The possible relationship between periodontal disease resulting from the infection of gingival tissue by the Gram-negative bacterium Porphyromonas gingivalis (P. gingivalis) and the development of neuroinflammation remains under investigation. Recently, P. gingivalis lipopolysaccharide (LPS) was reported in the human brain, thus suggesting it might activate brain microglia, a cell type participating in neuroinflammation. We tested the hypothesis of whether in vitro exposure to ultrapure P. gingivalis LPS may result in classical and alternative activation phenotypes of rat microglia, with the concomitant release of cytokines and chemokines, as well as superoxide anion (O2−), thromboxane B2 (TXB2), and matrix metalloprotease-9 (MMP-9). After an 18-h exposure of microglia to P. gingivalis LPS, the concentration-dependent responses were the following: 0.1–100 ng/mL P. gingivalis LPS increased O2− generation, with reduced inflammatory mediator generation; 1000–10,000 ng/mL P. gingivalis LPS generated MMP-9, macrophage inflammatory protein 1α (MIP-1α/CCL3), macrophage inflammatory protein-2 (MIP-2/CXCL2) release and significant O2− generation; 100,000 ng/mL P. gingivalis LPS sustained O2− production, maintained MMP-9, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) release, and triggered elevated levels of MIP-1α/CCL3, MIP-2/CXCL2, and cytokine-induced neutrophil chemoattractant 1 (CINC-1/CXCL-1), with a very low release of lactic dehydrogenase (LDH). Although P. gingivalis LPS was less potent than Escherichia coli (E. coli) LPS in stimulating TXB2, MMP-9, IL-6 and interleukin 10 (IL-10) generation, we observed that it appeared more efficacious in enhancing the release of O2−, TNF-α, MIP-1α/CCL3, MIP-2/CXCL2 and CINC-1/CXCL-1. Our results provide support to our research hypothesis because an 18-h in vitro stimulation with ultrapure P. gingivalis LPS resulted in the classical and alternative activation of rat brain microglia and the concomitant release of cytokines and chemokines.

BackgroundThe putative association between periodontal disease (PD), a chronic inflammatory disease affecting 64% of adults 65 years and older, and neuroinflammation, a condition which involves microglia, a cell of the brain innate immune system, remains relatively unexplored. We recently reported that lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg), a pathogenic bacteria implicated in periodontitis activates both classical (M1‐type) and alternative (M2‐type) activation of neonatal rat microglia (BMG) and concomitant release of superoxide anion, thromboxane B2, cytokines, and chemokines (Toxicological Sciences Supplement, 150 (1), Abstract # 2974, 2017). We hypothesized that classical activated microglia might also release the pro‐inflammatory matrix metalloproteinase‐9 (MMP‐9).MethodsP. gingivalis LPS (Pg LPS) was purchased from InvivoGen (San Diego, CA). E. coli LPS (Ec LPS) 026:B6 (from Difco Lab, Detroit, MI) was used as a positive control. BMG were isolated from neonatal rats, and treated in vitro with either Pg LPS or Ec LPS, in a concentration‐dependent manner, for 18 hours at 35.9°C. BMG were identified by confocal microscopy using the surface marker CD11b/c. Matrix metalloproteinase‐9 was determined with a rat total MMP‐9 DuoSet ELISA kit (R&D Systems).ResultsStatistically significant and concentration‐dependent release of pro‐inflammatory MMP‐9 was observed at > than 1ng/mL Ec LPS and 105 ng/mL Pg LPS.ConclusionsOur results provide support for our working hypothesis, because Pg LPS stimulated BMGs to release pro‐inflammatory MMP‐9 in a time‐ and concentration‐dependent manner. Thus, our current observations extend our studies on the effect of Pg LPS on rat brain microglia activation and contribute to further characterize both periodontitis, and the role of BMG in neuroinflammation and potential neurodegeneration.Support or Funding InformationSupport by College of Dental Medicine, Midwestern University is gratefully acknowledged.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

BackgroundThe putative association between periodontal disease (PD), a chronic inflammatory disease and neuroinflammation, a condition which involves brain microglia, remains under investigation. We have reported that Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS), a bacterium implicated in periodontitis, activates both classical (M1‐type) and alternative (M2‐type) activation of neonatal rat microglia (BMG) with concomitant release of superoxide anion, thromboxane B2, cytokines, and chemokines (Faseb Journal 32.1 Supplement: 702.2, 2018). We hypothesized that matrix metalloproteinase 9 (MMP‐9) release by Pg‐treated microglia might be detectable by both ELISA and visualized by confocal fluorescence imaging.MethodsP. gingivalis LPS (Pg LPS) 105 ng/mL from InvivoGen (San Diego, CA) and Escherichia coli LPS (Ec LPS) 026:B6 1 ng/mL from Difco Lab, Detroit, MI (positive control) were used to treat rat BMG for 18 hours at 35.9°C in vitro. MMP‐9 release was assessed with a rat MMP‐9 ELISA kit (R&D Systems). Confocal fluorescence imaging with a Nikon A1R laser confocal microscope was used to identify BMGs using a primary mouse anti‐rat CD11b/c antibody (Ab) (AbD SeroTec, Raleigh, NC), and secondary donkey anti‐mouse Ab (Thermo Fischer, Waltman, MA). MMP‐9 was visualized with a primary rabbit anti‐rat MMP‐9 polyclonal Ab, and secondary goat anti‐rabbit Ab, both from Abcam, Cambridge, MA.ResultsMMP‐9 presence in tissue culture supernates was detectable by ELISA in both Ec LPS and Pg LPS‐treated BMG. In contrast, MMP‐9 was observed by confocal fluorescence imaging only within Ec LPS‐treated BMG but not in Pg LPS‐treated BMG.ConclusionsBoth ELISA and confocal fluorescence imaging provide additional support for our working hypothesis, because Pg LPS stimulated BMGs to release MMP‐9. Thus, our current observations extend our studies on the effect of Pg LPS on rat brain microglia activation and contribute to further characterize the putative role of Pg LPS in both periodontitis, neuroinflammation and neurodegeneration.Support or Funding InformationSupport by College of Dental Medicine, College of Graduate Studies, and Chicago College of Osteopathic Medicine, Midwestern University are gratefully acknowledged.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background Microglia, the resident macrophages of the central nervous system, play an important role in immune defense by removing damaged neurons and cellular plaques. When activated microglia release pro‐inflammatory mediators (i.e. reactive oxygen intermediates, eicosanoids, cytokines, and metalloproteinases) which may contribute to the pathophysiology of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and HIV encephalopathy. Although rodent microglia have been used for immunological and pharmacological research, extrapolation of observations from rat microglia to human microglia has become challenging. Methods In 2017, a research collaboration was established between Neurosurgery at Cook County Hospital and the Pharmacology Department, College of Graduate Studies, Midwestern University. Several human brain biopsies were cultured in vitro and progressively developed into human brain tissue cultures. One of the human primary cultures (culture # 464) generated from a de‐identified human WHO grade I meningioma contained several cell types as determined by immunohistochemistry and described in 2019 (1). To further characterize microglia in brain culture # 464 three antibodies were used, namely against: A) cell surface integrin CD11b; B) the ionized calcium binding adaptor molecule 1 (Iba1), and C) the transmembrane proteinTMEM119. Results Immuno‐histochemistry observations appear to confirm the presence of microglia in de‐identified human WHO grade I meningioma brain culture # 464. Further studies will be required to confirm our current observations and determine a method to isolate viable microglia from culture # 464. Conclusions Microglia are present in de‐identified human brain culture # 464. Isolation of microglia would enable pharmacological and toxicological research with marine‐derived natural products and toxins in future studies (2). (1) T. Moazezi, et al. Characterization of de‐identified human brain culture by immunofluorescence. Chicago Chapter of the Society for Neuroscience, 4/19/2019, abstract F7, page 69. (2) Mayer, A.M.S., et al. Toxicological Sciences 149(2):484‐95, 2016. PMID: 26609141.