Kinetic profile of the transcriptome changesinduced in the choroid plexus by peripheralinflammation

Fernanda Marques1, Joao C Sousa1, Giovanni Coppola2, Ana M Falcao1,Ana Joao Rodrigues1, Daniel H Geschwind2, Nuno Sousa1, Margarida Correia-Neves1

and Joana A Palha1

1Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, CampusGualtar, Braga, Portugal; 2Department of Neurology, David Geffen School of Medicine-UCLA, Los Angeles,California, USA

The choroid plexus, being part of the blood–brain barriers and responsible for the production ofcerebrospinal fluid, is ideally positioned to transmit signals into and out of the brain. This study,using microarray analysis, shows that the mouse choroid plexus displays an acute-phase responseafter an inflammatory stimulus induced in the periphery by lipopolysaccharide (LPS). Remarkably,the response is specific to a restricted number of genes (out of a total of 24,000 genes analyzed, 252are up-regulated and 173 are down-regulated) and transient, as it returns to basal conditions within72 h. The up-regulated genes cluster into families implicated in immune-mediated cascades and inextracellular matrix remodeling, whereas those down-regulated participate in maintenance of thebarrier function. Importantly, several acute-phase proteins, whose blood concentrations rise inresponse to inflammation, may contribute to the effects observed in vivo after LPS injection, assuggested by the differential response of primary choroid plexus epithelial cell cultures to LPSalone or to serum collected from animals exposed to LPS. By modulating the composition of thecerebrospinal fluid, which will ultimately influence the brain parenchyma, the choroid plexusresponse to inflammation may be of relevance in brain homeostasis in health and disease.Journal of Cerebral Blood Flow & Metabolism (2009) 29, 921–932; doi:10.1038/jcbfm.2009.15; published online25 February 2009

Keywords: choroid plexus; cerebrospinal fluid; lipopolysaccharide; blood–brain barrier; inflammation; transcriptome

Introduction

The precise mechanisms through which peripheralinflammatory stimuli trigger brain inflammation arepoorly understood. Interest on the subject is increas-ing in light of the importance of inflammation in thecentral nervous system (CNS) in multiple sclerosis(MS), and the more recent implications of CNSinflammation in neurodegenerative diseases such asParkinson and Alzheimer diseases.

Although most studies addressing communicationbetween the periphery and the CNS focus on theblood–brain barrier (BBB) formed by endothelialcells of the brain capillaries, considerably less

attention has been paid to the involvement of theblood–cerebrospinal fluid (CSF) barrier (BCSFB),formed by the choroid plexus (CP) epithelial cells.The CP, located within the cerebral ventricles, iscomposed of a vascularized stroma surrounded by atight layer of epithelial cells that restrict cellular andmolecular traffic between the blood and the CSF(Redzic and Segal, 2004). The CP is best known forits role in the production of the CSF that fills thebrain ventricles and the subarachnoid space, and it isideally located to transmit signals into and out of thebrain (Emerich et al, 2005).

To date, studies published on the CP responseto peripheral inflammatory stimuli have mostlyfocused on single proteins or groups of proteins.Among these are immune mediators such as inter-leukin IL-1b and TNF (Nadeau and Rivest, 1999;Quan et al, 1999), enzymes such as prostaglandin D2synthase (Marques et al, 2007) and bacteriostaticproteins such as lipocalin 2 (LCN2) (Marques et al,2008). Accessory molecules important for leukocyte

Received 31 October 2008; revised 28 January 2009; accepted 29January 2009; published online 25 February 2009

Correspondence: Dr JA Palha, Life and Health Sciences ResearchInstitute (ICVS), School of Health Sciences, University of Minho,Campus Gualtar, Braga 4710-057, Portugal.E-mail: japalha@ecsaude.uminho.pt

Journal of Cerebral Blood Flow & Metabolism (2009) 29, 921–932& 2009 ISCBFM All rights reserved 0271-678X/09 $32.00

www.jcbfm.com

adhesion such as L-selectin, intercellular adhesionmolecule 1 (ICAM-1), and vascular cell adhesionmolecule 1 have also been described as up-regu-lated on CP epithelial cells during inflammation(Engelhardt et al, 2001), eventually facilitating accessof immune cells into the brain. Notably, it has beenrecently suggested that the CP may be the primaryroute of immune cells entry into the CSF in theexperimental allergic encephalomyelitis model ofMS (Brown and Sawchenko, 2007). Similarly, the CPmay facilitate entry of bacteria, as indicated in amouse model of infection with Streptococcus suis(Dominguez-Punaro et al, 2007). These observations,suggesting that the contribution of the CP to thebrain’s innate and adaptative immune responses isstill only poorly understood, prompted the presentdetailed investigation of the CP’s response to acuteperipheral inflammation. Specifically, we evaluatedthe overall kinetic response of the mouse CP toperipheral administration of the Gram-negative bac-teria cell wall lipopolysaccharide (LPS) and foundthat the CP, and particularly its epithelial cells,activates a rapid and transient acute-phase responseto LPS that is ultimately reflected in the compositionof the CSF. On the basis of complementary studies inCP epithelial cells in culture, we propose a signalingcascade that may underlie this response.

Materials and methods

Animals and Lipopolysaccharide Injection

All experiments were carried out using 8 to 9 weeks oldC57BL/6 male mice and 8 to 9 weeks old Wistar rats(Charles River, Barcelona, Spain), in accordance with theEuropean Community Council Directive 86/09/EEC guide-lines for the care and handling of laboratory animals.Animals were maintained under 12 h light/dark cycle at22.51C and 55% humidity and fed with regular rodent’schow and tap water ad libitum. To reduce the stress-induced changes in the hypothalamus-pituitary axis,animals were handled for 1 week before the beginning ofthe experiment. Animals were injected intraperitoneallywith LPS (5mg/g of body weight; Escherichia coli, sero-type O26:B6; Sigma, St Louis, USA) or vehicle alone(0.9% NaCl). Mice were anesthetized with ketaminehydrochloride (150 mg/kg) plus medetomidine (0.3 mg/kg), transcardially perfused with cold saline and killed 1,3, 6, 12, 24, or 72 h after LPS injection. For mRNA studies,the CP was rapidly removed from all ventricles, stored inRNA later (Ambion, Austin, TX, USA) and kept at �801C.The CP isolation was made under conventional lightmicroscopy (SZX7, Olympus, Hamburg, Germany). At leasttwo pools of CP (from three mice each) were prepared foreach time point. This experiment was performed twice: CPsamples from one experiment were used for microarrayanalysis; CPs collected in an independent experiment wereused to confirm, by quantitative real-time–polymerasechain reaction (qRT–PCR), the results obtained in the arraystudy. In this second experiment, five pools of CP wereprepared for each time point.

The CSF was collected from the cisterna magna andpooled from several mice. An aliquot of each pool was usedto verify the absence of blood contamination and theremainder immediately frozen until use.

Rats were similarly anesthetized and killed 3 and 6 hafter LPS injection. Blood was collected and used tostimulate primary cultures of rat CP epithelial cells.

Microarray Experimental Design and Data Analysis

Total RNA was isolated with Trizol (Invitrogen, Carlsbad,CA, USA) following manufacturer’s instructions. Afterquality assessment using the Agilent Bioanalyzer (AgilentTechnologies, CA, USA), 100 ng of total RNA from threepooled controls and two pooled samples from each timepoint were amplified and labeled with Illumina TotalPrepRNA Amplification Kit (Illumina Inc, San Diego, CA,USA). The labeled cRNA was then hybridized using therecommended protocol in a total of two Illumina Whole-genome Mouseref-8 expression Beadchips (Illumina Inc).This mouse beadchip contains eight arrays, each compris-ing a total of 24,000 well-annotated RefSeq transcripts.

After scanning, raw data from BeadStudio software(Illumina Inc) was read into R/Bioconductor and normal-ized using quantile normalization. A linear model wasapplied to the normalized data using Limma package in R/Bioconductor (Gentleman et al, 2004). A contrast analysiswas applied and differentially expressed genes wereselected using a Bayesian approach with a false discoveryrate of 5%.

The differentially expressed genes were categorizedusing Gene Ontology from Biomart (http://www.biomart.org/) or Ingenuity tools (Redwood City, CA, USA). Enrich-ment analysis was performed using the DAVID (http://david.niaid.nih.gov/david/ease.htm) and the Ingenuitysoftwares.

Gene Expression Measurements by QuantitativeReal-Time–Polymerase Chain Reaction

As described earlier, 500 ng of total RNA isolated wereamplified using a SuperScript RNA Amplification System(Invitrogen) according to the manufacturer’s instructions.After amplification, RNA was reverse transcribed into firststrand cDNA using random hexamers of the SuperscriptFirst-strand Synthesis System for RT–PCR (Invitrogen).

The qRT–PCR analysis was used to measure the expres-sion levels of selected mRNA transcripts. Primers weredesigned using the Primer3 software (Rozen and Skaletsky,2000) on the basis of the respective GenBank sequences.The expression level of the reference gene hypoxanthineguanine phosphoribosyl transferase (Hprt) (accessionnumber from GenBank: NM_013556) was used as internalstandard for normalization, as we have first confirmed thatits expression is not influenced by the experimentalconditions (Marques et al, 2007).

All other accession numbers and primer sequences areavailable on request. Reactions using equal amounts oftotal RNA from each sample were performed on a Light-Cycler instrument (Roche Diagnostics, Basel, Switzerland)

Choroid plexus acute inflammatory responseF Marques et al

922

Journal of Cerebral Blood Flow & Metabolism (2009) 29, 921–932

with the QuantiTect SYBR Green RT–PCR reagent kit(Qiagen, Hamburg, Germany) according to the manufac-turer’s instructions. Product fluorescence was detected atthe end of the elongation cycle. All melting curvesexhibited a single sharp peak at the expected temperature.

Cerebrospinal Fluid Analysis

Immune mediators (IL-6, IL-10, IL-12p70, TNF, IFNg, andCCL2) were measured using BD cytometric bead array (BDBiosciences, CA, USA) according to the manufacturer’sinstructions in 10 mL of pooled CSF samples obtained fromanimals injected with LPS at different time points. Three tofour pools of CSF from up to five animals each were usedfor the various time points studied. The detection limit forall proteins was 20 pg/mL.

Primary Cultures of Rat Choroid Plexus EpithelialCells

The CP is composed of a vascularized stroma surroundedby a tight layer of epithelial cells. Therefore, to study towhat extent the response observed in vivo was mediated byepithelial cells, we extended our study to the in vitroresponse of primary cultures of rat CP epithelial cells. Wechose to perform the primary cultures with rats for tworeasons: (1) the amount of tissue is considerable greaterwhen compared with that of the mouse, (2) as a secondanimal model for determining the CP response.

Epithelial cells from rat CP were prepared as describedpreviously by Strazielle and Ghersi-Egea (1999) with minormodifications. Briefly, neonates (postnatal day 3 or 4) werekilled and CP were dissected under conventional lightmicroscopy (SZX7, Olympus). The tissue was rinsed twicein phosphate buffered saline (without Ca2 + and Mg2 +)followed by a 25-min digestion with 0.1 mg/mL pronase(Sigma) at 371C. Predigested tissue was recovered bysedimentation and briefly shaken in 0.025% of trypsin(Invitrogen) containing 12.5 mg/ml DNAseI (Roche). Thesupernant was then withdrawn and kept on ice with 10%fetal bovine serum (Invitrogen). This step was repeated fivetimes. Cells were pelleted by centrifugation and ressup-ended in culture media consisting of Ham’s F-12 andDMEM (1:1) (Invitrogen) supplemented with 10% fetalbovine serum, 2 mmol/L glutamine (Invitrogen), 50 mg/mLgentamycin (Sigma), 5 mg/mL insulin, 5mg/mL transferrin,5 ng/mL sodium selenite (ITS, Sigma), 10 ng/mL epidermalgrowth factor (Sigma), 2 mg/mL hydrocortisone (Sigma),5 ng/mL basic fibroblast growth factor (Invitrogen). Forfurther enrichment, cells were incubated on plastic dishesfor 2 h at 371C. A differential attachment on plastic dishoccurred, and supernant containing mostly epithelial cellswas collected and placed for seeding on laminin (Boeh-ringer Ingelheim, GmbH, Germany) coated transwells(Corning, Lowell, MA, USA). To assess purity, the cellmonolayers were immunostained with an anti-transthyretinantibody (specific for CP epithelial cells) (kindly providedby Maria Joao Saraiva). Cell counting under the microscoperevealed that at least 95% of the cells stained positive fortransthyretin, thus confirming the purity of the cultures.

Stimulations were performed after the formation ofconfluent cell monolayers, approximately after 7 days inculture. The CP epithelial cells were stimulated with LPS(200 ng/mL) in the basal side (which corresponds to themembrane facing the blood in vivo) for 6, 12, and 24 h. Inanother set of experiments, CP epithelial cells weresimilarly stimulated for 6 h with serum collected from rats3 and 6 h after LPS or saline injection. Total RNA from CPepithelial cells cultures was extracted using a Micro ScaleRNA Isolation Kit (Ambion, Austin, TX, USA).

A total of 1,000 ng RNA were reverse-transcribed intofirst strand cDNA using Oligo-dt from the SuperscriptFirst-strand Synthesis System for RT–PCR (Invitrogen).The qRT–PCR analysis was used to measure the expressionlevels of selected mRNA transcripts.

Statistical Analysis

The values are reported as mean±s.e. Statisticalsignificance was determined using the nonparametricMann–Whitney test, with differences considered signifi-cant at P < 0.05.

Results

Reproducibility of the Gene Array Data

Two pooled CP samples from animals injected withLPS and killed at 1, 3, 6, 12, 24, or 72 h werecompared with three pooled CP samples from saline-injected animals. The data were analyzed in theR-Bioconductor. Quality control using inter-arrayPearson correlation and clustering based on varianceallowed us to ensure reproducibility between thereplicates (data not shown). After data normaliza-tion, differentially expressed genes were selectedbased on a false discovery rate of 5%. This analysisyielded a list of 252 up-regulated and 173 down-regulated genes at one or more of the experimentaltime points.

Kinetic Profile of the Choroid Plexus TranscriptomeAfter a Peripheral Inflammatory Stimulus

Figure 1 depicts the number of genes whose expres-sion was up-regulated or down-regulated throughoutthe experimental period; it shows that the CPdisplays a rapid (acute phase) response to LPS thatpeaked at 3 to 6 h after LPS administration and thatgradually returned to baseline by 72 h.

When genes were grouped with respect to the foldchanges (Table 1), it became evident that the foldchange was of higher magnitude for up-regulatedgenes (complete list of genes is available as Supple-mentary data Table S1).

Identification of Altered Gene Pathways

Gene ontology and biological pathway analyses ofdifferentially expressed genes, performed using the

Choroid plexus acute inflammatory responseF Marques et al

923

Journal of Cerebral Blood Flow & Metabolism (2009) 29, 921–932

Ingenuity software and the DAVID program, showedthat the biological pathways mostly altered areassociated with the innate immune response (Table 2).

Confirmation of Array Results by QuantitativeReal-Time–Polymerase Chain Reaction on a Set ofRelevant Genes

Within each pathway, and using RNA extracted fromCP pools of an independent experiment, a number ofgenes (Lcn2, Il6, Stat3, Saa3, Cxcl1, Timp1, Tlr2,Icam1, Cd14, Socs3, Stat1, Irf1, and Irf7 as up-regulated genes and Cldn5, Cldn11, Lama2, and Gjb6as down-regulated genes) were chosen for qPCRanalysis; this analysis confirmed the array data.Figure 2 exemplifies the kinetic expression profileof some of these genes.

Cerebrospinal Fluid Concentration of Cytokines

A significant correlation was found between the CPmRNA and the protein CSF concentration of thechemokine CCL2 (Figure 3A) and the cytokine IL-6(Figure 3B). The CSF levels of CCL2 (n = 4) and IL-6(n = 4) increased from 124.7±13.6 to 332.3±51.7 andfrom below detection to 29.4±7.7 pg/mL, respec-tively, within 1 h of LPS administration; 12 h later,the levels of both proteins returned to those of basalconditions and remained as such for all time pointsmeasured until 72 h. TNF levels also increased byLPS, as reported previously (Marques et al, 2007). Allother measured cytokines (IL-10, IL-12p70, and IFNg)were below the detection limit in the CSF fromcontrol and from LPS-treated animals; of relevance,no differences were observed in their gene expres-sion profile in the array.

Response of Primary Cultures of Rat Choroid PlexusEpithelial Cells to Various Stimuli

To study whether the observed response in vivo wasmediated by the CP epithelial cells, which is of

relevance given their role in producing most of theCSF, we extended our study to the in vitro responseof primary cultures of rat CP epithelial cells tovarious stimuli: LPS alone or serum collected fromrats treated with LPS. Figure 4 shows that, inresponse to both stimuli (LPS and serum from LPS-treated rats), the genes encoding for IL-1b, IL-6, andCXCL1 were up-regulated within 6 h. However, theobserved changes in the expression of Irf1 is unlikelyto result from LPS per se, but rather from othermediator(s) present in the serum of LPS-treated rats,as the up-regulation was confined to cells treatedwith the latter preparation. Figure 4 presents datafrom one of the three independent experiments.

Characterization of the Choroid Plexus Acute Response

Proteins Involved in Barrier Function and in CellAdhesion: Most of the genes differentially expressedafter the LPS injection were up-regulated but,interestingly, those encoding for proteins involvedin the formation of tight junctions were down-regulated (Table 2). Among the genes whose expres-sion was down-modulated were those encoding forclaudins 3, 5, and 11 (Cldn3, Cldn11, Cldn5) and forproteins of the extracellular matrix that participate incell-to-cell interactions such as endothelial cell-specific molecule 1 (Esm1), nidogens 1 and 2 (Nid1,Nid2), protocadherin 7 (Pcdh7), laminin alpha 2(Lama2), and decorin (Dcn). On the contrary, theexpression of genes encoding for proteins that mayfacilitate leukocyte migration, including Icam1,mucosal vascular addressin cell adhesion molecule 1(Madcam1), selectin platelet (Selp), and selectinendothelial cell (Sele), was up-regulated.

Degradation of the extracellular matrix is achievedthrough the action of proteases and the role of matrixmetalloproteinases has been extensively investigatedin this context (Flannery, 2006). We observed anup-regulation in the expression of genes encodingfor proteases such as collagenase 13 (Mmp13),

Figure 1 Kinetic profile of the CP response to LPS. The number of genes whose expression was found altered at 1, 3, 6, 12, 24, and72 h after the peripheral injection of LPS as compared with saline-injected mice. The genes whose expression was up-regulated arerepresented in black, and those that were down-regulated are represented in grey.

Choroid plexus acute inflammatory responseF Marques et al

924

Journal of Cerebral Blood Flow & Metabolism (2009) 29, 921–932

Table 1 Genes whose expression was most altered at each time point after LPS

Up-regulated Down-regulated

Accession Gene F. change Accession Gene F. change

1 hNM_008176 Chemokine C-X-C motif) ligand 1 (Cxcl1) 14.9 NM_029928 Protein tyrosine phosphatase, receptor type, B

(Ptprb)�1.7

NM_007707 Suppressor of cytokine signaling 3(Socs3)

5.4 NM_023612 Endothelial cell-specific molecule 1 (Esm1) �1.7

NM_013652 Chemokine (C-C motif) ligand 4 (Ccl4) 5.0 NM_009987 Chemokine (C-X3-C) receptor 1 (Cx3cr1) �1.6NM_007913 Early growth response 1 (Egr1) 4.4 NM_172411 RIKEN cDNA 2310007B03 gene

(2310007B03Rik)�1.5

NM_013654 Chemokine (C-C motif) ligand 7 (Ccl7) 4.1 NM_010171 Coagulation factor III (F3) �1.5NM_008361 Interleukin 1 beta (Il1b) 4.0 NM_026524 Mid1 interacting protein 1 (Mid1ip1) �1.5NM_009841 CD14 antigen (Cd14) 3.2 NM_010496 Inhibitor of DNA binding 2 (Idb2) �1.5NM_008416 Jun-B oncogene (Junb) 3.1 NM_001029934 Ubiquitin specific peptidase 32 (Usp32) �1.5NM_133662 Immediate early response 3 (Ier3) 3.0 NM_152804 Polo-like kinase 2 (Drosophila) (Plk2) �1.4NM_010234 FBJ osteosarcoma oncogene (Fos) 2.9 NM_020278 Leucine-rich repeat LGI family, member 1 (Lgi1) �1.4

3 hNM_008176 Chemokine (C-X-C motif) ligand 1

(Cxcl1)50.9 NM_013723 Podocalyxin-like (Podxl) �3.9

NM_011315 Serum amyloid A 3 (Saa3) 28.8 NM_023055 Solute carrier family 9, isoform 3 regulator 2(Slc9a3r2)

�3.0

NM_008491 Lipocalin 2 (Lcn2) 19.8 NM_029928 Protein tyrosine phosphatase, receptor type, B(Ptprb)

�2.8

NM_013652 Chemokine (C-C motif) ligand 4 (Ccl4) 18.4 NM_023612 Endothelial cell-specific molecule 1 (Esm1) �2.4NM_013654 Chemokine (C-C motif) ligand 7 (Ccl7) 15.1 NM_010171 Coagulation factor III (F3) �2.4NM_007707 Suppressor of cytokine signaling 3

(Socs3)14.9 NM_007409 Alcohol dehydrogenase 1 (class I) (Adh1) �2.3

NM_010493 Intercellular adhesion molecule (Icam1) 13.5 NM_009987 Chemokine (C-X3-C) receptor 1 (Cx3cr1) �2.2NM_015783 Interferon, alpha-inducible protein

(G1p2)11.3 NM_133249 Peroxisome proliferative activated receptor

gamma coactivator 1 b (Ppargc1b)�2.1

NM_011593 Tissue inhibitor of metalloproteinase 1(Timp1)

11.1 NM_010686 Lysosomal-associated protein transmembrane 5(Laptm5)

�2.1

NM_009252 Serine proteinase inhibitor, clade A,member 3N (Serpina3n)

10.6 NM_013805 Claudin 5 (Cldn5) �2.1

6 hNM_011315 Serum amyloid A 3 (Saa3) 31.1 NM_013723 Podocalyxin-like (Podxl) �2.6NM_008491 Lipocalin 2 (Lcn2) 26.7 NM_010686 Lysosomal-associated protein transmembrane 5

(Laptm5)�2.6

NM_008176 Chemokine (C-X-C motif) ligand 1(Cxcl1)

11.6 NM_029928 Protein tyrosine phosphatase, receptor type, B(Ptprb)

�2.5

NM_009252 Serine proteinase inhibitor, clade A,member 3N (Serpina3n)

9.8 NM_023612 Endothelial cell-specific molecule 1 (Esm1) �2.3

NM_011593 Tissue inhibitor of metalloproteinase 1(Timp1)

7.3 NM_008128 Gap junction membrane channel protein beta 6(Gjb6)

�2.2

NM_015783 Interferon, alpha-inducible protein(G1p2)

7.3 NM_009320 Solute carrier family 6, member 6 (Slc6a6) �2.1

NM_010493 Intercellular adhesion molecule (Icam1) 6.8 NM_032398 Plasmalemma vesicle associated protein (Plvap) �2.0NM_008161 Glutathione peroxidase 3 (Gpx3) 5.8 NM_001001309 Integrin alpha 8 (Itga8) �1.9NM_008620 Guanylate binding protein 4 (Gbp4) 5.5 NM_009349 Thioether S-methyltransferase (Temt) �1.9NM_013653 Chemokine (C-C motif) ligand 5 (Ccl5) 4.9 NM_020278 Leucine-rich repeat LGI family, member 1 (Lgi1) �1.8

12 hNM_008491 Lipocalin 2 (Lcn2) 52.7 NM_023612 Endothelial cell-specific molecule 1 (Esm1) �2.6NM_011315 Serum amyloid A 3 (Saa3) 46.9 NM_008973 Pleiotrophin (Ptn) �2.5NM_009252 Serine proteinase inhibitor, clade A,

member 3N (Serpina3n)18.1 NM_177470 Acetyl-Coenzyme A acyltransferase 2 (Acaa2) �2.3

NM_008176 Chemokine (C-X-C motif) ligand 1(Cxcl1)

11.8 NM_010686 Lysosomal-associated protein transmembrane 5(Laptm5)

�1.9

NM_008161 Glutathione peroxidase 3 (Gpx3) 10.5 NM_145434 Nuclear receptor subfamily 1, group D, member1 (Nr1d1)

�1.9

NM_009778 Complement component 3 (C3) 8.6 NM_007472 Aquaporin 1 (Aqp1) �1.9NM_008620 Guanylate nucleotide binding protein 4

(Gbp4)7.5 NM_008481 Laminin, alpha 2 (Lama2) �1.9

NM_013653 Chemokine (C-C motif) ligand 5 (Ccl5) 6.5 NM_146257 Solute carrier family 29 (nucleosidetransporters), member 4 (Slc29a4)

�1.8

NM_011593 Tissue inhibitor of metalloproteinase 1(Timp1)

6.1 NM_007833 Decorin (Dcn) �1.8

XM_355243.1 Proteoglycan 4 (Prg4) 5.9 NM_025953.1 Potassium channel, subfamily K, member 4(Kcnk4)

�1.8

24 hNM_008491 Lipocalin 2 (Lcn2) 19.2 NM_010382 Histocompatibility 2, class II antigen E beta (H2-

Eb1)�2.0

NM_011315 Serum amyloid A 3 (Saa3) 14.8 NM_207105 Response to metastatic cancers 1 (Rmcs1) �1.8NM_009252 Serine proteinase inhibitor, clade A,

member 3N (Serpina3n)7.6 NM_146257 Solute carrier family 29 (nucleoside

transporters), member 4 (Slc29a4)�1.6

Choroid plexus acute inflammatory responseF Marques et al

925

Journal of Cerebral Blood Flow & Metabolism (2009) 29, 921–932

metallopeptidase domain 7 (Adam7), and ADAMmetallopeptidase with thrombospondin type 1 and 4motifs (Adamts1, Adamts4). Notably, the expression ofthe gene encoding for the tissue inhibitor metallopro-teinase 1 (Timp1) was up-regulated as early as 3 h afterLPS injection, probably indicating a response againstdegradation of the extracellular matrix.

Taken together, these observations suggest that thebarrier function of the BCSFB may be transientlycompromised.

Acute-Phase Proteins: One of the most investigated,but still poorly understood, characteristics of theacute-phase response is the up-regulation and down-regulation of the positive and negative acute-phaseproteins, respectively. Adaptive changes to thestimulus in the synthetic and secretory liver machi-neries lead to alterations in the levels of theseproteins. Several well-described acute-phase liverproteins (Ceciliani et al, 2002; Gabay and Kushner,1999) were also found up-regulated in the CP soonafter the LPS injection. For instances, peak expres-sion of serum amyloid A1 (Saa1), serum amyloid A3(Saa3), lipocalin 2 (Lcn2), and Il6 were observed inthe CP at 3 and 12 h after the LPS injection. Theproduction of some of the peripheral acute-phaseproteins by the CP, and despite of specific character-istics of the CP response could represent a braindefence mechanism that is similar to that mediatedby the liver in the periphery.

Signaling Transduction Pathways: The expression ofgenes belonging to various well-described signalingtransduction pathways was up-regulated by LPS.These include several intermediate modulators andtranscription factors such as nuclear factor kappa B(NfkB1), the interferon regulatory factors 1, 2, 7, and9 (Irf1, Irf2, Irf7, Irf9), activated protein-1 (Junb andFos), Stat1 and Stat3 (Figure 2; Supplementary dataTable S1). Many of the proteins encoded by thesegenes have, as downstream targets, genes that encodefor cytokines such as Il1b, Il6, and chemokines such aschemokines (C-C motif) ligands 3 and 4 (Ccl3, Ccl4 ),all of which were seen to be up-regulated.

Interestingly, the CP is not only able to activatethese pathways but also to synthesize moleculesresponsible for the induction of inhibitory mechan-isms, as illustrated by the results for proteinsbelonging to the SOCS family. In our array, theexpression of genes encoding for the suppressors ofcytokine signaling 2 and 3 (Socs2, Socs3) was up-regulated.

Discussion

This study reveals that peripheral inflammatorystimuli can elicit an acute-phase response in theCP. As part of the BBBs, and because it determinesthe composition of the CSF, changes in the CPtranscriptome can potentially play an important role

Table 1 Continued

Up-regulated Down-regulated

Accession Gene F. change Accession Gene F. change

NM_009778 Complement component 3 (C3) 6.8 NM_172411 RIKEN cDNA 2310007B03 gene(2310007B03Rik)

�1.5

NM_008161 Glutathione peroxidase 3 (Gpx3) 6.8 NM_008128 Gap junction membrane channel protein beta 6(Gjb6)

�1.4

NM_029803 Interferon, alpha-inducible protein 27(Ifi27)

4.7 NM_145434 Nuclear receptor subfamily 1, group D, member1 (Nr1d1)

�1.4

NM_013654 Chemokine (C-C motif) ligand 7 (Ccl7) 4.4 NM_172802 Fascin homolog 2, actin-bundling protein,retinal (Fscn2)

�1.4

NM_008620 Guanylate nucleotide binding protein 4(Gbp4)

3.9 NM_008973 Pleiotrophin (Ptn) �1.4

NM_008176 Chemokine (C-X-C motif) ligand 1(Cxcl1)

3.8 NM_201639 Desmuslin (Dmn), transcript variant 1 �1.4

NM_025378 Interferon induced transmembraneprotein 3 (Ifitm3)

3.7 NM_080575 Acetyl-Coenzyme A synthetase 2 (AMPforming)-like (Acas2l)

�1.4

72 hNM_008161 Glutathione peroxidase 3 (Gpx3) 3.3 NM_011066 Period homolog 2 (Drosophila) (Per2) �1.6NM_017373 Nuclear factor, interleukin 3, regulated

(Nfil3)1.9 NM_010902 Nuclear factor, erythroid derived 2, like 2

(Nfe2l2)�1.4

NM_029803 Interferon, alpha-inducible protein 27(Ifi27)

1.8 NM_009427 Transducer of ErbB-2,1 (Tob1) �1.4

NM_013842.2 X-box binding protein 1 (Xbp1) 1.6 NM_177470 Acetyl-coenzyme A acyltransferase 2 (Acaa2) �1.4NM_198885.2 Scleraxis (Scx) 1.6 NM_207105 Response to metastatic cancers 1 (Rmcs1) �1.3NM_009778.1 Complement component 3 (C3) 1.6 NM_023671 Chloride channel, nucleotide-sensitive, 1A

(Clns1a)�1.3

NM_009252.1 Serine proteinase inhibitor, clade A,member 3N (Serpina3n)

1.6 NM_009895 Cytokine inducible SH2-containing protein(Cish)

�1.3

M69067 Histocompatibility 2, D region (H2l) 1.4 NM_009948 Carnitine palmitoyltransferase 1b, muscle(Cpt1b)

�1.3

NM_023612.3 Osteoclast inhibitory lectin (Ocil) 1.3 NM_013807 Polo-like kinase 3 (Drosophila) (Plk3) �1.3NM_023612.3 Endothelial cell-specific molecule 1

(Esm1)1.3 NM_011404 Solute carrier family 7 member 5 (Slc7a5) �1.3

Choroid plexus acute inflammatory responseF Marques et al

926

Journal of Cerebral Blood Flow & Metabolism (2009) 29, 921–932

in mediating the brain’s response to inflammation.Notably, the kinetics of the CP response proved to besimilar to that of the liver (Ceciliani et al, 2002;Gabay and Kushner, 1999) in terms of response onsetand shut-off; and in some of the acute-phase proteinssecreted (e.g., SAA, LCN2, IL-6, IL-1b) into the CSF.In addition, the liver and the CP acute responsesshare common signaling pathways, for example, NF-kB, JAK/STAT, and MAPK. Despite the abovecommonalities, the CP epithelium expresses a num-ber of distinct genes after being challenged with anacute inflammatory stimulus; these genes are im-plicated in its barrier function although, at leastwithin the time frame of the present experiments, wedid not observe any gross CP morphologic changes.

Changes in the functionality and integrity of theBBB are relevant to the pathogenesis of a variety ofdiseases of the CNS (e.g., HIV-1 encephalitis, Alzhei-mer disease, MS, Parkinson disease, ischemia, andtumors) (Persidsky et al, 2006). For example, proin-flammatory substances produced by microglia andspecific disease-associated proteins or cells (b-amy-

loid in Alzheimer disease and reactive T cells in MS)often mediate BBB dysfunction. Although it is notknown exactly how the vascular endothelial cellsthat form the BBB respond to an inflammatorystimulus, it is plausible that the CP response sharesmany of the mechanisms already described for theBBB. A number of studies support the idea thatchanges in endothelial cell tight junctions andcytoskeletal organization facilitate leukocyte migra-tion, through the interaction of chemokinesand adhesion molecules, such as E-selectin andvascular cell adhesion molecule 1 with leukocytes(Engelhardt, 2008). The herein presented data for theCP show decreased expression of tight junctionproteins and increased expression of proteins thatfacilitate migration. These changes are likely tounderlie the preferential migration of T lymphocyteacross the BCSFB that has been observed in animalmodels of MS (Brown and Sawchenko, 2007) andsuggest a role for the CP in cell entry into the CSFand ultimately, into the brain parenchyma. Togetherwith studies implying particular CP proteins in

Table 2 Clustering of the genes whose expression was altered in the choroid plexus upon peripheral LPS injection

Immune molecules Chemokines: Ccl4, Cxcl2, Cxcl13, Ccl4, Cxcl1, Ccl5, Ccl3, Cxcl16,Ccl7, Cxcl9, Ccl9, Ccl11, Ccl2, Ccl19, Cxcl10 m and Cxcl12 k

15m1k

Interleukins: Il1b, Il15, Il6 m 3mOther molecules with cytokine activity: Csf1, Csf3, Spp1 m 3m

Antigen presentation pathway Antigen presentation pathway: H2T23, H2K1, Psmb8, Psmb9,Tap2 m and H2Eb1 k

5m1k

Signaling pathways TLR and co-estimulatory molecules: Cd14, Tlr2 m 2mJAK/STAT signaling pathway: Socs3, Cish, Socs2, Stat1, Stat3 mand Pias3 k

5m1k

MAPK signaling pathway: Map3k1, Map3k6, Map3k3, Map3k8,Fos, Junb m and Mapk4 k

6m1k

NF-KB signaling pathway: Bcl3, Tnfrsf5, Egfr, Prkr, Il1b, Map3k3,Map3k8, Nfkb1, Nfkbia, Nfkbie, Ngfb, Relb, Ripk1, Tlr2, Tnfaip3m and Hdac2 k

15m1k

Complement signaling: C2, C3, C6, Slp, H2Bf, Serping1 m 6mInterferon signaling: Ifit3, Ifitm1, Ifngr2, Irf1, If2, If7, Isgf3 g, Mx1,Oas1 g, Psmb8, Stat1 m

11m

IL-10 signaling: Bcl3, Cd14, Fos, Il6, Il1b, Junb, Nfkbia, Nfkbie,Socs3, Stat3 m

10m

IL-6 signaling: A2 m, Bcl3, Cd14, Cebpb, Fos, Il6, Il1b, Junb,Nfkbia, Nfkbie, Stat3 m

11m

Acute phase response signaling Acute phase response: A2 m, Bcl3, C2, C3, Cebpb, H2Bf, Fos, Il6,Il1b, Junb, Map3k1, Nfkbia, Nfkbie, Ripk1, Saa1, Saa3,Serpina3n, Serping1, Socs2, Socs3, Stat3 m

21m

Glucocorticoid receptorsignaling

Glucocorticoid receptor signaling: A2 m, Bcl3, Ccl3, Ccl5, Ccl11,Cxcl13, Cdkn1a, Cebpb, Cxcl2, Dusp1, Fkbp5, Fos, Icam1, Il6,Il1b, Junb, Map3k1, Nfkb1, Nfkbia, Nfkbie, Sele, Stat1, Stat3 mand Tgfbr2 k

23m1k

Cell adhesion molecules Tight junctions: Cldn3, Cldn11, Cldn5 k 3kGap junctions: Gjb6 k 1kLeukocyte transendothelial migration: Icam1, Madcam1, Selp,Sele m and Pecam1 k

4m1k

Extracellular matrix: Pcdh7, Esm1, Ptn, Nid1, Nid2, Lama2, Dcn,Tgfbi, Emid2k

9k

Proteins that contribute tointegrity of ECM

Proteases/proteases inhibitors: Adamts1, Adamts4, Serping1,Serpina3n, Mmp13, Psmb8, Psmb9, Adam7, Usp18, Psme2b,Casp4, A2 m, Timp1m

13m

Transporters Solute carrier: Slc7a5, Slc43a3, Slc15a2, Slc31a2, Slc38a2,Slc7a11 m and Slc6a6, Slc23a2, Slc29a4, Slc7a10, Slc9a3r2,Slco2b1, Slc5a6, Slc9a3r2, Slco1a4, Slc22a5 k

6m10k

ABC transporter: Tap2 m 1m

m denotes up-regulated; k denotes down-regulated.

Choroid plexus acute inflammatory responseF Marques et al

927

Journal of Cerebral Blood Flow & Metabolism (2009) 29, 921–932

diseases such as Alzheimer disease (Sousa et al,2007), this study warrants further studies to examinethe CP transcriptome in neurologic and psychiatricdiseases.

Apart from being a potential site of cell entry intothe brain, the CP is also an active site of proteinsynthesis. Several inflammatory markers have beendescribed in the CSF of individuals with brainpathology (Andreasen and Blennow, 2005; Giovan-noni, 2006). The precise origin of these molecules isstill equivocal, but CP epithelial cells might beimportant for their trafficking/entry into the CSF.The nature of the proteins secreted by the CP maychange in response to disease or specific stimuli, asshown here for CCL2 and IL-6, and in previous

studies for various cytokines, carrier proteins, andiron-related proteins (Marques et al, 2007, 2008;Hughes et al, 2002; Thibeault et al, 2001). In fact,several inflammatory chemokines (CCL2, CCL5,CXCL10, CXCL12, and CXCL13) whose gene expres-sion is altered in the CP after peripheral LPSinjection are detectable in the CSF of MS subjects(Trebst and Ransohoff, 2001) and CCL2 mRNA levelsin the CP are increased after a single systemic bolusof LPS (Thibeault et al, 2001). The CP is known tosecrete proinflammatory and antiinflammatory mo-lecules, proteins that promote and inhibit extracel-lular matrix remodeling, and proteins to whichneuroprotective and/or toxic properties have beenascribed to. Their beneficial or detrimental effects

Figure 2 Analysis, by qRT–PCR, of the gene expression kinetic profile of selected genes. Confirming the array results, the expressionof Saa3, Cxcl1, Irf1, Irf7, Stat1, and Socs3 (A–F) was up-regulated and that of Cldn5, Cldn11, and Gjb6 (G–I) was down-regulated.As some genes were not found expressed in saline-injected animals, the mRNA expression profile is presented as the expression ratebetween the gene of interest and the reference gene.

Figure 3 The CSF levels of CCL2 and IL-6 after peripheral injection of LPS. The concentration of CSF CCL2 (A) and of IL-6 (B)increased 1 h after LPS administration and rapidly returned to basal levels. b.d., below detection (20 pg/mL).

Choroid plexus acute inflammatory responseF Marques et al

928

Journal of Cerebral Blood Flow & Metabolism (2009) 29, 921–932

may depend on other factors such as age and thepresence of pathogenic and/or disease conditions,which should next be investigated.

Proposed Model of the Choroid Plexus Response toPeripheral Inflammation

Irrespective of the final determinants of the balancebetween benefit and detriment, the present dataprovide interesting insight into the signaling trans-duction pathways that ultimately lead to theobserved gene expression profiles in response to a

pathogenic stimulus (Figure 5). Previous workshowed that serum concentrations of cytokines(e.g., IL-1b and IL-6) are increased after peripheraladministration of LPS (Ramadori and Christ, 1999).Receptors for these cytokines are present in CPepithelial cells (Chodobski and Szmydynger-Cho-dobska, 2001) and were detected by our arrayanalysis under basal conditions. Thus, LPS- andcytokine-mediated signaling transduction pathwaysmay be simultaneously engaged by peripheral in-flammation. In fact, the cytokine-mediated pathwaysmay play a general role in the CP response as thesecretome of LPS-treated cultured CP epithelial cells

Figure 4 Analysis, by qRT–PCR, of the gene expression profile from rat CP epithelial cells. Primary cultures of rat CP epithelial cellswere exposed to LPS alone for 6, 12, and 24 h; or for 6 h to serum obtained from rats 3 or 6 h after injection with LPS. Although bothLPS and serum from LPS-injected rats induced the expression of Il1b, Il6, and Cxcl1, only the serum from LPS-injected mice had aneffect on the expression of Irf1. The mRNA expression profiles are presented as the fold change increase in relation to the respectivecontrol (exposure to cell media for the stimulation with LPS, and exposure to serum collected from saline-injected rats for thestimulation with serum obtained from LPS-treated rats).

Choroid plexus acute inflammatory responseF Marques et al

929

Journal of Cerebral Blood Flow & Metabolism (2009) 29, 921–932

displays a much reduced number of altered proteins(Thouvenot et al, 2006). This interpretation isconsistent with that of the present study in whichthe up-regulation of Irf1 was not mediated by LPSalone, but by other molecules (e.g., IL-1b, IL-6, TNF)that are present in the serum on the peripheralinflammatory stimulus.

With respect to LPS signaling, we confirmed thatthe gene encoding for the Toll-like receptor 4 (Tlr4),the LPS cognate receptor, is expressed in the CP(Laflamme and Rivest, 2001; Chakravarty and Her-kenham, 2005). However, the expression of the geneencoding for TLR4 is not influenced by LPS whereasthat encoding for TLR2 is induced, as has beenreported previously (Laflamme et al, 2001). Ofnotice, the differential expression of TLRs in re-sponse to inflammation has been described in otherorgans (Ojaniemi et al, 2006). Interestingly, TLR2 isnot liganded by LPS. However, a mediatory role forproinflammatory cytokines (e.g., IL-1b and TNF) inthe up-regulation of Tlr2 has been proposed (Matsu-mura et al, 2003), as might as well be the case for theCP. Activation of TLR2-mediated signaling may alsoresult from the interaction of increased serum levelsof SAA, recently shown as a functional ligand forTLR2 (Cheng et al, 2008).

Activation of TLRs and of receptors for IL-1b, IL-6,and TNF results in the induction of several tran-scription factors, including interferon regulatoryfactor 3 (IRF3), activator protein-1, and NF-kB(Colonna, 2007; Honda and Taniguchi, 2006). Ouranalysis shows that several genes encoding forproteins belonging to the NF-kB, MAPK, STAT-JAK,and IRFs signaling pathways are induced in the CPduring the acute-phase response. All of these path-ways, together with others such as those mediated bycAMP (Reyes-Irisarri et al, 2008), participate in thedevelopment and regulation of innate and adaptativeimmune responses (Rawlings et al, 2004), and the CP,like the BBB endothelium (Laflamme et al, 1999;Laflamme and Rivest, 1999), responds to peripheralLPS by activating some of these pathways. Termina-tion of the CP response at 72 h may be, at least inpart, a consequence of the negative feedback inhibi-tion of STAT signaling by SOCS/CIS (Naka et al,2005), as some of the genes encoding for proteins inthis pathway are up-regulated after LPS administra-tion. A similar mechanism has been described in theBBB (Lebel et al, 2000).

In summary, this study shows that the CP canmount an acute-phase response. This responseincludes the release of proinflammatory and antiin-

Figure 5 Suggested pathways of the CP epithelial cells response to peripheral inflammation. The genes whose expression was, atone or more time points, down-regulated are represented in green, and whose expression was up-regulated at one or more time pointsare represented in red.

Choroid plexus acute inflammatory responseF Marques et al

930

Journal of Cerebral Blood Flow & Metabolism (2009) 29, 921–932

flammatory immune modulators into the CSF, and arelaxation of the CP barrier properties. The evidenceprovided for a potentially important role of the CP inthe cross talk between the immune system and theCNS warrants further studies that will add to ourunderstanding on how homeostasis is maintained inthe brain under normal and pathologic states.

Acknowledgements

This work was supported by grant POCTI/SAU-NEU/56618/2004 from the Portuguese Foundation forScience and Technology (FCT) /FEDER; and from agrant from the DANA foundation; Marques F, FalcaoAM, and Rodrigues AJ are recipients of fellowshipsfrom FCT/FEDER.

References

Andreasen N, Blennow K (2005) CSF biomarkers for mildcognitive impairment and early Alzheimer’s disease.Clin Neurol Neurosurg 107:165–73

Brown DA, Sawchenko PE (2007) Time course anddistribution of inflammatory and neurodegenerativeevents suggest structural bases for the pathogenesis ofexperimental autoimmune encephalomyelitis. J CompNeurol 502:236–60

Ceciliani F, Giordano A, Spagnolo V (2002) The systemicreaction during inflammation: the acute-phase proteins.Protein Pept Lett 9:211–23

Chakravarty S, Herkenham M (2005) Toll-like receptor 4 onnonhematopoietic cells sustains CNS inflammationduring endotoxemia, independent of systemic cyto-kines. J Neurosci 25:1788–96

Cheng N, He R, Tian J, Ye PP, Ye RD (2008) Cutting edge:TLR2 is a functional receptor for acute-phase serumamyloid A. J Immunol 181:22–6

Chodobski A, Szmydynger-Chodobska J (2001) Choroidplexus: target for polypeptides and site of their synth-esis. Microsc Res Tech 52:65–82

Colonna M (2007) TLR pathways and IFN-regulatoryfactors: to each its own. Eur J Immunol 37:306–9

Dominguez-Punaro MC, Segura M, Plante MM, LacoutureS, Rivest S, Gottschalk M (2007) Streptococcus suisserotype 2, an important swine and human pathogen,induces strong systemic and cerebral inflammatoryresponses in a mouse model of infection. J Immunol179:1842–54

Emerich DF, Skinner SJ, Borlongan CV, Vasconcellos AV,Thanos CG (2005) The choroid plexus in the rise, falland repair of the brain. Bioessays 27:262–74

Engelhardt B, Wolburg-Buchholz K, Wolburg H (2001)Involvement of the choroid plexus in central nervoussystem inflammation. Microsc Res Tech 52:112–29

Engelhardt B (2008) Immune cell entry into the centralnervous system: involvement of adhesion moleculesand chemokines. J Neurol Sci 274:23–6

Flannery CR (2006) MMPs and ADAMTSs: functionalstudies. Front Biosci 11:544–69

Gabay C, Kushner I (1999) Acute-phase proteins and othersystemic responses to inflammation. N Engl J Med340:448–54

Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M,Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K,Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C,Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G,Tierney L, Yang JY, Zhang J (2004) Bioconductor: opensoftware development for computational biology andbioinformatics. Genome Biol 5:R80

Giovannoni G (2006) Multiple sclerosis cerebrospinal fluidbiomarkers. Dis Markers 22:187–96

Honda K, Taniguchi T (2006) IRFs: master regulators ofsignalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat Rev Immunol 6:644–58

Hughes PM, Botham MS, Frentzel S, Mir A, Perry VH(2002) Expression of fractalkine (CX3CL1) and itsreceptor, CX3CR1, during acute and chronic inflamma-tion in the rodent CNS. Glia 37:314–27

Laflamme N, Lacroix S, Rivest S (1999) An essential role ofinterleukin-1beta in mediating NF-kappaB activity andCOX-2 transcription in cells of the blood-brain barrier inresponse to a systemic and localized inflammation butnot during endotoxemia. J Neurosci 19:10923–30

Laflamme N, Rivest S (1999) Effects of systemic immuno-genic insults and circulating proinflammatory cytokineson the transcription of the inhibitory factor kappaBalpha within specific cellular populations of the ratbrain. J Neurochem 73:309–21

Laflamme N, Rivest S (2001) Toll-like receptor 4: themissing link of the cerebral innate immune responsetriggered by circulating gram-negative bacterial cell wallcomponents. FASEB J 15:155–63

Laflamme N, Soucy G, Rivest S (2001) Circulating cellwall components derived from gram-negative, notgram-positive, bacteria cause a profound induction ofthe gene-encoding Toll-like receptor 2 in the CNS.J Neurochem 79:648–57

Lebel E, Vallieres L, Rivest S (2000) Selective involvement ofinterleukin-6 in the transcriptional activation ofthe suppressor of cytokine signaling-3 in the brain duringsystemic immune challenges. Endocrinology 141:3749–63

Marques F, Sousa JC, Correia-Neves M, Oliveira P, Sousa N,Palha JA (2007) The choroid plexus response to periph-eral inflammatory stimulus. Neuroscience 144:424–30

Marques F, Rodrigues AJ, Sousa JC, Coppola G, GeschwindDH, Sousa N, Correia-Neves M, Palha JA (2008)Lipocalin 2 is a choroid plexus acute-phase protein.J Cereb Blood Flow Metab 28:450–5

Matsumura T, Degawa T, Takii T, Hayashi H, Okamoto T,Inoue J, Onozaki K (2003) TRAF6-NF-kappaB pathwayis essential for interleukin-1-induced TLR2 expressionand its functional response to TLR2 ligand in murinehepatocytes. Immunology 109:127–36

Nadeau S, Rivest S (1999) Regulation of the gene encodingtumor necrosis factor alpha (TNF-alpha) in the rat brainand pituitary in response in different models ofsystemic immune challenge. J Neuropathol Exp Neurol58:61–77

Naka T, Fujimoto M, Tsutsui H, Yoshimura A (2005)Negative regulation of cytokine and TLR signalings bySOCS and others. Adv Immunol 87:61–122

Ojaniemi M, Liljeroos M, Harju K, Sormunen R, Vuoltee-naho R, Hallman M (2006) TLR-2 is upregulated andmobilized to the hepatocyte plasma membrane in thespace of Disse and to the Kupffer cells TLR-4 depen-dently during acute endotoxemia in mice. Immunol Lett102:158–68

Quan N, Stern EL, Whiteside MB, Herkenham M (1999)Induction of pro-inflammatory cytokine mRNAs in the

Choroid plexus acute inflammatory responseF Marques et al

931

Journal of Cerebral Blood Flow & Metabolism (2009) 29, 921–932

brain after peripheral injection of subseptic doses oflipopolysaccharide in the rat. J Neuroimmunol 93:72–80

Persidsky Y, Ramirez SH, Haorah J, Kanmogne GD (2006)Blood-brain barrier: structural components and functionunder physiologic and pathologic conditions. J Neu-roimmune Pharmacol 1:223–36

Ramadori G, Christ B (1999) Cytokines and the hepaticacute-phase response. Semin Liver Dis 19:141–55

Rawlings JS, Rosler KM, Harrison DA (2004) The JAK/STAT signaling pathway. J Cell Sci 117:1281–3

Redzic ZB, Segal MB (2004) The structure of the choroidplexus and the physiology of the choroid plexusepithelium. Adv Drug Deliv Rev 56:1695–716

Reyes-Irisarri E, Perez-Torres S, Miro X, Martinez E,Puigdomenech P, Palacios JM, Mengod G (2008) Differ-ential distribution of PDE4B splice variant mRNAs in ratbrain and the effects of systemic administration of LPSin their expression. Synapse 62:74–9

Rozen S, Skaletsky H (2000) Primer3 on the WWW forgeneral users and for biologist programmers. MethodsMol Biol 132:365–86

Sousa JC, Cardoso I, Marques F, Saraiva MJ, Palha JA (2007)Transthyretin and Alzheimer’s disease: where in thebrain? Neurobiol Aging 28:713–8

Strazielle N, Ghersi-Egea JF (1999) Demonstration of acoupled metabolism-efflux process at the choroidplexus as a mechanism of brain protection towardxenobiotics. J Neurosci 19:6275–89

Thibeault I, Laflamme N, Rivest S (2001) Regulation of thegene encoding the monocyte chemoattractant protein 1(MCP-1) in the mouse and rat brain in response tocirculating LPS and proinflammatory cytokines. J CompNeurol 434:461–77

Thouvenot E, Lafon-Cazal M, Demettre E, Jouin P, BockaertJ, Marin P (2006) The proteomic analysis of mousechoroid plexus secretome reveals a high protein secre-tion capacity of choroidal epithelial cells. Proteomics6:5941–52

Trebst C, Ransohoff RM (2001) Investigating chemokinesand chemokine receptors in patients with multiplesclerosis: opportunities and challenges. Arch Neurol58:1975–80

Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website (http://www.nature.com/jcbfm)

Choroid plexus acute inflammatory responseF Marques et al

932

Journal of Cerebral Blood Flow & Metabolism (2009) 29, 921–932