Secretion of the Phosphorylated Form of S100A9 from Neutrophils Is Essential for the Proinflammatory Functions of Extracellular S100A8/A9

Abstract

S100A8 and S100A9 are members of the S100 family of cytoplasmic EF-hand Ca2+-binding proteins and are abundantly expressed in the cytosol of neutrophils. In addition to their intracellular roles, S100A8/A9 can be secreted in the extracellular environment and are considered as alarmins able to amplify the inflammatory response. The intracellular activity of S100A8/A9 was shown to be regulated by S100A9 phosphorylation, but the importance of this phosphorylation on the extracellular activity of S100A8/A9 has not yet been extensively studied. Our work focuses on the impact of the phosphorylation state of secreted S100A9 on the proinflammatory function of neutrophils. In a first step, we characterized the secretion of S100A8/A9 in different stimulatory conditions and investigated the phosphorylation state of secreted S100A9. Our results on neutrophil-like differentiated HL-60 (dHL-60) cells and purified human neutrophils showed a time-dependent secretion of S100A8/A9 when induced by phorbol 12-myristoyl 13-acetate and this secreted S100A9 was found in a phosphorylated form. Second, we evaluated the impact of this phosphorylation on proinflammatory cytokine expression and secretion in dHL-60 cells. Time course experiments with purified unphosphorylated or phosphorylated S100A8/A9 were performed and the expression and secretion levels of interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor alpha, CCL2, CCL3, CCL4, and CXCL8 were measured by real-time PCR and cytometry bead array, respectively. Our results demonstrate that only the phosphorylated form of the complex induces proinflammatory cytokine expression and secretion. For the first time, we provide evidence that S100A8/PhosphoS100A9 is inducing cytokine secretion through toll-like receptor 4 signaling.

Loss of the adhesion G-protein coupled receptor ADGRF5 in mice induces airway inflammation and the expression of CCL2 in lung endothelial cells

“…8b). S100A8 and S100A9 have proinflammatory functions, and have been shown to induce CCL2 expression [44–48]. We found that S100a8 and S100a9 mRNA levels were significantly increased in Adgrf5 −/− lungs at 18.5 dpc (1.6- and 1.7-fold, respectively, p < 0.001), as well as thereafter (Fig.…”

Antimicrobial Peptides Derived from the Immune Defense Protein CAP37 Inhibit TLR4 Activation by S100A9

“…The increase of S100A9 protein correlates with the peak of neutrophil infiltration in the stroma that takes place at 12 to 18 hours post-wounding, 8 suggesting that the detected corneal S100A9 could be of neutrophil origin. 28 A careful investigation of the spatiotemporal expression of S100A9 following corneal abrasion will be necessary to estimate the contribution of the epithelial versus neutrophil origins of S100A9 in the cornea. The increase of S100A9 protein in the cornea between 6 and 24 hours post-wounding establishes that this protein can be a target for treatments with CAP37-derived peptides.…”

“…Elevated expression of S100A8 and S100A9 in the cornea has been correlated with inflammation, infection, and neovascularization. 19,28 For example, the expression levels of S100A8 and S100A9 are greatly induced in Gram-positive and Gram-negative induced bacterial keratitis. 34,35 Further, the knockdown of S100A8/S100A9 was found to decrease corneal inflammation and corneal perforation.…”

Memantine Displays Antimicrobial Activity by Enhancing Escherichia coli Pathogen-Induced Formation of Neutrophil Extracellular Traps

“…Further investigation of factors that modulate the cytoplasmic EF-hand Ca 2+ -binding protein S100A9-mediated signaling pathway may reveal more insights into the mechanisms responsible for enhancement of neutrophil antimicrobial activity. S100A9 could be regulated by phosphorylation, but the importance of this phosphorylation on the NET activity of this protein has not yet been extensively studied (Schenten et al, 2018). Along these lines, it is interesting to determine if the enhancement of NET release and bactericidal activity is phosphorylation-dependent.…”