ciągłą obecność bakterii i/lub cytokin prozapalnych. Rodziną enzymów kontrolujących acetylację są deacetylazy histonów (HDACs).Celem pracy było zbadanie profilu ekspresji i aktywności HDACs w ludzkich pierwotnych fibroblastach dziąsła (PHGFs) po infekcji P. gingivalis ATCC 33277 lub sty-mulacji cytokinami prozapalnymi (TNF (czynnik nekrozy nowotworu) i IL (interleuki-na)-1β). Stymulacja komórek mediatorami stanu zapalnego powodowała indukcję ekspresji kilku genów HDACs, m. in. HDAC1 oraz w największym stopniu – HDAC9. Jednakże dalsze doświadczenia nie potwierdziły tej regulacji na poziomie białka, co przełożyło się także na brak zmian w aktywności i poziomie acetylacji histonów po stymulacji TNF. Ekspresja tych samych genów ulegała indukcji pod wpływem infekcji. Co istotne, P. gingivalis powodował również podwyższenie aktywności enzymatycznej HDACs. W celu zbadania znaczenie indukcji mRNA HDAC1 i HDAC9 w patofizjolo-gii paradontozy, ekspresję tych genów wyciszono za pomocą siRNA. Jednak pomimo skutecznej transfekcji, stopień wyciszenia ekspresji badanych genów nie był wystarczający, aby zaobserwować zmiany w ekspresji mRNA cyklooksygenazy 2 oraz chemokin proza-palnych: CCL20 (ang. chemokine (C-C motif) ligand 20), CCL2 (ang. chemokine (C-C motif) ligand 2), CXCL10 (ang. C-X-C motif chemokine 10) i IL-8 w zastosowanych warunkach eksperymentalnych. Z tego względu sprawdzono poziom HDAC1 i HDAC9 w komórkach. Analiza Western blot pokazała, że P. gingivalis powodował degradację obu białek oraz nie wpływał na poziom acetylacji histonu H3.Podsumowując, badania pokazały, że zarówno cytokiny, jak i P. gingivalis wpływa-ją na profil ekspresji HDACs. Podwyższony poziom mRNA HDACs oraz podwyższona aktywność enzymatyczna po infekcji nie korelowały jednak z poziomem białka oraz nie były związane z globalnymi zmianami w acetylacji histonów. Wobec otrzymanych wyników zaproponowano następujące wyjaśnienie: indukcja ekspresji HDACs może być mechanizmem kompensacyjnym fibroblastów dziąsła w odpowiedzi na degradację białek spowodowaną aktywnością proteolityczną bakterii.Periodontitis is a chronic inflammatory disease caused by microbial imbalance and Porphyromonas gingivalis plays a crucial role in driving the inflammation. Although periodontitis is a well-studied disorder, still little is known about epigenetic regulation in that disease. Histone acetylation may be pathologically regulated by persistent presence of bacteria and/or pro-inflammatory cytokines. Are histone deacetylases (HDACs) represent a family of enzymes controlling acetylation. The aim of the study was to characterize HDAC expression profile and activity in primary human gingival fibroblasts (PHGFs) upon P. gingivalis infection or cytokine (TNF (tumor necrosis factor) and IL (interleukin)-1β) stimulation. Inflammatory cyto-kine stimulation caused an induction of several HDAC genes, including HDAC1 and HDAC9, induction of which was the most prominent. However, further experiments did not confirm that regulation at the protein level, which also did not correlate with changes in HDAC enzymatic activity and histone H3 acetylation level upon TNF stimu-lation. P. gingivalis up-regulated expression of the same genes in PHGFs. Importantly, HDAC enzymatic activity was also increased upon infection. To verify the role of the induction of HDAC1 and HDAC9 mRNA in the pathophysiology of periodontitis, the expression of these genes was silenced using siRNA. Although transfection was suc-cessful, the degree of gene silencing was insufficient to observe changes in COX2 (cyclooxygenase 2) and chemokines: CCL20 (chemokine (C-C motif) ligand 20), CCL2 (chemokine (C-C motif) ligand 2), CXCL10 (C-X-C motif chemokine 10) and IL-8 and expression. Based on these observations, the protein levels of HDAC1 and HDAC9 was estimated by Western blot analysis. The results showed that P. gingivalis degraded the proteins and did not influence the histone H3 acetylation level. To sum up, the presented study showed that both cytokines and P. gingivalis up-regulated the expression of several HDAC genes. However, the increased level of HDAC mRNA and the increased enzymatic activity upon infection did not correlate with protein expression. For that reason, the following explanation was proposed: induction of HDAC mRNA in PHGFs may be the compensating mechanism of the cells in response to the HDAC protein degradation caused by P. gingivalis proteolytic activity

Application of new cationic heptaprenol derivative in lipofection of DU145 and B16F10 cancer cells

Lipidy kationowe to amfifilowe cząsteczki zbudowane podobnie jak naturalnie występujące lipidy. Posiadają one ładunek dodatni, dzięki czemu oddziałują z ujemnie naładowanymi kwasami nukleinowymi tworząc lipopleksy. Ta właściwość pozwala na wykorzystanie ich jako wydajnych niewiusowych nośników DNA/RNA do komórek. Celem pracy było zbadanie potencjału lipofekcyjnego nowej kationowej pochodnej heptaprenolu z wykorzystaniem ludzkich i mysich komórek nowotworowych.Cationic lipids are molecules structurally similar to natural lipids. They contain positive charge and therefore are able to interact with negatively charged nucleic acids forming lipolexes. This feature allows to use them as effective non-viral vectors that transfer DNA/RNA into cells. The aim of the research was to estimate the lipofecting activity of the new cationic derivative of heptaprenol using human and murine cancer cells

TLR2 activation by Porphyromonas gingivalis requires both PPAD activity and fimbriae

Porphyromonas gingivalis, a keystone oral pathogen implicated in development and progression of periodontitis, may also contribute to the pathogenicity of diseases such as arthritis, atherosclerosis, and Alzheimer’s. P. gingivalis is a master manipulator of host immune responses due to production of a large variety of virulence factors. Among these, P. gingivalis peptidilarginine deiminase (PPAD), an enzyme unique to P. gingivalis, converts C-terminal Arg residues in bacterium- and host-derived proteins and peptides into citrulline. PPAD contributes to stimulation of proinflammatory responses in host cells and is essential for activation of the prostaglandin E2 (PGE2) synthesis pathway in gingival fibroblasts. Since P. gingivalis is recognized mainly by Toll-like receptor-2 (TLR2), we investigated the effects of PPAD activity on TLR2-dependent host cell responses to P. gingivalis, as well as to outer membrane vesicles (OMVs) and fimbriae produced by this organism. Using reporter cell lines, we found that PPAD activity was required for TLR2 activation by P. gingivalis cells and OMVs. We also found that fimbriae, an established TLR2 ligand, from wild-type ATCC 33277 (but not from its isogenic PPAD mutant) enhanced the proinflammatory responses of host cells. Furthermore, only fimbriae from wild-type ATCC 33277, but not from the PPAD-deficient strains, induced cytokine production and stimulated expression of genes within the PGE2 synthesis pathway in human gingival fibroblasts via activation of the NF-ĸB and MAP kinase-dependent signaling pathways. Analysis of ten clinical isolates revealed that type I FimA is preferable for TLR2 signaling enhancement. In conclusion, the data strongly suggest that both PPAD activity and fimbriae are important for TLR2-dependent cell responses to P. gingivalis infection

hTERT-immortalized gingival fibroblasts respond to cytokines but fail to mimic primary cell responses to Porphyromonas gingivalis

In periodontitis, gingival fibroblasts (GFs) interact with and respond to oral pathogens, significantly contributing to perpetuation of chronic inflammation and tissue destruction. The aim of this study was to determine the usefulness of the recently released hTERT-immortalized GF (TIGF) cell line for studies of host–pathogen interactions. We show that TIGFs are unable to upregulate expression and production of interleukin (IL)-6, IL-8 and prostaglandin E2 upon infection with Porphyromonas gingivalis despite being susceptible to adhesion and invasion by this oral pathogen. In contrast, induction of inflammatory mediators in TNFα- or IL-1β-stimulated TIGFs is comparable to that observed in primary GFs. The inability of TIGFs to respond directly to P. gingivalis is caused by a specific defect in Toll-like receptor-2 (TLR2) expression, which is likely driven by TLR2 promoter hypermethylation. Consistently, TIGFs fail to upregulate inflammatory genes in response to the TLR2 agonists Pam2CSK4 and Pam3CSK4. These results identify important limitations of using TIGFs to study GF interaction with oral pathogens, though these cells may be useful for studies of TLR2-independent processes. Our observations also emphasize the importance of direct comparisons between immortalized and primary cells prior to using cell lines as models in studies of any biological processes

Diabetes medication metformin inhibits osteoclast formation and activity in in vitro models for periodontitis

Diabetes and periodontitis are comorbidities and may share common pathways. Several reports indicate that diabetes medication metformin may be beneficial for the periodontal status of periodontitis patients. Further research using appropriate cell systems of the periodontium, the tissue that surrounds teeth may reveal the possible mechanism. Periodontal ligament fibroblasts anchor teeth in bone and play a role in the onset of both alveolar bone formation and degradation, the latter by inducing osteoclast formation from adherent precursor cells. Therefore, a cell model including this type of cells is ideal to study the influence of metformin on both processes. We hypothesize that metformin will enhance bone formation, as described for osteoblasts, whereas the effects of metformin on osteoclast formation is yet undetermined. Periodontal ligament fibroblasts were cultured in the presence of osteogenic medium and 0.2 or 1 mM metformin. The influence of metformin on osteoclast formation was first studied in PDLF cultures supplemented with peripheral blood leukocytes, containing osteoclast precursors. Finally, the effect of metformin on osteoclast precursors was studied in cultures of CD14(+) monocytes that were stimulated with M-CSF and receptor activator of Nf-κB ligand (RANKL). No effects of metformin were observed on osteogenesis: not on alkaline phosphatase activity, Alizarin red deposition, nor on the expression of osteogenic markers RUNX-2, Collagen I and Osteonectin. Metformin inhibited osteoclast formation and accordingly downregulated the genes involved in osteoclastogenesis: RANKL, macrophage colony stimulating factor (M-CSF) and osteoclast fusion gene DC-STAMP. Osteoclast formation on both plastic and bone as well as bone resorption was inhibited by metformin in M-CSF and RANKL stimulated monocyte cultures, probably by reduction of RANK expression. The present study unraveling the positive effect of metformin in periodontitis patients at the cellular level, indicates that metformin inhibits osteoclast formation and activity, both when orchestrated by periodontal ligament fibroblasts and in cytokine driven osteoclast formation assays. The results indicate that metformin could have a systemic beneficiary effect on bone by inhibiting osteoclast formation and activity