Aspergillus fumigatus conidia inhibit tumour necrosis factor- or staurosporine-induced apoptosis in epithelial cells

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1 Interntionl Immunology Advnce Access pulished Decemer 15, 2005 Interntionl Immunology 2005; 1 of 12 doi: /intimm/dxh356 ª The Jpnese Society for Immunology All rights reserved. For permissions, plese e-mil: journls.permissions@oxfordjournls.org Aspergillus fumigtus conidi inhiit tumour necrosis fctor- or sturosporine-induced poptosis in epithelil cells Ndi Berkov 1 *, Syille Lir-Fulleringer 1 *, Frncxoise Féméni 1, Dominique Huet 1, Mrie-Christine Wgner 2, Kmil Gorn 1, Frédéric Tournier 3, Oumïm Irhim-Grnet 4, Jcques Guillot 1,5, René Chermette 1,5, Pscl Boireu 1 nd Jen-Pul Ltgé 4 1 INRA, AFSSA, ENVA, UPVM, UMR 956; 22 rue Curie, Misons Alfort Cedex F-94700, Frnce 2 Plte-Forme de Cytométrie, Institut Psteur, Pris, Frnce 3 Lortoire de Cytophysiologie et Toxicologie Cellulire, Université Pris 7, Pris, Frnce 4 Unité des Aspergillus, Institut Psteur, Pris, Frnce 5 Service de Prsitologie-Mycologie, Ecole Ntionle Vétérinire d Alfort, 7 v de Générl de Gulle, Misons Alfort Cedex 94704, Frnce Keywords: poptosis, epithelil cells, fungi, progrmmed cell deth Astrct A mjor innte immune response to inhled conidi of the opportunistic pthogen Aspergillus fumigtus (Af) is the synthesis of pro-inflmmtory cytokines, which include tumour necrosis fctor (TNF)-, known inducer of poptosis. Modultion of host cell poptosis hs een reported to e one of the mechnisms wherey pthogens overcome host cell defences. Our study ws designed to investigte whether or not Af conidi could modulte poptosis induced y TNF- or sturosporine (STS). Exposure of epithelil cells treted y these inducers nd exposed to Af conidi decresed the numer of poptotic cells detected y Annexin V stining, nlysis of nucler morphology, terminl deoxynucleotidyl trnsferse-medited fluorescein dutp nick end-lelling rection nd immunolotting. Inhiition of poptosis y Af conidi ws seen in cells of the A549 pneumocyte II line, humn trchel epithelil 16HBE nd primry humn respirtory cells. Inhiition of poptosis y Af conidi ws lso oserved when poptosis ws induced y co-cultivting A549 cells with ctivted humn lveolr mcrophges. Unlike Af conidi, conidi of Cldosporium cldosporioides s well s ltex eds or killed Af conidi hve no inhiitory effect on TNF- or STS-induced poptosis. For TNF-induced poptosis, the oserved nti-poptotic effect of Af conidi ws found to e ssocited with significnt reduction of cspse-3. Introduction Aspergillus fumigtus (Af) is sprophytic mould, which is responsile for the mjority of invsive mycosis in ptients undergoing chemotherpy or orgn trnsplnttion (1). It propgtes through irorne conidi (spores), which re inhled into the smll irwys where they my germinte nd initite n infection. The invsion of irwy epithelil cells is therefore key step in the etiology of spergillosis, especilly since it hs een shown tht these cells re le to phgocytose Af conidi which cn survive t lest for some time in these cells (2 4). Pthogenic micro-orgnisms hve evolved different mechnisms to survive in the host environment: modultion of host cell poptosis is one of these mechnisms. Escherichi coli (5) nd Cndid licns (6) hve een found to induce poptosis in neutrophils, nd Cryptococcus neoformns hs een shown to induce poptosis in inflmmtory cells in grnuloms of rts with cryptococcl meningitis (7, 8). In contrst, inhiition of host cell poptosis y the yest form of Histoplsm cpsultum nd the protozo Theileri prv nd Toxoplsm my provide n intrcellulr niche for the pthogens y extending the life spn of infected host cells (9 11). Previous studies hve shown tht gliotoxin, the most undnt mycotoxin produced y Af, induces poptosis in mny types of cells (12). However, this secondry metolite Downloded from t Pennsylvni Stte University on My 12, 2016 *These uthors mde n equl contriution to this work. Correspondence to: N. Berkov; E-mil: nerkov@vet-lfort.fr Trnsmitting editor: A. Cooke Received 7 Decemer 2004, ccepted 17 Octoer 2005

2 2 Aspergillus fumigtus conidi inhiit poptosis is only produced y hyphe nd nothing is known out the influence of conidi of Af on host cell poptosis. Apoptotic cells show chrcteristic morphologicl chnges, including cell shrinkge, nucler/chromtin condenstion, internucleosoml clevge of DNA, memrne leing nd formtion of poptotic odies (13). Cspses, which re responsile for mny iochemicl nd morphologicl chnges ssocited with poptosis, re synthesized s inctive proenzymes, most of which hve to e ctivted y proteolytic clevge (14). There re vrious lterntive inducers of poptosis such s the proteins of the tumour necrosis fctor receptor (TNFR) fmily nd stimuli such s cytotoxic gents, cell metolites nd other pthologicl insults (15, 16). The receptor-medited (extrinsic) signlling pthwy involves the formtion of deth-inducing signlling complex (DISC), with susequent ctivtion of the inititor cspse, which either directly ctivtes the effector cspses or cleves cytosolic fctors, leding to the relese of cytochrome-c nd ctivtion of nother inititor cspse, nd susequently of effector cspses (17 19). The chemiclly medited (intrinsic) pthwy, which is clssiclly linked to mitochondril disturnce, is chrcterized y the relese of cytochrome-c from mitochondri, following the ctivtion of the inititor cspses, which in turn ctivte effector cspses (14, 16). Cspse-3 is one of the effector cspses which is situted t nodl points in oth poptotic pthwys. When ctivted y proteolytic clevge, cspse-3 cleves vitl cellulr proteins involved in the poptotic process (19, 20). However, cspseindependent poptotic pthwy hs lso een identified recently (21, 22). The studies descried in this report were designed to investigte whether Af conidi could induce or modify poptosis nd hence ffect host cell survivl. Exposure of lveolr mcrophges to Af conidi results in the synthesis of TNF- (23), which in turn my induce poptosis in epithelil cells. Here, we descrie in vitro experiments which hve een minly crried out with trnsformed humn type II pneumocyte cell line (A549) s model for lveolr epithelil cells, using TNF- nd sturosporine (STS) s inducers of poptosis. Cycloheximide (CXH), trnsltionl inhiitor, hs een used together with TNF, to fvour DISC-dependent ctivtion of inititor cspse (24 26). STS ws selected s n lterntive inducer of poptosis for the investigtion of the chemicl-medited pthwy. The results of the morphologicl nlysis of stined nuclei, flow cytometry, terminl deoxynucleotidyl trnsferse-medited fluorescein dutp nick end-lelling (TUNEL) ssy nd western lot nlysis demonstrted tht Af conidi only slightly induce poptosis in untreted A549 cells. In contrst, Af conidi drsticlly inhiit poptosis initited y either TNF- or STS. The mechnism of inhiition of poptosis following STS tretment remins unknown, while conidi-medited inhiition of cytokine-induced deth ppers to e cspse-3 dependent. The inhiition of poptosis y Af conidi ws lso oserved in the humn trchel epithelil 16HBE cell line, in irwy epithelil primry culture cells s well s in A549 cells co-cultivted with ctivted humn lveolr mcrophges (HAM) cells in the sence of the ddition of TNF or STS: this points to the iologicl significnce of our findings. Methods Fungl strins nd growth conditions Af CBS nd Cldosporium cldosporioides IP1232 (Institut Psteur, Pris, Frnce) were used throughout this study. Conidi of Af nd C. cldosporioides were otined from cultures grown on YM gr (0.3% yest extrct, 2% mlt extrct, 0.5% peptone nd 0.5% gr) for 3 dys t 37 C nd 25 C, respectively. Conidi were hrvested y flooding the pltes with sterile distilled wter nd then suspending the conidi in 0.15 M PBS. For some experiments, Af conidi were killed y incution in 3% PFA solution overnight followed y wshings with 0.2 M glycine nd PBS. Humn cells nd growth conditions Type II pneumocyte cell line A549 derived from humn lung crcinom ws otined from Americn Type Culture Collection [ATCC CCL 185 (27)] nd mintined in Kighn s modifiction of HAM s F12 medium supplemented with 10% of FCS (Invitrogen), pen/strep (16 mg ml ÿ1 penicillin nd 100 mg l ÿ1 streptomycin), 2 mm L-glutmine nd 1.5 g l ÿ1 sodium icronte. The cells were grown until confluent. Unless specified otherwise, ll incutions were t 37 C in n incutor with humidified tmosphere of 5% CO 2. Trypsin/ EDTA (Invitrogen) ws used to relese dherent cells for suculturing when this ws required. Humn trchel epithelil SV40-trnsformed cells (16HBE) were provided y Boisvieux-Ulrich (Cytophysiology nd Toxicology lortory, University of Pris 7, Frnce). 16HBE cells were mintined in Egle MEM medium (Invitrogen) with 2% UltroserG (Invitrogen), pen/strep, 2 mm L-glutmine (Sigm) nd 1.5 g l ÿ1 sodium icronte (Sigm) nd were grown until confluent (28). Primry epithelil cells were otined from humn nsl turintes (HNT) of ptients undergoing turinectomy (P. Hermn, CHU Lrioisière, Pris, Frnce) s descried previously (29). Briefly, HNT were wshed in Dulecco s modified Egle medium DMEM/F12 (Invitrogen) nd incuted with 2 mg ml ÿ1 pronse (Protese XIV; Sigm,) in DMEM/F12 supplemented with pen/strep, t 4 C for h under slow rotry gittion (80 r.p.m.). After wshing, ggregtes were discrded nd dissocited cells were filtered using 30-lm pore filter. The cell suspension ws then plted for 2 h t 37 C on plstic dishes (Flcon) to eliminte contminting firolsts. After centrifugtion, the superntnt contining the epithelil cells ws plted on collgen type I-coted wells nd cultivted in DMEM/F12 supplemented with 10% FCS nd pen/strep. HAM were otined from roncholveolr lvge of ptient who hd received lung trnsplnt (B. Philippe, Foch Hospitl, Suresnes, Frnce). Cells were hrvested y centrifugtion (400 3 g for 10 min t 4 C), re-suspended in RPMI complete medium contining 10% FCS nd seeded on cell culture pltes t 10 6 per well. Non-dherent cells were removed y wshing fter 2 h incution. Induction of poptosis Either A549 or 16HBE cells were seeded t cells per well in 1 ml of DMEM/F12 on 18-mm-dimeter cover slips Downloded from t Pennsylvni Stte University on My 12, 2016

3 (Mrienfeld, Germny) in 12-well pltes (Nunc, Nuclon TM Surfce) in triplicte nd grown for 16 h t 37 C. Primry epithelil cells were seeded t cells per well nd grown for 48 h. After wshing the cover slips with PBS BSA (PBS-5% BSA, Frction V, Sigm), the inducers of poptosis were dded to the wells in 1 ml of medium. To induce poptosis, two different types of inducers were used: (i) 1 lm STS (Sigm) nd (ii) 20 ng ml ÿ1 of TNF- (Sigm) with 2 lg of CHX (Sigm). After 7 h of incution conidi germinted, however, the poptotic fetures of cells, incuted with TNF- in the sence of CHX, ppered much lter (dt not shown). To efficiently induce poptosis nd decrese the time of incution, TNF- ws used together with CHX, since CHX hs een shown to suppress survivl signls induced y n enggement of TNFR (23). The presence of CHX is therefore only required to llow us to study the role of conidi on induced poptosis in the sence of conidil germintion. To investigte erly stges of poptosis y flow cytometry, the induction of poptosis ws performed for 1 h, period when mximl numers of poptotic cells nd miniml numer of necrotic cells were detected (dt not shown). The control cells were incuted either with CHX or medium lone. We did not detect ny difference etween CHX-treted cells (dt not shown) nd cells incuted with medium lone. None of the tretments inducing poptosis ffected the viility of conidi (dt not shown). To study the lte stges of poptosis, A549 cells were incuted with STS or TNF- for 6 h: this durtion of tretment ws defined y results reported y others (25, 30) nd on our own unpulished oservtion showing the sence of the 17-kD ctive form of cspse-3 fter 4 h of incution. HAM cells hve lso een used s source of TNF for poptosis induction. In order to estimte the optiml incution time, preliminry ssy of poptosis induction in A549 with 20 ng ml ÿ1 of TNF- during different periods (6 h, 24 h nd 48 h) ws performed. The highest numer of poptotic cells ws found fter 24 h incution (dt not shown). Therefore, for 24-h incution, different doses of TNF- were tested y morphologicl criteri for poptosis induction in A549 cells. Apoptotic fetures of cells were oserved t concentrtion of TNF- s low s 5 ng ml ÿ1 of TNF- (dt not shown). A549 cells were seeded t cells per well on 18-mm dimeter cover slips in 12-well pltes nd grown for 16 h. After wshing with PBS-5% BSA, the cover slips ering A549 cells were trnsferred onto the surfce of n 18-h-old culture of lveolr mcrophges stimulted with 1 lg ml ÿ1 of LPS for the sme time of culture. Under these culture conditions, HAM produced 10 ng ml ÿ1 of TNF-, s mesured in culture superntnt using commercil ELISA kit (BD Biosciences). Exposure to conidi Following wshing of A549, 16HBE or primry culture cells with PBS, Af conidi rnging from to 10 8 conidi per millilitre of medium were dded to the cells simultneously with the inducer of poptosis. In experiments when ctivted HAM hve een used s source of TNF, 10 7 conidi per millilitre hve een dded to the cells. The lower concentrtion of conidi ws chosen on the sis of the results of Wsylnk nd Moore (31); the higher concentrtions were chosen to Aspergillus fumigtus conidi inhiit poptosis 3 investigte dose dependence. After incution, unound conidi were removed y wshing wells with PBS-5% BSA. The resulting numer of poptotic cells ws compred with cultures incuted either with the inducers or the medium (control). To test for the specificity of the effect of live Af conidi, some wells received insted to 10 8 C. cldosporioides conidi, ltex eds or PFA-killed Af conidi. Flow cytometry nlysis A549 cells were lelled with n FITC Annexin V nd stined with propidium iodide (PI) using n poptosis detection kit (Sigm). The procedure consists of (i) the inding of FITC Annexin V to phosphtidylserine which trnsloctes from the interior to the exterior of the cell memrnes t the strt of the poptotic process nd () the inding of PI to DNA in cells where the memrne hs een totlly compromised (32). A549 cells were prepred s descried ove. Induction of poptosis ws performed for 1 h. Hrvested cells were centrifuged for 5 min t g nd fter wshing with PBS were re-suspended in inding uffer (10 mm HEPES/NOH, ph 7.5 contining 140 mm NCl nd 2.5 mm CCl 2 )t concentrtion of cells per millilitre. Five microlitres of 1:10 dilution of FITC Annexin V conjugte (50 lg ml ÿ1 in 50 mm Tris HCl, ph 7.5, contining 100 mm NCl) nd 10 ll of PI solution (100 lgml ÿ1 in 10 mm potssium phosphte uffer, ph 7.4, contining 150 mm NCl) were dded to 500 ll of cell suspension nd incuted in the drk t room temperture for 15 min. A suitle dilution of FITC Annexin V conjugte ws determined in preliminry experiments. The reltive level of poptotic cells ws detected using FACScn system (BD Biosciences) using CellQuest 3.3 softwre (33). Detection of poptotic ody formtion nd chromtin condenstion Nuclei of the control cells, of cells treted with the inducers of poptosis for 6 h nd of cells exposed to Af conidi in the presence or sence of inducers, together with untreted control cells, were stined with DNA inding dye Hoechst nd then nlysed using fluorescence microscope. The cover slips were wshed with PBS-5% BSA nd the cells fixed with PBS-4% (wt/vol) PFA (ph 7.4) for 1 h. Chromtin condenstion nd poptotic ody formtion ws ssessed y stining with Hoechst (1.5 lm; Sigm) for 30 min t room temperture. After wshing, the cover slips were mounted on slides with ProLong ntifde Vectshield (Vector Lortory). Smples were viewed with Zeiss fluorescence microscope using 3400 mgnifictions. For ech smple, cells from five rndom fields were counted nd the percentge of poptotic cells ws clculted s the numer of poptotic cells divided y the totl numer counted multiplied y 100. TUNEL ssy of DNA frgmenttion TUNEL technology, sed on lelling of DNA strnd reks, ws performed using n In Situ Cell Deth Detection kit (Roche Moleculr Biologicls) (34). After induction of poptosis for 6 h t 37 C, cover slips were wshed with PBS-5% BSA, the cells were fixed with PBS-4% (wt/vol) PFA (ph 7.4) for 1 h nd then permeilized in solution contining 0.1% Triton X-100/0.5% Downloded from t Pennsylvni Stte University on My 12, 2016

4 4 Aspergillus fumigtus conidi inhiit poptosis sodium citrte for 30 min t room temperture. Cover slips were wshed gin nd 50 ll of TUNEL rection mixture ws dded for 1 h nd incuted in humidified drk chmer t 37 C. After detection of incorported FITC y nti-fitc ntiody, conjugted with lkline phosphtse, sustrte ws dded nd stined cells were nlysed y light microscopy. Cspse nlysis In order to monitor the presence of oth pro-cspse-3 nd the ctive form of cspse-3, western lot nlysis of the cell lystes ws performed. Following the induction of poptosis, cells exposed (or not) to Af conidi nd untreted controls without conidi were simultneously hrvested y trypsinistion. After centrifugtion t g, the cell pellets were resuspended in 30 ll of uffer contining 150 mm NCl, 50 mm Tris HCl, ph 8.0, 0.1% triton, 0.1% SDS nd cocktil of protese inhiitors: 2.5 mm orthovndte, 10 mm prnitrophenylphosphte, 10 lg ml ÿ1 leupeptine nd 10 lg ml ÿ1 protinin. The resulting lystes were centrifuged for 10 min t g t 4 C. Protein concentrtion ws determined using icinchoninic cid-sed protein ssy (Interchim) nd equl mounts of protein were mixed with 23 Lemmli smple uffer (62.5 mm Tris HCL ph 6.8; 8.4% SDS, 5% mercptoethnol, 8.5% glycerol, 0.25% romophenol lue) nd mintined t 100 C for 7 min efore loding 20 lg of cell extrct per lne onto 15% SDS-polycrylmide gels (35). After electrophoresis, the seprted proteins nd moleculr weight stndrds were electrophoreticlly trnsferred onto 0.2 lm Immuno-Blot PVDF memrnes. The memrnes were locked with 10% skimmed milk in 0.1 M Tris uffer, 0.9% NCl, 0.05% Tween 20, ph 7.2, nd 3% norml got serum (Sigm), incuted for 3 h with polyclonl rit ntiody specific for humn cspse-3 (Acm, UK) diluted 1:250 in the locking solution nd fter wshing were incuted with got nti-rit ntiody (1:1000) coupled with peroxidse (Sigm) for 1 h. The memrnes were then wshed in PBS nd incuted with enhnced chemoluminescence regents (Amershm). The opticl density of the nds, with the reltive moleculr weights 32 nd 17 kd corresponding to procspse-3 nd the ctive form of cspse-3, ws estimted using the ImgeQunt system. To compre the intensities of the nds in the different lines, the vlues of the nds were divided y the vlue of the nd corresponding to procspse-3 in the control untreted cells without conidi. To confirm the involvement of cspse, different concentrtions of the cspse inhiitor Z-VAD.FMK or FMK negtive control rnging from to 0.5 mm were dded simultneously with the inducer of poptosis to the cells exposed to conidi. The percentge of poptosis ws ssessed ccording to the nucler morphology of the cells stined with 1.5 lm Hoechst Sttisticl nlysis At lest three different ssys were performed per experiment. The differences in the numer of poptotic cells for TNFnd STS-induced poptosis exposed or not to conidi were ssessed y nlysis of vrince. P-vlues <0.05 were considered to e significnt. Tukey s honestly significnt difference test ws pplied for comprison of mens etween groups. Mens followed y the sme letter re not significntly different. The vlues re expressed s men 6 SEM. Results Inhiition of poptosis in A549 cells exposed to Af conidi Flow cytometry nlysis. Flow cytometry of FITC Annexin V-lelled cells stined with PI llowed n nlysis of the erly stges of poptosis of the host cells. At the highest concentrtion, Af conidi inhiited TNF- nd STS-induced poptosis of A549 cells. The level of poptotic cells, fter 1 h incution with TNF or STS ws % nd %, respectively: n exmple of one replicte is shown in Fig. 1(A). TNF tretment of cells nd simultneous exposure to Af conidi leds to sttisticlly significnt (P < 0.05) dose-dependent decrese of the numer of poptotic cells from % in the controls to % with 10 8, to % with 10 7 nd to % with conidi (Fig. 1B). The equivlent effect in STS-treted cultures ws from % in controls to %, % nd %, respectively (P < 0.05). In n untreted control, % poptotic cells were found. No poptosis ws induced in the cultures following ddition of 10 7 or Af conidi. Spontneous poptosis ws wekly incresed to % when cells were exposed to 10 8 conidi (P < 0.05) (Fig. 1B). Detection of poptotic ody formtion nd chromtin condenstion The nti-poptotic role of Af conidi ws verified on dvnced stges of poptosis, when TNF- or STS-treted cells were simultneously exposed to conidi for 6 h (see Fig. 2(A) s representtive exmple of the inhiition of lte stges of poptosis y conidi). Quntifiction of the differences in the numer of poptotic cells for TNF- nd STS-induced poptosis showed tht % of TNF-treted nd % of STS-treted cells displyed fetures of poptosis, while CHX-treted cells (dt not shown) or untreted control cells were unffected y poptosis (Fig. 2B). Exposure of A549 cells to Af conidi inhiited the poptotic process: the numer of poptotic cells induced y oth inducers ws significntly decresed (P < 0.05) in the smples of cells treted with the inducers nd exposed to Af conidi (Fig. 2B). Figure 2 shows tht the nti-poptotic effect of Af conidi ws dose dependent. Exposure to , 10 7 nd 10 8 conidi decresed the numer of poptotic cells from % in control to %, % nd % for TNF-induced poptosis nd from % in control to %, % nd % (P < 0.05) for STS-induced poptosis, respectively. In cell cultures contining conidi without inducers of poptosis, 1.5% of the cells were poptotic. Detection of nucler DNA nicks using TUNEL technology The inhiitory effect of Af conidi on dvnced stge of poptosis ws confirmed y TUNEL. After 6 h incution, the cells treted either with TNF- or STS hve shown intense positive TUNEL stining in comprison with control cells or cells exposed only to Af conidi (Fig. 3). The numer Downloded from t Pennsylvni Stte University on My 12, 2016

5 Aspergillus fumigtus conidi inhiit poptosis 5 Fig. 1. (A) An exmple of contour digrm of FITC Annexin V/PI otined y flow cytometry of A549 cells exposed to Af conidi nd treted with the inducers of poptosis is shown. FITC Annexin V fluorescence is presented on the horizontl xis nd PI fluorescence on the verticl xis. The lower left qudrnt of the cytogrms represents the vile cells (FITC ÿ /PI ÿ ) nd the upper right qudrnt represents the necrotic cells (FITC + /PI + ), while poptotic cells were recorded in the lower right qudrnt (FITC + /PI ÿ ) nd ded cells in the upper left qudrnt (FITC ÿ /PI + ). In ech cse, the fluorescence of cells ws ssessed. Percentge of the erly-stge poptotic cells is mrked in the right lower qudrnt. Arevitions: (A549), control untreted A549 cells; (A549 + STS), A549 cells treted with 1 lm of STS; (A549 + STS conidi), A549 cells treted with 1 lm of STS nd exposed to 10 8 of conidi; (A conidi), A549 cells exposed to 10 8 of conidi; (A549 + TNF), A549 cells treted with 20 ng ml ÿ1 of TNF- nd 2 lg of CXH; (A549 + TNF conidi), A549 cells treted with 20 ng ml ÿ1 of TNF-, 2lg of CXH nd exposed to 10 8 of conidi. (B) Inhiition of poptosis is depending on the dose of the conidi. The percentge of poptotic cells ws clculted with dt from triplictes of four experiments. Mens followed y the sme letter re not significntly different. +, presence; ÿ, sence of TNF, STS or conidi. Downloded from t Pennsylvni Stte University on My 12, 2016 of poptotic cells fter TNF nd STS tretment reched % nd %, respectively. In contrst, control cells remined TUNEL negtive, while only 1% of cells exposed to 10 7 conidi per millilitre were TUNEL positive. The TUNEL ssy hs lso confirmed tht exposure to conidi inhiited TNF- nd STS-induced poptosis in A549 cells. Exposure to 10 7 conidi per millilitre decresed the numer of TUNEL-stined cells in TNF-treted culture from % to % (P < 0.05) nd in STS-treted culture from % to 5 6 2% (P < 0.05).

6 6 Aspergillus fumigtus conidi inhiit poptosis Fig. 2. (A) Conidil inhiition of poptotic ody formtion/chromtin condenstion induced y 6 h of STS or TNF tretment. After 6 h of tretment with conidi (the lte stge of poptosis), in the presence or sence of the inducers of poptosis (1 lm STS or 20 ng ml ÿ1 of TNF nd 2 lg of CXH per millilitre), cells were fixed with PBS-4% PFA for 1 h. Chromtin condenstion nd poptotic odies formtion were ssessed y stining with Hoechst Solid rrows indicte poptotic odies. The roken line rrows indicte Af conidi. One representtive experiment is shown. (B) Inhiition of lte stge of poptosis lso depends on the dose of the conidi. The percentge of cells with poptotic odies ws computed from triplictes of four experiments. Mens followed y the sme letter re not significntly different. +, presence; ÿ, sence of TNF, STS or conidi. Arevitions re s in Fig. 1. Impliction of cspse-3 in the inhiition of TNF-induced poptosis y Af conidi Since cspse-3 hs een implicted s generl effector of poptosis (36), we used western lot nlysis to test whether or not cspse-3 ctivtion took plce in cells treted with the inducers of poptosis nd Af conidi. Anti-cspse-3 ntiody detected 32-kD nd corresponding to pro-cspse-3 in the control cell lyste (Fig. 4). After 6 h of TNF tretment, the 32-kD nd disppered nd 17-kD nd corresponding to the ctive form of cspse-3 ppered. In TNF-treted cells exposed to Af conidi, the intensity of the 17-kD nd decresed, while the 32-kD pro-cspse-3 ws not detected. The effect ws dose dependent: the rtio of the intensity of the 17-kD nd in control to tht in TNF-treted cells ws 0.80, which dropped to 0.75 nd 0.30 for cultures treted with 10 7 nd 10 8 conidi, respectively. The intensity of the 17-kD nd in lyste of the cells exposed to conidi ws equl to those in TNF-treted cells without conidi (dt not shown). These results suggest tht conidil inhiition of poptosis in TNF-treted A549 cells ws due to the degrdtion of either cspse-3 or pro-cspse-3. In contrst, in the lystes of cells treted with STS or cells treted with STS nd 10 7 or 10 8 conidi, the 32-kD nd only ws oserved. The results showed tht under our experimentl conditions, STS-triggered poptosis of A549 cells ws cspse-3 independent. Involvement of cspse during TNF-induced poptosis ws confirmed y the use of the cspse inhiitor Z-VAD.FMK. Exposure to Z-VAD.FMK did not ffect STS-induced poptosis (Fig. 5). Supplementtion of conidi with to 0.5 mm Z-VAD. FMK did not increse the inhiition of poptosis induced y the conidi (Fig. 5). In contrst, Z-VAD. FMK inhiited TNF-induced poptosis in dose-dependent mnner, while Downloded from t Pennsylvni Stte University on My 12, 2016

7 Aspergillus fumigtus conidi inhiit poptosis 7 Fig. 3. Exmple of the inhiition of poptotic cell deth y Af conidi using TUNEL. The rrows indicte positive TUNEL stining. One representtive experiment is shown. Sme tretment s cells of Fig. 2; legend nd revitions s in Fig. 1 cption. Fig. 4. Western lot nlysis of pro-cspse-3 processing in TNF- nd STS-treted A549 cells. Control A549 cells, cells exposed to Af conidi in the presence or sence of inducers of poptosis (1 lm STS or 20 ng ml ÿ1 of TNF nd 2 lg of CXH per millilitre) nd cells treted only with the inducers of poptosis for 6 h (the lte stge of poptosis) were hrvested nd cell extrcts were used for western lot nlysis. Pro-cspse-3 nd cspse-3 were detected y nti-cspse-3 ntiody. At the left side, the moleculr weight stndrds re shown. The rrows indicte ctive 17-kD cspse-3. The vlues of the nd intensities were divided y the vlue of the nd corresponding to pro-cspse-3 in the control cells. The rtio is shown t the ottom of the figure. Arevitions re s in Fig. 1. Downloded from t Pennsylvni Stte University on My 12, 2016 control cultures without the regent showed no chnge in the numer of poptotic cells (Fig. 5). Furthermore, it ws shown tht the inhiitory effect of Z-VAD.FMK ws dditive to the effect induced y conidi, suggesting tht the inhiitions y Z-VAD.FMK nd conidi might oth trget cspse (Fig. 5). Inhiition of poptosis in TNF- nd STS-treted cells is induced specificlly y Af conidi As shown in Fig. 6, Af conidi strongly inhiited TNFinduced poptosis from % to % for STS-induced poptosis nd from % to % for STS-induced poptosis. Cldosporium cldosporioides conidi, even t doses s high s 10 8, hd no inhiitory effect. Percentge of poptotic cells exposed to C. cldosporioides ws % versus % in control cells for TNF-induced poptosis nd % versus % in control cells for STS-induced poptosis, respectively (P > 0.05). Moreover, exposure to 2-lm ltex eds or PFA-fixed conidi did not induce significnt reduction of poptosis induced y STS or TNF (Fig. 6). These results support the conclusion tht the inhiition of poptosis is specific nd depends on live conidi of Af.

8 8 Aspergillus fumigtus conidi inhiit poptosis A 60 STS C 10 c c c 0 Conidi - 5x10 6-5x x10 6 5x10 6 5x10 6 5x10 6 Z-VAD.FMK (mm) STS FMK. - (mm) Percentge of poptotic cells B TNF- Percentge of poptotic cells Conidi - 5x10 6-5x x10 6 5x10 6 5x10 6 5x10 6 Z-VAD.FMK (mm) TNF FMK. - (mm) Af conidi inhiit poptosis in cells other thn A549 Inhiition of poptosis following exposure to (10 7 ) conidi lso occurred in humn trchel epithelil 16HBE cells treted with TNF nd STS, (Fig. 7) lthough the 16HBE cells were less sensitive to the poptotic inducers thn A549 cells: for exmple, for TNF nd STS, 16HBE cells showed tht 16% nd 18% of cells, respectively, were poptotic, the reltive numers were 4% nd 6% fter exposure to conidi. There were no signs of poptosis in cultures exposed only to 10 7 conidi or in control cultures receiving no conidi. After 6 h of incution with TNF or STS, epithelil primry cells otined from HNT displyed poptotic fetures in % of TNF-treted cells nd % of STS-treted cells while untreted control cells were unffected y poptosis (Fig. 8). The exposure of HNT cells to Af conidi inhiited TNF- or STSinduced poptosis. Exposure to 10 7 conidi decresed the numer of poptotic cells from % to 4 6 3% (P < 0.05) for TNF-induced poptosis nd from % to % (P < 0.05) for STS-induced poptosis (Fig. 8). There were no signs of poptosis in cell cultures contining 10 7 conidi without inducers of poptosis or in control cultures receiving no conidi. Exposure to PFA-fixed conidi did not reduce the numer of poptotic cells significntly. C Fig. 5. Cspse inhiitor Z-VAD.FMK does not modulte STS-induced poptosis (A) ut increses conidil inhiition of TNF-induced poptosis (B). A549 cells were seeded t cells per well on cover slips nd grown for 16 h t 37 C. Cspse inhiitor Z-VAD.FMK or FMK negtive control rnging from to 0.5 mm ws dded simultneously with the inducer of poptosis to the cells exposed or not to conidi. Nucler morphology ws ssessed y stining with the nucler dye Hoechst The percentge of the lte-stge poptotic cells ws clculted from four experiments. Results re presented s mens 6 SEM. Mens followed y the sme letter re not significntly different. +, presence; ÿ, sence of TNF, STS, conidi or inhiitor. d e f f Conidi lso inhiited the poptosis of A549 cells induced y their co-culture with LPS-ctivted HAM (s the source of TNF for poptosis induction). In the co-culture growth conditions, the mount of poptotic cells ws low (6 6 1%). After exposure to Af conidi, the numer of poptotic cells ws significntly reduced (to %) (P < 0.05) (Fig. 9). Discussion The influence of Af conidi on host cell poptosis hs een investigted in vitro using epithelil cells since they re the likely primry trget of inhled Af conidi. We exmined the effect of Af conidi on poptosis of A549 cells treted with TNF-. TNF- ws selected s n inducer of poptosis since its role during Af infection hs een well estlished: the relese of TNF- hs een demonstrted ex vivo, using whole lood from helthy volunteers, stimulted y non-vile conidi nd hyphl frgments from Af (23). Furthermore, it hs een shown tht TNF- is produced in the lungs of norml nd immunocompromised mice (2 nd 6 ng ml ÿ1, respectively) in nimls responding to the intr-trchel dministrtion of Af conidi t concentrtions tht re comptile with those used in this study (37, 38). Resident lveolr g h Downloded from t Pennsylvni Stte University on My 12, 2016

9 Aspergillus fumigtus conidi inhiit poptosis Percentge of poptotic cells Conidi Ltex eds PFA-conidi TNF STS A. fumigtus A. fumigtus C C.cldosporioides c - 5x10 6-5x10 6 5x x10 6-5x10 6 5x x x x x Fig. 6. Af conidi specificlly inhiit TNF- nd STS-induced poptosis of A549 cells. A549 cells were treted with the inducers of poptosis (1 lm STS or 20 ng ml ÿ1 of TNF nd 2 lg of CXH per millilitre) for 6 h in the presence or sence of conidi of Af, Cldosporium cldosporioides, PFA-fixed conidi or ltex eds. Apoptotic cells were ssessed y stining with nucler dye Hoechst The percentge of poptotic cells ws clculted from triplictes of four experiments. Results re presented s mens 6 SEM. Mens followed y the sme letter re not significntly different. +, presence; ÿ, sence of TNF, STS or conidi. 16 HBE Percentge of poptotic cells Conidi TNF STS c - 5x10 6-5x10 6-5x Fig. 7. Af conidi decresed the numer of poptotic cells in 16HBE cells. HBE cells were treted for 6 h with conidi, in the presence or sence of the inducers of poptosis. Chromtin condenstion nd poptotic ody formtion were ssessed y stining with Hoechst The numer of poptotic cells ws compred with control nd with cultures incuted either with the inducers or with conidi. The percentge of poptotic cells ws clculted on the se of the dt from four experiments. Mens followed y the sme letter re not significntly different. +, presence; ÿ, sence of TNF, STS or conidi. A. fumigtus A. fumigtus c C.cldosporioides c Downloded from t Pennsylvni Stte University on My 12, 2016 mcrophges re responsile for the erly relese of TNF-, wheres the lter relese nd higher levels of this cytokine re produced y recruited neutrophils (37). Our study hs shown for the first time tht Af conidi do not promote poptosis of epithelil cells ut in contrst re le to inhiit erly, s well s lte stges of drug-induced poptosis. The iologicl significnce of this finding is emphsized y the inhiition of poptosis of A549 cells co-incuted with humn-ctivted mcrophges or humn respirtory epithelil primry culture cells. Altertion of poptosis could ply pivotl role in the first step of the interction etween Af nd the host since it would llow intrcellulr conidium survivl with susequent germintion nd host invsion. It hs een reported tht mny pthogenic gents interct t check(nodl)-points in the cscde of events leding to poptosis (39). For exmple, culovirus contins n ntipoptotic fctor tht inhiits cspses (40); EBV (41) nd herpes virus (42) encode proteins tht re homologues of the cellulr nti-poptotic protein Bcl-2. Thus, in the cse of Af conidi strong inhiition of induced poptosis yet lso show wek ugmenttion of spontneous poptosis. The complexity of the poptotic process is illustrted in our study where Af conidi re shown to hve n inhiitory effect nd the response to Af is morphotype specific. Such findings were repetedly oserved in immunologicl rections to conidi nd hyphe of Af (43, 44). In the cse of poptosis,

10 10 Aspergillus fumigtus conidi inhiit poptosis Fig. 8. Inhiition of poptosis in irwy primry culture cells exposed to Af conidi. Primry epithelil cells otined from the HNT were seeded t cells per well on cover slips nd grown for 48 h t 37 C. Induction of poptosis with STS nd TNF nd exposure to conidi hve een performed s descried ove. (A) Anlysis of nucler morphology. The rrows indicted poptotic odies. (B) Quntifiction of the poptotic inhiition. Mens followed y the sme letter re not significntly different. +, presence; ÿ, sence of either TNF, STS or conidi. A549+HAM Percentge of poptotic cells Conidi LPS - 5x10 6-5x Fig. 9. Af conidi inhiit poptosis induced y co-culture of HAM with A549 cells. HAM were stimulted with 1 lg ml ÿ1 of LPS nd grown for 18 h t 37 C. Susequently the cover slips contining A549 cells were trnsferred into the wells with HAM nd exposed or not to Af conidi. The numer of A549 poptotic cells ssessed y stining with dye Hoechst ws compred in co-culture contining either control HAM or non-stimulted HAM incuted with conidi. Mens followed y the sme letter re not significntly different. +: presence; ÿ: sence of either LPS or conidi. c the vrious fungl forms elicit different response: conidi inhiit poptosis wheres hyphe secrete gliotoxin, well known inducer of poptosis in mny cell types, including mcrophges nd monocytes (12, 45 47). Processing of pro-cspse-3 ws oserved in TNF-induced poptotic cells, resulting in the complete loss of n inctive 32-kD pro-cspse-3 nd the ppernce of n ctive 17- kd form. We hve shown tht the level of ctive cspse-3 s well s pro-cspse-3 ws inhiited y the exposure of TNF-treted cells to Af conidi. We do not know whether the inhiition of TNF-induced poptosis y Af conidi tkes plce upstrem or downstrem of cspse-3 ctivtion. A vriety of pthogens cn inhiit cspse-3, for exmple, the ntipoptotic effect of cytomeglovirus infection ws shown to e ssocited with significnt reduction of cspse-3 ctivity (48), nd the intrcellulr pthogen Leishmni mjor ws found to inhiit the spontneous poptosis of neutrophils vi mechnism involving the inhiition of cspse-3 ctivtion (49). Another intrcellulr pthogen, Chlmydophil pneumonie, inhiits poptosis induced y tretment with Downloded from t Pennsylvni Stte University on My 12, 2016

11 drugs or y deth receptor ligtion. The oserved inhiition ws ssocited with lck of cspse-3 ctivtion in infected cells: the protective effect ws confined to the cells inhited y Chlmydophil (25). It hs een reported tht herpes simplex virus (HSV-1) interferes with poptotic processes in infected cells: HSV-1 ws le to prevent poptosis which ws induced y vrious stimuli, including tretment with TNF- nd STS. A further prllel with the sitution seen fter Af conidi tretment is tht wild-type HSV-1 infection itself could induce low-level poptosis in cells (50, 29). It ws concluded tht HSV-1 cted t multiple metolic checkpoints. The other poptosis inducer used ws STS. If conidi similrly inhiit STS- nd TNF-induced poptosis, the mechnisms of induction nd inhiition re clerly different for these two drugs. TNF-dependent poptosis is induced through the cspse pthwy which is locked y Z-VAD.FMK which in contrst hs no effect on STS-induced poptosis nd procspse-3 remins uncleved. These oservtions indicte tht STS-induced poptosis in A549 cells does not require cspse ctivity. The lck of processing of pro-cspse-3 in cells exposed to STS ws lso reported for HeL H21 cells (30). While HeL H21 cells were efficiently killed fter TNF or STS tretment, extensive processing of cspse-3 ws only oserved fter TNF tretment. Recently, it ws shown tht STSinduced poptosis of some cncer cells, including A549 cells, is cspse independent (51). These results support the view tht vrious poptotic stimuli my ctivte different steps of poptosis. At this stge role for other proteolytic cspses cnnot e excluded. Using three different convergent methods, we hve shown tht Af conidi inhiited poptosis induced y two divergent pro-poptotic stimuli, TNF nd STS. The nture nd mode of ction of the conidil fctors tht lock poptosis re unknown: conidil molecules could e present in the irorne conidi nd relesed upon contct with the cell or synthesized de novo in the presence of the epithelil cells; two or severl molecules my inhiit the cspse-dependent nd cspseindependent poptotic pthwys or oth pthwys could e inhiited y the sme molecule: t this stge, we re unle to exclude dependence on the presence of conidi in erly stges of germintion, not distinguishle y stndrd light microscopy, relesing ctive products. Further experiments re required to fully understnd the role of conidi in the inhiition of poptosis. Acknowledgements This work ws supported in prt y grnt from The Institut Ntionl de l Recherche Agronomique (Trnsverslité INRA 2000 Mycotoxines AIP INRA A-263) nd y Fellowship (S.L-F.) from The University Pris XII Vl de Mrne, Pris, Frnce. We thnk Dvid Dresser (School of Biologicl Sciences, University of Edinurgh, UK) nd Svirshchevsky Elen (Institute of Bioorgnic Chemistry, Russi) for significnt discussions nd criticl reding of the mnuscript, Jcqueline Srfti (Unité des Aspergillus, Institut Psteur, Pris, Frnce) for technicl ssistnce nd Emmnuelle Boisieux-Ulrich (Université Pris 7) for the helpful suggestions in some experiments. We re lso thnkful to the reviewers for their suggestions. Arevitions Af Aspergillus fumigtus CHX cycloheximide DISC HAM HNT HSV-1 PI STS TNF TUNEL Aspergillus fumigtus conidi inhiit poptosis 11 deth-inducing signlling complex humn lveolr mcrophges humn nsl turintes herpes simplex virus propidium iodide sturosporine tumour necrosis fctor terminl deoxynucleotidyl trnsferse-medited fluorescein dutp nick end lelling References 1 Denning, D. W Invsive spergillosis. Clin. Infect. 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