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 ▲]

Hind limb unloading, a model of spaceflight conditions, leads to decreased B lymphopoiesis similar to aging

in FASEB Journal (2015), 29(2), 455-63

Within the bone marrow, the endosteal niche plays a crucial role in B-cell differentiation. Because spaceflight is associated with osteoporosis, we investigated whether changes in bone microstructure ... [more ▼]

Within the bone marrow, the endosteal niche plays a crucial role in B-cell differentiation. Because spaceflight is associated with osteoporosis, we investigated whether changes in bone microstructure induced by a ground-based model of spaceflight, hind limb unloading (HU), could affect B lymphopoiesis. To this end, we analyzed both bone parameters and the frequency of early hematopoietic precursors and cells of the B lineage after 3, 6, 13, and 21 d of HU. We found that limb disuse leads to a decrease in both bone microstructure and the frequency of B-cell progenitors in the bone marrow. Although multipotent hematopoietic progenitors were not affected by HU, a decrease in B lymphopoiesis was observed as of the common lymphoid progenitor (CLP) stage with a major block at the progenitor B (pro-B) to precursor B (pre-B) cell transition (5- to 10-fold decrease). The modifications in B lymphopoiesis were similar to those observed in aged mice and, as with aging, decreased B-cell generation in HU mice was associated with reduced expression of B-cell transcription factors, early B-cell factor (EBF) and Pax5, and an alteration in STAT5-mediated IL-7 signaling. These findings demonstrate that mechanical unloading of hind limbs results in a decrease in early B-cell differentiation resembling age-related modifications in B lymphopoiesis. [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 ▲]

iPLA 2 , a novel determinant in Ca2+ - and phosphorylation-dependent S100A8/A9 regulated NOX2 activity

in Biochimica et Biophysica Acta-Molecular Cell Research (2010), 1803(7), 840-847

The neutrophil NADPH oxidase (NOX2) is a key enzyme responsible for host defense against invading pathogens, via the production of reactive oxygen species. Dysfunction of NOX2 can contribute to ... [more ▼]

The neutrophil NADPH oxidase (NOX2) is a key enzyme responsible for host defense against invading pathogens, via the production of reactive oxygen species. Dysfunction of NOX2 can contribute to inflammatory processes, which could lead to the development of diseases such as atherosclerosis. In this paper, we characterize a pathway leading to NOX2 activation in which iPLA(2)-regulated p38 MAPK activity is a key regulator of S100A8/A9 translocation via S100A9 phosphorylation. Studies in cell-free or recombinant systems involved two Ca2+-binding proteins of the S100 family, namely S100A8 and S100A9, in NOX2 activation dependent on intracellular Ca2+ concentration ([Ca2+](i)) elevation. Using differentiated HL-60 cells as a model of neutrophils, we provide evidence that [Ca2+](i)-regulated S100A8/A9 translocation is mediated by an increase in [Ca2+](i) through intracellular Ca2+ store depletion. Moreover, we confirm that p38 MAPK induces S100A9 phosphorylation, a mandatory precondition for S100 translocation. Based on a pharmacological approach and an siRNA strategy, we identify iPLA(2) as a new molecular player aiding S100 translocation and NOX2 activity. Inhibition of p38 MAPK activity and S100A9 phosphorylation by bromoenol lactone, a selective inhibitor of iPLA(2), indicated that p38 MAPK-mediated S100A9 phosphorylation is dependent on iPLA(2). In conclusion, we have characterized a pathway leading to NOX2 activation in which iPLA(2)-regulated p38 MAPK activity is a key regulator of S100A8/A9 translocation via S100A9 phosphorylation. [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 ▲]