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Cutting Edge: Cyclic Polypeptide and Aminoglycoside Trigger IL-1 β Secretion by Activating the NLRP3 Inflammasome This information is current as of October 2, 2021. Ramanjaneyulu Allam, Murthy Narayana Darisipudi, Khader Valli Rupanagudi, Julia Lichtnekert, Jurg Tschopp and Hans-Joachim Anders J Immunol published online 28 January 2011

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 28, 2011, doi:10.4049/jimmunol.1002657

Cutting Edge: Cyclic Polypeptide and Aminoglycoside Antibiotics Trigger IL-1b Secretion by Activating the NLRP3 Inflammasome Ramanjaneyulu Allam,*,† Murthy Narayana Darisipudi,* Khader Valli Rupanagudi,* Julia Lichtnekert,* Jurg Tschopp,† and Hans-Joachim Anders* Clinical use of antibiotics is based on their capacity to Generally, some bacterial products can activate the host’s inhibit bacterial growth via bacteriostatic or bacterio- immunity through innate pathogen recognition receptors cidal effects. In this article, we show that the aminogly- such as TLR or the inflammasomes (1, 2). For example, coside , the cyclic lipopeptide anti- bacterial components activate TLR1, TLR2, TLR4, biotic B, and the cyclic antibiotics TLR6, and NLRP1; bacterial nucleic acids activate TLR9; gramicidin and can induce IL-1b secretion and the AIM2 inflammasome or bacterial activate Downloaded from in bone marrow dendritic cells and macrophages. LPS TLR5 and the NLRC4 inflammasome. TLR activation trig- priming was required to trigger the transcription and gers the secretion of multiple proinflammatory chemokines b or cytokines and induces pro–IL-1b (1). In contrast with all translation of pro–IL-1 butwasindependentof b TNFR or IL-1R signaling. All four antibiotics required other cytokines, the secretion of IL-1 requires inflamma- the NLRP3 inflammasome, the adaptor ASC, and some-mediated activation of caspase-1, also referred to as IL-1b–converting (2). Four different inflammasomes http://www.jimmunol.org/ caspase-1 activation to secrete IL-1b, a process that have been described to integrate the various endogenous and depended on efflux but was independent exogenous triggers of caspase-1 activation: NLRP1, NLRP3, of P2X7 receptor. All four antibiotics induced neutro- NLRC4, and AIM2 (2–5). Recently, distinct bacterial exo- phil influx into the peritoneal cavity of mice, which toxins have been reported to specifically trigger the NLRP1 required NLRP3 only in the case of . To- inflammasome-mediated (bacillus anthracis lethal toxin) or gether, certain antibiotics have the potential to directly the NLRP3 inflammasome-mediated caspase-1 activation (6– activate innate immunity of the host. The Journal of 8), including the bacterial nigericin (9, 10). We

Immunology, 2011, 186: 000–000. therefore questioned whether antibiotics, another class of bac- by guest on October 2, 2021 terial toxins, might have the potential to directly activate in- b he morbidity and mortality related to bacterial flammasome- and caspase-1–mediated release of IL-1 ,and infections has dramatically improved by the use of thereby enhance innate immunity as part of the host defense T antibiotics. Antibiotics are naturally occurring toxins against invading pathogens. that are secreted by microorganisms to suppress the growth of competitors in the same ecological niche. Antibiotics in clin- Materials and Methods ical use encompass semisynthetic compounds that have been Cell culture chemically optimized to improve biostability and efficacy. The Bone marrow-derived dendritic cells (BMDCs) and macrophages were iso- clinical use of antibiotics is based on their capacity to kill lated from 6-wk-old C57BL/6 mice (The Jackson Laboratory, Bar Harbor, bacteria (bactericidal effect) or to inhibit bacterial growth (bac- ME) by established protocols. In some experiments, BMDCs were prepared from NLRP3- (11), ASC- (9), P2X7- (12), TNFR1/2- (The Jackson Laboratory), teriostatic effect), which supports the host’s immune system or IL-1R1–deficient mice (The Jackson Laboratory). Human PBMCs were to eradicate the pathogen. As such, antibiotics and the host’s isolated from whole human blood of healthy, voluntary donors by Ficoll- immune defense have synergistic effects in controlling bac- Hypaque density-gradient centrifugation (Biochrom). Experiments involving human materials were in accordance with precepts established by the Helsinki terial infections. Currently, the clinical use of antibiotics is Declaration. Cells were cultured in RPMI medium 1640/GlutaMAX-I me- based on the concept that they reduce the bacterial load, dium (Invitrogen) supplemented with 10% FBS (v/v; Biochrom KG), 1% of enabling the host’s immune defense to control those that and streptomycin (PAA Laboratories). All cells were stimulated in 3 6 survive or that are resistant to the antibiotic drug. A direct serum-free RPMI 1640 medium at a density of 1 10 cells/ml. Cells were prestimulated for 3 h with ultrapure LPS (10 ng/ml; Invivogen); then later immunomodulatory effect of antibiotics on host defense is stimulated for 6 h with antibiotics (50 mg/ml; Sigma), ATP (5 mM; Inviv- speculative. ogen), and monosodium urate (250 mg/ml; Invivogen). In some experiments,

*Medizinische Poliklinik, Universita¨tMu¨nchen, Munich, Germany; and †Department The online version of this article contains supplemental material. of Biochemistry, University of Lausanne, Epalinges, Switzerland Abbreviations used in this article: BMDC, bone marrow-derived dendritic cell; ROS, Received for publication August 5, 2010. Accepted for publication January 4, 2011. reactive oxygen species. This work was supported by the Deutsche Forschungsgemeinschaft (Grant GRK1202). Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 Address correspondence and reprint requests to Dr. Hans-Joachim Anders, Medizinische Poliklinik-Innenstadt, LMU, Schillerstrasse 42, Mu¨nchen, 80336 Germany. E-mail ad- dress: [email protected]

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002657 2 CUTTING EDGE: ANTIBIOTICS ACTIVATE INFLAMMASOMES cells were prestimulated for 6, 12, and 24 h. For all conditions, cell-free duration of LPS priming augmented IL-1b secretion and supernatants were analyzed for cytokine secretion by ELISA. Supernatants caspase-1 activation (Supplemental Fig. 1B). However, 24-h were concentrated with Amicon ultrafilters (Millipore), or cells were lysed for k immunoblot analysis. Inhibitors such as Z-VAD-FMK (20 mM; Invivogen), LPS priming is toxic to cells (data not shown). NF- B in- cytochalasin D (5 mM), CA-074-Me (10 mM; Calbiochem), and N-acetyl hibition with Bay 11-7082 and inhibition of trans- cysteine (10 mM; Sigma) were added before 30 min of antibiotic stimulation. lation with cycloheximide abrogated secretion of mature IL- Cycloheximide (10 mM; Sigma) and Bay11-7082 (20 mM; Invivogen) were b added 1 h before LPS prestimulation. KCl (75 mM) was used to increase ex- 1 , as well as caspase-1 activation (Supplemental Fig. 1C), tracellular K+ concentration. NaCl (75 mM) was used in control experiments. suggesting that de novo transcription and translation of pro– IL-1b by LPS-driven NF-kB signaling as a mandatory step. Readout parameters Secondary TNF or IL-1 release was not required for this Cell supernatants were analyzed for IL-1b secretion by ELISA (BD Bio- process as polymyxin B, tyrothricin, gramicidin, and neo- sciences). Precipitated media supernatants or cell extracts were analyzed by mycin induced the secretion of similar IL-1b levels and standard immunoblot techniques as described previously (13). Primary Abs caspase-1 activation in TNFR1/2 or IL-1R1–deficient mice were polyclonal goat Ab to mouse–IL-1b (R&D Systems) and to anti– caspase-1 (sc-514; Santa Cruz Biotechnology). (Fig. 1B, Supplemental Fig. 1D) (14). Similar results were obtained in bone marrow-derived macrophages (Supplemen- Mouse peritonitis model tary Fig. 2A). However, tyrothricin and gramicidin showed Six-wk-old wild-type or NLRP3-deficient C57BL/6 mice received a single i.p. less induction of IL-1b secretion in human PBMCs (Sup- injection with 100 mg LPS (Invivogen). Three hours later, second injection plemental Fig. 2B). Interestingly, polymyxin B, tyrothricin, m m with 100 l of either PBS or 250 g of selected antibiotics was given through and gramicidin are cyclic polypeptide antibiotics, whereas the same route. After 6 h, all mice were sacrificed and peritoneal cavities were Downloaded from washed with 5 ml PBS. The lavage fluids were analyzed for neutrophil re- neomycin is an aminoglycoside antibiotic. Furthermore, struc- cruitment by FACS using Ly-6G and 7/4 (BD Biosciences and AbD Serotec). turally related antibiotics showed different capacity to induce IL-1b secretion, for example, cyclic polypeptides polymyxin Statistical analysis B and E () or the aminoglycosides neomycin and Data are expressed as the mean 6 SD. Comparison between two groups was gentamicin (Fig. 1A). performed by two-tailed t test. A p value ,0.05 was considered to be sta- tistically significant. All statistical analyses were calculated using GraphPad http://www.jimmunol.org/ Prism. Polymyxin B, tyrothricin, gramicidin, and neomycin trigger IL-1b release via the NLRP3 inflammasome Results Various compounds such as crystals, pore-forming toxins, Distinct polypeptide or aminoglycoside antibiotics activate dendritic or ATP induce IL-1b secretion by activating the NLRP3 b cells and macrophages to secrete IL-1 inflammasome (15). We therefore exposed polymyxin B, ty- To address a putative immunostimulatory potential of anti- rothricin, gramicidin, and neomycin to BMDCs isolated from biotics, we exposed LPS-primed BMDCs to selected members NLRP3-deficient or wild-type mice. Lack of NLRP3 sub- b b of commonly used classes of antibiotics and measured IL-1 stantially decreased IL-1 release on stimulation with all four by guest on October 2, 2021 production in cell culture supernatants after 6 h of stimula- antibiotics (Fig. 2A). However, polymyxin B and gramicidin tion. Among all antibiotics tested, only polymyxin B, tyro- elicited a partial response in NLRP3-deficient BMDCs as thricin, gramicidin, and neomycin induced IL-1b release in detected by an IL-1b ELISA, which might also detect extra- BMDCs (Fig. 1A). This effect was dependent on priming cellular pro–IL-1b (Fig. 2A). To address this possibility, we with LPS (Supplemental Fig. 1A), which provides the neces- performed immunoblotting of cell culture supernatants to sary signal for the induction of pro–IL-1b (2). Increased time dissect 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. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/

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 by guest on October 2, 2021 expressed as the mean 6 SD from three independent experiments all performed in triplicates.

NLRP3-deficient cells, cleaved p17 was completely absent all four antibiotics could induce IL-1b release in P2X7-de- also on polymyxin B and gramicidin exposure. In addition, ficient BMDCs (Fig. 3B). Potassium flux is obviously im- immunoblotting for the p10 subunit of caspase-1 in cell iso- portant for antibiotics-induced NLRP3 activation but is lates of wild-type and NLRP3-deficient cells was performed different from ATP because these antibiotics do not require to study caspase-1 activation. Stimulation with all four anti- P2X7. To test the role of the phagocytosis-dependent endo- biotics induced the caspase-1 cleaving product p10 in wild- somal leakage pathway of NLRP3 activation, we exposed type cells but much less in NLRP3-deficient cells, showing BMDCs to all four antibiotics in the presence or absence of that NLRP3 is required for antibiotics-induced caspase-1 the phagocytosis inhibitor cytochalasin D. Interestingly, cy- activation (Fig. 2B). NLRP3 needs the adaptor ASC to acti- tochalasin D reduced IL-1b release by tyrothricin, gramicidin, vate caspase-1 (15). In fact, lack of ASC, as well as the pan- and neomycin, but not by polymyxin B (Fig. 3C). In addi- caspase inhibitor Z-VAD-FMK, abrogated IL-1b release in tion, the cathepsin B inhibitor CA-074-Me and the antioxi- BMDCs on stimulation with all four antibiotics (Fig. 2C, dant N-acetylcysteine significantly suppressed tyrothricin- and 2D). Thus, IL-1b secretion induced by polymyxin B, tyro- neomycin-induced IL-1b secretion (Fig. 3D). These findings thricin, gramicidin, and neomycin involves the NLRP3/ASC/ suggest a contribution of the phagocytosis-cathepsin B–de- caspase-1 pathway. pendent and the reactive oxygen species (ROS)-dependent modes of NLRP3 activation for tyrothricin and neomycin. Antibiotics-induced NLRP3 activation depends on potassium flux but However, gramicidin and polymyxin B were independent of not on P2X7 cathepsin B- and ROS-induced NLRP3 activation. Together, Several models of NLRP3 activation have been described, that polymyxin B, gramicidin, tyrothricin, and neomycin activate is, P2X7- and potassium flux-dependent pore formation (9), the NLRP3 inflammasome through different pathways, but endosomal rupture involving cytosolic cathepsin B activity all were dependent on potassium efflux. (16), and oxidative stress (17). An extracellular potassium concentration of 75 mM suppressed potassium efflux and Antibiotics induce peritonitis after i.p. injection abrogated IL-1b secretion on ATP, as well as on polymyxin NLRP3 inducers can activate neurophil influx in in vivo B, gramicidin, tyrothricin, and neomycin (Fig. 3A). However, peritonitis models (11). Therefore, we tested the potential of ATP-induced IL-1b secretion requires the P2X7 receptor, but the antibiotics to induce neutrophil influx after LPS pre- 4 CUTTING EDGE: ANTIBIOTICS ACTIVATE INFLAMMASOMES Downloaded from http://www.jimmunol.org/

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 by guest on October 2, 2021 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. stimulation into the peritoneal cavity within 6 h after a of the antibiotic-producing microorganism itself. Third, when single i.p. injection. Polymyxin B, gramicidin, tyrothricin, used as an antibiotic drug clinically, the immunostimulatory and neomycin significantly increased neutrophil influx as potential might contribute to drug toxicity. In fact, poly- compared with LPS exposure alone (Supplemental Fig. 2C). myxin B’s, gramicidin’s, tyrothricin’s, and neomycin’s toxicity Without LPS priming these antibiotics did not induce neu- profiles do not recommend systemic treatment, and all four trophil influx (data not shown). Polymyxin B-induced neu- drugs are in topical use only (18–20). The cyclic polypeptides trophil influx was substantially reduced in Nlrp3-deficient and aminoglycosides have different toxicity profiles; therefore, mice, whereas NLRP3 was not required for the recruitment of it is unlikely that drug toxicity is solely mediated by IL-1b neutrophils on injection of gramicidin, tyrothricin, and neo- secretion. mycin (Supplemental Fig. 2C). Polymyxin B, gramicidin, ty- We found all four immunostimulatory antibiotics to acti- rothricin, and neomycin enhance neutrophil recruitment, but vate the NLRP3 inflammasome. This is not surprising be- this process involves additional pathways beyond NLRP3 cause NLRP3 integrates diverse classes of stimuli for IL-1b signaling. secretion. It is interesting that all antibiotics require potassium efflux to activate NLRP3, whereas they differ in terms of Discussion the involvement of additional ways to activate NLRP3. Tyro- Our data show that distinct cyclic polypeptide or amino- thricin, gramicidin, and neomycin, but not polymyxin B, glycoside antibiotics activate the NLRP3-inflammsome– and involve phagocytosis like monosodium urate crystals. Further- caspase-1–mediated release of IL-1b. IL-1b secretion will more, tyrothricin and neomycin, but not gramicidin or poly- elicit secondary effects after IL-1R activation, hence these an- myxin B, involve cathepsins and ROS production. 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