Inflammasome Activation by Adenylate Cyclase Directs Th17 Responses and Protection against pertussis

This information is current as Aisling Dunne, Pádraig J. Ross, Eva Pospisilova, Jiri Masin, of October 3, 2021. Aoife Meaney, Caroline E. Sutton, Yoichiro Iwakura, Jurg Tschopp, Peter Sebo and Kingston H. G. Mills J Immunol 2010; 185:1711-1719; Prepublished online 7 July 2010; doi: 10.4049/jimmunol.1000105 http://www.jimmunol.org/content/185/3/1711 Downloaded from

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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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Inflammasome Activation by Adenylate Cyclase Toxin Directs Th17 Responses and Protection against

Aisling Dunne,*,1 Pa´draig J. Ross,†,1 Eva Pospisilova,‡ Jiri Masin,‡ Aoife Meaney,† Caroline E. Sutton,† Yoichiro Iwakura,x Jurg Tschopp,{ Peter Sebo,‡ and Kingston H. G. Mills*,†

Inflammasome-mediated IL-1b production is central to the innate immune defects that give rise to certain autoinflammatory diseases and may also be associated with the generation of IL-17–producing CD4+ T (Th17) cells that mediate autoimmunity. However, the role of the inflammasome in driving adaptive immunity to has not been addressed. In this article, we demonstrate that inflammasome-mediated IL-1b plays a critical role in promoting Ag-specific Th17 cells and in generating protective immunity against Bordetella pertussis infection. Using a murine respiratory challenge model, we demonstrated that 2/2

the course of B. pertussis infection was significantly exacerbated in IL-1R type I-defective (IL-1RI ) mice. We found that Downloaded from adenylate cyclase toxin (CyaA), a key secreted by B. pertussis, induced robust IL-1b production by dendritic cells through activation of caspase-1 and the NALP3-containing inflammasome complex. Using mutant , we demonstrate that CyaA-mediated activation of caspase-1 was not dependent on adenylate cyclase enzyme activity but was dependent on the pore-forming capacity of CyaA. In addition, CyaA promoted the induction of Ag-specific Th17 cells in wild-type but not IL-1RI2/2 mice. Furthermore, the bacterial load was enhanced in IL-17–defective mice. Our findings demonstrate that CyaA, a virulence

factor from B. pertussis, promotes innate IL-1b production via activation of the NALP3 inflammasome and, thereby, polarizes http://www.jimmunol.org/ responses toward the Th17 subtype. In addition to its known role in subverting host immunity, our findings suggest that CyaA can promote IL-1b–mediated Th17 cells, which promote clearance of the from the respiratory tract. The Journal of Immunology, 2010, 185: 1711–1719.

ordetella pertussis is the causative agent of whooping effective at preventing severe disease, and studies in a mouse cough, a severe and debilitating respiratory infection of model suggested that a combination of Abs and Th1 and Th17 B infants and young children, which can have a lethal out- cells are involved (3–7). Protection against a primary infection come. Although efficacious whole-cell and acellular pertussis vac- with B. pertussis is mediated by innate and adaptive immune by guest on October 3, 2021 cines have been developed and are widely used, responses, and IFN-g secreted by NK and Th1 cells seems to be still accounts for almost 300,000 deaths annually worldwide critical (4, 8, 9). However, host immune responses are suppressed, (1). Furthermore, there is an increasing incidence of B. pertussis especially early in infection, because of the induction of regu- in adolescents and adults, possibly due to waning im- latory T cells (10), as well as by the various immune-subversion munity following the introduction of acellular vaccines into most strategies evolved by B. pertussis virulence factors to prolong sur- developed countries (2). Adaptive immunity generated by previ- vival in the host (2, 11, 12). ous infection or with the whole-cell vaccine is One virulence factor of B. pertussis, adenylate cyclase toxin (CyaA), belongs to the repeat in toxin family of pore-forming leu- kotoxins (13). It is unique among the repeat in toxin family of toxins *Immunology Research Centre and †Immune Regulation Research Group, School of in that the characteristic cytolysin moiety is fused to an enzymat- Biochemistry and Immunology, Trinity College, Dublin, Ireland; ‡Laboratory of ically active adenylate cyclase (AC) enzyme domain. CyaA targets Molecular Biology of Bacterial Pathogens, Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech Republic; xCenter for Exper- cells primarily via the integrin receptor CD11b/CD18, permeabil- imental Medicine and Systems Biology, Institute of Medical Science, University of izes cell membranes by forming small cation-selective pores, and { Tokyo, Tokyo, Japan; and Department of Biochemistry, University of Lausanne, delivers the amino terminal AC domain into host cells (14, 15). This Epalinges, Switzerland 1 subverts the host by catalyzing the unregulated A.D. and P.J.R. contributed equally to this work. conversion of ATP to cAMP, causing deregulated , Received for publication January 15, 2010. Accepted for publication May 22, 2010. inhibition of bactericidal functions and potassium efflux (16), and This work was supported by grants from Science Foundation Ireland (to K.H.G.M.) modulation of (DC) maturation and cytokine secre- and by Czech Science Foundation Grant GA310/08/0447 and Grant IAA500200914 from the Grant Agency of the Academy of Sciences of the Czech Republic (to P.S.). tion, inducing IL-10, while simultaneously inhibiting LPS-induced Address correspondence and reprint requests to Prof. Kingston Mills, School of IL-12 and CCL3 production (17, 18). Biochemistry and Immunology, Trinity College, Dublin 2, Ireland. E-mail address: NOD-like receptors (NLRs) are a family of intracellular pattern [email protected] recognition receptors capable of recognizing endogenous danger Abbreviations used in this paper: AC, adenylate cyclase; Bp, Bordetella pertussis; signals, such as monosodium urate crystals and ATP, and exoge- CyaA, adenylate cyclase toxin; DC, dendritic cell; KLH, keyhole limpet hemocyanin; MOI, multiplicity of infection; NLR, NOD-like receptor; PBFI/AM, 1,3- nous substances deemed foreign, such as silica particles, alum, benzenedicarboxylic acid, 4,49-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16- and asbestos (19–21). A subset of NLRs, termed NALPs, is also diylbis(5-methoxy-6,2-benzofurandiyl)]bis-,tetrakis [(acetyloxy)methyl] ester; WT, activated in response to bacterial pore-forming toxins. NALP1 is wild-type. activated by lethal toxin from Bacillus anthracis (22), whereas Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 NALP3 (NLRP3/cryopyrin) is activated by maitotoxin (23). Upon www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000105 1712 IL-1b PROTECTS AGAINST B. pertussis INFECTION exposure to these toxins, a large multiprotein complex, termed the repeatedly washed with 60% isopropanol to reduce the endotoxin content inflammasome, is formed, leading to the processing and subsequent of CyaA samples to ,300 IU/mg , as determined by the Limulus activation of caspase-1. Active caspase-1, in turn, catalyzes the amebocyte lysate assay QCL-1000 (Cambrex, East Rutherford, NJ). cleavage of pro–IL-1b, resulting in the secretion of biologically Animals and immunization active forms of this cytokine (24). The precise mechanism of 2 2 C57BL/6 (Harlan, Bicester, U.K.) and IL-1RI / mice, from an established inflammasome activation has yet to be determined; however, it colony originally from The Jackson Laboratory (Bar Harbor, ME), were was suggested that membrane disruption, generation of reactive bred and housed under specific pathogen-free conditions. Procedures were oxygen species, lysosomal acidification, and ionic perturbations performed on groups of four or five mice at 8–12 wk of age according to characterized by potassium efflux may be involved. regulations and guidelines of the Trinity College Dublin Ethics Committee and the Irish Department of Health. NALP3-deficient mice were provided The aim of this study was to examine the possible role of CyaA by Professor Jurg Tschopp. IL-17–deficient (IL-172/2) mice were provided in inducing the formation of an active inflammasome complex, by Dr. Yoichiro Iwakura (30). Mice were immunized (n = 5) s.c. in the hind culminating in IL-1b production, and to assess the role of IL-1b foot pads with combinations of depyrogenated keyhole limpet hemocyanin in protective adaptive immunity to B. pertussis. We demonstrate (KLH) (5 mg/mouse; Calbiochem, San Diego, CA), CyaA (1 mg/mouse), that CyaA is capable of driving robust IL-1b production by DCs YVAD-fmk (25 mg/mouse; Bachem), CyaA-E570K+ E581P, or PBS only. through activation of caspase-1 and the NALP3 inflammasome Cell culture complex, and using a set of CyaA mutants with altered pore- Bone marrow-derived immature DCs were prepared by culturing bone mar- forming capacity, we define the basis for this activity. Further- row cells obtained from the femur and tibia of C57BL/6 mice in RPMI 1640 more, we show that IL-1b is required for the generation of and 10% FCS supplemented with 40 ng/ml GM-CSF from a GM-CSF– Ag-specific Th17 cells and for effective bacterial clearance, dem- expressing cell line, as described previously (5). Cells were washed and re- onstrating for the first time that activation of the inflammasome is cultured with fresh medium containing 40 ng/ml GM-CSF every 3 d for 8 d. Downloaded from For assessment of IL-1b production, DCs were cultured (primed) with LPS involved in protective adaptive immunity to infection. (100 ng/ml) for 3 h prior to treatment for 1 h with CyaA. Supernatants were removed, after the indicated times poststimulation, and the concentrations of Materials and Methods IL-1b,TNF-a, and IL-6 were quantified by two-site ELISA (BD Biosciences, Reagents San Jose, CA and R&D Systems, Minneapolis, MN). J774A.1 cells (ATCC TIB 67) were maintained in RPMI 1640 medium containing 10% FCS.

1,3-benzenedicarboxylic acid, 4,49-[1,4,10,13-tetraoxa-7,16-diazacyclooct- http://www.jimmunol.org/ adecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-,tetrakis [(acety- B. pertussis respiratory challenge and in vitro infection loxy) methyl] ester (PBFI/AM) was purchased from Molecular Probes B. pertussis (strain Tohama I; 338) from a 48-h culture were concentrated to (Eugene, OR). Pluronic F-127, ATP, bafilomycin A1, and quinidine were 2 3 1010/ml in PBS with 1% casein (4). Aerosol challenge was administered obtained from Sigma-Aldrich (St. Louis, MO). Ultrapure LPS was over 15 min using a nebulizer (0.5 ml/min). The course of B. pertussis obtained from InvivoGen. Caspase-1 FLICA kit was from ImmunoChem- infection was followed by performing CFU counts on from groups istry Technologies (Bloomington, MN), and YVAD-fmk was from Bachem of four mice at various times after aerosol challenge. Lungs were aseptically (Weil am Rhein, Germany). removed and homogenized in 1 ml sterile physiological saline with 1% Production and purification of CyaA and its derivatives casein on ice. One hundred microliters of undiluted homogenate or serially diluted homogenate from individual lungs was spotted in triplicate onto Construction of CyaA-108OVA-AC2, CyaA-E509K+E516K, CyaA- Bordet-Gengou agar plates, and the number of CFU was estimated after E570K+E581P, CyaA-AC2, and CyaA-DAC was described previously 4 d of incubation at 37˚C. Results are reported as the mean number of by guest on October 3, 2021 (25–28). The intact CyaA and CyaA-derived were produced in B. pertussis CFU for individual lungs from four mice per experimental XL-1 Blue in the presence of the activating acyltransfer- group per time point. For in vitro infections, DCs (1 3 106/ml) were infected ase CyaC and purified close to homogeneity, as previously described (29). with wild-type (WT) strain Tohama I (338), or the WT strain 18323 when During the hydrophobic chromatography, the resin with bound toxin was CyaA-deficient B. pertussis strain 18HS19 was used in experiments (31), at

FIGURE 1. IL-1 is required for ef- fective clearance of B. pertussis infec- tion. A, DCs were infected in vitro with live B. pertussis (Tohama I; 338) at an MOI of 0.1, 1, 10, or 100 for the in- dicated times. IL-1b concentrations were quantified by ELISA. B–D,WT and IL-1RI2/2 mice were infected by aerosol challenge with B. pertussis. B, CFU were determined on individual homogenates. Results are ex- pressed as mean 6 SD CFU for four mice per group at each time point and are representative of two experiments. Lung (C) and spleen (D) cells were iso- lated from WT and IL-1RI 2/2 mice (n = 4) on day 14 and restimulated with heat-killed Bp (105/ml) or medium only. After 3 d, supernatants were tested for IL-17, IFN-g, and IL-5 by ELISA. Results are expressed as mean 6 SD and are representative of two independent experiments. ppp , 0.01, pppp , 0.001; WT versus IL-1R12/2. Bp, B. pertussis. The Journal of Immunology 1713 a multiplicity of infection (MOI) of 0.1, 1, 10, or 100. After 2, 6, or 24 h, We first demonstrated that infection of cultured murine bone supernatants were collected and tested for IL-1b by ELISA (R&D Systems). marrow-derived DCs with live B. pertussis induced robust IL-1b Ag-specific T cell responses production in a time- and dose-dependent manner (Fig. 1A). To determine whether IL-1b plays a role in the clearance of B. Lung cells were purified as described previously (32) and restimulated 2/2 with killed B. pertussis (105/ml), prepared as previously described (5). At pertussis infection in vivo, we challenged WT and IL-1RI mice day 14 of infection or 7 d after immunization, spleen or popliteal lymph with an aerosol of B. pertussis. WT mice exhibited a classical B. nodes were isolated and restimulated with increasing concentrations of pertussis infection, peaking between days 7 and 14, with successful KLH, heat-killed B. pertussis, or heat-inactivated CyaA (5 mg/ml). After bacterial clearance by day 35 (Fig. 1B). However, IL-1RI2/2 mice 3 d, supernatants were collected and analyzed for IL-17, IFN-g, IL-5, and IL-10 production by ELISA (BD Biosciences and R&D Systems). After exhibited a pronounced susceptibility to B. pertussis infection and 5 d, T cell cultures were restimulated with PMA (10 ng/ml) and ionomycin had a substantially increased bacterial burden compared with WT (1 mg/ml; Sigma-Aldrich) in the presence of brefeldin A (5 mg/ml; Sigma- mice at all time points following challenge. In addition, at day 35 Aldrich) for 5 h and then were analyzed by flow cytometry. when WT mice had successfully cleared the bacteria, IL-1RI2/2 mice Immunoblot analysis remained infected with bacteria, which detectable in the lungs for Cell-culture supernatants (500 ml) were precipitated with 500 ml methanol $56 d postchallenge. We next examined the cellular immune and 250 ml chloroform prior to vortexing and centrifugation at 13,000 rpm responses in the lung and spleen of infected mice on day 14; for 5 min. The upper phase was discarded, and 500 ml methanol was added infection was associated with the induction of Ag-specific IL-17 pro- to the protein-containing interphase. This mixture was centrifuged for duction in WT mice, but this was significantly reduced in IL-1RI2/2 5 min at 13,000 rpm, and the protein pellet was dried at 55˚C. The pellet was resuspended in 60 ml Laemmli buffer prior to electrophoresis on a 15% SDS-PAGE gel. Samples were transferred to polyvinylidene difluor- Downloaded from ide and blocked in 1% casein (Novagen, Madison, WI) prior to detection with anti–IL-1b (MAB4011; R&D Systems). Flow cytometry Active caspase-1 was detected using the caspase-1 FLICA kit from Immu- noChemistry Technologies. Briefly, DCs (1 3 106/ml) were treated with CyaA in the presence and absence of the unlabeled caspase-1 inhibitor YVAD-fmk for 90 min prior to treatment with the fluorescein-labeled http://www.jimmunol.org/ inhibitor FAM-YVAD-fmk (5-carboxyfluorescein-Tyr-Val-Ala-Asp- fluoromethyl ketone) for 1 h at 37˚C. The cells were washed three times and analyzed directly by flow cytometry on a CyAN (Dako, Carpinteria, CA). T cells were stained with an Ab against CD3 (Alexa-780 conjugate) and CD4 (PE-Cy5.5 conjugate; both from eBioscience, San Diego, CA) for 20 min before being washed and then stained for intracellular IL-17 (PE conjugate; BioLegend, San Diego, CA) using the Fix & Perm Cell Per- meabilization Kit (ADG Bio Research, Germany), according to the man- ufacturer’s instructions; analysis was done on a CyAN flow cytometer.

Data were analyzed by FlowJo software (Tree Star, Ashland, OR). by guest on October 3, 2021 Fluorescence measurement of cytosolic K+ J774A.1 cells (5 3 105) grown on glass coverslips were washed in mod- ified HBSS. Cells were loaded with 9.5 mM PBFI/AM (Molecular Probes) for 30 min at 25˚C in the presence of 0.05% (w/w) Pluronic F-127 (Sigma- Aldrich) in the dark. Ratiometric measurement was performed at 25˚C using the spectrofluorometer FluoroMax-3 (Jobin Yvon Horiba, Longju- meau, France) with DataMax software. Fluorescence intensity of PBFI (excitation wavelength 340, emission wavelengths 450 and 510 nm) was recorded every 15 s, and integration time for each wavelength was 3 s. Calibration experiments were performed in solution containing 50 mM HEPES (pH 7.2), with varying concentrations of potassium acetate (10, 30, 60, or 140 mM) and sodium acetate (5, 85, 115, or 135 mM). Cellular plasma membranes were permeabilized for potassium ions and protons by valinomycin and nigericin (3 mM; Sigma-Aldrich) for 30 min. Statistical analysis Cytokine levels were compared by one-way ANOVA. If significant differ- ences were found, the Tukey–Kramer multiple comparisons test was used to identify differences between individual groups. Results IL-1 is required for effective clearance of B. pertussis infection FIGURE 2. CyaA induces the processing and secretion of IL-1b in bone B. pertussis bacteria colonize the upper respiratory tract and se- marrow-derived DCs. A, Murine DCs were infected with WT (18323) and crete a number of virulence factors, including CyaA, which sub- CyaA-deficient B. pertussis (18HS19; CyaA KO) at an MOI of 1, 10, or vert host immune responses by inhibiting , promoting 100. After 24 h, the concentration of IL-1b was quantified in supernatants p , pp , ppp , IL-10, and generating T regulatory cells, all of which contribute to by ELISA. p 0.05, p 0.01, p 0.001; WT versus CyaA KO. B, DCs were primed with LPS (100 ng/ml) or medium only for 3 h prior to bacterial persistence and pathogenesis (2). Clearance of B. pertus- treatment with increasing doses of CyaA for 1 h. Supernatants were sis from the respiratory tract is mediated by IFN-g secreted by assessed for IL-1b production by ELISA. C, Proteins were extracted Th1 and NK cells, as well as Ag-specific Ab production (4, 8). from LPS-primed and unprimed DCs following treatment with ATP Furthermore, we demonstrated that adaptive immunity induced by (5 mM) or CyaA (1–100 ng/ml) and immunoblotted for IL-1b. Results was dependent on Ag-specific IL-17 production (5). In in A and B are mean 6 SE for triplicate cultures and are representative of this study, we examined the role of IL-1. three independent experiments. 1714 IL-1b PROTECTS AGAINST B. pertussis INFECTION mice (Fig. 1C, 1D). B. pertussis-specific IFN-g production was also virulence factors involved. The processing and secretion of IL-1b reduced in spleen and lungs of IL-1RI2/2 mice, but this was not as by and DCs requires two signals, one resulting in dramatic as the reduction in IL-17 production. The Th2 cytokine the transcriptional induction of pro–IL-1b in response, for IL-5 was only detected at low levels in Ag-stimulated spleen cells example, to a TLR agonist and a second signal culminating in from B. pertussis-infected mice, and there was no significant differ- cleavage and secretion of the mature cytokine through activation ence between WT and IL-1RI2/2 mice. These findings demonstrate of the inflammasome complex. Because CyaA has pore-forming a critical role for IL-1 in the generation of Ag-specific IL-17 and, to activity, we examined the possible role of this toxin in IL-1b a lesser extent, IFN-g production, as well as for successful clearance production by inducing the formation of an active inflammasome of a primary infection with B. pertussis. complex. Murine bone marrow-derived DCs were treated with WT or CyaA-deficient B. pertussis for 24 h at an MOI of 1, 10, b CyaA induces IL-1 release by DCs and 100, and IL-1b production was quantified by ELISA. The Having demonstrated that IL-1 plays a critical role in protective concentration of IL-1b induced by CyaA-deficient B. pertussis immunity to B. pertussis, we sought to identify the potential (strain:18HS19) was significantly lower than that induced by the corresponding WT B. pertussis (strain 18323) (Fig. 2A). This strain was less effective at inducing IL-1b than Tohama I, the more common strain used for the other aspects of this and other studies. Nevertheless, the data suggest that CyaA induces IL-1b production. To confirm and extend this finding, we examined the ability of purified CyaA to induce IL-1b production via in- flammasome activation. The secretion of mature active IL-1b is Downloaded from dependent first on the induction of pro–IL-1b through TLR- induced NF-kB activation and then on the activation of caspase- 1, which is required for cleavage of pro–IL-1b into mature IL-1b. Therefore, in these assays, DCs were first primed with a low concentration (100 ng/ml) of LPS for 3 h prior to treatment with increasing doses of CyaA. We found that CyaA or LPS alone did http://www.jimmunol.org/ not induce IL-1b secretion. However, CyaA induced robust IL-1b production by LPS-primed DCs in a dose-dependent manner (Fig. 2B). Furthermore, Western blotting showed that CyaA pro- moted efficient processing of pro–IL-1b into its mature and active form to the same extent as ATP, a known inducer of IL-1b processing (Fig. 2C). CyaA induces IL-1b production in a caspase-1–dependent

manner by guest on October 3, 2021 We next sought to determine whether CyaA drives the production of IL-1b through caspase-1, which is activated following assembly

FIGURE 3. CyaA activates caspase-1. A, DCs were treated with CyaA (100 ng/ml) in the presence or absence of the caspase-1 inhibitor YVAD- fmk (10 mM) or with medium only. Direct activation of caspase-1 in DCs by CyaA was assessed by flow cytometry using a fluorescein-labeled caspase-1 inhibitor. B, DCs were treated with medium only or LPS (100 ng/ml) for 3 h before the addition of ATP (5 mM) or CyaA (100 ng/ml) in the presence or absence of the caspase-1 inhibitor YVAD-fmk (10 mM) for 1 h; IL-1b and TNF-a concentrations were measured by ELISA. ppp , 0.01; pppp , 0.001; with versus without YVAD-fmk. C, FIGURE 4. CyaA induces IL-1b production in a NALP3-dependent DCs from WT and caspase-12/2 mice were primed with LPS (100 ng/ml) manner. DCs from WT or NALP32/2 mice were primed with LPS (100 or medium only for 3 h prior to treatment with ATP (5 mM) or CyaA (100 ng/ml) or medium only for 3 h prior to treatment with ATP (5 mM) or ng/ml) for 1 h. IL-1b was quantified in cell supernatants by ELISA. CyaA (100 ng/ml) for 1 h. A, IL-1b was quantified in cell supernatants by Results in B and C are mean 6 SE for triplicate cultures and are ELISA. B, Immunoblot analysis for the full-length and processed forms of representative of three independent experiments. IL-1b. Results are representative of two independent experiments. The Journal of Immunology 1715

of the inflammasome complex. The activation of caspase-1 by CyaA was assessed by flow cytometry analysis using a cell- permeable fluorescent probe that forms a covalent link with acti- vated caspase-1. The results revealed that CyaA alone induced robust caspase-1 activation and that this was reduced in the pres- ence of an unlabeled caspase-1 inhibitor (Fig. 3A). Pretreatment of DCs with a caspase-1 inhibitor, YVAD-fmk, significantly reduced CyaA-induced IL-1b production, but it had no effect on LPS- induced TNF-a (Fig. 3B), demonstrating a lack of off-target effects. Moreover, IL-1b production by CyaA was abrogated in DCs from caspase-12/2 mice (Fig. 3C), confirming a role for caspase-1 in CyaA-induced IL-1b. CyaA activates the NALP3 inflammasome via pore formation and K+ efflux Because CyaA induces pore formation and K+ release from cells (33), events that have been linked with assembly of a NALP3 containing inflammasome complex (23), we examined the hy- pothesis that CyaA may act through NALP3 to generate IL-1b. CyaA, like ATP, induced IL-1b secretion from LPS-primed DCs Downloaded from from WT, but not NALP32/2, mice (Fig. 4A). In contrast, the in- duction of pro–IL-1b was similar in DCs from WT and NALP32/2 mice (Fig. 4B), confirming that the defect in IL-1b production was due to a lack of processing and caspase-1 activation. These findings demonstrate that NALP3 plays an essential role in CyaA- induced maturation of IL-1b. http://www.jimmunol.org/ We next examined the possibility that CyaA activates the NALP3 inflammasome through modulation of ionic homeostasis. IL-1b release was completely abrogated when DCs were cultured in medium containing a high concentration of potassium to prevent efflux (Fig. 5A). Similarly, pretreatment of LPS-primed DCs with quinidine, a potassium channel blocker, significantly reduced CyaA-induced IL-1b production (Fig. 5B), but it had no effect on LPS-induced IL-6 production (data not shown). CyaA can by guest on October 3, 2021 translocate directly across the cytoplasmic membrane without the need for receptor-mediated (34). Consistent with this, we found that bafilomycin A1, an inhibitor of the vacuolar H+ ATPase, which was shown to play a role in particle-induced inflammasome activation, had no effect on CyaA-induced IL-1b release (Fig. 5B). These findings suggest that CyaA may activate the inflammasome through promotion of K+ efflux. We next examined the role of CyaA-induced pore formation in NALP3 activation using a number of CyaA mutants with reduced or enhanced pore-forming activity (25, 28). The extent of pore formation exhibited by these mutants is reflected in their ability to induce K+ efflux and was demonstrated in macrophages loaded with a K+-sensitive probe. WT CyaA caused a rapid decrease in

prior to treatment with CyaA (100 ng/ml). IL-1b was quantified in cell supernatants by ELISA. ppp , 0.01; with versus without quinidine. C, J774A.1 cells were loaded with potassium-sensitive probe PBFI/AM (9.5 mM final external concentration) at 25˚C for 45 min in the presence of Pluronic F-127 (12.5% [w/w]) and washed in HBSS before different CyaA mutants (3 mg/ml) or buffer control was added (arrow). Fluores- cence intensity ratio of PBFI (excitation wavelength 340, emission wave- lengths 450 and 510 nm) was recorded every 15 s. The scale on the right shows intracellular K+ values derived from calibration experiments. D, FIGURE 5. Inflammasome activation by CyaA is dependent on pore IL-1b production was assessed in DCs following treatment with WT or formation and potassium efflux. A, DCs from WT mice were primed with mutant versions of CyaA (100 ng/ml). pp , 0.05; ppp ,0.01 versus CyaA. LPS (100 ng/ml) for 2 h before being incubated in medium containing 50 (CyaA = WT; E509K+E516K = enhanced pore-forming activity; E570K+ mM KCl for 1 h prior to treatment with CyaA (100 ng/ml). IL-1b was E581K = reduced pore-forming activity; 108OVA-AC2 = no AC activity quantified in cell supernatants by ELISA. pppp , 0.001; with versus [with OVA insertion]; CyaA-DAC = no adenylate cyclase domain; CyaA- without KCl. B, DCs were primed with LPS (100 ng/ml) for 2 h before AC2 = no AC activity). Results are mean 6 SE for triplicate cultures and being treated with quinidine (250 mM) or bafilomycin A1 (250 nM) for 1 h are representative of three independent experiments. 1716 IL-1b PROTECTS AGAINST B. pertussis INFECTION intracellular K+ (Fig. 5C). Treatment of cells with a CyaA mutant specific responses over that seen in mice injected with PBS alone (E509K+E516K) with enhanced pore-forming activity, but defec- (Fig. 6A). In contrast, cells from mice immunized tive in AC domain translocation and cAMP accumulation, induced with KLH and CyaA produced IL-17 in response to Ag restim- greater reduction in K+ levels (Fig. 5C), and IL-1b release was ulation in vitro; however, this Ag-specific IL-17 production was similar to WT toxin (Fig. 5D), confirming that pore formation almost completely ablated in IL-1RI2/2 mice (Fig. 6A). CyaA did alone is sufficient to activate NALP3. In contrast, a mutant of not promote Ag-specific IFN-g production in WT or IL-1RI2/2 CyaA with defective pore-forming activity (E570K+E581P) (13) mice. In contrast, Ag-specific IL-10 was induced at significant did not affect K+ concentrations (Fig. 5C) and produced signifi- levels in IL-1RI2/2 and WT mice. Further evidence that CyaA cantly lower amounts of IL-1b from LPS-primed DCs (Fig. 5D). promotes the induction of Th17 cells was provided by intracellular The K+ efflux induced by CyaA is accompanied by an influx of cytokine staining on lymph node cells from immunized mice. A . An enzymatically inactive mutant of CyaA, which has substantially greater frequency of IL-17–secreting CD4+ T cells normal pore-forming activity and K+ efflux, but is defective in was detected in draining lymph nodes from mice immunized with mediating calcium influx into cells (108OVA-AC2) (35), induced KLH and CyaA compared with those immunized with KLH alone. IL-1b production similar to the WT toxin (Fig. 5D), suggesting that Furthermore, the frequency of IL-17–secreting CD4+CD3+ T cells calcium influx was not involved. Removal of the entire AC domain was closer to background in similarly immunized IL-1RI2/2 mice in CyaA-DAC mutant eliminated K+ efflux (Fig. 5C) and abrogated (Fig. 6B). the ability to induce IL-1b (Fig. 5D). Finally, mutation of the ATP- We next examined the role of caspase-1 activation by CyaA in binding site within the AC domain (CyaA-AC2), which abolishes mediating its ability to promote the induction of Th17 cells. Immu- cAMP production but retains pore formation, was capable of nization of mice with KLH and CyaA in the presence of the reducing intracellular K+ concentrations (Fig. 5C) and promoting caspase-1 inhibitor YVAD-fmk significantly reduced the induction Downloaded from IL-1b production (Fig 5D). Our findings demonstrate that of KLH-specific IL-17 (Fig. 7A). Furthermore, immunization with inflammasome activation by CyaA is dependent on pore KLH in the presence of the CyaA-E570K+E581P mutant, which is formation and K+ efflux but is independent of AC activity and unable to form pores, failed to induce Ag-specific IL-17 produc- cAMP accumulation mediated by CyaA. tion (Fig. 7A). These findings demonstrate that the promotion of Th17 cell induction by CyaA is dependent on pore formation and

b http://www.jimmunol.org/ CyaA drives IL-1 –dependant Ag-specific IL-17 production caspase-1 activation. in vivo Finally, we examined the hypothesis that CyaA may also pro- Having shown that IL-1 plays a key role in induction of Th17 mote B. pertussis-specific Th17 responses. Immunization of mice responses during B. pertussis infection and that CyaA promotes with CyaA generated T cells in lymph nodes that secreted IL-17 IL-1b production via inflammasome activation, we next sought to in response to in vitro restimulation with CyaA or killed B. per- determine whether CyaA can promote the induction of Th17 cells tussis. In contrast, B. pertussis-specific IL-17 was not detected in in vivo. Mice were immunized with a bystander Ag, KLH, using IL1RI2/2 mice (Fig. 7B). To show a direct link between IL-17 and CyaA as an adjuvant, and T cell responses were assessed. protection from B. pertussis, we infected IL-172/2 mice with B. Immunization of mice with KLH alone failed to induce Ag- pertussis and examined CFU in the lungs. The bacterial burden by guest on October 3, 2021

FIGURE 6. CyaA drives IL-1b-dependent induction of Th17 cells. WT and IL-1RI2/2 mice (n = 5) were immunized with KLH (5 mg), KLH + CyaA (1 mg), or PBS. A, After 7 d, popliteal lymph node cells were restimulated with KLH (2–50 mg/ml) for 3 d. IL-17, IFN-g, and IL-10 production was measured in supernatants by ELISA. Results are mean 6 SE for triplicate cultures and are representative of two independent experiments. B, Lymph node cells were cultured with KLH (10 mg/ml) for 5 d followed by restimulation with PMA (10 ng/ml) and ionomycin (1 mg/ml) in the presence of brefeldin A (5 mg/ml) for 5 h before staining for surface CD3 and CD4 and intracellular IL-17. Cells were first gated on the lymphocyte population on the basis of forward scatter and side scatter and then on CD3. The dot plots represent IL-17 versus CD4 expression on the CD3+ population. Results are representative of three independent experiments. The Journal of Immunology 1717

role in immunity to B. pertussis (4, 36). Although CyaA is a major virulence factor of B. pertussis and uses a variety of strategies to subvert host immune responses, our findings suggest that it may also be exploited by the host to activate protective innate and adaptive inflammatory responses against the pathogen. Dysregulated IL-1b production through inflammasome hyper- activation is central to the pathogenesis of certain autoinflammatory diseases, such as Muckle-Wells syndrome (37). IL-1 also plays a cen- tral role in the pathogenesis of autoimmune disease, where it is implicated in promoting the induction or expansion of pathogenic Th17 cells (38). The present study demonstrates that IL-1b,which is produced following activation of caspase-1 and the NALP3 inflam- masome by the B. pertussis virulence factor CyaA, plays a key role in the clearance of B. pertussis infection from the respiratory tract. Although it was demonstrated that NALP3 plays a crucial role in the host defense against the fungal pathogen Candida albicans (39), our study shows that inflammasome-mediated IL-1b may help to shape adaptive immune responses to infection. These findings pro- vide evidence that innate inflammatory responses activated through the inflammasome, considered to be pathogenic in many disease set- Downloaded from tings, may also promote protective adaptive immunity to pathogens. Therefore, drugs that target IL-1b or the inflammasome pathway, as well as controlling damaging pathology in inflammatory diseases, have the potential to suppress host protective immune responses and, thus, increase susceptibility to infection. Our findings also demonstrate that IL-1b induced by CyaA http://www.jimmunol.org/ promotes the induction of Th17 cells, a population of IL-17– secreting CD4+ effector T cells distinct from the classically de- fined Th1/Th2 lineage that are considered to play a key pathogenic role in inflammatory diseases. Studies in mice with targeted muta- tions in NALP3, leading to inflammasome hyperactivation, similar FIGURE 7. CyaA promotes the induction of Th17 cells, which play to that seen in Muckle-Wells syndrome, result in excessive IL-1b a role in protective immunity to B. pertussis. A, WT mice (n = 5) were production and enhanced Th17 cells (37). Although much of the immunized with CyaA, PBS + CyaA + YVAD-fmk (25 mg/ml), KLH + focus has been on their role in promoting chronic inflammation by guest on October 3, 2021 CyaA-E570K+E581P, or PBS. After 7 d, popliteal lymph node cells were and organ-specific autoimmunity, data are emerging to suggest that restimulated with KLH (2–50 mg/ml) for 3 d. IL-17 in supernatants was Th17 cells function with Th1 cells to mediate protective immunity quantified by ELISA. pppp , 0.001 versus KLH + CyaA. B, WT and 2 2 against a range of pathogens. For example, a dual role for IL-17 and IL-1RI / mice (n = 5) were immunized with PBS or CyaA. Lymph node cells were restimulated with heat-killed Bp (105/ml), heat- IFN-g was proposed for protective immunity to Mycobacterium inactivated CyaA (5 mg/ml), or medium only for 3 d. IL-17 in the super- tuberculosis following vaccination with pathogen-derived Ags natant was measured by ELISA. Results in A and B are mean 6 SE for (40). IL-17 was also shown to enhance clearance of Klebsiella pneu- triplicate cultures and are representative of two independent experiments. moniae via the induction of TNF-a,IL-1b, and MIP2 production in C, WT and IL-172/2 mice were infected with B. pertussis. After 2 h, 7 d, infected lung tissue (41). We highlighted a role for IL-17 in vaccine- and 14 d, CFU were determined on individual lung homogenates. Results induced immunity to B. pertussis (5). It was also demonstrated that are mean 6 SD CFU for four mice per group at each time point and are pertussis toxin promotes IL-17 production by T cells, macrophages, representative of two independent experiments. pp ,.0.05, ppp , 0.01; 2/2 and from B. pertussis-infected mice (7). Furthermore, IL-17 versus WT. Bp, B. pertussis. neutrophils, which are induced by IL-17, were shown to play a key role in protection against B. pertussis (6, 7, 11). In the current study, was identical after 2 h, but the CFU were significantly greater in we found that B. pertussis-specific IFN-g was also reduced, although 2/2 the lungs of IL-17 mice compared with WT mice 7 and 14 d not as dramatically as IL-17 in B. pertussis-infected IL-1RI2/2 mice. after challenge (Fig.7C). These findings demonstrate that CyaA Consistent with our demonstration in autoimmunity (38), this promotes the induction of B. pertussis-specific IL-17 production suggests that IL-1 is critical for the induction of Th17 responses and that IL-17 is required for optimum host protection against following infection, but it may also play a less significant role in B. pertussis. the induction of Th1 cells. Interestingly, the bacterial burden was exacerbated in IL-172/2 mice, although not as significantly as in IL- Discussion 1RI2/2 mice, suggesting that some, but not all, of the effects of IL-1b Our findings demonstrate that IL-1b is required for effective involve induction of IL-17 production. These findings are consistent clearance of B. pertussis infection and for the generation of Ag- with the well-established role of IFN-g (4, 36) and other effector specific Th17 cells, demonstrating, for the first time, a link be- cells and molecules, such as those induced through TLR4-activation tween inflammasome activation and IL-17 production following (5, 42, 43), in protection against B. pertussis and suggest that Th1 exposure to a pathogen. We demonstrate that CyaA promotes the and Th17 cells may cooperate to mediate bacterial clearance. induction of Th17 cells, an effect that is dependent on CyaA pore Consistent with previous reports on other toxins (22, 23), we formation, inflammasome activation, and IL-1b production. We found that the pore-forming activity of CyaA was essential also demonstrate that IL-1b plays a role in the induction of Ag- for promoting IL-1b via activation of the inflammasome in specific IFN-g, which was previously shown to play a protective a process that involves a reduction in intracellular potassium 1718 IL-1b PROTECTS AGAINST B. pertussis INFECTION levels. Interestingly, the AC activity of CyaA, which catalyses the III secretion system that subverts protective innate and adaptive immune responses. Infect. Immun. 76: 1257–1266. generation of superphysiological concentrations of intra- 13. Vojtova-Vodolanova, J., M. Basler, R. Osicka, O. Knapp, E. Maier, J. 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