An in-vitro investigation to determine the neuroinflammatory response of CNS cells to oral bacteria and their virulence factors.
Introduction: \ud Innate immune responses are important for brain tissue injury and repair. This study tested the innate responses of the neuroblastoma (IMR32) and the astrocytoma (SGVp12) cell lines and primary mixed glial CNS cells in vitro to virulence factors from Porphyromonas gingivalis ATCC 33277 and 53978 (W50). \ud \ud Methods:\ud Following treatment of each cell line, the morphological responses were examined using fluorescein-phalloidin labelling and recorded using the confocal microscope. Binding of two specific endotoxins to cells were determined by immunolabelling with anti-P. gingivalis (clones 1B5, and 1A1) antibodies to LPS, and its specific receptor CD14, and gingipains. The Bioplex magnetic bead array analysis from Bio-Rad was performed to measure cytokine release by the human cell lines (IMR32 and SVGp12). In addition a second ELISA assay specifically for TNF- release was also employed with the same samples. \ud Results: All cells abundantly expressed the CD14 receptor on their surface membrane and it responded to the P. gingivalis endotoxins rapidly by forming cell surface membrane “blebs” at multiple sites. Only prolonged incubation with the virulence factors displayed gingipains localised to perinuclear and lysosomal regions of SGVp12 cells and the primary mixed glial CNS cells. The fluorescein-phalloidin indicated considerable cell detachment and alterations in their actin cytoskeletal filaments from both W50 and 33277. IMR32 confirmed the presence of the CD14 receptor by immunoblotting and reflected changes in its protein levels with endotoxins from W50 and 33277. For example, treatment with P. gingivalis 33277 endotoxins, IMR32 cells completely lost their CD14 receptor, but was partially retained after exposure with endotoxins from P. gingivalis W50. Similarly, changes were noted with IL-8 chemokine secretion after SGVp12 cells were exposed to P. gingivalis W50 and not with P. gingivalis 33277. Proinflammatory cytokines IL-6 and TNF- remained at their physiological levels following treatment of SGVp12 cells to P. gingivalis W50 and 33277. Recurrent exposure of SGVp12 cells to endotoxins from P. gingivalis 33277 were tested whereby IL-8 acted as a marker of acute phase inflammation. However, the acute phase chemokine did not significantly change over and above the control samples. \ud \ud Conclusions:\ud All cell types responded to the LPS endotoxin, via the CD14 phagocytic receptor to bind LPS to the surface membrane and gingipains was generally internalised. IMR32 cells completely lost CD14 upon endotoxin treatment possibly cleaved and degraded by gingipains. Only P. gingivalis W50 was able to induce the secretion of IL-8 in SVGp12 cells. Literature suggests IL-8 mediates the recruitment and activation of neutrophils in inflamed tissues. In the same way, IL-8 induction following exposure to P. gingivalis W50 in astrocytes would result in the activation and recruitment of microglia to the site of infection/injury in the brain with the appropriate virulence of the microorganism. The results indicate P. gingivalis W50 which is more pathogenic in comparison to P. gingivalis 33277 use different means of innate immune mechanisms for survival in the host.