INFECTION AND IMMUNITY, Dec. 2009, p. 5369–5379 Vol. 77, No. 120019-9567/09/$12.00 doi:10.1128/IAI.00455-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

MyD88-Deficient Mice Exhibit Decreased Parasite-InducedImmune Responses but Reduced Disease Severity in a

Murine Model of Neurocysticercosis�

Bibhuti B. Mishra,1† Uma Mahesh Gundra,1† Kondi Wong,2 and Judy M. Teale1*Department of Biology, South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio,

One UTSA Circle, San Antonio, Texas 78249-1644,1 and Department of Pathology, Tampa General Hospital,One Tampa Circle, Tampa, Florida 336062

Received 22 April 2009/Returned for modification 29 May 2009/Accepted 18 September 2009

The symptomatic phase of neurocysticercosis (NCC), a parasitic disease of the central nervous system(CNS) in humans, is characterized by inflammatory responses leading to neuropathology and, in somecases, death. In an animal model of NCC in which mice were intracranially inoculated with the parasiteMesocestoides corti, the infection in mice lacking the myeloid differentiation primary response gene 88(MyD88�/�) resulted in decreased disease severity and improved survival compared with that in wild-type(WT) mice. The CNS of MyD88�/� mice was more quiescent, with decreased microgliosis and tissuedamage. These mice exhibited substantially reduced primary and secondary microglial nodule formationsand lacked severe astrogliotic reactions, which were seen in WT mice. Significantly reduced numbers ofCD11b� myeloid cells, �� T cells, �� T cells, and B cells were present in the brains of MyD88�/� mice incomparison with those of WT mice. This decrease in cellular infiltration correlated with a decrease inblood-brain barrier permeability, as measured by reduced fibrinogen extravasation. Comparisons ofcytokine expression indicated a significant decrease in the CNS levels of several inflammatory mediators,such as tumor necrosis factor alpha, gamma interferon, CCL2, and interleukin-6, during the course ofinfection in MyD88�/� mice. Collectively, these findings suggest that MyD88 plays a prominent role in thedevelopment of the hyperinflammatory response, which in turn contributes to neuropathology and diseaseseverity in NCC.

Neurocysticercosis (NCC) is the most common parasitic dis-ease of the central nervous system (CNS), occurring as a resultof infection of the brain with the larval stage of the tape wormparasite Taenia solium (56). In humans, the disease has a longasymptomatic phase, typically 3 to 5 years, followed by thesymptomatic phase, consisting of clinical signs such as epilepsy(43), increased intracranial (i.c.) pressure, obstructive hy-droencephalus, stroke, and encephalitis (55, 56). More than25% of all epileptic cases diagnosed in adults worldwide aredue to NCC (19). The sequential progression from asymptom-atic to symptomatic NCC depends upon the degeneration oflarvae, caused by either therapeutic treatment or normal attri-tion. This leads to the induction of a strong inflammatoryresponse, causing a chronic granulomatous reaction and themanifestation of symptoms of the disease (41, 57). The im-mune response in the CNS of symptomatic patients consists ofan overt TH1 phenotype (39) or a mixed TH1, TH2, and TH3phenotype, depending upon the absence or presence of gran-uloma formation (38). Specifically, the TH1 hyperinflamma-tory response prevailing in the CNS during the symptomaticphase is thought to be responsible for the severe neuropathol-ogy and mortality associated with NCC (55). Direct evidence

that the inflammatory/TH1 response contributes to the neuro-pathology and severity of NCC, however, is limited. Neverthe-less, along with antiparasitic drugs, the treatment of NCCpatients with immunosuppressive/anti-inflammatory factorssuch as corticosteroids helps to control the host inflammatoryresponse and associated neuropathology (32). Long-termtreatments with steroids, however, lead to problematic sideeffects that may become life-threatening. Therefore, despiterecent advances made in detection and therapy, effective treat-ment of NCC remains a major challenge, as cysticidal treat-ment itself results in the symptoms that one is trying to controland/or the manifestation of other complications. Therefore, itis important to understand the pathophysiological basis of theCNS inflammatory response in NCC and to identify criticalmolecules responsible for such responses.

The myeloid differentiation primary response gene 88(MyD88) is an important regulator of the host inflammatoryresponse (50, 51). The protein produced by the MyD88 gene isan adaptor molecule necessary for signal transductions orig-inating from the interleukin-1 receptor (IL-1R)/IL-18Rfamily of receptors and the Toll-like receptor (TLR) familyof proteins (35). Once engaged, TLRs signal through a com-mon pathway involving MyD88 (42), leading to the subse-quent downstream activation of the NF-�B and mitogen-activated protein (MAP) kinase pathways and inducing aTH1 proinflammatory response (28). Previous studies havedemonstrated that MyD88 knockout mice exhibit defectiveproinflammatory responses and display dramatic defects in

* Corresponding author. Mailing address: Department of Biology,The University of Texas at San Antonio, One UTSA Circle, SanAntonio, TX 78249-1644. Phone: (210) 458-7024. Fax: (210) 458-7025.E-mail: judy.teale@utsa.edu.

† B.B.M. and U.M.G. contributed equally to this work.� Published ahead of print on 28 September 2009.

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antimicrobial immunity in a variety of infectious diseasemodels, highlighting the importance of this molecule in in-fluencing a wide array of host responses and disease control(2, 8, 18, 45, 52). A contrasting situation occurs in onchocer-ciasis, an infection of the eye caused by another helminthparasite, Onchocerca volvulus. In this case, MyD88 plays apivotal role in the development of a persistent hyperinflam-matory response eventuating in corneal haze and hencecontributing to the development of river blindness (22, 26).An important unanswered question, therefore, is whetherMyD88-dependent mechanisms are involved in inflamma-tory responses that contribute to the observed pathologyand severity of NCC or play an essential role in the con-tainment of this disease.

The goal of this study was to identify the overall effect of theabsence of the MyD88-dependent signaling pathway, using awell-characterized murine model of NCC developed in ourlaboratory (14). In the present study, we compared the suscep-tibilities and immunopathology of MyD88�/� and wild-type(WT) mice infected i.c. with Mesocestoides corti. The contribu-tion of MyD88 signaling to CNS inflammation was assessed bymeasuring infiltration of various immune cells into the brain,blood-brain barrier (BBB) permeability, and proinflammatorycytokine responses in the CNS of MyD88�/� and WT mice.

MATERIALS AND METHODS

Mice. Female mice aged 3 to 5 weeks were used in this study. MyD88�/� micein the C57BL/6 background (originally from S. Akira, Osaka University, Osaka,Japan) were obtained from Michael Berton (Department of Microbiology andImmunology, University of Texas Health Science Center, San Antonio, TX).C57BL/6 mice were obtained from the National Cancer Institute animal program(Bethesda, MD) and used as WT controls. WT controls and MyD88�/� micewere bred in the University of Texas at San Antonio (UTSA) animal facility.Experiments were conducted under the guidelines of the IACUC, UTSA, Uni-versity of Texas System, the U.S. Department of Agriculture, and the NationalInstitutes of Health.

Antibodies. Phycoerythrin-conjugated antibodies purchased from BD Phar-mingen (San Diego, CA) included GL3 (pan-anti-��), H57-597 (pan-anti-��),M1/70 (anti-Mac1), 1D3 (anti-CD19), R35-95 (anti-rat immunoglobulin G2a[IgG2a], � chain), B81-3 (anti-hamster IgG2, � chain), and HA4/8 (anti-hamsterIgG2, � chain). Purified 2.4G2 (anti-CD16/32), anti-mouse fibrinogen, anti-mouse IL-6, anti-mouse glial fibrillary acidic protein (anti-mouse GFAP), andbiotinylated anti-mouse CD11b were also purchased from BD Pharmingen.Biotinylated anti-mouse neuronal nuclear protein was purchased from U.S. Bi-ological (Swampscott, MA). Anti-mouse GFAP was conjugated to Alexa Fluor488 (Molecular Probes, Eugene, OR) according to the manufacturer’s instruc-tions. For indirect immunofluorescence (IF), appropriate fluorescently conju-gated secondary antibodies (Jackson Immunoresearch Laboratories, WestGrove, PA) were used to detect specific primary antibodies against immunemediators and cell surface markers. Biotinylated primary antibodies were de-tected using Alexa Fluor 488-labeled streptavidin (Molecular Probes).

Murine model of NCC. In this study, we used a mouse model of NCC devel-oped in our laboratory (14, 15). M. corti metacestodes were maintained by serialintraperitoneal inoculation of 8- to 12-week-old female BALB/c mice. Metaces-todes were aseptically harvested, and murine NCC was induced by i.c. injectionof 50 �l of Hanks balanced salt solution (HBSS) containing about 40 parasitesinto 3- to 5-week-old mice under short-term anesthesia, as described previously.Mock-infected control mice were injected by the i.c. route with 50 �l sterileHBSS, using the same protocol. Before i.c. inoculation, mice were anesthetizedwith a 50-�l mixture of ketamine HCl and xylazine (30 mg/ml ketamine and 4mg/ml xylazine in phosphate-buffered saline [PBS]) given intramuscularly. Ani-mals were sacrificed at the indicated times after inoculation and analyzed forparasite burden and various immune parameters. Before sacrifice, animals wereanesthetized with 100 �l of the above mixture and perfused through the leftventricle with 10 ml cold PBS.

Tissue processing. The brain was immediately dissected from perfused ani-mals, embedded in optimal-cutting-temperature resin, and snap-frozen. Serial

horizontal cryosections of 10 �m in thickness were placed on silane prep slides(Sigma-Aldrich, St. Louis, MO). One in every four slides was fixed in formalin for10 min at room temperature and stained with hematoxylin and eosin (H&E). Theremainder of the slides were air dried overnight and fixed in fresh acetone for20 s at room temperature. Acetone-fixed sections were wrapped in aluminum foiland stored at �80°C or processed immediately for immunohistochemistry or IF.

H&E staining. After fixation in 10% formalin for 10 min at room temperature,slides were washed twice in deionized water, dehydrated for 30 s in 100%ethanol, stained for 30 s in hematoxylin, and washed in distilled water for 2 min.Tissue sections were stained with eosin for 15 s, followed by 2 min of treatment(each) with 95 and 100% ethanol. Slides were allowed to air dry and thensubmerged in xylene for 3 min and mounted using Cytoseal mounting medium(Stephens Scientific, Riverdale, NJ). The number and location of parasites weredetermined by microscopic examination of the stained tissues. Tissues were alsoanalyzed for the presence or absence of mononuclear infiltrates.

Luxol blue staining. Luxol blue staining of brain sections from mock- andparasite-infected WT and MyD88�/� mice was performed as previously de-scribed (27). Briefly, dissected brains were stored in 10% buffered formalinovernight for histological processing. Brains were dehydrated through gradedalcohol, cleared with xylene, and embedded in paraffin. Transverse sections werecut at a 5-�m thickness and were placed on silane prep slides. Sections weredeparaffinized in xylene for 5 min, rehydrated in 95% ethanol for 5 min at roomtemperature, and stained with preheated Luxol fast blue (Polysciences, War-rington, PA) at 60°C overnight. Slides were washed for 1 min in 95% ethanol inwater and differentiated in 0.05% lithium carbonate (Sigma-Aldrich, St. Louis,MO) followed by 70% ethanol for 3 to 4 min. These sections were rinsed indeionized water for 30 s, counterstained with cresyl etch violet (Sigma-Aldrich,St. Louis, MO), dehydrated through a 95% and 100% alcohol series for 5 mineach, cleared in xylene, and cover slipped with synthetic resin.

Brain mononuclear cell isolation. Leukocytes from mouse brains were isolatedas described by Irani and Griffin (24), with some modifications (13, 15). Eachperfused brain was gently minced through a fine 70-�m Nitex screen (SefarAmerica, Depew, NY) by use of a syringe plunger and collected in 10 ml HBSS(Invitrogen, Carlsbad, CA) containing 0.05% collagenase D (Roche Diagnostics,Indianapolis, IN), 0.1 �g/ml L-1-chloro-3,4-tosyl-amido-7-amino-2-heptanone-HCl (Sigma-Aldrich), 10 mg/ml DNase I (Sigma-Aldrich), and 10 mM HEPESbuffer, pH 7.4 (Invitrogen). The mixture was gently rocked at room temperaturefor 1 h and allowed to settle by gravity for 30 min to deplete undigested debris.The supernatant was collected, pelleted at 200 g for 5 min, and resuspendedin 3 ml Ca2/Mg2-free HBSS (Invitrogen) per brain. The suspension waslayered onto 10 ml of a density gradient containing 3 parts Ficoll-Hypaque(Amersham Pharmacia Biotech, Piscataway, NJ) and 1 part RPMI medium(Invitrogen) supplemented with 10% fetal bovine serum (Sigma), 10 mMHEPES (Invitrogen) and 50-�g gentamicin (Invitrogen)/ml. Each gradient wascentrifuged at 300 g for 30 min at room temperature. The overlaying mediumand interface of tissue debris were removed. The entire 10 ml of gradientmedium was then diluted fivefold with HBSS and centrifuged at 300 g for 10min. Cells were washed three times in 1 ml 0.1% bovine serum albumin (Sigma-Aldrich) in HBSS and counted. Cells were then diluted to 2 107 cells/ml andprocessed for IF staining of cell surface antigens for flow cytometric analysis(fluorescence-activated cell sorter [FACS] analysis).

FACS analysis. Our previous studies have demonstrated that infiltrating leu-kocytes consisting of macrophages, �� T cells, and NK cells are detected in theCNS during murine NCC by day 3 postinfection (p.i.), followed by �� T cells by1 week p.i. and B cells by 3 weeks p.i. (14). Additionally, the early infiltrating ��T cells appear to play a key role in amplifying further infiltration of leukocytes by1 week p.i. and afterward (13). Therefore, in the present study, leukocyte infil-tration in WT and MyD88�/� mice was analyzed by FACS analysis at 1 week p.i.and 3 weeks p.i. Single-cell suspensions of harvested mononuclear cells fromparasite-infected brains of WT and MyD88�/� mice were prepared at 2 107

cells/ml in staining buffer (10% fetal calf serum in PBS) and preincubated with1 �g of the 2.4G2 antibody for 5 to 10 min on ice prior to staining. Fiftymicroliters of cell suspension (equal to 106 cells) was dispensed into each tube orwell along with a previously determined optimal concentration of cell surface-specific antibody in 50 �l of staining buffer. Each test tube was mixed gently andincubated for 30 min in the dark in an ice bath. Cells were stained with fluoro-chrome-labeled antibodies of the appropriate isotype controlled for nonspecificbinding, while cells stained in the absence of primary antibodies served asnegative controls. After the incubation period, cells were washed by adding 2 mlof staining buffer followed by centrifugation for 7 min (300 to 400 g) at 4°C.The washing was repeated two more times, for a total of three washes. Cellpellets were suspended in 500 �l of staining buffer and analyzed on a BD LSR

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II flow cytometer (BD Biosciences). FlowJo (Three Star) software was used toanalyze the FACS data.

IF. IF microscopy was used to determine the presence of immune mediatorsand/or specific cell types in brains of parasite-infected mice at various timespostinfection (3 days, 1 week, and 3 weeks). Brains from mock-infected animalswere used as controls, and IF staining was done as described before (6, 13, 30).All steps were carried out at room temperature. Briefly, sections were incubatedwith specific primary antibodies in PBS with 3% host serum to prevent nonspe-cific binding. After 1 h, sections were washed seven times for 3 min each time andincubated with appropriate secondary antibodies for 30 min. Sections were thenwashed seven times for 3 min each time in 50 mM Tris-HCl, pH 7.6, with 0.1%Tween 20. For double-IF staining, the abovementioned procedures were sequen-tially repeated for each additional staining step. The sections were mountedusing FluorSave reagent (Calbiochem, La Jolla, CA) containing 0.3 �M 4�,6-diamidino-2-phenylidole (DAPI)–diacetate (Molecular Probes). Additional con-trol staining was performed to rule out any nonspecific staining. In each case,sections were blocked with saturating concentrations of appropriate host serumantibodies to eliminate false-positive staining due to FcR-mediated nonspecificbinding. Staining in the absence of primary antibodies provided additional neg-ative controls.

Analysis of fibrinogen extravasation. The integrity of the BBB was assessed 1week and 3 weeks after i.c. inoculation of M. corti metacestodes (4, 6). Adjacentbrain cryosections from infected and noninfected WT and MyD88�/� mice were

stained using antibodies against fibrinogen or CD11b. CD11b was used to pro-vide a frame of reference for the location of blood vessels, around which infil-tration of leukocytes takes place. At least five images of compromised pial bloodvessels (and control pial vessels from mock-infected mice) were captured usingidentical camera settings so that differences in the pixel intensities of the imageswere solely due to differences in fibrinogen extravasation. Disruption of the BBBwas determined by measuring the area (number of pixels) and fluorescenceintensity (average intensity of pixels) of fibrinogen surrounding vessels exhibitingleakage. This was done using the imaging software IP lab 4.0 (BD BiosciencesBioimaging, Rockville, MD). The relative extent of fibrinogen extravasation wascalculated by multiplying the number of pixels (area) by the average intensity ofpixels. Statistical analysis was performed with Student’s t test, using Sigma Plot8.0 (Systat Software, San Jose, CA).

Statistical analysis. We used Student’s t test for comparison of means ofdifferent groups in Sigma Plot 8.0 (Systat Software, San Jose, CA). A P value of�0.05 was considered to be statistically significant. Statistical differences in themortality of parasite-infected mice with NCC were analyzed using Kaplan-Meiersurvival analysis.

RESULTS

MyD88�/� mice exhibit reduced morbidity and mortality.WT and MyD88�/� mice were infected i.c. with 40 M. cortimetacestodes to evaluate the role of MyD88 in murine NCC.The development of disease severity in these animals was thencompared. CNS infection with M. corti metacestodes producessevere neurological signs, such as tilted head, walking in cir-cles, spinning when held from the tail, and weight loss between1 and 3 weeks p.i., that worsen at later time points (15). In thepresent study, the WT mice displayed these signs of infection.In contrast, the majority of MyD88�/� mice did not exhibitsevere neurological signs during the first 5 weeks p.i. By thistime point, 64.3% of M. corti-infected WT mice (9 of 14 mice)succumbed to the infection (Fig. 1). In contrast, only 21.4% ofMyD88�/� mice (3 of 14 mice) succumbed to the infection.Thus, the lack of MyD88 in mice resulted in reduced diseaseseverity and improved survival during murine NCC.

MyD88�/� mice exhibit reduced parasite loads in brainparenchyma. To examine whether the reduction in neurolog-ical symptoms and mortality of MyD88-deficient mice was as-sociated with effective clearance of parasites and/or their seg-regation to specific brain areas, serial horizontal sections ofinfected WT and MyD88�/� mouse brains were stained with

FIG. 1. MyD88�/� mice display reduced susceptibility during mu-rine NCC. Fourteen WT (C57BL/6) and 14 MyD88-deficient micewere infected i.c. with 40 M. corti organisms and assessed daily fordisease severity. The decreased susceptibility of MyD88�/� mice com-pared to WT mice is statistically significant, as determined by Kaplan-Meier survival curve statistical analysis (P � 0.05).

FIG. 2. CNS infection with M. corti in MyD88�/� mice is mostly localized in extraparenchymal regions. After 1 week and 3 weeks of infection,WT and MyD88�/� mice were sacrificed. The location and number of parasites were obtained by microscopic examination of serial H&E-stainedbrain sections. (A) Total numbers of parasites present in the brains of infected WT and MyD88�/� mice (n 4). (B) Total numbers of parasitespresent in parenchymal and extraparenchymal regions in individual brains were calculated (n 4). Parasites in MyD88�/� mice were localizedmostly in meninges, ventricles, and subarachnoid spaces. Significant differences in parasite distribution were observed in MyD88�/� versus WTanimals and are denoted by asterisks (***, P � 0.001).

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H&E, and the number of M. corti metacestodes was deter-mined by microscopic analysis. Brains of WT and MyD88�/�

mice harvested at 1 week and 3 weeks p.i. exhibited similarparasite counts (Fig. 2A). However, the distribution of organ-isms was altered. In WT mice, the majority of organisms hadinvaded the parenchyma at 1 and 3 weeks p.i., whereas inMyD88�/� mice the majority of organisms were located inextraparenchymal areas (Fig. 2B).

MyD88�/� mice display reduced CNS pathology after M.corti infection. We evaluated H&E- and Luxol fast blue-stained sections of infected brains to determine changes innervous tissue integrity (Fig. 3 and 4). Figure 3A-1 and A-2depict normal brain tissue morphology in control animals in-oculated i.c. with HBSS. In comparison, infected WT braintissue displayed a strong inflammatory response characterizedby the presence of a large number of infiltrating immune cells(Fig. 3B-1 and C-1), including infiltrates surrounding the par-asites in the parenchyma (Fig. 3D-1). Some areas containedsites of necrosis. In contrast, infected MyD88�/� brain tissueexhibited reduced CNS inflammation, with smaller numbers ofinfiltrating immune cells, compared to WT mice (Fig. 3B-2 andC-2). Smaller foci of immune cells surrounded parenchymalparasites in the MyD88�/� mice (Fig. 3D-2). In addition, whatappeared to be primary and secondary microglial nodule for-mation was apparent in brain parenchyma in infected WTanimals (Fig. 4B-1 and B-1�) but not in MyD88�/� mice (Fig.4B-2 and B-2�).

FIG. 3. Brain pathology associated with M. corti infection in WTand MyD88�/� mice. (A-1) H&E staining of brain cryosection showingnormal parenchymal tissue in an HBSS-inoculated WT mouse. (A-2)Normal parenchymal tissue in an HBSS-inoculated MyD88�/� mouse.(B-1) Parenchymal blood vessel in a WT mouse at 3 weeks p.i., asso-ciated with areas of infiltrating immune cells (*). (B-2) Parenchymalblood vessel in a MyD88�/� mouse at 3 weeks p.i., exhibiting a reducednumber of infiltrating immune cells (*). (C-1) Indusium griseum ofWT mouse at 1 week p.i., associated with massive infiltration of im-mune cells (*). The inset C-1� represents a 2 magnification of theselected area depicting immune cells with morphology indicative ofactivated cells. (C-2) Indusium griseum of MyD88�/� mouse at 1 weekp.i., associated with reduced infiltration of immune cells compared toWT mouse (*). The inset C-2� represents a 2 magnification of theselected area depicting immune cells of quiescent morphology com-pared to the WT mouse. (D-1) Parenchymal parasite in a WT mouseat 3 weeks p.i., associated with infiltrating immune cells. P, parasite.(D-2) Parenchymal parasite in a MyD88�/� mouse at 3 weeks p.i.,associated with reduced infiltrating immune cells. P, parasite. Resultsare from one representative experiment of three independent experi-ments (n 4 mice per time point), and the images were captured at amagnification of 200.

FIG. 4. Brain pathology associated with M. corti infection in WTand MyD88�/� mice. (A-1) Luxol blue staining of brain cryosectionshowing normal parenchymal tissue in an HBSS-inoculated WTmouse. (A-2) Normal parenchymal tissue in an HBSS-inoculatedMyD88�/� mouse. (B-1) Parenchyma of WT mouse at 3 weeks p.i.,exhibiting what appears to be microglial nodule formation associatedwith neuronal death. (B-1�) The boxed area in panel B-1 was magnified(4) to show details of microglial/macrophage nodule formation inWT mice. (B-2) Parenchyma of MyD88�/� mouse at 3 weeks p.i.,depicting reduced nodule formation compared to that in WT mouse.(B-2�) The boxed area in panel B-2 was magnified (4) to show theabsence of similar nodule formation in MyD88�/� mice. Results arefrom one representative experiment of three independent experiments(n 4 mice per time point), and the images were captured at amagnification of 200.

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MyD88�/� mice exhibit reduced numbers of infiltrating im-mune cells in the CNS. Our previous studies demonstratedthat the predominant immune cells initially recruited to thebrain after parasite inoculation are macrophages and �� T cells(3 to 5 days p.i.), followed by �� T cells (1 week p.i.) and B cells(3 weeks p.i.). The maximum infiltration of immune cells isusually detected between 2 and 5 weeks p.i. in the M. corti-infected mouse brain. To determine whether MyD88�/� micedisplayed any differences in infiltrating immune cells duringNCC, the total leukocytes from whole brains were isolated atthe peak of inflammation (3 weeks p.i.) and quantified bytrypan blue staining. Brains of MyD88-deficient mice at thattime exhibited significantly smaller numbers of leukocytes thandid those of WT mice (P � 0.005) (Fig. 5). In a mannerconsistent with our previous observations (5), the infiltratingleukocytes in brains of mock control mice were barely detect-able (Fig. 5).

To further elucidate the difference in recruitment of var-ious immune cell types in MyD88-deficient mice in NCC, weused flow cytometric analysis to determine the numbers ofCD11b myeloid cells, �� T cells, �� T cells, and B cells atboth an early stage of infection (1 week p.i.) and the peak ofinflammation (3 weeks p.i.) (Fig. 6). CD11b myeloid cellsrepresented the predominant leukocyte population at bothtimes p.i. in M. corti-infected brains of WT as well asMyD88�/� mice (Fig. 6A to C). In comparison with the WTinfected mice, the number of CD11b cells was significantlylower in MyD88�/� mice both at 1 week (Fig. 6A and C) andat 3 weeks p.i. (Fig. 6B and C). As seen for CD11b cells,there were significantly smaller numbers of �� T cells and�� T cells detected in MyD88�/� mice at 1 week (Fig. 6Aand C) as well as at 3 weeks p.i. (Fig. 6B and C) comparedto the WT mice. As reported previously, in murine NCCbrains B cells were detected only after 3 weeks p.i. (4).Likewise, CD19 B cells were detected at 3 weeks p.i. in WTor MyD88�/� infected mice (Fig. 6B), but not at 1 week p.i.(data not shown). However, the number of B cells detected

in MyD88�/� mouse brains was threefold lower than in theWT mice (Fig. 6B and C). Taken together, these data dem-onstrate that the absence of MyD88 leads to a significantreduction in leukocyte recruitment into the brain dur-ing NCC.

MyD88�/� mice display reduced BBB permeability afterM. corti infection. The deficiency in the number of immunecells in MyD88�/� mice could reflect a deficiency in leuko-cyte trafficking into the brain and/or a defect at subsequentstages of cell expansion. Leukocyte trafficking duringthe course of NCC has previously been correlated with thebreakdown of the BBB (3–7). Therefore, we investigated theimpact of MyD88 deficiency on BBB permeability in M.corti-infected brains by testing for the presence of the serumprotein fibrinogen in brain tissue by IF. Multiple brain cryo-sections of WT and MyD88�/� animals were analyzed.Mock-infected animals (both WT and MyD88�/�) exhibitedsmall amounts of fibrinogen staining (red) that was largelyconfined to the vessels (Fig. 7A-1 and A-2). Upon infection,pial vessels of infected WT animals displayed substantialextravasation of fibrinogen at 1 week p.i. (Fig. 7B-1), ac-companied by cellular infiltration (*). The extravasation re-mained high, as indicated by extensive staining for fibrino-gen in perivascular areas adjacent to the pial vessels, at 3weeks p.i. (Fig. 7C-1). In contrast, infected MyD88-deficientmice exhibited substantially less immunoreactivity for fibrin-ogen at both 1 and 3 weeks p.i. than did infected WT mice(Fig. 7B-2 and C-2). Measurement of the mean pixel inten-sity of fibrinogen staining confirmed that extravasation offibrinogen was significantly lower in parasite-infectedMyD88�/� mice than in the WT controls (Fig. 7D). Thesefindings suggest that the reduced number of immune cellsdetected in infected MyD88-deficient mice correlates withdecreased BBB permeability of CNS vasculatures.

MyD88�/� mice exhibit reduced IL-6 protein expressionin proximity to blood vessels. Our previous studies revealedthat IL-6 is detected in astrocytes proximal to the pial ves-sels that are actively involved in the infiltration of leukocytesinto the CNS during murine NCC (4). Thus, the productionof IL-6 was assayed in parasite-infected brain cryosectionsof WT and MyD88�/� mice by in situ IF microscopy (Fig. 8).In mock-infected/normal mouse brains, IL-6 was detected atlow basal levels (Fig. 8A-1 and A-2). Infected WT micedisplayed a progressive increase in the level of IL-6 protein,as measured by both the intensity of staining and number ofpositive cells. At 1 week p.i., an increased expression of IL-6was evident in periventricular and leptomeningeal areas ofthe brain (Fig. 8B-1). These induced effects were even morepronounced at 3 weeks p.i. (Fig. 8C-1). The upregulatedexpression was detected primarily in the astrocytes presentin periventricular and leptomeningeal areas of the brain,including the glia limitans, in proximity to pial vessels (Fig.8D-1). Although the infection in MyD88�/� mice inducedincreases in IL-6 protein expression in similar brain anatom-ical regions and cell types, the protein was detected at sub-stantially lower levels than those in WT mice at each timepoint analyzed (Fig. 8B-2, C-2, and D-2). Thus, the resultsfrom this study suggest that MyD88-deficient NCC miceexpress reduced levels of IL-6 in astrocytes present in prox-imity to the pial vessels.

FIG. 5. MyD88�/� mice exhibit reduced numbers of infiltratingimmune cells in the CNS. WT and MyD88�/� mice were infectedi.c. with M. corti and were sacrificed at 3 weeks p.i. Infiltratingimmune cells were isolated by Ficoll Hypaque density gradientcentrifugation as described in Materials and Methods. Total num-bers of viable immune cells in parasite-infected brains of both WTand MyD88�/� mice were counted by trypan blue exclusion staining(n 4). Significant differences in the numbers of immune cellsrecovered from MyD88�/� versus WT animals are denoted by as-terisks (**, P � 0.005).

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MyD88�/� mice express lower levels of inflammatory medi-ators after M. corti infection. As reported previously, infec-tion of the CNS by M. corti leads to an upregulation ofvarious proinflammatory cytokines/chemokines (4, 13–15),which appear to contribute to the immunopathogenesis ofthis disease. Among them, the Th1 cytokines tumor necrosis

factor alpha (TNF-�) and gamma interferon (IFN-�), pro-duced by �� T cells, correlated with exacerbating neuropa-thology and disease severity (15), while early induction ofCCL2 (�1 week) correlated with cellular infiltration intothe CNS during murine NCC (13). Thus, to determine theinvolvement of MyD88 in the CNS levels of these proin-

FIG. 6. MyD88�/� mice exhibit reduced numbers of various infiltrating immune cells in the CNS. WT and MyD88�/� mice were infected i.c.with M. corti and were sacrificed at 1 and 3 weeks p.i. Infiltrating immune cells were isolated by Ficoll Hypaque density gradient centrifugationas described in Materials and Methods. Total numbers of viable immune cells in parasite-infected brains of WT and MyD88�/� mice were countedby trypan blue staining. (A) In each experiment, brain leukocytes from parasite-infected WT and MyD88�/� mice (1 week) were isolated fromthree mice and pooled together. The percentages of immune cell types that were macrophages/microglia (CD11b), �� T cells (TCR �2 chain),and �� T cells (TCR � chain) were quantified by FACS analysis. A representative of three independent experiments is shown. The dark grayhistograms represent isotype control antibody-stained cells, and the light gray histograms represent positively stained cells. (B) Brain leukocytesfrom parasite-infected WT and MyD88�/� mice (3 weeks) were isolated from three mice and pooled together. FACS analysis displayed reducednumbers of macrophages and microglia (CD11b), �� T cells (TCR �2 chain), and �� T cells (TCR � chain) in MyD88�/� mice. Arepresentative of three independent experiments is shown. The dark gray histograms represent isotype control antibody-stained cells, and the lightgray histograms represent positively stained cells. (C) Results obtained from FACS analysis are expressed as means � standard errors of the meansfor three independent experiments. Significant differences in numbers of individual immune cells recovered from MyD88�/� versus WT animalsare denoted by asterisks (�, P � 0.05; ��, P � 0.005; ���, P � 0.005).

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flammatory cytokines after M. corti infection, enzyme-linkedimmunosorbent assay was performed on brain homogenatesfrom WT and MyD88�/� mice. In normal/mock-infectedmouse brains, TNF-�, IFN-�, and CCL2 were at low basal

levels in WT and MyD88�/� mice (Fig. 9). Upon parasiteinfection, WT mice exhibited increased levels of TNF-�,IFN-�, and CCL2 at 1 week p.i., which were further upregu-lated at 3 weeks p.i. (Fig. 9). M. corti infection in MyD88�/�

FIG. 7. Fibrinogen extravasation in WT and MyD88�/� mice. WT and MyD88�/� mice were infected i.c. with M. corti and were sacrificed at1 and 3 weeks p.i. After perfusion, brains were removed, frozen in optimal-cutting-temperature resin, and cryosectioned. Sections were thenanalyzed for expression of fibrinogen (red), using anti-fibrinogen primary antibody and rhodamine RXX-conjugated secondary antibody. Nucleiwere stained blue with DAPI. (A-1) Mock-infected WT control brain section stained with fibrinogen-specific antibody (magnification, 400). (A-2)Mock-infected MyD88�/� control brain section stained with fibrinogen-specific antibody (magnification, 400). (B-1) Indusium griseum area inthe brain of M. corti-infected WT mouse at 1 week p.i., showing abundant fibrinogen-positive staining. �, infiltrating immune cells; p, parasite.Magnification, 400. (B-2) Indusium griseum of M. corti-infected MyD88�/� mouse at 1 week p.i., exhibiting substantially reduced fibrinogen-positive staining. p, parasite. Magnification, 400. (C-1) Indusium griseum of M. corti-infected WT mouse at 3 weeks p.i., showing abundantfibrinogen-positive staining. Magnification, 400. (C-2) Reduced amount of fibrinogen staining observed in M. corti-infected MyD88�/� mousebrain at 3 weeks p.i. (D) Relative levels of fibrinogen expression in mock- and M. corti-infected MyD88�/� or WT animals, calculated as describedin Materials and Methods. Significant differences are denoted by asterisks (��, P � 0.005; ���, P � 0.005). Results are from one representativeexperiment of three independent experiments (n 3 mice per time point).

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FIG. 8. MyD88�/� mice present reduced brain expression of IL-6. In situ IF staining was performed on frozen sections of mock-infected andparasite-infected brains of WT and MyD88�/� mice at 1 and 3 weeks p.i. IL-6 expression was visualized in red (rhodamine RXX), astrocytes(GFAP) were visualized in green (Alexa Fluor 488), and nuclei acids (blue) were stained with DAPI. (A-1) IL-6 expression in brain of amock-infected WT mouse. (A-2) IL-6 expression in HBSS-inoculated MyD88�/� mouse brain. (B-1) IL-6 expression in brain of a parasite-infectedWT mouse at 1 week p.i. (B-2) IL-6 expression in brain of a parasite-infected MyD88�/� mouse at 1 week p.i. (C-1) IL-6 expression in brain ofa parasite-infected WT mouse at 3 weeks p.i. (C-2) IL-6 expression in brain of parasite-infected MyD88�/� mouse at 3 weeks p.i. (D-1) IL-6 ispredominantly expressed in brain astrocytes of a parasite-infected WT mouse at 3 weeks p.i. IL-6 is visualized in red (rhodamine RXX), andastrocytes are in green (Alexa Fluor 488). Arrowheads show colocalization of IL-6 and GFAP (yellow-orange). Magnification, 400. (D-2) IL-6expression is reduced in brain astrocytes of a MyD88�/� mouse at 3 weeks p.i. Arrowheads show colocalization (yellow-orange). Magnifica-tion, 400. Results are from one representative experiment of three independent experiments (n 4 mice per time point).

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mice resulted in marginal increases in the levels of thesecytokines over the basal control levels of the mock-infectedmice. However, compared to those in the WT infected mice,the levels of TNF-�, IFN-�, and CCL2 in MyD88�/� micewere significantly lower (P � 0.001), suggesting that induc-tion of these inflammatory mediators in NCC is largelydependent on MyD88 signaling.

DISCUSSION

The chronic stage of both human and murine NCC is asso-ciated with manifestation of the TH1 inflammatory response.The persistence of an inflammatory response in the CNS dur-ing NCC appears to play a prominent role in the severe neu-ropathology and mortality associated with this disease (57).The present study demonstrates a critical role played byMyD88-associated signaling in promoting the severity of NCC.MyD88�/� mice were less susceptible to infection and exhib-ited reduced pathological signs compared to infected WTmice. The presence of both primary and secondary microglialnodules that reflect engulfment of degenerating neurons andnecrotic brain tissue by glial cells appeared to be lacking in theinfected brains of MyD88�/� mice. These results strongly sup-port the role of MyD88-associated signaling in amplifying thedisease severity of murine NCC.

MyD88 is a primary adaptor molecule that interacts with manyhost pattern recognition receptors (35). Recruitment of MyD88

leads to the activation of MAP kinases and the transcriptionfactor NF-�B (28). This in turn allows the regulated expression ofa wide range of proinflammatory factors by macrophages andother cell types. Many of these mediators have important roles inthe amplification of acute inflammatory responses, innate im-mune function, and the development of acquired immunity. Thecytokines produced in the CNS during M. corti infection areindicative of a TH1 type of response (14, 15). Furthermore, adecreased expression of the TH1 cytokines TNF-� and IFN-� inM. corti-infected mice that lack �� T cells is associated with re-duced pathology and longer survival times (15). Thus, lower brainlevels of TH1 inflammatory responses that are associated withreduced CNS symptoms in M. corti-infected MyD88�/� micestrongly support the idea that TH1 cytokines such as TNF-� andIFN-� contribute to the CNS pathology during NCC. Within thatcontext, �� T cells are a major TH1 cytokine-producing cell typein the CNS after M. corti infection (15). The significant reductionin �� T-cell numbers in the CNS of MyD88-deficient mice afterM. corti infection correlates with the reduction in TH1 cytokineresponses. Moreover, �� T cells from M. corti-infected mice alsoexpress multiple TLRs (30). Indeed, �� T cells produce largeamounts of proinflammatory cytokines and chemokines uponstimulation with TLR ligands (unpublished results). Thus, it ispossible that MyD88-dependent responses in �� T cells directlycontribute to TH1 responses and inflammation-mediated pathol-ogy in NCC.

FIG. 9. MyD88�/� mice exhibit defects in the induction of several proinflammatory mediators. The brains from mock-infected control and M.corti-infected WT and MyD88�/� mice were harvested at 1 and 3 weeks p.i. and homogenized in PBS with protease inhibitors (100 mg/ml),whereupon TNF-�, IFN-�, and CCL2 protein levels were quantitated by sandwich enzyme-linked immunosorbent assay (data are means �standard errors). Results shown are averages for three infected and three mock-infected control mice for each time point. Significant differencesare denoted by asterisks (���, P � 0.001).

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The reduced neuropathology of MyD88-deficient (and �� Tcell�/�) mice coincides with a smaller number of parasites inbrain parenchyma. Although the exact mechanisms contribut-ing to tissue invasion by M. corti are unknown, we speculatethat the extent of inflammatory responses elicited by infiltrat-ing immune cells may influence the outcome. The initial im-mune response in WT mice is inflammatory and takes place inextraparenchymal spaces (meninges and subarachnoid spaces).Such responses likely cause tissue pathology, allowing easieraccess of the parasite to the parenchyma. A reduced level ofsuch responses in MyD88�/� mice may help to minimize in-vasion of the parasite. It is also likely that parenchymal para-sites would be more injurious, particularly in critical areas, ashas been reported for human patients (32, 55, 56).

Inflammation is a complex process consisting of various hostdeterminants. One of the important steps of inflammation isleukocyte migration to the site of infection or injury. In murineNCC, parasite infection results in infiltration of large numbersof macrophages, �� T cells, and �� T cells within the first weekof infection, followed by B cells by 3 weeks p.i. (4). The resultspresented here show a reduction in infiltration of immunecells, including macrophages, �� T cells, and �� T cells, afterM. corti infection in MyD88-deficient mice compared with WTmice. This reduction in cellular infiltration correlates with de-creased fibrinogen leakage compared to that in WT mice,reflecting reduced BBB breakdown in the MyD88-deficientmice. The BBB is a selective barrier formed by the endothelialcells that line cerebral microvessels (1, 40). It regulates thetransit of blood-borne molecules and immune cells into thebrain. During a pathological insult, the BBB frequently be-comes more permeable. Failure of the BBB is a critical eventin the development and progression of several neurologicalinflammatory diseases (12, 21, 36, 58). Although the mecha-nisms of BBB disruption during CNS diseases are not fullydeciphered, specific growth factors such as IL-6 and vascularendothelial growth factor have been shown to play crucial rolesin affecting the permeability of the BBB in various clinicalsettings (9, 11). Our previous studies have shown that IL-6expression by astrocytes in the glial limitans in the proximity ofpial vessels correlates with leukocyte migration through pialvessels during murine NCC (4). It is likely that significantlylower levels of IL-6 expression in astrocytes in MyD88-defi-cient mice contribute to decreased BBB permeability, eventu-ating in reduced leukocyte trafficking. In vitro studies are cur-rently under way to define such interactions.

The data presented here indicate a role for MyD88 in theproduction of proinflammatory responses that contribute to thedisease severity in murine NCC. This is in contrast to the well-established role for MyD88 signaling in mediating production ofproinflammatory cytokines leading to host resistance in variousinfections (31, 37, 44, 46, 49, 53, 54). Nevertheless, emergingfindings in parasite diseases such as river blindness and cerebralmalaria indicate that MyD88-mediated responses contribute todisease severity (17, 25). Similar functions of MyD88 in NCC andriver blindness are of interest since the associated immunopatho-genesis is similar in nature. In both cases, the infection does notproduce any detectable inflammatory response or disease syn-drome while the parasites are alive, suggesting an active suppres-sion of immune responses by the live parasite. However, thedeath/degeneration of the parasites results in the induction of

inflammatory responses that are the cause of the associated pa-thology and disease severity in river blindness (20) and NCC (32).Thus, in these extracellular helminth infections, the activation ofthe MyD88 signaling pathway results in the induction of inflam-matory responses and appears to be the cause of the associatedpathology and disease symptoms. However, the involvement ofany particular TLR in murine NCC has yet to be determined.Nonetheless, TLRs 1 to 13, except for TLR5 and -10, are differ-entially expressed in the CNS during murine NCC (29, 30). How-ever, MyD88 also interacts with several other TIR (Toll–IL-1R)domain-containing proteins within the IL-1R/IL-18R family, suchas the IL-1RI and IL-1R accessory protein (10). Besides, MyD88also has been reported to interact with several non-TIR domain-containing receptors, such as IFN-� receptor 1 (47), phosphati-dylinositol 3-kinase (34), IFN regulatory factor 1 (IRF1) (33),IRF5 (48), and IRF7 (23). Determining pattern recognition re-ceptors that are responsible for the induction of MyD88-depen-dent inflammatory responses in M. corti-infected mouse brains isof current interest in our laboratory.

An adequate amount of inflammation is required to mounta successful antimicrobial response to any kind of infection.However, sustained inflammation is the cause of tissue damageand associated pathology, particularly in CNS diseases (16).The common treatment for NCC consists of corticosteroids tosuppress inflammatory responses that accompany degenera-tion of the parasite after antihelminth drug treatment. Treat-ment of corticosteroids leads to unwanted and undesirable sideeffects. A better understanding of the exact MyD88 signaling-dependent pathways leading to unwanted inflammation couldpotentially lead to other therapeutic inhibitors, particularly ifthey could be targeted specifically to the CNS.

ACKNOWLEDGMENTS

We gratefully acknowledge the technical support of Pramod KumarMishra of the University of Texas Health Science Center at San Antonio.We thank Ashlesh Murthy for technical help with FACS analysis.

This work was supported by awards NS 35974, AI 59703, and P01 AI057986 from the National Institutes of Health.

We have no financial conflicts of interest.

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