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1 This information is current as of January 14, References Subscription Permissions Alerts Caspase-1 but Not Caspase-11 Is Required for NLRC4-Mediated Pyroptosis and Legionella Restriction of Infection by Flagellated Species in Mouse Macrophages and In Vivo Daiane M. Cerqueira, Marcelo S. F. Pereira, Alexandre L. N. Silva, Larissa D. Cunha and Dario S. Zamboni July J Immunol 2015; 195: ; Prepublished online ; doi: /jimmunol This article cites 47 articles, 19 of which you can access for free at: Why The JI? Submit online. Rapid Reviews! 30 days* from submission to initial decision No Triage! Every submission reviewed by practicing scientists Fast Publication! 4 weeks from acceptance to publication *average Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: Downloaded from by guest on January 14, 2019 The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD Copyright 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: Online ISSN:

2 The Journal of Immunology Caspase-1 but Not Caspase-11 Is Required for NLRC4-Mediated Pyroptosis and Restriction of Infection by Flagellated Legionella Species in Mouse Macrophages and In Vivo Daiane M. Cerqueira, Marcelo S. F. Pereira, Alexandre L. N. Silva, Larissa D. Cunha, and Dario S. Zamboni Gram-negative bacteria from the Legionella genus are intracellular pathogens that cause a severe form of pneumonia called Legionnaires disease. The bacteria replicate intracellularly in macrophages, and the restriction of bacterial replication by these cells is critical for host resistance. The activation of the NAIP5/NLRC4 inflammasome, which is readily triggered in response to bacterial flagellin, is essential for the restriction of bacterial replication in murine macrophages. Once activated, this inflammasome induces pore formation and pyroptosis and facilitates the restriction of bacterial replication in macrophages. Because investigations related to the NLRC4-mediated restriction of Legionella replication were performed using mice double deficient for caspase-1 and caspase-11, we assessed the participation of caspase-1 and caspase-11 in the functions of the NLRC4 inflammasome and the restriction of Legionella replication in macrophages and in vivo. By using several species of Legionella and mice singly deficient for caspase-1 or caspase-11, we demonstrated that caspase-1 but not caspase-11 was required for pore formation, pyroptosis, and restriction of Legionella replication in macrophages and in vivo. By generating F 1 mice in a mixed C57BL/6 background deficient (129 3 Casp-11 2/2 ) or sufficient (129 3 C57BL/6) for caspase-11 expression, we found that caspase-11 was dispensable for the restriction of Legionella pneumophila replication in macrophages and in vivo. Thus, although caspase-11 participates in flagellin-independent noncanonical activation of the NLRP3 inflammasome, it is dispensable for the activities of the NLRC4 inflammasome. In contrast, functional caspase-1 is necessary and sufficient to trigger flagellin/nlrc4-mediated restriction of Legionella spp. infection in macrophages and in vivo. The Journal of Immunology, 2015, 195: L egionella pneumophila is a Gram-negative bacterium that causes a severe pneumonia named Legionnaires disease. L. pneumophila is commonly found in soil and aquatic environments, where it infects a variety of protozoa, including several species of free-living amoebae (reviewed in Refs. 1, 2). Human contamination often occurs by inhalation of contaminated water droplets containing Legionella and/or infected amoebae. Legionnaires disease generally affects individuals in risk groups (i.e., patients with chronic lung diseases, immunocompromised Department of Cell Biology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo , Brazil Received for publication June 1, Accepted for publication July 7, This work was supported by the Pew Program in Biomedical Sciences, Conselho Nacional de Desenvolvimento Científico e Tecnológico/Instituto Nacional de Ciência e Tecnologia em Vacinas, Fundação de Amparo ao Ensino, Pesquisa e Assistência do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, and by Fundação de Amparo à Pesquisa do Estado de São Paulo Grants 2013/ and 2014/ (to D.S.Z.). D.M.C. (2012/ ), M.S.F.P. (2011/ ), A.L.N.S. (2012/ ), and L.D.C. (2012/ ) are supported by fellowships from the Fundação de Amparo à Pesquisa do Estado de São Paulo. D.S.Z. is a Research Fellow of the Conselho Nacional de Desenvolvimento Científico e Tecnológico. Address correspondence and reprint requests to Prof. Dario S. Zamboni, Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, Ribeirão Preto, São Paulo Brazil. address: dszamboni@fmrp.usp.br Abbreviations used in this article: BCYE, buffered charcoal yeast extract; BMDM, bone marrow derived macrophage; LCV, Legionella-containing vacuole; LDH, lactate dehydrogenase; MOI, multiplicity of infection; NLR, Nod-like receptor; WT, wild-type. Copyright Ó 2015 by The American Association of Immunologists, Inc /15/$25.00 individuals, and the elderly) (1, 2). Once it reaches the lung, L. pneumophila is able to infect alveolar macrophages using the same subset of genes used for amoeba infection. Immediately after encountering the alveolar macrophages, bacterial effector proteins are delivered into the macrophage cytoplasm through the type IV secretion system Dot/Icm. One of the many function of these effector proteins is the subversion of the normal trafficking of the Legionella-containing vacuole (LCV). Thus, vesicles from the endoplasmic reticulum are recruited to the LCV to create a favorable niche for bacterial replication inside the macrophages (reviewed in Refs. 3 5). In contrast to human cells, macrophages from most mouse strains are resistant to L. pneumophila infection, with the exception of the A/J mouse strain (6). The A/J strain has a mutation in the naip5/birc1e gene that encodes a receptor belonging to the Nod-like receptor (NLR) family (7, 8). These preliminary observations launched a successful line of investigations using L. pneumophila and its mutants as models to dissect the biology of the NLR family members (reviewed in Refs. 9, 10). The initial NLR report demonstrated that NAIP5 was linked to caspase-1 activation in a process that was similar to NLRC4, which is another member of the NLR family (11). These conclusions were validated by the generation of Naip5 2/2 mice (12). Additionally, studies with Salmonella Typhimurium and L. pneumophila revealed that the bacterial flagellin functioned as an agonist for the NAIP5/NLRC4 signaling platform (13 17). Moreover, this inflammasomewasreportedtobethekeyforrestrictionofl. pneumophila replication in mouse macrophages and in vivo (11, 13, 16 19). Once activated, this inflammasome promotes the formation of

3 2304 CASPASE-1 MEDIATED RESTRICTION OF LEGIONELLA INFECTION pores in the macrophage membrane (16, 17, 20); this process culminates in the induction of an inflammatory form of cell death termed pyroptosis (reviewed in Ref. 21). The mechanisms underlying NLRC4-mediated host resistance to L. pneumophila infection are still unclear, but pyroptosis has been proposed to be directly involved in host resistance (22). Furthermore, the NLRC4 inflammasome was reported to trigger the restriction of bacterial replication by additional processes dependent on flagellin but independent of caspase-1 and caspase-11 (18). Another pathway for caspase-1 activation has been reported in addition to the flagellin-mediated activation of the NLRC4 inflammasome. Several Gram-negative bacteria were demonstrated to be able to trigger caspase-1 activation via a process that required caspase-11 (the so-called noncanonical pathway for the activation of the NLRP3/ASC/caspase-1 inflammasome) (23, 24). This pathway effectively accounted for caspase-1 activation in response to L. pneumophila (25). Active caspase-11 triggers the formation of pores in the macrophage membranes independent of NLRC4, ASC, and NLRP3 (25). This action occurs concomitant with the cleavage of caspase-1 in a process that is dependent on NLRP3 and ASC (25). Paradoxically, another report indicated that caspase- 11 functioned as a component of the NLRC4 inflammasome and was required for the restriction of L. pneumophila infection in macrophages and in vivo (26). In this context, the participation of caspase-1 and caspase-11 in the physiological functions of the NLRC4 inflammasome and the restriction of L. pneumophila replication requires further investigation. Notably, most investigations related to the inflammasome field have been performed using a mouse strain that is double deficient for caspase-1 and caspase-11 (23). Using these double-deficient mice, researchers postulated that either one of these molecules (or both) were required for the mechanistic functions of the NLRC4 inflammasome. However, a comprehensive investigation using animals singly deficient for caspase-1 or caspase-11 has not been performed to evaluate the participation of these molecules in the restriction of L. pneumophila replication. Flagellated species of Legionella represent appropriate models for such an investigation because they are intracellular pathogens that fail to replicate extracellularly and encode a type IV secretion system that facilitates the delivery of flagellin to the host cell cytoplasm. Moreover, Legionella does not express a type III secretion system, which triggers the activation of NLRC4 via flagellin-independent pathways (reviewed in Ref. 27). In the present study, we systematically evaluated Casp-11 2/2, Casp-1/11 2/2, Casp1 2/2 /Casp-11 Tg, and Nlrc4 2/2 mice (and their macrophages) to assess the participation of caspase-1 and caspase-11 in the physiological functions of the NLRC4 inflammasome. Using L. pneumophila and three other flagellated species of Legionella, we found that caspase-1 but not caspase-11 was required for the functional activities of the NLRC4 inflammasome. These activities included caspase-1 cleavage, pore formation, induction of pyroptosis, and restriction of Legionella replication in macrophages and the lungs of infected mice. Materials and Methods Bacterial strains In this study, we used L. pneumophila strain JR32 and its isogenic mutant for flagellin (flaa). The non pneumophila Legionella strains used included Legionella micdadei (ATCC 33218) and Legionella bozemanii (ATCC 33217) as previously described (20). Additionally, a streptomycin mutant of Legionella gratiana (ATCC 49413) was generated (DZ571) and used in this study. All bacteria were grown in MOPS-buffered charcoal yeast extract (BCYE) agar (1% yeast extract, 1% MOPS [ph 6.9], 3.3 mm L-cysteine, 0.33 mm Fe(NO 3 ) 3, 1.5% Bacto agar, and 0.2% activated charcoal) at 37 C (28). For the in vivo and in vitro infections, bacteria were grown at 37 C in BCYE agar plates. After 48 h, they were diluted in sterile water and the OD at 600 nm was measured in a spectrophotometer (Bio- Photometer; Eppendorf, Hamburg, Germany) to determine the bacterial concentration in the solution. Western blot analysis Bone marrow-derived macrophages (BMDMs) were prepared from the indicated mice and differentiated in L929 cell conditioned medium as previously described (29). BMDMs were replated in RPMI 1640 medium supplemented with 10% (v/v) FBS and 5% (v/v) L929 cell conditioned medium in 48-well plates ( per well). These BMDMs were infected with bacterial strains at a multiplicity of infection (MOI) of 10 for the indicated times. Cell lysates and supernatants were subjected to SDS-PAGE analysis and Western blotting. Primary Abs used included a rat monoclonal against the p20 subunit of caspase-1 (Genentech, clone 4B4) and a rat monoclonal against caspase-11 (Abcam, Cambridge, MA), both at a 1:500 dilution. Loading control blot was performed using a rabbit polyclonal antiactin Ab (Sigma-Aldrich, St. Louis, MO, catalog no. A2066) at a 1:1000 dilution. An HRP-conjugated secondary Ab (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was used for detection at a 1:3000 dilution. The proteins were resuspended in SDS-containing loading buffer, separated on a 15% SDS-PAGE gel, and transferred (semidry transfer cell; Bio-Rad Laboratories, Hercules, CA) at 20 V for 40 min onto a nitrocellulose membrane (GE Healthcare, Piscataway, NJ) in transfer buffer (50 mm Tris, 40 mm glycine, and 10% methanol). The membranes were blocked for 1 h at 25 C in TBS with 0.1% Tween 20 and 5% nonfat dry milk and stained with primary Ab overnight at 4 C. The membranes were washed three times for 5 min in TBS with 0.1% Tween 20 and incubated for 1 h at 25 C with the appropriate HRP-conjugated secondary Ab. The Western blot luminol reagent (Santa Cruz Biotechnology, Santa Cruz, CA) was used for detection. Cytokine measurements For cytokine determination, BMDMs were seeded at a density of cells/well in 24-well plates and infected with WT L. pneumophila at an MOI of 10 for 9 h. The cytokines in the supernatant were measured with mouse IL-1b and IL-12p40 ELISA kits (OptEIA; BD Biosciences, Franklin Lakes, NJ) according to the manufacturer s instructions. Propidium iodide uptake assay and Ab generation Pore formation in BMDMs was determined by quantifying propidium iodide uptake. BMDMs were seeded into black 96-well plates ( cells/well). The cells were infected with bacterial strains at an MOI of 10 for 3 h. Infections were performed in RPMI 1640 media lacking phenol red with 15 mm HEPES and 0.38 g/l NaHCO 3 supplemented with 10% (v/v) FBS and 6 mg/ml propidium iodide. Throughout infection, the plates were incubated at 37 C in a FlexStation 3 microplate reader (Molecular Devices, Sunnyvale, CA), and propidium iodide fluorescence was measured every 5 min. To ensure greater efficiency of bacterial phagocytosis during the infection, the bacteria were opsonized with a rabbit polyclonal Ab (i.e., anti L. pneumophila, anti L. gratiana, anti L. micdadei, and anti- L. bozemanii; 1:1000 dilution). These polyclonal Abs were generated by injecting rabbits with heat-killed bacteria/rabbit. Animals were injected three times during a 15-d interval; then, the serum of each rabbit was tested for the presence of the specific Ab and stocked at 280 C. Lactate dehydrogenase release assay BMDMs were seeded into 24-well plates ( cells/well) and infected with bacterial strains at an MOI of 10. Infections were performed in RPMI 1640 media lacking phenol red with 15 mm HEPES and 2 g/l NaHCO 3 supplemented with 10% (v/v) FBS. After 4 h of infection, supernatants were harvested for analysis of lactate dehydrogenase (LDH) release by dying cells. Total LDH was determined by lysing the cultures with Triton X-100. LDH was quantified using the CytoTox 96 LDH-release kit (Promega, Madison, WI). Growth of L. pneumophila and L. gratiana in macrophages Growth curves were performed as previously described (30). To measure intracellular multiplication, BMDMs were seeded at a density of cells/well into 24-well tissue culture plates. Cultures were infected at an MOI of 0.15, followed by centrifugation for 5 min at g at room temperature and incubation at 37 C in a 5% CO 2 atmosphere. For CFU determination, the cultures were lysed in sterile water after 24, 48, and 72 h of infection; then, the cell lysates were combined with the cell culture supernatants from the respective wells. Lysates plus supernatants from each well were diluted in water, plated on BCYE agar plates, and incubated for 4 d at 37 C for CFU determination.

4 The Journal of Immunology 2305 Immunofluorescence and the number of bacteria per vacuole BMDMs were seeded at a density of cells/well onto 13-mm glass coverslips contained in 24-well tissue culture plates. The cells were infected with L. pneumophila at an MOI of 10, followed by centrifugation at g for 5 min and incubation for 1 h to allow phagocytosis. Then, the cells were washed three times with PBS at room temperature and incubated for an additional 9 h. The cultures were fixed and permeabilized for 10 min in ice-cold methanol. Coverslips were washed with PBS and blocked in PBS containing 5% BSA for 1 h. Bacteria were stained with a rabbit anti L. pneumophila polyclonal Ab (1:1000) (20), followed by an Alexa Fluor 488 conjugated goat anti-rabbit secondary Ab (1:3000 dilution; Invitrogen) or Alexa Fluor 594 conjugated goat anti-rabbit secondary Ab (1:3000 dilution; Invitrogen). The coverslips were mounted onto ProLong Gold antifade reagent (Invitrogen) on glass slides for fluorescence microscopy. Images were acquired with a Leica TCS SP2 SE inverted microscope using a 340, 1.25 numerical aperture oil objective (Leica HCX PL APO) and analyzed using Leica advanced fluorescence software. At least 100 infected cells were scored for each coverslip. Mice Mice were bred and maintained at the Animal Facility of the University of São Paulo at Ribeirão Preto. C57BL/6 and 129S6/SvEv mice were purchased from The Jackson Laboratory and breed and maintained in an institutional animal facility. Casp1/11 2/2 and Nlrc4 2/2 mice were generated as previously described (31, 32) and backcrossed to C57BL/6 mice for at least eight generations. Casp11 2/2 mice were generated in the C57BL/6 background (23). Casp1 2/2 /Casp-11 Tg mice are Casp1/11 2/2 mice expressing a transgene encoding a functional copy of the caspase-11 allele as previously described (23). To generate mixed genetic background mice, we crossed both 129 mice with C57BL/6 mice and 129 mice with Casp11 2/2 mice for one generation. The F 1 generation resultant mice from these crosses were named C57BL/6 and Casp11 2/2, respectively. The care of the mice was in compliance with the institutional guidelines on ethics in animal experiments approved by the Comissão de Ética em Experimentação Animal da Faculdade de Medicina de Ribeirão Preto (approved protocol no. 006/ 2008), which follows the Brazilian national guidelines recommended by the Conselho Nacional de Controle em Experimentação Animal. In vivo infections For in vivo infections, male or female mice aged wk were anesthetized with ketamine (90 mg/kg) (Agener União) and xylazine (5 mg/kg) (Dopaser) by i.p. administration, followed by intranasal inoculation with 40 ml PBS containing bacteria/ml. Mice were euthanized at the indicated time points for the determination of CFU per lung. The lungs were harvested and homogenized in 5 ml sterile water for 30 s using a tissue homogenizer (Fisher Scientific). Lung homogenates were diluted in water, plated on BCYE agar plates, and incubated for 4 d at 37 C for CFU determination. Statistical analysis Statistical analyses were performed using Prism 5.0 software (GraphPad Software, San Diego, CA). The unpaired Student t test was used to compare two groups. One-way ANOVA followed by multiple comparisons according to the Tukey procedure was used to compare three or more groups. Unless otherwise stated, data are expressed as the mean 6 SD. Differences were considered statistically significant at a p value, Results Caspase-1 activation and IL-1b production in response to L. pneumophila is NLRC4-dependent and caspase-11 independent WT L. pneumophila is able to infect C57BL/6 macrophages, leading to caspase-1 activation and IL-1b secretion in a process that is dependent on NLRC4 and bacterial flagellin (11, 16, 17, 20). Because the studies reporting these features were performed with mice double deficient for caspase-1 and caspase-11 (Casp1/11 2/2 ), we investigated whether flagellin-induced caspase-1 activation required caspase-11. We infected BMDMs from C57BL/6, Nlrc4 2/2, and Casp11 2/2 mice with WT L. pneumophila and measured caspase-1 cleavage and IL-1b secretion. Macrophages from the Casp1/11 2/2 mice were used as a control (31). We also used mice singly deficient for caspase-1; these mice were Casp1/11 2/2 mice expressing a functional caspase-11 allele to generate single caspase-1 deficient mice (23) (hereafter called Casp1 2/2 /Casp-11 Tg ). After 3 and 9 h of infection, cell supernatants were collected and subjected to Western blotting using a specific Ab against the p20 subunit of caspase-1. After 3 and 9 h of infection, C57BL/6 and Casp11 2/2 BMDMs were fully able to activate caspase-1 in response to flagellated L. pneumophila (Fig. 1A). In contrast, we observed a significant reduction in caspase-1 activation in the Nlrc4 2/2 BMDMs (Fig. 1A). The minor caspase-1 activation detected in the absence of NLRC4 was probably due to the noncanonical pathway for NLRP3 inflammasome activation (25). To certify that caspase-11 was appropriately expressed in these BMDMs, we analyzed caspase-11 expression after 9 h of infection with L. pneumophila and found that caspase-11 was efficiently upregulated in response to infection in BMDMs from C57BL/6, FIGURE 1. Caspase-11 is dispensable for caspase-1 activation and IL-1b secretion in response to flagellated L. pneumophila. BMDMs obtained from C57BL/6, Nlrc4 2/2, Casp1/11 2/2, Casp1 2/2 /Casp-11 Tg (Casp1 2/2 C11 Tg ), and Casp11 2/2 mice were left uninfected (NI) or stimulated with WT L. pneumophila. (A) Unprimed BMDMs were infected with WT L. pneumophila at an MOI of 10 for 3 and 9 h of infection. The supernatant was harvested and separated by SDS-PAGE, blotted, and probed with a monoclonal anti caspase-1 p20 subunit Ab. (B) Unprimed BMDMs were infected with WT L. pneumophila at an MOI of 10 for 9 h and cell lysates were separated by SDS-PAGE, blotted, and probed with a monoclonal anti caspase-11 p38/p43 Ab and with polyclonal rabbit anti-actin Ab. (C and D) Unprimed BMDMs were left uninfected (open bars) or stimulated with WT L. pneumophila (filled bars) at an MOI of 10. After 9 h of infection, the concentration of IL-1b (C) and IL-12p40 (D) in the culture supernatants was estimated by ELISA. Data show the averages 6 SD from triplicate wells. *p, 0.05 compared with C57BL/6 cultures infected with WT L. pneumophila. ND, not detected; NI, uninfected; WT Lp, WT L. pneumophila.

5 2306 CASPASE-1 MEDIATED RESTRICTION OF LEGIONELLA INFECTION Nlrc4 2/2,andCasp1 2/2 /Casp-11 Tg mice. As expected, Casp11 2/2 and Casp1/11 2/2 BMDMs did not express caspase-11 (Fig. 1B). Next, we investigated whether IL-1b secretion induced by WT L. pneumophila was dependent on caspase-11. C57BL/6, Nlrc4 2/2, Casp1/11 2/2, Casp1 2/2 /Casp-11 Tg,andCasp11 2/2 BMDMs were infected with WT L. pneumophila for 9 h, and the secretion of active IL-1b into the supernatant was evaluated. Notably, both C57BL/6 and Casp11 2/2 BMDMs secreted high levels of IL-1b, whereas BMDMs deficient in NLRC4 or caspase-1 failed to induce robust production of IL-1b (Fig. 1C). IL-12 secretion was measured in the same experiment to verify that all macrophages were able to produce a cytokine that was unrelated with inflammasome activation (Fig. 1D). The higher production of IL-1b detected in C57BL/6 than in Casp11 2/2 cultures was probably due to the noncanonical pathway for inflammasome activation, which requires caspase-11, ASC, and NLRP3 (23, 25). This pathway also occurs in response to flagellated bacteria such as L. pneumophila and Salmonella Thyphimurium via a flagellin/nlrc4-independent pathway (25, 33, 34). Collectively, these results demonstrate that caspase-1 activation and IL-1b production occurs via the flagellin/nlrc4 pathway independent of caspase-11. Macrophage pyroptosis triggered in response to L. pneumophila is NLRC4/caspase-1 dependent but caspase- 11 independent Pyroptosis is an inflammatory form of cell death that is readily induced in mouse macrophages infected with L. pneumophila in a process that is dependent on NLRC4 and flagellin (13, 16, 17, 20, 35). We previously demonstrated that caspase-11 can be activated by Legionella, but this secondary pyroptotic pathway is only detectable when the NLRC4 pathway is blocked by using a flagellin mutant bacteria or NLRC4-deficient macrophages (25). Thus, we used single knockout mice to investigate whether flagellin/nlrc4-mediated pore formation and pyroptosis required caspase-11 or caspase-1. Unprimed BMDMs from C57BL/6, Nlrc4 2/2, Casp11 2/2, Casp1 2/2 /Casp-11 Tg,andCasp1/11 2/2 mice were infected with WT L. pneumophila in medium containing propidium iodide. To assess NLRC4-induced pore formation, we quantified the influx of propidium iodide into the nuclei of the cells in real time during the initial 3 h of infection. We found that whereas C57BL/6 and Casp11 2/2 BMDMs readily triggered pore formation in response to flagellated L. pneumophila (Fig. 2A, 2B), Casp1 2/2 /Casp-11 Tg, Casp1/11 2/2, and Nlrc4 2/2 BMDMs failed to trigger pore formation (Fig. 2C E). Experiments performed with NLRC4-deficient cells confirmed that the pore formation measured in these conditions represented flagellin/nlrc4-induced pore formation. To further evaluate the role of caspase-1 and caspase-11 in NLRC4-induced pyroptosis, we quantified the release of LDH into the tissue culture supernatants. Unprimed C57BL/6, Nlrc4 2/2, Casp11 2/2, Casp1 2/2 /Casp-11 Tg,andCasp1/ 11 2/2 BMDMs were infected with WT L. pneumophila for 4 h, and then LDH was assessed in the tissue culture supernatants. Similar to the results from the pore formation assay, L. pneumophila triggered pyroptosis in C57BL/6 and Casp11 2/2 BMDMs, whereas cells deficient in Nlrc4, Casp1, andcasp1/11 failed to do so (Fig. 2F). Taken together, these studies indicate that caspase-1 but not caspase-11 is required for the pore formation and pyroptosis that occurs via the flagellin/nlrc4 pathway. The restriction of L. pneumophila replication in macrophages and in vivo is NLRC4/caspase-1 dependent but caspase-11 independent Next, we investigated the role of caspase-1 and caspase-11 in the restriction of L. pneumophila infection of BMDMs. This process FIGURE 2. Macrophage pyroptosis in response to flagellated L. pneumophila requires NLRC4 and caspase-1 but not caspase-11. BMDMs obtained from C57BL/6, Nlrc4 2/2, Casp1/11 2/2, Casp1 2/2 /Casp-11 Tg (Casp1 2/2 C11 Tg ), and Casp11 2/2 mice were left uninfected (NI) or stimulated with WT L. pneumophila. (A E) Unprimed BMDMs were left uninfected (s) or infected with WT L. pneumophila (d) at an MOI of 10. Pore formation was assessed fluorimetrically in real time by the uptake of propidium iodide (relative fluorescence units). Shown are pore formation in BMDMs from C57BL/6 (A), Casp11 2/2 (B), Casp1 2/2 C11 Tg (C), Casp1/ 11 2/2 (D), and Nlrc4 2/2 (E) mice.(f) Unprimed BMDMs were left uninfected (open bars) or stimulated with WT L. pneumophila (filled bars) at an MOI of 10 for 4 h prior to the assessment of extracellular LDH. Values represent the percentage of LDH released compared with cells lysed with Triton X-100. Data show the averages 6 SD from triplicate wells. *p, 0.05 compared with C57BL/6 cultures infected with WT L. pneumophila. NI, uninfected; RFU, relative fluorescence unit; WT Lp, WT L. pneumophila. has been reported by different groups to be dependent on flagellin and NLRC4 (11 13, 16 18, 35, 36). Initially, we evaluated bacterial replication by assessing the number of bacteria in the LCVs that formed after 10 h of infection. By infecting C57BL/6, Nlrc4 2/2, Casp1 2/2 /Casp-11 Tg, Casp11 2/2,andCasp1/11 2/2 BMDMs with WT L. pneumophila, we found that the C57BL/6 and Casp11 2/2 BMDMs restricted bacterial replication (i.e., most of the LCVs contained one or a few replicating bacteria) (Fig. 3A). In contrast, BMDMs from Nlrc4 2/2, Casp1 2/2 /Casp-11 Tg,andCasp1/11 2/2 mice contained significantly more bacteria per LCV (Fig. 3A). Representative images of the LCVs found in BMDMs illustrate the importance of NLRC4 and caspase-1 but not caspase-11 for limiting the intracellular replication of L. pneumophila (Fig. 3B). Next, we evaluated bacterial replication in BMDMs by performing growth curve assays during 3 d of infection. We infected C57BL/6, Nlrc4 2/2, Casp11 2/2, Casp1 2/2 /Casp-11 Tg,andCasp1/11 2/2 BMDMs with L. pneumophila and evaluated bacterial replication by scoring the total number of CFU present in the cultures after 24, 48, and 72 h of infection. We found a robust replication of WT L. pneumophila in Nlrc4 2/2, Casp1 2/2 /Casp-11 Tg,andCasp1/11 2/2

6 The Journal of Immunology 2307 L. pneumophila replication in the mouse lung. Collectively, these data indicate that flagellin recognition by the NLRC4 inflammasome employs responses that are dependent on caspase-1 but not caspase- 11 for the restriction of L. pneumophila replication in macrophages and in vivo. FIGURE 3. Early restriction of L. pneumophila replication within replicative vacuoles requires NLRC4 and caspase-1 but not caspase-11. BMDMs obtained from C57BL/6, Nlrc4 2/2, Casp1/11 2/2, Casp1 2/2 / Casp-11 Tg (Casp1 2/2 C11 Tg ), and Casp11 2/2 mice were infected with WT L. pneumophila at an MOI of 10 for 1 h, washed, and further incubated for an additional 9 h. Cells were fixed, the nuclei were stained with DAPI (blue), and the bacteria were stained with a rabbit anti L. pneumophila polyclonal Ab (red). The numbers of bacteria contained in each LCV were scored under an epifluorescence microscope. At least 100 LCVs were scored in each of the triplicate coverslips. Shown are the frequencies of the LCVs containing 1, 2 3, 4 10, or more then 10 (+10) bacteria (A). Representative images of LCVs containing one or a few bacteria are shown (B). Original magnification Data show the average 6 SD from the assessment in triplicate. *p, 0.05 compared with the same group of C57BL/6 BMDMs. BMDMs. In contrast, Casp11 2/2 and C57BL/6 BMDMs were able to limit intracellular bacterial infection (Fig. 4A). The increased replication of WT L. pneumophila in NLRC4 compared with Casp1/11 2/2 was previously reported and was thought to occur via an unknown pathway that was flagellin- and NLRC4- dependent but caspase-1/11 independent (18). A flagellindeficient strain of L. pneumophila (flaa) was used as a control; this strain is known to be able to bypass the NLRC4-mediated growth restriction (17). The flaa mutants effectively replicated in all macrophages used, indicating that all of the BMDMs were able to support bacterial replication in the absence of the NLRC4 inflammasome (Fig. 4B). Taken together, these data suggest that the flagellin/nlrc4-mediated growth restriction occurs via a process that is dependent on caspase-1 but independent of caspase-11. Finally, we tested whether caspase-11 was important for the restriction of L. pneumophila infection in vivo by employing a murine model of Legionnaires disease. C57BL/6, Nlrc4 2/2, Casp1 2/2 /Casp-11 Tg, Casp11 2/2,andCasp1/11 2/2 mice were infected intranasally with WT L. pneumophila. The bacterial CFU were enumerated from lung homogenates after 4, 48, and 72 h. The Casp-11 2/2 mice were as restrictive as the WT C57BL/6 mice at both 48 and 72 h postinfection (Fig. 4C). In contrast, the Nlrc4 2/2, Casp1 2/2 /Casp-11 Tg, and Casp1/11 2/2 mice supported higher pulmonary replication of WT L. pneumophila during the acute replication phase (48 h) than C57BL/6 mice and were less efficient at clearing pulmonary bacteria at 72 h (Fig. 4C). These results confirm the important role of NLRC4 and caspase-1 but not caspase-11 in the restriction of Caspase-11 is dispensable for restriction of L. pneumophila infection in macrophages and in vivo regardless of the mouse genetic background Macrophages from the 129 strain are more susceptible to L. pneumophila infection than are those from the C57BL/6 strain (8). Additionally, cells from the 129 strain were demonstrated to contain a naturally occurring 5-bp deletion that interfered with exon splicing and consequently generated cells with nonfunctional caspase-11 (23). Therefore, we tested whether the increased susceptibility of the 129 strain was caused by the deficiency in caspase-11. To address this question, we generated a mouse in a 129 and C57BL/6 mixed background. By crossing 129 with C57BL/6, we generated F 1 mice that were fully complemented for all of the mutations present in the parental strains, including caspase-11 (hereafter called C57BL/6). In contrast, the cross of 129 with Casp11 2/2 (fully generated in the C57BL/6 background) resulted in F 1 mice that were similarly complemented for all mutations except for caspase-11 (hereafter called Casp-11 2/2 ). To verify that caspase-11 was appropriately expressed in these mouse strains, we generated BMDMs from C57BL/6, Casp11 2/2, C57BL/6, and Casp11 2/2 mice and infected them with WT L. pneumophila. After 9 h of infection, cells from both the parental C57BL/6 and the C57BL/6 F 1 mice were able to express caspase-11. In contrast, cells from the parental 129 and Casp11 2/2 mice did not express detectable caspase-11 (Fig. 5A). Next, we used macrophages from these mice to evaluate the effect of caspase-11 on the restriction of intracellular bacterial replication. BMDMs obtained from C57BL/6, 129, C57BL/6, and Casp11 2/2 mice were infected with WT L. pneumophila, and the bacterial multiplication within LCVs was evaluated after 10 h of infection. BMDMs from 129 mice were slightly more susceptible than those from C57BL/6, as demonstrated by the higher proportion of LCVs containing 4 10 bacteria per LCV (Fig. 5B). However, these differences were unlikely to be caused by the absence of caspase-11, because Casp11 2/2 and C57BL/6 BMDMs showed a similar frequency of the number of bacteria per LCV (Fig. 5B). To further evaluate bacterial replication in BMDMs, we performed growth curve assays in cells from C57BL/6, 129, C57BL/6, and Casp11 2/2 mice. Bacterial growth was evaluated at 24, 48, and 72 h postinfection in BMDMs infected with WT L. pneumophila or the flaa mutant. All of the BMDMs used were permissive to flaa replication and restrictive to the WT bacteria (Fig. 6A, 6B). These data further suggest that flagellin-mediated activation of the NLRC4 inflammasome is critical for macrophage resistance to L. pneumophila infection, whereas caspase-11 is not required for bacterial growth regardless of the mouse genetic background. To determine whether caspase-11 was dispensable for caspase-1 activation, we used cells from these mice to evaluate caspase-1 cleavage in BMDMs infected with WT L. pneumophila. Caspase-1 was effectively activated in BMDMs from C57BL/6 or 129 backgrounds and cells from the F 1 mice, regardless of the presence or absence of caspase-11 (Fig. 6C). Finally, we used these F 1 mice to evaluate the role of caspase-11 in the restriction of L. pneumophila infection in vivo. By infecting C57BL/6, 129, C57BL/6, and Casp11 2/2 mice with WT L. pneumophila, we found that the 129 mice were more susceptible to infection than were the C57BL/6, C57BL/6, and

7 2308 CASPASE-1 MEDIATED RESTRICTION OF LEGIONELLA INFECTION FIGURE 4. The restriction of L. pneumophila replication in macrophages and in vivo is dependent on NLRC4 and caspase-1 but not caspase-11. (A and B) BMDMs obtained from C57BL/6 (d), Nlrc4 2/2 ( ), Casp1/11 2/2 (N), Casp1 2/2 /Casp-11 Tg (Casp1 2/2 C11 Tg ; s), and Casp11 2/2 (n) mice were infected with WT L. pneumophila (A) or with a flagellin-deficient mutant (flaa) (B) at a concentration of bacteria per well (MOI of 0.15). Cultures were incubated for 24, 48, and 72 h for CFU determination. Shown are the averages 6 SD from triplicate wells. (C) C57BL/6,Nlrc4 2/2, Casp1/11 2/2, Casp1 2/2 C11 Tg,andCasp11 2/2 mice were infected intranasally with WT L. pneumophila. The mice were sacrificed 4, 48, and 72 h postinfection, and diluted lung homogenates were added to BCYE agar plates for CFU determination. Each symbol represents a single animal, and the horizontal lines represent the average (approximately seven mice per group were used). *p, 0.05 compared with C57BL/6. WT Lp, WT L. pneumophila Casp11 2/2 mice after 48 and 72 h of infection (Fig. 6D). Importantly, the C57BL/6 mice did not differ from the Casp11 2/2 mice in terms of the restriction of L. pneumophila replication at 48 and 72 h (Fig. 6D), thereby supporting our previous data indicating that caspase-11 was dispensable for the restriction of L. pneumophila replication in macrophages and in vivo. Caspase-1 but not caspase-11 is involved in the NLRC4- mediated restriction of infection by flagellated species of Legionella We previously demonstrated that several flagellated species of Legionella triggered NAIP5/NLRC4 inflammasome activation in macrophages and underwent NLRC4-mediated restriction of bacterial replication in the mouse lung (19, 20). Therefore, we used some of these flagellated species of Legionella to evaluate the effect of caspase-1 and caspase-11 on the induction of pyroptosis and restriction of bacterial replication in macrophages and in vivo. First, we assessed the requirement for caspase-1 and caspase-11 for the restriction of replication of L. gratiana. By infecting C57BL/6, Nlrc4 2/2, Casp11 2/2, Casp1 2/2 /Casp-11 Tg, and Casp1/11 2/2 mice and their macrophages with L. gratiana, we found that caspase-1 but not caspase-11 was required for the restriction of L. gratiana replication in macrophages (Fig. 7A) and in vivo (Fig. 7B). As previously demonstrated (19), Nlrc4 2/2 and Casp1/11 2/2 mice were susceptible to L. gratiana replication (Fig. 7A, 7B). These data support our observations with L. pneumophila infection and indicate that NLRC4 and caspase-1, but not caspase-11, play an important role in growth restriction in mouse cells. Next, we used additional species of Legionella to assess the role of caspase-1 and caspase-11 in the induction of macrophage pyroptosis. BMDMs obtained from C57BL/6, Nlrc4 2/2, Casp11 2/2, Casp1 2/2 /Casp-11 Tg,andCasp1/11 2/2 mice were left uninfected or FIGURE 5. Caspase-11 is dispensable for the early restriction of L. pneumophila replication in macrophages from genetically defined F 1 mice from the crossing of C57BL/ mice. BMDMs obtained from C57BL/6, 129, F 1 mice from C57BL/6 crossing, and F 1 mice from Casp11 2/2 crossing were left uninfected (NI) or stimulated with WT L. pneumophila for 9 h. (A) Cell lysates were separated by SDS- PAGE, blotted, and probed with the monoclonal anti caspase-11 p43/p38 Ab and with polyclonal rabbit anti-actin Ab. (B) BMDMs obtained from C57BL/6, 129, C57BL/6, and Casp11 2/2 mice were infected with WT L. pneumophila at an MOI of 10 for 1 h, washed, and further incubated for an additional 9 h. Cells were fixed, the nuclei were stained with DAPI (blue), and the bacteria were stained with a rabbit anti L. pneumophila polyclonal Ab (green). The numbers of bacteria contained in each LCV were scored under an epifluorescence microscope. At least 100 LCVs in each of the triplicate coverslips were scored. Shown are the frequencies of the LCVs containing 1, 2 3, 4 10, or.10 (+10) bacteria. Data show the average 6 SD from triplicate samples. *p, 0.05 compared with the same group of the C57BL/6 BMDMs. NI, uninfected; WT Lp, WT L. pneumophila. infected with L. gratiana, L. bozemanii, and L. micdadei. L. pneumophila was included as a control in this experiment. By evaluating propidium iodide uptake in real time during the first 3 h of infection, we found that L. gratiana, L. bozemanii, L. micdadei, and L. pneumophila induced pore formation in a process that was dependent on NLRC4 and caspase-1 but not caspase-11 (Fig. 7C G). Pore formation was similar in C57BL/6 and Casp11 2/2 cells for all species tested, indicating that the NLRC4 inflammasome could fully function to induce pore formation during the early stages of infection in a process that required caspase-1 but not caspase-11. Collectively, these data support the findings that flagellated Legionella triggers a process that culminates with macrophage pyroptosis and the restriction of bacterial replication in a caspase-1 dependent and caspase-11 independent manner. Discussion Activation of the NAIP5/NLRC4 inflammasome in response to flagellated bacteria is pivotal for the restriction of microbial replication in murine macrophages and in vivo (reviewed in Ref. 37). However, mouse strains that are double deficient for caspase-1 and caspase-11 have been extensively used to evaluate the functions of the NLRC4 inflammasome (11 13, 15 18, 22, 33, 35). Therefore, either or both of these molecules may be required for the mechanistic functions of the NLRC4 inflammasome. Using mouse strains that are singly deficient for caspase-1, caspase-11, and both, we determined that caspase-1 but not caspase-11 was essential for the functional activities of the NLRC4 inflammasome, including the induction of pyroptosis and restriction of bacterial replication in mouse macrophages and in vivo. These data are in contrast with those from previous reports showing that caspase-11

8 The Journal of Immunology 2309 FIGURE 6. Caspase-11 is dispensable for the restriction of L. pneumophila replication in macrophages and in vivo in genetically defined F 1 mice from the crossing of C57BL/ mice. (A and B) BMDMs obtained from C57BL/6 (d), 129 ()), F 1 mice from C57BL/6 crossing ( ), and F 1 mice from Casp11 2/2 crossing (O) were infected with the flaa mutant (A)orWTL. pneumophila (B). Cultures were infected with bacteria per well (MOI 0.15) and incubated for 24, 48 and 72 h for CFU determination. Shown are the averages 6 SD from triplicate wells. (C) Unprimed BMDMs from C57BL/6, 129, C57BL/6, and Casp11 2/2 mice were left uninfected (NI) or stimulated with WT L. pneumophila. After 9 h of infection, the supernatant was harvested and separated by SDS-PAGE, blotted, and probed with the monoclonal anti caspase-1 p20 subunit Ab. (D) C57BL/6, 129, C57BL/6, and Casp11 2/2 mice were infected intranasally with WT L. pneumophila. The mice were sacrificed 4, 48, and 72 h postinfection, and diluted lung homogenates were added to BCYE agar plates for CFU determination. Each symbol represents a single animal, and the horizontal lines represent the averages (approximately seven mice per group were used). *p, 0.05 compared with C57BL/6. NI, uninfected; WT Lp, WT L. pneumophila. was required for the restriction of L. pneumophila replication in macrophages and in vivo (26). Notably, the caspase-11 deficient mice used in the previous study were different from those used herein. The casp-11 2/2 mouse strain used in our study was generated in a fully C57BL/6 genetic background (23), whereas the previous strain was generated in a 129 mixed background and further backcrossed to C57BL/6 (38). Additionally, the strains of L. pneumophila used also differed. These discrepancies may be relevant to the explanation for these differences given that the lysis of LCV and release of bacterial LPS into the macrophage cytoplasm has been proposed to underlie caspase-11 activation (39), and this process may be variable among different strains of L. pneumophila. Thus, it is possible that the differences in mouse strains, bacterial strains, or bacterial cultivation may explain the contrasting results found herein and elsewhere (26). In the present study, we used several species of flagellated Legionella in combination with caspase-11 deficient mice in an isogenic C57BL/6 background or in a mixed (F 1 C57BL/ ) genetic background. Our data strongly indicated that caspase-11 was not involved in the flagellin/nlrc4-mediated restriction of L. pneumophila replication. Nonetheless, it is still possible that caspase-11 is involved in the modulation of actin polymerization in response to L. pneumophila, FIGURE 7. Pore formation induced in response to flagellated species of Legionella are independent of caspase-11, and caspase-11 is not required for the restriction of L. gratiana replication in macrophages and in vivo. (A) BMDMs obtained from C57BL/6 (d), Nlrc4 2/2 ( ), Casp1/11 2/2 (N), Casp1 2/2 /Casp-11 Tg (Casp1 2/2 C11 Tg ; s), and Casp11 2/2 (n) mice were infected with L. gratiana per well (MOI of 0.15). Cultures were incubated for 24, 48, and 72 h for CFU determination. Shown are the averages 6 SD from triplicate wells. (B) C57BL/6, Nlrc4 2/2, Casp1/11 2/2, Casp1 2/2 C11 Tg,andCasp11 2/2 mice were infected intranasally with WT L. gratiana. The mice were sacrificed 4 and 48 h postinfection, and diluted lung homogenates were added to BCYE agar plates for CFU determination. Each symbol represents a single animal, and the horizontal lines represent the averages (approximately seven mice per group were used). *p, 0.05 compared with C57BL/6. (C G) Unprimed BMDMs from C57BL/6 (d), Nlrc4 2/2 ( ), Casp1/11 2/2 (N), Casp1 2/2 C11 Tg (s), and Casp11 2/2 (n) mice were left uninfected (C) or infected with L. gratiana (D), L. bozemanii (E), L. micdadei (F), and L. pneumophila (G) atanmoiof 10. Pore formation was assessed fluorimetrically in real time by the uptake of propidium iodide (relative fluorescence units) into the nucleus of the permeabilized BMDMs. RFU, relative fluorescence unit. a process that can facilitate the restriction of bacterial replication in macrophages (26). Of note, the caspase-1 single-deficient mice used in the present study were the Casp1/11 2/2 mice expressing a functional caspase-11 allele (23). In this strain, caspase-11 complementation does not restore the caspase-11 expression to the WT levels (Fig. 1B), possibly because the transgene encoding Casp-11 is only present in one chromosome. Although we did not detected an effect of caspase-11 complementation in the restriction of L. pneumophila replication, we cannot exclude the possibility that a full caspase-11 complementation enhances the bacterial killing in the Casp1/11 2/2. In this context, further experiments using single Casp-1 2/2 mice will be important to test this hypothesis. Nonetheless, our data generated with the Casp- 11 2/2 in C57BL/6 background and with the F 1 mice (129 3 Casp- 11 2/2 and C57BL/6) does not support a role of caspase-11 in restriction of L. pneumophila replication. Regardless of the redundant role of caspase-11 in the restriction of L. pneumophila replication, work by different groups has recently established that L. pneumophila triggers the activation of caspase-11 in mouse macrophages (25, 26, 39 41). Moreover, L. pneumophila triggers caspase-11 in a process that is dependent

9 2310 CASPASE-1 MEDIATED RESTRICTION OF LEGIONELLA INFECTION on the Dot/Icm type IV secretion system but independent of the flagellin/nlrc4 pathway (25, 40, 41). Accordingly, caspase-11 was reported to be broadly activated in response to LPS from several species of Gram-negative bacteria (24, 39, 41 45). Therefore, we envisage caspase-11 in a pathway that is triggered by Legionella LPS and leads to pore formation and induction of the noncanonical activation of the NLRP3/ASC inflammasome (25). Our data support the hypothesis that this pathway does not play a role in the restriction of L. pneumophila and L. gratiana infection in vivo (Figs. 4C, 6D, and 7B); this result is supported by previous observations that NLRP3 and ASC are dispensable for the restriction of L. pneumophila replication in vivo (18, 19, 35). Notably, caspase-11 facilitated the recruitment of neutrophils to the sites of infection via the induction of IL-1a (40). This feature should favor a role for caspase-11 in the restriction of L. pneumophila replication in vivo. However, caspase-11 independent production of IL-1a and caspase-11 independent recruitment of neutrophils have also been reported (46). In support of these findings, we did not detect differences in neutrophil recruitment to the lungs of infected mice in the absence of caspase-11 (data not shown). Therefore, the caspase- 11 independent mechanisms of neutrophil recruitment may explain our data regarding the unnecessary role of caspase-11 in the restriction of L. pneumophila replication in vivo. Caspase-11 plays a critical role in host responses to Gramnegative bacteria and in the induction of damaging host responses, including LPS-induced septic shock (23, 38, 39, 42, 43, 47). In this context, it will be important to determine the physiological consequences of caspase-11 activation and identify their activation partners in the context of Legionella infection. This information will provide us with a more complex view of the molecular composition of the inflammasomes that participate in the host responses to intracellular bacteria while contributing to our understanding of the molecular mechanisms underlying host resistance and the generation of the inflammatory process in response to intracellular pathogens. Acknowledgments We are grateful to Maira Nakamura for technical assistance and to Vishva Dixit (Genentech, South San Francisco, CA) and Richard Flavell (Yale University, New Haven, CT) for providing the genetically deficient mice used in this study. Disclosures The authors have no financial conflicts of interest. References 1. Newton, H. J., D. K. Ang, I. R. van Driel, and E. L. Hartland Molecular pathogenesis of infections caused by Legionella pneumophila. Clin. Microbiol. Rev. 23: Swanson, M. S., and B. K. Hammer Legionella pneumophila pathogesesis: a fateful journey from amoebae to macrophages. Annu. Rev. Microbiol. 54: Asrat, S., D. 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