NLRP3 Inflammasome the by Leptospiral Glycolipoprotein

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Downregulation of the Na/K-ATPase Pump by Leptospiral Glycolipoprotein Activates the NLRP3 Inflammasome

This information is current as Sonia Lacroix-Lamandé, Martine Fanton d'Andon, Eric of September 25, 2021. Michel, Gwenn Ratet, Dana J. Philpott, Stephen E. Girardin, Ivo G. Boneca, Alain Vandewalle and Catherine Werts J Immunol 2012; 188:2805-2814; Prepublished online 8 February 2012;

doi: 10.4049/jimmunol.1101987 Downloaded from http://www.jimmunol.org/content/188/6/2805

References This article cites 47 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/188/6/2805.full#ref-list-1 http://www.jimmunol.org/

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Downregulation of the Na/K-ATPase Pump by Leptospiral Glycolipoprotein Activates the NLRP3 Inﬂammasome

Sonia Lacroix-Lamande´,*,†,‡ Martine Fanton d’Andon,*,† Eric Michel,*,†,x Gwenn Ratet,*,† Dana J. Philpott,{ Stephen E. Girardin,‖ Ivo G. Boneca,*,† Alain Vandewalle,#,** and Catherine Werts*,†

Leptospira interrogans is responsible for a zoonotic disease known to induce severe kidney dysfunction and inflammation. In this work, we demonstrate that L. interrogans induces NLRP3 inflammasome-dependent secretion of IL-1b through the alteration of potassium transport in bone marrow-derived macrophages. Lysosome destabilization also contributed to the IL-1b production upon stimulation with live, but not dead, bacteria. Using bone marrow-derived macrophages from various TLRs and nucleotide-binding oligomerization domain-deficient mice, we further determined that IL-1b production was dependent on TLR2 and TLR4, suggesting a participation of the leptospiral LPS to this process. Hypokaliemia in leptospirosis has been linked to the presence of glycolipo- Downloaded from protein, a cell wall component of L. interrogans that is known to inhibit the expression and functions of the Na/K-ATPase pump. We show in this study that glycolipoprotein activates the inflammasome and synergizes with leptospiral LPS to produce IL-1b, mimicking the effect of whole bacteria. These results were confirmed in vivo, as wild-type mice expressed more IL-1b in the kidney than TLR2/4-deficient mice 3 d postinfection with L. interrogans. Collectively, these findings provide the first characterization, to our knowledge, of bacteria-induced activation of the NLRP3 inflammasome through the downregulation of a specific host potassium transporter. The Journal of Immunology, 2012, 188: 2805–2814. http://www.jimmunol.org/

eptospira interrogans is a spirochete responsible for a hallmark of the infection in susceptible mice is a deleterious worldwide zoonosis transmitted by rodents, which shed TLR-independent inflammation (2). Among the candidate re- L the bacteria in their urine. Humans can be infected ceptors that could be involved in this inflammation are the NLRs, through skin contact with contaminated water and develop acute either recognizing muropeptides released by the peptidoglycan leptospirosis, a mild to severe flulike infection that can sometimes (PG; in the case of Nod1 or Nod2) or responsible for the in- lead to a life-threatening disease with liver, lung, and kidney flammasome activation (for NLRP, NLRC, and NAIP proteins) failure (1). Conserved components of the bacterial cell wall (the leading to the secretion of the potent proinflammatory cytokine by guest on September 25, 2021 so-called microbe-associated molecular patterns) are recognized IL-1b (3). IL-1b is a pivotal inflammatory cytokine that plays an by TLRs and nucleotide-binding oligomerization domain (Nod)- important role in the innate immune defense against pathogens like receptors (NLRs) from the host innate immune system, which (4). The main mechanism that allows the pro–IL-1b to be secreted trigger host defense. We recently set up a new murine model to and active is its cleavage by caspase-1, itself activated by an in- study acute leptospirosis; by opposition to asymptomatic control flammasome. The inflammasome is a platform of proteins com- mice, C57BL6/J mice deficient for both TLR2 and TLR4 die of posed of an NLR family member, procaspase-1, and the adaptor acute leptospirosis, as a result of impaired bacterial clearance due protein ASC. Once activated, NLR proteins oligomerize and re- to the lack of sensing of the atypical leptospiral LPS (2). A cruit adaptor proteins, which can bind procaspase-1, and allow for its dimerization and cleavage, licensing the further processing *Institut Pasteur, G5 Biologie et Geńe´tique de la Paroi Bacte´rienne, Paris 75724, b b France; †INSERM Groupe Avenir, Paris 75015, France; ‡Institut National de la of the pro–IL-1 in a mature secreted IL-1 cytokine. Different Recherche Agronomique, Infectiologie Animale et Sante´ Publique, Nouzilly inflammasomes exist. NLRP1 recognizes toxins (5), whereas the 37380, France; xInstitut de Biologie de l’Ecole Normale Superieure-Unite´ Mixte de Recherche 7618 Bioemco, Paris 75230, France; {Department of Immunology, NLRC4 inflammasome is either stimulated upon recognition by University of Toronto, Toronto, Ontario M5S 1A1, Canada; ‖Department of Labora- NAIP5 of flagellin motifs from bacteria or recognition by NAIP2 tory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A1, of a conserved component of the bacterial type three secretion Canada; #INSERM U773, Paris 75018, France; and **Universite´ Paris Diderot Sor- bonne Paris Cite´, Paris 75013, France system (6–8). The best-characterized NLRP is NLRP3, which can Received for publication July 8, 2011. Accepted for publication January 3, 2012. be activated by different stimuli, such as reactive oxygen species This work was supported by the Institut Pasteur and European Research Council (ROS), lysosome destabilization (9), and potassium efflux or low Grant PGN from SHAPE to VIR 202283 (to I.G.B.). intracellular potassium concentration (10, 11). AIM2, which does Address correspondence and reprint requests to Dr. Catherine Werts, Institut Pasteur, not belong to the NLR family, also forms an inflammasome G5 Biologie et Geńe´tique de la Paroi Bacte´rienne, 28 rue du Dr Roux, 75724 Paris Cedex 15, France. E-mail address: [email protected] sensing microbial DNA (12). In this study, we aimed to charac- terize the inflammasome response to L. interrogans in bone Abbreviations used in this article: APDC, (2R, 4R)-4-aminopyrrolidine-2,4-dicarboxylate; BMDM, bone marrow-derived macrophage; DPI, diphenylene iodonium; GLP, marrow-derived macrophages (BMDMs). We demonstrate that a + + 2 glycolipoprotein; MOI, multiplicity of infection; NKCC2, Na -K -Cl ;NLR, bacterial cell wall component of Leptospira, called glycolipo- nucleotide-binding oligomerization domain-like receptor; Nod, nucleotide-binding oligomerization domain; PG, peptidoglycan; qPCR, quantitative PCR; qRT- protein (GLP), activates the NLRP3 inflammasome through PCR, quantitative real-time PCR; ROS, reactive oxygen species; TRIF, Toll/ modulation of potassium transport and, together with LPS, con- IL-1R domain-containing adapter inducing IFN-b; WT, wild-type. tributes to the IL-1b production induced by L. interrogans in- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 fection. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101987 2806 SYNERGY BETWEEN LEPTOSPIRAL LPS AND GLP INDUCES IL-1b

Materials and Methods Generation of mouse BMDMs and in vitro stimulation L. interrogans and in vivo infection experiments BMDMs were isolated as described previously and cultured for 7 d in 10% 3 5 m L. interrogans interrogans serovar Copenhageni strain Fiocruz L1-130 was L929-conditioned medium (17). Mouse BMDMs (2 10 cells in 200 l) 3 6 used in this study as described elsewhere (13). Five days after i.p. infection or (3 10 cells in 3 ml) were, respectively, seeded in 96-well and 6-well plates, and 3 h later were stimulated with live or heat-killed L. interrogans of 3-wk-old guinea pigs with 1 3 108 Leptospira in PBS, blood from at different multiplicities of infection (MOIs). Stimulations were stopped at infected guinea pig was serially diluted (1/20) in liquid EMJH medium different time points, supernatants were removed, and cells and super- (Bio-Rad) and incubated at 28˚C without agitation. Three weeks after, the natants were stored at 220˚C until further use. In some experiments, m last dilution tube leading to Leptospira’s growth was submitted to 0.22 M mouse BMDMs were preincubated 30 min before stimulation with 40 nM filtration, which let the thin Leptospira in but restrained contaminants. This bafilomycin A1, 70 mM KCl, 25 mM (2R, 4R)-4-aminopyrrolidine-2,4- culture was expanded in liquid EMJH until the stationary phase, aliquoted dicarboxylate (APDC; Tebu-Bio), 0.2 mM hexa-N-acetyl-chitohexaose (1 ml) in 15% glycerol, frozen, and stored in liquid nitrogen until further (Seikagaku Biobusiness), 2 mM diphenylene iodonium (DPI), 0.5 mM use. Five to 2 wk before the experiment, the strain was put back (dilution ouabain, 25 mM glibenclamide, and 5 mM colchicine, all from Sigma- 1/20) in EMJH liquid medium and weekly passaged at 28˚C without ag- Aldrich. The reagents used had no effect on bacterial growth or cell via- itation. Just before infection, the bacteria in early stationary phase (∼5 3 bility (data not shown). 8 3 10 Leptospira/ml) were centrifuged (4000 g, 25 min, 23˚C), resus- Real-time and RT-PCR pended in endotoxin-free PBS, and counted using a Petroff-Hauser chamber. Mice were infected with 2 3 108 L. interrogans in 200 ml Total RNA was extracted from whole organs using the RNeasy mini kit PBS by the i.p. route and sacrificed 3 d postinfection. The kidneys of (Qiagen) and from BMDMs using the RNeasy micro kit (Qiagen). RNA m infected and naive mice were removed, frozen in liquid nitrogen, and concentration was determined by measuring OD at 260 nm. A total of 2 g m m further stored at 280˚C for nucleic acid preparations or dosage of proteins. RNAwas incubated in a final volume of 20 l containing 1 l oligo(dT) (100 mM) (Fermentas), 1 ml 2’-deoxynucleoside 5’-triphosphate (10 mM each), 2 Downloaded from For in vitro stimulation, Leptospira in PBS were killed by incubation at ml DTT (0.1 M), 0.5 ml Superscript II reverse transcriptase (200 U/ml), and 4 56˚C for 30 min. ml53 first-strand buffer (Invitrogen). RNAwith oligo(dT) was first denatured Mice at 65˚C for 5 min, then the enzyme and other reagents were added and maintained at 42˚C for 1 h and then heat-inactivated at 70˚C for 15 min. The Female C57BL/6/J mice (8–10 wk old) were purchased from Janvier and generated cDNA was stored at 220˚C. After RT, quantitative PCR (qPCR) used as control mice. Mice deficient for TLR2 (TLR22/2) or TLR4 was performed using cDNA combined with primers, probes, and reaction mix 2/2

(TLR4 ), originally provided by Shizuo Akira (Osaka University, Osaka, according to the manufacturer’s recommendations (Applied Biosystems). http://www.jimmunol.org/ Japan), have been further backcrossed eight times into C57BL/6J mice, qPCR reactions were run on a Step one Plus RT PCR apparatus (Applied before being crossed and genotyped to get double-deficient TLR22/2/ Biosystems), with the following conditions for FAM TAMRA probes: 50˚C TLR42/2 mice (TLR2/42/2) (13). TLR9-deficient mice were provided by for 2 min, 95˚C for 10 min, followed by 40 cycles with denaturation at 95˚C Prof. Shizuo Akira to Dr. Fabrice Laurent (Institut National de la Recherche for 15 s, and annealing temperature 60˚C for 1 min. For SYBR Green reac- Agronomique, Nouzilly, France). TLR5 and MyD882/2 mice with a tions (Applied Biosystems), a melting curve stage was added according to the C57BL/6J background were provided by Michel Chignard (Institut Pasteur), manufacturer’s instructions. All qPCRs displayed efficiency between 90 and C57BL6/J Lps2 (Toll/IL-1R domain-containing adapter inducing IFN-b 110%. PCR data were reported as the relative increase in mRNA transcripts [TRIF]2/2) mice were kindly given by Dr. Beutler (The Scripps Research versus that found in respective tissues from naive mice or nonstimulated Institute, La Jolla, CA). Mice deficient for Nod1 (Nod12/2) or Nod2 BMDMs, corrected by the respective levels of hypoxanthine phospho- (Nod22/2), respectively, given by John Bertin (Millenium, Cambridge, ribosyltransferase mRNA used as an internal standard. The sequences pri-

MA) and Jean-Pierre Hugot (Hoˆpital Robert Debre´, Paris, France), have mers and probes are depicted in Table I, except for NLRP3 primers and by guest on September 25, 2021 been further backcrossed eight times into C57BL6/J mice, before being probes (186456959; Applied Biosystems). crossed and genotyped to get double Nod12/2/Nod22/2 (Nod1/22/2). NLRP32/2 and ASC2/2 mice were given to D. Philpott by Jurg Tschopp ELISA ´ (Universite de Lausanne, Lausanne, Switzerland). All protocols were The production of IL-1b, CCL5, and IL-6 in the cell-free supernatants of reviewed by the Institut Pasteur, the competent authority, for compliance BMDM cells and in kidneys was determined using ELISA kits (R&D with the French and European regulations on Animal Welfare and with Systems) according to the manufacturer’s instructions. Public Health Service recommendations. Western blotting Media and reagents Lysates from cells grown in six-well plates in OPTIMEM medium (Invitrogen) Endotoxin-free PBS was from BioWhittaker (Verviers, Belgium). All cell- and corresponding supernatants were prepared as described (18). Cells were culture products were from Invitrogen. All agonists and media were con- lysed in 200 ml Triton lysis buffer (20 mM Tris CL [pH 7.4], 1% Triton X-100, Limulus trolled for lack of endotoxin content by the amebocyte gelation 10% glycerol, 137 mM NaCl, and 2 mM EDTA) supplemented just before use assay performed using the QCL-1000 from BioWhittaker. Repurified with protease inhibitor (complete with EDTA) and phosphatase inhibitor Escherichia coli Staphylococcus aureus LPS and PG from were from mixture tablets (Roche) as specified by the manufacturer. Proteins were pre- L. interrogans InvivoGen. LPS from strain Fiocruz was purified as de- cipitated from supernatants (3 ml) using an equal volume of methanol and 0.25 scribed previously (14). volume of chloroform, further mixed, and centrifuged at 20,000 3 g for GLP extraction 10 min. The upper phase was discarded, and another equal volume of methanol was added to the interphase, followed by mixing and centrifugation at GLP from strain Fiocruz was purified according to published procedures 20,000 3 g for 5 min. The protein pellets were dried at 55˚C and resuspended (15, 16). L. interrogans were grown at 28˚C in EMJH (Bio-Rad) medium in 50 ml PBS complemented with protease inhibitor (complete with EDTA) without agitation. At the end of the exponential phase, bacteria were and phosphatase inhibitor mixture (Roche). Protein contents were measured pelleted at 9000 3 g in an endotoxin-free 50 ml polypropylene tube and with the Bradford assay (Bio-Rad), and samples from cell lysates (80 mg) and kept frozen at 280˚C. Resuspended pellet in endotoxin-free Tris/HCl 0.01 supernatants (20 mg) were mixed with Laemmli’s SDS-sample buffer, heated M (pH 7.4) was digested overnight at 34˚C with lysozyme (50 mg/ml) and to 100˚C for 5 min prior to loading on 15% acrylamide-bis 40:1 gel, and then then ultracentrifuged (30 min, 18˚C, 20,000 3 g). Supernatant was treated transferred onto polyvinylidene difluoride membrane 0.45 (0.8 mA/cm2,1h) by RNase and DNase (50 mg/ml each for 3 h at 37˚C), then dialyzed for or 0.22 mM(0.8mA/cm2, 1 h 30 min; Millipore) to detect the cleaved caspase 24 h at 4˚C against Tris/HCl buffer 0.1 M (pH 7.4). After acidification to p10. All subsequent steps were done following the Cell Signaling Technology pH 3.7 with acetic acid 1 M at 4˚C, GLP was precipitated by ultracen- Western immunoblotting protocol. After blocking, the blots were incubated trifugation (37000 3 g, 30 min, 4˚C), washed twice with 0.1 M acetic acid, overnight at 4˚C with rabbit polyclonal anti-murine caspase-1 p10 (sc-514) or and lyophilized. GLP was resuspended in PBS (1 mg/ml) and kept frozen IL-1b (sc-7884, lot 2811), or GAPDH (sc-25778; all from Santa Cruz Bio- at 220˚C. For proteinase K digestion assay, GLP and proteinase K (Euro- technology), all diluted 1:150 and further probed with a secondary goat anti- medex) (200 mg/ml each) were incubated overnight at 37˚C under agitation rabbit IgG, coupled to HRP (Cell Signaling Technology; diluted 1:2000). in PBS supplemented with 1 mM CaCl2. Proteinase K was inactivated by Because of lack of consistency between different lots of sc7884, the purified 10 min boiling. As a control for proteinase K activity, BSA was digested goat IgG anti–murine IL-1b (AB-401-NA; R&D Systems) was also used in the same conditions, and the digestion products were separated on a (1:1000), along with the secondary rabbit anti-goat IgG coupled to HRP (Bio- 4–15% acrylamide SDS-PAGE and further stained with silver nitrate. Rad; 1:1000). An ECL kit (Pierce) and Supersignal Femto Western blotting The Journal of Immunology 2807 substrate were used for Ab detection (Thermo Scientific). If needed, mem- infection with L. interrogans at the MOI of 100 was first moni- branes were stripped with the Restore buffer (Thermo Scientific) and reprobed tored both by immunodetection of the pro–IL-1b in cellular with Abs as described above. Coomassie-stained gels showing residual high extracts and by ELISA of the secreted protein in corresponding molecular proteins remaining after transfer are shown as a loading control of immunodetection of IL-1b p17 in the precipitated supernatants. supernatants at 2, 4, 8, and 24 h after stimulation (Fig. 1B). Pro– IL-1b was clearly detected by Western blot at 4 h and progres- Statistical analysis sively increased until 24 h after stimulation with live or heat-killed Statistical differences were analyzed using unpaired, two-tailed nonpara- bacteria, consistent with the IL-1b secretion measured by ELISA metric tests (Mann–Whitney or Kruskal–Wallis). The p values ,0.05 were (Fig. 1B). IL-1b secretion results from the cleavage of the proform considered to be significant and indicated with asterisks. of IL-1b in active IL-1b p17 peptide by caspase-1, itself activated by the inflammasome in caspase-1 p10 and p20 peptides. There- Results fore, we also monitored by Western blot the kinetics of caspase-1 b L. interrogans infection induces IL-1 secretion through p10 and IL-1b p17 appearance after stimulation or infection with NLRP3 inflammasome activation L. interrogans at the MOI of 100 (Fig. 1C). The caspase-1 BMDMs from C57BL6 mice were stimulated for 24 h with L. cleavage into p10 peptide was detected 2 h after stimulation, in- interrogans, either alive or heat-killed by 30 min at 56˚C. The creased until 8 h, and was no longer visible at 24 h postinfection. stimulation resulted in a dose-dependent production of IL-1b in In contrast to the caspase-1 cleavage occurring early in cellular cell supernatants that interestingly was higher with live than heat- extracts, IL-1b p17 was only detectable at 24 h in precipitated killed L. interrogans (Fig. 1A). This higher production of IL-1b supernatants of cells stimulated with live or heat-killed L. inter- was unlikely due to the division of live L. interrogans, resulting in rogans (Fig. 1C). Because the Western blot technique of IL-1b Downloaded from a higher number of bacteria activating the cells, because the IL-6 p17 detection appeared to be less sensitive than the ELISA, IL-1b production was equivalent with live and killed bacteria (Fig. 1A). secretion was measured by ELISA 24 h poststimulation with The kinetic of IL-1b production upon BMDMs stimulation or L. interrogans at the MOI of 100 in all subsequent experiments. http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. IL-1b production and inﬂammasome activation by L. interrogans-infected macrophages. (A) BMDMs from C57BL/6 mice were incubated for 24 h with live or heat-killed L. interrogans at different MOIs, and IL-1b and IL-6 productions were measured in supernatants by ELISA. Statistical differences over medium condition with p , 0.05 were represented with an asterisk (*). (B) BMDMs from C57BL/6 mice were stimulated with live or heat- killed L. interrogans (MOI 100:1) for 2, 4, 8, and 24 h. Supernatants were collected for IL-1b measurement by ELISA, and cellular extracts were used for Western blotting and detection of the pro–IL-1b (sc 7884) and GAPDH. (C) BMDMs from C57BL/6 mice were stimulated with live or heat-killed L. interrogans (MOI 100:1) for 2, 4, 8, and 24 h. Cellular extracts were used for Western blotting and detection of the caspase-1. Supernatants (SUP) were collected and precipitated for IL-1b p17 detection by Western blot (murine IL-1b Ab from R&D Systems). As a loading control, Coomassie-stained gels showing residual high molecular proteins remaining after transfer are shown. (D) BMDMs from C57BL/6 mice (WT), ASC2/2, and NLRP32/2 were incubated for 24 h with live or heat-killed L. interrogans (MOI 100:1). IL-1b and IL-6 were measured by ELISA in the supernatants of BMDMs. Statistical differences between L. interrogans-stimulated BMDMs from WT and 2/2 mice were represented with an asterisk (*). All data presented in the different panels are representative of three independent experiments, each involving a minimum of n = 3 mice. 2808 SYNERGY BETWEEN LEPTOSPIRAL LPS AND GLP INDUCES IL-1b

To find out the inflammasome protein involved in the secretion of compared with WT BMDMs (Fig. 1D), demonstrating that NLRP3 IL-1b by BMDMs infected with L. interrogans,wefirsttestedthe is the prominent inflammasome receptor involved in IL-1b pro- contribution of ASC, the adaptor of inflammasomes, important for duction induced by both live and heat-killed L. interrogans. cytokine secretion (19). In contrast to IL-6 secretion that was com- parable in L. interrogans-stimulated wild-type (WT) and ASC-defi- L. interrogans-induced NLRP3 inflammasome is triggered by cient BMDMs, IL-1b secretion upon L. interrogans infection or potassium fluxes stimulation with heat-killed bacteria was almost abrogated in ASC- Several strategies or inhibitors were then used to decipher the deficient BMDMs compared with WT BMDMs (Fig. 1D). Lepto- different pathways that can possibly induce the NLRP3 inflam- spirosis is often associated to renal failure with hypokaliemia and masome upon L. interrogans activation. Two different inhibitors sodium wasting (20), and a defective Na+-K+-Cl2 (NKCC2) co- of ROS production, DPI and APDC, which inhibit the NADPH transport has been measured in patients with leptospirosis (21). oxidases, were used and did not have any effect on the induction Therefore, we next tested NLRP3, which uses ASC as an adaptor of IL-1b in BMDMs infected with L. interrogans (Fig. 2A). We and is known to be triggered by potassium efflux (10, 11). IL-1b verified that DPI was active because it inhibited L. interrogans- secretion upon L. interrogans infection or stimulation with heat- induced NO production in the same experiment (data not shown). killed bacteria was almost abrogated in NLRP3-deficient BMDMs In contrast, high extracellular potassium concentration and gli- Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. Leptospiral PG does not participate in the IL-1b production. (A) BMDMs from WT mice were infected with L. interrogans (MOI 10 and 100) in presence of KCl (70 mM), glibenclamide (25 mM), APDC (25 mM), and DPI (2 mM). IL-1b and IL-6 productions were measured in the supernatant 24 h later. Statistical differences over infected but untreated cells with p , 0.05 were represented with an asterisk (*). (B) BMDMs from WT mice were incubated at different MOIs with live or heat-killed bacteria in presence or absence of bafilomycin A1 (40 nM) and IL-1b production was measured in the supernatant 24 h later. Statistical differences between medium and bafilomycin conditions with p , 0.05 were represented with an asterisk (*). (C) BMDMs from WT mice were stimulated for 24 h with S. aureus PG (PG Staph; 20 mg/ml) or L. interrogans (Lepto; MOI 100:1) with or without hexa-N-acetylchitohexaose (Inhibitor; 0.2 mM), and IL-1b secretion was determined in the supernatants. (D) BMDMs from WT and Nod1/Nod22/2 mice (Nod1/22/2) were incubated for 24 h with medium or live L. interrogans (MOI 100:1), and IL-1b secretion was measured in the supernatants. (E) BMDMs from WT mice stimulated for 24 h with live (Lepto) or heat-killed L. interrogans (HK Lepto; MOI 100:1) with or without colchicine (5 mM). Dosages of IL-1b and IL-6 were determined in the supernatants by ELISA. Statistical differences between stimulated BMDMs with or without inhibitors with p , 0.05 were represented with an asterisk (*). All data presented in the different panels are representative of three independent experiments, each involving a minimum of n = 3 mice. The Journal of Immunology 2809 benclamide, a drug that inhibits ATP-sensitive potassium chan- contrast, TRIF2/2 BMDMs produced more IL-1b upon infection nels and is a specific inhibitor of the NLRP3 inflammasome (22), with live bacteria than WT BMDMs (Fig. 3A). This phenomenon both decreased IL-1b production, although IL-6 secretion was not wasnotobservedwithkilledbacteriaforwhichnodifferenceinIL- altered in these conditions (Fig. 2A). Similar results were ob- 1b production was measured between WT and TRIF2/2 BMDMs. served by stimulation with killed bacteria (data not shown). In- We further stimulated BMDMs from different TLR-deficient mice terestingly, the use of bafilomycin A1, which inhibits lysosomal with live or killed bacteria (Fig. 3A). As a whole, no significant acidification, resulted in a decrease of IL-1b secretion when live difference was observed among WT, TLR5-, and TLR9-deficient L. interrogans were used (Fig. 2B), but did not show any effect BMDMs postinfection with live or stimulation with killed bacteria upon stimulation with heat-killed bacteria (Fig. 2B). Together, (Fig. 3A). However, when stimulated with live or heat-killed L. these results show that heat-killed L. interrogans stimulates the interrogans, BMDMs from TLR22/2 and TLR42/2 produced sig- NLRP3 inflammasome and induces IL-1b secretion through nificantly less IL-1b compared with WT BMDMs (Fig. 3A). These a potassic pathway, whereas live L. interrogans stimulates the results showed that TLR2 and TLR4 are crucial effectors of the NLRP3-induced IL-1b production through both potassic- and IL-1b production induced by L. interrogans in BMDMs. lysosome-dependent pathways. Leptospiral LPS provides the first signals for inflammasome Leptospiral PG does not contribute to the IL-1b production priming Some reports have highlighted a relationship between Nod1- and Because LPS from L. interrogans has been shown to stimulate Nod2-dependent pathways and the NLRP3 inflammasome (23). In both TLR2 and TLR4 in murine cells (13), we determined by addition, recent work showed that the degradation of S. aureus PG quantitative real-time PCR (qRT-PCR) whether LPS could trigger Downloaded from by lysozyme in lysosomes was able to trigger IL-1b production IL-1b mRNA expression in BMDMs. IL-1b mRNA expression (24). We therefore used hexachitohexaose, a lysozyme inhibitor, was upregulated by leptospiral LPS stimulation in WT BMDMs to check if leptospiral PG could be involved. As expected (24), the (Fig. 3B). Reduced IL-1b mRNA upregulation was observed in 2/2 2/2 2/2 induction of IL-1b secretion by PG from S. aureus was inhibited MyD88 , TLR2 , and TLR4 BMDMs (Fig. 3B). In 2/2 by the lysozyme inhibitor, although no effect was observed on IL- contrast, a marked increase was observed in the TRIF BMDMs

1b secretion after L. interrogans infection (Fig. 2C). We also (Fig. 3B). Therefore, LPS induced IL-1b mRNA expression http://www.jimmunol.org/ stimulated BMDMs from Nod1 and Nod2 double-deﬁcient mice through a TLR2/TLR4/MyD88-dependent pathway, but indepen- with L. interrogans. Compared to WT BMDMs, no difference was dent of TRIF, which correlates with IL-1b production upon in- observed, suggesting that the Nod receptors were not involved in fection with the whole bacteria (Fig. 3A). These results suggest the L. interrogans-induced IL-1b production (Fig. 2D). Together, that leptospiral LPS is an important trigger of IL-1b mRNA up- these results suggest that the leptospiral PG does not participate in regulation induced by the whole L. interrogans and explain the 2/2 IL-1b production triggered by the infection of BMDMs with L. lack of IL-1b production in the MyD88 BMDMs. Interest- 2/2 interrogans. ingly, the higher induction of IL-1b mRNA in TRIF BMDMs compared with WT (Fig. 3B) suggests a negative regulatory effect

b by guest on September 25, 2021 Crystal-induced cellular stress is not involved in IL-1 of TRIF upon MyD88-dependent TLR4 signaling important for secretion upon L. interrogans infection IL-1b mRNA upregulation. We further determined if a danger signal produced by BMDMs Recent studies have shown that besides pro–IL-1b induction, upon infection with live L. interrogans could be involved in IL-1b a priming of NLRP3 mRNA expression by an NF-kB signal is also induction. For example, uric acid is a danger signal produced upon required to get a functional protein able to transmit the inflam- cell necrosis. Elevated levels of circulating uric acid have been masome signal (28). Interestingly, stimulation of BMDMs with measured in sera from Leptospira-infected bovines (25). Uric acid LPS resulted in a MyD88-independent upregulation of NLRP3 2 2 can crystallize, and monosodium urate crystals have been shown mRNA (Fig. 3C), whereas this effect was impaired in TRIF / to destabilize the lysosomes, resulting in NLRP3 activation (26). mice (Fig. 3C). Accordingly to the fact that MyD88 is the adaptor This phenomenon is blocked by colchicine, a microtubule inhib- of TLR2 and TLR4, although TRIF is the adaptor of TLR4 but not 2 2 itor used in the treatment of gouty arthritis (26). However, even TLR2, NLRP3 upregulation by LPS was altered in TLR4 / 2 2 high concentrations of colchicine did not inhibit IL-1b production BMDM but not in TLR2 / BMDMs (Fig. 3C). upon L. interrogans stimulation or infection (Fig. 2E). These data Ouabain is a natural ligand of the sodium potassium ATPase suggested that IL-1b secretion by BMDMs is not linked to a enzyme (Na/K-ATPase), acting as a blocker of the pump and crystal-like danger signal produced by dying cells infected with allowing potassium efflux (29) and inflammasome activation (30). L. interrogans. Costimulation of WT BMDM with leptospiral LPS and ouabain showed an increase in the IL-1b production compared with stim- b IL-1 production induced by L. interrogans is dependent on ulation with leptospiral LPS alone (Fig. 3D). Compared to E. coli TLR2, TLR4, and MyD88 LPS, for which IL-1b production in presence of ouabain was al- To get a deeper understanding of how IL-1b secretion was induced tered in TLR42/2 cells, IL-1b responses following stimulation with by L. interrogans, we sought to determine the L. interrogans leptospiral LPS and ouabain treatment were only slightly decreased components responsible for IL-1b production in BMDMs. IL-1b in TLR42/2 and TLR22/2 BMDMs, consistent with the dual rec- secretion requires two signals, one to activate the inflammasome and ognition of leptospiral LPS by TLR2 and TLR4 in murine cells a second one to induce pro–IL-1b expression (27). This last signal (13). Taken together, these results suggest that LPS from L. inter- usually emanates from TLR- or TNF-dependent signaling. Therefore, rogans is a major signal for the upregulation of IL-1b and NLRP3 we stimulated BMDMs deficient for the TLR adaptor proteins mRNA and that another leptospiral component, able to alter the MyD88, which is involved in the signaling of every TLR except potassium fluxes, contributes to promoting IL-1b secretion. TLR3, and TRIF, the only adaptor of TLR3 and an important adaptor of TLR4. Compared to WT, no IL-1b could be detected in the GLP activates the inflammasome supernatants of MyD882/2 BMDMs, showing a major contribution Several in vitro and in vivo studies have shown that L. interrogans of the TLR/MyD88 pathway in IL-1b production (Fig. 3A). In downregulates potassic ion transporters in renal epithelial cell 2810 SYNERGY BETWEEN LEPTOSPIRAL LPS AND GLP INDUCES IL-1b

FIGURE 3. IL-1b production by L. interrogans-stimulated macrophages is MyD88, TLR2, and TLR4 dependent. (A) BMDMs from C57BL/6 WT, TLR22/2, TLR42/2, TLR52/2,TLR92/2, MyD882/2, and TRIF2/2 mice were incubated for 24 h with live or heat- killed L. interrogans (MOI 100:1). Pro- duction of IL-1b was measured by ELISA in the supernatants. Statistical differences between stimulated BMDMs 2/2 from WT over mice with p , 0.05 Downloaded from were represented with an asterisk (*). (B and C) BMDMs from C57BL/6 WT, TLR22/2, TLR42/2, MyD882/2, and TRIF2/2 mice were incubated for 24 h with L. interrogans LPS (1 mg/ml) and total RNA was extracted for analysis of

IL-1b mRNA (B) and NLRP3 mRNA http://www.jimmunol.org/ (C) expression by qRT-PCR. PCR data are presented as relative expression over nonstimulated cells. Statistical differences between stimulated BMDMs from WT over 2/2 mice were represented with an asterisk: *p , 0.05. (D)WT, TLR22/2, and TLR42/2 BMDMs were stimulated with L. interrogans LPS (1 mg/ml) or E. coli LPS (100 ng/ml) in by guest on September 25, 2021 presence of ouabain (0.5 mM), and the production of IL-1b was measured by ELISA in the supernatant 24 h later. Statistical differences between stimulated BMDMs from WT over 2/2 mice with p , 0.05 were represented with an asterisk (*). All data presented in the different panels are representative of three independent experiments, each involving a minimum of n = 3 mice.

lines, especially the Na/K-ATPase (31, 32) and NKCC2, a sym- fraction of L. interrogans, which was shown to inhibit in a dose- porter that imports one sodium ion, one potassium, and two dependent manner the ion transport activities of Na/K-ATPase (31, chloride (21). The Na/K-ATPase is composed of two subunits (a 32). Therefore, we puriﬁed the GLP from the L. interrogans strain and b1) that for each ATP imports two potassium ions and exports Fiocruz according to published procedures (15, 16). Interestingly, three sodium ions, therefore establishing concentration gradients stimulation of BMDMs with GLP downregulated the expression of K+ and Na+ ions across the plasma membrane. Interestingly, we of the Na/Kb1-ATPase (Fig. 4A). Like the heat-killed L. inter- observed that L. interrogans infection downregulated the expres- rogans, the GLP heated 30 min at 56˚C also downregulated the sion of the b1 subunit of the Na/K-ATPase in BMDMs (Fig. 4A). Na/Kb1 mRNA expression (Fig. 4A), as well as proteinase K- In contrast, no regulation of the Na/Ka subunit was observed (data treated GLP, which has been further boiled (Fig. 4B), suggesting not shown). Glycolipoprotein (GLP) is an outer cell membrane that the inhibitory effects of the GLP on the Na/K-ATPase did not The Journal of Immunology 2811 Downloaded from http://www.jimmunol.org/

FIGURE 4. LPS synergizes with GLP to produce IL-1b.(A) Na/Kb1 mRNA expression was measured via qRT-PCR in BMDMs from WT mice stimulated for 24 h with live or heat-killed L. interrogans (MOI 100:1) or native or heated GLP from L. interrogans (10 mg/ml). (B) Na/Kb1 mRNA expression was measured by qRT-PCR in BMDM from WT mice stimulated for 24 h with GLP (10 mg/ml), inactivated proteinase K alone, and GLP (10 mg/ml) treated with proteinase K and inactivated. (C) BMDM from C57BL/6 mice were stimulated with or without LPS from Leptospira (1 mg/ml). Three hours later, cells were stimulated with or without GLP (10 mg/ml) for additional 3 h. Supernatants were collected and precipitated for IL-1b p17 detection by Western blot (sc 7884). The Coomassie-stained gel showing residual high molecular proteins remaining after transfer is shown as a loading control.(D) GLP and LPS from L. interrogans were incubated at different doses, separately or in combination, with BMDMs from WT mice for 24 h, and IL-1b was by guest on September 25, 2021 measured by ELISA in the supernatants. All data presented in the different panels are representative of three independent experiments, each involving a minimum of n = 3 mice. rely on the peptidic moiety but rather on some lipid component, as channel, and the sodium-chloride symporter NCCT, a cotransporter previously published (32, 33). We next tested whether the GLP, that reabsorbs Na+ and Cl2 in the kidney of L. interrogans-infected through its action on the potassium pump, could be the signal mice (Table I). Compared to noninfected mice, L. interrogans in- required for the inflammasome activation. Therefore, BMDMs fection resulted in the downregulation of all transporters tested (Fig. from WT mice were sequentially stimulated with leptospiral LPS 5A), suggesting that the infection can deeply alter the potassium and for 3 h, then with GLP for additional 3 h (Fig. 4C). IL-1b p17 was sodium fluxes in the kidney, as it was shown in kidneys of patients immunodetected in the precipitated supernatant of cells only when with leptospirosis (21). Interestingly, the extent of downregulation of LPS and GLP were present together (Fig. 4C), showing that GLP the different transporters was similar in all mice tested, suggesting can activate the inflammasome. Also, compared with the stimu- that TLR2 and TLR4 are not involved in the downregulation of ionic lation with L. interrogans measured 24 h poststimulation by transporters induced by L. interrogans infection (Fig. 5A). ELISA, GLP alone was not able to trigger IL-1b production (Fig. IL-1b and NLRP3 mRNA expressions were measured 3 d 4D). LPS from L. interrogans induced IL-1b production only at postinfection in the kidneys of WT, TLR22/2, TLR42/2, and high concentrations .1 mg/ml. When combined, LPS and GLP double TLR2/42/2 mice. Compared to naive mice, induction of from L. interrogans synergized to produce IL-1b (Fig. 4D). These IL-1b mRNA was observed in kidneys of infected mice but was results show that GLP, through its action on Na/K-ATPase, acti- significantly decreased in TLR-deficient mice compared with WT vates the inflammasome and together with LPS promotes IL-1b mice. In addition, NLRP3 mRNA upregulation was observed in production in BMDMs. the kidneys of L. interrogans-infected WT and TLR22/2 mice, but was impaired in TLR42/2 and TLR2/42/2 mice compared with L. interrogans infection downregulates ion transporters in naive mice (Fig. 5B). Consistent with the above results, more IL- kidney 1b protein was measured in infected WT mouse kidneys com- The potential physiological relevance of our in vitro findings was pared with naive mice (Fig. 5C), but the increased production of determinedby analyzing the kidneys ofWT,TLR22/2, TLR42/2,and IL-1b protein was blunted in infected TLR-deficient mice. As double TLR2/42/2 mice 3 d post i.p. infection with 2 3 108 bacteria. a control, we measured the mRNA expression and protein levels We determined by qRT-PCR the mRNA expression of ion trans- of the chemokine CCL5 (RANTES), which, as already shown in porters and ion channels, including Na/K-ATPase and NKCC2, both our previous study (2), were upregulated in all infected mice and of which have been shown to be downregulated by L. interrogans even increased in the kidneys of TLR2/42/2 compared with the (31), the epithelial sodium channel, the kidney-specific chloride other mice (Fig. 5B, 5C), showing that kidneys from TLR2/42/2 2812 SYNERGY BETWEEN LEPTOSPIRAL LPS AND GLP INDUCES IL-1b

Table I. Primer and probe sequences and associated references

Primer Name Primer Sequence Probe Sequence (FAM-TAMRA) Sequence Accession No. HPRT 59-CTGGTGAAAAGGACCTCTCG-39 59-TGTTGGATACAGGCCAGACTTTGTTGGAT-39 NM_013556.2 59-TGAAGTACTCATTATAGTCAAGGGCA-39 IL-1b 59-CAACCAACAAGTGATATTCTCCATG-39 59-CTGTGTAATGAAAGACGG CACACCCACC-39 NM_008361 59-GATCCACACTCTCCAGCTGCA-39 Na/Kb159-CTGCCTGGCTGGCATCTT-39 59-CCATCCAAGTAATGCTGC TAACCATC-39 NM_009721 59-TGGTATGTGGGCTTCAGTTCAC-39 Na/Ka 59-CGTGATGGAGATGAGGGAGAA-39 59-CAGCAAGATAGTGGAGATTCCCTTCAACTCC-39 NM_144900 59-TGTGAATGGACAACTGGTACTTGTT-39 ENaC 59-ACCGCATGAAGACGGCC-39 59-CGTGGCGGTGCTGTGGCTCTG-39 NM_011324 59-CCAGTACATCATGCCGAAGGT-39 NKCC2 59-AGATTGGCGTGGTCATAGTCAGAA-39 U20975 59-TGCTGCTGATGTTGCCGTCTT-39 NCCT 59-CTCACCCTCCTCATCCCCTATCT-39 NM_019415 59-AGAGCAGCATCCCGAGAGTAATC-39 ClC-Kb 59-CAGACCCTGGTGGGCGTTGTAA-39 NM_019701 59-TGGTCAGGGTTGAAATTGCTT-39 ClC-Kb, kidney-specific chloride channel; ENaC, epithelial sodium channel; HPRT, hypoxanthine phosphoribosyltransferase. Downloaded from were still able to produce cytokines. These results show that TLR2 we showed that L. interrogans infection induced IL-1b production and TLR4 play an important role in vivo in the priming of IL-1b in BMDMs and that this secretion was dependent on the NLRP3 mRNA and TLR4 in the priming of the NLRP3 inflammasome, inflammasome and TLR2 and TLR4 activation. We further deter- which is consistent with the results found in the BMDMs. mined that leptospiral LPS provides the first signals for inflammasome priming, upregulating both the pro–IL-1b mRNA through Discussion a TLR2/TLR4/MyD88 pathway and the NLRP3 mRNA in a http://www.jimmunol.org/ In this work, we demonstrated that bacteria can induce IL-1b se- TLR4/TRIF-dependent manner. We aimed to identify the bacterial cretion through dysregulation of host potassium transport. Indeed, component that could promote NLRP3 inflammasome activation by guest on September 25, 2021

FIGURE 5. IL-1b production and downregulation of ion channel expression in L. interrogans-infected mice. WT, TLR22/2, TLR42/2, and TLR2/42/2 adult mice were infected by i.p. route with 2 3 108 L. interrogans for 3 d, and kidneys were removed for mRNA and protein analysis. mRNA expressions of transporters and channels (A) and of IL-1b, CCL5, and NLRP3 (B) were measured by qRT-PCR. Data are represented as a ratio of infected over naive (fold increase) (n = 2 for naive mice and n = 6–8 for infected mice). Statistical differences between stimulated WT over 2/2 mice with p , 0.05 were represented with an asterisk (*). (C) Protein productions of IL-1b and CCL5 were measured in the kidney by ELISA (n = 2 for naive mice and n = 6–8 for infected mice). Statistical differences between infected over naive mice with p , 0.05 were represented with an asterisk (*). The Journal of Immunology 2813 through potassium dysregulation. We found that leptospiral GLP, involved in the NLRP3 mRNA priming, as this is exclusively which has been shown by others to inhibit the functions of the TLR4/TRIF dependent. Na/K-ATPase, could activate the inflammasome and that costim- Recently, the NLRP3 inflammasome has been shown to promote ulation of BMDM with leptospiral LPS and purified GLP resulted renal inflammation and chronic kidney disease after experimental in production of active IL-1b, mimicking the L. interrogans in- renal injury in mice (37). In this study, we showed that NLRP3 fection. The important role of TLR2 and TLR4 in the IL-1b pro- mRNA was upregulated in the kidneys of L. interrogans-infected duction was further confirmed in vivo in the mouse kidney, where WT mice, but this effect was decreased in the kidneys of TLR42/2 L. interrogans is inducing a profound ion transport dysregulation. and TLR2/42/2 mice, which are susceptible mice that will suc- GLP is an outer cell membrane fraction of L. interrogans, cumb to the L. interrogans infection (2). The fact that the IL-1b composed of ∼20% protein, 40% carbohydrate, and 33% lipid response in the kidney relied on TLR2 and TLR4 activation upon (15). GLP, and not the LPS fraction, was shown to inhibit in L. interrogans infection was not expected because in our previous a dose-dependent manner the transport activity of the Na/K- study, an important IL-1b mRNA upregulation was measured in ATPase (31, 32). The inhibitory properties of GLP have been the liver of TLR2/42/2 mice (2). These results reinforce the view studied in details, and it was shown that GLP did not change the of a compartmentalized and differential response to L. interrogans affinity of Na/K-ATPase for potassium, as ouabain did, but in- infection occurring in different target organs such as the liver and creased the affinity for sodium (32). Moreover, the inhibition of kidneys (38). The NLRP3-dependent IL-1b secretion in the kid- the Na/K-ATPase by GLP is specific because GLP does not in- ney seems to be part of the host protective response, whereas the hibit the H/K-ATPase, and the outer cell fraction of E. coli does IL-1b production in the liver of TLR2/42/2 would participate in not inhibit the Na/K-ATPase (31). Finally, the lipid fraction of a deleterious inflammatory response. Downloaded from GLP (i.e., unsaturated fatty acids) was shown to be responsible In addition to NLRP3-dependent IL-1b production triggered for the inhibition of the Na/K-ATPase, which is in accordance by both live and killed bacteria through the potassium pathway, with the thermostability of GLP’s inhibitory activity (34). In this only live bacteria were able to trigger IL-1b production through study, we purified L. interrogans GLP and observed its ability a lysosomal damage pathway that was blocked by the use to downregulate the Na/K-ATPase mRNA expression in the of bafilomycin. Examples of lysosome-induced IL-1b secretion

BMDMs, even after heating and proteinase K digestion. These include crystals or particulate that can damage the lysosomes, http://www.jimmunol.org/ data, together with the similar effect of live and heat-killed L. favoring the release of cathepsins or other important medi- interrogans on the downregulation of the pump, suggest that ators of NLRP3 activation in the cytosol (39, 40). We showed GLP does not contribute to the difference in IL-1b production that neither crystals nor PG were involved in this IL-1b pro- upon macrophage stimulation with live and heat-killed bacteria. duction. The fact that both LPS and GLP were extracted from the bac- To our knowledge, the mechanism of activation of NLRP3 terial cell wall raised the issue of contaminations. Although through downregulation of transporters such as the Na/K-ATPase purification protocols are quite different between GLP and LPS has never been described for any bacteria. No precise mechanisms preparation, frequent leptospiral LPS contaminations of GLP of NLRP3 inflammasome activation have been provided for Sal- were observed on gel after specific staining of LPS with silver monella (41), Listeria (42) and Chlamydiae, even if in the latter by guest on September 25, 2021 nitrate (data not shown) and confirmed by the fact that this case the activation was shown to rely on both ROS and potassium partially purified GLP was activating by itself the IL-1b secre- pathways (43). However, pathogenic Vibrio activated NLRP3 via tion (data not shown). Therefore, we only used for the current cytotoxins and hemolysins (44). Several hemolysins with cyto- study GLP preparations that produced ,50 pg/ml of IL-1b after toxic activities have been described in L. interrogans (45–47), but BMDM stimulation with 10 mg of GLP. We also observed that, at the lack of genetic tools (48) to inactivate these genes and obtain higher concentrations than the usual 1 mg/ml used to stimulate targeted mutants make it difficult to test the hypothesis that macrophages, the leptospiral LPS was able to stimulate by itself injected toxins or pore-forming hemolysins could be responsible the IL-1b production, suggesting that contaminants, most prob- for the bafilomycin-sensitive pathway. ably GLP traces, were present. Our results suggest that an active process linked to the bacterial Besides the Na/K-ATPase, the mRNA expression of other ion viability may be involved in the lysosome-dependent IL-1b pro- channels and transporters, never tested in the context of L. inter- duction. This pathway could be instrumental in the detection of rogans infection, was also found downregulated in mouse kidney potentially infectious bacteria. The question remains to understand upon L. interrogans infection. This in vivo dysregulation is most how this NLRP3-dependent pathway is activated by viable Lep- probably due to GLP, but others have shown that bacterial LPS, tospira. though TLR4 activation, and Pam3cys synthetic lipoprotein, To conclude, we demonstrated that Leptospira infection trig- through TLR2 activation, both mediated inhibition of HCO3 ab- gered IL-1b secretion predominantly through NLRP3- and TLR2/ sorption in medullary thick ascending limb (35), suggesting that 4-dependent pathways and that leptospiral GLP activated the bacterial compounds may act directly through TLRs to impair inflammasome through a unique mechanism dependent upon the the systemic electrolyte balance. Most probably, both GLP and downregulation of the Na/K-ATPase transporter. TLR agonists from L. interrogans contribute collectively to the profound electrolyte dysregulation occurring in L. interrogans- infected kidneys. Besides inflammasome activation, stimulation Acknowledgments of BMDM with Pam3cysSK4, a synthetic agonist of TLR2, also We thank Fabrice Laurent and Dominique Buzoni-Gatel for critical reading upregulated IL-1b mRNA expression (data not shown), suggesting of the manuscript and Ivan Tattoli and Fraser Soares for preparation of that other compounds of Leptospira, such as LipL32, a major li- bone marrow cells. poprotein of pathogenic Leptospira signaling through TLR2 (14, 36), could also participate in IL-1b production. Therefore, both LipL32 and LPS would be responsible for IL-1b mRNA upreg- Disclosures ulation. However, according to our results, LipL32 would not be The authors have no financial conflicts of interest. 2814 SYNERGY BETWEEN LEPTOSPIRAL LPS AND GLP INDUCES IL-1b

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