Innate immune-stimulatory activity of Porphyromonas gingivalis fimbriae is eliminated by phase separation using Triton X-114

J Immunol Methods. 2017 Feb:441:31-38. doi: 10.1016/j.jim.2016.11.012. Epub 2016 Nov 30.

Abstract

Fimbriae are virulence factors of Porphyromonas gingivalis (P. gingivalis). In this study, the action of fimbriae on neutrophil respiratory burst and cytokine production by mononuclear cells (MNC) were investigated. Native or denatured form of purified P. gingivalis fimbriae contained endotoxin at an equivalence of 1-3μglipopolysaccharides(LPS)/mg protein. The endotoxin could be reduced to the equivalent of 1ng-LPS/mg protein by phase separation using Triton X-114. Unfractionated fimbriae caused serum-dependent priming of neutrophils for enhanced respiratory burst, but both native and denatured forms of Triton X-114-fractionated fimbriae were not active at 100μg/mL. Unfractionated fimbriae induced serum-dependent production of IL-1β by MNC. Triton X-114-fractionated fimbriae (10μg/mL)-induced production of IL-1β, IL-8 or TNF-α was much lower than that induced by unfractionated fimbriae or 10ng/mL P. gingivalis-LPS preparation. Triton X-114-fractionated fimbriae immobilized on polystyrene tubes induced adhesion-stimulated superoxide release by LPS-primed neutrophils in a β2 integrin-dependent manner. P. gingivalis cells caused priming of neutrophils; however, Toll-like receptor (TLR) 4 antagonists did not affect this response. Thus, P. gingivalis fimbriae were ineffective in inducing innate immune response in leukocytes; however, they induced β2 integrin-mediated response by neutrophils. Immune-stimulatory components of P. gingivalis might be recognized by receptors other than TLR4.

Keywords: Cytokine; Fimbriae; Neutrophil priming; Phase separation; Porphyromonas gingivalis; Triton X-114.

MeSH terms

  • CD18 Antigens / immunology
  • Chemical Fractionation
  • Cytokines / biosynthesis
  • Endotoxins / immunology
  • Fimbriae, Bacterial / chemistry*
  • Fimbriae, Bacterial / immunology*
  • Humans
  • Immunity, Innate*
  • Lipopolysaccharides / immunology
  • Neutrophils / immunology*
  • Octoxynol
  • Polyethylene Glycols
  • Porphyromonas gingivalis / immunology*
  • Porphyromonas gingivalis / pathogenicity
  • Respiratory Burst
  • Signal Transduction
  • Toll-Like Receptor 4 / immunology

Substances

  • CD18 Antigens
  • Cytokines
  • Endotoxins
  • Lipopolysaccharides
  • TLR4 protein, human
  • Toll-Like Receptor 4
  • Polyethylene Glycols
  • Octoxynol
  • Nonidet P-40