Neurobiology of Disease 64 (2014) 16–29

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Neurobiology of Disease

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Complex regulation of acute and chronic neuroinflammatory responsesin mouse models deficient for nuclear factor kappa B p50 subunit

Taisia Rolova a,1, Lakshman Puli a,1, Johanna Magga a,b, Hiramani Dhungana a, Katja Kanninen a,Sara Wojciehowski a, Antero Salminen c, Heikki Tanila a, Jari Koistinaho a,⁎, Tarja Malm a

a Department of Neurobiology, A.I. Virtanen Institute, University of Eastern Finland, Finlandb Institute of Biomedicine, University of Oulu, Finlandc Department of Neurology, Institute of Clinical Medicine, University of Eastern Finland, Finland

Abbreviations: Aβ, β-amyloid; AD, Alzheimer's disprotein; APdE9, transgenic mice expressing APP695 prothuman presenilin 1 gene with exon 9 deletion; BM, bondeoxyuridine; CCL, CC chemokine ligand; CCR, CC chemdifferentiation; c/EBP, CCAAT-enhancer-binding proteihigh level; Iba1, ionized calcium-binding adapter molecsynthase; IL, interleukin; int, intermediate level; ko,lipopolysaccharide; MCP, monocyte chemotactic protein;Oct, octamer; PS1, presenilin 1; RANTES, regulated on actiand secreted; SSC, side scatter; tg, transgenic; TNF, tumor⁎ Corresponding author at: A.I. Virtanen Institute for M

Eastern Finland, P.O. Box 1627, FIN-70211, Kuopio, FinlanE-mail address: Jari.Koistinaho@uef.fi (J. Koistinaho).Available online on ScienceDirect (www.sciencedir

1 The authors have contributed equally.

0969-9961/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.nbd.2013.12.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 17 August 2013Revised 11 November 2013Accepted 4 December 2013Available online 15 December 2013

Keywords:MicrogliaBeta-amyloidInflammationCytokinesAlzheimer's disease

Inflammation is a major mechanism of acute brain injury and chronic neurodegeneration. This neuroinflamma-tion is known to be substantially regulated by the transcription factorNF-κB,which is predominantly found in theform of heterodimer of p65 (RelA) and p50 subunit, with p50/p50 homodimers being also common. The p65subunit has a transactivation domain, whereas p50 is chiefly involved in DNA binding. Binding of the p65/p50heterodimers is thought to induce expression of numerous proinflammatory genes in microglia. Here we showthat cultured microglia deficient for the gene (Nfkb1) encoding p50 subunit show reduced induction of proin-flammatory mediators, increased expression of anti-inflammatory genes, and increased expression of CD45, animmunoregulatory molecule, in response to lipopolysaccharide (LPS) exposure, but increased capacity to takeup β-amyloid (Aβ) which is associated with enhanced release of tumor necrosis factor alpha (TNFα). However,Nfkb1 deficiency strongly increases leukocyte infiltration and the expression of proinflammatory genes in re-sponse to intrahippocampal administration of LPS. Also, when crossing Nfkb1 deficient mice with APdE9 trans-genic mice the expression of proinflammatory genes was strongly enhanced, whereas Aβ burden was slightlybut significantly reduced. These alterations in expression of inflammatory mediators in Nfkb1 deficient micewere associatedwith reduced expression of CD45. Our data demonstrates a crucial and complex role p50 subunitof NF-κB in brain inflammation, especially in regulating the phenotype of microglia after acute and chronic in-flammatory insults relevant to clinical conditions, contributing to both pro-inflammatory and anti-inflammatory responses of microglia, infiltration of leukocytes, and clearance of Aβ in Alzheimer's disease.

© 2013 Elsevier Inc. All rights reserved.

Introduction

Nuclear factor kappa B (NF-κB) is an ubiquitously expressed mem-ber of the Rel family of transcription factors, which is essential for the

ease; APP, amyloid precursorein with Swedish mutation ande marrow; BrdU, 5-Bromo-2′-okine receptor; CD, cluster ofn; FBS, fetal bovine serum; hi,ule iNOS, inducible nitric oxideknockout; lo, low level; LPS,NF-κB, nuclear factor kappa B;vation, normal T cells expressednecrosis factor; wt, wild-type.olecular Sciences, University ofd.

ect.com).

ghts reserved.

proper function of the immune system (for review see Baeuerle andHenkel, 1994; Baldwin, 1996). In the central nervous system (CNS),some neurons exhibit constitutive NF-κB activity (Gabriel et al., 1999;Kaltschmidt et al., 1994b), and this activity is implicated in synapticplasticity (Kaltschmidt et al., 2006) and neurogenesis (Denis-Doniniet al., 2008). However, NF-κB can be dramatically up regulated in resi-dent glial cells, astrocytes andmicroglia, and also in neurons in responseto CNS injury and inflammation (Gabriel et al., 1999; Kaltschmidt et al.,1994a, 1997; Soos et al., 2004). Induced neuronal NF-κB activity in CNSdisorders is considered to mediate cell death/survival (Clemens et al.,1997; Duckworth et al., 2006; Kaltschmidt et al., 1999; Zhang et al.,2005),while NF-κB in glial cells is a key orchestrator of inflammatory re-sponse. NF-κB can be induced in response to various stimuli, such asproinflammatory cytokines, adenosine triphosphate, reactive oxygenspecies (ROS), physical stress and microbial products (Baeuerle andHenkel, 1994; Baldwin, 1996; Ferrari et al., 1997; Laflamme andRivest, 1999; Rosenstiel et al., 2001; Schreck et al., 1991; Zhang andGhosh, 2001). Canonical NF-κB activation pathway involves degrada-tion of inhibitory protein IκB and translocation of free NF-κB dimerfrom cytoplasm into the nucleus where it can bind to the DNA and

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activate transcription of the target genes (Baldwin, 1996; DiDonatoet al., 1997; Jacobs and Harrison, 1998; Urban et al., 1991; Zhang andGhosh, 2001).

In the brain, NF-κB is predominantly found in the form of heterodi-mer of p65 (RelA) and p50 subunits (Kaltschmidt et al., 1994b), withp50/p50 homodimers being also common. The p65 subunit has atransactivation domain, whereas p50 is chiefly involved in DNA binding(Baldwin, 1996; Kunsch et al., 1992). P50 is generated by the proteolyticcleavage of the precursor protein p105 encoded by the Nfkb1 gene. Un-like p65 knockoutmice that die during embryogenesis (Beg et al., 1995),the mice lacking Nfkb1 gene (Nfkb1 ko) develop normally, but exhibitsome defects in B and T-cell mediated immune responses (Sha et al.,1995), and cytokine-stimulated granulopoiesis (Wang et al., 2009).It is considered that the early phase of inflammatory response is con-tributed by the accumulation of p50/p65 dimers, known to activatetranscription of proinflammatory genes in microglia, such as cytokinestumor necrosis factor (TNF) α, interleukin (IL)-1β and IL-6, proteolyticenzymes, and inducible nitric oxide synthase (iNOS) (Kreutzberg,1996; Tanaka et al., 2006). In contrast, at the resolution phase of inflam-mation p50/p65 heterodimers are substituted by p50/p50 homodimers,which lack transcriptional transactivation domain and therefore arethought to have inhibitory function (Driessler et al., 2004; Elsharkawyet al., 2010; Kastenbauer and Ziegler-Heitbrock, 1999). Given the factsthat p50 can have opposite effect on the activation of NF-κB targetgenes depending on its binding partner and that NF-κB subunits can in-teract with other cellular signaling systems, such as AP-1 (Dendorferet al., 1994; Roebuck et al., 1999; Stein et al., 1993), Oct-1/Oct-2 (delaPaz et al., 2007; Lu et al., 2007; Meade et al., 2012), c/EBP-α/PU.1(Paz-Priel et al., 2009; Ponomarev et al., 2011), c/EBP-β (Dunn et al.,1994; Kunsch et al., 1994; Xia et al., 1997), IRF (Honda and Taniguchi,2006) and CREB (Martin et al., 2005), it is not surprising that the studiesusing Nfkb1 ko mice in various models of CNS injury have reportedsomewhat conflicting results.

Alzheimer's disease (AD) is characterized by the deposition of β-amyloid (Aβ) peptides (Dickson, 1997), hyperphosphorylation andaggregation of neuronal tau protein (Iqbal et al., 1998), and subsequentneuronal loss (Braak and Braak, 1991; Gomez-Isla et al., 1996; Priceet al., 1998). Aβ deposits termed “plaques” are surrounded by reactivemicroglia and astrocytes, and these cells are believed to contribute todisease pathogenesis and exacerbate brain damage (Akiyama et al.,2000). There have been several in vitro studies showing that microgliacan respond to Aβ directly by the release of cytotoxic inflammatoryme-diators, and thus promote neuronal degeneration (Bianca et al., 1999;Chen et al., 2005; Combs et al., 2001; Giulian et al., 1996; Meda et al.,1995). On the other hand, numerous in vitro and in vivo studies havedemonstrated that microglia can actually clear Aβ deposits in responseto active (Ard et al., 1996; Janus et al., 2000; Morgan et al., 2000; Nicollet al., 2003) and passive (Bard et al., 2000; Strohmeyer et al., 2005;Wilcock et al., 2004) immunization with Aβ or even after stimulationwith lipopolysaccharide (LPS) (DiCarlo et al., 2001; Malm et al., 2005).Importantly, exposure to Aβ also triggers activation of NF-κB in microg-lia (Bonaiuto et al., 1997; Chen et al., 2005).

LPS is probably the best-studied inducer of the canonical NF-κBpathway acting via CD14 receptors and, depending on the type ofLPS, also via toll-like receptor 2 and/or 4 (TLR2/4) on macrophages/microglia (Chakravarty and Herkenham, 2005; Dong et al., 2006;Heumann and Roger, 2002; Zhang and Ghosh, 2001). Whenadministered directly into the hippocampus, LPS causes widespreadmicrogliosis and production of inflammatorymediators without degen-eration of hippocampal neurons (Malm et al., 2005). On the other hand,APdE9 transgenic (tg) mouse overexpressing mouse amyloid precursorprotein (APP) gene with a human Aβ-encoding sequence and Swedishmutation, and human presenilin 1 (PS1) gene with exon 9 deletion(Jankowsky et al., 2001) is a widely used animalmodel of AD, especiallyfor studies on Aβ deposition. These mice exhibit age-related accumula-tion of Aβ (Jankowsky et al., 2004), reactive astrogliosis and

microgliosis, and memory impairment (Gimbel et al., 2010; Kanninenet al., 2009; Malm et al., 2007).

Here, we demonstrate a crucial and complex role of p50, the mostcommon NF-κB subunit in the brain, in acute and chronic neuroinflam-mation by utilizing Nfkb1 ko and APdE9 tg mice, primary microgliacultures and LPS administration. We chose to use transgenic ADmouse model as AD is the most common neurodegenerative diseasewith neuroinflammation. We show that Nfkb1 deficiency significantlyincreases Aβ clearance by microglia in vitro and ex vivo and reduces in-soluble Aβ burden in the brain.Moreover, whileNfkb1 deficiency in cul-tured microglia is associated with LPS-induced induction of CD45 and ashift in cytokine expression towards reduced inflammation, Nfkb1 defi-ciency in the brain strongly increases the LPS-induced infiltration ofleukocytes as well as LPS or Aβ-induced expression of proinflammatorymolecules, which are associated with reduced CD45 expression inmicroglia. The study emphasizes the complex role of NF-κB in neuroin-flammation, and shows that deficiency of Nfkb1may reduce inflamma-tion in vitro but enhance it in vivo.

Experimental procedures

Animals

Nfkb1−/− (p50/p105 knockout, hereafter referred as Nfkb1 ko)mice (Sha et al., 1995) were purchased from Jackson Laboratories (BarHarbor, ME, USA), back-crossed to the C57BL/6J strain for 6 generationsand maintained as homozygotes. APdE9 mice were obtained as de-scribed (Kanninen et al., 2009). The Nfkb1 ko mice were crossed withAPdE9 mice to establish a mouse line carrying mutated APP and PS1transgene on Nfkb1 null background. Non-transgenic littermates wereused as wild-type controls (wt). The animals were kept in the Lab Ani-mal Center at the University of Eastern Finland, on a 12 h light/darkcycle, and providedwith standard laboratory food andwater ad libitum.All animal work was conducted according to the national regulationsand the Council of Europe (Directive 86/609) of the usage and welfareof laboratory animals and approved by the Animal Experiment Commit-tee in State Provincial Office of Southern Finland.

Behavioral test methods

12-month-oldmale (14 wtmice, 8 ApdE9/Nfkb1 komice, 9Nfkb1 ko,and 15 ApdE9)mice from the ApdE9 tg × Nfkb1 komouse breedingweretested for their anxiety level and spatial learning capability byelevated plus maze and Morris water maze tests, respectively. Micewere caged individually andwere allowed to adapt to laboratory condi-tions for about a week prior to testing.

Elevated plus maze comprised a plus-shaped elevated platform(40 cm above floor), with two opposite arms open (length 30 cm,width 5 cm, ledge 0.25 cm) and two opposite arms enclosed withhigh walls (30 cm × 5 cm × 15 cm). All four arms merged into asquare platform (5 cm × 5 cm) in the center. This test was carried outin a quiet, dimly lit room. A camera mounted on top allowed the inves-tigator sitting in adjacent room to monitor the movements of mice onthe maze. The mouse was gently placed in the maze center and let tofreely explore the maze for 5 min. The number of arm entries and thetime spent on each arm was monitored with a semi-automaticcustom-made software. The number of visits and percentage of totaltime spent on open and closed arms were calculated and reported.Data from only those mice making 5 or more moves between thearms during the 5-min testing period were included into the statisticalanalysis.

Morris water maze comprised a black cylindrical plastic water tankwith a diameter of 120 cm located in a well-lit room. A black squareescape platform (14 cm × 14 cm) was located 1 cm below the watersurface. The platform location was kept constant and the startingposition varied between four constant locations at the pool rim. The

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temperature of thewater wasmaintained at 21 °C. Two days before theactual test, the mice were pre-trained to find the platform and climbonto it. The platform was placed half-way inside the black plastic alley(1 m × 14 cm × 25 cm) andwas hidden 1 cmbelow thewater surface.Micewere placed in thewaterwith their nose pointing towards thewallat one of the starting points in a randommanner. If the mouse failed tofind the platformwithin themaximum time (60 s), it was gently guidedtowards it. After reaching the platform, themouse was left to stay therefor 10 s. Each mouse got at least 5 min of rest between the trials. Thetest for the task acquisition consisted of four consecutive days of testing,with five trials per day. On day 5, the first and the 5th trial were probetrials without the platform to assess the search bias of the mice for60 s. A target zone was defined as a 30 cm diameter circle centered onthe previous platform location. Since target zone comprised 6.25% ofthe total surface area of the tank, in a probe trial of 60 s a randomlyswimming mouse would spend 3.75 s in the target area. If this timewas significantly exceeded, the mouse was considered to have learntsearch bias. Swim pattern was recorded and analyzed by video tracker(HVS Image, Hampton, UK) connected to a ceiling camera. Escape laten-cy, swimpath length and swimming speedwere determined by theHVSsoftware.

Intrahippocampal LPS injection

Themicewere injected intrahippocampally with 4 μg of LPS (4 μg/μlin physiological saline; LPS from Salmonella typhimurium; Sigma, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) or physiologicalsaline at the following coordinates: M/L ± 2.5 mm, A/P −2.7 mm,and D/V −2.5 mm from the bregma point under halothane (NicholasPiramal India Limited, Ennore, Chennai, India) or isoflurane (BaxterOy, Helsinki, Finland) anesthesia (the rest of the experiments). The in-jections were performed in a stereotactic apparatus (David Kopf,model 940, Tujunga, CA, USA) using a 5-μl syringe (Hamilton, Reno,NV, USA) over a period of 10 min (5 min each hippocampus). Afterthe injection, the skull was cleaned with saline and incision was closedwith silk sutures. In one experiment, in which brains were collected forhistological analysis 7 days after the LPS injection, 5-Bromo-2′-deoxyuridine (Sigma) was administered 4 h after the operation andthen every 24 h for 6 days as described in (Malm et al., 2008).

Immunohistochemistry and histochemical staining

The mice were anesthetized with an overdose of tribromoethanol(Avertin, Sigma) diluted in tert-amyl alcohol and flushed transcardiallywith heparinized saline (2.5 IU/ml). The brains were removed andpostfixed in 4% paraformaldehyde at 4 °C overnight. The fixed brainswere cryoprotected in 30% sucrose for 2 days, snap frozen in liquidnitrogen and cut with a cryostate into 20-μm-thick coronal sections(LPS-injection studies) at the dorsal hippocampal level starting1.3 mm from bregma or sagittal sections (uninfected APdE9 mice).Four to six sections at 400-μm intervals per animal were analyzedimmunohistochemically using the following antibodies: Iba1 (1:250;Wako, Richmond, VA, USA), CD68 (macrosialin; 1:200; Serotec, Oxford,UK), CD45 (1:100; Serotec), neutrophil (1:5000; Serotec), CCL5(RANTES; 1:9; R&D Systems; Minneapolis, MN, USA), BrdU (1:50;Boehringer Mannheim) and pan-Aβ (1:250; Biosource, Invitrogen, LifeTechnologies Corporation, Carlsbad, CA, USA). After overnight incuba-tion with the primary antibody, brain sections were incubated withappropriate Alexa Fluor 568-conjugated secondary antibodies (1:200;Molecular Probes, Eugene, OR, USA) or appropriate biotinylated second-ary antibodies (Vector Laboratories, Burlingame, CA, USA) followed byVectastain ABC peroxidase system (Vector) with Ni-enhanced 3,3'-di-aminobenzidine development. In each experiment a negative controlwas included, in which incubation with primary antibody was omitted.For congo red staining, sections mounted on slides were incubated insaturated alcoholic alkaline NaCl solution for 20 min followed by

incubation in alkaline 0.2% alcoholic congo red (Sigma) solution for an-other 30 min. The slideswere then rinsed quickly in 95% and 100% alco-hol solutions, dehydrated, cleared in xylene and coverslipped. To assessapoptosis, tissue sectionswere stainedwith ApopTagRed in situ apopto-sis detection kit (Chemicon, Millipore Corporation, Billerica, MA, USA)according to the manufacturer's instructions. Immunostained cellswere imaged under appropriate filter sets with Olympus AX70 micro-scope (Olympus, NY, USA) connected to ColorView camera (Soft Imag-ing System, Münster, Germany) (LPS-injection studies) or withOlympus BX40 microscope connected to Olympus optical DP50 camera(uninjected APdE9 mice). Immunoreactive areas were quantified withImagePro Plus (Media Cybernetics, Silver Spring, MD, USA) (LPS-injec-tion studies) or Photoshop CS3 extended version software (uninjectedAPdE9 mice). The area of interest (hippocampus) was outlined asshown in Fig. 3, and immunoreactive signal within the area of interestwas segmented by applying constant threshold. Data are expressed asthe percentage of immunoreactive area per whole area of interest. Thenumber of animals in each experiment is specified in figure legend.

Quantitative Real-Time PCR

RNA was extracted from frozen hippocampi by using TRIzolreagent (Invitrogen). RNA concentration and purity were measuredwith Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific,Waltham, MA, USA). cDNA was synthesized from 500 ng of total RNAusing randomhexamer primers as a template andMaxima reverse tran-scriptase (all reagents from Fermentas, Fermentas Finland, Helsinki,Finland). The relative expression levels of mRNA encoding mouseTNFα, IL-1β, IL-6, MCP-1 (CCL2), IL-10, TGFβ, iNOS, Iba1, and CD11bwere measured with quantitative RT-PCR StepOnePlus machine(Applied Biosystems, Life Technologies Corporation, Carlsbad, CA,USA) by using specific assays-on-demand (Applied Biosystems) targetmixes according to the manufacturer's instructions. The expressionlevels were normalized to ribosomal RNA and presented as fold changein the expression. The number of animals in each experiment is speci-fied in figure legend.

Protein isolation

Cytosolic and nuclear proteins were isolated from fresh hippocam-pal and cortical samples according to a protocol modified from (Ogitaet al., 2004). Hippocampi and frontal cortices were quickly dissectedout of freshly removed brains and placed on ice or snap frozen. Thesamples were homogenized in 10 volumes of homogenizing buffer con-taining 10 mM Tris–HCl buffer (pH 7.5), 0.32 M sucrose, 1 mM EDTA,1 mM EGTA, 5 mM dithiothreitol (DTT) and protease inhibitor mixture(Complete; Roche Applied Science, Mannheim, Germany), followed bythe addition of Nonidet P-40 (Roche Applied Science) at a final concen-tration of 0.5% (wt/vol). Homogenates were centrifuged at 6000 ×g at4 °C for 10 min, and cytosolic fractions were collected. The pelletswere washed twice with homogenizing buffer and resuspended in 10volumes of extraction buffer containing 50 mM Tris–HCl buffer(pH 7.5), 10% (vol/vol) glycerol, 400 mM NaCl, 1 mM EDTA, 1 mMEGTA, 5 mM DTT, 0.5% (wt/vol) Nonidet P-40 and the aforementionedprotease inhibitor mixture. The samples were incubated on ice for30 min with slow agitation and centrifuged at 16,000 ×g at 4 °C for10 min to obtain supernatants as nuclear extracts. Nuclear extractswere stored at−70 °C until used. Protein concentrations in the sampleswere determined byusing the Bio-Rad Protein Assay (Bio-Rad Laborato-ries; Hercules, CA, USA).

Equal expression of APP in Nfkb1 wt and ko mice was verified byWestern blotting. SDS/PAGE electrophoresis and blotting were per-formed as described previously (Kanninen et al., 2009). Briefly, cytosolicfractions from frontal cortical samples (6 samples in each group) wereresolved on a 10% SDS/PAGE gel, blotted onto polyvinyl difluoridemem-branes (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and

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probed overnight with anti-APP (A4) antibody (clone 22C11; 1:1000;Chemicon, Millipore), recognizing all isoforms of human and mouseAPP. To ensure equal protein loading, the same membranes wereprobed with anti-β-actin antibody (Sigma).

Analysis of NF-κB transcriptional activity

Electrophoretic mobility shift assay (EMSA) was performed asdescribed previously in detail (Helenius et al., 1996). Double-strandedconsensus oligonucleotides for NF-κB binding site were from Promega(Madison, WI, USA), mutated oligonucleotides were from SantaCruz Biotechnology (Santa Cruz, CA, USA). Probes were labeled withT4 polynucleotide kinase (Promega) and unspecific binding wasblocked by 2 μg of poly(dI-dC):poly(dI-dC) (Roche Applied Science).In supershift assays, binding mixtures were incubated with specific an-tibodies (Santa Cruz Biotechnology) to p50 (sc-1192X), p65 (sc-372X),and YY1 (sc-281X) for 1 h. Bound and free probes were separated on anative 4% polyacrylamide gel, and radioactive bands were visualizedwith Typhoon 9400 imager (GE Healthcare). Pixel volumes of specificbands (indicated as specific in representative pictures 2 B and D) werecalculated with ImageQuaNT software (GE Healthcare). The data wereexpressed as percentage of the band density in the non-injected wtsample. Analysis of p65 DNA-binding activity in the cortical samplesfrom APdE9 tg × Nfkb1 komice was done using NF-κB (p65) Transcrip-tion Factor Assay Kit (Cayman Chemical Company; Ann Arbor, MI, USA)according to the manufacturer's instructions, n = 6.

MCP-1 and Aβ ELISA

MCP-1 expression in cytosolic fractions of hippocampal sampleswasanalyzed by ELISA using CCL2/JE/MCP-1 Quantikine ELISA Kit (R&DSystems). Colorimetric reactionwas quantified by using the LabsystemsMultiscan MS Plate Reader (Thermo Fisher Scientific).

The content of soluble and insoluble Aβ species in the frontal corti-ces of APdE9 mice was assessed as described previously (Kanninenet al., 2009). Briefly, freshly frozen frontal cortices were homogenizedin PBS and centrifuged for 1 h at 48,000 ×g at 4 °C. The supernatantwas used to assess the level of soluble Aβ species. Insoluble Aβ wasextracted from the remaining pellet by using 5 M guanidine-HCl/50 mMTris · HCl, pH 8.0. The levels of Aβ42 and Aβ40were measuredby Human Aβ42 and Human Aβ40 ELISA kits, respectively (Biosource),and normalized to tissue weight. There were 10–11 samples in eachgroup.

Primary cultures

Mixed astrocyte/microglia cell cultures were prepared from fore-brains of newborn Nfkb1 ko or wt mice. The cells were cultured inDMEM medium supplemented with 10% FBS, 1% penicillin–streptomy-cin and 2 mM L-glutamine (all from Gibco, Invitrogen) in poly-L-lysinecoated 75 cm2

flasks. Type 1 microglia were harvested on day 12 andday 19 by shaking culture flasks for 15 min at 120 rpm at 37 ºC. Thecells were plated at the density of 90,000 cells per well on a 48-wellplate and incubated for 2 days to let them settle down in the restingstate. The cells were then stimulated with 100 ng/ml LPS (Escherichiacoli; Sigma) for 6, 24 or 48 h. Untreated wells were used as a control.The conditioned media were stored at −70 °C until further analysis.The cells were fixed with 10% formalin for 20 min and stored in PBS at4 °C.

To evaluate CD45 immunoreactivity, fixedmicroglia cells were incu-bated overnight with rat anti-mouse CD45 antibody (1:100; Serotec)followed by a 2 h-incubation with goat anti-rat Alexa Fluor 568-conjugated secondary antibody (1:200; Molecular Probes). Nucleiwere counterstained with bisbenzimide solution (5 μg/ml; Sigma). Im-munostained cells were visualized under appropriate filter sets withfluorescence microscope Olympus IX71 (Olympus) equipped with

color camera. Cell images were taken at 10× magnification, and the in-tensity of the staining was quantified using ImagePro Plus software.

The levels of IL-6, IL-10, MCP-1 (CCL2), TNFα, IFNγ and IL-12p70weremeasured from conditionedmedium samples with CBAMouse In-flammation Kit (BD Biosciences, San Jose, CA, USA) according to themanufacturer's instructions.

For some experiments, mouse bone marrow (BM)-derived mono-cytes were isolated with EasySep mouse monocyte enrichment kit(StemCell Technologies, Vancouver, British Columbia, Canada) accord-ing to the manufacturer's instructions and cultured, when these cellsadhered the plastic and differentiated towards microglia-like cells(Magga et al., 2012).

In vitro Aβ degradation and intracellular cytokine staining

For the Aβ uptake assay, cells were incubated in IMDM, 10% FBS,2 mM L-glutamine and penicillin–streptomycin (all from Invitrogen)overnight, followed by incubation with 0.5 μg/ml Aβ42 (HiLyte Fluor488, AnaSpec, Fremont, CA, USA) for 4 h and analyzed on a flowcytometer. A minimum of 10,000 events was acquired on FACSCaliburflow cytometer equipped with a 488 laser (BD Biosciences). The dataanalysis was performed using Cellquest Pro software (BD Biosciences),n = 3. For ex vivo Aβ degradation assay, brain sections were obtainedfrom 21-month-old APdE9 mice as described (Magga et al., 2012).Cells were applied onto the brain sections in IMDM medium supple-mented with 10% FBS, 2 mM L-glutamine, penicillin–streptomycin and10 ng/ml MCSF (R&D Systems). After 4 days of incubation, sectionswere collected and Aβ quantification performed with Aβ42 ELISA kit(Biosource). The data represent averages of 2 independent experiments(n = 9–12). The concentration of TNFα in themediumwas determinedwith ELISA kit (R&D Systems).

Statistical analysis

The data are expressed as mean ± SEM. The data were analyzedwith SPSS software (SPSS Inc. Chicago, IL, USA). The difference betweendata sets was considered statistically significant if p b 0.05. The detailsof statistical analysis for each experiment are specified in figure legend.

Results

Nfkb1 modulates cytokine secretion from LPS-stimulated microglial cells

Transcriptional factor NF-κB plays a central role in microglial func-tions. Therefore, we first determined the role of p50/p105 subunit ofNF-κB in inflammatory microglial responses in vitro. For this purposewe prepared primarymicroglia cell cultures fromwt andNfkb1 ko new-born mice, stimulated the cells with 100 ng/ml LPS and measuredcytokine release into the medium at 6, 24 and 48 h of the stimulation(see Fig. 1). Despite slightly higher levels of secreted TNFα in the ko cul-ture after 24 h stimulation (p b 0.01), the levels of IL-6 and MCP-1(CCL2) were dramatically reduced (p b 0.001) in comparison to thewt microglia (Fig. 1). Moreover, significant levels of IL-10, an anti-inflammatory cytokine, were detected only in the wt, but not Nfkb1 komicroglia cell cultures at 24 h and 48 h (Fig. 1). Neither wt nor ko mi-croglia produced any detectable levels of IFNγ and IL-12p70 (data notshown). Thus, the Nfkb1 deficient microglia demonstrated a clearly al-tered inflammatory response to bacterial LPS resembling to someextenttheM2 inflammatory responses inNfkb1 deficientmacrophages report-ed earlier (Cao et al., 2006; Porta et al., 2009).

LPS-induced NF-κB up regulation in hippocampus is blocked in Nfkb1ko mice

Having shown that the Nfkb1 deficiency modulates cytokineproduction in pure and isolated microglia cultures, we used our

Fig. 1.Nfkb1 komicroglia exhibited altered cytokine response to LPS. Cytokine secretionwasmeasuredwith cytometric bead array inflammation kit in culture supernatants from primarymurinemicroglia stimulatedwith 100 ng/ml LPS. The data represent averages of 2 experiments (n = 3 + 3) and are shown asmean ± SEM. P values are derived from one-way ANOVAwith Tukey's post hoc tests. ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001.

Fig. 2. Nfkb1 deficiency prevented LPS-induced NF-κB activation. (A) EMSA quantificationdata showing dynamics of NF-κB activation in the LPS-injected hippocampus. Wt micewere injected bilaterally with LPS and hippocampal samples from the left hemispherewere collected at different time points, n = 3–4. (B) A representative image of EMSAgel demonstrating specificity of the assay with the sample collected 48 h after the LPS in-jection.WhenDNA-bound complexeswere incubatedwith antibody specific for theNF-κBp50 subunit, but not NF-κB p65 subunit or unrelated transcription factor YY1, the assayshowed considerable supershift (ss), indicating that active NF-κB dimers contained p50subunit, but not as much p65 subunit, at 48 h time point. Sp, specific binding band; ns,non-specific band. (C, D) Quantification data (C) and a representative image of EMSA gel(D) showing up regulation of NF-κB DNA-binding activity in the wt, but not Nfkb1 ko hip-pocampi at 6 h time point, when one hippocampus was injectedwith LPS and the contra-lateral one with saline as a control, n = 3–4. Data in the graphs represent densitometricquantification of EMSA band indicated as specific (sp) in representative pictures B and Dand expressed as percentage of the band density in the non-injected wt sample. Dataare shown as mean ± SEM. P values are derived from one-way (A) or two-way (C)ANOVA followed by the Bonferroni's post hoc tests. In the graph (A), ⁎p b 0.05 in compar-ison to 0 h control; in the graph (C), ⁎⁎p b 0.01 in comparison to the wt LPS-injectedsamples.

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intrahippocampal LPS injection model to verify the role of NF-κB inbrain inflammation in vivo. When we injected wt mice bilaterally withLPS or saline into the hippocampi (4 μg each hippocampus) and ana-lyzed these tissues with electromobility shift assay, we found that LPSinduced a dramatic up regulation of NF-κB activity, reaching 5 timesthe uninjected control level already 5 h after the injection (p b 0.05)and staying at that high level for at least 2 days (Fig. 2A). At 2-daytime point most DNA-binding NF-κB dimers contained p50 subunit asdemonstrated by supershift assay (Fig. 2B). To confirm that there wasno NF-κB activation in Nfkb1 ko mice, we injected the ko and wt miceintrahippocampallywith LPS and evaluatedNF-κBDNA-binding activityin the hippocampi at 6 h time point. As expected, wt mice exhibitedstrong NF-κB binding activity at this time point, which was preventedby Nfkb1 deficiency (Figs. 2C, D).

NF-κB deficiency increases neutrophil infiltration after the LPS injectionin vivo

Activated microglia and infiltrated leukocytes, including neutro-phils, are the major constituents of LPS-induced neuroinflammation.Therefore, we next investigated whether Nfkb1 deficiency alters theresponse of these cells to the LPS injected into the hippocampus. InLPS-injected hippocampi, low levels of CD45 were expressed by en-dogenous microglia at 5 h time point, whereas at 14 h and 2-daytime points the injection site was dominated by newly infiltrated,strongly CD45-immunoreactive unramified leukocytes. At 5-daytime point CD45 immunoreactivity was still high, but was foundonly in ramified, microglia-like cells (Fig. 3A). In accordance withCD45 staining, LPS-induced infiltration of neutrophils peaked at 2-day time point (Fig. 3B), confirming the previously demonstratedtime-course of LPS-induced infiltration of these cells (Ji et al.,2007). Practically, no neutrophils were seen in saline-injected ani-mals at this time point. When neutrophil infiltration was comparedbetween the LPS-injected Nfkb1 deficient and wt mice, the LPS-induced neutrophil infiltration was found to be 34% higher in theLPS-injected ko mice compared to the corresponding wt mice(p b 0.05) (Fig. 4A). Similarly, there tended to be more CD45hi ex-pressing leukocytes in Nfkb1 ko animals at this time point

Fig. 3. Dynamics of leukocyte infiltration in LPS-injected wt hippocampi. Wt mice were injected bilaterally with LPS and histological samples of the right hemisphere were collected atdifferent time points. (A) Representative pictures and quantification data of CD45 staining. Arrows, CD45lo ramified cells; arrow heads, CD45hi cells. (B) Representative pictures and quan-tification data of the neutrophil staining (clone 7/4). Arrows, specifically stained cells. Immunoreactivity was quantified from 4 to 6 hippocampal sections as shown in the lower left paneland expressed as the percentage of the whole hippocampal area. Data in the graphs are shown as mean ± SEM. There were 3–6 animals per group. P values are derived from one-wayANOVA with Bonferroni's post hoc tests. ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 in comparison to the 5 h time point. Scale bar 50 μm.

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(p = 0.08, Student's t-test; Fig. 4A). We next used Iba1 (ionizedcalcium binding adaptor molecule 1) to evaluate the inflammationstatus of microglia. Iba1 is a macrophage/microglia-specific actin-cross-linking protein, which is essential for membrane ruffling,phagocytosis, migration and proliferation (Ohsawa et al., 2000).We found that neutrophil immunoreactivity inversely correlatedwith Iba1 immunoreactivity in the whole hippocampus at 2-daytime point (Fig. 4B) (p b 0.05). In fact, LPS injection caused a signifi-cant loss of Iba1-expressing cells around the injection site in both ge-notypes, but significantly more so in the ko animals (Fig. 4C). Inaddition, the increased number of infiltrated neutrophils was accom-panied by significantly increased expression of RANTES (CCL5), achemokine responsible for the recruitment of blood monocytes intothe brain parenchyma, around the blood vessels of the LPS-injectedko mice (Fig. 4D–F). These results indicate that Nfkb1 expression,most likely through p50-mediated NF-κB binding activity, protectsthe brain against the invasion of blood-derived leukocytes upon

brain inflammation. These findings also suggest that p50-mediatedNF-κΒ binding activity may be needed to maintain mobility andphagocytic properties of microglia.

Proinflammatory phenotype of microglia is enhanced in Nfkb1 deficientmice after LPS injection

Based on the immunohistochemical staining with anti-neutrophiland CD45 antibodies, no leukocytes were observed in the LPS-injectedhippocampi anymore at day 5 (Fig. 3A). Instead, numerous ramifiedmicroglia-like cells expressed strong CD45 immunoreactivity, suggest-ing suppression of pro-inflammatory activation of microglia at thatlate time point after LPS stimulation. To compare cytokine productionby activatedmicroglia between thewt and komice, and to avoid the ef-fects of infiltrating neutrophils, we measured mRNA levels of some in-flammatory cytokines and other inflammatory molecules by qRT-PCRin the hippocampi at 5 days after the LPS injection (Fig. 5A). The

Fig. 4. Nfkb1 deficiency enhanced neutrophil infiltration and disappearance of Iba1+ cells in LPS-injected hippocampi. Quantification data of neutrophil and CD45 (A), Iba1 (B, C), andRANTES (CCL5; D) -immunopositive areas from 4 to 6 hippocampal sections (A, B, D) or from only 3 middle sections including the injection site (C). The animals were injected bilaterallywith LPS or saline. Panel (A) shows data only from the LPS-injected animals because no neutrophils were seen in the saline-injected animals at this time point. Data are shown asmean ±SEM. There were 4–6 animals per group. P values are derived from Student's t-test (A) or two-way ANOVA followed by the Bonferroni's post hoc tests (B, C, D). ⁎p b 0.05, ⁎⁎p b 0.01 incomparison to the wt LPS group; #p b 0.05, ##p b 0.01 in comparison to the corresponding saline-injected controls. Panels (E) and (F) show representative pictures of RANTES staining inLPS-injected hippocampus. There were two types of staining: vascular (E) and microglial-like (F). Scale bar 50 μm.

Fig. 5. Nfkb1 deficiency modulated inflammatory gene expression and microglial phenotype in LPS-injected hippocampi. (A) Expression of cytokines and microglial cell markers in thehippocampus at 5 days after the LPS injection wasmeasured by qRT-PCR, n = 9–11. The contralateral hippocampi were injectedwith saline as a control. (B) Immunohistochemical anal-ysis of Iba1 and CD45 expression, and BrdU incorporation in the hippocampal region at 7-day time point after the LPS injection. There were 7–9 animals per group. 4–6 hippocampal sec-tions per animal were quantified as shown in Fig. 3. Data in the graphs are shown as mean ± SEM. P values are derived from two-way ANOVA (linear mixed model in panel (B)) withBonferroni's post hoc tests. ⁎p b 0.05, ⁎⁎⁎p b 0.001 in comparison to the wt LPS group; #p b 0.05 in comparison to the corresponding saline controls.

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mRNA levels of all proinflammatory cytokines measured (TNFα, IL-1β,IL-6 and MCP-1) and iNOS were 5–9-fold higher in the LPS-injected komice in comparison to the wt mice (p b 0.001). Interestingly, themRNA levels for anti-inflammatory cytokine TGFβwere also up regulat-ed in the ko hippocampi, but only 1.7-fold, while IL-4was below detect-able level in all the groups (data not shown). In contrast toproinflammatory cytokines, anti-inflammatory cytokine IL-10 wasexpressed at an 18-times lower level in the LPS-injected ko hippocampiin comparison to the corresponding wt controls. Thus, microglial re-sponse to LPS was clearly shifted towards pro-inflammatory phenotypeinNfkb1 komouse brain.We then further checked the expression ofmi-croglia markers Iba1 and CD11b at the same 5-day time point (Fig. 5A)in LPS-injected wt and Nfkb1 ko mice using qRT-PCR. CD11b is a part ofintegrin complex expressed on the cells of myeloid origin and is impli-cated in cell migration and complement-mediated phagocytosis(Choucair-Jaafar et al., 2011; Ross, 2000). We found that Iba1 was 36%down regulated, while CD11b was 37% up regulated in the LPS-injected Nfkb1 ko hippocampi compared to corresponding wt tissue.The down regulation of LPS-induced expression of Iba1 in Nfkb1 komicewas also confirmed by immunohistochemistry (Fig. 5B). Immuno-histochemical analysis also revealed that the LPS-induced expression ofCD45 at this 5 day time point was blocked in Nfkb1 ko mouse brain(Fig. 5B). Considering that CD45 is thought to negatively regulate pro-inflammatory cytokine receptor-mediated signaling, it was not surpris-ing that we observed a remarkable, almost 4-fold increase in CCR2mRNA level after LPS injection inNfkb1 ko hippocampi when comparedto wt hippocampi (Fig. 5A). The expression of CCR2 receptor is critical

Fig. 6.Nfkb1deficiencymodulated inflammatory response andAβ phagocytosis in 12-month-olTNFα, IL-1β and MCP-1 in the hippocampus of the double-transgenic AD × Nfkb1 ko and AD ×mate controls, n = 6. (B) NF-κB p65DNA-binding activity in the frontal cortex expressed as theical analysis of microglial markers (C) and amyloid deposits (E, pan-Aβ and Congo red staining)Six 20-μm thick sagittal sections from eachmouse were analyzed. (D) The content of PBS-insoluwt controls as measured by ELISA, n = 10–11. (F) Total APP expression was not affected byas mean ± SEM. P values are derived from one-way ANOVA followed by the Bonferroni's postAD × Nfkb1wt mice; #p b 0.05, ##p b 0.01 in comparison to the corresponding non-AD contro

for the infiltration of peripheral monocytes into the brain parenchymaand their differentiation into microglia (Mildner et al., 2007), as wellas for resident microglial activation (Zhang et al., 2007). Importantly,mRNA levels of CCR2 correlated well with the mRNA levels of its ligandMCP-1 (CCL2) in the hippocampus. These data confirmed that the mi-croglia phenotype was strongly more pro-inflammatory, displayedincreased properties of phagocytosis and migration, and favored infil-tration of peripheral inflammatory cells in the absence of p50/p105 sub-unit compared to wt mouse brain. Based on the normal gross anatomyand veryminor apoptosis staining (TUNEL) at 7 days after LPS injection,the LPS-induced expression of proinflammatory cytokines failed to leadto neuronal degeneration (data not shown). The barely detectableTUNEL-positivity was apparently caused by the mechanical damage bythe needle and did not differ between the genotypes. Total cell prolifer-ation indicated by BrdU incorporation, was 30% higher in the LPS-injected animals compared to saline-injected mice irrespective of thegenotype (Fig. 5B).

Nfkb1 deficiency enhances Aβ-induced inflammation in AD transgenicmice

Microglial inflammatory responses are associated with AD brainpathology. Aβ peptides and deposits, in particular, have been reportedto activate NF-κB in microglia (Bonaiuto et al., 1997; Chen et al.,2005). Therefore, we investigated whether Nfkb1 deficiency alters Aβ-induced microglial activation. For this purpose we crossed APPswe/PS1dE9 (APdE9) transgenic mice (further referred to as AD mice) withNfkb1 ko mice and established a mouse line carrying mutated APP and

d double-transgenic APdE9 (AD) × Nfkb1 komice. (A)QRT-PCRdata showing expression ofNfkb1wt (harboring wt Nfkb1 gene) mice and their non-AD (no APdE9 expression) litterpercentage of the activity in non-AD × Nfkb1wtmice, n = 5–6. (C, E) Immunohistochem-in the hippocampal area of AD × Nfkb1 komice and AD × Nfkb1wt controls, n = 11–12.ble (guanidine-soluble) Aβ42 in the frontal cortex of AD × Nfkb1 komice and AD × Nfkb1Nfkb1 deficiency in AD mice as demonstrated by Western blot, n = 6. Data are shownhoc tests (A, B) or from Student's t-test (C–F). ⁎p b 0.05, ⁎⁎p b 0.01 in comparison to thels.

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PS1 transgenes on NF-κB p50/p105 null background. We first evaluatedwhether APdE9 genotype affects NF-κB binding activity. As expected,NF-κB p65 DNA-binding activity was induced in 12-month-old ADmice and this induction of NF-κB p65 DNA-binding activity was blockedin AD × Nfkb1 komice, whichwere thus deficient forNF-κB p50 subunit(Fig. 6B). We next assessed whether cytokine expression in the hippo-campus of AD mice was altered and was dependent on Nfkb1. Whilethe mRNA levels of TNFα and to some extent also MCP-1, but not IL-1β, were significantly increased in AD mice compared to the corre-sponding wt controls, the AD × Nfkb1 ko mice showed a 1.9-fold in-crease in TNFα (p b 0.001) and IL-1β (p b 0.05) and a 2.5-foldincrease in MCP-1 expression (p b 0.05), when compared to the non-AD Nfkb1 ko mice (Fig. 6A). Moreover, AD × Nfkb1 ko mice expressed2.4 times more TNFα and 4.4 times more MCP-1 (p b 0.01), when com-pared to the normal AD mice. As Nfkb1 deficiency clearly regulated ex-pression of inflammatory mediators, we next analyzed expression ofmarkers of microglial activity Iba1, CD68 and CD45 in the hippocampusof these mice. Immunohistochemical analysis showed that while therewas no significant difference between normal AD and AD × Nfkb1 komice in Iba1 and CD68 protien expression levels in the hippocampus,CD45 immunoreactivity was decreased by 58% (Fig. 6C). Thus, the ex-tent of microgliosis was not altered in 12-month-old AD × Nfkb1 komice compared to AD mice. However, strongly reduced expression ofCD45 protein, a key immunomodulatory molecule, indicated that NF-κB may modulate microglial activation status at least partially viaCD45 when regulating the expression of pro-inflammatory cytokines.

We next determinedwhethermore pro-inflammatory phenotype ofmicroglia in AD × Nfkb1 ko mice affected the Aβ burden. At the age ofover 12 months about 98.5% of the Aβ42 is in insoluble form and issecreted about 5 times more than Aβ40 (Timmer et al., 2010). In thefrontal cortices, we detected a small (14%), but significant decrease(p b 0.05) in the level of Aβ42 in AD × Nfkb1 ko mice as compared tonormal AD mice as measured by ELISA (Fig. 6D). In the hippocampus,pan-Aβ immunostaining, which recognizes all species of Aβ, did notdemonstrate any difference between AD and AD × Nfkb1 ko mice(Fig. 6E). However, there was on average a 10% decrease, although notsignificant, in the congophilic amyloid deposits (dense core plaques)in AD × Nfkb1 ko mice. Aβ40 levels did not show significant changes(data not shown). Because the expression of APP protein was notaltered in AD × Nfkb1 ko brain when compared to AD brain (Fig. 6F),these results suggest that Nfkb1 deficiency slightly increases Aβclearance.

To determine whether more pro-inflammatory brain milieu togeth-er with slightly reduced insoluble Aβ42 burden affected learning abilityof themice, we performedMorriswatermaze test.Morris watermaze isa classic test for assessing spatial learning and memory in rodents. Es-cape latency is the time needed for a mouse to escape out of the openwater by reaching the hidden platform. On the first day, it took the

Fig. 7. Nfkb1 deficiency did not have an effect on spatial memory formation, but significantly rekomice. (A,B) Time needed to reach the hiddenplatform (A) and search bias (B, time spent in tharms expressed as the percentage of total time spent on the elevated plusmaze. Data are shownAD vs. non-AD mice; ⁎p b 0.05 (in C) Nfkb1 ko vs. Nfkb1wt mice.

mice long to reach the platform, but by day 5 all the mice learnt to es-cape by taking the shortest route (Fig. 7A). AD mice exhibited signifi-cantly increased escape latencies, suggesting impaired learning,(p = 0.007), irrespective of the Nfkb1 genotype (p = 0.40). Therewas no interaction between the two genotypes (p = 0.74). Probetrial (day 5, trial 5) did not reveal a significant effect of the Nfkb1genotype on search bias either (Fig. 7B, p = 0.49). Thus, more pro-inflammatory brain milieu associated with the Nfkb1 deficiency didnot worsen cognitive deficits in the AD mice.

In contrast to the Morris water maze test, Nfkb1 deficiency had asignificant impact on mouse behavior in the elevated plus maze test(see Fig. 7C). Elevated plus maze is a test for fear and anxiety in mice.Typically mice prefer to spend time on the closed arms of the mazewith only occasional visits to the open arms. The less anxious andstressed the mice are, the more they venture onto the open arms (e.g.,under the influence of anxiolytics). Nfkb1 deficiency significantly in-creased the time themice spent on the open arms (p = 0.015), suggest-ing they had lower anxiety level, irrespective of the APdE9 transgene.

Increased inflammation with Nfkb1 deficiency is associated with sustainedAβ phagocytosis and altered CD45 expression in microglia

Wenext further tested the role of p50 subunit of NF-κB in phagocyticactivity of microglia. As obtaining large numbers of primary microgliacells is challenging, and because monocytes represent the same cell lin-eage asmicroglia and differentiate towardsmicroglia in culture (Maggaet al., 2012), wemade use ofmonocytes isolated frommouse bonemar-row (BM) by depleting T cells, B cells, NK cells, dendritic cells,granulocytes, and hematopoietic progenitors. Wt or Nfkb1 ko mono-cytes were incubated on top of the brain sections showing heavy Aβburden (cut from aged APdE9 animals) for 4 days. ELISA measurementof Aβ42 content at the end of the incubation period demonstratedthat the ko monocytes phagocytosed Aβ42 about 30% more efficientlythanwtmonocytes (Fig. 8A). This ex vivo findingwas further confirmedwith fluorochrome-conjugated Aβ42 uptake assay using flow cytome-try. In this assay, after 4 h of incubation Aβ42 was found inside thecells in endosome-like vesicles (Fig. 8B). Again, the microglia-like cellsderived from Nfkb1 ko BM monocytes internalized Aβ42 about 50%more efficiently than the corresponding wt cells (Fig. 8C). When thesecultured cells were further characterized by flow cytometry, weobserved that the cells taking up fluorescent Aβ had high side scatterproperties (SSChi) and high CD11b expression (CD11bhi), whereas thecells that did not contain fluorescent Aβ showed low side scatter prop-erties (SSClo) and only intermediate CD11b expression (CD11bint).These data supported the idea that the Aβ clearing cells were the cellsthat had differentiated into macrophages whereas the cells not able totake up Aβ were presumably undifferentiated monocytes (Rogleret al., 1998) (Fig. 8D–F). Thus, these data support the idea that Nfkb1

duced anxiety level in 12-month-old Nfkb1 ko and double-transgenic APdE9 (AD) × Nfkb1e target zone during trial 5 on day 5) in theMorriswatermaze test. (C) Time spent on openasmean ± SEM, n = 8–15. P values are derived from two-way ANOVA. ⁎⁎p b 0.01 (in A)

Fig. 8. Nfkb1 deficiencymodulated Aβ phagocytosis and cytokine secretion in BM-derived monocytic cells. (A) BM-derivedmonocytes/macrophages reduced Aβ content in the brain sec-tions from ADmice ex vivo. The data are shown as percentages of Aβ42 content in control sections in the absence of added cells as measured by ELISA and represent averages of 2 inde-pendent experiments (n = 9–12). (B–G) fluorescent Aβ42 uptake assay evaluated by flow cytometry. (B) A representative picture of a cell containing internalized fluorescent Aβ42, scalebar 10 μm. (C) Higher percentage of untreated monocytic cells obtained from the Nfkb1 ko mouse BM was associated with Aβ42 after 4 h incubation as compared to the wt BM cells,n = 3; nt, no treatment. (D–G) BM-derived monocytic cells differed in their SSC characteristics. (E) SSClo cells exhibited intermediate levels of CD11b expression and predominantlydid not contain internalized Aβ42. (F) The most of SSChi cells expressed higher levels of CD11b and internalized Aβ42. (G) SSChi to SSClo ratio was increased on Nfkb1 null backgroundand by LPS treatment, n = 3. Data in the graphs are shown asmean ± SEM. P values are derived from one-way ANOVAwith Bonferroni's post hoc tests. ⁎p b 0.05, ⁎⁎p b 0.01 in compar-ison to the wt cells with the same treatment; ###p b 0.001 in comparison to the corresponding untreated control cells.

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deficient cells of monocyte lineage, including microglia, are indeedmore efficient in taking up and clearing Aβ when compared to corre-sponding wt cells. Moreover, our results suggest that p50 subunit ofNF-κB negatively regulates the differentiation of the cells of monocytelineage towards phagocytes. This hypothesis was further supported bythe findings that monocyte differentiation into macrophages (SSChi toSSClo cell ratio) was enhanced in monocyte cultures prepared fromNfkb1 ko cells compared to the cultures prepared from correspondingwt cells (p b 0.05), and this differentiation was strongly induced bythe stimulation with 10 ng/ml LPS in either genotype cells (p b 0.001;Fig. 8G). Interestingly, the increased Aβ phagocytosis by Nfkb1 ko cellswas associated with substantially increased TNFα secretion (16 ± 2and 107 ± 11 pg/ml (p b 0.001, n = 5–7) into the medium of Nfkb1ko and wt monocytes, respectively). Finally, because CD45 has beenreported to regulate various immune responses of microglia and theclearance of at least oligomeric form of Aβ, and we found that themicroglial CD45 induction observed in AD mice was completelyprevented when these mice were made Nfkb1 deficient, we analyzedCD45 protein expression in cultured Nfkb1 deficient microglia. Interest-ingly,while CD45 expressionwas slightly less inNfkb1 deficientmicrog-lia compared to wt microglia without any stimulation, LPS treatmenttime-dependently down regulated CD45 immunoreactivity in wtmicroglia, but up regulated CD45 immunoreactivity in Nfkb1 deficient

microglia, reaching a 76% difference to corresponding wt cells at 48 h(p b 0.001; Fig. 9). Altogether, our results show thatNfkb1 deficiency in-creases Aβ clearance by microglia both ex vivo and in vitro, and is asso-ciated with increased TNFα secretion but reduced CD45 expression.Moreover, LPS treatment of cultured microglia, which increases Aβclearance of wtmicroglia but notNfkb1 deficientmicroglia, correspond-ingly reduces CD45 expression in wt but not Nfkb1 deficient microglia,indicating that CD45 may at least partially mediate the NF-κB-regulated alterations of phagocytic activity in these cells.

Discussion

This study demonstrates a crucial and complex role of p50/p105subunit of NF-κB in brain inflammation, especially in regulating thephenotype of microglia after acute and chronic inflammatory insultsrelevant to clinical conditions. First, in pure cultured microglia cellsthe lack of p50/p105 subunit clearly reduced the LPS-induced pro-inflammatory phenotype but accelerated differentiation of monocyticcells into microglia/macrophages with enhanced ability to phagocytoseAβ. However, the lack of p50/p105 subunit in vivo resulted in strong en-hancement of LPS-induced neutrophil infiltration and expression ofpro-inflammatory cytokines as well as slightly reduced accumulationof Aβ in transgenic ADmice. Because p50 subunits mediate the binding

Fig. 9. LPS stimulation induced CD45 expression in the Nfkb1 ko, but not wt, primary mi-croglia in vitro. (A) Representative pictures of CD45-positive Nfkb1wt and ko microglia at0 h and 48 h of the stimulation with 100 ng/ml LPS. Scale bar 100 μm. (B) Quantificationof the CD45 staining in microglia expressed as mean fluorescence intensity. The data rep-resent averages of 2 experiments (n = 5–6) and are shown asmean ± SEM. P values arederived from one-way ANOVAwith Tukey's post hoc tests. ⁎⁎⁎p b 0.001 in comparison tothe wt cells with the same treatment; #p b 0.05, ###p b 0.001 in comparison to the corre-sponding 0 h control cells.

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of p65/p50 heterodimers that activate NF-κB-regulated genes, whilep50/p50 homodimers inhibit the same NF-κB-regulated genes, thelack of p50 subunit prevents both activation and inhibition of these tar-get genes via canonical NF-κB pathway. The expression of most NF-κB-regulated genes is also dependent on other transcription factors such asAP-1, Oct-1/Oct-2, c/EBP-α/PU.1, c/EBP-β, IRF and CREB. Together withthe previously published data on peripheral cell types, our results indi-cate that while the absence of p50/p105 subunit in microglia shifts theirphenotype from M1-type towards M2-type, corresponding to a switchfrompro-inflammatory status to anti-inflammatory one, p50/p105defi-ciency in the brain shifts the phenotype of microglia to the opposite di-rection, most likely due to the presence of other inflammatory cells andrelated signaling pathways that become activated upon exposure to LPSor Aβ-related pathology.

Good candidate cells to cause the difference in LPS-induced inflam-mation between the brain and microglia cultures of Nfkb1 deficientmice are neutrophils, which circulate in larger numbers in LPS-treatedNfkb1 ko mice (Mizgerd et al., 2004) and showed increased infiltrationto the brain in our study. CD45 is a master switch in guiding the inflam-matory response to M2 type (Tan et al., 2000a, 2000b). As neutrophilsgenerate enhanced and long-lasting production of ROS that can leadto (additional) release of cytokines such as TNFα and IL-6 via JNK path-way and inactivate Oct-1 thereby preventing p50-driven alterations inCD45 expression (Paz-Priel et al., 2009; Ponomarev et al., 2011; Wanget al., 2009), the overall expression of pro-inflammatory genes could re-main high and exacerbated in the brain in the absence of silencing p50subunits, whereas in culturedmicroglia the opposite outcomewould be

observed. This hypothesis is supported by our findings of opposite cyto-kine expression in culturedmicroglia and in the brain and that after LPStreatment CD45 expression was increased in Nfkb1 deficient microgliacultures but decreased in the brain of Nfkb1 deficient mice. While theexact mechanisms behind the difference in inflammatory response be-tween pure cultured microglia and the brain after LPS stimulation inNfkb1 deficient mice remain uncertain, our results indicate that studieson cell culture models alone are clearly insufficient for evaluating theimpact of transcription factors on inflammation in live animals.

Previous studies on the role of NF-κB and its subunit p50 in inflam-mation and leukocyte infiltration to the injured tissue are not consis-tent. Pharmacological inhibition of NF-κB activity or genetic deficiencyof p50 (Nfkb1 gene) has been reported to result in neuroprotection inbrain ischemia models (Nurmi et al., 2004a, 2004b). In line with thesestroke studies,Nfkb1 deficientmice show reduced infiltration of neutro-phils to the lung after LPS stimulation (Mizgerd et al., 2004). However,LPS-induced infiltration of monocytes and T cells is increased in the kid-ney of Nfkb1 deficient mice (Panzer et al., 2009). Similarly, injection ofliving E. coli into the lung (Mizgerd et al., 2003) or IL-1β into the stria-tum (Campbell et al., 2008) results in increased infiltration of neutro-phils to the corresponding tissues in these mice. While the findingsindicating protection by Nfkb1 deficiency have been explained bypreventing p65/p50 heterodimer from acting as a major inducer ofpro-inflammatory genes, the results showing detrimental effects ofthe Nfkb1 deficiency have been explained by the lack of repressingp50/p50 homodimers in regulation of proinflammatory genes. Consid-ering that Nfkb1 deficient mice lack both the proinflammatory effect ofp65/p50 heterodimers and the repressing effect of p50/p50homodimers, it is evident that the interaction of p50 subunit withother transcription factors is likely to play a role in regulation and espe-cially in induction of pro- and anti-inflammatory genes.

In AD, NF-κB is activated in neurons and glia around Aβ plaque-surrounding areas. In addition, strong evidence indicates that Aβ caninduce NF-κB binding activity in microglia directly as well as via in-creased expression of cytokines, ROS and advanced glycosylatedendproducts (AGEs) (Bonaiuto et al., 1997; Chen et al., 2005). Eventhough numerous studies have shown that enhanced NF-κB activityleads to increased expression and release of pro-inflammatory cyto-kines, there are only a few studies addressing the role of NF-κB in Aβphagocytosis. While pharmacological inhibitors of NF-κB have been re-ported not to affect Aβ phagocytosis in microglia, defective phagocyto-sis has been observed in macrophages of mice deficient for both c-Reland p50 (Courtine et al., 2012). Also in agreement with previous studies(Kassed and Herkenham, 2004), we found Nfkb1 deficiency to reduceanxiety-like behavior, but found no effect of Nfkb1 deficiency on learn-ing andmemory. It is thus surprising that we found improved phagocy-tosis of Aβ in NF-κB deficient microglia both in vitro and ex vivo as wellas reduced levels of insoluble Aβ in Nfkb1 deficient AD mice comparedto normal AD mice. The reduction of Aβ levels in Nfkb1 deficient ADmice was not caused by reduced expression of APP gene, as APP proteinlevels were not altered. The increased Aβ clearancewas associated withincreased expression of TNFα, whereas MCP-1 and IL6 as well as CD45,an immunoregulatory molecule reported to contribute to Aβ phagocy-tosis in AD mice (Zhu et al., 2011), showed opposite alteration in vitroand in vivo. While exact mechanism of enhanced Aβ clearance byNfkb1 deficient microglia and mice remains unclear, our findings showcorrelation of increased CD45 expression and increased Aβ phagocyto-sis in cultured microglia, which is in agreement with previous studies.Similarly to the acute effect of LPS treatment, the chronically increasedAβ production in the brain resulted in enhanced expression of pro-inflammatory genes in the absence ofNfkb1. Thus, it is likely that the in-teraction of microglia with other cells present in the brain as well as in-terplay between p50 and other transcription factors determine theinflammatory status also in the AD brain. The interacting cells thatwould result in increased inflammation in the brain cannot be neutro-phils, as these cells do not penetrate into CNS in AD or in AD mouse

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models. Instead, one may hypothesize that the perivascular macro-phages, which accumulate from the periphery in AD mouse models,may contribute to Aβ clearance, regulate inflammation and also expressthe sameNADPHoxidase as a source of ROS, could be a key cell type thatresults in aggravated inflammation in our ADmice.While these hypoth-eses remain highly speculative, further analysis of the interactionbetween microglia and perivascular macrophages or neutrophilswill be needed to clarify the role of p50 and NF-κB pathways in CNSinflammation.

Fig. 10. Schematic representation of the effect of Nfkb1 deletion onmicroglial phenotype in vitrscription of p65/p50 target genes cannot be activated in response to LPS, thus leading to a decreaCD45 and increased phagocytosis. Lower panel: in the brain,microglia are surrounded byother ior just perivascular macrophages in AD. In the Nfkb1 ko mice, LPS injection induces increased iproduced by neutrophils via JNK pathway changemicroglial phenotype towards more proinflamand c/EBP-α, are involved in the regulation ofmicroglial genes. The lack of inhibitory p50 homodCD45 expression.

Nfkb1 deficiency resulted in complex changes in the expression ofmicroglia markers. CD11b, a leukocyte-specific protein implicated incell migration and complement-mediated phagocytosis, showed in-creased up regulation, whereas Iba1, a macrophage/microglia-specificprotein essential for phagocytosis and proliferation, showed significantdown-regulation inNfkb1 deficientmice 5 days after LPS stimulation. Inaddition, CD45, a receptor type protein tyrosine phosphatase and a reg-ulator of immune responses expressed by all leukocytes, showedreduced expression in Nfkb1 deficient mice after LPS stimulation and

o and in vivo. Upper panel: in the absence of p50 subunit encoded by the Nfkb1 gene, tran-se in the production of proinflammatory cytokines IL-6 andMCP-1, increased expression ofmmune cells, such as neutrophils andperivascularmacrophages in the case of LPS injectionnfiltration of neutrophils into the brain parenchyma. ROS and proinflammatory cytokinesmatory. In addition to NF-κB, multiple transcription factors, such as IRF, CREB, AP1, Oct-1imers inNfkb1 ko animals increases expression of inflammatory cytokines, while reducing

28 T. Rolova et al. / Neurobiology of Disease 64 (2014) 16–29

inNfkb1 deficient ADmice. As Iba1 ismacrophage/microglia specific,weconfirmed its reduced induction in Nfkb1 deficientmice by immunohis-tochemistry at 2 days and 7 days after LPS stimulation.While the num-ber of Iba1-immunoreactivemicroglia was reduced also inwtmice afterLPS stimulation, the reduction was significantly more pronounced inNfkb1 deficient mice and inversely correlated with the number of infil-trated neutrophils. This finding is in agreement with a previous studyshowing that LPS-induced infiltrating neutrophils kill residentmicrogliaand that a substantial portion of CD11b-immunoreactive cells in thebrain soon after LPS treatment is in fact infiltrating neutrophils andmonocytes (Ji et al., 2007). This observation of enhanced toxicity of in-filtrating neutrophils in Nfkb1 deficient mice after LPS stimulation sup-ports the role of neutrophils as a primary source of mediatorsresulting in signals that promote inflammation in the absence of p50subunit of NF-κB.

In sum, Nfkb1 gene encoding for p50/p105 subunit of NF-κB hascomplex functions in regulation of acute and chronic neuroinflamma-tion, contributing to both pro-inflammatory and anti-inflammatory re-sponses of microglia cells, infiltration of leukocytes, and clearance ofAβ in AD. Even though NF-κB activity is thought to promote inflamma-tion in the brain, our data show that this is eventually true only in cellculture conditions, and in fact, genetic deficiency of p50 NF-κB subunitresulting in inactivation of NF-κB increases inflammation in vivo(Fig. 10).

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