A r t i c l e s In vivo imging of inflmmsome ctivtion revels sucpsulr mcrophge urst response tht moilizes innte nd dptive immunity Pervinder Sgoo 1,, Zcris Grci 1,, Betrice Brert 1,, Frice Lemître 1,, Dvid Michonneu 1,, Mtthew L Alert 3,, Yves Levy 5 8 & Philippe Bousso 1,,5 npg 16 Nture Americ, Inc. All rights reserved. The inflmmsome is ctivted in response to vriety of pthogens nd hs n importnt role in shping dptive immunity, yet the sptiotemporl orchestrtion of inflmmsome ctivtion in vivo nd the mechnisms y which it promotes n effective immune response re not fully understood. Using n in vivo reporter to visulize inflmmsome ssemly, we estlish the distriution, kinetics nd propgtion of the inflmmsome response to locl virl infection. We show tht modified vccini Ankr virus induces inflmmsome ctivtion in sucpsulr sinus (SCS) mcrophges, which is immeditely followed y cell deth nd relese of extrcellulr ASC specks. This trnsient inflmmsome signling in the lymph node genertes roust influx of inflmmtory cells nd moilizes T cells from the circultion to increse the mgnitude of T cell responses. We propose tht fter infection, SCS mcrophges deliver urst response of inflmmsome ctivity nd cell deth tht trnsltes into the rodening of T cell responses, identifying n importnt spect of inflmmsome-driven vccintion strtegies. Innte immune sensing of pthogens or dmge is chrcterized y ctivtion of memrne nd cytosolic pttern-recognition receptors including Toll-like receptors (TLRs), retinoic cid inducile gene I like helicse receptors, cytosolic DNA sensors nd NOD-like receptors 1. The inflmmsome forms prt of this first-line response, medited primrily y phgocytic ntigen-presenting cells such s mcrophges nd dendritic cells, nd refers to hetero-multimeric protein scffold ssemled upon ctivtion of cytosolic pttern-recognition receptors. Multiple inflmmsome complexes exist, ech triggered y severl cytosolic sensor fmilies with specificities for distinct pttern-recognition motifs. Once ctivted, mny of these sensors ssocite with poptosis-ssocited speck-like protein with CARD domin (ASC), common dptor protein tht promotes recruitment nd proximity-induced utoctlytic mturtion of cspse-1 (ref. 3). These processes occur through prionlike polymeriztion of ASC into filments nd ultimtely result in the formtion of micrometer-sized structures referred to s ASC specks. Inflmmtory effector mechnisms unleshed y ctive cspse-1 include the proteolytic processing nd relese of interleukin-1β (IL-1β) nd IL-18 nd induction of cellulr pyroptotic deth,5. In vitro, ASC speck formtion cn coincide with cell deth nd cytokine relese 6,7, ut the reltionship etween inflmmsome ctivtion in vivo nd induction of cell deth remins to e documented. Recent studies hve provided evidence tht ASC specks cn exist s ctive extrcellulr structures, rising interest in the fte nd dynmics of ASC specks fter cell deth 8,9. Despite n incresing knowledge of effector mechnisms induced y the inflmmsome response, understnding of how inflmmsome ctivtion unfolds in vivo is still limited. In prticulr, the loction nd timing of inflmmsome ctivity in tissue is uncler, s re the immedite effects of cellulr inflmmsome ctivity. Although severl fluorescent proes hve een developed to study inflmmsome ctivtion in vitro 1 1, their use for in vivo pplictions hs remined chllenging, lcking trnsltion to single-cell resolution t the orgn level 1,1,13. Another question in inflmmsome iology is how its ctivity regultes innte nd dptive immunity. Proinflmmtory IL-1 nd IL-18 hve een shown to hve roles in locl nd peripherl recruitment nd priming of the immune response 1 17, nd the sence of inflmmsome ctivity hs een ssocited with poor pthogen clernce nd incresed disese severity 17 1. Mechnisms tht link the inflmmsome pthwy to T cell responses include dnger-ssocited moleculr pttern (DAMP)-medited dendritic cell (DC) ctivtion, effects of inflmmtory cytokines on T cell polriztion 3 nd ctivtion or noncognte induction of interferon-γ (IFN-γ) in memory T cells 16,. In this respect, understnding the sptiotemporl dynmics of inflmmsome ctivtion my help clrify the downstrem effects on the T cell response. Here we estlish n in vivo reporter system to visulize inflmmsome ctivtion in rel time nd dissect its influence on the immune response fter locl virl infection. 1 Dynmics of Immune Responses Unit, Institut Psteur, Pris, Frnce. Institut Ntionl de l Snté et de l Recherche Médicle U668, Pris, Frnce. 3 Lortory of Dendritic Cells Immunoiology, Institut Psteur, Pris, Frnce. Institut Ntionl de l Snté et de l Recherche Médicle U818, Pris, Frnce. 5 Vccine Reserch Institute, Créteil, Frnce. 6 Institut Ntionl de l Snté et de l Recherche Médicle U955, Créteil, Frnce. 7 Fculté de Médecine, Université Pris Est, Créteil, Frnce. 8 Service d Immunologie Clinique et Mldies Infectieuses, Hôpitl H. Mondor-A. Chenevier, Créteil, Frnce. Correspondence should e ddressed to P.B. (philippe.ousso@psteur.fr). Received 9 Octoer; ccepted Novemer; pulished online 1 Decemer 15; doi:1.138/nm.16 6 VOLUME NUMBER 1 JANUARY 16 nture medicine
r t i c l e s npg 16 Nture Americ, Inc. All rights reserved. RESULTS MVA induces inflmmsome ctivtion in mcrophges To exmine locl virl infection s n inflmmsome trigger, we selected MVA, repliction-deficient group I doule-strnded DNA poxvirus used in clinicl vccintion progrms, with strong immunogenicity through ctivtion of endosoml TLRs nd cytosolic melnom differentition-ssocited protein 5 (MDA-5), Asent in melnom (AIM) nd NOD-like receptor NLRP3 (refs. 5 7). As such, it delivers temporlly defined stimulus for inflmmsome ctivtion, llowing model for direct monitoring of downstrem cellulr events of infection. Using oth wild-type () nd cspse-1 deficient (Csp1 / ; Csp / ) mouse strins, we confirmed tht MVA infection induced inflmmsome ctivity in one mrrow derived mcrophges (BMDMs) y detection of cspse-1 ctivity (Fig. 1 nd Supplementry Fig. 1) nd IL-1β (Fig. 1). Addition of the cspse- 1 specific inhiitor Z-YVAD-FMK prevented IL-1β relese induced y ATP tretment of lipopolyscchride (LPS)-primed BMDMs or MVA-infected BMDMs, demonstrting tht IL-1β relese depends on the conventionl inflmmsome pthwy 8,9. To detect inflmmsome ctivtion in rel time, we stly trnsduced BMDMs to express fusion protein of ASC nd GFP (ASC- GFP) s red-out for inflmmsome ssemly. As nticipted, MVA infection induced formtion of ASC specks in vitro (Fig. 1c,d), which could e visulized y redistriution of ASC-GFP s it oligomerizes to form single mcromoleculr complex 3 (Supplementry Movie 1). Concomitnt with inflmmsome ctivity, MVA infection lso induced cspse-1 dependent mcrophge deth (Fig. 1e). In vitro, ASC-GFP expression in BMDMs did not lter cellulr levels of cspse-1 ctivity, IL-1β relese or the kinetics or mount of cell deth (Supplementry Fig. 1 f). These oservtions correspond with recent report highlighting the crucil role of the levels of upstrem inflmmsome sensor proteins in determining the rte of speck formtion. In vivo, fter locl virl infection (sucutneous footpd injection) with MVA, we found tht virus primrily trgeted mcrophges within the sucpsulr (SCS-M) nd medullry regions of FLICA-1 MOI MOI 1 MOI 5 MOI 5 IL-1β (pg/ml) 8 6 1 BMDM + Z-YVAD-FMK 1 5 5 5 MVA (MOI) LPS + ATP c ASC-GFP min min min d Numer of ASC-speck per field the drining lymph node, consistent with previous reports of their role in sequestering lymph-orne pthogens 31 33 (Supplementry Fig. ). MVA infection resulted in rpid nd specific loss of lymph node mcrophges, most notly CD169 + F/8 SCS-Ms (Fig. 1f,g). These findings suggest tht inflmmsome signling nd pyroptotic deth my e n importnt component of the immune response to MVA. SCS mcrophges initite the inflmmsome response to MVA Detection of inflmmsome ctivity in vivo t single-cell resolution hs so fr een restricted to sttic imging on fixed tissue sections 3, limiting n understnding of the identity, dynmics nd fte of cells undergoing inflmmsome ctivtion. This is due to the lck of mouse models to detect inflmmsome ctivtion nd in prt to the inherently trnsient nture of cell signling tht my ultimtely progress to cellulr demise. To circumvent this limittion, we developed strtegy for dynmic two-photon imging of inflmmsome ssemly y in vivo detection of ASC speck formtion. Mixed one mrrow chimeric (BM chimeric) mice were generted y trnsfer of cycling one mrrow derived hemtopoietic stem cells (BM-HSCs) retrovirlly trnsduced to express ASC-GFP. ASC-GFP-expressing BM-HSCs derived from Rg / mice were mixed with either or cspse-1 deficient BM-HSCs nd used to reconstitute recipient mice (Fig. ). This strtegy served to restrict reporter expression to the myeloid comprtment nd limit cspse-1 dependent functionl inflmmsome signling to cells expressing the reporter. Within lymph nodes of chimeric mice, ASC-GFP ws expressed y myeloid cells, with norml distriution throughout the lymph node volume (Supplementry Fig. 3,). Intrvitl imging of chimeric mice reveled ASC-GFP expression y cells loclized to the sucpsulr sinus region of resting lymph nodes (Fig. ). These cells formed dense network t the lymph node periphery, displyed mcrophge-like morphology nd were stined fter in vivo injection of CD169-specific ntiody (Fig. c), indicting myeloid-derived reconstitution of lymph node sucpsulr sinus mcrophges, s previously descried 35. 6 5 5 LPS + ATP MVA (MOI) e Percentge of dying cells 15 1 5 1 5 MVA (MOI) 5 5 f CD169 g Cell numer per LN 6 18 1 1 5 SCS-M Med-M Mcrophge DC Figure 1 MVA infection induces inflmmsome ctivtion, speck formtion nd rpid loss of SCS mcrophges in 1 1 drining lymph nodes. () Detection of cspse-1 ctivity (FLICA-1 + stining) in nd cspse-1 deficient (Csp1 / ) 1 1 3 BMDMs 6 h fter infection with MVA t different multiplicities of infection (MOIs). Dt re representtive of more thn three experiments performed in triplicte. () IL-1β relese y BMDMs cultured with MVA or LPS nd ATP (lck 1 1 rs) is inhiited y ddition of cspse-1 inhiitor Z-YVAD-FMK (white rs). Dt re representtive of more thn 6 18 three experiments performed in triplicte. (c) Three consecutive imges from time-lpse microscopy of BMDMs (dshed lines) expressing ASC-GFP (green), showing ASC-GFP protein redistriution leding to speck formtion 6 h fter infection with MVA (MOI = 5). Scle r, 1 µm. Representtive imges from more thn 1 independent experiments. (d) Detection of ASC specks in cultures of BMDMs expressing ASC-GFP, 15 h fter tretment with MVA or LPS + ATP. (e) Percentge of dying cells (ssessed y flow cytometry for propidium iodide stining) in nd Csp1 / BMDM cultures 6 h fter MVA infection. Dt re representtive of more thn three experiments performed in triplicte. (f) Immunofluorescence stining of CD169 + ntigen in poplitel lymph nodes from mice up to 18 h fter sucutneous injection of MVA (5 1 6 plque-forming units (p.f.u.)) showing loss of SCS-mcrophges (SCS-Ms) from sucpsulr sinus regions of lymph nodes. Scle r, 1 µm. Representtive of four experiments, n = 3 mice per group. (g) Flow cytometry detection of numers of lymph node SCS-M, medullry mcrophge (med-m), mcrophge nd dendritic cell (DC) susets in mice fter injection of MVA. Representtive of more thn 6 experiments, n = 6 mice per time point. Error rs, men ± s.e.m. P <.1, P <.1, one-wy nlysis of vrince (ANOVA) followed y Tukey s post hoc test for multiple comprison (d) or Student s t-test (e,g). nture medicine VOLUME NUMBER 1 JANUARY 16 65
A r t i c l e s npg 16 Nture Americ, Inc. All rights reserved. Figure Dynmic imging of inflmmsome ctivtion in ASC-GFP retrogenic mice revels rpid kinetics of cellulr inflmmsome signling. () Chimers generted y trnsfer of Rg / BM-HSCs trnsduced to express ASC-GFP into irrdited recipient mice with or Csp1 / BM cells. () Intrvitl microscopy of the poplitel lymph node in ASC-GFP BM chimers, showing GFP (green) expression in cells loclized to the lymph node collgen cpsule (white). Dshed lines indicte lymph node edge. Scle r, 3 µm. SHG, second hrmonics genertion. (c) Intrvitl view of ASC-GFP BM chimeric mouse lymph node fter in vivo stining with phycoerythrin (PE)-conjugted nti-cd169 showing GFP + cell nd CD169 colocliztion in the sucpsulr sinus region. Scle r, 3 µm. (d,e) Intrvitl imges of ASC-GFP BM chimeric mouse lymph node over time (t) intervl of 1 h fter injection with MVA (d) or (e). Arrows indicte ASC speck formtion events; dshed lines indicte lymph node edge nd delinete individul ASC-GFP expressing cells. Scle rs, 3 µm. (f) Intrvitl imges of ASC speck formtion in lymph node mcrophge 3 h fter locl infection with MVA. t is reltive to strt of ASC-GFP redistriution during speck formtion. Scle r, 1 µm. (g) Kinetics of speck formtion in vivo detected y redistriution of ASC-GFP protein (chnge in men GFP intensity) from cell cytoplsm to the speck region in single lymph node mcrophge on locl MVA infection. Dt re representtive of 36 individul mice ( f) or representtive of more thn independent events (g). When ASC-GFP chimeric mice were injected sucutneously with MVA, we detected ASC speck formtion primrily within these mcrophges in drining lymph nodes (Fig. d nd Supplementry Movie ). Of note, we did not oserve ASC speck formtion in chimeric mice injected with (Fig. e nd Supplementry Movie 3). Single-cell nlysis of the redistriution of ASC-GFP leding to speck ssemly reveled rpid nd dynmic cellulr process (Fig. f,g), with the mjority of cells undergoing complete ASC oligomeriztion within 1 min (mode,.8 min; medin, 6 min; rnge, 99 min) (Fig. 3 nd Supplementry Fig. 3c). We next quntified ASC speck formtion occurring during the first 1 h fter locl MVA injection nd identified n cute inflmmsome response, detectle within 1 h fter infection nd peking over the susequent h (Fig. 3). For these nlyses we counted ASC specks tht formed during imging (referred to s forming ASC specks) nd ASC specks tht hd lredy formed (referred to s formed ASC specks) t the time of imging. Inflmmsome ctivtion then stedily declined until neither forming nor formed specks were detected t 1 h. The numer of ASC specks detected fter infection corresponded with the kinetics of SCS-M loss oserved y histologicl nd lymph node cell-suspension nlysis methods descried ove (Fig. 1f,g). Thus the stedy decline in the numers of specks detected fter infection proly reflects the loss of SCS-Ms. Throughout the imging period, ASC specks were not detected in lymph nodes of -injected controls or within deeper regions of the lymph node ccessile y two-photon imging fter MVA injection (Supplementry Movie ). Further nlysis of the reltive sptil rrngement of specks in the lymph node reveled tht the mjority were loclized to within 3 µm of the sucpsulr region, s indicted y the presence of cpsulr collgen fiers (Fig. 3c nd Supplementry Fig. 3d,e). When compiled, these dt revel rpid triggering of ASC speck formtion, generting Rg / BM-HSC f Retrovirl trnsduction ASC-GFP or BM-HSC c CD169-PE ASC-GFP Collgen-SHG min Myeloid-specific ASC-GFP expression d Mixed BM chimer t MVA No speck Speck forming Persisting speck 5 min ASC-GFP Collgen-SHG Composite t + 1 h e 76 min 1 3 trnsient nd sptilly confined wve of inflmmsome ctivity spnning the first 7 h fter infection. Cell deth nd relese of extrcellulr ASC specks in vivo Pyroptosis is cspse-1 dependent form of cell deth 36 tht, unlike other modes of cell deth, is n inflmmtory event ssocited with cell swelling, memrne lysis nd nucler condenstion 37. Using timelpse microscopy, we exmined infection (MVA-GFP), inflmmsome ctivtion (ASC-mCherry) nd cell deth (DNA-inding SytoxBlue) events in BMDMs in prllel (Fig. nd Supplementry Movie 5). Dynmic nlysis of virlly encoded GFP nd nucler stin intensities reveled tht ASC speck formtion in BMDMs ws closely followed y loss of cell integrity nd cellulr demise (Fig. ). This process occurred within minutes fter inflmmsome ssemly (mode, 3 min; medin, 7 min; rnge, min) nd ws lrgely intrinsic to cell infection, with no pprecile cell-to-cell trnsctivtion of the inflmmsome response to infection (Supplementry Fig.,). We next exmined the process of ASC speck formtion nd cell deth y in vivo leling of lymph nodes with the cell-permele nucler DNA inding dye Hoechst 333 (Hoechst), enling detection of cell-deth events y trcking nucler condenstion. Intrvitl imging reveled tht mcrophges undergoing inflmmsome ctivtion died rpidly fter speck formtion, progressing first to nucler condenstion nd then to disppernce of the nucleus (Fig. c nd Supplementry Fig. c). While exmining in vivo Hoechst leling of drining lymph nodes, we noted tht the mjority of formed specks we detected did not hve ny ssocited nuclei, suggesting they were remnnts of cells fter inflmmsome ctivtion. To more firmly estlish this hypothesis, we injected fluorescence-conjugted ntiody to ASC into MVAinfected ASC-GFP chimeric mice. Formed specks were counterstined t g Men GFP intensity 5 15 1 5 t + 1 h Speck region Cell cytoplsm Time (min) 66 VOLUME NUMBER 1 JANUARY 16 nture medicine
r t i c l e s npg 16 Nture Americ, Inc. All rights reserved. Figure 3 Sucpsulr mcrophges provide sptiotemporlly defined inflmmsome response to locl virl infection. () Intrvitl trcking of ASC speck formtion nd its kinetics in the poplitel lymph node fter locl MVA infection. Ech row represents n individul ASC-GFP + cell; ech squre represents 1-min period. Dt re compiled from more thn independent events. () Quntifiction of inflmmsome ctivity in drining lymph node 1 h fter locl injection of or MVA. Numers of formed nd ctively forming ASC specks detected y intrvitl microscopy per imging field re shown. Ech dot represents the men ± s.e.m. numer of events detected t ech experimentl time point (n = 8 33 movies cquired from t lest three mice for ech time point). Dt re fit to log Gussin curve. (c) Percentge of ASC-GFP specks detected t distnces up to 1 µm from the lymph node cpsule; µm ws identified y second hrmonics signl genertion y collgen fiers. Dt re men ± s.e.m. of 35 speck events detected within 11 imging fields (5 independent experiments), ech cquired t imging depths up to 1 µm. with the ASC ntiody, confirming their extrcellulr nture (Fig. d). Intrvitl trcking of formed specks over severl hours reveled tht mny specks re long-lived entities, persisting for more thn h in the lymph node microenvironment (Fig. e). Moreover, specks generted in vivo remined s long-lsting structures, grdully diminished in size efore eventully disppering, suddenly disppered or were frgmented into multiple specks (Fig. e,f nd Supplementry Movie 6). In ddition, on severl occsions we oserved the cquisition of specks y other cells within the lymph node (Supplementry Movie 7). These in vivo oservtions re consistent with two recent reports descriing the ility of mcrophges to phgocytize specks in vitro, with the potentil to precipitte inflmmsome ctivtion within the engulfing cell 8,9. 1:3 1: 6:5 6:55 MVA-GFP ASC-mCherry SytoxBlue GFP intensity c. 1.8 1.6 1. 1. 1. ASC speck formed 1 18 15 1 9 6 3 Time (min) 3 69 1 8 min 1 min 3 min 59 min 5 3 1 SytoxBlue intensity d f 6 1 18 3 Time fter infection (min) No speck Speck prtilly formed Speck fully formed Speck diminishes Speck disppers Formed specks/field. 1.5 1..5 1 3 5 6 7 8 9 1 11 c 1 8 6 Speck events (%) 1 1 3 5 67 8 91 1 Distnce from cpsule (µm) MVA In vivo nlysis of inflmmsome ctivity lso reveled phenomenon wherey ASC speck formtion nd susequent cell deth resulted in rpid loclized recruitment nd clustering of resident cells towrd newly generted specks (Fig. g nd Supplementry Movie 8). Such recruitment occurred within minutes fter speck formtion nd cell deth nd could e detected y Hoechst stining nd y trcking myeloid cells (GFP + ) in ASC-GFP chimers (Supplementry Movie 8). In summry, our results descrie the rpid nd loclized initition of inflmmsome ctivtion in the lymph node on virl infection, resulting in mcrophge deth, persistence of extrcellulr ASC specks nd locl cell recruitment. ASC-GFP ASC-PE ma Collgen (SHG) ASC-GFP Speck persisting Merge ASC- GFP Speck fully formed Speck prtilly formed Speck diminishes Speck disppers Speck frgments Figure ASC ggregtion ASC-GFP Hoechst 98 min 16 min 5 min precedes rpid cell deth relesing long-lived extrcellulr ASC specks in vivo. () Time-lpse microscopy of ASC-mCherry (top nd middle), 1 3 5 5.6 Speck disppering nucler stining (SytoxBlue, middle) nd GFP expression (middle) in 3 min 35 min min BMDMs fter infection with MVA-GFP. Trnsmitted light imges g confirm cell deth (ottom). Scle rs, 1 µm. Numers indicte time (h:min) fter infection. Representtive imges from more thn Speck frgmenting 5 min 7 min 55 min 5 independent experiments. () Chnges in BMDM cytoplsmic 3 min 36 min 6 min GFP intensity nd nucler SytoxBlue fter infection with MVA-GFP show rpid loss of cell integrity reltive to time of full ASC speck formtion (red line). Plot shows men fluorescence intensity ± s.e.m. for 5 cellulr events. (c) In situ nucler (Hoechst) stining of lymph node mcrophges from ASC-GFP BM chimeric mice shows MVA-induced ASC speck formtion is ssocited with rpid nucler condenstion. Scle r, 1 µm. Dshed white lines indicte detectle cell cytoplsm; dshed lue lines indicte nucler regions. Time points re from strt of imging. Imges re representtive of five independent experiments. (d) Intrvitl imging of lymph nodes from ASC-GFP BM chimeric mice fter MVA tretment (6 h) showing colocliztion (rrows) of loclly injected ASC-specific PE monoclonl ntiody (ma) with formed ASC-GFP specks. Scle rs, µm (left) or µm (inset). SHG, second hrmonics genertion. Imges re representtive of three independent experiments. (e) Intrvitl trcking of formed ASC specks induced y MVA infection. Rows represent individul formed specks trcked in vivo; ech squre shows chnges oserved over 1 min. Imges representtive of more thn 8 independent events (n = 8 mice). (f) Intrvitl imges of speck ftes. Scle rs, 1 µm. Imges representtive of more thn 8 independent events (n = 8 mice). Time points re from strt of imging. (g) In situ nucler stining (Hoechst) of lymph node resident cells nd intrvitl imging of ASC speck formtion (green) in lymph nodes of ASC-GFP BM chimeric mice fter MVA infection. Time points re from strt of imging. Scle r, 1 µm. Imges re representtive of five independent experiments. Hoechst ASC-GFP ASC-PE ma e.5..3..1 Forming specks/field nture medicine VOLUME NUMBER 1 JANUARY 16 67
A r t i c l e s Normlized mount 1 8 6 Neutrophils per LN ( 1 ) 15 1 5 CCL11 6 CCL * * 5 3 1 CXCL c Monocytes per LN ( 1 ) + + MVA + MVA 3 15 5 15 1 5 1 CXCL1 3 1 CCL19 1 5 Il1r1 / + MVA d XCL-1 NK cells per LN ( 1 ) 8 6 3 1 LIF 15 1 5 CCL Il1r1 / npg 16 Nture Americ, Inc. All rights reserved. 6 18 Figure 5 Inflmmsome ctivtion regultes the mgnitude of the inflmmtory innte infiltrte into drining lymph nodes. () Whole lymph node chemokine profiling in, cspse-1 deficient (Csp1 / ) nd IL-1R1 deficient (Il1r1 / ) mice h fter MVA or injection. Chemokine levels re normlized to totl lymph node protein content (n = 3 mice per group). ( d) Asolute numers of CD11 hi Ly6G + neutrophils, Ly6C hi CD11 + monocytes nd Inflmmsome signling leds to influx of inflmmtory cells Our in vivo nlysis reveled tht inflmmsome ctivtion induced y MVA ws restricted to the lymph node. Therefore, we next ssessed how inflmmsome signling trnsltes to ctivtion of the innte nd dptive immune response y performing whole lymph node screening of chemokine meditors expressed erly ( h) fter locl virl infection (Fig. 5). We oserved rpid production of chemokines induced y MVA, mny of which were reduced in the sence of cspse-1 or IL-1 receptor type 1 (IL-1R1) signling (CCL11, CCL, CXCL, CXCL1 nd CCL19). This shows tht trnsient wve of inflmmsome ctivtion elicited y MVA is potent modultor of chemokine relese in drining lymph nodes. Consistent with these dt nd previous reports on chemokine induction y MVA 5,38, this wve of chemokine relese ws ssocited with rpid nd roust infiltrtion of neutrophils, monocytes nd nturl killer (NK) cells to the lymph node. Innte immune cell recruitment ws sustntilly reduced in the sence of cspse-1 or IL-1R1 signling (Fig. 5 d nd Supplementry Fig. 5). To identify which cellulr comprtment ws responsile for responding to inflmmsome-derived IL-1 signls, we generted BM chimeric mice in which only hemtopoietic cells ( BM > Il1r1 / ) or only nonhemtopoieticlly derived cells (Il1r1 / BM > ) expressed IL-1R1. Infection-induced recruitment of neutrophils nd monocytes ws olished in the sence of hemtopoietic cellulr e Neutrophils per LN ( 1 ) 1 8 6 * 6 18 1 8 6 * * * * Monocytes per LN ( 1 ) 8 6 1 6 18 * MVA MVA MVA MVA > Il1r1 / > > Il1r1 / NK cells in lymph nodes s ssessed y flow cytometry t vrious time points fter sucutneous MVA infection in (lck circles), Csp1 / (open circles) nd Il1r1 / (gry circles) mice. Dt re comined from more thn 6 independent experiments. (e,f) Asolute numers of neutrophils (e) nd monocytes (f) in lymph nodes of BM chimeric mice (Il1r1 / >, red; > Il1r1 /, gry; control group >, lck rs) s ssessed y flow cytometry, h (left) or 18 h (right) fter MVA infection or control () injection. Dt re representtive of two independent experiments with n = 5 mice per group. Error rs, men ± s.e.m. *P <.5, P <.1, P <.1, one-wy ANOVA with Tukey s multiple comprison test ( f) or Mnn- Whitney U-test (). LN, lymph node. f sensing of IL-1 nd ws prtilly diminished y nonhemtopoietic cell expression of IL-1R (Fig. 5e,f). This indictes tht cells of predominntly hemtopoietic origin rely IL-1 signls in the lymph node to promote infiltrtion of innte cells to the drining lymph node. Inflmmsome signls roden composition of T cell responses To ssess the contriution of inflmmsome ctivtion to the dptive immune response, we focused on the genertion of the ntigenspecific CD8 + T cell response directed ginst the immunodominnt vccini virl peptide (VVp) B8R 7 expressed y MVA (Fig. 6). Lck of cspse-1 ctivity or IL-1R1 expression resulted in modertely lower percentges of ntigen-specific T cells induced y MVA infection (Fig. 6). However, oth cspse-1 nd IL-1R1 deficient mice showed reduced solute numers of VVp-specific cells (Fig. 6c). We hypothesized tht n overll increse in leukocyte infiltrtion induced y inflmmsome ctivity my led to more nive T cells eing recruited to the drining lymph node for priming nd ctivtion. Indeed, functionl inflmmsome nd IL-1R1 signling promoted the overll ccumultion of CD8 + T cells in the drining lymph node s erly s 18 h fter MVA injection (Fig. 6d,e). To test whether inflmmsome ctivtion fvors the recruitment of T cells tht were not initilly present in the lymph node, we trnsferred T cells into nd cspse-1 nd IL-1R1 deficient recipients nd 68 VOLUME NUMBER 1 JANUARY 16 nture medicine
r t i c l e s npg 16 Nture Americ, Inc. All rights reserved. CD 7.1 Gted on CD8 + T cells.5 7. 93.. 66. HK -TSY pentmer MVA 6.8.1 CD8 + HK -TSY + cells (%) 6 immeditely dministered locl MVA infection (Fig. 6f) so tht the mjority of trnsferred T cells were present in the circultion t the time of infection. We trnsferred comintion of T cells tht could e susequently re-trced in vivo, ech t different rtio to provide minor circulting T cell popultions nd simulte n in vivo limiting-dilution ssy. We nlyzed the specific contriution of trnsferred T cells in the ulk of the VVp-specific T cell pool t the pek of the response to evlute the moiliztion of T cells tht were not initilly present in the lymph node. In mice, lrge frction of the VVp response ws derived from the trnsferred T cell popultions. However, this contriution ws reduced in cspse- 1 deficient nd even more so in IL-1R1 deficient mice (Fig. 6g,h). In prticulr, the CFP + T cells (which were trnsferred in the lowest numers) were virtully sent from the VVp-specific response in cspse-1 nd IL-1R1 deficient mice. We confirmed tht this effect ws ttriuted to the defective erly recruitment of doptively trnsferred T cells from the circultion to infection-drining lymph nodes in the sence of inflmmsome signling (Supplementry Fig. 6). As expected, delying MVA injection until 8 h fter T cell trnsfer to llow time for trnsferred T cells to equilirte within lymphoid orgns resulted in n overll T cell response tht ws less dependent on erly inflmmsome-driven T cell recruitment, with trnsferred T cells mking similr contriutions towrd the overll VVp-specific T cell response in mice nd mice with disrupted inflmmsome signling (Supplementry Fig. 6). In line with these findings, locking T cell trfficking to the drining lymph node during MVA infection in mice using CD6L-specific locking ntiody resulted in significnt reduction in the numer of VVp-specific T cells (Supplementry Fig. 7,). Furthermore, nlysis of IFN-γ T cell responses to other H-K -restricted peptide epitopes for MVA (A3L, K3L, A19L nd A8R) showed tht locking T cell recruitment lso reduced the diversity of the T cell response (Supplementry Fig. 7c,d) further highlighting the importnce of T cell trfficking to the drining lymph node for optiml responses. These dt indicte tht inflmmsome ctivtion increses the mgnitude Il1r1 / c HK -TSY specific CD8 + T cells per LN ( 1 ) 1 8 6 Il1r1 / d CD3 + T cells per LN ( 1 5 ) 1 8 6 Il1r1 / CFP 18 e CD8 + T cells per LN ( 1 5 ) 3 1 18 nd rodens the potentil repertoire of the T cell response y moilizing ntigen-specific T cell clones from the periphery. DISCUSSION Secondry lymphoid orgns re key loctions for initition of innte host defense nd priming of the dptive response. Here we provide evidence of host immune circuit linking ctivtion of these two rms of the response to deliver effective immunity, which is coordinted through inflmmsome signling initited y sucpsulr mcrophge sensing of infection. Methods to detect inflmmsome ctivtion in situ hve so fr een very limited, relying primrily on tissue histologicl stining for speck protein components, ctive cspse-1 or mture IL-1β 8,39 1. This tsk is further complicted y the close link etween inflmmsome ctivtion nd cell deth nd the inherent difficulty in detecting such trnsient events. Our pproch circumvents these limittions y proing the inflmmsome in rel-time nd in single cells using intrvitl imging. A potentil cvet of our strtegy my e tht ASC overexpression lters the timing of inflmmsome ctivtion. However, this ws not oserved in vitro, nd furthermore, the kinetics of the immune response to MVA nd SCS mcrophge deth in our chimers ws similr to tht oserved in mice, suggesting tht overexpression of the proe hs no ovious effect on the oserved response. With the ility to detect the cell type, loction nd timing of inflmmsome pthwy, we provide the first glimpse into the dynmics of inflmmsome ctivtion in the context of lymph-orne virus. As recent in vitro studies hve demonstrted tht the kinetics of cspse-1 ctivtion re essentilly the sme irrespective of the intensity or types of stimulus,3, our results my e directly relevnt to other types of pthogenic chllenges in vivo. A recent study y Kstenmüller et l. hs indicted role for the inflmmsome pthwy in innte immune cell ctivtion nd recruitment in response to lymph-orne pthogens, highlighting n importnt role for SCS mcrophges in the context of IL-18 nd IL-1β relese 16. In ddition, this report nd previous work show tht SCS f T cell trnsfer CFP + CD5.1 + 15 min CD5. + recipients Il1r1 / Percentge trnsferred CD8 + T cells within HK -TSY + MVA dy 6 LN nlysis Figure 6 Inflmmsome signls recruit circulting ntigen-specific Gted on CD8 g HK -TSY + cells T cells to the drining lymph node. ( c) Flow cytometry nlysis of h Il1r1 / 5 pentmer (HK -TSY) stining for detection of percentge of (inset numers) 3.6.8.5 MVA vccini B8R peptide-specific T cells in drining lymph nodes 6 d fter 9. 5.3 1. infection () nd quntifiction (,c) s percentges of totl CD8 + T cells () nd * (c) solute cell numers in, cspse-1 deficient (Csp1 / ) nd 3 IL-1R1 deficient (Il1r1 / ) mice. n = 5 mice per group nd re representtive CD5.1 dt from independent experiments. (d,e) Asolute numers of CD3 + (d) nd CD8 + (e) T cells detected in lymph nodes y flow cytometry up to 18 h 1 fter sucutneous MVA infection. (f) Schemtic of trnsfer of CFP + or CD5.1 + T cells into CD5. + recipient mice (1: rtio) nd drining lymph node (dln) CD5. + CFP + CD5.1 + Il1r1 / nlysis. (g,h) Composition of HK -TSY specific CD8 + T cells in drining lymph nodes 6 d fter MVA infection showing trnsferred CFP + (lue) nd CD5.1 + (red) T cells. Inset numers indicte percentge of popultion. Percentges s proportion of totl pentmer-specific T cells re represented in the pie chrts (g, ottom) nd summrized in h. Comined dt from three independent experiments (n = more thn 7 mice per group). Error rs, men ± s.e.m. *P <.5, P <.1, P <.1, one-wy ANOVA with Tukey s multiple comprison test. nture medicine VOLUME NUMBER 1 JANUARY 16 69
A r t i c l e s npg 16 Nture Americ, Inc. All rights reserved. depletion lters neutrophils nd NK cell recruitment in the drining lymph node upon virl chllenge 16,33. The present results extend these findings y demonstrting directly tht mcrophges in the sucpsulr re of the lymph node rpidly undergo inflmmsome ctivtion, which is immeditely followed y pyroptotic cell deth. Although our dt support the ide tht SCS mcrophges re the mjor popultion undergoing inflmmsome ctivtion during the erly phse of the response, it is likely tht other cell types, such s inflmmtory monocytes or neutrophils, my lso contriute to the inflmmsome response during lter stges of infection,5. Our results provide in vivo support for the ide tht the inflmmsome cts s digitl switch, wherey pyroptosis coincides with urstrelese of proinflmmtory cytokines, which re rpidly processed y concomitnt cspse-1 ctivtion, phenomenon recently descried in vitro,3. Thus, in vivo, inflmmsome signling y sucpsulr sinus mcrophges proly delivers concentrted nd loclized inflmmtory stimulus to surrounding cells, defining discrete ntomicl comprtmentliztion of the inflmmsome response. Extrcellulr ASC specks re ctive when injected in vivo 8,9, ut their genertion in situ hs not, to our knowledge, een chrcterized previously. We now directly demonstrte tht most ASC specks persist for severl hours s extrcellulr structures efore showing signs of degrdtion. Detection of speck frgmenttion nd engulfment in vivo suggests tht these events my provide potentil mechnisms of mplifying the inflmmsome response nd wrrnt further exmintion of the functionl relevnce of these phenomen in contriuting towrd the immune response. One potentil consequence of lymph-orne infection is the ltertion of the SCS mcrophge lyer. Neutrophil or dendritic cell infiltrtion cn contriute to disruption nd/or destruction of this lyer 6,7. Our results estlish pyroptosis s n dditionl, direct mechnism ccounting for the clernce of CD169 + SCS mcrophges oserved during infection 7. Locl recruitment of myeloid cells triggered y inflmmsome ctivtion nd pyroptosis is reminiscent of neutrophil clustering oserved round dying cells 6,8,9. Although this immedite response is proly due to the relese of locl meditors upon cell deth, such s extrcellulr ATP or other DAMPs 5 5, our results suggest tht susequent IL-1 sensing y hemtopoietic cells lrgely mplifies the initil inflmmsome signls. This second level of signl propgtion resulting in chemokine production nd cellulr recruitment to the drining lymph node my therefore work in concert with other mechnisms of lymph node remodeling induced y infection, such s vsculr nd lymph ngiogenesis leding to incresed orgn volume 53. Emerging dt re highlighting the importnce of inflmmsome regultion of the dptive T cell response in the context of vccine djuvnticity, infection nd utoimmune disorders 5 58. In severl studies, lck of inflmmsome ctivity hs een shown to result in poorer T cell response to infection 19,57,58. Triggering of the inflmmsome pthwy my increse the qulity of ntigen presenttion or ct on T cell differentition nd expnsion through the direct ction of IL-1 (refs.,59). Here we hve uncovered n dditionl mechnism wherey inflmmsome ctivtion enhnces the mgnitude nd redth of composition of T cell responses y promoting recruitment of T cells from the periphery. MVA nd other vccine vectors tht engge the inflmmsome pthwy my therefore e dvntgeous in moiliztion of mximl numer of ntigen-specific T cell clones to the drining lymph node. In summry, our results revel n immune circuit operting in lymph nodes in which sptilly comprtmentlized wve of inflmmsome ctivtion trnsltes into the mssive recruitment of innte nd dptive immune cells tht ultimtely mximizes T cell responses. Our pproch to proing inflmmsome ctivity in vivo will llow dissecting the contriution of this pthwy in vccintion, infection or inflmmtory disese. Methods Methods nd ny ssocited references re ville in the online version of the pper. Note: Any Supplementry Informtion nd Source Dt files re ville in the online version of the pper. Acknowledgments We wish to thnk O. Schwrtz, N. Mnel nd memers of P.B. s lortory for criticl review of the mnuscript; A. Cumno (Institut Psteur) for helpful discussions in generting BM chimeric mice; nd the Centre d Immunologie Humine (Institut Psteur) for support with flow cytometry. This work ws supported y Institut Psteur (P.B.), Institut Ntionl de l Snté et de l Recherche Médicle (P.B.), the Vccine Reserch Institute (P.B.), Fondtion pour l Recherche Médicle (P.B.) nd Strting Grnt from the Europen Reserch Council (P.B.). AUTHOR CONTRIBUTIONS P.B. nd P.S. designed the experiments, nlyzed the dt nd wrote the mnuscript; P.S. nd B.B. conducted the experiments with essentil support from Z.G., F.L.; D.M., M.L.A. nd Y.L. provided scientific input nd regents. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Reprints nd permissions informtion is ville online t http://www.nture.com/ reprints/index.html. 1. Monie, T.P., Moncrieffe, M.C. & Gy, N.J. Structure nd regultion of cytoplsmic dpter proteins involved in innte immune signling. Immunol. Rev. 7, 161 175 (9).. Frnchi, L., Muñoz-Plnillo, R. & Núñez, G. Sensing nd recting to microes through the inflmmsomes. Nt. Immunol. 13, 35 33 (1). 3. 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USA 16, 7119 71 (9). nture medicine VOLUME NUMBER 1 JANUARY 16 71
npg 16 Nture Americ, Inc. All rights reserved. ONLINE METHODS Mice. Wild-type () C57Bl/6 mice were purchsed from Chrles River Frnce. Cspse-1 deficient (Csp1 / Csp/11 /, B6N.19S-Csp1 tm1flv /J) 6, IL- 1R1 deficient (Il1r1 /, B6.19S7-Il1r1 tm1imx /J), C57Bl/6 Rg /, CD5.1 + (B6.SJL-Ptprc Pepc /BoyJ), C57Bl/6-GFP (expressing GFP under the uiquitin promoter) nd C57Bl/6-CFP (expressing CFP under the ctin promoter) mice were red in the niml fcility of Institut Psteur under specific pthogen free conditions, nd littermtes were rndomly ssigned to experimentl tretment groups. Experimentl protocols were ethiclly pproved y the Institutionl Committees on Animl Welfre t Institut Psteur nd conducted in strict ccordnce with French guidelines for niml cre nd use. Tretments nd regents. Recominnt MVA-B (expressing full-length HIV Gg, fused to three Pol nd two Nef frgments) 61 ws provided y the Agence Ntionle de Recherche sur le Sid (ANRS), nd recominnt MVA-B strin expressing enhnced GFP ws provided y Trnsgene Lortories (Strsourg, Frnce). Mice were injected sucutneously (s.c.) in the footpd with 5 1 6 p.f.u. of MVA. In vivo leling of CD169 + cells ws performed y s.c. injection of.5 µg PE-conjugted nti-cd169 (Biolegend, clone 3D6.11) pproximtely 1 h efore imging. In vivo stining of extrcellulr ASC specks ws performed y injection of.5 µg AF555-conjugted nti-asc ntiody (Bioss Antiodies, s-671r-a555) 6 h fter MVA or injection, nd imging commenced within 3 min. Whole lymph node leling with Hoechst 333 (333 trihydrochloride, trihydrte, Invitrogen) ws performed y s.c. injection of 1 µl of.1 µm solution prepred in, nd imging commenced within 3 min. The investigtors were not linded to group lloction during experiments or dt nlysis. In vivo lockde of CD6L ws performed y intrvenous (i.v.) injection of 1 µg locking ntiody (eioscience, MEL-1) 6 h efore MVA injection nd h fter MVA injection. mice were treted with IL-1β neutrlizing ntiody (R&D systems, AF-1-NA) y dministrtion of 1 µg intrperitonelly (i.p.) 1 h efore T cell trnsfer or MVA injection. Flow cytometry. To detect cspse ctivity, mcrophges (. 1 6 cells) were seeded in ultr-low ttchment -well pltes (Corning) overnight nd then stimulted with MVA expressing fluorescent protein to monitor infection t multiplicity of infection (MOI) of 1, 5, 5 or 5 for 6 1 h. Cells were gently hrvested nd stined with FLICA-1 FITC (cspse-1 proe FAM-YVAD- FMK) nd FLICA-3/7 66 (cspses-3/7 FAM-DEVD-FMK) kits ccording to the mnufcturer s instructions (Immunochemistry Technologies). Control cultures were treted with 1 µm sturosporine (STS) (Sigm) to induce cspse-3 ctivity. To detect tissue infection y MVA nd inflmmtory cell recruitment, poplitel lymph nodes were tesed prt using forceps nd digested in RPMI 16 contining 1 mg/ml collgense D nd 5 ng/ml DNse (Sigm) for 15 min t 37 C, nd then pssed through 7-µm cell striner. Virlly expressed GFP ws detectle directly y flow cytometry. Cell-surfce stining ws performed with the following ntiodies, ll from Biolegend nd t dilution of 1: (.5 µg/ml) unless otherwise specified, in the presence of locking nti-cd16/3 (ebioscience, clone 93) nd fter exclusion of doulets nd ded cells (live/ded fixle dyes, Invitrogen): nti-cd3 (17A), nti-nk.1.1 (PK136), nti-cd8 (53-6.7), nti-cd19 (1D3), nti-cd169 (3D6.11/ MOMA-1, AD Serotec,), nti-cd11 (M1/7, BD), nti-f/8 (BM8), nti-cd5.1 (A), nti-cd5. (1), nti-cd11c (N18), (1:5) nti-i-a/e (M5/11.15.), (1:) nti-ly-6g (1A8), nti-ly-6c (AL-1). Cell suset nlysis ws performed on the sis of the following phenotypic gting definitions: CD3 CD11 + CD169 + F/8 for SCS-M, CD3 CD11 + CD169 + F/8 + for Med-M, CD3 Ly6G CD169 CD11 + for mcrophges, CD3 I-A/E hi CD11c + for DCs, CD3 Ly6G Ly6C hi CD11 + for inflmmtory monocytes, CD11 hi Ly6G + for neutrophils nd CD11 + NK1.1 + for NKs cells. Asolute cell numers were quntified using AccuCheck ed microspheres (Life Technologies) during flow cquisition. Intrcellulr stining ws performed using the Cytofix/Cytoperm kit (BD Bioscience) using APCconjugted nti-grnzyme B (GB11, BD iosciences) nd FITC-conjugted nti-ifnγ (XMG1., eioscience). Anlyses were performed using n LSRII Cytometer (BD Biosciences) nd nlyzed using FlowJo softwre version 1.6 (Tree Str). Cell deth induced y MVA ws detected in BMDM cultures infected with MVA-GFP y counterstining with.5 µg/ml propidium iodide (Sigm). ELISA. For ELISA detection of IL-1β in cell-free superntnts, BMDMs were cultured overnight with MVA (MOI 1 5) or 1 µg/ml LPS (serotype 6:B6, Sigm) in the presence or sence of 75 µm cspse-1 inhiitor Z-YVAD-FMK (R&D Systems). The next dy, LPS-treted cells were pulsed with 5 mm ATP for 1 h. Superntnts were hrvested nd nlyzed using IL-1β DuoSet ELISA kit ccording to the mnufcturer s instructions (R&D Systems). Inflmmsome ASC proe genertion. ASC-GFP (enhnced GFP) or ASCmCherry (monomeric cherry) 6 constructs encoding the full sequence of ASC with fluorescent protein tg on the 5 sequence end were cloned into murine stem cell virl (MSCV) vector using stndrd techniques. Retrovirus ws generted y co-trnsfection of ASC-MSCV (3 µg) nd pci-eco (1 µg) plsmid DNA into HEK93T cells using Lipofectmine trnsfection regent (6 µl) (Life Technologies) in Opti-MEM (Gico). Trnsfection ws quenched fter 8 h with DMEM contining 1% FBS, 1% penicillin/ streptomycin. After overnight incution t 37 C, culture medium ws replced with either complete DMEM or Iscove s Modified Dulecco s Medium (IMDM) for trnsduction of BMDMs or hemtopoietic stem cells (HSCs), respectively. Retrovirus contining superntnt ws hrvested from HEK93T cell cultures during the next d, filtered (.5 µm) nd used to perform sequentil rounds of BMDM nd HSC trnsduction s descried elow. Bone mrrow derived mcrophge genertion nd trnsduction. Murine one mrrow derived mcrophges (BMDMs) from or Csp1 / mice were differentited from one mrrow (1 1.5 1 6 cells seeded in 9.5-cm wells) y culture in DMEM, 1% FBS, 1% penicillin/streptomycin, 1% HEPES (ll from Gico) with ng/ml mcrophge-colony stimulting fctor (M-CSF, ebioscience) or % L99 superntnt, nd used t dy 7. BMDMs were stly trnsduced to express ASC-GFP or mcherry y dding retrovirlconditioned medium to mcrophge cultures on dys 3 nd during differentition. Retrovirl superntnt ws supplemented with fresh DMEM, 1% FBS, 1 µg/ml polyrene (Sigm), 1% HEPES nd ng/ml M-CSF (ebioscience) to prepre retrovirl conditioned medium, which ws used to replce medium on BMDM cultures nd incuted t 37 C overnight. On dy 5, retrovirl medium ws replced with fresh culture medium, nd cultures were mintined until dy 7 for use. Trnsduction efficiencies of >7% were routinely chieved. Time-lpse videomicroscopy. Mcrophges trnsduced to express ASC-GFP or ASC-mCherry (. 1 6 cells) were seeded in 35-mm FluoroDish cell culture dishes (World Precision Instruments) in phenol red free RPMI supplemented with 1% FBS, 1% penicillin/streptomycin nd 1 ng/ml M-CSF for 6 h, with dditionl 1 µg/ml LPS where indicted. Immeditely efore imging, cell medium ws replced with medium contining MVA, MVA-GFP or 5 mm ATP nd supplemented with.1 µm SytoxBlue (Invitrogen). Time-lpse microscopy ws performed using DMI6B inverted microscope (Leic Microsystems) equipped with heted chmer for temperture, humidity, nd CO control, with 1 /.75 NA dry ojective (Olympus) nd CoolSNAP HQ Roper cmer (Photometrics) for 15 h fter induction. Videos were nlyzed using ImgeJ or FiJi softwres (http://rs.info.nih.gov/ij/). Bone mrrow chimers. To generte mixed BM chimeric mice for intrvitl imging of inflmmsome ctivity, lethlly irrdited mice were reconstituted with BM-derived HSCs retrovirlly trnsduced to stly express ASC-GFP protein. Donor HSCs used for reconstitution were negtively selected (Miltenyi Linege Cell Depletion Kit) from C57Bl/6 Rg / BM (to chieve myeloidrestricted proe expression) nd cultured overnight ( 3 1 6 cells per 9.5-cm well) in complete IMDM supplemented with 1% FBS, 1% HEPES,.1% -mercptoethnol, ng/ml stem cell fctor/c-kit lignd (SCF) nd 1 ng/ml thromopoietin (TPO). After culture overnight, HSC cultures were resuspended in ASC-GFP retrovirl superntnt (supplemented with 1% FBS, 1% HEPES,.1% -mercptoethnol, ng/ml SCF, 1 ng/ml TPO, 1 µg/ml polyrene) nd spin-trnsduced t 1, g nd 3 C for 9 min. Fresh complete IMDM ws dded fter further 6-h incution t 37 C. This ws repeted the following dy, with the exception tht 6 h fter spin-trnsduction, HSCs were hrvested, wshed in fresh medium, nd seeded onto pre-prepred 1-cm dishes of OP-9 feeder cells (ATCC) 6 in complete IMDM. Cultures nture medicine doi:1.138/nm.16