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Abstract: TH-PO548

Inorganic Polyphosphate Amplifies the Macrophage-Inflammatory Response Induced by Lipopolysaccharide

Session Information

Category: Bone and Mineral Metabolism

  • 401 Bone and Mineral Metabolism: Basic

Authors

  • Ito, Toru, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
  • Yamamoto, Suguru, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
  • Kaneko, Yoshikatsu, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
  • Goto, Shin, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
  • Gejyo, Fumitake, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
  • Narita, Ichiei, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
Background

Recent studies have revealed that inorganic polyphosphate (polyP), a linear polymer of orthophosphate, is involved in various physiological events like blood clotting, inflammation and energy metabolism. Infection, particularly sepsis, is a major cause of death in patients with chronic kidney disease (CKD) and is associated with high levels of phosphate in the serum. However, the role of phosphate in the induction of an inflammatory response during CKD is largely unknown. In this study, we examined the effect of polyP on lipopolysaccharide (LPS)-induced macrophage proinflammatory signaling in vitro.

Methods

A reaction of THP-1 derived macrophages with LPS (1.0 ng/mL) from Escherichia coli and polyP of various chain lengths (1, 2, 3, 15, 65, 100, 700 mer) and concentrations (1-200 µM, calculated as an orthophosphate monomer) was carried out. The levels of inflammatory cytokines released into the culture medium were measured using ELISA. The expressions of proinflammatory cytokine mRNAs of IL-1β, TNFα and IL-6 and signaling proteins like NF-κβ and MAPK were analyzed by real-time PCR and western blotting, respectively. The effect of polyP on LPS binding to the Toll-like receptor (TLR) 4 on cells and its structure was examined by quartz crystal microbalance (QCM), flow cytometry and fluorescence microscopy visualization, and isothermal titration calorimetry (ITC) and dynamic light scattering spectroscopy (DLS), respectively.

Results

PolyP amplified the inflammatory cytokine production induced by LPS (IL-1β, 50 µM polyP-65: 23.0 ± 2.9 pg/mL; 1 ng/mL LPS: 440.8 ± 45.7 pg/mL; 1 ng/mL LPS + 50 µM polyP-65: 1666.6 ± 162.4 pg/mL, p<0.05) in a dose- and chain length-dependent manner, whereas orthophosphate had no such effect. It also enhanced the expression levels of proinflammatory cytokine mRNAs, LPS-induced macrophage signaling (indicated by an increased expression of NF-κβ, MAPK) and LPS micelle formation. Results of QCM, flow cytometry and fluorescence microscopy showed that polyP bound directly to LPS, enhancing its interaction with TLR4.

Conclusion

This study suggests that polyP may be important in promoting an LPS-induced inflammatory response in macrophages and could be a form of phosphate associated with acute inflammation in patients with CKD.