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 ▲]

miRNAs Regulate Cytokine Secretion Induced by Phosphorylated S100A8/A9 in Neutrophils.

in International journal of molecular sciences (2019), 20(22),

The release of cytokines by neutrophils constitutes an essential process in the development of inflammation by recruiting and activating additional cells. Neutrophils are also able to secrete a complex of ... [more ▼]

The release of cytokines by neutrophils constitutes an essential process in the development of inflammation by recruiting and activating additional cells. Neutrophils are also able to secrete a complex of S100A8 and S100A9 proteins (S100A8/A9), which can amplify the general inflammatory state of the host and is involved in the pathogenesis of several chronic inflammatory diseases, such as rheumatoid arthritis (RA). S100A8/A9 have received renewed attention due to their susceptibility to several function-altering post-translational modifications. In that context, it has been recently demonstrated that only the phosphorylated form of S100A8/A9 (S100A8/A9-P) is able to induce the secretion of several cytokines in neutrophils. Here, we investigate the mechanism by which this post-translational modification of S100A8/A9 can regulate the extracellular activity of the protein complex and its impact on the inflammatory functions of neutrophils. We found that S100A8/A9-P are present in large amounts in the synovial fluids from RA patients, highlighting the importance of this form of S100A8/A9 complex in the inflammation process. Using miRNA-sequencing on S100A8/A9-P-stimulated differentiated HL-60 cells, we identified a dysregulation of miR-146a-5p and miR-155-5p expression through TRL4 signaling pathways. Our data reveal that overexpression of these miRNAs in neutrophil-like cells reduces S100A8/A9-P-mediated secretion of pro-inflammatory cytokines. [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 ▲]

An essential role of syntaxin 3 protein for granule exocytosis and secretion of IL-1α, IL-1β, IL-12b, and CCL4 from differentiated HL-60 cells

in Journal of Leukocyte Biology (2014), 97

Besides their roles in the killing of pathogens, neutrophils have the capacity to package a variety of cytokines into cytoplasmic granules for subsequent release upon inflammatory conditions. Because the ... [more ▼]

Besides their roles in the killing of pathogens, neutrophils have the capacity to package a variety of cytokines into cytoplasmic granules for subsequent release upon inflammatory conditions. Because the rapid secretion of cytokines orchestrates the action of other immune cells at the infection site and thus, can contribute to the development and chronicity of inflammatory diseases, we aimed to determine the intracellular SNARE machinery responsible for the regulation of cytokine secretion and degranulation. From a constructed gene-expression network, we first selected relevant cytokines for functional validation by the CBA approach. We established a cytokine-secretion profile for human neutrophils and dHL-60 cells, underlining their similar ability to secrete a broad variety of cytokines within proinflammatory conditions mimicked by LPS stimulation. Secondly, after screening of SNARE genes by microarray experiments, we selected STX3 for further functional studies. With the use of a siRNA strategy, we show that STX3 is clearly required for the maximal release of IL-1α, IL-1β, IL-12b, and CCL4 without alteration of other cytokine secretion in dHL-60 cells. In addition, we demonstrate that STX3 is involved in MMP-9 exocytosis from gelatinase granules, where STX3 is partly localized. Our results suggest that the secretion of IL-1α, IL-1β, IL-12b, and CCL4 occurs during gelatinase degranulation, a process controlled by STX3. In summary, these findings provide first evidence that STX3 has an essential role in trafficking pathways of cytokines in neutrophil granulocytes. [less ▲]

Role of the Neutrophil NADPH Oxidase and S100A8/A9 in the Pathophysiology of Chronic Inflammation

in Khatami, Mahin (Ed.) Inflammation, Chronic Diseases and Cancer - Cell and Molecular Biology, Immunology and Clinical Bases (2012)

An essential role of STIM1, Orai1, and S100A8-A9 proteins for Ca2+ signaling and FcγR-mediated phagosomal oxidative activity

in Journal of Immunology (2011), 186(4), 2182-2191

Phagocytosis is a process of innate immunity that allows for the enclosure of pathogens within the phagosome and their subsequent destruction through the production of reactive oxygen species (ROS ... [more ▼]

Phagocytosis is a process of innate immunity that allows for the enclosure of pathogens within the phagosome and their subsequent destruction through the production of reactive oxygen species (ROS). Although these processes have been associated with increases of intracellular Ca(2+) concentrations, the mechanisms by which Ca(2+) could regulate the different phases of phagocytosis remain unknown. The aim of this study was to investigate the Ca(2+) signaling pathways involved in the regulation of FcγRs-induced phagocytosis. Our work focuses on IgG-opsonized zymosan internalization and phagosomal ROS production in DMSO-differentiated HL-60 cells and neutrophils. We found that chelation of intracellular Ca(2+) by BAPTA or emptying of the intracellular Ca(2+) store by thapsigargin reduced the efficiency of zymosan internalization. Using an small interfering RNA strategy, our data establish that the observed Ca(2+) release occurs through two isoforms of inositol 1,4,5-triphosphate receptors, ITPR1 and ITPR3. In addition, we provide evidence that phagosomal ROS production is dependent on extracellular Ca(2+) entry. We demonstrate that the observed Ca(2+) influx is supported by ORAI calcium release-activated calcium modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1). This result suggests that extracellular Ca(2+) entry, which is required for ROS production, is mediated by a store-operated Ca(2+) mechanism. Finally, our data identify the complex formed by S100A8 and S100A9 (S100 calcium-binding protein A8 and A9 complex), two Ca(2+)-binding proteins, as the site of interplay between extracellular Ca(2+) entry and intraphagosomal ROS production. Thus, we demonstrate that FcγR-mediated phagocytosis requires intracellular Ca(2+) store depletion for the internalization phase. Then phagosomal ROS production requires extracellular Ca(2+) entry mediated by Orai1/STIM1 and relayed by S100A8-A9 as Ca(2+) sensor. [less ▲]

STIM1 but not STIM2 is an essential regulator of Ca2+ influx-mediated NADPH oxidase activity in neutrophil-like HL-60 cells

in Biochemical Pharmacology (2009), 78(5), 504-513

Extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), is known to be a critical event for NADPH oxidase (NOX2) regulation in neutrophils. While defective NOX2 activity has ... [more ▼]

Extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), is known to be a critical event for NADPH oxidase (NOX2) regulation in neutrophils. While defective NOX2 activity has been linked to various inflammatory diseases, regulatory mechanisms that control Ca2+ influx-induced NOX2 activation are poorly understood in SOCE. The role of STIM1, a Ca2+ sensor that transduces the store depletion signal to the plasma membrane, seems well established and supported by numerous studies in non-phagocytic cells. Here, in neutrophil-like HL-60 cells we used a siRNA approach to delineate the effect of STIM1 knock-down on NOX2 activity regulated by Ca2+ influx. Because the function of the STIM1 homolog, STIM2, is still unclear, we determined the consequence of STIM2 knock-down on Ca2+ and NOX2. STIM1 and STIM2 knock-down was effective and isoform specific when assayed by real-time PCR and Western blotting. Consistent with a unique role of STIM1 in the regulation of SOCE, STIM1, but not STIM2, siRNA significantly decreased Ca2+ influx induced by fMLF or the SERCA pump inhibitor thapsigargin. A redistribution of STIM1, originally localized intracellularly, near the plasma membrane was observed by confocal microscopy upon stimulation by fMLF. Inhibition of STIM1-induced SOCE led to a marked decrease in NOX2 activity while STIM2 siRNA had no effect. Thus, our results provide evidence for a role of STIM1 protein in the control of Ca2+ influx in neutrophils excluding a STIM2 involvement in this process. It also places STIM1 as a key modulator of NOX2 activity with a potential interest for anti-inflammatory pharmacological development. [less ▲]

Regulation of superoxide production in neutrophils: role of calcium influx

in Journal of Leukocyte Biology (2008), 84(5), 1223-1237

Upon stimulation, activation of NADPH oxidase complexes in neutrophils produces a burst of superoxide anions contributing to oxidative stress and the development of inflammatory process. Store-operated ... [more ▼]

Upon stimulation, activation of NADPH oxidase complexes in neutrophils produces a burst of superoxide anions contributing to oxidative stress and the development of inflammatory process. Store-operated calcium entry (SOCE), whereby the depletion of intracellular stores induces extracellular calcium influx, is known to be a crucial element of NADPH oxidase regulation. However, the mechanistic basis mediating SOCE is still only partially understood, as is the signal-coupling pathway leading to modulation of store-operated channels. This review emphasizes the role of calcium influx in the control of the NADPH oxidase and summarizes the current knowledge of pathways mediating this extracellular calcium entry in neutrophils. Such investigations into the cross-talk between NADPH oxidase and calcium might allow the identification of novel pharmacological targets with clinical use, particularly in inflammatory diseases. [less ▲]

Ca2+-dependent regulation of NOX2 activity via MRP proteins in HL-60 granulocytes

in Calcium Binding Proteins (2008), 3(1), 25-30

Recently, two proteins of the S100 protein family, the myeloid-related calcium-binding proteins MRP-8 and MRP-14 have been implicated in the Ca2+-induced activation of the neutrophil NADPH oxidase (NOX2 ... [more ▼]

Recently, two proteins of the S100 protein family, the myeloid-related calcium-binding proteins MRP-8 and MRP-14 have been implicated in the Ca2+-induced activation of the neutrophil NADPH oxidase (NOX2) but the mechanism underlying this process remains unclear. In this study, the role of MRP-8/14 in the Ca2+-dependent regulation of NOX2 activity was characterized in neutrophil-like HL-60 cells using small interfering RNAs (siRNAs) to knock-down endogenous MRP-8 and/or MRP-14 expression. Real-time PCR and Western blot revealed that MRP-8 and MRP-14 expression was 20 times higher in dimethylsulfoxide-differentiated neutrophil-like HL-60 cells compared to quiescent HL-60 cells. Knock-down of MRP-8 and MRP-14 in differentiated HL-60 cells decreased protein levels by 30 and 45% respectively. The impact of the reduced MRP-8/14 protein expression on NOX2 activity was investigated by measuring fMLF-induced H2O2 production. In cells simultaneously transfected with MRP-8 and MRP14 siRNAs, H2O2 production was reduced by 50%, suggesting that both MRP-8 and MRP-14 are required for NOX2 activity; single knock-downs were inefficient. To elucidate the role of Ca2+ in MRP8/14, and consequently in NOX2 activation, siRNA-transfected cells were treated with the Ca2+ ionophore ionomycin prior to stimulation with PMA, a Ca2+-independent protein kinase C activator. PMA-induced H2O2 production was enhanced by ionomycin. This amplification of NOX2 activity was abolished by MRP8/14 knock-down, indicating that both MRP-8 and MRP-14 are necessary to regulate Ca2+-induced NOX2 activation. Taken together, our results suggest that the mechanism of MRPs activation is highly dependent on the increase of intracellular Ca2+ level for a full activation of NOX2. [less ▲]

Interleukin-8 primes oxidative burst in neutrophil-like HL-60 through changes in cytosolic calcium

in Cell Calcium (2005), 37(6), 531-40

In response to a variety of stimuli, neutrophils release large amount of reactive oxygen species (ROS) generated by NADPH oxidase. This process known as the respiratory burst is dependent on cytosolic ... [more ▼]

In response to a variety of stimuli, neutrophils release large amount of reactive oxygen species (ROS) generated by NADPH oxidase. This process known as the respiratory burst is dependent on cytosolic free calcium concentration ([Ca(2+)](i)). Proinflammatory cytokines such as interleukin-8 (IL-8) may modulate ROS generation through a priming phenomenon. The aim of this study was to determine the effect of human IL-8 on ROS production in neutrophil-like dimethylsulfoxide-differentiated HL-60 cells (not equalHL-60 cells) and further to examine the role of Ca(2+) mobilization during the priming. IL-8 at 10 nM induced no ROS production but a [Ca(2+)](i) rise (254 +/- 36 nM). IL-8 induced a strongly enhanced (2 fold) ROS release during stimulation with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF). This potentiation of ROS production is dependent of extracellular Ca(2+) (17.0+/-4.5 arbitrary units (A.U.) in the absence of Ca(2+) versus 56.6 +/- 3.9 A.U. in the presence of 1.25 mM of Ca(2+)). Also, IL-8 enhanced fMLF-stimulated increase in [Ca(2+)](i) (375 +/- 35 versus 245 +/- 21 nM, 0.1 microM of fMLF). IL-8 had no effect on not equalHL-60 cells in response to 1 microM of thapsigargin (472 +/- 66 versus 470 +/- 60 nM). In conclusion, Ca(2+) influx is necessary for a full induction of neutrophil priming by IL-8. [less ▲]