cortisol on p.gingivalis

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influence of cortisol on p.gingivalis.

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Exposure of Porphyromonas gingivalis to cortisol increases bacterial growth Aliye Akcal a,*, Olivier Huck b,c, Nurcan Buduneli a, Jean-Luc Davideau b,c, Timur Kose d, Henri Tenenbaum

Exposure of Porphyromonas gingivalis to cortisol increases bacterial growth Aliye Akcal a,*, Olivier Huck b,c, Nurcan Buduneli a, Jean-Luc Davideau b,c, Timur Kose d, Henri Tenenbaum . Archives of oral biology 2014.

ABSTRACTObjective: Psychological stress is considered as a risk factor for periodontal diseases. The stress-related hormone, cortisol is one of the main molecules released during human stress response and is found in plasma and gingival crevicular fluid. This hormone has been suggested to modify composition of subgingival biofilms. The aim of this study was to investigate the effect of exposure to cortisol on Porphyromonas gingivalis (P. gingivalis) growth.

Materials and methods: P. gingivalis ATCC strain 33277 was cultured under strict anaerobic conditions at 37 8C in Brain Heart Infusion medium supplemented with hemin (5 mg ml1) and menadione (1 mg ml1). Bacterial cultures were incubated with or without hydrocortisone (0.0410 mg ml1) at 370C for 12, 24 and 48 h and bacterial growth was evaluated by spectrophotometric method (OD600 nm). Cortisol consumption has been followed by HPLC. Results: Cortisol significantly increased P. gingivalis growth in the first 24 h peaking at 12 h but this increase was not related to the concentration used. During the time period, no consumption of cortisol was observed.

Conclusions: This study provides further support for the idea that stress-induced hormone; cortisol may influence the growth of P. gingivalis. This specific effect may be involved in the relationship between stress and periodontal diseases. abbrevationsHPLC High performance liquid chromatographyGCF Gingival crevicular fluid

contentsIntroductionGeneral reviewReview of literatureAimMaterials and methodsResultsDiscussionConclusionCritical evaluation INTRODUCTION Periodontal diseases are inflammatory diseases of tooth supporting tissues induced by oral bacterial biofilms1. Development of these diseases involves several bacterial species such as Porphyromonas gingivalis (P. gingivalis), Prevotella intermedia, Treponema denticola or Aggregatibacter actinomycetemcomitans within subgingival biofilm as well as host response2 . Several local, systemic and environmental risk factors such as oral hygiene, smoking, diabetes mellitus are known to affect subgingival biofilms composition and increase the severity of periodontal destruction3 Additionally, consequences of stress on lifestyle (less oral hygiene, high-fat diet) are indirect effects that could worsen different types of manifestations such as plaque accumulation and gingival inflammation4.In response to stressful events, several stress markers such as cortisol, catecholamines, chromogranin A, salivary alpha-amylase are released after the stimulation of central and autonomous nervous system5

These stress markers are found in blood, saliva and gingival crevicular fluid (GCF) and may influence the inflammatory processes and development of periodontal diseases6.

Cortisol is one of the most extensively studied stress-related hormone. Cortisol is a glucocorticoid hormone and is secreted by the stimulation of hypothalamus-pituitary- adrenal (HPA) axis in response to psychological stress. Salivary level of cortisol reliably reflects HPA axis activity and is used as a biological marker of stress in human psychological studies7. In a recent clinical study including individuals with salivary cortisol levels varying between 0.006 and 0.015 mg/mL depending on the severity of periodontitis and depression, it was suggested that a high salivary cortisol level may show an increased risk for chronic periodontitis8. Analysis of the effect of catecholamines9 and cortisol10 on the growth of periodontal bacteria has demonstrated the direct role of stress hormones on bacterial growth in subgingival biofilm. Nor-adrenalin reduces growth of P. gingivalis and Aggregatibacter actinomycetemcomitans but increases growth of other species such as Eikenella corrodens9The concept of microbial endocrinology11 was introduced several decades after this recognition and possible correlations between elevated hormone levels and composition of subgingival microbiota .General reviewStress:Stress is defined as a state of physiological or psychological strain caused by adverse stimuli such as physiological, mental, emotional, internal or external that tends to disturb the functioning of an organism and which the organism desires to avoid.

Stress and immune system: Stress can result in the down regulation of the immune system, mediated primary through the hypothalamus- pituitary- adrenal and sympathetic adrenal medullary axis.

The hypothalamic pituitary adrenocortical axis (HPA) and its end product, cortisol is thought to be important mediator of its relationship between stressful life experience and health outcome.

HPA response is a component of the organisms adaptive system for maintaining function under changing environmental circumstances

The function of cortisol is to depress the immune system by diminishing the IgA and IgG secretions

CORTISOL: When activated by stress, hypothalamus releases corticotrophin releasing hormones into the level that commence the hypothalamus to the anterior pituitary. This triggers the anterior pituitary to secrete ACTH hormone in to the blood stream. Once it reaches the adrenal cortex ACTH stimulates the release of glucocorticoid in the form of cortisol. The HPA axis is regulated in a complete negative feedback system with circulatory glucocorticoid inhibitory action .Base line level of cortisol in humans is 10-6g, but in morning hours 20 g.In people who have normal routine of normal sleep and day time activity, cortisol level is 5 g between 10 p.m. to 4 a.m. Cortisol detection kit:CorticountCoat-A-Coat kitCortisol decreases lymphocyte proliferative responses, interleukin-2 (IL-2) production and neutrophil functions.ACTH-induced increase in cortisol caused a leukocytosis, primarily of mature and immature neutrophils and decrease in NK cell activityStress and periodontitis:Stress influences the immune system and along with changes the behaviour . Stress decreases the saliva flow and increases the plaque formation. Studies have proven that the psychosocial factors are predisposing factors for the development of periodontitis.Neglected oral hygiene Cigarette smoking Alcohol consumption Disturbed sleeping pattern decrease in growth hormone- impairs the tissue repair response.

Effect of pro-inflammatory cytokines and glucocorticoids in stressful condition : IL-1 is also known to activate the HPA leads to a feedback loop. Increase level of IL-1, IL6, cortisol and decrease level of salivary IgA found in gingival crevicular fluid.

Alteration in T helper I cell/ T helper II cell ratio: The effect of the T cell: T helper 1 cell: stimulates cellular immunity through production of interferon and IL-2. T helper 2 cell: promote B cell dysfunction and humoral immunity through the release of IL-4, 5, 6, and 10. Marshall et al demonstrated, a T helper 2 cell responses in medical students during stressful working periods.

p.gingivalis and periodontitis

P.Gingivalis is a gram-negative, anaerobic, nonmotile asaccharolytic rods that usually exhibit coccal to short rod morphologies.

P.gingivalis is a member of the much investigated black-pigmented Bacteroides group

Adults having a healthy and minimally diseased periodontium reveal subgingival P. gingivalis

P.Gingivalis has been associated with chronic and aggressive periodontitis. The virulence of P.Gingivalis are LipopolysaccharideProteinases.The primary function of proteases and peptidases secreted by asaccharolytic bacteria such as P. gingivalis is, most likely, to provide nutrients for growth. The best studied are the cysteine proteinases, or the gingipains, with specificities for cleavage after arginine and lysine residues - Kgp, Rgp .Gingipains contribute to the virulence potential of P. gingivalis in a multifactorial way, especially by influencing the binding of the bacterium to host tissues .

Review of literatureSaito et al12 evaluated the effects of noradrenaline on P. gingivalis. P. gingivalis was incubated in the presence of 25 mM, 50 mM, or 100 mM adrenaline or noradrenaline at 37 0C for 12, 24 or 36 h and growth was evaluated by OD660. Auto-inducer-2 (AI-2) was measured by luminescence of Vibrio harveyi BB 170. Expression of P. gingivalis genes was evaluated using a microarray and RT-PCR. Rgp activity of arg-gingipainA and B (Rgp) was measured with a synthetic substrate. Growth of P. gingivalis FDC381 was inhibited by noradrenaline at 24 and 36 h. Growth inhibition by noradrenaline increased dose-dependently. Inhibition of growth partially recovered with addition of propranolol. AI-2 production from P. gingivalis showed a marked decrease with addition of noradrenaline compared with peak production levels in the control group. Expression of rgpB showed a significant increase with addition of noradrenaline, which was partially reduced by addition of propranolol. Results suggest that stressors influence the expression of the virulence factors of P. gingivalis via noradrenaline.Hilgert et al13 evaluated the extent and severity of chronic periodontitis and its association with the levels of salivary cortisol and the scores obtained with a stress questionnaire in a population aged 50 years and over. They studied 235 individuals in a crosssectional study. They answered the Lipps Inventory of Stress Symptoms for Adults, were instructed to collect three saliva samples for cortisol analysis, and were examined for evaluation for periodontitis. Based on logistic regression, cortisol levels were positively associated with the following outcomes: means of clinical attachment level (CAL) > = 4 mm [OR = 5.1, 95%CI (1.2, 20.7)]; 30% of sites with CAL > = 5 mm [OR = 6.9, 95%CI (1.7, 27.1)]; and 26% of sites with probing depth > = 4 mm [OR = 10.7, 95%CI (1.9, 54.1)] after adjustment for confounding variables. The results suggest that cortisol levels were positively associated with the extent and severity of periodontitis. AIMThe aim of the present study was to investigate the effect of exposure to cortisol on P. gingivalis growth over time MATERIALS AND METHODSBacterial strain :The P. gingivalis strain ATCC 33277 was purchased from ATCC and was cultivated under strict anaerobic conditions at 37 oC in Brain Heart infusion supplemented with hemin (5 mg ml1) and menadione (1 mg ml1).Influence of cortisol on bacterial growth :

Prior to the growth test, P. gingivalis culture was evaluated by OD600 nm. First, a pilot study was conducted in order to determine the optimum hydrocortisone concentrations to be used. A total of 15 different hydrocortisone concentrations (0.00520 mg ml1) were tested in this pilot study. Hydrocortisone concentrations that resulted in similar growth rates were omitted and a single concentration that could reflect the group of neighbouring concentrations was chosen, and when a large difference between neighbouring concentration was ob-served, new groups between these ranges were formed in order to detect a clear effect of hydrocortisone.

Based on the findings of this pilot study (data not shown), a total of 8 different hydrocortisone concentrations (0.0410 mg ml1) were decided to be used in the present study. Bacterial culture was incubated with hydrocortisone at different concentrations (0.0410 mg ml1) at 37 0C in anaerobic conditions for 12, 24 and 48 h.

Growth variations were evaluated in comparison with P. gingivalis culture without hydrocortisone (as control). The growth test was performed in triplicate at all time points.

Time-course consumption of cortisol :Time-course consumption of hydrocortisone was analyzed by high-performance liquid chromatography (HPLC). This system consists of a Dionex 3000 with a Macherey Nagel 300-5C18 column (4 mm 250 mm, bead size 5 mm and the pore 100 A) at room temperature. Samples were centrifuged for 5 min at 15,000 g at room temperature and supernatants were diluted at concentration 1/10 in a solution of trifluoroacetic acid (TFA)1 0.1% in water. 500 mL of this dilution was loaded in the column. . The eluents used for the gradient were: A- 0.1% (vol/vol) TFA, 99.9% H2O (vol/vol). B- 70% (vol/vol) acetonitrile, 0.09% (vol/ vol) TFA and 29.91% (vol/vol) H2OThe gradient is reported on each chromatogramFlow rate was set at 0.7 mL/min with a wavelength of 242 nm. The retention time was approximately 37 min for hydrocortisone.

Comparison between peaks corresponding to bacterial medium supplemented with hydrocortisone with or without P. gingivalis was done at each time point (12, 24 and 48 h).

Statistical analysis :A statistical software package was used for the statistical analysis. One-way repeated measures of variance (ANOVA) was used to test the significance of the different cortisol levels between test and control groups. In case of no significance, the time differences were tested for significance using Students t-test. The level of significance was p < 0.05. RESULTS Influence of cortisol on bacterial growth : First, they assessed effect of cortisol on bacterial growth. P. gingivalis cultures were challenged by hydrocortisone at different concentrations. At 12-h, all the hydrocortisone concentrations tested (0.0410 mg ml1) resulted in an increase in the bacterial growth in comparison with the control. This increase was between 24% and 45% depending on the hydrocortisone concentration at this time. Hydrocortisone increased bacterial growth in a dose-independent manner ( p = 0.967

P. gingivalis growth in the presence of different concentrations of hydrocortisone (0.0410 mg mlS1) at 12 , 24 , 48hr Effect of hydrocortisone on P. gingivalis growth decreases with time :The effect of hydrocortisone on P. gingivalis growth was evaluated at different time-point (12 h, 24 h and 48 h). The maximum bacterial growth was detected at 12 h ( p = 0.005 vs 24 h; p < 0.001 vs 48 h) and decreased with time. At 24 h a significant increase was still detectable in comparison with control but at 48 h, no significant effect could be observed

P. gingivalis growth according to different time-points (12, 24 and 48 h). *Significantly higher at 12 h compared with 24 h. #Significantly higher at 12 h compared with 48 h. zSignificantly higher at 24 h compared with 48 h Time-course consumption of cortisol :To assess if P. gingivalis used hydrocortisone as a nutrient during the bacterial growth, we analyzed the hydrocortisone concentration in each sample by HPLC. Comparison between P. gingivalis culture with hydrocortisone and medium with hydrocortisone alone revealed no significant difference in hydrocortisone concentration between the test and control groups at any time-point tested (12, 24 and 48 h)

Hydrocortisone consumption in medium was followed by HPLC. No differences were found between peaks related to hydrocortisone observed at 37 min in the medium with or without P. gingivalis. This observation was done at each time point (12, 24 and 48 h). DISCUSSIONIn the present study, they investigated possible effects of cortisol on the growth of P. gingivalis. The growth rate of P. gingivalis was significantly increased after addition of cortisol in the culture medium at 12 h and 24 h in a dose-independent manner. This finding suggests that stress-induced hormone cortisol may have a specific effect on the growth of P. gingivalis. Despite the increase in growth rate, HPLC experiments demonstrated that total amount of cortisol was stable at all time-points of the study (12, 24 and 48 h). They selected 48 h as the maximum incubation time for P. gingivalis growth in an attempt to exclude possible effects of decrease in available nutrients.Cortisol shows circadian rhythms, and its level is the highest in early morning and lowest at night14 and has already been shown to be positively associated with the severity of periodontal disease, systemic disease, age, gender and life style such as smoking and stress4

Psychological stress has been described as a risk factor for periodontal disease progression but the mechanisms largely remain unknown.

Cortisol levels in saliva were correlated with periodontal destruction particularly in patients with aggressive periodontitis13 In an experimental model, increased cortisol level deteriorated alveolar bone loss induced by oral challenge with P. gingivalis15Catecholamines were tested in an in vitro model to demonstrate potential role of stress-related hormones on growth rate of a large number of oral bacterial species and increased and/or decreased growth responses of different species were described9, 12, 16 .

In the present study, cortisol was found to induce an increase in the growth-rate of P. gingivalis. This finding provides further support for the potential role of psychological stress on microbiological periodontal ecology.

Moreover, it can be speculated that tissue destruction will progress particularly when considering the immune suppression, which can be induced by cortisol17 Interestingly, noradrenalin has been reported to modify virulence of P. gingivalis12. Results of this in vitro study demonstrated an increase in ArgX protease activity of P. gingivalis in the presence of noradrenalin. Another mechanism involving auto-inducer has been demonstrated in Escherichia coli model and catecholamine increased auto-inducer activation and consequently stimulated the growth of wide range of bacterial species of the 43 species biofilm18 Enhanced levels of corticosteroids have been reported in saliva, which provide selective nutrient advantage to certain specific oral microorganisms10Clinical studies suggest that stress-induced hormones in gingival crevicular fluid could provide nutrients that favour the subgingival growth of pathogenic microbiota19.In the present study, cortisol concentration was not modified in the culture medium. They performed HPLC analysis to evaluate whether P. gingivalis growth can be explained by cortisol consumption. However, the present finding of unchanged hydrocortisone consumption does not indicate that cortisol is a direct nutritional source for P. gingivalis .Cortisol may have an indirect effect. It is possible that microorganisms in dental plaque can survive in saliva and can utilize various salivary components as a substrate. Salivary glands are targets for glucocorticoid action and increased levels of stress hormone chromogranin A levels may be influenced by cortisol20. It is likely that the decreased growth with time is due to the decreased concentration of some critical nutrients, or the accumulation of some toxic materials. CONCLUSIONIn conclusion, it may be suggested that stress-induced hormone cortisol may have a specific effect on growth of P. gingivalis. This specific effect may be one of the mechanisms involved in the interaction between stress and periodontal diseases. Critical evaluationThis study was done on in vitro and further studies are needed to prove cortisol facilitate growth of periodontopathogens. bibliography1. Colombo AP, Boches SK, Cotton SL, Goodson JM, Kent R, Haffajee AD, et al. Comparisons of subgingival microbial profiles of refractory periodontitis, severe periodontitis, and periodontal health using the human oral microbe identification microarray. J Periodontol 2009;80: 142132.2. Castillo DM, Sanchez-Beltran MC, Castellanos JE, Sanz I, Mayorga-Fayad I, Sanz M, et al. Detection of specific periodontal microorganisms from bacteraemia samples after periodontal therapy using molecular-based diagnostics. J Clin Periodontol 2011;38:41827. 3. Pihlstrom BL, Michalowicz BS, Johnson NW. Periodontal diseases. Lancet 2005;19:180920. 4. Leresche L, Dworkin SF. The role of stress in inflammatory disease, including periodontal disease: review of concepts and current findings. Periodontol 2000 2002;30:91103 5. Akcali A, Huck O, Tenenbaum H, Davideau JL, Buduneli N. Periodontal diseases and stress: a brief review. J Oral Rehabil 2013;40:608. 6. Pradeep AR, Raj S, Aruna G, Chowdhry S. Gingival crevicular fluid and plasma levels of neuropeptide Substance-P in periodontal health, disease and after nonsurgical therapy. J Periodontal Res 2009;44:2327.

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