of November 2, 2018.This information is current as Networks

Dependent Monocyte−Activator Protein-1 and −BκExpression and Secretion via NF-

-3Microglial Matrix Metalloproteinase-1 and UpregulatesMycobacterium tuberculosis

Edwards and Jon S. FriedlandAnwen Bullen, Joanna C. Porter, Dan Agranoff, Dylan R.Federico Roncaroli, Shruti Dholakia, Rachel C. Moores, Justin A. Green, Paul T. Elkington, Caroline J. Pennington,

http://www.jimmunol.org/content/184/11/6492doi: 10.4049/jimmunol.09038112010;

2010; 184:6492-6503; Prepublished online 5 MayJ Immunol 

MaterialSupplementary

1.DC1http://www.jimmunol.org/content/suppl/2010/05/05/jimmunol.090381

Referenceshttp://www.jimmunol.org/content/184/11/6492.full#ref-list-1

, 14 of which you can access for free at: cites 52 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

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

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

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2010 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on Novem

ber 2, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

by guest on N

ovember 2, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

The Journal of Immunology

Mycobacterium tuberculosis Upregulates Microglial MatrixMetalloproteinase-1 and -3 Expression and Secretion viaNF-kB– and Activator Protein-1–Dependent MonocyteNetworks

Justin A. Green,* Paul T. Elkington,* Caroline J. Pennington,† Federico Roncaroli,‡

Shruti Dholakia,* Rachel C. Moores,* Anwen Bullen,* Joanna C. Porter,x

Dan Agranoff,* Dylan R. Edwards,† and Jon S. Friedland*

Inflammatory tissue destruction is central to pathology in CNS tuberculosis (TB). We hypothesized that microglial-derived matrix

metalloproteinases (MMPs)have akey role indriving suchdamage.Analysis of all of theMMPsdemonstrated that conditionedmedium

fromMycobacterium tuberculosis-infected humanmonocytes (CoMTb) stimulated greaterMMP-1, -3, and -9 gene expression in human

microglial cells than direct infection. In patients with CNS TB, MMP-1/-3 immunoreactivity was demonstrated in the center of brain

granulomas.Concurrently, CoMTbdecreased expression of the inhibitors, tissue inhibitor ofmetalloproteinase-2, -3, and -4.MMP-1/-3

secretion was significantly inhibited by dexamethasone, which reduces mortality in CNS TB. Surface-enhanced laser desorption

ionization time-of-flight analysis of CoMTb showed that TNF-a and IL-1b are necessary but not sufficient for upregulating MMP-1

secretion and act synergistically to drive MMP-3 secretion. Chemical inhibition and promoter-reporter analyses showed that NF-kB

and AP-1 c-Jun/FosB heterodimers regulate CoMTb-inducedMMP-1/-3 secretion. Furthermore, NF-kB p65 and AP-1 c-Jun subunits

were upregulated in biopsy granulomas from patients with cerebral TB. In summary, functionally unopposed, network-dependent

microglial MMP-1/-3 gene expression and secretion regulated by NF-kB and AP-1 subunits were demonstrated in vitro and, for the

first time, in CNS TB patients. Dexamethasone suppression of MMP-1/-3 gene expression provides a novel mechanism explaining the

benefit of steroid therapy in these patients. The Journal of Immunology, 2010, 184: 6492–6503.

Tuberculosis (TB) of the CNS is a devastating disease.Mortality is ∼30%, rising to nearly 70% when coinfectedwith HIV (1). CNS TB is characterized by an extensive

inflammatory response and marked tissue destruction, in both themeninges and the brain parenchyma (2, 3). Therapy for CNS TB isprolonged, lasting a minimum of 9 mo. Concurrent administration

of dexamethasone decreases mortality by 30% (4), but novel ap-proaches to therapy are needed because even with optimal treat-ment significant mortality and morbidity occurs. Furthermore,rates of multidrug and extensively drug resistant Mycobacteriumtuberculosis continue to rise (4, 5).Matrixmetalloproteinases (MMPs),a familyof23zinc-containing

endopeptidases, are implicated as effectors of extracellular matrixdestruction in CNS disorders including HIV dementia, Alzheimer’sdisease, and multiple sclerosis (6–8). MMPs may modulate in-flammatory cytokine activity, cleave cell surface receptors, activatecaspase-3, and regulate other MMP family members (9–13). Reg-ulatory feedback loops exist; for example, IL-1b stimulates MMP-3secretion, which degrades biologically active IL-1b (14). MMPactivity is controlled at the level of gene transcription, by com-partmentalization, by release as proforms that require proteolyticcleavage for activation and through 1:1 stoichiometric inhibition bytissue inhibitors of metalloproteinases (TIMPs) (15).MMPs are secreted by cells in an organ- and agonist-specific

manner (16). In work delineating the role of MMPs in pulmonaryTB, we have demonstrated that MMP-9 is upregulated in M. tu-berculosis infection by several cell types. However, upregulation ofMMP-1 (collagenase-1) and MMP-3 (stromelysin-1, whose func-tions include MMP-1 activation) appeared relatively TB-specific(17–19). In CNS TB, we demonstrated that astrocytes secrete onlyMMP-9 (gelatinase B) in response to monocyte-dependent net-works, despite upregulation of other MMP transcripts (20). Clini-cally elevated cerebrospinal fluid MMP-9 concentrations relatedto physical signs of local tissue damage and death in TB meningitis(21, 22). Furthermore, in TB meningitis dexamethasone suppressedcerebrospinal fluid MMP concentrations (23). MMP-1 and MMP-3

*Department of Infectious Diseases and Immunity, Hammersmith Campus, ImperialCollege London; ‡Department of Clinical Neuroscience, Charing Cross Campus,Imperial College London; xMedical Research Council Laboratory of Molecular CellBiology, University College London, London; and †School of Biological Sciences,University of East Anglia, Norwich, United Kingdom

Received for publication November 30, 2009. Accepted for publication March 27,2010.

J.A.G. was supported by the James Maxwell Grant Prophit Tuberculosis Fellowshipfrom The Royal College of Physicians London, U.K., and by a Medical ResearchCouncil (U.K.) Clinical Training Fellowship. S.D. was supported by a bursary fromThe Pathology Society, U.K. P.T.E. is funded by a U.K. National Institutes of HealthResearch Intermediate Fellowship. R.C.M. is supported by a Wellcome Trust ClinicalResearch Training Fellowship. J.S.F., D.A., and P.T.E. are grateful for support fromthe National Institutes of Health Research Biomedical Research Centre fundingscheme. The funders had no role in study design, data collection and analysis, de-cision to publish, or preparation of the manuscript.

Address correspondence and reprint requests to Dr. Justin A. Green, Department ofInfectious Diseases and Immunity, Imperial College London, Hammersmith Campus,Du Cane Road, London W12 0NN, U.K. E-mail address: justin.green@imperial.ac.uk

The online version of this article contains supplemental material.

Abbreviations used in this paper: CoATb, conditioned medium from astrocytes in-fected with Mycobacterium tuberculosis; CoMCont, conditioned medium from un-infected monocytes; CoMTb, conditioned medium from monocytes infected withMycobacterium tuberculosis; Ct, cycle threshold; MMP, matrix metalloproteinase;MOI, multiplicity of infection; SELDI-TOF, surface-enhanced laser desorption ion-ization time-of-flight; TB, tuberculosis; TIMP, tissue inhibitor of metalloproteinase.

Copyright� 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903811

by guest on Novem

ber 2, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

are both implicated in CNS inflammation (9, 24). MMP-9 (gelati-nase B) is also activated by MMP-3 and degrades collagenase lysisproducts; hence MMP-1, -3, and -9 act in a coordinated manner todegrade matrix proteins (25). The major source of CNS MMP-1 islikely to be cells of the monocyte lineage because astrocytes, oli-godendrocytes, and neurons do not secrete MMP-1 (26). Microglia,the resident CNSmacrophages, may be a critical source ofMMPs inresponse to inflammatory stimuli, but their role in CNS TB has neverbeen investigated (27).Control of MMP gene expression is via cis-acting elements in

the promoter, often shared between different MMPs, leading to theircoexpression in response to inflammatory signals (15). NF-kB isa key transcriptional regulator of many proinflammatory genes, in-cluding some MMPs (28). There are five subunits of NF-kB: p65(RelA), p50, p52,RelB, and c-Rel.Deletionof each subunit leads to acharacteristic phenotype in mice (29). The MMP-1, but not MMP-3,promoter has one active NF-kB consensus binding site (30). AP-1 isa second major transcription factor regulating MMP secretion(31). TheMMP-1 and -3 promoters havemultiple AP-1 binding sitesupstream of the transcription start site (32). AP-1 is composed ofheterodimers, formed from a family of seven related proteins derivedfrom the fos and jun gene families: JunB, c-Jun, JunD, FosB, c-Fos,Fra-1, and Fra-2. In TB, AP-1 subunits JunB, c-Jun, and FosB havea role in control of CCL5, MMP-9, and IFN-g gene expression (21,33, 34). In addition, corticosteroids may regulate gene transcription,both via nuclear localization and binding to glucocorticoid responseelements and by inhibition of NF-kB DNA binding, or trans-repression. There are no published data concerning control ofMMP-1 and -3 secretion in CNS TB.We investigated the hypothesis that microglia are central to the

MMP response in CNS TB. We investigated gene expression andsecretion following direct M. tuberculosis infection of microgliaand, in the context of CNS cellular networks, in a cellular model ofCNS TB. Next, we studied for the first time both MMP and tran-scription factor expression in brain biopsies of patientswithCNSTB.We demonstrate that M. tuberculosis-driven monocyte-microglialnetworks increase MMP-1 and -3 but not TIMP-1 to -4 gene ex-pression and secretion. Networks are driven by the proinflammatorycytokines TNF-a and IL-1b and regulated by NF-kB and AP-1 bothin vitro and in CNS TB patients. These effects are opposed bydexamethasone, which may partly explain the beneficial effect ofcorticosteroids in the treatment of CNS TB.

Materials and MethodsReagents

Chemicals were from Sigma-Aldrich (Gillingham, U.K.), tissue culturematerials were from Invitrogen (Paisley, U.K.), and tissue culture plasticwas from Techno Plastic Products (Trasadingen, Switzerland) unless oth-erwise stated. Recombinant TNF-a, IL-1b, anti–TNF-a neutralizing Abs,and IL-1Ra were from PeproTech (Rocky Hill, NJ). Helenalin (BIOMOL,Exeter, U.K.) and SC-514 (Merck, Nottingham, U.K.) were used in NF-kBinhibition experiments.

M. tuberculosis culture

The virulent M. tuberculosis strain H73Rv Pasteur and a GFP-expressingvariant (gift of Brian Robertson, Imperial College London) were culturedin Middlebrook 7H9 broth (BD Biosciences, Oxford, U.K.) with 10% oleicacid, albumin, dextrose, catalase enrichment medium, 0.2% glycerol, and0.02% Tween 80 with agitation. M. tuberculosis at OD 0.6 (Biowave CellDensity Meter; Biochrom, Cambridge, U.K.), corresponding to 108 CFUper milliliter was used for infection. Endotoxin level was,0.03 ng/ml LPS(Amoebocyte Lysate Assay; Associates of Cape Cod, Falmouth, MA).

Cell culture and M. tuberculosis infection

Monocytes were isolated from healthy blood donors (U.K. Blood Trans-fusion Service) by Ficoll-Paque (GE Healthcare, Little Chalfont, U.K.)

density gradient centrifugation and adherence purification (17). Monocyteswere infected with M. tuberculosis at a multiplicity of infection (MOI) of1. At 24 h, cell culture medium was collected and 0.2-mm sterile-filtered(Nalgene, Hereford, U.K.). Conditioned medium from M. tuberculosis-infected monocytes was termed CoMTb, and conditioned medium fromuninfected monocytes was termed CoMCont. Initial experiments demon-strated a dose–response effect for CoMTb stimulation, and after this 1:5dilution was used to stimulate cells in all of the experiments.

Human CHME3 microglial cells (used with permission of Marc Tardieu,Paris, France, and kindly provided by Nicola Woodroofe, Sheffield HallamUniversity, Sheffield, U.K.) were maintained in DMEM with 10% FCS(Biosera, Ringmer, U.K.) and 3 mM glutamine. Cells were seeded at 35–50,000 cells per square centimeter for experiments, which were performedin macrophage serum-free medium (Invitrogen). In direct infection ex-periments, samples were sterilized by filtration through a 0.2-mm Duraporemembrane (Millipore, Watford, U.K.) that does not remove MMPs (35).Preparation of conditioned medium from infected microglia termedCoMicTb (and control medium CoMicCont) was as per CoMTb above,except cells were infected at a MOI of 10 in macrophage serum-freemedium. Trypan blue was used to assess cell viability.

Human fetal astrocytes (a gift of V. Wee Yong, University of Calgary,Calgary, Canada) were obtained as described (36) and maintained in MEMcontaining 0.1% dextrose, 1 mM sodium pyruvate, 0.1 mM nonessentialamino acids, 0.2 mM glutamine, and 10% FCS. Flasks were coated with10 mg/ml polyornithine to aid adhesion. Cultures were .99% pure asshown by immunostaining with Ab to glial fibrillary acidic protein (36).Preparation of conditioned medium from infected primary human as-trocytes (CoATb) was as for CoMTb above, except serum-free MEM anda MOI of 10 was used.

Gene expression analysis by real-time quantitative PCR

Microglia were lysed with TRI Reagent (Sigma-Aldrich), and total RNAwas extracted. cDNA was prepared from 1 mg RNA, reverse-transcribedusing 2 ml random primers and 200 U SuperScript III reverse transcriptase(both Invitrogen), according to the supplier’s instructions. QuantitativePCRs were performed as described (37). The cycle threshold (Ct) at whichamplification entered the exponential phase was determined, and thisnumber was used to indicate the amount of target RNA in each sample.MMP Cts were normalized to ribosomal 18S mRNA.

Casein zymography

A total of 20 ml cell culture medium with 5 ml loading buffer [0.25M Tris(pH 6.8), 50% glycerol, 5% SDS, and bromophenol blue crystals] wasloaded on 12% casein gels (Invitrogen). Gels were run at 125 V for 2.5 h(buffer 25 mM Tris, 190 mM glycine, and 0.1% SDS). After 1 h incubationin 2.5% Triton X-100, gels were transferred to collagenase buffer [55 mMTris (pH 7.6), 200 mM NaCl, 5 mM CaCl2, and 0.02% Brij] for 40 h at37˚C. Gels were stained with 0.2% Coomassie blue (Pharmacia Biotech,Uppsala, Sweden) for 1 h before destaining in acetic acid/methanol/water(1:3:6). A total of 5 ng MMP-1 standard (Merck, Frankfurt, Germany) wasrun on each gel. Gels were analyzed densitometrically by Scion ImageAnalysis (National Institutes of Health Image, version 1.61, National In-stitutes of Health, Bethesda, MD) normalized to standard.

Western blotting

Standard Western blotting was performed as described (18). Overnightincubation in 1:1000 MMP-1 primary Ab (The Binding Site, Birmingham,U.K.) was followed by washing and incubation with 1:2000 peroxidase-conjugated secondary Ab for an hour (The Binding Site). Bands werevisualized by chemiluminescence with the ECL Plus system (GE Health-care) according to the manufacturer’s instructions. For cytoplasmic IkBa,Western cytoplasmic extracts were prepared (see below), and 15 mg pro-tein aliquots were processed as above and probed with 1:1000 primaryAb (Millipore) and 1:2000 HRP-linked rabbit secondary Ab (Cell SignalingTechnology, Danvers, MA). For cytoplasmic b-actin IkBa probing, mem-branes were stripped for 15 min with Restore Plus Western Blot StrippingBuffer (Pierce Biotechnology, Rockford, IL), reblocked with 5% milkprotein/0.1% Tween 20, and probed with 1:1000 mouse anti–b-actin pri-mary Ab (Sigma-Aldrich), then 1:5000 anti-mouse secondary Ab (JacksonImmunoResearch Laboratories, West Grove, PA).

Luminex analysis

MMP-1 concentrations were analyzed by Fluorokine multianalyte profilingkit according to the manufacturer’s protocol (R&D Systems, Abingdon,U.K.) on the Luminex platform (Bio-Rad, Hemel Hempstead, U.K.). Theminimum level of detection for MMP-1 was 10 pg/ml. CoMTb cytokine

The Journal of Immunology 6493

by guest on Novem

ber 2, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

and chemokine concentrations were analyzed using Bio-Rad cytokinemultiplex beads according to the manufacturer’s instructions to constructan eight-point low reading standard curve with a lower level of sensitivityof 1 pg/ml for all of the analytes.

MMP-3, TIMP-1, and TIMP-2 ELISAs

MMP-3, TIMP-1, and TIMP-2 concentrations in cell culture medium weremeasured by ELISA (R&D Systems) according to the manufacturer’s in-structions. The lower level of detection was 30 pg/ml.

Size exclusion chromatography fractionation of CoMTb

Fractionation of CoMTb by 10-kDa Micron YM-10 centrifugal filter units(Millipore) was performed according to the manufacturer’s instructions.

Surface-enhanced laser desorption ionization time-of-flightmass spectroscopy of CoMTb

CoMTb was concentrated 320 with a Micron YM-3 size exclusion columnfollowed by reconstitution in RPMI. CoMTb was thawed on ice and de-natured for 30 min on ice in 9 M urea, 2% w/v CHAPS, 2% v/v DTT,and 50 mM Tris. Samples were applied to equilibrated CM10 chips (Ci-phergen, Fremont, CA), incubated for 1 h at 500 rpm, washed five times,rinsed, and air-dried. A saturated solution of sinapinic acid (Sigma-Aldrich)was applied to each spot on the chip and allowed to dry. Chips were read ona PBS II mass spectrometer (Ciphergen). Laser intensity was set at 215(range 0–300), detector sensitivity was set at 7 (range 0–10), and spectrawere optimized between 2000 and 20,000 Da. The mass spectrometer wascalibrated with the all-in-one peptide calibrant (Ciphergen) according to themanufacturer’s instructions. Spectra were collected and analyzed in Pro-teinchip software, version 3.2.1 (Ciphergen), and peaks were detected usingthe software’s automatic peak detection and detection of multicharge peaksfunctions.

Confocal microscopy

Microglial cells and monocytes were plated onto four-well Lab-Tek Per-manox chamber slides (Nunc, Roskilde, Denmark) and infected with GFP-expressing M. tuberculosis. After 2 h, cells were fixed in 4% para-formaldehyde and permeabilized for 10 min in 0.2% Triton X-100 in PBS.After three washes, cells were incubated for 10 min with a tetrame-thylrhodamine isothiocyanate–phalloidin conjugate (Invitrogen) at a con-centration of 250 ng/ml to bind F-actin. After three further washes, cellswere mounted with 700 ml S3023 mountant (Dako, Glostrup, Denmark) andcovered with a 22 mm 3 50 mm coverslip and left overnight to harden.

Confocal microscopy was performed as described (38), using a LSM 510laser-scanning microscope equipped with a 633 oil immersion objective(Zeiss, Thornwood, NY). Laser excitation at 488 and 532 nm was used.Images were collected as horizontal sections taken at 0.1-mm intervalsthrough whole-cell volumes. Images from representative areas of in-dividual cells were taken, and y–z projections were compiled using ImageJsoftware (National Institutes of Health). Composites were imported intoAdobe Photoshop CS3 (Adobe Systems, San Jose, CA) and recreated aspseudocolor overlays (phalloidin staining in red and GFP-M. tuberculosisstaining in green).

Immunohistochemistry

Tissue samples were provided by the Human Biomaterials Resource Centreat Imperial College Healthcare National Health Service Trust. Ethicalconsent for the study of anonymized paraffin-embedded sections from thehistopathology archive was obtained from the Hammersmith HospitalsResearch Ethics Committee in accordance with The Human Tissue Act2004. Five nonimmunosuppressed patients with biopsy-proven CNSM. tuberculosis infection were analyzed (39). Two positive control pa-tients were analyzed who had undergone temporal lobectomy for in-tractable epilepsy. Tissue from beyond the epileptic focus at the excisionmargin was examined. Two negative control tissues examined were frompostmortem, macroscopically normal brains, from subjects with no his-tory of neurologic or infectious disease. Sections were immunostained forIba-1, a macrophage/microglial marker (rabbit polyclonal, WAKO PureChemical Industries, Osaka, Japan), MMP-1 (mouse monoclonal, Merck),c-Jun (rabbit polyclonal, AbD Serotec, Kidlington, U.K.), p65 (rabbitpolyclonal, Santa Cruz Biotechnology, Santa Cruz, CA), and phospho-p38(mouse monoclonal, Sigma-Aldrich). Sections of 5 mm in thickness weredewaxed in xylene and rehydrated. Endogenous peroxidase activity wasblocked with 0.3% hydrogen peroxide for 30 min. Sections were steamedfor 20 min [0.01 M citrate (pH 6.5)] and blocked with 10% normal goatserum for 10 min (Vector Laboratories, Burlingame, CA). The primary

Abs (Iba-1 at 1:400, MMP-1 at 1:150, p65 at 1:100, c-Jun at 1:200, andp38 at 1:200 dilutions) were applied for 16–20 h at 4˚C. Staining wasvisualized by the Vectastain Elite ABC kit (Vector Laboratories). Thereaction product was revealed with 2 mg/ml 3,39-diaminobenzidine and0.0075% hydrogen peroxide in PBS. p65 and c-Jun were developed usingcomponents of the Super Sensitive Polymer-HRP IHC Detection System(BioGenex Laboratories, Dan Ramon, CA). Finally, the slides werecounterstained with hematoxylin, dehydrated in graded ethanol, andcovered with a coverslip in di-n-butyl-phthalate. CNS TB biopsy negativecontrols included both full staining and omission of the primary Ab.

Immunofluorescence

Deparaffinized and rehydrated 5-mm tissue sections were steamed in citratebuffer (pH 6.0). Sections were blocked with 10% normal goat serum (VectorLaboratories). After 16–20 h incubation with anti–MMP-1 Ab at 4˚C, slideswere incubated for 1 h with secondary Ab conjugated to a fluorescent dye(1:200 dilution, Alexa Fluor 488, Invitrogen). After incubation with 10%normal goat serum inPBS, the anti–Iba-1Abwas applied for 2 h at 4˚C. Slideswere incubated with the secondary Ab conjugated to a second fluorescentdye (1:200 dilution, Alexa Fluor 555, Invitrogen) for 1 h. Between each step,sections were washed with PBS three times for 5 min. To reduce auto-fluorescence, tissue sections were treated with 0.3% Sudan black B (BDHChemicals, Poole, U.K.) in 70% ethanol. Images were obtained usinga E1000M imaging fluorescence microscope (Nikon, Tokyo, Japan) anda QICAM digital camera (QImaging, Surrey, Canada) and analyzed usingImage Pro Plus software (Media Cybernetics, Bethesda, MD). For negativecontrols, primary Abs were omitted.

Detection of NF-kB and AP-1 nuclear binding

Nuclear and cytoplasmic extracts were prepared with a kit according to themanufacturer’s instructions (Active Motif, Rixensart, Belgium). Nuclearextracts were analyzed by the TransAM kit (Active Motif). Primary Abswere used to detect p50, p52, p65, RelB, or RelC. Competition experi-ments demonstrated specificity of binding by adding 20 pM per well ofeither wild-type or mutated NF-kB oligonucleotide before assaying withthe p65 Ab. Primary AP-1 Abs were to JunB, c-Jun, JunD, FosB, c-Fos,Fra-1, and Fra-2.

Promoter-reporter analysis

Microglial cells were cotransfected with MMP-1 promoter constructs (giftofIanClarke,UniversityofEastAnglia,Norwich,U.K.,andMatthewVincenti,Dartmouth Medical School, Hanover, NH) and a control reporter plasmid,constitutively expressingRenilla luciferase, with FuGENEHD (Roche, BaselSwitzerland). Cells were stimulated after 12 h, and luciferase activity wasanalyzed 24 h later by Dual-Luciferase Reporter Assay System (Promega,Madison, WI) on a luminometer (Medical Devices, Wokingham, U.K.). Re-nilla luciferase activity was used to normalize firefly activity to control fortransfection efficiency. For the NF-kB, site a mutation construct was madebetween 22886 and 22878 bp altering 59-ATGGAAAAA-39 to 59-ATG-GAACCC-39 In addition, the AP-1 site at21949 to21955wasmutated from59-TGAGTTA-39 to 59-TGATTTA-39 (40).

Statistical analysis

Data are presented as means (6SD) of three samples and represent ex-periments performed in triplicate on at least two occasions, unless other-wise stated. Paired groups were compared with the Student t test. Multipleintervention experiments were compared by one-way ANOVA and Tukey’scorrection for multiple pairwise comparisons with equal variance tested byLevene’s test. p , 0.05 was taken as significant. All of the analyses weredone using SPSS software (version 15.0; SPSS, Chicago, IL).

ResultsGlobal analysis of MMP gene expression inM. tuberculosis-infected and CoMTb-stimulated humanmicroglia

First, we analyzed gene expression of all 23 humanMMPs and fourTIMPs in unstimulated (control) microglia at 24 h compared withdirect M. tuberculosis infection and stimulation with media fromuninfected monocytes (CoMCont) or M. tuberculosis-infectedmonocytes (CoMTb). There were relative increases in MMP-1, -3,and -9 gene expression normalized to 18S RNA of 3.66-, 3.34-, and2.8-fold, respectively, after CoMTb stimulation compared with

6494 MMPs IN CNS TB

by guest on Novem

ber 2, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

CoMCont stimulation (all p , 0.05, summarized in SupplementalFig. 1). PCR Ct data were also used to compare absolute levels ofgene expression presented as value (SD, and fold change over

CoMCont). Low Ct values indicate a high level of gene expression(Fig. 1A). MMP-1 and -3 were the most highly upregulated byCoMTb, with mean Ct thresholds of 22.9 (60.6, 4.1-fold) and 24.8

FIGURE 1. CoMTb upregulates MMP-1 and -3 gene

expression and secretion in human microglia. A, PCR

Cts for MMP and TIMP gene expression in human mi-

croglia stimulated by M. tuberculosis and CoMTb. The

mean Ct at which PCR amplification enters the loga-

rithmic phase is shown with increasing grayscale from

unshaded through to black reflecting increasing mRNA

expression. Of the MMPs upregulated by CoMTb,

MMP-1 and -3 were the most highly expressed. B,

CoMTb drives greater MMP-1 secretion in microglia

than M. tuberculosis, analyzed by casein zymography,

confirmed by Western blotting. C, Kinetics of MMP-1

and -3 gene expression. Microglia were stimulated with

CoMCont or CoMTb, and mRNA accumulation was

analyzed by real-time PCR (mRNA levels normalized to

18S RNA, fold change relative to baseline control).

Mean values 6 SD from three experiments are pre-

sented.D, MicroglialMMP-3 secretion after stimulation

by CoMTb or direct M. tuberculosis infection analyzed

by ELISA. E, TIMP-1 secretion, analyzed by ELISA, is

unaffected byCoMTb.F, TIMP-2 secretion, analyzed by

ELISA, is suppressed byCoMTb.G,MMP-3 secretion is

upregulated in response to CoMTb but not CoATb. For

all of the experiments, except C, bars represent mean

values6 SD of three samples, representative of at least

duplicate experiments performed in triplicate. pp ,0.05; ppp , 0.01.

The Journal of Immunology 6495

by guest on Novem

ber 2, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

(61.4, 3.7-fold) respectively (p , 0.01 and p , 0.05), whereasMMP-9 and -10 had moderate levels with Ct values.25 (2.8-fold,p = 0.05, and 2.4-fold, p, 0.01, respectively).M. tuberculosis aloneupregulated only MMP-27 (p , 0.05). MMP-7, -20, -21, -26, and-28 expression were not detected (data not shown). TIMP-1 to -4gene expression was not altered by direct infection or by CoMTb.

Monocyte-microglial networks drive greater MMP-1 and -3secretion from human microglia than direct M. tuberculosisinfection

M. tuberculosis upregulated MMP-1 in a dose-dependent fashion toa maximum of 4.1-fold over control at a MOI of 10 (p, 0.01; Fig.1B). Confocal microscopy confirmed that microglia were infectedby M. tuberculosis at a MOI of 10 (data not shown). CoMTbstimulation caused a much greater 20.6-fold increase in MMP-1secretion (p , 0.01; Fig. 1B). Western blotting confirmed thatbands on casein zymography were MMP-1 (Fig. 1B, lower panel).Kinetic studies demonstrated that MMP-1 mRNA accumulates by6 h and progressively increased until 48 h with a 23.4 (64.7)-foldupregulation of gene expression (p , 0.01; Fig. 1C). MMP-3gene expression increased more rapidly and peaked at 24 h witha 34.8 (64.8)-fold upregulation of mRNA (p , 0.01; Fig. 1C).CoMTb resulted in 92.1 6 13.0 ng/ml MMP-3 secretion at 72 h,which represented a 25.3 (614.1)-fold increase over CoMCont(p , 0.01), with direct infection having minimal effect (Fig. 1D).CoMTb stimulation did not alter TIMP-1 secretion (Fig. 1E) andsuppressed TIMP-2 secretion 29% compared with that of CoM-Cont-stimulated cells (p , 0.05; Fig. 1F). Thus, the net effectof CoMTb on MMP and TIMP secretion will be a shift towardmatrix degradation.CoATb did not stimulate microglial MMP-1 or -3 secretion (Fig.

1G and data not shown), demonstrating that astrocyte networks arenot key in driving microglial MMP secretion. Conditioned mediumfrom microglia infected with TB (CoMicTb) similarly did not up-regulate MMP secretion by microglial cells (data not shown).

MMP-1 and -3 are expressed by microglia within granulomasof patients with CNS TB

To investigate the clinicopathological significance of cellular data,immunohistochemistry of brain biopsies from five patients withproven CNS TB was performed. Iba-1 immunoreactivity, a markerof microglia/macrophages, was strong in both parenchymal braintissue and TB granulomas (Fig. 2A). MMP-1 immunoreactivitywas greatest in the center of TB granulomas and also presentassociated with microglia outside granulomas (Fig. 2B). As-trocytes, oligodendrocytes, and neurons did not stain for Iba-1 orMMP-1. Dual immunofluorescence staining confirmed colocali-zation of MMP-1 with microglia (Fig. 2D–F). MMP-3 immuno-reactivity was predominantly localized to the perigranulomaregion (Fig. 2G–I). No such positive staining was seen in the twocontrol normal brains.

Dexamethasone suppresses MMP-1 and -3 secretion inCoMTb-stimulated microglia

Dexamethasone is of proven benefit in treating patients with CNSTB, although its mechanism of action is unknown (4, 41). Pre-incubation with 0.01 mM dexamethasone decreased CoMTb-induced MMP-1 mRNA expression to basal levels (p , 0.01; Fig.3A). Similarly, dexamethasone decreased MMP-1 gene expressionto undetectable levels in M. tuberculosis-infected cells. Dexa-methasone also decreased CoMTb-induced MMP-3 expression tobasal levels (p , 0.05; Fig. 3B). Dexamethasone had no effect onTIMP-1 and TIMP-2 gene expression (Fig. 3C). MMP-1 secretionwas suppressed 19.0-fold, and MMP-3 secretion was suppressed

3.1-fold (p , 0.01; Fig. 3D, 3E). Preincubation with dexameth-asone did not alter CoMTb-stimulated TIMP-1 secretion or furthersuppress TIMP-2 secretion. In the presence of 0.01 mM dexa-methasone, TIMP-1 secretion was 29.5 6 1.0 ng/ml, and TIMP-2secretion was 14.76 1.1 ng/ml (p = NS, comparison with CoMTbalone; see Fig. 1E, 1F for comparison).

Investigation of the mediators in CoMTb driving microglialMMP-1 and MMP-3 secretion

To identify mediators in CoMTb stimulating MMP-1 and -3 se-cretion, microglia were stimulated with spin column fractionationCoMTb aliquots of,10 and.10 kDa. Only the.10-kDa fractionupregulated MMP-1 (Fig. 4A). With surface-enhanced laser de-sorption ionization time-of-flight (SELDI-TOF) mass spectros-copy, a 17-kDa peak was identified that was retained in the .10-kDa fraction and disappeared with preincubation of CoMTb with1 mg/ml anti-TNF (Fig. 4B). rTNF-a reproduced this 17-kDapeak. Preincubation of CoMTb with anti–TNF-a neutralizing Absabolished the effect of CoMTb on microglial MMP-1 secretion,confirming a critical role for TNF-a (Fig. 4C). Because we havepreviously shown IL-1b to be critical for MMP-1 secretion instromal cells (42), we preincubated cells with IL-1Ra, whichcaused a 63% decrease in CoMTb-induced MMP-1 activity (p ,0.01; Fig. 4D).Next, we measured TNF-a and IL-1b concentrations in CoMTb

(Supplemental Fig. 2) and stimulated microglia with recombinantcytokines. TNF-a (100 ng/ml, 62.5-fold more than in 1:5 dilutedCoMTb) only stimulated 32.9% of the level of MMP-1 secretiongenerated in response to CoMTb, and IL-1b alone had no effect(Supplemental Fig. 3A, 3B). Addition of TNF-a and IL-1b to-gether at concentrations in excess of those in CoMTb showed anadditive effect only (Fig. 4E). Similar data were observed forMMP-3 secretion after pretreatment with anti–TNF-a and IL-1Ra

FIGURE 2. Microglia express MMP-1 and -3 in granulomas of patients

with CNS TB. Brain biopsies from five patients with TB and two positive

and negative controls were immunostained for the microglial/macrophage

marker Iba-1, MMP-1, and MMP-3 as described inMaterials and Methods.

Areas of immunoreactivity appear as brown against the blue counterstain. In

CNS TB granulomas, microglial cells are immunoreactive for (A) Iba-1 and

(B) MMP-1. Arrow heads delineate granuloma. C, Negative control (A–C,

original magnification 320). D, Iba-1 immunofluorescence. E, MMP-1

immunofluorescence. F, Merged Iba-1 and MMP-1 image demonstrates

colocalization of staining. Images shown are representative of five biopsies

studied (D–F, original magnification 340). G and H, MMP-3 immunore-

activity in a typical CNS TB granuloma (G, original magnification 320;

H, original magnification340). In both panels, there is a transition between

the heavily immunoreactive granuloma center (top right of panel) and more

lightly stained perigranulomatous fibrosis (left of panel). I, MMP-3 control

(omission of primary Ab; original magnification320).

6496 MMPs IN CNS TB

by guest on Novem

ber 2, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

use (Supplemental Fig 4A–D). In contrast to the effect on MMP-1,synergy between these proinflammatory cytokines on MMP-3secretion was observed (Fig. 4F).

NF-kB and AP-1 regulate CoMTb-induced microglial MMP-1secretion in vitro and in patients

In a kinetic analysis of DNA binding activity, NF-kB p50 subunitbinding inCoMTb-stimulatedmicroglia increased 2.0-fold comparedwith that ofCoMCont-stimulated cells at 1 h,withmaximal activity at4 h (p , 0.05; Fig. 5A). p65 DNA binding increased 3.1-fold at 1 h(p , 0.05; Fig. 5B). RelB binding reached a maximal 2.0-fold in-crease at 4 h (p,0.05; Fig. 5C).A sustained increase inDNAbindingof RelB subunits was still present at 24 h (p, 0.05). No changes inc-Rel or p52 activity were detected (Fig. 5D, 5E). Degradation ofcytoplasmic IkBa occurred by 10 min in CoMTb-stimulated cells,with resynthesis by 60 min (Fig. 5F). Helenalin, an irreversible p65alkylator, blocked nuclear translocation of p65 and p50 to belowbasal levels (Fig. 5G). Additionally, helenalin completely inhibitedMMP-1 secretion in a dose-dependent manner (Fig. 5H). Inhibitor ofNF-kB kinase 2 blockade by SC-514 suppressed MMP-1 mRNAaccumulation (Fig. 5I) and secretion (Supplemental Fig. 5). Nochange in cell viability or basalMMP secretionwas observed in theseexperiments.Next,wedemonstrated, for thefirst time in the humanCNS, that in

TB patients nuclear p65 staining is present in an increasing gradient

toward the center of the granuloma. Specifically, nuclear p65 wasmost strongly expressed in granuloma-associated microglia andnot seen in negative control biopsies (Fig. 6A, 6B). To investigatetranscriptional regulation further, a series of MMP-1 promoter-reporter constructs were transfected into microglial cells (Fig. 6C).CoMTb increased full-length promoter activity above CoMContlevels within 10 h (p, 0.01), peaking at 24 h (p, 0.01; Fig. 6D).With a series of 59 truncationmutant constructs, a 70.4%decrease inpromoter activity between 22942 and 22001 bp upstream of thetranscription start site was demonstrated (p , 0.01), with a further13.9% decrease below baseline between 22001 and 21551 (p ,0.05; Fig. 6E). In experiments using constructs with site-specificmutation of the single consensus NF-kB site (at 22886 to 22878bp) CoMTb-stimulated promoter activity decreased to 22.56 4.7%of wild type (p, 0.01; Fig. 6F). Furthermore, with a construct witha mutation in the AP-1 site at 21949 to 21955 bp, such promoteractivity decreased similarly to 30.4 6 6.4% (p , 0.01; Fig. 6F).Therefore, we investigated AP-1 activity further.After CoMTb stimulation of microglial cells in vitro, binding of

AP-1 subunits c-Jun and FosB increased at 1 h 111.5 and 83.4%,respectively (both p , 0.05; Fig. 7A). Findings at a 6 h time pointwere similar (data not shown). To investigate whether our in vitrofindings in the cellular model reflected transcriptional regulation inpatients with active CNS TB, we performed immunohistochemistryfor AP-1 activation. We demonstrated that in CNS TB, but not

FIGURE 3. Dexamethasone suppresses CoMTb-

inducedMMP-1 and -3 gene expression. Microglia were

infected withM. tuberculosis (MOI 10, gray shading) or

stimulated with CoMTb (filled bars), with or without

preincubation with 0.01 mM dexamethasone for 2 h.

Control (openbars) andCoMCont (diagonal shading) are

also shown. A, Dexamethasone suppressedMMP-1 gene

expression at 24 h in infected microglia to below control

levels and to basal levels in CoMTb-stimulated cells. B,

Dexamethasone suppressed MMP-3 gene expression to

basal levels in CoMTb-stimulated cells.C,Mean PCRCt

for MMP-1, MMP-3, TIMP-1, and TIMP-2 gene ex-

pression. A low Ct indicates high mRNA levels. MMP-1

and -3 were the most highly suppressed MMPs by

dexamethasone, and TIMP gene expression was un-

altered. Mean values 6 SEM are given for three ex-

periments performed on separate vials of cells. D and E,

Dexamethasone suppresses CoMTb-induced MMP-1

and -3 secretion. Data were analyzed by one-way

ANOVA, followed by Tukey’s multiple comparison.

pp , 0.05; ppp, 0.01.

The Journal of Immunology 6497

by guest on Novem

ber 2, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

negative controls, biopsies stained positively for c-Jun. The c-Jundetected was almost exclusively in the perigranuloma region, whichis rich in microglia and macrophages (Fig. 7B–E).

DiscussionCNS TB has a very high morbidity and mortality even if optimallytreated. Because outcome is improved by adjuvant dexamethasone,excessive neuroinflammation might contribute to poor outcome. Inthis study, we define a potential mechanism for this CNS tissuedestruction and for the protective effect of dexamethasone. Im-portantly, we confirm key in vitro findings of unopposed, NF-kB/AP-1–driven upregulation of MMP-1/-3 expression and secretionin CNS biopsies from TB patients. We first investigated our hy-pothesis that infiltrating monocytes and microglia, the residentCNS innate cell, are key drivers of the intense tissue-destructiveprocess observed in patients with CNS TB. MMP-1 and -3 were

the principal MMPs upregulated by microglia in TB as a result ofactivation of monocyte-dependent networks. MMP-1 and -3 geneexpression and secretion were dependent in part on IL-1b andTNF-a activity, which may act in synergy. NF-kB and AP-1transcription factors binding at specific sites in the MMP-1 pro-moter are key regulators of gene expression. Dexamethasone

suppressed MMP-1 and -3, but not TIMP-1 and -2, gene expres-

sion and secretion, which is the first time that a potential mech-

anism explaining the beneficial effect of steroids in the treatment

of CNS TB has been identified. The significance of findings in the

cellular model were strengthened by investigation of MMP se-

cretion and NF-kB and AP-1 transcription factor activation in

the invaluable clinical resource of biopsy specimens from CNS

TB patients.In the comprehensive analysis of MMP and TIMP gene expres-

sion in human microglia, M. tuberculosis-dependent networks

FIGURE 4. TNF-a and IL-1b in CoMTb are nec-

essary, but not sufficient, for MMP-1 and -3 secretion.

A, CoMTb was fractionated by m.w. exclusion spin

columns into ,10- and .10-kDa fractions. Only

the .10 kDa fraction upregulated MMP-1 secretion.

Densitometric analysis of MMP-1 secretion was con-

firmed by Western blotting. B, SELDI-TOF mass

spectroscopy of CoMTb shows a unique 17-kDa peak

in CoMTb that is abolished by anti–TNF-a treatment

and replicated by rTNF-a. C and D, Anti–TNF-a (C)

and IL-1Ra preincubation (D) with CoMTb for 2 h

reduced MMP-1 secretion by CoMTb-stimulated cells.

Data shown are mean 6 SD of three samples plus

a representative casein zymogram. E, TNF-a and IL-

1b have an additive effect on MMP-1 secretion. F, IL-

1b or TNF-a alone do not cause MMP-3 secretion but

together act synergistically. Bars are mean values 6SD of three samples, representative of at least dupli-

cate experiments performed in triplicate. Data were

analyzed by one-way ANOVA, followed by Tukey’s

multiple comparison. pp , 0.05; ppp , 0.01.

6498 MMPs IN CNS TB

by guest on Novem

ber 2, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

markedly increased MMP-1, -3, and -9 gene expression and moder-ately increased MMP-8 and -10 expression. MMP-1, -3, and -9 mayact on substrates specific to the CNS: MMP-1 degrades brevicanand tenascin, MMP-3 and -9 can degrade myelin and tight junctionproteins, andMMP-1 and -3 are both proapoptotic to neurons (9, 43).The expression profile was relatively M. tuberculosis-specific, be-cause LPS-stimulated human microglia express MMP-1, -3, -8, -10,and -12 genes (27). Similarly, in rat microglia, LPS upregulated onlyMMP-1, -3, and -8 expression (44).

CoMTb, but not conditioned media fromM. tuberculosis-infectedastrocytes ormicroglia, stimulated high level secretion ofMMP-1 and-3, demonstrating the importance of monocyte-microglial networks.Direct M. tuberculosis infection of microglia only caused moderateincreases inMMP-1and -3 secretion anddid not altergene expression.Direct infection did not upregulate MMPs in microglia other thanMMP-27, whose Ct valuewas very low, suggesting that this was onlya moderate effect. M. tuberculosis was internalized with microglialcells on confocal microscopy, and our data are comparable with

FIGURE 5. CoMTb stimulation upregulates NF-kB DNA binding in microglial cells. Microglial cells were stimulated by CoMCont (gray lines) and CoMTb

(black lines).Nuclear extractswere analyzedbyELISA forNF-kB subunits. CoMTb stimulates (A)maximal nuclear p50binding at 4 h, (B)maximal p65 binding at

1 h, and (C) nuclear RelB accumulation sustained beyond 24 h, but no change in binding of (D) c-Rel or (E) p52. Th y-axis is fold change inDNAbinding (OD) over

baseline.Means6SD of data from three independent experiments were compared by Student t test. pp, 0.05; ppp, 0.01.F, RepresentativeWestern blot of three

experiments demonstrating the kinetics of cytoplasmic IkBa degradation.Reprobing of themembrane forb-actin data confirms equal loading.G, Helenalin (5mM)

decreasesp50 (graybars) and p65 (blackbars)DNAbinding activity.H, NF-kBblockade suppressesMMP-1 secretion (analyzedbyLuminex).Bars aremean6SD

of three samples, representative of at least duplicate experiments performed in triplicate. I, MMP-1 gene expression is suppressed by NF-kB inhibition. mRNA

accumulation was analyzed by real-time PCR at 24 h, normalized to 18S RNA, and expressed as fold change relative to 24 h CoMContmRNA (mean6 SD, n = 3

experiments). Data were analyzed by one-way ANOVA, followed by Tukey’s multiple comparison. For H and I, pp, 0.05; ppp, 0.01.

The Journal of Immunology 6499

by guest on Novem

ber 2, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

FIGURE 6. NF-kB is a critical regulator of MMP-1. A, NF-kB p65 is expressed in nuclei of microglia at the center of CNS TB granulomas. Areas of

immunoreactivity appear brown against the blue counterstain (original magnification 340). Arrow demonstrates an example of strong nuclear staining. In

comparison to a negative control, normal postmortem brain fully stained in the same way demonstrates no positive staining (original magnification320). C,

Schematic representation of full-length MMP-1 promoter and deletion constructs. D, MMP-1 promoter kinetics. Microglial cells were transiently transfected

with the full-length MMP-1 promoter and stimulated with CoMCont (gray line) or CoMTb (black line). Firefly luciferase activity, normalized to Renilla lu-

ciferase activity, was analyzed over 48 h. E, MMP-1 promoter deletion constructs. Microglial cells were transfected with full-length MMP-1 promoter ex-

pression plasmid (black bars) or deletion constructs (gray bars) and stimulated with CoMCont or CoMTb for 24 h. CoMTb-stimulated full-length MMP-1

promoter was designated as maximal luciferase activity (100%), and other results are expressed as a percentage of this maximum. F, MMP-1 promoter site-

specificmutation constructs.Microglial cells were transiently transfected with full-length promoter (black bars) or mutation constructs of a NF-kBorAP-1 site

(gray bars), stimulated and analyzed as perD. Bars are mean6 SD from three samples and represent at least three separate experiments performed in triplicate.

Data were analyzed by Student t test (D) or one-way ANOVA, followed by Tukey’s multiple comparison (E, F) pp, 0.05; ppp, 0.01.

6500 MMPs IN CNS TB

by guest on Novem

ber 2, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

previous data (45). The effect of CoMTb on MMP-1 and -3 geneexpression was prolonged and remained detectable at 48 h.We demonstrated that microglia express MMP-1 and -3 at the

site of tissue destruction in patients with active, biopsy-provenCNSTB. Microglial markers and MMP-1 colocalized in both brain pa-renchyma and granulomas, indicating that microglia, as effectorcells of the CNS immune response, contribute to MMP-dependentCNS tissue destruction. However, the marker used Iba-1 (nor anyother available) cannot differentiate between peripheral and cen-trally derived monocytic cells, making interpretation of the relativeroles of these cells more difficult (39). MMP-3 immunoreactivitywas confined to areas of the granuloma rich in microglia, althoughdual staining was not performed. In contrast to the upregulation ofMMPs, microglial TIMP gene expression and secretion were eithernot affected (TIMP-1) or decreased by CoMTb (TIMP-2). Thus, inCNS TB, there is relatively increased secretion ofMMPs comparedwith that of TIMPs suggesting a shift toward matrix degradation.We have reported loss of neuronal TIMP staining in biopsies fromCNS TB patients previously, and macrophages similarly do notupregulate TIMPs in response to M. tuberculosis (18, 20).Dexamethasone demonstrated differential effects upon both

MMP and TIMP gene expression and secretion. MMP-1 and -3were suppressed by preincubation with steroids, but TIMP-1 and -2

remained unchanged. This key finding may explain why steroidshave a beneficial effect clinically in the treatment of CNS TB.MMP-9 is downregulated by dexamethasone in an animal model ofbacterial meningitis, and we have previously described the dif-ferential effect of dexamethasone onMMPs in other model systems(18, 20, 46).Intercellular networks were a greater stimulus toMMP expression

and secretion thandirect infectionwithM. tuberculosis. Fractionationand SELDI-TOF analysis revealed that TNF-a and IL-1b werenecessary, but not sufficient, to reproduce the effect of CoMTb onMMP-1 and -3 secretion. Stimulation with TNF-a and IL-1-b atconcentrations in considerable excess of those in CoMTb were re-quired to cause MMP-1 and -3 secretion similar to that driven byCoMTb. TNF-a and IL-1-b synergized to stimulate MMP-3 but notMMP-1 secretion. TNF-a is known to stimulate MMP-1 secretionfrom various cells but often requires costimulation. We reported thatoncostatin M plus TNF-a drives fibroblast MMP-1 secretion, andZhou et al. (47) showed thatmonocytes requiredGM-CSF in additionto TNF-a (19). IL-1b only stimulated microglial MMP-1 secretionwhen combined with TNF-a. In contrast, IL-1-b alone stimulatedMMP-1 secretion in rabbit synovial fibroblasts or gastric epithelialcells, demonstrating cell-specific regulation (31, 48). Inmicroglia,wedid not identify any other factors responsible for MMP secretion,although in other stromal cells we found that pathogen-derived fac-tors were important (49).CoMTbupregulatednuclearbindingof thep50,p65,andRelBNF-

kB subunits accompanied by rapid, transient IkBa degradation.Helenalin blocked p65 and p50 translocation into the nucleus, in-dicating that p65–p50 heterodimers activate MMP gene transcrip-tion in CNS TB, data supported by using SC-514, an inhibitor ofNF-kB2 found to be highly specific at the concentrations used (50).RelB was upregulated by CoMTb and may form heterodimers withp50, because its other major dimer partner p52 was unchanged byCoMTb. The role of RelB in both TB immunopathology and MMPsecretion are unknown, but RelB null mice have defects in innateresponses to pathogens, such as Toxoplasma gondii (51). Promoter-reporter analysis confirmed a key role for NF-kB.Marked reductionof promoter activity was observed with deletion or mutation of theNF-kB site at 22886 to 22878 bp. Importantly, the data demon-strate p65 activation in biopsies from patients with CNS TB. Al-though no dual stainingwas performed, we observed that toward thecenter of the granuloma, where MMP-1 secretion is detected, p65immunoreactivity was predominantly nuclear.Where inflammationwas less severe, there was reduced NF-kB nuclear localization.In addition, a distal AP-1 site at 21955 to 21945 bp was a key

regulator of promoter activity (40, 52). In microglia, c-Jun andFosB DNA binding were elevated in response to CoMTb stimu-lation. c-Jun and FosB appear central in MMP responses to M.tuberculosis and are upregulated in pulmonary epithelial cells(with c-Fos) and astrocytes (with JunB) (21, 33). In microglia, c-Fos expression was nonsignificantly elevated, and JunB was de-creased. AP-1 has a central role in lipoarabinomannan-inducedMMP-9 secretion, but there are no data on M. tuberculosis-in-duced AP-1–regulated MMP-1 secretion (53). Therefore, our ob-servation that nuclear c-Jun is clearly detected in patient CNS TBgranulomas, where microglia reside, is a key, new finding.In summary, by investigating a combination of patient biopsies and

a cellularmodel of CNSTB,we demonstrated thatMMP-1 and -3 areupregulated in patientswithCNSTB.Monocyte-dependent networksinvolving TNF-a and IL-1 are more important than either directM. tuberculosis infection or glial-dependent networks in drivingMMP-1 and -3 gene expression and secretion from humanmicroglialcells. TIMPs were not upregulated, suggesting that M. tuberculosisuncouples MMP secretion, creating a matrix-degrading phenotype.

FIGURE 7. AP-1 subunit nuclear translocation is upregulated in TB

invitro and in vivo.A, Microgliawere stimulatedwith CoMCont (diagonally

hatched bars) or CoMTb (black bars) for 1 h. Nuclear extracts were assayed

for JunB, c-Jun, JunD, FosB, c-Fos, Fra-1, and Fra-2 activity. The y-axis

represents percentage increase in DNA binding activity over 0 h value. Each

bar is mean6 SEM of data from three independent experiments. pp, 0.05

by Student t test. B, Microglia express c-Jun in patients with CNS TB. Brain

biopsies from five patients with CNS TB and positive and negative controls

were immunostained for c-Jun as described inMaterials andMethods. Areas

of immunoreactivity appear brown against the blue counterstain (original

magnification 310). C, CNS TB granuloma associated microglia/macro-

phages demonstrate strong nuclear c-Jun reactivity (arrows, original mag-

nification 320). D, Negative control, incubated with secondary Ab alone

(original magnification 310). E, Negative control normal brain, incubated

with both primary and secondary Ab (original magnification320).

The Journal of Immunology 6501

by guest on Novem

ber 2, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

We also demonstrated, for the first time in patients, that in CNS TBNF-kB and AP-1 are activated in the same parts of the granulomawhere MMP-1 and -3 expression is seen in human microglial cells.Dexamethasone downregulatedMMP, but notTIMP, gene expressionand secretion, suggesting a potential mechanism of action to limithost tissue destruction. Although dexamethasone improves outcomein CNS TB, significant morbidity andmortality persist, and thereforemore specific targeting of host proinflammatory pathwaysmay revealnovel therapeutic targets in the future.

DisclosuresThe authors have no financial conflicts of interest.

References1. Torok, M. E., T. T. Chau, P. P. Mai, N. D. Phong, N. T. Dung, L. V. Chuong,

S. J. Lee, M. Caws, M. D. de Jong, T. T. Hien, and J. J. Farrar. 2008. Clinical andmicrobiological features of HIV-associated tuberculous meningitis in Vietnam-ese adults. PLoS One 3: e1772.

2. Simmons, C. P., G. E. Thwaites, N. T. Quyen, E. Torok, D. M. Hoang,T. T. Chau, P. P. Mai, N. T. Lan, N. H. Dung, H. T. Quy, et al. 2006. Pretreatmentintracerebral and peripheral blood immune responses in Vietnamese adults withtuberculous meningitis: diagnostic value and relationship to disease severity andoutcome. J. Immunol. 176: 2007–2014.

3. Thwaites, G. E., J. Macmullen-Price, T. H. Tran, P. M. Pham, T. D. Nguyen,C. P. Simmons, N. J. White, T. H. Tran, D. Summers, and J. J. Farrar. 2007.Serial MRI to determine the effect of dexamethasone on the cerebral pathologyof tuberculous meningitis: an observational study. Lancet Neurol. 6: 230–236.

4. Thwaites, G. E., D. B. Nguyen, H. D. Nguyen, T. Q. Hoang, T. T. Do,T. C. Nguyen, Q. H. Nguyen, T. T. Nguyen, N. H. Nguyen, T. N. Nguyen, et al.2004. Dexamethasone for the treatment of tuberculous meningitis in adolescentsand adults. N. Engl. J. Med. 351: 1741–1751.

5. Gandhi, N. R., A. Moll, A. W. Sturm, R. Pawinski, T. Govender, U. Lalloo,K. Zeller, J. Andrews, and G. Friedland. 2006. Extensively drug-resistant tu-berculosis as a cause of death in patients co-infected with tuberculosis and HIVin a rural area of South Africa. Lancet 368: 1575–1580.

6. Conant, K.,N.Haughey,A.Nath, C. StHillaire,D. S.Gary, C.A. Pardo, L.M.Wahl,M. Bilak, E. Milward, and M. P. Mattson. 2002. Matrix metalloproteinase-1 acti-vates a pertussis toxin-sensitive signaling pathway that stimulates the release ofmatrix metalloproteinase-9. J. Neurochem. 82: 885–893.

7. Fainardi, E., M. Castellazzi, T. Bellini, M. C. Manfrinato, E. Baldi, I. Casetta,E.Paolino, E.Granieri, andF.Dallocchio. 2006.Cerebrospinal fluid and serum levelsand intrathecal production of activematrixmetalloproteinase-9 (MMP-9) asmarkersof disease activity in patients with multiple sclerosis.Mult. Scler. 12: 294–301.

8. McGeer, P. L., and E. G. McGeer. 1999. Inflammation of the brain in Alz-heimer’s disease: implications for therapy. J. Leukoc. Biol. 65: 409–415.

9. Choi, D. H., E. M. Kim, H. J. Son, T. H. Joh, Y. S. Kim, D. Kim, M. Flint Beal,and O. Hwang. 2008. A novel intracellular role of matrix metalloproteinase-3during apoptosis of dopaminergic cells. J. Neurochem. 106: 405–415.

10. Kawasaki, Y., Z. Z. Xu, X. Wang, J. Y. Park, Z. Y. Zhuang, P. H. Tan, Y. J. Gao,K. Roy, G. Corfas, E. H. Lo, and R. R. Ji. 2008. Distinct roles of matrix met-alloproteases in the early- and late-phase development of neuropathic pain. Nat.Med. 14: 331–336.

11. Serratı, S., M. Cinelli, F. Margheri, S. Guiducci, A. Del Rosso, M. Pucci, G. Fibbi,L.Bazzichi, S. Bombardieri,M.Matucci-Cerinic, andM.Del Rosso. 2006. Systemicsclerosis fibroblasts inhibit in vitro angiogenesis by MMP-12-dependent cleavageof the endothelial cell urokinase receptor. J. Pathol. 210: 240–248.

12. Parks, W. C., C. L. Wilson, and Y. S. Lopez-Boado. 2004. Matrix metal-loproteinases as modulators of inflammation and innate immunity. Nat. Rev.Immunol. 4: 617–629.

13. Boire, A., L. Covic, A. Agarwal, S. Jacques, S. Sherifi, and A. Kuliopulos. 2005.PAR1 is a matrix metalloprotease-1 receptor that promotes invasion and tu-morigenesis of breast cancer cells. Cell 120: 303–313.

14. Schonbeck, U., F. Mach, G. K. Sukhova, C. Murphy, J. Y. Bonnefoy,R. P. Fabunmi, and P. Libby. 1997. Regulation of matrix metalloproteinase ex-pression in human vascular smooth muscle cells by T lymphocytes: a role forCD40 signaling in plaque rupture? Circ. Res. 81: 448–454.

15. Yan, C., and D. D. Boyd. 2007. Regulation of matrix metalloproteinase geneexpression. J. Cell. Physiol. 211: 19–26.

16. Crocker, S. J., R. Milner, N. Pham-Mitchell, and I. L. Campbell. 2006. Cell andagonist-specific regulation of genes for matrix metalloproteinases and their tis-sue inhibitors by primary glial cells. J. Neurochem. 98: 812–823.

17. Elkington, P. T., J. E. Emerson, L. D. Lopez-Pascua, C. M. O’Kane,D. E. Horncastle, J. J. Boyle, and J. S. Friedland. 2005. Mycobacterium tuber-culosis up-regulates matrix metalloproteinase-1 secretion from human airwayepithelial cells via a p38 MAPK switch. J. Immunol. 175: 5333–5340.

18. Elkington, P. T., R. K. Nuttall, J. J. Boyle, C. M. O’Kane, D. E. Horncastle,D. R. Edwards, and J. S. Friedland. 2005. Mycobacterium tuberculosis, but notvaccine BCG, specifically upregulates matrix metalloproteinase-1. Am. J. Respir.Crit. Care Med. 172: 1596–1604.

19. O’Kane, C. M., P. T. Elkington, and J. S. Friedland. 2008. Monocyte-dependentoncostatin M and TNF-alpha synergize to stimulate unopposed matrix metal-

loproteinase-1/3 secretion from human lung fibroblasts in tuberculosis. Eur. J.Immunol. 38: 1321–1330.

20. Harris, J. E., R. K. Nuttall, P. T. Elkington, J. A. Green, D. E. Horncastle,M. B. Graeber, D. R. Edwards, and J. S. Friedland. 2007. Monocyte-astrocyte net-works regulate matrix metalloproteinase gene expression and secretion in centralnervous system tuberculosis in vitro and in vivo. J. Immunol. 178: 1199–1207.

21. Harris, J. E., J. A. Green, P. T. Elkington, and J. S. Friedland. 2007. Monocytesinfected with Mycobacterium tuberculosis regulate MAP kinase-dependent as-trocyte MMP-9 secretion. J. Leukoc. Biol. 81: 548–556.

22. Price, N. M., J. Farrar, T. T. Tran, T. H. Nguyen, T. H. Tran, and J. S. Friedland.2001. Identification of a matrix-degrading phenotype in human tuberculosisin vitro and in vivo. J. Immunol. 166: 4223–4230.

23. Green, J. A., C. T. Tran, J. J. Farrar, M. T. Nguyen, P. H. Nguyen, S. X. Dinh,N. D. Ho, C. V. Ly, H. T. Tran, J. S. Friedland, and G. E. Thwaites. 2009.Dexamethasone, cerebrospinal fluid matrix metalloproteinase concentrations andclinical outcomes in tuberculous meningitis. PLoS One 4: e7277.

24. Haorah, J., S. H. Ramirez, K. Schall, D. Smith, R. Pandya, and Y. Persidsky.2007. Oxidative stress activates protein tyrosine kinase and matrix metal-loproteinases leading to blood-brain barrier dysfunction. J. Neurochem. 101:566–576.

25. Rosenberg, G. A., L. A. Cunningham, J. Wallace, S. Alexander, E. Y. Estrada,M. Grossetete, A. Razhagi, K. Miller, and A. Gearing. 2001. Immunohisto-chemistry of matrix metalloproteinases in reperfusion injury to rat brain: acti-vation of MMP-9 linked to stromelysin-1 and microglia in cell cultures. BrainRes. 893: 104–112.

26. Ben-Hur, T., Y. Ben-Yosef, R. Mizrachi-Kol, O. Ben-Menachem, and A. Miller.2006. Cytokine-mediated modulation of MMPs and TIMPs in multipotentialneural precursor cells. J. Neuroimmunol. 175: 12–18.

27. Nuttall, R. K., C. Silva, W. Hader, A. Bar-Or, K. D. Patel, D. R. Edwards, andV. W. Yong. 2007. Metalloproteinases are enriched in microglia compared withleukocytes and they regulate cytokine levels in activated microglia. Glia 55:516–526.

28. Li, Q., and I. M. Verma. 2002. NF-kappaB regulation in the immune system. Nat.Rev. Immunol. 2: 725–734.

29. Gerondakis, S., R. Grumont, R. Gugasyan, L. Wong, I. Isomura, W. Ho, andA. Banerjee. 2006. Unravelling the complexities of the NF-kappaB signallingpathway using mouse knockout and transgenic models. Oncogene 25: 6781–6799.

30. Cortez, D. M., M. D. Feldman, S. Mummidi, A. J. Valente, B. Steffensen,M. Vincenti, J. L. Barnes, and B. Chandrasekar. 2007. IL-17 stimulates MMP-1expression in primary human cardiac fibroblasts via p38 MAPK- and ERK1/2-dependent C/EBP-beta, NF-kappaB, and AP-1 activation. Am. J. Physiol. HeartCirc. Physiol. 293: H3356–H3365.

31. Barchowsky, A., D. Frleta, and M. P. Vincenti. 2000. Integration of the NF-kappaB and mitogen-activated protein kinase/AP-1 pathways at the collagenase-1promoter: divergence of IL-1 and TNF-dependent signal transduction in rabbitprimary synovial fibroblasts. Cytokine 12: 1469–1479.

32. Vincenti, M. P., and C. E. Brinckerhoff. 2007. Signal transduction and cell-typespecific regulation of matrix metalloproteinase gene expression: can MMPs begood for you? J. Cell. Physiol. 213: 355–364.

33. Wickremasinghe, M. I., L. H. Thomas, C. M. O’Kane, J. Uddin, andJ. S. Friedland. 2004. Transcriptional mechanisms regulating alveolar epithelialcell-specific CCL5 secretion in pulmonary tuberculosis. J. Biol. Chem. 279:27199–27210.

34. Samten, B., J. C. Townsend, S. E. Weis, A. Bhoumik, P. Klucar, H. Shams, andP. F. Barnes. 2008. CREB, ATF, and AP-1 transcription factors regulate IFN-gamma secretion by human T cells in response to mycobacterial antigen.J. Immunol. 181: 2056–2064.

35. Elkington, P. T., J. A. Green, and J. S. Friedland. 2006. Filter sterilization ofhighly infectious samples to prevent false negative analysis of matrix metal-loproteinase activity. J. Immunol. Methods 309: 115–119.

36. Vecil, G. G., P. H. Larsen, S. M. Corley, L.M. Herx, A. Besson, C. G. Goodyer, andV. W. Yong. 2000. Interleukin-1 is a key regulator of matrix metalloproteinase-9expression in human neurons in culture and following mouse brain trauma in vivo.J. Neurosci. Res. 61: 212–224.

37. Nuttall, R. K., C. J. Pennington, J. Taplin, A. Wheal, V. W. Yong, P. A. Forsyth,and D. R. Edwards. 2003. Elevated membrane-type matrix metalloproteinases ingliomas revealed by profiling proteases and inhibitors in human cancer cells.Mol. Cancer Res. 1: 333–345.

38. Porter, J. C., M. Falzon, and A. Hall. 2008. Polarized localization of epithelialCXCL11 in chronic obstructive pulmonary disease and mechanisms of T cellegression. J. Immunol. 180: 1866–1877.

39. Imai, Y., I. Ibata, D. Ito, K. Ohsawa, and S. Kohsaka. 1996. A novel gene iba1 inthe major histocompatibility complex class III region encoding an EF handprotein expressed in a monocytic lineage. Biochem. Biophys. Res. Commun. 224:855–862.

40. Rutter, J. L., U. Benbow, C. I. Coon, and C. E. Brinckerhoff. 1997. Cell-type specificregulation of human interstitial collagenase-1 gene expression by interleukin-1 beta(IL-1 beta) in human fibroblasts and BC-8701 breast cancer cells. J. Cell. Biochem.66: 322–336.

41. Simmons, C. P., G. E. Thwaites, N. T. Quyen, T. T. Chau, P. P. Mai, N. T. Dung,K. Stepniewska, N. J. White, T. T. Hien, and J. Farrar. 2005. The clinical benefitof adjunctive dexamethasone in tuberculous meningitis is not associated withmeasurable attenuation of peripheral or local immune responses. J. Immunol.175: 579–590.

42. O’Kane, C. M., P. T. Elkington, M. D. Jones, L. Caviedes, M. Tovar,R. H. Gilman, G. Stamp, and J. S. Friedland. 2009. STAT3, p38 MAP Kinase and

6502 MMPs IN CNS TB

by guest on Novem

ber 2, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

NF-kappaB Drive Unopposed Monocyte-dependent Fibroblast MMP-1 Secretionin Tuberculosis. Am. J. Respir. Cell Mol. Biol. In press.

43. Vos, C. M., L. Sjulson, A. Nath, J. C. McArthur, C. A. Pardo, J. Rothstein, andK. Conant. 2000. Cytotoxicity by matrix metalloprotease-1 in organotypic spinalcord and dissociated neuronal cultures. Exp. Neurol. 163: 324–330.

44. Woo, M. S., J. S. Park, I. Y. Choi, W. K. Kim, and H. S. Kim. 2008. Inhibition ofMMP-3 or -9 suppresses lipopolysaccharide-induced expression of proin-flammatory cytokines and iNOS in microglia. J. Neurochem. 106: 770–780.

45. Rock, R. B., S. Hu, G. Gekker, W. S. Sheng, B. May, V. Kapur, andP. K. Peterson. 2005. Mycobacterium tuberculosis-induced cytokine and che-mokine expression by human microglia and astrocytes: effects of dexametha-sone. J. Infect. Dis. 192: 2054–2058.

46. Liu, X., Q. Han, R. Sun, and Z. Li. 2008. Dexamethasone regulation of matrixmetalloproteinase expression in experimental pneumococcal meningitis. BrainRes. 1207: 237–243.

47. Zhou, M., Y. Zhang, J. A. Ardans, and L. M. Wahl. 2003. Interferon-gammadifferentially regulates monocyte matrix metalloproteinase-1 and -9 throughtumor necrosis factor-alpha and caspase 8. J. Biol. Chem. 278: 45406–45413.

48. Pillinger, M. H., N. Marjanovic, S. Y. Kim, J. U. Scher, P. Izmirly, S. Tolani,V. Dinsell, Y. C. Lee, M. J. Blaser, and S. B. Abramson. 2005. Matrix metal-

loproteinase secretion by gastric epithelial cells is regulated by E prostaglandinsand MAPKs. J. Biol. Chem. 280: 9973–9979.

49. Elkington, P. T., J. A. Green, J. E. Emerson, L. D. Lopez-Pascua, J. J. Boyle,C. M. O’Kane, and J. S. Friedland. 2007. Synergistic up-regulation of epithelialcell matrix metalloproteinase-9 secretion in tuberculosis. Am. J. Respir. CellMol. Biol. 37: 431–437.

50. Kishore, N., C. Sommers, S. Mathialagan, J. Guzova, M. Yao, S. Hauser,K. Huynh, S. Bonar, C. Mielke, L. Albee, et al. 2003. A selective IKK-2 inhibitorblocks NF-kappa B-dependent gene expression in interleukin-1 beta-stimulatedsynovial fibroblasts. J. Biol. Chem. 278: 32861–32871.

51. Caamano, J., J. Alexander, L. Craig, R. Bravo, and C. A. Hunter. 1999. The NF-kappa B family member RelB is required for innate and adaptive immunity toToxoplasma gondii. J. Immunol. 163: 4453–4461.

52. Vincenti, M. P., and C. E. Brinckerhoff. 2002. Transcriptional regulation ofcollagenase (MMP-1, MMP-13) genes in arthritis: integration of complex sig-naling pathways for the recruitment of gene-specific transcription factors. Ar-thritis Res. 4: 157–164.

53. Rivera-Marrero, C. A., W. Schuyler, S. Roser, J. D. Ritzenthaler, S. A. Newburn,and J. Roman. 2002. M. tuberculosis induction of matrix metalloproteinase-9:the role of mannose and receptor-mediated mechanisms. Am. J. Physiol. LungCell. Mol. Physiol. 282: L546–L555.

The Journal of Immunology 6503

by guest on Novem

ber 2, 2018http://w

ww

.jimm

unol.org/D

ownloaded from