Shauna Culshaw

Peptidylarginine deiminase from Porphyromonas gingivalis citrullinates human fibrinogen and α-enolase: Implications for autoimmunity in rheumatoid arthritis

OBJECTIVE To investigate protein citrullination by the periodontal pathogen Porphyromonas gingivalis as a potential mechanism for breaking tolerance to citrullinated proteins in rheumatoid arthritis (RA). METHODS The expression of endogenous citrullinated proteins was analyzed by immunoblotting of cell extracts from P gingivalis and 10 other oral bacteria. P gingivalis-knockout strains lacking the bacterial peptidylarginine deiminases (PADs) or gingipains were created to assess the role of these enzymes in citrullination. Citrullination of human fibrinogen and α-enolase by P gingivalis was studied by incubating live wild-type and knockout strains with the proteins and analyzing the products by immunoblotting and mass spectrometry. RESULTS Endogenous protein citrullination was abundant in P gingivalis but lacking in the other oral bacteria. Deletion of the bacterial PAD gene resulted in complete abrogation of protein citrullination. Inactivation of arginine gingipains, but not lysine gingipains, led to decreased citrullination. Incubation of wild-type P gingivalis with fibrinogen or α-enolase caused degradation of the proteins and citrullination of the resulting peptides at carboxy-terminal arginine residues, which were identified by mass spectrometry. CONCLUSION Our findings demonstrate that among the oral bacterial pathogens tested, P gingivalis is unique in its ability to citrullinate proteins. We further show that P gingivalis rapidly generates citrullinated host peptides by proteolytic cleavage at Arg-X peptide bonds by arginine gingipains, followed by citrullination of carboxy-terminal arginines by bacterial PAD. Our results suggest a novel model where P gingivalis-mediated citrullination of bacterial and host proteins provides a molecular mechanism for generating antigens that drive the autoimmune response in RA.

A Role for IL-18 in Neutrophil Activation

IL-18 expression and functional activity has been identified in several autoimmune and infectious diseases. To clarify the potential role of IL-18 during early innate immune responses, we have explored the capacity of IL-18 to activate neutrophils. Human peripheral blood-derived neutrophils constitutively expressed IL-18R (α and β) commensurate with the capacity to rapidly respond to IL-18. IL-18 induced cytokine and chemokine release from neutrophils that was protein synthesis dependent, up-regulated CD11b expression, induced granule release, and enhanced the respiratory burst following exposure to fMLP, but had no effect upon the rate of neutrophil apoptosis. The capacity to release cytokine and chemokine was significantly enhanced in neutrophils derived from rheumatoid arthritis synovial fluid, indicating differential responsiveness to IL-18 dependent upon prior neutrophil activation in vivo. Finally, IL-18 administration promoted neutrophil accumulation in vivo, whereas IL-18 neutralization suppressed the severity of footpad inflammation following carrageenan injection. The latter was accompanied by reduction in tissue myeloperoxidase expression and suppressed local TNF-α production. Together, these data define a novel role for IL-18 in activating neutrophils and thereby promoting early innate immune responses.

A Novel Therapy of Murine Collagen-Induced Arthritis with Soluble T1/ST2

Rheumatoid arthritis is characterized by chronic inflammatory infiltration of the synovium, leading to eventual cartilage and bone destruction. Previously, we have reported that soluble T1/ST2 (sST2), a member of the IL-1R gene family, inhibits LPS-induced macrophage proinflammatory cytokine production. In this study, we report the therapeutic effect of sST2-Fc in the murine model of collagen-induced arthritis. A short term administration of sST2-Fc fusion protein significantly attenuated disease severity compared with controls treated with normal IgG. Histological examination revealed that while control IgG-treated mice developed severe cellular infiltration in the joints, synovial hyperplasia, and joint erosion, this pathology was profoundly reduced in sST2-Fc-treated animals. Treatment of sST2-Fc also down-regulated serum levels of IL-6, IL-12, and TNF-α. Spleen cells from the sST2-Fc-treated mice produced significantly less IFN-γ, TNF-α, IL-6, and IL-12 compared with cells from the control mice when cultured with collagen in vitro. Finally, pretreatment with ST2-Fc markedly inhibited the ability of human monocytic THP1 cells to release TNF-α when cocultured with peripheral blood T cells from rheumatoid patients. Together these results demonstrate that sST2-Fc may provide a novel approach in treating chronic autoimmune conditions by inhibiting the release of proinflammatory cytokines.

Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase: a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis

Background Rheumatoid arthritis (RA) is characterised by autoimmunity to citrullinated proteins, and there is increasing epidemiologic evidence linking Porphyromonas gingivalis to RA. P gingivalis is apparently unique among periodontal pathogens in possessing a citrullinating enzyme, peptidylarginine deiminase (PPAD) with the potential to generate antigens driving the autoimmune response. Objectives To examine the immune response to PPAD in patients with RA, individuals with periodontitis (PD) and controls (without arthritis), confirm PPAD autocitrullination and identify the modified arginine residues. Methods PPAD and an inactivated mutant (C351A) were cloned and expressed and autocitrullination of both examined by immunoblotting and mass spectrometry. ELISAs using PPAD, C351A and another P gingivalis protein arginine gingipain (RgpB) were developed and antibody reactivities examined in patients with RA (n=80), individuals with PD (n=44) and controls (n=82). Results Recombinant PPAD was a potent citrullinating enzyme. Antibodies to PPAD, but not to Rgp, were elevated in the RA sera (median 122 U/ml) compared with controls (median 70 U/ml; p<0.05) and PD (median 60 U/ml; p<0.01). Specificity of the anti-peptidyl citrullinated PPAD response was confirmed by the reaction of RA sera with multiple epitopes tested with synthetic citrullinated peptides spanning the PPAD molecule. The elevated antibody response to PPAD was abolished in RA sera if the C351A mutant was used on ELISA. Conclusions The peptidyl citrulline-specific immune response to PPAD supports the hypothesis that, as a bacterial protein, it might break tolerance in RA, and could be a target for therapy.

Influence of periodontal disease, Porphyromonas gingivalis and cigarette smoking on systemic anti-citrullinated peptide antibody titres.

BACKGROUND Anti-citrullinated protein antibody (ACPA) responses may precede clinical onset of rheumatoid arthritis. Porphyromonas gingivalis peptidylarginine deiminase can citrullinate proteins possibly inducing autoimmunity in susceptible individuals. AIM To determine whether periodontitis, carriage of P. gingivalis, smoking and periodontal therapy influence ACPA titres. METHODS Serum and plaque samples were collected from 39 periodontitis patients before and after non-surgical periodontal treatment, and from 36 healthy subjects. Carriage of P. gingivalis was determined by PCR of plaque DNA. ACPA was determined by anti-cyclic citrullinated peptide (CCP) enzyme-linked immunosorbent assay (ELISA). Anti-P. gingivalis titres were determined by ELISA. RESULTS Untreated periodontitis patients had higher anti-CCP antibody titres than healthy controls [three patients (8%) greater than manufacturer suggested assay diagnostic threshold (5 Assay Units/AU) versus none (0%); mean ± SEM: 1.37 ± 0.23 versus 0.40 ± 0.10 AU, p < 0.0001]. Periodontitis patients who smoked demonstrated lower anti-P. gingivalis (15956 ± 4385 versus 2512 ± 1290 Units/ml, p < 0.05), but similar anti-CCP than non-smoking periodontitis patients (smokers: 1.31 ± 0.35; non-smokers: 1.41 ± 0.32 AU). Healthy smokers demonstrated elevated anti-CCP titres (0.75 ± 0.19 AU), at levels between healthy non-smokers (0.15 ± 0.05 AU) and non-smoker periodontitis patients. Six months after periodontal treatment, there were significant reductions in anti-CCP (non-smokers p < 0.05) and anti-P. gingivalis (all participants p < 0.01). CONCLUSION In subjects with periodontitis, P. gingivalis infection may be responsible for inducing autoimmune responses that characterize rheumatoid arthritis.

Murine neutrophils present Class II restricted antigen

Neutrophils were originally described as short lived, terminally differentiated phagocytes that contribute only to the innate immune response. Recent evidence of neutrophil cytokine production and expression of numerous cell surface proteins has suggested that neutrophils are likely to influence adaptive responses and may satisfy the criteria of antigen presenting cells. Under certain inflammatory conditions human neutrophils express major histocompatibilty complex (MHC) Class II and the costimulatory molecules CD80 and CD86. We have employed a murine T cell hybridoma with a transgenic T cell receptor specific for ovalbumin peptide 323–339 (OVA323–339), and a green fluorescent reporter of T cell receptor ligation, to directly investigate neutrophil-T cell interactions. These cells provide an ideal model system, allowing precise identification of antigen specificity and a clear readout of T cell activation. Additionally, whilst murine neutrophils have previously been shown to stimulate MHC Class I-dependent CD8+ T cell activation, CD4+ T cells stimulation via MHC Class II-expressing neutrophils has not been investigated. We addressed this by isolating murine neutrophils, loading with OVA323–339 and co-culturing with T cells specific for the OVA323–339/MHC Class II complex, and this resulted in T cell activation, as determined by expression of the green-fluorescent protein reporter. Antigen-pulsed neutrophils were also able to prime naïve OVA-specific CD4+ T cells in a contact-dependent manner, as shown by proliferation and cytokine production. Activation of lymphocytes was not due to contaminating macrophages. These studies demonstrate that murine neutrophils present MHC Class II-restricted peptides and induce T cell proliferation, confirming findings in human neutrophils, and demonstrate a novel pro inflammatory effect of murine neutrophils.

Periodontitis, Porphyromonas , and the pathogenesis of rheumatoid arthritis

Epidemiological data indicate a link between rheumatoid arthritis (RA) and periodontal disease (PD). In vitro and in vivo studies have sought to dissect potential mechanisms by which PD may contribute to initiation and progression of RA. However, these are both multifactorial, chronic diseases, and their complex etiologies and pathogenesis themselves remain incompletely understood. Could there really be an etiological link or does this simply represent a statistical coincidence muddied by common risk factors? This review seeks to provide background on these two diseases in the context of recent discoveries suggesting that their pathogenesis may be related. In particular, the process of citrullination, a post-translational protein modification, has been highlighted as a process common to both diseases. The evidence for a relationship between the diseases is explored and its potential mechanisms discussed.

Antifungal, cytotoxic, and immunomodulatory properties of tea tree oil and its derivative components: potential role in management of oral candidosis in cancer patients

Candida albicans forms oral biofilms that cause disease and are difficult to treat with conventional antifungal agents. Tea tree oil (TTO) is a natural compound with reported antimicrobial and immunomodulatory activities. The aims of the study were to evaluate the antifungal efficacy of TTO and key derivatives against C. albicans biofilms, to assess the toxicological effects of TTO on a clinically relevant oral cell line, and to investigate its impact on inflammation. TTO and its derivatives were examined against 100 clinical strains of C. albicans. Planktonic minimum inhibitory concentrations (MICs) were determined using the CLSI M-27A broth microdilution method. Sessile MICs were determined using an XTT reduction assay. Inhibition, time-kill, and mode of action studies were performed. OKF6-TERT2 epithelial cells were used for cytotoxicity and cytokine expression assays. Planktonic C. albicans isolates were susceptible to TTO, terpinen-4-ol (T-4-ol), and α-terpineol, with an MIC50 of 0.5, 0.25, and 0.25%, respectively. These three compounds also displayed potent activity against the 69 biofilm-forming strains, of which T-4-ol and α-terpineol displayed rapid kill kinetics. For all three compounds, 1 × MIC50 effectively inhibited biofilm growth when C. albicans were treated at 0, 1, and 2 h post adhesion. By scanning electron microscopy analysis and PI uptake, TTO and derivative components were shown to be cell membrane active. TTO and T-4-ol were cytotoxic at 1 × MIC50, whereas at 0.5 × MIC50 T-4-ol displayed no significant toxicity. Transcript and protein analysis showed a reduction of IL-8 when treated with TTO and T-4-ol. These data provide further in vitro evidence that TTO and its derivative components, specifically T-4-ol, exhibit strong antimicrobial properties against fungal biofilms. T-4-ol has safety advantages over the complete essential oil and may be suitable for prophylaxis and treatment of established oropharyngeal candidosis. A clinical trial of T-4-ol is worthy of consideration.

Are we any closer to beating the biofilm: novel methods of biofilm control.

Purpose of review A multidisciplinary approach to the treatment and management of biofilms has resulted from the growing appreciation of the role that biofilms play in modern medicine. Conventional antimicrobial agents are generally ineffective against biofilms, and as a result novel laboratory-based and clinical strategies have emerged. The purpose of this review is to analyse the recent literature relating to novel treatment strategies targeting the growing spectrum of clinically relevant biofilms. Recent findings Microscopy and molecular techniques have provided greater insights into identifying the key bacterial and fungal biofilm pathogens. Knowledge of these microorganisms has provided a foundation for the development of specific molecules, often microbial derived, with antimicrobial and/or biofilm disruptive properties, augmenting conventional antibiotics treatments. The validity of some such rationally designed therapeutics has been explored in clinical trials. Summary Biofilms are inherently difficult to treat, and mechanical disruption is the mainstay of clinical management. With scientific progress in molecular microbiology, there is an abundance of newly discovered molecules and pathways, providing novel therapeutic and prophylactic targets.

What can the periodontal community learn from the pathophysiology of rheumatoid arthritis

AIM The aim of this paper is to provide a narrative review of the aetiopathogeneis and treatments of rheumatoid arthritis (RA), focusing on aspects that may share commonality with periodontitis. RESULTS A myriad of cell types, cytokines and pathways have been investigated in both periodontitis and RA. Chronic inflammatory diseases, including RA, psoriatic arthritis, ankylosing spondylitis and periodontitis are likely to share pathogenic mechanisms of inflammation-mediated solid tissue destruction. The aetiopathogenesis of these diseases has been extensively researched over the last several decades and advances in understanding have revolutionized arthritis therapeutics. CONCLUSION The rational, targeted inhibition of mediators in RA has provided clinically useful therapeutics and shed light on mechanisms underpinning disease pathogenesis. RA should be considered a prototypic disease revealing how understanding disease pathogenesis may transform therapeutic options and patient outcomes.