ARTICLES. Dectin-1 is required for b-glucan recognition and control of fungal infection

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1 Dectin- is required for -glucn recognition nd control of fungl infection Philip R Tylor,5, S Vicky Tsoni,5, Jnet A Willment, Kevin M Dennehy, Mrcel Ross, Helen Findon 3, Ken Hynes 3, Chd Steele, Mrin Botto 3, Simon Gordon & Gordon D Brown -Glucn is one of the most undnt polyscchrides in fungl pthogens, yet its importnce in ntifungl immunity is uncler. Here we show tht deficiency of dectin-, the myeloid receptor for -glucn, rendered mice susceptile to infection with Cndid licns. Dectin--deficient leukocytes demonstrted significntly impired responses to fungi even in the presence of opsonins. Impired leukocyte responses were mnifested in vivo y reduced inflmmtory cell recruitment fter fungl infection, resulting in sustntilly incresed fungl urdens nd enhnced fungl dissemintion. Our results estlish fundmentl function for -glucn recognition y dectin- in ntifungl immunity nd demonstrte signling non Toll-like pttern-recognition receptor required for the induction of protective immune responses. Infections with normlly nonpthogenic fungi such s Cndid licns re n emerging prolem resulting from modern medicl interventions nd the incresing prevlence of cquired immunodeficiency. The high incidence of moridity nd mortlity ssocited with such diseses, especilly once the fungus hs disseminted, demonstrtes deficiencies in oth present ntifungl therpies nd understnding of the host immune response. Protection ginst such orgnisms is medited minly y phgocytic cells tht recognize, ingest nd kill the invding pthogen, inducing T helper type immune response, which in turn ctivtes fungicidl effector mechnisms, such s the respirtory urst. Although cells such s neutrophils nd mcrophges re thought to e crucil in tht process, the mechnism underlying the recognition nd initition of the protective responses to these pthogens remins uncler. The cell wlls of fungi consist minly of crohydrtes, including mnnose-sed structures (the mnnoproteins), -glucn nd chitin. For immune systems of infected hosts, such polyscchrides serve s pthogen-ssocited moleculr ptterns (PAMPs) tht cn e recognized y vriety of host-expressed pttern-recognition receptors, including the Toll-like receptors (TLRs), lthough the precise functions of ech of the myrid receptors tht cn respond to these pthogens nd contriute to the induction of protective responses hve not een fully elucidted. Historiclly, the cell wlls of fungi were shown to e covered y lyer of mnnoproteins, which prompted much interest in mnnose-sed recognition systems. Susequent evidence hs suggested tht this model my e too simplistic nd tht other PAMPs, prticulrly -glucns, re exposed on the cell surfce nd therefore re potentilly importnt in immune recognition 3. In fungi such s C. licns nd Scchromyces cerevisie, -glucns cn comprise up to 5% of the dry weight of the fungl cell wll nd re essentil structurl components tht provide elsticity nd mechnicl strength.inisoltedform,-glucns re known to stimulte immune function, hving vriety of eneficil effects, including protection ginst tumor development or infection with fungl, cteril, virl or protozol pthogens,5. Host-cell recognition of -glucn is medited minly y dectin-, myeloid-expressed type II trnsmemrne C-type lectin like receptor tht contins n immunoreceptor tyrosine-sed ctivtion motif in its cytoplsmic til 6,7. Dectin- inds to mny fungl species, including scchromyces 8, cndid, coccidioides 9, pneumocystis nd spergillus 3. In vitro, dectin- hs een shown to medite vriety of oth TLR-dependent nd TLRindependent ntifungl cellulr responses, including the respirtory urst,5, phgocytosis 5,6 nd the production of mny cytokines nd chemokines 8,,,7,8. Here we hve ssessed the contriution of -glucn recognition to the outcome of fungl infection in vivo y using dectin--deficient mice. We found tht recognition of these crohydrtes hd n essentil function in ntifungl immunity nd host survivl y promoting myeloid cell ctivtion nd regulting the susequent inflmmtory response. Our studies demonstrte signling non Toll-like pttern-recognition receptor required for the induction of protective immune responses nd provide new insights into the innte sensing of fungl pthogens. Sir Willim Dunn School of Pthology, University of Oxford, Oxford OX3RE, UK. Institute of Infectious Disese nd Moleculr Medicine, University of Cpe Town, Cpe Town 795, South Afric. 3 Imperil College School of Medicine, Hmmersmith Cmpus, London W NN, UK. Deprtment of Peditrics, Division of Pulmonology, Children s Hospitl of Pittsurgh, University of Pittsurgh School of Medicine, Pittsurgh, Pennsylvni 53, USA. 5 These uthors contriuted eqully to this work. Correspondence should e ddressed to G.D.B. (gordon.rown@mwe.co.z). Received July; ccepted Octoer; pulished online Decemer 6; doi:.38/ni8 NATURE IMMUNOLOGY VOLUME 8 NUMBER JANUARY 7 3

2 Bone mrrow Dectin--WT Dectin--KO +/ +/+ Neu Neu 5.6 k Mo Mo k Gr- hi 7/ hi 7/ Dectin- Dectin- Gr- Figure Dectin--deficient mice. () Southern lot showing the wild-type -kilose nd in nontrgeted emryonic stem cell clone (+/+) nd the presence of n dditionl 5.6-kilose nd in heterozygous Clec7-trgeted emryonic stem cell clone (+/ ). () Flow cytometry of mouse one mrrow, with gting on Gr- hi 7/ hi neutrophils (Neu) nd monocytes (Mo; left), for nlysis of dectin- expression y dectin--wild-type mice (dectin--wt; middle) nd their dectin--knockout littermtes (dectin--ko; right). Plots re representtive of dt otined from three mice per group. RESULTS Dectin--deficient mice show no gross normlities To exmine the function of -glucn recognition in ntifungl immunity, we generted mice deficient in dectin- (clled dectin-- knockout mice here) using conventionl gene-trgeting vector (Supplementry Fig. online). We confirmed deletion of exons 3 of the gene encoding dectin- (Clec7), corresponding to the cytoplsmic til, trnsmemrne nd stlk regions, y Southern lot with two externl proes (Fig. ). Flow cytometry of peripherl leukocytes, done s descried efore 9, confirmed tht Clec7 expression ws rogted (Fig. ). Mice with heterozygous deficiency showed intermedite protein expression (Supplementry Fig. ), suggesting gene-dosge effect. The dectin--knockout mice were vile, hd no gross normlities nd hd norml peripherl leukocyte counts (Tle ), nd thioglycollte-elicited peritonel mcrophges from dectin--knockout nd dectin-- wild-type mice hd similr ntigen phenotype nd no normlities were evident other thn the lck of expression of dectin- (Supplementry Fig. ). Impired myeloid cell ctivtion y fungl prticles We next ssessed the ility of dectin--knockout mcrophges to recognize nd respond to zymosn, -glucn-rich prticle derived from the cell wll of S. cerevisie. Consistent with dectin- s eing the min -glucn receptor on mcrophges, thioglycollte-elicited mcrophges from dectin--knockout mice hd considerly impired recognition of zymosn. The extent of recognition in dectin--knockout mcrophges ws similr to tht otined y tretment of dectin--wild-type cells with competing -glucn (Fig.,). Prior opsoniztion with mouse serum restored the inding of these prticles y promoting -glucnindependent recognition through complement receptors. However, the loss of dectin- lso resulted in filure to mount n inflmmtory response to zymosn, ssessed y relese of tumor necrosis fctor (TNF), which ws not restored y serum opsoniztion (Fig. ). Tht defect ws specific for fungl prticles, s dectin--knockout mcrophges showed no impirment in their response to other microil stimuli, including lipopolyscchride nd the TLR gonist Tle Differentil splenocyte counts Pm 3 CSK (Fig. c). Thus, dectin- is required for inflmmtory responses to oth opsonized nd unopsonized prticles. As dectin- hs een ssocited with the respirtory urst response to zymosn in mcrophges, we exmined tht response in peritonel-elicited mcrophges. Although low mounts of respirtory urst re induced in those cells, there ws no pprent defect in the respirtory urst fter stimultion with serum-opsonized zymosn in the genetrgeted mcrophges (Fig. d). Although we otined significnt difference with unopsonized zymosn, tht could e ttriuted to the lck of prticle recognition of these cells (Fig. ). Thus dectin- seems to hve redundnt function in the respirtory urst in mcrophges. Dendritic cells produce interleukin (IL-) nd IL- in response to unopsonized yest, through mechnism dependent on the kinse Syk nd the dptor CARD9, which is thought to involve dectin- (refs.,7,3). However, we did not detect ny sustntil defect in the production of those cytokines in dectin--knockout one mrrow derived dendritic cells cultured with zymosn (Supplementry Fig. online). In contrst, the production of those cytokines ws impired sustntilly in elicited dectin--knockout mcrophges (Supplementry Fig. ). As dendritic cells re known to express other receptors involved in the recognition of yest, these dt suggested tht dectin- is dispensle for fungl recognition in those cells nd tht the phenotypes of cells with Syk or CARD9 deficiency my not e restricted to specific lockde of the dectin- pthwy. We lso exmined the effect of dectin- deficiency on neutrophils, which re essentil phgocytes in ntifungl immunity. In thioglycollte-elicited neutrophils, the sence of dectin- resulted in loss of the -glucn-dependent recognition of unopsonized zymosn noted in dectin--wild-type cells (Fig. 3). Although dectin--knockout neutrophils showed n ttenuted respirtory urst when cultured together with unopsonized yest, which my lso e ttriutle to the lck of prticle recognition, the response ws not fully restored when we used opsonized zymosn prticles (Fig. 3). Thus, dectin- Cell type Mrker WT cells (n) KO cells (n) B cells B +.5 ± 6.99 ().58 ±.85 () CD + T cells CD3 + CD +.33 ± 3.35 () 3.85 ±.3 () CD + CD5 + T cells CD3 + CD + CD ±.8 ().9 ±.7 () CD8 + T cells CD3 + CD ±.9 () ±.6 () CD8 + CD5 + T cells CD3 + CD8 + CD ±.9 ().35 ±.8 () Nturl killer cells CD9 + CD3.3 ±.3 ().39 ±.3 () Dendritic cells CDc hi. ±.35 ().69 ±.56 () Red pulp mcrophges F/8 hi.97 ±.3 ().8 ±.8 () Neutrophils Gr- hi CD +.3 ±.3 ().9 ±. () Eosinophils Gr- int CD + F/ ±.33 ().33 ±. () Resident monocytes Gr- CD + F/ ±.9 ().58 ±.85 () Inflmmtory monocytes Gr- + CD + F/8 +. ±.7 ().33 ±.6 () Splenocytes were isolted from mtched dectin--knockout mice (KO) nd dectin--wild-type mice (WT) nd cell types were identified with mrkers nd flow cytometry. Red pulp mcrophges were identified with utofluorescence nd F/8 stining; eosinophils nd monocytes distinguished y forwrd- nd side-sctter profiles. n ¼ numer of mice. Dt represent cell numers 6 (men ± s.e.m.). 3 VOLUME 8 NUMBER JANUARY 7 NATURE IMMUNOLOGY

3 Pm c 3 CSK d Dectin--WT Dectin--KO recognition (RFU) TNF (reltive units) medites recognition nd cellulr responses to fungl prticles in neutrophils. Enhnced fungl dissemintion in dectin--knockout mice Becuse cndid is one of the leding cuses of nosocomil fungl infections nd is well studied in nimls,5 7, we exmined the function of dectin- in vivo y infecting mice intrvenously with vrious doses of C. licns SC53 s model of systemic cndidisis 6. Dectin--knockout mice hd much lower survivl thn wildtype mice fter infection with sulethl dose ( colonyforming units (CFU)) or lethl dose ( 5 CFU) of C. licns (Fig. ). Dectin--knockout mice tht succumed to infection showed evidence of gstrointestinl involvement resulting mcroscopiclly in considerle enlrgement of the stomch (Fig. nd dt not shown). Histologicl exmintion showed no sustntil inflmmtion of the stomch nd intestinl tissues, nd the fungus seemed to e locted minly in the lumen (dt not shown). However, the stomchs of infected dectin--knockout mice were full of food (dt not shown), suggesting ostruction of the gstrointestinl trct Control Op-zymosn TNF (ng/ml) TNF (ng/ml) 5 LPS Op-zymosn.. 6 TLR gonist (ng/ml) Time (min) Figure Impired -glucn recognition y dectin--knockout mcrophges. () Flse-color photomicrogrphs of thioglycollte-elicited dectin--wild-type nd dectin--knockout mcrophges leled with fluorescein isothiocynte tgged zymosn (green), fter incution for 3 min t 37 C (5 zymosn prticles per mcrophge). Originl mgnifiction,. () Fluorimetry for zymosn recognition (top) y dectin--wild-type mcrophges (lck rs) nd dectin--knockout mcrophges (gry rs), expressed s reltive fluorescent units (RFU), nd ELISA quntifiction of TNF production (ottom) fter removl of zymosn nd incution for further 3 h t 37 C. Experiments were done in the presence (+) or sence ( ) of competing -glucns. Dt represent men (± s.e.m.) of four pooled, normlized experiments. (c) ELISA of TNF production y dectin--wild-type (filled circles) nd dectin--knockout (open circles) thioglycollte-elicited mcrophges fter tretment with the solule TLR gonist Pm 3 CSK or TLR gonist lipopolyscchride. Dt represent men (± s.e.m.) of replictes from one representtive experiment of two. (d) Respirtory urst of dectin--wild-type (filled circles) nd dectin--knockout (open circles) thioglycollte-elicited mcrophges in response to zymosn nd complement-opsonized zymosn (Op-zymozn), s ssessed y dihydrorhodmine 3. Dotted lines, sl H O production in unstimulted cells. Dt represent men (± s.e.m.) of pooled normlized men fluorescent intensity dt (presented s cumultive H O ) from three independent experiments., P o.5;, P o.; nd, P o.. (ANOVA with Bonferroni post-test (); Student s t-test (d)). Figure 3 Impired -glucn recognition y dectin--knockout neutrophils. () Flourimetry of 9/Sv dectin--wild-type neutrophils (lck rs) nd dectin--knockout neutrophils (gry rs) incuted with fluorescein isothiocynte leled zymosn or serum-opsonized zymosn (Op-zymosn), expressed s reltive inding index (RBI), s descried 6. Experiments were done in the presence (+) or sence ( ) of competing -glucns. Dt represent men (± s.e.m.) of triplictes of one representtive experiment from two. () Respirtory urst of dectin--knockout neutrophils (open circles) nd dectin--wild-type neutrophils (filled circles) in response to opsonized zymosn (right) nd unopsonized zymosn (left). Dotted lines, sl H O production in unstimulted cells. Dt represent men (± s.e.m.) of triplictes of one representtive experiment of two., P o.5;, P o.; nd, P o. (ANOVA with Bonferroni post-test (); Student s t-test ()) Cumultive H O Cumultive H O To elucidte the development of disese, we lso exmined dectin-- knockout mice efore the onset of deth. At dy 9 fter infection with CFU C. licns, dectin--knockout mice hd significntly higher fungl urdens thn those of dectin--wild-type mice throughout the gstrointestinl trct, lthough renl fungl lods were similr to those of wild-type mice t this time point (Fig.,c). Enhnced systemic dissemintion of C. licns in the dectin--knockout mice ws evident even within h of infection (dt not shown), consistent with defect in the innte recognition nd control of the fungl pthogen. However, we were unle to detect significnt differences in cytokine production erly in infection in systemiclly infected dectin--knockout nd dectin--wild-type mice (dt not shown). As Cumultive H O ( ) recognition (RBI) Time (min) Op-zymosn Op-zymosn 6 Time (min) NATURE IMMUNOLOGY VOLUME 8 NUMBER JANUARY 7 33

4 CFU/g Survivl (%) 8 6 n = 7 n = Time (d) < < Stomch Smll intestine Cecum Lrge intestine Kidney gstrointestinl dissemintion of C. licns fter intrvenous chllenge hs not een widely reported (lthough it hs een noted in other models; L. Romni, personl communiction), we re-exmined fungl distriution in dectin--wild-type mice infected with lethl dose ( 5 CFU) of C. licns. Those mice hd gstrointestinl involvement similr to tht of dectin--knockout mice, including enlrgement of the stomch (Supplementry Fig. 3 online), which suggested tht enlrgement of the stomch ws norml pthologicl process in lethlly infected mice. Although the kidneys re the chief trget orgn of cndid 8,we never found difference in renl fungl lods in dectin--knockout versus dectin--wild-type mice erly in infection (Fig. c). However, the kidneys of dectin--knockout mice otined t the time of deth hd enhnced coloniztion with the C. licns, prticulrly in the pelvic region, with extension up the renl tuules. Such coloniztion ws not evident in the kidneys of the dectin--wild-type mice, which hd mostly clered the infection y the end of the experiment (Fig. 5). Foclly, the fungl hyphe extended through the tuulr epithelium into the interstitium nd were surrounded y cute neutrophilic 5 n = 8 n = 8 Figure Dectin--knockout mice re more susceptile thn dectin--wildtype mice to live C. licns. () Survivl curves of 9/Sv dectin--wildtype mice (filled circles) nd dectin--knockout mice (open circles) infected intrvenously with CFU (left) or 5 CFU (right) C. licns SC53. P ¼.66 (left) nd P ¼.35 (right; log-rnk test). (,c) Quntifiction of the live C. licns fungl urden in the gstrointestinl trct () or kidneys(c) of dectin--wild-type mice (filled circles) nd dectin--knockout mice (open circles) t 9 d fter intrvenous infection with CFU., P o.5, nd, P o. (two-tiled Mnn-Whitney test). Dt re representtive of two independent experiments. c CFU/g inflmmtion, indictive of invsive cndidisis (Fig. 5 nd dt not shown). Thus, dectin- deficiency results in enhnced fungl dissemintion nd susceptiility to infection. Leukocyte responses to fungl pthogens require dectin- To etter understnd the in vivo phenotype of dectin--deficient mice, we next exmined the interction of dectin--knockout leukocytes with C. licns in vitro (Fig. 6). Although immune recognition of nd response to C. licns is thought to involve mny pttern-recognition receptors 5,7, we found tht recognition of unopsonized yest y dectin--knockout mcrophges ws much lower thn tht of dectin- -wild-type mcrophges (Fig. 6). The lower recognition corresponded to significnt impirment in the inflmmtory response, s ssessed y TNF relese (Fig. 6). Furthermore, the inflmmtory response to the yest ws not restored fter opsoniztion of the yest with serum, despite its conferring norml fungl recognition to the dectin--deficient cells (Fig. 6). Once recognized, dectin--knockout mcrophges did not show ny defect in their ility to kill serum-opsonized or unopsonized yest (Fig. 6c nd dt not shown), which ws consistent with our oservtion of norml respirtory urst response to ound zymosn prticles (Fig. d) nd suggested tht fungl recognition nd killing occurred through different mechnism(s). Dectin--deficient neutrophils, however, hd significntly less ility thn dectin--wild-type neutrophils to kill unopsonized C. licns in coculture, lthough killing ws restored when the yest were opsonized with serum (Fig. 6d). In ddition to studying the response to C. licns, we lso exmined the inflmmtory response of elicited mcrophges to unopsonized live Aspergillus fumigtus nd found tht TNF production ws lso significntly lower in the dectin--knockout cells thn in dectin--wild-type cells (Fig. 6e). Thus, dectin- is required for norml leukocyte responses to live fungl prticles. Dectin- required for inflmmtion in cndid infection As our dt from systemic infection with C. licns suggested tht the susceptiility of the dectin--knockout mice ws due to defect in the erly innte response, we further explored how the loss of -glucn recognition y leukocytes ffected inflmmtion in response to fungi in vivo using peritonel infection model. We injected mice intrperitonelly with 5 C. licns yest nd ssessed inflmmtory leukocytes in the peritoneum fter h y flow cytometry, s Dectin--KO Dectin--WT Figure 5 Kidney disese in dectin--knockout mice tht succum to infection. (,) Photomicrogrphs of fungl growth in periodic cid Schiff stined kidneys of representtive dectin--knockout mouse (, left) or dectin--wild-type mouse (, right) otined t deth fter intrvenous chllenge with CFU C. licns SC53. Middle (), enlrgement of oxed re t left., hyphl tissue invsion in the kidney of dectin-- knockout mouse. Originl mgnifiction, (, left, right) nd (, middle, nd ). (c) Quntifiction of the fungl urden in dectin-- wild-type kidneys (filled circles) nd dectin--knockout kidneys (open circles) t the time of deth or 8 d fter intrvenous infection with CFU C. licns. Mice with fungl urden of less thn CFU hd clered the infection y the end of the experiment (dy 8). Ech symol represents one mouse., P o.5 (Mnn-Whitney nonprmetric t-test). Dt re representtive of two independent experiments. c CFU/g < Kidney 3 VOLUME 8 NUMBER JANUARY 7 NATURE IMMUNOLOGY

5 C. licns recognition (RFU) TNF (pg/ml) 5 c d e Killing (%) Cont C. l Killing (%) 8 6 Op C. licns recognition (RFU) TNF (pg/ml). 6 descried 9. Flow cytometry with the grnulocyte nd monocyte mrkers Gr- nd 7/ nd the mcrophge mrker F/8 showed tht dectin--knockout mice hd mny fewer recruited cells thn did dectin--wild-type mice, including Gr- hi 7/ hi neutrophils, Gr- + 7/ hi F/8 + inflmmtory monocytes nd Gr- int 7/ lo F/8 + side-sctter-high eosinophils, nd lso hd reproducile trend of less resident mcrophge emigrtion (Fig. 7,). The lower inflmmtory cell recruitment ws lso ssocited with mny defects in the production of specific cytokines nd growth fctors in the inflmmtory lesion, with considerle reproducile defects evident in the production of IL-6, the chemokines CCL nd CCL3, nd grnulocyte nd grnulocyte-monocyte colony-stimulting fctors (Fig. 7c). A similr impirment in inflmmtion ws lso evident fter intrperitonel.. Op C. licns/mcrophge Unopsonized Opsonized TNF (ng/ml) 6 8 Cont A. fum Figure 6 The function of dectin- in the recognition nd killing of live fungl prticles. () Fluorimetry (top) of dectin--wild-type mcrophges (lck rs) nd dectin--knockout mcrophges (gry rs) incuted for 3 min t 37 C with live rhodmine green-x leled C. licns (C. l;.5:, cndid/mcrophge rtio), expressed s reltive fluorescent units (RFU), nd ELISA of TNF production (ottom) fter removl of unound fungi, determined during the next 3 h t 37 C. Cont, control (medium lone). Dt represent men (± s.e.m.) of replictes from one representtive ssy of two. () Fluorimetry (top) of dectin--wild-type mcrophges (filled circles) nd dectin--knockout mcrophges (open circles) incuted for 3 min t 37 C with serum-opsonized C. licns (Op C. licns; yest/ cell rtio, horizontl xis), nd ELISA of TNF production (ottom). Dt represent men (± s.e.m.) of replictes from one representtive ssy of two. (c,d) Killing ssy of dectin--wild-type (lck rs) nd dectin--knockout (gry rs) mcrophges (c) nd neutrophils (d) incuted with opsonized C. licns (c) or either unopsonized or opsonized C. licns (d). Dt represent men (± s.e.m.) of replictes from one representtive ssy of two (c) or from four independent pired dt sets (d). (e) ELISA of TNF production y dectin--wild-type mcrophges (lck rs) nd dectin-- knockout mcrophges (gry rs) in response to live A. fumigtus (A. fum). Dt represent men (± s.e.m.) from one representtive experiment of two., P o.5;, P o.; nd, P o. (Student s t-test). injection of mice with zymosn prticles ut not fter injection of thioglycollte roth (Supplementry Fig. online). Thus, dectin-- knockout mice hve n intrinsic defect in their inflmmtory response to fungl prticles in vivo. DISCUSSION Here we hve shown tht the recognition of -glucn y dectin- is n essentil component of the innte immune response to fungl pthogens. Recognition of those crohydrtes y dectin-, long with collortive TLR signling 8,, induces proinflmmtory response tht results in the rpid recruitment of inflmmtory leukocytes, Gr- Dectin--WT % Dectin--KO 3%.9%.7% Figure 7 Anorml ntifungl inflmmtory response in vivo in the sence of dectin-. () Flow cytometry for Gr- hi 7/ hi neutrophils nd Gr- + 7/ hi inflmmtory monocytes in dectin--wild-type mice (left) nd dectin-- knockout mice (right) fter h of intrperitonel infection with 5 live C. licns. Numers djcent to outlined res indicte percent neutrophils (top) nd monocytes (ottom). Dt re representtive of three experiments with seven to nine mice in ech. () Sctter plots of myeloid cell susets in the peritonel cvities of dectin--wild-type mice (filled circles ) nd dectin- -knockout mice (open circles) fter h of intrperitonel infection with 5 live C. licns. Ech symol represents n individul mouse; horizontl rs, medins. Neut, neutrophil; Mono, monocyte; Eos, eosinophil; Res. Mf, resident mcrophge. Dt represent one of three experiments. (c) Bio-Plex ssys for cytokines, chemokines nd growth fctors in lvge fluid from the inflmed peritonel cvities of dectin--wildtype mice (filled circles ) nd dectin--knockout mice (open circles) fter h of intrperitonel infection with 5 live C. licns. Horizontl rs, medins. Only inflmmtory meditors with reproducile differences in seprte experiments re presented. MCP-, chemokine CCL; MIP-, chemokine CCL3; G-CSF, grnulocyte colony-stimulting fctor; GM-CSF, grnulocyte-monocyte colony-stimulting fctor. Dt re representtive of three independent experiments., P o., nd, P o. (Mnn-Whitney nonprmetric t-test). c Cytokine (ng/ml) 5 3 7/ 7/ Cells ( 6 ) 3 Neut Mono Eos Res. Mφ IL-6 MCP- MIP-α G-CSF GM-CSF NATURE IMMUNOLOGY VOLUME 8 NUMBER JANUARY 7 35

6 including neutrophils, nd the continment nd killing of the fungus. In the sence of dectin-, defective ctivtion of tissue resident mcrophges impeded the susequent inflmmtory response, leding to impired recruitment of myeloid cells, which is key in controlling the pthogen. Tht occurred in the context of deficiency in inflmmtory meditors nd growth fctors, which would normlly enhnce neutrophil ctivtion nd neutrophil-medited killing 3. Ultimtely, the sence of dectin- nd of norml inflmmtory response resulted in considerly enhnced dissemintion of the pthogen nd incresed host susceptiility. Our study hs lso emphsized the importnce of the non Toll-like receptors in immune responses. Pttern-recognition receptors such s CD (ref. 3) nd CD36 (ref. 3) re importnt in the recognition of other pthogens nd their components, lthough the min function of those receptors seems to e the cpture of PAMPs, rendering them ccessile to the TLRs. As TLRs seem to recognize fungl PAMPs other thn -glucn, t lest on zymosn, dectin- my lso serve similr function in PAMP presenttion. However, the inility to restore TLRmedited inflmmtory responses fter cellulr trgeting of fungl prticles y serum opsoniztion indicted tht the signling pthwys induced y dectin- (refs. 8,3) re essentil for those responses. Furthermore, mny of the dectin--medited ctivities, including phgocytosis 6, the respirtory urst,5 nd the production of certin cytokines 7, re TLR independent. Thus, our dt hve demonstrted the requirement of cell surfce, signling, non Toll-like pttern-recognition receptor for the induction of protective immune responses. Given the importnce of dectin- in the recognition of -glucn, it is relevnt to consider the function of CR3, lctosylcermide nd scvenger receptors, ll of which hve een proposed to e -glucn receptors involved in fungl recognition 8. CR3 hs lso een linked to the -glucn-medited enhncement of ntitumor monoclonl immunotherpy 33 nd ws initilly proposed to e the min -glucn receptor on myeloid cells. As our dt hve demonstrted tht dectin- hs nonredundnt function in -glucn recognition, the function of those other receptors is uncler. It is possile tht they re involved in promoting intrcellulr signling 33,3, nd they my lso contriute to -glucn recognition in cell types lcking dectin- (ref. 35). As recognition of -glucns is centrl to ntifungl immunity, it is not unexpected tht fungi my hve evolved to void immune recognition y hiding these crohydrtes. Although yest forms of cndid nd scchromyces cn induce inflmmtory responses y mens of exposed -glucns, most of those polyscchrides re conceled y mnnoprotein lyer tht is thought to prevent excessive immune ctivtion 36. In ddition, oth A. fumigtus 3 nd C. licns 3 seem to completely msk their -glucns fter trnsition from the yest to the hyphl form, morphogenic trit proposed to e linked to virulence 37,38. -Glucns my lso e msked y encpsultion, s occurs in Cryptococcus neoformns 39, or my e lower in undnce in the cell wll, s occurs with Prcoccidioides rsiliensis fter infection of the lung.avoidnceof-glucn recognition my therefore e common immune evsion strtegy in fungi, supporting the hypothesis tht trgeting the unmsking of cell-wll -glucns my led to new ntimycotic gents 36. In ddition to recognizing -glucns, dectin- cn recognize n unidentified lignd on lymphocytes nd cn modulte T cell function 6,,. Dectin- is expressed y mcrophges nd dendritic cells in the T cell res of the spleen nd lymph nodes 3, suggesting involvement in the interctions etween ntigen-presenting cells nd T cells. However, fter exmining the ntiody response to ovlumin, we found tht the dectin--deficient mice hd norml T cell dependent immune responses, with no evident T helper type type is or effect on isotype switching (dt not shown). Thus, the function of dectin- in the dptive response, if ny, remins to e clrified. In summry, our studies of dectin--deficient mice hve emphsized the fundmentl function of -glucn recognition in the development of n ntifungl inflmmtory response nd the restriction of fungl dissemintion in vivo. Our studies, therefore, hve demonstrted previously unknown mechnism underlying the recognition nd initition of the protective responses to these pthogens nd distinguish dectin- s cell surfce non-tlr necessry for ntifungl immunity. In ddition, given the similrity etween the mouse nd humn systems, our studies suggest tht modultion of dectin- (ref. 5) my lso represent n lterntive pproch for the tretment of fungl infections. METHODS Genertion of dectin--deficient mice. The genertion of the dectin--knockout mice is descried in the Supplementry Methods online. Mice were mintined in ccordnce with institutionl guidelines t the University of Oxford, London (Imperil College) nd the University of Cpe Town. Clec7 +/+ nd Clec7 / mice were on mixed 9/Sv C57BL/6 genetic ckground unless stted otherwise. Fungus propgtion, leling nd opsoniztion. C. licns SC53 ws streked onto yest peptone dextrose (YPD) or Sourud gr pltes for isoltion of individul colonies. Colonies were cultured in shking incutor for 6 h t 37 C in 5 ml of YPD roth, for in vitro ssys, or t 3 C in Sourud roth for in vivo infections. A. fumigtus isolte 373 ws cultured nd used s descried. For leling with Rhodmine Green-X (Invitrogen), live C. licns ws wshed extensively in PBS efore eing resuspended t density of 3. 8 yest per ml. Rhodmine Green-X ws dded to finl concentrtion of mg/ml nd cells were rotted gently for 3 min t 5 C. Then, C. licns ws wshed extensively with PBS to remove free Rhodmine Green-X efore use. For opsoniztion of yest prticles, mouse lood ws collected y crdic puncture nd ws immeditely plced t C. Blood cells were removed y centrifugtion,g for 5 min t C nd serum ws stored in liquots t 8 C s soon s possile fter lood ws collected, to preserve complement ctivity. Aliquots were defrosted efore use nd were discrded fter use. Yest prticles, typiclly up to 8 zymosn or C. licns, were resuspended in ml RPMI medium with % (volume/volume) hetinctivted FCS nd then were mixed for 5 3 min t 37 C withml freshly defrosted mouse serum with frequent gittion. Yest prticles were then wshed t lest four times to remove residul complement nd serum proteins efore susequent use. In vitro fungus-recognition ssys. These ssys were done essentilly s descried 7,8,,6 8, with some modifictions. For in vitro recognition of zymosn nd live fungl prticles y mcrophges, peritonel exudte cells were isolted y lvge with ice-cold 5 mm EDTA in PBS from mice tht hd een treted intrperitonelly d efore with thioglycollte roth (Difco). The thioglycollte roth used does not contin yest extrct. Mcrophges were plted in -well pltes t density of.5 5 cells per well (for zymosnrecognition ssys) or 6 cells per well (for live C. licns recognition ssys) in RPMI medium with % (volume/volume) het-inctivted FCS. Cells were wshed three times with medium efore the ddition of yest prticles. Fluorescein isothiocynte leled zymosn (Invitrogen) or Rhodmine Green-X leled live C. licns SC53 were used in recognition ssys t mcrophge/prticle rtios of 5: for zymosn nd : to.: for live C. licns. After incution for 3 min t 37 C to llow efficient recognition of the fungl prticles y oth isoforms of dectin- (ref. 7), unound yest cells were wshed wy y four wshes with RPMI medium plus % (volume/ volume) het-inctivted FCS. The medium ws replced nd cells were cultured for 3 h t 37 C for nlysis of production of proinflmmtory cytokines or for h t 37 C for mesurement of IL-p nd IL-. After incution, superntnts were stored t 8 C until use, cells were lysed in 3% 36 VOLUME 8 NUMBER JANUARY 7 NATURE IMMUNOLOGY

7 (volume/volume) Triton X-, ph 7.5, nd fluorescence ws mesured with Titer-Tek Fluoroskn II (Lsystems). Inflmmtory responses to A. fumigtus (multiplicity of infection, 5:) were mesured fter h of fungus mcrophge coculture, s descried. For in vitro recognition of zymosn y neutrophils, peritonel inflmmtory cells were collected with 5 mm EDTA in PBS t 6 8 h fter thioglycollte dministrtion. Cells were pretreted for 3 min on ice with mg/ml of -glucn. Inflmmtory cells (5 5 ) were mixed with fluorescein isothiocynte leled zymosn prticles ( 6 prticles; : zymosn/ inflmmtory cell rtio) in ml of RPMI medium plus % (volume/ volume) het-inctivted FCS in 5-ml polystyrene tues nd were centrifuged t 35g for 5 min t C. Inflmmtory cells nd zymosn were then incuted for h t 37 C. Cellulr recognition of zymosn ws determined y flow cytometry. Where required, zymosn ws opsonized with complement s descried ove. For in vitro recognition of zymosn y one mrrow derived dendritic cells, the cells were incuted with vrious concentrtions of unleled zymosn (Sigm) for h t 37 C, then superntnts were collected nd were stored t 8 C until use for cytokine nlysis. Cytokine production nd respirtory urst. Cytokine, chemokine nd growth fctor concentrtions were mesured with the Bio-Plex Protein Arry System (Bio-Rd) s directed y the mnufcturer. TNF, IL-p, nd IL- were lso mesured y commercil enzyme-linked immunosorent ssy (ELISA; OptEIA cytokine ELISA sets; BD Phrmingen). For nlysis of hydrogen peroxide (H O ) genertion, inflmmtory cells were loded with dihydrorhodmine 3 t finl concentrtion of mm. After incution with zymosn, cells were detched nd the conversion of dihydrorhodmine 3 ws ssessed y flow cytometry nd is expressed s men fluorescent intensity or ritrry units. Cells loded with dihydrorhodmine 3 ut not treted with zymosn were used to ssess ckground H O production. In vitro C. licns killing ssy. This ssy ws sed on pulished ssys 9 with some modifictions. Where required, complement opsonistion ws done s descried ove. Thioglycollte-elicited peritonel mcrophges, pooled from three to five mice per group, were plted t density of 6 mcrophges per well of -well plte on the dy efore the ssy. Cells were wshed three times with medium efore the ddition of live C. licns t rtio of. yest per mcrophge. Cells were llowed to interct for 3 min t 37 C with C. licns, then unound prticles were removed y four wshes with medium nd then cells were returned to the incutor for h to llow fungl killing. Control pltes were kept t C to provide mesure of live fungi in the wells. After incution, medium ws removed nd cells were lysed, with scrping, y incution for 5 min t 5 C with wter t ph of, s descried 5. Lysis uffer ws neutrlized with excess PBS nd CFU C. licns ws determined y plting on YPD gr nd incution overnight t 37 C. Thioglycollte-elicited peritonel exudte cells were collected from t lest two mice per group y peritonel lvge with ice-cold RPMI medium plus % (volume/volume) het-inctivted FCS t h fter the dministrtion of thioglycollte nd were pooled. Those cells were used s source of inflmmtory grnulocytes (minly neutrophils) nd monocytes. Inflmmtory leukocytes (.6 6 ) were mixed with live C. licns (.3 to ) in the wells of 8-well plte nd were kept for 6 min t C to llow the cells to settle, efore eing trnsferred to n incutor t 37 C for further 6 min. Control pltes were kept in prllel t C during tht incution. After incution, cells were mixed, with scrping, nd were plted on YPD gr for counting of vile C. licns fter incution overnight t 37 C. In vivo model of systemic cndidisis. Cultures of C. licns SC53 grown for h in Sourud roth were wshed twice in PBS nd were resuspended t the required concentrtion. Femle mice ( 5 weeks old; 9/Sv genetic ckground) were nesthetized intrperitonelly (% (volume/volume) ketmine nd 8% (volume/volume) xylzine in PBS) nd were weighed efore infection. Live C. licns ws dministered intrvenously in finl volume of ml in PBS. Mice were weighed nd monitored dily with n niml welfre scoring sheet. Mice were killed y CO sphyxition fter certin score ws chieved, s determined y the niml welfre scoring sheet, or when they hd lost % of their ody weight. Experiments continued for mximum of 8 d, when ll surviving mice were killed nd nlyzed. In vivo inflmmtory response models. Mice were injected intrperitonelly with 6 zymosn or 5 live C. licns nd were killed fter h. The inflmmtory infiltrte ws collected y lvge with ice-cold 5 mm EDTA in PBS. Inflmmtory cell popultions were counted nd then were nlyzed y flow cytometry to determine the leukocyte composition s descried 9,9. Cytokines in peritonel lvge fluid were determined s descried ove. In vivo immune response study. For nlysis of ntiody responses, lood ws otined from mice on dy for preimmune serum, nd then mice were immunized intrperitonelly with 5 mg ovlumin in lum on dys nd. On dy, lood ws gin otined from mice for immune serum. Ovlumin-specific ntiodies in ser from dys nd were mesured y ELISA with isotype-specific ntiodies (Southern Biotechnology). Antiodies. The following ntiodies, long with the pproprite isotype controls nd streptvidin complexes, were used: A (ntiody to dectin- (nti-dectin-) 9, ); fluorescein isothiocynte conjugted 7/ (ntiody to neutrophils nd monocytes), phycoerythrin-conjugted nti-cd9 (clone 6D5) nd iotin-conjugted nti-f/8 (Serotec); CyChrome-conjugted nti CD3 moleculr complex (clone 7A), peridinine chlorophyll protein nd cynin 5.5 conjugted nti-cd (clone M/7), phycoerythrin-conjugted nti- CDc (clone HL3), fluorescein isothiocynte conjugted nti-cd,cd35 (clone 7G6), phycoerythrin-conjugted nti-cd3 (clone B3B), iotinconjugted nti-cd35 (clone 8C), peridinine chlorophyll protein nd cynin 5.5 conjugted nti-b (clone RA3-6B), phycoerythrin-conjugted nti-cd9 (clone DX5), phycoerythrin-conjugted nti-gr- (nti-ly6g/c; clone RB6-8C5) nd streptvidin conjugted to fluorescein isothiocynte, phycoerythrin or llophycocynin (BD Phrmingen); fluorescein isothiocynte conjugted nti-cd3 (clone HM3), phycoerythrin-conjugted nti-cd (clone RM-5), fluorescein isothiocynte conjugted nti-cd8 (clone 5H) nd iotin-conjugted nti-cd5 (clone PC6 5.3; Cltg Lortories); nd streptvidin AlexFluor 88 (Invitrogen). Sttisticl nlysis. One-wy nlysis of vrince (ANOVA) with Bonferroni post-tests ws used when multiple groups were nlyzed nd the two-tiled Student s t-test ws used for nlysis of two groups, with pired nlysis when pproprite. For the nlysis of nonprmetriclly distriuted dt, the twotiled Mnn-Whitney test ws used. Survivl dt were nlyzed with the logrnk test. Results were considered sttisticlly significnt with P vlues of less thn.5. Note: Supplementry informtion is ville on the Nture Immunology wesite. ACKNOWLEDGMENTS We thnk the niml fcility stff of for cre of the nimls; nd A. Bygrve, P. Norsworthy, L. Fick, M. Tyler, D. Willims, C. Huysmen, L. Grhm nd C. Mske for help, regents nd technicl ssistnce with the genertion nd study of the gene-trgeted mice nd histology. We dedicte this pper to the memory of Alert Beyers. S.G. nd G.D.B. shre senior uthorship. Supported y the Wellcome Trust (559, 7579, 767 nd 7; Reserch Creer Development Fellowship, P.R.T.; Senior Fellowship in Biomedicl Science in South Afric, G.D.B.), the Cncer Assocition of South Afric, the Medicl Reserch Council South Afric, the Ntionl Institutes of Helth (ROHL837) nd the Biotechnology nd Biologicl Sciences Reserch Council (/B/P/8, 6/P7835 nd BBS/B/33). AUTHOR CONTRIBUTIONS P.R.T., S.V.T., J.A.W., K.M.D., M.R., H.F., K.H. nd C.S. did the experiments; M.B., G.D.B., P.R.T. nd J.A.W. generted the knockout mice; P.R.T., K.H., S.G., C.S. nd G.D.B. conceived nd directed the experiments; nd P.R.T. nd G.D.B. wrote the pper. COMPETING INTERESTS STATEMENT The uthors declre tht they hve no competing finncil interests. NATURE IMMUNOLOGY VOLUME 8 NUMBER JANUARY 7 37

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