cAMP Signaling of Adenylate Cyclase Blocks the Oxidative Burst of through Epac-Mediated Inhibition of Phospholipase C Activity This information is current as of September 27, 2021. Ondrej Cerny, Karen E. Anderson, Len R. Stephens, Phillip T. Hawkins and Peter Sebo J Immunol published online 30 December 2016 http://www.jimmunol.org/content/early/2016/12/30/jimmun ol.1601309 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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 30, 2016, doi:10.4049/jimmunol.1601309 The Journal of Immunology

cAMP Signaling of Adenylate Cyclase Toxin Blocks the Oxidative Burst of Neutrophils through Epac-Mediated Inhibition of Phospholipase C Activity

Ondrej Cerny,* Karen E. Anderson,† Len R. Stephens,† Phillip T. Hawkins,† and Peter Sebo*

The adenylate cyclase toxin- (CyaA) plays a key role in immune evasion and virulence of the agent pertussis. CyaA penetrates the complement receptor 3–expressing and ablates their bactericidal capacities by catalyzing unregulated conversion of cytosolic ATP to the key second messenger molecule cAMP. We show that signaling of CyaA-generated cAMP blocks the oxidative burst capacity of neutrophils by two converging mechanisms. One involves cAMP/

kinase A–mediated activation of the Src homology region 2 domain–containing phosphatase-1 (SHP-1) and limits the Downloaded from activation of MAPK ERK and p38 that are required for assembly of the NADPH oxidase complex. In parallel, activation of the exchange protein directly activated by cAMP (Epac) provokes inhibition of the phospholipase C by an as yet unknown mecha- nism. Indeed, selective activation of Epac by the cell-permeable analog 8-(4-chlorophenylthio)-29-O-methyladenosine-39,59-cyclic monophosphate counteracted the direct activation of phospholipase C by 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzene- sulfonamide. Hence, by inhibiting production of the protein kinase C–activating lipid, diacylglycerol, cAMP/Epac signaling blocks

the bottleneck step of the converging pathways of oxidative burst triggering. Manipulation of membrane composition http://www.jimmunol.org/ by CyaA-produced signaling of cAMP thus enables B. pertussis to evade the key innate host defense mechanism of –mediated killing of by neutrophils. The Journal of Immunology, 2017, 198: 000–000.

hooping cough, or pertussis, is a major respiratory parapertussis bacteria. Both species produce a Repeat in ToXin infectious disease that is on the rise in the most de- (RTX) adenylate cyclase (AC) toxin-hemolysin (CyaA, ACT, or W veloped countries, such as Australia, the United States, AC-Hly) that plays a key role in suppression of innate immune the Netherlands, or the U.K., which have switched to the use of defense of the host in the early stages of bacterial colonization of acellular pertussis vaccines (1). This highly contagious illness is the airway (2–4). CyaA is a 1706-residue-long bifunctional pro- caused mostly by the Gram-negative coccobacillus Bordetella tein and consists of an N-terminal cell-invasive AC enzyme (∼384 by guest on September 27, 2021 pertussis and less frequently by the human-adapted Bordetella residues) that is fused to an ∼1300-residue-long pore-forming RTX cytolysin (Hly) moiety (5–7). The Hly moiety binds host myeloid cells through the complement receptor (CR) 3 (the aMb2 *Laboratory of Molecular Biology of Bacterial Pathogens, Institute of Microbiology integrin CD11b/CD18, or Mac-1), hence targeting the key sentinel † of the CAS, v.v.i., 142 20 Prague, Czech Republic; and Signalling Programme, cells of the innate , such as neutrophils, macro- Babraham Institute, Babraham, Cambridge CB22 3AT, United Kingdom phages, or dendritic cells (8–10). In parallel to permeabilizing ORCID: 0000-0002-9755-7715 (P.S.). phagocytes by forming cation-selective (cytolytic) pores in their Received for publication July 28, 2016. Accepted for publication November 16, 2016. plasma membrane, the toxin translocates across the plasma membrane its AC domain that is activated by cytosolic calmod- This work was supported by Czech Science Foundation Grant GA13-14547S (to P.S.), Ministry of Education, Youth, and Sports of the Czech Republic Project Grant ulin, and catalyzes massive and unregulated conversion of cellular LM2015064 (Czech National Node to EATRIS), institutional support through Grants ATP into the key second messenger molecule cAMP (6, 7, 11, 12). RVO 61388971 and tSVV260317, and Biotechnology and Biological Sciences Research Council UK Grant BB/J004456/1 (to L.R.S. and P.T.H.). Accumulation of cAMP then subverts cellular signaling mecha- nisms and provokes a near-instantaneous paralysis of bactericidal Address correspondence and reprint requests to Dr. Peter Sebo, Institute of Micro- biology of the CAS, v.v.i., Vı´denska ´ 1083, 142 20 Prague 4, Czech Republic. E-mail functions of phagocytes, such as oxidative burst and opsonopha- address: [email protected] gocytic uptake and killing of bacteria (8, 13–18). The CyaA- The online version of this article contains supplemental material. provoked inhibition of reactive oxygen species (ROS) production Abbreviations used in this article: AC, adenylate cyclase; BGA, Bordet-Gengou prevents of neutrophils and limits their capacity to form agar; 6-Bnz-cAMP, N6-benzoyladenosine-39,59-cyclic monophosphate; cAMPS, extracellular traps (19). CyaA action hence ablates virtually all 8-bromoadenosine-39,59-cyclic monophosphorothioate; 8-CPT-cAMP, 8-(4-chloro- phenylthio)-29-O-methyladenosine-39,59-cyclic monophosphate; CR, complement bactericidal activities of neutrophils and provokes their impotence receptor; CyaA, adenylate cyclase toxin-hemolysin; CyaA-KK-AC2, CyaA-E509K+ 2 in controlling the early phases of B. pertussis (20). At E516K-AC ; DAG, diacylglycerol; db-cAMP, N-6,29-O-dibutyryladenosine-39,59-cyclic later stages of infection, the CyaA action is expected to dampen monophosphate; Epac, exchange protein directly activated by cAMP; IP3, inositol 1,4,5- trisphosphate; m-3M3FBS, 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfona- TLR-triggered activation of and to skew the matu- mide; MOI, multiplicity of infection; o-3M3FBS, 2,4,6-trimethyl-N-[2-(trifluoromethyl) ration of CR3-expressing dendritic cells, thus impairing their ca- phenyl]benzenesulfonamide; PFA, paraformaldehyde; PIP2, phosphatidylinositol 4,5- pacity to present Ags to T cells and induce appropriate adaptive bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PKA, ; PKC, protein kinase C; PLC, phospholipase C; ROS, reactive oxygen species; RTX, immune responses (21). Repeat in ToXin; SHP-1, Src homology domain 2–containing protein tyrosine phospha- Neutrophils undergo oxidative burst and produce ROS in re- tase 1; SS, Stainer-Scholte; Syk, spleen tyrosine kinase; WT, wild-type. sponse to multiple stimuli, such as the tripeptide fMLF, derived Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 from bacterial lipoproteins (22), the complement component 5a

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601309 2 cAMP THROUGH Epac INHIBITS PLC AND PMN OXIDATIVE BURST

(23), or after engagement of the opsonin receptors by Ab- or were incubated at 37˚C under continuous gentle shaking. At indicated time complement-opsonized bacteria, viruses, fungi, or parasites (24). postinfection, aliquots were removed and centrifuged at 15,800 3 g for 10 All of these stimuli trigger signaling pathways that converge to- min to pellet neutrophils and bacteria. The pellet was resuspended in dH2O for 5 min to lyse neutrophils, diluted in SS medium, and plated on BGA for ward the activation of PI3K and phospholipase C (PLC) (25, 26). CFU counting after 5 d of growth at 37˚C in a humidified atmosphere The PI3K phosphorylates phosphatidylinositol 4,5-bisphosphate containing 5% CO2. Results are shown as the ratio of CFU per neutrophil, (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3) that ac- using the initial count of neutrophils at the beginning of the experiment. cumulates in the inner leaflet of the plasma membrane and recruits CyaA activity determinations were performed in phosphate-free HBSS buffer containing 2 mM Ca2+. the cytosolic small GTPase Rac. This serves as one of the subunits of the forming ROS-producing NADPH oxidase complex in the Production and purification of CyaA variants phagocytic membrane (27). In parallel, the activated PLC cleaves Intact CyaA toxin, an enzymatically inactive CyaA-AC2 toxoid (34), and 2 PIP2 into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol toxoid with increased pore-forming capacity CyaA-E509K+E516K-AC (DAG), which leads to activation of the subfamily of typical (CyaA-KK-AC2) were produced in XL1-Blue (Stratagene) isoforms of protein kinase C (PKC). These bring about the transformed with the pCACT3- or pT7CACT1-derived constructs, as ap- phosphorylation of the other subunits of NADPH oxidase com- propriate (34). Exponential-phase 500-ml cultures were grown at 37˚C and PHOX PHOX PHOX induced by isopropyl-1-thio-b-d-galactopyranoside (1 mM) for 4 h before plex, such as p40 , p47 , or p67 (28), which is crucial the cells were washed in 50 mM Tris-HCl (pH 8), 150 mM NaCl, for the capacity of the NADPH oxidase complex to produce ROS. resuspended in 50 mM Tris-HCl (pH 8), 0.2 mM CaCl2, and disrupted by The catalytic activity of the complex can then also be potentiated sonication. The insoluble cell debris was resuspended in 8 M urea, 50 mM by phosphorylation of NADPH oxidase subunits by Akt (protein Tris-HCl (pH 8), 50 mM NaCl, and 0.2 mM CaCl2 to extract the CyaA . Upon centrifugation at 25,000 3 g for 20 min, clarified urea ex- Downloaded from kinase B) and MAPKs, ERK, and p38 (29). tracts were loaded onto a DEAE-Sepharose column equilibrated with 8 M The exact mechanism by which signaling of CyaA-produced urea, 50 mM Tris-HCl (pH 8), 120 mM NaCl. After washing, the CyaA was cAMP blocks oxidative burst and ROS production by neutro- eluted with 8 M urea, 50 mM Tris-HCl (pH 8), 2 M NaCl, diluted four times phils is, however, poorly understood. We show that it primarily with 50 mM Tris-HCl (pH 8), 1 mM CaCl2, 1 M NaCl buffer, and further involves cAMP-exchange protein directly activated by cAMP purified on a phenyl-Sepharose column equilibrated with the same buffer. Unbound proteins were washed out with 50 mM Tris-HCl (pH 8), and the (Epac)-mediated inhibition of DAG production by PLC. CyaA was eluted with 8 M urea, 50 mM Tris-HCl (pH 8), and 2 mM EDTA,

and stored at 220˚C. The protein concentration was determined by the http://www.jimmunol.org/ Materials and Methods Bradford assay (Bio-Rad, Hercules, CA) using BSA as a standard. Abs and reagents Determination of ROS production Zymosan, poly-RGD, anti-BSA Ab, N-6,29-O-dibutyryladenosine-39,59- ROS production was measured using a luminol-based assay as described cyclic monophosphate (db-cAMP), HRP, fMLF, and luminol were previously (33). In brief, 5 3 105 primary human neutrophils in HBSS obtained from Sigma-Aldrich; N6-benzoyladenosine-39,59-cyclic mono- supplemented with 1% glucose, 2 mM MgCl2,and2mMCaCl2 were in- phosphate (6-Bnz-cAMP), 8-(4-chlorophenylthio)-29-O-methyladenosine- cubated as indicated in the figure legends at 37˚C with 150 mM luminol and 39,59-cyclic monophosphate (8-CPT-cAMP), and 8-bromoadenosine-39,59- 18.75 U of HRP, before the cells were transferred to wells containing the cyclic monophosphorothioate (cAMPS) were from BIOLOG Life Science given activator such as fMLF, human complement-opsonized zymosan,

Institute; and NSC87877 and H-89 were purchased from Calbiochem. human complement-opsonized E. coli,orB. pertussis cultivated as de- by guest on September 27, 2021 2,4,6-Trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide (m-3M3FBS) scribed earlier, or upon adhesion to MaxiSorp ELISA plates (Nunc) that and 2,4,6-trimethyl-N-[2-(trifluoromethyl)phenyl]benzenesulfonamide were coated either with BSA and then opsonized with rabbit polyclonal (o-3M3FBS) were from Tocris. Abs were purchased from anti-BSA serum or were coated with poly-RGD. Luminescence was Technology (phospho-p44/42 MAPK [20G11] and phospho-p38 [D3F9]) recorded using a Safire2 microplate reader (Tecan). Corresponding amounts and Abcam (anti–b-actin). of appropriate solvents (e.g., HBSS for zymosan and fMLF; H2O for db- cAMP, 6-BNZ-cAMP, 8-CPT-cAMP, cAMPS, and NSC87877; and DMSO Bacterial strains and growth conditions for H-89, m-3M3FBS, o-3M3FBS, and PMA) were added as solvent control B. pertussis strains were grown on Bordet-Gengou agar (BGA) plates into all samples to which the indicated activators or inhibitors were not containing 15% of defibrinated sheep blood at 37˚C in a humidified at- added. ROS production in time was plotted and the area under the curve was calculated, with the effect of treatment being calculated as percentage of mosphere containing 5% CO2. Liquid cultures were grown at 37˚C in Stainer-Scholte (SS) medium (30) supplemented with Ca2+ to a final ROS production compared with the positive control taken as 100%. concentration of 2 mM, to assure proper CyaA secretion and folding (31). Determination of PIP amounts in cell membranes The used strains were derived from B. pertussis Tohama I (B. pertussis 3 cyaA-wt) that was obtained as strain CIP 81.32 from the Collection of The amount of PIP3 in membranes of neutrophils was determined as de- Institute Pasteur (Paris, France). The mutant strains carrying an in-frame scribed elsewhere (35). In brief, purified neutrophils were preincubated deletion of the cyaA open reading frame on the chromosome (B. pertussis with 100 ng/ml CyaA for 5 min at 37˚C in a chloroform-resistant tube. DcyaA), or secreting an enzymatically inactive CyaA-AC2 toxoid 2 Neutrophils were subsequently activated by the indicated concentration of (B. pertussis cyaA-AC ) because of insertion of a GlySer dipeptide be- fMLF for 1 min. The PIP3 production was then stopped by the addition of a tween residues 188 and 189 of CyaA (32), were described previously (13). mixture of 64.5% methanol, 32.3% chloroform, and 3.14% 1M HCl. Ten nanograms of C16/17 PIP3 was added as an internal standard per sample. Isolation and infection of neutrophils To the mixture, an equal volume of chloroform was added and the mixture Primary human neutrophils were purified from whole blood obtained at the was incubated for 5 min at room temperature, leading to separation of transfusion unit of the Thomayer Hospital in Prague as described elsewhere aqueous and organic phases. The organic phase was removed and washed (33). In brief, blood was centrifuged (300 3 g, 20 min, 17˚C, without brakes) with the equal amount of a mixture of 53.3% methanol, 26.6% chloroform, to remove platelet-rich plasma. Pellet was resuspended in 0.72% Dextran and 20% 0.01M HCl. The washed organic phase was derivatized with T500 in 0.9% NaCl and left to sediment RBCs (30 min, room temperature, in 133.3 mM (diazomethyl)trimethylsilan (Sigma-Aldrich) for 10 min, and the dark). After sedimentation, the upper layer containing leukocytes was derivatization was stopped by the addition of glacial acetic acid to the final centrifuged (300 3 g, 6 min, 17˚C), and the pellet was resuspended in concentration of 0.8%. The derivatized organic phase was washed twice platelet-poor plasma obtained by centrifugation of platelet-rich plasma with a mixture of 53.3% methanol, 26.6% chloroform, and 20% water, (2000 3 g, 30 min, 17˚C). Granulocytes were obtained by centrifugation of followed by addition of 0.125 vol of the mixture of methanol and water leukocytes through Percoll gradient from the 37.8%/45.9% interface. (9:1). The mixture was dried under vacuum and resuspended in 80% For in vitro infection experiments, purified human neutrophils (2 3 106 methanol. The amount of PIP3 was determined by mass spectrometry (36). 2+ cells/ml) in HBSS medium containing 10 mM glucose and 1.9 mM Ca Determination of DAG in cell membranes were mixed with suspensions of B. pertussis cells (grown to OD600 =1in liquid SS medium [30] containing 2 mM CaCl2) to reach a multiplicity of The amount of DAG was determined as described elsewhere (37). In brief, infection (MOI) of 100:1 (15% of SS medium final), and the cocultures purified neutrophils were preincubated with 10 ng/ml of CyaA for 10 min The Journal of Immunology 3 at 37˚C before activation by 3 mM fMLF for 1 min. The reaction was Results stopped by the addition of ice-cold PBS, and the cells were subsequently The cAMP-elevating CyaA toxin enables B. pertussis to escape lysed by the mixture of hexane with isopropanol (3:2), and the amount of DAG in the mixture was determined. ROS-mediated killing Although B. pertussis bacteria are sensitive to killing by ROS, the Opsonization of zymosan with human complement CyaA toxin was previously shown to inhibit oxidative burst and Anonymous fresh human blood was purchased at the transfusion unit of the bactericidal ROS production by neutrophils (8, 13). We thus tested Thomayer Hospital in Prague, and complete human sera were obtained by whether the CyaA-generated cAMP signaling alone accounts for centrifugation at 300 3 g, for 20 min at 17˚C. Before use, the sera were controlled by ELISA for presence of any detectable amounts of Abs rec- the inhibition of oxidative burst and escape of B. pertussis to ROS- ognizing the pertussis toxin, CyaA, and filamentous hemagglutinin Ags of mediated killing. Therefore, wild-type (WT) B. pertussis Tohama B. pertussis. For opsonization by human complement, 1 3 108 E. coli I(B. pertussis cyaA-wt), the B. pertussis cyaA-AC2 mutant pro- cells, or 1 mg of zymosan, were incubated with 50% human serum for ducing a hemolytic but catalytically inactive CyaA-AC2 toxoid 30 min at 37˚C under gentle shaking, and the suspensions were washed D twice with serum-free HBSS. (unable to convert ATP to cAMP) and the B. pertussis cyaA bacteria, not producing CyaA at all, were cocultured with purified FACS analysis of neutrophil apoptosis human neutrophils at MOI 100:1. As revealed by monitoring of bacterial viability (CFU) in time (Fig. 1A), the growth of Apoptosis of neutrophils was determined using double staining with Annexin V 2 and Hoechst 33258. Cells were preincubated with 100 ng/ml of either CyaA or B. pertussis cyaA-AC bacteria was inhibited in the presence of 2 CyaA-AC for 5 min before stimulation with 1 mg/ml of human serum- neutrophils. In contrast, the parental B. pertussis WT strain mul- opsonized zymosan for 1 h at 37˚C under continuous gentle shaking. Cells tiplied even more (p , 0.01) in the presence of neutrophils than in were next washed three times with ice-cold HBSS to remove any excess of the Downloaded from unbound toxin. Subsequently, the cells were stained by 10 mlAnnexinVFITC in their absence. In line with that, the capacity of neutrophils to in- Annexin buffer for 15 min in the dark at room temperature. Five minutes crease ROS production over the basal level was lost already within before analysis, 1 mg/ml Hoechst 33258 was added to cells on ice and 20,000 5 min from mixing with the B. pertussis WT bacteria (Fig. 1B), cells per sample were analyzed by flow cytometry (LSR II instrument; BD whereas addition of paraformaldehyde (PFA)-killed B. pertussis Biosciences). The data were analyzed by FlowJo (version 7.6.1). cells or infection with live B. pertussis cyaA-AC2 or B. pertussis cAMP ELISA DcyaA bacteria provoked oxidative burst of neutrophils (Fig. 1B). Hence, the capacity of secreted CyaA to invade neutrophils and http://www.jimmunol.org/ The accumulation of cAMP in primary human neutrophils was measured using a competition ELISA as described earlier (15). In brief, 0.5 3 106 cells catalyze conversion of ATP into the signaling molecule cAMP were incubated for the indicated times with CyaA at concentrations indi- was critical for the suppression of oxidative burst of neutrophils cated in the figure legends. After the time indicated in the figure legends, by B. pertussis. the reaction was stopped by the addition of ice-cold PBS, and the cells were pelleted by centrifugation and lysed with 0.2% Tween 20 in 50 mM HCl. CyaA-produced cAMP blocks diverse oxidative The samples were boiled for 15 min at 100˚C, neutralized with 150 mM burst-activating cascades unbuffered imidazole, and cAMP concentration was determined by ELISA. MaxiSorp 96-well ELISA plates (Nunc) were coated with cAMP-BSA Oxidative burst of neutrophils can be triggered by agonists of conjugate (1:6000 dilution) in 100 mM sodium carbonate buffer (pH 9.5) diverse G protein–coupled, Ig (Fc receptor), or complement- overnight at 4˚C. Plates were blocked for 3 h at room temperature with binding receptors (Fc receptor and CR), which activate the three by guest on September 27, 2021 0.1% Tween 20 (TBST) and 2% BSA in TBS (TBST-BSA). Cell lysates and known signaling pathways converging to NADPH complex as- rabbit anti-cAMP polyclonal Ab (1:3000 dilution; kind gift of Agnes Ull- sembly and ROS production. We thus examined whether cAMP mann, Institute Pasteur, Paris, France) were incubated in the coated and blocked plate overnight at 4˚C. The unbound primary Ab was washed out signaling elicited by CyaA action interferes with induction of ROS by three washing steps, and the secondary goat anti-rabbit HRP-conjugated by various stimuli. The sensitivity of neutrophils to CyaA- Ab (1:1000 dilution; GE Healthcare) was added. After three washes, the triggered incapacitation exhibited the expected interdonor varia- reactions were revealed using o-phenylenediamine as substrate. tion, but toxin at 10 ng/ml strongly and reproducibly suppressed Immunodetection of proteins (Western blotting) ROS production triggered by the bacterial tripeptide fMLF, or by adhesion to IgG- or poly-RGD–coated surface, respectively 3 6 Freshly isolated neutrophils (0.75 10 per sample) were incubated in (Fig. 2). Higher toxin concentrations of up to 100 ng/ml were then complete HBSS at 37˚C. Inhibitors were added at the indicated time before the addition of CyaA and incubated for the indicated times. Cellular processes required for full suppression of oxidative burst by serum- were stopped with addition of ice-cold PBS (137 mM NaCl, 2.7 mM KCl, opsonized zymosan and in particular by opsonized E. coli cells 10 mM Na2HPO4,2mMKH2PO4 [pH 7.4]), and cells were lysed immedi- (Fig. 2). Signaling of CyaA-produced cAMP thus interfered with ately with 1% Nonidet P-40 in 20 mM Tris-HCl (pH 8) buffer containing ROS induction through all the three known triggering pathways, 100mMNaCl,10mMEDTA,10mMNa4P2O7,1mMNa3VO4,50mM NaF, 10 nM Calyculin A, and Complete Mini inhibitors (Roche). and as noted previously (19), the loss of ROS production by Upon separation by SDS-PAGE and transfer onto nitrocellulose membranes, CyaA-treated neutrophils was not due to induction of apoptosis proteins were probed by the indicated mAbs and revealed by corresponding (Supplemental Fig. 1). peroxidase-conjugated secondary Ab (1/5000; GE Healthcare) using the West Femto Maximum Sensitivity Substrate (Pierce). Chemiluminescence signals The cAMP-elevating activity of CyaA is sufficient for were quantified using ImageQuant LAS 4000 imaging system instrument suppression of ROS production (Fuji), and images were analyzed using the AIDA two-dimensional densi- tometry software (version 3.28; Raytest Isotopenmessgeraete). To analyze in more detail the mechanism by which the CyaA- elicited signaling inhibits ROS induction, we further focused on Ethics statement the most sensitive and best characterized fMLF-triggered ROS Human peripheral blood neutrophils were purified from the blood of induction pathway. CyaA is a trifunctional toxin, which, besides anonymous healthy human donors who gave written informed consent for conversion of cytosolic ATP into cAMP, promotes also Ca2+ influx the use of their blood for research purposes. All studies complied with the into cells and permeabilizes the cytoplasmic membrane of cells Declaration of Helsinki. through cation-selective pores that promote efflux of cytosolic Statistical analysis potassium (K+). To examine whether the pore-forming activity of The significance of differences in values was assessed by one-way ANOVA CyaA synergized with cAMP signaling in bringing about the followed by Tukey’s honest significant difference test using GraphPad suppression of ROS production in neutrophils (Fig. 1B), we used Prism 7 software. two purified and previously characterized CyaA constructs that 4 cAMP THROUGH Epac INHIBITS PLC AND PMN OXIDATIVE BURST Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Growth of B. pertussis in the presence of neutrophils. For infection of primary human neutrophils, all B. pertussis strains were grown in liquid SS medium supplemented with 2 mM Ca2+.(A) Primary human neutrophils in HBSS buffer were mixed with B. pertussis WT or with mutant bacteria lacking the CyaA enzymatic activity of B. pertussis cyaA-AC2, or with bacteria defective in production of the entire CyaA toxin B. pertussis DcyaA at MOI 100:1. At the indicated times, aliquots of cultures were diluted and plated on BGA. CFUs of recovered bacteria were counted 5 d later. Survival of B. pertussis WT in the absence of neutrophils (B. pertussis WT; no neutrophils) was monitored as a control. Values show CFU counts per neutrophil (per initial neutrophil count) and represent one representative result of five independent experiments performed in triplicates. (B) Primary human neutrophils were activated either with nonopsonized and PFA-inactivated B. pertussis cells or with live B. pertussis WT, B. pertussis cyaA-AC2,orB. pertussis DcyaA bacteria at MOI 100:1. The left panel represents mean values from three independent experiments (n = 3). The signals were integrated over time (106 min), and ROS production was normalized to the ROS levels produced in response to PFA-killed B. pertussis cell control. The right panel shows the time dependence of ROS production in one representative experiment of three performed. ‡p , 0.001 versus B. pertussis WT starting inoculum, †p , 0.001 versus B. pertussis WT at the indicated time point, **p , 0.001 versus PFA-killed B. pertussis cells. have selective defects of one or two of the three biological ac- permeabilizing CyaA-AC2 or CyaA-KK-AC2 toxoids. Hence, the tivities of CyaA, respectively (Table I and references cited inhibition of oxidative burst capacity of neutrophils was solely due therein). The used enzymatically inactive CyaA-AC2 toxoid is to the signaling effects of CyaA-generated cAMP. unable to convert ATP into cAMP, but still elicits Ca2+ influx into To dissect whether the cAMP-activated protein kinase A (PKA), cells and permeabilizes cellular membrane for K+ efflux like the the Epac, or signaling through both pathways in parallel was in- intact CyaA (34, 38). The other construct, CyaA-E509K+E516K- volved in CyaA-triggered inhibition of ROS production, we used AC2 (CyaA-KK-AC2), is devoid of the enzymatic AC and Ca2+ the cell-permeable and PKA-specific (6-Bnz-cAMP) or Epac- influx-promoting activities, whereas its cell-permeabilizing (K+ specific (8-CPT-cAMP) cAMP analogs. As shown in Fig. 4A, efflux-eliciting) capacity is strongly enhanced (34, 38). As then signaling of either of the selective activators interfered with shown in Fig. 3, action of the intact CyaA (10 ng/ml) increased the fMLF-stimulated ROS production, but at an equal (1 mM) con- intracellular concentration of cAMP in neutrophils already within centration, the PKA-specific activator (6-Bnz-cAMP) was less 5 min (Fig. 3A) and blocked the fMLF-stimulated ROS production potent. To ascertain the involvement of the PKA pathway, we next (Fig. 3B). In contrast, even 100 ng/ml of the CyaA-AC2, or of the used the PKA-selective inhibitors cAMPS and H-89 at their CyaA-KK-AC2 toxoid, did not cause any inhibition of ROS commonly applied concentrations. The impact of CyaA action on production. Moreover, at 1 mM concentration of db-cAMP, a fMLF-induced ROS production was, indeed, reduced but was not membrane-permeable analog of cAMP, a strong inhibition of ROS abolished in neutrophils that were pretreated for 20 min with production was observed, whereas the impact of db-cAMP sig- 1 mM cAMPS or 10 mM H-89, respectively (Fig. 4B). These data naling was not potentiated anymore by the presence of the cell- thus show that, despite some parallel contribution of PKA signaling, The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. CyaA inhibits ROS production induced in neutrophils by opsonized-zymosan, fMLF, , or attachment. (A) Primary human neutrophils were preincubated with CyaA at the indicated concentrations for 5 min before stimulation with 1 mg/ml serum-opsonized zymosan. ROS production was measured over time. (B) Primary human neutrophils were preincubated with CyaA at the indicated concentration for 5, 10, or 20 min before stimulation with 300 nM fMLF, and ROS production was measured for 330 s. Mean values for relative ROS production (left panel; three independent experiments) and time course of ROS production by neutrophils preincubated with CyaA for 10 min (right panel; one result (Figure legend continues) 6 cAMP THROUGH Epac INHIBITS PLC AND PMN OXIDATIVE BURST

Table I. Comparison of biological activities of used CyaA variants

Toxoid cAMP Production, % Ca2+ Influx,a % Pore Formation, % References CyaA 100 100 100 (32, 69–72) CyaA-AC2 0 ∼100 100 (32, 69–72) CyaA-KK-AC2 0 ,5 .500 (69–71) aThe course of Ca2+ influx elicited by CyaA and CyaA-AC2 differs in time (70). the CyaA-generated cAMP provoked inhibition of ROS production Similarly, exposure to the Epac-specific activator, 8-CPT-cAMP in neutrophils primarily through activation of Epac. (1 mM), provoked inhibition of fMLF-stimulated DAG produc- Previously, we found that signaling of CyaA-produced cAMP tion, whereas no effect of the PKA activator 6-Bnz-cAMP was through the PKA pathway yields activation of the tyrosine phos- observed. The levels of the other product of PLC-mediated PIP2 phatase Src homology domain 2–containing protein tyrosine transformation, IP3, followed similar trends, albeit inhibition of IP3 phosphatase 1 (SHP-1) that inhibits ERK activity in macrophages production was not statistically significant, likely because of the (13, 39). Therefore, we assessed whether CyaA action on fMLF- low dynamic range of the assay (data not shown). These data hence stimulated neutrophils interferes with the activating phosphory- reveal that signaling of CyaA-generated cAMP through Epac lation of the ERK and p38 kinases that are known to be involved caused inhibition of PLC. Moreover, cAMP-activated Epac still

in NADPH oxidase assembly. As shown in Fig. 4C, the action of inhibited m-3M3FBS–activated PLC by an as yet unknown Downloaded from CyaA, but not of CyaA-AC2, caused dephosphorylation of both mechanism. ERK and p38 (because of interdonor variation, the level of sta- PLC inhibition is expected to decrease the activity of the DAG- tistical significance of p , 0.001 was not reached for p38 de- dependent downstream PKC. To corroborate that the CyaA-elicited phosphorylation). Indeed, inhibition of SHP-1 by the NSC87877 cAMP signaling inhibited ROS production upstream of the PKC inhibitor blocked the effect of CyaA (10 ng/ml), suggesting that step in the signaling cascade, the impact of CyaA action on ROS

activation of SHP-1 plays a role in CyaA-elicited inhibition of production was examined in the presence of 1 mM PMA, a PKC- http://www.jimmunol.org/ ROS production in neutrophils. activating analog of DAG. Indeed, ROS production in PMA- activated neutrophils was completely resistant to toxin-mediated CyaA blocks ROS production in neutrophils through inhibition inhibition even at 100 ng/ml of CyaA (Fig. 5D), and only a modest of PLC inhibition of ROS production was observed when a reduced Induction of NADPH complex assembly and ROS production concentration (0.1 mM) of the PKC activator was used (Supplemental requires the activity of PI3K and the activation of PLC. To assess Fig. 2). Taken together, these results suggest that the major mecha- the impact of CyaA treatment on class I PI3K activity, we pre- nism by which CyaA-elicited cAMP signaling inhibits the oxidase is incubated the neutrophils for 5 min with 100 ng/ml CyaA before through Epac-mediated inhibition of PLC and hence inhibition of addition of fMLF for 1 min and extraction of PIP3. The fMLF- PKC. by guest on September 27, 2021 stimulated increase in PIP3 was relatively unaffected by the presence of CyaA (Fig. 5A), showing that the toxin-provoked Discussion inhibition of ROS production was not mediated by PI3K inhibi- We show that activation of Epac by CyaA-generated cAMP me- tion. These results also suggest there was little impact of CyaA on diates the near-instantaneous annihilation of the oxidative burst upstream signaling via the fMLF receptor and activation of Gi/o. capacity of neutrophils through inhibition of the PLC, thus enabling Therefore, the impact of CyaA action on PLC-dependent pro- the pathogen to escape ROS-mediated killing. The involvement of duction of ROS was next examined in neutrophils treated by the pertussis toxin in this activity could be excluded, because pertussis PLC activator m-3M3FBS, using the o-3M3FBS analog as a toxin was produced by all three used B. pertussis strains and its control unable to activate PLC (40). As shown in Fig. 5B, the action would take ∼4 h to generate sufficient levels of cAMP that m-3M3FBS-triggered production of ROS was significantly inhibi- would impair ROS production in neutrophils (41). As summarized ted in neutrophils that were preincubated with 10 or 100 ng/ml in the model shown in Fig. 6, besides the Epac-mediated block of CyaA, albeit the inhibition was reproducibly less complete than in PLC activity by an as yet unknown pathway, the mechanism of fMLF-activated neutrophils. Pretreatment with the 8-CPT-cAMP inhibition of ROS production by CyaA action may, to some extent, activator of Epac (1 mM) impaired m-3M3FBS–stimulated ROS involve also the activation of PKA signaling. It remains to be production to a similar extent, whereas no decrease of ROS pro- explored whether the previously observed cAMP-PKA–dependent duction was observed upon activation of PKA with 1 mM 6-Bnz- activation of the tyrosine phosphatase SHP-1 (13, 39) does also cAMP, indicating that activation of Epac provoked the inhibition of play a role in the inhibition of ROS production in CyaA-treated PLC. In line with that, exposure of neutrophils to 100 ng/ml CyaA neutrophils. SHP-1 activity was indeed required for CyaA- for 20 min at 37˚C ablated the fMLF-triggered conversion of PIP2 triggered dephosphorylation of ERK and p38 (Fig. 4C). Hence into DAG by PLC, whereas the effect of 10 ng/ml CyaA was not activation of SHP-1 by CyaA-elicited cAMP signaling might in- statistically significant due to high interdonor variation (Fig. 5C). hibit MAPKs involved in NADPH complex assembly. This sug-

representative of three independent experiments) are shown. (C) Primary human neutrophils were preincubated with CyaA variants at the indicated concentration for 5 min before stimulation with the indicated agent. ROS production was measured for 1 h. Mean values for relative (left panel) and absolute (right panel) ROS production, summarizing three independent experiments (n = 3), are shown. Values for IgG-BSA + 10 or 100 ng/ml CyaA and for poly-RGD + 10 or 100 ng/ml CyaA were all under detection limit of the assay. The left panels represent values normalized to ROS production to serum- opsonized zymosan control that were integrated over duration of the experiment (59 min) and obtained in three independent experiments (n 5 3). In the right panels, the time dependence of ROS production is shown, and the values represent the results of one representative experiment of three performed. **p , 0.001 versus activator-only–treated control. The Journal of Immunology 7 Downloaded from

FIGURE 3. CyaA-provoked cAMP accumulation is sufficient for inhibition of ROS production by neutrophils. (A) Primary human neutrophils (2 3 106 cells/ml) were incubated with CyaA for the indicated time. The total amount of cAMP in cell lysates was measured using a competitive ELISA assay. Mean values for one representative experiment of three independent experiments performed in triplicates are given. (B) Primary human neutrophils were pre- incubated with CyaA variants at the indicated concentrations in the presence or absence of 1 mM db-cAMP for the indicated time before stimulation with

300 nM fMLF. ROS production was measured over time as shown in the right panel (330 s). Mean values for relative ROS production by neutrophils http://www.jimmunol.org/ preincubated with CyaA for 10 min summarize three independent experiments (n = 3). **p , 0.001 versus fMLF-treated control. gests the existence of an additional and so far undescribed sig- hibition of the ROS-inducing pathways also upstream of PLC, at naling pathway hijacked by the action of CyaA. the level of signaling of opsonin receptors. Indeed, we have re- The concentrations of CyaA used in this study are likely to be cently found that CyaA-generated cAMP signaling provokes in- physiologically relevant. Comparable amounts of CyaA (∼10 ng/ml) activation of Syk in human , thereby interfering with were, indeed, detected in nasal washes and aspirates of mucosal the iC3b-activated prophagocytic and ROS-inducing signaling of fluids from B. pertussis–infected infants and experimentally CR3 through Syk (10). challenged baboons (42). This indicates that the real local con- We further show that it is the capacity of the AC enzyme domain by guest on September 27, 2021 centration of CyaA on the mucosal surface in vivo, before dilution of CyaA to convert ATP into cAMP, which is central to the capacity in the wash solution or the mucosal secretion fluids, may be of the toxin to inhibit the oxidative burst of neutrophils. Somewhat approaching up to ∼100 ng/ml. Moreover, the AC enzyme activity unexpectedly, neither the Ca2+ influx-promoting activity of CyaA, of CyaA found in this study in the culture supernatants of neu- which accompanies AC domain translocation into cells, nor the trophils, infected with B. pertussis at the MOI 100:1 (Fig. 1B), pore-forming activity of the toxin, which yields corresponded well to the activity of the added purified toxin used permeabilization and K+ efflux from target cells, were found to in our experiments (Fig. 2). Importantly, in cells from some do- synergize with cAMP signaling in bringing about the inhibition of nors, even as little as 1 ng/ml CyaA reduced the magnitude of the the oxidative burst in toxin-treated neutrophils (Fig. 3B). Such oxidative burst of fMLF-activated neutrophils (Fig. 2B, right synergy would have been expected to result from the impact of panel). toxin-provoked Ca2+ influx on PKC activity and from perturbation Previously, the capacity of CyaA to inhibit oxidative burst of of ion homeostasis upon cell permeabilization by the inserted neutrophils stimulated by serum-opsonized zymosan (8, 17) or the CyaA pores, which activates MAPK signaling (46–48). The PKC activator PMA (19) was observed, but the mechanism behind impact of cAMP signaling thus overrode the impact of the pore- was not addressed. We analyzed in this study the interference of forming activity. This likely reflects the adaptation of the pore- CyaA signaling with ROS production elicited by fMLF, a natu- forming RTX moiety of CyaA for the purpose of delivery of the rally produced tripeptide released from bacterial lipoproteins in AC enzyme into target cells, at the expense of the pore-forming the course of bacterial infection. We took advantage of the activity. Indeed, CyaA forms smaller membrane pores and ex- available knowledge on signaling pathways activated by this G hibits a significantly lower specific cell-permeabilizing activity protein–coupled receptor agonist (43). Further, fMLF-provoked than do the bona fide pore-forming RTX leukotoxins (49). This signaling involves similar PLC and PI3K activation steps as the would explain why the HlyA toxin of E. coli could trigger an signaling pathways involved in activation of the oxidase by oxidative burst at low concentrations (50), whereas the CyaA- complement or Ab-opsonized bacteria. Production of the second AC2 toxoid failed to trigger ROS induction (Fig. 3B). messengers DAG and PIP3 by PLC and PIP3 is then involved in It remains enigmatic by which mechanism and signaling path- both signaling cascades. Thus, the focus on CyaA-mediated in- ways the binding of CyaA-generated cAMP to Epac translates into hibition of fMLF-triggered ROS induction allowed us to examine inhibition of PLC activity, because the described function of Epac the key signaling steps that are downstream of a broad range of is to serve as a guanine exchange factor for the Ras family GTPase various immunoreceptor-activated tyrosine kinases, including Lyn Rap1 (51). In the absence of data, a plausible hypothesis, worthy and spleen tyrosine kinase (Syk) (44, 45). It is worth noting that, of exploration, would be that activation of Rap1 by cAMP-Epac besides interfering with the bottleneck step at the level of PLC might lead to activation of the tyrosine phosphatase SHP-1 (52), activation, the CyaA-generated cAMP signaling can provoke in- and thereby cause dephosphorylation of ERK and potentially of 8 cAMP THROUGH Epac INHIBITS PLC AND PMN OXIDATIVE BURST Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. Both PKA and Epac inhibit ROS production in neutrophils. (A) Primary human neutrophils were preincubated with 10 ng/ml CyaA or with 1 mM cAMP analogs for 20 min at 37˚C before stimulation with 300 nM fMLF. ROS production was measured over time as documented in the right panel (330 s). The left panel represents values normalized to ROS production to fMLF control integrated during whole experiment (330 s) and obtained in three independent experiments (n = 3). In the right panel, the time dependence of ROS production is shown, and the values are from one result that was representative of the outcome of three independent experiments. (B) Neutrophils were pretreated with PKA inhibitors cAMPS (1 mM) or H-89 (10 mM dissolved in DMSO) for 20 min before the addition of CyaA. ROS production was subsequently induced by the addition of 300 nM fMLF and measured over 330 s. DMSO solvent controls were performed by adding equal amounts of DMSO also into samples to which H-89 was not added. The results represent mean relative values from three or two independent experiments for cAMPS (n = 3) and H-89 (n = 2), respectively. (C) Primary human neutrophils were preincubated with or without 500 nM NSC-87877 for 20 min before exposure to CyaA at the indicated concentration for 10 min at 37˚C and before subsequent stimulation with 300 nM fMLF for 1 min. p38 and ERK phosphorylation were detected by immunoblotting, using b-actin as a loading control. One representative blot of three independent experiments is shown. Mean 6 SD from densitometric analysis of three experiments is shown (n = 3). *p , 0.01 versus fMLF-treated control, **p , 0.001 versus fMLF-treated control, †p , 0.001 versus each other. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. Inhibition of ROS production caused by CyaA is due to the Epac-dependent block of PLC activity. (A) Primary human neutrophils were preincubated with 100 ng/ml of CyaA for 5 min before stimulation with the indicated concentration of fMLF for 1 min. The total amount of PIP3 in cells was determined by mass spectrometric analysis after the extraction of lipids. Values in the left panel represent relative PIP3 production from two inde- pendent experiments. Values in the right panel represent the total PIP3 production in one representative of two independent experiments. (B and D) Primary human neutrophils were preincubated with CyaA variants at the indicated concentration or with 1 mM cAMP analogs for 20 min at 37˚C before stimulation with either 12.5 mM m-3M3FBS (solubilized in DMSO) for direct PLC stimulation (B) or with 1 mM PMA (solubilized in DMSO) for direct PKC stimulation (D). DMSO solvent controls were performed by adding equal amounts of DMSO also into samples to which PMA and m-3M3FBS were not added. ROS production was measured over 5 min (B)or1h(D). Mean values for relative ROS production compared with positive control are given (n = 3). (C) Primary human neutrophils were preincubated with 10 ng/ml CyaA for 20 min before stimulation with 300 nM fMLF for 1 min. The total amount of DAG in cells was determined by mass spectrometry of extracted lipids. The values show one representative result of three independent experiments. **p , 0.001 versus activator-only–treated control. 10 cAMP THROUGH Epac INHIBITS PLC AND PMN OXIDATIVE BURST

mediated activation of the oxidase, but that both Epac and PKA may be involved in the effects of the toxin on the fMLF response. Indeed, we observed (Fig. 4C) that the contribution of PKA activation to the inhibition of ROS production in response to fMLF could be indirect and might potentially result from inhibition of MAPK (ERK and p38) signaling toward NADPH complex assembly. This would be in line with our previous reports that activation of PKA signaling by CyaA-generated cAMP activates, by an as yet unknown mechanism, the tyrosine phosphatase SHP-1, which is known to deactivate ERK by dephosphorylation of its Thr202 and Tyr204 residues (13, 39, 54). We have measured fMLF-stimulated phosphorylation of ERK on these two sites and found it to be significantly reduced by incubation with active CyaA toxin (Fig. 4C). Dephosphorylation of p38 caused by CyaA action was also observed, albeit the result did not reach the level of statistical significance in repeated experiments because of interdonor variation. Moreover, the effect of 10 ng/ml toxin wassignificantlyreversedbytreatmentwiththeSHP-1in- hibitor NSC87877 for both ERK and p38 (Fig. 4C). These

results are consistent with the hypothesis that CyaA toxin mediates Downloaded from some of its inhibitory effects on ROS production in response to fMLF via SHP-1–mediated dephosphorylation of ERK and p38. When ROS production was triggered by direct activation of PLC with m-3M3FBS (40), the oxidative burst could still be inhibited by 10 ng/ml CyaA. In contrast, when ROS production was induced FIGURE 6. Scheme of CyaA-provoked signaling that blocks ROS pro- duction in neutrophils. After binding of CyaA and delivery of the AC do- through direct activation of PKC by 1 mM PMA, it could not be http://www.jimmunol.org/ main into the cytosol of neutrophils, the toxin is activated by binding of inhibited even at increased concentrations of CyaA (Fig. 5D). This , and its enzymatic AC domain catalyzes uncontrolled conver- suggests that blocking of ROS production by cAMP occurs primarily sion of cytosolic ATP into cAMP, thus activating PKA and Epac signaling. through inhibition of PLC and, consequently, an indirect inhibition By an as yet uncharacterized mechanism, activation of PKA probably leads of PKC. That induction of the oxidative burst through PKC activa- to the enhancement of activity of the SHP-1 phosphatase, which yields tion by PMA might be rather insensitive to cAMP signaling of CyaA dephosphorylation and loss of activity of MAPK ERK. Simultaneously, was also indicated by the recent work of Eby et al. (19), who focused activated Epac yields inhibition of PLC by an as yet uncharacterized on analysis of NETosis. The authors used a lower concentration of mechanism, thus preventing activation of PKC. Inhibition of ERK by PKA PMA (0.02 mM) that triggered modest levels of ROS production

and of PLC/PKC by Epac then prevents assembly of NADPH oxidase, thus by guest on September 27, 2021 (Supplemental Fig. 2), yet still permitted inhibition of NETosis by blocking ROS production and killing of invading bacteria. CyaA. This goes well with our observation that ROS induction by low levels of PKC activation could be partly reversed by CyaA p38 as well, the inhibition of which was reported to cause inhibition action (Supplemental Fig. 2). The activation of the oxidase by PMA of ROS production in neutrophils (53). ERK is, indeed, known to be involves multiple indirect pathways, not just direct phosphorylation regulated by the SHP-1 phosphatase (54). For p38 activity regula- of the oxidase subunits by PKC (e.g., GTP/GDP exchange on Rac). tion, the involvement of SHP-1 is indicated by data shown in It is thus plausible to assume that CyaA can inhibit the actions of low Fig. 4C, but it remains to be conclusively demonstrated. Intrigu- doses of PMA through targets other than PLC/DAG. ingly, we found previously that the CyaA/cAMP-elicited activation In contrast with the reports on cAMP signaling effects on the of SHP-1 and inhibition of ERK in monocytes was mediated by the activity of the PI3K isoforms in other cells (64–68), we did not cAMP-triggered activity of PKA and not by Epac (13, 39, 55). The observe a strong impact of the CyaA-generated cAMP signaling involvement of cAMP-activated PKA signaling in promoting de- on the levels of PI3K-produced PIP3 (Fig. 5) or on Akt phos- phosphorylation of ERK and p38 has previously been reported in phorylation in fMLF-activated neutrophils (data not shown). Our human neutrophils (56, 57). However, in contrast with the report of study, however, involved only the PI3K isoforms acutely regulated Yue and colleagues (58), we did not observe any phosphorylation of by 1-min stimulation of human neutrophils with fMLF, PI3Kg, and PLC by PKA upon CyaA-mediated elevation of cAMP in neutro- PI3Kd (22). The impact of CyaA action on the activity of other phils (data not shown). Those authors based their hypothesis that PI3K isoforms thus deserves further exploration. PKA phosphorylates and inhibits PLC on the usage of a cell- Taken together, in this article, the reported Epac-mediated in- permeable 8-[4-chlorophenythio]-cAMP analog. This is, however, hibition of oxidative burst of neutrophils via inhibition of PLC not strictly specific for PKA and was reported to activate Epac as activity reveals that the manipulation of the composition of neu- well (59). Moreover, 8-[4-chlorophenythio]-cAMP was also found trophil membranes is a novel cellular target of B. pertussis CyaA to activate to some extent the cGMP-dependent protein kinase G toxin and plays a key role in evasion of the whooping cough agent (60), the cGMP-specific phosphodiesterase (61), as well as the Epac to ROS-mediated killing by neutrophils. protein, which was discovered only after the work of Yue et al. (58) was published (62) (the 8-[4-chlorophenythio]-cAMP compound Acknowledgments used by Yue and colleagues [58] would actually have been Epac- We gratefully acknowledge V.M. Bierhanzl for generous help with determi- specific if it was modified by a methyl group at the 29 position [63]). nation of DAG. Our data indicate that CyaA action had a lower impact on m-3M3FBS-induced ROS production than on fMLF-induced ROS Disclosures production (Figs. 5B, 6). 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70

60

50

40 % of cells of % 30

20

10

0 Zymosan - + + +

CyaA [ng/ml] - - - 100

CyaA-AC- [ng/ml] - - 100 -

Fig. S1: CyaA inhibits spontaneous apoptosis in neutrophils. Primary human neutrophils were pre-incubated with 100 ng/ml of either CyaA or CyaA-AC- for 5 minutes prior to stimulation with 1 mg/ml of serum-opsonized zymosan. Apoptosis was detected by FACS using Hoechst 33258 and Annexin V staining. The values represent data for one representative experiment out of three performed in triplicates. 140000000 120

120000000

100

100000000 80 ** 80000000 ** 60 60000000 Control Control 40 40000000 [%] production ROS ROS production [A.U.] production ROS 100 ng/ml 100 ng/ml 20000000 CyaA 20 CyaA

0 0 0 0,05 0,1 0,15 0,2 0,25 0,3 0 0,1 0,2 0,3 PMA concentration [μM] PMA concentration [μM]

Fig. S2: PMA stimulates ROS production in a dose-dependent manner. Primary human neutrophils were pre-incubated for 20 minutes with 100 ng/ml CyaA prior to stimulation with indicated concentrations of PMA. The ROS production was measured for 1 hour. Mean values represent the total amount of ROS produced (left panel) and the normalized ROS production (right panel) for one out of three independent experiments performed in triplicates (n=9). **, p<0,001 versus control