The Female Brain Response to Bacterial and Viral Pathogens In

Estradiol Is Required for a Proper Immune Response to Bacterial and Viral Pathogens in the Female Brain

This information is current as Geneviève Soucy, Guy Boivin, Fernand Labrie and Serge of September 28, 2021. Rivest J Immunol 2005; 174:6391-6398; ; doi: 10.4049/jimmunol.174.10.6391 http://www.jimmunol.org/content/174/10/6391 Downloaded from

References This article cites 43 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/174/10/6391.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

by guest on September 28, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Estradiol Is Required for a Proper Immune Response to Bacterial and Viral Pathogens in the Female Brain1

Genevie`ve Soucy,* Guy Boivin,† Fernand Labrie,* and Serge Rivest2*

Although the neuroprotective effects of estrogens are well recognized, the exact mechanisms involved in the ability of these sex steroids to protect the cerebral tissue still remain unclear. We tested in our study the hypothesis that estradiol (E2) modulates the innate immune response and expression of genes encoding proteins that a provide survival signal to neurons during infection. Mice received a single systemic or cerebral injection of LPS to trigger a robust but transient inflammatory reaction in the brain. The endotoxin increased transcriptional activation of genes encoding TLR2, TNF-␣, and IL-12 in microglial cells. Expression of these transcripts was largely inhibited in the brain of ovariectomized mice at time 24 h postchallenge. E2 replacement therapy totally rescued the ability of the endotoxin to trigger microglial cells and these permissive effects of E2 are mediated via the estrogen ␣ ␣ ␤ receptor (ER) . Indeed, ER -deficient mice exhibited an inappropriate reaction to LPS when compared with ER -deficient and Downloaded from wild-type mice. This defective innate immune response was also associated with a widespread viral replication and neurodegeneration in ovariectomized mice inoculated intranasally with HSV-2. These data provide evidence that interaction of E2 with their nuclear ER␣ plays a critical role in the control of cytokines involved in the transfer from the innate to adaptive immunity. This transfer is deviant in mice lacking E2, which allows pathogens to hide from immune surveillance and exacerbates neuronal damages during viral encephalitis. The Journal of Immunology, 2005, 174: 6391–6398. http://www.jimmunol.org/ n infected host recognizes invading microorganisms via sion in monocytes (11). Anti-inflammatory properties have also elements called pathogen-associated molecular patterns been attributed to these steroids (12–15) and such effects on the A (1). Interaction between pathogen-associated molecular immune system have been suggested to contribute to the neuro- patterns with their cognate receptors expressed on the surface of protective roles of estrogens (16–18). However, the exact mech- specific cells of the immune system leads to an innate immune anisms involved in the ability of estrogens to modulate the innate response that is characterized by transcriptional activation genes immune response in the brain still remain poorly understood. 3 encoding cytokines, chemokines, and many other proteins in- Estradiol (E2) is the main secreted estrogen. It acts via two volved in the acute-phase response. In this regard, Gram-negative nuclear receptors, namely estrogen receptor (ER)␣ and ER␤. cell wall component LPS is a useful tool to trigger the events that These two receptors are highly homologous and they share many by guest on September 28, 2021 take place during the innate immune and inflammatory response functional characteristics, but their transcriptional activities are (2). The endotoxin injected either systemically or centrally also different and complementary (19). They are both expressed in the triggers proinflammatory signaling and gene expression in micro- brain, but the anatomic distribution and subcellular colocalization glial cells throughout the CNS (3–8). The main role of this rapid are highly different between the receptor subtypes (20). The inflammatory response is to eliminate the pathogen and prepare the present series of experiments tested the hypothesis that E2 plays a transfer from the innate to adaptive immunity. critical role in the control of the innate immunity in the female This innate immune response is under the control of numerous brain. We subsequently determined the receptor involved in these factors including glucocorticoids that are the most potent endog- events in ER␣-deficient (ERKO) and ER␤-deficient (BERKO) enous inhibitory feedback on proinflammatory signal transduction mice. Finally, a mouse model of viral encephalitis (21) was used to pathways (9). Although more subtle, sex steroids have been shown investigate the role of E2 in the mechanisms that control the trans- to modulate the immune system. Estrogen replacement therapy fer from innate to adaptive immune reactions. decreased the number of circulating monocytes in postmenopausal We provide in our study compelling evidence that E2 is required women (10) and estrogen treatment increased Fas ligand expres- for the rapid production of key cytokines involved in the transfer from the innate to adaptive immunity. Alteration of this positive feedback by these sex steroids on microglial cells has direct con- *Laboratory of Molecular Endocrinology and †Infectious Diseases, Centre Hospitalier sequences on the cerebral tissue. de l’Universite´Laval Research Center and Department of Anatomy and Physiology, Universite´Laval, Que´bec City, Que´bec, Canada Received for publication November 19, 2004. Accepted for publication February Materials and Methods 28, 2005. Animals The costs of publication of this article were defrayed in part by the payment of page Adult intact and ovariectomized (OVX) CD1 mice (ϳ25 g of body weight; charges. This article must therefore be hereby marked advertisement in accordance Charles River Breeding Laboratories) and C57BL/6J mice (ϳ35–45 g of with 18 U.S.C. Section 1734 solely to indicate this fact. body weight) were acclimated to standard laboratory conditions (14 h light, 1 This work is supported by the Canadian Institutes in Health Research (CIHR). S.R. 10 h dark cycle; lights on at 0600 h and off at 2000 h) with free access to is a CIHR Scientist and holds a Canadian Research Chair in Neuroimmunology. G.S. mouse chow and water. Knockout mice were provided by Prof. P. Cham- is supported by a Master of Science Studentship from Les Fonds de la Recherche en bon (Colle`ge de France, Strasbourg, France) (22). Appropriate housing Sante´du Que´bec (FRSQ). G.B. is a FRSQ Senior Scholar. 2 Address correspondence and reprint requests to Dr. Serge Rivest, Laboratory of 3 Molecular Endocrinology, Centre Hospitalier de l’Universite´Laval Research Center, Abbreviations used in this paper: E2, estradiol; NeuN, neuronal nuclei; CVO, cir- 2705 boulevard Laurier, Sainte-Foy, G1V 4G2 Que´bec, Canada. E-mail address: cumventricular organ; ER, estrogen receptor; iba1, anti-ionized calcium binding [email protected] adapter molecule 1; OVX, ovariectomized.

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 6392 E2 IN CEREBRAL INNATE AND ADAPTIVE IMMUNITY conditions (microisolators) and surveillance procedures were used to main- MEM (Invitrogen Life Technologies) containing either no virus (vehicle) tain a disease-free state within the colony. Mice heterozygous for the mu- or 103 PFU of HSV-2 strain was inoculated intranasally in the animals. The tated ER␣ gene were bred and the first generation offspring were genotyped viral strain was plaque-purified three times from the original isolate of an by PCR analysis of tail genomic DNA. For ERKO mice, the following HSV-infected subject. Mice were deeply anesthetized and perfused at dif- PCR primers were used: P1, 5Ј-TTGCCCGATAACAATAACAT-3Ј; P2, ferent days after the HSV-2 inoculation (3, 5, 7, 8, and 10 days). 5Ј-ATTGTCTCTTTCTGACAC-3Ј; P3, 5Ј-GGCATTACCACTTCTCCT GGGAGTCT-3Ј; and P4, 5Ј-TCGCTTTCCTGAAGACCTTTCATAT-3Ј. Brain preparation and histology The sizes of the P1-P2 and P3-P4 PCR fragments in wild-type mice are 387 and 815 bp, respectively. The size of the P3-P4 fragment amplified from Rapidly after the perfusions, brains were removed from the skull, postfixed the ERKO allele is 255 bp. Tail DNA genotyping of BERKO mice was for 1–3 days, and then placed in 10% sucrose diluted in the solution of 4% performed using the primers: P1, 5Ј-AAGTGTAAGCCAGCCCAAGG-3Ј; paraformaldehyde-borax buffer overnight at 4°C. The frozen brains were P2, 5Ј-AGCTCCCTGTGATCTGATAT-3Ј; and P3, 5Ј-AAGCGCATGCT mounted on a microtome (Reichert-Jung; Cambridge Instruments) and cut CCAGACTGC-3Ј, which yield ϳ380 bp fragments for wild-type mice into 25-␮m coronal sections from the olfactory bulb to the end of the (ϩ/ϩ) (P1 and P2 primers), ϳ240 bp fragments for homozygous animals medulla. The slices were collected in a cold cryoprotectant solution (0.05 (Ϫ/Ϫ) (P1 and P3 primers), and both fragments for heterozygotes (ϩ/Ϫ). M sodium phosphate buffer, 30% ethylene glycol, 20% glycerol) and stored Animal breeding and experiments were conducted according to Canadian at Ϫ20°C. Plasmids were linearized and sense and antisense riboprobes Council on Animal Care guidelines, as administered by the Laval Univer- were synthesized as described in Table I. Hybridization histochemical lo- sity Animal Care Committee. A total of 150 mice were assigned to four calization of the different transcripts and their colocalization within micro- protocols that contained different treatments and postinjection times (3, 9, glial cells was conducted on every twelfth section of the whole rostrocau- 35 and 24 h and 3, 5, 7, 8, and 10 days). dal extent of each brain using S-labeled cRNA probes as previously described (23, 24). Surgeries and treatments

Immunoﬂuorescence and confocal laser scan microscopy Downloaded from Intact and OVX mice receiving intracerebral injections of LPS (2.5 ␮g/␮l from Escherichia coli, serotype O55:B5, product no. L-2880; Sigma-Al- Brain slices were washed in 50 mM potassium PBS and preincubated for drich) or sterile saline solution (0.9%) were anesthetized with an i.p. in- 30 min at room temperature in potassium PBS containing 0.4% Triton jection (10 ml/kg body weight) of a mixture of ketamine hydrochloride (15 X-100, 1% BSA, and 4% goat serum. The slices were then incubated over- mg/ml) and xylazine (1 mg/ml) and placed in a stereotaxic apparatus night at 4°C with a polyclonal sheep HSV Ab FITC-conjugated (1/200) and (David Kopf Instruments). The striatum was then reached (Ϫ2.0 mm lat- a neuronal nuclei (NeuN) Ab (1/800; Chemicon International) diluted in eral and Ϫ3.0 mm dorsoventral to the bregma) with a 33-gauge stainless potassium PBS containing 0.4% Triton X-100, 1% BSA, and 4% goat steel cannula (Plastics One) that was connected to a 50-␮l Hamilton sy- serum. After incubation with the primary Abs, brain slices were washed http://www.jimmunol.org/ ringe with an intramedic polyethylene tubing (PE-50; Clay Adams). A and incubated for 90 min at room temperature in the secondary Ab solution volume of 1 ␮l was infused over 2 min by means of a microinjection pump (for NeuN), which consisted of a Alexa Fluor 594-conjugated goat anti- (model A-99; Razel Scientiﬁc Instruments). These coordinates were se- rabbit (Molecular Probes) diluted 1/500 in potassium PBS containing 0.4% lected on the basis of previous data showing a robust hybridization signal Triton X-100 and 1% BSA. Sections were then rinsed, mounted on gelatin- and reliable pattern of cytokine gene expression over the ipsilateral cere- coated slides, washed with water and cover-slipped with 1,4- bral cortex, hippocampus, corpus callosum, and basal ganglia following a diazabicyclo(2.2.2)octane (DABCO; Sigma-Aldrich). Laser scanning con- single bolus of LPS (3). These coordinates were therefore used to represent focal microscopy studies were performed with a ϫ20 and ϫ100 Plan-Apo a general mechanism of immune response in the cerebral tissue. At differ- oil immersion objective, numerical apertures 0.8 and 1.35, ϫ2 numerical ent times after the injection (24 h, and 3 and 7 days), mice were deeply zoom with a BX-61 microscope (Olympus America). anesthetized via an i.p. injection (100 ␮l) of a mixture of ketamine hydro- by guest on September 28, 2021 chloride (91 mg/ml) and xylazine (9.1 mg/ml) and then rapidly perfused Data analysis transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M borax buffer (pH 9.5 at 4°C). Semiquantitative analysis of the hybridization signal was conducted as pre-

A second protocol was developed to verify the ability of E2 to modulate viously described (4, 25, 26). The number of cytokine-expressing cells was the inflammatory reaction by microglial cells in response to systemically quantified in serial sections ipsilateral to the injection site (see Figs. 1–5 for injected LPS. One group of OVX mice was anesthetized with isoflurane details). Data are reported as mean Ϯ SEM values for experimental and ␤ and were implanted s.c. with a silastic implant of E2 (cholesterol to 17 -E2, control mice. Statistical analysis was performed by a two- or three-way 100:1). The other group of OVX mice was sham operated. Seven days after ANOVA (StatView 4.5, for Macintosh) followed by a Bonferroni-Dunn the surgery, animals (intact, OVX-sham, OVX-E2) received an i.p. injec- test procedure as posthoc comparisons for each time postinjection. tion of LPS (1 mg/kg) or saline solution and were killed 3, 9, and 24 h afterward as described. Results In the third protocol, wild-type, ERKO, and BERKO mice received an i.p. injection of LPS (1 mg/kg) or saline solution and were killed 24 h after Innate immune reaction in response to intrastriatal LPS infusion the injection. Fig. 1 depicts representative hybridization signals in the brain of Finally, intranasal inoculation of HSV-2 was used as a model of viral encephalitis, which causes strong innate and adaptive immune responses in mice that received a single bolus of sterile saline (vehicle) or LPS the brain (21). Intact and OVX CD1 mice were anesthetized for a few into the dorsal basal ganglia. As expected, implantation of the seconds by inhalation of isoflurane (Janssen-Ortho). A total of 20 ␮lof cannula and stress of infusion caused a low expression of TNF-␣

Table I. Plasmids and enzymes used for the synthesis of the cRNA probes

Plasmid Vector Insert Antisense Probe Sense Probe Source

Mouse IL-12p40 pCL-Neo 1050 bp XhoI/T3 NotI/T7 Dr. K. Pahan (University of Nebraska Medical Center, Lincoln, NE) Mouse IFN pGEMEX 550 bp HindIII/T3 EcoRI/SP6 Dr. I. Campbell (Scripps Research Institute, La Jolla, CA) Mouse TLR2a PCR blunt II 2.278 kb EcoRV/SP6 SpeI/T7 PCR ampliﬁcation Mouse TNF Bluescript K IIϩ 1300 bp PstI/T3 BamHI/T7 Subcloned from a PUc19 plasmid provided by Dr. D. Radzioch (McGill University Montre´al, Quebec, Canada)

a The DNA fragment of 2.278 kb corresponding to the almost complete coding sequence (2.355 kb) of the reported mouse TLR2 mRNA (nucleotide 307–2661, GenBank accession no. AF185284) was ampliﬁed by PCR from a cDNA macrophage B10R cell line library using a pair of 23 bp oligonucleotide primers complementary to nucleotides 323–345 (5Ј-GGCTCTTCTGGATCTTGGTGGCC-3Ј) and to 2579–2601 (5Ј-GGGCCACTCCAGGTAGGTCTTGG-3Ј). The Journal of Immunology 6393

FIGURE 1. Intracerebral LPS infusion causes a profound expression wave of TNF-␣ mRNA in the mouse brain. A, Intraparenchymal injection of the vehicle solution (sterile saline) induces a localized inflammatory reaction at the level of the cannula track (top row). This result contrasts with the robust and widespread signal for the proinflammatory cytokine transcript in the ipsilateral side 24 h and 3 days after a single infusion with the bacterial cell wall component (2.5 ␮g of LPS/␮l, second row). B, Dual-la- beling combining immunocytochemistry with in situ hybridization to identify the phenotype of IL-12-, TNF-␣-, and TLR2-expressing cells. Microglia were labeled by immuno- Downloaded from histochemistry using an Ab directed against iba1, brown ramified cells). The different transcripts were thereafter hybridized separately on adjacent coronal sections via a radioactive in situ hybridization technique (silver

grains). Black arrows indicate double- http://www.jimmunol.org/ labeled cells (agglomeration of silver grains within brown ramified cells). All (100%) IL-12-, TNF-␣-, and TLR2-expressing cells were localized with the iba1-immunoreactive signal, but not all microglia contained positive messages for the immune genes. by guest on September 28, 2021 mRNA (Fig. 1A, top row). This localized pattern of positive TNF- TLR2 was robust across the ipsilateral side of intact mice, no pos- ␣-expressing cells lining the cannula track contrasts with the in- itive cells were found in this region of OVX mice after the endo- tense and widespread transcriptional activation of TNF-␣ across toxin treatment. The number of positive IL-12 cells and the OD the hemisphere of the ipsilateral side. The cerebral cortex, hip- values for TNF-␣ and TLR2 signal were not significantly different pocampus, corpus callosum, and basal ganglia exhibited an intense for OVX-LPS, OVX-vehicle, and intact-vehicle groups of mice TNF-␣ mRNA signal 24 h after the single bolus of LPS (Fig. 1, (Fig. 2B, 24 h). The endotoxin was therefore only able to rapidly 24 h LPS). The message spread throughout the entire hemisphere trigger microglial cells in the brain of intact animals. However, the at 3 days and largely declined 7 days following the endotoxin brains of OVX mice responded to the single bolus of LPS at 3 and administration. The pattern for the mRNA encoding TLR2 was 7 days postadministration. TNF-␣ and TLR2 mRNA signals were similar, but the signal for IL-12p40 was more localized to the similar and significantly higher in the brain of both LPS-treated infusion site and vanished 3 days after the intrastriatal LPS infu- groups than in vehicle-administered mice 3 days following the sion (Fig. 2). In this particular case, numerous IL-12p40-express- intracerebral treatment (Fig. 2B). The gene encoding IL-12p40 still ing cells were found in the cerebral cortex of mice killed 24 h after remained undetectable at 3 days post LPS infusion in intact and the single LPS treatment (Fig. 2, top left panels). In contrast, TLR2 OVX mice. Therefore, OVX completely abolished the synthesis of mRNA signal remained strong up to 7 days post LPS infusion (Fig. this particular cytokine in response to intraparenchymal endotoxin 2B, bottom). infusion. To ascertain the phenotype of IL-12p40-, TNF-␣-, and TLR2- We have consequently verified whether these effects were spe- expressing cells, immunocytochemistry was combined with in situ cific to a model of intracerebral endotoxin insult and performed hybridization. We used in this study an Ab directed against anti- another series of experiments in intact and OVX mice challenged ionized calcium binding adapter molecule 1 (iba1) to label cells of with a systemic bolus of LPS. We have previously shown that this myeloid lineage (macrophages and microglia) (27, 28). All three dose of LPS (1 mg/kg) causes a profound increase in the transcrip- transcripts colocalized with iba1-immunoreactive cells providing tion of the gene encoding TLR2 first with the circumventricular clear anatomic evidence that microglia are the key cells responsi- organs (CVOs), choroid plexus, leptomeninges, and microcapillar- ble for the cerebral innate immune response in the presence of a cell ies and thereafter across the brain parenchyma in a migratory-like wall component derived from Gram-negative bacteria (Fig. 1B). pattern (24). Fig. 3 depicts representative examples of such a phe- nomenon in the choroid plexus/adjacent regions and microcapil- Role of E2 in the control of cerebral innate immunity laries/adjacent areas. A very strong signal was detected in these Of interest is the aberrant innate immune reaction in the brain of structures 24 h after the single i.p. LPS bolus. Scattered TLR2- OVX mice (Fig. 2). Indeed, OVX mice failed to respond to a single expressing cells were also found in the cortex and in various re- bolus of LPS at 24 h; although the signal for IL-12, TNF-␣, and gions of the CNS (Fig. 3, arrows). Save for the groups of cells 6394 E2 IN CEREBRAL INNATE AND ADAPTIVE IMMUNITY

FIGURE 2. The innate immune response is altered in the brain of ovariectomized (OVX) mice. A, Photomi- crographs depicting hybridization signal of IL-12p40 (top), TNF-␣ (middle), and TLR2 (bottom) mRNA 24 h after the intrastriatal LPS infusion. The coronal sections were taken from the x-ray ﬁlm (Biomax; Kodak), whereas darkﬁeld photomicrographs were taken from the same section dipped into the nuclear emulsion milk (NTB-2; Kodak). B, Quantitative

analysis of the different cytokine tran- Downloaded from scripts in the ipsilateral side. The number of IL-12-expressing cells (top) in four coronal sections of each animal with OD measurements (middle and bottom) are shown. Values are mean Ϯ SEM of three to four mice per group, and statistical analysis was http://www.jimmunol.org/ performed using a two-way ANOVA followed by a Bonferroni-Dunn -Signif ,ء .(posthoc test (StatView 4.5 icantly different (p Ͻ 0.05) from their corresponding vehicle-treated groups. Scale bar represents 500 ␮m. IL, intact LPS; OL, OVX LPS; IV, intact vehicle; OV, OVX vehicle. by guest on September 28, 2021

associated with the choroid plexus and CVOs, TLR2 transcript Role of E2 in a mouse model of viral infection levels remained low to undetectable in parenchymal microglia of The next series of experiments investigated the consequences of an OVX animals challenged with the bacterial cell wall component altered innate immune response in a model of viral infection, (Fig. 3, middle column). Replacement therapy with E completely 2 which involves both innate and adaptive immunities. The HSV-2 rescued the ability of LPS to trigger TLR2 gene expression is neurotrophic and its replication in the CNS is associated with a throughout the cerebral tissue (Fig. 3, right column). robust inflammatory response in the hindbrain of male mice (21). These data therefore clearly support the concept that E is re- 2 Female mice inoculated with HSV-2 also exhibited a strong hy- quired for expression of the normal complement of proinflamma- bridization signal for numerous inflammatory genes and the viral tory proteins in response to both intracerebral and systemic LPS treatments in the mouse brain. To determine the receptor involved thymidine kinase (index of HSV replication) in the pons and me- in these effects, we used mutant mice deficient in either ER␣ dulla 7–10 days postinoculation. However, the signals were low or (ERKO mice) or ER␤ (BERKO mice) that were challenged with a barely detectable in more rostral regions, such as the hypothala- single i.p. LPS bolus. Similar to OVX animals, ERKO mice were mus, mammillary bodies, and thalamic structures of intact female less sensitive to the endotoxin and hybridization signal for the gene mice (Fig. 5). In contrast, a widespread pattern of HSV-immuno- encoding TLR2 was significantly lower in ERKO mice than their reactive neurons and TLR2-expressing cells was found across the wild-type littermates. In contrast, a strong increase in TLR2 gene hypothalamus of OVX mice killed 10 days after being inoculated expression took place in the CNS of both BERKO and wild-type with HSV-2. This microglial reactivity (TLR2-positive cells) (Fig. mice. Semiquantitative analysis in the region of the median emi- 5C) is a direct consequence of an increased neurovirulence (Fig. nence/arcuate nucleus indicated that TLR2 mRNA levels were sig- 5A) and HSV-induced neuronal damage (Fig. 5B). The latter was nificantly lower in ERKO mice than the other LPS-treated groups estimated by immunohistochemistry using a primary Ab directed (Fig. 4, bottom). Actually, the OD value was four times lower in against the neuronal marker NeuN, which vanishes rapidly in de- ERKO mice compared with their wild-type littermates 24 h after generating neurons (29). As depicted in Fig. 5B, HSV-immunore- the systemic LPS challenge. active neurons no longer contained nuclear NeuN staining in the The Journal of Immunology 6395

␤ FIGURE 3. Role of 17 -E2 on systemic LPS-induced innate immune response in the brain. Mice received a single i.p. injection of either vehicle (sterile saline) or LPS (1 mg/kg) and were killed 24 h afterward. The coronal sections depict representative patterns of TLR2 hybridization signal in intact, OVX, or OVX mice that received replacement therapy with 17␤-

E2. Please note the robust expression of TLR2 mRNA ϩ in the brain of intact (left column) and OVX E2 (right column) mice 24 h following the i.p. LPS administration, whereas OVX mice exhibited an attenuated response to the endotoxin (middle column). Coronal sections were taken from x-ray ﬁlms (Biomax; Kodak), whereas the same sections dipped into nuclear emulsion (NTB-2; Kodak) were used for the darkﬁeld photomicrographs (bottom). SH, sham implant. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

␤ FIGURE 4. The permissive influence of 17 -E2 on the CNS innate immunity is dependent on ER␣. Both ER␣ (ERKO, top panels) and ER␤ (BERKO, middle panels) groups of mice were killed 24 h following a single i.p. injection of LPS (1 mg/kg). TLR2 gene expression was used as a general index of innate immune response in the brain that was stimulated by the endotoxin and significantly prevented in the brain of ERKO mice (KOϩLPS). The semiquantitative analysis was performed in the region depicted by the rectangle illus- trated on the coronal section (top left). Values are mean Ϯ SEM and statistical analysis was performed using a two-way ANOVA followed by a Bonferroni- -Significantly dif ,ء .(Dunn posthoc test (StatView 4.5 ferent (p Ͻ 0.05) from their corresponding vehicle- treated groups. †, Significantly different (p Ͻ 0.05) from all the others LPS-treated groups. Note that vehicle-treated mice exhibited very low TLR2 mRNA signal, which explains the nondetectable OD levels in most of the control groups (f). 6396 E2 IN CEREBRAL INNATE AND ADAPTIVE IMMUNITY Downloaded from http://www.jimmunol.org/ FIGURE 5. Widespread viral replication and inflammation in the brain of OVX mice. Both intact and OVX groups were inoculated intranasally with 20 ␮l of MEM containing 103 PFU of HSV-2. A, HSV-immunoreactive (ir) neurons in the hypothalamic paraventricular nucleus and the anterior/lateral hypothalamic area of OVX mice 10 days after the intranasal inoculation. B, Laser scanning confocal microscopic image of HSV-immunoreactive (ir) neurons (green, FITC) and the neuronal marker NeuN (red, Alexa Fluor 594). Laser scanning confocal microscopy studies were performed with a ϫ20 and ϫ100 Plan-Apo oil immersion objective, numerical apertures 0.8 and 1.35, ϫ2 numerical zoom with a BX-61 microscope (Olympus America). Emissions were recorded by photomultipliers preset, respectively, for FITC (green pseudo color) and Alexa Fluor 594 (red pseudo color) fluorescent dyes in Fluoview SV500 imaging software. Thirteen 0.5-␮m confocal z-series were acquired for each area and corrected by two Kahlman low speed scans. Acquired z-series images were then flattened into one image and exported in 24 bit TIFF format. C, Expression of TLR2 (used as a general index of macrophage/microglia activation) mRNA during the course of HSV-2 replication in the brain of intact and OVX mice is shown. Coronal sections were taken from x-ray films (Biomax; Kodak). Note the strong immunoreactive signal for HSV-2 in the hypothalamic nuclei and areas of OVX animals (A, right column), but not in by guest on September 28, 2021 the rostral brain of intact mice inoculated with the virus (A, left column). These stressed neurons no longer contain nuclear NeuN staining (B, right column). A robust TLR2 transcriptional activation also took place in the mediobasal forebrain of HSV-treated mice devoid of estrogen (C, right column). Such a macrophage/microglial reactivity is a consequence of HSV-induced neuronal damage in the rostral brain of OVX mice (see Results).

hypothalamus of OVX mice. Intact mice inoculated with the virus (20). These data are of great interest because these regions are had very few (if any) positive HSV-immunoreactive neurons in considered to be immune sentinels of the brain during systemic ϩ Ϫ these rostral regions, but numerous HSV /NeuN neurons were infection. found across the hindbrain of both intact and OVX mice (data not The innate immune response is driven through specific recog- shown). These data indicate that an inadequate innate immune re- nition systems, such as interaction between LPS and its receptors action to HSV-2 is associated with an increased neurovirulence CD14 and TLR4. Both CD14 and TLR4 receptors are constitu- and a more widespread neurodegeneration in the brain of tively expressed in the CVOs, and circulating LPS is able to cause OVX mice. a rapid transcriptional activation of genes encoding proinflammatory molecules in these organs (6, 8, 30). Constitutive expression Discussion of CD14, TLR4, and ER␣ in CVOs provides direct anatomical The present study shows that E2 plays an essential role in modu- evidence that estrogen may enhance the LPS signaling events lating the proinflammatory signaling and gene expression in mi- within these regions that are devoid of blood-brain barrier. Al- croglial cells of mice. An aberrant reaction took place in the brain though we have yet to determine whether ER␣ is colocalized of OVX mice challenged with LPS either centrally or systemically. within CD14- and TLR4-expressing cells, it is tempting to propose These permissive properties of E for the inflammatory responses 2 that interaction between E and ER␣ in the CVOs is required for are mediated via ER␣, because ERKO and OVX mice had similar 2 altered microglial reactivity to LPS. The neuroanatomical organi- a proper immune response to infection. zation of ER␣ and ER␤ across the rodent brain has indicated a The ability of E2 to modulate the innate immune response is widespread distribution of target sites for estrogen to influence however not specific to the CVOs because centrally injected LPS multiple cellular functions in a site- and receptor-dependent man- is able to activate microglial cells in a sex steroid-dependent manner. Indeed, several regions were found to express both receptor ner in female mice. Although in situ hybridization and immuno- subtypes, but fine differences were reported in terms of intensity histochemistry may not be sensitive enough to detect ER␣ in mi- and positive signal (20). Moreover, the mRNA encoding ER␣ has croglia, E2 was found to modulate the effects of LPS on primary a more widespread distribution than the ER␤, and ER␣ is the only cultures of microglial cells (12). This effect is also the case for transcript expressed at different levels of all the sensorial CVOs TLR4 transcript that is undetectable in microglial cells by in situ The Journal of Immunology 6397

hybridization (30), but these cells respond to LPS in a TLR4- cells are actually believed to play a critical role in multiple scle- dependent manner (26, 31, 32). It can therefore be concluded that rosis, especially during the demyelinating episodes (see review

LPS and E2 can target their respective cognate receptors in micro- Ref. 42). Of interest is that multiple sclerosis occurs more com- glial cells throughout the brain. monly in females than males indicating that sex steroids may play Interaction between LPS and TLR4 triggers signaling pathways a role in this autoimmune disease. Our data ﬁt very well with this

that are similar to those activated by IL-1. Indeed, type I IL-1R has mechanism and the permissive properties of E2 on the innate im- the same Toll/IL-1R homology domain capable of interacting with munity may explain the susceptibility of females to autoimmune

MyD88, an adaptor protein that is recruited upon activation of type diseases. In contrast, alteration of circulating E2 levels during ce- I IL-1R or TLRs. This leads to NF-␬B signaling and transcriptional rebral infection may have profound consequences for the CNS if T activation of numerous proinﬂammatory genes involved in the cells are not properly activated by APCs in the brain microglia. control of the innate immune process. A critical question at this The fact that these cells no longer express IL-12 mRNA in OVX point is how ER␣ interferes with this pathway and whether this mice treated with LPS clearly support the concept that appropriate effect is direct or indirect? ER␣ belongs to a superfamily of ligand- transfer from innate to acquired immunity is dependent on estro- activated transcription factors comprising steroids, thyroid hor- gens in the female brain. mones, retinoids, and vitamin D characterized by a central DNA- Interestingly, neurovirulence and neuronal damage spread into binding domain capable of targeting the receptor to speciﬁc DNA the regions of OVX mice not previously found in male (21) and sequences known as hormone response elements (33). ER␣ has a intact female mice (as found in our study). Numerous hypotha- modular structure consisting of distinct functional domains allow- lamic areas and nuclei exhibited a strong immunoreactive signal

ing the binding on the consensus site AGGTCA referred to as the for HSV only in OVX mice. These groups of infected and degen- Downloaded from estrogen response element. The consensus estrogen response ele- erating neurons were also associated with microglial reactivity as ment is present in a number of genes expressed in the brain, which indicated by the robust hybridization signal for the gene encoding are known to be under the inﬂuence of estrogen. In this regard, TLR2 in the mediobasal forebrain (Fig. 5). It can therefore be Wissink et al. (34) have shown that estrogen can up-regulate ex- concluded that inappropriate transfer from innate to adaptive im- pression of the serotonin-1A receptor via a new mechanism in- munity allows HSV-2 to replicate more aggressively within neu-

volving synergistic activation by NF-␬B with ER␣. These authors rons of the CNS. In this regard, deficient IFN-␥ production was http://www.jimmunol.org/ have proposed that NF-␬B complexes cooperate with ER␣ to re- reported to be directly related to HSV-2 neurovirulence (43). The cruit cofactors into the complex and thereby synergistically acti- low IL-12 production by microglia/macrophages may then be a vate the promoter through nonclassical estrogen response elements key mechanism for the pathogens to hide from the immune sur- by a mechanism that does not involve direct receptor binding to veillance resulting in increased viral replication in the brain of DNA (34). It would be interesting to investigate whether such a OVX mice. mechanism takes place in microglial cells in vivo. Estrogen replacement therapy has been associated with neuro- It is nevertheless important to mention that numerous studies protection and the current dogma is that these effects are attribut- have provided evidence that estrogens inhibit NF-␬B and suppress able to the immunosuppressive properties of these hormones. In proinflammatory signaling and cytokine gene expression. Binding this study we propose that it is actually the opposite. A defective by guest on September 28, 2021 ␣ of E2 to its nuclear ER was found to interfere with cytokine- innate immune response takes place in the brain of OVX mice in induced activation of a NF-␬B reporter in HepG2 cells (35). Tran- an ER␣-dependent manner. This may render the host more vul- ␬ scriptional activity of the RelA subunit of NF- B can be sup- nerable to infection in the presence of low circulating E2 levels and ␤ pressed in a hormone-dependent manner (36, 37), whereas 17 -E2 exacerbate neuronal damages due to an inappropriate innate im- is able to decrease LPS-induced NF-␬B binding activity and IL- mune reaction by microglia. Obviously, these neuroprotective ef- 1␣, IL-6, and TNF-␣ production, but not IL-10, IL-12, and MIP fects of estrogens are likely to differ between models in which production by splenic macrophages (38). Inducible NO synthase proinflammatory cytokines are generally associated with adverse

expression is attenuated by E2, though the latter failed to alter outcomes, such as in ischemia reperfusion injuries and chronic NF-␬B activation in primary rat microglia and N9 microglial cell demyelinating diseases. lines (15). Ospina et al. (39) have reported that estrogen decreases the inﬂammatory response to IL-1 in the brain in OVX animals and Acknowledgments that sex hormone replacement in OVX rats attenuated LPS fever We thank Dr. D. Radzioch (McGill University, Montreal, Quebec, Canada) (40), but it potentiated IL-1␤-induced fever and cyclooxygenase-2 for the cDNA containing the mouse TNF-␣, Dr. K. Pahan (University of

expression in the brain (41). E2 was also found to significantly Nebraska Medical Center, Lincoln, NE) for the mouse IL-12p40 cDNA, prevent LPS-induced microglial reactivity in an ER␣-dependent Dr. Y. Imai (National Institute of Neuroscience, Kodaira, Japan) for the manner (13, 14). The latter studies reached these conclusions anti-iba1 Ab, and Dr. P. Chambon (University of Strasbourg, Strasbourg, France) for the ERKO and BERKO breeding mouse pairs. based on the changes in inducible NO synthase, PGE2, and met- alloproteinase-9 levels, which have not been assayed in the present study. It is therefore difficult to explain the discrepancies between Disclosures these studies and the present set of data, but the effects of sex The authors have no financial conflict of interest. steroids may depend on the target genes. In the present series of experiments using various approaches and models in vivo, expres- References sion of genes involved in the control of the innate immune re- 1. Anderson, K. V. 2000. Toll signaling pathways in the innate immune response. Curr. Opin. Immunol. 12: 13–19. sponse was clearly attenuated in microglial cells of OVX mice 2. Wright, S. D. 1999. Toll, a new piece in the puzzle of innate immunity. J. Exp. challenged with a Gram-negative cell wall component. Med. 189: 605–610. 3. Soulet, D., and S. Rivest. 2003. Polyamines play a critical role in the control of Among the genes assessed in the present study, IL-12 is of par- the innate immune response in the mouse CNS. J. Cell Biol. 162: 257–268. ticular interest. IL-12 is a key cytokine involved in the transfer 4. Nadeau, S., and S. Rivest. 2003. Glucocorticoids play a fundamental role in between the innate and adaptive immunity. Macrophage-derived protecting the brain during innate immune response. J. Neurosci. 23: 5536–5544. IL-12 stimulates the differentiation of a subset of T lymphocytes 5. Nadeau, S., and S. Rivest. 2002. Endotoxemia prevents the cerebral inflammatory ϩ wave induced by intraparenchymal lipopolysaccharide injection: role of glu- (CD4 ) into Th1 cells that produce IFN-␥. These activated Th-1 cocorticoids and CD14. J. Immunol. 169: 3370–3381. 6398 E2 IN CEREBRAL INNATE AND ADAPTIVE IMMUNITY

6. Nadeau, S., and S. Rivest. 2000. Role of microglial-derived tumor necrosis factor induction of the gene-encoding Toll-like receptor 2 in the CNS. J. Neurochem. in mediating CD14 transcription and NF-␬B activity in the brain during endo- 79: 648–657. toxemia. J. Neurosci. 20: 3456–3468. 25. Lacroix, S., and S. Rivest. 1997. Functional circuitry in the brain of immune- 7. Nadeau, S., and S. Rivest. 2001. The complement system is an integrated part of challenged rats: partial involvement of prostaglandins. J. Comp. Neurol. 387: the natural innate immune response in the brain. FASEB J. 15: 1410–1412. 307–324. 8. Nguyen, M. D., J. P. Julien, and S. Rivest. 2002. Innate immunity: the missing 26. Glezer, I., H. Zekki, C. Scavone, and S. Rivest. 2003. Modulation of the innate link in neuroprotection and neurodegeneration? Nature Rev. Neurosci. 3: immune response by NMDA receptors has neuropathological consequences. 216–227. J. Neurosci. 23: 11094–11103. 9. McKay, L. I., and J. A. Cidlowski. 1999. Molecular control of immune/inflam- 27. Ito, D., Y. Imai, K. Ohsawa, K. Nakajima, Y. Fukuuchi, and S. Kohsaka. 1998. matory responses: interactions between nuclear factor-␬B and steroid receptor- Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain signaling pathways. Endocr. Rev. 20: 435–459. Res. Mol. Brain. Res. 57: 1–9. 10. Ben-Hur, H., G. Mor, V. Insler, I. Blickstein, Y. Amir-Zaltsman, A. Sharp, 28. Imai, Y., I. Ibata, D. Ito, K. Ohsawa, and S. Kohsaka. 1996. A novel gene iba1 A. Globerson, and F. Kohen. 1995. Menopause is associated with a significant in the major histocompatibility complex class III region encoding an EF hand increase in blood monocyte number and a relative decrease in the expression of protein expressed in a monocytic lineage. Biochem. Biophys. Res. Commun. 224: estrogen receptors in human peripheral monocytes. Am. J. Reprod. Immunol. 34: 855–862. 363–369. 29. Blais, V., and S. Rivest. 2004. Effects of TNF-␣ and IFN-␥ on nitric oxide- 11. Mor, G., E. Sapi, V. M. Abrahams, T. Rutherford, J. Song, X.-Y. Hao, induced neurotoxicity in the mouse brain. J. Immunol. 172: 7043–7052. S. Muzaffar, and F. Kohen. 2003. Interaction of the estrogen receptors with the 30. Laflamme, N., and S. Rivest. 2001. Toll-like receptor 4: the missing link of the Fas ligand promoter in human monocytes. J. Immunol. 170: 114–122. cerebral innate immune response triggered by circulating Gram-negative bacterial 12. Vegeto, E., G. Pollio, P. Ciana, and A. Maggi. 2000. Estrogen blocks inducible cell wall components. FASEB J. 15: 155–163. nitric oxide synthase accumulation in LPS-activated microglia cells. Exp. Ger- 31. Lehnardt, S., L. Massillon, P. Follett, F. E. Jensen, R. Ratan, P. A. Rosenberg, ontol. 35: 1309–1316. J. J. Volpe, and T. Vartanian. 2003. Activation of innate immunity in the CNS 13. Vegeto, E., C. Bonincontro, G. Pollio, A. Sala, S. Viappiani, F. Nardi, triggers neurodegeneration through a Toll-like receptor 4-dependent pathway. A. Brusadelli, B. Viviani, P. Ciana, and A. Maggi. 2001. Estrogen prevents the Proc. Natl. Acad. Sci. USA 100: 8514–8519. 32. Lehnardt, S., C. Lachance, S. Patrizi, S. Lefebvre, P. L. Follett, F. E. Jensen,

lipopolysaccharide-induced inflammatory response in microglia. J. Neurosci. 21: Downloaded from 1809–1818. P. A. Rosenberg, J. J. Volpe, and T. Vartanian. 2002. The Toll-like receptor TLR4 is necessary for lipopolysaccharide-induced oligodendrocyte injury in the 14. Vegeto, E., S. Belcredito, S. Etteri, S. Ghisletti, A. Brusadelli, C. Meda, A. Krust, CNS. J. Neurosci. 22: 2478–2486. S. DuPont, P. Ciana, P. Chambon, and A. Maggi. 2003. Estrogen receptor-␣ 33. Mangelsdorf, D. J., C. Thummel, M. Beato, P. Herrlich, G. Schu¨tz, K. Umesono, mediates the brain antiinflammatory activity of estradiol. Proc. Natl. Acad. Sci. B. Blumberg, P. Kastner, M. Mark, P. Chambon, and R. M. Evans. 1995. The USA 100: 9614–9619. nuclear receptor superfamily: the second decade. Cell 83: 835–839. 15. Bruce-Keller, A. J., J. L. Keeling, J. N. Keller, F. F. Huang, S. Camondola, and 34. Wissink, S., B. van der Burg, B. S. Katzenellenbogen, and P. T. van der Saag. M. P. Mattson. 2000. Antiinflammatory effects of estrogen on microglial activa- 2001. Synergistic activation of the serotonin-1A receptor by nuclear factor-␬B tion. Endocrinology 141: 3646–3656.

and estrogen. Mol. Endocrinol. 15: 543–552. http://www.jimmunol.org/ 16. Garcia-Segura, L. M., I. Azcoitia, and L. L. DonCarlos. 2001. Neuroprotection by 35. Harnish, D. C., M. S. Scicchitano, S. J. Adelman, C. R. Lyttle, and estradiol. Prog. Neurobiol. 63: 29–60. S. K. Karathanasis. 2000. The role of CBP in estrogen receptor cross-talk with 17. Wise, P. M., D. B. Dubal, M. E. Wilson, S. W. Rau, and M. Bottner. 2001. nuclear factor-␬B in HepG2 cells. Endocrinology 141: 3403–3411. Minireview: neuroprotective effects of estrogen-new insights into mechanisms of 36. Valentine, J. E., E. Kalkhoven, R. White, S. Hoare, and M. G. Parker. 2000. action. Endocrinology 142: 969–973. Mutations in the estrogen receptor ligand binding domain discriminate between 18. Jover, T., H. Tanaka, A. Calderone, K. Oguro, M. V. Bennett, A. M. Etgen, and hormone-dependent transactivation and transrepression. J. Biol. Chem. 275: R. S. Zukin. 2002. Estrogen protects against global ischemia-induced neuronal 25322–25329. death and prevents activation of apoptotic signaling cascades in the hippocampal 37. McKay, L. I., and J. A. Cidlowski. 1998. Cross-talk between nuclear factor-␬B CA1. J. Neurosci. 22: 2115–2124. and the steroid hormone receptors: mechanisms of mutual antagonism. Mol. En- 19. McDonnell, D. P., and J. D. Norris. 2002. Connections and regulation of the docrinol. 12: 45–56. human estrogen receptor. Science 296: 1642–1644. 38. Deshpande, R., H. Khalili, R. G. Pergolizzi, S. D. Michael, and M. D. Chang.

20. Laflamme, N., R. E. Nappi, G. Drolet, C. Labrie, and S. Rivest. 1998. Expression 1997. Estradiol down-regulates LPS-induced cytokine production and NF␬B ac- by guest on September 28, 2021 and neuropeptidergic characterization of estrogen receptors (ER␣ and ER␤) tivation in murine macrophages. Am. J. Reprod. Immunol. 38: 46–54. throughout the rat brain: anatomical evidence of distinct roles of each subtype. 39. Ospina, J. A., H. N. Brevig, D. N. Krause, and S. P. Duckles. 2004. Estrogen J. Neurobiol. 36: 357–378. suppresses IL-1␤-mediated induction of COX-2 pathway in rat cerebral blood 21. Boivin, G., Z. Coulombe, and S. Rivest. 2002. Intranasal herpes simplex virus vessels. Am. J. Physiol. Heart Circ. Physiol. 286: H2010–H2019. type 2 inoculation causes a profound thymidine kinase dependent cerebral in- 40. Mouihate, A., and Q. J. Pittman. 2003. Neuroimmune response to endogenous flammatory response in the mouse hindbrain. Eur. J. Neurosci. 16: 29–43. and exogenous pyrogens is differently modulated by sex steroids. Endocrinology 22. DuPont, S., A. Krust, A. Gansmuller, A. Dierich, P. Chambon, and M. Mark. 144: 2454–2460. 2000. Effect of single and compound knockouts of estrogen receptors ␣ (ER␣) 41. Mouihate, A., X. Chen, and Q. J. Pittman. 1998. Interleukin-1␤ fever in rats: and ␤ (ER␤) on mouse reproductive phenotypes. Development 127: 4277–4291. gender difference and estrous cycle influence. Am. J. Physiol. 275(5 Pt. 2): 23. Laflamme, N., H. Echchannaoui, R. Landmann, and S. Rivest. 2003. Cooperation R1450–R1454. between Toll-like receptor 2 and 4 in the brain of mice challenged with cell wall 42. Hemmer, B., J. J. Archelos, and H. P. Hartung. 2002. New concepts in the im- components derived from Gram-negative and Gram-positive bacteria. Eur. J. Im- munopathogenesis of multiple sclerosis. Nat. Rev. Neurosci. 3: 291–301. munol. 33: 1127–1138. 43. Lewandowski, G., M. Hobbs, and A. Geller. 1998. Evidence that deficient IFN-␥ 24. Laflamme, N., G. Soucy, and S. Rivest. 2001. Circulating cell wall components production is a biological basis of herpes simplex virus type-2 neurovirulence. derived from Gram-negative and not Gram-positive bacteria cause a profound J. Neuroimmunol. 81: 66–75.