Martin R. Ling

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.

Proteomic analysis of a noninvasive human model of acute inflammation and its resolution: the twenty-one day gingivitis model

The 21-day experimental gingivitis model, an established noninvasive model of inflammation in response to increasing bacterial accumulation in humans, is designed to enable the study of both the induction and resolution of inflammation. Here, we have analyzed gingival crevicular fluid, an oral fluid comprising a serum transudate and tissue exudates, by LC−MS/MS using Fourier transform ion cyclotron resonance mass spectrometry and iTRAQ isobaric mass tags, to establish meta-proteomic profiles of inflammation-induced changes in proteins in healthy young volunteers. Across the course of experimentally induced gingivitis, we identified 16 bacterial and 186 human proteins. Although abundances of the bacterial proteins identified did not vary temporally, Fusobacterium outer membrane proteins were detected. Fusobacterium species have previously been associated with periodontal health or disease. The human proteins identified spanned a wide range of compartments (both extracellular and intracellular) and functions, including serum proteins, proteins displaying antibacterial properties, and proteins with functions associated with cellular transcription, DNA binding, the cytoskeleton, cell adhesion, and cilia. PolySNAP3 clustering software was used in a multilayered analytical approach. Clusters of proteins that associated with changes to the clinical parameters included neuronal and synapse associated proteins.

Impaired neutrophil directional chemotactic accuracy in chronic periodontitis patients.

Aim To investigate the chemotactic accuracy of peripheral blood neutrophils from patients with chronic periodontitis compared with matched healthy controls, before and after non-surgical periodontal therapy. Material & Methods Neutrophils were isolated from patients and controls (n = 18) by density centrifugation. Using the Insall chamber and video microscopy, neutrophils were analysed for directional chemotaxis towards N-formyl-methionyl-leucyl-phenylalanine [fMLP (10 nM), or CXCL8 (200 ng/ml)]. Circular statistics were utilized for the analysis of cell movement. Results Prior to treatment, neutrophils from patients with chronic periodontitis had significantly reduced speed, velocity and chemotactic accuracy compared to healthy controls for both chemoattractants. Following periodontal treatment, patient neutrophils continued to display reduced speed in response to both chemoattractants. However, velocity and accuracy were normalized for the weak chemoattractant CXCL8 while they remained significantly reduced for fMLP. Conclusions Chronic periodontitis is associated with reduced neutrophil chemotaxis, and this is only partially restored by successful treatment. Dysfunctional neutrophil chemotaxis may predispose patients with periodontitis to their disease by increasing tissue transit times, thus exacerbating neutrophil-mediated collateral host tissue damage.

Peripheral blood neutrophil cytokine hyper-reactivity in chronic periodontitis:

Pro-inflammatory cytokine release (IL-8, IL-6, TNF-α, IL-1β) by peripheral blood neutrophils, isolated from periodontitis patients (before/after therapy) and matched controls, was determined after 18 h culture in the presence/absence of Escherichia coli LPS, opsonised Staphylococcus aureus, heat-killed Fusobacterium nucleatum and Porphyromonas gingivalis. All cultures demonstrated differences in the amounts of each cytokine detected (P < 0.0001), with a clear release pattern (IL-8 > IL-6 > TNF-α = IL-1β). Median cytokine release from unstimulated patient neutrophils was consistently, but non-significantly, higher than from control cells. Stimulated cytokine release from untreated patient neutrophils was also consistently higher than from control cells. This hyper-reactivity was significant for all tested cytokines when data for all stimuli were combined (P < 0.016). In terms of individual stimuli, significant hyper-reactivity was detected with LPS (IL-8), F. nucleatum (IL-8, TNF-α), opsonised S. aureus (IL-8, TNF-α, IL-1β) and P. gingivalis (IL-8, IL-1β). Cytokine production by patient neutrophils did not reduce following successful non-surgical periodontal therapy and, except for responses to F. nucleatum, the cytokine hyper-reactivity detected pre-therapy was retained. These data demonstrate that chronic periodontitis is characterised by neutrophils that constitutively exhibit cytokine hyper-reactivity, the effects of which could modulate local and systemic inflammatory-immune responses and influence the risk and severity of periodontitis-associated systemic inflammatory diseases.

Neutrophil superoxide production in the presence of cigarette smoke extract, nicotine and cotinine

AIM To determine the effect of cigarette smoke extract, nicotine and cotinine on lucigenin-detectable neutrophil superoxide production. MATERIALS & METHODS Neutrophils from periodontally healthy individuals were treated with aqueous smoke extract, nicotine and cotinine, prior to stimulation or at the same time as stimulation with Fusobacterium nucleatum, IgG-opsonized Staphylococcus aureus and Escherichia coli Lipopolysaccharide (LPS). Superoxide generation was determined by lucigenin chemiluminescence. RESULTS Smoke extract induced superoxide release from neutrophils (p <0.0001) in a dose-dependent manner. By contrast, superoxide generation by neutrophils in response to pathologically relevant stimuli was inhibited by pre-treatment with smoke extract (p <0.01). This inhibition did not require the continued presence of the extract. A similar reduction in stimulated superoxide production by smoke extract was detected when neutrophils were simultaneously exposed to the extract and stimuli. Nicotine and cotinine (0-10 μg/ml) had no effect on superoxide release from unstimulated or stimulated neutrophils. CONCLUSIONS Stable water-soluble components of cigarette smoke directly induce superoxide generation by otherwise unstimulated neutrophils, but reduce superoxide responses of cells to pathologically relevant stimuli. These data suggest potential neutrophil-mediated mechanisms by which smoking may initiate and maintain oxidative stress at periodontally healthy sites and participate in disease progression, by reducing innate immune responses.

Effects of C-reactive protein on the neutrophil respiratory burst in vitro.

This study determined the influence of physiologically relevant concentrations of C-reactive protein (CRP) on reactive oxygen species (ROS) production by neutrophils. Neutrophils from healthy individuals were incubated with soluble pentameric CRP prior to TLR stimulation with Fusobacterium nucleatum, or FcγR stimulation with IgG-opsonised Staphylococcus aureus or heat-aggregated IgG. ROS generation by unstimulated cells and those after stimulation were determined using luminol, isoluminol and lucigenin chemiluminescence, detecting predominantly intracellular hypochlorous acid (HOCl), extracellular hydrogen peroxide (detected as HOCl) and extracellular superoxide respectively. Baseline (unstimulated) neutrophil ROS generation and release was reduced compared with vehicle control by 10 µg/ml CRP. There was no consistent effect of CRP on FcγR-stimulated HOCl production, but the extracellular superoxide response was reduced by 10 µg/ml CRP. By contrast, CRP reduced intracellular (10 µg/ml) and extracellular (3 and 10 µg/ml) HOCl generation, but increased superoxide release (1–10 µg/ml) in response to TLR stimulation. Physiologically relevant concentrations of CRP inhibited baseline ROS generation and reduced FcγR-stimulated extracellular superoxide and TLR-stimulated HOCl release, suggesting that CRP may offer some degree of host protection from neutrophil-associated, low-level oxidative stress. However, CRP enhanced TLR-mediated superoxide release from neutrophils, potentially increasing oxidative stress but aiding host protection from infection.

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.

Oral Epithelial Cell Culture Model for Studying the Pathogenesis of Chronic Inflammatory Disease

The interactions between bacteria, epithelium, and neutrophilic polymorphonuclear leukocytes (neutrophils) are the key to the initiation and progression of many chronic inflammatory-immune diseases. In addition, all can be influenced by external factors, such as micronutrients, thereby providing potentially novel approaches to therapy. This chapter will therefore provide detailed methods for core techniques involved in studying cellular and molecular epithelial responses to a bacterial challenge in relation to chronic inflammatory disease pathogenesis and therapy.

Antioxidant Micronutrients and Oxidative Stress Biomarkers

Chronic inflammatory diseases are the major causes of mortality in humans and recent research has improved our understanding of the major impact of life-style factors upon inflammatory diseases and conditions. One of the most influential of these is nutrition, which may drive both pro-inflammatory as well as anti-inflammatory cascades at molecular and cellular levels. There are a variety of model systems that may be employed to investigate the impact of micronutrients and macronutrients upon inflammatory pathways, many of which operate through oxidative stress, either at the level of controlling the redox state of the cell and downstream redox-regulated gene transcription factors, and other acting as free radical generating or scavenging agents. This chapter focuses upon biological sample preparation prior to assay and details methods for analyzing certain antioxidant micronutrients and biomarkers of oxidative stress.