IL‐1β enhances the antibacterial activity of astrocytes by activation of NF‐κB

Astrocytes have important immune functions in CNS, and astrocytes stimulated by interferon‐γ were showed to have direct antimicrobial function. However whether astrocytes without the stimulation of cytokines have antibacterial function, and how this function is regulated are still largely unknown. In this study, we found that primary cultured astrocytes inhibited the growth of both gram‐negative and gram‐positive bacteria. Further more, we showed that interleukin‐1β (IL‐1β) enhanced the antibacterial effect in a dose‐dependent manner, and the antibacterial effect of astrocytes from IL‐1β receptor‐deficient mice failed to be enhanced by IL‐1β. IL‐1β stimulated IκBα degradation, NF‐κB nuclear translocation, and transactivation in astrocytes. NF‐κB inhibitors blocked NF‐κB activation and the enhanced antibacterial effect induced by IL‐1β. In addition, overexpression of dominant negative IκBα in astrocytes inhibited IκBα degradation and NF‐κB transactivation, and also inhibited the enhanced antibacterial effect induced by IL‐1β. All these data demonstrated that IL‐1β enhanced the antibacterial activity of astrocytes by activation of NF‐κB. © 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.     

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.     

Lack of Oestrogenic Inhibition of the Nuclear Factor‐κB Pathway in Somatolactotroph Tumour Cells

Activation of nuclear factor (NF)‐κB promotes cell proliferation and inhibits apoptosis. We have previously shown that oestrogens sensitise normal anterior pituitary cells to the apoptotic effect of tumour necrosis factor (TNF)‐α by inhibiting NF‐κB nuclear translocation. In the present study, we examined whether oestrogens also modulate the NF‐κB signalling pathway and apoptosis in GH3 cells, a rat somatolactotroph tumour cell line. As determined by Western blotting, 17β‐oestradiol (E₂) (10^?9 m ) increased the nuclear concentration of NF‐κB/p105, p65 and p50 in GH3 cells. However, E₂ did not modify the expression of Bcl‐xL, a NF‐κB target gene. TNF‐α induced apoptosis of GH3 cells incubated in either the presence or absence of E₂. Inhibition of the NF‐kB pathway using BAY 11‐7082 (BAY) (5 μm ) decreased the viability of GH3 cells and increased the percentage of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)‐positive GH3 cells. BAY also increased TNF‐α‐induced apoptosis of GH3 cells, an effect that was further increased by an inhibitor of the c‐Jun N‐terminal protein kinase pathway, SP600125 (10 μm ). We also analysed the role of the NF‐κB signalling pathway on proliferation and apoptosis of GH3 tumours in vivo. The administration of BAY to nude mice bearing GH3 tumours increased the number of TUNEL‐positive cells and decreased the number of proliferating GH3 cells. These findings suggest that GH3 cells lose their oestrogenic inhibitory action on the NF‐κB pathway and that the pro‐apoptotic effect of TNF‐α on these tumour pituitary cells does not require sensitisation by oestrogens as occurs in normal pituitary cells. NF‐κB was required for the survival of GH3 cells, suggesting that pharmacological inhibition of the NF‐κB pathway could interfere with pituitary tumour progression.     

Inflammatory cytokine tumor necrosis factor α suppresses neuroprotective endogenous erythropoietin from astrocytes mediated by hypoxia‐inducible factor‐2α

Interest in erythropoietin (EPO) as a neuroprotective mediator has grown since it was found that systemically administered EPO is protective in several animal models of disease. However, given that the blood–brain barrier limits EPO entry into the brain, alternative approaches that induce endogenous EPO production in the brain may be more effective clinically and associated with fewer untoward side‐effects. Astrocytes are the main source of EPO in the central nervous system. In the present study we investigated the effect of the inflammatory cytokine tumor necrosis factor α (TNFα) on hypoxia‐induced upregulation of EPO in rat brain. Hypoxia significantly increased EPO mRNA expression in the brain and kidney, and this increase was suppressed by TNFα in vivo. In cultured astrocytes exposed to hypoxic conditions for 6 and 12 h, TNFα suppressed the hypoxia‐induced increase in EPO mRNA expression in a concentration‐dependent manner. TNFα inhibition of hypoxia‐induced EPO expression was mediated primarily by hypoxia‐inducible factor (HIF)‐2α rather than HIF‐1α. The effects of TNFα in reducing hypoxia‐induced upregulation of EPO mRNA expression probably involve destabilization of HIF‐2α, which is regulated by the nuclear factor (NF)‐κB signaling pathway. TNFα treatment attenuated the protective effects of astrocytes on neurons under hypoxic conditions via EPO signaling. The effective blockade of TNFα signaling may contribute to the maintenance of the neuroprotective effects of EPO even under hypoxic conditions with an inflammatory response.     

Cooperative induction of CXCL10 involves NADPH oxidase: Implications for HIV dementia

With the increasing prevalence of HIV‐associated neurocognititve disorders (HAND), understanding the mechanisms by which HIV‐1 induces neuro‐inflammation and subsequent neuronal damage is important. The hallmark features of HIV‐encephalitis, the pathological correlate of HIV‐associated Dementia (HAD), are gliosis, oxidative stress, chemokine dysregulation, and neuronal damage/death. Since neurons are not infected by HIV‐1, the current thinking is that these cells are damaged indirectly by pro‐inflammatory chemokines released by activated glial cells. CXCL10 is a neurotoxic chemokine that is upregulated in astroglia activated by HIV‐1 Tat, IFN‐γ, and TNF‐α. In this study we have demonstrated that HIV‐1 Tat increases CXCL10 expression in IFN‐γ and TNF‐α stimulated human astrocytes via NADPH oxidase. We have shown that the treatment of astrocytes with a mixture of Tat and cytokines leads to a respiratory burst that is abrogated by apocynin, an NADPH oxidase inhibitor. Pretreatment of Tat, IFN‐γ, and TNF‐α stimulated astrocytes with apocynin also resulted in concomitant inhibition of CXCL10 expression. Additionally, apocynin was also able to reduce Tat and cytokine‐mediated activation of the corresponding signaling molecules Erk1/2, Jnk, and Akt with a decrease in activation and nuclear translocation of NF‐κB, important regulators of CXCL10 induction. Understanding the mechanisms involved in reducing both oxidative stress and the release of pro‐inflammatory agents could lead to the development of therapeutics aimed at decreasing neuro‐inflammation in patients suffering from HAD. © 2009 Wiley‐Liss, Inc.     

Increased levels of proinflammatory cytokines in the aged rat brain attenuate injury‐induced cytokine response after excitotoxic damage

In order to evaluate proinflammatory cytokine levels and their producing cell types in the control aged rat brain and after acute excitotoxic damage, both adult and aged male Wistar rats were injected with N‐methyl‐D ‐aspartate in the striatum. At different survival times between 6 hr and 7 days after lesioning, interleukin‐1 beta (IL‐1β), interleukin‐6 (IL‐6), and tumor necrosis factor alpha (TNF‐α) were analyzed by enzyme‐linked immunosorbent assay and by double immunofluorescence of cryostat sections by using cell‐specific markers. Basal cytokine expression was attributed to astrocytes and was increased in the normal aged brain showing region specificity: TNF‐α and IL‐6 displayed age‐dependent higher levels in the aged cortex, and IL‐1β and IL‐6 in the aged striatum. After excitotoxic striatal damage, notable age‐dependent differences in cytokine induction in the aged vs. the adult were seen. The adult injured striatum exhibited a rapid induction of all cytokines analyzed, but the aged injured striatum showed a weak induction of cytokine expression: IL‐1β showed no injury‐induced changes at any time, TNF‐α presented a late induction at 5 days after lesioning, and IL‐6 was only induced at 6 hr after lesioning. At both ages, in the lesion core, all cytokines were early expressed by neurons and astrocytes, and by microglia/macrophages later on. However, in the adjacent lesion border, cytokines were found in reactive astrocytes. This study highlights the particular inflammatory response of the aged brain and suggests an important role of increased basal levels of proinflammatory cytokines in the reduced ability to induce their expression after damage. © 2009 Wiley‐Liss, Inc.     

Proinflammatory cytokine regulation of cyclic AMP‐phosphodiesterase 4 signaling in microglia in vitro and following CNS injury

Cyclic AMP suppresses immune cell activation and inflammation. The positive feedback loop of proinflammatory cytokine production and immune activation implies that cytokines may not only be regulated by cyclic AMP but also conversely regulate cyclic AMP. This study examined the effects of tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β on cyclic AMP‐phosphodiesterase (PDE) signaling in microglia in vitro and after spinal cord injury (SCI) or traumatic brain injury (TBI). TNF‐α or IL‐1β stimulation produced a profound reduction (>90%) of cyclic AMP within EOC2 microglia from 30 min that then recovered after IL‐1β but remained suppressed with TNF‐α through 24 h. Cyclic AMP was also reduced in TNF‐α‐stimulated primary microglia, albeit to a lesser extent. Accompanying TNF‐α‐induced cyclic AMP reductions, but not IL‐1β, was increased cyclic AMP‐PDE activity. The role of PDE4 activity in cyclic AMP reductions was confirmed by using Rolipram. Examination of pde4 mRNA revealed an immediate, persistent increase in pde4b with TNF‐α; IL‐1β increased all pde4 mRNAs. Immunoblotting for PDE4 showed that both cytokines increased PDE4A1, but only TNF‐α increased PDE4B2. Immunocytochemistry revealed PDE4B nuclear translocation with TNF‐α but not IL‐1β. Acutely after SCI/TBI, where cyclic AMP levels are reduced, PDE4B was localized to activated OX‐42⁺ microglia; PDE4B was absent in OX‐42⁺ cells in uninjured spinal cord/cortex or inactive microglia. Immunoblotting showed PDE4B2 up‐regulation from 24 h to 1 wk post‐SCI, the peak of microglia activation. These studies show that TNF‐α and IL‐1β differentially affect cyclic AMP‐PDE signaling in microglia. Targeting PDE4B2 may be a putative therapeutic direction for reducing microglia activation in CNS injury and neurodegenerative diseases. © 2012 Wiley Periodicals, Inc.     

NOV/CCN3 upregulates CCL2 and CXCL1 expression in astrocytes through β1 and β5 integrins

Increasing evidence suggests that CCN matricellular proteins play important roles in inflammation. One of the major cell types that handle inflammation in the brain is the astrocyte, which, upon activation, dramatically increases its production of cytokines and chemokines. Here, we report that NOV/CCN3, added to primary cultured rat brain astrocytes, markedly increased the expression of CCL2 and CXCL1 chemokines, as indicated by ELISA and RT‐qPCR assays. This effect was selective, as the production of thirteen other cytokines and chemokines was not affected by NOV. NOV expression by astrocytes was demonstrated by immunocytochemistry and Western blot analysis, and astrocyte transfection with NOV small interfering RNA (siRNA) markedly decreased CXCL1 and CCL2 production, indicating that endogenous NOV played a major role in the control of astrocytic chemokine synthesis. NOV was shown to mediate several of its actions through integrins. Here, we observed that siRNAs against integrins β1 and β5 decreased basal and abrogated NOV‐stimulated astrocyte expression of CCL2 and CXCL1, respectively. Using a panel of kinase inhibitors, we demonstrated that NOV action on CCL2 and CXCL1 production involved a Rho/ROCK/JNK/NF‐κB and a Rho/qROCK/p38/NF‐κB pathway, respectively. Thus, distinct integrins and signaling mechanisms are involved in NOV‐induced production of CCL2 and CXCL1 in astrocytes. Finally, astrocytic expression of NOV was detected in rat brain tissue sections, and NOV intracerebral injection increased CCL2 and CXCL1 brain levels in vivo. Altogether, our data shed light on the signaling pathways operated by NOV and strongly suggest that NOV mediates astrocyte activation and, therefore, might play a role in neuroinflammation. © 2010 Wiley‐Liss, Inc.     

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     

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.     

Synaptotagmin‐11 inhibits cytokine secretion and phagocytosis in microglia

Cytokine secretion and phagocytosis are key functions of activated microglia. However, the molecular mechanisms underlying their regulation in microglia remain largely unknown. Here, we report that synaptotagmin‐11 (Syt11), a non‐Ca²⁺‐binding Syt implicated in Parkinson disease and schizophrenia, inhibits cytokine secretion and phagocytosis in microglia. We found Syt11 expression in microglia in brain slices and primary microglia. Interestingly, Syt11‐knockdown (KD) increased cytokine secretion and NO release in primary microglia both in the absence and presence of lipopolysaccharide. NF‐κB was activated in untreated KD microglia together with enhanced synthesis of IL‐6, TNF‐α, IL‐1β, and iNOS. When the release capacity was assessed by the ratio of extracellular to intracellular levels, only the IL‐6 and TNF‐α secretion capacity was increased in Syt11‐KD cells, suggesting that Syt11 specifically regulates conventional secretion. Consistently, Syt11 localized to the trans‐Golgi network and recycling endosomes. In addition, Syt11 was recruited to phagosomes and its deficiency enhanced microglial phagocytosis. All the KD phenotypes were rescued by expression of an shRNA‐resistant Syt11, while overexpression of Syt11 suppressed cytokine secretion and phagocytosis. Importantly, Syt11 also inhibited microglial phagocytosis of α‐synuclein fibrils, supporting its association with Parkinson disease. Taken together, we propose that Syt11 suppresses microglial activation under both physiological and pathological conditions through the inhibition of cytokine secretion and phagocytosis.     

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.     

Cell autonomous and noncell‐autonomous role of NF‐κB p50 in astrocyte‐mediated fate specification of adult neural progenitor cells

In previous work, we demonstrated that NF‐κB p50 acts as crucial regulator of adult hippocampal neural progenitor cells (ahNPC). Indeed, NF‐κB p50 knockout (KO) mice are characterized by remarkably reduced hippocampal neurogenesis. As a follow up to that work, herein we show that when cultured in vitro, ahNPC from wild type (WT) and p50KO mice are not significantly different in their neurogenic potential. This observation prompted us to investigate cell‐autonomous and noncell‐autonomous consequences of p50 absence on neuronal fate specification of ahNPC. In particular, we focused our attention on astrocytes, known to provide soluble proneurogenic signals, and investigated the influence of WT and p50KO astrocyte conditioned media (ACM) on WT and p50KO ahNPC differentiation. Interestingly, while WT ACM promoted both neuronal and astroglial differentiations, p50KO ACM only supported astroglial differentiation of WT ahNPC. By using a LC–MS/MS approach, we identified some proteins, which are significantly upregulated in p50KO compared with WT astrocytes. Among them, lipocalin‐2 (LCN‐2) was recognized as a novel astroglial‐derived signal regulating neuronal fate specification of ahNPC. Interestingly, LCN‐2 proneurogenic effect was greatly reduced in p50KO NPC, where LCN‐2 receptor gene expression appeared downregulated. In addition to that, we demonstrated p50KO NPC unresponsiveness to both neuronal and astroglial fate specification signals from WT and p50KO ACM, and we identified a reduced expression of α2δ1, a thrombospondin‐1 receptor, as another phenotypic change occurring in ahNPC in the absence of p50. Altogether, our data suggest that dysregulated NPC‐astrocyte communication may contribute to a reduced hippocampal neurogenesis in p50KO mice in vivo. GLIA 2016 GLIA 2017;65:169–181