Multifaceted role of nitric oxide in an in vitro mouse neuronal injury model: transcriptomic profiling defines the temporal recruitment of death signalling cascades.

Nitric oxide is implicated in the pathogenesis of various neuropathologies characterized by oxidative stress. Although nitric oxide has been reported to be involved in the exacerbation of oxidative stress observed in several neuropathologies, existent data fail to provide a holistic description of how nitrergic pathobiology elicits neuronal injury. Here we provide a comprehensive description of mechanisms contributing to nitric oxide induced neuronal injury by global transcriptomic profiling. Microarray analyses were undertaken on RNA from murine primary cortical neurons treated with the nitric oxide generator DETA-NONOate (NOC-18, 0.5 mM) for 8-24 hrs. Biological pathway analysis focused upon 3672 gene probes which demonstrated at least a ±1.5-fold expression in a minimum of one out of three time-points and passed statistical analysis (one-way anova, P < 0.05). Numerous enriched processes potentially determining nitric oxide mediated neuronal injury were identified from the transcriptomic profile: cell death, developmental growth and survival, cell cycle, calcium ion homeostasis, endoplasmic reticulum stress, oxidative stress, mitochondrial homeostasis, ubiquitin-mediated proteolysis, and GSH and nitric oxide metabolism. Our detailed time-course study of nitric oxide induced neuronal injury allowed us to provide the first time a holistic description of the temporal sequence of cellular events contributing to nitrergic injury. These data form a foundation for the development of screening platforms and define targets for intervention in nitric oxide neuropathologies where nitric oxide mediated injury is causative.

The helminth product ES-62 protects against septic shock via Toll-like receptor 4-dependent autophagosomal degradation of the adaptor MyD88.

Sepsis is one of the most challenging health problems worldwide. Here we found that phagocytes from patients with sepsis had considerable upregulation of Toll-like receptor 4 (TLR4) and TLR2; however, shock-inducing inflammatory responses mediated by these TLRs were inhibited by ES-62, an immunomodulator secreted by the filarial nematode Acanthocheilonema viteae. ES-62 subverted TLR4 signaling to block TLR2- and TLR4-driven inflammatory responses via autophagosome-mediated downregulation of the TLR adaptor-transducer MyD88. In vivo, ES-62 protected mice against endotoxic and polymicrobial septic shock by TLR4-mediated induction of autophagy and was protective even when administered after the induction of sepsis. Given that the treatments for septic shock at present are inadequate, the autophagy-dependent mechanism of action by ES-62 might form the basis for urgently needed therapeutic intervention against this life-threatening condition.

A global transcriptomic view of the multifaceted role of glutathione peroxidase-1 in cerebral ischemic-reperfusion injury.

Transient cerebral ischemia often results in secondary ischemic/reperfusion injury, the pathogenesis of which remains unclear. This study provides a comprehensive, temporal description of the molecular events contributing to neuronal injury after transient cerebral ischemia. Intraluminal middle cerebral artery occlusion (MCAO) was performed to induce a 2-h ischemia with reperfusion. Microarray analysis was then performed on the infarct cortex of wild-type (WT) and glutathione peroxidase-1 (a major antioxidant enzyme) knockout (Gpx1(-/-)) mice at 8 and 24h postreperfusion to identify differential gene expression profile patterns and potential alternative injury cascades in the absence of Gpx1, a crucial antioxidant enzyme, in cerebral ischemia. Genes with at least ±1.5-fold change in expression at either time point were considered significant. Global transcriptomic analyses demonstrated that 70% of the WT-MCAO profile overlapped with that of Gpx1(-/-)-MCAO, and 28% vice versa. Critical analysis of the 1034 gene probes specific to the Gpx1(-/-)-MCAO profile revealed regulation of additional novel pathways, including the p53-mediated proapoptotic pathway and Fas ligand (CD95/Apo1)-mediated pathways; downplay of the Nrf2 antioxidative cascade; and ubiquitin-proteasome system dysfunction. Therefore, this comparative study forms the foundation for the establishment of screening platforms for target definition in acute cerebral ischemia intervention.

Gene profiling reveals hydrogen sulphide recruits death signaling via the N-methyl-D-aspartate receptor identifying commonalities with excitotoxicity.

Recently the role of hydrogen sulphide (H(2) S) as a gasotransmitter stimulated wide interest owing to its involvement in Alzheimer's disease and ischemic stroke. Previously we demonstrated the importance of functional ionotropic glutamate receptors (GluRs) by neurons is critical for H(2) S-mediated dose- and time-dependent injury. Moreover N-methyl-D-aspartate receptor (NMDAR) antagonists abolished the consequences of H(2) S-induced neuronal death. This study focuses on deciphering the downstream effects activation of NMDAR on H(2) S-mediated neuronal injury by analyzing the time-course of global gene profiling (5, 15, and 24 h) to provide a comprehensive description of the recruitment of NMDAR-mediated signaling. Microarray analyses were performed on RNA from cultured mouse primary cortical neurons treated with 200 µM sodium hydrosulphide (NaHS) or NMDA over a time-course of 5-24 h. Data were validated via real-time PCR, western blotting, and global proteomic analysis. A substantial overlap of 1649 genes, accounting for over 80% of NMDA global gene profile present in that of H(2) S and over 50% vice versa, was observed. Within these commonly occurring genes, the percentage of transcriptional consistency at each time-point ranged from 81 to 97%. Gene families involved included those related to cell death, endoplasmic reticulum stress, calcium homeostasis, cell cycle, heat shock proteins, and chaperones. Examination of genes exclusive to H(2) S-mediated injury (43%) revealed extensive dysfunction of the ubiquitin-proteasome system. These data form a foundation for the development of screening platforms and define targets for intervention in H(2) S neuropathologies where NMDAR-activated signaling cascades played a substantial role.

Up-regulation of endoplasmic reticulum stress-related genes during the early phase of treatment of cultured cortical neurons by the proteasomal inhibitor lactacystin.

Inhibition of proteasome degradation pathway has been implicated in neuronal cell death leading to neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. We and others demonstrated that treatment of cortical neurons with the proteasomal inhibitor lactacystin leads to apoptosis. We discovered by microarray analysis that lactacystin treatment modulates the expression of both potentially neuroprotective as well as pro-apoptotic genes in neurons. However, the significance of the genes which upon transcriptional modulation contributed to proteasomal inhibition-induced apoptosis, remained unidentified. By employing microarray analysis to decipher the time-dependent changes in transcription of these genes in cultured cortical neurons, we discovered different groups of genes were transcriptionally regulated in the early and late phase of lactacystin-induced cell death. In the early phase, several neuroprotective genes such as those encoding the proteasome subunits and ubiquitin-associated enzymes, as well as the heat-shock proteins (HSP) were up-regulated. However, the pro-apoptotic endoplasmic reticulum (ER) stress-associated genes were also up-regulated at the early phase of lactacystin-induced neuronal cell death. In the late phase, genes encoding antioxidants and calcium-binding proteins were up-regulated while those associated with cholesterol biosynthesis were down-regulated. The data suggest that ER stress may participate in mediating the apoptotic responses induced by proteasomal inhibition. The up-regulation of the neuroprotective antioxidant genes and calcium-binding protein genes and down-regulation of the cholesterol biosynthesis genes in the later phase are likely consequences of stimulation of the pro-apoptotic signaling pathways in the early phase of lactacystin treatment.

New developments on the TNFα-mediated signalling pathways.

TNFα (tumour necrosis factor α) is an extensively studied pleiotropic cytokine associated with the pathogenesis of a variety of inflammatory diseases. It elicits a wide spectrum of cellular responses which mediates and regulates inflammation, immune response, cell survival, proliferation and apoptosis. TNFα initiates its responses by binding to its receptors. TNFα-induced effector responses are mediated by the actions and interactions among the various intracellular signalling mediators in the cell. TNFα induces both survival and apoptotic signal in a TRADD (TNF receptor-associated DD)-dependent and -independent way. The signals are further transduced via a variety of signalling mediators, including caspases, MAPKs (mitogen-activated protein kinases), phospholipid mediators and miRNA/miR (microRNA), whose roles in specific functional responses is not fully understood. Elucidating the complexity and cross talks among signalling mediators involved in the TNFα-mediated responses will certainly aid in the identification of molecular targets, which can potentially lead to the development of novel therapeutics to treat TNFα-associated disorders and in dampening inflammation.

Temporal transcriptomic profiling reveals cellular targets that govern survival in HOCl-mediated neuronal apoptosis.

AIMS: With the identification of hypochlorous acid (HOCl) as a biomarker in diseased brains and endogenous detection of its modified proteins, HOCl might be implicated in the development of neurodegenerative disorders. However, its effect on neuronal cell death has not yet been investigated at gene expression level. MAIN METHODS: Therefore, DNA microarray was performed for screening of HOCl-responsive genes in primary mouse cortical neurons. Neurotoxicity caused by physiological relevant HOCl (250µM) exhibited several biochemical markers of apoptosis. KEY FINDINGS: The biological processes affected during HOCl-mediated apoptosis included cell death, response to stress, cellular metabolism, and cell cycle. Among them, mRNAs level of cell death and stress response genes were up-regulated while expression of metabolism and cell cycle genes were down-regulated. SIGNIFICANCE: Our results showed, for the first time, that HOCl induces apoptosis in cortical neurons by upregulating apoptotic genes and gene expression of stress response such as heat shock proteins and antioxidant proteins were enhanced to provide protection. These data form a foundation for the development of screening platforms and define targets for intervention in HOCl neuropathologies where HOCl-mediated injury is causative.

SphK1 regulates proinflammatory responses associated with endotoxin and polymicrobial sepsis.

During sepsis, activation of phagocytes leads to the overproduction of proinflammatory cytokines, causing systemic inflammation. Despite substantial information regarding the underlying molecular mechanisms that lead to sepsis, several elements in the pathway remain to be elucidated. We found that the enzyme sphingosine kinase 1 (SphK1) is up-regulated in stimulated human phagocytes and in peritoneal phagocytes of patients with severe sepsis. Blockade of SphK1 inhibited phagocyte production of endotoxin-induced proinflammatory cytokines. We observed protection against sepsis in mice treated with a specific SphK1 inhibitor that was enhanced by treatment with a broad-spectrum antibiotic. These results demonstrated a critical role for SphK1 in endotoxin signaling and sepsis-induced inflammatory responses and suggest that inhibition of SphK1 is a potential therapy for septic shock.

Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells.

High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces growth, and in combination with reduced serum, or with the growth factors FGF and platelet-derived growth factor-AB, S1P has an enhancing effect on growth. We also show that the MSCs cultured in S1P-supplemented media are able to maintain their differentiation potential for at least as long as that for cells grown in the usual serum-containing media. This is shown by the ability of cells grown in S1P-containing media to be able to undergo osteogenic as well as adipogenic differentiation. This is of interest, since S1P is a relatively inexpensive natural product, which can be obtained in homogeneous high-purity batches: this will minimize costs and potentially reduce the unwanted side effects observed with serum. Taken together, S1P is able to induce proliferation while maintaining the multipotency of different human stem cells, suggesting a potential for S1P in developing serum-free or serum-reduced defined medium for adult stem cell cultures.

Phospholipase D1 mediates TNFalpha-induced inflammation in a murine model of TNFalpha-induced peritonitis.

BACKGROUND: Tumor Necrosis Factor alpha (TNFalpha) is a pleiotropic cytokine extensively studied for its role in the pathogenesis of a variety of disease conditions, including in inflammatory diseases. We have recently shown that, in vitro, that TNFalpha utilizes PLD1 to mediate the activation of NFkappaB and ERK1/2 in human monocytes. The aim of this study was to investigate the role(s) played by phospholipase D1 (PLD1) in TNFalpha-mediated inflammatory responses in vivo. METHODOLOGY/FINDINGS: Studies were performed in vivo using a mouse model of TNFalpha-induced peritonitis. The role of PLD1 was investigated by functional genomics, utilizing a specific siRNA to silence the expression of PLD1. Administration of the siRNA against PLD1 significantly reduced PLD1 levels in vivo. TNFalpha triggers a rapid pyrogenic response, but the in vivo silencing of PLD1 protects mice from the TNFalpha-induced rise in temperature. Similarly TNFalpha caused an increase in the serum levels of IL-6, MIP-1alpha and MIP-1beta: this increase in cytokine/chemokine levels was inhibited in mice where PLD1 had been silenced. We then induced acute peritonitis with TNFalpha. Intraperitoneal injection of TNFalpha triggered a rapid increase in vascular permeability, and the influx of neutrophils and monocytes into the peritoneal cavity. By contrast, in mice where PLD1 had been silenced, the TNFalpha-triggered increase in vascular permeability and phagocyte influx was substantially reduced. Furthermore, we also show that the TNFalpha-mediated upregulation of the cell adhesion molecules VCAM and ICAM1, in the vascular endothelium, were dependent on PLD1. CONCLUSIONS: These novel data demonstrate a critical role for PLD1 in TNFalpha-induced inflammation in vivo and warrant further investigation. Indeed, our results suggest PLD1 as a novel target for treating inflammatory diseases, where TNFalpha play key roles: these include diseases ranging from sepsis to respiratory and autoimmune diseases; all diseases with considerable unmet medical need.

Substance P in polymicrobial sepsis: molecular fingerprint of lung injury in preprotachykinin-A-/- mice.

Deletion of mouse preprotachykinin-A (PPTA), which encodes mainly for neuropeptide substance P, has been shown to protect against lung injury and mortality in sepsis. This study explored microarray-based differential gene expression profiles in mouse lung tissue 8 h after inducing microbial sepsis and the effect of PPTA gene deletion. A range of genes differentially expressed (more than two-fold) in microarray analysis was assessed, comparing wild-type and PPTA-knockout septic mice with their respective sham controls, and the data were further validated. Genetic deletion of substance P resulted in a significantly different expression profile of genes involved in inflammation and immunomodulation after the induction of sepsis, compared with wild-type mice. Interestingly, apart from the various proinflammatory mediators, the antiinflammatory cytokine interleukin-1 receptor antagonist gene (IL1RN) was also elevated much more in PPTA(-/-) septic mice. In addition, semiquantitative RT-PCR analysis supported the microarray data. The microarray data imply that the elevated levels of inflammatory gene expression in the early stages of sepsis in PPTA-knockout mice are possibly aimed to resolve the infection without excessive immunosuppression. As scientists are divided over the effects of pro- and antiinflammatory mediators in sepsis, it seems prudent to define the status depending on a complete genome profile. This is the first report exploring pulmonary gene expression profiles using microarray analysis in PPTA-knockout mice subjected to cecal ligation and puncture-induced sepsis and providing additional biological insight into the protection received against lung injury and mortality.

Mast cells express IL-17A in rheumatoid arthritis synovium.

The proinflammatory cytokine IL-17A is considered a crucial player in rheumatoid arthritis (RA) pathogenesis. In experimental models of autoimmune arthritis, it has been suggested that the cellular source of IL-17A is CD4(+) T cells (Th17 cells). However, little is known about the source of IL-17 in human inflamed RA tissue. We explored the cellular sources of IL-17A in human RA synovium. Surprisingly, only a small proportion of IL-17-expressing cells were T cells, and these were CCR6 negative. Unexpectedly, the majority of IL-17A expression colocalized within mast cells. Furthermore, we demonstrated in vitro that mast cells produced RORC-dependent IL-17A upon stimulation with TNF-alpha, IgG complexes, C5a, and LPS. These data are consistent with a crucial role for IL-17A in RA pathogenesis but suggest that in addition to T cells innate immune pathways particularly mediated via mast cells may be an important component of the effector IL-17A response.

IL-33 exacerbates autoantibody-induced arthritis.

Rheumatoid arthritis pathogenesis comprises dysregulation in both innate and adaptive immunity. There is therefore intense interest in the factors that integrate these immunologic pathways in rheumatoid arthritis. In this paper, we report that IL-33, a novel member of the IL-1 family, can exacerbate anti-glucose-6-phosphate isomerase autoantibody-induced arthritis (AIA). Mice lacking ST2 (ST2(-/-)), the IL-33 receptor alpha-chain, developed attenuated AIA and reduced expression of articular proinflammatory cytokines. Conversely, treatment of wild-type mice with rIL-33 significantly exacerbated AIA and markedly enhanced proinflammatory cytokine production. However, IL-33 failed to increase the severity of the disease in mast cell-deficient or ST2(-/-) mice. Furthermore, mast cells from wild-type, but not ST2(-/-), mice restored the ability of ST2(-/-) recipients to mount an IL-33-mediated exacerbation of AIA. IL-33 also enhanced autoantibody-mediated mast cell degranulation in vitro and in synovial tissue in vivo. Together these results demonstrate that IL-33 can enhance autoantibody-mediated articular inflammation via promoting mast cell degranulation and proinflammatory cytokine production. Because IL-33 is derived predominantly from synovial fibroblasts, this finding provides a novel mechanism whereby a host tissue-derived cytokine can regulate effector adaptive immune response via enhancing innate cellular activation in inflammatory arthritis.

Role of sphingosine kinase and sphingosine-1-phosphate in inflammatory arthritis.

The importance of sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) in inflammation has been extensively demonstrated. As an intracellular second messenger, S1P plays an important role in calcium signaling and mobilization, and cell proliferation and survival. Activation of various plasma membrane receptors, such as the formyl methionyl leucyl phenylalanine receptor, C5a receptor, and tumor necrosis factor α receptor, leads to a rapid increase in intracellular S1P level via SphK stimulation. SphK and S1P are implicated in various chronic autoimmune conditions such as rheumatoid arthritis, primary Sjögren's syndrome, and inflammatory bowel disease. Recent studies have demonstrated the important role of SphK and S1P in the development of arthritis by regulating the pro-inflammatory responses. These novel pathways represent exciting potential therapeutic targets.

Environmental toxicogenomics: a post-genomic approach to analysing biological responses to environmental toxins.

Environmental genomics has revolutionised how researchers can study the molecular basis of adverse effects of environmental toxicants. It is expected that the new discipline will afford efficient and high-throughput means to delineate mechanisms of action, risk assessment, identify and understand basic pathogenic mechanisms that are critical to disease progression, predict toxicity of unknown agents and to more precisely phenotype disease subtypes. Previously, we have demonstrated the potential of environmental genomics in a toxicant exposure model and, perhaps, this might become a crucial tool in biological response marker or biomarker discovery. To illustrate how toxicogenomics can be useful, we provide here an overview of some of the past and potential future aspects of environmental genomics. The present article also reviews the principles and technological concerns, and the standards and databases of toxicogenomics. In addition, applications of toxicogenomics in drug target identifications and validation strategies are also discussed.

Distinct roles of sphingosine kinase 1 and 2 in murine collagen-induced arthritis.

Sphingosine kinase (SphK) phosphorylates sphingosine into sphingosine-1-phosphate (S1P). S1P plays a critical role in angiogenesis, inflammation, and various pathologic conditions. To date, two mammalian isoenzymes, SphK1 and SphK2, have been identified. Although both SphK1 and SphK2 share overall homology and produce the common product, S1P, it has been proposed they display different unique and separate functions. In this study, we examined the role of SphK1 and SphK2 in a murine collagen-induced arthritis model by down-regulating each isoenzyme via specific small interfering RNA (siRNA). Prophylactic i.p. administration of SphK1 siRNA significantly reduced the incidence, disease severity, and articular inflammation compared with control siRNA recipients. Treatment of SphK1 siRNA also down-regulated serum levels of S1P, IL-6, TNF-alpha, IFN-gamma, and IgG2a anti-collagen Ab. Ex vivo analysis demonstrated significant suppression of collagen-specific proinflammatory/Th1 cytokine (IL-6, TNF-alpha, IFN-gamma) release in SphK siRNA-treated mice. Interestingly, mice received with SphK2 siRNA develop more aggressive disease; higher serum levels of IL-6, TNF-alpha, and IFN-gamma; and proinflammatory cytokine production to collagen in vitro when compared with control siRNA recipients. Together, these results demonstrate the distinct immunomodulatory roles of SphK1 and SphK2 in the development of inflammatory arthritis by regulating the release of proinflammatory cytokines and T cell responses. These findings raise the possibility that drugs which specifically target SphK1 activity may play a beneficial role in the treatment of inflammatory arthritis.

VAMP8 is essential in anaphylatoxin-induced degranulation, TNF-alpha secretion, peritonitis, and systemic inflammation.

VAMP8, a member of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) family of fusion proteins, initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. VAMP8 physiological function in inflammation has not been elucidated. In this paper, we show that deficiency of VAMP8 protects mice from anaphylatoxin (C5a)-induced neutropenia, peritonitis, and systemic inflammation. We show that, in vivo, VAMP8 deletion inhibits neutropenia and phagocyte recruitment. We also show that in macrophages, VAMP8 localizes on secretory granules and degranulation is inhibited in VAMP8-deficient macrophages. Moreover, VAMP8(-/-) mice show reduced systemic inflammation with inhibition of serum TNF-alpha levels, whereas IL-1beta, IL-6, and MIP1alpha release are not affected. In wild-type macrophages, TNF-alpha colocalizes with VAMP8-positive vesicles, and in VAMP8-deficient macrophages, the TNF-alpha release is inhibited. Furthermore, VAMP8 regulates the release of TNF-alpha and beta-hexosaminidase triggered by fMLP, and VAMP8(-/-) mice are protected from fMLP-induced peritonitis. These data demonstrate that the VAMP8 vesicle-associated-SNARE is required for the proper trafficking of secretory lysosomal granules for exocytosis in macrophages and for the release of the potent proinflammatory cytokine, TNF-alpha.

The cytokine interleukin-33 mediates anaphylactic shock.

Anaphylactic shock is characterized by elevated immunoglobulin-E (IgE) antibodies that signal via the high affinity Fc epsilon receptor (Fc epsilonRI) to release inflammatory mediators. Here we report that the novel cytokine interleukin-33 (IL-33) potently induces anaphylactic shock in mice and is associated with the symptom in humans. IL-33 is a new member of the IL-1 family and the ligand for the orphan receptor ST2. In humans, the levels of IL-33 are substantially elevated in the blood of atopic patients during anaphylactic shock, and in inflamed skin tissue of atopic dermatitis patients. In murine experimental atopic models, IL-33 induced antigen-independent passive cutaneous and systemic anaphylaxis, in a T cell-independent, mast cell-dependent manner. In vitro, IL-33 directly induced degranulation, strong eicosanoid and cytokine production in IgE-sensitized mast cells. The molecular mechanisms triggering these responses include the activation of phospholipase D1 and sphingosine kinase1 to mediate calcium mobilization, Nuclear factor-kappaB activation, cytokine and eicosanoid secretion, and degranulation. This report therefore reveals a hitherto unrecognized pathophysiological role of IL-33 and suggests that IL-33 may be a potential therapeutic target for anaphylaxis, a disease of considerable unmet medical need.