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1.  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
2.  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
3.  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
4.  Periodontitis: a host-mediated disruption of microbial homeostasis. Unlearning learned concepts 
Periodontology 2000  2013;62(1):10.1111/j.1600-0757.2012.00450.x.
PMCID: PMC3692012  PMID: 23574467
5.  Natural resolution of inflammation 
Periodontology 2000  2013;63(1):149-164.
PMCID: PMC4022040  PMID: 23931059
6.  Co-culture of human bone marrow stromal cells with endothelial cells alters gene expression profiles 
The intricate relationship between angiogenesis and osteogenesis in vivo must be replicated in bone tissue engineering constructs to ensure the formation of a functional vascular network to support successful bone formation. Although communication between bone marrow stromal cells (MSC) and endothelial cells (EC) is recognized as one of the most important cellular interactions in bone regeneration, the underlying mechanisms of this biological process are not well understood. The purpose of this study was to analyze global gene expression associated with intercellular communication between MSC and EC using HumanWG-6 v3.0 expression BeadChips with a one-channel platform system (Illumina, San Diego, CA, USA). Each array contains more than 48,000 probes derived from human genes. A global map of MSC gene expression was generated following co-culture of MSC with EC for 5 and 15 days, in a direct-contact model. The map was used to determine relative alterations in functional processes and pathways. Co-culturing EC with MSC up-regulated genes related to angiogenesis as von Willebrand factor, platelet/endothelial cell adhesion molecule-1, cadherin 5, angiopoietin-related protein 4, and cell surface antigen CD34, and genes playing important roles in osteogenesis as alkaline phosphatase, FK506 binding protein 5, and bone morphogenetic protein. These findings clearly demonstrated that EC had a significant impact on MSC, particularly the bidirectional regulation of angiogenesis and osteogenesis. Moreover, cell-matrix interactions and TGF-β signal pathways were implicated for a crucial role in endothelial, cell-induced gene regulation in MSCs. A detailed study of the functional correlates of the microarray data is warranted to explore cellular and molecular interactions of importance in bone tissue engineering.
PMCID: PMC4022041  PMID: 23918270
7.  Impact of Periodontal Therapy on the Subgingival Microbiota of Severe Periodontitis: Comparison between Good Responders and “Refractory” Subjects by the Human Oral Microbe Identification Microarray (HOMIM) 
Journal of periodontology  2012;83(10):1279-1287.
This study compared the changes on the subgingival microbiota of subjects with “refractory” periodontitis (RP) or treatable periodontitis (GR) before and after periodontal therapy by using the Human Oral Microbe Identification Microarray (HOMIM).
Individuals with chronic periodontitis were classified as RP (n=17) based on mean attachment loss (AL) and/or >3 sites with AL ≥2.5 mm after scaling and root planing, surgery and systemically administered amoxicillin and metronidazole or as GR (n=30) based on mean attachment gain and no sites with AL ≥2.5 mm after treatment. Subgingival plaque samples were taken at baseline and 15 months after treatment and analyzed for the presence of 300 species by HOMIM analysis. Significant differences in taxa before and after therapy were sought using the Wilcoxon test.
The majority of species evaluated decreased in prevalence in both groups after treatment; however, only a small subset of organisms was significantly affected. Species that increased or persisted in high frequency in RP but were significantly reduced in GR included Bacteroidetes sp., Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella spp., Tannerella forsythia, Dialister spp., Selenomonas spp., Catonella morbi, Eubacterium spp., Filifactor alocis, Parvimonas micra, Peptostreptococcus sp. OT113, Fusobacterium sp. OT203, Pseudoramibacter alactolyticus, Streptococcus intermedius or Streptococcus constellatus and Shuttlesworthia satelles. In contrast, Capnocytophaga sputigena, Cardiobacterium hominis, Gemella haemolysans, Haemophilus parainfluenzae, Kingella oralis, Lautropia mirabilis, Neisseria elongata, Rothia dentocariosa, Streptococcus australis and Veillonella spp. were more associated with therapeutic success.
Persistence of putative and novel periodontal pathogens, as well as low prevalence of beneficial species was associated with chronic “refractory” periodontitis.
PMCID: PMC3971922  PMID: 22324467
Refractory Periodontal Disease; Non-surgical periodontal therapy; antimicrobials; DNA microarrays; Microbiology
8.  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
9.  Resolvin E1 and Chemokine-like Receptor 1 ChemR23 Mediate Bone Preservation 
The polyunsaturated ω-3 fatty acid EPA-derived resolvin E1 (RvE1) enhances resolution of inflammation, prevents bone loss and induces bone regeneration. While the inflammation-resolving actions of RvE1 are characterized, molecular mechanism of its bone-protective actions are of interest. To test the hypothesis that receptor-mediated events impact bone changes, we prepared transgenic mice over expressing the RvE1 receptor chemR23 on leukocytes. In zymosan-initiated peritonitis, neutrophil polymorphonuclear leukocyte infiltration in response to RvE1 was limited requiring log order lower doses in chemR23tg mice. Ligature-induced alveolar bone loss was diminished in chemR23tg mice. Local RvE1 treatment of uniform craniotomy in the parietal bone significantly accelerated regeneration of the bone defect. In in vitro bone cultures, RvE1 significantly enhanced expression of osteoprotegerin (OPG) without inducing change in RANKL levels, while the osteogenic markers alkaline phosphatase, bone sialoprotein and Runt-related transcription factor 2 (RunX2) remained unchanged. These results indicate that RvE1 modulates osteoclast differentiation and bone remodeling by direct actions on bone, rescuing OPG production and restoring a favorable RANKL/OPG ratio, in addition to known anti-inflammatory and pro-resolving actions.
PMCID: PMC3538964  PMID: 23241890
10.  Unraveling the Link Between Periodontitis and Cardiovascular Disease 
PMCID: PMC3886775  PMID: 24342998
Editorials; cardiovascular disease; infection; inflammation; periodontitis
11.  Comparisons of Subgingival Microbial Profiles of Refractory Periodontitis, Severe Periodontitis and Periodontal Health using the Human Oral Microbe Identification Microarray (HOMIM) 
Journal of periodontology  2009;80(9):1421-1432.
This study compared the subgingival microbiota of subjects with refractory periodontitis (RP) to those in subjects with treatable periodontitis (GR) or periodontal health (PH) using the Human Oral Microbe Identification Microarray (HOMIM).
At baseline, subgingival plaque samples were taken from 47 periodontitis and 20 PH individuals, and analyzed for the presence of 300 species by HOMIM. The periodontitis subjects were classified as RP (n=17) based on mean attachment loss (AL) and/or >3 sites with AL ≥2.5 mm after SRP, surgery and systemically administered amoxicillin and metronidazole or as GR (n=30) based on mean attachment gain and no sites with AL ≥2.5 mm after treatment. Significant differences in taxa among groups were sought using the Kruskal Wallis and Chi-square tests.
More species were detected in diseased patients (GR or RP) than those without disease (PH). RP subjects were distinguished from GR and PH by a significantly high frequency of putative periodontal pathogens such as, Parvimonas micra, Campylobacter gracilis, Eubacterium nodatum, Selenomonas noxia, Tannerella forsythia, Porphyromonas gingivalis, Prevotella spp., Treponema spp., Eikenella corrodens, as well as “unusual” species (Pseudoramibacter alactolyticus, TM7 spp. oral taxon (OT) 346/356, Bacteroidetes spp. OT 272/274, Solobacterium moorei, Desulfobulbus sp. OT 041, Brevundimonas diminuta, Sphaerocytophaga sp. OT 337, Shuttleworthia satelles, Filifactor alocis, Dialister invisus/pneumosintes, Granulicatella adiacens, Mogibacterium tidmidum, Veillonella atypica, Mycoplasma salivarium, Synergistes sp. cluster II, Acidaminococcaceae [G-1] sp. OT 132/150/155/148/135) [p<0.05]. Species that were more prevalent in PH than in periodontitis patients included Actinomyces sp. OT 170, Actinomyces spp. cluster I, Capnocytophaga sputigena, Cardiobacterium hominis, Haemophilus parainfluenzae, Lautropia mirabilis, Propionibacterium propionicum, Rothia dentocariosa/mucilagenosa, Streptococcus sanguinis (p<0.05).
RP patients present a distinct microbial profile compared to patients in the GR and PH groups as determined by HOMIM.
PMCID: PMC3627366  PMID: 19722792
Refractory periodontitis; subgingival microbiota; periodontal pathogen; HOMIM; periodontal therapy
12.  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
13.  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
14.  Role of Suppressors of Cytokine Signaling 3 in Bone Inflammatory Responses 
Suppressor of cytokine signaling 3 (SOCS3) is a potent regulator of cytokine signaling in macrophages and T cells. In recent studies, evidence has been provided for SOCS3 activation in all major bone cells including osteoclasts, chondrocytes, synoviocytes, and osteoblasts. The investigation of SOCS3 function in bone remodeling systems implicates SOCS3 as a key signaling molecule in bone cell-mediated inflammatory responses. Both pro- and anti-inflammatory functions of SOCS3 have been demonstrated in different types of bone cells. This review provides an overview of the important role of SOCS3 in inflammatory responses of various bone cells and in bone inflammatory disorders such as periodontal disease and arthritis. Understanding the roles of SOCS3 in inflammatory diseases of bone and joints such as arthritis, osteomyelitis, and periodontal diseases is critical to revealing insights into signaling pathways that can be manipulated in potential therapeutic approaches.
PMCID: PMC3887271  PMID: 24454312
SOCS3; bone; inflammation
15.  iNOS-Derived Nitric Oxide Stimulates Osteoclast Activity and Alveolar Bone Loss in Ligature-Induced Periodontitis in Rats 
Journal of Periodontology  2011;82(11):1608-1615.
Inflammatory stimuli activate inducible nitric oxide synthase (iNOS) in a variety of cell types, including osteoclasts (OC) and osteoblasts, resulting in sustained NO production. In this study, we evaluate the alveolar bone loss in rats with periodontitis under long-term iNOS inhibition, and the differentiation and activity of OC from iNOS-knockout (KO) mice in vitro.
Oral aminoguanidine (an iNOS inhibitor) or water treatment was started 2 weeks before induction of periodontitis. Rats were sacrificed 3, 7, or 14 days after ligature placement, and alveolar bone loss was evaluated. In vitro OC culture experiments were also performed to study the differentiation of freshly isolated bone marrow cells from both iNOS KO and wild-type C57BL/6 mice. OC were counted 6 days later after tartrate-resistant acid phosphatase staining (a marker of osteoclast identity), and bone resorption activity was assessed by counting the number of resorption pits on dentin disks.
Rats with ligature showed progressive and significant alveolar bone loss compared to sham animals, and aminoguanidine treatment significantly inhibited ligature-induced bone loss at 7 and 14 days after the induction. In comparison to bone marrow cells from wild-type mice, cells from iNOS KO mice showed decreased OC growth and the resulting OC covered a smaller culture dish area and generated fewer resorption pit counts.
Our results demonstrate that iNOS inhibition prevents alveolar bone loss in a rat model of ligature-induced periodontitis, thus confirming that iNOS-derived NO plays a crucial role in the pathogenesis of periodontitis, probably by stimulating OC differentiation and activity.
PMCID: PMC3361509  PMID: 21417589
Animal experimental use; bone resorption; iNOS enzyme; nitric oxide; osteoclasts; periodontitis
16.  Local and cardiorenal effects of periodontitis in nitric oxide-deficient hypertensive rats 
Archives of Oral Biology  2010;56(1):41-47.
In this study we have assessed the renal and cardiac consequences of ligature-induced periodontitis in both normotensive and nitric oxide (NO)-deficient (L-NAME-treated) hypertensive rats.
Materials and methods
Oral L-NAME (or water) treatment was started two weeks prior to induction of periodontitis. Rats were sacrificed 3, 7 or 14 days after ligature placement, and alveolar bone loss was evaluated radiographically. Thiobarbituric reactive species (TBARS; a lipid peroxidation index), protein nitrotyrosine (NT; a marker of protein nitration) and myeloperoxidase activity (MPO; a neutrophil marker) were determined in the heart and kidney.
In NO-deficient hypertensive rats, periodontitis-induced alveolar bone loss was significantly diminished. In addition, periodontitis-induced cardiac NT elevation was completely prevented by L-NAME treatment. On the other hand L-NAME treatment enhanced MPO production in both heart and kidneys of rats with periodontitis. No changes due to periodontitis were observed in cardiac or renal TBARS content.
In addition to mediating alveolar bone loss, NO contributes to systemic effects of periodontitis in the heart and kidney.
PMCID: PMC3359128  PMID: 20864089
Periodontitis; Nitric oxide; Neutrophils; Nitrotyrosine; Lipid peroxidation; Heart; Kidney
17.  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
18.  Resolvin E1 Regulates ADP Activation of Human Platelets 
Resolvin E1 (RvE1) is an eicosapentaenoic acid (EPA)-derived specialized pro-resolving mediator generated during resolution of acute inflammation. RvE1 exhibits potent organ-protective actions in vivo and acts on specific cell types including platelets. Here, we investigated the ability of RvE1 to regulate adenosine diphosphate (ADP) activation of platelets via specific receptors because RvE1 reduces platelet aggregation with certain agonists including ADP.
Methods and Results
RvE1 (0.1nM–100nM) incubated with platelets gave reduced ADP-stimulated P-selectin mobilization (IC50 ~1.6×10−12 M) and polymerized actin content compared to control platelets. RvE1 (1–100nM) did not stimulate or block intracellular calcium mobilization. Using a new P2Y12-β-arrestin-coupled cell system, ADP-activated P2Y12 with an EC50 of 5×10−6 M and RvE1 did not directly stimulate P2Y12 or block ADP-P2Y12 signals. In this system, another eicosanoid LTE4 (EC50 1.3×10−11 M) dose dependently activated P2Y12. When recombinant P2Y12-expressing cells were transiently transfected with an RvE1 receptor, human ChemR23 (present on human platelets), addition of RvE1 (0.1nM-10.0nM) blocked ADP signals (IC50 ~1.6×10−11 M) in P2Y12-ChemR23-expressing cells compared to mock transfections.
These results demonstrate that RvE1’s regulatory actions (i.e reducing ADP-stimulated P-selectin mobilization and actin polymerization) are hChemR23-dependent. Moreover, they document specific platelet actions of RvE1 selectively engaged with ADP-activated platelets that illuminate a new cellular mechanism and impact of omega-3 EPA that may contribute to both resolution of vascular inflammation and ADP-dependent platelet activation relevant in pathologic cardiovascular events.
PMCID: PMC2982748  PMID: 20702811
Eicosapentaenoic acid (EPA); Resolvin E1; inflammation; platelet; adenosine diphosphate (ADP)
19.  Impaired Phagocytosis in Localized Aggressive Periodontitis: Rescue by Resolvin E1 
PLoS ONE  2011;6(9):e24422.
Resolution of inflammation is an active temporally orchestrated process demonstrated by the biosynthesis of novel proresolving mediators. Dysregulation of resolution pathways may underlie prevalent human inflammatory diseases such as cardiovascular diseases and periodontitis. Localized Aggressive Periodontitis (LAP) is an early onset, rapidly progressing form of inflammatory periodontal disease. Here, we report increased surface P-selectin on circulating LAP platelets, and elevated integrin (CD18) surface expression on neutrophils and monocytes compared to healthy, asymptomatic controls. Significantly more platelet-neutrophil and platelet-monocyte aggregates were identified in circulating whole blood of LAP patients compared with asymptomatic controls. LAP whole blood generates increased pro-inflammatory LTB4 with addition of divalent cation ionophore A23187 (5 µM) and significantly less, 15-HETE, 12-HETE, 14-HDHA, and lipoxin A4. Macrophages from LAP subjects exhibit reduced phagocytosis. The pro-resolving lipid mediator, Resolvin E1 (0.1–100 nM), rescues the impaired phagocytic activity in LAP macrophages. These abnormalities suggest compromised resolution pathways, which may contribute to persistent inflammation resulting in establishment of a chronic inflammatory lesion and periodontal disease progression.
PMCID: PMC3173372  PMID: 21935407
20.  Association of salivary lysozyme and C-reactive protein with metabolic syndrome 
Salivary lysozyme (SLZ) is a proteolytic enzyme secreted by oral leukocytes and contains a domain that has an affinity to advanced glycation end products (AGE). Thus, we hypothesized that SLZ would be associated with metabolic syndrome (metS), a pro-inflammatory state.
Utilizing cross-sectional data from 250 coronary artery disease (CAD) and 250 non-CAD patients, the association of SLZ with metS was tested by logistic regression analyses controlling for age, sex, smoking, total cholesterol and CRP levels. The analyses were stratified by CAD status to control for the possible effects of CAD.
MetS was found in 122 persons. The adjusted Odds Ratio (OR) for metS associated with the highest quartile of SLZ was 1.95 with 95% confidence interval (CI) 1.20 - 3.12, p-value = 0.007, compared with the lower three quartiles combined. Among the 40 subjects with metS but without CAD, the OR was 1.63 (CI: 0.64 - 4.15, p=0.31), while in the CAD group, SLZ was significantly associated with metS [OR=1.96 (1.09 - 3.52), p= 0.02]. In both subgroups, CRP was not significantly associated with metS.
Salivary lysozyme was significantly associated with metS (OR=1.95) independent of CRP level. Future longitudinal research is warranted.
PMCID: PMC2923268  PMID: 20666873
inflammation; metabolic syndrome; Salivary lysozyme; C-reactive protein; atherosclerosis
21.  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
23.  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
24.  Results From the Periodontitis and Vascular Events (PAVE) Study: A Pilot Multicentered, Randomized, Controlled Trial to Study Effects of Periodontal Therapy in a Secondary Prevention Model of Cardiovascular Disease 
Journal of periodontology  2009;80(2):190-201.
In the Periodontitis and Vascular Events (PAVE) pilot study, periodontal therapy was provided as an intervention in a secondary cardiac event prevention model through five coordinated cardiac– dental centers.
Subjects were randomized to either community care or protocol provided scaling and root planing to evaluate effects on periodontal status and systemic levels of high-sensitivity C-reactive protein (hs-CRP).
After 6 months, there was a significant reduction in mean probing depth and extent of 4- or 5-mm pockets. However, there were no significant differences in attachment levels, bleeding upon probing, or extent of subgingival calculus comparing subjects assigned to protocol therapy (n = 151) to those assigned to community care (n = 152). Using intent-to-treat analyses, there was no significant effect on serum hs-CRP levels at 6 months. However, 48% of the subjects randomized to community care received preventive or periodontal treatments. Secondary analyses demonstrated that consideration of any preventive or periodontal care (i.e., any treatment) compared to no treatment showed a significant reduction in the percentage of people with elevated hs-CRP (values >3 mg/l) at 6 months. However, obesity nullified the periodontal treatment effects on hs-CRP reduction. The adjusted odds ratio for hs-CRP levels >3 mg/l at 6 months for any treatment versus no treatment among non-obese individuals was 0.26 (95%confidence interval: 0.09 to 0.72), adjusting for smoking, marital status, and gender.
This pilot study demonstrated the critical role of considering obesity as well as rigorous preventive and periodontal care in trials designed to reduce cardiovascular risk.
PMCID: PMC2778200  PMID: 19186958
C-reactive protein; cardiovascular diseases; controlled clinical trial; obesity; periodontitis; prevention
25.  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

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