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

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.

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

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.

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

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-1ß, 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.

Regulation of Neutrophil Degranulation and Cytokine Secretion: A Novel Model Approach Based on Linear Fitting.

Neutrophils participate in the maintenance of host integrity by releasing various cytotoxic proteins during degranulation. Due to recent advances, a major role has been attributed to neutrophil-derived cytokine secretion in the initiation, exacerbation, and resolution of inflammatory responses. Because the release of neutrophil-derived products orchestrates the action of other immune cells at the infection site and, thus, can contribute to the development of chronic inflammatory diseases, we aimed to investigate in more detail the spatiotemporal regulation of neutrophil-mediated release mechanisms of proinflammatory mediators. Purified human neutrophils were stimulated for different time points with lipopolysaccharide. Cells and supernatants were analyzed by flow cytometry techniques and used to establish secretion profiles of granules and cytokines. To analyze the link between cytokine release and degranulation time series, we propose an original strategy based on linear fitting, which may be used as a guideline, to (i) define the relationship of granule proteins and cytokines secreted to the inflammatory site and (ii) investigate the spatial regulation of neutrophil cytokine release. The model approach presented here aims to predict the correlation between neutrophil-derived cytokine secretion and degranulation and may easily be extrapolated to investigate the relationship between other types of time series of functional processes.

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

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.

Three weeks of murine hindlimb unloading induces shifts from B to T and from th to tc splenic lymphocytes in absence of stress and differentially reduces cell-specific mitogenic responses.

Extended space missions are known to induce stress and immune dysregulation. Hindlimb unloading is a ground-based model used to reproduce most spaceflight conditions. The aim of this study was to better characterize the consequences of prolonged exposure to hindlimb unloading on murine splenic lymphocyte sub-populations. To ensure that the observed changes were not due to tail restraint but to the antiorthostatic position, three groups of mice were used: control (C), orthostatic restrained (R) and hindlimb unloaded (HU). After 21 days of exposure, no difference in serum corticosterone levels nor in thymus and spleen weights were observed between HU mice and their counterparts, revealing a low state of stress. Interestingly, flow cytometric analyses showed that B cells were drastically reduced in HU mouse spleens by 59% and, while the T cells number did not change, the Th/Tc ratio was decreased. Finally, the use of a fluorescent dye monitoring lymphoproliferation demonstrated that lymphocyte response to mitogen was reduced in Th and Tc populations and to a greater extent in B cells. Thus, we showed for the first time that, even if restraint has its own effects on the animals and their splenic lymphocytes, the prolonged antiorthostatic position leads, despite the absence of stress, to an inversion of the B/T ratio in the spleen. Furthermore, the lymphoproliferative response was impaired with a strong impact on B cells. Altogether, these results suggest that B cells are more affected by hindlimb unloading than T cells which may explain the high susceptibility to pathogens, such as gram-negative bacteria, described in animal models and astronauts.

Modulation of Pleurodeles waltl DNA polymerase mu expression by extreme conditions encountered during spaceflight.

DNA polymerase µ is involved in DNA repair, V(D)J recombination and likely somatic hypermutation of immunoglobulin genes. Our previous studies demonstrated that spaceflight conditions affect immunoglobulin gene expression and somatic hypermutation frequency. Consequently, we questioned whether Polµ expression could also be affected. To address this question, we characterized Polµ of the Iberian ribbed newt Pleurodeles waltl and exposed embryos of that species to spaceflight conditions or to environmental modifications corresponding to those encountered in the International Space Station. We noted a robust expression of Polµ mRNA during early ontogenesis and in the testis, suggesting that Polµ is involved in genomic stability. Full-length Polµ transcripts are 8-9 times more abundant in P. waltl than in humans and mice, thereby providing an explanation for the somatic hypermutation predilection of G and C bases in amphibians. Polµ transcription decreases after 10 days of development in space and radiation seem primarily involved in this down-regulation. However, space radiation, alone or in combination with a perturbation of the circadian rhythm, did not affect Polµ protein levels and did not induce protein oxidation, showing the limited impact of radiation encountered during a 10-day stay in the International Space Station.

Gravity changes during animal development affect IgM heavy-chain transcription and probably lymphopoiesis.

Our previous research demonstrated that spaceflight conditions affect antibody production in response to an antigenic stimulation in adult amphibians. Here, we investigated whether antibody synthesis is affected when animal development occurs onboard a space station. To answer this question, embryos of the Iberian ribbed newt, Pleurodeles waltl, were sent to the International Space Station (ISS) before the initiation of immunoglobulin heavy-chain expression. Thus, antibody synthesis began in space. On landing, we determined the effects of spaceflight on P. waltl development and IgM heavy-chain transcription. Results were compared with those obtained using embryos that developed on Earth. We find that IgM heavy-chain transcription is doubled at landing and that spaceflight does not affect P. waltl development and does not induce inflammation. We also recreated the environmental modifications encountered by the embryos during their development onboard the ISS. This strategy allowed us to demonstrate that gravity change is the factor responsible for antibody heavy-chain transcription modifications that are associated with NF-κB mRNA level variations. Taken together, and given that the larvae were not immunized, these data suggest a modification of lymphopoiesis when gravity changes occur during ontogeny.

Sphingosine kinases regulate NOX2 activity via p38 MAPK-dependent translocation of S100A8/A9.

Neutrophils play a fundamental role in host defense by neutralizing pathogens through the generation of ROS by NOX2. In nonexcitable cells, Ca(2+) influx is essentially mediated via SOCE, a complex mechanism in which depletion of intracellular Ca(2+) stores from the ER results in Ca(2+) entry through Ca(2+) SOCs at the plasma membrane. In this regard, it is well established that extracellular Ca(2+) entry participates to NOX2 activation. S1P, produced by SphKs, has been involved in Ca(2+) homeostasis and thus, could intervene in NOX2 regulation. The aim of this study was to characterize the importance of SphKs in NOX2 activation and the signaling cascade involved in this mechanism. Treatment of neutrophil-like dHL-60 cells by DHS, a SphK inhibitor, and SphK siRNA inhibited fMLF-induced NOX2 activity. Sequential activation of cells by thapsigargin and the phorbol ester PMA revealed that SphK-regulated NOX2 activity relies on intracellular Ca(2+) store depletion. Confocal microscopy and immunoblot analysis showed that stimulation by thapsigargin and PMA mediated S100A8/A9 recruitment to the plasma membrane and p38 MAPK activation. S100A8/A9 translocation decreased when SphK activity was blocked. This result was confirmed in purified human neutrophils, which were physiologically stimulated by fMLF. In addition, p38 MAPK was found to be regulated by SphKs. These results define a pathway leading to NOX2 activation, in which p38 MAPK-mediated S100A8/A9 translocation is regulated by Ca(2+) store depletion-dependent SphK activation.

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

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.

iPLA2, a novel determinant in Ca2+- and phosphorylation-dependent S100A8/A9 regulated NOX2 activity.

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.

Ageing is associated with increased expression but decreased activity of CYP2E1 in male Wistar rats.

The effect of ageing on CYP2E1 activity and its protein and mRNA contents was investigated in both adult (9 months) and senescent (24 months) male Wistar rats. The CYP2E1 activity (as measured by chlorzoxazone hydroxylation) was significantly decreased by 36% in senescent rats as compared to adult rats. However, this decrease of activity was not associated with a loss of protein content because the amount of both CYP2E1 protein and CYP2E1 mRNA did not decrease in senescent rats but rather increased, by 79% and 64% respectively, as compared to adult rats. Lipid peroxidation was increased significantly by 140% with ageing. The decrease in CYP2E1 activity could be explained by post-translational modification of CYP2E1 proteins, due to an increase in oxidative stress in senescent animals, leading to a loss of their functionality. However, no changes in the extent of protein carbonyls were observed in the adult versus senescent rats (16.2 +/- 9.6 vs. 12.7 +/- 7.3 nmol/mg prot) and the major proteasome activity remained unchanged. With regards to the increase of CYP2E1 expression, our results showed that the amount of hepatocyte nuclear factor 1alpha mRNA, a transcription factor that positively regulates CYP2E1, was strongly increased (154%) in senescent rats.