Abstract
Porphyromonas gingivalis, a major opportunistic pathogen in the etiology of chronic periodontitis, successfully survives in human gingival epithelial cells (GECs). P. gingivalis abrogates the effects of a host danger molecule, extracellular ATP (eATP)/P2X7 signaling, such as the generation of reactive oxygen species (ROS) via the mitochondria and NADPH oxidases (NOX) from primary GECs. However, antimicrobial functions of ROS production are thoroughly investigated in myeloid-lineage immune cells and have not been well-understood in epithelial cells. Therefore, this study characterizes antibacterial NOX2 generated ROS and host downstream effects in P. gingivalis infected human primary GECs. We examined the expression of NOX isoforms in the GECs and demonstrate eATP stimulation increased the mRNA expression of NOX2 (p < 0.05). Specific peptide inhibition of NOX2 significantly reduced eATP-mediated ROS as detected by DCFDA probe. The results also showed P. gingivalis infection can temporally modulate NOX2 pathway by reorganizing the localization and activation of cytosolic molecules (p47phox, p67phox, and Rac1) during 24 h of infection. Investigation into downstream biocidal factors of NOX2 revealed an eATP-induced increase in hypochlorous acid (HOCl) in GECs detected by R19-S fluorescent probe, which is significantly reduced by a myeloperoxidase (MPO) inhibitor. MPO activity of the host cells was assayed and found to be positively affected by eATP treatment and/or infection. However, P. gingivalis significantly reduced the MPO product, bactericidal HOCl, in early times of infection upon eATP stimulation. Analysis of the intracellular levels of a major host-antioxidant, glutathione during early infection revealed a substantial decrease (p < 0.05) in reduced glutathione indicative of scavenging of HOCl by P. gingivalis infection and eATP treatment. Examination of the mRNA expression of key enzymes in the glutathione synthesis pathway displayed a marked increase (p < 0.05) in glutamate cysteine ligase (GCL) subunits GCLc and GCLm, glutathione synthetase, and glutathione reductase during the infection. These suggest P. gingivalis modulates the danger signal eATP-induced NOX2 signaling and also induces host glutathione synthesis to likely avoid HOCl mediated clearance. Thus, we characterize for the first time in epithelial cells, an eATP/NOX2-ROS-antibacterial pathway and demonstrate P. gingivalis can circumvent this important antimicrobial defense system potentially for successful persistence in human epithelial tissues.
Highlights
Epithelial cells, as the first line of defense in the oral cavity and other mucosal regions in the body, are continuing to unfold as a critical cell type central to the host immune system for appropriate recognition and responses against invading microbes and pathogens (Sandros et al, 2000; Dale, 2002, 2003; Sugawara et al, 2002; Artis, 2008)
We have shown that P. gingivalis infection and extracellular ATP (eATP) induce reactive oxygen species (ROS) in primary gingival epithelial cells (GECs), largely generated by NADPH oxidases (NOX) inside the cell (Choi et al, 2013)
It has been previously determined that eATP treatment of primary GECs results in sustained ROS levels as early as 30 min after treatment (Choi et al, 2013), we initially examined the mRNA expression of the different NOX isoforms with eATP stimulation to evaluate the NOX isoform important for eATP-induced ROS
Summary
Epithelial cells, as the first line of defense in the oral cavity and other mucosal regions in the body, are continuing to unfold as a critical cell type central to the host immune system for appropriate recognition and responses against invading microbes and pathogens (Sandros et al, 2000; Dale, 2002, 2003; Sugawara et al, 2002; Artis, 2008) Part of this wellorchestrated host innate response to stress or infection stimuli includes the release of small “danger signaling molecules” such as ATP which functions as a sensor against colonizing pathogens through activation of the purinergic receptor P2X7 (Gordon, 1986; Schwiebert and Zsembery, 2003; Trautmann, 2009; Yilmaz et al, 2010; Almeida-da-Silva et al, 2016). The H2O2 is converted to hypochlorous acid (HOCl) by myeloperoxidase enzyme (MPO) to neutralize the phagocytosed bacteria (Hampton et al, 1998; Cross and Segal, 2004; Nauseef, 2007, 2014)
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