Introduction

1. Social Order In Male Mice Social Disruption Stress (SDR) Dominant (alpha) Aggressor + 2 hours For 6 nights Materials and Methods Results Figures Conclusions Submissive Influence of  -adrenergic receptors on P. gingivalis LPS-induced cytokine production Jessica Shepherd1, Rebecca G. Allen3, Megan Miller1, Mark L.Hanke3, Jeffrey D. Galley1, David A. Padgett1,2,3, John F. Sheridan1,2,3, and Binnaz Leblebicioglu4, Michael T. Bailey1,2 1Section of Oral Biology, College of Dentistry 2 Institute for Behavioral Medicine Research, College of Medicine, 3Integrated Biomedical Sciences Graduate Program, College of Medicine 4Section of Periodontology, College of Dentistry, The Ohio State University, Columbus, OH 43210 Introduction • The Social Disruption stressor increased the levels of IL-1, IL-6, and TNF-. • Only the mean level of IL-6 was reduced in supernatants from cells from stressed mice treated with propranolol. • Propranolol administration did not reduce stress-induced increases in IL-1 or TNF-. • Varying doses of NE on LPS-stimulated macrophages had no effect on IL-6 cytokine production. Periodontitis is an inflammatory disease involving tissues which surround and support the teeth. The disease is prevalent among 15-25% of the U.S. population and is one of the major causes of tooth loss in adults. The destruction that occurs during periodontitis is induced in part by an overreaction of immune response to periodontal pathogens. Periodontitis can be initiated by many pathogens, with Porphyromonas gingivalis considered one of the primary etiologic factors. This oral bacterium induces an inflammatory response which includes the production of cytokines IL-1 , IL-6, and TNF- . Excess and prolonged cytokine production can cause damage to healthy tissue leading to the breakdown of gingival connective tissue and bone loss. Emotional or psychosocial stress is defined as a possible risk indicator for periodontal disease, but the mechanisms through which stress affects this disease are unknown. Our previous research has demonstrated exposure to a social stressor called social disruption enhances IL-1 , IL-6, and TNF- cytokine production by splenic macrophages stimulated with lipopolysaccharide (LPS) derived from P. gingivalis. The purpose of this study was to determine whether stress-induced increases in IL-1, IL-6, and TNF- from P. gingivalis LPS-treated splenocytes is mediated by the sympathetic nervous system through -adrenergic receptors. Repeated social defeat enhances P. gingivalis induced IL-6 production • Stress-induced increases inIL-6 levels are influenced by the sympathetic nervous system through -adrenergic receptor signaling. • Additional studies are necessary to determine whether the sympathetic nervous system is involved in psychological stress-periodontal association. • There is no evidence to indicate that adrenergic signaling in macrophages directly affects IL-6 production. Figure 1. Mice were treated with the nonspecific -adrenergic receptor antagonist, propranolol (10 mg/kg subcutaneously), or vehicle 30 minutes prior to exposure to SDR. After 6 pairings of SDR and propranolol, on 6 consecutive days, mice were sacrificed and splenocytes were isolated. Cells were cultured with LPS from P. gingivalis (10µg/ml), and cytokine levels in the culture supernatants were assessed after 18 hrs using ELISA. • Animals: Out bred, male CD-1 mice between the ages of 6 and 8 weeks were purchased from Charles • River Laboratories (Wilmington, MA) and housed 3 per cage for one week prior to experiments. • Mice were kept on a 12:12 hour light:dark schedule with lights on at 6 am with food and water • available ad libitum. The Ohio State University’s Animal Care and Use Committee approved all experimental procedures. • Methods: • Mice were treated with the nonspecific -adrenergic receptor antagonist, propranolol • (10 mg/kg subcutaneously), or vehicle 30 minutes prior to exposure to social disruption (SDR). After 6 • pairings of SDR and propranolol, on 6 consecutive days, mice were sacrificed and splenocytes were • isolated. Cells were cultured with LPS from P. gingivalis (10µg/ml), and cytokine levels in the culture • supernatants were assessed after 18 hrs using enzyme linked immunosorbent assays (ELISA), (BD • Biosciences, San Diego, CA). Analyses of variance were used to determine statistical significance. • Social Disruption:The social disruption (SDR) stressor took place at the beginning of the dark (i.e. • active) cycle (1700 hr), for a 2 hour period over 6 consecutive days. SDR began with the introduction • of an aggressor mouse into the home cage of the resident mice. After each 2 hour SDR session the • aggressor was removed and the mice were left undisturbed until the next day. Following the sixth • day of SDR the repeatedly defeated home cage residents were sacrificed and checked for wounding. • Macrophage cells cultured in vitro (1,000,000 cells/well in a 96 well plate) were treated with doses of Norepinephrine (0M, 1x10-9 M, 1x10-7 M, 1x10-5 M) for 2 hours. The cells were then stimulated with LPS from P. gingivalis for 18 hours. Cytokine levels of IL-6 in the culture supernatants were assessed using ELISA. Repeated social defeat enhances P. gingivalis induced TNF-  production Repeated social defeat enhances P. gingivalis induced IL-1  production Figure 2. Mice were treated with the nonspecific -adrenergic receptor antagonist, propranolol (10 mg/kg subcutaneously), or vehicle 30 minutes prior to exposure to SDR. After 6 pairings of SDR and propranolol, on 6 consecutive days, mice were sacrificed and splenocytes were isolated. Cells were cultured with LPS from P. gingivalis (10µg/ml), and cytokine levels in the culture supernatants were assessed after 18 hrs using ELISA. Figure 3. Mice were treated with the nonspecific -adrenergic receptor antagonist, propranolol (10 mg/kg subcutaneously), or vehicle 30 minutes prior to exposure to SDR. After 6 pairings of SDR and propranolol, on 6 consecutive days, mice were sacrificed and splenocytes were isolated. Cells were cultured with LPS from P. gingivalis (10µg/ml), and cytokine levels in the culture supernatants were assessed after 18 hrs using ELISA. 0 1x10-9 1x10-7 1x10-5 Figure 4. Macrophage cells in culture were treated with doses of Norepinephrine (0M, 1x10-9 M, 1x10-7 M, 1x10-5 M) for 2 hours. The cells were then stimulated with LPS from P. gingivalis for 18 hours. Cytokine levels of IL-6 in the culture supernatants were assessed using ELISA.