selective sensitization of human neutrophils to lukgh mediated cytotoxicity by staphylococcus aureus and il 8

Accepted Manuscript Selective Sensitization of Human Neutrophils to LukGH Mediated Cytotoxicity by Staphylococcus aureus and IL-8 Philipp Janesch, Harald Rouha, Susanne Weber, Stefan Malafa, Karin Gross, Barbara Maierhofer, Adriana Badarau, Zehra C Visram, Lukas Stulik, Eszter Nagy PII: S0163-4453(17)30053-1 DOI: 10.1016/j.jinf.2017.02.004 Reference: YJINF 3887 To appear in: Journal of Infection Received Date: 25 October 2016 Revised Date: 14 February 2017 Accepted Date: 17 February 2017 Please cite this article as: Janesch P, Rouha H, Weber S, Malafa S, Gross K, Maierhofer B, Badarau A, Visram ZC, Stulik L, Nagy E, Selective Sensitization of Human Neutrophils to LukGH Mediated Cytotoxicity by Staphylococcus aureus and IL-8, Journal of Infection (2017), doi: 10.1016/ j.jinf.2017.02.004 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Selective Sensitization of Human Neutrophils to LukGH Mediated Cytotoxicity by Staphylococcus aureus and IL-8 RI PT Philipp Janesch, Harald Rouha, Susanne Weber, Stefan Malafa*, Karin Gross, Barbara Maierhofer, Adriana Badarau, Zehra C Visram, Lukas Stulik, Eszter Nagy# # M AN U Running Head: PMN sensitization to LukGH toxicity SC Arsanis Biosciences GmbH, Vienna, Austria Address correspondence to Eszter Nagy, eszter.nagy@arsanis.com; P: +4317990-117-10; TE D Address: Helmut-Qualtinger-Gasse 2, 1030 Vienna, Austria *Present address: Stefan Malafa, Department of Virology, Medical University of Vienna, EP Vienna, Austria AC C P J and H R contributed equally to this work ACCEPTED MANUSCRIPT ABSTRACT Objectives: Staphylococcus aureus produces up to five bi-component leukocidins - LukSF-PV, gamma-hemolysins AB and CB, LukGH (LukAB) and LukED - to evade innate immunity by lysing phagocytic cells Species specificity of these leukocidins limits the relevance of animal models, therefore we assessed their individual contribution using human neutrophils Methods: Human polymorphonuclear leukocytes (PMNs) were activated with stimuli relevant during bacterial infections and sensitivity to recombinant leukocidins was measured in cell- viability assays Leukocidin receptor expression was quantified by flow cytometry SC RI PT Results: We observed greatly variable sensitivities of different PMN preparations towards 11 LukGH Activation of PMNs by lipopolysaccharide (LPS) or S aureus culture supernatant (CS) 12 lacking all leukocidins resulted in higher surface expression of CD11b, the LukGH receptor, and 13 greatly enhanced the sensitivity towards LukGH, eliminating the variability observed with 14 unstimulated cells In contrast, CS induced a decrease in sensitivity of PMNs to the other four 15 leukocidins and reduced surface staining for their cognate receptors (CXCR1, CXCR2, C5aR, 16 C5L2) Delta-toxin and peptidoglycan mimicked the effect of CS Moreover, IL-8, an important 17 cytokine in neutrophil activation, also selectively increased LukGH sensitivity Deletion of 18 lukGH, but not other leukocidin genes, prevented PMN killing upon infection with USA300 CA- 19 MRSA 20 Conclusion: Inflammatory signals enhance the susceptibility of human PMNs to lysis by 21 LukGH rendering this toxin dominant among the S aureus leukocidins in vitro AC C EP TE D M AN U 10 22 23 24 ACCEPTED MANUSCRIPT 25 INTRODUCTION 26 Phagocytic cells, especially neutrophils are the cornerstones of host defense against 28 Staphylococcus aureus [1,2] S aureus has developed multiple mechanisms to avoid its 29 elimination by phagocytic cells The most direct one is killing of neutrophils, monocytes and 30 macrophages by secreted leukocidins These cytolytic toxins – the two gamma-hemolysins 31 (HlgAB, HlgCB), the Panton-Valentine leukocidin (LukSF-PV), LukED and LukGH (LukAB) - 32 belong to the bi-component beta-barrel pore forming toxin family [3,4] It is rather enigmatic 33 that S aureus has evolved to employ five different leukocidins and little is known about their 34 individual contribution to human S aureus infections The lukSF-PV gene is carried on 35 prophages in 95 % determined microscopically by Giemsa staining as well as flow ACCEPTED MANUSCRIPT cytometric analysis of forward- versus side-scatter properties Cell viability was > 98% based on 78 Trypan blue (Thermo Fisher Scientific) exclusion Cells at a concentration of 1x106/mL were 79 pre-incubated for hour at 37°C, 5%CO2 with LPS (purified from E coli O111, List 80 Laboratories), S aureus CS, S aureus peptidoglycan (Sigma-Aldrich), S aureus delta-toxin 81 (synthetic peptide from AnaSpec) or human IL-8 (amino acids Ser28-Ser99, BioLegend) at 82 concentrations indicated in the figures prior to use in cell-based toxicity assays or surface 83 staining for leukocidin receptor expression by flow cytometry SC 84 RI PT 77 Recombinant leukocidins Monomers of HlgAB, HlgCB, LukSF-PV and LukED, as well as 86 the co-expressed LukGH dimer (all based on the TCH1516 genome sequences) were 87 recombinantly expressed in E coli as described previously [10,13] Briefly, LukS-PV, LukF- 88 PV, HlgA, HlgC and HlgB were expressed in soluble form with an N-terminal NusA/His6 tag, 89 which was removed proteolytically after the first purification step Purification typically 90 involved three chromatographic steps: IMAC (immobilized metal affinity column); cation 91 exchange or IMAC; and size exclusion chromatography [13] 92 expressed without tag and purified by cation exchange (SP-Sepharose FF, ml, GE Healthcare) 93 and gel filtration (Superdex 75 16/60, GE Healthcare) The LukGH complex was generated by 94 co-transformation of E coli with two plasmids containing different antibiotic resistance 95 markers, carrying either the lukH or the lukG gene LukG was expressed as a fusion protein 96 with NusA/His6 at the N-terminus to allow metal ion affinity purification of the complex, while 97 LukH was expressed in un-tagged form The two proteins were co-purified by IMAC from the 98 soluble fraction The NusA/His6 tag was removed proteolytically with enterokinase giving the 99 un-tagged, mature LukGH complex which was further purified by cation exchange 100 chromatography using the SP FF column For the cell binding studies, proteins were labeled 101 with the amino reactive reagent Sulfo-NHS-LC biotin (Thermo Scientific), according to the 102 manufacturer’s instructions, yielding biotin levels of – biotin/protein LukE and LukD were AC C EP TE D M AN U 85 ACCEPTED MANUSCRIPT The proteins were assayed for purity by SDS-PAGE, monomeric state by size exclusion 104 chromatography (except for LukGH where dynamic light scattering was used), primary structure 105 integrity by mass spectrometry and secondary structure by circular dichroism, and for 106 functionality in in vitro toxin potency assays Endotoxin content was measured for several toxin 107 batches using the chromogenic Limulus Amebocyte Lysate (LAL Chromogenic Endotoxin 108 Quantification kit, Pierce or ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit, 109 Genscript) Values of up to 700 ng LPS per mg toxin for LukGH and between and 250 ng/mg 110 for the other leukocidins were measured In the experiments where the concentration-dependent 111 effect of LPS was tested, the corresponding LukGH batch contained ~ 0.001 ng/mL LPS at nM 112 LukGH concentration M AN U SC RI PT 103 113 In vitro leukocidin potency assays PMNs were used for measuring cytotoxicity induced by 115 recombinant leukocidins as described previously [10,13] Briefly, 2.5x104 cells/well were 116 exposed to equimolar mixtures of S- and F-components for the bi-component toxins LukSF-PV, 117 HlgAB, HlgCB, LukED or the co-expressed LukGH dimer in neutrophil medium (RPMI-1640 118 with 10% FCS (Sigma-Aldrich), mM L-Glutamine (Thermo Fisher Scientific)), for h at 119 37°C, 5% CO2 Cell viability was measured by determining cellular ATP levels with the Cell 120 Titer-Glo® Luminescent Cell Viability Assay Kit (Promega) according to manufacturer’s 121 instructions Percent cell viability after leukocidin exposure was calculated relative to control 122 cells pre-incubated with the same stimulating agent in the absence of toxin Data were analyzed 123 by non-linear regression using Prism (Graph Pad) The toxin monomers alone did not affect 124 PMN viability in the concentration range tested AC C EP TE D 114 125 126 Flow cytometry based surface staining PMNs (1x105/well) were incubated with 127 fluorochrome-labeled antibodies (BioLegend, if not stated otherwise) specific for CD11b (clone 128 ICRF44, Brilliant Violet 421), CD18 (clone TS1/18, PE), CXCR1 (clone 8F1/CXCR1, ACCEPTED MANUSCRIPT AlexaFluor488), CXCR2 (clone 5E8/CXCR2, AlexaFluor647), C5L2 (clone 1D9-M12, PE), 130 C5aR (clone S5/1, PE) and the corresponding isotype control antibodies in Hanks' Balanced Salt 131 Solution (HBSS, Thermo Fisher Scientific) supplemented with 0.5 % BSA (Biomol) and 0.01 % 132 NaN3 for 30 at °C Samples were measured with a CytoFLEX flow cytometer (Beckman 133 Coulter) and data analyzed with FCS express version 4.0 (De Novo Software) Results are 134 expressed as relative change in signal intensity compared to unstimulated control cells unless 135 stated otherwise RI PT 129 For the detection of LukGH binding to PMNs, 1x106 cells/well were re-suspended in 137 neutrophil medium with or without 500 ng/mL LPS for 30 min, washed in HBSS + 0.5% BSA 138 and incubated with 27.25 nM biotinylated LukGH for 30 on ice After washing in HBSS, 139 cell-bound toxin was detected with Alexa 488-labeled Streptavidin (Molecular Probes) by flow 140 cytometry Biotinylated LukS-PV was included as a control Toxin binding is expressed as 141 median fluorescence intensity (MFI) TE D 142 M AN U SC 136 PMN infection assays with live bacteria Overnight cultures of S aureus grown in RPMI-CAS 144 were diluted 1:100 and grown to mid-log phase (OD600nm: 0.5) at 37°C Bacteria were harvested, 145 washed with PBS to remove secreted toxins, re-suspended in RPMI-1640 with 10% FCS, mM 146 L-glutamine and 10 mM HEPES (Sigma-Aldrich) and added to 2.5x104 PMNs/well at different 147 MOIs Reactions were incubated for 90 at 37°C and 5% CO2, followed by addition of 148 Calcein-AM fluorescent viability dye (eBioscience) at a final concentration of µM for 30 149 PMN viability was quantified using a fluorescence plate reader (λex=485 nm, λem=528 nm) 150 Percent viability was calculated relative to mock-treated cells incubated in the same medium 151 (100% viability) AC C EP 143 152 153 RESULTS 154 ACCEPTED MANUSCRIPT Human PMNs from different donors display highly variable sensitivities towards LukGH 156 We determined the cytotoxic potencies of the five S aureus leukocidins - LukSF-PV, LukGH, 157 HlgAB, HlgCB and LukED- towards freshly isolated human PMNs over a broad concentration 158 range by measuring cellular ATP levels All five leukocidins were highly potent with mean EC50 159 values ranging from ~ 0.05 to nM (data not shown) RI PT 155 However, we observed a substantial variability in LukGH sensitivity of PMNs isolated 161 from different blood donors, with EC50 values in the ~ 0.05-25 nM range (individual and 162 cumulative response curves shown in Fig 1A and B, respectively) Some donors were tested 163 multiple times and their PMNs displayed variable LukGH sensitivities on different days (data not 164 shown) Importantly, PMN susceptibility to other leukocidins did not show this variability This 165 is exemplified with LukSF-PV that was tested with all the 25 donors and consistently displayed 166 high potencies with EC50 values < 0.5 nM and > 95% PMN killing (Fig 1) 169 M AN U 168 These data suggest that human PMNs from different isolations have greatly different sensitivities to LukGH, a unique observation among S aureus leukocidins TE D 167 SC 160 LukGH susceptibility correlates with PMN activation and surface density of CD11b, the 171 LukGH receptor Neutrophils can be activated by various microbial stimuli for example 172 lipopolysaccharide (LPS) of Gram-negative bacteria, which results in increased surface 173 expression of CD11b that is widely used as an activation marker of phagocytic cells [14,15] 174 Since it also acts as the receptor for LukGH we hypothesized that the different LukGH 175 sensitivities of the human donors may originate from differences in the PMN activation status 176 This was also supported by our notion that PMNs from the same blood donor can display 177 different LukGH sensitivities when measured on different days AC C EP 170 178 Therefore, we assessed whether LPS induced activation of PMNs renders them more 179 sensitive to LukGH In these experiments a substantial increase in LukGH sensitivity was 180 detectable with LPS-treated cells (Fig 2A) in a concentration dependent manner (Fig S1A and ACCEPTED MANUSCRIPT S2), while susceptibility to LukSF-PV, included as control, did not change (Fig 2B, S1B and 182 S2) Interestingly, the high variation in LukGH sensitivity seen with unstimulated cells (EC50 183 values ranging from 0.1 – 6.2 nM) was largely abolished after LPS exposure, as all PMN 184 preparations displayed comparably high sensitivity (EC50 values ranging from 0.01 – 0.03 nM) 185 irrespective of their sensitivity without LPS activation (Fig 2A) RI PT 181 Consistent with the increased LukGH sensitivity upon LPS exposure, we also observed 187 upregulation of CD11b and CD18 on activated cells by flow cytometry (Fig 3A) This increase 188 was approximately 2-fold, and occurred at higher LPS concentrations than required to increase 189 LukGH sensitivity (decrease in EC50, Fig S2) LPS-treatment also led to an approximately 2- 190 fold increase in surface binding of biotin-labeled LukGH, not observed with biotinylated LukS- 191 PV, the cell binding entity of LukSF-PV (Fig 3B) M AN U SC 186 To delineate how PMN activation by LPS affects the activity of the other leukocidins, we 193 performed further experiments where changes in sensitivity were measured in parallel with 194 receptor surface expression levels for all five leukocidins (Fig and Fig S3) These experiments 195 confirmed the increase in LukGH sensitivity (Fig 4A) and CD11b/CD18 upregulation (Fig 4B) 196 However, PMN activation did not increase the sensitivity towards HlgAB, HlgCB, LukED and 197 LukSF-PV This observation was in line with the surface staining results (Fig 4B and Fig S3), 198 where no increase in signal intensity was detected for CXCR1/CXCR2 (HlgAB, LukED 199 receptors), C5L2 and C5aR (LukSF-PV and HlgCB receptors) 201 EP AC C 200 TE D 192 Taken together these results suggest that PMN activation by LPS results in a selective increase in LukGH sensitivity 202 203 PMNs exposed to S aureus culture supernatant are sensitized to LukGH mediated 204 cytotoxicity and become less sensitive to other leukocidins S aureus culture supernatant (CS) 205 was shown previously to induce increased surface density of CD11b on human PMNs [16] It 206 was therefore tempting to speculate that S aureus itself increases LukGH sensitivity We tested ACCEPTED MANUSCRIPT 460 27 Haas CJC de, Veldkamp KE, Peschel A, et al Chemotaxis Inhibitory Protein of 461 Staphylococcus aureus, a Bacterial Antiinflammatory Agent J Exp Med 2004; 462 199(5):687–695 463 28 Laarman AJ, Mijnheer G, Mootz JM, et al Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis EMBO J 2012; 31(17):3607– 465 3619 467 468 29 Hanzelmann D, Joo H-S, Franz-Wachtel M, et al Toll-like receptor activation depends on lipopeptide shedding by bacterial surfactants Nat Commun 2016; 7:12304 SC 466 RI PT 464 30 Wang R, Braughton KR, Kretschmer D, et al Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA Nat Med 2007; 470 13(12):1510–1514 471 M AN U 469 31 Somerville GA, Cockayne A, Durr M, Peschel A, Otto M, Musser JM Synthesis and Deformylation of Staphylococcus aureus -Toxin Are Linked to Tricarboxylic Acid Cycle 473 Activity J Bacteriol 2003; 185(22):6686–6694 TE D 472 474 32 Ikeda S, Hanaki H, Yanagisawa C, et al Identification of the active component that 475 induces vancomycin resistance in MRSA J Antibiot (Tokyo) 2010; 63(9):533–538 33 Langevelde P van, Dissel JT van, Ravensbergen E, Appelmelk BJ, Schrijver IA, EP 476 Groeneveld PH Antibiotic-induced release of lipoteichoic acid and peptidoglycan from 478 Staphylococcus aureus: quantitative measurements and biological reactivities Antimicrob 479 Agents Chemother 1998; 42(12):3073–3078 AC C 477 480 34 Qazi BS, Tang K, Qazi A Recent Advances in Underlying Pathologies Provide Insight 481 into Interleukin-8 Expression-Mediated Inflammation and Angiogenesis Int J Inflamm 482 2011; 2011:1–13 483 484 35 Berube B, Wardenburg J Staphylococcus aureus α-Toxin: Nearly a Century of Intrigue Toxins 2013; 5(6):1140–1166 19 ACCEPTED MANUSCRIPT 485 36 Heyer G Staphylococcus aureus agr and sarA Functions Are Required for Invasive 486 Infection but Not Inflammatory Responses in the Lung Infect Immun 2002; 70(1):127– 487 133 488 37 Garcia JE, Rodriguez FM, Cabo MR de, et al Evaluation of inflammatory cytokine secretion by human alveolar macrophages Mediators Inflamm 1999; 8(1):43–51 490 38 Utgaard JO, Jahnsen FL, Bakka A, Brandtzaeg P, Haraldsen G Rapid secretion of RI PT 489 prestored interleukin from Weibel-Palade bodies of microvascular endothelial cells J 492 Exp Med 1998; 188(9):1751–1756 493 SC 491 39 Drost EM, MacNee W Potential role of IL-8, platelet-activating factor and TNF-α in the sequestration of neutrophils in the lung: effects on neutrophil deformability, adhesion 495 receptor expression, and chemotaxis Eur J Immunol 2002; 32(2):393–403 496 M AN U 494 40 Roberts PJ, Pizzey AR, Khwaja A, Carver JE, Mire-Sluis AR, Linch DC The effects of interleukin-8 on neutrophil fMetLeuPhe receptors, CD11b expression and metabolic 498 activity, in comparison and combination with other cytokines Br J Haematol 1993; 499 84(4):586–594 500 TE D 497 41 Detmers PA, Powell DE, Walz A, Clark-Lewis I, Baggiolini M, Cohn ZA Differential effects of neutrophil-activating peptide 1/IL-8 and its homologues on leukocyte adhesion 502 and phagocytosis J Immunol Baltim Md 1950 1991; 147(12):4211–4217 504 505 506 507 508 509 510 42 Dumont AL, Nygaard TK, Watkins RL, et al Characterization of a new cytotoxin that AC C 503 EP 501 contributes to Staphylococcus aureus pathogenesis Mol Microbiol 2011; 79(3):814–825 43 Malachowa N, Whitney AR, Kobayashi SD, et al Global changes in Staphylococcus aureus gene expression in human blood PloS One 2011; 6(4):e18617 44 Malachowa N, Kobayashi SD, Braughton KR, et al Staphylococcus aureus leukotoxin GH promotes inflammation J Infect Dis 2012; 206(8):1185–1193 45 Cheung GYC, Otto M The potential use of toxin antibodies as a strategy for controlling acute Staphylococcus aureus infections Expert Opin Ther Targets 2012; 16(6):601–612 20 ACCEPTED MANUSCRIPT 511 512 46 Aman MJ, Adhikari RP Staphylococcal bicomponent pore-forming toxins: targets for prophylaxis and immunotherapy Toxins 2014; 6(3):950–972 47 Sause WE, Buckley PT, Strohl WR, Lynch AS, Torres VJ Antibody-Based Biologics and 514 Their Promise to Combat Staphylococcus aureus Infections Trends Pharmacol Sci 2016; 515 37(3):231–241 RI PT 513 516 517 SC 518 M AN U 519 520 521 522 526 527 528 529 530 531 EP 525 AC C 524 TE D 523 532 533 534 535 536 21 ACCEPTED MANUSCRIPT 537 FIGURE LEGENDS 538 Figure Human PMNs from different donors display variable sensitivity towards LukGH 540 PMNs freshly isolated from whole blood of healthy adult donors were co-incubated with LukGH 541 or LukSF-PV for hours in the indicated concentration range Susceptibility to these toxins was 542 assessed in a luminescent cell viability assay measuring cellular ATP content (A) Representative 543 examples from three individual donors, each performed in duplicate (B) Cumulative dose- 544 response from 25 independent experiments with different PMN preparations and donors Error 545 bars indicate SEM SC RI PT 539 M AN U 546 Figure PMN activation via LPS increases sensitivity to LukGH Purified human PMNs 548 were stimulated with 500 ng/mL LPS for hour and then tested for susceptibility towards 549 LukGH (A) and LukSF-PV (B) at the indicated concentration ranges in a luminescent cell 550 viability assay measuring cellular ATP content Data represent three independent experiments 551 using different PMN donors, error bars indicate SEM 552 TE D 547 Figure LPS upregulates CD11b/CD18 expression on human PMNs and selectively 554 increases cell binding of LukGH (A) Purified human PMNs exposed to LPS at the indicated 555 concentrations for hour were stained for CD11b and CD18 and expression levels measured by 556 flow cytometry Results are expressed as relative change compared to untreated cells (B) 557 Binding of LukGH to cells pre-treated with LPS (500 ng/mL) was measured with biotinylated 558 toxin and a streptavidin-conjugated detection reagent by flow cytometry Biotinylated LukS-PV 559 was included as a control Data represent three independent experiments using different PMN 560 donors, error bars indicate SEM AC C EP 553 561 22 ACCEPTED MANUSCRIPT Figure LPS mediated PMN activation increases sensitivity towards LukGH but not to 563 other leukocidins (A) Human PMNs were pre-treated with 500 ng/mL LPS followed by 564 exposure to individual leukocidins in the 0.005 – 100 nM dose range Cell viability was 565 determined after hours in an ATP-based viability assay Potency is shown as nM toxin 566 concentration at the EC50 (B) The effect of LPS (500 ng/mL) on leukocidin receptor staining 567 intensity on human PMNs was assessed with receptor-specific antibodies as indicated Surface 568 expression levels are expressed as relative change compared to untreated cells Data are shown 569 as mean + range from three independent experiments using different donors SC RI PT 562 570 Figure S aureus culture supernatant inversely affects the sensitivity of PMNs to 572 LukGH and the other leukocidins (A) Human PMNs from three different donors were pre- 573 treated with a 5x diluted, sterile-filtered, non-cytolytic bacterial culture supernatant (CS) of a 574 USA300 CA-MRSA mutant lacking the genes for alpha-hemolysin and all bi-component 575 toxins (TCH1516∆hla-lukSF-lukED-hlgABC-lukGH), followed by exposure to LukGH at the 576 indicated concentration range Cell viability was determined after hours in ATP-based 577 viability assays Error bars indicate SEM (B) CS effect on the sensitivity to all other 578 leukocidins was tested in parallel, toxin potency is expressed as nM concentration at the EC50 579 (C) Effect of CS on leukocidin receptor staining intensity was measured by flow cytometry and 580 expressed as relative change compared to untreated cells Data in (B) and (C) are shown as 581 mean + range from three independent experiments using different donors TE D EP AC C 582 M AN U 571 583 Figure S aureus peptidoglycan and delta-toxin sensitize PMNs to LukGH mediated cell 584 death PMNs from three donors were exposed to increasing concentrations of S aureus 585 peptidoglycan and delta-toxin, followed by measurement of LukGH and LukSF-PV sensitivity 586 and flow cytometric analysis of CD11b receptor expression Full titration-curves from ATP 587 viability assays are shown for one representative donor (A, LukGH; B, LukSF-PV); EC50 values 23 ACCEPTED MANUSCRIPT 588 for LukGH and LukSF-PV and changes in CD11b surface staining signals from all three donors 589 are summarized in (C); n.d., no EC50 determined (less than 50% viability observed at lowest 590 toxin concentration tested); Data in (C) are shown as mean + range from three independent 591 experiments using different donors RI PT 592 Figure IL-8 changes the sensitivity of human PMNs to the different leukocidins PMNs 594 from donors were exposed to increasing concentrations of IL-8 for hour and then tested for 595 toxin mediated cell lysis with all five leukocidins (A) Toxin sensitivities, summarized as EC50 596 values for all tested IL-8 concentrations (B) Relative change in leukocidin receptor staining 597 intensity upon PMN exposure to 12.5 nM IL-8 Data are shown as mean + range from three 598 independent experiments using different donors M AN U SC 593 599 Figure LukGH is the dominant cytotoxin during PMN infection by S aureus Purified 601 human PMNs were infected with a USA300 CA-MRSA wild-type strain (black bars) and 602 isogenic toxin gene deletion mutants carrying (grey bars) or lacking (white bars) the gene for 603 LukGH at MOI 10 (A), 50 (B) and 100 (C) for hours PMN viability was measured with a 604 Calcein-AM viability dye and is expressed as percentage relative to non-infected cells Data are 605 presented as mean +/- SEM of two independent experiments with different donors EP AC C 606 TE D 600 607 Figure S1 Activation of human PMNs with LPS results in increased sensitivity to LukGH 608 Purified human PMNs were stimulated with different LPS concentrations for hour and then 609 tested for susceptibility towards LukGH (A) and LukSF-PV (B) in a luminescent cell viability 610 assay measuring cellular ATP content Results for the full LPS concentration range for one 611 representative donor are shown 612 24 ACCEPTED MANUSCRIPT Figure S2 LPS-concentration dependent changes in LukGH sensitivity and CD11b 614 expression PMNs from three donors were exposed to increasing concentrations of LPS, 615 followed by measurement of LukGH and LukSF-PV sensitivity in a luminescent cell viability 616 assay and flow cytometric analysis of CD11b receptor expression EC50 values for LukGH and 617 LukSF-PV and changes in CD11b surface staining signals from all three donors are summarized 618 Error bars indicate SEM RI PT 613 619 Figure S3 LPS mediated PMN activation results in increased CD11b surface expression 621 levels 622 fluorochrome labeled antibodies specific for the leukocidin receptors as indicated Black: 623 unstimulated cells; red: LPS activated cells Filled histograms represent cells stained with 624 receptor specific antibody, open histograms represent cells stained with the respective isotype 625 control antibodies Representative histograms from one PMN donor are shown SC 620 M AN U Neutrophils were exposed to 500 ng/mL LPS for hours and then stained with 629 630 631 632 EP 628 AC C 627 TE D 626 25 AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ... human PMNs by S aureus components selectively increases LukGH mediated cytotoxicity ACCEPTED MANUSCRIPT 233 IL- 8 selectively sensitizes human PMNs for LukGH mediated cytotoxicity IL- 8 (CXCL8) is 235...ACCEPTED MANUSCRIPT Selective Sensitization of Human Neutrophils to LukGH Mediated Cytotoxicity by Staphylococcus aureus and IL- 8 RI PT Philipp Janesch, Harald Rouha, Susanne... impact of IL- 8 on leukocidin toxicity towards 2 38 human PMNs We did not observe changes in LukSF-PV and HlgCB toxicity, but susceptibility 239 towards LukGH greatly increased at low nanomolar IL- 8