Denis F. Kinane

Host‐response: understanding the cellular and molecular mechanisms of host‐microbial interactions – Consensus of the Seventh European Workshop on Periodontology

BACKGROUNDnMajor challenges in periodontology include understanding the pathophysiology, the interplay between various components of the host response, parallels with other diseases and identifying biomarkers of the disease.nnnOBJECTIVESnFour reviews were compiled with the aim of better understanding: (1) the role of polymorphic nuclear leucocytes (PMNs), i.e. neutrophils; (2) the function of cytokine networks in the host response; (3) whether parallels exist with rheumatoid arthritis (RA); and (4) whether useful biomarkers currently exist to help in the management of periodontal disease.nnnMATERIAL AND METHODSnBased on the focused questions, electronic and manual searches were conducted for human, animal and cellular studies on the above topics.nnnRESULTSnPapers fulfilling the inclusion criteria were selected and reviews were written and reviewed and corrected before the academy meeting to produce consensus statements.nnnCONCLUSIONnThe following consensus statements were produced. PMNs are important in the pathophysiology of periodontal disease but there is limited evidence on their much quoted destructive potential. Cytokine networks are enormously complex and we are really at the beginning of understanding their role in the disease process. RA has both similarities and marked differences to periodontal disease although the existing utilization of anti-cytokine therapies and other molecules in its treatment may have importance in periodontal disease therapy. Biomarkers for periodontal disease have yet to be completely defined but the ratio of receptor activator of NF-κB ligand to osteoprotegerin appears to be a biomarker test with utility for detecting bone destruction.

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

BACKGROUNDnAnti-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.nnnAIMnTo determine whether periodontitis, carriage of P. gingivalis, smoking and periodontal therapy influence ACPA titres.nnnMETHODSnSerum 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.nnnRESULTSnUntreated 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).nnnCONCLUSIONnIn subjects with periodontitis, P. gingivalis infection may be responsible for inducing autoimmune responses that characterize rheumatoid arthritis.

Chlorhexidine decreases the risk of ventilator-associated pneumonia in intensive care unit patients: a randomized clinical trial.

BACKGROUND AND OBJECTIVEnThe aim was to evaluate whether oral swabbing with 0.2% chlorhexidine gluconate (CHX) decreases the risk of ventilator-associated pneumonia (VAP) in intensive care unit (ICU) patients.nnnMATERIAL AND METHODSnSixty-one dentate patients scheduled for invasive mechanical ventilation for at least 48 h were included in this randomized, double-blind, controlled study. As these patients were variably incapacitated, oral care was provided by swabbing the oral mucosa four times/d with CHX in the CHX group (29 patients) and with saline in the control group (32 patients). Clinical periodontal measurements were recorded, and lower-respiratory-tract specimens were obtained for microbiological analysis on admission and when VAP was suspected. Pathogens were identified by quantifying colonies using standard culture techniques.nnnRESULTSnVentilator-associated pneumonia developed in 34/61 patients (55.7%) within 6.8 d. The VAP development rate was significantly higher in the control group than in the CHX group (68.8% vs. 41.4%, respectively; p = 0.03) with an odds ratio of 3.12 (95% confidence interval = 1.09-8.91). Acinetobacter baumannii was the most common pathogen (64.7%) of all species identified. There were no significant differences between the two groups in clinical periodontal measurements, VAP development time, pathogens detected or mortality rate.nnnCONCLUSIONnThe finding of the present study, that oral care with CHX swabbing reduces the risk of VAP development in mechanically ventilated patients, strongly supports its use in ICUs and indeed the importance of adequate oral hygiene in preventing medical complications.

Innate Cellular Responses to the Periodontal Biofilm

This chapter addresses the host responses to the microbial biofilm that constitutes the subgingival dental plaque. The host response to infection draws upon the innate, inflammatory and adaptive immune systems, whose role is to provide the appropriate response to the offending microorganisms. In some cases, this will be little or no response when encountering commensals, and in other cases a gradated response depending very much on the hosts own determination of the pathogenic nature of the microbial insult: and herein lies the root of variation in host responses that govern individual susceptibility. In some individuals and with some bacteria this will be an innate-only response, others will need to invoke the inflammatory response, and yet others will require the adaptive immune response - be it cellular, humoral or both - to reduce or remove the challenge from the microbes. Of course these responses would be somewhat easier to predict with a single pathogen challenge, and become infinitely more complex as the biofilm increases in complexity. Oral infections, in particular gingival inflammation, originate from not just one but many microorganisms. This polymicrobial infection may result in chronic inflammation, which may lead to tissue destruction, as evident in chronic periodontitis. Although many organisms are present in the subgingival biofilm, interestingly, the putative pathogens associated with gingivitis and periodontitis may comprise very small fractions of the total biomass. An understanding of the interaction of structural and defensive host cells with the biofilm is pivotal to understanding periodontal disease etiology and to developing tailored therapeutics. Thus, this chapter addresses the main structural cells, i.e. epithelial cells, exposed to the biofilm.

Host response in aggressive periodontitis

It is critical to understand the underlying host responses in aggressive periodontitis to provide a better appreciation of the risk and susceptibility to this disease. Such knowledge may elucidate the etiology and susceptibility to aggressive periodontitis and directly influence treatment decisions and aid diagnosis. This review is timely in that several widely held tenets are now considered unsupportable, namely the concept that Aggregatibacter actinomycetemycomitans is the key pathogen and that chemotactic defects in polymorphonuclear leukocytes are part of the etiopathology. This review also serves to put into context key elements of the host response that may be implicated in the genetic background of aggressive periodontitis. Furthermore, key molecules unique to the host response in aggressive periodontitis may have diagnostic utility and be used in chairside clinical activity tests or as population screening markers. It is becoming increasingly appreciated that the microbial etiology of aggressive periodontitis and the histopathology of this disease are more similar to than different from that of chronic periodontitis. An important therapeutic consideration from the lack of support for A.xa0actinomycetemycomitans as a critical pathogen here is that the widely held belief that tetracycline had a role in aggressive periodontitis therapy is now not supported and that antibiotics such as those used effectively in chronic periodontitis (metronidazole and amoxicillin) are not contraindicated. Furthermore, A.xa0actinomycetemycomitans-related molecules, such as cytolethal distending toxin and leukotoxin, are less likely to have utility as diagnosis agents or as therapeutic targets.

Periodontal therapy in chronic periodontitis lowers gingival crevicular fluid interleukin-1beta and DAS28 in rheumatoid arthritis patients.

To evaluate clinical outcomes and effects of non-surgical periodontal therapy on serum, gingival crevicular fluid (GCF) interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels in chronic periodontitis patients with/without rheumatoid arthritis (RA), fifteen RA patients with chronic periodontitis (RA-P) and 15 systemically healthy non-RA chronic periodontitis patients (H-P) were recruited. Clinical periodontal recordings, GCF, and blood samples were obtained at baseline, 1, 3, and 6xa0months after periodontal treatment. GCF, serum IL-1β, TNF-α levels were analyzed by ELISA. Disease activity score 28 (DAS28) was used to assess RA clinical morbidity. Study groups were compared by Mann–Whitney U test. Wilcoxon test was used to compare the data at baseline, 1, 3, and 6xa0months after periodontal therapy within the same group. DAS28 decreased significantly after periodontal therapy in RA-P group (pxa0<xa00.01). Serum TNF-α concentrations of H-P group were significantly higher than those of RA-P group (pxa0<xa00.01), whereas IL-1β levels were similar. No significant change was observed in serum levels of these cytokines after periodontal therapy. GCF IL-1β amounts decreased significantly in both groups following treatment (pxa0<xa00.01). At 6-months, H-P GCF IL-1β concentrations were significantly lower than baseline. DAS28 and GCF IL-1β correlated with clinical periodontal indices (pxa0<xa00.01). Significant decreases in DAS28 and GCF IL-1β amounts after periodontal treatment suggest that periodontal therapy synergizes with systemic RA therapy to improve RA status.

Porphyromonas gingivalis influences actin degradation within epithelial cells during invasion and apoptosis

Porphyromonas gingivalis, a Gram‐negative oral pathogen, has been shown to induce apoptosis in human gingival epithelial cells, yet the underlining cellular mechanisms controlling this process are poorly understood. We have previously shown that the P.u2003gingivalis proteases arginine and lysine gingipains, are necessary and sufficient to induce host cell apoptosis. In the present study, we demonstrate that ‘P.u2003gingivalis‐induced apoptosis’ is mediated through degradation of actin leading to cytoskeleton collapse. Stimulation of human gingival epithelial cells with P.u2003gingivalis strains 33277 and W50 at moi:100 induced β‐actin cleavage as early as 1u2003h and human serum inhibited this effect. By using gingipain‐deficient mutants of P.u2003gingivalis and purified gingipains, we demonstrate that lysine gingipain is involved in actin hydrolysis in a dose and time‐dependent manner. Use of Jasplakinolide and cytochalasin D revealed that P.u2003gingivalis internalization is necessary for actin cleavage. Further, we also show that lysine gingipain from P.u2003gingivalis can cleave active caspase 3. Taken together, we have identified actin as a substrate for lysine gingipain and demonstrated a novel mechanism involved in microbial host cell invasion and apoptosis.

P. gingivalis modulates keratinocytes through FOXO transcription factors.

P. gingivalis is a prominent periodontal pathogen that has potent effects on host cells. In this study we challenged gingival epithelial cells with P. gingivalis with the aim of assessing how mRNA levels of key target genes were modulated by P. gingivalis via the transcription factors FOXO1 and FOXO3. Primary mono- and multi-layer cultures of gingival epithelial cells were challenged and barrier function was examined by fluorescent dextran and apoptosis was measured by cytoplasmic histone associated DNA. Gene expression levels were measured by real-time PCR with and without FOXO1 and FOXO3 siRNA compared to scrambled siRNA. P. gingivalis induced a loss of barrier function and stimulated gingival epithelial cell apoptosis in multilayer cultures that was in part gingipain dependent. P. gingivalis stimulated an increase in FOXO1 and FOXO3 mRNA, enhanced mRNA levels of genes associated with differentiated keratinocyte function (keratin-1, -10, -14, and involucrin), increased mRNA levels of apoptotic genes (BID and TRADD), reduced mRNA levels of genes that regulate inflammation (TLR-2 and -4) and reduced those associated with barrier function (integrin beta-1, -3 and -6). The ability of P. gingivalis to modulate these genes was predominantly FOXO1 and FOXO3 dependent. The results indicate that P. gingivalis has pronounced effects on gingival keratinocytes and modulates mRNA levels of genes that affect host response, differentiation, apoptosis and barrier function. Moreover, this modulation is dependent upon the transcription factors FOXO1 or FOXO3. In addition, a new function for FOXO1 was identified, that of suppressing TLR-2 and TLR-4 and maintaining integrin beta -1, beta -3 and beta -6 basal mRNA levels in keratinocytes.

Mapping biological to clinical phenotypes during the development (21 days) and resolution (21 days) of experimental gingivitis

AIMnTo characterize and map temporal changes in the biological and clinical phenotype during a 21-day experimental gingivitis study.nnnMATERIALS AND METHODSnExperimental gingivitis was induced over 21 days in healthy human volunteers (n = 56), after which normal brushing was resumed (resolution phase). Gingival and plaque indices were assessed. Gingival crevicular fluid was collected from four paired test and contra-lateral control sites in each volunteer during induction (Days 0, 7, 14 and 21) and resolution (Days 28 and 42) of experimental gingivitis. Fluid volumes were measured and a single analyte was quantified from each site-specific, 30s sample. Data were evaluated by analysis of repeated measurements and paired sample tests.nnnRESULTSnClinical indices and gingival crevicular fluid volumes at test sites increased from Day 0, peaking at Day 21 (test/control differences all p < 0.0001) and decreased back to control levels by Day 28. Levels of four inflammatory markers showed similar patterns, with significant differences between test and control apparent at Day 7 (substance P, cathepsin G, interleukin-1β, elastase: all p < 0.03) and peaking at Day 21 (all p < 0.002). Levels of α-1-antitrypsin showed no pattern.nnnCONCLUSIONSnLevels of substance P, cathepsin G, interleukin-1β and neutrophil elastase act as objective biomarkers of gingival inflammation induction and resolution that typically precede phenotypical changes.