Abstract
Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate units that are linked by phosphoanhydride bonds and is involved in various pathophysiological processes. However, the role of polyP in immune cell dysfunction is not well-understood. In this study, using several biochemical and cell biology approaches, including cytokine assays, immunofluorescence microscopy, receptor-binding assays with quartz crystal microbalance, and dynamic light scanning, we investigated the effect of polyP on in vitro lipopolysaccharide (LPS)-induced macrophage inflammatory response. PolyP up-regulated LPS-induced production of the inflammatory cytokines, such as tumor necrosis factor α, interleukin-1β, and interleukin-6, in macrophages, and the effect was polyP dose- and chain length-dependent. However, orthophosphate did not exhibit this effect. PolyP enhanced the LPS-induced intracellular macrophage inflammatory signals. Affinity analysis revealed that polyP interacts with LPS, inducing formation of small micelles, and the polyP-LPS complex enhanced the binding affinity of LPS to Toll-like receptor 4 (TLR4) on macrophages. These results suggest that inorganic polyP plays a critical role in promoting inflammatory response by enhancing the interaction between LPS and TLR4 in macrophages.
Highlights
Inorganic polyphosphate is a linear polymer of orthophosphate units that are linked by phosphoanhydride bonds and is involved in various pathophysiological processes
Recent studies have revealed that polyP exhibits various physiological effects, such as activating blood procoagulant cascades [4], eliciting pro-inflammatory response in endothelial cells [5, 6], and promoting amyloid fibril formation [7, 8], and it is possible that the reaction of polyP with inflammatory immune cells is associated with the pathology of infection [6, 9, 10]
We report that inorganic polyP amplified LPSinduced macrophage inflammatory response in vitro by enhancing the binding affinity of LPS to Toll-like receptor 4 (TLR4) and promoting LPS micelle formation
Summary
To investigate the effect of polyP on LPS-induced macrophage inflammatory response, THP-1– derived macrophages were reacted with LPS isolated from Escherichia coli with or without polyP-65 (chain length 60 –70-mer). LPS induced the expression of inflammatory cytokine genes TNF␣, IL-1, and IL-6 in macrophages, as reported previously [14]. The addition of polyP-65 markedly enhanced cytokine expression (TNF␣: polyP and LPS (7.16 Ϯ 1.98) versus LPS alone (1.14 Ϯ 0.55); IL-1: polyP and LPS (5.70 Ϯ 1.49) versus LPS alone (1.38 Ϯ 0.22); IL-6: polyP and LPS (16.11 Ϯ 8.63) versus LPS alone (0.92 Ϯ 0.60), p Ͻ 0.05), whereas polyP-65 itself did not induce macrophage inflammatory response in the absence of LPS (Fig. 1, A–C).
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