Nerve Growth Factor modulates LPS - induced microglial glycolysis and inflammatory responses

Author’s Accepted Manuscript Nerve Growth Factor modulates LPS - induced microglial glycolysis and inflammatory responses Georgia Fodelianaki, Felix Lansing, Prabesh Bhattarai, Maria Troullinaki, Maria Alejandra Zeballos, Ioannis Charalampopoulos, Achille Gravanis, Peter Mirtschink, Triantafyllos Chavakis, Vasileia Ismini Alexaki

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S0014-4827(19)30088-6 https://doi.org/10.1016/j.yexcr.2019.02.023 YEXCR11331

To appear in: Experimental Cell Research Received date: 16 December 2018 Revised date: 22 February 2019 Accepted date: 24 February 2019 Cite this article as: Georgia Fodelianaki, Felix Lansing, Prabesh Bhattarai, Maria Troullinaki, Maria Alejandra Zeballos, Ioannis Charalampopoulos, Achille Gravanis, Peter Mirtschink, Triantafyllos Chavakis and Vasileia Ismini Alexaki, Nerve Growth Factor modulates LPS - induced microglial glycolysis and inflammatory responses, Experimental Cell Research, https://doi.org/10.1016/j.yexcr.2019.02.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Nerve Growth Factor modulates LPS - induced microglial glycolysis and inflammatory responses

Georgia Fodelianaki1,#, Felix Lansing1, Prabesh Bhattarai1, Maria Troullinaki1, Maria Alejandra Zeballos1, Ioannis Charalampopoulos2, Achille Gravanis2, Peter Mirtschink1, Triantafyllos Chavakis1, Vasileia Ismini Alexaki1,#

1. Institute of Clinical Chemistry and Laboratory Medicine, University Clinic Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany 2. Department of Pharmacology, Medical School, University of Crete, Heraklion, Greece

#

Correspondence:

Dr. V.I. Alexaki, Institute for Clinical Chemistry and Laboratory Medicine, Fetscherstrasse 74, 01307 Dresden, Germany [email protected]

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Abstract Microglia, the parenchymal immune cells of the central nervous system, orchestrate neuroinflammation in response to infection or damage, and promote tissue repair. However, aberrant microglial responses are integral to neurodegenerative diseases and critically contribute to disease progression. Thus, it is important to elucidate how microglia - mediated neuroinflammation is regulated by endogenous factors. Here, we explored the effect of Nerve Growth Factor (NGF), an abundant neurotrophin, on microglial inflammatory responses. NGF, via its high affinity receptor TrkA, downregulated LPS - induced production of proinflammatory cytokines and NO in primary mouse microglia and inhibited TLR4 - mediated activation of the NF-κB and JNK pathways. Furthermore, NGF attenuated the LPS - enhanced glycolytic activity in microglia, as suggested by reduced glucose uptake and decreased expression of the glycolytic enzymes Pfkβ3 and Ldhα. Consistently, 2DG - mediated glycolysis inhibition strongly downregulated LPS - induced cytokine production in microglial cells. Our findings demonstrate that NGF attenuates pro-inflammatory responses in microglia and may thereby contribute to regulation of microglia - mediated neuroinflammation.

Keywords: microglia; neuroinflammation; NGF; neurotrophin; cell metabolism; glycolysis.

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Introduction Neuroinflammation is a feature of several Central Nervous System (CNS) diseases, such as Multiple Sclerosis (MS), Alzheimer’s Disease (AD), Parkinson’s Disease (PD) and Amyotrophic Lateral Sclerosis (ALS) (Glass et al. 2010; Ransohoff 2016). Microglia, the resident immune cells of the CNS have a central role in neuroinflammation (Kierdorf and Prinz 2017; Li and Barres 2018). In homeostasis, microglia coordinate neuronal development and plasticity (Kierdorf and Prinz 2017). They constantly patrol their microenvironment, provide trophic support to surrounding cells and orchestrate synapse formation (synaptic pruning) (Kierdorf and Prinz 2017; Li and Barres 2018). In response to tissue damage or infection, microglia get activated and release pro-inflammatory cytokines, reactive oxygen species (ROS) and nitric oxide (NO) (Saijo and Glass 2011). On the other hand, they also promote resolution of inflammation, tissue repair and remodeling through debris clearance by phagocytosis and neurotrophic support (Li and Barres 2018; Saijo and Glass 2011). However, aberrant microglial responses are a pathophysiological hallmark of many neurodegenerative diseases, since excessive microglial activation and associated overproduction of proinflammatory mediators can cause a neurotoxic environment, which critically contributes to disease progression (Block et al. 2007; Salter and Stevens 2017). Therefore, it is fundamental to gain a better understanding of the regulation of microglia – mediated neuroinflammation (Li and Barres 2018; Salter and Stevens 2017). Nerve Growth Factor (NGF) is an abundant neurotrophin, which is essential for neural development and survival (Bibel and Barde 2000). It is predominantly produced by neurons and astrocytes, but also oligodendrocytes, endothelial cells and microglia (Biane et al. 2014; Biernacki et al. 2005; Du and Dreyfus 2002; Elkabes et al. 1996; Ridet et al. 1997; Zhang et al. 2007). NGF binds with high affinity to the Tropomyosin receptor kinase A (TrkA), a receptor with tyrosine kinase activity, and with lower affinity to the pan-neurotrophin p75NTR 3

(Bibel and Barde 2000). Here, we investigate the effects of NGF on microglial inflammatory responses. We previously showed that microglia express TrkA, which can be activated by the neurosteroid dehydroepiandrosterone (DHEA), triggering anti-inflammatory responses (Alexaki et al. 2018). Here we show that the prototype ligand of TrkA, NGF, also negatively regulates lipopolysaccharide (LPS) - induced inflammation in a TrkA - dependent manner. This anti-inflammatory effect of NGF in microglia is associated with metabolic alterations, as shown by reduced glucose uptake and expression of glycolytic enzymes. These data indicate that NGF is an endogenous homeostatic factor modulating microglia - mediated neuroinflammation.

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Materials and Methods Primary microglial culture and treatments Primary microglial cells were isolated from brains of adult C57BL/6 mice as previously described (Alexaki et al. 2018). Cells were cultured in DMEM/F12 with Glutamax, supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin (all from Invitrogen, Darmstadt, Germany) and 5 ng/ml of murine recombinant Granulocyte and Macrophage Colony Stimulating Factor (GM-CSF) (Peprotech, United States of America, USA) in poly-L-lysine (Sigma-Aldrich, USA)-coated flasks at 37 °C and 5 % CO2. Before treatments, microglial cells were cultured for 2 days in the aforementioned medium without GM-CSF. Thereafter, cells were treated for 24 hours with 100 ng/ml NGF (Merck Millipore, Darmstadt, Germany) or vehicle control (PBS) and then incubated again with NGF or PBS together with either 100 ng/ml LPS (Ultrapure LPS, E. coli K12, Invivogen, USA) alone or 100 ng/ml LPS and 20 ng/ml murine Interferon gamma (IFNγ) (Peprotech, USA), as described in the figure legends. In some experiments, microglial cells were pre-treated for 30 min with 1 μM TrkA inhibitor, which inhibits the kinase activity of TrkA (Merck Millipore, Darmstadt, Germany), or control solution containing the same dilution of DMSO. For glycolysis inhibition, microglia were incubated in medium without or with 5 mM 2-deoxy-Dglucose (2DG) (Sigma-Aldrich, USA) for 30 min. All treatments were performed in reducedserum (1 % FBS) DMEM/F12 supplemented with Glutamax and antibiotics.

Bone marrow - derived macrophage (BMDM) culture and treatments BMDM were prepared from C57BL/6 mice, as previously described (Chung et al. 2017). After flushing the bone marrow and lysis of erythrocytes using red blood cell lysis buffer (eBioscience), bone marrow cells were cultured in RPMI-1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10 ng/ml GM-CSF for 7 days. Thereafter, 5

differentiated BMDM were treated for 24 hours with 100 ng/ml NGF or PBS, and then incubated again with NGF or PBS together with either 100 ng/ml LPS alone or 100 ng/ml LPS and 20 ng/ml IFNγ. All treatments were performed in reduced - serum (1% FBS) RPMI1640 supplemented with antibiotics.

Cytokine measurements Primary microglial cells were plated at a density 0.5x106 cells/well in 24-well cell culture plates coated with poly-L-lysine and treated with NGF or vehicle control (PBS) as well as with LPS or vehicle control (PBS). Cytokines were then measured in cell culture supernatants using the Meso Scale Discovery (MSD) V-Plex Pro-inflammatory Mouse Panel (Meso Scale Diagnostics, Rockville, Maryland, USA) in a MSD plate reader (QuickPlex SQ 120, Meso Scale Diagnostics, Rockville, Maryland, USA) according to the manufacturer’s protocol. Secretion of TNF was measured using the mouse TNF-alpha DuoSet ELISA kit (R&D Systems, Germany) in a Biotek Synergy HT multi-detection microplate reader (Biotek, Germany) following manufacturer’s instructions.

NO measurement Nitrite and nitrate production was measured in cell lysates using the NO Assay Kit (Fluorometric) (Abcam, Cambridge, United Kingdom) according to manufacturer’s instructions. Briefly, primary microglial cells were plated at a density of 1x106 cells/well in 12-well cell culture plates pre-coated with poly-L-lysine and were incubated with NGF or PBS and then with LPS or PBS, as described in the respective figure legend. Cells were lysed in 200 µl Assay Buffer and total nitrite and nitrate concentration was measured in 2-fold diluted samples following manufacturer’s instructions using a Biotek Synergy HT multidetection microplate reader (Biotek, Germany).

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2-Deoxy-D-glucose (2DG) uptake assay To measure glucose uptake, the Glucose Uptake-Glo Assay (Promega, Mannheim, Germany) was used. Primary mouse microglial cells were plated at a density of 8x104 cells/well in 96well cell culture poly-L-lysine-coated plates. Cells were treated or not for 18 hours with NGF or vehicle control (PBS) in the presence of LPS or vehicle control (PBS), as described in the figure legend. Cells were washed twice with glucose-free Agilent Seahorse XF Base Medium (102353-100, Agilent, USA) supplemented with 1 mM L-glutamine (Invitrogen, Darmstadt, Germany), pH 7.4 and incubated in the same medium for 45 min at 37 oC. 2DG was added to the medium at a final concentration of 10 mM and cells were incubated for 10 min at 37 oC. 2DG uptake was measured following the manufacturer’s instructions and the luminescence signal was measured using a Biotek Synergy HT multi-detection microplate reader (Biotek, Germany).

BV2 culture and treatments for Western Blot analysis The murine brain - derived microglial cell line BV2 (ICLC ATL03001) was purchased from Interlab Cell Line Collection (ICLC, Genova, Italy). Cells were maintained in RPMI medium (InVitrogen, Darmstadt, Germany) supplemented with 10 % FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C and 5 % CO2. Cells were treated or not for different time intervals with NGF (100 ng/ml), LPS (100 ng/ml) or NGF + LPS (both at 100 ng/ml) in RPMI medium with 1 % FBS. Western blot analysis was performed as previously described (Alexaki et al. 2018). Briefly, cells were washed twice with ice - cold PBS, lysed in lysis buffer (10 mM Tris pH 7.4, 1 % SDS, 1 mM sodium orthovanadate, 1:400 benzonase (SigmaAldrich, Munich, Germany)), supplemented with Mini Protease Inhibitor and Phosphatase Inhibitor Cocktail (Roche, Mannheim, Germany) and cell lysates were denatured by heating at 95 °C for 3 min. Protein samples (60 μg) were subjected to SDS-PAGE electrophoresis and transferred onto Hybond nitrocellulose membranes (Amersham Biosciences, Germany). 7

Membranes were blocked for one hour in 5% Bovine Serum Albumin (BSA) (Sigma-Aldrich, USA) or 5 % non-fat milk diluted in TBS-T buffer (0.15 M NaCl, 2.7 mM KCl, 24.8 mM Trisbase, 0.1 % Tween-20), and immunoblotted overnight at 4 °C. Antibodies used were rabbit anti-IkB-a (Cell Signaling, #9242, 1:500), mouse anti-phospho-SAPK/JNK (Thr183/Tyr185) (Cell Signaling, #9255, 1:500), rabbit anti-Vinculin (Cell Signaling, #4650, 1:1000) and mouse anti-Actin (Abcam, ab3280, 1:1000). Anti-mouse and anti-rabbit HRPconjugated secondary antibodies (R&D, Wiesbaden-Nordenstadt, Germany) were used at a dilution of 1:2000. After washes with TBS-T, membranes were developed using SuperSignal West Pico Chemiluminescent Substrate (Life Technologies) or SuperSignal West Fempto Chemiluminescent Substrate (Life Technologies) and the luminescent image analyzer LAS3000 (Fujifilm, Dusseldorf, Germany). The intensity of the bands was quantified with the Fiji software (Schindelin et al. 2012).

RNA extraction and gene expression analysis Total RNA was isolated from cells using the NucleoSpin RNA isolation kit (Macherey-Nagel, Duren, Germany). RNA was reverse transcribed with the iScript cDNA Synthesis kit (Biorad, Munich, Germany). cDNA was analyzed by qPCR using the SsoFast Eva Green Supermix (Bio-Rad, Munich, Germany), a CFX384 real-time System C1000 Thermal Cycler (Bio-Rad) and the Bio-Rad CFX Manager 3.1 software. The relative amount of the different mRNAs was quantified with the ΔΔCt method and using 18S rRNA for normalization (Alexaki et al. 2018; Chung et al. 2017). Primers used are described in Table 1.

Statistical analysis All values are presented as the mean ± standard errors of the means (SEM). Comparison between groups was made with Mann-Whitney U test, unpaired Student's t test or one-way

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ANOVA with p≤ 0.05 as a significance level using GraphPad Prism 6.0 Software (GraphPad Software, CA, USA). Results NGF attenuated pro-inflammatory responses of microglial cells in a TrkA – dependent manner. We first examined the effect of NGF on microglial inflammatory responses induced by LPS. To this end, mouse primary microglial cells pre-treated with either NGF or vehicle control (PBS) were stimulated with LPS or vehicle control (PBS) and analysed for cytokine production. NGF pre-treatment alone did not influence cytokine release (not shown), while it significantly downregulated LPS - induced tumour necrosis factor (TNF), interleukin (IL)6 and IL1beta release (Figure 1A-C) and LPS - induced Tnf, Il6 and Il1beta gene expression (Figure 1D-F). Furthermore, NO production in LPS - stimulated microglia was significantly reduced by NGF pre-treatment (Figure 1G). NGF did not affect cytokine expression or NO production in the absence of LPS (not shown). On the contrary, NGF did not influence cytokine expression in BMDM treated with LPS (Supplementary figure 1A-B) or LPS and IFNγ (Supplementary figure 1C-D). Microglia express the NGF receptor TrkA (Alexaki et al. 2018; Rizzi et al. 2018). Hence, we next examined whether the anti-inflammatory effect of NGF was mediated through TrkA. Cells were pre-treated with NGF in the presence of a TrkA inhibitor or vehicle control and were subsequently activated with LPS. Inhibition of TrkA abolished the inhibitory effect of NGF on LPS - stimulated Il6 expression in microglia cells (Figure 1H). The antiinflammatory effect of NGF was also observed in microglia activated with LPS and IFNγ. NGF pre-treatment down-regulated Tnf and Il6 expression in cells stimulated with LPS and IFNγ and these inhibitory effects of NGF were reversed by TrkA inhibition (Figure 1I,J). Our

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findings collectively indicate that NGF, acting through TrkA, reduced inflammatory reactions of microglial cells.

NGF down-regulated TLR4 signalling in microglial cells. Next, we questioned how NGF mediates its anti-inflammatory effects in LPS - stimulated microglial cells. LPS activates through Toll-like receptor 4 (TLR4) nuclear factor kappa-lightchain-enhancer of activated B cells (NF-κB), as evidenced by inhibitor of κB alpha (IκBα) degradation, and JUN N-terminal Kinase (JNK), which both mediate pro-inflammatory effects in microglia (Okun et al. 2009). BV2 cells were treated with NGF and LPS for different time intervals and the activation of these two pathways was examined. LPS-induced degradation of IκBα was reversed by co-incubation with NGF (Figure 2A). Similarly, NGF also inhibited LPS - induced JNK phosphorylation (Figure 2B). These data suggest that NGF regulated early TLR4 signalling in microglia thereby limiting microglial inflammatory activation.

NGF reduced LPS - driven glycolytic reprogramming in microglia. Previous reports demonstrated that LPS stimulation of microglia leads to increased glycolysis (Orihuela et al. 2016; Gimeno-Bayon et al. 2014). We questioned whether inhibition of glycolysis affects pro-inflammatory activation of microglia. To this end, mouse primary microglial cells were stimulated with LPS in the presence of the non-metabolisable glucose analogue 2DG for restriction of glycolysis or control solution and analysed for proinflammatory gene expression. 2DG significantly reduced LPS - induced release of IL6 and IL1β (Figure 3A,B), and expression of Il6, Il1β and nitric oxide synthase 2 (Nos2) (Figure 3D,E,G), as compared to control treated cells. On the contrary, 2DG did not influence LPS induced TNF release or Tnf expression (Figure 3C,F), suggesting that TNF expression is regulated by LPS independently of glycolysis.

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These findings prompted us to investigate whether NGF affects LPS - driven glycolytic activity in microglial cells. First, we addressed the effect of NGF on glucose uptake of LPS activated microglia. Mouse primary microglial cells were treated for 18 hours with LPS in the presence of NGF or vehicle control (PBS) and then glucose uptake was determined using 2DG. NGF significantly reduced LPS - induced 2DG uptake (Figure 4A). Since the glycolytic rate is controlled by rate-limiting glycolytic enzymes (TeSlaa and Teitell 2014), we then examined, whether NGF influences the expression of the rate-limiting glycolytic enzymes Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3) and Lactate dehydrogenase A (Ldhα). Consistently with its inhibitory effect on LPS-induced glucose uptake, NGF downregulated the LPS - enhanced expression of Pfkfb3 (Figure 4B) and Ldhα (Figure 4C). Collectively, these data suggest that NGF exerts anti-inflammatory effects and attenuates inflammation - associated upregulation of glycolysis in LPS - stimulated microglia.

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Discussion Neurodegenerative diseases are characterized by microglia - mediated neuroinflammation (Glass et al. 2010). Aberrant microglial activation contributes to neurotoxicity and neuronal destruction and thereby to the pathology of neurodegenerative CNS diseases (Li and Barres 2018; Ransohoff 2016; Saijo and Glass 2011). Microglial cells are highly sensitive to their microenvironment: they constantly surveil their surrounding with their moving processes seeking for potential insults, while they get rapidly activated by infectious or inflammatory stimuli (Li and Barres 2018). Conceivably, similarly to their responsiveness to inflammatory signals, microglial cells could also be sensitive to factors present in their microenvironment conferring regulation of microglial responses. Neurotrophins constitute a family of growth factors with important roles in neuronal growth, differentiation and survival (Bibel and Barde 2000). NGF is one of the best - studied neurotrophins, which, besides its neuroprotective potential, also exerts immunomodulatory properties (Minnone et al. 2017). Its receptor TrkA is expressed in a number of peripheral immune cells, such as B- and T- lymphocytes, monocytes/macrophages and dendritic cells (Minnone et al. 2017), as well as microglial cells (Alexaki et al. 2018). Both pro- and antiinflammatory effects were described for NGF (Minnone et al. 2017). For instance, it promotes survival of immune cells, activates chemotaxis and phagocytosis in neutrophils and macrophages, induces degranulation of mast cells and promotes differentiation and immune functions of B cells (Minnone et al. 2017). Thus, with few exceptions, such as its antiinflammatory effects in monocytes (Prencipe et al. 2014), NGF in the periphery acts in a rather pro-inflammatory fashion. On the contrary, in the CNS, NGF functions are predominantly anti-inflammatory. Intraventricular administration of NGF in Experimental Autoimmune Encephalitis (EAE), a widely used model of MS, reduced inflammatory cell infiltration and demyelination, decreased IFNγ and increased IL10 levels, delayed disease 12

onset and decreased disease severity (Arredondo et al. 2001; Flugel et al. 2001; Villoslada et al. 2000). (Arredondo et al. 2001) In accordance, transfer of myelin basic protein (MBP) specific CD4+T cells transduced to overexpress NGF failed to induce EAE, while it efficiently suppressed induction of EAE by non-transduced MBP-specific T cell transfer (Flugel et al. 2001). Furthermore, NGF depletion was associated with enhanced brain inflammation in rats subjected to EAE (Micera et al. 2000). Moreover, NGF - mediated engulfment of Aβ by microglia dampened Aβ - induced microglial inflammation in a TrkA - dependent manner and conferred neuronal protection against Aβ-induced loss of dendritic spines (Rizzi et al. 2018; Zhu et al. 2016). Here we studied the effect of NGF on LPS - induced microglial inflammatory responses. In accordance to its reported anti-inflammatory role in Αβ - stimulated microglia (Rizzi et al. 2018), NGF attenuated TLR4 - mediated pro-inflammatory activation. This effect was mediated through TrkA and was likewise observed in microglia activated with both LPS and IFNγ. Interestingly, NGF inhibited early TLR4 – mediated activation of the NFκB and JNK pathways, suggesting that TrkA signalling interferes with TLR4 signalling in microglia. However, the exact mechanism of this interference remains to be discovered in a future study. Moreover, NGF attenuated the LPS - induced glycolytic shift in microglia. In macrophages, the counterparts of microglia in the periphery, inflammation is associated with increased glycolysis, in order to cover the enhanced energy demands of inflammatory cells (Kelly and O'Neill 2015). Similarly, microglial inflammatory activation is also linked to enhanced glycolysis (Gimeno-Bayon et al. 2014; Orihuela et al. 2016; Holland et al. 2018). Here we showed that restriction of glycolysis by 2DG downregulated the LPS - induced expression of Il6, Il1β and Nos2 in microglia. In contrast, LPS - induced Tnf expression was not influenced by 2DG - mediated inhibition of glycolysis, similarly to what was reported in macrophages (Tannahill et al. 2013). These observations collectively indicate that glycolysis critically

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facilitates microglial inflammation and that NGF attenuates the latter at least partially through modulation of glycolysis. Thus, NGF emerges as a factor of therapeutic interest in the context of neurodegenerative diseases, not only due to its neuroprotective role, but also due to its role in regulation of neuroinflammation. However, therapeutic NGF administration is limited by its inability to cross the Blood Brain Barrier (BBB) and its poor pharmacokinetic properties (Faustino et al. 2017). Therefore, systemic administration of small BBB permeable molecules targeting TrkA appears as an attractive perspective. In this context, we have recently shown that systemically administered DHEA, as well as a non-metabolisable analogue of it, negatively regulated microglial - mediated neuroinflammation through TrkA activation (Alexaki et al. 2018; Pediaditakis et al. 2016). The present study together with our previous study therefore clearly establish the anti-inflammatory role of TrkA in microglia. Thus, modulation of TrkA signalling might represent an interesting way to therapeutically interfere with microglia mediated neuroinflammation.

Acknowledgments We would like to thank Mrs Sylvia Grossklaus and Mrs Christine Mund for excellent technical assistance.

Funding Supported by grants from the Deutsche Forschungsgemeinschaft (AL1686/3-1 and AL1686/22 to V.I.A. and SFB-TR 205 to V.I.A. and to T.C.). The funding source(s) had no involvement in the study design, collection, analysis and interpretation of data, writing of the manuscript and decision to submit the article for publication.

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Declaration of interest None

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18

Table 1: Real-time qPCR primers Gene name 18s Il6 Il1β Nos2 Tnf Pfkfb3 Ldhα

Forward primer

Reverse primer

5’-GTTCCGACCATAAACGATGCC-3’ 5’-CCTTCCTACCCCAATTTCCAAT-3’ 5’-TGGGATGATGATGATAACCTGC-3’ 5‘-ACCTTGTTCAGCTACGCCTT-3’ 5’-AGCCCCCAGTCTGTATCCTTCT-3’ 5’-AGAACTTCCACTCTCCCACCCAAA-3’ 5’-TGCCTACGAGGTGATCAAGCT-3’

5‘-TGGTGGTGCCCTTCCGTCAAT-3’ 5’-AACGCACTAGGTTTGCCGAGTA-3’ 5’-TCGTTGCTTGGTTCTCCTTGTA-3’ 5’-CATTCCCAAATGTGCTTGTC-3’ 5’-AAGCCCATTTGAGTCCTTGATG-3’ 5’-AGGGTAGTGCCCATTGTTGAAGGA-3’ 5’-GCACCCGCCTAAGGTTCTTC-3’

19

Figure legends Figure 1. NGF attenuated LPS-induced inflammatory responses in microglia. (A-C) Mouse primary microglial cells were pre-treated for 24 hours with 100 ng/ml NGF or vehicle control (PBS) and incubated again with NGF or PBS in the presence of 100 ng/ml LPS or PBS for 6 hours. Cytokine concentrations were measured in culture supernatants. Data are presented as mean ± SEM (n=3); * P≤ 0.05. ND: non-detectable (D-F) Microglia were pre-treated as in (A-C) and treated again with NGF or PBS in the presence of 100 ng/ml LPS for 4 hours. Cytokine gene expression was analyzed by qPCR. Relative gene expression of cells treated with PBS+LPS was set in each experiment as 1. Data are presented as mean ± SEM (n=4); * P≤ 0.05. (G) Microglial cells were treated as in (A-C) and nitric oxide was measured in cell lysates. Data are presented as mean ± SEM (n=4); ** P≤ 0.01; * P≤ 0.05. (H) Primary microglia were pre-treated for 24 hours with 100 ng/ml NGF or PBS in the presence of 1 μM TrkA inhibitor or control DMSO solution. Then they were treated again with NGF or PBS in the presence of TrkA inhibitor or DMSO and with 100 ng/ml LPS for 4 hours. Il6 gene expression was analyzed by qPCR and relative gene expression of cells treated with DMSO+PBS+LPS was set in each experiment as 1. Data are presented as mean ± SEM (n=5); * P≤ 0.05; n.s.: non-significant. (I-J) Microglia were pre-treated as in (H) and then they were treated again with NGF or PBS in the presence of TrkA inhibitor or DMSO together with 100 ng/ml LPS and 20 ng/ml IFNγ for 4 hours. Cytokine gene expression was analysed by qPCR. Relative gene expression of cells treated with DMSO+PBS+LPS/IFNγ was set in each experiment as 1. Data are shown as mean ± SEM (n=4); * P≤ 0.05; n.s.: non-significant.

Figure 2. NGF downregulated TLR4 signaling in microglia. (A) BV2 cells were treated or not for 60 min with vehicle control (PBS), 100 ng/ml LPS and PBS, 100 ng/ml NGF or the combination of LPS and NGF (both 100 ng/ml) and lysates were 20

analysed by western blot for IκB. Actin was used as loading control. Representative blots from one out of five independent experiments are shown. (B) BV2 cells were treated with 100 ng/ml NGF, 100 ng/ml LPS and PBS, or NGF and LPS (each at 100 ng/ml) for the indicated time intervals and lysates were analysed by western blot for phosphoJNK. Vinculin was used as loading control. The intensity of phosphoJNK and Vinculin bands was quantified at 30 min in 4 independent experiments and in each experiment the ratio phosphoJNK1(46kDa) / Vinculin and phosphoJNK2(54kDa) / Vinculin was set as 1 for samples treated with PBS+LPS. Data are mean ± SEM (n=4); * P≤ 0.05.

Figure 3. Glycolysis inhibition reduced LPS - induced inflammation in inflammatory microglia. (A-C) Mouse primary microglial cells were stimulated for 6 hours with 100 ng/ml LPS in the presence of 5 mM 2DG or vehicle control (PBS) and cytokine levels were measured in culture supernatants. Data are presented as mean ± SEM (n=3); * P≤ 0.05; n.s.: non-significant. (DG) Microglia were treated with 100 ng/ml LPS together with 5 mM 2DG or PBS for 4 hours and gene expression was analyzed by qPCR. Relative gene expression of cells treated with PBS+LPS was set in each experiment as 1. Data are presented as mean ± SEM (n=5); ** P≤ 0.01; * P≤ 0.05; n.s.: non-significant.

Figure 4. NGF attenuated LPS - induced glycolysis in microglia. (A) Mouse primary microglial cells were stimulated for 18 hours with 100 ng/ml LPS in the presence of 100 ng/ml NGF or PBS and then 2DG uptake was examined. Data are shown relative to samples treated with PBS+LPS; in each experiment the mean luminescence intensity of samples treated with PBS+LPS was set as 1. Data are presented as mean ± SEM (n=5); ** P≤ 0.01. (B,C) Primary microglia were pre-treated for 24 hours with 100 ng/ml NGF or PBS and treated again with NGF or PBS in the presence of 100 ng/ml LPS for 4 21

hours. Relative gene expression of cells treated with PBS+LPS was set in each experiment as 1. Data are presented as mean ± SEM (n=4); * P≤ 0.05.

Supplementary figure 1. –No effect of NGF on BMDM inflammatory responses. (A-B) Mouse primary BMDMs were pre-treated for 24 hours with 100 ng/ml NGF or vehicle control (PBS) and incubated again with NGF or PBS in the presence of 100 ng/ml LPS or PBS for 4 hours. Cytokine gene expression was analyzed by qPCR. Relative gene expression of cells treated with PBS+LPS was set in each experiment as 1. Data are presented as mean ± SEM (n=6); * P≤ 0.05; n.s.: non-significant. (C-D) BMDM were pre-treated as in (A-B) and were then treated again with NGF or PBS together with 100 ng/ml LPS and 20 ng/ml IFNγ for 4 hours. Cytokine gene expression was analysed by qPCR. Relative gene expression of cells treated with PBS+LPS/IFNγ was set in each experiment as 1. Data are shown as mean ± SEM (n=6); n.s.: non-significant.

22

DM Relative gene expression SO + DM PB SO S+L + P Trk NGF S Ai+ +LP Trk PBS S Ai+ +LP NG S F+ LP S 1.5

1.0

0.5

0.0

Tnf

0.0

H Il6

*

n.s. 1.5

1.0

1.5

1.0

0.5

0.0

E Il6

*

0.5

0.0

I

*

n.s. 0.5 2000

Tnf nM

*

30000 20000 10000

30 15 0 2

0

1.0

1.5

1.0

0.5

0.0

PB S+ LP S NG F+ LP S

0.5 40000

PB S+ LP S NG F+ LP S

ND

IL6

PB S

1.0

* 50000

PB S

300

pg/ml

**

DM SO DM +PB Relative gene expression SO S+L + P Trk NGF S/IFN Ai+ +LP γ Trk PBS S/IF Ai+ +LP Nγ NG S/I F+ FN LP γ S/I FN γ

1.5

B

PB S+ LP S NG F+ LP S

200

pg/ml

TNF

Relative gene expression

D PB S+ LP S NG F+ LP S

0

PB S

100

PB S+ LP S NG F+ LP S

400

Relative gene expression

PB S+ LP S NG F+ LP S

PB S

pg/ml

A

DM SO DM +PB Relative gene expression SO S+L + P Trk NGF S/IFN Ai+ +LP γ Trk PBS S/IF Ai+ +LP Nγ NG S/I F+ FN LP γ S/I FN γ

PB S+ LP S NG F+ LP S

Relative gene expression

Figure 1

Figure 1

C IL1β

8

*

6

4

ND

F G

Il1β Nitric Oxide

1.5

* 4000 3000

0.0

J

*

**

Il6

n.s.

*

1000 0

Figure 2

Figure 2

NGF+LPS

NGF

PBS+LPS

PBS

A

IkB Ac�n

B

NGF 0

pJNK

30

PBS+LPS 60

0

30

NGF+LPS

60 120

0

54kDa 46kDa

*

0.5

0.0

1.5

1.0

*

0.5

0.0

PB S+ LP S NG F+ LP S

1.0

pJNK 54kDa / Vinculin

1.5

PB S+ LP S NG F+ LP S

pJNK 46kDa / Vinculin

Vinculin

30

60 120

0

Il6

E

1.5

**

0.5

0.0 1.0

*

Il1β

1.5

**

0.5

0.0

pg/ml 800

F Tnf

1.5

n.s.

1.0

0.5

0.0

Relative gene expression

10000

10 9 8 7 6 5 4 3 2 1 0

PB S+ LP S 2D G+ LP S

20000

pg/ml

40000

C

PB S+ LP S 2D G+ LP S

1.0

* IL1β

PB S+ LP S 2D G+ LP S

D B

Relative gene expression

30000

PB S+ LP S 2D G+ LP S

IL6

Relative gene expression

PB S+ LP S 2D G+ LP S

pg/ml

A

PB S+ LP S 2D G+ LP S

PB S+ LP S 2D G+ LP S

Relative gene expression

Figure 3

Figure 3

TNF n.s.

600

400

200 0

G Nos2

1.5

1.0

**

0.5

0.0

1.5

1.0

**

0.5

0.0

Relative gene expression

2DG Uptake Pfkfb3

1.5

1.0

*

0.5

0.0

PB S+ LP S NG F+ LP S

B

Relative gene expression

PB S+ LP S NG F+ LP S

A

PB S+ LP S NG F+ LP S

Relative mean luminesence

Figure 4

Figure 4

C Ldhα

1.5

1.0

*

0.5

0.0