of April 6, 2018.This information is current as

Toxoplasmosis Monocytes Control CerebralhighLy6C

DunayRitaJeron, Matthias Mack, Markus M. Heimesaat and Ildiko

Aindrila Biswas, Dunja Bruder, Susanne A. Wolf, Andreas

http://www.jimmunol.org/content/194/7/3223doi: 10.4049/jimmunol.1402037February 2015;

2015; 194:3223-3235; Prepublished online 20J Immunol 

MaterialSupplementary

7.DCSupplementalhttp://www.jimmunol.org/content/suppl/2015/02/20/jimmunol.140203

Referenceshttp://www.jimmunol.org/content/194/7/3223.full#ref-list-1

, 32 of which you can access for free at: cites 82 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2015 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on April 6, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

by guest on April 6, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

The Journal of Immunology

Ly6Chigh Monocytes Control Cerebral Toxoplasmosis

Aindrila Biswas,* Dunja Bruder,*,† Susanne A. Wolf,‡ Andreas Jeron,* Matthias Mack,x

Markus M. Heimesaat,{ and Ildiko Rita Dunay*

Cerebral infection with the parasite Toxoplasma gondii is followed by activation of resident cells and recruitment of immune cells

from the periphery to the CNS. In this study, we show that a subset of myeloid cells, namely Ly6ChighCCR2+ inflammatory

monocytes that infiltrate the brain upon chronic T. gondii infection, plays a decisive role in host defense. Depletion of this

monocyte subset resulted in elevated parasite load and decreased survival of infected mice, suggesting their crucial role. Notably,

Ly6ChighCCR2+ monocytes governed parasite control due to production of proinflammatory mediators, such as IL-1a, IL-1b,

IL-6, inducible NO synthase, TNF, and reactive oxygen intermediate. Interestingly, Ly6ChighCCR2+ monocytes were also able to

produce the regulatory cytokine IL-10, revealing their dual feature. Moreover, we confirmed by adoptive transfer that the

recruited monocytes further develop into two distinct subpopulations contributing to parasite control and profound host defense.

The differentiated Ly6CintCCR2+F4/80int subset upregulated MHC I and MHC II molecules, suggesting dendritic cell properties

such as interaction with T cells, whereas the Ly6CnegF4/80high cell subset displayed elevated phagocytic capacity while upregu-

lating triggering receptor expressed on myeloid cells-2. Finally, we have shown that the recruitment of Ly6Chigh monocytes to the

CNS is regulated by P-selectin glycoprotein ligand-1. These results indicate the critical importance of recruited Ly6Chigh mono-

cytes upon cerebral toxoplasmosis and reveal the behavior of further differentiated myeloid-derived mononuclear cell subsets in

parasite control and immune regulation of the CNS. The Journal of Immunology, 2015, 194: 3223–3235.

Toxoplasmosis is a common worldwide zoonotic infectioncaused by the opportunistic intracellular parasite Toxo-plasma gondii (1). After oral uptake, the parasites cross

the intestinal epithelium as well as the blood-brain barrier; whiletraveling within mononuclear cells, they reach immune-privilegedsites such as the CNS (2–5).The primary infection with T. gondii is followed by the re-

cruitment of neutrophil granulocytes, monocytes, and dendriticcells (DC) to the site of infection (6–8). We have recently shownthat, in the acute stage of infection, DCs secrete high levels of IL-12, triggering adaptive immunity (9). The proinflammatory cyto-kine IFN-g produced mainly by activated innate lymphoid cells,NK, and T cells is the major driving factor for host protectionagainst this intracellular pathogen (10, 11). In our previous stud-ies, we detected that Ly6ChighGr1+ monocytes were crucial duringthe acute stage of T. gondii infection, producing high amounts ofTNF, inducible NO synthase (iNOS), and reactive oxygen inter-mediates (ROS), which directly contributed to the control of the

parasite burden in the host (6, 7, 12, 13). In addition, we haveshown that neutrophil granulocytes rather contributed to the ileal

pathology in the acute stage (7).After reaching the brain, the parasites persist lifelong within

tissue cysts, strictly controlled by the host immune system. IFN-g–

dependent immune response during the chronic stage suggests

ongoing basal inflammation associated with resident cell activa-

tion in the CNS (14, 15). Upon immunosuppression, the latent

infection can reactivate and develop into life-threatening en-

cephalomyelitis (1, 6, 7, 16–18). Within the inflamed brain, res-

ident immune cells, such as microglia and astrocytes, become

activated, displaying significant antiparasitic properties (19–21).

Despite limited access, the CNS is inundated by immune cells

from the periphery during the chronic stage of T. gondii infection.

The adaptive immune responses in the CNS have been extensively

investigated, describing an important contribution to the local

parasite control (22–25). Besides, several studies have highlighted

the function and phenotype of certain mononuclear cells such as

microglia, brain DCs, and macrophages upon cerebral toxoplas-

mosis; however, the role of recruited newly described myeloid cell

subpopulations remains undefined (19, 26, 27).Monocytes are generated frommacrophage and DC precursors in

the bone marrow and are released in the bloodstream in a CCR2-

dependent manner (28–30). On the basis of chemokine receptor

expression and specific surface molecules (31), monocytes are

divided into two major populations: CX3CR1lowCCR2+Ly6Chigh

and CX3CR1highCCR22Ly6Clow (6, 30). A short-lived CX3CR1

low

CCR2+Ly6Chigh inflammatory monocyte subset that is actively

recruited to sites of infection and inflammation (32) serves as an

immediate precursor for Ag-presenting DCs and macrophages (32,

33). CX3CR1highCCR22Ly6Clow patrolling resident monocytes

adhere and migrate to luminal surfaces of blood vessels in the

noninflamed state (34). Recent reports indicate that the recruit-

ment of inflammatory Ly6Chigh monocytes and their macrophage

or DC progeny reflects the changing needs of the affected tis-

sue along the course of inflammation, which includes cytokine

*Institute of Medical Microbiology, University of Magdeburg, 39120 Magdeburg,Germany; †Immune Regulation Group, Helmholtz Centre for Infection Research, 38124Braunschweig, Germany; ‡Department of Cellular Neuroscience, Max-Delbrueck-Center for Molecular Medicine, 13092 Berlin, Germany; xUniversitatsklinikumRegensburg, Innere Medizin II/Nephrologie-Transplantation, 93042 Regensburg,Germany; and {Department of Microbiology and Hygiene, Charite-University Med-icine Berlin, 12203 Berlin, Germany

Received for publication August 11, 2014. Accepted for publication January 16,2015.

This work was supported by the German Research Foundation (Deutsche For-schungsgemeinschaft DU1112/3/1 and SFB854 to I.R.D.).

Address correspondence and reprint requests to Dr. Ildiko Rita Dunay, Instituteof Medical Microbiology, University of Magdeburg, Leipziger Strasse 44, 39120Magdeburg. E-mail address: ildikodunay@gmail.com

The online version of this article contains supplemental material.

Abbreviations used in this article: DC, dendritic cell; iNOS, inducible NO synthase;PSGL-1, P-selectin glycoprotein ligand-1; ROS, reactive oxygen intermediate; TREM2,triggering receptor expressed on myeloid cells-2.

Copyright� 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402037

by guest on April 6, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

production, Ag presentation, and phagocytosis of cellular debris(12, 13, 35, 36). In the CNS, functional macrophage heterogeneityhas been demonstrated in several models of autoimmune pathol-ogy as well as in neurodegenerative and infectious diseases (19,26, 27, 36, 37), but the precise role of the recruited mononuclearcell subsets in cerebral toxoplasmosis remains to be addressed.Subsequently, the availability of new surface markers and fatemapping led to a paradigm shift describing the behavior of distinctmonocyte-derived macrophages and their difference to tissue-resident mononuclear cells (26, 33, 38, 39).In this study, we set out to investigate the phenotype and par-

ticular function of the recruited Ly6ChighCCR2+ monocytes alongthe course of a low-cyst dose-induced chronic T. gondii infection.We demonstrate that, during cerebral T. gondii infection, Ly6Chigh

CCR2+ inflammatory monocytes are recruited to the brain andperform crucial antiparasitic functions. Their specific ablationresulted in decreased survival, suggesting pivotal defense functionagainst the invaders. The infiltrated Ly6ChighCCR2+ monocytespromoted parasite control through production of proinflammatorymolecules, such as IL-1a, IL-1b, IL-6, iNOS, TNF, and ROS.Furthermore, we describe that the recruited Ly6ChighCCR2+ mono-cytes differentiated and changed their phenotype and function intotwo distinct mononuclear cell subpopulations. One subset down-regulated Ly6C, CCR2 and upregulated CD11c, MHC I as well asMHC II (Ly6CintCCR2intCD11c+), adopting DC properties. Theother myeloid cell subpopulation did not exhibit Ly6C expression,but upregulated F4/80 and triggering receptor expressed on myeloidcells-2 (TREM2), presenting potent phagocytic capacity (Ly6Cneg

CCR2intF4/80high), respectively. Finally, we show that P-selectinglycoprotein ligand-1 (PSGL-1) plays a role in the recruitment ofLy6Chigh monocytes to the CNS. Our results indicate the criticalimportance of the recruited Ly6Chigh monocytes in the CNS uponchronic T. gondii infection, thus further exposing the dynamics andbehavior of antiparasitic and inflammatory properties of the mono-nuclear cell subsets in a murine model of cerebral toxoplasmosis.

Materials and MethodsAnimals

All animal experiments were approved by the local authorities according toGerman and European legislation. Experiments were conducted with adultC57BL/6 female mice (8 wk old; purchased from Janvier, Saint Berthevin,France). Four to five animals per group in up to four independent exper-iments were investigated.

Infection

T. gondii cysts of type II ME49 strain were harvested from the brains offemale NMRI mice strain infected i.p. with T. gondii cysts 5–6 mo earlier,as described previously (15). Brains obtained from infected mice weremechanically homogenized in 1 ml sterile PBS, and the cyst numbers weredetermined using a light microscope. Three cysts were administered i.p.into mice in a total volume of 200 ml. Control mice were mock infectedwith sterile PBS.

Histopathology

Brains were removed and immersed in 4% paraformaldehyde for severaldays and sectioned coronally at 4 mm. Paraffin-embedded, 4-mm–thicksections were deparaffinized and conventionally stained with H&E. Pri-mary Abs against macrophage 1 Ag (1:200; Abcam), to label microgliaand mononuclear cells, and anti-Toxoplasma (1:200; Dianova DLN-16734),to label T. gondii, were used. Slides were developed using the BondPolymer Refine Detection kit (Menarini/Leica). For the evaluation, wholetissue sections were digitized at 230 nm resolution using a MiraxMidiSlide Scanner (Zeiss MicroImaging).

Parasite number

To determine the total T. gondii cyst burden in the brains of infected andanti-CCR2–treated mice, full brains were obtained and mechanically ho-

mogenized in 1 ml sterile PBS and the total cyst numbers were determinedusing light microscope. To confirm T. gondii cyst numbers, five to sixcoronal slides per mouse (same brain region) were analyzed by micros-copy; n = 4 mice per group. The blinded analysis was performed by tworesearchers.

Ab treatment

Ablation of monocytes was performed by administration of anti-CCR2mAb(clone MC-21; provided by M. Mack, University of Regensburg). Micewere injected with 75 mg (40) Ab i.p. on days 20, 22, 24, 26, and 28postinfection. Twenty-four hours after the last treatment on day 29, themice were sacrificed. Rat IgG (eBioscience, San Diego, CA) was used asa control mAb.

Cell isolation

Brains were homogenized in a buffer containing 1 M HEPES (pH 7.3) and45% glucose and then sieved through a 70 mm strainer. The cell suspensionwas washed and fractionated on 25–75% Percoll gradient (GE Healthcare)for 25 min at 8003 g without brake. The cells in the interphase comprisedof mononuclear cells, which were washed with PBS and used immediatelyfor further experiments. Peripheral blood obtained by cardiac puncture wastreated with erythrocyte (RBC) lysis buffer (eBioscience) and then washedtwice with ice-cold PBS at 300 3 g for 10 min, followed by staining withthe desired fluorescent conjugated Abs.

Flow cytometry

Mononuclear cells isolated from different organs were incubated with ananti-FcgRIII/II Ab (clone 93) to block unspecific staining. Thereafter,cells were stained with fluorochrome-conjugated Abs against cell surfacemarkers, as follows: CD45 (30-F11), CD11b (M1/70), CD11c (N418),Ly6C (HK1.4), F4/80 (BM8), MHCI H-2Db (28-14-8), MHCII I-A/I-E(M5/114.15.2), VLA-4 (R1-2), and LFA-1 (M17/4), all purchased fromeBioscience; Ly6G (1A8), PSGL-1 (2PH1), and CD62-L (MEL-14) fromBD Biosciences; and CX3CR1 (polyclonal goat), Trem2 (237920), andCCR2 (475301) from R&D Systems for 30 min on ice, and then washedand fixed in 4% paraformaldehyde for 10 min. Matched isotype controlswere used to assess the level of unspecific staining.

For intracellular cytokine staining, single-cell suspensions from thebrains (5 3 105 cells/well) were stimulated in 24-well flat-bottom plates inthe presence of Toxoplasma lysate Ag (5 mg/ml), brefeldin A (10 mg/ml,GolgiPlug; BD Biosciences), and monensin (10 mg/ml; BioLegend). After6 h, cells were incubated with FcR-blocking Ab (clone 93) for 15 min onice and surface stained for CD45 (30-F11), CD11b (M1/70), Ly6G (1A8),Ly6C (HK1.4), and CD11c (N418) in FACS buffer for 30 min on ice;washed twice in FACS buffer; and fixed in 4% paraformaldehyde. Cellswere then permeabilized using BD Perm/Wash Buffer. To measure cyto-kine expression, cells were stained with the following Abs: TNF (MP6-XT22), IL-12p40 (C17.8), IL-1a (ALF-161), IL-1b (NJTEN3), IL-6(MP5-20F3), and IL-10 (JES5-16E3) from eBioscience and iNOS (M-19)from Santa Cruz for 45 min in BD Perm/Wash Buffer.

A total of 100,000 cells was acquired using a flow cytometer (BD FACSCanto II), and flow cytometric data were analyzed using FlowJo software(Version 9.6.4; Tree Star). Matched isotype controls were used to subtractthe nonspecific background staining.

Detection of reactive oxygen species

Single-cell suspensions from brain after blocking their FcgRs were surfacestained for CD45 (30-F11), CD11b (M1/70), Ly6G (1A8), Ly6C (HK1.4),and CD11c (N418) in FACS buffer for 30 min in ice. ROS production wasmeasured by Total ROS Detection Kit (ENZO, 51011), according to themanufacturer’s instructions.

In vitro phagocytosis assay

The isolated mononuclear cells were cultured in 12-well chambers (NalgeNunc International) at a density of 4 3 105 cells/ml. A total of 50 mlFluoSpheres latex beads were added (Molecular Probes) after 1-h pre-treament with cytoclasin D (Sigma-Aldrich), and cells were incubatedunder standard culture conditions for 8 h. Samples were acquired on flowcytometer (BD FACS Canto II) and analyzed with FlowJo software (Ver-sion 9.6.4; Tree Star).

Immunofluorescence

Frozen sections were acetone fixed for 10 min and then rehydrated andblocked by incubation for 10 min in diluent (PBS containing 2.0% normalgoat serum, 1% BSA, 0.1% gelatin, 0.05% Tween 20, and 0.05% sodium

3224 MONOCYTES IN CEREBRAL TOXOPLASMOSIS

by guest on April 6, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

azide). Sections were incubated in primary Abs (Ly6C clone HK1.4,eBioscience; CCR2, GenWay Biotech) in diluent for 60 min, rinsed in washbuffer (PBS containing 0.5% FBS), and incubated for 60 min with sec-ondary Abs conjugated to Alexa 488 or Alexa 594 (Molecular Probes). Fornegative controls, primary Abs were substituted by isotype-matched con-trols of the same species. Sections were rinsed in wash buffer and mountedin Vectashield containing DAPI (Vector Labs, Burlingame, CA) or ProLongGold containing DAPI (Molecular Probes). Slides were examined witha Zeiss epifluorescence microscope equipped with an AxioCam CCDcamera and Axiovision v4.0 software for image capture (Carl Zeiss,Peabody, MA). Images were processed with similar linear adjustments forall samples in Photoshop 4.0 (Adobe, San Jose, CA).

Adoptive transfer of CD11b+Ly6Chigh monocytes

Bone marrow cells were harvested from femora and tibiae of wild-typenoninfected mice, lysed with erythrocyte (RBC) lysis buffer (eBiosci-ence), and washed twice with ice-cold PBS at 300 3 g for 10 min, beforestaining with the desired fluorescent conjugated Abs. CD11b+Ly6Chigh

monocytes were sorted using BD FACS Diva with Digital Vantage Optionfrom wild-type C57BL/6 bone marrow cells and labeled with 5 mM CFSE(Life Technologies), and 1 3 106 cells were injected i.v. into 4-wkT. gondii (ME49)–infected C57BL/6 mice. Forty-eight hours later, the re-cipient mice were sacrificed and the brain mononuclear cells were isolatedand analyzed by flow cytometry.

Anti–PSGL-1 treatment

To assess the transmigration capacity of the cells, 2 mg/kg rat anti-mousePSGL-1 (4RA10; BioXCell) was administered i.p. every alternate day fromday 16 to day 26 postinfection. Twenty-four hours after the last treatment onday 27, the mice were sacrificed. Isotype-matched rat IgG (eBioscience)was administered to the controls.

Statistical analysis

Data were analyzed by Student t test for two groups or one-way ANOVAfor several groups, followed by Tukey’s posttest with GraphPad Prism 6(San Diego, CA). In all cases, results were presented as mean 6 SD andwere considered significant with p , 0.05.

ResultsMyeloid cells are recruited to the brain upon chronic T. gondiiinfection

Cerebral toxoplasmosis is associated with activation of residentimmune cells and peripheral cell recruitment to the CNS; however,the heterogeneity of infiltrating immune cell subsets has not beenfully characterized. Accordingly, we investigated the characteristicfeatures and fate of the newly described Ly6Chigh monocytesfollowing extravasation in the brains of infected mice. We ob-served an ingress of CD45high populations in the brains of C57BL/6mice after 4 wk with a low-dose T. gondii infection (Fig. 1B),whereas, in uninfected controls, the major leukocyte populationin the brain were resting resident microglia, (CD45lowCD11b+;Fig. 1A), corroborating previous reports (19, 27, 38). Furtherphenotyping of the cell subsets (Fig. 1B) subdivided the CD45high

population into a CD45highCD11b2 subset (ungated) comprisedmainly of recruited lymphocytes and a CD45highCD11b+ pop-ulation (upper gate; 8.0 6 1.06% of the parent population)encompassing myeloid-derived cells, namely monocytes, neu-trophils, macrophages, and DCs. Brain resident–activated micro-glia expressed elevated levels of CD45 upon the infection(CD45intCD11b+gated, lower; 4.1 6 0.31% of the parent pop-ulation).The Ly6G (1A8) Ab (7, 40) was used to distinguish between

Ly6G2 monocytes (Fig. 1C; 96.0 6 2.11% of the CD45high

CD11b+) and Ly6G+ neutrophils (4.0 6 2.02% of the CD45high

CD11b+). Relative expression of Ly6C and F4/80 was used tofurther differentiate between the myeloid cell subsets (Fig. 1D).Based on these surface markers, we identified three distinctmyeloid cell subpopulations, as follows: Ly6ChighF4/80int, Ly6CintF4/80int, and Ly6CnegF4/80high. These data suggest that, during chronic

T. gondii infection, alongside with the activation of residentmicroglia, a heterogeneous population of myeloid cells infiltratesthe CNS that can be further divided into three distinct subsetsbased on their Ly6C and F4/80 expression.

Ly6ChighCCR2+ cells localize in the parenchyma duringcerebral toxoplasmosis

Immunofluorescence analysis of brain sections of T. gondii–infected mice revealed accumulation of Iba1-positive cells(Fig. 2E) in the meninges as well as in the cortex compared withnoninfected controls (Fig. 2A). Closer examination suggestedrobust cell activation of Iba-positive microglia with less ramifi-cation, bigger soma, marked increase of average surface area, andrather amoeboid morphology (Fig. 2I). Importantly, inflammatoryfoci of infected brains contained Ly6C (Fig. 2F, 2J)- and CCR2(Fig. 2G, 2K)-positive cells, suggesting Ly6ChighCCR2+ mono-cytes (Fig. 2H, 2L). These amoeboid-shaped inflammatory cellswere mainly located adjacent to the lesions in the cortex close tothe vessels. These observations further indicate that Ly6Chigh

CCR2+ monocytes are recruited to the CNS upon T. gondii in-fection, which is in accordance with previous studies describingrapid influx of monocytes in the CNS upon inflammatory pro-cesses (16, 38, 41).

Ablation of CCR2+Ly6Chigh monocytes upon cerebraltoxoplasmosis is detrimental

To evaluate the contribution of CCR2+Ly6Chigh monocytes in theparasite control, we took advantage of the newly available de-pleting anti-CCR2 Ab (MC-21) (35, 42, 43). Thus, we appliedeither anti-CCR2 Ab or isotype control IgG mAb, on alternatingdays from 20 d postinfection to 28 d postinfection, to two groupsof infected mice, respectively. Twenty-four hours after the last Abtreatment, mice were sacrificed and the successful depletion ofCCR2+Ly6Chigh monocytes in the blood was confirmed by flowcytometry by staining for Ly6C and CD11b (2.0 6 0.23–0% ofperipheral blood leukocytes; Fig. 3A, 3B, 3D).Importantly, we noticed a reduction of recruited myeloid cells

(8.0 6 1.5% to 2.0 6 0.28%) in the brains of anti-CCR2–treatedmice (Fig. 3C, upper panel). This observation was further con-firmed with a compelling decrease of Ly6Chigh monocytes (lowerpanel; 66.5 6 8.6% to 11.2 6 6.2%) and a slight decrease in theLy6Cint population (30.3 6 2.7% to 24.2 6 1.6%). In addition,a rise of the Ly6Cneg population was detected (4.0 6 0.7% to65.0 6 4.8%) that might have entered the brain before depletionoccurred (before day 20). Alterations in the absolute cell numbersrevealed a significant reduction of Ly6Chigh (p , 0.001) andLy6Cint (p , 0.01) cell subsets. Moreover, there was an increasein the Ly6Cneg (p , 0.001) cell subset, whereas the microgliacompartment remained unaltered (Fig. 3E).Next, we investigated whether the selective ablation of CCR2+

Ly6Chigh monocytes had an impact on brain pathology and sur-vival of chronically infected mice. Histological examination ofinfected anti-CCR2 Ab-depleted mice revealed a higher frequencyof inflammatory foci (Fig. 4B, 4D), macrophage 1 Ag+ microglia,and mononuclear cells, and infiltration of immune cells into thecortex (Fig. 4D, 4F). Notably, we observed increased numbers ofT. gondii cyst numbers (Fig. 4H, 4I) when compared with non-treated infected control brains. Most importantly, depletion ofLy6Chigh monocytes was followed by decreased survival rates, asby day 60 all anti-CCR2–treated mice succumbed due to the in-fection, whereas all nontreated infected control mice survived(Fig. 4J). Together these results demonstrate that Ly6ChighCCR2+

monocytes carry out fundamental functions in parasite control

The Journal of Immunology 3225

by guest on April 6, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

during cerebral toxoplasmosis, and their depletion exacerbates theoutcome of the infection.

Characterization of mononuclear cell subsets upon cerebralT. gondii infection

The heterogeneity of monocyte–macrophage populations and theirmultifunctionality in the CNS have been intensively studied re-cently (33, 35, 38, 44). The different mononuclear cell subsets canbe distinguished by their pattern of .50 characteristic surfacemarkers, making their discrimination between the subpopulationsa complex assignment (45, 46). Therefore, to perceive the distinctmyeloid cell subsets in the brain upon T. gondii infection, we firstcompared the expression of specific surface markers. Brainmononuclear cells from infected and control mice were isolated,and comprehensive flow cytometry analysis was performed.Microglia from noninfected and infected mice did not expressLy6C, in contrast to monocytes (Fig. 5A, 5E). CCR2, which isa receptor for monocyte chemokine protein-1 (or CCL2), has beenestablished to play a determining role for inflammatory monocyteegress from bone marrow (6, 32). As expected, Ly6Chigh mono-cytes notoriously expressed high levels of CCR2, and its expres-sion on Ly6Cint and Ly6Cneg population was lower, whereas CCR2was absent on microglia. This observation initiated our hypothesis

that the Ly6Cint and Ly6Cneg subpopulations might have differ-entiated from the infiltrating Ly6Chigh monocyte subset (Fig. 5I,5M).Next, we measured the expression of the chemokine receptor

CX3CR1. Fractalkine, the ligand of the CX3CR1 receptor, isexpressed by neurons along with other cells and plays a role inmaintaining certain microglial function (47–49). Microglia ex-pressed high amounts of CX3CR1, as described previously (30,34, 35, 38, 50, 51), whereas the receptor on recruited myeloid cellswas present in low levels (Fig. 5B, 5F). The F4/80 Ag, which isexhibited by mature macrophages, was expressed predominantlyby activated microglia and by myeloid-derived Ly6Cneg cells. Theexpression intensity of F4/80+ within the Ly6Chigh and Ly6Cint

population was weaker (Fig. 5J, 5N). We observed a significantupregulation of a common DC marker CD11c on activatedmicroglia, in line with previous studies highlighting their activa-tion status (27). Furthermore, expression of CD11c was high onLy6Cint and Ly6Cneg myeloid-derived cells (Fig. 5C, 5G). Thesurface marker TREM2 was strongly upregulated on activatedmicroglia and myeloid-derived Ly6Cneg cells (Fig. 5K, 5O),pointing toward a potentially elevated phagocytic capacity. TheLy6Chigh and Ly6Clow subpopulations expressed low levels ofTREM2, implying little phagocytic activity. Parallel to their ele-

FIGURE 1. Myeloid cell recruitment and activation of microglia cells. Leukocytes were isolated from brains of noninfected (A) and T. gondii–infected

(B–D) C57BL/6 mice and analyzed by flow cytometry. Following the basic forward light scatter–side light scatter gating, the singlet cells were selected for

further characterization. (A) Shows resident microglia (CD11b+CD45low, lower gate), and (B) shows the percentage of activated microglia (CD11b+CD45int,

lower gate) and the myeloid population (CD11b+CD45high, upper gate). (C) The myeloid population consists of neutrophils (CD11b+Ly6G+, upper gate)

and monocytes (CD11b+Ly6G2, lower gate). (D) The monocytes can be further divided according to their expression of Ly6C and F4/80: Ly6ChighF4/80int,

Ly6CintF4/80int, and Ly6CnegF4/80high. Numbers represent percentage of parent population. Data shown are representative of four individual experiments

(n = 4); results are shown as mean 6 SD .

FIGURE 2. Immunofluorescence staining

of microglia and monocytes in brain slides.

We show the cortex and the cortical me-

ninges in noninfected control (A–D) and

infected (E–L) brains. In the noninfected

brain (A), Iba+ microglia have a ramified

morphology, whereas in the infected brain

(E) they display a rather amoeboid mor-

phology with bigger soma. The Ly6C and

CCR2 staining are negative in the control

brains, but positive in the parenchyma of the

infected brains (F and G), displaying the

recruitment of inflammatory monocytes (H)

during T. gondii infection. Five to six cor-

onal slides per mouse were analyzed; n = 4

mice per group. This experiment was re-

peated three times. Scale bars, 100 mm in

(A)–(H) and 25 mm in (I)–(L).

3226 MONOCYTES IN CEREBRAL TOXOPLASMOSIS

by guest on April 6, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

vated CD11c expression, Ly6Cint cells upregulated primarily theactivation markers MHC I and II, indicating their efficacy to ini-tiate adaptive immune responses by Ag presentation. Activatedmicroglia, Ly6Chigh, and Ly6Cneg cells also expressed MHC I andII, but with less intensity (Fig. 5D, 5H, 5L, 5P). Collectively, theextensive phenotypic characterization of the myeloid cell subsetsrevealed that these cells are distinct from microglia, and theyexpress different levels of specific surface markers, suggestingdefinite functions.Next, we elucidated the surface markers of the mononuclear cell

subsets in the blood before they entered the infected CNS at 4 wkafter the T. gondii infection. As anticipated, the Ly6Chigh mono-cytes expressed high amount of CCR2, whereas the residentmonocytes were CCR2neg (Supplemental Fig. 1E, 1F). CX3CR1and F4/80 were predominantly expressed on resident monocytes

(Supplemental Fig. 1G–J). The surface Ag CD11c was weaklyexpressed on all investigated cell populations in the blood; how-ever, the highest appearance was observed on Ly6Chigh monocytes(Supplemental Fig. 1K, 1L). Similarly, MHC I was upregulated onLy6Chigh inflammatory monocytes upon T. gondii infection, andMHC II was mainly expressed on resident monocytes during in-fection (Supplemental Fig. 1M–P).

Unique cytokine profile of myeloid cell subsets in cerebraltoxoplasmosis

Consequently, to find out the particular cytokine production ofthese three distinct myeloid-derived cell subsets, we performedintracellular flow cytometry analysis. We detected that Ly6Chigh

monocytes were able to produce high amounts of proinflammatorymediators such as IL-1a, IL-1b, IL-6, TNF, and iNOS (Fig. 6A–

FIGURE 3. Depletion of CCR2+Ly6Chigh monocytes.

C57BL/6 mice were infected with T. gondii and from day

20 to day 28 postinfection alternatively treated with either

IgG mAb or anti-CCR2 to deplete inflammatory mono-

cytes. After the standard forward light scatter–side light

scatter gating, single cells were selected for further char-

acterization. (A left, and B) displays the representative

plots to define Ly6Chigh inflammatory monocytes (upper

gate), Ly6Cint neutrophils (middle gate), and Ly6Cneg

resident monocytes (lower gate) in the blood. The coex-

pression of Ly6C and CCR2 of the inflammatory mono-

cytes (Ly6Chigh) is shown further (A, right). (D) The bar

graph represents the percentage of Ly6Chigh in the blood.

(C) After the basic forward light scatter–side light scatter

and singlet gating, upper plots show the gating of activated

microglia (CD11b+CD45int) and the myeloid population

(CD11b+CD45high) in the brain. Lower plots display the

monocyte subsets (from the myeloid gate): Ly6Chigh,

Ly6Cint, and Ly6Cneg. (E) The bar graphs represent the

total cell number of the respective subset in the brain.

Numbers in the plots represent percentage of parent pop-

ulation. Data shown are representative of five individual

experiments (with n = 4 individual mice); results shown as

mean 6 SD. Significant differences (**p , 0.01, ***p ,0.001) were determined using the Student t test. Act.

microglia, activated microglia.

FIGURE 4. Increased parasite load and decreased

survival in anti-CCR2–treated T. gondii–infected mice

(A–H). Immunohistological representation of control

(left panel) and anti-CCR2 (right panel) brains shows

more inflammation by H&E staining (D, arrows), in-

creased habitation of mononuclear cells (F), and more

parasites (H) in the anti-CCR2 brain. (I) The bar graph

is the quantification of the total cyst burden in the

control and anti-CCR2 brains. (J) Survival curve of

the T. gondii–infected control and anti-CCR2–treated

mice was monitored from day 0 until day 60. Five to

six coronal slides per mouse were analyzed; n = 4

mice per group. Total cyst load was determined from

brain lysates; n = 4 mice per group. The survival ex-

periment was repeated twice for a total of n = 8–10

mice per experiment. Significant differences (*p ,0.05) were determined using Student t test.

The Journal of Immunology 3227

by guest on April 6, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

6C, 6F–6H, 6J, 6K, 6N, 6O). ROS secretion was measured ex-plicitly by Ly6Chigh monocytes, suggesting their strong potentialto eliminate the parasites (Fig. 6L, 6P). The Ly6Cint and Ly6Cneg

population produced lesser amounts of cytokines; however, IL-12p40 was mainly produced by Ly6Cint cells, suggesting theirrole to shape the adaptive immune system (Fig. 6D, 6I). Activatedmicroglia contributed with low levels of cytokine production tothe host defense in this model with low-dose T. gondii–inducedchronic infection in the brain. Interestingly, alongside theirproinflammatory and antiparasitic functions, Ly6Chigh cells alsoexpressed the immunoregulatory cytokine IL-10 upon in vitrostimulation, implicating their possible dual nature to maintain

tissue homeostasis and counterbalance the ongoing CNS inflam-mation (Fig. 6M, 6Q).

Ly6Cneg myeloid cells perform strong phagocytic capacity

To examine the phagocytic properties of microglia and recruitedmyeloid cell subsets, we performed ex vivo phagocytosis assay. To

this end, the respective cell subsets were isolated, sorted, and then

incubated with fluorescent latex beads. Whereas recruited Ly6Chigh

and Ly6Cint cells exhibited low phagocytic capacity (31.1 6 3.6%

and 25.0 6 2.2%, respectively), Ly6Cneg cells demonstrated

prominent phagocytic ability (80.2 6 5.6%), alongside with those

of activated resident microglia (60.2 6 1.2%; Fig. 6E).

FIGURE 5. Phenotypic characterization of mononuclear cell populations in the brain. (A–P). To measure the expression of surface and activation markers

by leukocytes in the brain, infected and noninfected controls were analyzed by flow cytometry. A similar gating strategy was followed as in Fig. 1A–D.

(A–D and I–L) Representative histograms showing expression levels of surface markers by cell population. Bars mark the cells positive for the particular

marker. Numbers above bars display the percentage of cells positive for the marker of the concerned population: resident microglia (CD11b+CD45low),

activated microglia (CD11b+CD45int), inflammatory monocytes (CD11b+CD45highLy6G2Ly6Chigh), Ly6Cint monocytes (CD11b+CD45highLy6G2Ly6Cint),

Ly6Cneg monocytes (CD11b+CD45highLy6G2Ly6Cneg), isotype control, tinted (B, D, K, and L). (E–H and M–P) Bar graphs represent the median fluores-

cence intensity for the specific marker median fluorescence intensity (MFI) 6 SD (n = 4). Data are representative of four independent experiments. One-

way ANOVA analysis followed by Tukey’s post hoc test was performed for multiple comparisons (*p , 0.05, **p , 0.01, ***p , 0.001). Act. microglia,

activated microglia; ns, not significant; Res. microglia, resident microglia.

3228 MONOCYTES IN CEREBRAL TOXOPLASMOSIS

by guest on April 6, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

FIGURE 6. Cytokine production and phagocytic capacity of the cell subsets in the brain. (A–D and F–Q) Intracellular cytokine production was analyzed

in cells isolated from brains of noninfected and infected mice after in vitro stimulation with Toxoplasma lysate Ag. The cells were gated as shown in the

representative plots of Fig. 1A–D. (A–D and J–M) Representative histograms showing cytokine expression by cell population. Bars mark the cells positive

for the particular cytokine. Numbers above bars display the percentage of cells positive for the cytokine of the respective population: resident microglia

(CD11b+CD45low), activated microglia (CD11b+CD45int), inflammatory monocytes (CD11b+CD45highLy6G2Ly6Chigh), Ly6Cint monocytes (CD11b+

CD45highLy6G2Ly6Cint), Ly6Cneg monocytes (CD11b+CD45highLy6G2Ly6Cneg), isotype control, tinted. (F–I and N–Q) Bar graphs represent the median

fluorescence intensity (MFI) of the respective fluorochrome for a particular cytokine, MFI 6 SD (n = 4). Data are representative of four independent

experiments. (E) The uptake of fluorescent latex beads by brain mononuclear cells ex vivo was measured by flow cytometry. After the standard forward light

scatter–side light scatter gating and singlet gating, CD11b+ cells were further gated, as shown in Fig. 1. Bar graph shows the percentage of phagocytosis of

the latex beads by the respective population. Data are representative of three experiments with n = 4; results are shown as mean 6 SD. One-way ANOVA

analysis followed by Tukey’s post hoc test was performed for multiple comparisons (*p , 0.05, **p , 0.01, ***p , 0.001). Act. microglia, activated

microglia; ns, not significant; Res. microglia, resident microglia.

The Journal of Immunology 3229

by guest on April 6, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Ly6Chigh cells infiltrate and differentiate in the brain uponadoptive transfer

To confirm that recruited Ly6Chigh monocytes further differentiateto the previously described Ly6Cint and Ly6Cneg cells followingextravasation in the CNS, we conducted adoptive transfer ex-periments. To address this, we injected 1 3 106 sorted, CFSE-labeled Ly6ChighCD11b+ cells isolated from the bone marrow ofwild-type mice i.v. into T. gondii–infected recipient mice at day 28postinfection. The cells were injected when the ongoing inflam-mation had already affected the blood-brain–barrier permeabilityand the recruitment of the inflammatory cells to the CNS reachedits peak (data not shown). Notably, CFSE-labeled Ly6Chigh cellswere found in the CNS as early as 24 h after the transfer (data notshown). Forty-eight hours after the transfer, Ly6Chigh cells (27.263.2% of CD11b+CFSE+ gate) downregulated Ly6C and apparentlydifferentiated into Ly6Cint (32.0 6 1.6% of CD11b+CFSE+ gate)and Ly6Cneg cells (41.0 6 2.0% of CD11b+CFSE+ gate; Fig. 7B).These data suggest that Ly6Chigh monocytes are recruited to thebrain upon T. gondii infection, in which a subset of them losestheir Ly6C expression and further generates the Ly6Cint andLy6Cneg subsets.

Migration of CCR2+Ly6Chigh monocytes to the brain isPSGL-1 dependent

Selective leukocyte homing to the site of inflammation has beenshown to be dependent on chemokines and distinct adhesionmolecules (52–55). To evaluate whether any particular adhesionmolecule plays a role in CCR2+Ly6Chigh monocyte recruitment tothe CNS in toxoplasmosis, we measured the expression of Lselectin (CD62L), LFA-1, and PSGL-1 on Ly6Chigh monocytes.CD62L, which is known to be important for leukocyte rolling

on the inflamed endothelium (56, 57), was highly expressed onLy6Chigh inflammatory monocytes in the periphery, but wasdownregulated upon entry to the brain. Resident monocytes in theblood expressed only low levels of CD62L (Fig. 8A, 8D, 8G, 8J).LFA-1 was expressed both in the periphery and in the brain by therecruited monocytes as well as on Ly6Cint and Ly6Cneg cells in theCNS. Resident monocytes in the blood upregulated LFA-1 uponT. gondii infection, similarly to resident microglia cells in thebrain (Fig. 8B, 8E, 8H, 8K). PSGL-1 expression on the Ly6Chigh

inflammatory monocytes was significantly higher compared withresident cells in the blood and in the CNS (Fig. 8C, 8F, 8I, 8L).Hence, to test whether the high PSGL-1 levels detected on the

surface of Ly6Chigh monocytes had a functional role in their mi-gration to the CNS, mice were treated i.p. with anti–PSGL-1 Ab

from day 16 to day 26 after T. gondii infection. After 24 h of thelast Ab application, we observed an increased proportion of

Ly6Chigh monocytes in the peripheral blood (Fig. 9A, 9B; 3.1 60.6% to 5.46 0.92%) of the parent population of the treated mice,

implying that, in the absence of PSGL-1, sufficient transmigration

does not occur and cells accumulate in the blood. In the brains of

infected anti PSGL-1–treated mice, we observed a reduction in

the percentage (40.6 6 1.67% to 25.2 6 2.1% of the CD45high

CD11b+Ly6G2 population) and in the total cell numbers within

the Ly6Chigh compartment (Fig. 9C [lower panel], 9D, respec-

tively). No significant differences were detected in the frequencies

and total numbers of other cell subsets (Fig. 9C, lower panel). The

inhibition of Ly6Chigh cell recruitment to the brain was markedly

diminished (p , 0.01; Fig. 9E), confirming that PSGL-1 is an

important mediator for monocyte homing to the CNS.

DiscussionDue to the recent discovery of several novel characteristic markersand transcription factors, our knowledge of the myeloid cell het-

erogeneity has advanced extensively (26, 30, 33, 36, 38, 41, 45, 58,

59). Previous studies have suggested that, alongside the resident

microglia, recruited mononuclear cells are important to control

CNS inflammation and (different) CNS infections (16, 27, 36, 37,

39). However, differences in the experimental setups have made it

difficult to precisely correlate results and determine the relative

importance of the definitive cell subpopulations during the in-

flammatory processes and infections.The role of bone marrow–derived CCR2+Ly6Chigh monocytes

was investigated under various infectious conditions in the pe-

riphery, as well as in the CNS. We have previously shown that

inflammatory monocytes control acute T. gondii infection in the

ileum, by producing antimicrobial mediators (6, 7). Also, dur-

ing Listeria monocytogenes infection, the absence of Ly6Chigh

monocytes leads to rapid death of mice, demonstrating their im-

portant role in host defense (32, 60, 61). Furthermore, we recently

observed that Ly6Chigh monocytes are substantially involved in

brain inflammation and immune cell recruitment to the CNS,

leading to experimental cerebral malaria upon Plasmodium ANKA

infection (our unpublished observations). In cases of viral en-

cephalitis, Ly6Chigh monocytes infiltrate the CNS and, although

contributing to viral clearance, they also induce significant im-

munopathology (16, 62). The opposing beneficial and detrimental

nature of Ly6Chigh monocytes, which seemingly depends on the

type of infection, warrants further characterization to understand

their intricate behavior.In the chronic phase of T. gondii infection, parasites persist in

cysts within immune-privileged sites (15). The latent stage is as-

sociated with marginal inflammation and cell recruitment to the

CNS, which is necessary to provide adequate IFN-g levels, the

major driving force for parasite control. In the murine models of

chronic infection, the characteristics of T cell subsets (24, 63–65),

specific mononuclear cells, for example, resident microglia and

APCs, have been extensively studied. In contrast, the role of the

newly described myeloid cell subsets is controversial and thus

requires further investigation (19–21). Microglia cells have been

shown to eliminate parasites in an IFN-g–dependent manner, in

addition to their efficient phagocytic capacity and cytokine pro-

duction (19, 20, 66, 67). Some early studies suggest that brain DCs

differentiate from local resident cells, whereas others have proposed

a peripheral hematopoietic cell origin upon cerebral toxoplasmosis

(27, 39, 68).In this study, we have elucidated the nature of the recruited

myeloid cells within the brain upon cerebral toxoplasmosis. We

FIGURE 7. Adoptively transferred CFSE+Ly6Chigh monocytes are

recruited to the brain in chronic T. gondii infection. Sorted CFSE+CD11b+

Ly6Chigh monocytes from wild-type bone marrow were injected i.v. into

T. gondii–infected mice. Forty-eight hours later, brain mononuclear cells

were isolated. After the standard forward light scatter–side light scatter and

singlet gating, CFSE+CD11b+ cells were analyzed in infected mice that

did not [Control; (A)] or did receive CFSE-labeled cells (B). CFSE+ cells

showed heterogeneous Ly6C expression; numbers represent percentage of

the parent population. Data shown are representative of two individual

experiments (n = 4); results shown as mean 6 SD.

3230 MONOCYTES IN CEREBRAL TOXOPLASMOSIS

by guest on April 6, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

found that, upon low-dose infection with T. gondii, myeloid-derived Ly6Chigh cells infiltrated the brain and expressed spe-cific surface markers CD45highCD11b+Ly6G2Ly6ChighF4/80int. Inaddition, based on their Ly6C and F4/80 expression, there weretwo more CD45+ myeloid populations present in the CNS, namelyLy6CintF4/80int and Ly6CnegF4/80high.In our previous studies, we detected that CCR22/2 mice were

extremely sensitive to T. gondii infection, with .90% of micesuccumbing in the acute phase of infection. We therefore con-cluded that CCR2+Ly6Chigh monocytes were necessary to controlthe acute T. gondii infection and to limit the small intestinal pa-thology (6). The remaining 10% of infected mice displayed ele-vated parasite numbers in the CNS, suggesting that these cellsmight also be involved in the chronic phase of the infection (6).Thus, to study the role of CCR2+Ly6Chigh monocytes in thechronic stage, the depletion of this particular cell subset wasnecessary to perform after the infection overcame the acute phase

and parasites were present in the CNS. Previously, we comparedthe effect of commonly used Abs to ablate inflammatory mono-cytes and addressed the concerns surrounding the expression ofGr1, Ly6C, and Ly6G, using the available depletion strategies ofneutrophil granulocytes and inflammatory monocytes (7). In theseearlier studies, we used the Gr-1–specific RB6-8C5 Ab; however,the application of this Ab led to a depletion of both Ly6Chigh

Ly6G2 monocytes, as well as Ly6G+Ly6C+ neutrophils (7). Ascontrol, we used the 1A8 Ab that reacts specifically to Ly6G anddepletes solely neutrophils (40). Importantly, our results con-firmed that Ly6Chigh monocytes are crucial in the control of theacute T. gondii infection, and that Ly6G+ neutrophils rather con-tributed to the immunopathology (7).In the current study, taking advantage of a new depletion strategy

of Ly6ChighCCR2+ monocytes, we applied anti-CCR2 Ab (MC-21), which selectively depletes CCR2+ inflammatory monocytes(69, 70). We observed an increase in immunopathology and ele-

FIGURE 8. CCR2+Ly6Chigh monocytes express PSGL-1. Cells from the peripheral blood (A–F) (gating strategy: Supplemental Fig. 1) and brains (G–L)

(gating strategy: Fig. 1) of noninfected and infected mice were measured for the expression of adhesion molecules. (A–C and G–I) Representative his-

tograms showing adhesion molecule expression by cell population. Bars mark the cells positive for the particular adhesion molecule. Numbers above bars

display the percentage of cells positive for the marker of the concerned population. Blood cell subsets (D–F) are indicated as follows: resident monocytes,

resident monocytes from infected mice, inflammatory monocytes, inflammatory monocytes from infected mice, isotype control, tinted. Brain leukocyte

subsets (J–L) are as follows: resident microglia (CD11b+CD45low), activated microglia (CD11b+CD45int), inflammatory monocytes (CD11b+CD45high

Ly6G2Ly6Chigh), Ly6Cint monocytes (CD11b+CD45highLy6G2Ly6Cint), Ly6Cneg monocytes (CD11b+CD45highLy6G2Ly6Cneg), isotype control, tinted.

(D–F and J–L) Bar graphs represent the median fluorescence intensity for the Ab, median fluorescence intensity (MFI)6 SD (n = 5). Data are representative

of two independent experiments. One-way ANOVA analysis followed by Tukey’s post hoc test was performed for multiple comparisons (*p , 0.05, **p ,0.01, ***p , 0.001). Act. microglia, activated microglia; Inf. Ly6Chigh, infected Ly6Chigh; Inf. res. mono., infected resident monocytes; ns, not significant;

Res. microglia, resident microglia; Res. mono., resident monocytes.

The Journal of Immunology 3231

by guest on April 6, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

vated parasite numbers in the CNS during cerebral toxoplasmosis.Most importantly, mice were unable to control the parasites andsuccumbed to infection. This finding strongly suggests thatLy6ChighCCR2+ monocytes play a critical role in governing thechronic phase of Toxoplasma infection.Due to the differential expression of Ly6C and F4/80 among

the myeloid compartment, we characterized the phenotype ofthe recruited CCR2+Ly6Chigh monocytes, Ly6Cint and Ly6Cneg

myeloid-derived cells, and resident microglia. Therefore, weconducted comprehensive surface and intracellular stainings usinghighly specific markers. CCR2 expression was the highest on thenewly recruited Ly6Chigh cells, and was lower on Ly6Cint andLy6Cneg cells. The chemokine receptor CX3CR1 was predomi-nantly expressed on microglia and was absent on Ly6Chigh

monocytes, in accordance with previous studies (31, 33, 34, 38,71). The mature macrophage marker F4/80 was expressed mainlyby activated microglia and by Ly6Cneg cells, suggesting theirmacrophage-like nature. Microglia upregulated CD11c uponinfection-induced activation, as previously reported (27). CD11cexpression was also elevated in Ly6Cint and Ly6Cneg cells,pointing toward their DC phenotype. Interestingly, MHC I and IIwere most intensely expressed on the surface of Ly6Cint cells,implying that these cells may represent the previously character-ized brain DCs (27). These results proved that the recruited my-eloid cell populations are different from the resident microglia.Comparing the cytokine profile between the resident and

recruited mononuclear cell subsets in the CNS revealed thatLy6Chigh monocytes produced the highest levels of proin-

flammatory molecules, such as IL-1a, IL-1b, IL-6, TNF, andiNOS. These results indicate that monocytes are essential tocontrol T. gondii infection in the brain, similar to our previousfindings in the periphery (6, 7). Additionally, the elevated ROSproduction was observed exclusively by the Ly6Chigh monocytes,further suggesting their critical role in the host defense arsenal.This proinflammatory signature of Ly6Chigh cells was reaffirmed incerebral viral infections (62), and in other parasitic models (72, 73).IL-12p40 secretion was the highest by Ly6Cint cells, well in linewith their CD11c and MHC expression, suggesting their capabilityto initiate adaptive immune responses. In comparison with CD45+

CD11b+ myeloid-derived cells, the activated resident microgliacontributed to lesser extent to the secretion of proinflammatorymediators in cerebral toxoplasmosis. Remarkably, in addition totheir antiparasitic capacity, Ly6Chigh cells were also able to secretethe regulatory cytokine IL-10, suggesting a dual function of lim-iting pathogen expansion and regulating detrimental immunopa-thology in the CNS. Our observations are consistent with a recentstudy by Grainger et al. (72), reporting similar dual features ofLy6Chigh cells in the acute T. gondii infection model. Additionally,this study also described that monocytes regulate neutrophil func-tion by secreting PGE2, hence contributing to our understandingof their complex functions. Such decisive roles played by theLy6Chigh cells during the resolution of inflammation, tissue re-generation, and debris clearance were also seen in models of au-toimmunity and neurodegeneration (26, 36, 37).We also found that activated microglia alongside with Ly6Cneg

myeloid-derived cells displayed phagocytic potential, whereas

FIGURE 9. PSGL-1 modulates Ly6Chigh monocyte migration to the CNS. Anti–PSGL-1 (2 mg/kg; anti–PSGL-1, right) or rat IgG (control, left) was

administered to T. gondii–infected mice i.p. every alternate day from day 16 to day 26 postinfection . Flow cytometric analysis of blood and brain was

performed 24 h after the last treatment. After the forward light scatter–side light scatter gating and doublet exclusion, cells were further analyzed. (A)

Representative plots are shown to define Ly6Chigh inflammatory monocytes (upper gate), Ly6Cint neutrophils (middle gate), and Ly6Cneg resident

monocytes (lower gate) in the blood. (B) The bar graphs represent the percentage of singlets of the respective population in the peripheral blood. (C)

Representative plots to illustrate the analysis of Ly6Chigh inflammatory monocytes in the brain. Upper plots show the gating of microglia (CD11b+CD45int)

and the myeloid population (CD11b+CD45high) in the brain. The myeloid gate is further characterized for the monocyte subsets (lower plots): Ly6Chigh,

Ly6Cint, and Ly6Cneg. (D) The bar graphs represent the total cell number of the respective population in the brain. (E) The relative inhibition in the re-

cruitment of Ly6Chigh monocytes following anti–PSGL-1 treatment was compared with the IgG-treated controls. Numbers were normalized on control

brains. The numbers in the representative contour plots are the percentage of the parent population. Data are representative of two independent experiments

with n = 5 mice per group; results shown as mean 6 SD. Significant differences (*p , 0.05, **p , 0.01, ***p , 0.001) in the bar graphs were determined

using the Student t test. Act. microglia, activated microglia; Res.monocytes., resident monocytes.

3232 MONOCYTES IN CEREBRAL TOXOPLASMOSIS

by guest on April 6, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Ly6Chigh and Ly6Cint cells were less capable in this activity. TheF4/80 and particularly the TREM2 expression of those cells fol-lowed the same pattern, as described recently for the latter mol-ecule (49, 74–77), suggesting that the activated microglia hasthe capacity to engulf invading microorganisms and dead tissueremains during the ongoing inflammation (49, 76–79). The currentparadigm suggests that monocytes do not substantially contributeto tissue-resident macrophages under steady state conditions;rather, resident macrophages and microglia in the CNS develop inthe embryonic stage (38). However, during infection and inflam-mation, macrophage-like cells can differentiate from infiltratingLy6Chigh inflammatory monocytes. The diverse behavior of themonocyte-derived macrophages was demonstrated in experimen-tal autoimmune uveitis, in which the kinetics of CX3CR1

lowLy6C+

and CX3CR1highLy6C2 changed along the course of the disease

(36). Such heterogeneity was also shown in an acute model ofcolitis, in which the recruited CX3CR1

intLy6Chigh monocytespromoted inflammation, but over time gave rise to a CX3CR1

int

Ly6Clow population that displayed all the trademarks of a DC (35).In cerebral toxoplasmosis, the conversion of monocytes to brainDCs upon recruitment was also proposed by a previous study(27). Confirming their plasticity, we also detected that recruitedmonocytes in the brain further differentiate into two distinctpopulation CD11c+Ly6Cint brain DCs and Ly6CnegF4/80+ mac-rophages and carry out particular functions in parasite clearance.Blood monocytes are comprised of two distinct populations,

the inflammatory CX3CR1lowCCR2+Ly6Chigh and the patrolling

CX3CR1highCCR22Ly6Clow cells. Correspondingly, in our cur-

rent experiments, CX3CR1lowCCR2+Ly6Chigh monocytes and

CX3CR1highCCR22Ly6Clow were present in the blood, and, upon

Toxoplasma infection, the common DC marker CD11c and theMHC I molecule were upregulated on the surface of Ly6Chigh

monocytes, suggesting modified activation status even beforereaching the site of infection.Leukocyte transmigration in the blood through the blood-brain

barrier upon cerebral inflammation is dependent on adhesionmolecules and their receptors. Inflammatory monocytes use theCCR2–CCL2 axis to egress from the bone marrow, but themechanism of crossing the blood-brain barrier is poorly under-stood (6, 78). Therefore, we investigated the involvement of cer-tain key molecules in the recruitment of Ly6Chigh inflammatorymonocytes to the CNS. CD62L (L-selectin), which assists immunecells to enter different tissues (56, 57), was highly expressed onLy6Chigh monocytes in the blood, but was downregulated upontheir entry into the CNS. Consistent with previous studies, theCD62L expression was low on resident monocytes and microglia(38). LFA-1, which can interact with P- and E-selectin on theactivated endothelial cells (prompting recruitment), was presentboth in the periphery and in the CNS on Ly6Chigh monocytes, aswell as on differentiated Ly6Cint and Ly6Cneg monocytes. Previ-ous studies have described elevated LFA-1 expression on residentmicroglia in cerebral toxoplasmosis and their functional role onrecruited. Additionally, the functional role of LFA-1 on recruitedDCs is well characterized (27). We measured highest PSGL-1expression on the surface of Ly6Chigh monocytes, which led usto the hypothesis that this molecule might play a role in monocyteentry.The dependence on PSGL-1 for lymphocyte migration is well

established (56, 57, 79–82). However, to date, the function ofPSGL-1 in monocyte recruitment has not been sufficientlyaddressed. One previous study described the role of PSGL-1 inLy6Chigh monocyte homing to the site of atherosclerosis in bloodvessels of mice (54). This study revealed that Ly6Chigh monocytes,which are PSGL-1high and CD62L+, preferentially interacted with

P- and E-selectin on activated endothelium or with CD62L ona rolling/adherent leukocyte under flow by secondary tethering.However, the authors speculated that other adhesion moleculessuch as LFA-1 and VCAM-1 (ligand VLA-4) may not be keyfactors in monocyte homing, as their ligands were expressed atlower levels on Ly6Chigh cells. Furthermore, other studies detectedPSGL-1–dependent monocyte migration in the periphery duringLeishmania major infection, tumor metastasis, and thrombusformation (48, 81, 82). Thus, to our knowledge, we investigatedfor the first time PSGL-1–dependent monocyte recruitment to theCNS. Treatment of mice with anti–PSGL-1 Ab revealed a signifi-cant inhibition of Ly6Chigh cell recruitment to the brain uponchronic T. gondii infection, confirming that PSGL-1 is critical formonocyte homing to the CNS.Altogether, our findings combined with emerging evidence from

other murine models further highlight the plasticity of recruitedLy6Chigh monocytes. Cerebral T. gondii infection leads to cytokineproduction by the inundated Ly6Chigh monocytes, which playan influential role in the protection of the inflamed brain. TheLy6Chigh monocytes further give rise to Ly6Cint and Ly6Cneg

subsets and perform divergent functions such as Ag presentationand phagocytosis. Thus, monocytes and their descendants playmultifaceted functions to control cerebral toxoplasmosis. In con-clusion, our findings indicate that, during cerebral T. gondii in-fection, Ly6Chigh inflammatory monocytes infiltrate the CNS anddifferentiate into phenotypically and functionally distinct cellsubsets and carry out pivotal functions to control the chronicstage.

AcknowledgmentsWe thank Dana Zabler for excellent technical assistance.

DisclosuresThe authors have no financial conflicts of interest.

References1. Munoz, M., O. Liesenfeld, and M. M. Heimesaat. 2011. Immunology of Toxo-

plasma gondii. Immunol. Rev. 240: 269–285.2. Barragan, A., and L. D. Sibley. 2002. Transepithelial migration of Toxoplasma

gondii is linked to parasite motility and virulence. J. Exp. Med. 195: 1625–1633.3. Da Gama, L. M., F. L. Ribeiro-Gomes, U. Guimaraes, Jr., and A. C. V. Arnholdt.

2004. Reduction in adhesiveness to extracellular matrix components, modulationof adhesion molecules and in vivo migration of murine macrophages infectedwith Toxoplasma gondii. Microbes Infect. 6: 1287–1296.

4. Courret, N., S. Darche, P. Sonigo, G. Milon, D. Buzoni-Gatel, and I. Tardieux.2006. CD11c- and CD11b-expressing mouse leukocytes transport single Toxo-plasma gondii tachyzoites to the brain. Blood 107: 309–316.

5. Lambert, H., N. Hitziger, I. Dellacasa, M. Svensson, and A. Barragan. 2006.Induction of dendritic cell migration upon Toxoplasma gondii infection poten-tiates parasite dissemination. Cell. Microbiol. 8: 1611–1623.

6. Dunay, I. R., R. A. Damatta, B. Fux, R. Presti, S. Greco, M. Colonna, andL. D. Sibley. 2008. Gr1(+) inflammatory monocytes are required for mucosalresistance to the pathogen Toxoplasma gondii. Immunity 29: 306–317.

7. Dunay, I. R., A. Fuchs, and L. D. Sibley. 2010. Inflammatory monocytes but notneutrophils are necessary to control infection with Toxoplasma gondii in mice.Infect. Immun. 78: 1564–1570.

8. Denkers, E. Y., B. A. Butcher, L. Del Rio, and S. Bennouna. 2004. Neutrophils,dendritic cells and Toxoplasma. Int. J. Parasitol. 34: 411–421.

9. Mashayekhi, M., M. M. Sandau, I. R. Dunay, E. M. Frickel, A. Khan,R. S. Goldszmid, A. Sher, H. L. Ploegh, T. L. Murphy, L. D. Sibley, andK. M. Murphy. 2011. CD8a(+) dendritic cells are the critical source ofinterleukin-12 that controls acute infection by Toxoplasma gondii tachyzoites.Immunity 35: 249–259.

10. Klose, C. S. N., M. Flach, L. Mohle, L. Rogell, T. Hoyler, K. Ebert, C. Fabiunke,D. Pfeifer, V. Sexl, D. Fonseca-Pereira, et al. 2014. Differentiation of type 1 ILCsfrom a common progenitor to all helper-like innate lymphoid cell lineages. Cell157: 340–356.

11. Schulthess, J., B. Meresse, E. Ramiro-Puig, N. Montcuquet, S. Darche,B. Begue, F. Ruemmele, C. Combadiere, J. P. Di Santo, D. Buzoni-Gatel, andN. Cerf-Bensussan. 2012. Interleukin-15-dependent NKp46+ innate lymphoidcells control intestinal inflammation by recruiting inflammatory monocytes.Immunity 37: 108–121.

The Journal of Immunology 3233

by guest on April 6, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

12. Dunay, I. R., and L. D. Sibley. 2010. Monocytes mediate mucosal immunity toToxoplasma gondii. Curr. Opin. Immunol. 22: 461–466.

13. Karlmark, K. R., F. Tacke, and I. R. Dunay. 2012. Monocytes in health anddisease: Minireview. Eur. J. Microbiol. Immunol. 2: 97–102.

14. Hermes, G., J. W. Ajioka, K. A. Kelly, E. Mui, F. Roberts, K. Kasza, T. Mayr,M. J. Kirisits, R. Wollmann, D. J. P. Ferguson, et al. 2008. Neurological andbehavioral abnormalities, ventricular dilatation, altered cellular functions, in-flammation, and neuronal injury in brains of mice due to common, persistent,parasitic infection. J. Neuroinflammation 5: 48.

15. Parlog, A., L.-A. Harsan, M. Zagrebelsky, M. Weller, D. von Elverfeldt,C. Mawrin, M. Korte, and I. R. Dunay. 2014. Chronic murine toxoplasmosis isdefined by subtle changes in neuronal connectivity. Dis. Model. Mech. 7: 459–469.

16. Getts, D. R., R. L. Terry, M. T. Getts, M. M€uller, S. Rana, B. Shrestha,J. Radford, N. Van Rooijen, I. L. Campbell, and N. J. C. King. 2008. Ly6c+“inflammatory monocytes” are microglial precursors recruited in a pathogenicmanner in West Nile virus encephalitis. J. Exp. Med. 205: 2319–2337.

17. Mohle, L., A. Parlog, J. Pahnke, and I. R. Dunay. 2014. Spinal cord pathology inchronic experimental Toxoplasma gondii infection. Eur. J. Microbiol. Immunol.4: 65–75.

18. Kung, D. H., E. A. Hubenthal, J. Y. Kwan, S. A. Shelburne, J. C. Goodman, andJ. S. Kass. 2011. Toxoplasmosis myelopathy and myopathy in an AIDS patient:a case of immune reconstitution inflammatory syndrome? Neurologist 17: 49–51.

19. Strack, A., V. C. Asensio, I. L. Campbell, D. Schl€uter, and M. Deckert. 2002.Chemokines are differentially expressed by astrocytes, microglia and inflam-matory leukocytes in Toxoplasma encephalitis and critically regulated by inter-feron-gamma. Acta Neuropathol. 103: 458–468.

20. Schl€uter, D., L.-Y. Kwok, S. L€utjen, S. Soltek, S. Hoffmann, H. Korner, andM. Deckert. 2003. Both lymphotoxin-alpha and TNF are crucial for control ofToxoplasma gondii in the central nervous system. J. Immunol. 170: 6172–6182.

21. Drogem€uller, K., U. Helmuth, A. Brunn, M. Sakowicz-Burkiewicz, D. H. Gutmann,W. Mueller, M. Deckert, and D. Schl€uter. 2008. Astrocyte gp130 expression iscritical for the control of Toxoplasma encephalitis. J. Immunol. 181: 2683–2693.

22. Nishanth, G., M. Sakowicz-Burkiewicz, U. Handel, S. Kliche, X. Wang,M. Naumann, M. Deckert, and D. Schl€uter. 2010. Protective Toxoplasma gondii-specific T-cell responses require T-cell-specific expression of protein kinaseC-theta. Infect. Immun. 78: 3454–3464.

23. Dupont, C. D., D. A. Christian, E. M. Selleck, M. Pepper, M. Leney-Greene,G. Harms Pritchard, A. A. Koshy, S. Wagage, M. A. Reuter, L. D. Sibley, et al.2014. Parasite fate and involvement of infected cells in the induction of CD4+and CD8+ T cell responses to Toxoplasma gondii. PLoS Pathog. 10: e1004047.

24. Suzuki, Y., X. Wang, B. S. Jortner, L. Payne, Y. Ni, S. A. Michie, B. Xu,T. Kudo, and S. Perkins. 2010. Removal of Toxoplasma gondii cysts from thebrain by perforin-mediated activity of CD8+ T cells. Am. J. Pathol. 176: 1607–1613.

25. Stumhofer, J. S., J. S. Silver, and C. A. Hunter. 2013. IL-21 is required foroptimal antibody production and T cell responses during chronic Toxoplasmagondii infection. PLoS One 8: e62889.

26. Shechter, R., and M. Schwartz. 2013. Harnessing monocyte-derived macro-phages to control central nervous system pathologies: no longer ‘if’ but ‘how.’ J.Pathol. 229: 332–346.

27. John, B., B. Ricart, E. D. Tait Wojno, T. H. Harris, L. M. Randall,D. A. Christian, B. Gregg, D. M. De Almeida, W. Weninger, D. A. Hammer, andC. A. Hunter. 2011. Analysis of behavior and trafficking of dendritic cells withinthe brain during toxoplasmic encephalitis. PLoS Pathog. 7: e1002246.

28. Serbina, N. V., T. M. Hohl, M. Cherny, and E. G. Pamer. 2009. Selective ex-pansion of the monocytic lineage directed by bacterial infection. J. Immunol.183: 1900–1910.

29. Varol, C., L. Landsman, D. K. Fogg, L. Greenshtein, B. Gildor, R. Margalit,V. Kalchenko, F. Geissmann, and S. Jung. 2007. Monocytes give rise to mucosal,but not splenic, conventional dendritic cells. J. Exp. Med. 204: 171–180.

30. Geissmann, F., S. Jung, and D. R. Littman. 2003. Blood monocytes consist oftwo principal subsets with distinct migratory properties. Immunity 19: 71–82.

31. Palframan, R. T., S. Jung, G. Cheng, W. Weninger, Y. Luo, M. Dorf,D. R. Littman, B. J. Rollins, H. Zweerink, A. Rot, and U. H. von Andrian. 2001.Inflammatory chemokine transport and presentation in HEV: a remote controlmechanism for monocyte recruitment to lymph nodes in inflamed tissues. J. Exp.Med. 194: 1361–1373.

32. Geissmann, F., M. G. Manz, S. Jung, M. H. Sieweke, M. Merad, and K. Ley.2010. Development of monocytes, macrophages, and dendritic cells. Science327: 656–661.

33. Mildner, A., S. Yona, and S. Jung. 2013. A close encounter of the third kind:monocyte-derived cells. Adv. Immunol. 120: 69–103.

34. Auffray, C., D. Fogg, M. Garfa, G. Elain, O. Join-Lambert, S. Kayal, S. Sarnacki,A. Cumano, G. Lauvau, and F. Geissmann. 2007. Monitoring of blood vesselsand tissues by a population of monocytes with patrolling behavior. Science 317:666–670.

35. Zigmond, E., C. Varol, J. Farache, E. Elmaliah, A. T. Satpathy, G. Friedlander,M. Mack, N. Shpigel, I. G. Boneca, K. M. Murphy, et al. 2012. Ly6C himonocytes in the inflamed colon give rise to proinflammatory effector cells andmigratory antigen-presenting cells. Immunity 37: 1076–1090.

36. London, A., I. Benhar, M. J. Mattapallil, M. Mack, R. R. Caspi, andM. Schwartz. 2013. Functional macrophage heterogeneity in a mouse model ofautoimmune central nervous system pathology. J. Immunol. 190: 3570–3578.

37. Shechter, R., A. London, C. Varol, C. Raposo, M. Cusimano, G. Yovel, A. Rolls,M. Mack, S. Pluchino, G. Martino, et al. 2009. Infiltrating blood-derived mac-

rophages are vital cells playing an anti-inflammatory role in recovery from spinalcord injury in mice. PLoS Med. 6: e1000113.

38. Ginhoux, F., and S. Jung. 2014. Monocytes and macrophages: developmentalpathways and tissue homeostasis. Nat. Rev. Immunol. 14: 392–404.

39. Fischer, H.-G., and G. Reichmann. 2001. Brain dendritic cells and macrophages/microglia in central nervous system inflammation. J. Immunol. 166: 2717–2726.

40. Daley, J. M., A. A. Thomay, M. D. Connolly, J. S. Reichner, and J. E. Albina.2008. Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice.J. Leukoc. Biol. 83: 64–70.

41. London, A., E. Itskovich, I. Benhar, V. Kalchenko, M. Mack, S. Jung, andM. Schwartz. 2011. Neuroprotection and progenitor cell renewal in the injuredadult murine retina requires healing monocyte-derived macrophages. J. Exp.Med. 208: 23–39.

42. Br€uhl, H., J. Cihak, J. Plachy, L. Kunz-Schughart, M. Niedermeier, A. Denzel,M. Rodriguez Gomez, Y. Talke, B. Luckow, M. Stangassinger, and M. Mack.2007. Targeting of Gr-1+,CCR2+ monocytes in collagen-induced arthritis. Ar-thritis Rheum. 56: 2975–2985.

43. Avraham-Davidi, I., S. Yona, M. Grunewald, L. Landsman, C. Cochain,J. S. Silvestre, H. Mizrahi, M. Faroja, D. Strauss-Ayali, M. Mack, et al. 2013.On-site education of VEGF-recruited monocytes improves their performance asangiogenic and arteriogenic accessory cells. J. Exp. Med. 210: 2611–2625.

44. Ransohoff, R. M., and A. E. Cardona. 2010. The myeloid cells of the centralnervous system parenchyma. Nature 468: 253–262.

45. Ransohoff, R. M., P. Kivisakk, and G. Kidd. 2003. Three or more routes forleukocyte migration into the central nervous system. Nat. Rev. Immunol. 3: 569–581.

46. Chan, W. Y., S. Kohsaka, and P. Rezaie. 2007. The origin and cell lineage ofmicroglia: new concepts. Brain Res. Brain Res. Rev. 53: 344–354.

47. Mizuno, T., J. Kawanokuchi, K. Numata, and A. Suzumura. 2003. Productionand neuroprotective functions of fractalkine in the central nervous system. BrainRes. 979: 65–70.

48. Zujovic, V., J. Benavides, X. Vige, C. Carter, and V. Taupin. 2000. Fractalkinemodulates TNF-a secretion and neurotoxicity induced by microglial activation.Glia 29: 305–315.

49. Takahashi, K., C. D. P. Rochford, and H. Neumann. 2005. Clearance of apoptoticneurons without inflammation by microglial triggering receptor expressed onmyeloid cells-2. J. Exp. Med. 201: 647–657.

50. Drevets, D. A., M. J. Dillon, J. S. Schawang, N. Van Rooijen, J. Ehrchen,C. Sunderkotter, and P. J. M. Leenen. 2004. The Ly-6Chigh monocyte subpop-ulation transports Listeria monocytogenes into the brain during systemic infec-tion of mice. J. Immunol. 172: 4418–4424.

51. Yona, S., K. W. Kim, Y. Wolf, A. Mildner, D. Varol, M. Breker, D. Strauss-Ayali,S. Viukov, M. Guilliams, A. Misharin, et al. 2013. Fate mapping reveals originsand dynamics of monocytes and tissue macrophages under homeostasis. Im-munity 38: 79–91.

52. Butcher, E. C. 1991. Leukocyte-endothelial cell recognition: three (or more)steps to specificity and diversity. Cell 67: 1033–1036.

53. Ley, K., C. Laudanna, M. I. Cybulsky, and S. Nourshargh. 2007. Getting to thesite of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immu-nol. 7: 678–689.

54. An, G., H. Wang, R. Tang, T. Yago, J. M. McDaniel, S. McGee, Y. Huo, andL. Xia. 2008. P-selectin glycoprotein ligand-1 is highly expressed on Ly-6Chimonocytes and a major determinant for Ly-6Chi monocyte recruitment to sites ofatherosclerosis in mice. Circulation 117: 3227–3237.

55. McEver, R. P. 2001. Adhesive interactions of leukocytes, platelets, and the vesselwall during hemostasis and inflammation. Thromb. Haemost. 86: 746–756.

56. Sperandio, M., M. L. Smith, S. B. Forlow, T. S. Olson, L. Xia, R. P. McEver, andK. Ley. 2003. P-selectin glycoprotein ligand-1 mediates L-selectin-dependentleukocyte rolling in venules. J. Exp. Med. 197: 1355–1363.

57. Leon, B., and C. Ardavın. 2008. Monocyte migration to inflamed skin and lymphnodes is differentially controlled by L-selectin and PSGL-1. Blood 111: 3126–3130.

58. Satpathy, A. T., C. G. Briseno, J. S. Lee, D. Ng, N. A. Manieri, W. Kc, X. Wu,S. R. Thomas, W.-L. Lee, M. Turkoz, et al. 2013. Notch2-dependent classicaldendritic cells orchestrate intestinal immunity to attaching-and-effacing bacterialpathogens. Nat. Immunol. 14: 937–948.

59. Epelman, S., K. J. Lavine, and G. J. Randolph. 2014. Origin and functions oftissue macrophages. Immunity 41: 21–35.

60. Serbina, N. V., T. P. Salazar-Mather, C. A. Biron, W. A. Kuziel, and E. G. Pamer.2003. TNF/iNOS-producing dendritic cells mediate innate immune defenseagainst bacterial infection. Immunity 19: 59–70.

61. Serbina, N. V., T. Jia, T. M. Hohl, and E. G. Pamer. 2008. Monocyte-mediateddefense against microbial pathogens. Annu. Rev. Immunol. 26: 421–452.

62. Terry, R. L., D. R. Getts, C. Deffrasnes, C. van Vreden, I. L. Campbell, andN. J. C. King. 2012. Inflammatory monocytes and the pathogenesis of viralencephalitis. J. Neuroinflammation 9: 270.

63. Schl€uter, D., T. Meyer, A. Strack, S. Reiter, M. Kretschmar, O. D. Wiestler,H. Hof, and M. Deckert. 2001. Regulation of microglia by CD4+ and CD8+T cells: selective analysis in CD45-congenic normal and Toxoplasma gondii-infected bone marrow chimeras. Brain Pathol. 11: 44–55.

64. Schl€uter, D., T. Meyer, L.-Y. Kwok, M. Montesinos-Rongen, S. L€utjen,A. Strack, M. L. Schmitz, and M. Deckert. 2002. Phenotype and regulation ofpersistent intracerebral T cells in murine Toxoplasma encephalitis. J. Immunol.169: 315–322.

65. Clark, R. T., J. P. Nance, S. Noor, and E. H. Wilson. 2011. T-cell production ofmatrix metalloproteinases and inhibition of parasite clearance by TIMP-1 duringchronic Toxoplasma infection in the brain. ASN Neuro 3: e00049.

3234 MONOCYTES IN CEREBRAL TOXOPLASMOSIS

by guest on April 6, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

66. Deckert, M., J. D. Sedgwick, E. Fischer, and D. Schl€uter. 2006. Regulation ofmicroglial cell responses in murine Toxoplasma encephalitis by CD200/CD200receptor interaction. Acta Neuropathol. 111: 548–558.

67. Suzuki, Y., J. Claflin, X. Wang, A. Lengi, and T. Kikuchi. 2005. Microglia andmacrophages as innate producers of interferon-gamma in the brain followinginfection with Toxoplasma gondii. Int. J. Parasitol. 35: 83–90.

68. Deshpande, P., I. L. King, and B. M. Segal. 2007. Cutting edge: CNS CD11c+cells from mice with encephalomyelitis polarize Th17 cells and support CD25+CD4+ T cell-mediated immunosuppression, suggesting dual roles in the diseaseprocess. J. Immunol. 178: 6695–6699.

69. Mack, M., J. Cihak, C. Simonis, B. Luckow, A. E. Proudfoot, J. Plachy,H. Br€uhl, M. Frink, H. J. Anders, V. Vielhauer, et al. 2001. Expression andcharacterization of the chemokine receptors CCR2 and CCR5 in mice. J.Immunol. 166: 4697–4704.

70. Hammond, M. D., R. A. Taylor, M. T. Mullen, Y. Ai, H. L. Aguila, M. Mack,S. E. Kasner, L. D. McCullough, and L. H. Sansing. 2014. CCR2+ Ly6C(hi)inflammatory monocyte recruitment exacerbates acute disability following in-tracerebral hemorrhage. J. Neurosci. 34: 3901–3909.

71. Auffray, C., M. H. Sieweke, and F. Geissmann. 2009. Blood monocytes: de-velopment, heterogeneity, and relationship with dendritic cells. Annu. Rev.Immunol. 27: 669–692.

72. Grainger, J. R., E. A. Wohlfert, I. J. Fuss, N. Bouladoux, M. H. Askenase,F. Legrand, L. Y. Koo, J. M. Brenchley, I. D. C. Fraser, and Y. Belkaid. 2013.Inflammatory monocytes regulate pathologic responses to commensals duringacute gastrointestinal infection. Nat. Med. 19: 713–721.

73. Sponaas, A. M., A. P. Freitas do Rosario, C. Voisine, B. Mastelic, J. Thompson,S. Koernig, W. Jarra, L. Renia, M. Mauduit, A. J. Potocnik, and J. Langhorne.2009. Migrating monocytes recruited to the spleen play an important role incontrol of blood stage malaria. Blood 114: 5522–5531.

74. Melchior, B., A. E. Garcia, B.-K. Hsiung, K. M. Lo, J. M. Doose, J. C. Thrash,A. K. Stalder, M. Staufenbiel, H. Neumann, and M. J. Carson. 2010. Dual in-

duction of TREM2 and tolerance-related transcript, Tmem176b, in amyloid

transgenic mice: implications for vaccine-based therapies for Alzheimer’s dis-

ease. ASN Neuro 2: e00037.75. Takahashi, K., M. Prinz, M. Stagi, O. Chechneva, and H. Neumann. 2007.

TREM2-transduced myeloid precursors mediate nervous tissue debris clearance

and facilitate recovery in an animal model of multiple sclerosis. PLoS Med. 4:

e124.76. Turnbull, I. R., S. Gilfillan, M. Cella, T. Aoshi, M. Miller, L. Piccio,

M. Hernandez, and M. Colonna. 2006. Cutting edge: TREM-2 attenuates mac-

rophage activation. J. Immunol. 177: 3520–3524.77. Piccio, L., C. Buonsanti, M. Cella, I. Tassi, R. E. Schmidt, C. Fenoglio, J. Rinker,

II, R. T. Naismith, P. Panina-Bordignon, N. Passini, et al. 2008. Identification of

soluble TREM-2 in the cerebrospinal fluid and its association with multiple

sclerosis and CNS inflammation. Brain 131: 3081–3091.78. Shi, C., and E. G. Pamer. 2011. Monocyte recruitment during infection and

inflammation. Nat. Rev. Immunol. 11: 762–774.79. Engelhardt, B. 2009. PSGL-1—the hidden player in T cell trafficking into the

brain in multiple sclerosis? J. Leukoc. Biol. 86: 1023–1025.80. Sathiyanadan, K., C. Coisne, G. Enzmann, U. Deutsch, and B. Engelhardt. 2014.

PSGL-1 and E/P-selectins are essential for T-cell rolling in inflamed CNS

microvessels but dispensable for initiation of EAE. Eur. J. Immunol. 44: 2287–

2294.81. Hoos, A., D. Protsyuk, and L. Borsig. 2014. Metastatic growth progression

caused by PSGL-1-mediated recruitment of monocytes to metastatic sites.

Cancer Res. 74: 695–704.82. Inoue, T., Y. Tsuzuki, K. Matsuzaki, H. Matsunaga, J. Miyazaki, R. Hokari,

Y. Okada, A. Kawaguchi, S. Nagao, K. Itoh, et al. 2005. Blockade of PSGL-1

attenuates CD14+ monocytic cell recruitment in intestinal mucosa and amelio-

rates ileitis in SAMP1/Yit mice. J. Leukoc. Biol. 77: 287–295.

The Journal of Immunology 3235

by guest on April 6, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from