Helen J. Wright

Hypochlorous acid regulates neutrophil extracellular trap release in humans

Neutrophil extracellular traps (NETs) comprise extracellular chromatin and granule protein complexes that immobilize and kill bacteria. NET release represents a recently discovered, novel anti‐microbial strategy regulated non‐exclusively by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generation of reactive oxygen intermediates (ROIs), particularly hydrogen peroxide. This study aimed to characterize the role of ROIs in the process of NET release and to identify the dominant ROI trigger. We employed various enzymes, inhibitors and ROIs to record their effect fluorometrically on in vitro NET release by human peripheral blood neutrophils. Treatment with exogenous superoxide dismutase (SOD) supported the established link between hydrogen peroxide and NET production. However, treatment with myeloperoxidase inhibitors and direct addition of hypochlorous acid (HOCl; generated in situ from sodium hypochlorite) established that HOCl was a necessary and sufficient ROI for NET release. This was confirmed by the ability of HOCl to stimulate NET release in chronic granulomatous disease (CGD) patient neutrophils which, due to the lack of a functional NADPH oxidase, also lack the capacity for NET release in response to classical stimuli. Moreover, the exogenous addition of taurine, abundantly present within the neutrophil cytosol, abrogated NET production stimulated by phorbol myristate acetate (PMA) and HOCl, providing a novel mode of cytoprotection by taurine against oxidative stress by taurine.

Neutrophil Hyper-responsiveness in Periodontitis

Peripheral neutrophil hyper-responsiveness in chronic periodontitis leads to excessive reactive oxygen species (ROS) production. We aimed to determine whether neutrophil hyper-responsiveness was constitutive or reactive, and to discover the effect of non-surgical therapy. Peripheral blood neutrophils from patients (n = 19), before and 3 months after therapy, and matched control individuals were Fcγ-receptor-stimulated with/without priming with P. gingivalis and F. nucleatum. Total and extracellular ROS were determined by luminol/isoluminol chemiluminescence. The high total ROS generation of patients’ neutrophils compared with that of control individuals (P = 0.016) continued at a reduced level post-therapy (P = 0.059). Reduced activity post-therapy was also seen with priming. Unstimulated total ROS levels did not differ between patients and control individuals before or after therapy. However, the high unstimulated, extracellular ROS production by patients’ neutrophils compared with control individuals (P < 0.05) continued post-therapy and was unaffected by priming. Therapy reduced Fcγ-receptor-stimulated total ROS production, but not unstimulated extracellular radical release, suggesting that constitutive and reactive mechanisms underlie neutrophil hyper-responsiveness.

Differential activation of NF-κB and gene expression in oral epithelial cells by periodontal pathogens

To investigate the molecular effects of the periodontopathogens Fusobacterium nucleatum (FN) and Porphyromonas gingivalis (PG) on the oral epithelium, the H400 oral epithelial cell line was cultured in the presence of non‐viable bacteria. Following confirmation of the presence of transcripts for the bacterial pattern recognition receptors in H400 cells, Toll‐like receptors ‐2, ‐4 and ‐9, and components of the NF‐κB signalling pathway, immunocytochemical analyses were performed showing that NF‐κB was activated within 1 h of exposure to both periodontopathogens. A significantly greater number of NF‐κB nuclear translocations were apparent following H400 cell exposure to FN as compared with PG. Gene expression analyses indicated that transcripts known to be regulated by the NF‐κB pathway, including cytokines/chemokines TNF‐α, IL‐1β, IL‐8, MCP‐1/CCL2 and GM‐CSF, were up‐regulated following 4 and 24 h of exposure to both periodontopathogens. In addition, H400 periodontopathogen exposure resulted in differential regulation of transcripts for several cytokeratin gene family members. Consistent with the immunocytochemical data, microarray results indicated that FN induced a greater number of gene expression changes than PG following 24 h of exposure, 609 and 409 genes, respectively. Ninety‐one genes were commonly differentially expressed by both periodontopathogens and represented biological processes commonly associated with periodontitis. Gene expression analyses by reserve transcriptase‐polymerase chain reaction (RT‐PCR) of molecules identified from the microarray data sets, including Heme oxygenase‐1, lysyl oxidase, SOD2, CCL20 and calprotectin components, confirmed their differential expression profiles induced by the two periodontopathogens. FN and PG have clearly different molecular effects on oral epithelial cells, potentially highlighting the importance of the composition of the plaque biofilm in periodontitis pathogenesis.

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 FcγR-stimulated ROS production was subsequently achieved by priming control neutrophils with IFN-α/-β/-γ, but not LPS, and gene expression analysis indicated that exposure to the type I IFN (in particular IFN-α) better replicated the mRNA profile observed in vivo. Further studies demonstrated that plasma levels of IFN-α were significantly higher in samples from patients relative to unaffected controls. Following successful periodontitis treatment, plasma IFN-α levels, neutrophil ISG expression, and FcγR-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-α may be one determinant of the distinct molecular phenotype and hyperresponsivity exhibited by patients’ peripheral blood neutrophils.

Extracellular deoxyribonuclease production by periodontal bacteria.

BACKGROUND AND OBJECTIVE Whilst certain bacteria have long been known to secrete extracellular deoxyribonuclease (DNase), the purpose in microbial physiology was unclear. Recently, however, this enzyme has been demonstrated to confer enhanced virulence, enabling bacteria to evade the hosts immune defence of extruded DNA/chromatin filaments, termed neutrophil extracellular traps (NETs). As NETs have recently been identified in infected periodontal tissue, the aim of this study was to screen periodontal bacteria for extracellular DNase activity. MATERIAL AND METHODS To determine whether DNase activity was membrane bound or secreted, 34 periodontal bacteria were cultured in broth and on agar plates. Pelleted bacteria and supernatants from broth cultures were analysed for their ability to degrade DNA, with relative activity levels determined using an agarose gel electrophoresis assay. Following culture on DNA-supplemented agar, expression was determined by the presence of a zone of hydrolysis and DNase activity related to colony size. RESULTS Twenty-seven bacteria, including red and orange complex members Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Parvimonas micra, Prevotella intermedia, Streptococcus constellatus, Campylobacter rectus and Prevotella nigrescens, were observed to express extracellular DNase activity. Differences in DNase activity were noted, however, when bacteria were assayed in different culture states. Analysis of the activity of secreted DNase from bacterial broth cultures confirmed their ability to degrade NETs. CONCLUSION The present study demonstrates, for the first time, that DNase activity is a relatively common property of bacteria associated with advanced periodontal disease. Further work is required to determine the importance of this bacterial DNase activity in the pathogenesis of periodontitis.

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.

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.

Fusobacterium nucleatum regulation of neutrophil transcription

BACKGROUND AND OBJECTIVE Abnormal neutrophil responses have been observed in periodontitis patients, including hyper-reactivity in terms of production of reactive oxygen species (ROS) following exposure to the key quorum-sensing plaque bacterium, Fusobacterium nucleatum. This study was designed to characterize the transcriptional response of neutrophils to F. nucleatum. MATERIAL AND METHODS Peripheral blood neutrophils were exposed to F. nucleatum, and gene expression was analysed using high-throughput transcriptomics. RESULTS Microarray technology demonstrated differential expression of 208 genes (163 increased and 43 decreased relative to control genes), which identified regulation of several ontological classes, including signal transduction (13%), transcription regulation (7%) and ROS response (14%). Individual gene expression analysis of selected transcripts, including CSF, CXCL3, FOS, HMOX1, HSP40, SOD2, NFKB2 and GP91, in individual and pooled RNA samples from control and F. nucleatum-exposed neutrophils corroborated microarray data. Analysis of ROS generation, combined with transcript analysis, in response to a panel of proinflammatory stimuli (F. nucleatum, Porphyromonas gingivalis, Escherichia coli lipopolysaccharide and opsonized Staphylococcus aureus) identified significant differences in ROS and transcript regulatory control. Further analyses of neutrophils from periodontitis patients and periodontally healthy control subjects stimulated with F. nucleatum indicated significant differential induction of several ROS response-related transcripts. CONCLUSION These data demonstrate that neutrophils are transcriptionally active in response to the periodontal pathogen F. nucleatum and that these changes in gene expression are likely to affect neutrophil function. The differential response of neutrophils to a range of stimuli combined with data demonstrating differences between patient and control neutrophils indicate the importance of this cell and its interaction with the local tissue environment in the pathogenesis of periodontitis.

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.

Alteration of Neutrophil Reactive Oxygen Species Production by Extracts of Devil’s Claw (Harpagophytum)

Harpagophytum, Devils Claw, is a genus of tuberiferous xerophytic plants native to southern Africa. Some of the taxa are appreciated for their medicinal effects and have been traditionally used to relieve symptoms of inflammation. The objectives of this pilot study were to investigate the antioxidant capacity and the content of total phenols, verbascoside, isoverbascoside, and selected iridoids, as well as to investigate the capacity of various Harpagophytum taxa in suppressing respiratory burst in terms of reactive oxygen species produced by human neutrophils challenged with phorbol myristate acetate (PMA), opsonised Staphylococcus aureus, and Fusobacterium nucleatum. Harpagophytum plants were classified into different taxa according to morphology, and DNA analysis was used to confirm the classification. A putative new variety of H. procumbens showed the highest degree of antioxidative capacity. Using PMA, three Harpagophytum taxa showed anti-inflammatory effects with regard to the PBS control. A putative hybrid between H. procumbens and H. zeyheri in contrast showed proinflammatory effect on the response of neutrophils to F. nucleatum in comparison with treatment with vehicle control. Harpagophytum taxa were biochemically very variable and the response in suppressing respiratory burst differed. Further studies with larger number of subjects are needed to corroborate anti-inflammatory effects of different taxa of Harpagophytum.