Social stress enhances IL-1β and TNF-α production by Porphyromonas gingivalis lipopolysaccharide-stimulated CD11b+ cells
Introduction
The body responds to tissue damage and infection by mounting an inflammatory response that serves to protect the affected tissue from further insult. If unresolved, however, inflammation can have consequences such as causing extensive damage to healthy tissue near the site of the original insult or even in other distal organ systems. For example, a growing body of literature is beginning to validate a link between chronic periodontitis, caused by unresolved inflammation that is initially localized in the gingival epithelium and underlying connective tissue [1], and an increased risk of coronary artery disease where inflammation appears to be the main culprit [2], [3], [4]. Interestingly, these same diseases have been associated with the stress response. Periodontal symptoms can be exacerbated during periods of stress, and stress can increase the risk of developing coronary disease [5]. As a result, a thorough understanding of the many ways through which inflammation can be regulated is imperative for learning how to control and to treat inflammatory diseases.
Porphyromonas gingivalis is an oral bacterial pathogen that is consistently associated with the development of chronic periodontitis. Although P. gingivalis possesses virulence factors that alone can induce tissue damage, such as Arg-X and Lys-X specific extracellular cysteine proteinases [6], the accumulation of inflammatory cytokines is a major contributor to the breakdown of periodontal tissues. The production of inflammatory cytokines is initiated when pattern recognition receptors, such as Toll-like receptors (TLR) 2, 4, and 5 bind to and are activated by pathogen associated molecular patterns (PAMPs) such as lipoproteins, lipopolysaccharide (LPS), or fimbriae [7], [8]. While there is some debate as to whether P. gingivalis derived LPS activates both TLR2 and TLR4, several studies indicate that both receptors can be stimulated by highly purified LPS or lipid A molecules, and by stimulation with the intact bacterium [9], [10], [11]. The cytokines that are produced upon TLR ligation are aimed at enhancing the immune response to ultimately eradicate the pathogen. However, when they are produced in excess, inflammatory cytokines will also facilitate the degradation of host tissue. For example, it is well known that cytokines like IL-1β and TNF-α are key players in tissue destruction and bone resorption during experimental periodontitis in monkeys; blocking these cytokines significantly reduced disease progression [12], [13]. As such, tissue damage during periodontitis is greatest when cytokine levels are highest.
As infection with P. gingivalis progresses in the oral cavity, the inflammatory response can result in ulceration and enhanced vascular permeability at the loci of infection. As a result, the infectious bacteria can enter into the bloodstream to cause a transient bacteremia. Although the bacteria have been found in coronary plaques [14], [15], colonization of systemic organs is not necessary for this chronic oral disease to have systemic effects. Cytokine producing cells, such as CD11b+ macrophages in reticuloendothelial organs (i.e., the spleen, liver, and lungs), are capable of producing high levels of inflammatory cytokines upon encountering P. gingivalis or its lipopolysaccharide (LPS). In fact, patients with periodontal disease often have higher systemic levels of C-reactive protein (CRP), IL-6, IL-1, and TNF-α [16]. And, these inflammatory mediators are known to be involved in the development and progression of many systemic diseases. If the stress response is able to enhance the production of these inflammatory mediators, it could have a significant impact on systemic health.
The field of PsychoNeuroImmunology (PNI) has clearly shown that an individual's emotional state or exposure to psychological stressors can significantly affect the immune response [17]. Most studies have focused on the ability of stressors to suppress the immune response, and many of the mechanisms through which this occurs are already known. In general, suppression of immunity is due to the anti-inflammatory effects of adrenal glucocorticoid (GC) hormones, such as corticosterone in rodents or cortisol in humans [18], [19]. Ligation of GC receptors on mononuclear cells suppresses the expression of cytokines, chemokines, and adhesion molecules in part through a negative regulation of NF-κB activation and function [20], [21]. After exposure to the social stressor SDR, the GC receptor is no longer able to translocate to the nucleus of macrophages [22]. This renders the cells resistant to the suppressive effects of GCs and results in increased cell viability, even when high levels of corticosterone are added to ex vivo cultures [23], [24]. In addition, the production of IL-1α/β, TNF-α, and IL-6 is significantly increased in macrophages from mice exposed to SDR, in comparison to the production by macrophages from non-stressed home cage control mice [25], [26], [27], [28]. It is not known whether this enhanced cytokine production only occurs when the macrophages are stimulated with LPS derived from enteric Gram-negative bacteria, such as E. coli, or whether the enhanced cytokine production will also occur when the cells are stimulated with the LPS from an oral Gram-negative bacterium that has been associated with systemic inflammation (i.e., P. gingivalis). The purpose of this study was to test the hypothesis that exposure to a social stressor would result in increased production of IL-1β and TNF-α by macrophages stimulated with the LPS from P. gingivalis. If true, the findings could help to explain the interrelatedness of stress, inflammation, and oral pathogens.
Section snippets
Mice
Male CD-1 mice (aged 6–8 weeks) were purchased from Charles River Laboratories (Hollister, CA) and allowed to acclimate to the animal facility for at least 1 week prior to experimentation. Mice were kept in an AAALAC approved vivarium with food and water available ad libitum. The lights were maintained on a 12:12 h light:dark schedule with lights on at 0600. All procedures were approved by The Ohio State University's Animal Care and Use Committee.
Social disruption
The social stressor, social disruption (SDR),
Exposure to SDR changed behavioral responses to an open field and to a novel object
In concordance with previous studies, exposure to the social stressor, SDR, significantly affected behavior in the open field test. Mice exposed to SDR spent significantly less time in the center of the open field than did the non-stressed control mice (t(16) = 2.99, p < 0.01; Fig. 1A). This reduced amount of time was not related to a reduction in overall activity, since SDR did not affect overall locomotor activity as assessed by the total distance traveled (t(16) = 0.49, p > 0.05, not significant;
Discussion
Exposure to the social stressor, SDR, led to the development of anxiety-like behavior as evidenced by two widely used measures to assess anxiety in rodents (i.e., the open field test and the novel object/neophobia test) [39], [40]. This is in concurrence with our previous study showing that SDR enhances anxiety like behavior in the open field and light dark preference test, with the current study adding neophobia to the list of SDR-induced anxiety-like behaviors. Several studies have shown that
Acknowledgments
The authors gratefully acknowledge technical assistance from Rebecca Allen and Jeremy Fairborn. This work was funded by Public Health Service grants1R03AI069097 (MB) from the National Institute of Allergy and Infectious Diseases and 5R01MH046801-16 (JS, DP) from the National Institute of Mental Health and an OSU College of Dentistry Seed Grant (MB) and T32DEO14320-6 (JS, DP) from the National Institute of Dental and Craniofacial Research.
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- 1
These authors contributed equally.
- 2
Current affiliation: Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, United States.