© 2009 Nature America, Inc. All rights reserved. ([email protected]). ([email protected]). University School of Medicine, New York, New York, USA (K.J.M.). of Neuropathology, Berlin, Berlin, Germany Charité-Universitätsmedizin (A.H.) and Department of Medicine, The Leon H. Charney Division of Cardiology, New York Immunology & Inflammatory Diseases, General Massachusetts Hospital, Harvard Medical School, USA. Charlestown, Massachusetts, USA. Massachusetts, Harvard Medical School, Boston, USA. Massachusetts, 1 this of breadth the for account might TLRs the among signaling TLR2-TLR6 and ( TLR2-TLR1 of identification early the Although RNA viral and DNA CpG flagellin, (LPS), lipopolysaccharide ing of range wide a includ protozoa, and fungi viruses, bacteria, from recognize ligands to pathogen receptors of group restricted this of understood. poorly remain TLRs the by recognition facilitating mechanisms the defined, well been regulated link to conserved signaling pathways activating NF- adaptor moleculesTIR-containing suchof as recruitment MyD88, by propagated TIRAP is (also signaling called transduction MAL), signal TRIF and initiates TRAM that that domain (TIR) Toll–interleukin-1 cytoplasmic a and domain (LRR) repeat by leucine-rich this of family are an characterized extracellular members 12 The pathogens. against defense host for essential genes of regulation transcriptional in the role critical their illuminated has motifs ger and Toll-like receptors (TLRs), which recognize conserved nonself receptors, such recognition as pattern the scaven germ line–encoded host. by is mediated ligands healthy endogenous or modified microbial of the Sensing in absent normally are that structures molecular discriminating by organism the protects system immune innate The events. signaling coreceptor by amyloid- and atherogenic which by mechanism molecular a common as activation CD36-TLR4-TLR6 identify results Our ligands. disparate these for receptor a common CD36, receptor scavenger the from signals by regulated is heterodimer identified newly this of Assembly amyloid- and LDL oxidized that show we Here unclear. remain activation of mechanisms molecular the diseases, these of pathology the underlie to believed is amyloid- and (LDL) low-density oxidized components self altered the of deposition disease, Alzheimer’s and In T Douglas Golenbock Katey J Rayner Cameron R Stewart assembly of a Toll-like receptor 4 and 6 heterodimer CD36 ligands promote sterile through nature immunology nature Received 3 November 2008; accepted 7 December 2009; published online 27 December 2009; Lipid Unit, Department of Medicine and A A notable aspect of the mammalian TLR family is the ability ability the is family TLR mammalian the of aspect notable A 0 0 2 2 1 . Over the last decade, intense study of the mammalian TLRs TLRs mammalian the of study intense decade, last .the Over 9 8 ) heterodimers led to the speculation that combinatorial combinatorial that speculation the to led heterodimers )  2 triggers a protracted sterile inflammatory response. Although chronic stimulation of the innate immune system system immune innate the of stimulation chronic Although response. inflammatory sterile a protracted triggers . While these downstream signaling pathways have have pathways signaling downstream these While . 1 4 , Laurent Boyer Department Department of Biochemistry and Molecular Biophysics, School of Medicine, Washington University, St. Louis, Missouri, USA.

1 , 7 aDV , Lynda M Stuart 3 & J Kathryn Moore A NCE ONLINE PUBLIC ONLINE NCE  stimulate sterile inflammation and suggest a new model of TLR heterodimerization triggered triggered heterodimerization TLR of model a new suggest and inflammation sterile stimulate 2 , Ruiqin Zhong  trigger inflammatory signaling through a heterodimer of Toll-like receptors 4 and 6. 4 and receptors Toll-like of a heterodimer through signaling inflammatory trigger 2 . Upon activation, TLR TLR activation, Upon . 2 Laboratory Laboratory of Immunology,Developmental Department of Pediatrics, General Massachusetts Hospital, 3 2 Department Department of Infectious Diseases and Immunology, University of Medical Massachusetts School, Worcester, , 7 κ , Kim Wilkinson B- and interferon- A 1 TION , 6 7 These These authors contributed equally to this work. should Correspondence be addressed to K.J.M. 4

, William A Frazier 2 - - .

1 , 7 accumulate in atherosclerosis and Alzheimer’s disease plaques. disease Alzheimer’s and atherosclerosis in accumulate that ligands sterile to response immune innate the activate to family us to invest ligands endogenous modified these of CD36-dependent signa tissues, and circulation the from molecules cells host apoptotic , amyloid and oxidized self including altered of uptake components, endocytic the in roles established has CD36 Mycoplasma for as coreceptor a function TLR2-TLR6 to its ascribed addition recently In origin. self and pathogen both of ligands polyanionic binds that TLR2 agonists integrin LPS-binding LPS as recognize to MD-2 such and components soluble ( with TLR4 acts which well-studied CD14, the bridge is to this of molecules example prime A surface responses. signaling of initiation and other recognition microbe with which in together paradigm act emerging TLRs an suggests that function coreceptors TLR of enhance identification recent the However, described. repertoire , Janine M van Gils The The class B scavenger receptor CD36 ( tpyoocs aureus Staphylococcus l ing has also been implicated in the pro-inflammatory effects effects pro-inflammatory the in implicated been also has ing β 3 4 doi:10.1038/ni.183 3 and mannose binding lectin, can coordinate responses to responses coordinate can lectin, binding mannose and , Adam Lacy-Hulbert i , no other functional TLR heterodimers have yet been been yet have heterodimers TLR functional other no , gate whether CD36 cooperates with members of the TLR of members with cooperates CD36 gate whether arpaeatvtn lpppie2 (MALP-2) lipopeptide-2 -activating 4–8 . receptor CD36 recognition is pattern an archetypal 9 . While important for the clearance of these these of clearance the for important While . 1 , Jiusheng Deng 6 –derived lipoteichoic acid (LTA) and and (LTA) acid lipoteichoic –derived 1 2 . , Joseph El Khoury 10–13 A 6 0 Present Present addresses: Department 0 0 . These observations led led observations These . 1 5 , Annett Halle 6 A 0 0 ), as well as Dectin-1, 0 2 ticles e l c i rt A 2 9 5 Center Center for 6 ) coreceptor coreceptor ) 5 , 3 , 6 , 4,5  ,

© 2009 Nature America, Inc. All rights reserved. I-, MIP-1 MIP-2, chemokines these of expression ( increase to failed TLR2, not but ( Gro1 ing ( encod mRNAs in increases 30-fold to 3- showed cells these oxLDL, ( wild-type in by oxLDL induced but LDL ( not native genes four identified chemokines of panel complete a of screening Array oxLDL. plaques, atherosclerotic of component inflammatory key a with TLRs various in deficient macrophages stimulated we duction, for the upstream TLRs required for proatherosclerotic chemokine pro Toll– common interleukin-1the receptor requires signaling adaptor MyD88mediators (refs. 15,16).inflammatory To these search of tion of influx an initiate that chemokines of expression the events, particularly pro-inflammatory of cascade a off setting cholesterolemia, in hyper wall during the abnormally accumulate artery TLR6 and TLR4 of signaling Cooperative RESULTS coreceptors. relevant the with coexpressed are TLRs that suggest others may if,be identified as herein, we demonstrate the and heterodimers, TLR certain of assembly facilitate can coreceptors amyloid- and oxLDL from signals CD36 as acting receptor the common for by ligand-binding regulated heterodimerization, TLR of model new a suggest data Moreover, our disease. Alzheimer’s and atherosclerosis in activation identify a common molecular mechanism innate underlying immune results These disease. Alzheimer’s of pathognomonic is that toxicity of oxidized LDL (oxLDL) and amyloid- effects deleterious the in implicated mediators pro-inflammatory of gated by both the MyD88 and TRIF adaptors, leading to the induction is propa TLR4 of and signaling TLR TLR6. heterodimer TLR4-TLR6 Src through which to signals kinases induce undescribed a previously recognition, CD36-mediated rather but TLRs the of ligation not is note, we determine that the earliest event in this inflammatory cascade CD36, and particular TLR6. of TLR4 Of composed complex trimeric and (LDL) amyloid- units. AU, arbitrary of an are experimental representative All data are mean NF- ( TLRs. in the indicated cells HEK293 expressing of an NF- (50 Myd88 IL-1 encoding ( to compared wild-type). cells is (* to reported unstimulated cells (50 stimulated PCR and the in fold increase mRNA in oxLDL (qRT)- reverse by quantitative was analyzed expression macrophages. Myd88 in (WT), wild-type ( the macrophage inflammatory response to oxLDL. 1 Figure s e l c i rt A  the required mRNA RANTES of induction oxLDL whereas MyD88, e a Fig. 1 Fig. i. 1 Fig.

) Effect ) of Effect MD-2 soluble on oxLDL-induced , b We demonstrate that recognition of oxidized low density lipoprotein κ ) OxLDL-induced chemokine gene expression chemokine ) OxLDL-induced µ B luciferase reporter gene expression. gene B expression. reporter luciferase g/ml, g/ml, 12 h). ( −/− −/− a a

). Transcriptional upregulation of mRNAs encoding Gro1, Gro1, encoding mRNAs of upregulation Transcriptional ). ). By contrast, macrophages deficient in TLR4 or TLR6, TLR6, or TLR4 in deficient macrophages contrast, By ). κ TLR4 and TLR6 cooperatively mediate mediate TLR4 and TLR6 cooperatively and macrophages stimulated with oxLDL stimulated macrophages B-dependent luciferase reporter gene reporter luciferase B-dependent Cxcl1 1 4 β . Although this is inflammation sterile in nature, produc Trif in wild-type, in wild-type, γ ), MIP-2 ( was similarly abolished in macrophages lacking lacking macrophages in abolished similarly was −/− ± Tlr2 d s.d. and samples of triplicate ) OxLDL-induced expression expression ) OxLDL-induced , , also known as µ g/ml, g/ml, 12 h) compared β −/− β peptide by CD36 triggers assembly of a of by assembly hetero peptide CD36 triggers , , c . These observations support the notion that that notion the support observations These . ) Expression ) of mRNA Expression Tlr4 Cxcl2 Tlr4 −/− Supplementary Table 1 Supplementary −/− , , Tlr6 ), MIP-1 P , , Ticam1 < 0.05, Tlr6 n −/− of 3 or 4. −/− ( a β

−/− and ) ) and γ

in in vivo

(

( Ccl9 b )

Supplementary Fig. 1 Supplementary ) and RANTES ( ) RANTES and , including the neuro c a Il1b (fold induction) mRNA (fold induction) 100 ). In response to to response In ). 10 75 25 50 0 2 4 6 8 0 2 4 6

WT WT Tlr4 –/ – Ccl5

Tlr6 Tlr2 –/ ) ) ­ - - - - - ­ –

–/

Myd88 – expression of the mRNA encoding the cytokine interleukin (IL)-1 interleukin cytokine the encoding mRNA the of expression upregulated for essential also were MyD88 and TLR6 TLR4, kines, ( TRIF adaptor TLR alternative which showed a corresponding decrease in atherosclerotic lesion lesion atherosclerotic in decrease corresponding a showed which much lower in the descending aorta of the mice lacking CD36 was ( expression mRNA RANTES and MIP-2 Gro1, weeks. 12 for diet atherosclerosis-prone mediators inflammatory these of induction for required also is CD36 whether Todetermine ( oxLDL to response in transcription RANTES and MIP-2 did macrophages not Gro1, upregulate macrophages, CD36-deficient ( cells ing NF- NF- oxLDL-induced augment not did CD14 coreceptor TLR4 Notably,prototypic the coreceptors. whereas TLR- be to known receptors lipid-binding two CD36, and CD14 of roles the tested we agonist, TLR a as acts oxLDL how Tounderstand coreceptor a TLR4-TLR6 is CD36 heterodimer. TLR undescribed hitherto a of existence the invoking for macrophage required chemokine production in response to atherogenic lipids, are pathways, signaling TRIF-dependent and MyD88 These data were not due to LPS contamination. our that observations indicating the block the ( MD-2 that complex TLR4 ( LPS recognizes the of cofactor essential an MD-2, of absence NF- TLR4-TLR6-mediated Notably, or ( TLR2-TLR4 TLR2-TLR6, TLR4-TLR9 cells coexpressing NF- increase not did oxLDL as ( in increase NF- sixfold a observed we individually, TLR6 or TLR4 TLR2, expressing NF- an by driven and ­construct TLR4 via signaling oxLDL a of reporter TLR6. to expression Whereas oxLDL induce was unable for requirements the determine NF- regulator transcriptional the of activation the is signaling MyD88 of ( Fig. Fig. 1 Il1b –/ – * Tlr4

presence of polymyxin B or the MD-2 inhibitor B-1287, which which B-1287, inhibitor MD-2 the or B polymyxin of presence κ κ * i rsos t oLL ( oxLDL to response in ) B–driven reporter expression threefold in TLR4-TLR6 express in TLR4-TLR6 threefold expression reporter B–driven . e sd cl-ae NF- cell-based a used We B. * –/ d – * Fig. ). The signaling cooperation of TLR4 and TLR6 was specific, specific, was TLR6 and TLR4 of cooperation ). signaling The d

TLR9 TLR6 TLR4 TLR2 Fig. 2 Fig.

biological effects of LPS ( LPS of effects biological NF-κB activity (AU)

aDV Ccl Ccl Cxcl Cxcl * demonstrate demonstrate that TLR4 and TLR6, acting together through 100 120 140 Tlr

20 40 60 80 1 * 5 9 6 e * 2 1 –/ ). Moreover, TLR4-TLR6 signaling was not altered in in altered not was signaling Moreover,TLR4-TLR6 ). A κ – a * – – – – – – – – NCE ONLINE PUBLIC ONLINE NCE B activation in both TLR4 B and cells expressing TLR6 activation Fig. 1 Fig. ). Furthermore, as observed in in observed as Furthermore, ). + – – – Apoe + d b

), and was not augmented by the addition of of addition the by augmented not was ), and mRNA (fold induction) + + – – – 10 15 20 25 0 5 −/− n vivo in Cxcl1 + + – – – – κ and * B response element in HEK293 cells cells HEK293 in element response B κ + + – – – Cxcl2 Fig. 1 Fig. B-dependent reporter expression in in expression reporter B-dependent i. 1 Fig. * , we profiled gene expression in in expression gene profiled we , Cd36 + * κ

A Ccl9 Supplementary Fig. 1 Fig. Supplementary Myd8 WT b + + B–luciferase reporter assay to to assay reporter B–luciferase κ TION * κ ). In addition to these chemo these to ). addition In B activation occurred in the the in occurred activation B −/− B activation, CD36 increased increased CD36 B activation, c Ccl5 ). A notable consequence consequence notable A ). 8 –/ Apoe

e NF- B activity (AU) –

nature immunology nature κ 100 200 300 400 500 TLR6 TLR4 20 25 30 −/− 0 2 4 6 8 Cxcl1 Tlr4 mice fed a Western + + – Tr Cxcl2 WT −/− if –/ and and – + Ccl9 ), further further ), Fig. 2 Fig. Fig. Fig. 2 Tlr6 + MD-2 – MD- Ccl5 *

1 −/− b d c 2 ). ). ), β ­ -

© 2009 Nature America, Inc. All rights reserved. TLR4 and TLR6 was required for maximal induction of these these of induction maximal for required was TLR6 and TLR4 heter ( end signaling TLR2-TLR6-dependent vate CD36, of ligands by bacterial the used that from differs way. cooperation note, this Of of A nition recog CD36 that indicate data These shown). not (data TLR4-TLR9 or TLR2-TLR4 TLR2-TLR6, including pairs TLR other coexpressing NF- an of expression NF- of ( CD14 activation by augmented not however, was TLR4-TLR6-signaling; of fication ampli threefold to two- a caused cells these in CD36 of expression A reverse ( TLR6 and TLR4 coexpressing NF- an of activation A CD36. through signaling TLR4-TLR6 amyloid- lar (ref. CD36 bind to shown amyloid- disease, Alzheimer’s of component matory the of cells by microglia in called lineage, the brain driven response inflammatory pro a tracted by characterized is disease Alzheimer’s atherosclerosis, Like Amyloid- oxLDL. to responses inflammatory phage pathway act in receptors to macro these a initiate common signaling phenocopy macrophages ( oxLDL described previously as zymosan, to response in burst respiratory competent a Whereas burst. ( LDL native ( to oxLDL exposed macrophages (ROS). Wild-type species oxygen reactive of production the in complex signaling this of requirement the measured we oxLDL, to responses inflammatory macrophage the CD36, role explore of and in TLR4 TLR6 promoting the upregulates that chemokines proatherosclerotic these of expression pathway signaling the of component key a is ( formation and wild-type ( TLR6. and NF- an of expression oxLDL-induced on CD14 2 Figure nature immunology nature macromolecular a that suggest data our responses, inflammatory a single experiment and are representative of an experimental experimental an of representative are and experiment a single (50 oxLDL with ( Cd36 d a

) Reactive oxygen species production in wild-type, wild-type, in production species oxygen ) Reactive NF-κB activity (AU) 1,00 o CD14 CD36 TLR6 TLR4 200 400 600 800 −/− genous ligands of CD36 specifically trigger the TLR4-TLR6 TLR4-TLR6 the trigger specifically CD36 of ligands genous o 0 0 dimer. Moreover, because simultaneous expression of CD36, CD36, of expression simultaneous because Moreover, dimer. Apoe

Fig. 2 Fig. The coreceptor CD36 is required for TLR4-TLR6-dependent responses. ( responses. TLR4-TLR6-dependent for required is CD36 coreceptor The β 8,11  + + + + – – – – β b 42–1 peptide triggers CD36-TLR4-TLR6 signaling CD36-TLR4-TLR6 triggers peptide Supplementary Fig. 2 Supplementary * −/− , 1–42 c , these cells failed , to failed with cells produce these ROS stimulated when β Cd36 Supplementary Fig. 2 Fig. Supplementary + + ) Chemokine gene expression analyzed by quantitative reverse transcription (qRT)-PCR in (qRT)-PCR transcription reverse quantitative by analyzed expression gene ) Chemokine mice ( mice peptide, amino acids 1–42 (A 1–42 acids amino peptide, d µ was unable to activate NF- activate to unable was ). Collectively, these data demonstrate that that demonstrate data these Collectively, ). g/ml) or zymosan (1 (1 zymosan or g/ml) and oxLDL triggers TLR signaling via a via path shared TLR signaling and oxLDL triggers Cd36 + + −/− . aureus S. macrophages stimulated with oxLDL (50 (50 oxLDL with stimulated macrophages n κ b κ −/− = 3) fed a high-fat diet for 12 weeks to induce atherosclerotic lesion formation ( formation lesion atherosclerotic induce to weeks 12 for diet a high-fat fed = 3) B–dependent luciferase gene in HEK293 cells cells HEK293 in gene luciferase B–dependent

B-dependent luciferase gene in HEK293 cells cells HEK293 in gene luciferase B-dependent mRNA (fold induction)

10 12 , 0 2 4 6 8 aDV Tlr4 Tlr4 11). We therefore tested whether fibril whether tested therefore We 11). Cxcl1 n LA wih rfrnily acti preferentially which LTA, and * Cd3 WT A −/− −/− Fig. 3 Fig. NCE ONLINE PUBLIC ONLINE NCE Fig. 3 Fig. 6 and and ). Thus CD36, like MyD88 (ref. and and Cxcl2 –/ µ – * g/ml) for 45 min. Data are the mean mean the are Data min. 45 for g/ml) 1 ), showed a robust respiratory respiratory robust a showed ), c a 7 ). ). A . Moreover, inflam the known ), whereas the control peptide peptide control the ), whereas Tlr6 Tlr6 Ccl * β κ β i. 3 Fig. 5 1–42 −/− B luciferase reporter gene in HEK293 cells expressing TLR4 TLR4 expressing cells HEK293 in gene reporter B luciferase −/− 1–42 κ B ( B macrophages showed showed macrophages cells, suggesting that that suggesting cells, β was unable to induce induce to unable was c induced a 3–5-fold 3–5-fold a induced 1–42 mRNA (relative expression) d Tlr4 Fig. 3 Fig. 10 10 10 10 10 ), suggesting that that suggesting ), in in vivo –4 –8 –7 –6 –5 A ), could activate activate could ), Fig. 2 Fig. −/− Apo TION β , has also been been , also has

, , e b –/ – Tlr6 ). The extra extra The ). Cxcl1 n . To further

d Cd36

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), but not ), not but Apo −/−

g/ml, 6 h; 6 h; g/ml, –/ Cd36 – e –/

and and – 15), P −/− κ 0.00 0.010 0.015 0.020 0.025

Cd36 ≤ B ------­ 0.05. AU, arbitrary units. arbitrary AU, 0.05.

0 5 b ± Apo ) and aortas of of aortas ) and s.d. of triplicate samples in samples triplicate of s.d. −/− immune response to endogenous host ligands. host endogenous to response innate immune the mediates molecules these containing complex signaling whereas TRIF was not required for A for required not was TRIF whereas to signaling leading the in cascade components these for role essential an indicating treated similarly in sion ( A to exposed Wild-type response. this of signaling initiation the in TLR4-TLR6 involved is whether determine to sought we thus and by A for affected required individuals disease of fluid Alzheimer’s cerebrospinal and brains the in response IL-1 inflammatory amyloid- microglial the to of component critical A IL-1 for microglia primes activation TLR4-TLR6 to A response immune innate microglial the ing regulat in complex signaling CD36-TLR4-TLR6 the of importance ( LPS with stimulated when production NO impaired showed whereas cells microglia, these wild-type in that to comparable was microglia A deficient in CD36, TLR4 or TLR6, but not TLR2 ( microglia in abrogated were responses inflammatory Notably,these cells ( oxide microglial tion nitric and of ROS by wild-type and ( ref. ligands microbial to responses TLR priate logy, expression of cell surface markers and function, including appro they were similar to primary mouse microglial preparations that showed cell lines in microglial these of morpho characterization Functional cell in lines deficient CD36, mouse microglial TLR2, TLR4 and TLR6. by amyloid- microglia of is involved in activation the disease nitrogen and this of is that pathognomonic oxygen the promote neurotoxicity that species reactive and cytokines including mediators, inflammatory secrete and morphology activated an acquire plaques amyloid- surrounding In microglia disease, Alzheimer’s activation microglial promotes signaling TLR4-TLR6 e Fig. 4 Fig. –/

β – macrophages stimulated stimulated macrophages Cxcl2 1–42

a Cd36 β ) Effect of CD36 and and CD36 of ) Effect Apo

Fig. 4 Fig. –/ (ref. (ref.

induced secretion of NO in in NO of secretion induced – d 1 e –/ 7

18). Consistent with published 18). reports, Consistent with published A – ). Notably, we observed no increase in in increase no observed we Notably, ). . To determine whether signaling through CD36-TLR4-TLR6 0.0004 0.0008 0.0012 0.0016 b 18). Elevated amounts of this cytokine are detected detected are cytokine this of amounts Elevated 18). Apoe ). These findings provide functional evidence of the the of evidence functional provide findings These ). β β is the secretion of the inflammatory cytokine cytokine inflammatory the of secretion the is β 0 1–42 1–42

−/− Apo

Il1b e

and and –/ showed a fourfold increase in in increase fourfold a showed

upregulation of IL-1 of upregulation – 1 Ccl 7 Cd36 . We previously established that CD36 is is CD36 that established previously We . mRNA upregulation ( upregulation mRNA 5

c Apo

). ). –/

Tlr4 – e –/ – −/− d

, , ROS (AU) ROS (AU) 1.0 1.5 2.0 2.5 3.0 Tlr6 10 0 2 4 6 8 0 Md2 −/− WT WT * −/− β and and 1–42 β (also known as as known (also mRNA by microglia by mRNA Tlr4 Tlr4 Supplementary Fig. 3 Fig. Supplementary upregulation of of upregulation Myd88 Fig. 4 Fig. –/ –/ β Fig. 4a Il1b β – – 1–42 1–42 ticles e l c i rt A Tlr6 Tlr mRNA expres mRNA  d β . induced secre 6 −/− production , we generated , we generated ). In ). –/ –/ , β c – – Il1b ). Moreover, -containing -containing microglia, microglia, Cd3

Cd3 Fig. Fig. 4a OxLDL Unstimulated Zymosan Unstimulated microglia microglia

contrast, contrast, Ly96 mRNA mRNA 6 6 –/ –/ – – Il1b −/− – c 1 1 ).  ) - - - - ­ ,

© 2009 Nature America, Inc. All rights reserved. Tlr6 experimental an of representative are and wild-type, in PCR experimental an of representative are A with stimulated genotype indicated the of microglia in production species wild-type, 4 Figure IL-1 produce to microglia these of failure the with containing A of cocultures effects neurotoxic no contrast, ( cells microglial wild-type and neurons of tures shown) not (data A However, effects morphological previ minimal only A reported with induced line cell As neuronal mouse a dysfunction. of incubation ously, neuronal promote would glia A whether tested we amyloid- of effects cytotoxic the A by RANTES chemokine the of induction ( mRNA * experimental an of representative mean the are Data measured. was activity (10 LTA (1 with stimulated were Cells heterodimers. TLR2-TLR6 and TLR4-TLR6 expressing cells NF- an activate to CD36 of ligands microbial and endogenous of ability the of Comparison TLR4 and cells HEK293 expressing TLR6 ( NF- an A on CD14 and ( measured. was activity (revA ( A with stimulated were Cells receptors. indicated the expressing cells HEK293 NF- an of ( signaling. amyloid- 3 Figure s e l c i rt A  survival. neuronal percentage of quantification Right, a P

) or the control peptide reverse A reverse peptide control the or )

Because IL-1 Because d a ≤ κ −/− µ mRNA (fold induction) Nitric oxide (µM) units. arbitrary AU, 0.05. B luciferase reporter gene in HEK293 HEK293 in gene reporter luciferase B β 10 15 20 25 30 M) or oxLDL (50 (50 oxLDL or M) ± 0 1 2 3 4 5 6 0 5 ) ( ) microglial cells stimulated with A with stimulated cells microglial κ µ s.d. of triplicate samples and are are and samples triplicate of s.d. B luciferase reporter gene in in gene reporter luciferase B

g/ml), g/ml), β a CD36-TLR4-TLR6 signaling induces microglial inflammatory responses that promote neurotoxicity. ( neurotoxicity. promote that responses inflammatory microglial induces signaling CD36-TLR4-TLR6 κ The Alzheimer’s disease peptide peptide disease Alzheimer’s The , activates CD36-TLR4-TLR6 CD36-TLR4-TLR6 activates i. 4 Fig. *

Tlr2 WT b B luciferase reporter gene in in gene reporter luciferase B a ) (10 (10 ) * Il1b , β

Tlr2 b * 1–42 −/− –/ Amyloid- ) S. aureus S. * β

Myd8 Tlr6 Tlr4 WT – * 1–42 , , β d µ Tlr4 Tlr Tlr4 induced widespread neuronal cell death in cocul in death cell neuronal widespread induced , as well as NO and ROS, has been implicated in in implicated been has ROS, and NO as well as , ), it was essential for TLR4-TLR6–dependent TLR4-TLR6–dependent for essential was it ), –/ –/ 4 –/ luciferase and h) 5 M, 8 – –

– of expression -induced –/ −/− – −/−

Tlr µ β g/ml) and luciferase luciferase and g/ml) c 10 15 20 , ,

6 A (10:1),

–/ 1–42 β , , 0 5

– CD36 of Effect ) Tlr6 induced expression expression induced Tlr4 Tlr6 Cd36 A Control β 1–42 -CD36-TLR4-TLR6 signaling in micro in signaling -CD36-TLR4-TLR6 Il1b –/ −/−

−/− – −/− Tri WT , , , , Myd88 f and and Cd36 n β –/ β b of 3. 3. of – 1–42 Nitric oxide (µM) 42–1 e n

β 10 15 20 of 3. * 3. of 0 5

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* –/ microglia, consistent consistent microglia, *

– β ) ) −/− –/ –/ µ Md2 1–42 8 – – M, 72 h) and stained with anti–neuronal class III III class anti–neuronal with stained and h) 72 M, macrophages stimulated with A with stimulated macrophages

–/ Tlr4 Fig. 4 Fig. a d – –/ A Control P

– ( β −/− NF-κB activity (AU) NF-κB activity (AU) 10 12 1–42 < 0.05. ( < 0.05. CD36 β TLR6 TLR4 Fig. 4 Fig. 200 100 200 300 400 500 600 700 800 120 160 0 2 4 6 8 CD36 TLR6 TLR4 microglia stimulated with A with stimulated microglia 40 80 , NO and ROS ROS and NO , 0 f ). In marked marked ). In 100 120 140 β Ccl5 20 40 60 80 – – – – Tri WT 1–42 e 0 * ). f oxLDL A S. aureu LTA + – f + – – –/ ) Left, CAD mouse neuronal cells cocultured with wild-type, wild-type, with cocultured cells neuronal mouse CAD ) Left, β – WT alone 1– 42 * 18,19 + – – –

f Md2 1 + s – Amyloid-β Control –/ 8 - - -

– . ,

TLR4-TLR6 β – + + – + + – 1–42 ( Tlr4 A induced THP-1of rapidly CD36 with ligands Stimulation monocytes or oxLDL with stimulated monocytes THP-1 in TLR6 and we examined by the immunoprecipitation of association CD36, TLR4 assembles, complex signaling To heterotrimeric how this understand formation complex TLR4-TLR6 regulates CD36 signaling TLR6. and TLR4 common CD36, a of composed of complex activation the to disease Alzheimer’s atherosclerosis in and Alzheimer’s inflammation with sterile link and associated pathology disease responses inflammatory promotes signaling microglial TLR4-TLR6 that indicate data A these from together, protected not were glia d LPS Control Fig. 4 Fig. WT + + –/ – , – e (10 (10 ) ) + + – * Il1b β + + f µ + + + * 1–42 ). Notably, neurons in cocultures containing containing cocultures in neurons Notably, ). β M) for 45 min. Data are the mean mean the are Data min. 45 for M) ( 1–42 d aDV c ) and ) and (10 (10 Tlr2 (10 (10

ROS (AU) b 1.0 2.0 0.5 1.5 NF-κB activity (AU) 0 A + + + –/ 120 160 CD36 µ TLR6 TLR4 – NCE ONLINE PUBLIC ONLINE NCE Ccl5 40 80 M, 6 h). Data are the mean mean the are Data 6 h). M, µ 0

M) or LPS (100 ng/ml) for 24 h. ( h. 24 for ng/ml) (100 LPS or M) WT *

(encoding RANTES; RANTES; (encoding Tlr β + 4 –/ – – – revA A

-tubulin (green) and DAPI nuclear stain (blue). (blue). stain nuclear DAPI and (green) -tubulin – β Tlr4 1– Tlr6 * β 42 –/ –/ NF-κB activity (AU) – – A Control 100 150 200 250 300 350 CD36 TLR6 TLR2 a 50 Cd36 β + + , 1–42 b

) Production of nitric oxide in ( in oxide nitric of ) Production –/

– * β 1–42 Tlr6 A oxLDL A S. aureu LTA + + + + – – TION 10 12 β 0 2 4 6 8 1–42 –/ idcd el et. Taken death. cell -induced ± e – ) mRNA measured by qRT-by measured ) mRNA s.d. of triplicate samples and and samples triplicate of s.d.

± WT

* s s.d. of triplicate samples samples triplicate of s.d. c nature immunology nature

TLR2-TLR6 NF- B activity (AU) Tlr2 κ – + + – CD14 CD36 1,00 Neuronal survival (%) Tlr2 TLR6 TLR4 200 400 600 800

100 120 140 160 –/ – * 20 40 60 80 c −/− 0 0 0 ) Reactive oxygen oxygen ) Reactive

, , Tlr4

Tlr4 –/ A Control Tlr2

Tlr Tlr Tlr WT – + + – – – + + β 1–42 6 4 2 * −/− –/ –/ –/ – – – −/− + + + and and a * micro ) + + + β + + + – * 1–42 - .

© 2009 Nature America, Inc. All rights reserved. proteins from THP-1 monocytes treated with oxLDL (50 oxLDL with treated monocytes THP-1 from proteins ( measured. 5 (genistein, (50 oxLDL with treated were Cells cells. HEK293 expressing in gene CD36-TLR4-TLR6 reporter luciferase NF- an of expression oxLDL-induced on ( (460–472). CD36 of acids amino 13 9 or 6, terminal the to corresponding competitors peptide soluble of absence or presence the in peptide C-terminal CD36 a His-tagged by precipitated proteins of Lyn immunoblot (top). cells HEK293 CD36-transfected or vector empty from proteins ( ( TLR4 or TLR6 to antibody with (IB) immunoblotted were proteins (IP) precipitated (50 oxLDL with CD36 or CD36 wild-type and TLR6 (YFP), protein a fluorescent with TLR4-tagged with transfected cells HEK293 from proteins TLR4-precipitated ( cells. HEK293 TLR4-TLR6–expressing in gene reporter NF- an of expression induced (CD36 CD36 of mutants C-terminal-domain or ( CD36. by initiated event signaling proximal a membrane by 6 Figure internalization. ligand upon triggered is complex TLR6 ( of upregulation abolished microglia of ment Moreover,endocytosis. requires dynamin-dependent Dynasore treat also complex CD36-TLR4-TLR6 the from signaling that suggesting ( expression chemokine of induction the and uptake oxLDL itor Dynasore dependent endocytosis that can be specifically blocked using the inhib dynamin- through membrane plasma the from internalized are both A or oxLDL of endocytosis whether test to us prompted colocalization dominantly in intracellular compartments ( oxLDL, with pre CD36, upon treatment TLR4 and TLR6 colocalized cells, but in resting seen was CD36, TLR6 and TLR4 of colocalization Minimal receptors. the with transfected cells HEK293 in microscopy and TLR6, we examined the localization of these receptors by confocal and TLR6 ( TLR4 with CD36 of coprecipitation complexes yielding of formation A (50 oxLDL with stimulated macrophages in expression gene of RT-PCR analysis Quantitative microglia. bar, 10 Scale panels. bottom in shown is receptors individual of Expression (arrowheads). white of areas as appears (green) CD36 and (red) TLR6 (blue), TLR4 of colocalization triple image, merged false-colored the In antibody. 647–conjugated Alexa anti-CD36 an with stained and min 30 for (right) oxLDL with treated or (left) unstimulated were TLR6-CFP. and Cells TLR4-YFP tagged fluorescently and CD36 with transfected cells ( CD36. and TLR6 TLR4, to antibodies with (IB) immunoblotted were proteins Precipitated A or (50 oxLDL with treated monocytes THP-1 from proteins (IP) CD36-precipitated of ( formation. complex 5 Figure nature immunology nature in Data experiments. of separate three representative c c Fig. 5 β β ) Fluorescence microscopy of HEK293 HEK293 of microscopy ) Fluorescence ) CD36 immunoblot of Src- or Lyn-precipitated Lyn-precipitated or Src- of immunoblot ) CD36 1–42 1–42 β 1–42 Y463F Ala (10 (10 e was required for signaling via this complex. CD36 and TLR4 TLR4 and CD36 complex. this via signaling for required was ), consistent with the idea that signaling from the CD36-TLR4-

, , CD36 (50 (50 TLR4-TLR6 activation is triggered triggered is activation TLR4-TLR6 TLR4-TLR6 induced CD36-ligand . Cells were untreated or treated treated or untreated were . Cells µ e Fig. 5a ) Effect of kinase inhibition on coprecipitation of CD36 with TLR4 and TLR6 in THP-1 monocytes. Immunoblot analysis of CD36-precipitated of CD36-precipitated analysis Immunoblot of and TLR4 in with TLR6 monocytes. CD36 THP-1 on coprecipitation inhibition ) of kinase Effect M, 6 h; 6 h; M, µ d µ M; PP1, M; 10 PP1, ) Effect of kinase inhibition inhibition kinase of ) Effect 20,21 g/ml) for the indicated times. times. indicated the for g/ml) Y463F µ b g/ml) for 15 min and TLR4- and min 15 for g/ml) ) Immunoblot analysis of analysis ) Immunoblot , . Treatment of macrophages with Dynasore blocked a b a e , , CD36 ) Effect of wild-type wild-type of ) Effect ). To further define the interaction of CD36, TLR4 , ) in the presence and absence of the dynamin inhibitor Dynasore (80 (80 Dynasore inhibitor dynamin the of absence and presence the ) in b ) Immunoblot analysis analysis ) Immunoblot

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µ Il1b M); wortmannin (10 nM) or inactive analogs (daidzen, 5 (daidzen, analogs (10 nM) or inactive M); wortmannin c a oxLDL (min a d ). This intracellular , Inhibitor d a mRNA by A by mRNA d NF- B activity (AU) NF-κB activity (AU) CD36 κ µ IB: CD36 TLR6 TLR4 IB: TLR6 IB: TLR4 A are the mean CD36 mRNA TLR4 TLR6 g/ml) in the absence or presence of kinase inhibitors described in described inhibitors of kinase or in presence g/ml) the absence 100 150 200 250 300 350 120 160 200 TION 50 40 80 (relative expression) 10 12 14 16 18 0 20 40 60 80 0 0 0 0 0 0 ) Cxcl1 + + –

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100 150 200 250 IB: CD36 50 IB: TLR6 IB: TLR4 b A PP1 0 β cell surface localization of CD36, selective replacement of residues residues of replacement altering selective CD36, with of not While localization surface interact cell signaling. to oxLDL-induced postulated abrogated domain kinases, a terminus, C sequences cytoplasmic regulatory CD36 the identify of Notably, mutation activation. to TLR4-TLR6 for essential CD36 mutated we formation, and signaling. and dimerization TLR4-TLR6 of regulator critical a as CD36 of Tyr463 ( TLR6 with TLR4 of association induce CD36, CD36 wild-type of coexpressing coexpression cells HEK293 in TLR6 and TLR4 ( coprecipitation NF- TLR4-TLR6 for required also was CD36 of ligand-induced Tyr463 blocked CD36, of domain this within Tyr463, of (Cys464) but not the adjacent ( TLR6 and TLR4 coexpressing cells NF- induce (CD36 alanines with 460–472 + + + e (min) Fig. 6 Fig. WT mRNA + + To further explore how CD36 regulates TLR4-TLR6 complex complex TLR4-TLR6 regulates CD36 how explore further To PP3 + + + + (relative expression) 0.5 1.0 1.5 2.0 2.5 C464S IP 0 + + : – b LY CD36 + + + Il1b Dynasore Ve ). Whereas oxLDL markedly augmented coprecipitation of of coprecipitation augmented markedly oxLDL ). Whereas * Wo hicle 5 + + rt b κ TLR4 µ IB: . IB: B–dependent luciferase gene expression in HEK293 HEK293 in expression gene luciferase B–dependent M). ox

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≤ µ 0.005). AU, arbitrary units. AU, arbitrary 0.005). + + IP: – Y463F Unstimulated g/ml, 6 h; 6 h; g/ml, – : CD3 µ GFP(TLR4) Y463F µ g/ml, 5 h) in the presence of kinase inhibitors 5 inhibitors g/ml, of h) kinase in the presence + + – – TLR6 + – M; PP3, M; 10 PP3, 6 with these TLRs was insufficient to to insufficient was TLRs these with WT IP: + + – Ala CD36 Y463F + + + + PP1 ) impaired the ability of CD36 to to CD36 of ability the impaired ) d + ) or microglia stimulated with with stimulated microglia ) or CD3 Fig. 6 Fig. WT + + PP3 + 6 µ M) and cellular luciferase was luciferase M) and cellular Fig. 6 Fig. d Competitor c . Data in all experiments are . in Data all experiments a IB: IB: Ly TLR4 κ ). Furthermore, mutation mutation ). Furthermore, Inhibitor CD36 CD36: atvto ( activation B ox

Integrated density Integrated density TLR4 b LD - 0.2 0.4 0.6 0.8 1.2 1.0 0.4 0.8 1.2 1.6 n TLR6 ). These data identify identify data ). These IP: 0 0 – L – - ticles e l c i rt A Src CD36 + IP: – – MISYCACRSKTIK ox CD36peptide TLR6 Ly TLR4 LD TLR6 + + + n L PP1 Src + – Fig. PP3 Ly CD36 – n

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© 2009 Nature America, Inc. All rights reserved. ­identifiescommona means for chronic mononuclear cell activation in ligandmimeticsandTLRendogenousthese how as act ligands of can and IL-1 encodingpro-IL-1 signaling in macrophages and microglia results in reactivetranscriptionoxygennitrogenandspecies. Additionally, of CD36-TLR4-TLR6 mRNA componentsmodifiedself these of effectsdeleterious the in implicateddirectlymediators ­inflammatory this ­proximal signaling event required for TLR4-TLR6 activation. Together, amyloid- and ­lipids glia.Notably, thispathway triggeredis through atherogenicbinding micro of and macrophages of activation immune innate initiates and ligands endogenous these senses that TLR6 and TLR4 of ­composed cells. mononuclear of activation and recruitment the fuel to believed are and respectively, brain and wall artery the of amyloid- and LDL oxidized tissues: in affected components self modified of deposition is the anomalous these of pathology the diseases underlie that plaques accumu in lineage prominently monocyte late the of cells which in response matory inflam sterile protracted a by characterized are disease Alzheimer’s and Atherosclerosis understood. well less remain inflam conditions matory sterile infection. underlying to mechanisms response molecular the immune contrast, By innate the of expanded understanding greatly our has patterns molecular pathogen-associated ing detect in role critical their and TLRs mammalian of discovery The DISCUSSION promote TLR4-TLR6 signaling in a similar manner. we postulate that CD36 delivery of Lyn during complex formation maytransduction signal TLR4 of activation and domain TIR TLR4 the of phosphorylation the in implicatedLyn is initiation.Because signal Tyr463initiatedat facilitatingTLR4-TLR6heterodimerizationin and Collectively, these data support a role for CD36-Lyn kinase interactions oxLDL-treated( inmonocytes TLR6 and TLR4 with CD36 on CD36-TLR4-TLR6 signaling and blocked the physical association of tor PP1, but not the inactive analog PP3, genereplicated expression ( these inhibitory effects NF- of activation CD36-TLR4-TLR6–dependent blocked wortmannin), and (LY294002 kinase phosphatidylinositol-3-OH of inhibitors or daidzen analog inactive its not but genistein, inhibitor and CD36-TLR4-TLR6 complex formation. TheNF- on generalinhibitors kinase tyrosine tyrosine kinase of effect the assessed motif (amino acids 460–463) encompassing Tyr463 ( and, as shown by peptide competition, this binding required the MISY C terminus of CD36 was sufficient to mediate the interaction with Lyn, kinasetypic in the Src family ( immunoprecipitated preferentially with Lyn, but not with Src,binding proteinthe protoas the kinase Lyn. As observed in macrophages expressing CD36 as a model system, we identified this ~60-kDa CD36- approximately 60 kDa ( a scrambled version: FAK (125 kDa), Paxillin (68 kDa) and a protein of tyrosine proteins that bound the native CD36 C-terminalproteins peptide precipitated but notwith these peptides identified three main terminusphospho (residues 460–472) or a scrambled version. Immunoblottingdomain using His-tagged of peptides corresponding to the native CD36 C s e l c i rt A  two important, age-related, chronic inflammatory diseases.

u wr ietfe a rvosy necie TR heterodimer TLR undescribed previously a identifies work Our To test the functional role of Lyn in CD36-TLR4-TLR6 signaling, we this for partners interacting potential identify to sought next We

heterotrimericsignalingcomplexregulates pro-expression the of 14,17 β secretion. This work thus provides the molecular mechanism . A common characteristic of these age-related diseases diseases age-related these of characteristic common A . Fig. 6 β , priming these cells for inflammasomeactivationforcells these priming , β o sae rcpo, D6 ta poie the provides that receptor, CD36, shared a to d Supplementary Fig. 4 ). Notably, treatment with the Lyn kinase inhibi Fig. 6 in vivo in c β ). A peptide corresponding to the peptide accumulate in plaques plaques in accumulate peptide , including,chemokines and ). Using HEK293 cells over Fig. 6 κ B activation B κ c B reporter B ). Fig.6d 1 2 , CD36 , e 2 ). 2 ------­ ­ ,

to its mature form dependent signaling is triggered during atherogenesis. during triggered is signaling dependent MyD88- how into insight mechanistic provides thus LDL oxidized to responses inflammatory these regulates that complex TLR4-TLR6 CD36- a of identification Our activation. ligands macrophage chronic lipoprotein and oxidized of generation the promoting thereby wall, in the artery stress oxidative amplifies macrophages of function defense innate This species. oxygen reactive of production and burst respiratory macrophage the activates by CD36-TLR4-TLR6 initiated signaling MyD88-dependent expression, chemokine oxLDL-induced to In addition signaling. TLR4-TLR6 in this coreceptor of facilitating lipidemic hyper of aortas the in reduced was chemokines these of expression TRIF-dependent chemokine RANTES. Consistent with these findings, observed that to similar signature chemokine MyD88-dependent a triggered phages macro in signaling CD36-TLR4-TLR6 oxLDL-induced that found we Notably, known. not were responsible ligands the and triggered are pathways sensing microbial these which by mechanism ever,the wall artery the of inflammation MyD88-dependent this the aorta in expression chemokine decreased to part in attributable size, lesion atherosclerotic in reduction severe a causes atherosclerosis of ing ing led to the expression of both MyD88- and genes, TRIF-dependent signal This CD14. and MD-2 as such complex, homodimeric TLR4 the of components known of independent was and TLR6, and TLR4 and lipids atherogenic heterodimer amyloid- that TLR found We a TLR4-TLR6. identified of work composed our First, into insights recognition. applicable TLR generally some yielded also has work this identified been have CD36 for ligands microbial eral sev as patterns, molecular pathogen-associated unidentified yet as recognize also may heterodimer TLR this processes, inflammatory oxide nitric of production microglial and TLR4 the in of blocks increase IL-1 cerebral geted deletion disease Alzheimer’s of model a mouse in increased is brain the in expression TLR4 death. induced neurons from amyloid- ROS and surrounding protects production IL-1 abrogates in microglia signaling TLR4-TLR6 and cytokine, tion regulated this of we highly of show that disruption induc the in hit’ ‘first the as complex CD36-TLR4-TLR6 identified IL-1 pro- for microglia and macrophages primes that pathway signaling the of components crucial as TLR4-TLR6 identifies now work Our IL-1 impaired in show CD36 deficient Monocytes and disease. Alzheimer’s sclerosis pro-IL-1 of regulation the understanding of importance the emphasized has inflammasome NALP-3 the of tors as activa (E. communication) Latz, personal crystals and cholesterol pro-IL-1 processes that inflammasome the called complex NF- by transcription IL-1 of disease of markers reduces cytokine this of tion plaques disease Alzheimer’s and sclerotic atherosclerosis, acute coronary events and Alzheimer’s disease Alzheimer’s and events coronary acute atherosclerosis, of risk decreased with associated is (D299G) signaling receptor ates versial IL-1 Targeted deletion of the TLR adaptor MyD88 in a mouse model model mouse a in MyD88 adaptor TLR the of deletion Targeted Although we focus on two endogenous ligands involved in sterile sterile in involved ligands endogenous two on focus we Although IL-1 cytokine The inflammatory β β 1 and in neuroinflammation Alzheimer’s disease remains contro expression. These data identify signaling through the newly newly the through signaling identify data These expression. 5 3 . Upstream of TLR4 or activation TLR2 has in been implicated β 0 , it is noteworthy that a TLR4 polymorphism that attenu that polymorphism TLR4 a that noteworthy is it , is a two-step process requiring both upregulation of of upregulation both requiring process two-step a is β Apoe aDV initiated signaling that required coexpression of both both of coexpression required that signaling initiated β −/− A expression in response to oxLDL in and amyloid- response expression NCE ONLINE PUBLIC ONLINE NCE in vivo in mice lacking CD36, emphasizing the important role 2 7 . The recent of amyloid- identification κ B and activation of a cytosolic multiprotein multiprotein cytosolic a of activation and B , and furthermore elicited expression of the the of expression elicited furthermore and , 2 8 , and, consistent with our model, tar model, our with , consistent and, β A is found prominently in athero is prominently found TION 14,17 28,29

nature immunology nature β expression in athero in expression , and targeted inhibi targeted and , . Although the role of of role the .Although 18,25,26 β , nitric oxide, ,and nitric . The secretion . secretion The 4,5 16,23,24 . Moreover, . β β peptide β amounts amounts protein protein ; ; how 31–33 β 10,11 Il1b β 1 . ------­ ­ 8 .

© 2009 Nature America, Inc. All rights reserved. A.L.-H. interpret helped the data and the write manuscript; L.M.S. and K.J.M. the performed precipitation peptide assays; J.E.K. assisted with A neurotoxicity and experiments provided the TLR constructs; R.Z. and W.A.F. assays; A.H. and D.T.G. generated microglia,the immortalized the performed and L.B. confocal performed microscopy; J.D. and K.J.R. the performed luciferase data; K.W. immunoprecipitation and performed gene expression studies; J.M.v.G. C.R.S. the using performed experiments oxLDL and analyzed and interpreted the AUTH to (068089/Z/02/Z L.M.S.). Health Assistance Foundation to(A2008-130 K.J.M.) and the Wellcome Trust to K.J.M. and J.E.K.), the Ellison Medical Foundation (K.J.M.), the American Health of (R01AG20255 to K.J.M.; toR01NS059005 J.E.K.; R01 AG032349 Technology Agency) for knockout mice. Supported by the US National Institutes We thank S. Akira (Osaka University) and K. Miyake Science (Japan and A is availableNote: information on the Supplementary s codes. Accession online the in http://www.nature.com/natureimmunology/. at paper the of version available are references associated any and Methods M coreceptors. relevant their with coexpressed are TLRs the herein, demonstrate we as if, identified be may that other TLR and heterodimers suggests signaling TLR4-TLR6 in coreceptor of function the triggering importance model highlights phosph TLR6 and/or TLR4 promote Lyn of may CD36 similarly kinase, recruitment that, through suggest signaling LPS for required is TLR4 of domain TIR the of tion ROS of production the including NF- of amyloid- to responses impaired show activation macrophages Lyn-deficient and association TLR4-TLR6 kinase; Lyn third, chemical with inhibitors of Lyn kinase interaction blocked CD36 ligand–inducedits regulated CD36 of Tyr463 NF- second, and heterodimerization TLR4-TLR6 induce to ability its blocked CD36 of Tyr463 of following mutation the first, by observations: supported is model This kinase. Lyn inter with CD36 action by initiated event membrane proximal a by triggered for described signaling ‘inside-out’ integrins the to this compare to us ing lead cell, the within CD36 of heterodimer terminus C the from TLR4-TLR6 occurred zation induce that signals The transduction. signal TLR4-TLR6 modulating in CD36 for role active more a cate complexes molecules to pattern-recognition them deliver and microbial concentrate to suggested been have receptor, vitronectin the and as such CD14 coreceptors, TLR several Although activation. its for required was which coreceptor, CD36, relevant with the coexpression of because possible made only was heterodimer ligands have since been described. Our of a identification TLR4-TLR6 ascribed with heterodimers other no that noteworthy is However, it repertoire TLR the diversify further might heterodimerization that speculation the to led heterodimers TLR2-TLR6 and TLR2-TLR1 of TLR6, requiring expression of functional CD36. The early description and TLR4 of association physical ligand-dependent a showed assays Coimmunoprecipitation CD36. coreceptor, surface cell a from ated initi signaling by regulated is activation and assembly dimer which in heterodimerization TLR of model a new suggest data our Second, pathways. adaptor these of both engages TLR4-TLR6 that indicating nature immunology nature conceived the ideas, the designed research and wrote the manuscript. i ck g ethods n no a l O i n wledgme R R C g - 3 g 4 ON . Our data are consistent with TLR4-TLR6 signaling being being signaling . TLR4-TLR6 with Our are data consistent a t e TRIBUTI w a y n . CDNtr Sgaig aea ( Gateway Signaling UCSD-Nature o ts r g ON ): A

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6. 12. 11. 10. 9. 8. 7. 5. 4. 3. 2. 1. reprintsandpermissions/. R Published online at http://www.nature.com/natureimmunology/. 34. 33. 32. 31. 30. 29. 28. 27. 26. 25. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. eprints and permissions information is available online at http://npg.nature.com/ at online available is information permissions and eprints

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© 2009 Nature America, Inc. All rights reserved. blue tetrazolium (NBT) reduction assay and nitric oxide was measured by by measured described as reaction was Griess oxide nitric and assay reduction (NBT) tetrazolium blue ROS and NO production. (deconvolution)with Velocitywas performed software. 4.4.1 objective and immersion appr ×60, (CSU-X1, aperture, oil disc a Perkin Plan-apo 1.4 numerical with Elmer) spinning Ultraview with microscope Ti Exlipse Nikon a with visualized was Alexa with Fluor stained 647–anti-CD36 (1:100 and dilution, HM36, saponin Biolegend). Immunofluorescence0.5% with treated methanol, ice-cold with 50 with stimulated and described as plasmids CD36 and TLR4-YFP, with TLR6-CFP transfected and coverslips was provided D & (R ELISA by Systems). In some supernatants experiments, soluble MD-2 culture from cells stably cell secreting MD-2 in measured was IL-8 Secreted (Promega) System activity Reporter Assay gene Luciferase Dual the reporter using and measured was lysed were cells h, 5 for ligand with stimulation an with NF- cotransfected described as cytometry flow by confirmed was and mutants C-terminal of CD36 wild-type (CD36 (100 tomycin changed to DMEM (Invitrogen) containing penicillin (100 units/ml) and strep described as (Invitrogen) 2000 Lipofectamine ing with pcDNA3.1 vectors containing the cDNAs overnight in OptiMEM contain CD36, streptomycin. For of CD14 and TLRs, expression were cells transfected and penicillin lacking medium in transfection before h 24 for well) per cells assays. cell HEK293 mRNA expression was calculated using the comparative cycle method ( Relative (Bio-Rad). System Detection PCR Real-Time iQ an using performed Ccl5 0.3 with used was ng) (25 cDNA Biosystems). (Applied RNAreverse using MultiScribe transcriptase was reverse transcribed expression. cytokine and chemokine of Analysis or dimethylsulfoxide (control) for 30 min (ref.(Sigma-Aldrich) medium.To free 80 with were pretreated dynamin, cells inhibit interferon- U/ml 100 with primed were cells mixed glial cultures using J2 recombinant retrovirus as described described as isolated were microglia and macrophages peritoneal Primary Collection. Mononuclear cell culture. (CD36 CD36 of CD36 mutants C-terminal and CD36 wild-type ( reporter ( CFP containing vectors pcDNA3.1 Sigma-Aldrich. from described as prepared were Company Peptide was oxidized as described and plasmids. reagents Ligands, on Research Care. Subcommittee Animal Hospital General Massachusetts the Health Survey Policy for Public the Humane the Care and and Use of Act Laboratory WelfareAnimals and Animal USDA the with accordance in done was procedures all for use and care Animal conditions. pathogen-free under bred for 12 weeks and were aortas as isolated described (Tekladcholesterol) fat, (wt/wt) 0.15% 88137; 21% (wt/wt) Adjusted Calories female week-old Agency. Technology Apoe and Science Japan and Miyake K. Myd88 Mice. METHODS ONLINE nature immunology nature YFP , fusion constructs between human human between constructs fusion −/− Il1b ) or cyan fluorescent protein ( protein fluorescent cyan or ) C57BL/6 mice C57BL/6 were from Jackson Laboratories. C464S −/− and ) or control ( 3 and h 5 ) have been described been have ) t . Immortalized microglial cell lines were generated from primary primary from generated were lines cell microglial Immortalized . t Cd36 p 4 1 µ : Trif / . For immunofluorescence, cells HEK293T were plated on glass / g/ml) and 0%–2% FBS. Equivalent cell surface expression of of expression surface cell Equivalent FBS. 0%–2% and g/ml) a Apoe d −/− −/− d g HEK293 cells were cultured in six-well plates (6 × 10 × (6 plates six-well in cultured were cells HEK293 Gapdh Apoe mice were provided by mice were S.provided Akira. e S. aureus S. −/− n e 3 . and and o The THP-1 cell line was from American Type Culture 5 −/− κ Reactive oxygen production was measured by was nitro measured oxygen Reactive production . A r B response element–luciferase reporter gene. reporter B After element–luciferase response ) oligonucleotide primers. Quantitative RT-PCR was g 18,35 mice were generated in our laboratory mice were generated / β o ; plasmids 13016, 13018, 13021, 13029, 13642), 13642), 13029, 13021, 13018, 13016, plasmids ; 1–42 Cd36 TLR2-YFP priate filters.priate Image and acquisition processing and zymosan were prepared as described as prepared were zymosan and . 5 Human Technologies LDL from Biomedical . and reverse A µ −/− g/ml oxLDL for 30 min. Cells were fixed fixed were Cells min. 30 for oxLDL g/ml CFP Apoe TLR ) sequences, and NF- and sequences, ) , TLR4-YFP −/− and yellow fluorescent protein protein fluorescent yellow and γ 5 mice were fed a Western diet diet Western a fed were mice 3 1 h and stimulated in serum serum in stimulated and h 1 µ . NF- For 8 β M target ( target M . 3 42–1 5 7 S. aureus S. Ala . After 24 h, the media was was . h,media 24 the After . All mice were housed and Five micrograms of total total of micrograms Five peptides from American , CD36 Tlr2 , Md2 TLR6-CFP κ −/− −/− B assays, cells were were cells assays, B Cd14 Cxcl1 –derived LTA was –derived Y463F , mice were from Tlr4 Ala 39,40 µ 20). κ −/− , CD36 , , CD36 M Dynasore Dynasore M B-luciferase B-luciferase Cxcl2 and and −/− . Microglial Cd36 , 35–37 , Tlr6 ∆ TLR9- , C464S ∆ Y463F . Six- Ccl9 Ct). −/− −/− 5,7 5 ) - - 5 , , . , . ,

TLR4 (HTA125, eBioscience), TLR6 (hPer6, eBioscience), Lyn (H-6,Cruz), (H300,SantaSanta CD36 Cruz)toantibodies immunoblotted (Novagen)with and washedthree times inRIPA buffer described and eluted withLaemmli SDS sample buffer as antibodies secondary conjugated Science) or control IgG (I5381, Sigma) overnight at anti–human4CD36(FA6, °C,Sciences),Cell anti-GFP13.1Rochethen(7.1and Applied for 2 h with agarose 4 incubatedwith proteinwaslysate of mg 1phosphatase inhibitorsand sodiumpyrophosphate, 1mM sodium orthovanadate) containing protease and buffer (50 mM Tris-HCl, pH 7.5, 125 mM NaCl, 1% NP-40, 5 mM NaF, 1.5 mM Immunoprecipitation. A with treated wells in nuclei neuronal of number the by comparing calculated was survival neuronal percentage The conditions. blinded under in condition field per per fields nuclei 15 of number quantifying by assessed was viability cell and microscope confocal scanning TCS SP2 AOBS a laser Leica with imaged were cells Neuronal (Invitrogen). stain nuclear and Hoechst Invitrogen) (A-11001, III β class anti–neuronal mouse with stained and fixed were neurons vehicle, A with stimulation after hours Seventy-two cultures. neuronal of top 5 (Corning; inserts Transwell into seeded ( knockout or wild-type and h) (48 withdrawal serum by induced was differentiation Neuronal FCS. and 10% (Cellgro) ciprofloxacin containing (Invitrogen) in slips F12/DMEM cells neuronal CAD mouse assay. Neurotoxicity 42. 41. 40. 39. 38. 37. 36. 35. A (HSD). Difference groups of three or more with Student’s by analyzed analysis. Statistical stimulation. before h 1 for Technology) Signaling (Cell LY294002 or wortmannin Biosciences), (EMD inhibitors. Kinase or RSKTIK before addition of the His lysates were preincubated with soluble peptides MISYCACRSKTIK, and CACRSKTIKimmunoblotted for Lyn as described above. In imidazolepeptide in competitionRIPA. Boundassays, proteins werecell eluted with Laemmli SDS sample buffer TheHis Ni-NTA resin (Qiagen) and incubated with cell lysates (1 mg/ml)Nucleic and atAcidLaboratory.Chemistry 4 His°C for 1.5 h. CACRSKTIK,RSKTIK) were synthesized by the WashingtonUniversity Protein His scrambled, to the cyt Hyperfilm (Amersham). For peptide binding experiments,or Src peptides(Santa Cruz)corresponding as described -tubulin (clone Tuj1) followed by Alexa Fluor 488–goat anti–mouse IgG IgG anti–mouse 488–goat Fluor Alexa by followed Tuj1) (clone -tubulin

i Y, ag JK, cila, . Ciaasi DM Caatrzto o a CNS a of Characterization D.M. Chikaraishi, & M. McMillian, J.K., Wang, Y., Qi, large a is MD-2 Secreted D.M. Segal, & J.A. Spitzer, A., Mazzoni, A., Visintin, of F.Immortalization Bistoni, & R. Mazzolla, V., Bocchini, R., Barluzzi, E., Blasi, Roberson, S.M. & Walker, W.S. Immortalization of cloned mouse splenic macrophages I.S. Coraci, K.J. Moore, K.J. Moore, V.V.Kunjathoor, el ie CD i wih opooia dfeetain s ntae b serum by initiated is differentiation morphological which in deprivation. CAD, line, cell 4. receptor Toll-like to sensitivity lipopolysaccharide confers efficiently that protein polymeric (1990). 229–237 retrovirus. carrying v-raf/v-myc a by cells microglial murine (1988). 341–351 with a retrovirus containing the v-raf/mil and v-myc oncogenes. fibrils. b-amyloid to response in in Alzheimer mice. Invest. Clin. hyperlipidemic J. in atherosclerosis ameliorate not does pathways CD36 or A macrophages. macrophages. in loading lipid to leading lipoprotein density low modified of uptake the for responsible receptors 6 -peptideNi-NTA resin was washed sequentially withRIPA or150 mM o plasmic C-terminal domain of CD36 (native, His Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. et al. et β ′ t al. et J. Neurosci. J. t al. et 6 s disease brains and can mediate production of reactive oxygen species 1–42 KTRCYK; n slbe etds MISYCACRSKTIK, peptides, soluble CKITSRICAYMKS; and J. Immunol. J.

115 et al. et Divergent response to LPS and bacteria in CD14-deficient murine CD14-deficient in bacteria and LPS to response Divergent Cells were pretreated with genistein, daidzen, PP1, PP3 PP3 PP1, daidzen, genistein, with pretreated were Cells D6 a ls B cvne rcpo, s xrse o microglia on expressed is receptor, scavenger B class a CD36, and with vehicle. with and os f eetrmdae lpd pae i saegr receptor scavenger via uptake lipid receptor-mediated of Loss The difference between two groups was statistically statistically was groups two between difference The , 2192–2201 (2005). 2192–2201 , P For assessment of microglia-induced neurotoxicity, neurotoxicity, microglia-induced of assessment For -value of <0.05 was considered significant. considered was <0.05 of -value Cells were lysed in radioimmuneprecipitation(RIPA)in lysed were Cells Scavenger receptors class A-I/II and CD36 are the principal the are CD36 and A-I/II class receptors Scavenger t -test or analysis of variance (one-way ANOVA) for for ANOVA) (one-way variance of analysis or -test Tlr2

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