Interleukin‐1β protects astrocytes against oxidant‐induced injury via an NF‐κB‐Dependent upregulation of glutathione synthesis

Astrocytes produce and export the antioxidant glutathione (GSH). Previously, we found that interleukin‐1β (IL‐1β) enhanced the expression of astrocyte system x_c^?, the transporter that delivers the rate‐limiting substrate for GSH synthesis—cyst(e)ine. Herein, we demonstrate directly that IL‐1β mediates a time‐dependent increase in extracellular GSH levels in cortical astrocyte cultures, suggesting both enhanced synthesis and export. This increased GSH production was blocked by inhibition of nuclear factor‐κB (NF‐κB) activity but not by inhibition of p38 MAPK. To determine whether this increase could provide protection against oxidative stress, the oxidants tert‐butyl hydroperoxide (tBOOH) and ferrous sulfate (FeSO₄) were employed. IL‐1β treatment prevented the increase in reactive oxygen species produced in astrocytes following tBOOH exposure. Additionally, the toxicity induced by tBOOH or FeSO₄ exposure was significantly attenuated following treatment with IL‐1β, an effect reversed by concomitant exposure to l ‐buthionine‐S,R‐sulfoximine (BSO), which prevented the IL‐1β‐mediated rise in GSH production. IL‐1β failed to increase GSH or to provide protection against t‐BOOH toxicity in astrocyte cultures derived from IL‐1R1 null mutant mice. Overall, our data indicate that under certain conditions IL‐1β may be an important stimulus for increasing astrocyte GSH production, and potentially, total antioxidant capacity in brain, via an NF‐κB‐dependent process. GLIA 2015;63:1568–1580     

Nuclear factor‐κB/p65 responds to changes in the Notch signaling pathway in murine BV‐2 cells and in amoeboid microglia in postnatal rats treated with the γ‐secretase complex blocker DAPT

Microglial cells constitutively express Notch‐1 and nuclear factor‐κB/p65 (NF‐κB/p65), and both pathways modulate production of inflammatory mediators. This study sought to determine whether a functional relationship exists between them and, if so, to investigate whether they synergistically regulate common microglial cell functions. By immunofluorescence labeling, real‐time polymerase chain reaction (RT‐PCR), flow cytometry, and Western blot, BV‐2 cells exhibited Notch‐1 and NF‐κB/p65 expression, which was significantly up‐regulated in cells challenged with lipopolysaccharide (LPS). This was coupled with an increase in expression of Hes‐1, tumor necrosis factor‐α (TNF‐α), and interleukin‐1β (IL‐1β). In BV‐2 cells pretreated with N‐[N‐(3,5‐difluorophenacetyl)‐1‐alany1]‐S‐phenyglycine t‐butyl ester (DAPT), a γ‐secretase inhibitor, followed by LPS stimulation, Notch‐1 expression level was enhanced but that of all other markers was suppressed. Additionally, Hes‐1 expression and NF‐κB nuclear translocation decreased as shown by flow cytometry. Notch‐1's modulation of NF‐κB/p65 was also evidenced in amoeboid microglial cells (AMC) in vivo. In 5‐day‐old rats given intraperitoneal injections of LPS, Notch‐1, NF‐κB/p65, TNF‐α, and IL‐1β immunofluorescence in AMC was markedly enhanced. However, in rats given an intraperitoneal injection of DAPT prior to LPS, Notch‐1 labeling was augmented, but that of TNF‐α and IL‐1β was reduced. The results suggest that blocking of Notch‐1 activation with DAPT would reduce the level of its downstream end product Hes‐1 along with suppression of NF‐κB/p65 translocation, resulting in suppressed production of proinflammatory cytokines. It is concluded that Notch‐1 signaling can trans‐activate NF‐κB/p65 by amplifying NF‐κB/p65‐dependent proinflammatory functions in activated microglia. © 2010 Wiley‐Liss, Inc.     

Tripchlorolide protects neuronal cells from microglia‐mediated β‐amyloid neurotoxicity through inhibiting NF‐κB and JNK signaling

Recent research has focused on soluble oligomeric assemblies of β‐amyloid peptides (Aβ) as the proximate cause of neuroinflammation, synaptic loss, and the eventual dementia associated with Alzheimer's disease (AD). In this study, tripchlorolide (T₄), an extract of Tripterygium wilfordii Hook. F (TWHF), was studied as a novel agent to suppress neuroinflammatory process in microglial cells and to protect neuronal cells against microglia‐mediated oligomeric Aβ toxicity. T₄ significantly attenuated oligomeric Aβ(1‐42)‐induced release of inflammatory productions such as tumor necrosis factor‐α, interleukin‐1β, nitric oxide (NO), and prostaglandin E₂. It also downregulated the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) in microglial cells. Further molecular mechanism study demonstrated that T₄ inhibited the nuclear translocation of nuclear factor‐κB (NF‐κB) without affecting I‐κBα phosphorylation. It repressed Aβ‐induced JNK phosphorylation but not ERK or p38 MAPK. The inhibition of NF‐κB and JNK by T₄ is correlated with the suppression of inflammatory mediators in Aβ‐stimulated microglial cells. These results suggest that T₄ protects neuronal cells by blocking inflammatory responses of microglial cells to oligomeric Aβ(1‐42) and that T₄ acts on the signaling of NF‐κB and JNK, which are involved in the modulation of inflammatory response. Therefore, T₄ may be an effective agent in modulating neuroinflammatory process in AD. © 2009 Wiley‐Liss, Inc.     

Impairment of nuclear factor‐κB activation increased glutamate excitotoxicity in a motoneuron–neuroblastoma hybrid cell line expressing mutant (G93A) Cu/Zn‐superoxide dismutase

Mutations in the superoxide dismutase 1 (SOD1) gene are linked to glutamate excitotoxicity in familial amyotrophic lateral sclerosis (fALS), but the underlying mechanism remains unclear. We investigated whether nuclear factor‐κB (NF‐κB) activation is involved in glutamate excitotoxicity by using motor neuron–neuroblastoma hybrid cells that expressed a mutant (G93A) SOD1 (mtSOD1) or wild‐type SOD1 (wtSOD1). MtSOD1 cells were more vulnerable to glutamate excitotoxicity than wtSOD1 cells and showed higher NF‐κB activity, higher nuclear cRel expression, and lower nuclear RelA expression under basal conditions. Glutamate treatment increased NF‐κB activation along with nuclear expressions of RelA and cRel in wtSOD1 cells but induced only weak nuclear RelA expression in mtSOD1 cells. Suppression of NF‐κB activation using transfection of the superrepressive mutant form of IκBα (mIκBα) inhibited nuclear RelA expression in both types of SOD1 cells, which increased glutamate excitotoxicity in wtSOD1 cells but not in mtSOD1 cells. Furthermore, immunohistochemistry confirmed stronger RelA immunoreactivity in the nuclei of motor neurons of spinal cord in wild‐type SOD1 transgenic mice than in those in SOD1 G93A transgenic mice. In addition, we found that glutamate treatment decreased XIAP expression and increased caspase‐3 activity in mtSOD1 cells and mIκBα‐overexpressing wtSOD1 cells. Our results suggest that glutamate excitotoxicity in motor neurons of SOD1‐linked fALS is attributable, at least in part, to the impairment of IκBα‐dependent RelA activation and subsequent apoptosis mediated by XIAP inhibition and caspase‐3 activation. © 2010 Wiley‐Liss, Inc.     

Differential contributions of microglial and neuronal IKKβ to synaptic plasticity and associative learning in alert behaving mice

Microglia are CNS resident immune cells and a rich source of neuroactive mediators, but their contribution to physiological brain processes such as synaptic plasticity, learning, and memory is not fully understood. In this study, we used mice with partial depletion of IκB kinase β, the main activating kinase in the inducible NF‐κB pathway, selectively in myeloid lineage cells (mIKKβKO) or excitatory neurons (nIKKβKO) to measure synaptic strength at hippocampal Schaffer collaterals during long‐term potentiation (LTP) and instrumental conditioning in alert behaving individuals. Resting microglial cells in mIKKβKO mice showed less Iba1‐immunoreactivity, and brain IL‐1β mRNA levels were selectively reduced compared with controls. Measurement of field excitatory postsynaptic potentials (fEPSPs) evoked by stimulation of the CA3‐CA1 synapse in mIKKβKO mice showed higher facilitation in response to paired pulses and enhanced LTP following high frequency stimulation. In contrast, nIKKβKO mice showed normal basic synaptic transmission and LTP induction but impairments in late LTP. To understand the consequences of such impairments in synaptic plasticity for learning and memory, we measured CA1 fEPSPs in behaving mice during instrumental conditioning. IKKβ was not necessary in either microglia or neurons for mice to learn lever‐pressing (appetitive behavior) to obtain food (consummatory behavior) but was required in both for modification of their hippocampus‐dependent appetitive, not consummatory behavior. Our results show that microglia, through IKKβ and therefore NF‐κB activity, regulate hippocampal synaptic plasticity and that both microglia and neurons, through IKKβ, are necessary for animals to modify hippocampus‐driven behavior during associative learning. GLIA 2015;63:549–566     

Redox regulation of NF‐κB p50 and M1 polarization in microglia

Redox‐signaling is implicated in deleterious microglial activation underlying CNS disease, but how ROS program aberrant microglial function is unknown. Here, the oxidation of NF‐κB p50 to a free radical intermediate is identified as a marker of dysfunctional M1 (pro‐inflammatory) polarization in microglia. Microglia exposed to steady fluxes of H₂O₂ showed altered NF‐κB p50 protein–protein interactions, decreased NF‐κB p50 DNA binding, and augmented late‐stage TNFα expression, indicating that H₂O₂ impairs NF‐κB p50 function and prolongs amplified M1 activation. NF‐κB p50^?/? mice and cultures exhibited a disrupted M2 (alternative) response and impaired resolution of the M1 response. Persistent neuroinflammation continued 1 week after LPS (1 mg/kg, IP) administration in the NF‐κB p50^?/? mice. However, peripheral inflammation had already resolved in both strains of mice. Treatment with the spin‐trap DMPO mildly reduced LPS‐induced 22 h TNFα in the brain in NF‐κB p50^+/+ mice. Interestingly, DMPO failed to reduce and strongly augmented brain TNFα production in NF‐κB p50^?/? mice, implicating a fundamental role for NF‐κB p50 in the regulation of chronic neuroinflammation by free radicals. These data identify NF‐κB p50 as a key redox‐signaling mechanism regulating the M1/M2 balance in microglia, where loss of function leads to a CNS‐specific vulnerability to chronic inflammation. GLIA 2015;63:423–440     

Sulfoglucuronosyl paragloboside is a ligand for T cell adhesion: Regulation of sulfoglucuronosyl paragloboside expression via nuclear factor κB signaling

Inflammatory cytokines such as tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β stimulate glucuronosyltransferase genes (S and P) in endothelial cells (ECs) and up‐regulate sulfoglucuronosyl paragloboside (SGPG) expression, which serves as a ligand for T cell adhesion. However, the mechanism of cytokine‐mediated gene up‐regulation has not been elucidated. To evaluate the precise mechanism of SGPG up‐regulation, we have specifically inhibited the SGPG synthesis in the cerebromicrovascular EC line (SV‐HCECs), a transformed brain ECs of human origin. SV‐HCECs were transfected with small interfering RNA designed to mimic the human natural killer epitope‐1 sulfotransferase (HNK‐1ST), the ultimate enzyme that transfers the sulfate group to glucuronic acid for SGPG synthesis. An inhibition of SGPG expression along with a reduction of human CD4⁺ cell adhesion was observed in siRNA HNK‐1ST (siHNK‐1)‐transfected cells after TNFα stimulation. A thorough screening of the signaling system confirmed that TNFα/IL‐1β stimulation up‐regulated nuclear factor κB (NFκB) signaling in SV‐HCECs. siHNK‐1 transfection interfered with the SGPG up‐regulation after TNFα/IL‐1β stimulation in transfected cells and reduced the T cell adhesion. Hence, our study indicates that T cell‐SGPG adhesion in SV‐HCECs may proceed through NFκB activation. In addition, siHNK‐1 transfection reduced the NFκB activity compared with cells that were transfected with scrambled siRNA, before and after TNFα/IL‐1β stimulation. This is the first report indicating that NFκB signaling is involved in SGPG gene expression in brain ECs by an unknown mechanism. Its down‐regulation by inhibiting HNK‐1ST expression may have a potential use in preventing the T cell invasion and consequently nerve damage during inflammation. © 2009 Wiley‐Liss, Inc.     

α‐Synuclein activates innate immunity but suppresses interferon‐γ expression in murine astrocytes

Glial activation and neuroinflammation contribute to pathogenesis of neurodegenerative diseases, linked to neuron loss and dysfunction. α‐Synuclein (α‐syn), as a metabolite of neuron, can induce microglia activation to trigger innate immune response. However, whether α‐syn, as well as its mutants (A53T, A30P, and E46K), induces astrocyte activation and inflammatory response is not fully elucidated. In this study, we used A53T mutant and wild‐type α‐syns to stimulate primary astrocytes in dose‐ and time‐dependent manners (0.5, 2, 8, and 20 μg/ml for 24 hr or 3, 12, 24, and 48 hr at 2 μg/ml), and evaluated activation of several canonical inflammatory pathway components. The results showed that A53T mutant or wild‐type α‐syn significantly upregulated mRNA expression of toll‐like receptor (TLR)2, TLR3, nuclear factor‐κB and interleukin (IL)‐1β, displaying a pattern of positive dose–effect correlation or negative time–effect correlation. Such upregulation was confirmed at protein levels of TLR2 (at 20 μg/ml), TLR3 (at most doses), and IL‐1β (at 3 hr) by western blotting. Blockage of TLR2 other than TLR4 inhibited TLR3 and IL‐1β mRNA expressions. By contrast, interferon (IFN)‐γ was significantly downregulated at mRNA, protein, and protein release levels, especially at high concentrations of α‐syns or early time‐points. These findings indicate that α‐syn was a TLRs‐mediated immunogenic agent (A53T mutant stronger than wild‐type α‐syn). The stimulation patterns suggest that persistent release and accumulation of α‐syn is required for the maintenance of innate immunity activation, and IFN‐γ expression inhibition by α‐syn suggests a novel immune molecule interaction mechanism underlying pathogenesis of neurodegenerative diseases.     

Role of ERK map kinase and CRM1 in IL‐1β‐stimulated release of HMGB1 from cortical astrocytes

Reactive astrocytes are traditionally thought to impede brain plasticity after stroke. However, we previously showed that reactive astrocytes may also contribute to stroke recovery, partly via the release of a nuclear protein called high‐mobility group box 1 (HMGB1). Here, we investigate the mechanisms that allow stimulated astrocytes to release HMGB1. Exposure of rat primary astrocytes to IL‐1β for 24 h elicited a dose‐dependent HMGB1 response. Immunostaining and western blots of cell lysates showed increased intracellular levels of HMGB1. Western blots confirmed that IL‐1β induced a release of HMGB1 into astrocyte conditioned media. MAP kinase signaling was involved. Levels of phospho‐ERK were increased by IL‐1β, and the MEK/ERK inhibitor U0126 decreased HMGB1 upregulation in the stimulated astrocytes. Since HMGB1 is a nuclear protein, the role of the nuclear protein exporter, chromosome region maintenance 1 (CRM1), was assessed as a candidate mechanism for linking MAP kinase signaling to HMGB1 release. IL‐1β increased CRM1 expression in concert with a translocation of HMGB1 from nucleus into cytoplasm. Blockade of IL‐1β‐stimulated HMGB1 release with the ERK inhibitor U0126 was accompanied by a downregulation of CRM1. Our findings reveal that IL‐1β stimulates the release of HMGB1 from activated astrocytes via ERK MAP kinase and CRM1 signaling. These data suggest a novel pathway by which inflammatory cytokines may enhance the ability of reactive astrocytes to release prorecovery mediators after stroke. © 2010 Wiley‐Liss, Inc.     

Induction of interleukin‐1β by activated microglia is a prerequisite for immunologically induced fatigue

We previously reported that an intraperitoneal (i.p.) injection of synthetic double‐stranded RNA, polyriboinosinic:polyribocytidylic acid (poly‐I:C), produced prolonged fatigue in rats, which might serve as a model for chronic fatigue syndrome. The poly‐I:C‐induced fatigue was associated with serotonin transporter (5‐HTT) overexpression in the prefrontal cortex (PFC), a brain region that has been suggested to be critical for fatigue sensation. In the present study, we demonstrated that microglial activation in the PFC was important for poly‐I:C‐induced fatigue in rats, as pretreatment with minocycline, an inhibitor of microglial activation, prevented the decrease in running wheel activity. Poly‐I:C injection increased the microglial interleukin (IL)‐1β expression in the PFC. An intracerebroventricular (i.c.v.) injection of IL‐1β neutralising antibody limited the poly‐I:C‐induced decrease in activity, whereas IL‐1β (i.c.v.) reduced the activity in a dose‐dependent manner. 5‐HTT expression was enhanced by IL‐1β in primary cultured astrocytes but not in microglia. Poly‐I:C injection (i.p.) caused an increase in 5‐HTT expression in astrocytes in the PFC of the rat, which was inhibited by pretreatment with minocycline (i.p.) and rat recombinant IL‐1 receptor antagonist (i.c.v.). Poly‐I:C injection (i.p.) led to a breakdown of the blood–brain barrier and enhanced Toll‐like receptor 3 signaling in the brain. Furthermore, direct application of poly‐I:C enhanced IL‐1β expression in primary microglia. We therefore propose that poly‐I:C‐induced microglial activation, which may be at least partly caused by a direct action of poly‐I:C, enhances IL‐1β expression. Then, IL‐1β induces 5‐HTT expression in astrocytes, resulting in the immunologically induced fatigue.     

Selective estrogen receptor modulators decrease the production of interleukin‐6 and interferon‐γ‐inducible protein‐10 by astrocytes exposed to inflammatory challenge in vitro

Expression of proinflammatory molecules by glial cells is involved in the pathophysiological changes associated with chronic neurological diseases. Under pathological conditions, astrocytes release a number of proinflammatory molecules, such as interleukin‐6 (IL‐6) and interferon‐γ‐inducible protein‐10 (IP‐10). The ovarian hormone estradiol exerts protective effects in the central nervous system that, at least in part, may be mediated by a reduction of local inflammation. This study was designed to assess whether estradiol affects the production of IL‐6 and IP‐10 by primary cultures of newborn mice astrocytes exposed to lipopolysaccharide (LPS), a bacterial endotoxin known to cause neuroinflammation. In addition, the possible anti‐inflammatory effect of several selective estrogen receptor modulators (SERMs) was also assessed. LPS induced an increase in the expression of IL‐6 and IP‐10 mRNA levels in astrocytes and an increase in IL‐6 and IP‐10 protein levels in the culture medium. These effects of LPS were impaired by estradiol and by the four SERMs tested in our study: tamoxifen, raloxifene, ospemifene, and bazedoxifene. All SERMs tested showed a similar effect on IL‐6 and IP‐10 mRNA levels, but raloxifene and ospemifene were more effective than tamoxifen and bazedoxifene in reducing protein levels in LPS‐treated cultures. Finally, we report that news SERMs, ospemifene and bazedoxifene, exert anti‐inflammatory actions by a mechanism involving classical estrogen receptors and by the inhibition of LPS‐induced NFκB p65 transactivation. The results suggest that estrogenic compounds may be candidates to counteract brain inflammation under neurodegenerative conditions by targeting the production and release of proinflammatory molecules by astrocytes. © 2009 Wiley‐Liss, Inc.     

Tumor necrosis factor‐alpha mediates activation of NF‐κB and JNK signaling cascades in retinal ganglion cells and astrocytes in opposite ways

Tumor necrosis factor‐alpha (TNF) is an important mediator of the innate immune response in the retina. TNF can activate various signaling cascades, including NF‐κB, nuclear factor kappa B (NF‐κB) and c‐Jun N‐terminal kinase (JNK) pathways. The harmful role of these pathways, as well as of TNF, has previously been shown in several retinal neurodegenerative conditions including glaucoma and retinal ischemia. However, TNF and TNF‐regulated signaling cascades are capable not only of mediating neurotoxicity, but of being protective. We performed this study to delineate the beneficial and detrimental effects of TNF signaling in the retina. To this end, we used TNF‐treated primary retinal ganglion cell (RGC) and astrocyte cultures. Levels of expression of NF‐κB subunits in RGCs and astrocytes were evaluated by quantitative RT‐PCR (qRT‐PCR) and Western blot (WB) analysis. NF‐κB and JNK activity in TNF‐treated cells was determined in a time‐dependent manner using ELISA and WB. Gene expression in TNF‐treated astrocytes was measured by qRT‐PCR. We found that NF‐κB family members were present in RGCs and astrocytes at the mRNA and protein levels. RGCs failed to activate NF‐κB in the presence of TNF, a phenomenon that was associated with sustained JNK activation and RGC death. However, TNF initiated the activation of NF‐κB and mediated transient JNK activation in astrocytes. These events were associated with glial survival and increased expression of neurotoxic pro‐inflammatory factors. Our findings suggest that, in the presence of TNF, NF‐κB and JNK signaling cascades are activated in opposite ways in RGCs and astrocytes. These events can directly and indirectly facilitate RGC death.     

Phosphatidylinositol 3‐kinase/protein kinase Cδ activation induces close homolog of adhesion molecule L1 (CHL1) expression in cultured astrocytes

Upregulation of expression of the close homolog of adhesion molecule L1 (CHL1) by reactive astrocytes in the glial scar reduces axonal regeneration and inhibits functional recovery after spinal cord injury (SCI). Here, we investigate the molecular mechanisms underlying upregulation of CHL1 expression by analyzing the signal transduction pathways in vitro. We show that astrogliosis stimulated by bacterial lipopolysaccharide (LPS) upregulates CHL1 expression in primary cultures of mouse cerebral astrocytes, coinciding with elevated protein synthesis and translocation of protein kinase δ (PKCδ) from cytosol to the membrane fraction. Blocking PKCδ activity pharmacologically and genetically attenuates LPS‐induced elevation of CHL1 protein expression through a phosphatidylinositol 3‐kinase (PI3K) dependent pathway. LPS induces extracellular signal‐regulated kinases (ERK1/2) phosphorylation through PKCδ and blockade of ERK1/2 activation abolishes upregulation of CHL1 expression. LPS‐triggered upregulation of CHL1 expression mediated through translocation of nuclear factor κB (NF‐κB) to the nucleus is blocked by a specific NF‐κB inhibitor and by inhibition of PI3K, PKCδ, and ERK1/2 activities, implicating NF‐κB as a downstream target for upregulation of CHL1 expression. Furthermore, the LPS‐mediated upregulation of CHL1 expression by reactive astrocytes is inhibitory for hippocampal neurite outgrowth in cocultures. Although the LPS‐triggered NO‐guanylate cyclase‐cGMP pathway upregulates glial fibrillary acid protein expression in cultured astrocytes, we did not observe this pathway to mediate LPS‐induced upregulation of CHL1 expression. Our results indicate that elevated CHL1 expression by reactive astrocytes requires activation of PI3K/PKCδ‐dependent pathways and suggest that reduction of PI3K/PKCδ activity represents a therapeutic target to downregulate CHL1 expression and thus benefit axonal regeneration after SCI. © 2009 Wiley‐Liss, Inc.