The immune system, by and large, plays a primary role to minimize and/or prevent infection, in general, and periodontal disease, in particular. We propose that perturbation of both innate and adaptive immune responses contribute to the pathogenesis of and susceptibility to periodontal disease. The fundamental hypothesis of our studies is that periodontal pathogens produce immunomodulatory proteins that alter either innate or adaptive immunity, thereby enhancing their own virulence and/or that of other opportunistic microorganisms. Our investigations have demonstrated that Aggregatibacter actinomycetemcomitans produces an immunotoxin, the cytolethal distending toxin (Cdt), that impairs human T-cells by inducing cell cycle arrest and apoptosis. In this proposal we focus on a novel, nonlethal, effect of Cdt on two critical cells that comprise the innate immune system. Specifically, we propose the Cdt induces a pro-inflammatory response by virtue of its ability to act as a PIP3 phosphatase, deplete cells of this critical signaling lipid and in turn block PI-3K signaling leading to decreased pAkt to activation of GSK3?. Preliminary studies indicate that treatment of the human macrophage cell line, THP1, and human macrophages with Cdt results in the synthesis and release of pro-inflammatory cytokines (TNF? and IL-1?) as well as enhancement of TLR agonist-induced cytokine release. Cdt also augments de novo synthesis of cytokines (IL-6 and IL-13) following mast cell activation. These effects on both macrophages and mast cells are consistent with PIP3 depletion, blockade of the PI-3K signaling pathway and the role of this pathway as a negative regulator of the pro-inflammatory response. We now plan to extend these preliminary studies to define the extent and mechanism by which Cdt alters the synthesis and release of inflammatory mediators by human macrophages and mast cells. The study is divided into three specific aims: (1) To define the pro-inflammatory effects of Cdt on the innate immune response of macrophages and mast cells;( 2) To determine if the Cdt-mediated pro-inflammatory effects on the innate immune system ais dependent upon PIP3 phosphatase activity and altered PI-3K/PIP3/Akt signaling;and (3) To determine the role of cellugyrin and, possibly GSK3?, in Cdt-mediated altered glucose uptake and utilization and its relationship to the pro-inflammatory response. It is anticipated that these studies will lead to a more detailed understanding of Cdt mode of action and contribute to a greater understanding of the pathogenesis of disease caused by Cdt-producing bacteria. In addition to advancing our understanding of Cdt mode of action, these studies will advance our understanding of the mechanism(s) by which the PI-3K signaling pathway regulates TLR-mediated cytokine production. Thus these studies will identify new targets for future in vivo studies involving pharmacologic and immunologic intervention in disease caused by bacteria that produce Cdt.

Public Health Relevance

The fundamental goal of the proposed study is to determine the potential of abacterial-derivedtoxin, the cytolethal distending toxin (Cdt), to perturb host defenses. Specifically, this study focuses on a novel role for this toxin in inducing and/or augmenting a pro-inflammatory cytokine response in human macrophages and mast cells. We propose that this response contributes to the pathogenesis of inflammatory disorders such as periodontal disease.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Research Project (R01)
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Oral, Dental and Craniofacial Sciences Study Section (ODCS)
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Lunsford, Dwayne
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University of Pennsylvania
Schools of Dentistry/Oral Hygn
United States
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Boesze-Battaglia, Kathleen; Walker, Lisa P; Dhingra, Anuradha et al. (2017) Internalization of the Active Subunit of the Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Is Dependent upon Cellugyrin (Synaptogyrin 2), a Host Cell Non-Neuronal Paralog of the Synaptic Vesicle Protein, Synaptogyrin 1. Front Cell Infect Microbiol 7:469
Scuron, Monika D; Boesze-Battaglia, Kathleen; Dlaki?, Mensur et al. (2016) The Cytolethal Distending Toxin Contributes to Microbial Virulence and Disease Pathogenesis by Acting As a Tri-Perditious Toxin. Front Cell Infect Microbiol 6:168
Shenker, Bruce J; Boesze-Battaglia, Kathleen; Scuron, Monika Damek et al. (2016) The toxicity of the Aggregatibacter actinomycetemcomitans cytolethal distending toxin correlates with its phosphatidylinositol-3,4,5-triphosphate phosphatase activity. Cell Microbiol 18:223-43
Boesze-Battaglia, Kathleen; Walker, Lisa P; Zekavat, Ali et al. (2015) The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Active Subunit CdtB Contains a Cholesterol Recognition Sequence Required for Toxin Binding and Subunit Internalization. Infect Immun 83:4042-55
Shenker, Bruce J; Ojcius, David M; Walker, Lisa P et al. (2015) Aggregatibacter actinomycetemcomitans cytolethal distending toxin activates the NLRP3 inflammasome in human macrophages, leading to the release of proinflammatory cytokines. Infect Immun 83:1487-96
Shenker, Bruce J; Walker, Lisa P; Zekavat, Ali et al. (2014) Blockade of the PI-3K signalling pathway by the Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces macrophages to synthesize and secrete pro-inflammatory cytokines. Cell Microbiol 16:1391-404