Characterization, Quantification, and Visualization of Neutrophil Extracellular Traps.

Following the discovery of neutrophil extracellular traps (NETs) in 2004 by Brinkmann and colleagues, there has been extensive research into the role of NETs in a number of inflammatory diseases, including periodontitis. This chapter describes the current methods for the isolation of peripheral blood neutrophils as well as of oral neutrophils for subsequent NET experiments, including approaches to quantify and visualize NET production, the ability of NETs to entrap and kill bacteria, and the removal of NETs by nuclease-containing plasma.

Activation of the neutrophil respiratory burst by plasma from periodontitis patients is mediated by pro-inflammatory cytokines.

AIM: To determine the effect of periodontitis patients' plasma on the neutrophil oxidative burst and the role of albumin, immunoglobulins (Igs) and cytokines. MATERIALS AND METHODS: Plasma was collected from chronic periodontitis patients (n=11) and periodontally healthy controls (n=11) and used with/without depletion of albumin and Ig or antibody neutralization of IL-8, GM-CSF or IFN-α to prime/stimulate peripheral blood neutrophils, isolated from healthy volunteers. The respiratory burst was measured by lucigenin-dependent chemiluminescence. Plasma cytokine levels were determined by ELISA. RESULTS: Plasmas from patients were significantly more effective in both directly stimulating neutrophil superoxide production and priming for subsequent formyl-met-leu-phe (fMLP)-stimulated superoxide production than plasmas from healthy controls (p<0.05). This difference was maintained after depletion of albumin and Ig. Plasma from patients contained higher mean levels of IL-8, GM-CSF and IFN-α. Individual neutralizing antibodies against IL-8, GM-CSF or IFN-α inhibited the direct stimulatory effect of patients' plasma, whereas the ability to prime for fMLP-stimulated superoxide production was only inhibited by neutralization of IFN-α. The stimulating and priming effects of control plasma were unaffected by antibody neutralization. CONCLUSIONS: This study demonstrates that plasma cytokines may have a role in inducing the hyperactive (IL-8, GM-CSF, IFN-α) and hyper-reactive (IFN-α) neutrophil phenotype seen in periodontitis patients.

Effect of nicotine, cotinine and cigarette smoke extract on the neutrophil respiratory burst.

AIMS: To determine the effect of nicotine, cotinine and cigarette smoke extract (CSE) on the neutrophil respiratory burst and their effect on activation of the nuclear factor-κB (NFκB) pathway in oral epithelium. MATERIALS AND METHODS: Neutrophils from periodontally healthy individuals were treated with nicotine, cotinine and CSE before stimulation with Fusobacterium nucleatum, IgG-opsonized Staphylococcus aureus and Escherichia coli lipopolysaccharide. Total and extracellular reactive oxygen species (ROS) generation was determined by luminol/isoluminol chemiluminescence. Activation of NFκB in oral epithelial cells was determined by immunocytochemistry. RESULTS: Smoke extract alone caused increased neutrophil extracellular isoluminol-dependent chemiluminescence, not detectable with luminol. However, pre-treatment with smoke extract reduced both total and extracellular ROS generation in response to all stimuli. Nicotine and cotinine had no effect on the neutrophil respiratory burst. Smoke extract, nicotine and cotinine did not induce oral epithelial cell NFκB activation. CONCLUSIONS: These data demonstrate that smoke extract reduces the ability of neutrophils to generate ROS after stimulation with F. nucleatum and IgG-opsonized S. aureus but, at high concentrations, stimulates extracellular ROS generation. During periodontitis, cigarette smoking may differentially affect neutrophil function, generally preventing elimination of periodontal pathogens but, in heavy smokers, also stimulating ROS release and oxidative stress mediated tissue damage.

Periodontitis associates with a type 1 IFN signature in peripheral blood neutrophils.

Peripheral blood neutrophils from periodontitis patients exhibit a hyperreactive and hyperactive phenotype (collectively termed hyperresponsivity) in terms of production of reactive oxygen species (ROS). The molecular basis for this phenomenon, however, has yet to be determined. Our objectives were to identify genes differentially expressed in hyperresponsive peripheral blood neutrophils from chronic periodontitis patients relative to periodontally healthy controls and use these data to identify potential contributory pathways to the hyperresponsive neutrophil phenotype. Using microarray technology we demonstrated differential expression of 163 genes (149 increased, 14 decreased) representing a range of ontological classes. There was increased expression of a significant number of IFN-stimulated genes (ISG). RT-PCR analysis of ISG transcripts in individual and pooled samples further corroborated these data, and indicated that levels decreased to near those of controls following successful therapy. Significantly enhanced FcgammaR-stimulated ROS production was subsequently achieved by priming control neutrophils with IFN-alpha/-beta/-gamma, but not LPS, and gene expression analysis indicated that exposure to the type I IFN (in particular IFN-alpha) better replicated the mRNA profile observed in vivo. Further studies demonstrated that plasma levels of IFN-alpha were significantly higher in samples from patients relative to unaffected controls. Following successful periodontitis treatment, plasma IFN-alpha levels, neutrophil ISG expression, and FcgammaR-stimulated neutrophil ROS output of patients, all decreased to levels comparable with those of controls. In conclusion, although chronic periodontitis is a complex disease, raised IFN-alpha may be one determinant of the distinct molecular phenotype and hyperresponsivity exhibited by patients' peripheral blood neutrophils.

Neutrophil Extracellular Traps Exert Potential Cytotoxic and Proinflammatory Effects in the Dental Pulp.

INTRODUCTION: Neutrophil extracellular traps (NETs) are an important innate immune mechanism aimed at limiting the dissemination of bacteria within tissues and localizing antibacterial killing mechanisms. There is significant interest in the role of NETs in a range of infectious and inflammatory diseases; however, their role in diseased pulp has yet to be explored. Our aim was to determine their relevance to infected pulp and how their components affect human dental pulp cell (HDPC) responses. METHODS: Diseased pulp tissue was stained for the presence of extracellular DNA and elastase to detect the presence of NETs. Bacteria known to infect pulp were also assayed to determine their ability to stimulate NETs. Coculture studies and NET component challenge were used to determine the effect of extracellular NET release on HDPC viability and inflammatory response. NET-stimulated HDPC secretomes were assessed for their chemotactic activity for lymphocytes and macrophages. RESULTS: Data indicate that NETs are present in infected pulp tissue and whole NETs, and their histone components, particularly H2A, decreased HDPC viability and stimulated chemokine release, resulting in an attraction of lymphocyte populations. CONCLUSIONS: NETs are likely important in pulpal pathogenesis with injurious and chronic inflammatory effects on HDPCs, which may contribute to disease progression. Macrophages are chemoattracted to NET-induced apoptotic HDPCs, facilitating cellular debris removal. NETs and histones may provide novel prognostic markers and/or therapeutic targets for pulpal diseases.

Characterization, Quantification, and Visualization of Neutrophil Extracellular Traps.

Following the discovery of neutrophil extracellular traps (NETs) in 2004 by Brinkmann and colleagues, there has been extensive research into the role of NETs in a number of inflammatory diseases, including periodontitis. This chapter describes the current methods for the isolation of peripheral blood neutrophils for subsequent NET experiments, including approaches to quantify and visualize NET production, the ability of NETs to entrap and kill bacteria, and the removal of NETs by nuclease-containing plasma.

Crevicular fluid levels of TGFbeta1 in drug-induced gingival overgrowth.

OBJECTIVE: To determine if local, gingival crevicular fluid (GCF) levels of TGFbeta1 were altered in drug-induced gingival overgrowth. PATIENTS AND METHODS: GCF samples were collected on Periopaper strips from 45 renal transplant recipients who had been medicated with cyclosporin or cyclosporin in combination with other putative overgrowth-inducing drugs for a minimum of 6 months. Twenty-two subjects had gingival overgrowth while the other 23 patients showed no signs of gingival changes and constituted the medicated control group. Non-medicated controls consisted 20 periodontally healthy individuals who had never taken overgrowth-inducing drugs. GCF levels of TGFbeta1 and alkaline phosphatase, a marker of inflammation, were determined by enhanced chemiluminescence ELISA and enzyme activity assays, respectively. RESULTS: TGFbeta1 levels in GCF from overgrowth and non-overgrowth sites in overgrowth sufferers did not differ. However, there were significant differences in median concentration (P = 0.001) and GCF levels of TGFbeta1 per sample (P = 0.05) between study groups with overgrowth patients having higher amounts per sample and lower concentrations than medicated and healthy controls. Median levels of alkaline phosphatase per GCF sample differed between site (P = 0.01) with higher levels present at overgrowth sites. Despite this, the concentration of enzyme in GCF did not differ between site or patient group. CONCLUSIONS: GCF TGFbeta1 detected in overgrowth patients could reflect a higher level of gingival inflammation because of difficulties in plaque control consequent on the development of overgrowth. However, the higher local levels of total TGFbeta1 in overgrowth patients could indicate that it is a risk factor for developing gingival overgrowth.

Ascorbate and α-tocopherol differentially modulate reactive oxygen species generation by neutrophils in response to FcγR and TLR agonists.

Periodontitis, a ubiquitous chronic inflammatory disease, is associated with reduced antioxidant defences and neutrophil hyperactivity in terms of reactive oxygen species (ROS) generation. Its phenotype is thus characterized by oxidative stress. We have determined the effect of antioxidant micronutrients ascorbate and α-tocopherol on neutrophil ROS generation. Peripheral neutrophils from periodontally-healthy individuals (n = 20) were challenged with phorbol myristate acetate, IgG-opsonised Staphylococcus aureus, Fusobacterium nucleatum or PBS in the presence and absence of micronutrients (50 µM). Total and extracellular ROS were measured by luminol and isoluminol chemiluminescence respectively. Total and extracellular unstimulated, baseline ROS generation was unaffected by α-tocopherol, but inhibited by ascorbate and a combination of both micronutrients. Fcγ-receptor (Fcγ-R)-stimulated total or extracellular ROS generation was not affected by the presence of individual micronutrients. However, the combination significantly reduced extracellular FcγR-stimulated ROS release. Neither micronutrient inhibited TLR-stimulated total ROS, but the combination caused inhibition. Ascorbate and the micronutrient combination, but not α-tocopherol, inhibited extracellular ROS release by TLR-stimulated cells. Such micronutrient effects in vivo could be beneficial in reducing collateral tissue damage in chronic inflammatory diseases, such as periodontitis, while retaining immune-mediated neutrophil function.