of March 25, 2010 This information is current as

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2009;183;5537-5547; originally publishedJ. Immunol. Shuliang Liu and Tammy Kielian  

MyD88-Dependent Pathways Is Mediated by TLR4- andkoseri

CitrobacterMicroglial Activation by

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Microglial Activation by Citrobacter koseri Is Mediated byTLR4- and MyD88-Dependent Pathways1

Shuliang Liu* and Tammy Kielian2†

Citrobacter koseri is a Gram-negative bacterium that can cause a highly aggressive form of neonatal meningitis, which oftenprogresses to establish multifocal brain abscesses. Despite its tropism for the brain parenchyma, microglial responses to C. koserihave not yet been examined. Microglia use TLRs to recognize invading pathogens and elicit proinflammatory mediator expressionimportant for infection containment. In this study, we investigated the importance of the LPS receptor TLR4 and MyD88, anadaptor molecule involved in the activation of the majority of TLRs in addition to the IL-1 and IL-18 receptors, for their rolesin regulating microglial activation in response to C. koseri. Proinflammatory mediator release was significantly reduced in TLR4mutant and MyD88 knockout microglia compared with wild-type cells following exposure to either live or heat-killed C. koseri,indicating a critical role for both TLR4- and MyD88-dependent pathways in microglial responses to this pathogen. However,residual proinflammatory mediator expression was still observed in TLR4 mutant and MyD88 KO microglia following C. koseriexposure, indicating a contribution of TLR4- and MyD88-independent pathway(s) for maximal pathogen recognition. Interest-ingly, C. koseri was capable of surviving intracellularly in both primary microglia and macrophages, suggesting that these cellsmay serve as a reservoir for the pathogen during CNS infections. These results demonstrate that microglia respond to C. koseriwith the robust expression of proinflammatory molecules, which is dictated, in part, by TLR4- and MyD88-dependentsignals. The Journal of Immunology, 2009, 183: 5537–5547.

I n the United States, �30–40% of bacterial meningitis casesare caused by Gram-negative organisms (1, 2). One exampleof a Gram-negative pathogen that exhibits a remarkable de-

gree of tropism for the brain is Citrobacter koseri (formerly C.diversus) (3–9). One intriguing feature of C. koseri meningitis isthe propensity of organisms to disseminate into the CNS paren-chyma and establish multifocal abscesses (6–9). Indeed, infantsthat become infected with C. koseri either by transmission via aninfected mother during parturition or hospitalization experience analarming frequency of brain abscess development (i.e., �77%),which far exceeds the occurrence by any other meningitis-causingbacteria (7). A significant percentage of infants infected with C.koseri experience a high mortality rate (i.e., 30–50%) and amongthose that survive, �75% experience long-term neurological def-icits, including seizures, cognitive disabilities, and hearing loss (8).Therefore, although the incidence of C. koseri infections is ratherlow, the serious impact of this disease highlights the need to un-derstand the pathogenic mechanisms induced by this organism.Although several studies have examined the pathogenesis of C.koseri-induced meningitis and brain abscess in animal models(10–15), to date, the responses of resident microglia to C. koseri

have not yet been examined, despite the tropism of this organismfor the CNS parenchyma.

Microglia are the resident mononuclear phagocyte population inthe CNS parenchyma and represent an important component of theinnate immune response against invading pathogens (16–19). Be-cause of the high predilection of C. koseri for the brain, it is likelythat resident microglia play a key role in sensing CNS colonizationand mounting an initial antibacterial immune response before therecruitment of peripheral immune cells. Microglia are equippedwith a repertoire of pattern recognition receptors (PRRs),3 includ-ing TLRs that play a pivotal role in detecting invariant motifsexpressed by pathogens (known as pathogen-associated molecularpatterns or PAMPs) (20, 21). Gram-negative bacteria such as C.koseri contain an abundance of LPS in their outer cell wall thatexerts potent proinflammatory activity (22, 23). The LPS recogni-tion complex is formed by a tripartite set of proteins, namelyCD14, TLR4, and MD-2. The current consensus is that LPS bindsto CD14, which subsequently triggers TLR4 activation and down-stream signaling pathways, culminating in the release of a widearray of inflammatory mediators that participate in antibacterialimmunity (20, 21, 24, 25). MyD88 is a pivotal downstream sig-naling adaptor molecule recruited by the TLR4 receptor complexthat leads to proinflammatory gene expression by NF-�B andMAPK signaling pathways (25, 26). However, TLR4 can alsotransduce signals via MyD88-independent pathways through itsassociation with other adaptors such as TIR domain-containingadaptor protein and TIR domain-containing adaptor inducingIFN-� that lead to the induction of IFN-inducible gene products(21, 25). Microglia express CD14, TLR4, and MyD88 that rep-resent important players in the LPS-sensing machinery of these

*Department of Neurobiology and Developmental Sciences, University of Arkansasfor Medical Sciences, Little Rock, AR 72205; and †Department of Pathology andMicrobiology, University of Nebraska Medical Center, Omaha, NE 68198

Received for publication January 9, 2009. Accepted for publication August 26, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the National Institute of Neurological Disorders andStroke, National Institutes of Health Grant R01 NS055385 (to T.K.) and the NationalInstitute of Neurological Disorders and Stroke supported Core Facility at UAMS (P30NS047546). S.L. was the recipient of a Graduate Student Research Funds award fromthe University of Arkansas for Medical Sciences Graduate School.2 Address correspondence and reprint requests to Dr. Tammy Kielian, University ofNebraska Medical Center, Department of Pathology and Microbiology, 985900 Ne-braska Medical Center, Omaha, NE 68198-5900. E-mail address: tkielian@unmc.edu

3 Abbreviations used in this paper: PRR, pattern recognition receptor; CSF, cerebro-spinal fluid; GPA, gentamicin protection assay; KO, knockout; MOI, multiplicity ofinfection; WT, wild type.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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cells (27). Since C. koseri is a Gram-negative pathogen, wepredicted that this organism would trigger TLR4-dependentpathways; however, a role for MyD88-dependent vs -indepen-dent signaling was uncertain. Because microglial responses toC. koseri, let alone the involvement of TLR4 and MyD88 inbacterial recognition has never been reported, one objective ofthe current study was to evaluate the functional importance ofthese molecules using primary microglia isolated from MyD88knockout (KO) animals in addition to mice that possess a pointmutation in the cytoplasmic tail of TLR4 (hereafter referred toas TLR4 mutant), resulting in its inability to signal.

Phagocytosis refers to the process whereby particulate mate-rial is engulfed by phagocytic cells via opsonic or nonopsonicpathways. Phagocytes, such as macrophages and neutrophils,utilize this mechanism to internalize and kill microbes via theaction of reactive oxygen and nitrogen intermediates and deg-radative enzymes (28, 29). However, many pathogens, such asMycobacterium tuberculosis (30) and Leishmania species (31),have evolved strategies to evade microbial killing mechanismsand survive intracellularly in phagocytes. It has been proposedthat the infiltration of Citrobacter-harboring monocytes/macro-phages into the brain ventricles and periventricular parenchymais important in the pathogenesis of brain abscess formation bythis organism (10, 14). Ancillary evidence to support this pos-sibility was provided in studies where C. koseri intracellularsurvival and replication was demonstrated in the human mono-cyte cell line U937 (14). Although resident microglia areequipped with numerous phagocytic receptors (16, 32), to date,no studies have examined whether C. koseri is capable of in-tracellular survival and/or replication in this CNS phagocytepopulation. We envision that microglia may harbor viable C.koseri intracellularly and serve as a reservoir for bacterial sur-vival and replication within the brain parenchyma, which maycontribute to disease pathogenesis.

In the current study, we demonstrated that C. koseri is a potentstimulus of microglial activation typified by the synthesis ofnumerous proinflammatory mediators, including NO, TNF-�,IL-1�, CXCL2 (MIP-2), and CCL2 (MCP-1). The induction ofthese mediators by C. koseri was found to be mediated primar-ily via TLR4 and MyD88, because their production was signif-icantly attenuated in primary microglia deficient for either mol-ecule. However, a small residual response to C. koseri was stillobserved in TLR4 mutant and MyD88 KO microglia, suggest-ing a minor contribution for pathways independent of bothmolecules.

Analysis of intracellular survival by gentamicin protection as-says revealed that although C. koseri replicated efficiently in ahuman monocyte cell line (U937), microglia did not support sig-nificant bacterial proliferation. However, C. koseri was capable ofsurviving intracellularly in primary microglia since organismswere not eradicated from cells during the 168 h time course eval-uated in these studies. Collectively, these results suggest that mi-croglia may play a dual role during brain colonization by C. koseri.On the one hand, C. koseri is a potent inducer of numerous proin-flammatory molecules by primary microglia, which likely par-ticipate in the genesis of a protective antibacterial immune re-sponse. Conversely, microglia demonstrate poor intracellularkilling of C. koseri and the persistence of bacteria within mi-croglia may facilitate the spread of infection or maladaptivemicroglial responses. Further studies are needed to addressthese questions that may impact the pathogenesis of these dev-astating infections caused by C. koseri colonization of the CNS.

Materials and MethodsC. koseri clinical isolates and preparation of heat-killedbacteria

The C. koseri isolates used in this study (strains 4036 and 4277) wereprovided by Dr. J. G. Vallejo (Baylor College of Medicine, Houston, TX).Strain 4036 was originally isolated from the cerebrospinal fluid (CSF) of aninfant with meningitis and multiple brain abscesses, whereas strain 4277was recovered from a tracheostomy of a chronically ventilator-dependentchild without signs or symptoms of respiratory infection (12). On the basisof the antibiotic sensitivity profile of both strains (performed by the Clin-ical Microbiology laboratory at Arkansas Children’s Hospital, Little Rock,AR), isolates were propagated in the presence of ampicillin (20 �g/ml; MPBiomedicals) for positive selection of C. koseri. C. koseri glycerol stockswere streaked onto blood agar plates and incubated for 20 h one day beforeinitiating broth cultures. C. koseri strains 4036 and 4277 were propagatedin brain-heart infusion broth with constant agitation (250 rpm) and recov-ered at mid-log-phase by washing twice with ice-cold PBS (pH 7.4). Thenumbers of colony-forming units used for infection were initially estimatedby OD620 readings before microglial infection and subsequently deter-mined by plating out serial dilutions on blood agar plates. To prepareheat-killed C. koseri stocks, bacteria were incubated at 56°C for 1 h withfrequent vortexing. The timing regimen for preparing C. koseri workingstocks was held constant throughout these experiments to avoid potentialconfounds by changes in virulence factor expression that could occur atvarious growth phases.

Primary microglia cultures

Neonatal TLR4 mutant and TLR4 WT (C3H/HeJ and C3H/FeJ, respec-tively, The Jackson Laboratory) as well as MyD88 KO mice (provided byDr. S. Akira, Osaka University, Japan) (33) were used to prepare primarymicroglial cultures as described previously (34, 35). Microglia isolatedfrom C57BL/6 mice were used as wild-type (WT) controls for MyD88 KOcells since the latter had been backcrossed to C57BL/6 mice for a total of10 generations. The C3H/HeJ strain harbors a missense mutation in thethird exon of the TLR4 gene, rendering it incapable of signaling (36).C3H/FeJ mice are on the same genetic background as the TLR4 mutantstrain and are used as WT controls for comparison. The purity of microglialcultures in these studies was routinely �98%.

Collection of thioglycollate-elicited peritoneal macrophages andU937 monocyte cell line maintenance

C57BL/6 mice (5–7 wk old) were used to procure thioglycollate-elicitedperitoneal macrophages. Briefly, each mouse received an i.p. injection of2–3 ml of sterile 4% thioglycollate broth (Brewer’s modified medium;Baltimore Biological Laboratory). On day 4 following thioglycollate in-jections, mice were euthanized using an overdose of inhaled isoflurane, andperitoneal macrophages were harvested by vigorous lavage of the perito-neal cavity with 10 ml of ice-cold sterile PBS. When necessary, any con-taminating RBC were eliminated by hypotonic lysis (BD PharmLyse) ac-cording to the manufacturer’s instructions. Elicited macrophages werewashed extensively, resuspended in RPMI 1640 medium (Mediatech), sup-plemented with 10% FBS (HyClone), and seeded into 24-well tissue cul-ture plates at 6 � 105 cells/well for gentamicin protection assays.

The human monocyte cell line U937 was provided by Dr. S. Barger(University of Arkansas for Medical Sciences, Little Rock, AR). Cells werecultivated in RPMI 1640 medium (Mediatech) supplemented with 2 mML-glutamine, 1 mM sodium pyruvate, and 10% FBS. U937 cells wereseeded in 24-well plates at 5 � 105 cells/well in medium containing 0.1�g/ml PMA (Sigma-Aldrich) for 1 day before gentamicin protection as-says, to induce cell adherence and maturation. Incubation of U937 cellswith PMA was discontinued 24 h before the initiation of gentamicin pro-tection assays to avoid any potential activation artifacts.

ELISA

Quantitation of TNF-� and IL-1� (mouse CytoSet; BioSource Interna-tional), CXCL2 (mouse DuoSet; R&D Systems), and CCL2 (mouseOptEIA; BD Biosciences) levels in conditioned supernatants from micro-glia treated with C. koseri strains was performed using standard sandwichELISA according to the manufacturer’s instructions. The lower limit ofsensitivity for these assays was 15.6 pg/ml.

Nitrite assay

Levels of nitrite, a stable end product of NO following its reaction with O2,were quantitated in conditioned supernatants of microglia treated with

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C. koseri using the Griess reagent (0.1% naphtyletylenediamine dihydro-chloride, 1% sulfanilamide, and 2.5% phosphoric acid; all from Sigma-Aldrich). The absorbance at 550 nm was measured on a plate reader (Spec-tra Max 190; Molecular Devices), and nitrite concentrations weredetermined using a standard curve with sodium nitrite (NaNO2; Sigma-Aldrich; level of sensitivity 0.4 �M).

Cell viability assays

The effect of C. koseri on microglial viability was evaluated by the abilityof mitochondria to convert the substrate (MTT) into formazan crystals asdescribed previously (37).

Gentamicin protection assay (GPA)

To evaluate the ability of C. koseri clinical isolates to survive and/or rep-licate in microglia or macrophages, GPAs were performed. Gentamicinkills extracellular C. koseri, but because its ability to permeate the eukary-otic cell membrane is limited, intracellular organisms are protected from itsbactericidal activity. Primary microglia and thioglycollate-elicited perito-neal macrophages from C57BL/6 mice and the human monocyte cell lineU937 were plated into 24-well tissue culture plates in antibiotic-free me-dium for 24 h. Subsequently, cells were infected with live C. koseri strainsat a multiplicity of infection (MOI) of 10 (10 bacteria to 1 microglia/macrophage) for 1 h at 37°C. After the 1-h incubation, all wells werewashed extensively with PBS to remove extracellular organisms and in-cubated with medium supplemented with 100 �g/ml gentamicin for oneadditional hour at 37°C. Finally, all wells were washed twice and receivedfresh medium supplemented with 20 �g/ml gentamicin (exceeding theminimal inhibitory concentration for both C. koseri strains; data not shown)and incubated at 37°C until the appropriate time point for analysis. Thecontinued presence of gentamicin ensured effective killing of any residualextracellular bacteria. Microglia, macrophages, and U937 monocytes werelysed anywhere from 24 to 168 h after C. koseri infection with PBS con-taining 0.5% Triton X-100 (Fisher Scientific), whereupon cell lysates wereserially diluted and plated onto blood agar plates to quantitate the numbersof viable intracellular bacteria. In some experiments, microglia and mac-rophages were pretreated with LPS (Escherichia coli O111:B4; List Bio-logical Laboratories) for 6 h before live C. koseri exposure to determinewhether the activation status of cells would impact bacterial survival and/orreplication. Results are presented as the number of colony-forming unitsper well after normalization to the actual MOI of the infectious inoculumas determined on blood agar plates. This was required because OD620 read-ings can only provide an estimate of bacterial density on the day of infec-tion. The number of viable bacteria in wells devoid of microglia or mac-rophages served as a control to confirm adequate killing of extracellular C.koseri by gentamicin. All assays were performed in triplicate with resultsrepeated in a minimum of four independent experiments.

Protein extraction and Western blotting

Protein extracts were prepared from primary microglia as previously described(38) and quantified using a standard protein assay (bicinchoninic acid proteinassay reagent; Bio-Rad). CD14 and MyD88 expression was evaluated byWestern blot using rat anti-mouse CD14 (BD Biosciences) and rabbit anti-mouse MyD88 (eBioscience) Abs. Blots were developed using the ImmobilonWestern substrate (Millipore) and visualized using an Alpha Innotech imager(Alpha Innotech). Blots were stripped and reprobed with a rabbit anti-actinpolyclonal Ab (Sigma-Aldrich) for verification of equivalent protein loading.

Statistics

Significant differences across experimental groups were determined with aone way ANOVA followed by the Holm-Sidak method for pairwise mul-tiple comparisons using SigmaStat 3.0 (SPSS Science).

ResultsPrimary microglia are capable of recognizing theGram-negative pathogen C. koseri and respond withrobust proinflammatory mediator release

Microglia are the resident innate immune cells in the brain paren-chyma (16) and produce numerous proinflammatory mediators uponexposure to bacterial pathogens, including but not limited to NO,TNF-�, IL-1�, CXCL2, and CCL2 (18, 39–44). Many of these proin-flammatory molecules are important for inducing the influx and sub-sequent activation of peripheral immune cells into the infected CNS(45–47). Despite the fact that C. koseri is capable of colonizing the

brain as evident by its propensity to establish meningitis and multi-focal brain abscesses (7, 8), the effector responses of primary micro-glia to this pathogen have not yet been described. Both C. kosericlinical isolates evaluated in this study induced microglial activationin a dose-dependent manner typified by NO, IL-1�, TNF-�, CXCL2,and CCL2 production within 24 h following bacterial exposure (Fig.1). There were no significant differences between the stimulatory ef-fects of the two C. koseri strains examined despite the divergent sitesfrom which these organisms were originally recovered (CSF and tra-chea). Cell viability assays revealed that these C. koseri isolates werenot toxic to primary microglia at any of the doses examined (Fig. 1F).Similar effects were observed with live C. koseri strains except thetime course of microglial activation was accelerated and could not beexamined past 6 h following C. koseri exposure due to overwhelmingbacterial replication (Fig. 2).

Since C. koseri is a Gram-negative pathogen, we next examinedthe effects of bacterial stimulation on the expression of CD14 andMyD88, two molecules known to play a pivotal role in transducingactivation signals in response to LPS (48). We also attempted toexamine microglial TLR4 expression; however, we were unable tosuccessfully detect TLR4 by Western blotting with any of the com-mercially available Abs tested. Low levels of CD14 were detectedon unstimulated microglia; however, following treatment with ei-ther C. koseri strain receptor expression increased dramatically(Fig. 3A). In contrast, little change in MyD88, a central adaptormolecule for TLR signaling, was observed in response to bacterialtreatment (Fig. 3B). These results demonstrate that microglia ex-press a repertoire of molecules involved in mediating cell activa-tion in response to Gram-negative pathogens.

TLR4-mediated signal(s) are pivotal for C. koseri-inducedmicroglial activation

Since C. koseri is a Gram-negative organism, it was envisionedthat its LPS motifs would trigger microglial activation via TLR4.However, despite the tropism of this pathogen for the CNS paren-chyma, no studies to date have examined the importance of TLRsignals in eliciting microglial activation in response to C. koseri. Itwas also possible that alternative bacterial moieties, such as li-poproteins, flagellin, and bacterial DNA, may also contribute tomicroglial activation via engagement of TLR2 (49), TLR5 (50),and TLR9 (51), respectively. To assess the functional importanceof TLR4 in eliciting inflammatory mediator release in response toC. koseri, we compared microglia isolated from TLR4 mutant andWT mice. The production of several molecules, including NO,IL-1�, TNF-�, CXCL2, and CCL2 was significantly attenuated inTLR4 mutant microglia compared with WT cells in response toheat-inactivated C. koseri (Fig. 4). Despite a pivotal role for TLR4in C. koseri-dependent microglial activation, TLR4-independenteffects were also evident since low but significant levels of inflam-matory mediator production were still detected in TLR4 mutantmicroglia. The reduction in proinflammatory mediator expressionin TLR4 mutant microglia following C. koseri exposure was notthe result of cytotoxic effects or differences in cell density (Fig.4F). Since the production of virulence factors released by viablebacteria may conceivably affect microglial activation via TLR4-dependent pathways, we also examined responses to viable C. ko-seri strains. The results still demonstrated a pivotal role for TLR4-dependent signaling in microglial responses to live C. koseri (Fig.5). Collectively, these data suggest that TLR4-mediated path-way(s) are pivotal for C. koseri-dependent microglial activationand the generation of a robust proinflammatory response. How-ever, the residual activation detected in TLR4 mutant microgliasuggests that alternative receptors besides TLR4 are required toachieve maximal microglial activation.

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C. koseri elicits microglial activation primarily via aMyD88-dependent pathway

MyD88 is the central adaptor molecule involved in the majority ofTLR signal transduction pathways as well as the IL-1R and

IL-18R. TLR4 engagement by LPS facilitates the activation ofboth MyD88-dependent and MyD88-independent signalingpathways, leading to the expression of several gene products,including inflammatory cytokines and chemokines (21). The

FIGURE 1. Microglia produce proinflammatory mediators in response to heat-killed C. koseri strains in a dose-dependent manner. Primary microgliawere plated at 2 � 105 cells/well in 96-well plates and exposed to heat-killed C. koseri strains at 104 –107 per well for 24 h. NO (A), IL-1� (B), TNF-�(C), CXCL2 (D), and CCL2 (E) release was quantitated by nitrite assay (A) and ELISA (B–E). Microglial viability was assessed using a MTT assay andthe raw OD570 absorbance values are reported (F). Significant differences in inflammatory mediator release between unstimulated and C. koseri-treatedmicroglia are denoted with asterisks (�, p � 0.05; ��, p � 0.001). Similar results were obtained in four independent experiments.

FIGURE 2. Live C. koseri is a potent inducer of microglial activation. Primary microglia were plated at 2 � 105 cells/well in 96-well plates and exposedto live C. koseri strains at the indicated MOI for 6 h. IL-1� (A), TNF-� (B), CXCL2 (C), and CCL2 (D) release was quantitated by ELISA. Microglialviability was assessed using a MTT assay, and the raw OD570 absorbance values are reported (E). Significant differences in inflammatory mediator releasebetween unstimulated and C. koseri-treated microglia are denoted with asterisks (�, p � 0.05; ��, p � 0.001). Similar results were obtained in fourindependent experiments.

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relative importance of MyD88-dependent vs -independent mech-anisms in microglial responses to C. koseri has not yet been ex-amined and could reveal interesting insights into the role of alter-native pathways of microglial activation. The expression ofnumerous proinflammatory mediators, including NO, IL-1�,TNF-�, CXCL2, and CCL2, was significantly attenuated inMyD88 KO microglia following C. koseri exposure comparedwith WT cells (Fig. 6). Despite the pivotal role of MyD88-dependentsignals in regulating inflammatory mediator release, residual produc-tion of these molecules was still detected in MyD88 KO microglia,suggesting that MyD88-independent mechanisms also contribute toC. koseri recognition and cytokine signaling in microglia. The inabil-ity of MyD88 KO microglia to respond to C. koseri was not attrib-utable to differences in cell number or bacterial cytotoxicity (Fig. 6F).Similar responses were obtained with MyD88 KO microglia in re-

sponse to live C. koseri (Fig. 7). The fact that C. koseri-mediatedmicroglial activation was more attenuated with the loss of MyD88-compared with TLR4-dependent signaling implicates additionalMyD88-driven pathways for inducing maximal responses to thisGram-negative pathogen.

Intracellular survival of C. koseri in primary microglia andmacrophages

Several findings have suggested that intracellular survival of C. koseriin macrophages may play a role in primary colonization and pathogendissemination in the CNS. First, macrophages harboring C. koseriwere found infiltrating the periventricular parenchyma in an infant ratmodel of infection (10). Second, intracellular survival and replicationof C. koseri has been demonstrated in the human monocyte cell lineU937 (14). However, despite its tropism for the CNS parenchyma, nostudies have examined whether C. koseri can persist in primary mi-croglia or macrophages, or whether these cells types are permissivefor intracellular replication of bacteria. To examine these possibilities,we performed gentamicin protection assays using primary mouse mi-croglia and macrophages. Although neither cell type was capable ofsupporting C. koseri replication over the initial 72-h interval exam-ined, microglia and macrophages did provide a permissive environ-ment for bacterial survival as evident by the failure to completelyclear viable intracellular organisms (Fig. 8, A and B). Although aslight increase in intracellular burdens was observed over time withstrain 4277 in microglia, these differences did not achieve statisticalsignificance (Fig. 8A). The number of surviving extracellular bacteriain the absence of microglia or macrophages was negligible, indicatingeffective killing of extracellular organisms by gentamicin (data notshown). Interestingly, the C. koseri isolate recovered from the CSF(strain 4036) demonstrated enhanced intracellular survival comparedwith the tracheal pathogen (strain 4277). The human U937 monocyte

FIGURE 3. Effect of C. koseri strains on CD14 and MyD88 expression inmicroglia. Primary microglia were plated in 6-well plates at 4 � 106 cells/wellovernight and the following day exposed to 107 of either C. koseri strain 4036or 4277. Whole-cell lysates were prepared 24 h following bacterial stimula-tion, whereupon CD14 (A) and MyD88 (B) expression were evaluated byWestern blotting. Subsequently, the same membrane was stripped and re-probed with a �-actin Ab to demonstrate uniformity in gel loading.Similar results were obtained in three independent experiments.

FIGURE 4. C. koseri-induced microglial activation is partially TLR4-dependent. TLR4 mutant or WT microglia were plated at 2 � 105 cells/well in96-well plates and exposed to heat-killed C. koseri strains at 107 per well for 24 h. NO (A), IL-1� (B), TNF-� (C), CXCL2 (D), and CCL2 (E) levels werequantitated by nitrite assay (A) and ELISAs (B–E). Microglial viability was assessed using a MTT assay, and the raw OD570 absorbance values are reported(F). Significant differences in inflammatory mediator release between unstimulated and C. koseri-treated microglia are denoted with asterisks (�, p � 0.05;��, p � 0.001), whereas changes between C. koseri treated TLR4 mutant vs WT microglia are indicated by a hatched sign (##, p � 0.001). Similar resultswere obtained in three independent experiments.

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cell line supported intracellular replication of both C. koseri isolates,in agreement with previous reports (Fig. 8C) (14, 52).

In the setting of C. koseri CNS infection, resident microglia andmacrophages likely encounter a complex milieu of molecules, both

host- and bacterial-derived, that may subsequently modify theirantibacterial responses. For example, cell wall components areshed during normal bacterial growth and as such, LPS would beavailable to exert its proinflammatory effects that could feed back

FIGURE 5. Microglial activation in response to live C. koseri is partially mediated by TLR4. TLR4 mutant or WT microglia were plated at 2 � 105 cells/wellin 96-well plates and exposed to live C. koseri strains at the indicated MOI for 6 h. IL-1� (A), TNF-� (B), CXCL2 (C), and CCL2 (D) release was quantitatedby ELISA. Microglial viability was assessed using a MTT assay, and the raw OD570 absorbance values are reported (E). Significant differences in inflammatorymediator release between unstimulated and C. koseri-treated microglia are denoted with asterisks (�, p � 0.05; ��, p � 0.001), whereas changes between C.koseri-treated TLR4 mutant vs WT microglia are indicated by a hatched sign (#, p � 0.05; ##, p � 0.001). Similar results were obtained in two independent experiments.

FIGURE 6. C. koseri stimulates microglial production of proinflammatory mediators primarily via a MyD88-dependent pathway. Primary microglia recoveredfrom MyD88 WT or KO mice were plated at 2 � 105 cells/well in a 96-well plate and exposed to heat-killed C. koseri strains at 107 per well for 24 h. The levelsof NO (A), IL-1� (B), TNF-� (C), CXCL2 (D), and CCL2 (E) produced by microglia were quantitated by nitrite assay (A) and ELISA (B–E). Microglial viabilitywas assessed using a MTT assay, and the raw OD570 absorbance values are reported (F). Significant differences in inflammatory mediator release betweenunstimulated and C. koseri-treated microglia are denoted with asterisks (�, p � 0.05; ��, p � 0.001), whereas changes between C. koseri-treated MyD88 KO vsWT microglia are indicated by a hatched sign (##, p � 0.001). Similar results were obtained in three independent experiments.

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in an autocrine/paracrine manner to influence the ability of micro-glia/macrophages to regulate bacterial growth and/or survival. Toevaluate this possibility, primary microglia, macrophages, and theU937 cell line were treated with 100 ng/ml LPS 6 h before theaddition of live C. koseri strains for GPA. This LPS dose andtiming regimen is sufficient to elicit detectable proinflammatorymediator release (i.e., IL-1�, CXCL2) from cells. Unexpectedly,the intracellular survival and persistence of strain 4277 (trachealisolate) was augmented in all three cell types over the 72-h intervalexamined (Fig. 8, C–E). Interestingly, with LPS pretreatment, theintracellular survival of strain 4036 (CSF isolate) was decreased inmacrophages (Fig. 8E) but enhanced in microglia (Fig. 8D). Thesefindings suggest that activated macrophages are more efficient thanmicroglia in killing C. koseri strain 4036 upon phagocytosis.

To more fully define the duration of C. koseri intracellular sur-vival in microglia, we extended our analysis out to 168 h postin-fection to determine whether intracellular bacteria persist or de-cline over time. Interestingly, although the number of viableintracellular C. koseri eventually declined over the first 120 h fol-lowing infection, bacterial burdens showed an upward trend at thelatest time point examined (i.e., 168 h), which occurred with bothC. koseri strains tested (Fig. 9). We also investigated whether theeffect of LPS pre-treatment on intracellular C. koseri survival inmicroglia was dose-dependent. In general, we were unable to dem-onstrate any differential effects of LPS dose on the numbers ofintracellular bacteria (Fig. 9). However, a 6 h LPS pretreatmentperiod, regardless of dose, led to a dramatic increase in the numberof phagocytized bacteria compared with non-stimulated microglia.This disparity in intracellular bacteria between LPS pretreated andnontreated microglia persisted throughout the 168 h time intervalexamined (Fig. 9).

We next examined the extent of proinflammatory mediator ex-pression by microglia during intracellular C. koseri infection. Thelevels of CXCL2 and CCL2 were highest at 24 h following bac-terial exposure, decreased dramatically from 48 to 120 h, and be-gan to rise at 168 h following infection (Fig. 10, C and D, respec-tively), which coincided with the upward trend in viableintracellular bacteria (Fig. 9). In contrast to chemokine production,IL-1� and TNF-� were only detected during the first 72 h follow-ing C. koseri exposure and decreased dramatically in a time-de-pendent manner (Fig. 10, A and B, respectively). Overall, theseresults suggest that microglia may serve as a reservoir for C. koserisurvival/replication for at least a 1-week period and that continuedchemokine production may be triggered by an as of yet unidenti-fied intracellular PRR.

DiscussionAmong the Gram-negative enteric bacilli responsible for menin-gitis in neonates, C. koseri is an infrequent but formidable etio-logical agent based on its propensity to disseminate and establishmultifocal brain abscesses (7, 8). Several studies have focused onthe pathogenesis of this devastating disease using an infant ratmodel of C. koseri infection. However, despite the tropism of C.koseri for the CNS, no studies to date have examined microglialresponsiveness to this pathogen or the receptor signaling pathwaysleading to cell activation and subsequent proinflammatory media-tor release. In the current report we demonstrate that TLR4- andMyD88-dependent pathways are critical for microglial activationin response to C. koseri and that microglia can serve as an intra-cellular reservoir for bacterial survival.

TLRs are type I integral membrane glycoproteins that facilitatepathogen recognition of motifs conserved across broad classes of

FIGURE 7. MyD88-dependent signaling is pivotal for microglial responses to live C. koseri. Primary microglia recovered from MyD88 WT or KO micewere plated at 2 � 105 cells/well in 96-well plates and exposed to live C. koseri strains at the indicated MOI for 6 h. IL-1� (A), TNF-� (B), CXCL2 (C),and CCL2 (D) release was quantitated by ELISA. Microglial viability was assessed using a MTT assay, and the raw OD570 absorbance values are reported(E). Significant differences in inflammatory mediator release between unstimulated and C. koseri-treated microglia are denoted with asterisks (�, p � 0.05;��, p � 0.001), whereas changes between C. koseri-treated MyD88 KO vs WT microglia are indicated by a hatched sign (##, p � 0.001). Similar resultswere obtained in two independent experiments.

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microorganisms. Pertinent to C. koseri, we were interested in ex-amining the functional importance of TLR4 based on its well-described role in LPS recognition. Interestingly, although micro-glia utilized TLR4 signaling to elicit the production of numerousproinflammatory mediators, including NO, IL-1�, TNF-�,CXCL2, and CCL2, evidence for a TLR4-independent recognitionpathway was also apparent based on the residual expression ofthese molecules by TLR4 mutant microglia. One explanation thatmay account for this finding is the triggering of alternative TLR byantigenic motifs contained within C. koseri including lipoproteins,

flagellin, and bacterial DNA that could engage TLR2, TLR5, andTLR9, respectively. Indeed, recent studies have identified TLR2 asan important TLR4-driven sensor of Gram-negative bacterial in-fection both in the CNS and the periphery (53, 54), and an earlierstudy has revealed an important role for the C. koseri flagellarmolecule fliP in regulating IL-10 production and virulence (15).This scenario does appear plausible because the residual activationobserved in MyD88 KO microglia in response to C. koseri wasbarely detectable compared with the significant levels of inflam-matory mediators still produced by TLR4 mutant microglia. The

FIGURE 8. C. koseri survives intracellularly in primary microglia and macrophages. Primary microglia (A and D), macrophages (B and E), and the U937monocyte cell line (C and F) were exposed to live C. koseri strains at a MOI of 10 and intracellular survival was determined by gentamicin protection assaysas described in Materials and Methods. D–F, Cells were pretreated with 100 ng/ml LPS for 6 h before C. koseri infection to determine the effects of cellactivation on bacterial persistence, whereas A–C did not receive LPS pretreatment. Cell lysates were prepared at the indicated time points postinfection,whereupon the number of viable bacteria was determined by quantitative culture (mean cfu � SD). Significant differences between the number of viableorganisms recovered from cells at the various time points following infection are denoted with asterisks (�, p � 0.05; ��, p � 0.01; ANOVA). Similarresults were obtained in three independent experiments.

FIGURE 9. Viable C. koseri persist in microglia for extended time intervals. Primary microglia were exposed to live C. koseri strains 4036 (A) or 4277(B) at a MOI of 10 and intracellular survival was determined by gentamicin protection assays as described in Materials and Methods. A subset of microgliawere pretreated with either 1 or 100 ng/ml LPS (E and Œ, respectively) for 6 h before C. koseri exposure to determine the effects of cell activation onbacterial persistence, whereas some microglia did not receive LPS pretreatment (F). Cell lysates were prepared at the indicated time points postinfection,whereupon the number of viable bacteria was determined by quantitative culture. Scatter plots representing the values from three independent samples areshown for each experimental treatment. Similar results were obtained in three independent experiments.

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low levels of inflammatory mediator production remaining inMyD88 KO microglia in response to C. koseri are similar to aprevious study by our laboratory demonstrating residual responsesto LPS (55). A recent report (56) provides added evidence to sup-port our findings, where LPS stimulation was found to induce sev-eral chemokine and cytokine genes such as CCL3, CCL4, CXCL2,and TNF-� in MyD88-deficient cells as evaluated by microarrayanalysis. This report demonstrated that the TIR domain-containingadaptor inducing IFN-�-dependent pathway activated TNF-� pro-duction and secretion via a NF-�B-independent manner and sug-gested that secreted TNF-� acts in an autocrine manner to inducedelayed NF-�B activation. Since depletion of IRF-3 was found toimpair NF-�B activation, IRF-3 has been implicated as a mediatorof TNF-� activation via a MyD88-independent pathway. A morerecent study (57) has demonstrated an alternative pathway ofTLR4 signaling in response to Gram-negative bacteria in humanbladder epithelial cells involving cAMP. However, it is uncertainwhether a similar mechanism exists in microglia, making this pos-sibility highly speculative.

An alternative explanation to account for the residual proinflam-matory mediator production in TLR4 mutant and MyD88 KO mi-croglia is that C. koseri also engages additional PRR to trigger cellactivation. This possibility is not unexpected given the essentialnature of an effective CNS antibacterial response to contain infec-tion and the fact that natural polymorphisms in TLR4 have beenreported in the human population and are proposed to result inaberrant cytokine responses and increased susceptibility to Gram-negative infections (58). Some candidates include members of theintracellular NOD-like receptor family of proteins, scavenger re-ceptors, and C-type lectin receptors, but these wait testing in futurestudies to assess their contributions toward microglial cytokinesignaling in response to C. koseri. The former appears to representa viable possibility based on our findings that chemokine produc-tion by microglia harboring intracellular C. koseri began to in-

crease at 168 h following bacterial exposure, which coincided withthe amplification of viable intracellular bacteria.

During CNS infections with C. koseri, macrophages appear toharbor a significant number of intracellular bacteria, suggestingthat they may be exploited by the organism for intracellular rep-lication and/or survival. Since microglia may also serve as an ob-vious target during C. koseri infection in the brain, we wanted toexamine whether these cells would enable bacterial propagation orexert antimicrobial activity. We were able to confirm earlier find-ings by Townsend et al. (14) where the human monocyte cell lineU937 was capable of supporting the intracellular replication of C.koseri. However, C. koseri did not multiply to a considerable ex-tent in primary microglia during the initial 72 h following bac-terial exposure, although the organism was capable of survivalas evidenced by the persistence of viable intracellular bacteria.We next extended the time intervals following microglial C.koseri exposure out to 168 h to ascertain whether intracellularbacteria were eventually cleared or persisted. Interestingly, in-tracellular C. koseri burdens declined between 72 and 120 hafter bacterial exposure, whereupon a rebound in viable bacteriawas detected suggestive of ongoing replication. The reason forthis delayed increase in intracellular C. koseri is uncertain butmay be influenced by the lengthy infection period where max-imal microglial antibacterial responses may have begun towane. Importantly, this organism is capable of surviving withinthe phagolysosome (14), which is an important virulence de-terminant for other intracellular pathogens such as Leishmania(59) and Coxiella (60).

Next, we were interested in determining whether the intracel-lular persistence of C. koseri was unique to microglia by com-paring responses with primary macrophages. Similar resultswere obtained with macrophages, suggesting that C. koseri doesnot co-opt either cell type to replicate to a significant extent;however, our results do indicate that the organism is capable of

FIGURE 10. Persistence of intra-cellular C. koseri is associated withproinflammatory mediator release bymicroglia. Primary microglia wereexposed to live C. koseri strains at aMOI of 10 for gentamicin protectionassays, whereupon conditioned super-natants were collected at the indicatedintervals following bacterial exposurefor quantitation of IL-1� (A), TNF-�(B), CXCL2 (C), and CCL2 (D) byELISA. A subset of microglia werepre-treated with either 1 or 100 ng/mlLPS for 6 h as described in Fig. 9,whereas some microglia did not re-ceive LPS pretreatment. Similar re-sults were obtained in three indepen-dent experiments.

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thwarting antibacterial effector responses to prolong intracellu-lar survival. Currently, the reason(s) to account for the prefer-ential replication of C. koseri in U937 monocytes but not mi-croglia or macrophages are not known; however, severalexplanations can be considered. Differences in C. koseri iso-lates, immortalized cell lines vs primary cells, species of origin,or maturational state of the target cell may all impact whethermicroglia, macrophages, or monocytes can support C. koseriintracellular replication. For example, compared with more ter-minally differentiated cell types (i.e., microglia and macro-phages) monocytes (as represented by U937 cells) signify amore immature state and possibly exert less antimicrobial ac-tivity, which might make them more permissive for intracellularbacterial replication. This is supported by earlier studies dem-onstrating that overexpression of inducible NO synthase re-sulted in enhanced bactericidal activity to Leishmania and Bru-cella in U937 monocytes (52, 61). Although microglial andmacrophage antibacterial responses to C. koseri are not highlyeffective, as demonstrated by the failure to clear intracellularburdens by 168- and 72-h postinfection, respectively, their re-sponse is sufficient to limit bacterial replication. With regard topathology, it is possible that during C. koseri sepsis, monocytesharboring intracellular bacteria serve as a “Trojan horse” tointroduce organisms into the CSF and periventricular paren-chyma following CSF extravasation. Once CNS infection is es-tablished, resident microglia and newly recruited macrophagescan internalize C. koseri, but antimicrobial killing mechanismsare ineffective, permitting bacterial persistence. Therefore, anendogenous reservoir for C. koseri may be present within theCNS to facilitate the dissemination and persistence of infection.

One intriguing finding that surfaced during the course of thesestudies relates to the differential effects of cell activation on theability of C. koseri to survive intracellularly in microglia vs mac-rophages. Specifically, preactivation of macrophages with LPS ledto a more robust killing of the C. koseri CSF isolate, whereas thisorganism persisted in activated microglia. In contrast, the trachealC. koseri strain was capable of intracellular replication in LPSactivated microglia, whereas the bacteria survived in macrophages.Overall, it appeared that LPS treatment resulted in enhanced bac-terial clearance/containment in macrophages compared with mi-croglia, which is in agreement with the more potent bactericidalactivity of the former. Importantly, the dose of LPS used to treatmicroglia/macrophages before C. koseri exposure (100 ng/ml) wellexceeds that reported to induce LPS hyporesponsiveness or toler-ance (62), suggesting that a tolerogenic mechanism is unlikely tobe involved in the differential abilities of microglia and macro-phages to respond to live C. koseri. This is supported by the factthat similar responses were observed with a lower concentration ofLPS (i.e., 1 ng/ml). A final note that should be acknowledged isthat we use the term “persistence” to reflect a relatively consistentnumber of intracellular bacteria over time. However, in reality thisfinding could also represent a net balance between bacterial killingand de novo replication, a possibility that was not examined in thecurrent study.

C. koseri was a potent stimulus of microglial activation as typ-ified by the ability of both live and heat-killed organisms to inducethe expression of numerous inflammatory mediators, includingNO, TNF-�, IL-1�, CXCL2, and CCL2. With regard to the po-tential roles of these factors in disease pathogenesis, NO is a potentantibacterial mediator that induces DNA damage, lipid peroxida-tion, and protein nitrosylation. The importance of NO scavengingfrom the bacterial perspective is apparent from the fact that manybacterial species encode for NO metabolizing enzymes (63, 64).TNF-� and IL-1� are important for activating the vascular endo-

thelium of the blood-brain barrier as well as inducing the expres-sion of antibacterial effector molecules such as NO and TLRs (35).Both CXCL2 and CCL2 are chemokines that are important forneutrophil and monocyte/macrophage/lymphocyte recruitment, re-spectively. Since C. koseri infection in the brain is typified by thepresence of all these immune cell populations, it is likely thatchemokines produced by activated microglia participate in cell re-cruitment into the infected CNS. By virtue of its ability to inducemonocyte migration, CCL2 production by microglia could inad-vertently enhance C. koseri colonization to the brain by recruitingadditional cells harboring viable intracellular bacteria. Interest-ingly, both CXCL2 and CCL2 production were increased in mi-croglia as the number of intracellular C. koseri began to increaseslightly at 168 h following bacterial exposure, suggesting that che-mokine induction may serve as a means to elicit additional mono-cytes/macrophages to continue a productive CNS infection.

In summary, the results presented in this study demonstrate thatTLR4- and MyD88-dependent pathways play an essential role insignaling proinflammatory mediator release by microglia in re-sponse to C. koseri. The ability of bacteria to survive within mi-croglia and infiltrating macrophages could represent a pathogenicmechanism to ensure C. koseri dissemination and persistence inthe CNS.

AcknowledgmentsWe thank Dr. Shizuo Akira for providing the MyD88 KO mice and GailWagoner and Teresa Fritz for maintaining the TLR4 mutant and MyD88KO mouse colonies.

DisclosuresThe authors have no financial conflict of interest.

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