Role of S100A8/A9 for Cytokine Secretion, Revealed in Neutrophils Derived from ER-Hoxb8 Progenitors

in International Journal of Molecular Sciences (2021)

S100A9, a Ca2+-binding protein, is tightly associated to neutrophil pro-inflammatory functions when forming a heterodimer with its S100A8 partner. Upon secretion into the extracellular environment, these ... [more ▼]

S100A9, a Ca2+-binding protein, is tightly associated to neutrophil pro-inflammatory functions when forming a heterodimer with its S100A8 partner. Upon secretion into the extracellular environment, these proteins behave like damage-associated molecular pattern molecules, which actively participate in the amplification of the inflammation process by recruitment and activation of pro-inflammatory cells. Intracellular functions have also been attributed to the S100A8/A9 complex, notably its ability to regulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. However, the complete functional spectrum of S100A8/A9 at the intracellular level is far from being understood. In this context, we here investigated the possibility that the absence of intracellular S100A8/A9 is involved in cytokine secretion. To overcome the difficulty of genetically modifying neutrophils, we used murine neutrophils derived from wild-type and S100A9-/- Hoxb8 immortalized myeloid progenitors. After confirming that differentiated Hoxb8 neutrophil-like cells are a suitable model to study neutrophil functions, our data show that absence of S100A8/A9 led to a dysregulation of cytokine secretion after lipopolysaccharide (LPS) stimulation. Furthermore, we demonstrate that S100A8/A9-induced cytokine secretion was regulated by the nuclear factor kappa B (NF-κB) pathway. These results were confirmed in human differentiated HL-60 cells, in which S100A9 was inhibited by shRNAs. Finally, our results indicate that the degranulation process could be involved in the regulation of cytokine secretion by S100A8/A9. [less ▲]

Calcium signaling and regulation of neutrophil functions: Still a long way to go.

in Journal of leukocyte biology (2019)

Neutrophils are the most abundant leukocytes in blood and disruption in their functions often results in an increased risk of serious infections and inflammatory autoimmune diseases. Following recent ... [more ▼]

Neutrophils are the most abundant leukocytes in blood and disruption in their functions often results in an increased risk of serious infections and inflammatory autoimmune diseases. Following recent discoveries in their influence over disease progression, a resurgence of interest for neutrophil biology has taken place. The multitude of signaling pathways activated by the engagement of numerous types of receptors, with which neutrophils are endowed, reflects the functional complexity of these cells. It is therefore not surprising that there remains a huge lack in the understanding of molecular mechanisms underlining neutrophil functions. Moreover, studies on neutrophils are undoubtedly limited by the difficulty to efficiently edit the cell's genome. Over the past 30 years, compelling evidence has clearly highlighted that Ca(2+) -signaling is governing the key processes associated with neutrophil functions. The confirmation of the role of an elevation of intracellular Ca(2+) concentration has come from studies on NADPH oxidase activation and phagocytosis. In this review, we give an overview and update of our current knowledge on the role of Ca(2+) mobilization in the regulation of pro-inflammatory functions of neutrophils. In particular, we stress the importance of Ca(2+) in the formation of NETs and cytokine secretion in the light of newest findings. This will allow us to embrace how much further we have to go to understand the complex dynamics of Ca(2+) -dependent mechanisms in order to gain more insights into the role of neutrophils in the pathogenesis of inflammatory diseases. The potential for therapeutics to regulate the neutrophil functions, such as Ca(2+) influx inhibitors to prevent autoimmune and chronic inflammatory diseases, has been discussed in the last part of the review. [less ▲]

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

in Frontiers in immunology (2018), 9

S100A8 and S100A9 are members of the S100 family of cytoplasmic EF-hand Ca(2+)-binding proteins and are abundantly expressed in the cytosol of neutrophils. In addition to their intracellular roles, S100A8 ... [more ▼]

S100A8 and S100A9 are members of the S100 family of cytoplasmic EF-hand Ca(2+)-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)-1alpha, IL-1beta, 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. [less ▲]