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InflammasomeSecretion by Activating the NLRP3

βAminoglycoside Antibiotics Trigger IL-1Cutting Edge: Cyclic Polypeptide and

and Hans-Joachim AndersKhader Valli Rupanagudi, Julia Lichtnekert, Jurg Tschopp Ramanjaneyulu Allam, Murthy Narayana Darisipudi,

ol.1002657http://www.jimmunol.org/content/early/2011/01/28/jimmun

published online 28 January 2011J Immunol 

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Cutting Edge: Cyclic Polypeptide and AminoglycosideAntibiotics Trigger IL-1b Secretion by Activating theNLRP3 InflammasomeRamanjaneyulu Allam,*,† Murthy Narayana Darisipudi,* Khader Valli Rupanagudi,*Julia Lichtnekert,* Jurg Tschopp,† and Hans-Joachim Anders*

Clinical use of antibiotics is based on their capacity toinhibit bacterial growth via bacteriostatic or bacterio-cidal effects. In this article, we show that the aminogly-coside antibiotic neomycin, the cyclic lipopeptide anti-biotic polymyxin B, and the cyclic peptide antibioticsgramicidin and tyrothricin can induce IL-1b secretionin bone marrow dendritic cells and macrophages. LPSpriming was required to trigger the transcription andtranslation of pro–IL-1b but was independent ofTNFR or IL-1R signaling. All four antibiotics requiredthe NLRP3 inflammasome, the adaptor ASC, andcaspase-1 activation to secrete IL-1b, a process thatdepended on potassium efflux but was independentof P2X7 receptor. All four antibiotics induced neutro-phil influx into the peritoneal cavity of mice, whichrequired NLRP3 only in the case of polymyxin B. To-gether, certain antibiotics have the potential to directlyactivate innate immunity of the host. The Journal ofImmunology, 2011, 186: 000–000.

The morbidity and mortality related to bacterialinfections has dramatically improved by the use ofantibiotics. Antibiotics are naturally occurring toxins

that are secreted by microorganisms to suppress the growth ofcompetitors in the same ecological niche. Antibiotics in clin-ical use encompass semisynthetic compounds that have beenchemically optimized to improve biostability and efficacy. Theclinical use of antibiotics is based on their capacity to killbacteria (bactericidal effect) or to inhibit bacterial growth (bac-teriostatic effect), which supports the host’s immune systemto eradicate the pathogen. As such, antibiotics and the host’simmune defense have synergistic effects in controlling bac-terial infections. Currently, the clinical use of antibiotics isbased on the concept that they reduce the bacterial load,enabling the host’s immune defense to control those thatsurvive or that are resistant to the antibiotic drug. A directimmunomodulatory effect of antibiotics on host defense isspeculative.

Generally, some bacterial products can activate the host’simmunity through innate pathogen recognition receptorssuch as TLR or the inflammasomes (1, 2). For example,bacterial cell wall components activate TLR1, TLR2, TLR4,TLR6, and NLRP1; bacterial nucleic acids activate TLR9;and the AIM2 inflammasome or bacterial proteins activateTLR5 and the NLRC4 inflammasome. TLR activation trig-gers the secretion of multiple proinflammatory chemokinesor cytokines and induces pro–IL-1b (1). In contrast with allother cytokines, the secretion of IL-1b requires inflamma-some-mediated activation of caspase-1, also referred to asIL-1b–converting enzyme (2). Four different inflammasomeshave been described to integrate the various endogenous andexogenous triggers of caspase-1 activation: NLRP1, NLRP3,NLRC4, and AIM2 (2–5). Recently, distinct bacterial exo-toxins have been reported to specifically trigger the NLRP1inflammasome-mediated (bacillus anthracis lethal toxin) orthe NLRP3 inflammasome-mediated caspase-1 activation (6–8), including the bacterial ionophore nigericin (9, 10). Wetherefore questioned whether antibiotics, another class of bac-terial toxins, might have the potential to directly activate in-flammasome- and caspase-1–mediated release of IL-1b, andthereby enhance innate immunity as part of the host defenseagainst invading pathogens.

Materials and MethodsCell culture

Bone marrow-derived dendritic cells (BMDCs) and macrophages were iso-lated from 6-wk-old C57BL/6 mice (The Jackson Laboratory, Bar Harbor,ME) by established protocols. In some experiments, BMDCs were preparedfrom NLRP3- (11), ASC- (9), P2X7- (12), TNFR1/2- (The Jackson Laboratory),or IL-1R1–deficient mice (The Jackson Laboratory). Human PBMCs wereisolated from whole human blood of healthy, voluntary donors by Ficoll-Hypaque density-gradient centrifugation (Biochrom). Experiments involvinghuman materials were in accordance with precepts established by the HelsinkiDeclaration. Cells were cultured in RPMI medium 1640/GlutaMAX-I me-dium (Invitrogen) supplemented with 10% FBS (v/v; Biochrom KG), 1% ofpenicillin and streptomycin (PAA Laboratories). All cells were stimulated inserum-free RPMI 1640 medium at a density of 13 106 cells/ml. Cells wereprestimulated for 3 h with ultrapure LPS (10 ng/ml; Invivogen); then laterstimulated for 6 h with antibiotics (50 mg/ml; Sigma), ATP (5 mM; Inviv-ogen), and monosodium urate (250 mg/ml; Invivogen). In some experiments,

*Medizinische Poliklinik, Universitat Munchen, Munich, Germany; and †Departmentof Biochemistry, University of Lausanne, Epalinges, Switzerland

Received for publication August 5, 2010. Accepted for publication January 4, 2011.

This work was supported by the Deutsche Forschungsgemeinschaft (Grant GRK1202).

Address correspondence and reprint requests to Dr. Hans-Joachim Anders, MedizinischePoliklinik-Innenstadt, LMU, Schillerstrasse 42, Munchen, 80336 Germany. E-mail ad-dress: hjanders@med.uni-muenchen.de

The online version of this article contains supplemental material.

Abbreviations used in this article: BMDC, bone marrow-derived dendritic cell; ROS,reactive oxygen species.

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

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002657

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cells were prestimulated for 6, 12, and 24 h. For all conditions, cell-freesupernatants were analyzed for cytokine secretion by ELISA. Supernatantswere concentrated with Amicon ultrafilters (Millipore), or cells were lysed forimmunoblot analysis. Inhibitors such as Z-VAD-FMK (20 mM; Invivogen),cytochalasin D (5 mM), CA-074-Me (10 mM; Calbiochem), and N-acetylcysteine (10 mM; Sigma) were added before 30 min of antibiotic stimulation.Cycloheximide (10 mM; Sigma) and Bay11-7082 (20 mM; Invivogen) wereadded 1 h before LPS prestimulation. KCl (75 mM) was used to increase ex-tracellular K+ concentration. NaCl (75 mM) was used in control experiments.

Readout parameters

Cell supernatants were analyzed for IL-1b secretion by ELISA (BD Bio-sciences). Precipitated media supernatants or cell extracts were analyzed bystandard immunoblot techniques as described previously (13). Primary Abswere polyclonal goat Ab to mouse–IL-1b (R&D Systems) and to anti–caspase-1 (sc-514; Santa Cruz Biotechnology).

Mouse peritonitis model

Six-wk-old wild-type or NLRP3-deficient C57BL/6 mice received a single i.p.injection with 100 mg LPS (Invivogen). Three hours later, second injectionwith 100 ml of either PBS or 250 mg of selected antibiotics was given throughthe same route. After 6 h, all mice were sacrificed and peritoneal cavities werewashed with 5 ml PBS. The lavage fluids were analyzed for neutrophil re-cruitment by FACS using Ly-6G and 7/4 (BD Biosciences and AbD Serotec).

Statistical analysis

Data are expressed as the mean 6 SD. Comparison between two groups wasperformed by two-tailed t test. A p value ,0.05 was considered to be sta-tistically significant. All statistical analyses were calculated using GraphPadPrism.

ResultsDistinct polypeptide or aminoglycoside antibiotics activate dendriticcells and macrophages to secrete IL-1b

To address a putative immunostimulatory potential of anti-biotics, we exposed LPS-primed BMDCs to selected membersof commonly used classes of antibiotics and measured IL-1bproduction in cell culture supernatants after 6 h of stimula-tion. Among all antibiotics tested, only polymyxin B, tyro-thricin, gramicidin, and neomycin induced IL-1b release inBMDCs (Fig. 1A). This effect was dependent on primingwith LPS (Supplemental Fig. 1A), which provides the neces-sary signal for the induction of pro–IL-1b (2). Increased time

duration of LPS priming augmented IL-1b secretion andcaspase-1 activation (Supplemental Fig. 1B). However, 24-hLPS priming is toxic to cells (data not shown). NF-kB in-hibition with Bay 11-7082 and inhibition of protein trans-lation with cycloheximide abrogated secretion of mature IL-1b, as well as caspase-1 activation (Supplemental Fig. 1C),suggesting that de novo transcription and translation of pro–IL-1b by LPS-driven NF-kB signaling as a mandatory step.Secondary TNF or IL-1 release was not required for thisprocess as polymyxin B, tyrothricin, gramicidin, and neo-mycin induced the secretion of similar IL-1b levels andcaspase-1 activation in TNFR1/2 or IL-1R1–deficient mice(Fig. 1B, Supplemental Fig. 1D) (14). Similar results wereobtained in bone marrow-derived macrophages (Supplemen-tary Fig. 2A). However, tyrothricin and gramicidin showedless induction of IL-1b secretion in human PBMCs (Sup-plemental Fig. 2B). Interestingly, polymyxin B, tyrothricin,and gramicidin are cyclic polypeptide antibiotics, whereasneomycin is an aminoglycoside antibiotic. Furthermore, struc-turally related antibiotics showed different capacity to induceIL-1b secretion, for example, cyclic polypeptides polymyxinB and E (colistin) or the aminoglycosides neomycin andgentamicin (Fig. 1A).

Polymyxin B, tyrothricin, gramicidin, and neomycin trigger IL-1brelease via the NLRP3 inflammasome

Various compounds such as crystals, pore-forming toxins,or ATP induce IL-1b secretion by activating the NLRP3inflammasome (15). We therefore exposed polymyxin B, ty-rothricin, gramicidin, and neomycin to BMDCs isolated fromNLRP3-deficient or wild-type mice. Lack of NLRP3 sub-stantially decreased IL-1b release on stimulation with all fourantibiotics (Fig. 2A). However, polymyxin B and gramicidinelicited a partial response in NLRP3-deficient BMDCs asdetected by an IL-1b ELISA, which might also detect extra-cellular pro–IL-1b (Fig. 2A). To address this possibility, weperformed immunoblotting of cell culture supernatants todissect the pro–IL-1b from the cleaved IL-1b (p17). In

FIGURE 1. Polymyxin B, tyrothricin, gramicidin, and neomycin trigger IL-1b secretion. A, Murine BMDCs were primed with LPS and exposed to members of

five different classes of antibiotics at a concentration of 50 mg/ml, and IL-1b secretion was measured in supernatants after 6 h of stimulation. ATP was used as

a positive control. Note that only polymyxin B, tyrothricin, gramicidin, and neomycin induced IL-1b secretion. B, LPS primed BMDCs from TNFR1/2 and IL-

1R1–deficient mice were exposed to 50 mg/ml of polymyxin B, tyrothricin, gramicidin, and neomycin. IL-1b secretion was measured in supernatants after 6 h of

stimulation. Data are expressed as the mean 6 SD from three independent experiments, all performed in triplicates.

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NLRP3-deficient cells, cleaved p17 was completely absentalso on polymyxin B and gramicidin exposure. In addition,immunoblotting for the p10 subunit of caspase-1 in cell iso-lates of wild-type and NLRP3-deficient cells was performedto study caspase-1 activation. Stimulation with all four anti-biotics induced the caspase-1 cleaving product p10 in wild-type cells but much less in NLRP3-deficient cells, showingthat NLRP3 is required for antibiotics-induced caspase-1activation (Fig. 2B). NLRP3 needs the adaptor ASC to acti-vate caspase-1 (15). In fact, lack of ASC, as well as the pan-caspase inhibitor Z-VAD-FMK, abrogated IL-1b release inBMDCs on stimulation with all four antibiotics (Fig. 2C,2D). Thus, IL-1b secretion induced by polymyxin B, tyro-thricin, gramicidin, and neomycin involves the NLRP3/ASC/caspase-1 pathway.

Antibiotics-induced NLRP3 activation depends on potassium flux butnot on P2X7

Several models of NLRP3 activation have been described, thatis, P2X7- and potassium flux-dependent pore formation (9),endosomal rupture involving cytosolic cathepsin B activity(16), and oxidative stress (17). An extracellular potassiumconcentration of 75 mM suppressed potassium efflux andabrogated IL-1b secretion on ATP, as well as on polymyxinB, gramicidin, tyrothricin, and neomycin (Fig. 3A). However,ATP-induced IL-1b secretion requires the P2X7 receptor, but

all four antibiotics could induce IL-1b release in P2X7-de-ficient BMDCs (Fig. 3B). Potassium flux is obviously im-portant for antibiotics-induced NLRP3 activation but isdifferent from ATP because these antibiotics do not requireP2X7. To test the role of the phagocytosis-dependent endo-somal leakage pathway of NLRP3 activation, we exposedBMDCs to all four antibiotics in the presence or absence ofthe phagocytosis inhibitor cytochalasin D. Interestingly, cy-tochalasin D reduced IL-1b release by tyrothricin, gramicidin,and neomycin, but not by polymyxin B (Fig. 3C). In addi-tion, the cathepsin B inhibitor CA-074-Me and the antioxi-dant N-acetylcysteine significantly suppressed tyrothricin- andneomycin-induced IL-1b secretion (Fig. 3D). These findingssuggest a contribution of the phagocytosis-cathepsin B–de-pendent and the reactive oxygen species (ROS)-dependentmodes of NLRP3 activation for tyrothricin and neomycin.However, gramicidin and polymyxin B were independent ofcathepsin B- and ROS-induced NLRP3 activation. Together,polymyxin B, gramicidin, tyrothricin, and neomycin activatethe NLRP3 inflammasome through different pathways, butall were dependent on potassium efflux.

Antibiotics induce peritonitis after i.p. injection

NLRP3 inducers can activate neurophil influx in in vivoperitonitis models (11). Therefore, we tested the potential ofthe antibiotics to induce neutrophil influx after LPS pre-

FIGURE 2. IL-1b secretion triggered by polymyxin B, tyrothricin, neomycin, and gramicidin requires all components of the NLRP3 inflammasome. A, Wild-

type and NLRP3-deficient mice derived BMDCs were primed with LPS and exposed 50 mg/ml of polymyxin B, tyrothricin, gramicidin, and neomycin. IL-1b

secretion was measured in supernatants after 6 h of stimulation. ATP was used as a positive control. B, Immunoblot analysis of mature IL-1b (p17) and caspase-1

cleavage product (p10) in wild-type and NLRP3-deficient BMDCs stimulated with antibiotics. C, IL-1b ELISA for wild-type and ASC-deficient BMDCs

stimulated with antibiotics. Note that IL-1b secretion was dependent on the presence of NLRP3 and ASC for all drugs. D, Wild-type BMDCs were stimulated

with antibiotics in the presence or absence of the caspase inhibitor Z-VAD-FMK. IL-1b secretion was measured in supernatants after 6 h of stimulation. Data are

expressed as the mean 6 SD from three independent experiments all performed in triplicates.

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stimulation into the peritoneal cavity within 6 h after asingle i.p. injection. Polymyxin B, gramicidin, tyrothricin,and neomycin significantly increased neutrophil influx ascompared with LPS exposure alone (Supplemental Fig. 2C).Without LPS priming these antibiotics did not induce neu-trophil influx (data not shown). Polymyxin B-induced neu-trophil influx was substantially reduced in Nlrp3-deficientmice, whereas NLRP3 was not required for the recruitment ofneutrophils on injection of gramicidin, tyrothricin, and neo-mycin (Supplemental Fig. 2C). Polymyxin B, gramicidin, ty-rothricin, and neomycin enhance neutrophil recruitment, butthis process involves additional pathways beyond NLRP3signaling.

DiscussionOur data show that distinct cyclic polypeptide or amino-glycoside antibiotics activate the NLRP3-inflammsome– andcaspase-1–mediated release of IL-1b. IL-1b secretion willelicit secondary effects after IL-1R activation, hence these an-tibiotics have the potential to trigger innate immunity. This isremarkable in three ways. First, these microbial toxins areknown to inhibit bacterial growth directly (bactericidal orbacteriostatic effect). As a new entry, they also may suppressbacterial growth indirectly via activating host defense. Second,the immunostimulatory effects might also affect the growth

of the antibiotic-producing microorganism itself. Third, whenused as an antibiotic drug clinically, the immunostimulatorypotential might contribute to drug toxicity. In fact, poly-myxin B’s, gramicidin’s, tyrothricin’s, and neomycin’s toxicityprofiles do not recommend systemic treatment, and all fourdrugs are in topical use only (18–20). The cyclic polypeptidesand aminoglycosides have different toxicity profiles; therefore,it is unlikely that drug toxicity is solely mediated by IL-1bsecretion.We found all four immunostimulatory antibiotics to acti-

vate the NLRP3 inflammasome. This is not surprising be-cause NLRP3 integrates diverse classes of stimuli for IL-1bsecretion. It is interesting that all antibiotics require potassiumefflux to activate NLRP3, whereas they differ in terms ofthe involvement of additional ways to activate NLRP3. Tyro-thricin, gramicidin, and neomycin, but not polymyxin B,involve phagocytosis like monosodium urate crystals. Further-more, tyrothricin and neomycin, but not gramicidin or poly-myxin B, involve cathepsins and ROS production. The reasonsfor these diverse mechanisms remain unclear. It is reportedthat gramicidin dimers can form membrane-spanning pores,which may explain its dependency on potassium flux (19).Cyclic polypeptide and aminoglycoside antibiotics act as

antibacterial toxins; this is why they are in clinical use asantibiotics. Notably, all of these antibiotics cannot be used

FIGURE 3. Polymyxin B, tyrothricin, gramicidin, and neomycin activate NLRP3 through different ways. A, LPS-primed wild type BMDCs were stimulated

with antibiotics in serum-free buffer with or without 75 mM KCl and NaCl. IL-1b secretion was measured in supernatants after 6 h (B). Wild-type and P2X7-

deficient BMDCs were primed with LPS and exposed to antibiotic. IL-1b secretion was measured in supernatants after 6 h of stimulation. ATP was used as

control. C and D, LPS-primed wild type BMDCs were treated with cytochalasin D, N-acetyl cysteine (NAC), and CA-07-Me for 30 min. Pretreated cells were

stimulated with antibiotics. IL-1b secretion was measured in supernatants after 6 h of stimulation. Data are expressed as the mean 6 SD from three independent

experiments all performed in triplicate.

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systemically because of drug toxicity, but the role of IL-1binduction in this context remains speculative. At least i.p. in-jection caused neutrophil influx, suggesting that these anti-biotics can, in fact, elicit proinflammatory effects in vivo.Polymyxin-driven neutrophil recruitment partially dependedon NLRP3 also in vivo, whereas this was not the case forthe other antibiotics, suggesting that the process of antibioticdrug-induced neutrophil recruitment involves additional mech-anisms. In addition, the LPS-binding capacity of polymyxinB is much appreciated by researchers to neutralize LPS con-tamination, but IL-1b induction might affect the resultsof such in vitro experiments. Together, our data documentthat polymyxin B, gramicidin, tyrothricin, and neomycinhave the potential to trigger IL-1b secretion by activating theNLRP3 inflammasome through potassium efflux. Thus, theseantibiotics add on to the list of microbial toxins that directlyactivate innate immunity of the host.

AcknowledgmentsWe thank Dr. M. Schnurr (University of Munich, Munich, Germany) for pro-

viding the P2X7-deficient mice, Dr. V. Vielhauer (University of Munich) for

providing TNFR-deficient mice, and Dr. V.M. Dixit (Genentech, San Fran-

cisco, CA) for providing the ASC-deficient mice.

DisclosuresThe authors have no financial conflicts of interest.

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