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1.  Alternative Splicing Generates a Diacylglycerol Kinase α Transcript That Acts as a Dominant-Negative Modulator of Superoxide Production in Localized Aggressive Periodontitis 
Journal of periodontology  2013;85(7):934-943.
Diacylglycerol (DAG), levels of which are tightly regulated by diacylglycerol kinases (DGKs), is a lipid mediator linked to key biologic functions. Members of the DGK family undergo alternative splicing, generating the protein diversity necessary to control different intracellular DAG pools. DGKα function is altered in polymorphonuclear neutrophils (PMNs) of patients with localized aggressive periodontitis (LAgP), suggesting a genetic basis. Here, the authors assess DGKα spliced transcripts in human LAgP neutrophils.
In an expression library of a patient with LAgP, PMNs were screened for different DGKα transcripts. Real-time polymerase chain reaction and in vitro expression assays were performed to assess the fate of different transcripts on protein translocation and superoxide production in human leukemia cells (HL-60) and COS-7 cells.
A DGKα transcript that lacks exon 10 (DGKαΔ10) and generates a premature stop codon and a truncated protein was identified as being upregulated in LAgP neutrophils. In vitro assays revealed that DGKαΔ10 translocation occurred even in the absence of important regulatory motifs. Transfection of HL-60 neutrophil-like cells with the DGKαΔ10 spliced variant induced an increase in the stimulated production of su-peroxide anion replicating the phenotype of LAgP PMNs.
DGKαΔ10 can act as a dominant-negative transcript that can modulate superoxide production and provides an example of genetic regulation of the inflammatory response that may be relevant to human inflammatory diseases such as LAgP. J Periodontol 2014;85:934-943.
PMCID: PMC4527154  PMID: 24171497
Aggressive periodontitis; diacylglycerol kinase; inflammation; neutrophils; phosphoprotein phosphatases; protein kinases
2.  Impact of Resolvin E1 on Murine Neutrophil Phagocytosis in Type 2 Diabetes 
Infection and Immunity  2014;83(2):792-801.
Diabetic complications involve inflammation-mediated microvascular and macrovascular damage, disruption of lipid metabolism, glycosylation of proteins, and abnormalities of neutrophil-mediated events. Resolution of inflamed tissues to health and homeostasis is an active process mediated by endogenous lipid agonists, including lipoxins and resolvins. This proresolution system appears to be compromised in type 2 diabetes (T2D). The goal of this study was to investigate unresolved inflammation in T2D. Wild-type (WT) and genetically engineered mice, including T2D mice (db/db), transgenic mice overexpressing the human resolvin E1 (RvE1) receptor (ERV1), and a newly bred strain of db/ERV1 mice, were used to determine the impact of RvE1 on the phagocytosis of Porphyromonas gingivalis in T2D. Neutrophils were isolated and incubated with fluorescein isothiocyanate-labeled P. gingivalis, and phagocytosis was measured in a fluorochrome-based assay by flow cytometry. Mitogen-activated protein kinase (MAPK) (p42 and p44) and Akt (Thr308 and Ser473) phosphorylation was analyzed by Western blotting. The mouse dorsal air pouch model was used to evaluate the in vivo impact of RvE1. Results revealed that RvE1 increased the neutrophil phagocytosis of P. gingivalis in WT animals but had no impact in db/db animals. In ERV1-transgenic and ERV1-transgenic diabetic mice, phagocytosis was significantly increased. RvE1 decreased Akt and MAPK phosphorylation in the transgenic animals. In vivo dorsal air pouch studies revealed that RvE1 decreases neutrophil influx into the pouch and increases neutrophil phagocytosis of P. gingivalis in the transgenic animals; cutaneous fat deposition was reduced, as was macrophage infiltration. The results suggest that RvE1 rescues impaired neutrophil phagocytosis in obese T2D mice overexpressing ERV1.
PMCID: PMC4294250  PMID: 25486994
3.  Early Cytokine Response to Infection with Pathogenic vs Non-Pathogenic Organisms in a Mouse Model of Endodontic Infection 
PLoS ONE  2015;10(7):e0132752.
Using the subcutaneous chamber model of infection, we showed previously that a mixture of four endodontic pathogens (EP: P. intermedia, F. nucleatum, S. intermedius and P. micra) are able to persist without clearance for up to seven days, while a non-pathogenic oral species, S. mitis, was substantially cleared in this time. Here we have compared the cytokine response inside the chambers against these microorganisms. A majority of cytokines tested (17/24) showed different patterns of expression. Several cytokines had a peak of expression at 2 h after infection in response to the EP, while none showed this pattern in S. mitis infections. Chemokines were uniformly present at similar or higher levels in response to S. mitis, with redundant expression of CXCR2 ligands, while several growth/survival factors were present at higher levels in EP infections. Protease activity expressed by EP may be responsible for the lower levels of some chemokines. T-cell associated cytokines were in general expressed at extremely low levels, and did not differ between the two infections. The inflammatory markers IL-6, IL-1α and IL1-β were expressed at similar levels in both infections at early times, while TNFα was preferentially present in S. mitis infections. In EP infected chambers, reciprocal changes in levels of IL-6 and IL-1α were observed at later times suggesting a switch in the inflammatory response. Analysis of the cytokine response to infection with the individual species from the EP mix suggests that P. intermedia drives this inflammatory switch. Together these results show a surprising level of divergence of the host response to pathogenic and non-pathogenic organisms associated with oral infections, and supports a dominant effect of P. intermedia in polymicrobial endodontic infections.
PMCID: PMC4501552  PMID: 26171605
5.  A Molecular Defect in Intracellular Lipid Signaling in Human Neutrophils in Localized Aggressive Periodontal Tissue Damage1 
Host defense mechanisms are impaired in patients with congenital neutrophil (polymorphonuclear neutrophils (PMN)) defects. Impaired PMN chemotaxis is observed in localized aggressive periodontitis (LAP), a familial disorder characterized by destruction of the supporting structures of dentition. In the present studies, we sought evidence for molecular events underlying this aberrant human PMN phenotype. To this end, PMN transendothelial migration and superoxide anion generation were assessed with LAP patients and asymptomatic family members, as well as patients with other chronic mucosal inflammation. PMN from LAP patients showed decreased transmigration across vascular endothelial monolayers (18 ± 12% of control, n = 4) and increased superoxide anion generation (358 ± 37%, p = 0.003). Gene expression was analyzed using oligonucleotide microarrays and fluorescence-based kinetic PCR. cDNA microarray and kinetic-PCR analysis revealed diminished RNA expression of leukocyte-type diacylglycerol (DAG) kinase α in PMN from LAP patients (4.6 ± 1.7 relative units, n = 6, p = 0.007) compared with asymptomatic individuals (51 ± 27 relative units, n = 7). DAG kinase activity was monitored by DAG phosphorylation and individual DAG molecular species were quantified using liquid chromatography and tandem mass spectrometry-based lipidomics. DAG kinase activity was also significantly decreased (73 ± 2%, p = 0.007) and correlated with increased accumulation of 1,2-diacyl-sn-3-glycerol substrates (p = 0.01). These results implicate defects in both PMN transendothelial migration and PMN DAG kinase α signaling as disordered functions in LAP. Moreover, they identify a potential molecular lesion in PMN signal transduction that may account for their aberrant responses and tissue destruction in this disease.
PMCID: PMC4318492  PMID: 14734770
6.  Impact of metronidazole and amoxicillin combination on matrix metalloproteinases-1 and tissue inhibitors of matrix metalloproteinases balance in generalized aggressive periodontitis 
European Journal of Dentistry  2015;9(1):53-59.
Generalized aggressive periodontitis (GAgP) is a complex periodontal disease affecting the entire dentition with a rapid destruction of the periodontium and resulting in loss of teeth. We hypothesized that better clinical healing of adjunctive use of amoxicillin plus metronidazole combination may be related to the effect of this combination therapy to restore imbalance between matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP) which is associated with connective tissue and alveolar bone destruction in patients with GAgP.
Materials and Methods:
Twenty-eight subjects diagnosed with GAgP were recruited. Patients were randomly assigned to test or control groups. MMP-1/TIMP-1 ratio was compared between groups receiving scaling and root planning (SRP) alone (control) or in combination with amoxicillin plus metronidazole (test). Clinical periodontal variables were measured. Gingival crevicular fluid samples were obtained and analyzed for MMP-1 and TIMP-1. Measurements were taken at baseline and repeated at 3 and 6 months after therapy.
Total MMP-1 levels were significantly decreased in both groups (P < 0.05) at 3 and 6 months. MMP-1 concentration levels showed a similar pattern to MMP-1 total levels decreasing significantly at 3 months (P < 0.05). TIMP-1 concentration levels increased in the test group throughout the study period, while the difference did not reach statistical significance (P > 0.05). TIMP-1/MMP-1 balance was restored in test group at 6 months significantly better than the control group (P < 0.05).
The results of this study suggest that metronidazole and amoxicillin combination as an adjunct to SRP results in better clinical healing through restoring TIMP-1/MMP-1 balance.
PMCID: PMC4319300  PMID: 25713485
Aggressive periodontitis; amoxicilline; matrix metalloproteinases-1; metronidazole; scaling and root planing; tissue inhibitors of matrix metalloproteinases-1
7.  Porphyromonas gingivalis Lipid A Phosphatase Activity Is Critical for Colonization and Increasing the Commensal Load in the Rabbit Ligature Model 
Infection and Immunity  2014;82(2):650-659.
Periodontitis is a disease of polymicrobial etiology characterized by inflammation, degradation of host tissue, and bone that irreversibly destroys the supporting apparatus of teeth. Porphyromonas gingivalis contains lipid A with structural heterogeneity that has been postulated to contribute to the initiation of dysbiosis in oral communities by modulating the host response, thereby creating a permissive environment for its growth. We examined two P. gingivalis lipid A phosphatase mutants which contain different “locked” lipid A structures that induce different host cellular responses for their ability to induce dysbiosis and periodontitis in rabbits. Lipopolysaccharide (LPS) preparations obtained from these strains were also examined. After repeated applications of all strains and their respective LPS preparations, P. gingivalis wild type, but not the lipid A mutants, had a significant impact on both the oral commensal microbial load and composition. In contrast, in rabbits exposed to the mutant strains or the LPS preparations, the microbial load did not increase, and yet significant changes in the oral microbial composition were observed. All strains and their respective LPS preparations induced periodontitis. Therefore, the ability to alter the lipid A composition in response to environmental conditions by lipid A phosphatases is required for both colonization of the rabbit and increases in the microbial load. Furthermore, the data demonstrate that multiple dysbiotic oral microbial communities can elicit periodontitis.
PMCID: PMC3911377  PMID: 24478080
8.  Metabolic Disease Risk in Children by Salivary Biomarker Analysis 
PLoS ONE  2014;9(6):e98799.
The study of obesity-related metabolic syndrome or Type 2 diabetes (T2D) in children is particularly difficult because of fear of needles. We tested a non-invasive approach to study inflammatory parameters in an at-risk population of children to provide proof-of-principle for future investigations of vulnerable subjects.
Design and Methods
We evaluated metabolic differences in 744, 11-year old children selected from underweight, normal healthy weight, overweight and obese categories by analyzing fasting saliva samples for 20 biomarkers. Saliva supernatants were obtained following centrifugation and used for analyses.
Salivary C-reactive protein (CRP) was 6 times higher, salivary insulin and leptin were 3 times higher, and adiponectin was 30% lower in obese children compared to healthy normal weight children (all P<0.0001). Categorical analysis suggested that there might be three types of obesity in children. Distinctly inflammatory characteristics appeared in 76% of obese children while in 13%, salivary insulin was high but not associated with inflammatory mediators. The remaining 11% of obese children had high insulin and reduced adiponectin. Forty percent of the non-obese children were found in groups which, based on biomarker characteristics, may be at risk for becoming obese.
Significantly altered levels of salivary biomarkers in obese children from a high-risk population, suggest the potential for developing non-invasive screening procedures to identify T2D-vulnerable individuals and a means to test preventative strategies.
PMCID: PMC4051609  PMID: 24915044
9.  Resolvin D1 and Lipoxin A4 Improve Alveolarization and Normalize Septal Wall Thickness in a Neonatal Murine Model of Hyperoxia-Induced Lung Injury 
PLoS ONE  2014;9(6):e98773.
The critical fatty acids Docosahexaenoic Acid (DHA) and Arachidonic Acid (AA) decline in preterm infants within the first postnatal week and are associated with neonatal morbidities, including bronchopulmonary dysplasia (BPD). DHA and AA are precursors to downstream metabolites that terminate the inflammatory response. We hypothesized that treatment with Resolvin D1 and/or Lipoxin A4 would prevent lung injury in a murine model of BPD.
To determine the effect of Resolvin D1 and/or Lipoxin A4 on hyperoxia-induced lung injury.
C57/BL6 pups were randomized at birth to Room Air, Hyperoxia (>90% oxygen), Hyperoxia + Resolvin D1, Hyperoxia + Lipoxin A4, or Hyperoxia + Resolvin D1/Lipoxin A4. Resolvin D1 and/or Lipoxin A4 (2 ng/g) were given IP on days 0, 3, 6, and 9. On day 10, mice were sacrificed and lungs collected for morphometric analyses including Mean Linear Intercept (MLI), Radial Alveolar Count (RAC), and Septal Thickness (ST); RT-PCR analyses of biomarkers of lung development and inflammation; and ELISA for TGFβ1 and TGFβ2.
The increased ST observed with hyperoxia exposure was normalized by both Resolvin D1 and Lipoxin A4; while, hyperoxia-induced alveolar simplification was attenuated by Lipoxin A4. Relative to hyperoxia, Resolvin D1 reduced the gene expression of CXCL2 (2.9 fold), TIMP1 (6.7 fold), and PPARγ (4.8 fold). Treatment with Lipoxin A4 also led to a reduction of CXCL2 (2.4 fold) while selectively increasing TGFβ2 (2.1 fold) and Smad3 (1.58 fold).
The histologic and biochemical changes seen in hyperoxia-induced lung injury in this murine model can be reversed by the addition of DHA and AA fatty acid downstream metabolites that terminate the inflammatory pathways and modulate growth factors. These fatty acids or their metabolites may be novel therapies to prevent or treat lung injury in preterm infants.
PMCID: PMC4043836  PMID: 24892762
10.  Blocking Pro-Inflammatory Cytokine Release Modulates Peripheral Blood Mononuclear Cell Response to Porphyromonas Gingivalis 
Journal of periodontology  2012;84(9):1337-1345.
Chronic periodontitis is an inflammatory disease in which cytokines play a major role in the progression of disease. Anti-inflammatory cytokines (IL-4 and IL-10) were reported to be absent or reduced in diseased periodontal tissues, suggesting an imbalance between the pro- and anti-inflammatory mediators. We have tested the hypothesis that there is cellular cross-talk mediated by pro- and anti-inflammatory cytokines and that blocking pro-inflammatory cytokine (TNF-α and IL-1) production will enhance anti-inflammatory cytokine (IL-4 and IL-10) production from peripheral blood mononuclear cells (PBMC) in response to P. gingivalis.
PBMC were isolated from individuals diagnosed with chronic periodontitis or healthy individuals and cultured for 24 hours. Concanavalin-A (ConA) was used as an activator of lymphocyte function. Live and heat-killed P .gingivalis or lipopolysaccharide from P. gingivalis was used as the bacterial stimulants. TNF-α and IL-1 production was neutralized by specific antibodies against TNF-α and IL-1α or β. Culture supernatants were evaluated by ELISA for TNF-α, IL-1β, IL-4, and IL-10 production.
Live P. gingivalis did not result in any significant IL-10 or IL-4 release while heat-killed P. gingivalis led to a significant increase in IL-10 levels compared to unstimulated or live P. gingivalis-stimulated cells from both healthy and periodontitis individuals. Overall, PBMC from patients with chronic periodontitis produced significantly lower IL-10 in response to ConA and P. gingivalis suggesting chronic suppression of the anti-inflammatory cytokine production. Blocking the pro-inflammatory cytokine response did not result in any substantial change in IL-10 or IL-4 response to live P. gingivalis. Blocking the pro-inflammatory cytokine response restored IL-10 production by cells from chronic periodontitis in response to P. gingivalis LPS.
These findings suggest that PBMC from patients with chronic periodontitis have suppressed anti-inflammatory cytokine production that can, in part, be restored by neutralizing pro-inflammatory cytokines. Monocytes are an important source of IL-10 production and monocyte-derived IL-10 might play a regulatory role in the pathogenesis of chronic periodontitis.
PMCID: PMC3935330  PMID: 23173823
IL-4; IL-10; monocytes; Porphyromonas gingivalis; Periodontitis
11.  Arginine deiminase inhibits Porphyromonas gingivalis surface attachment 
Microbiology  2013;159(Pt 2):275-285.
The oral cavity is host to a complex microbial community whose maintenance depends on an array of cell-to-cell interactions and communication networks, with little known regarding the nature of the signals or mechanisms by which they are sensed and transmitted. Determining the signals that control attachment, biofilm development and outgrowth of oral pathogens is fundamental to understanding pathogenic biofilm development. We have previously identified a secreted arginine deiminase (ADI) produced by Streptococcus intermedius that inhibited biofilm development of the commensal pathogen Porphyromonas gingivalis through downregulation of genes encoding the major (fimA) and minor (mfa1) fimbriae, both of which are required for proper biofilm development. Here we report that this inhibitory effect is dependent on enzymic activity. We have successfully cloned, expressed and defined the conditions to ensure that ADI from S. intermedius is enzymically active. Along with the cloning of the wild-type allele, we have created a catalytic mutant (ADIC399S), in which the resulting protein is not able to catalyse the hydrolysis of l-arginine to l-citrulline. P. gingivalis is insensitive to the ADIC399S catalytic mutant, demonstrating that enzymic activity is required for the effects of ADI on biofilm formation. Biofilm formation is absent under l-arginine-deplete conditions, and can be recovered by the addition of the amino acid. Taken together, the results indicate that arginine is an important signal that directs biofilm formation by this anaerobe. Based on our findings, we postulate that ADI functions to reduce arginine levels and, by a yet to be identified mechanism, signals P. gingivalis to alter biofilm development. ADI release from the streptococcal cell and its cross-genera effects are important findings in understanding the nature of inter-bacterial signalling and biofilm-mediated diseases of the oral cavity.
PMCID: PMC3709564  PMID: 23242802
12.  Photobiomodulation accelerates orthodontic alignment in the early phase of treatment 
Numerous strategies have been proposed to decrease the treatment time a patient requires in orthodontic treatment. Recently, a number of device-accelerated therapies have emerged in orthodontics. Photobiomodulation is an emerging area of science that has clinical applications in a number of human biological processes. The aim of this study was to determine if photobiomodulation reduces the treatment time in the alignment phase of orthodontic treatment.
This multicenter clinical trial was performed on 90 subjects (73 test subjects and 17 controls), and Little's Index of Irregularity (LII) was used as a measure of the rate of change of tooth movement. Subjects requiring orthodontic treatment were recruited into the study, and the LII was measured at regular time intervals. Test subjects used a device which produced near-infrared light with a continuous 850-nm wavelength. The surface of the cheek was irradiated with a power density of 60 mW/cm2 for 20 or 30 min/day or 60 min/week to achieve total energy densities of 72, 108, or 216 J/cm2, respectively. All subjects were fitted with traditional orthodontic brackets and wires. The wire sequences for each site were standardized to an initial round alignment wire (014 NiTi or 016 NiTi) and then advanced through a progression of stiffer arch wires unit alignment occurred (LII < 1 mm).
The mean LII scores at the start of the clinical trial for the test and control groups were 6.35 and 5.04 mm, respectively. Multi-level mixed effect regression analysis was performed on the data, and the mean rate of change in LII was 0.49 and 1.12 mm/week for the control and test groups, respectively.
Photobiomodulation produced clinically significant changes in the rates of tooth movement as compared to the control group during the alignment phase of orthodontic treatment.
PMCID: PMC4384947  PMID: 24326198
Orthodontic tooth movement; Photobiomodulation; Alignment; Accelerates
13.  Performance of Multiplex Cytokine Assays in Serum and Saliva among Community-Dwelling Postmenopausal Women 
PLoS ONE  2013;8(4):e59498.
Multiplexing arrays increase the throughput and decrease sample requirements for studies employing multiple biomarkers. The goal of this project was to examine the performance of Multiplex arrays for measuring multiple protein biomarkers in saliva and serum. Specimens from the OsteoPerio ancillary study of the Women’s Health Initiative Observational Study were used. Participants required the presence of at least 6 teeth and were excluded based on active cancer and certain bone issues but were not selected on any specific condition. Quality control (QC) samples were created from pooled serum and saliva. Twenty protein markers were measured on five multiplexing array panels. Sample pretreatment conditions were optimized for each panel. Recovery, lower limit of quantification (LLOQ) and imprecision were determined for each analyte. Statistical adjustment at the plate level was used to reduce imprecision estimates and increase the number of usable observations. Sample pre-treatment improved recovery estimates for many analytes. The LLOQ for each analyte agreed with manufacturer specifications except for MMP-1 and MMP-2 which were significantly higher than reported. Following batch adjustment, 17 of 20 biomarkers in serum and 9 of 20 biomarkers in saliva demonstrated acceptable precision, defined as <20% coefficient of variation (<25% at LLOQ). The percentage of cohort samples having levels within the reportable range for each analyte varied from 10% to 100%. The ratio of levels in saliva to serum varied from 1∶100 to 28∶1. Correlations between saliva and serum were of moderate positive magnitude and significant for CRP, MMP-2, insulin, adiponectin, GM-CSF and IL-5. Multiplex arrays exhibit high levels of analytical imprecision, particularly at the batch level. Careful sample pre-treatment can enhance recovery and reduce imprecision. Following statistical adjustments to reduce batch effects, we identified biomarkers that are of acceptable quality in serum and to a lesser degree in saliva using Multiplex arrays.
PMCID: PMC3618114  PMID: 23577067
14.  A critical role for suppressors of cytokine signaling 3 in regulating LPS-induced transcriptional activation of matrix metalloproteinase-13 in osteoblasts 
PeerJ  2013;1:e51.
Suppressor of cytokine signaling 3 (SOCS3) is a key regulator of cytokine signaling in macrophages and T cells. Although SOCS3 seems to contribute to the balance between the pro-inflammatory actions of IL-6 family of cytokines and anti-inflammatory signaling of IL-10 by negatively regulating gp130/Jak/Stat3 signal transduction, how and the molecular mechanisms whereby SOCS3 controls the downstream impact of TLR4 are largely unknown and current data are controversial. Furthermore, very little is known regarding SOCS3 function in cells other than myeloid cells and T cells. Our previous study demonstrates that SOCS3 is expressed in osteoblasts and functions as a critical inhibitor of LPS-induced IL-6 expression. However, the function of SOCS3 in osteoblasts remains largely unknown. In the current study, we report for the first time that LPS stimulation of osteoblasts induces the transcriptional activation of matrix metalloproteinase (MMP)-13, a central regulator of bone resorption. Importantly, we demonstrate that SOCS3 overexpression leads to a significant decrease of LPS-induced MMP-13 expression in both primary murine calvariae osteoblasts and a mouse osteoblast-like cell line, MC3T3-E1. Our findings implicate SOCS3 as an important regulatory mediator in bone inflammatory diseases by targeting MMP-13.
PMCID: PMC3628613  PMID: 23638389
Inflammation; Periodontitis; Cytokine; Osteoblasts
15.  Oral Inflammatory Diseases and Systemic Inflammation: Role of the Macrophage 
Inflammation is a complex reaction to injurious agents and includes vascular responses, migration, and activation of leukocytes. Inflammation starts with an acute reaction, which evolves into a chronic phase if allowed to persist unresolved. Acute inflammation is a rapid process characterized by fluid exudation and emigration of leukocytes, primarily neutrophils, whereas chronic inflammation extends over a longer time and is associated with lymphocyte and macrophage infiltration, blood vessel proliferation, and fibrosis. Inflammation is terminated when the invader is eliminated, and the secreted mediators are removed; however, many factors modify the course and morphologic appearance as well as the termination pattern and duration of inflammation. Chronic inflammatory illnesses such as diabetes, arthritis, and heart disease are now seen as problems that might have an impact on the periodontium. Reciprocal effects of periodontal diseases are potential factors modifying severity in the progression of systemic inflammatory diseases. Macrophages are key cells for the inflammatory processes as regulators directing inflammation to chronic pathological changes or resolution with no damage or scar tissue formation. As such, macrophages are involved in a remarkably diverse array of homeostatic processes of vital importance to the host. In addition to their critical role in immunity, macrophages are also widely recognized as ubiquitous mediators of cellular turnover and maintenance of extracellular matrix homeostasis. In this review, our objective is to identify macrophage-mediated events central to the inflammatory basis of chronic diseases, with an emphasis on how control of macrophage function can be used to prevent or treat harmful outcomes linked to uncontrolled inflammation.
PMCID: PMC3353263  PMID: 22623923
innate immune system; macrophage; oral disease; inflammation; resolution
16.  TLR Cross-Talk Specifically Regulates Cytokine Production by B Cells from Chronic Inflammatory Disease Patients1 
Chronic systemic inflammation links periodontal disease and diabetes to increased incidence of serious comorbidities. Activation of TLRs, particularly TLR2 and TLR4, promotes chronic systemic inflammation. Human B cells have been generally thought to lack these TLRs. However, recent work showed that an increased percentage of circulating B cells from inflammatory disease patients express TLR2 and TLR4, and that TLR engagement on B cells resulted in unexpected changes in gene expression. New data show that B cells from inflammatory disease patients secrete multiple cytokines in response to different classes of TLR ligands. Furthermore, the B cell response to combinations of TLR ligands is cytokine- and ligand-specific. Some cytokines (IL-1β and IL-10) are predominantly regulated by TLR4, but others (IL-8 and TNF-α) are predominantly regulated by TLR2, due in part to TLR-dictated changes in transcription factor/promoter association. TLR2 and TLR9 also regulate B cell TLR4 expression, demonstrating that TLR cross-talk controls B cell responses at multiple levels. Parallel examination of B cells from periodontal disease and diabetes patients suggested that outcomes of TLR cross-talk are influenced by disease pathology. We conclude that disease-associated alteration of B cell TLR responses specifically regulates cytokine production and may influence chronic inflammation.
PMCID: PMC2851147  PMID: 19917698
17.  Immune response of macrophages from young and aged mice to the oral pathogenic bacterium Porphyromonas gingivalis 
Periodontal disease is a chronic inflammatory gum disease that in severe cases leads to tooth loss. Porphyromonas gingivalis (Pg) is a bacterium closely associated with generalized forms of periodontal disease. Clinical onset of generalized periodontal disease commonly presents in individuals over the age of 40. Little is known regarding the effect of aging on inflammation associated with periodontal disease. In the present study we examined the immune response of bone marrow derived macrophages (BMM) from young (2-months) and aged (1-year and 2-years) mice to Pg strain 381. Pg induced robust expression of cytokines; tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10, chemokines; neutrophil chemoattractant protein (KC), macrophage colony stimulating factor (MCP)-1, macrophage inflammatory protein (MIP)-1α and regulated upon activation normal T cell expressed and secreted (RANTES), as well as nitric oxide (NO, measured as nitrite), and prostaglandin E2 (PGE2) from BMM of young mice. BMM from the 2-year age group produced significantly less TNF-α, IL-6 and NO in response to Pg as compared with BMM from 2-months and 1-year of age. We did not observe any difference in the levels of IL-1β, IL-10 and PGE2 produced by BMM in response to Pg. BMM from 2-months and 1-year of age produced similar levels of all chemokines measured with the exception of MCP-1, which was reduced in BMM from 1-year of age. BMM from the 2-year group produced significantly less MCP-1 and MIP-1α compared with 2-months and 1-year age groups. No difference in RANTES production was observed between age groups. Employing a Pg attenuated mutant, deficient in major fimbriae (Pg DPG3), we observed reduced ability of the mutant to stimulate inflammatory mediator expression from BMMs as compared to Pg 381, irrespective of age. Taken together these results support senescence as an important facet of the reduced immunological response observed by BMM of aged host to the periodontal pathogen Pg.
PMCID: PMC3001696  PMID: 21114831
18.  Diabetes-Induced Oxidative Stress Is Mediated by Ca2+-Independent Phospholipase A2 in Neutrophils 
Neutrophils from people with poorly controlled diabetes present a primed phenotype and secrete excessive superoxide. Phospholipase A2 (PLA2)-derived arachidonic acid (AA) activates the assembly of NADPH oxidase to generate superoxide anion. There is a gap in the current literature regarding which PLA2 isoform regulates NADPH oxidase activation. The aim of this study was to identify the PLA2 isoform involved in the regulation of superoxide generation in neutrophils and investigate if PLA2 mediates priming in response to pathologic hyperglycemia. Neutrophils were isolated from people with diabetes mellitus and healthy controls, and HL60 neutrophil-like cells were grown in hyperglycemic conditions. Incubating neutrophils with the Ca2+-independent PLA2 (iPLA2) inhibitor bromoenol lactone (BEL) completely suppressed fMLP-induced generation of superoxide. The nonspecific actions of BEL on phosphatidic acid phosphohydrolase-1, p47phox phosphorylation, and apoptosis were ruled out by specific assays. Small interfering RNA knockdown of iPLA2 inhibited superoxide generation by neutrophils. Neutrophils from people with poorly controlled diabetes and in vitro incubation of neutrophils with high glucose and the receptor for advanced glycation end products ligand S100B greatly enhanced superoxide generation compared with controls, and this was significantly inhibited by BEL. A modified iPLA2 assay, Western blotting, and PCR confirmed that there was increased iPLA2 activity and expression in neutrophils from people with diabetes. AA (10 μM) partly rescued the inhibition of superoxide generation mediated by BEL, confirming that NADPH oxidase activity is, in part, regulated by AA. This study provides evidence for the role of iPLA2 in enhanced superoxide generation in neutrophils from people with diabetes mellitus and presents an alternate pathway independent of protein kinase C and phosphatidic acid phosphohydrolase-1 hydrolase signaling.
PMCID: PMC2847507  PMID: 20053941
19.  Priming of neutrophil oxidative burst in diabetes requires preassembly of the NADPH oxidase 
Journal of leukocyte biology  2008;84(1):292-301.
Hyperglycemia associated with diabetes mellitus results in the priming of neutrophils leading to oxidative stress that is, in part, responsible for diabetic complications. p47phox, a NADPH oxidase cytosolic subunit, is a key protein in the assembly of the NADPH oxidase leading to superoxide generation. Little is known about the priming mechanism of oxidative pathways in neutrophils of people with diabetes. In this study, the kinetics of p47phox activation was investigated by comparing neutrophils from diabetic and healthy subjects, and the mechanism of hyperglycemia-induced changes was studied by using neutrophil-like HL-60 cells as a model. In resting neutrophils from diabetic subjects, p47phox prematurely translocates to the cell membrane and preassembles with p22phox, a NADPH oxidase membrane subunit. This premature p47phox translocation and preassembly with p22phox were also observed in HL-60 cells cultured with high glucose (HG; 25 mM) and with the specific ligand for the receptor for advanced glycation end products (RAGE), S100B. Phosphorylation of ERK1/2, but not p38 MAPK, was the primary signaling pathway, as evidenced by PD98059 suppressing the translocation of p47phox in HL-60 cells incubated with HG and S100B. HL-60 cells cultured in HG and S100B exhibited a 1.8-fold increase in fMLP-induced superoxide generation compared with those cultured in normal glucose (5.5 mM). These data suggest that HG and increased AGE prime neutrophils and increase oxidative stress inducing the translocation of p47phox to the cell membrane and preassembly with p22phox by stimulating a RAGE-ERK1/2 pathway.
PMCID: PMC2774791  PMID: 18390927
inflammation; signal transduction; cell activation
20.  Ceruloplasmin Induces Polymorphonuclear Leukocyte Priming in Localized Aggressive Periodontitis 
Journal of periodontology  2009;80(8):1300-1306.
Polymorphonuclear leukocytes (PMNs) from subjects with localized aggressive periodontitis (LAgP) present multiple functional abnormalities associated with a phenotypically primed PMN phenotype. Local inflammation is characterized by hypoxia, which leads to increased production of superoxide (O2−) by PMNs. Ceruloplasmin (CP) is also induced by hypoxia and inflammation. The aim of this study was to investigate the role of CP in O2− generation in PMNs from healthy subjects and patients with LAgP.
PMNs were isolated from healthy subjects and those with LAgP (N = 36). Superoxide was measured by cytochrome-C reduction at 550 nm. Intracellular CP expression was analyzed by real-time polymerase chain reaction and Western blotting. Serum levels of CP were measured by enzyme-linked immunosorbent assay. Intracellular iron ion conversion was spectrophotometrically determined by measuring the absorbance of σ-phenanthroline at 510 nm.
O2− generation was significantly higher in LAgP PMNs before and after stimulation with formyl-methionylleucyl-phenylalanine (100 nM). CP expression in PMNs and CP levels in serum were significantly higher in subjects with LAgP compared to the PMNs and serum samples from matched healthy donors (P <0.05). LAgP PMNs also had significantly higher levels of Fe3 + and lower levels of Fe2 + compared to healthy PMNs (P <0.05), suggesting increased iron conversion. Exogenous CP treatment of healthy PMNs resulted in significant increases in O2− generation and iron ion conversion similar to LAgP PMNs.
LAgP PMNs are primed to express higher levels of CP, leading to hypoxia-mediated O2− generation in PMNs and increased oxidative stress and neutrophil-mediated tissue injury in LAgP.
PMCID: PMC2774796  PMID: 19656030
Ceruloplasmin; hypoxia; iron ion; priming; superoxide
21.  Chronic Hyperglycemia Predisposes to Exaggerated Inflammatory Response and Leukocyte Dysfunction in Akita Mice1 
The role of polymorphonuclear neutrophils (PMN) in mediating diabetic tissue damage to the periodontium was investigated in a novel model of chronic hyperglycemia, the Akita mouse. Induction of acute peritoneal inflammation in wild-type (WT) and Akita mice resulted in exaggerated IL-6 response in Akita mice (2.9-fold increase over WT values) and a markedly increased chemokine response (KC, 2.6-fold; MCP-1, 2.6-fold; and MIP-1α, 4.4-fold increase over WT values). Chemotaxis to both fMLP and WKYMVm was significantly reduced in isolated Akita PMN compared with WT PMN as measured in a Boyden chamber. Superoxide release in contrast was significantly increased in Akita PMN as measured with cytochrome c reduction. Bone marrow-derived Akita PMN showed partial translocation of p47phox to the cell membrane without external stimulation, suggesting premature assembly of the superoxide-producing NADPH oxidase in hyperglycemia. In vivo studies revealed that ligature-induced periodontal bone loss is significantly greater in Akita mice compared with WT. Moreover, intravital microscopy of gingival vessels showed that leukocyte rolling and attachment to the vascular endothelium is enhanced in periodontal vessels of Akita mice. These results indicate that chronic hyperglycemia predisposes to exaggerated inflammatory response and primes leukocytes for marginalization and superoxide production but not for transmigration. Thus, leukocyte defects in hyperglycemia may contribute to periodontal tissue damage by impairing the innate immune response to periodontal pathogens as well as by increasing free radical load in the gingival microvasculature.
PMCID: PMC2774801  PMID: 17082643
22.  Modeling of Trabecular Bone and Lamina Dura Following Selective Alveolar Decortication in Rats 
Journal of periodontology  2008;79(9):1679-1688.
Modifying the balance between resorption and apposition through selectively injuring the cortical plate of the alveolus has been an approach to speed tooth movement and is referred to as periodontally accelerated osteogenic orthodontics. The aim of this study was to investigate the alveolar response to corticotomy as a function of time and proximity to the surgical injury in a rat model.
Maxillary buccal and lingual cortical plates were injured in 36 healthy adult rats adjacent to the upper left first molars. Twenty-four animals were euthanized at 3, 7, or 11 weeks. In one group, the maxillae were removed and stripped of soft tissues, and histomorphometric analysis was performed to study alveolar spongiosa and periodontal ligament (PDL) modeling dynamics. Catabolic activity was analyzed with tartrate-resistant acid phosphatase–positive osteoclasts and preosteoclasts. Anabolic actions were measured using a fluorescent vital bone stain series followed by sacrifice at 30 and 51 days. To further analyze the new bone formation, a separate group of animals were fed with calcein fluorescent stain and processed for non-decalcified fluorescent stain histology.
At 3 weeks, the surgery group had significantly (P <0.05) less calcified spongiosa bone surface, greater periodontal ligament surface, higher osteoclast number, and greater lamina dura apposition width. The catabolic activity (osteoclast count) and anabolic activity (apposition rate) were three-fold greater, calcified spongiosa decreased by two-fold, and PDL surface increased by two-fold. Surgical injury to the alveolus that induced a significant increase in tissue turnover by week 3 dissipated to a steady state by postoperative week 11. The impact of the injury was localized to the area immediately adjacent to the decortication injury.
Selective alveolar decortication induced increased turnover of alveolar spongiosa, and the activity was localized; dramatic escalation of demineralization-remineralization dynamics is the likely biologic mechanism underlying rapid tooth movement following selective alveolar decortication.
PMCID: PMC2563959  PMID: 18771369
Bone formation; osteoclast; tooth movement
23.  Activation of RAGE induces elevated O2- generation by mononuclear phagocytes in diabetes 
Journal of leukocyte biology  2006;81(2):520-527.
Oxidative stress has been found to play a role in the pathogenesis of diabetic complications. The aim of this study was to define the oxidative burst of diabetic monocytes to characterize the phenotype associated with poor diabetic control. Superoxide (O2-) is the first molecule generated during the respiratory burst of phagocytes by NADPH oxidase, and its generation by monocytes from 26 controls and 34 diabetic subjects was evaluated in this study. Under resting states or stimulation by PMA or opsonized zymosan, diabetic monocytes produce significantly more O2- than nondiabetic cells. The increased O2- generation was found to be correlated with glycemic control (glycated hemoglobin) of patients. To clarify the effects of hyperglycemia on O2- generation, normal human monocytes were treated with receptor for advanced glycation endproducts (RAGE) ligands (AGE protein and S100B) or high glucose media before stimulation. RAGE ligands and high glucose concentration increased O2- generation from human mononuclear phagocytes. RAGE ligands, specifically and potently, increased O2- generation from mononuclear phagocytes, and high-glucose effects were associated with correspondingly increased osmotic pressure. Differentiated THP-1 cells, from a human monocytic cell line, were used as a model of human monocytes to study the effects of S100B, the RAGE ligand. It was confirmed that RAGE is involved in the priming of O2- generation by S100B. This study demonstrates that RAGE ligands can contribute significantly to the hyper-responsive phenotype of diabetic monocytes, which might be reversible by blocking the RAGE or controlling the presence of RAGE ligands by controlling hyperglycemia.
PMCID: PMC2533418  PMID: 17095613
24.  Phosphorylation of Pleckstrin Increases Proinflammatory Cytokine Secretion by Mononuclear Phagocytes in Diabetes Mellitus1 
The protein kinase C (PKC) family of intracellular enzymes plays a crucial role in signal transduction for a variety of cellular responses of mononuclear phagocytes including phagocytosis, oxidative burst, and secretion. Alterations in the activation pathways of PKC in a variety of cell types have been implicated in the pathogenesis of the complications of diabetes. In this study, we investigated the consequences of PKC activation by evaluating endogenous phosphorylation of PKC substrates with a phosphospecific PKC substrate Ab (pPKC(s)). Phosphorylation of a 40-kDa protein was significantly increased in mononuclear phagocytes from diabetics. Phosphorylation of this protein is downstream of PKC activation and its phosphorylated form was found to be associated with the membrane. Mass spectrometry analysis, immunoprecipitation, and immunoblotting experiments revealed that this 40-kDa protein is pleckstrin. We then investigated the phosphorylation and translocation of pleckstrin in response to the activation of receptor for advanced glycation end products (RAGE). The results suggest that pleckstrin is involved in RAGE signaling and advanced glycation end product (AGE)-elicited mononuclear phagocyte dysfunction. Suppression of pleckstrin expression with RNA interference silencing revealed that phosphorylation of pleckstrin is an important intermediate in the secretion and activation pathways of proinflammatory cytokines (TNF-α and IL-1β) induced by RAGE activation. In summary, this study demonstrates that phosphorylation of pleckstrin is up-regulated in diabetic mononuclear phagocytes. The phosphorylation is in part due to the activation of PKC through RAGE binding, and pleckstrin is a critical molecule for proinflammatory cytokine secretion in response to elevated AGE in diabetes.
PMCID: PMC2150995  PMID: 17579087
25.  Epithelial and Connective Tissue Cell CTGF/CCN2 expression in Gingival Fibrosis 
The Journal of pathology  2006;210(1):59-66.
Gingival overgrowth and fibrosis is a side effect of certain medications and occurs in non-drug induced forms either as inherited (human gingival fibromatosis) or idiopathic gingival overgrowth. The most fibrotic drug-induced lesions develop in response to therapy with phenytoin, the least fibrotic lesions are caused by cyclosporin A, and intermediate fibrosis occurs in nifedipine-induced gingival overgrowth. Connective tissue growth factor (CTGF/CCN2) expression is positively related to the degree of fibrosis in these tissues. In the present study, the hypothesis was investigated that CTGF/CCN2 is expressed in human gingival fibromatosis tissues and contributes to this form of non-drug-induced gingival overgrowth. Histopathology/immunohistochemistry studies show that human gingival fibromatosis lesions are highly fibrotic, similar to phenytoin-induced lesions. Connective tissue CTGF/CCN2 levels were equivalent to the expression in phenytoin-induced gingival overgrowth. The additional novel observation was made that CTGF/CCN2 is highly expressed in the epithelium of fibrotic gingival tissues. This finding was confirmed by in situ hybridization. Real time PCR analyses of RNA extracted from control and drug-induced gingival overgrowth tissues for CTGF/CCN2 were fully consistent with these findings. Finally, normal primary gingival epithelial cell cultures were analyzed for the basal and TGF-β1 or lysophosphatidic acid stimulated CTGF/CCN2 expression at the protein and RNA levels. Data indicate that fibrotic human gingival tissues express CTGF/CCN2 in both the epithelium and connective tissues and cultured gingival epithelial cells express CTGF/CCN2, and lysophosphatidic acid further stimulates CTGF/CCN2 expression. These findings suggest that interactions between epithelial and connective tissues could contribute to gingival fibrosis.
PMCID: PMC1557679  PMID: 16841303
gingival overgrowth; fibrosis; connective tissue growth factor; epithelium; fibroblast; epithelial-mesenchymal transition

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