[Show abstract][Hide abstract] ABSTRACT: Material Supplementary 3.DCSupplemental.html http://www.jimmunol.org/content/suppl/2015/01/19/jimmunol.140189 Subscriptions http://jimmunol.org/subscriptions is online at: The Journal of Immunology Information about subscribing to
Journal of immunology (Baltimore, Md. : 1950). 02/2015;
[Show abstract][Hide abstract] ABSTRACT: Abstract The objective of this study was to evaluate the impact of smoking on the early molecular events involved in peri-implant healing at either a micro-roughened or a micro-roughened with superimposed nanofeatures surface implants in humans. Twenty-one subjects, 10 smokers and 11 non-smokers received 4 mini-implants (2.2 x 5.0 mm; 2 of each surface). After 3 and 7 days, paired mini-implants were retrieved by reverse threading and RNA isolated from implant adherent cells. Whole genome microarrays were used interrogate the gene expression profiles. The study failed to identify differences in the gene expression profiles of implant adherent cells at this early stage of osseointegration (up to day 7) comparing smokers and non-smokers individuals.
Journal of Oral Implantology 09/2014; · 0.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) regulate the synthesis of cytokines in response to Toll-like receptor (TLR) activation. Our recent microarray study comparing normal and inflamed human dental pulps showed that miRNA-181 (miR-181) family is differentially expressed in the presence of inflammation. Prior studies have reported that the dental pulp, which is composed primarily of TLR4/2+ fibroblasts, expresses elevated levels of cytokines including interleukin-8 (IL-8) when inflamed. In this study, we employed an in-vitro model to determine the role of the miRNA-181 family in the TLR agonist-induced response in human fibroblasts. TLR4/2+ primary human dental pulp fibroblasts were stimulated with lipopolysaccharide from Porphyromonas gingivalis (Pg LPS), a known oral pathogen, and IL-8 and miR-181 expression measured. An inversely proportional relationship between IL-8 and miR-181a was observed. In-silico analysis identified a miR-181a-binding site on the 3' untranslated region (UTR) of IL-8, which was confirmed by dual-luciferase assays. MiR-181a directly binds to the 3'UTR of IL-8, an important inflammatory component of the immune response, and modulates its levels. This is the very first report demonstrating miR-181a regulation of IL-8.Genes and Immunity advance online publication, 22 May 2014; doi:10.1038/gene.2014.24.
[Show abstract][Hide abstract] ABSTRACT: Objective: Over a twelve day period of osteoclast differentiation from monocytes, differentially expressed microRNAs (miRNAs) were investigated and bioinformatics analysis performed on miRNAs predicted to target genes linked to osteoclastogenesis. We aim to gain a deeper understanding of osteoclastogenesis, which may have future therapeutic implications upon diseases where bone remodeling/resorption play a role in pathogenesis.
Method: Leukophoresed buffy coats were obtained from healthy human donors (N=4) and CD14+ monocytes isolated. Monocyte-to-osteoclast differentiation was induced by culturing cells with sRANKL and M-CSF. Differentiation was confirmed by microscopy, TRAP+ staining and osteolysis. Freshly isolated monocytes and monocytes cultured in the presence of M-CSF alone were utilized as controls. On days 0, 3, 6, 9 and 12, cells were lysed, total RNA isolated and miRNA levels interrogated using miRCURY LNA™ microRNA Arrays (N=36). Bioinformatic analysis for predicted miRNA-mRNA target interactions was performed on significant, differentially expressed miRNAs observed during the differentiation process using publically available algorithms.
Result: MiRNA profiling of osteoclast differentiation revealed conserved and differentially expressed miRNAs. These included miRNAs previously reported known to be involved in osteoclastogenesis, such as miR-29b and miR-223, and novel miRNAs, such as miR-378e with predicted target, ELK4, a member of the Ets family of transcription factors. Further, a number of differentially expressed miRNAs were found to be present in both monocyte- to -osteoclast, and monocyte -to -macrophage differentiation; however, the magnitude of the expression change varied between the two groups.
Conclusion: This study profiled miRNA expression during monocyte-to-osteoclast and monocyte-to-macrophage differentiation and identified novel miRNAs not previously described. Our in-silico analysis reveals potential mRNA targets which serve as candidates for future investigation. This study highlights the role of miRNA in osteoclastogenesis and has implications in physiologic and pathophysiology processes affecting humans, such as periodontal disease, peri-implant disease, osteoporosis and rheumatoid arthritis.
[Show abstract][Hide abstract] ABSTRACT: miRNAs are small noncoding RNAs which act to post-transcriptionally silence gene expression through translational inhibition and mRNA destabilization. It is clear that miRNA impact the function of undifferentiated stem cells. Also, several miRNAs are known to regulate NF-&kappaB signaling. One unexplored avenue is the effect of NF-&kappaB transactivation on miRNA expression.
Objective: The goal of this study was to define by a gene profiling approach the spectrum of miRNA that are expressed in human mesenchymal stem cells (hMSCs) that express constitutively active NF-&kappaB.
Method: hMSCs were transduced with adenoviral vectors encoding NF-&kappaB and GFP to induce transgene expression. Cell lysates were collected after 4, 12, 24, and 48 hours. RNA was isolated, quantified and RNA integrity was checked. Real Time PCR (RT-PCR) was done for a known NF-&kappaB miRNA target gene (miR-146a) and miR-891 was selected as a control miRNA, which is not regulated by NF-&kappaB. RNU6 was used as the housekeeping gene. Also the miRNA expression profiles were checked by microarray using LNA-based arrays (Exiqon). ANOVA and t-test were used for statistical analysis.
Result: miR-146a expression levels were significantly induced after 12, 24 and 48 hours, reaching more than 10-fold high for NF-&kappaB samples compared to GFP control. For the microarray analysis, Benjamini- Hochberg test between the NF-&kappaB and GFP samples showed no differences in miRNA profiles for time points 4 and 12 hours, whereas 17 and 180 differentially expressed miRNAs were identified for time points 24 and 48 hours, respectively. Amongst the differently expressed miRNAs, miR-155, miR-93, miR-30b, miR-214, miR-15b have targets like Osx and Runx2, which are transcription factors related to bone formation and regeneration.
Conclusion: NF-&kappaB predominantly induced miRNA expression in undifferentiated hMSCs.
[Show abstract][Hide abstract] ABSTRACT: Antioxidants possess significant therapeutic potential for the treatment of inflammatory disorders. One such disorder is periodontitis characterised by an antimicrobial immune response, inflammation, and irreversible changes to the supporting structures of the teeth. Recognition of conserved pathogen-associated molecular patterns is a crucial component of innate immunity to Gram-negative bacteria such as Escherichia coli, as well as the periodontal pathogen Aggregatibacter actinomycetemcomitans. In this study, we investigated the antioxidants Phloretin, Silymarin, Hesperetin, and Resveratrol to ascertain whether they altered the production of inflammatory mediators by innately-activated leukocytes. Peripheral blood mononuclear cells were stimulated with lipopolysaccharide purified from Aggregatibacter actinomycetemcomitans, and the production of cytokines, chemokines, and differentiation factors was assayed by enzyme-linked immunosorbent assay, cytometric bead array, and RT-PCR. Significant inhibition of these factors was achieved upon treatment with Phloretin, Silymarin, Hesperetin, and Resveratrol. These data further characterise the potent anti-inflammatory properties of antioxidants. Their ability to inhibit the production of inflammatory cytokines, chemokines, and differentiation factors by a heterogeneous population of leukocytes has clear implications for their therapeutic potential in vivo.
Mediators of Inflammation 01/2014; 2014:938712. · 2.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apical periodontitis is an inflammatory disease of the periradicular tissues caused by the host's immune response to infection of the root canal system. MicroRNAs (miRNAs) have been shown to play an important role in the regulation of inflammation and the immune response; however, their role in the pathogenesis of endodontic periapical disease has not been explored. The purpose of this study was to examine the differential expression of miRNAs in diseased periapical tissues as compared with healthy controls.
We first compared miRNA profiles in diseased periapical tissues collected from patients undergoing endodontic surgery with those of healthy pulps by using microarray analyses. The target genes of the differentially expressed miRNAs were identified by using miRWalk and PubMed. Selected miRNAs linked to inflammation and the immune response were then confirmed in a separate cohort of diseased and healthy tissues by using quantitative reverse transcription-polymerase chain reaction. Healthy pulps and periodontal ligaments were used as controls. Data were normalized to the level of SNORD 44, which served as an endogenous control.
Of the 381 miRNAs identified by using microarray, 24 miRNAs were down-regulated in diseased periapical tissues compared with controls (n = 13) (P < .003). The down-regulation of 7 miRNAs was confirmed from 9 selected miRNAs by using quantitative real-time polymerase chain reaction (n = 19) (P < .05). Target genes of these miRNAs include key mediators in the immune and inflammatory response such as interleukin-6, matrix metalloproteinase-9, and transforming growth factor-β.
These findings offer new insight into the pathogenesis of endodontic disease and have the potential to impact the development of new methods for prevention, diagnosis, and treatment of apical periodontitis.
Journal of endodontics 12/2013; 39(12):1498-503. · 2.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine the early temporal-wide genome transcription regulation by the surface topography at the bone-implant interface of implants bearing microroughened or superimposed nanosurface topology.
Four commercially pure titanium implants (2.2 × 5.0 mm) with either a moderately roughened surface (TiOblast) or super-imposed nanoscale topography (Osseospeed) were placed (n = 2/surface) in edentulous sites of eleven systemically healthy subjects and subsequently removed after 3 and 7 days. Total RNA was isolated from cells adherent to retrieved implants. A whole-genome microarray using the Affymetrix Human gene 1.1 ST Array was used to describe the gene expression profiles that were differentially regulated by the implant surfaces.
There were no significant differences when comparing the two implant surfaces at each time point. However, the microarray identified several genes that were differentially regulated at day 7 vs. day 3 for both implant surfaces. Functionally relevant categories related to the extracellular matrix (ECM), collagen fibril organization, and angiogenesis were upregulated at both surfaces (day7 vs. day3). Abundant upregulation of several differential markers of alternative activated macrophages was observed (e.g., MRC1, MSR1, MS4A4A, SLC38A6, and CCL18). The biological processes involved with the inflammatory/immune response gene expression were concomitantly downregulated.
Gene regulation implicating collagen fibrillogenesis and ECM organization as well as the inflammatory/immune responses involving the alternative activated pathway are observed in implant adherent cells at early (3-7 days) after implantation. These gene expression events may indicate a pivotal role of collagen fibrillogenesis as well as immunomodulation in altering bone accrual and biomechanical physical properties of the implant-bone interface.
Clinical Oral Implants Research 09/2013; · 3.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are a class of small, noncoding RNAs that regulate post-transcriptional expression of their respective target genes and are responsive to various stimuli, including LPS. Here we examined the early (4 h) miRNA responses of THP1-differentiated macrophages challenged with LPS derived from the periodontal pathogens, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis or environmentally-modified LPS obtained from P. gingivalis grown in cigarette smoke extract. Predicted miRNA-gene target interactions for LPS-responsive miR-29b and let-7f were confirmed using dual-luciferase assays and by transfection experiments using miRNA mimics and inhibitors. Convergent and divergent miRNA profiles were observed in treated samples where differences in miRNA levels related to the type, concentration and incubation times of LPS challenge. Dual-luciferase experiments revealed miR-29b targeting of interleukin-6 receptorα (IL-6Rα) and IFN-γ inducible protein 30 and let-7f targeting of suppressor of cytokine signaling 4 and thrombospondin-1. Transfection experiments confirmed miR-29b and let-7f modulation of IL-6Rα and SOCS4 protein expression levels, respectively. Thus, we have demonstrated convergent/divergent miRNA responses to wild type LPS and its environmentally-modified LPS, and demonstrate miRNA targeting of key genes linked to inflammation and immunity. Our data indicate that these LPS-responsive miRNAs may play a key role in fine-tuning the host response to periodontal pathogens.
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: This pilot study evaluated the molecular, histologic, and radiographic healing of bone to instrumentation with piezoelectric or high speed rotary (R) devices over a 3-week healing period. MATERIAL AND METHODS: Fourteen Sprague-Dawley rats (Charles River Laboratories International, Inc., Wilmington, MA, USA) underwent bilateral tibial osteotomies prepared in a randomized split-leg design using Piezotome® (P1) (Satelec Acteon, Merignac, France), Piezotome 2® (P2) (Satelec Acteon), High-speed R instrumentation, or sham surgery (S). At 1 week, an osteogenesis array was used to evaluate differences in gene expression while quantitative analysis assessed percentage bone fill (PBF) and bone mineral density (BMD) in the defect, peripheral, and distant regions at 3 weeks. Qualitative histologic evaluation of healing osteotomies was also performed at 3 weeks. RESULTS: At 1 week, expression of 11 and 18 genes involved in bone healing was significantly (p < .05) lower following P1 and P2 instrumentation, respectively, relative to S whereas 16 and 4 genes were lower relative to R. No differences in PBF or BMD were detected between groups within the osteotomy defect. However, significant differences in PBF (p = .020) and BMD (p = .008) were noted along the peripheral region between P2 and R groups, being R the group with the lowest values. Histologically, smooth osteotomy margins were present following instrumentation using P1 or P2 relative to R. CONCLUSIONS: Piezoelectric instrumentation favors preservation of bone adjacent to osteotomies while variations in gene expression suggest differences in healing rates due to surgical modality. Bone instrumented by piezoelectric surgery appears less detrimental to bone healing than high-speed R device.
Clinical Implant Dentistry and Related Research 06/2013; · 3.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: miRNA regulates immunity by modulating the expression of key genes involved in leukocyte development and function. Furthermore, immunity includes innate and adaptive systems comprising myeloid-derived cells and lymphocytes which mediate nonspecific and pathogen-specific responses. Innate immunity encompasses the generation of myeloid-derived cells and their ability to mount a rapid and appropriate response to pathogen-associated danger. Here we highlight common components of research investigating the role of miRNA in innate immunity. The identification of miRNAs involved in immunity generally begins with the profiling of samples generated ex vivo or in vitro which include the cell types and immunological conditions of interest. From here a core set of methods and technologies are utilised. Various technologies exist for quantifying miRNA expression, including qRT-PCR and microarray platforms capable of profiling all known human miRNAs as listed by miRBASE. miRNA array expression data may be confirmed by qRT-PCR before in silico analysis of predicted miRNA targets, and these miRNA-target interactions validated by dual luciferase assays. Finally, the biological significance of any miRNA is dependent upon the degree of modulation it exerts combined with the importance of it target within the relevant system. A widely used approach for ascertaining this is to perform experiments in which the miRNA is constitutively expressed or knocked-down and altered function or experimental outcome tested. To date these methods have revealed several important roles for miRNA in the functioning of the immune system. As the technologies and techniques described here continue to develop so will our understanding of the function
[Show abstract][Hide abstract] ABSTRACT: Inflammation of the pulp and periradicular tissues is estimated to affect approximately 22 million people in the United States. Pulpitis represents an immune response to bacteria in root canal systems and is the most common reason for patients seeking emergency dental care. MicroRNAs (miRNAs) play a pivotal role in the regulation of immune responses, including the acute inflammatory response triggered by activation of toll-like receptors (TLR); however, their role in pulpal immune response is still being explored. Our recent microarray study showed that the hsa-mir 181 family is differentially expressed in inflamed pulps and in macrophages stimulated with TLR agonists. At present, no study has characterized the expression of miR181 family in pulpal fibroblasts. Objectives: The objectives of this study are: 1) to characterize the expression of hsa-mir-181 family in primary human fibroblasts; and 2) to investigate if their expression level is influenced by TLR agonists. Methods: Primary cultures of human pulpal fibroblasts (HPF) were exposed at 1, 4 and 8 hours to 1μg, 100 ng and 10 ng per milliter of P. gingivalis LPS. Unchallenged HPF served as controls. Expression of the miR181 family was quantified by qRT-PCR using RNU-6b as the endogenous control. Relative quantification values were calculated together with the statistical significance using one-way ANOVA. P value was set at ‹0.05. Potential target messenger RNAs for the miR181 family were identified using the databases PUBMED and miRWalk. Results: Hsa-miR181 family is expressed in HPF. Our preliminary data suggest that TLR activation modulates expression of hsa-mir 181a and 181b. Bioinformatics search shows that miR181 family targets inflammatory mediators such as IL-2 and MMP-9. Conclusion: This is the very first report on the potential role of mir181 in regulation of pulpal immune response. Further functional studies are warranted to explore the role of miRNAs in pulpal response.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVES: Implant surface topography is a key determinant affecting osteoblastic differentiation and cell-cell signaling of implant-adherent cells. MATERIALS AND METHODS: To assess the early osteoinductive and cell-cell signaling events in adherent cells, commercially pure titanium implants (2.2 × 5 mm) with nanotopography (HF-treated TiO(2) grit-blasted) were compared with micron-scale topography TiO(2) grit-blasted (micron-scale, control) implants in vivo. Six implants (n = 3/surface) were placed in 10 systemically healthy subjects and removed by reverse threading at 1, 3, and 7 days. Gene expression profiles of adherent cells were interrogated using low-density RT-PCR arrays. RESULTS: Osteoinduction was not observed at day 1 on either surface. At 3 days, elevated levels of BMP6, osteopontin, and osterix (OSX) were observed in RNA of cells adherent to both micron-scale and nanotopography surfaces. Both surfaces supported osteoinductive gene expression at 7 days; however, modest elevations of most mRNAs and significantly higher OSX mRNA levels were measured for cells adhered to nanotopography implants. Further, chemokine and cytokine profiles including CXCL10, CXCL14, IL-9, IL-22, and TOLLIP were upregulated on nanotopographic surfaces as compared with microtopographic surfaces. CONCLUSIONS: Implants with superimposed nanoscale topography generate a greater induction of genes linked to osteogenesis and cell-cell signaling during the early phases of osseointegration.
Clinical Oral Implants Research 10/2012; · 3.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective: Macrophages recognize pathogen-associated molecular patterns (PAMPs) through Toll-like receptors (TLRs) and mobilize transcriptional programs aimed at host defense. MicroRNAs (miRNA) are small, ~21nucleotide, single-stranded, non-coding RNAs that regulate diverse biological processes, including inflammation and immunity. However, little is known about the role of these key mediators in the host response against periodontal pathogens. Here we dissect the early mechanisms underlying effector-specific responses by comparing the miRNA profiles of macrophages challenged with LPS from periodontal pathogens recognized by TRL4 or TLR2.
Method: Macrophages were differentiated from the monocytic cell line THP-1 and challenged for 4 hours with 100ng/µl purified LPS from Aggregatibacter actinomycetemcomitans, Y4 serotype B, Porphyromonas gingivalis, strain 33277 (Pg), or Pg grown in cigarette smoke extract at a concentration of 4000 ng nicotine equivalents/ml. miRNA profiles were interrogated using Nanostring technology and expression confirmed by RT-PCR. mRNA targets for differentially expressed miRNAs were identified using PUBMED and miRWalk. Candidate mRNAs were selected if identified as miRNA targets in at least 5/8 databases and linked to immunity/inflammation by GO Biological Process. mRNA/miRNA interactions were confirmed using a dual-luciferase assay.
Result: Our results show that of the 653 human miRNA probes approximately 200 (~30%) miRNAs were responsive to LPS. We identified convergent/divergent expression profiles including a “core” response of 11 upregulated and 20 down-regulated miRNAs common to the three LPS’s as well as LPS-specific miRNA expression. Our dual-luciferase assays confirmed targeting of thrombospondin1 (THBS1), a potent activator of TGF-β and macrophage chemoattractant by LPS-responsive let-7f.
Conclusion: Our data indicates that LPS from periodontal pathogens induces convergent/divergent miRNA expression in human macrophages and identifies key targets linked to inflammation and immunity. We also confirm let-7f targeting of THBS1. These pathogen-effector responsive miRNAs will be useful in our understanding of host-pathogen interactions that may be exploited therapeutically.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression via posttranscriptional repression. They are critical to normal cellular function, and bioinformatic predictions indicate that at least one third of all messenger RNAs might be regulated by miRNAs. Although both the innate and adaptive immune responses are known to be regulated by miRNAs, their role in regulating endodontic disease has yet to be explored. The purpose of this study was to examine the differential expression of miRNAs in normal and inflamed human dental pulps and to explore their functional gene targets.
After obtaining informed consent, we collected normal and inflamed human pulps (N = 30). Microarray and molecular biology techniques were then used for gene profiling and identifying functional gene targets.
Of the 335 human miRNAs identified in the pulp tissues, 3 miRNAs, miR-150∗, miR-584, and miR-766, were significantly up-regulated in inflamed pulps as compared with normal pulps (P < .003). Thirty-three miRNAs were down-regulated in the inflamed pulps (P < .003). The false discovery rate for these findings is estimated to be approximately 5%. The potential gene targets for these miRNAs include proinflammatory cytokines as well as other key mediators of the immune and inflammatory response to infection.
Our data identify differential expression of miRNAs in healthy and diseased human dental pulps. These findings highlight the intricate and specific roles of miRNA in inflammation and immunity, both of which are key aspects of pulpal pathology.
Journal of endodontics 06/2012; 38(6):746-52. · 2.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In periodontitis, a common chronic inflammatory condition, gram-negative-rich bacterial biofilms trigger, in susceptible individuals, perpetuating inflammation that results in extensive tissue damage of tooth supporting structures. To delineate immune cell-dependent mechanisms whereby bacterial challenge drives persistent destructive inflammation in periodontitis and other inflammatory diseases, we studied involved tissues ex vivo and investigated host cell responses to the periodontal pathogen Porphyromonasgingivalis, in vitro. Diseased lesions were populated by abundant Th17 cells, linked to infection, chronic inflammation/autoimmunity and tissue pathology. In vitro, P. gingivalis, particularly the more virulent strain W83, stimulated myeloid antigen presenting cells (APC) to drive Th17 polarization. Supernatants from myeloid APC exposed to P. gingivalis were capable of enhancing Th17 but not Th1 polarization. P. gingivalis favored the generation of Th17 responses by stimulating the production of Th17 related cytokines IL-1β, IL-6 and IL-23, but not Th1 related IL-12. By inducing NFκB activation, P. gingivalis promoted IL-1β, IL-6 and IL-12p40 production, but not IRF3 phosphorylation, connected to generation of the IL-12p35 chain, ultimately restricting formation of the intact IL-12 molecule. Promotion of Th17 lineage responses was also aided by P. gingivalis proteases, which appeared to differentially degrade pivotal cytokines. In this regard, IL-12 was largely degraded by P. gingivalis, whereas IL-1β was more resistant to proteolysis. Our data unveil multiple pathways by which P. gingivalis may orchestrate chronic inflammation, providing insights into interventional strategies.
Journal of Autoimmunity 05/2012; · 7.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective. This preclinical pilot study evaluated the systemic, radiographic, and histological responses to bone putty containing lidocaine in a canine tooth extraction model. Methods. In five beagle dogs the right mandibular premolars were extracted and sockets grafted with (1) xenograft particulate bone and a collagen sponge plug (control), (2) bone putty alone, (3) bone putty mixed with xenograft (3 : 1), or (4) xenograft sandwiched between bone putty. At 6 weeks post-op, the systemic and local responses were evaluated using a blood chemistry panel, micro-CT, and histological analyses. Results. No significant differences in blood chemistries were noted at 6 weeks postgrafting compared to baseline. Sockets grafted with either bone putty formulation demonstrated comparable radiographic and histologic evidence of bone healing compared to control sockets. Conclusions. Our preclinical results indicate that this bone putty appears to be a safe biocompatible device that may be useful in the postoperative management of tooth extractions.
International Journal of Dentistry 01/2012; 2012:894815.
[Show abstract][Hide abstract] ABSTRACT: The aim of this pilot investigation was to determine if microRNA expression differed in the presence or absence of obesity, comparing gingival biopsies obtained from patients with or without periodontal disease. Total RNA was extracted from gingival biopsy samples collected from 20 patients: 10 non-obese patients (BMI < 30 kg/m(2)) and 10 obese patients (BMI > 30 kg/m(2)), each group with 5 periodontally healthy sites and 5 chronic periodontitis sites. MicroRNA expression patterns were assessed with a quantitative microRNA PCR array to survey 88 candidate microRNA species. Four microRNA databases were used to identify potential relevant mRNA target genes of differentially expressed microRNAs. Two microRNA species (miR-18a, miR-30e) were up-regulated among obese individuals with a healthy periodontium. Two microRNA species (miR-30e, miR-106b) were up-regulated in non-obese individuals with periodontal disease. In the presence of periodontal disease and obesity, 9 of 11 listed microRNAs were significantly up-regulated (miR-15a, miR-18a, miR-22, miR-30d, miR-30e, miR-103, miR-106b, miR-130a, miR-142-3p, miR-185, and miR-210). Predicted targets include 69 different mRNAs from genes that comprise cytokines, chemokines, specific collagens, and regulators of glucose and lipid metabolism. The expression of specific microRNA species in obesity, which could also target and post-transcriptionally modulate cytokine mRNA, provides new insight into possible mechanisms of how risk factors might modify periodontal inflammation and may represent novel therapeutic targets.
Journal of dental research 01/2012; 91(1):33-8. · 4.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective: To study the alteration of DNA methylation in the TNFA promoter region in a human monocytic cell line (THP.1) upon Campylobacter rectus (C. rectus) challenge and in gingival biopsies with periodontal diseases. Methods: THP.1 cells were challenged with C. rectus at different time points. Gingival biopsies were collected from sites exhibiting chronic periodontitis, experimental gingivitis in induced and resolved phases, and periodontal health. The methylation status of 10 CpG methylation loci within the TNFA promoter region were analyzed by pyrosequencing, and the transcription of TNFA was studied by Real-time PCR. Luciferase reporter assay was utilized to study the effect of methylation on the TNFA promoter activity. Results: After 96 hours C. rectus (MOI 100) challenge, the overall methylation level of TNFA in THP.1 cells was significantly lower than the mock challenged [14% (9.75- 25.75%) vs. 21% (16.75-27.25%), respectively, shown as median and interquartile range, p=0.028)], while the mRNA level showed 4-fold induction in the C. rectus challenged cells (p=0.008). One CG locus at -244bp exhibited a lower methylation level in the C. rectus challenged (43.7%) as compared to mock challenged cells (53%, p=0.04) and a significantly reduced methylation level in periodontitis biopsies in comparison to samples with periodontal health (45.8% vs.51.8%, p=0.04). The methylation level at the same site in the resolved phase of experimental gingivitis (52.4%), which is comparable to the level of periodontal health, significantly increased as compared to biopsies from the induction phase (46.7%, p=0.01). The similar change in methylation status was also present at CpG locus -73bp. In vitro methylation of the TNFA promoter decreased the luciferase activity to 49% that of the unmethylated promoter (p=0.03). Conclusion: Although C rectus challenge of THP.1 Cells decresaed TNFA methylation and increased TNFA transcription, different CpG loci were targeted in naturally occurring periodontal diseases. (Supported by U54 RR0243831/RR00046)