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of September 6, 2018. This information is current as Production in Human Macrophages β Type I IFN Signaling To Regulate IL-1 Triggers Host Mycobacterium tuberculosis Alan Sher and Carl G. Feng Timothy G. Myers, Ronald L. Rabin, Giorgio Trinchieri, Shenderov, Kevin D. Kirschman, Katrin D. Mayer-Barber, Aleksey Novikov, Marco Cardone, Robert Thompson, Kevin http://www.jimmunol.org/content/187/5/2540 doi: 10.4049/jimmunol.1100926 July 2011; 2011; 187:2540-2547; Prepublished online 22 J Immunol References http://www.jimmunol.org/content/187/5/2540.full#ref-list-1 , 26 of which you can access for free at: cites 49 articles This article average * 4 weeks from acceptance to publication Fast Publication! Every submission reviewed by practicing scientists No Triage! from submission to initial decision Rapid Reviews! 30 days* Submit online. ? The JI Why Subscription http://jimmunol.org/subscription is online at: The Journal of Immunology Information about subscribing to Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. All rights reserved. 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on September 6, 2018 http://www.jimmunol.org/ Downloaded from by guest on September 6, 2018 http://www.jimmunol.org/ Downloaded from

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of September 6, 2018.This information is current as

Production in Human MacrophagesβType I IFN Signaling To Regulate IL-1

Triggers HostMycobacterium tuberculosis

Alan Sher and Carl G. FengTimothy G. Myers, Ronald L. Rabin, Giorgio Trinchieri,Shenderov, Kevin D. Kirschman, Katrin D. Mayer-Barber, Aleksey Novikov, Marco Cardone, Robert Thompson, Kevin

http://www.jimmunol.org/content/187/5/2540doi: 10.4049/jimmunol.1100926July 2011;

2011; 187:2540-2547; Prepublished online 22J Immunol 

Referenceshttp://www.jimmunol.org/content/187/5/2540.full#ref-list-1

, 26 of which you can access for free at: cites 49 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. All rights reserved.1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Mycobacterium tuberculosis Triggers Host Type I IFNSignaling To Regulate IL-1b Production in HumanMacrophages

Aleksey Novikov,*,†,‡ Marco Cardone,x Robert Thompson,* Kevin Shenderov,*

Kevin D. Kirschman,{ Katrin D. Mayer-Barber,* Timothy G. Myers,‖ Ronald L. Rabin,{

Giorgio Trinchieri,*,x Alan Sher,*,1 and Carl G. Feng*,1

Mycobacterium tuberculosis is a virulent intracellular pathogen that survives in macrophages even in the presence of an intact

adaptive immune response. Type I IFNs have been shown to exacerbate tuberculosis in mice and to be associated with disease

progression in infected humans. Nevertheless, the mechanisms by which type I IFNs regulate the host response to M. tuberculosis

infection are poorly understood. In this study, we show that M. tuberculosis induces an IFN-related gene expression signature in

infected primary human macrophages, which is dependent on host type I IFN signaling as well as the mycobacterial virulence

factor, region of difference-1. We further demonstrate that type I IFNs selectively limit the production of IL-1b, a critical

mediator of immunity to M. tuberculosis. This regulation occurs at the level of IL1B mRNA expression, rather than caspase-1

activation or autocrine IL-1 amplification and appears to be preferentially used by virulent mycobacteria since avirulent M. bovis

bacillus Calmette-Guerin (BCG) fails to trigger significant expression of type I IFNs or release of mature IL-1b protein. The latter

property is associated with decreased caspase-1–dependent IL-1b maturation in the BCG-infected macrophages. Interestingly,

human monocytes in contrast to macrophages produce comparable levels of IL-1b in response to either M. tuberculosis or BCG.

Taken together, these findings demonstrate that virulent and avirulent mycobacteria employ distinct pathways for regulating IL-

1b production in human macrophages and reveal that in the case of M. tuberculosis infection the induction of type I IFNs is

a major mechanism used for this purpose. The Journal of Immunology, 2011, 187: 2540–2547.

Mycobacterium tuberculosis is a highly virulent patho-gen that infects approximately one-third of the world’spopulation and is responsible for almost 2 million deaths

annually. M. tuberculosis establishes a life-long persistent infectionin most infected individuals despite the development of an ongoingimmune response. It has been proposed that the organism does soby subverting innate defense programs in macrophages that typi-cally eliminate invading microbes (1, 2). However, the mechanismsby which virulent mycobacteria manipulate host innate signalingpathways are not fully understood.

Studies in mice have revealed that a significant proportion of thetranscriptional response to M. tuberculosis in vivo and in macro-phages in vitro is MyD88 independent but type I IFN signalingdependent (3–5). The mechanisms of host–pathogen interaction thatregulateM. tuberculosis-induced type I IFNs have yet to be defined.However, studies on Listeria monocytogenes and Francisellatularensis infections have suggested that type I IFN inductionrequires the recognition of bacterial products in the host cell cy-tosol (6). A mycobacterial secretion system, ESX1, has been sug-gested to play a key role in mycobacterial virulence by facilitatingthe secretion of M. tuberculosis products into the host cytoplasm(7, 8). ESX1 is partially encoded by the region of difference-1(RD1), a gene locus in the M. tuberculosis genome that is de-leted in all strains of M. bovis bacillus Calmette-Guerin (BCG)vaccines (9). Importantly, ESX1 has been implicated in type I IFNinduction as well as activation of the inflammasome, a cytosolicprotein complex that cleaves pro–IL-1b into its mature bioactiveform, in virulent mycobacteria-infected macrophages (4, 10–13).The IL-1 and type I IFN signaling pathways play opposite roles

in host resistance toM. tuberculosis. Mice deficient in IL-1R or IL-1b show increased susceptibility to the pathogen (14, 15), sug-gesting that the cytokine signaling is essential for the host controlof the infection. Interestingly, a recent study has shown that a M.tuberculosis Zn2+ metalloprotease, Rv0198c (zmp1), required forfull virulence of the pathogen in mice, suppresses IL-1b pro-cessing by inhibiting inflammasome activation in murine macro-phages (16). This observation has led to the hypothesis thatmycobacteria regulate IL-1b maturation as part of their survivalstrategy in macrophages (17). In contrast to IL-1b, type I IFNshave been shown to promote infection by M. tuberculosis in mice.Thus, virulent clinical isolates of M. tuberculosis induce high

*Immunobiology Section, Laboratory of Parasitic Diseases, National Institute ofAllergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;†Howard Hughes Medical Institute–National Institutes of Health Research ScholarsProgram, Howard Hughes Medical Institute, Chevy Chase, MD 20892; ‡TheWarren Alpert Medical School of Brown University, Providence, RI 02912; xCancerand Inflammation Program, Center for Cancer Research, National Cancer Institute,National Institutes of Health, Bethesda, MD 20892; {Laboratory of Immunobio-chemistry, Center for Biologics Evaluation and Research, U.S. Food and Drug Admi-nistration, Bethesda, MD 20892; and ‖Research and Technologies Branch, NationalInstitute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,MD 20892

1A.S. and C.G.F. contributed equally to this work.

Received for publication April 4, 2011. Accepted for publication June 20, 2011.

This research was supported by the Intramural Research Program of the NationalInstitute of Allergy and Infectious Diseases, National Institutes of Health, Depart-ment of Health and Human Services.

Address correspondence and reprint requests to Dr. Carl Feng, Immunobiology Sec-tion, Laboratory of Parasitic Diseases, National Institute of Allergy and InfectiousDiseases, National Institutes of Health, Room 6142, Building 50, 50 South DriveMSC-8003, Bethesda, MD 20892-8003. E-mail address: [email protected]

Abbreviations used in this article: BCG, bacillus Calmette-Guerin; CT, cycle thresh-old; IFNABR2, IFN-a/b receptor 2; RD1, region of difference-1; TB, tuberculosis;WT, wild-type.

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levels of type I IFNs in vivo, and in most studies mice deficient intype I IFN signaling display significantly reduced bacterial loadsfollowing infection (18–20). Similarly, intranasal administrationof polyriboinosinic-polyribocytidylic acid, a type I IFN-inducingagent, exacerbates pulmonary tuberculosis (TB) in wild-type (WT)but not IFN-a/b receptor-deficient mice (21).The role of type I IFNs in the regulation of human M. tuber-

culosis infection is poorly understood. Nevertheless, a recentclinical study revealed that IFN-inducible genes are highly ex-pressed in the leukocytes of active but not latent TB patients (22),strongly arguing for a contributing role of type I IFNs in TBprogression in humans. Previous in vitro studies have also shownthat M. tuberculosis infection significantly upregulates the ex-pression of a number of type I IFN-inducible genes (23, 24). Inthis study, we report that this induction of a type I IFN signature inhuman macrophages is a property of live virulent mycobacteriaand is not triggered by either BCG or an RD1-deficient M. tu-berculosis mutant. Additionally, we report that IL-1b, also pref-erentially induced by virulent M. tuberculosis, is negativelyregulated by type I IFNs. These findings demonstrate a functionalintersection between type I IFN and IL-1b signaling in M. tu-berculosis-infected human macrophages.

Materials and MethodsCell cultures and infection

Peripheral blood-derived monocytes were isolated from healthy donorsby counterflow centrifugal elutriation under protocols approved by theInstitutional Review Boards of both the National Institute of Allergy andInfectious Diseases and the Department of Transfusion Medicine of theNational Institutes of Health for these studies after appropriate informedconsent. Monocytes were differentiated as previously described (23).Briefly, cells were cultured in antibiotic-free RPMI 1640 medium con-taining 10% FCS, 2 mM glutamate, 50 mM 2-ME, and 10 mM HEPESbuffer in 24-well plates (Corning) at a density of 1 3 106 cells/ml for 7 d.Recombinant human M-CSF (10 ng/ml; PeproTech) was added on days 0,2, and 4. For differentiation of THP-1 cells, the cell line (1 3 106 cells/well) was stimulated with PMA (10 ng/ml) in 24-well plates for 2 d. Thecells were then rested in fresh complete medium for 24 h before infection.

For stimulation with mycobacteria, differentiated macrophages were ex-posed to live BCG or RD1 mutant (gift from Dr. S. Derrick, U.S. Food andDrug Administration) or WT M. tuberculosis H37Rv at a multiplicity ofinfection of 5, or with irradiated M. tuberculosis at 50 mg/ml. In someexperiments, a mouse mAb to human IFN-a/b receptor-2 (10 mg/ml) wasincluded. Recombinant human IFN-b or IFN-g was purchased from Pepro-Tech, and IL-27 was obtained from R&D Systems. All cytokines were used ata final concentration of 10 ng/ml. In some experiments, IL-27R/Fc (2 mg/ml;R&D Systems) and anakinra (30 mg/ml; Amgen) were added to the cultures.

Measurement of gene expression

Total RNA was isolated using the RNAeasy Mini kit (Qiagen, Valencia,CA), and residual DNA was digested using RNAse-free DNAse (Qiagen).The individual RNA samples were reversely transcribed using SuperScriptII reverse transcriptase (Invitrogen, Carlsbad, CA). Unless otherwise in-dicated, gene expression was measured using a previously described SYBRGreen-based real-time quantitative PCR (25) in which 18S mRNAwas usedas the housekeeping gene. The oligonucleotide primers used were: 18S,forward, 59-CACGGCCGGTACAGTGAAAC-39 and reverse, 59-CCCGT-CGGCATGTATTAGCT-39; IL-1b, forward, 59-TTCTTCGACACATGG-GATAACG-39 and reverse, 59-TGGAGAACACCACTTGTTGCT-39; IL-6,59-TAATGGGCATTCCTTCTTCT-39 and 59-TGTCCTAACGCTCATAC-TTTT-39; TNF, forward, 59-CAGCCTCTTCTCCTTCCTGAT-39 and re-verse, 59-GCCAGAGGGCTGATTAGAGA-39; CXCL10, forward, 59-AT-TTGCTGCCTTATCTTTCTG-39 and reverse 59-TCTCACCCTTCTTTT-TCATTGTAG-39; IFN-b, forward, 59-AGC AGT TCC AGA AGG AGGAC-39 and reverse, 59-TGA TAG ACA TTA GCC AGG AGG TT-39; IL-1RI, 59-AGCTGGCTGGGTGGTTCAT-39 and 59-CGATTCTGGCATTT-TCTCATAGTC-39; IL-1RII, 59-GGGGGAAATGATCACAGGAATGGT-39and 59-CCCATGAAGGCCAGCAATACAACA-39; IL-1RACP, 59-CCTC-TCGGGGCAACATCAAC-39 and 59-GACCGCCTGGGACTTTTCTTC-39;and IL-1Ra, forward, -39GGA AGA TGT GCC TGT CCT GT-39 and re-verse, 59-TCT CGC TCA GGT CAG TGA TG-39.

Analysis of expression of the 12 subtypes of IFN-a in M. tuberculosis-infected macrophages was performed using a TaqMan-based PCR witheither molecular beacons or locked nucleic acid probes that enhancedspecificity for each subtype (P. Hillyer, V.P. Mane, L.M. Schramm, A.G.Chen, Z. Zhao, M.B. Navarro, K.D. Kirschman, S. Bykadi, R.G. Jubin, andR.L. Rabin, submitted for publication). Data were analyzed with SDSsoftware (version 2.3; Applied Biosystems) and cycle threshold (CT) val-ues were exported to a spreadsheet (Microsoft Excel, Redmond, WA) tocalculate both standard curves and foldchange in IFN expression relative tothe housekeeping gene 18S (DCT). Standard curves and RNA input amountwere used to solve for IFN copy number per microgram of RNA.

The nCounter analysis system (NanoString Technologies) was used toscreen for the expression of signature genes associated with NF-kB andIFN signaling pathways as previously described (26). Two specific probes(capture and reporter) for each gene of interest were employed. Briefly, 5 mlcell lysate (2000 cells/ml in RLT buffer; Qiagen) was hybridized with cus-tomized Reporter CodeSet and Capture ProbeSet (NanoString Technologies)according to the manufacturer’s instructions for direct labeling of mRNAs ofinterest with molecular barcodes without the use of reverse transcription oramplification. The hybridized samples were then recovered in the Nano-String Prep Station and the mRNA molecules were counted with theNanoString nCounter. For analysis of expression, raw counts (+1) weretransformed to log base 2 and each sample profile was median-normalized tothe RPS18 internal control (housekeeping gene). A mixed effects ANOVAmodel was used to calculate expression differences including the blooddonor subject as a random effect, comparable to a paired t test (JMPGenomics software; SAS software, Cary, NC). Statistical significance wascalculated using a false discovery rate adjustment of the raw p value (27).

Cytokine measurement and immunoblotting

Tissue culture supernatants were screened using a commercial multiplexELISA assay for human IL-1a, IL-1b, IL-6, IL-8, IL-10, IL-12p70, IL-23,TNF, and CXCL10 (Quansys Biosciences). Commercially availableELISA kits (R&D Systems) were also used to determine the concentrationsof IL-1b in culture supernatants.

For immunoblotting, supernatants were precipitated with methanol/chloroform and the resulting protein pellet was resuspended in reducingsample buffer (Thermo Fisher Scientific). Cells were lysed using cell lysisbuffer (Cell Signaling Technology) supplemented with a protease inhibitormixture (Calbiochem) and PMSF (Sigma-Aldrich). The samples were nextseparated by SDS-PAGE on a 12% polyacrylamide gel and then transferredonto a polyvinylidene difluoride membrane. The membranes were thansequentially probed with polyclonal Abs for caspase-1 (Cell SignalingTechnology) or IL-1b (R&D Systems or Cell Signaling Technology).

Statistics

A Wilcoxon ranked sum test was used in experiments involving pairwisecomparisons. A p value of ,0.05 was considered statistically significant.

ResultsVirulent M. tuberculosis induces a subset of type I IFNs inhuman macrophages

We first determined whether M. tuberculosis triggers IFNB mRNAexpression in human macrophages at 4 and/or 24 h postinfectionusing a SYBR Green-based quantitative PCR assay and found thatthe gene expression was significantly elevated in infected primaryhuman macrophages (Fig. 1A) and THP-1 cells (Fig. 1B). Asexpected, the induction of IFNB was closely correlated with theexpression of the IFN-inducible gene CXCL10 in M. tuberculosis-infected primary macrophages (Fig. 1C) and THP-1 cells (Fig.1D). In addition to IFN-b, there are 12 closely related IFN-asubtypes in the human type I IFN family (28). To profile thesubtypes of IFN-a induced by M. tuberculosis in infected mac-rophages, we next quantified the copy number of IFN-b andIFN-a family members using a TaqMan-based PCR at 24 hpostinfection (Fig. 1E, 1F). This independent assay confirmed theinduction of IFNB expression and further revealed that among allIFN-a subtypes examined, only IFNA1 is specifically upregulatedby M. tuberculosis, suggesting a restricted IFN-a subtype usage inM. tuberculosis-infected human macrophages.

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Regulation of M. tuberculosis-induced cytokine expression inhuman macrophages by type I IFNs

To identify innate inflammatory cytokines that are regulated bytype I IFN signaling, we treated M. tuberculosis-infected macro-phages with a mouse mAb specific for IFN-a/b receptor 2 (IFN-ABR2), thereby blocking signaling of all members of the type I IFNfamily.We then analyzed the effect of the signaling blockade on theexpression of 64 genes that are known to be critical in mediatingNF-kB– or IFN-dependent cellular responses to microbial stimu-lation using a novel nCounter analysis system. We found that M.tuberculosis strongly induced both NF-kB– and IFN-related genes(Fig. 2A, left panel). Importantly, the expression of IFN-induciblegenes such as IFIT1, IFIT2, MX1, MX2, and IL27 was significantlyreduced in the presence of the mAb to IFNABR2 (Fig. 2A, rightpanel), thus establishing that the induction of IFN-related genesin M. tuberculosis-infected macrophages is dependent on endo-genously produced type I IFNs. Additionally, we observed thatblocking type I IFN signaling led to increased expression in a smallgroup of genes (Fig. 2A, right panel). Among these, IL1B and IL1Awere the most highly upregulated.To further determine whether type I IFNs are a regulator of IL1B

mRNA expression in M. tuberculosis infection, we extended ourstudy to a larger cohort of healthy donors and compared the ex-pression of IL1B in the infected macrophage cultures with orwithout IFNABR2 blockade. In addition to reducing the expres-sion of the IFN-dependent CXCL10 (data not shown), blockade oftype I IFN signaling significantly increased IL1B mRNA levelswhen compared with the cultures infected with M. tuberculosisalone (Fig. 2B). In contrast, expression of other NF-kB–dependentgenes, such as IL6 and TNF, was not significantly altered by themAb, suggesting a selective regulation of IL1B expression bytype I IFNs.

Downregulation of M. tuberculosis-induced IL-1b by type IIFNs occurs independently of alterations in IL-1R signaling,IL-27R signaling, and caspase-1 activation

Type I IFNs have been shown to modulate IL-1 receptor expression(29) and it is well established that IL-1 can autoamplify its pro-duction (30, 31) through interaction with its own receptors.

Therefore, it was possible that the increase in IL1B expression inour mAb blocking experiments was due to the release of type IIFN-dependent inhibition of IL-1R signaling and subsequent au-tocrine stimulation of IL-1 production. To test this, we first ana-lyzed the effect of endogenous type I IFNs on the expression ofthe signaling receptors IL1RI and IL1RAcP, the inhibitory recep-tor ILRII, and the receptor antagonist IL1Ra in M. tuberculosis-infected primary macrophages. We found that relative to unin-fected controls, M. tuberculosis-infected macrophages displayedreduced expression of the IL1RI but increased levels of IL1RAcp,IL1RII, and IL1Ra (Fig. 3A). Nevertheless, blockade of type I IFNsignaling failed to significantly alter the expression of any IL-1Rspecies.To further exclude the possibility that endogenous type I IFNs

regulate IL-1–induced IL1B expression, we compared gene ex-pression in M. tuberculosis-infected macrophages cultured withmAb to IFNABR2 or anakinra, a recombinant IL-1Ra competingwith IL-1 for IL-1RI binding, or both. Addition of anakinra did notsignificantly reduce IL1B expression in macrophages infectedwith M. tuberculosis alone or in combination with the mAb toIFNABR2 (Fig. 3B). Taken together, these findings suggest thatthe IL-1R–dependent positive feedback loop does not appear tocontribute significantly to M. tuberculosis-induced IL-1b pro-duction in human macrophages and that the augmented IL1Bexpression following blockade of type I IFN signaling is not dueto enhanced IL-1R signaling.Because IL-27, an IFN-inducible regulatory cytokine, has also

been implicated in suppressing IL-1b production in macrophagesthrough the regulation of IL-1 receptor expression (32), we ex-amined its possible involvement in regulating M. tuberculosis-induced IL-1b production. We found that the addition of exoge-nous IL-27 or neutralization of endogenous IL-27 activity witha soluble IL-27R/Fc chimera did not significantly alter M. tuber-culosis-induced IL-1b production (Fig. 3C), thus arguing againsta role for this cytokine in regulating IL-1b production in infectedmacrophages.In addition to mRNA induction, mature IL-1b release requires

caspase-1 activation and cleavage of pro–IL-1b. To determinewhether type I IFNs regulate caspase-1 activation in M. tubercu-

FIGURE 1. Induction of type I IFN in M. tuberculosis-infected human macrophages. Expression of type I IFNs and CXCL10 mRNA was analyzed in

macrophages unstimulated (open symbols) or infected with M. tuberculosis (filled symbols) using a quantitative PCR. Levels of IFNB (A, B) and IFN-

inducible CXCL10 (C, D) in primary human macrophages (A, C) at 4 and 24 h postinfection or in PMA-differentiated THP-1 cells (B, D) at 24 h post-

infection were measured by a SYBR Green-based PCR. The data shown are relative fold increase of genes of interest over 18S RNA. The normalized gene

expression is calculated as 22DCT where DCT equals treated CT minus 18S CT. E, A representative expression profile of type I IFNs in uninfected or M.

tuberculosis-infected primary macrophages from a single donor and (F) summary expression data of IFNB and IFNA1 of all donors (n = 14) were de-

termined at 24 h using a TaqMan-based quantitative PCR. The data are expressed as mRNA copy number (per microgram of RNA). Each paired data set

represents an individual donor (A, C, F) or an independent experiment (B, D).

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losis-infected macrophages, IL1B mRNA as well as the cleavageof caspase-1 and IL-1b in culture supernatants of mycobacterial-infected THP-1 cells were analyzed. Addition of exogenous type IIFNs decreased the expression of IL1B mRNA, whereas supple-mentation of cultures with mAb specific for IFNABR2 increasedIL1B expression (Fig. 3D). Comparable effects of type I IFN orIFNABR2 mAb were observed when the levels of mature IL-1bprotein in the same cultures were analyzed by Western blotting. Incontrast, the extent of caspase-1 cleavage was affected minimallyby either culture condition, indicating that type I IFNs play a mi-nor role in regulating inflammasome activation and in the pro-cessing of pro–IL-1b in M. tuberculosis-infected human macro-phages.

M. tuberculosis-induced IL-1b production is suppressed bytype I but not type II IFNs and this regulation occurs in humanmacrophages but not monocytes

Thus far our results showed that the induction of type I IFNs byM. tuberculosis negatively regulates IL1B expression. We nextquantified the levels of IL-1b protein in the infected cultures andfound that consistent with the IL1B mRNA expression pattern(Fig. 2B), blockade of endogenous type I IFN signaling signifi-cantly increased the amount of IL-1b protein released into thecultures (Fig. 4A). Conversely, addition of exogenous IFN-bmarkedly suppressed IL-1b protein secretion in M. tuberculosis-

infected macrophages (Fig. 4B). Interestingly, IFN-g, an IFNfamily member known to activate macrophages, had a minimaleffect on M. tuberculosis-induced IL-1b secretion in the sameinfected cultures, suggesting that type I and type II IFNs playdistinct roles in regulating IL-1b production. Finally, we de-termined the effect of exogenous IFN-b and IFN-g on the IL-1bresponse of human monocytes to M. tuberculosis and observedthat neither IFN suppressed the production of IL-1b in theinfected monocyte cultures (Fig. 4B), indicating that the IL-1b

FIGURE 3. Regulation of IL-1b by M. tuberculosis-induced type I IFN

is independent of altered IL-1R and IL-27R signaling and caspase-1 ac-

tivation. A, Expression of IL-1R components and IL-1Ra in the presence or

absence of IFNABR2 mAb and (B) IL1B levels in the presence of anakinra

and/or IFNABR2 mAb was measured in M. tuberculosis-infected human

macrophages at 24 h using quantitative PCR. Data shown are fold change

over noninfected cultures. Each symbol represents an individual donor. C,

IL-1b secretion in macrophage cultures incubated with recombinant IL-27

or IL-27R/Fc protein measured by ELISA at 24 h postinfection. Each line

represents an individual donor. D, IL1B mRNA expression, caspase-1 ac-

tivation, and IL-1b processing in THP-1 cells infected withM. tuberculosis

alone or in combination with exogenous IFN-b or IFNABR2 mAb at 24 h.

IL1B mRNA in lysates was measured using quantitative PCR, whereas

cleaved caspase-1 and mature IL-1b in culture supernatants were assayed

by Western blotting involving sequential probing of the same membrane

with Abs against each product. The mRNA data shown are fold change

over nonstimulated cultures and data shown are the means (6SD) of three

independent experiments with similar results.

FIGURE 2. Regulation of the macrophage cytokine response to M. tu-

berculosis by type I IFN signaling.A, Primary humanmacrophages fromfive

individual healthy donors were left unstimulated or infected with M. tu-

berculosis for 24 h in the presence or absence of a blocking mAb to

IFNABR2 and gene expression analyzed using an nCounter gene expression

assay. Data are expressed as the log2 ratio (fold change) of mRNA counts.

Left panel, M. tuberculosis infected relative to uninfected. Right panel, M.

tuberculosis plus IFNABR2 mAb relative to M. tuberculosis alone. Known

IFN-dependent genes are shown in boxed areas. *False discovery rate, 0.5.

B, Increased IL1B expression in human macrophages infected with M. tu-

berculosis following blockade of endogenous type I IFN measured by

quantitative PCR at 24 h. The data shown are fold change over noninfected

cultures. Each paired data set represents an individual donor.

FIGURE 4. Differential effects of type I IFN on IL-1b production in M.

tuberculosis-infected macrophages and monocytes. A, Increased IL-1b

protein secretion by M. tuberculosis-infected primary human macrophages

cultured with mAb to IFNABR2. Each paired data set represents an in-

dividual donor. B, IFN-b limits M. tuberculosis-induced IL-1b secretion

by macrophages but not monocytes. Primary human macrophages and

monocytes from the same donors were infected with M. tuberculosis in the

presence or absence of exogenous IFN-b or INF-g. The cytokines (10 ng/

ml) were added to the cultures at the beginning of the infection. The

quantity of secreted IL-1b protein was measured at 24 h by ELISA. Each

symbol represents an individual donor.

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response of monocytes and macrophages to M. tuberculosis isdifferentially regulated by IFNs.

Virulent mycobacteria induce type I IFN expression in anRD1-dependent fashion

To determine whether the ability to induce type I IFNs is a commonproperty of all mycobacteria, we compared expression of type IIFN-related genes in macrophages exposed to virulent versusavirulent or inactivated mycobacteria. We found that BCG andirradiated M. tuberculosis stimulated significantly weaker ex-pression of IFNA1, IFNB, and CXCL10 compared with live M.tuberculosis (Fig. 5A), indicating a preferential induction of type IIFNs by virulent mycobacteria in human macrophages.Because the RD1 locus, which is present only in virulent M.

tuberculosis, has been implicated in the induction of type I IFNsin mice (4, 13), we compared IFN-related gene expression in pri-mary human macrophages infected with an RD1-deficient mutant(ΔRD1) or WTM. tuberculosis. Compared to WTM. tuberculosis,the ΔRD1 mutant was significantly less effective at inducing theexpression of the IFN-inducible IFIT1 and MX1 while being ca-pable of upregulating TNF, IL6, and IL1B mRNA (Fig. 5B), in-dicating a requirement for the mycobacterial virulence deter-minant RD1 in inducing type I IFNs in human macrophages.

Virulent M. tuberculosis induces higher levels of CXCL10 andIL-1b protein compared with BCG

To compare the cytokine response of human macrophages tovirulent and avirulent mycobacteria at the protein level, primaryhuman macrophages were infected with either M. tuberculosisor BCG, and secreted inflammatory cytokines/chemokines werequantified using a multiplex or a conventional ELISA assay.Whereas a low level of IFN-b protein was detected in M. tuber-culosis-infected macrophage cultures of some but not all donors(data not shown), large quantities of IFN-inducible CXCL10were induced (Fig. 6A) in every donor tested. Consistent with

the analysis of mRNA expression (Fig. 5A), multiplex cytokinemeasurements showed that M. tuberculosis-infected human mac-rophages secreted a significantly higher quantity of CXCL10 aswell as IL-1b when compared with those infected with BCG. Incontrast, the levels of other NF-kB–dependent proinflammatorycytokines were comparable. This difference in IL-1b protein se-cretion was subsequently confirmed using a conventional IL-1bELISA assay showing that M. tuberculosis stimulated primarymacrophages release ∼10-fold more IL-1b protein than eitherBCG- or irradiated M. tuberculosis-stimulated cells (Fig. 6B, leftpanel). The difference was not due to a failure of avirulentmycobacteria to activate the innate system since all bacteria in-duced a comparable level of IL1B mRNA in the same infectedmacrophage cultures (Fig. 6B, right panel). Similar to primarymacrophages, M. tuberculosis-infected differentiated THP-1 cellsalso consistently produced higher levels of secreted IL-1b proteinthan those stimulated with BCG (Fig. 6C).

FIGURE 5. Induction of type I IFNs by virulent mycobacteria in human

macrophages. A, Primary macrophages were exposed to live or irradiated

M. tuberculosis or BCG for 24 h and the expression of IFNA, IFNB, and

CXCL10 was analyzed by quantitative PCR. The data shown are fold

change over nonstimulated cultures (n = 8–14). B, Human macrophages

from two individual healthy donors were infected with either WT (filled

symbols) or RD1-deficient (open symbols) H37Rv M. tuberculosis and the

expression of the indicated genes was analyzed using an nCounter gene

expression assay at 4 and 24 h. The data shown indicate fold change over

nonstimulated cultures after gene expression was normalized against

RPS18.

FIGURE 6. Virulent but not avirulent mycobacteria stimulate IL-1b

protein secretion in human macrophages. A and B, Expression of cytokines

in mycobacterial-stimulated primary macrophage cultures was analyzed at

24 h using a multiplex cytokine assay (A; M. tuberculosis, filled symbols;

BCG, open symbols; n = 7), ELISA assay (B, left panel), or quantitative

PCR (B, right panel). C, Levels of IL-1b were determined in mycobac-

terial-infected THP-1 cell cultures by ELISA, and each paired data set

represents an independent experiment. D, Expression of caspase-1 and IL-

1b in infected primary human macrophage cultures was analyzed at 24 h

by Western blotting involving sequential probing of the same membrane

with the corresponding Abs. E, M. tuberculosis- and BCG-infected THP-1

cell cultures were analyzed at 24 h postinfection by Western blotting as

described in D. IFN-b (10 ng/ml) was added to some cultures at the be-

ginning of the infection. Data shown are representative of two indepen-

dent experiments with similar results. F, Levels of secreted ΙL-1b protein

in BCG-infected primary macrophage cultures with or without exogenous

IFN-b were determined at 24 h by ELISA. G, Levels of secreted IL-1b

protein (left panel) and IL1B mRNA (right panel) in mycobacterial-stim-

ulated monocyte cultures at 24 h were measured by ELISA or quantitative

PCR, respectively. Monocytes isolated from the same donors shown in B

were stimulated with M. tuberculosis, BCG, or irradiated M. tuberculosis.

mRNA data shown are fold change over noninfected cultures. Each symbol

in B, F, and G represents an individual donor.

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M. tuberculosis is more efficient than BCG in activatingcaspase-1 in infected macrophages

Given that type I IFNs negatively regulate IL1B mRNA expressionin M. tuberculosis-infected macrophages and BCG fails to in-duce type I IFN expression, it was surprising that BCG-infectedmacrophages produce decreased rather than increased IL-1bproduction. To determine whether M. tuberculosis and BCG dif-ferentially regulate IL-1b production at the posttranscriptionallevel, we analyzed levels of cleaved caspase-1 and IL-1b in theculture supernatants as well as procaspase-1 in the cell lysates ofinfected primary human macrophages. We found that virulentM. tuberculosis is more efficient than avirulent BCG and RD1-deficeint M. tuberculosis in activating caspase-1 and triggering thesecretion of mature IL-1b protein (Fig. 6D).Type I IFNs have been suggested to activate the inflammasome

in the Francisella infection model (33). Therefore, it was possiblethat compared with M. tuberculosis-infected cells, the weakercaspase-1 activation observed in BCG-infected culture is due tothe lower level of type I IFNs expressed (Fig. 5A). However, theaddition of exogenous IFN-b failed to increase caspase-1 cleavageto the level observed in M. tuberculosis-infected cells (Fig. 6E).Moreover, consistent with our earlier finding with M. tuberculosis(Fig. 4B), exogenous IFN-b reduced the quantity of BCG-inducedIL-1b to undetectable levels in primary macrophage cultures (Fig.6F), arguing that type I IFNs do not regulate mycobacteria-induced IL-1b production by altering caspase-1 activation. Takentogether, these observations suggested that the difference in IL-1b production by BCG- versus M. tuberculosis-infected humanmacrophages is due to differential caspase-1 activation rather thantype I IFN induction by these bacteria.

Monocytes, in contrast to macrophages, mount equivalentIL-1b response to M. tuberculosis and BCG

We next examined the IL-1b response of human monocytes tomycobacterial infection, as it has been reported previously that theproduction of this cytokine is differentially regulated in the twomononuclear cell populations (34, 35). In contrast to macrophages(Fig. 6B), the levels of IL1B mRNA and secreted IL-1b proteinwere comparable in monocyte cultures infected with M. tuber-culosis, BCG, or irradiated M. tuberculosis (Fig. 6G), arguing thatonly differentiated macrophages are capable of distinguishingvirulent from avirulent mycobacteria with regard to IL-1b release.Therefore, the IL-1b response to mycobacteria is influenced bothby properties of the pathogen as well as the host cell type that isinfected.

DiscussionAlthough essential for resistance to viral infections, type I IFNshave been shown to contribute to pathogenesis of intracellularbacterial infections (36, 37). In the case of M. tuberculosis, thecytokines have been shown to impair host resistance in mice (4,18) and to be associated with TB disease progression in infectedhumans (22). However, the mechanisms by which type I IFNsregulate the host response to M. tuberculosis infection are poorlyunderstood in both mice and humans. In this report, we show thattype I IFNs are preferentially induced by virulent mycobacteriaand function as regulators of IL-1b production in infected humanmacrophages through suppression of IL1B mRNA expression.This host cytokine-mediated, transcriptional regulation of IL-1bproduction is thus distinct from a previously described mechanismin murine macrophages involving direct inhibition of inflamma-some activation by the mycobacterial product zmp1 (16). Takentogether, our findings reveal a second pathway through which M.

tuberculosis can manipulate host IL-1b production indirectly byinducing type I IFNs (Fig. 7). Although the molecular mechanismsrequired for the induction of type I IFNs by M. tuberculosis haveyet to be fully elucidated, it is interesting to note that live M.tuberculosis selectively induces only a subset of type I IFNs inmacrophages, as shown in this study, and dendritic cells (24).Therefore, a detailed comparison of the M. tuberculosis-inducedIFN signature with that known to be triggered by viral infectionmay provide important clues in understanding the “probacterial”versus antiviral effects of this important cytokine family.Our findings reveal that type I IFNs selectively inhibit IL-1b

mRNA induction but not caspase-1 activation or IL-1R signalingin M. tuberculosis-infected human macrophages. A similar sup-pressive effect of IFN-b on IL-1b gene expression has been re-ported in dendritic cells isolated from multiple sclerosis patientsbefore treatment (38). Nevertheless, type I IFNs have also beenshown to regulate IL-1b production at the posttranscriptionallevel. For example, it has been reported that type I IFNs positivelyregulate IL-1b release by enhancing inflammasome activation inF. tularensis-infected murine macrophages (33), although in vivotype I IFN signaling has been shown to impair host control ofFrancisella infection (39). Moreover, although a previous studyindicated that type I IFN/Tyk2 signaling regulates LPS-inducedIL-1b production at the translational level in murine macrophages(40), a more recent paper reported that type I IFNs regulate LPS

FIGURE 7. Schematic diagram illustrating the regulation of IL-1b in

human macrophages infected with either M. tuberculosis or BCG. A,

Whereas M. tuberculosis and BCG trigger equivalent levels of IL1B

mRNA, only M. tuberculosis can efficiently activate the inflammasome to

process pro–IL-1b leading to release of mature IL-1b. Additionally, M.

tuberculosis is able to simultaneously limit the production of IL-1b by at

least two independent pathways (indicated in red lines). The first mecha-

nism involves the previously described direct inhibition of caspase-1 ac-

tivation via zmp1 (1) whereas the second, revealed in the present study,

entails the suppression of IL1B mRNA expression indirectly through the

induction of host type I IFN secretion (2). Dotted lines indicate hypo-

thetical pathways necessary for sensing bacterial products critical for

triggering caspase-1 activation and type I IFN production. These two

pathways are not effectively triggered by BCG or other RD1-deficient

mycobacteria since they involve cytosolic sensors that these bacteria

cannot access because they lack the ESX-1 secretion system. B, Table

summarizing the levels of key elements in the pathways described in A in

infected macrophages. The effects of IFN blockade on the expression of

these components in M. tuberculosis-infected macrophages are also de-

scribed. PRR, pattern recognition receptor.

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and alum triggered IL-1b production at the levels of both pro–IL-1b protein synthesis and inflammasome activation (41). Thesediscordant findings have yet to be reconciled. One possibility isthat the different mechanisms observed may result from variationsin the strength, duration, and/or the combination of stimuli usedfor triggering pro–IL-1b synthesis and mature IL-1b release.Differences in the timing and duration of exogenous type I IFNexposure employed in the different studies may also have con-tributed to the distinct results obtained.Interestingly, although IFN-a/b and IFN-g share related sig-

naling pathways, analyses performed on a subset of donors sug-gest that IFN-g does not exhibit the same effects on IL-1bproduction by M. tuberculosis-infected human macrophages.Moreover, IFN-g, a known macrophage-stimulating cytokine, isunable to antagonize the suppressive effects of endogenous type IIFNs on infected human macrophages, suggesting a dominanteffect of type I over type II IFN signaling in regulating the IL-1bresponse to M. tuberculosis in human macrophages. This dis-tinction may be due partly to the known ability of M. tuberculosisto interfere with IFN-g signaling in macrophages, which has beenproposed as a mechanism for evasion of adaptive immunity by thepathogen (42, 43).The functions of the type I IFN and IL-1b pathways in human TB

are poorly understood. Studies in the murine model have suggestedthat two cytokines play opposite roles in host resistance to M. tu-berculosis, and the present study demonstrates that type I IFNsclearly limit IL-1b production in human macrophages infected withthe pathogen. Because IL-1b is host protective in murine TB, re-striction of IL-1b production by type I IFNs might be expected toprevent host clearance of the organism. However, due to the knownproinflammatory effects of IL-1b on cell recruitment, it is alsopossible that downregulation of its activity in some settings couldbe host protective through dampening excessive IL-1b–mediatedtissue pathology induced by virulent mycobacteria (44) and/or evenpreventing the establishment of the infection by restricting macro-phage recruitment to granulomas, a process that has recently beenproposed to support rather than limit mycobacterial survival in vivo(45). The impact of such regulation on the establishment andmaintenance of chronic M. tuberculosis infection in humans re-mains to be elucidated.Regardless of the exact mechanisms involved, the balance be-

tween IL-1b and type I IFN responses may be pivotal in ensuringthe survival of host as well as pathogen, thereby contributing to themaintenance of persistent M. tuberculosis infection. Disturbingthis balance may be detrimental for host control of the infection,as evidenced by our previous finding that polyriboinosinic-poly-ribocytidylic acid induction of type I IFN results in increasedbacterial growth and host pathology in mice (21) and the obser-vation of TB reactivation in chronic hepatitis C patients undergoingtherapy with pegylated IFN-a and ribavirin (46, 47).In agreement with previous studies with murine macrophages,

avirulent mycobacteria (in this case BCG) trigger substantially lesstype I IFNs than does virulent M. tuberculosis in human macro-phages. Given the negative regulatory role of type I IFNs in M.tuberculosis infection, one might predict from these observationsthat IL-1b production would be increased in BCG-infected mac-rophages due to the release in type I IFN-mediated suppression.Nevertheless, in both human and mouse macrophages (10–12)only low levels of mature IL-1b are triggered by avirulent my-cobacteria. As indicated in this study, in human macrophages, thisdecreased IL-1b production is not due to a defect in the inductionof IL1B mRNA message by BCG but rather the inability of theorganism to trigger caspase-1–dependent processing and secretionof mature IL-1b. A likely explanation for this caspase-1 activation

defect is the different intracellular trafficking of virulent versusavirulent mycobacteria in macrophages. Thus, virulent M. tuber-culosis (which expresses the ESX-1/RD1 secretion system) hasbeen proposed to translocate bacterial products from the phag-olysosome into the cytoplasm where, as previously demonstratedin murine macrophages, they trigger host inflammasome activa-tion and mature IL-1b protein release (10–12, 44). In contrast,avirulent BCG and other RD1-deficient mycobacteria lack thissecretion system and are retained within the phagolysosomalcompartment (Fig. 7). Therefore, regardless of the regulation ofIL1B transcription by type I IFNs, avirulent mycobacteria remainincapable of stimulating the production of mature bioactive IL-1bprotein. For this reason, it can be concluded that in macrophagesinfected with virulent versus avirulent mycobacteria IL-1b pro-duction is governed by both overlapping and distinct mechanisms.In addition to highlighting distinctions between M. tubercu-

losis and BCG, our data reveal major differences between humanmacrophages and monocytes in the regulation of IL-1b productionfollowing mycobacterial infection. It has been previously dem-onstrated that following pattern recognition receptor stimulation,human monocytes do not require a second signal for IL-1b releasebecause caspase-1 is constitutively activated (34, 35). The latterdistinction is the likely explanation of our observation that, indirect contrast to infected macrophages,M. tuberculosis- and BCG-infected monocytes secrete comparable levels of IL-1b. This cell-specific difference in IL-1b regulation in mononuclear phagocytesmay have important in vivo implications. Thus, if the mycobac-terial infection is not contained locally within tissue macrophagesand disseminates systemically, interaction of circulating mono-cytes with mycobacteria will trigger the robust production of IL-1b without the requirement for additional inflammasome activa-tion signals, thereby preventing further spread of the pathogen.M. tuberculosis has evolved multiple strategies to counteract

host defense mechanisms in infected macrophage by regulatinghost cytokine signaling, cell survival, Ag presentation, and micro-bicidal activities (48, 49). In this study, we have demonstrateda cytokine regulatory loop by which M. tuberculosis can mani-pulate host IL-1 production through the induction of type I IFNs.These findings have important implications for the understandingof M. tuberculosis virulence, persistence, and pathogenesis. Be-cause type I IFNs are readily triggered by numerous viral andother pathogens, these pathways may be of particular relevance indelineating the mechanisms by which coinfections may contributeto TB progression. The extent to which type I IFN and IL-1binduction and signaling influence the outcome of M. tuberculosisinfection in humans is thus an important topic for future in-vestigation.

AcknowledgmentsWe thank Dr. Lyudmila Lyakh (National Cancer Institute) for initial help in

measuring gene expression using the nCounter analysis system and David

Kugler for assistance with graphics. We also thank Drs. Siamon Gordon and

Kathryn Zoon for invaluable discussions and Drs. Dragana Jankovic and

Alena Srinivasan for critical reading of the manuscript.

DisclosuresThe authors have no financial conflicts of interest.

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