NF-κB In Astrocytes Research Articles

Droplet-based functional CRISPR screening of cell-cell interactions by SPEAC-seq.

Cell-cell interactions are essential for the function and contextual regulation of biological tissues. We present a platform for high-throughput microfluidics-supported genetic screening of functional regulators of cell-cell interactions. Systematic perturbation of encapsulated associated cells followed by sequencing (SPEAC-seq) combines genome-wide CRISPR libraries, cell coculture in droplets and microfluidic droplet sorting based on functional read-outs determined by fluorescent reporter circuits to enable the unbiased discovery of interaction regulators. This technique overcomes limitations of traditional methods for characterization of cell-cell communication, which require a priori knowledge of cellular interactions, are highly engineered and lack functional read-outs. As an example of this technique, we describe the investigation of neuroinflammatory intercellular communication between microglia and astrocytes, using genome-wide CRISPR-Cas9 inactivation libraries and fluorescent reporters of NF-κB activation. This approach enabled the discovery of thousands of microglial regulators of astrocyte NF-κB activation important for the control of central nervous system inflammation. Importantly, SPEAC-seq can be adapted to different cell types, screening modalities, cell functions and physiological contexts, only limited by the ability to fluorescently report cell functions and by droplet cultivation conditions. Performing genome-wide screening takes less than 2 weeks and requires microfluidics capabilities. Thus, SPEAC-seq enables the large-scale investigation of cell-cell interactions.

The impact of astrocytic NF-κB on healthy and Alzheimer’s disease brains

Astrocytes play a role in healthy cognitive function and Alzheimer’s disease (AD). The transcriptional factor nuclear factor-κB (NF-κB) drives astrocyte diversity, but the mechanisms are not fully understood. By combining studies in human brains and animal models and selectively manipulating NF-κB function in astrocytes, we deepened the understanding of the role of astrocytic NF-κB in brain health and AD. In silico analysis of bulk and cell-specific transcriptomic data revealed the association of NF-κB and astrocytes in AD. Confocal studies validated the higher level of p50 NF-κB and phosphorylated-p65 NF-κB in glial fibrillary acidic protein (GFAP)+-astrocytes in AD versus non-AD subjects. In the healthy mouse brain, chronic activation of astrocytic NF-κB disturbed the proteomic milieu, causing a loss of mitochondrial-associated proteins and the rise of inflammatory-related proteins. Sustained NF-κB signaling also led to microglial reactivity, production of pro-inflammatory mediators, and buildup of senescence-related protein p16INK4A in neurons. However, in an AD mouse model, NF-κB inhibition accelerated β-amyloid and tau accumulation. Molecular biology studies revealed that astrocytic NF-κB activation drives the increase in GFAP and inflammatory proteins and aquaporin-4, a glymphatic system protein that assists in mitigating AD. Our investigation uncovered fundamental mechanisms by which NF-κB enables astrocytes' neuroprotective and neurotoxic responses in the brain.

Astrocytic phagocytosis of myelin debris and reactive characteristics in vivo and in vitro.

Persistent myelin debris can inhibit axonal regeneration, thereby hindering remyelination. Effective removal of myelin debris is essential to eliminate the interference of myelin debris in oligodendrocyte progenitor cell (OPC) activation, recruitment to demyelinating sites and/or differentiation into mature oligodendrocytes (OLs). In addition to microglia, it has been reported that astrocytic phagocytosis of myelin debris is a feature of early demyelination. In the present study, astrocytes effectively phagocytized myelin debris in vitro and in vivo. On the 5th day after injecting myelin debris into the brain, astrocytes were enriched in the area injected with myelin debris compared with microglia, and their ability to engulf myelin debris was stronger than that of microglia. When exposed to myelin debris, astrocytes phagocytizing myelin debris triggered self-apoptosis, accompanied by the activation of NF-κB, down-regulation of Nrf2, and the increase of ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (bFGF). However, the activation of astrocytic NF-κB did not influence the inflammatory cytokines IL-1β, IL-6, and TNF-α, and the anti-inflammatory factor IL-10. The proliferation of astrocytes and mobilization of OPCs in the subventricular zone were elevated on the 5th day after intracerebral injection of myelin debris. The results suggested that myelin phagocytosis of astrocytes should help improve the microenvironment and promote myelin regeneration by increasing CNTF and bFGF within the central nervous system. However, the molecular interaction of astrocytes acting as phagocytes remains to be further explored. Therefore, an improvement of astrocytes to phagocytize myelin debris may be a promising treatment measure to prevent demyelination and promote remyelination in MS and other diseases with prominent myelin injury.

Neuroprotective action of α-Klotho against LPS-activated glia conditioned medium in primary neuronal culture

The α-Klotho is an anti-aging protein that, when overexpressed, extends the life span in humans and mice. It has an anti-inflammatory and protective action on renal cells by inhibiting NF-κB activation and production of inflammatory cytokines in response to TNF-α. Furthermore, studies have shown the neuroprotective effect of α-Klotho against neuroinflammation on different conditions, such as aging, animal models of neurodegenerative diseases, and ischemic brain injury. This work aimed to evaluate the effects of α-Klotho protein on primary glial cell culture against the proinflammatory challenge with LPS and how this could interfere with neuronal health. Cortical mixed glial cells and purified astrocytes were pretreated with α- α-Klotho and stimulated with LPS followed by TNFα, IL-1β, IL-6, IFN-γ levels, and NF-κB activity analysis. Conditioned medium from cortical mixed glia culture treated with LPS (glia conditioned medium (GCM) was used to induce neuronal death of primary cortical neuronal culture and evaluate if GCM-KL (medium from glia culture pretreated α-Klotho followed by LPS stimulation) or GCM + LPS in the presence of KL can reverse the effect. LPS treatment in glial cells induced an increase in proinflammatory mediators such as TNF-α, IL-1β, IL-6, and IFN-γ, and activation of astrocyte NF-κB. GCM treated-cortical neuronal culture induced a concentration-dependent neuronal death. Pretreatment with α-Klotho decreased TNF-α and IL-6 production, reverted NF-κB activation, and decreased neuronal death induced by GCM. In addition, KL incubation together with GCM + LPS completely reverts the neuronal toxicity induced by low concentration of GCM-LPS. These data suggest an anti-inflammatory and neuroprotective effect of α-Klotho protein in the CNS. This work demonstrated the therapeutic potential of α-Klotho in pathological processes which involves a neuroinflammatory component.

Widespread brain parenchymal HMGB1 and NF-κB neuroinflammatory responses upon cortical spreading depolarization in familial hemiplegic migraine type 1 mice

Neuroinflammatory changes involving neuronal HMGB1 release and astrocytic NF-κB nuclear translocation occur following cortical spreading depolarization (CSD) in wildtype (WT) mice but it is unknown to what extent this occurs in the migraine brain. We therefore investigated in familial hemiplegic migraine type 1 (FHM1) knock-in mice, which express an intrinsic hyperexcitability phenotype, the extent of neuroinflammation without and after CSD. CSD was evoked in one hemisphere by pinprick (single CSD) or topical KCl application (multiple CSDs). Neuroinflammatory (HMGB1, NF-κB) and neuronal activation (pERK) markers were investigated by immunohistochemistry in the brains of WT and FHM1 mutant mice without and after CSD. Effects of NMDA receptor antagonism on basal and CSD-induced neuroinflammatory changes were examined by, respectively, systemically administered MK801 and ifenprodil or topical MK801 application. In FHM1 mutant mice, CSD caused enhanced neuronal HMGB1 release and astrocytic NF-κB nuclear translocation in the cortex and subcortical areas that were equally high in both hemispheres. In WT mice such effects were only pronounced in the hemisphere in which CSD was induced. Neuroinflammatory responses were associated with pERK expression indicating neuronal activation. Upon CSD, contralateral cortical and striatal HMGB1 release was reduced by topical application of MK801 in the hemisphere contralateral to the one in which CSD was induced. This study reveals that neuroinflammatory activation after CSD is widespread and extends to the contralateral hemisphere, particularly in brains of FHM1 mutant mice. Effective blockade of CSD-induced neuroinflammatory responses in the contralateral hemisphere in FHM1 mice by local NMDA receptor antagonism suggests that neuronal hyperexcitability-related neuroinflammation is relevant in migraine pathophysiology, but possibly also other neurological disorders in which spreading depolarization is involved.

The neuronal (pro)renin receptor and astrocyte inflammation in the central regulation of blood pressure and blood glucose in mice fed a high-fat diet.

We report here that the neuronal (pro)renin receptor (PRR), a key component of the brain renin-angiotensin system (RAS), plays a critical role in the central regulation of high-fat-diet (HFD)-induced metabolic pathophysiology. The neuronal PRR is known to mediate formation of the majority of angiotensin (ANG) II, a key bioactive peptide of the RAS, in the central nervous system and to regulate blood pressure and cardiovascular function. However, little is known about neuronal PRR function in overnutrition-related metabolic physiology. Here, we show that PRR deletion in neurons reduces blood pressure, neurogenic pressor activity, and fasting blood glucose and improves glucose tolerance without affecting food intake or body weight following a 16-wk HFD. Mechanistically, we found that a HFD increases levels of the PRR ligand (pro)renin in the circulation and hypothalamus and of ANG II in the hypothalamus, indicating activation of the brain RAS. Importantly, PRR deletion in neurons reduced astrogliosis and activation of the astrocytic NF-κB p65 (RelA) in the arcuate nucleus and the ventromedial nucleus of the hypothalamus. Collectively, our findings indicate that the neuronal PRR plays essential roles in overnutrition-related metabolic pathophysiology.

NADPH oxidase1 inhibition leads to regression of central sensitization during formalin induced acute nociception via attenuation of ERK1/2-NFκB signaling and glial activation

Role of NADPH oxidase1 in the development of inflammatory pain has been demonstrated by gene knockout studies. Nevertheless, pharmacological inhibition of NOX1 is a requisite approach for therapeutic utility. Recently, we have reported the anti-nociceptive effect of newly identified NOX1 specific inhibitor ML171 (2-acetylphenothiazine). Inhibition of NOX1 resulted in attenuation of nociceptive sensitization during acute inflammatory pain via inhibition of ROS generation and its downstream ERK1/2 activation. However, glial activation accompanying inflammation is closely related to the initiation and maintenance of pain. Peripheral nociceptive inputs activate the primary afferents via release of various chemical mediators which are potentially capable of mediating signals from neuron to glia in DRG and subsequently in spinal cord dorsal horn. The subsequent interactions between neuron and glia contribute to pain hypersensitivity. Thus, the present study was focused to investigate the effect of ML171 on ERK1/2 signaling, glial activation, and crosstalk between neuron and glia in a mouse model of formalin induced acute nociception. Thus, the present study was focused to investigate the effect of ML171 on ERK1/2 signaling, glial activation, and crosstalk between neuron and glia in DRG and dorsal horn of the spinal cord of lumbar region (L3-L5) in a mouse model of formalin induced acute nociception.Intraperitoneal administration of ML171 decreased nociceptive behavioral responses, i.e. the flinch and lick counts, in formalin induced nociceptive mice. Immunofluorescence and Western blot analysis demonstrated decreased levels of nociceptive mediators like p-ERK1/2, p-NFκB p65, Iba1 and GFAP in DRG as well as in spinal cord dorsal horn; supporting anti-nociceptive potential of ML171. Further, co-localization studies showed the neuron-glia crosstalk in tissue dependent manner. ERK1/2 was found to be activated in glia and NFκB in neurons in DRG; whereas in case of spinal cord ERK1/2 was activated in neurons and NFκB in astrocytes. Decrease in nociceptive behavioral response and activation of nociceptive mediators after intraperitoneal administration of ML171 strongly advocate anti-nociceptive potential of ML171.This is the first report demonstrating modulation of ERK1/2-NFκB signaling pathway, glial activation and regulation of neuron-glia crosstalk by NADPH oxidase1 inhibition towards its anti-nociceptive action.

Mechanisms of activation induced by antiphospholipid antibodies in multiple sclerosis: Potential biomarkers of disease?

Multiple sclerosis (MS) is a chronic, multifactorial, inflammatory disease of the central nervous system where demyelination leads to neurodegeneration and disability. The pathogenesis of MS is incompletely understood, with prevalence of antiphospholipid antibodies (aPL) speculated to contribute to MS pathogenesis. In fact, MS shares common clinical features with the Antiphospholipid Syndrome (APS) such as venous thromboembolism. Consequently, the presence of aPL which are associated with blood clot formation in the APS need to be further investigated for a possible pro-coagulant role in the development of thrombosis in MS. The effects of IgG aPL from patients with MS upon astrocyte activation has never been characterized. We purified IgG from 30 subjects. A human astrocytic cell line was treated with 100 μg/ml IgG for 1 h, and cell extracts were examined by immunoblot using antibodies to p38 MAPK and NFκB to further examine intracellular signaling pathways induced by these IgGs. Only IgG from patients who are positive for aPL caused phosphorylation of p38 MAPK and NFκB in astrocytes. These effects were not seen with IgG from patients with MS but with no aPL or healthy controls. Understanding the intracellular mechanism of aPL-mediated astrocyte activation may help to establish new therapeutic approaches, such as selective inhibition of the mitogen-activated protein kinases, to control MS activity or possible thrombotic states.

Interleukin 4 inhibits high mobility group box-1 protein-mediated NLRP3 inflammasome formation by activating peroxisome proliferator-activated receptor-γ in astrocytes

High mobility group box-1 protein (HMGB-1) is one of the most important DAMPs and has been previously shown to promote the formation of the NOD-like receptor with pyrin domain containing-3 (NLRP3) inflammasome in microglia. Interleukin 4 (IL4) is a Th2-derived cytokine that plays a significant role in the function of various immune cells. However, the underlying molecular mechanism by which IL4 signaling antagonizes NLRP3 inflammasome is poorly characterized. In particular, whether IL4 could modulate NLRP3 inflammasome in astrocytes remains unknown. In the present study, we elucidated this phenomenon and the mechanism by which IL4 inhibits HMGB1-mediated NLRP3 inflammasome formation in astrocytes. For this purpose, we cultured and extracted primary astrocytes, setup different concentrations of HMGB1, and used immunofluorescence and western blotting to detect NLRP3 inflammasome formation, including NLRP3, ASC and caspase-1, and signaling changes in the nuclear factor κB (NF-κB). Meanwhile, BAY 11–7082 and IL4 were added with HMGB1 to observe the NLRP3 inflammasome and changes in NF-κB expression. Our data showed that HMGB1 could effectively promote NLRP3 inflammasome formation by activating NF-κB in astrocytes. This effect can be inhibited by BAY 11–7082, a NF-κB inhibitor. Meanwhile, IL4 could activate PPARγ via the STAT6 singling pathway and inhibit NF-κB activation, significantly decreasing formation of the NLRP3 inflammasome complex. Our study demonstrated that the NLRP3 inflammasome complex is also expressed in astrocytes, and IL4 could inhibit HMGB1-mediated NLRP3 inflammasome formation, through negative regulation of NF-κB activity and promotion of PPARγ activation.

Astrocytic NF-κB brings the best and worst out of microglia.

The role of astrocytes and microglia in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) remains incompletely understood. In this issue of The EMBO Journal , Alami et al (2018) employ a sophisticated genetic system that allows precise temporal control of NF‐κB activation in astrocytes to demonstrate that the timing of astrocyte activation is a key determinant of disease progression. Their results suggest that astrocyte activation drives microglia proliferation and that this can amplify not only the protective microglial effects in the presymptomatic phase of ALS, but also the detrimental microglial effects in the symptomatic phase.

Celastrol suppresses expression of adhesion molecules and chemokines by inhibiting JNK-STAT1/NF-κB activation in poly(I:C)-stimulated astrocytes.

In the central nervous system, viral infection can induce inflammation by up-regulating pro-inflammatory mediators that contribute to enhanced infiltration of immune cells into the central nervous areas. Celastrol is known to exert various regulatory functions, including anti-microbial activities. In this study, we investigated the regulatory effects and the mechanisms of action of celastrol against astrocytes activated with polyinosinic-polycytidylic acid (poly(I:C)), a synthetic dsRNA, as a model of pro-inflammatory mediated responses. Celastrol significantly inhibited poly(I:C)-induced expression of adhesion molecules, such as ICAM-1/VCAM-1, and chemokines, such as CCL2, CXCL8, and CXCL10, in CRT-MG human astroglioma cells. In addition, celastrol significantly suppressed poly(I:C)-induced activation of JNK MAPK and STAT1 signaling pathways. Furthermore, celastrol significantly suppressed poly(I:C)-induced activation of the NF-κB signaling pathway. These results suggest that celastrol may exert its regulatory activity by inhibiting poly(I:C)-induced expression of pro-inflammatory mediators by suppressing activation of JNK MAPK-STAT1/NF-κB in astrocytes.

Activation of NF-κB Signal Pathway and Downstream IL-1β Expression in Hippocampal Astrocytes of LPS-Induced Aged Rats

Background: The activation of nuclear factor-kappa B (NF-κB) signal pathway and downstream expression of IL-1β may play a pivotal role in neuroinflammation and cognitive dysfunction. We previously reported that a single lipopolysaccharide (LPS) dose induces prolonged neuroinflammation which is associated with astrocytic NF-κB signal pathways in aged rats. Blockade of the pathway by pyrrolidine dithiocarbamate (PDTC) in astrocytes could markedly suppress them, which may provide innovative ideas for clinical improvement for postoperative cognitive dysfunction (POCD). Methods: Rats were randomly assigned to vehicle, LPS, LPS+PDTC groups (n = 8 per group, per time point). 1) vehicle control (0.9% NaCl i.p.), 2) LPS (2 mg/kg i.p), 3) LPS + PDTC (LPS and PDTC50 mg/kg i.p.). After injection, whole brain tissues acquired at a series of time points (days 1, 3,7, 15 and 30) was prepared to carry out immunofluorescence, isolated hippocampal tissues were used for enzyme-linked immunosorbent assay (ELISA). NF-κB p65, p-IκBα and IL-1β were detected respectively by immunofluorescence and IL-1β protein levels were determined by ELISA. A separate cohort of rats (n=8-10/group) were tested in a Morris water maze (MWM) for spatial learning and memory. Results: The PDTC treatment suppressed the LPS-induced canonical NF-κB signaling pathway- nuclear translocation of NF-κB p65 and IκBα phosphorylation, positive expression of GFAP and IL-1β in hippocampal astrocytes of aged rats by immunofluorescence and reduced cognitive dysfunction by MWM test; The LPS-induced IL-1β protein increase in hippocampus of aged rats was attenuated by PDTC with ELISA. Conclusion: These findings suggest that NF-κB signaling pathway and downstream IL-1β in senescent astrocytes may play important roles in age-related neuroinflammation and cognitive dysfunction and provide potential discovery tool and therapeutic methods with PDTC for verifying the NF-κB signal pathway and treating cognitive impairment, especially POCD. (Funded by National Clinical Research for Geriatric Disorders of China, and Beijing Municipal Administration of Hospitals' Ascent Plan.) Citation: Min-Hui Kan, Hui-Qun Fu, Long Fan, Yan Wu, Tian-Long Wang. Activation of NF-κB Signal Pathway and Downstream IL-1β Expression in Hippocampal Astrocytes of LPS-Induced Aged Rats. J Anesth Perioper Med 2017; 4 : 149-61. doi: 10.24015/ JAPM.2017.0051 This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Effect of Huperzine A on Aβ-induced p65 of astrocyte in vitro.

Alzheimer's disease (AD) is the most common cause of dementia. Its pathology often accompanies inflammatory action, and astrocytes play important roles in such procedure. Rela(p65) is one of significant message factors in NF-κB pathway which has been reported high expression in astrocyte treated by Aβ. HupA, an alkaloid isolated from Chinese herb Huperzia serrata, has been widely used to treat AD and observations reflected that it improves memory and cognitive capacity of AD patients. To reveal its molecular mechanisms on p65, we cultured astrocytes, built Aβ-induced AD model, treated astrocytes with HupA at different concentrations, assayed cell viability with MTT, and detected p65 expression by immunohistochemistry and PCR. Our results revealed that treatment with 10μM Aβ1-42 for 24h induced a significant increase of NF-κB in astrocytes; HupA significantly down-regulated p65 expression induced by Aβ in astrocytes. This study infers that HupA can regulate NF-κB pathway to treat AD.

Regulating effect of activated NF-κB on edema induced by traumatic brain injury of rats.

To observe the effect of nuclear transcription factor-κB (NF-κB) on cerebral edema in rats with traumatic brain injury (TBI). Male SD rats with fluid percussion injury (FPI) were selected. After separation and culture, rats' astrocytes all suffered FPI. The expression of NF-κB and the water content were detected at the animal and cellular levels, while the activity of NOX was evaluated at the cellular level. According to the results, the positive expression of NF-κB and expression of mRNA were significantly increased and the water content was increased for rats after TBI, while NF-κB inhibitor BAY11-7082 could significantly reduce the effect of TBI. 1 and 3h after FPI of astrocytes, the activation of NF-κB was increased and BAY 11-7082 could significantly improve the injury-induced swelling of astrocytes. After the injury of astrocytes, the activity of NOX was also increased, while BAY 11-7082 could reduce the activity of NOX. The results show that the activation of NF-κB in astrocytes is a key factor in the process of cerebral edema after TBI of rats.

Signaling pathways regulating neuron-glia interaction and their implications in Alzheimer's disease.

Astrocytes are the most abundant cells in the central nervous system. They play critical roles in neuronal homeostasis through their physical properties and neuron-glia signaling pathways. Astrocytes become reactive in response to neuronal injury and this process, referred to as reactive astrogliosis, is a common feature accompanying neurodegenerative conditions, particularly Alzheimer's disease. Reactive astrogliosis represents a continuum of pathobiological processes and is associated with morphological, functional, and gene expression changes of varying degrees. There has been a substantial growth of knowledge regarding the signaling pathways regulating glial biology and pathophysiology in recent years. Here, we attempt to provide an unbiased review of some of the well-known players, namely calcium, proteoglycan, transforming growth factor β, NFκB, and complement, in mediating neuron-glia interaction under physiological conditions as well as in Alzheimer's disease. This review discusses the role of astrocytic NFκB and calcium as well as astroglial secreted factors, including proteoglycans, TGFβ, and complement in mediating neuronal function and AD pathogenesis through direct interaction with neurons and through cooperation with microglia.

Nuclear factor-kappa B regulates pain and COMT expression in a rodent model of inflammation.

Nuclear factor-kappa B (NF-κB) is a ubiquitously expressed protein complex regulating the transcription of genes involved in inflammation and pain. Increased NF-κB activity in immune and nervous system cells is linked to several chronic pain conditions in humans as well as inflammation and nerve injury-evoked pain in animals. A recent in vitro study further demonstrates that increased NF-κB activity in astrocytes decreases transcription of catechol-o-methyltransferase (COMT), an enzyme that inactivates catecholamines that cause pain. The purpose of the present study was to examine the relationship between systemic and astrocytic NF-κB activity, pain, and COMT expression in an animal model of inflammation. Results demonstrated that administration of the inflammatory stimulant complete Freund's adjuvant (CFA) led to increased pain and decreased COMT protein expression in an NF-κB-dependent manner. Specifically, we found that rats and mice receiving intraplantar CFA exhibited increased behavioral responses to mechanical and thermal heat stimuli. CFA-evoked pain was blocked in rats receiving a pre-emptive systemic dose of the NF-κB inhibitor MG132 and exacerbated in IKKca mice with constitutive NF-κB activity in astrocytes. Furthermore, we observed NF-κB-linked reductions in COMT expression in midbrain at 6h and 1d following CFA in rats and at 1h and 1d in forebrain and midbrain following CFA in IKKca mice. Collectively, these results demonstrate that systemic and astrocytic NF-κB activity drive inflammatory pain and regulate the expression of COMT in forebrain and midbrain structures.

A Commentary On: "NFκB-Activated Astroglial Release of Complement C3 Compromises Neuronal Morphology and Function Associated with Alzheimer's Disease". A cautionary note regarding C3aR.

GENERAL COMMENTARY published: 07 May 2015 doi: 10.3389/fimmu.2015.00220 A commentary on: “NF κ B-activated astroglial release of complement C3 compromises neuronal morphology and function associated with Alzheimer’s disease”. A cautionary note regarding C3aR Trent M. Woodruff 1 * and Andrea J. Tenner 2 School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia, 2 Department of Molecular Biology and Biochemistry, Institute for Memory Impairment and Neurological Disorders, University of California-Irvine, Irvine, CA, USA Keywords: complement, C3aR, Alzheimer’s disease, SB290157 A commentary on Edited by: Peter F. Zipfel, Leibniz Institute for Natural Product Research and Infection Biology, Germany Reviewed by: Daniel Ricklin, University of Pennsylvania, USA Josh Thurman, University of Colorado, USA *Correspondence: Trent M. Woodruff t.woodruff@uq.edu.au Specialty section: This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology Received: 30 March 2015 Accepted: 23 April 2015 Published: 07 May 2015 Citation: Woodruff TM and Tenner AJ (2015) A commentary on: “NFκB-activated astroglial release of complement C3 compromises neuronal morphology and function associated with Alzheimer’s disease”. A cautionary note regarding C3aR. Front. Immunol. 6:220. doi: 10.3389/fimmu.2015.00220 NFκB-activated astroglial release of complement C3 compromises neuronal morphology and function associated with Alzheimer’s disease by Lian H, Yang L, Cole A, Sun L, Chiang AC, Fowler SW, et al. Neuron (2015) 85:101–15. doi: 10.1016/j.neuron.2014.11.018 Introduction A recent study by Zheng and colleagues in Neuron (1) shows convincing evidence that activation of NF-κB in astrocytes induces expression of the complement component 3 (C3) and impairs neuronal function [summarized by Yates (2)]. The authors also state that the detrimental effects of astrocyte- derived C3 are due to the interaction of the C3 cleavage fragment, C3a, with its receptor (C3aR) on neurons (although no evidence of neuronal C3aR expression, nor generation of the cleavage product C3a, was provided). There are a number of caveats with this latter interpretation due to the use of the so-called C3aR-antagonist SB290157 (3), upon which they base this conclusion. These are summarized below. Off-Target Activity In 2004, Proctor and colleagues (4) reported that when SB290157 was administered to rats (at the same dose as used by Zheng and colleagues), it caused rapid neutropenia. Although SB290157 could block ischemia-induced intestinal pathology in rats, this therapeutic effect was shown not to be due to inhibition of the C3aR, but rather, due to the non-specific effect on circulating leukocyte concentrations (4). This finding was later replicated in mice by an independent group (5). These authors summarized that “C3aR antagonism does not appear to be responsible for the anti-inflammatory actions of this C3aRA” (4). Agonist Versus Antagonist Activity In 2005, Mathieu and colleagues (6) reported that SB290157 surprisingly had full agonist (not antagonist) activity at C3aR in many cell systems. They found that SB290157 completely activated Frontiers in Immunology | www.frontiersin.org May 2015 | Volume 6 | Article 220

Withaferin A Inhibits Nuclear Factor-κB-Dependent Pro-Inflammatory and Stress Response Pathways in the Astrocytes.

Several lines of evidence suggest that astrocytes play a key role in modulating the immune responses of the central nervous system (CNS) to infections, injuries, or pathologies. Yet, their contribution to these processes remains mostly elusive. Astroglia are endowed with a wide range of toll-like receptors (TLR) by which they can sense infectious agents as well as endogenous danger signals released by damaged cells. Here we demonstrate that the activation of astrocytic TLR4 by bacterial lipopolysaccharide (LPS) challenge can promote nuclear factor κB (NF-κB)-dependent induction of pro-inflammatory and stress response mediators, particularly Tumor Necrosis Factor α (TNFα), cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS). Since the steroid lactone Withaferin A was described to inhibit NF-κB activity in different cell types, we next determined the impact of this natural compound towards the identified astrocytic signalling pathway. Innate immune activation was induced by stimulation of the LPS/TLR4 axis in spinal cord astrocytes. We provide evidence that both pre-treating and post-treating the cells with Withaferin A attenuate astrocytic NF-κB activity as well as the consequent production of TNFα, COX-2, and iNOS induced by stimulation of the LPS/TLR4 pathway. This study suggests that Withaferin A may be an eligible candidate for the treatment of neuroinflammatory and stress conditions characterized by an important astrocytic input.

Activation of NF-κB Mediates Astrocyte Swelling and Brain Edema in Traumatic Brain Injury

Brain edema and associated increased intracranial pressure are major consequences of traumatic brain injury (TBI). While astrocyte swelling (cytotoxic edema) represents a major component of the brain edema in the early phase of TBI, its mechanisms are unclear. One factor known to be activated by trauma is nuclear factor-κB (NF-κB). Because this factor has been implicated in the mechanism of cell swelling/brain edema in other neurological conditions, we examined whether NF-κB might also be involved in the mediation of post-traumatic astrocyte swelling/brain edema. Here we show an increase in NF-κB activation in cultured astrocytes at 1 and 3 h after trauma (fluid percussion injury, FPI), and that BAY 11-7082, an inhibitor of NF-κB, significantly blocked the trauma-induced astrocyte swelling. Increased activities of nicotinamide adenine dinucleotide phosphate-oxidase and the Na(+), K(+), 2Cl(-) cotransporter were also observed in cultured astrocytes after trauma, and BAY 11-7082 reduced these effects. We also examined the role of NF-κB in the mechanism of cell swelling by using astrocyte cultures derived from transgenic (Tg) mice with a functional inactivation of astrocytic NF-κB. Exposure of cultured astrocytes from wild-type mice to in vitro trauma (3 h) caused a significant increase in cell swelling. By contrast, traumatized astrocyte cultures derived from NF-κB Tg mice showed no swelling. We also found increased astrocytic NF-κB activation and brain water content in rats after FPI, while BAY 11-7082 significantly reduced such effects. Our findings strongly suggest that activation of astrocytic NF-κB represents a key element in the process by which cytotoxic brain edema occurs after TBI.

Myoloid-Related Protein 8, an Endogenous Ligand of Toll-Like Receptor 4, Is Involved in Epileptogenesis of Mesial Temporal Lobe Epilepsy Via Activation of the Nuclear Factor-κB Pathway in Astrocytes

The role of Toll-like receptor 4 (TLR4) in the activation of innate immunity has been extensively studied in the past several years. Here, we are the first to report that myeloid-related protein 8 (MRP8), an endogenous TLR4 ligand, is involved in the epileptogenesis of mesial temporal lobe epilepsy (MTLE). We find that the expression of MRP8, TLR4, and interleukin 1-β (IL-1β) was upregulated in a MTLE model during both acute and chronic disease stages. We next investigated the possible roles played by astrocytes, which have been shown to be the major source of IL-1β during epilepsy. Stimulation via MRP8 led to the induction of IL-1β in astrocytes in vitro, accompanied by the activation of Nuclear Factor-κB, while knockdown of TLR4 or inhibition of NF-κB in astrocytes prevented this IL-1β induction. Thus, MRP8 may potentiate the perpetuation of MTLE by activating the NF-κB pathway in astrocytes, and could be a new target for anticonvulsant therapies.