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1.  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.  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
4.  The hidden ‘mycobacteriome’ of the human healthy oral cavity and upper respiratory tract 
Journal of Oral Microbiology  2015;7:10.3402/jom.v7.26094.
The incidence of opportunistic non-tuberculous mycobacteria (NTM) infections has increased considerably in the past decades causing an array of infections, including respiratory and soft-tissue infections. NTM are ubiquitous and can be found in numerous environments, including households and water plants. However, NTM have not been reported to be associated with the healthy human oral microbiome. Since the oral cavity and upper respiratory track are the main ports of entry of microorganisms into the human body, elucidating NTM diversity and prevalence will assist in the assessment of the potential risks of infection elicited by these opportunistic pathogens. Here, we report the identification of a ‘non-tuberculous mycobacteriome’ in healthy individuals. We employed a modified DNA extraction procedure in conjunction with mycobacterial-specific primers to screen niches in the oral cavity (buccal mucosa and dental plaque) and upper respiratory tract (nostrils and oropharynx) of 10 healthy subjects. A total of 50 prevalent operational taxonomic units sequenced on MiSeq (Illumina) using 16S rRNA V3–V4 region were detected across all screened niches, showing the presence of diverse NTM communities. NTM DNA was detected in the nostrils of all 10 subjects, in buccal mucosa of 8 subjects, in the oropharynx of 7 subjects, and in the dental plaques of 5 subjects. Results from quantitative PCR showed each individual harbored 103–104 predicted NTM per each screened niche. The modification of standard DNA isolation methods to increase sensitivity toward mycobacterial species represents an important step to advance the knowledge of the oral as well as the overall human microbiome. These findings clearly reveal for the first time that healthy individuals harbor a ‘non-tuberculous mycobacteriome’ in their oral cavity and upper respiratory tract and may have important implications in our understanding of infections caused by NTM.
PMCID: PMC4329316  PMID: 25683180
oral microbiome; Mycobacterium; non-tuberculous-mycobacteria; periodontal disease
5.  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
6.  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
7.  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
8.  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
9.  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
10.  Can Salivary Phosphate Levels Be an Early Biomarker to Monitor the Evolvement of Obesity? 
Contributions to nephrology  2013;180:138-148.
Phosphate is an essential nutrient required for important biological reactions that maintain the normal homoeostatic control of the cell. The adverse effects of phosphate metabolism in obesity have not been studied in detail, chiefly because such an association is thought to be uncommon. However, in some animal models of obesity, serum phosphate levels were noted to be higher than the nonobese controls. For example, leptin-deficient (ob/ob) mice become severely obese and have high serum phosphate levels. In this study, we analyzed the phosphate content in saliva collected from children (n = 77; 10.5 ± 1.8) to evaluate association with body mass index; there is a significant increase of salivary phosphate content in obese compared to normal-weight children (ANOVA p < 0.001). The correlation coefficient (r) between BMI and phosphate was 0.33 (p = 0.0032). Our results suggest that the human salivary phosphate level may be an early biomarker of the genesis of obesity in children. The diagnostic importance lies in the fact that the salivary phosphate level could provide a noninvasive predictive marker in the development of obesity. Further studies will be required to understand the underlying mechanism of increased salivary phosphate accumulation in obese and overweight children. Nevertheless, its occurrence without systemic changes could be of diagnostic value, particularly in monitoring evolvement of obesity.
PMCID: PMC3896993  PMID: 23652556
11.  Obesity and Dental Decay: Inference on the Role of Dietary Sugar 
PLoS ONE  2013;8(10):e74461.
To evaluate the relationship of children’s obesity and dental decay.
We measured parameters related to obesity and dental decay in 8,275 4th and 5th grade Kuwaiti children (average age = 11.36 years) in a cross-sectional study. First to determine body weight, height, age for computation of BMI . Second, to determine numbers of teeth, numbers of fillings and numbers of untreated decayed teeth to determine extent and severity of dental disease. From these measurements, we computed measures of dental decay in children from four body weight categories; obese, overweight, normal healthy weight and underweight children.
The percentage of children with decayed or filled teeth varied inversely with the body weight category. The percentage of decayed or filled teeth decreased from 15.61% (n=193) in underweight children, to 13.03% (n=4,094) in normal healthy weight children, to 9.73% (n=1,786) in overweight children to 7.87% (n=2,202) in obese children. Differences between all groups were statistically significant. Male children in this population had more dental decay than female children but the reduction of tooth decay as a function of BMI was greater in male children.
The finding of an inverse obesity-dental decay relationship contradicts the obesity-sugar and the obesity-dental decay relationship hypotheses. Sugar is well recognized as necessary and sufficient for dental decay. Sugar is also hypothesized to be a leading co-factor in obesity. If the later hypothesis is true, one would expect dental decay to increase with obesity. This was not found. The reasons for this inverse relationship are not currently clear.
PMCID: PMC3795155  PMID: 24130667
12.  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
13.  Deep Sequencing of the Oral Microbiome Reveals Signatures of Periodontal Disease 
PLoS ONE  2012;7(6):e37919.
The oral microbiome, the complex ecosystem of microbes inhabiting the human mouth, harbors several thousands of bacterial types. The proliferation of pathogenic bacteria within the mouth gives rise to periodontitis, an inflammatory disease known to also constitute a risk factor for cardiovascular disease. While much is known about individual species associated with pathogenesis, the system-level mechanisms underlying the transition from health to disease are still poorly understood. Through the sequencing of the 16S rRNA gene and of whole community DNA we provide a glimpse at the global genetic, metabolic, and ecological changes associated with periodontitis in 15 subgingival plaque samples, four from each of two periodontitis patients, and the remaining samples from three healthy individuals. We also demonstrate the power of whole-metagenome sequencing approaches in characterizing the genomes of key players in the oral microbiome, including an unculturable TM7 organism. We reveal the disease microbiome to be enriched in virulence factors, and adapted to a parasitic lifestyle that takes advantage of the disrupted host homeostasis. Furthermore, diseased samples share a common structure that was not found in completely healthy samples, suggesting that the disease state may occupy a narrow region within the space of possible configurations of the oral microbiome. Our pilot study demonstrates the power of high-throughput sequencing as a tool for understanding the role of the oral microbiome in periodontal disease. Despite a modest level of sequencing (∼2 lanes Illumina 76 bp PE) and high human DNA contamination (up to ∼90%) we were able to partially reconstruct several oral microbes and to preliminarily characterize some systems-level differences between the healthy and diseased oral microbiomes.
PMCID: PMC3366996  PMID: 22675498
14.  Elevated Proinflammatory Cytokine Production by a Skewed T Cell Compartment Requires Monocytes and Promotes Inflammation in Type 2 Diabetes 
An appropriate balance between proinflammatory (Th17 and Th1) and anti-inflammatory (regulatory T cells [Tregs] and Th2) subsets of T cells is critical to maintain homeostasis and avoid inflammatory disease. Type 2 diabetes (T2D) is a chronic inflammatory disease promoted by changes in immune cell function. Recent work indicates T cells are important mediators of inflammation in a mouse model of T2D. These studies identified an elevation in the Th17 and Th1 subsets with a decrease in the Treg subset, which culminates in inflammation and insulin resistance. Based on these data, we tested the hypothesis that T cells in T2D patients are skewed toward proinflammatory subsets. Our data show that blood from T2D patients has increased circulating Th17 cells and elevated activation of Th17 signature genes. Importantly, T cells required culture with monocytes to maintain Th17 signatures, and fresh ex vivo T cells from T2D patients appeared to be poised for IL-17 production. T cells from T2D patients also have increased production of IFN-γ, but produce healthy levels of IL-4. In contrast, T2D patients had decreased percentages of CD4+ Tregs. These data indicate that T cells in T2D patients are naturally skewed toward proinflammatory subsets that likely promote chronic inflammation in T2D through elevated cytokine production. Potential therapies targeted toward resetting this balance need to be approached with caution due to the reciprocal relationship between Th17 cells and Tregs. Understanding the unique aspects of T2D T cells is essential to predict outcomes of such treatments.
PMCID: PMC3089774  PMID: 21169542
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.  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
17.  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
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.  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
22.  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
23.  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
24.  CD38 Expression in Neutrophils From Patients With Localized Aggressive Periodontitis 
Journal of periodontology  2005;76(11):1960-1965.
Localized aggressive periodontitis (LAgP) is associated with neutrophil dysfunction including decreased chemotaxis and reduced calcium entry. It has been suggested that CD38 is involved in chemotaxis. Little is known, however, about the relationship of CD38 and LAgP patients. In this study, we focused on the level of CD38 expression between LAgP and normal subjects and examined the involvement of CD38 in abnormal neutrophil chemotaxis of LAgP patients.
Neutrophils from 12 normal subjects and 12 LAgP patients were isolated from peripheral venous blood. Membrane associated proteins were extracted from cells with or without N-formylmethionine leucyl-phenylalanine (fMLP) stimulation. CD38 expression was measured using Western blotting. Band density was measured using an imaging densitometer.
There was no statistical difference between normal subjects and LAgP patients in resting CD38 expression (basal level). However, the fMLP-stimulated neutrophils exhibited a significant decrease of CD38 expression in LAgP subjects compared to normal subjects. The decrease of CD38 was positively correlated with the defect in chemotactic migration to fMLP.
These data suggest that the lower expression of CD38 in neutrophils may be related to altered neutrophil function in LAgP.
PMCID: PMC1351027  PMID: 16274316
Calcium; CD38; chemotaxis; neutrophil; periodontitis
25.  Effect of Neutrophil Apoptosis on Monocytic Cytokine Response to Porphyromonas gingivalis Lipopolysaccharide 
Journal of periodontology  2005;76(6):964-971.
Neutrophil apoptosis may play a critical role in the resolution of inflammation by stimulating anti-inflammatory cytokine generation from monocytes. In this study, we investigated the effect of apoptotic neutrophils on interleukin (IL)-10 and IL-1β production from monocytes in response to Porphyromonas gingivalis lipopolysaccharide.
Peripheral blood neutrophils from healthy individuals were isolated by sodium diatrizoate density gradient centrifugation. In order to induce apoptosis, neutrophils were cultured for 24 hours in modified Dulbecco’s medium supplemented with 10% autologous serum. Cell apoptosis was quantified by Annexin V positivity and loss of CD16 expression on the cell surface. Peripheral blood mononuclear cells were isolated from the same subjects; monocytes were purified by magnetic cell sorting and cultured with or without apoptotic or fresh neutrophils. Lipopolysaccharide from Porphyromonas gingivalis was used for cell stimulation. IL-1β and IL-10 levels in supernatants were determined by enzyme-linked immunosorbent assay (ELISA).
IL-10 generation was significantly increased in monocytes cultured with apoptotic neutrophils compared to monocytes alone or cocultured with fresh neutrophils (P <0.05). IL-1β was suppressed both in resting and lipopolysaccharide-stimulated monocytes in the presence of apoptotic neutrophils compared to monocytes alone or monocytes cultured with fresh neutrophils at all time points (P <0.05).
Neutrophil apoptosis provides a signal to monocytes, changing the phenotype of the monocyte resulting in the production of anti-inflammatory cytokines and suppression of proinflammatory cytokines in response to lipopolysaccharide.
PMCID: PMC1224731  PMID: 15948692
Apoptosis; cytokines; interleukin-1; interleukin-10; lipopolysaccharides; monocytes; neutrophils; Porphyromonas gingivalis

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