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Helicobacter pylori−Induced IL-1β Secretion in Innate Immune Cells Is Regulated by the NLRP3 Inflammasome and Requires the Cag Pathogenicity Island This information is current as of September 27, 2021. Raphaela P. Semper, Raquel Mejías-Luque, Christina Groß, Florian Anderl, Anne Müller, Michael Vieth, Dirk H. Busch, Clarissa Prazeres da Costa, Jürgen Ruland, Olaf Groß and Markus Gerhard

J Immunol published online 29 August 2014 Downloaded from http://www.jimmunol.org/content/early/2014/08/28/jimmun ol.1400362 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2014/08/28/jimmunol.140036 Material 2.DCSupplemental

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 29, 2014, doi:10.4049/jimmunol.1400362 The Journal of Immunology

Helicobacter pylori–Induced IL-1b Secretion in Innate Immune Cells Is Regulated by the NLRP3 Inﬂammasome and Requires the Cag Pathogenicity Island

Raphaela P. Semper,* Raquel Mejı´as-Luque,* Christina Groß,† Florian Anderl,* Anne Muller,€ ‡ Michael Vieth,x Dirk H. Busch,*,{ Clarissa Prazeres da Costa,* Jurgen€ Ruland,†,{ Olaf Groß,† and Markus Gerhard*,{

Infection with the gram-negative bacterium Helicobacter pylori is the most prevalent chronic bacterial infection, affecting ∼50% of the world’s population, and is the main risk factor of gastric cancer. The proinflammatory cytokine IL-1b plays a crucial role in the development of gastric tumors and polymorphisms in the IL-1 gene cluster leading to increased IL-1b production have been associated with increased risk for gastric cancer. To be active, pro–IL-1b must be cleaved by the inflammasome, an intracellular Downloaded from multiprotein complex implicated in physiological and pathological inflammation. Recently, H. pylori was postulated to activate the inflammasome in murine bone marrow–derived dendritic cells; however, the molecular mechanisms as well as the bacterial virulence factor acting as signal 2 activating the inflammasome remain elusive. In this study, we analyzed the inflammasome complex regulating IL-1b upon H. pylori infection as well as the molecular mechanisms involved. Our results indicate that H. pylori–induced IL-1b secretion is mediated by activation of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain–containing 3 inflammasome. We also show that reactive oxygen species, potassium efflux, and lysosomal destabi- http://www.jimmunol.org/ lization are the main cellular mechanisms responsible of nucleotide-binding oligomerization domain family, pyrin domain–containing 3 inflammasome activation upon H. pylori infection, and identify vacuolating cytotoxin A and cag pathogenicity island as the bacterial virulence determinants involved. Moreover, in vivo experiments indicate an important role for the inflammasome in the onset and establishment of H. pylori infection and in the subsequent inflammatory response of the host. The Journal of Immunology, 2014, 193: 000–000.

elicobacter pylori infection remains a highly prevalent presence of the cag pathogenicity island (cagPAI) and vacuolating

infection worldwide (1) and the chronic inflammation cytotoxin A (VacA) has been associated with more severe gastritis by guest on September 27, 2021 H elicited by the bacterium is one of the main causes of and increased risk of gastric ulcer and stomach cancer (2–5). gastric cancer. Progression to gastric cancer has been shown to be The inflammatory response toward H. pylori is characterized related to the severity of the host’s inflammatory response, which by the recruitment of different immune cells, mainly dendritic cells is influenced by bacterial virulence factors. Among them, the (DCs), neutrophils, macrophages, and B and T lymphocytes to the site of infection (6). Cells of the innate immune system recognize pathogens via conserved structures known as pathogens associated *Institut fur€ Medizinische Mikrobiologie, Immunologie und Hygiene, Technische molecular patterns and through different pattern recognition recep- Universita¨tMunchen,€ 81675 Munich, Germany; †Institut fur€ Klinische Chemie und Pathobiochemie, Klinikum Rechts der Isar, Technische Universita¨tMunchen,€ 81675 tors, such as extracellular or endosomal membrane bound TLRs, Munich, Germany; ‡Institute of Molecular Cancer Research, University of Zurich,€ cytosolic nucleotide-binding oligomerization domain (NOD)-like 8057 Zurich,€ Switzerland; xInstitut fur€ Pathologie, Klinikum Bayreuth, 95445 Bayr- { receptors (NLRs) and intracellular RIG-like receptors. The recog- euth, Germany; and German Centre for Infection Research, Partner Site Munich, 81675 Munich, Germany nition of H. pylori by TLRs has been extensively studied, whereas Received for publication February 6, 2014. Accepted for publication July 30, 2014. the role of NLRs is mostly unknown. H. pylori was reported to be This work was supported by a research scholarship from Elite Network of Bavaria (to recognized via NOD1 in epithelial cells (7) and via NOD2 in bone R.P.S.) and a Bavarian Molecular Biosystems Research Network (BioSysNet) grant marrow–derived DCs (BMDCs) (8). Many NLRs are involved in the from the Bavarian Ministry of Sciences, Research and Arts (to O.G.). assembly of a multiprotein complex known as inflammasome. The Address correspondence and reprint requests to Prof. Markus Gerhard, Institut fur€ inflammasome consists of a cytoplasmic sensor protein (e.g., NLR Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universita¨t Munchen,€ Trogerstrasse 30, 81675 Munich, Germany. E-mail address: markus. family, pyrin domain–containing 1 [NLRP1], NLRP3, or NLR [email protected] family, CARD domain–containing 4 [NLRC4] of the NLR family), The online version of this article contains supplemental material. the adaptor protein apoptosis-associated speck-like domain con- Abbreviations used in this article: APDC, (2R, 4R)-4-aminopyrrolidine-2, 4-dicar- taining a caspase recruitment domain (ASC) and the effector protein boxylate; ASC, apoptosis-associated speck-like domain containing a caspase recruit- procaspase-1. Pathogens as well as noninfectious stimuli can acti- ment domain; BabA, blood group Ag–binding adhesin; BMDC, bone marrow– derived DC; BMM, bone marrow–derived macrophage; Cag, cytotoxin-associated vate the inflammasome. Two signals are required for inflamma- gene; DC, dendritic cell; gGT, g-glutamyltranspeptidase; LDH, lactate dehydroge- some oligomerization. A first signal, often referred to as the nase; MOI, multiplicity of infection; NLR, NOD-like receptor; NLRC4, NLR family, CARD domain–containing 4; NLRP3, NLR family, pyrin domain–containing 3; priming signal, leads to the activation of NF-kB and thereby NOD, nucleotide-binding oligomerization domain; PAI, pathogenicity island; PFA, transcription of pro–IL-1b and pro–IL-18. This signal can be paraformaldehyde; ROS, reactive oxygen species; SabA, sialic acid–binding adhesin; induced by TLR agonists like LPS or peptidoglycan, NOD2 ago- T4SS, type IV secretion system; VacA, vacuolating cytotoxin A. nists like muramyldipeptide or by cytokines like TNF. Formation of Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 the inflammasome complex is induced by a second, independent

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400362 2 H. PYLORI ACTIVATES THE NLRP3 INFLAMMASOME signal that is specific for the different types of inflammasomes. and NK cell isolation kit (Miltenyi Biotec), according to manufacturer’s Assembly and thereby the activation of the inflammasome induces instructions. the cleavage and activation of caspase-1, which promotes the pro- Cell infection and treatments cessing and secretion of the active IL-1b and IL-18 (9). Besides, caspase-1 also induces pyroptosis, an inflammation-associated form Cells were plated in antibiotic free medium and stimulated with LPS (Sigma-Aldrich) 3 h prior to treatment unless otherwise indicated. LPS of cell death, in different cell types including DCs (10). (10 ng/ml) was used for BMDCs and BMMs prestimulation, whereas IL-1b and IL-18 have been extensively linked to gastric car- neutrophils were stimulated with 50 ng/ml LPS. PBMCs, B cells, T cells, cinogenesis. Both cytokines were also shown to be important in and NK cells were incubated with 5 ng/ml LPS. the context of H. pylori infection. Polymorphisms in the IL-1 and Infections were carried out at the indicated time points with H. pylori at multiplicity of infection (MOI) 1, 5, 10, and 50. Stimulation with 5 mM IL-18 genes inducing elevated levels of these inflammasome- ATP for 1 h and infection with S. typhimurium at MOI 10 for 6 h were used regulated cytokines increase the predisposition to the develop- as controls. ment of gastric cancer (11–13). Stomach-specific expression of Chemical inhibitors were added 30 min before cell infection or stimu- human IL-1b in a transgenic mouse model resulted in inflam- lation. The caspase-1 inhibitor Z-YVAD-FMK (Calbiochem) was used at m mation, dysplasia and gastric cancer (14) and more recently, IL-1b 10 M, KCl (Roth) at 50 mM, cytochalasin D (Sigma-Aldrich) at 2.5 and 5 mM, whereas the ROS inhibitors Ebselen (ALX-270-097; Enzo) and induced by H. pylori was shown to enhance gastric carcinogenesis (2R, 4R)-4-aminopyrrolidine-2, 4-dicarboxylate (APDC) (A7361; Sigma- in mice (15), whereas IL-18 has been linked to increased metas- Aldrich) were used at 10 and 50 mM, and 50, 250, and 500 mM, respectively. tasis and immune escape of gastric tumor cells (16). IL-1b enables To discriminate effects induced by dead bacteria, H. pylori was either H. pylori to colonize the gastric corpus by inhibiting acid secretion heat-inactivated for 5 min at 95˚C or fixed in 4% paraformaldehyde (PFA) 2 2 for 4 h at 4˚C. (17) and IL-1 receptor knockout mice (IL-1R / ) were found to Downloaded from be protected against Helicobacter felis infection–induced gastritis. Bacterial uptake assay 2/2 In contrast, IL-18 mice showed enhanced immunopathology H. pylori uptake was evaluated by the gentamicin protection assay. Briefly, associated with lower H. felis colonization levels (18), suggesting BMDCs were infected for 60 min and then incubated for 45 min at 37˚C in a dual role for the inflammasome during Helicobacter infection. medium containing gentamicin (100 mg/ml) to kill extracellular bacteria. In a recent report, H. pylori cagPAI and the interaction between Cells were then washed in PBS and lysed in 0.5% saponin/PBS, and the number of intracellular bacteria was determined by plating on Wilkins– TLR2/NOD2 and NLRP3 were reported to regulate IL-1b pro- http://www.jimmunol.org/ Chalgren blood agar plates. Supernatants were plated in parallel as control duction in H. pylori–infected DCs (8). However, the cellular for the antibiotic treatment. mechanisms implicated in the activation of the inflammasome, as well as the H. pylori factor acting as a second signal activating ELISA, Western blot, and lactate dehydrogenase assay NLRP3 remain elusive. Cell supernatants and stomach extracts were analyzed for cytokine (IL-1b, In the current study, we show that H. pylori exhibits a complex IL-18, IL-10, and TNF) secretion by ELISA (eBioscience), according to intracellular pattern of NLRP3 inflammasome activation involv- supplier’s instructions. Cell lysates were obtained by lysing cells in in 13 SDS buffer (62.5 mM ing reactive oxygen species (ROS) production, lysosomal de- Tris-HCl [pH 6.8], 2% SDS [w/v], 10% glycerine [v/v], 50 mM DTT, and + stabilization and potassium efflux (K ). Inflammasome activation 0.01% bromphenol blue [w/v]). Proteins from cell-free supernatants were depends on several bacterial virulence factors, namely an intact extracted by TCA precipitation, resuspended in Laemmli buffer and by guest on September 27, 2021 cagPAI and the presence of VacA. Finally, we show that NLRP3 is 53 SDS buffer. Equal amounts of lysates were loaded on 12 or 15% important for H. pylori colonization and inflammation-associated SDS-PAGE gels. Separated proteins were transferred to polyvinylidene difluoride membranes that were incubated overnight with the specific Ab pathology in vivo. according to manufacturer’s instructions. Anti-mouse-IL-1b (R&D Sys- tems), anti-mouse caspase-1 (Adipogene), anti-human IL-1b (Cell Sig- Materials and Methods naling Technology), anti-human caspase-1 (Santa Cruz Biotechnology) and anti–b-actin (Sigma-Aldrich) were used as primary Abs. HRP-coupled Bacterial strains and culture conditions secondary Abs were from Promega. The H. pylori strains G27 (19), PMSS1, and SS1 (20) were grown on The release of lactate dehydrogenase (LDH) was measured in super- Wilkins–Chalgren blood agar plates supplemented with 10% horse serum natants using the CytoTox 96 kit (Promega), according to manufacturer’s instructions. and Dent supplement (Oxoid) under microaerobic conditions (10% CO2, 5% O2, 85% N2; 37˚C). The PMSS1 isogenic mutant strains PMSS1DgGT (21), PMSS1DVacA Strains of mice (21) and PMSS1DCagE (22), G27DBabA, G27DSabA, G27DBabASabA, Specific-pathogen-free C57BL/6 mice were originally purchased from G27DgGT (23), G27DVacA, G27DCagA, and G27DCagE were grown on Harlan Laboratories, Nlrp3-deficient mice (24), and Asc knockout mice Wilkins–Chalgren blood agar plates containing 20 mg/ml kanamycin or 15 mg/ (25) were provided by J. Tschopp (University of Lausanne, Lausanne, ml chloramphenicol, respectively, for selection under the same conditions. Switzerland) and V. Dixit (Genentech, South San Francisco, CA). Nlrc4 Salmonella typhimurium was grown on Columbia blood agar plates (BD knockout mice (25) were provided by F. Greten (II. Medizinische Klinik Biosciences). und Poliklinik, Klinikum rechts der Isar der Technische Universita¨t € Isolation of immune cells Munchen, Munchen, Germany) and Caspase-1 deficient mice by Richard Flavell through The Jackson Laboratory (26). Wild-type littermates or For generation of murine BMDCs and bone marrow–derived macrophages C57BL/6 cohoused animals were used as controls to exclude an influence (BMMs), bone marrow was obtained from femur and tibia of donor mice. of difference in the gut flora of independently bred mice. Mice were bred Cellswereculturedfor7dat37˚CinRPMI1640mediumorDMEM, in a pathogen-free animal facility at the Institute for Medical Microbiol- respectively, containing 10% FCS (Sigma-Aldrich), 1% penicillin/ ogy, Immunology and Hygiene (Technische Universita¨tMunchen).€ streptomycin (Life Technologies), 50 mM 2-ME (Sigma-Aldrich), and 20 ng/ml GM-CSF (Miltenyi Biotec) to generate BMDCs or 15% Animal experimentation, assessment of H. pylori colonization, M-CSF containing supernatant (v/v) from M-CSF–secreting L929 and gastric histopathology fibroblasts (L cell conditioned medium) to generate BMMs. Neutrophils were isolated from bone marrow cells by magnetic cell All animal experiments were approved by local authorities (Regierung von labeling (Miltenyi Biotec) with an anti-Ly6G Ab (Hycult Biotech). Oberbayern [55.2-1-54-2532-155-12]). Human PBMCs were obtained, after informed consent, from whole For in vivo experiments, 6- to 8-wk-old female C57BL/6 and C57BL/6 blood of H. pylori negative donors by biocoll (Biochrom) density gradi- DNlrp3 were infected with an orogastric dose of 109 CFUs of H. pylori ent separation. strain PMSS1. Infection was repeated on days 3 and 5. Monocytes, B cells, T cells, and NK cells were isolated from PBMCs After mice were sacrificed, the stomach was removed, opened along the with monocyte isolation kit II, B cell isolation kit II, T cell isolation kit II, lesser curvature, washed with PBS, and dissected longitudinally into equal The Journal of Immunology 3 strips. Of every stomach, the same section was assigned to the same downstream (Supplemental Fig. 1A, 1B), its processing was not detected until processing to reduce sampling error. 3 h post-H. pylori infection, whereas H. pylori alone had no effect For quantitative assessment of H. pylori colonization, one stomach at this short interval (Supplemental Fig. 1C). At later time points, section was homogenized in Brucella broth and serial dilutions were plated on WC dent agar plates and supplemented with bacitracin (200 mg/ml), H. pylori induced the release of IL-1b but always at lower levels nalidixic acid (10 mg/ml), and polymyxin B (3 mg/ml). when compared with LPS-prestimulated cells, confirming that H. For assessment of gastric histopathology, paraffin-embedded stomach pylori provides the second signal for inflammasome activation. sections were H&E stained and scored for H. pylori colonization, gastritis For being biologically active, IL-1b is cleaved by caspase-1, the degree of atrophy, and intestinal metaplasia, according to the updated Sydney score system (27). effector protease of the inflammasome. Thus, activation of the inflammasome after H. pylori infection was confirmed by the pres- Real-time PCR and cytokine ELISAs ence of the active caspase-1 subunits p20 or p10 (Fig. 1B, 1D). Gastric IFN-g and IL-17 expression was determined by RT-PCR. Total Furthermore, when PBMCs (Supplemental Fig. 1F) or BMDCs gastric RNA was isolated using RNA isolation kit (Quiagen). Total RNA (Supplemental Fig. 1G) were incubated with a caspase-1–specific (1–2 mg) was retrotranscribed (reverse transcriptase from Promega) and used inhibitor, the secretion of IL-1b was significantly reduced, whereas g g for real-time PCR (CFX Bio-Rad). Absolute values of IFN- (IFN- forward no changes on the levels of the inflammasome-independent cyto- 59-TCAAGTGGCATAGATGTGGAAGAA-39/IFN-g reverse 59-TGGC- TCTGCAGGATTTTCATG-39) and IL-17 (IL-17 forward 59-GCT CCA GAA kine TNF-a or intracellular pro–IL-1b were detected (Supplemental GGC CCT CAG A-39/IL-17 reverse 59-AGC TTT CCC TCC GCA TTG Fig. 1F–H). These results demonstrate that IL-1b secretion during A-39) expression were normalized to GAPDH expression (GAPDH forward H. pylori infection is mediated by caspase-1. 59-GCA CAG TCA AGG CCG AGA AT-39/ GAPDH reverse 59-GCC TTC Because the activated inflamamsome is also responsible for TCC ATG GTG GTG AA-39). To determine IL-1b, IL-18, IL-10, and TNF-a protein levels in stomach inducing pyroptosis, we analyzed cell viability by measuring the Downloaded from extracts, a piece of stomach was weighted and homogenized in PBS with release of LDH. PBMCs (Fig. 1E) or BMDCs (Fig. 1F) infected proteinase inhibitor (Roche). After determining the whole protein content with H. pylori did not secrete more LDH compared with unin- of the supernatant by Pierce BCA protein assay (Thermo), 5 mg protein fected cells, indicating that H. pylori does not induce pyroptosis at were used for measuring cytokine levels by ELISA (eBioscience or R&D the time points investigated. Systems, respectively). H. pylori mediates IL-1b secretion in an NLRP3 and ASC-

Preparation of gastric single-cell suspensions and flow http://www.jimmunol.org/ cytometry dependent manner Inflammasomes are multiprotein complexes consisting of the For the isolation of gastric immune cells, a stomach section was digested in 1 mg/ml collagenase (Sigma-Aldrich) 6 200 mg/ml Dnase I (Roche) for 30 min adaptor protein ASC and a sensor protein, often of the NLR family at 37˚C with shaking. Single-cell suspensions were filtered and stained for (NLRP1, NLRP3, and NLRC4) in addition to caspase-1. To CD4 and CD45 (eBioscience). Flow cytometry was performed on a CyanADP confirm that H. pylori induces inflammasome activation and to instrument (DakoCytomation) and analyzed using FlowJo software (Tree Star). elucidate which sensor protein is engaged, BMDCs from Cas- 2 2 2 2 2 2 2 2 Statistical analysis pase-1 / , Asc / , Nlrp3 / , and Nlrc4 / mice were cocultured with H. pylori. In the absence of Caspase-1, the release of IL-1b Data are presented as mean 6 SD unless otherwise stated. Data were analyzed for normal distribution before nonparametric tests (Mann– was significantly dampened, whereas no changes in TNF-a se- by guest on September 27, 2021 Whitney U test) or parametric tests (Student t test or ANOVA with Bon- cretion was detected (Fig. 2A). Likewise, the lack of Asc or Nlrp3 ferroni correction for multiple comparisons) were used to determine sta- was translated into a reduction of IL-1b levels (Fig. 2B), whereas tistical significance using GraphPad Prism software (GraphPad). Statistical no significant changes in the IL-1b release was detected in Nlrc4- significance was established when p # 0.05. deficient mice (Fig. 2C). As expected, TNF-a production was not affected in the absence of Asc, Nlrp3 (Fig. 2B), or Nlrc4 (Fig. 2C). Results In addition, cleavage of caspase-1 and IL-1b was not detected H. pylori induces inflammasome activation and IL-1b release when Nlrp3 and Asc-deficient BMDCs were infected with H. pylori in innate immune cells (Fig. 2D). These results indicate that H. pylori primarily activates Activation of the inflammasome by H. pylori has been shown in the NLRP3 inflammasome. murine BMDCs, whereas human cells have not been investigated. Therefore, we initially infected human PBMCs with the H. pylori Activation of NLRP3 inflammasome by H. pylori involves strain G27 and measured the release of IL-1b in the supernatants. potassium efflux, lysosomal destabilization, and ROS We observed mature IL-1b secretion after infecting the cells with production H. pylori (Fig. 1A, 1B), suggesting that H. pylori is able to provide NLRP3 inflammasome has been described to be activated by K+ the priming and, more importantly, the second signal required for efflux in different infection models. To depict whether NLRP3 inflammasome stimulation. By prestimulating the cells with LPS activation upon H. pylori infection involves K+ efflux, we blocked we aimed at discriminating the effect of H. pylori as second sig- it by incubating BMDCs with 50 mM KCl 30 min prior infection. nal, since LPS alone lead to the production of pro–IL-1b but not to H. pylori–induced IL-1b production was inhibited by blocking K+ the cleavage to mature IL-1b (Supplemental Fig. 1A). Higher IL- efflux (Fig. 3A), whereas the levels of TNF-a were not signifi- 1b secretion was observed when cells were prestimulated with cantly altered (Supplemental Fig. 2A). Similar results were obtained LPS as priming signal, clearly showing that H. pylori can provide when human PBMCs were treated with KCl prior to H. pylori the second signal required for inflammasome activation. infection (Supplemental Fig. 3A). Increased IL-1b secretion was also detected upon infection of Phagocytosis as well as subsequent lysosomal damage with murine BMDCs (Fig. 1C, 1D), BMDMs (Supplemental Fig. 1D) release and activation of cathepsin B are additional mechanisms and bone marrow–derived neutrophils (Supplemental Fig. 1E), in- associated with the activation of the NLRP3 inflammasome. dependently of the H. pylori strain used, indicating that H. pylori Therefore, we next explored whether endocytic processes were activates the inflammasome in different innate immune cells. To necessary for H. pylori–induced IL-1b production. For this further characterize the effect of H. pylori infection on the release of purpose, BMDCs were incubated with cytochalasin D to inhibit IL-1b, time-course experiments were performed in BMDCs. Even actin polymerization and therefore phagocytosis. Following in- though pro–IL-1b was produced after 3 h LPS prestimulation hibition of phagocytosis, H. pylori–induced IL-1b secretion was 4 H. PYLORI ACTIVATES THE NLRP3 INFLAMMASOME Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. H. pylori induces IL-1b secretion via inﬂammasome activation. (A) Human PBMCs were infected with the H. pylori strain G27 at MOI 5. Cells were prestimulated with 5 ng/ml LPS where indicated. After overnight culture, IL-1b secretion was measured in the supernatants by ELISA. Data are presented as mean 6 SD from nine different healthy donors. H. pylori–infected cells were compared with uninfected control cells, whereas infected and LPS-prestimulated PBMCs were compared with only LPS-stimulated control cells (asterisks shown on top of the bars) (p , 0.0001 ANOVA,*p # 0.05, **p # 0.01, ***p # 0.001, Bonferroni-corrected for pairwise comparison). (B) The protein levels of caspase-1 p10, IL-1b p17, and b-actin were measured in the supernatants or cell lysates of human PBMCs infected overnight with H. pylori G27 at MOI 5. Cells were prestimulated with 5 ng/ml LPS where indicated. One representative blot of three independent experiments is shown. (C) Murine BMDCs were infected with the H. pylori strains PMSS1 or G27 overnight at MOI 10 and 50, and the secretion of IL-1b was measured in the supernatants by ELISA. For prestimulation, cells were incubated with 10 ng/ml LPS. As a positive control, cells were stimulated with ATP (5 mM) for 1 h. Data are presented as mean 6 SD of three independent experiments. H. pylori–infected BMDCs were compared with uninfected control cells, whereas infected and LPS-prestimulated cells were compared with only LPS- stimulated control cells (asterisks shown on top of the bars) (p , 0.0001 ANOVA, **p # 0.01, ***p # 0.001, Bonferroni-corrected for pairwise comparison). (D) The protein levels of caspase-1 p20 and IL-1b were detected in the supernatants, and b-actin in cell lysates of BMDCs infected overnight with H. pylori PMSS1 or G27 at MOI 10 and 50. Cells were prestimulated with 10 ng/ml LPS where indicated. Stimulation with 5 mM ATP for 1 h was used as a positive control. One representative blot is shown. (E) Cytotoxicity was measured by LDH release in the supernatant of human PBMCs infected overnight with H. pylori G27 at MOI 5. Cells were prestimulated with 5 ng/ml LPS where indicated. Absorption was measured at 490 nm. Results are presented as mean 6 SD from four different healthy donors. (F) To determine cytotoxicity, LDH release was measured in the supernatants of BMDCs infected overnight with H. pylori PMSS1 at MOI 10 and 50. Cells were prestimulated with 10 ng/ml LPS where indicated. Absorption was measured at 490 nm. Data are presented as mean 6 SD of three independent experiments. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. H. pylori activates the inflammasome in an NLRP3- and ASC-dependent manner. (A) BMDCs from wild-type and caspase-1–deficient mice were infected overnight with H. pylori PMSS1 (MOI 10 and 50), and the levels of IL-1b and TNF-a were analyzed in the supernatants by ELISA. For prestimulation, 10 ng/ml LPS were used. Results are presented as mean 6 SD of three independent experiments. Cells from caspase-1 knockout mice were compared with wild-type cells (p , 0.0001 ANOVA, ***p # 0.001, Bonferroni-corrected for pairwise comparison). (B) The levels of IL-1b and TNF-a were analyzed in the supernatants of BMDCs from wild-type, Nlrp32/2, and Asc2/2 mice infected overnight with H. pylori PMSS1 (MOI 10 and 50). LPS (10 ng/ml) were used for prestimulation. As a positive control, cells were stimulated for 1 h with ATP (5 mM). Results are presented as mean 6 SD of four independent experiments. Cells from Nlrp3 and Asc knockout mice were compared with wild-type cells (p , 0.0001 ANOVA, *p # 0.05, ***p # 0.001, Bonferroni-corrected for pairwise comparison). (C) BMDCs from wild-type and Nlrc42/2 mice were infected overnight with H. pylori PMSS1 (MOI 10 and 50), and the levels of IL-1b and TNF were analyzed in the supernatants by ELISA. Cells were prestimulated with 10 ng/ml LPS. S. typhimurium (MOI 10), which was previously reported to activate the NLRC4 inflammasome (25), was used as a control. Results are presented as mean 6 SD of four independent experiments. Cells from Nlrc4 knockout mice were compared with wild-type cells (p , 0.0001 ANOVA, ***p # 0.001, Bonferroni-corrected for pairwise comparison). (D) The protein levels of active caspase-1 p20 and IL-1b p17 were measured in the supernatants and b-actin in cell lysates of wild-type, Nlrp32/2, and Asc2/2 BMDCs infected overnight with H. pylori PMSS1 at MOI 10 and 50. For prestimulation, 10 ng/ml LPS were used. ATP (5 mM) was used as a positive control. One representative blot is shown. 6 H. PYLORI ACTIVATES THE NLRP3 INFLAMMASOME Downloaded from

FIGURE 3. H. pylori activates the NLRP3 inflammasome through potassium efflux, lysosomal destabilization and K efflux. BMDCs were infected 6 h http://www.jimmunol.org/ with H. pylori PMSS1 (MOI 50), and IL-1b secretion was measured in the supernatants by ELISA. For prestimulation, 10 ng/ml LPS were used. KCl (A), cytochalasin D (B), ebselen (C), or APDC (D) were added at the indicated concentrations 30 min before infection. Results are presented as mean 6 SD of three independent experiments. (p , 0.0001 ANOVA, *p , 0.05, ***p # 0.001, Bonferroni-corrected for pairwise comparison). Inhibitor-treated cells were compared with nontreated cells. decreased (Fig. 3B). To rule out a deleterious effect of the in- H. pylori virulence factor VacA and the CagPAI are important hibitor used, we assessed cellular toxicity. No adverse effects for inflammasome activation were detected when using cytochalasin D (Supplemental Fig. To elucidate whether activation of the inflammasome was actively by guest on September 27, 2021 2B). Comparable results were obtained for human PBMCs induced by bacterial secreted factors, the bacterium was subjected (Supplemental Fig. 3B). to heat inactivation or PFA fixation. A slightly reduced IL-1b secre- H. pylori It is well-known that induces ROS; however, it is tion was detected when cells were incubated with PFA-fixed H. pylori yet unclear whether ROS activate the inflammasome or rather (Fig. 4A). Significantly lower levels of the cytokine were observed act as priming signal leading to increased expression of pro– after heat-inactivating the bacterium, suggesting that heat-sensitive IL-1b and NLRP3 proteins. Nevertheless, we analyzed whether H. pylori virulence factors might actively regulate IL-1b secretion. ROS could be involved in the H. pylori–mediated activation To identify the H. pylori virulence determinants involved of the NLRP3 inflammasome. To minimize the effect on priming, in inflammasome activation, LPS-prestimulated BMDCs were the cells were prestimulated with LPS before inhibitor addition. infected with the H. pylori strain PMSS1 deficient for either of the To prevent ROS induction, the inhibitors ebselen and (2R, 4R)- virulence factors, CagA, CagE, VacA, or g-Glutamyltranspeptisdase 4-aminopyrrolidine-2, 4-dicarboxylate (APDC) were used. (gGT) and the SS1 strain, which expresses VacA (30) CagA and Preincubation of BMDCs with ebselen and APDC resulted in CagE (31) but has a nonfunctional cagPAI. Absence of CagE, significant lower levels of IL-1b secretion, showing that H. pylori–mediatedROSproductionisinvolvedininflamma- a VirB4 ATPase of the type IV secretion system (T4SS) essential for some activation (Fig. 3C, 3D). The secretion of the inflamma- both the assembly of the system and CagA transfer, resulted in some independent cytokines TNFa and IL-6, as well as the lower levels of IL-1b (Fig. 4B). Similar results were detected when phagocytic bacterial uptake was analyzed to exclude off-target cells were stimulated with the SS1 strain. Interestingly, the H. pylori effects. No changes in TNFa or IL-6 levels (Supplemental Fig. PMSS1 strain lacking CagA was still capable of inducing IL-1b, 2C) were detected when using ebselen, whereas APDC induced indicating that other components of the PAI are responsible for a reduction in the levels of IL-6. This effect could be due to the inflammasome activation. Also, a PMSS1 mutant lacking the pore- agonistic activation of glutamate receptors expressed on DCs forming toxin VacA induced lower amounts of IL-1b secretion. (28) by APDC, leading to inhibition of cAMP signaling and IL- Conversely, the absence of gGT did not influence the release of IL- 6 production as reported previously (29). Importantly, no dif- 1b. These results correlated with caspase-1 activation. Thus, active ferences in bacterial uptake were observed after using the caspase-1 p20 was not detected after heat inactivation of the bac- inhibitors (Supplemental Fig. 2E). When analyzing the effect of terium or when infected the cells with bacterial strains lacking these inhibitors on the secretion of IL-1b by human PBMCs, we a functional T4SS or VacA (Fig. 4C, 4D). Levels of TNF-a se- observed lower levels of IL-1b secretion after ebselen treatment, cretion were measured to exclude inflammasome independent whereas APDC completely abolished IL-1b release (Supplemental effects. No changes in TNF-a release were observed (Supplemental Fig. 3C, 3D). Thus, our results show that H. pylori activates the Fig. 4A, 4B). In addition, no influence on bacterial uptake was NLRP3 inflammasome by several distinct mechanisms, involving detected (Supplemental Fig. 4C), indicating that all the H. pylori K+ efflux, phagocytosis, and ROS production. strains used were equally phagocytized (Supplemental Fig. 4C). The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. H. pylori virulence factors cagPAI and VacA are involved in inflammasome activation. (A) BMDCs were incubated overnight with viable, heat-inactivated (h.i.), or PFA-fixed H. pylori PMSS1 (MOI 50), and IL-1b secretion was measured in the supernatants by ELISA. For prestimulation, 10 ng/ml LPS were used. Results are presented as mean 6 SD of three independent experiments. Cells stimulated with treated-H. pylori were compared with cells incubated with live bacteria. Asterisks on top of the bars indicate significance relative to only LPS-stimulated control cells (p , 0.0001, ANOVA. *p , 0.05, **p # 0.01, ***p # 0.001, Bonferroni-corrected for pairwise comparison). (B) IL-1b secretion levels were measured in the supernatants of BMDCs infected overnight with the H. pylori wild-type strains PMSS1 or SS1 or the PMSS1 isogenic mutant strains deficient for CagA, CagE, VacA, or gGT at MOI 50. Cells were prestimulated with LPS (10 ng/ml). Data (mean 6 SD) from three independent experiments are shown. Cells stimulated with H. pylori knockout strains were compared with cells infected with the wild-type strain. Asterisks on top of the bars indicate significance relative to only LPS-stimulated control cells (p , 0.0001, ANOVA. *p # 0.05, **p # 0.01, ***p # 0.001, Bonferroni-corrected for pairwise comparison). (C) The protein levels of active caspase-1 p20 were measured in the supernatants and b-actin in the cell lysate of LPS-prestimulated BMDCs incubated overnight with viable, h.i., or PFA-fixed H. pylori PMSS1 (MOI 50). For prestimulation, 10 ng/ml LPS were used. One representative blot is shown. (D) The protein levels of active caspase-1 p20 were measured in the supernatants and b-actin in the cell lysate of LPS-prestimulated BMDCs infected overnight with the H. pylori wild-type strains PMSS1 or SS1 or the PMSS1 isogenic mutant strains deficient for CagA, CagE, VacA, or gGT at MOI 50. For prestimulation, 10 ng/ml LPS were used. One representative blot is shown. (E) PBMCs were infected overnight with H. pylori G27 or the indicated isogenic mutants at MOI 5, and IL-1b secretion was measured in the supernatants by ELISA. For prestimulation, 5 ng/ml LPS were used. Results from four different healthy donors are presented as mean 6 SD. Cells stimulated with H. pylori knockout strains were compared to the infected with the wild-type strain. Asterisks on top of the bars indicate significance relative to the only LPS-stimulated control cells (p , 0.0001, ANOVA. *p # 0.05, **p # 0.01, (Figure legend continues) 8 H. PYLORI ACTIVATES THE NLRP3 INFLAMMASOME

When corroborating these results in human PBMCs, we ob- tivation of the inflammasome, and we identify NLRP3 as the inflam- served reduced levels of IL-1b upon infection with a CagE defi- masome mainly activated in response to the bacterium. Our results are cient strain, whereas only slightly reduced IL-1b levels were in agreement with recent data showing that IL-1b secretion by observed when infecting with VacA or gGT mutants (Fig. 4E). H. pylori–infected murine DCs depended on the cooperative inter- Importantly, CagA also seemed not important for induction of IL- action between TLR2/NOD2 and NLRP3 inflammasome (8). How- 1b in human PBMCs. Interestingly, we observed that adhesion ever, we provide direct evidence that H. pylori provides the second of the bacterium to the cells is important for inflammasome ac- signal inducing the assembly and activation of the inflammasome and tivation, because infection with strains deficient in the adhesins identify the main virulence factors and cellular mechanisms involved. blood group Ag–binding adhesin (BabA) or sialic acid–binding Because of the broad spectrum of stimuli, which are able to adhesin (SabA) led to lower IL-1b secretion. No changes in the activate the NLRP3 inflammasome, NLRP3 has been considered levels of the inflammasome-independent cytokine TNF-a were to be a sensor of the disruption of host physiology. H. pylori found (Supplemental Fig. 4D). To identify the major source of possesses several virulence factors able to profoundly change IL-1b in human PBMCs, we performed negative selection to cellular physiology, and therefore, it is not surprising that we found isolate monocytes/macrophages, NK cells, B cells, and T cells. a number of molecular mechanisms involved in the H. pylori– After incubation of these cell subsets with H. pylori,weob- mediated activation of the NLRP3 inflammasome. Common served that inflammasome activation and IL-1b secretion NLRP3 inflammasome–activating mechanisms include potassium occurred mainly in monocytes/macrophages (Fig. 4F). efflux, lysosomal destabilization, and generation of ROS. We ob- These data indicate that in murine BMDCs and human PBMCs, served that blocking of these three stimuli in immune cells sub- the cagPAI, but not CagA, is the main H. pylori virulence factor stantially reduced IL-1b secretion upon H. pylori infection. The Downloaded from involved in the activation of the inflammasome. p58 subunit of H. pylori VacA was shown to induce potassium efflux from liposomes at acid pH values (32), whereas induction of NLRP3 inflammasome is involved in the adaptive immune ROS by VacA has been demonstrated in some studies (33–35). In response to H. pylori addition, VacA was described to lower mitochondrial transmem- Our in vitro data indicated that IL-1b secretion upon H. pylori brane potential (36), another mechanism by which the NLRP3

infection depended on the activation of the NLRP3 inflamma- inflammasome can be activated (37). These observations suggest http://www.jimmunol.org/ some. Therefore, we investigated whether the inflammasome plays that VacA might be involved in inflammasome stimulation. Indeed, a role in the immune response toward H. pylori infection in vivo. we observed that VacA-deficient bacteria were less efficient in For this purpose we infected Nlrp3-deficient mice with H. pylori inducing IL-1b release in murine DCs as well as in human PBMCs, PMSS1. After 1 mo of infection, reduced levels of IL-1b and confirming an involvement of VacA in inflammasome activation. IL-18 were detected in Nlrp3-deficient mice corroborating our This observation is in contrast to data from Kim et al. (8) who in vitro findings (Fig. 5A). We measured the levels of TNF-a and could not detect changes in IL-1b secretion after infecting cells IL-10, both cytokines important in the context of H. pylori in- with H. pylori strains deficient for VacA. One possible explanation fection but independent on the inflammasome, as control. No for this discrepancy is our strategy of using LPS as priming signal differences were detected between wild type and Nlrp3-deficient to dissect the bacterial factor acting as second signal triggering the by guest on September 27, 2021 mice as expected. Notably, the expression of pro–IL-1b was not inflammasome, which was not included in the previous study. affected (Supplemental Fig. 4E), indicating inflammasome- Although other virulence factors such as gGT can induce ROS specific effects. Interestingly, higher colonization of the gastric production, we did not observe significant effects on IL-1b se- mucosa was detected in Nlrp3-deficient mice when compared with cretion when using gGT-deficient bacteria. We speculate that in wild-type animals (Fig. 5B), which was accompanied by a lower the absence of gGT, other virulence factors such as VacA can elicit degree of inflammation (Fig. 5C). In addition, lower infiltration of the cellular mechanisms responsible of inflammasome activation. + + CD45 and CD4 cells was observed in the stomach of Nlrp3 Our data further show that cagPAI, but not CagA, is important for knockout mice compared with wild-type animals (Fig. 5D). IL-1b secretion. This observation is in line with the previous To assess the influence of the inflammasome in the adaptive results by Kim et al. (8); however, we show that a functional immune response toward H. pylori, we measured the levels of IL- T4SS, encoded by H. pylori cagPAI, is important to provide the 17 and IFN-g in the gastric mucosa of infected mice. The ex- second signal triggering assembly and activation of the inflam- pression of both cytokines was reduced in Nlrp3-deficient mice masome. Many bacterial species have been described to activate when compared with wild-type (Fig. 5E). primarily the NLRC4 inflammasome via T3SS and T4SS (38). Altogether, these results indicate that the NLRP3 inflammasome Interestingly, the delivery of effector molecules through the T4SS influences H. pylori colonization of the gastric mucosa as well as has been recently shown to be required for NLRP3 inflammasome the subsequent immune response and gastric pathology. activation as well (39), indicating that the presence of an active T4SS might represent an alternative mechanism of NLRP3 acti- Discussion vation during bacterial infection. Apart from CagA, H. pylori The proinflammatory cytokine IL-1b has been extensively reported peptidoglycan is also delivered into host cells through the T4SS. to play an essential role in H. pylori infection and H. pylori–associ- The particulate nature of peptidoglycan was found to be essential ated gastric pathology, but little is known about the mechanisms for activation of NLRP3 inflammasome and alteration of pepti- triggering its expression and release from immune cells upon infec- doglycan by Staphylococcus aureus strongly reduces production tion. In the current study, we show that IL-1b secretion induced by of IL-1b in response to infection (40). Therefore, we speculate H. pylori in human and murine innate immune cells depends on ac- that H. pylori peptidoglycan injected into the host cell might be

Bonferroni-corrected for pairwise comparison). (F) IL-1b release was measured in the supernatants of monocytes/macrophages, T cells, B cells, and NK cells isolated from total PBMCs of three different healthy donors. Cells were infected overnight with H. pylori G27 at MOI 5. For prestimulation, 5 ng/ml LPS were used. Results are presented as mean 6 SD. Asterisks on top of the bars indicate signiﬁcance relative to only LPS-stimulated control cells (p , 0.0001, ANOVA. ***p # 0.001 Bonferroni-corrected for pairwise comparison). The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. NLRP3 influences the adaptive immune response to H. pylori.(A)IL-1b and IL-18 protein levels detected in gastric mucosa extracts of wild- type and Nlrp32/2 mice infected 1 mo with H. pylori PMSS1. Data from one representative experiment are shown. Horizontal lines indicate the mean (*p # 0.05, **p # 0.01, t test). (B) Colony forming units per milligram stomach of wild-type and Nlrp32/2 mice after 1 mo of infection. Horizontal lines indicate the median. Data from two pooled experiments are shown (**p # 0.01, Mann–Whitney U test). (C) Inflammatory score assessed in the gastric mucosa of wild-type and Nlrp3-deficient mice after 1 mo of infection. Data from two pooled experiments are shown. Horizontal lines indicate the median (*p # 0.05, Mann– Whitney U test). Representative micrographs of H&E-stained stomach sections are shown. Scale bar, 50 mm. (D) Percentage of leukocytes (CD45+) and CD4+ T cells infiltrating the gastric mucosa of wild-type and Nlrp3-deficient mice infected for 1 mo with H. pylori PMSS1. Cells were pregated on live cells based on forward and side scatter and gated on CD45+ and CD4+ cells. Horizontal lines indicate the mean. Data from two pooled experiments are shown (*p # 0.05, t test). (E) Relative IL-17 and IFN-g mRNA expression in the stomach mucosa of H. pylori–infected wild-type and Nlrp3-deficient mice. Results were normalized to GAPDH. Horizontal lines indicate the median. Results from two pooled experiments are shown (*p # 0.05, Mann–Whitney U test). involved in the activation of the inflammasome. Further experi- adhesion mediated T4SS-dependent secretion of a factor indepen- ments are needed to substantiate this hypothesis. dent of CagA induces activation of the inflammasome. Thus, binding In this context, it was interesting to note the markedly reduced IL- of the bacterium to the host cells represents a crucial event for 1b secretion when human PBMCs were infected with H. pylori inflammasome activation. This might also involve phagocytosis of strains lacking either of the adhesins BabA and SabA, suggesting that H. pylori, which is delayed and reduced in adhesion-deficient strains. 10 H. PYLORI ACTIVATES THE NLRP3 INFLAMMASOME

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