The NLRP3 Inflammasome Contributes to Brain Injury in Pneumococcal Meningitis and Is Activated...

The Journal of Immunology

The NLRP3 Inflammasome Contributes to Brain Injury inPneumococcal Meningitis and Is Activated throughATP-Dependent Lysosomal Cathepsin B Release

Tobias Hoegen,*,1 Nadin Tremel,*,1 Matthias Klein,* Barbara Angele,* Hermann Wagner,†

Carsten Kirschning,‡ Hans-Walter Pfister,* Adriano Fontana,x Sven Hammerschmidt,{

and Uwe Koedel*

Streptococcus pneumoniae meningitis causes brain damage through inflammation-related pathways whose identity and mecha-

nisms of action are yet unclear. We previously identified caspase-1, which activates precursor IL-1 type cytokines, as a central

mediator of inflammation in pneumococcal meningitis. In this study, we demonstrate that lack of the inflammasome components

ASC or NLRP3 that are centrally involved in caspase-1 activation decreases scores of clinical and histological disease severity as

well as brain inflammation in murine pneumococcal meningitis. Using specific inhibitors (anakinra and rIL-18–binding protein),

we further show that ASC- and NLRP3-dependent pathologic alterations are solely related to secretion of both IL-1b and IL-18.

Moreover, using differentiated human THP-1 cells, we demonstrate that the pneumococcal pore-forming toxin pneumolysin is

a key inducer of IL-1b expression and inflammasome activation upon pneumococcal challenge. The latter depends on the release

of ATP, lysosomal destabilization (but not disruption), and cathepsin B activation. The in vivo importance of this pathway is

supported by our observation that the lack of pneumolysin and cathepsin B inhibition is associated with a better clinical course

and less brain inflammation in murine pneumococcal meningitis. Collectively, our study indicates a central role of the NLRP3

inflammasome in the pathology of pneumococcal meningitis. Thus, interference with inflammasome activation might be a prom-

ising target for adjunctive therapy of this disease. The Journal of Immunology, 2011, 187: 5440–5451.

Streptococcus pneumoniae is a leading cause of pneumonia,bacteremia, and meningitis and is responsible for .1.5million deaths each year worldwide. Meningitis has the

worst prognosis of any pneumococcal diseases, with a mortalityrate of 15–30%. Moreover, up to half of the survivors are left withlong-term sequelae (1). The poor prognosis of meningitis is re-lated to weaknesses of the host’s immune system inside the ce-rebrospinal fluid (CSF) space, which includes the absence ofsoluble pattern recognition receptors and the presence of immu-nosuppressive factors such as TGF-b (2). As a consequence, once

having entered the CSF, S. pneumoniae multiplies easily, reachingsimilar high titers as under bacterial culture conditions (3). Asa result of the unrestrained pneumococcal proliferation and au-tolysis, large quantities of subcapsular bacterial components arereleased into the CSF (4). Their presence is recognized by residentimmunocompetent cells by means of surface and intracellularpattern recognition receptors such as TLR2 and TLR4 (5). TLRactivation results in MyD88-dependent production of high levelsof cytokines and chemokines (6), which facilitates the accumulationof large amounts of blood-borne leukocytes (predominantly neu-trophils) inside the CSF. The resultant excessive neutrophilic in-flammation causes collateral damage to the brain, thus contributingsubstantially to the unfavorable outcome of meningitis (7, 8).Among the cytokines that induce and perpetuate meningeal

inflammation are IL-1 cytokine family members. Concentrations of

IL-1b and IL-18 are elevated in CSF samples from patients with

bacterial meningitis. Moreover, CSF IL-1b (but not IL-18) levels

correlate significantly with CSF leukocyte counts and clinical

outcome (9, 10). In rats and rabbits, intracisternal application of

rIL-1b was sufficient to induce meningitis (11, 12) and neutral-

izing Abs directed against IL-1b prevented meningeal inflamma-

tion after intracisternal inoculation with S. pneumoniae (13). In

line with the latter finding, mice lacking IL-1R1 showed less

profound inflammatory infiltrates around the meninges and lower

amounts of brain cytokines and chemokines as compared with

wild-type mice in a model of hematogenous pneumococcal men-

ingitis (14). Consistently, our group demonstrated that mice lack-

ing caspase-1, which activates pro–IL-1 type cytokines, showed a

strongly diminished inflammatory host response to pneumococci

in the CSF (15). Accordingly, Braun et al. (16) reported that the

pan-caspase inhibitor z-VAD-fmk prevented hippocampal injury

*Department of Neurology, Clinic of the University of Munich, D-81377 Munich,Germany; †Institute of Medical Microbiology, Immunology, and Hygiene, TechnicalUniversity of Munich, D-81675 Munich, Germany; ‡Institute for Medical Microbi-ology, University of Essen, D-45122 Essen, Germany; xInstitute for ExperimentalImmunology, University of Zurich, CH-8044 Zurich, Switzerland; and {DepartmentGenetics of Microorganisms, Interfaculty Institute for Genetics and FunctionalGenomics, Ernst Moritz Arndt University, D-17487 Greifswald, Germany

1T.H. and N.T. contributed equally to this work.

Received for publication March 18, 2011. Accepted for publication September 14,2011.

This work was supported by German Research Foundation Grants Pf 246/7-1 (toU.K. and H.-W.P.) and SFB576-TPA5 (to U.K.) and by Else-Kroner-Fresenius StiftungGrant A84/08 (to U.K.). A.F. is the Hertie Senior Research Professor of Neuroscienceof the Gemeinnuetzige Hertie-Stiftung.

Address correspondence and reprint requests to Prof. Uwe Koedel, Clinic Grossha-dern of the University of Munich, Marchioninistrasse 15, D-81377 Munich, Germany.E-mail address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: ASC, apoptosis-associated speck-like protein;BBB, blood–brain barrier; BMDM, bone marrow-derived macrophage; CSF, cere-brospinal fluid; DPI, diphenylene iodonium; ICP, intracranial pressure; LDH, lactatedehydrogenase; ox-ATP, oxidized ATP; rIL-18BP, rIL-18–binding protein; RIP,receptor-interacting protein; ROS, reactive oxygen species; WT, wild-type.

Copyright� 2011 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/11/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100790

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and leukocyte influx into the CSF compartment of rabbits withpneumococcal meningitis. These data of others and us indicate akey role of the IL-1/caspase-1 pathway in pneumococcal menin-gitis. However, the molecular mechanisms through which IL-1 isproduced during pneumococcal meningitis are still not resolved.In general, IL-1b is produced in a two-step process that firstinvolves generation of the biologically inactive precursor pro–IL-1b, typically in response to TLR activation (17, 18). In a secondstep, pro–IL-1b is then cleaved by caspase-1 (or further proteasessuch as neutrophil-derived serine proteases) into an active cytokineand secreted. Activation of caspase-1 is controlled by a large mul-tiprotein complex called the inflammasome. The inflammasomecontains a nucleotide-binding domain and leucine-rich repeat con-taining gene product family receptor (NLR) protein (such asNLRP3) and an adaptor protein called apoptosis-associated speck-like protein (ASC), which links the NLR protein to the proformof caspase-1 (17, 18).In this study, we analyzed inflammation upon pneumococcal

infection by applying a murine meningitis model and differentiatedhuman monocytoid cells, and we identified the NLRP3 inflam-masome as central driver of S. pneumoniae-induced brain pa-thology.

Materials and MethodsEthics statement

This study was carried out in strict accordance with the recommendationsin the Guide for the Care and Use of Laboratory Animals (Institute ofLaboratory Animal Resources, National Research Council) and with theGerman Animal Protection Act. The study protocol was approved by theCommittee on the Ethics of Animal Experiments of the Government ofUpper Bavaria (permit nos. 55.2-1-54-2531-32-04 and 55.2-1-54-2531-47-08). All surgery was performed under ketamine/xylazine anesthesia, andall efforts were made to minimize suffering.

Mouse meningitis model

The model used in this study has been described previously (5, 8). Briefly,mice were weighed and clinically examined. The clinical score usedconsists of: presence of tremor, piloerection, and seizures; spontaneousmotor activity; vigilance; proprioception; a beam balancing test; and apostural reflex test. In healthy animals, the score was 0; infected animalsthat died within the observation period received 13 points. Meningitis wasintroduced by transcutaneous injection of 15 ml bacterial suspensioncontaining 107 (or 108) CFU/ml S. pneumoniae serotype 2 strain D39 (orits isogenic pneumolysin mutant D39DPly) (19) into the cisterna magnaunder short-term anesthesia with halothane. Next, animals were allowed towake up and food and water were supplied ad libitum. At 24 h postin-fection, animals were weighed again, scored clinically, and body temper-ature was taken. Subsequently, mice were anesthetized with 100 mg/kgketamine and 10 mg/kg xylazine and a catheter was placed into the cis-terna magna, and intracranial pressure (ICP) was measured. Thereupon,CSF and blood samples were withdrawn for the determination of CSF andblood leukocyte counts as well as blood bacterial titers. After deep anes-thesia with ketamine, animals were perfused transcardially with 15 ml ice-cold PBS containing 10 U/ml heparin. The brain was removed and frozenimmediately.

Experimental groups

To analyze the role of the NLRP3 inflammasome, ASC-deficient mice(n = 10; ASC2/2; provided by Prof. V.M. Dixit, San Francisco, CA) andNLRP3-deficient mice (n = 10; NLRP32/2; a gift from Prof. J. Tschopp,Lausanne, Switzerland) were infected with pneumococcal strain D39 andcompared with infected wild-type (WT) mice (n = 12; C57BL/6). Becausereceptor-interacting protein (RIP)2 was reported to compete with ASCbinding to caspase-1 (20) and contribute to NOD1 and NOD2 signaling,we also studied mice lacking RIP2 (n = 10; RIP22/2; provided by Prof.V.M. Dixit). For the evaluation of the role of IL-1 family cytokines in theimmunopathogenesis of pneumococcal meningitis, WT mice were treatedeither with the IL-1 receptor antagonist anakinra (n = 6; from Amgen; 100mg/kg given i.p. prior to infection) (21) or with anakinra in combinationwith mouse rIL-18–binding protein (rIL-18BP; n = 6; from Sino Biolog-ical; 5 mg/kg given i.p. prior to infection) (22); controls received 0.5 ml

and 1.0 ml PBS i.p., respectively (n = 6 in each group). Additionally, weevaluated the role of cathepsin B in pneumococci-induced meningeal in-flammation. In these series of experiments, WT mice were treated with5 mg/kg Ca-074Me (diluted in 5% DMSO-containing PBS) (23) or DMSO-PBS (given i.p. immediately before and 6 h postinfection; n = 7 in eachgroup). The influence of pneumolysin on the clinical course was assessedby infecting WT and ASC-deficient mice either with the pneumolysin-deficient strain D39DPly (n = 6 and n = 4, respectively) or the WT D39strain (n = 10 and n = 4, respectively). Finally, the role of granulocytes inpneumococcal-induced inflammation was investigated by rendering miceneutropenic with anti-GR1 Abs prior to infection (n = 6) (8).

Determination of bacterial titers in blood and brain

Cerebella were dissected and homogenized in sterile saline. Blood samplesand cerebellar homogenates were diluted serially in sterile saline, plated onblood agar plates, and cultured for 24 h at 37˚C under 5% CO2.

Analysis of the blood–brain barrier integrity

For the determination of the blood–brain barrier (BBB) integrity, frozenmouse brain extracts were examined for diffusion of albumin using ELISAas described previously (6).

Analysis of cerebral bleeding

Mice brains were cut in a frontal plane into 10-mm-thick sections. Be-ginning from the anterior parts of the lateral ventricles, 10 serial sectionswere photographed with a digital camera in 0.3-mm intervals throughoutthe ventricle system. Hemorrhagic spots were counted and the bleedingarea was measured (ImageTool; University of Texas Health Science Centerat San Antonio, San Antonio, TX).

Assessment of brain pathology

For better comparison, the degree of BBB disruption and the number ofcerebral hemorrhages were combined in a neuropathological score (neu-roscore). The degree of BBB disruption was scored as follows: 0, 1, and2 points were given if the brain albumin concentration was ,30 ng/mg,between 31 and 90 ng/mg, and .90 ng/mg brain protein, respectively. Thenumber of hemorrhagic spots was scored as follows: a score of 0 indicated0–1 cerebral bleeding spots, whereas scores of 1 and 2 indicated 2–12 and.12 cerebral bleeding spots per 10 investigated brain sections, respec-tively. The maximum neuropathological score was 4 and indicated severebrain injury, whereas a score of 0 stood for no pathological alterations.

Measurement of mouse brain IL-1b levels

Mouse brain concentrations of IL-1b were assessed by ELISA (R&DSystems), according to the manufacturer’s instructions.

Cell culture experiments

Human THP-1 cells were maintained in RPMI 1640 supplemented with10% heat-inactivated low-endotoxin FCS (PAA Laboratories) and 10 mg/mlpenicillin/streptomycin. For experiments, cells were plated in 24-wellplates (5 3 105 cells/well) and differentiated for 48 h with 100 nM PMA(Sigma-Aldrich) in RPMI 1640 supplemented with FCS. Then, the culturemedium was replaced by RPMI 1640 supplemented with 10% normalhuman serum or 10% C5-depleted human serum (in selected experiments;both from TECOmedical), and cells were exposed to S. pneumoniae se-rotype 2 strain D39 (106, 53 106, or 107 CFU/ml; in selected experiments,to its isogenic pneumolysin mutant D39DPly [107 CFU/ml] or GFP-expressing D39 [107 CFU/ml]) (24) for 3, 6, or 18 h. In separate experi-ments, the following compounds were added to the culture medium: z-YVAD-fmk (50 mM; BIO-CAT), Ca-074-Me (50 mM), bafilomycin A1(250 nM), diphenylene iodonium (DPI, 10 mM; all from Merck Chem-icals), cytochalasin D (1 mM), oxidized ATP (ox-ATP, 1 mM) and potas-sium chloride (65 mM; all from Sigma-Aldrich).

Bone marrow-derived macrophages (BMDMs; from WT and ASC-deficient mice) were prepared from bone marrow cells isolated from thefemur. Bones were flushed with HBSS and the cell suspension was forcedthrough a 70-mm mesh. Collected cells were resuspended in completemacrophage medium (containing DMEM, 50 ng/ml rM-CSF, 10% FCS, 10mM HEPES, 10 mM L-glutamine, and 10 mg/ml penicillin/streptomycin)and cultured at 37˚C in 5% CO2. After 7 d, virtually 100% of the cellsexpressed the macrophage marker CD11b. The culture medium was re-placed by complete macrophage medium lacking penicillin/streptomycin.Again 24 h later, cells were exposed to S. pneumoniae serotype 2 strainD39 (107 CFU/ml) or its isogenic pneumolysin mutant D39DPly (107

CFU/ml).

The Journal of Immunology 5441

Determination of IL-1b and TNF-a in cell culture supernatantsand lysates

IL-1b and TNF-a levels were measured in cell culture supernatants andcell lysates using a commercially available ELISA (R&D Systems) inaccordance with the instructions of the manufacturer.

Western blot analysis of IL-1b and caspase-1

THP-1 cells were lysed in a hypotonic buffer containing 10 mM HEPES,10 mM KCl, 1.5 mM MgCl2, 2 mM DTT, and a protease and phosphataseinhibitor mixture (consisting of aprotinin, PMSF, leupeptin, pepstatin A).For Il-1b immunoblotting, lysates and precollected supernatants werefurther processed separately, whereas both samples were mixed and pro-cessed together for caspase-1 detection. To concentrate IL-1b and caspase-1, 15 ml StrataClean resin was added to 300 ml sample. The resin-boundproteins were recovered by centrifugation, washed with PBS, separated ona 4–12% NuPage Tris-Bis gel (Invitrogen), transferred to a polyvinylidenedifluoride membrane (Millipore), and probed either with a rabbit poly-clonal Ab to Casp1 p10 (sc-515, 1:500 dilution) or IL-1b (sc-7884, 1:1000dilution; both from Santa Cruz Biotechnology). For all blots, bound pri-mary Abs were detected using a peroxidase-conjugated Ab against rabbitIgG (1:2000 dilution; Sigma-Aldrich) and the FemtoMax supersensitivechemiluminescence substrate kit (Rockland Immunochemicals). Blotswere visualized and digitalized using a Doc-ItLS image analysis system(UVP).

Immunocytochemical detection of IL-1b, caspase-1, LAMP-2,and cathepsin B

Differentiated THP-1 cells adherent to round glass coverslips (2 3 105

cells/well, 24-well plate) were fixed with 4% buffered paraformaldehydeand permeabilized with 0.1% Triton X-100. Then, cells were stained usingrabbit anti–IL-1b (1:100), rabbit anti-caspase-1 (1:100, both from SantaCruz Biotechnology), mouse anti–LAMP-2 (2 mg/ml; BD Pharmingen), ormouse anti-cathepsin B Abs (4 mg/ml; Merck Chemicals), followed bysecondary anti-rabbit or anti-mouse Alexa Fluor 546-coupled Abs (Invi-trogen) and a DAPI nucleic acid stain. Additionally, caspase-1–like activitywas detected using the FAM FLICA caspase-1 kit following the manu-facturer’s protocol (AbD Serotec). Pictures were recorded using a cooled,high-resolution Moticam 5000 CCD camera mounted on an Olympus B51fluorescence microscope.

Labeling of lysosomes with LysoSensor Green DND-189

The evaluation of the stability of lysosomal membranes was performed bymeans of an acidotropic fluorescent probe LysoSensor Green DND-189,which accumulates in acidic organelles. Briefly, THP-1 cells were loadedwith 1 mM LysoSensor Green DND-189 (Molecular Probes/Invitrogen) for15 min. After replacing the loading medium with fresh medium, cells weremonitored on a Leica DM IL inverted fluorescence microscope equippedwith a cooled CCD camera (VarioCam; PCO Computer Optics).

Assessment of cellular caspase-1 and cathepsin B activity

Caspase-1 and cathepsin B activities were determined by measuring thecleavage of enzyme-specific, fluorogenic substrates in cell lysates usingcommercially available assay kits (BioVision).

Determination of ATP and lactate dehydrogenase

Release of ATP from cells into the supernatant was monitored using abioluminescence assay kit (Molecular Probes). The lactate dehydrogenase(LDH) activity was determined in centrifuged culture supernatants (S) andin cell pellets (P) of THP-1 control wells after lysis with an equal volume ofRPMI 1640 containing 0.1% Triton X-100, using a colorimetric assay kit(BioVision). The cytotoxicity was calculated as percentage LDH release bythe ratio of P/(S + P).

Statistical analysis

SYSTAT 9 (SPSS) was used for statistical analysis. The principal statisticaltest was an unpaired Student t test (combined with Bonferroni a adjustmentin case of multiple comparisons) and the Mantel log-rank test for survival.Differences were considered significant at p , 0.05. Data are displayed asmeans 6 SD.

ResultsAmelioration of pneumococcal meningitis by ASC and NLRP3deficiency

Previously, we demonstrated that depletion of caspase-1 improvesclinical outcome of pneumococcal meningitis (15). To clarify themechanisms underlying meningitis-associated caspase-1 activa-tion, we first infected WT (C57BL/6n) and ASC2/2 mice withhigh doses of S. pneumoniae D39 (108 CFU/ml). More than 70%of WT mice (8 of 11 mice) died within 24 h postinfection whereasthe death rate of ASC-deficient mice was merely 20% (2 of 10mice; p = 0.017). We next inoculated mice with lower doses ofS. pneumoniae (107 CFU/ml). By 24 h postinfection, all WT micedeveloped clinical signs of infection, which manifested in an in-creased clinical score (Fig. 1A), loss of body weight, and hypo-thermia, but only 2 of 12 mice succumbed within the observationperiod. Compared to WT mice, ASC2/2 mice developed less severedisease. This was reflected by lower clinical scores and a less pro-nounced loss of body weight and change of temperature (data notshown). Lethality of infected ASC2/2mice was 10% (1 of 10 mice).Because intracranial complications are major determinants of an

unfavorable clinical outcome in meningitis (25), we next investi-gated the impact of ASC deficiency on meningitis-associated brainpathology. Intrathecal infection with S. pneumoniae D39 signifi-cantly increased ICP in WT mice. At 24 h after pneumococcalinoculation, ASC-deficient mice had significantly lower ICP val-ues than did infected WT mice (Fig. 1B). Additionally, BBBbreaching and cerebral bleeding were less pronounced in brains ofinfected ASC2/2 mice than in those of WT mice, as indicated by asignificantly reduced neuropathologic score (Fig. 1C, 1D). The re-duction in brain pathology correlated with an attenuated accumu-lation of neutrophils, major contributors to meningitis-associatedbrain damage, in the CSF of ASC2/2 mice as compared withinfected WT mice (Fig. 1E). In contrast, pneumococcal outgrowthwithin the brain and blood was not significantly altered in ASC2/2

mice compared with WT mice (Fig. 1F, 1G).Caspase-1 activation by bacterial muramyl dipeptide was re-

ported to require the NLRs NOD2 and NLRP3, which recruitRIP2 and ASC upon their activation (26). Experimental workalso demonstrated that 1) NOD2–RIP2 signaling contributes topneumococci-induced cell activation (27), and 2) the pneumo-coccal toxin pneumolysin promotes caspase-1 activation in anNLRP3-dependent manner (28). We thus used mice lacking eitherNLRP3 or RIP2. Similar to ASC2/2 mice, infected NLRP32/2

mice showed statistically significant amelioration of both diseaseseverity and brain pathology, as evidenced by lower clinical scoresand ICP values as well as less hemorrhagic spots and lowerneuropathologic scores (Fig. 1A–D). The alleviation of diseasewas again associated with a reduction in CSF pleocytosis (Fig.1E). In contrast to the NLRP3 deficiency, the lack of RIP2 hadno impact on the clinical course, meningitis-associated brain pa-thology, and meningeal leukocyte infiltration (Fig. 1A–E). Neitherthe genetic depletion of RIP2 nor that of NLRP3 resulted in sig-nificant alterations of pneumococcal titers in the brain and theblood (Fig. 1F, 1G). Lethality of infected RIP2 and NLRP32/2

was 20% (2 of 10 mice) and 10% (1 of 10 mice). Our data assigna central role to the NLRP3 inflammasome, but not to NOD/RIP2signaling, in the pathogenesis of pneumococcal meningitis.

Blockade of IL-1 family cytokine signaling is protective inpneumococcal meningitis

To determine the contribution of NLRP3 inflammasome-dependentIL-1 signaling to pneumococcal meningitis, infected WT micewere pretreated with the rIL-1R antagonist anakinra (shown to be

5442 NLRP3 INFLAMMASOME IN PNEUMOCOCCAL MENINGITIS

effective in mice; Refs. 21, 29) alone or in combination withmouse rIL-1BP (22). IL-1R blockade significantly attenuated themeningitis-induced rise in intracranial pressure as well as men-ingeal inflammation. However, the improvement of the clinicalstatus and brain pathology was not significant (Fig. 2). Thecombination of anakinra with rIL-1BP resulted in a more pro-nounced reduction of CSF pleocytosis (by 64.5%, compared with47.9% in anakinra-treated mice), which was also paralleled bya significant amelioration of the disease, as evidenced by signifi-cant lower clinical and neuropathological scores (Fig. 2C, 2D). Asin mice lacking ASC or NLRP3 expression, mice receiving IL-1Rcompetitor or the combination of the competitor with rIL-18BPshowed nearly identical bacterial titers in the brain as comparedwith PBS-treated mice (data not shown). Thus, we conclude thatthe ASC2/2 and NLRP32/2 phenotypes are related to impairedIL-1b and IL-18 signaling.

Cathepsin B activity is required for S. pneumoniae–inducedcaspase activation and IL-1b release by differentiated THP-1macrophages

To further investigate S. pneumoniae-induced caspase-1 activationand IL-1b production, we performed experiments in differentiated

THP-1, as macrophages are the predominant source of IL-1b inpneumococcal meningitis (30). First, we challenged the cells with

increasing amounts of live S. pneumoniae D39. Infection with 107

CFU/ml (but not with 106 or 5 3 106 CFU/ml) pneumococci

markedly elevated IL-1b concentrations in cell culture super-

natants 6 h later (data not shown). Next, we characterized the

impact of pneumococcal challenge on the release of IL-1b and

ATP (a well-known stimulator of Il-1b production), on the acti-

vation of caspase-1 and cathepsin B (a potential caspase-1 acti-

vator), as well as on LDH release (a widely used cell death

indicator) over time (Supplemental Fig. 1). Significantly elevated

IL-1b levels in cell culture supernatants were found 6 and 18 h

after pneumococcal stimulation. Prior to the secretion of IL-1b,

increases in both caspase-1 and cathepsin B activities (.10- and

5-fold, respectively) were detectable in cell lysates. The time ki-

netic of caspase-1 activity equalled that of the release of ATP into

the supernatant, suggesting involvement of both ATP release and

cathepsin B activation in pneumococci-induced pro–IL-1b pro-

cessing. Raised LDH concentrations in the supernatant were seen

at late time points during infection (18 h after challenge), and no

temporal relationships were found between LDH levels and

caspase-1 activity (Supplemental Fig. 1). These data argue against

FIGURE 1. ASC and NLRP3 (but not RIP2) defi-

ciency is protective in murine pneumococcal meningi-

tis. A, Infected ASC-deficient and NLRP3 mice (n = 9

analyzed mice/group) showed a reduction in disease

severity at 24 h postinfection, as indicated by lower

clinical score values compared with those of infected

WT mice (n = 10). The amelioration of disease was

paralleled by (B) a less pronounced rise in ICP and (C,

D) milder brain pathology. This is reflected by (C) less

macroscopically visible hemorrhages in brains obtained

from ASC- and NLRP3-deficient mice (compared with

those of infected WT and RIP2-deficient mice) as well

as (D) significantly lower neuroscore values. The neu-

roscore comprises two items: the degree of BBB dis-

ruption and the number of cerebral hemorrhages. E,

The reduction in brain pathology was associated with

lower WBC counts in the CSF of ASC- and NLRP3-

deficient mice, compared with those of WT and RIP2-

deficient mice. F and G, The lack of ASC, NLRP3, and

RIP2 had no effect on bacterial outgrowth in the brain

blood. Data are given as means 6 SD. *p , 0.05

compared with uninfected WT controls (n = 8), #p ,0.05 compared with infected WT mice using an un-

paired Student t test and Bonferroni correction for

multiple comparisons.


a significant role of caspase-1–dependent inflammatory cell death(31, 32) in pneumococcal infection.Recent studies suggested that extracellular ATP induces ca-

thepsin B activation (33) and accelerates caspase-1–dependentpro–IL-1b processing (34). Cathepsin B activity, in turn, mightcontribute to NLRP3 inflammasome activation or circumvent it bydirect IL-1 family proform cytokine cleavage (35). We thereforeexamined the effect of inhibiting ATP signaling and cathepsin Bon pneumococci-induced caspase-1 activation and IL-1b release.Moreover, we comparatively analyzed the caspase-1 antagonist z-YVAD-fmk and purposeful increase of extracellular potassiumconcentration for their potential to interfere with IL-1 activation(36). The addition of potassium chloride to the culture mediuminhibited pneumococci-induced caspase-1 activation and IL-1brelease (Fig. 3A–D) to a similar degree as did the caspase-1 in-hibitor z-YVAD-fmk. Both treatments had no effect on ATP lib-eration, cathepsin B activation, as well as the production of theinflammasome-independent cytokine TNF-a (Fig. 3E–G). Addi-tionally, neither z-YVAD nor the increase in extracellular potas-sium affected macrophage cell death in response to pneumococcalinfection (Fig. 3H).Next, we treated THP-1 macrophages either with ox-ATP, a

P2X7 purinoreceptor antagonist (37), or the cathepsin B inhibitorCa-074-Me (38), and exposed them to S. pneumoniae. Supple-mentation of the culture medium with ox-ATP resulted in a sig-nificant inhibition of caspase-1 activation and IL-1b release. Incontrast to potassium chloride substitution, however, ox-ATP alsoblocked cathepsin B activation (Fig. 3A–F). Similarly, S. pneu-moniae-induced cathepsin B activation, caspase-1 activation, andIL-1b secretion were blunted in THP-1 pretreated with Ca-074-Me (Fig. 3A–F), suggesting a dominant role of cathepsin B inpneumococci-induced IL-1b release by macrophages. Cathepsin Binhibition was also associated with an attenuation of TNF-a andLDH release into the cell supernatant (Fig. 3G, 3H). These find-ings are in line with recent studies that demonstrated an in-volvement of cathepsin B in caspase-1–independent necrosis as

well as in the trafficking of TNF-a containing vesicles to theplasma membrane and its subsequent liberation (38, 39).It has been proposed that the NLRP3 inflammasome is activated

either by lysosomal damage induced by the cargo taken up byphagocytosis and the subsequent release of cathepsin B (35) or byreactive oxygen species (ROS) generated by a NADPH oxidaseupon particle phagocytosis (40). Thus, we assessed whethertreatment of cells with 1) cytochalasin D, which impairs actinfilament assembly and thus prevents phagocytosis, 2) bafilomycinA, which blocks the vacuolar H+-ATPase system required for ly-sosomal acidification, or 3) DPI, a well characterized NADPHoxidase inhibitor, interferes with IL-1b release. Neither the addi-tion of cytochalasin D nor that of bafilomycin A or DPI to theculture medium resulted in a significant inhibition of pneumococci-induced caspase-1 and cathepsin B activation as well as IL-1b andATP liberation (Fig. 3A–F). Furthermore, immunohistochemistryof differentiated THP-1 macrophages using an Ab against LAMP-2, which is known to stain late endosomes as well as lysosomes(38), showed a splotchy staining that did not recede after exposureto S. pneumoniae. This indicated that the lysosomal membranecompartment remained intact upon infection. Moreover, bacteriathat constitutively express GFP were virtually exclusively presentin the extracellular space, but hardly inside the lysosomes (Fig.4A). Using an anti-cathepsin B Ab, we found a splotchy staining inunchallenged THP-1 macrophages similar to the LAMP-2 stain-ing, which is in agreement with its known lysosomal localization.However, translocation of cathepsin B to the cytoplasm was ob-served 3 h after S. pneumoniae stimulation. At later times (6 h) thestaining seemed to be entirely cytoplasmic (Fig. 4B). Similarly,when cells were preloaded with LysoSensor Green D-189, wefound a splotchy staining that disappeared after pneumococcalchallenge, which is suggestive of lysosomal leakage (Fig. 4C) andrelease of the lysosomal content such as cathepsin B. This wasparalleled by an increase in caspase-1 activity and also an in-creased intracellular staining for IL-1b and caspase-1, reflectinginduction of protein expression by pneumococcal challenge (Fig.

FIGURE 2. IL-1R and IL-18 antagonism is protective in pneumococcal meningitis. Pretreatment with the IL-1R antagonist anakinra (100 mg/kg i.p.; n =

6) only tended to improve the clinical status (A) and neuropathological alterations (quantified by means of a neuroscore; C) in pneumococcal meningitis,

whereas the combined administration of anakinra and rIL-18 (5 mg/kg) led to a significant amelioration of disease severity and brain pathology in mice with

pneumococcal meningitis. Anakinra and anakinra plus rIL-18BP significantly attenuated the meningitis-induced rise in ICP (B) as well as meningeal

inflammation (D, as indicated by increased CSF WBC counts; compared with vehicle-injected, infected mice), although the reduction of CSF WBC counts

was greater in mice that received the combination therapy than in anakinra-treated mice. A total of six mice were used in each group. One mouse in each

infected control group died spontaneously during the experiment, whereas all treated, infected mice survived. Data are given as means 6 SD. *p , 0.05

compared with infected WT mice using an unpaired Student t test and Bonferroni correction for multiple comparisons.


4D–F). Taken together, our data provide evidence for a pneumo-cocci-induced ATP-dependent lysosomal release of cathepsin B inthe absence of bacterial phagocytosis and subsequent lysosomalrupture, which is at least partly dependent on ATP liberation andleads to caspase-1–dependent IL-1b release.

S. pneumoniae-induced IL-1b release by THP-1 macrophagesdepends on the presence of terminal complement factors andpneumolysin

Recent studies demonstrated that the pore-forming pneumococcaltoxin pneumolysin induces caspase-1 activation and IL-1b se-cretion in murine peritoneal macrophages (41, 42) through acti-vation of the NLRP3 inflammasome (28). Moreover, we observed

in a previous study that complement activation is a key factor inIL-1b production during pneumococcal meningitis (43). Both theanaphylatoxin C5a and the terminal complement complex C5b-9,which can form pores in host membranes (44), were also reportedto stimulate IL-1b release by human mononuclear cells (45, 46).In this study, we contribute to clarification of the role of pneu-molysin and complement in pneumococci-induced IL-1b releaseby exposing cells to a pneumolysin-deficient strain and challengingcells in the absence of C5.The pneumolysin-deficient mutant failed to induce IL-1b release

by both murine BMDMs and THP-1 macrophages (Fig. 5A, 5B,5K). In BMDMs, IL-1 b production was dependent on the pres-ence of ASC. In THP-1 cells, the impairment of IL-1b secretion

FIGURE 3. S. pneumoniae promotes caspase-1–dependent IL-1b release in an ATP- and cathepsin B-dependent manner. Differentiated human THP-1

cells (5 3 105 cells/well) were exposed to 107 CFU/ml bacteria for 6 h. A and B, Activation of caspase-1 in THP-1 cells by S. pneumoniae. Mixtures of cell

lysates and precollected supernatants were analyzed for caspase-1 p10 expression by Western blot (A) as well as caspase-1 activity by measuring the

cleavage of enzyme-specific, fluorogenic substrates using a commercially available assay kit (B). C and D, IL-1b release into the cell culture supernatant

upon pneumococcal challenge. Cell supernatants were examined for the presence of IL-1b p17 immunoreactivity by Western blot (C) as well as IL-1 b

levels by ELISA (D). Additionally, the liberation of ATP (E) and cathepsin B activity (F) were determined using a bioluminescence assay kit and by

measuring the cleavage of enzyme-specific, fluorogenic substrates using a specific assay kit, respectively. G and H, TNF-a and LDH release into the cell

culture supernatant upon pneumococcal challenge. TNF-a concentrations were measured by ELISA in cell culture supernatants collected 6 and 18 h

postinfection. Immediately before bacterial challenge, cells were treated either with medium, the caspase-1 inhibitor zYVAD-fmk (50 mM), potassium

chloride (65 mM), the cathepsin B inhibitor Ca-074me (50 mM), the purinoreceptor antagonist ox-ATP (1 mM), the NADPH oxidase inhibitor DPI (10

mM), the phagocytosis inhibitor cytochalasin D (1 mM), and bafilomycin A1 (250 nM). All data are given as means 6 SD for two independent experiments

performed in triplicate. *p, 0.05 compared with D39-stimulated cells using an unpaired Student t test and Bonferroni correction for multiple comparisons.


was paralleled by a reduced ATP release as well as repressedcathepsin B and caspase-1 activation (Fig. 5C–F, 5K). These datasuggest a key role for the inflammasome in pneumolysin-dependent IL-1b production. Additionally, an attenuated upregu-lation of IL-1b (and caspase-1) protein expression in THP-1macrophages upon exposure to the pneumolysin-deficient mutantwas also evident, suggesting a cytokine-inducing potency of pneu-molysin (47, 48) (Fig. 5G, 5H). Accordingly, the pneumolysin-deficient mutant was less potent in inducing TNF-a release thanthe WT strain (Fig. 5I).The increase in IL-1b and caspase-1 protein expression was also

dampened in THP-1 macrophages challenged with WT S. pneumo-niae when C5 was depleted. Additionally, the absence of C5 in theculture medium resulted in a reduction of IL-1b and TNF-a release,but it did not affect pneumococci-induced ATP liberation as well ascathepsin B and caspase-1 activation (Fig. 5A–I). Taken together,these data suggest that pneumolysin contributes to IL-1b release by

macrophages upon exposure to S. pneumoniae in two ways: it en-hances the expression of both caspase-1 and IL-1b, and it activatescaspase-1 in a cathepsin B-dependent manner. The terminal com-plement factors only augmented caspase-1 and IL-1b upregulation.

Role of pneumolysin and cathepsin B in the inflammatory hostresponse in pneumococcal meningitis

To extend our analysis of pneumolysin and cathepsin B in S.pneumoniae infection, we next assessed their role in murine pneu-mococcal meningitis. Intracisternal inoculation of the pneumolysin-deficient mutant caused less severe disease than did the WT strain.This was reflected by lower clinical score values (Fig. 6A) as wellas a less pronounced loss of body weight and temperature (datanot shown). The amelioration of disease was accompanied bya reduction in intracranial complications as exemplified by lowerICP values in mutant strain-infected than in WT strain-infectedmice (Fig. 6B). Additionally, infection with the pneumolysin-de-ficient mutant was associated with a reduction in CSF pleocytosis(by ∼50%; Fig. 6C) as well as in blood bacterial titers (3.06 60.69 and 2.02 6 0.51 log10 CFU/ml in mutant strain-infected andWT strain-infected mice, respectively; p = 0.007). Bacterial out-growth in the brain, however, was equal in mice infected eitherwith the mutant or the WT strain. To investigate the significance ofpneumolysin-induced inflammasome activation in vivo, we nextinfected ASC-deficient mice with both pneumococcal strains.ASC-deficient mice developed milder clinical signs of meningitis,irrespective of the bacterial strain inoculated. Moreover, ICPvalues and CSF leukocyte numbers were quite similar in ASC-deficient mice infected with the pneumolysin-deficient mutant andthe WT strain (Fig. 6). Thus, the inflammasome appears to play acritical role in mediating immune responses to pneumolysin-expressing S. pneumoniae.In a next series of experiments, we inhibited cathepsin B in vivo.

The administration of Ca-074-Me prior to pneumococcal infectionresulted in a reduction of disease severity, ICP, as well as CSFpleocytosis without altering brain and blood bacterial titers (Fig. 6).In an attempt to gain insight into the role of pneumolysin and

cathepsin B in pneumococci-induced IL-1b production in vivo,we also measured IL-1b concentrations in brain homogenates byELISA. We observed a strong induction of IL-1b expression inbrains obtained from WT strain-infected mice (97 6 34 pg/mgprotein versus not detectable in PBS-injected mice). Brain IL-1blevels were significantly lower in mice infected with thepneumolysin-deficient mutant (33 6 42 pg/mg brain protein; p =0.017) and in mice infected with the WT strain and treated withCa-074-Me (44 6 16 pg/mg brain protein; p = 0.012). Thus, theamelioration of disease observed in mice infected with thepneumolysin-deficient strain or treated with the cathepsin B in-hibitor might be attributable to an attenuated IL-1b generation.Observing that the effects of pneumolysin deficiency or ca-

thepsin B inhibition on IL-1b production were weaker in vivo thanin vitro, we further assessed the role of neutrophils in meningitis-induced IL-1b generation. Neutrophils are the predominant cellpopulation within the meningeal infiltrate (8) and can produce IL-1b in a caspase-1–independent manner (49). Granulocyte deple-tion by pretreatment with a monoclonal anti-GR1 Ab led to adramatic reduction in neutrophil counts in the blood (52 6 14cells/ml, compared with 2196 6 501 cells/ml in isotype controlAb-treated mice) and CSF at 24 h postinfection (SupplementalFig. 2). The elimination of neutrophils was paralleled by a sig-nificantly attenuated brain pathology (Supplemental Fig. 2), butincreased bacterial titers in the blood (data not shown) and brain(Supplemental Fig. 2). Additionally, granulocyte depletion resul-ted in a significant reduction in brain IL-1b levels (41 6 26 and

FIGURE 4. S. pneumoniae induces cathepsin translocation to the cyto-

plasm and caspase-1 activation in the absence of lysosomal rupture. Dif-

ferentiated human THP-1 cells (5 3 105 cells/well) were exposed to 107

CFU/ml bacteria for 3 (middle column) or 6 h (right column) or to medium

for 6 h (left column). At these time points, cells were processed for im-

munocytochemistry and stained with anti–LAMP-2 (A), anti-cathepsin B

(B), anti–IL-1b (E), and anti–caspase-1 (F) Abs as described in Materials

and Methods. Lysosomal deacidification (an indicator for lysosomal

leakage) was monitored using the acidotropic fluorescent probe Lyso-

Sensor Green DND-189, which accumulates in acidic organelles (C).

Caspase-1–like activity was detected using the FAM FLICA caspase-1 kit

following the manufacturer’s protocol (D). Specimens stained with the

mentioned Abs or incubated with FLICA solution were evaluated using

a cooled, high-resolution Moticam 5000 CCD camera mounted on an

Olympus B51 fluorescence microscope (original magnification, 31000).

Cells loaded with LysoSensor were monitored in vivo on a Leica DM IL

inverted fluorescence microscope equipped with a cooled CCD camera

(original magnification 3400).


91 6 25 pg/mg brain protein in anti–GR1-treated and isotype Ab-treated mice, respectively; p = 0.034), suggesting a possiblecontribution of neutrophil-dependent (caspase-1–independent) path-ways to meningitis-induced IL-1b production.

DiscussionData from patients (9, 10) and animal experiments (13, 14) indi-cate that excessive production of IL-1b plays a key role in thepathogenesis of pneumococcal meningitis. Furthermore, we pre-viously demonstrated that caspase-1 is an essential mediator inthis disease (15). Our present data provide evidence that S.pneumoniae induces caspase-1 activation via the NLRP3 inflam-

masome and that NLRP3 and ASC deficiencies are associatedwith significantly decreased clinical and histological disease se-verity as well as brain inflammation. This observation is in linewith the concept that meningitis-related brain damage is largelydue to a massive neutrophilic inflammatory reaction and the con-comitant release of cytotoxic host factors (2). Initial evidence wasraised with blockade of adhesion-promoting receptors of neu-trophils. For instance, i.v. injection of anti-CD18 Abs effectivelyprotected against meningitis-related brain damage and death(7, 16). This finding was strengthened by results of recent mousestudies that demonstrated a nearly complete abrogation of braintissue injury when neutrophils were depleted by using neutrophil-specific Abs (8). In addition to the toxic effects of the host

FIGURE 5. S. pneumoniae-induced IL-1b release depends on pneumolysin and complement, whereas caspase-1 activation requires only pneumolysin.

Differentiated human THP-1 cells were exposed to pneumolysin-producing (D39) or isogenic pneumolysin-deficient (D39DPly) S. pneumoniae in the

absence or presence of complement factor C5. A and B, IL-1b liberation into the cell culture supernatant in response to pneumococcal challenge. Cell

supernatants were examined for the presence of both IL-1b p31 (proform) and p17 (mature form) immunoreactivity by Western blot (A) as well as IL-1b

levels by ELISA (B). C and D, Activation of caspase-1 in THP-1 cells by S. pneumoniae. Mixtures of cell lysates and precollected supernatants were

analyzed for caspase-1 p45 (proform) and p10 expression by Western blot (C) as well as caspase-1 activity by measuring the cleavage of enzyme-specific,

fluorogenic substrates using a commercially available assay kit (D). ATP release (E) and cathepsin B activity (F) were determined using a bioluminescence

assay kit and by measuring the cleavage of enzyme-specific, fluorogenic substrates using a specific assay kits, respectively. THP-1 cells were also processed

for immunocytochemistry and stained with anti–IL-1b (G) and anti–caspase-1 (H) Abs as described in Materials and Methods. Specimens were viewed

using a cooled, high-resolution CCD camera mounted on a fluorescence microscope (original magnification 31000). I, TNF-a release into the cell culture

supernatant upon pneumococcal challenge. TNF-a concentrations were measured by ELISA in cell culture supernatants collected 6 and 18 h postinfection.

K, Murine WT and ASC-deficient BMDMs were exposed to pneumolysin-producing D39 or isogenic pneumolysin-deficient D39DPly. IL-1b secretion into

the cell culture supernatant upon pneumococcal challenge was determined by ELISA. All data are given as means 6 SD for two independent experiments

performed in triplicate. *p, 0.05 compared with D39-stimulated cells (in normal human serum) using an unpaired Student t test and Bonferroni correction

for multiple comparisons.


response, direct bacterial toxicity was implicated as an additionalfactor driving brain damage in pneumococcal meningitis. How-ever, this aspect is of minor relevance for the protective phenotypeassociated with the lack of NLRP3 or ASC, as neither was asso-ciated with different bacterial loads of the brain as compared withWT controls.Recently, at least two pathways have been proposed for the

activation of the NLRP3 inflammasome (50). According to the firsthypothesis, NLRP3 activators trigger the production of ROS,which are sensed directly or indirectly by NLRP3, leading to itsactivation (40, 51, 52). Support for this hypothesis comes fromexperiments demonstrating that mitochondrial ROS scavengerssuch as Mito-TEMPO or NADPH oxidase inhibitors such as DPIattenuated caspase-1 activation (40, 51). However, we did not

observe any alterations in pneumococci-induced IL-1b generationby differentiated THP-1 cells following pretreatment with DPIor MnTBAP (an intracellular ROS scavenger that also interfereswith mitochondrial ROS; data not shown). The second hypothesisplaces protease activity upstream of NLRP3 inflammasome acti-vation. In this model, NLRP3 activators induce lysosomal dam-age, which leads to the release of lysosomal proteases such ascathepsin B into the cytosol. The lysosomal proteases, in turn,could either degrade a putative NLRP3 inhibitor or cleave a sub-strate in the cytosol that would generate a NLRP3 ligand (53).This model has been consolidated by the demonstration that di-verse crystals and protein aggregates disrupt lysosomal integrityand activate NLRP3 (35, 54). Accordingly, IL-1b secretion uponinfection with the intracellular pathogen Listeria monocytogeneswas found to be largely dependent on phagolysosomal rupture(induced by the pore-forming bacterial toxin listeriolysin O) andcathepsin B release (55). Moreover, nigericin, thought to activatethe NLRP3 inflammasome solely through acting as a potassiumionophore, has been demonstrated to induce lysosomal leakage ofcathepsin B and subsequent caspase-1 activation, noteworthily inthe absence of obvious lysosomal desintegration (38). In line withthe latter finding is our observation that pneumococcal challengedoes not lead to disruption of the lysosomal membrane compart-ment, but to lysosomal deacidification and cathepsin B release intothe cytosol, and that cathepsin B inhibition prevents pneumococci-induced IL-1b secretion.We further observed an increase in ATP levels in the supernatant

of THP-1 cells upon exposure to S. pneumoniae. Moreover, pre-treatment with the purinoreceptor P2X7 antagonist oxidized ATPresulted in an attenuated activation of cathepsin B and caspase-1as well as in reduced IL-1b generation. Thus, pneumococcalchallenge might result in secretion of ATP that activates P2X7

receptors through an autocrine loop and triggers a series of sig-naling events culminating in IL-1b release. This is in line witha previous study demonstrating endogenous ATP release frommonocytes as an early step in the inflammasome activation cas-cade induced by a variety of pathogen- or danger-associatedmolecular patterns including muramyl dipeptide or monosodiumurate (56). Moreover, hyperoxia exposure has also been reportedto lead to ATP release, which further stimulated P2X7-mediatedpotassium efflux and inflammasome activation (57). In bothstudies, the ATP concentrations measured in the cell culturesupernatants were quite similar to that we found in our experi-ments, but well below the threshold required to stimulate P2X7

receptors. This discrepancy may be explained by a significantunderestimation of the ATP amount released at the cell surface,due to a fast diffusion and rapid hydrolysis of cell-derived ATPby ectonucleotidases expressed by host cells and/or bacteria (58).Similar to the mode of action to the potassium ionophore niger-icin, a major event triggered by extracellular ATP is the releaseof cathepsin B (33). The ATP-induced protease release was notpreventable by high extracellular potassium concentrations, sug-gesting its independence of potassium ion efflux (33). Accord-ingly, we did not detect a significant reduction in pneumococci-induced cathepsin B activity in THP-1 cells in the presence ofhigh potassium levels. Instead of the potassium efflux, a rise inlysosomal pH has been proposed as the trigger of the lysosomalsecretion of cathepsin B. This hypothesis is based on the obser-vation that compounds capable of increasing lysosomal pH, in-cluding ammonium chloride, hydrogen ionophores, or vacuolarH+-ATPase inhibitors such as bafilomycin A, can induce the re-lease of lysosomal content (59). This concept may also explain thelack of effect of bafilomycin A pretreatment on pneumococci-induced cathepsin B and caspase-1 activation by us.

FIGURE 6. Lack of pneumolysin and cathepsin B inhibition are pro-

tective in murine pneumococcal meningitis. Intracisternal inoculation of

the pneumolysin-deficient mutant (n = 6 analyzed mice) caused less severe

disease than did the WT strain (n = 9 analyzed mice) and ASC-deficient

mice (n = 4 analyzed mice). This was reflected by lower clinical score

values (A). The amelioration of disease was accompanied by a reduction in

ICP (B) and in CSF WBC counts (C). There were no significant differences

in all these parameters between ASC-deficient mice infected with the

pneumolysin-deficient (D39DPly) and the WT (D39) strain. Similarly,

pretreatment of mice with cathepsin inhibitor Ca-075-Me (5 mg/kg i.p.;

n = 7 analyzed mice in each group) led to an improved clinical course (A)

and an attenuated rise in ICP (B), which was paralleled by a less pro-

nounced infiltration of leukocytes into the CSF (C) as compared with

infected mice treated with the vehicle of Ca-074-Me (PBS containing 5%

DMSO). Data are given as means 6 SD. *p , 0.05 compared with WT

mice infected with the D39 strain, #p , 0.05 compared with vehicle-

treated, infected mice using an unpaired Student t test and Bonferroni

correction for multiple comparisons.


Furthermore, we demonstrated in this study that differentiatedTHP-1 cells release significantly less ATP into cell culture super-natants upon challenge with a pneumolysin-deficient as comparedwith a pneumolysin-producing pneumococcal strain. This wasparalleled by reduced cathepsin B and caspase-1 activity as well asIL-1b release. Our data are in agreement with a recent study (28)that showed an amplification of TLR agonist-induced IL-1b se-cretion from murine dendritic cells by pneumolysin (28). Thiseffect was dependent on cathepsin B, NLRP3, and caspase-1.Moreover, live S. pneumoniae was found to promote IL-1b re-lease. This release also required NLRP3 and pneumolysin (28).Similarly, Shoma et al. (41) reported a critical involvement ofpneumolysin in IL-1b generation by murine peritoneal macro-phages after exposure to S. pneumoniae. Besides activatingcaspase-1, pneumolysin was shown to induce the cellular pro-duction of IL-1 family cytokines in a TLR4-dependent manner.This observation is in line with the study of Malley et al. (48) whofirst described a role of TLR4 in pneumolysin recognition, but itcontrasts with the results from studies of McNeela et al. (28) andWitzenrath et al. (42). In the latter study, stimulation of IL-1bproduction in S. pneumoniae-infected human monocytes andmurine BMDMs involved signals dependent on TLR2 (but notTLR4) and the NLRP3 inflammasome (42). In the study byMcNeela et al. (28), pneumolysin was not capable of induc-ing cytokine secretion by murine dendritic cells. By using livepneumolysin-producing and pneumolysin-deficient bacteria, wedemonstrated in this study that pneumolysin is required for bothinduction of cytokine expression (such as IL-1b and TNF-a) andactivation of caspase-1. The latter was evidenced by 1) the re-pression of caspase-1 activation and IL-1b release by humanTHP-1 cells upon exposure to the pneumolysin-deficient strain,and 2) the secretion of similar amounts of IL-1b from WT andASC-deficient BMDMs following challenge with pneumolysin-deficient and WT bacteria. The activation of caspase-1 seems tobe at least partly related to the pneumolysin-triggered liberationof ATP from infected THP-1 cells. However, further studies arenecessary to identify which pattern recognition receptors are in-volved in sensing pneumolysin-producing versus pneumolysin-deficient pneumococcal strains. Collectively, our experimentssuggest the following model of inflammasome activation by S.pneumoniae: the pneumococcal toxin pneumolysin may inducethe release of ATP. Extracellular ATP, in turn, might be the majortrigger of the lysosomal secretion of cathepsin B as activator of theNLRP3 inflammasome upon S. pneumoniae challenge.Furthermore, our data show involvement of pneumolysin and

cathepsin B in the immunopathogenesis of pneumococcal menin-gitis. Both inoculation of pneumolysin-deficient pneumococci andinhibition of cathepsin B were associated with a reduction in brainIL-1b levels, CSF leukocyte counts, and disease severity. In linewith our results are reports on the protective effects of cathepsin Binhibition in animal models of acute and chronic neurodegenerativedisorders, including cerebral ischemia and Alzheimer’s disease(60, 61). Moreover, high cathepsin B activity was detected in CSFsamples from patients with neuroinflammatory diseases such asmultiple sclerosis (62). Inflammasome activation plays an importantrole in the three diseases listed above by exacerbating brain in-flammation (54, 63, 64). Therefore, it is conceivable that cathepsinB release and activation may represent a more widespread mech-anism for the propagation of inflammasome-dependent immuneresponses in the brain.The role of pneumolysin in meningitis has been evaluated in

previous studies, which, however, gave inconclusive results withregard to its impact on clinical outcome, bacterial outgrowth, aswell as meningeal inflammation (65–68). By using an adult mouse

model, in this study we found that pneumolysin-deficient S. pne-umoniae evoked merely ameliorated disease. The milder clinicalcourse was paralleled by lower bacterial numbers in the blood,suggesting that the effect on the clinical picture is partly due toless severe sepsis, a typical systemic complication of pneumo-coccal meningitis (1, 6). This observation is in agreement withprevious studies in which adult rodents were applied (66, 67).Additionally, we observed less pronounced neuropathological al-terations in adult mice infected with the mutant strain, whichwas also reported for adult rats (67) and neonatal rats (68). Thismight be owing to the absence of a direct toxic effect of the bac-terial cytolysin on brain cells. Also, it is conceivable that a mil-der inflammatory reaction upon confrontation with pneumolysin-deficient bacteria contributes to the observed reduction in braininjury. This statement is supported by our observation of signifi-cantly lower CSF leukocyte counts and brain IL-1b levels in micesubjected to meningitis with pneumolysin-deficient pneumococci.This reduction might be related to the lack of the cytokine-inducing and cytokine-activating potency of pneumolysin be-cause bacterial outgrowth in the brain (and thus the amounts otherproinflammatory pneumococcal molecules) was similar postin-fection with the pneumolysin-deficient and the WT strain. In ac-cordance with our observation, a reduction in CSF neutrophil/monocyte counts in adult rats challenged with pneumolysin-deficient bacteria has been reported (67). Results from applica-tion of mouse models of pneumococcal bacteremia or pneumoniain which pneumolysin-deficient bacteria were applied lend furthersupport to this idea (69, 70). However, in none of the three otherstudies (65, 66, 68) did pneumolysin deficiency result in significantlylower meningeal inflammation. The causes for these differing resultsare unclear, but they might be attributable to different experimentalsetups (e.g., animal species used).Meningitis due to S. pneumoniae is a serious disease with high

mortality and morbidity rates. Therefore, new therapies based onan understanding of pathophysiology are warranted. In this study,we show that pneumococcal infection of the CSF leads to in-flammasome activation that enhances the inflammatory reactionand contributes to brain injury and adverse outcome. We alsodemonstrate that pneumolysin is a key player in meningitis-induced IL-1b generation by both inducing IL-1b protein ex-pression and inflammasome activation. Inflammasome activationupon S. pneumoniae challenge depends on the release of ATP,lysosomal destabilization, and cathepsin B activation. We con-clude that interference with inflammasome activation might be apromising target for adjunctive therapy in pneumococcal menin-gitis.

DisclosuresThe authors have no financial conflicts of interest.

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The NLRP3 Inflammasome Contributes to Brain Injury in Pneumococcal Meningitis and Is Activated...

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