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Bacterial RNACutting Edge: TLR13 Is a Receptor for

DalpkeAsa Hidmark, Antonia von Saint Paul and Alexander H.

ol.1200898http://www.jimmunol.org/content/early/2012/08/15/jimmun

published online 15 August 2012J Immunol 

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Cutting Edge: TLR13 Is a Receptor for Bacterial RNAAsa Hidmark, Antonia von Saint Paul, and Alexander H. Dalpke

Bacterial RNA (bRNA) can induce cytokine productionin macrophages and dendritic cells (DCs) through a pre-viously unidentified receptor. Gene expression analysisof murine DCs showed that bRNA induced gene regu-lation similar to that induced by stimulation of TLR7with R848. Although TLR7 was dispensable for cyto-kine induction by bRNA, TLR-associated proteinsMyD88 and UNC93B were required. TLR13 is anendosomal murine TLR that has been described tointeract with UNC93B with, so far, no characterizedligand. Small interfering RNA against TLR13 reducedcytokine induction by bRNA in DCs. Moreover, Chi-nese hamster ovary cells transfected with TLR13, butnot with TLR7 or 8, could activate NF-kB in responseto bRNA or Streptococcus pyogenes in an RNA-specificmanner. TLR7 antagonist IRS661 could, in addition,inhibit TLR13 signaling and reduced recognition ofwhole Gram-positive bacteria by DCs, also in the ab-sence of TLR7. The results identify TLR13 as a recep-tor for bRNA. The Journal of Immunology, 2012,189: 000–000.

Receptors of the innate immune system detect mo-lecular patterns during the life cycles of infectingpathogens, depending on the localization and avail-

ability of these patterns. TLRs recognize a wide range ofmicrobial components and are important for raising innateimmune defenses against bacterial infections. Whereas LPSfrom Gram-negative bacteria commonly activates the innateimmune system via TLR4 (1), it is less well understood howGram-positive bacteria are recognized. Although TLR adaptormolecule MyD88 is important for activation of the innateimmune system by Staphylococcus aureus (2) and strains ofEnterococcus and Lactobacillus (3), activation by group AStreptococcus can occur independently of TLR2, 4, and 9 (4).Several studies have shown that bacterial RNA (bRNA) acti-vates the innate immune system (5–8). RNA receptor TLR7,which recognizes single-stranded viral RNA (9), has beensuggested to induce IFN-b (8) or IL-12 (3) production bymyeloid dendritic cells (DCs) in response to bRNA. However,

RNA of Streptococcus pyogenes induces IFN-b in myeloid DCsin an MyD88-dependent but TLR7-independent manner (6).In addition, RNA-dependent induction of TNF-a by group BStreptococcus in macrophages has been shown to be indepen-dent of nucleic acid-sensing TLR3, 7, and 9, although re-quiring MyD88 and UNC93B (10), needed for the function ofendosomal TLRs (11). Thus, the receptor for bRNA, whichmay play an important role in recognizing Gram-positivebacteria, has not yet been identified.We found that murine TLR13, an UNC93B-interacting

orphan receptor (12, 13), could mediate the recognition ofbRNA and whole Gram-positive bacteria when expressed inChinese hamster ovary (CHO) cells. When TLR13 expressionwas targeted in DCs, using small interfering RNA (siRNA),cytokine induction by bRNA was reduced. Our results indi-cate that TLR13 is required and sufficient for the induction ofcytokines in response to bRNA.

Materials and MethodsMice

Wild-type (wt) C57BL/6 mice were purchased from Charles River Labora-tories. MyD882/2 mice were provided by H. Wagner (Munich, Germany).TLR72/2 mice were provided by S. Bauer (Marburg, Germany). UNC93Bmice harboring H412R missense mutation (3D) (11) were supplied byM. Freudenberg (Freiburg, Germany). Animal experiments were approved bylocal authorities.

Cells

Bone marrow-derived DCs were generated as previously described (14). GM-CSF was provided to the culture by adding supernatant of 363-Ag8 cells,supplied by M. Lutz (Wurzburg, Germany). DCs were harvested at day 7 andseeded in 24-well plates (8 3 105 DCs per well). CHO cells (DSMZ,#ACC110) were grown in Ham’s F12 medium. Media were supplementedwith 10% heat-inactivated FCS, penicillin G, and streptomycin sulfate(100 IU/ml) (Life Technologies).

Bacteria and stimulations

Staphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 6051), Listeriamonocytogenes (ATCC 15313), Enterococcus faecalis (ATCC 29212), Lacto-bacillus sp. (patient isolate), and Streptococcus pyogenes (ATCC 12344) weregrown to midlog phase. S. pyogenes was heat killed for 45 min at 70˚C. RNAwas purified from lysozyme-treated bacteria or eukaryotic cells (THP-1 orRAW 264.7), using TRIzol (Invitrogen) followed by High Pure RNAIsolation Kit (Roche) with DNase I digest. RNase A (Invitrogen) digestionof bRNA was performed at room temperature. bRNA (3 mg/ml fromL. monocytogenes unless otherwise stated) was packaged with 2.5 ml DOTAP

Department of Infectious Diseases, Medical Microbiology and Hygiene, University ofHeidelberg, 69120 Heidelberg, Germany

Received for publication March 22, 2012. Accepted for publication July 16, 2012.

This work was supported by a grant from the Medical Faculty of the University ofHeidelberg (to A.H.).

The microarray data presented in this article have been submitted to the ArrayExpressdatabase (www.ebi.ac.uk/arrayexpress/experiments) under accession number E-MTAB-1139.

Address correspondence and reprint requests to Dr. Asa Hidmark, Department of In-fectious Diseases, Medical Microbiology and Hygiene, University of Heidelberg, Im

Neuenheimer Feld 324, 69120 Heidelberg, Germany. E-mail address: asa.hidmark@med.uni-heidelberg.de

The online version of this article contains supplemental material.

Abbreviations used in this article: bRNA, bacterial RNA; CHO, Chinese hamster ovary;DC, dendritic cell; pI:C, polyinosinic–polycytidylic acid; siRNA, small interfering RNA;wt, wild-type.

Copyright� 2012 by TheAmerican Association of Immunologists, Inc. 0022-1767/12/$16.00

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per microgram of RNA, as previously described (5). Ultrapure LPS fromSalmonella minnesota (100 ng/ml) was provided by U. Seydel (Borstel,Germany). Other agents used were R848 (InvivoGen), polyinosinic–poly-cytidylic acid (pI:C, 50 mg/ml; Sigma-Aldrich), Pam3Csk4 (InvivoGen),and CpG ODN 1668 (MWG-Biotech). Phosphorothioate oligonucleo-tide inhibitor IRS661 (59-TGCTTGCAAGCTTGCAAGCA-39; EurofinsMWG Operon) was added 1 h before stimulation.

TLR expression vectors

Murine TLR13 from (Mammalian Gene Collection; http://mgc.nci.nih.gov/)pCR-Blunt II-TOPO-TLR13 (I.M.A.G.E. IRAMp995J1823Q) was clonedinto pcDNA3.1 (Invitrogen) using ApaI/XhoI. TLR8, from pCR4-TOPO-TLR8 (I.M.A.G.E. IRCLp5011B0512D), was digested with SpeI/FspI andligated into pcDNA3.1 cut with NheI/XbaI. TLR7 in pcDNA3 was a giftfrom T. Espevik (Trondheim, Norway).

DNA transfection, selection, and luciferase reporter assay

CHO cells were cotransfected with vectors expressing TLR and NF-kB luciferasereporter pGL4.32[luc2P/NF-kB-RE/Hygro] (Promega), using Lipofectamine2000 (Invitrogen) in 100-mm plates and incubated overnight. Bulk transfectedcells were distributed in 24-well plates and stimulated upon seeding. Cloneswere generated by selection with G-418 sulfate (400 mg/ml; PAA Laboratories)and hygromycin B (100 mg/ml; InvivoGen) and distributed at 0.5 3 105 perwell in 48-well plates for stimulation.

Quantitative RT-PCR

RNA was purified 6 h post treatment with the High Pure RNA Isolation Kit(Roche), including DNase I digestion. cDNA was generated using the HighCapacity cDNAReverse Transcription Kit (Applied Biosystems) and measuredwith ABsolute SYBR Green Rox Mix Kit (ABgene House), using ABI PRISM7700 (Applied Biosystems). Primer sequences are available on request.

siRNA transfection

siRNA against TLR13 (target sequence: 59-CACCTATGTTCTTGTAAG-TAA-39) or Negative Control-N3 siRNA (Riboxx) was delivered with theNucleofector Kit VPA-1011 (Lonza) or riboxx FECT Transfection Reagent(Riboxx) according to the manufacturer’s protocols. The riboxx FECTtransfection was performed upon seeding 13 105 DCs per 48 wells in 150 mlmedia. Each nucleofection was divided into 96 wells. Media were changedafter 24 h, and the cells were stimulated 48 h post siRNA transfection.

ELISA

Supernatants were analyzed using BD OptEIA Mouse ELISA Set IL-12p40 orIL-6 (BD Biosciences).

FIGURE 1. Induction of cytokines by bRNA requires MyD88 and

UNC93B but is independent of TLR7. Induction of cytokines in myeloid DCs

from wt or knockout mice upon treatment with bRNA or RNase A-digested

bRNA (bRNA RNase), pI:C, LPS, or R848 (1 mg/ml). (A) IL-12p40 mRNA

after treatment of wt DCs with RNA from L. monocytogenes (L.m.), B. subtilis(B.s.), E. faecalis (E.f.), S. aureus (S.a.), or RNase A digests of respective RNA

(RNA RNase) or eukaryotic RNA (n.t., Not tested). (B and C) Cytokine

induction in DCs from wt or MyD882/2 mice treated with bRNA or bRNA

RNAse; induction of (B) IL-12p40 mRNA and (C) IL-12p40 protein in

supernatants at 17 h. (D) Fold induction of IFN-b mRNA over samples

treated with bRNA RNase. (E) IL-12p40 mRNA in DCs from wt and

TLR72/2 mice. (F) IL-12p40 mRNA in DCs from wt and UNC93B-mu-

tated 3D mice treated with bRNA, bRNA RNase, pI:C, LPS, R848, CpG

DNA (1 mM), or Pam3Csk4 (1 mg/ml) (Pam3C). n $ 3 mice from at least

three independent experiments. Error bars show mean 6 SD. **p # 0.01,

***p # 0.001.

FIGURE 2. bRNA and R848 induce similar gene regulation in DCs.

Microarray analysis of gene regulation in DCs treated with bRNA, RNase A

digest of bRNA (bRNA RNase), R848 (1 mg/ml), or mock (untreated); log2

of signal intensity values of (A) bRNA RNase versus bRNA, (B) bRNA

RNase versus R848, (C) R848 versus bRNA, or (D) untreated versus bRNA

RNase. Diagonal lines indicate 2-fold changes. n = 4 mice.

2 CUTTING EDGE: TLR13 IS A RECEPTOR FOR BACTERIAL RNA

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FACS

Brefeldin A (2 mg/ml; Sigma-Aldrich) was added at 5 h post stimulation. At16 h, cells were fixed and stained using Cytofix/Cytoperm (BD Biosciences)with APC-conjugated anti-mouse IL-12 (p40/p70) Ab (BD #554480) andassayed using a FACSCanto (BD Biosciences).

Microarray

RNA was purified 6 h post stimulation, as described for quantitative RT-PCR,hybridized to Mouse Microarray Ref-8 v2.0 Expression BeadChips (Illumina)at the Genomics and Proteomics Core Facility at the Deutsches Krebsfor-schungszentrum (Heidelberg, Germany) and analyzed as described in thesupplemental information online. The microarray data from this publicationhave been submitted to the ArrayExpress database, www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-1139, and assigned the identifier E-MTAB-1139.

Statistical analysis

Two-tailed unpaired Student t tests were used. Statistical significance indi-cated: *p # 0.05, **p # 0.01, and ***p # 0.001. Error bars show mean 6SD.

Results and DiscussionCytokine induction by bRNA requires MyD88 and UNC93B but isindependent of TLR7

RNA from Gram-positive bacteria, but not from eukaryoticcells, has been shown to induce cytokines in myeloid DCswhen delivered to the endosome (5, 8, 10). We found thatbRNA from a number of bacterial species induced IL-12p40

FIGURE 3. TLR13 is activated by bRNA and required

for full DC activation by Gram-positive bacteria. (A)

CHO cells cotransfected with vectors expressing TLR13,

TLR7, or TLR8 and NF-kB luciferase reporter were

treated with L. monocytogenes (L.m.) or S. aureus (S.a.)bRNA, RNase A-digested bRNA (bRNA RNase), or

R848. Stable CHO clones transfected with NF-kB lu-

ciferase reporter and TLR13 or empty vector were

selected and (B) treated with 3 mg/ml eukaryotic RNA,

bRNA (wedge represents 3, 1.5, or 0.75 mg/ml), or LPS;

(C) treated with 100 CFU per cell of S. pyogenes (S.p.),heat-killed S.p. (hk S.p.), or RNase-treated heat-killed

S.p. (hk S.p. RNase); (D) pretreated with IRS661 (wedge

represents 0.05, 0.1, 0.2, or 0.4 pmol/ml) and stimulated

with bRNA (S.a) or LPS. n $ 3 independent experi-

ments; luciferase was assayed 18 h post stimulation and

normalized to respective untreated cells. DCs from wt (E)

or TLR72/2 (F) mice were pretreated with 0.5 pmol/ml

of IRS661 and stimulated with S. aureus bRNA, 10 CFU

per cell of S. aureus (S.a.), E. faecalis (E.f.), S. pyogenes(S.p.), or Lactobacillus sp. (L.), R848 (0.1 mg/ml), CpG

(200 nM), or Pam3Csk4 (5 mg/ml) (Pam3C). The per-

cent inhibition by IRS661 is shown for IL-12p40 mRNA

(top panels) or secreted IL-12 (bottom panels). n = 3 mice.

Error bars show mean 6 SD. n.d., No induction de-

tected. The p values are shown only for inhibition in re-

lation to CpG; *p # 0.05, **p # 0.01, ***p # 0.001.

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mRNA in murine bone marrow-derived myeloid DCs (Fig.1A). RNase A digestion of the bRNA significantly reduced IL-12p40 induction. Previous studies have been contradictoryregarding the ability of bRNA to induce cytokines in theabsence of MyD88. We showed that induction of IL-12p40mRNA (Fig. 1B), secreted IL-12p (Fig. 1C), and IFN-bmRNA (Fig. 1D) by bRNA from Gram-positive bacteria re-quired MyD88. TLR7 has been suggested to mediate IFN-b(8) or IL-12p40 (3) production in response to bRNA inmyeloid DCs, whereas other studies have shown TLR7 to benonessential for bRNA recognition (5, 6, 10). Using knock-out cells, we found TLR7 to be dispensable for induction ofIL-12p40 mRNA in response to bRNA (Fig. 1E).UNC93B has previously been reported to be required for

induction of TNF-a by bRNA in macrophages (10). We foundthat induction of IL-12p40 mRNA by bRNA was abolishedin DCs from UNC93B defect 3D mice (11) (Fig. 1F).

bRNA induces signaling similar to that induced by TLR7in myeloid DCs

To characterize gene regulation induced by bRNA in DCs,we performed a microarray analysis (Fig. 2, SupplementalTable I). Both bRNA and TLR7 ligand R848 induced morethan 2-fold regulation of several hundred genes, comparedwith the RNase-digested control (Fig. 2A, 2B). However,gene regulation by bRNA and R848 showed a very high de-gree of similarity: Only seven genes were induced differently(Fig. 1C). Treatment with RNase A-digested bRNA andDOTAP did not induce any gene regulation, compared withuntreated control, showing that the bRNA preparations didnot contain any other components, in addition to RNA, ca-pable of DC activation (Fig. 2D). The microarray resultsupported the hypothesis that bRNA induced signaling via anendosomal, MyD88-dependent TLR.

TLR13 activates NF-kB in response to bRNA and mediatesrecognition of Gram-positive bacteria

TLR13 is a murine orphan TLR expressed by myeloid DCs,B cells, and macrophages (13). To investigate whether TLR13could mediate bRNA signaling, CHO cells were cotransfectedwith an NF-kB luciferase reporter and vectors expressingTLR13, 7, or 8, or empty vector, before treatment withbRNA (Fig. 3A). Treatment with bRNA induced NF-kBactivation specifically in TLR13-transfected cells (Fig. 3A). Aclone stably transfected with TLR13 and NF-kB luciferasereporter induced up to 6-fold NF-kB activation in a dose-dependent response to bRNA (Fig. 3B). CHO cells havea frameshift mutation rendering TLR2 defective, whereas theyare capable of responding to LPS (15). Responses to bRNAwere of the same magnitude as responses to stimulation byLPS. The TLR13-expressing CHO cell clone induced NF-kBluciferase reporter activity in response to both live and heat-killed S. pyogenes, but not in response to RNase A-treatedbacteria (Fig. 3C), showing that TLR13 can contribute torecognition of group A streptococci. TLR7-inhibitory oligo-nucleotide IRS661 has previously been shown to inhibitbRNA signaling (3). We show that IRS661 inhibited TLR13-mediated activation of NF-kB luciferase reporter in responseto bRNA, but not to LPS (Fig. 3D). We proceeded to in-vestigate if TLR7/13 antagonist IRS661 could inhibitbRNA-induced signaling in DCs. Pretreatment with IRS661

completely inhibited IL-12p40 induction by bRNA in wt andTLR72/2 DCs (Fig 3E, 3F). The IL-12p40 production inresponse to several Gram-positive bacteria was significantlyinhibited by IRS661 treatment of wt and TLR72/2 DCs,compared with induction by CpG via TLR9 or Pam3Csk4 viaTLR2, which were not inhibited. Thus, inhibition of TLR13reduced the innate immune induction by several Gram-positive bacteria independently of TLR7 (4, 6).

FIGURE 4. siRNA targeting TLR13 reduces cytokine production in re-

sponse to bRNA treatment. DCs were transfected with TLR13 or control

siRNA, using riboxx FECT and treated with OptiMEM (Mock), S. aureusRNA (bRNA) (wedge represents 0.25 and 0.5 mg/ml), or CpG (100 nM).

(A) Relative expression of TLR13 mRNA was assayed in mock-treated

samples. The samples were analyzed for (B) IL-12p40 mRNA and (C) IL-6

mRNA. (D) Supernatants (6 h) were assayed for IL-6 by ELISA. (E) DCs

were harvested at 16 h post treatment, stained with an anti–IL-12-APC Ab,

and assayed by FACS. IL-12 is displayed as mean fluorescence intensity

(MFI). DCs were transfected with TLR13 or control siRNA using nucleo-

fection and treated with OptiMEM (Mock); S. aureus RNA (bRNA), 3 mg/ml;

or CpG. The samples were analyzed for (F) IL-6 mRNA and (G) IL-12p40

mRNA. n = 5 mice. Error bars show mean 6 SD. *p # 0.05, **p # 0.01,

***p # 0.001.

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Decreasing TLR13 expression reduces induction of cytokines in responseto bRNA

To determine whether TLR13 was required for bRNA sig-naling in DCs, TLR13 expression was targeted with siRNA.The TLR13 mRNA expression in cells treated with TLR13siRNA was reduced to less than half of that in cells treatedwith a control siRNA (Fig. 4A). DCs transfected with siRNAagainst TLR13 produced significantly less IL-12p40 mRNAin response to bRNA than did DCs transfected with controlsiRNA, whereas IL-12p40 mRNA production in response toCpG did not differ (Fig. 4B). Production of IL-6 mRNA(Fig. 4C) and secreted IL-6 (Fig. 4D) was specifically reducedin TLR13 siRNA-transfected DCs in response to bRNA.Induction of secreted IL-12 could not be detected at 6 h (notshown). Instead, we stained the siRNA-transfected, bRNA-treated DCs for intracellular IL-12 production at 16 h andfound that mean fluorescence intensity was significantly re-duced in DCs treated with siRNA against TLR13. A similarreduction of the cytokine response to bRNA could be ob-served when the TLR13 siRNA was delivered by nucleopo-ration (Fig. 4F, 4G).We show in this article for the first time, to our knowledge,

that RNA from Gram-positive bacteria can activate previousorphan receptor TLR13 and that TLR13 activation contrib-utes to the innate response to Gram-positive bacteria. Al-though TLR13 and TLR7 do not display a high degree ofsequence similarity (16, 17), bRNA induces a gene expressionprofile similar to that induced by TLR7 ligand R848. Micelacking MyD88 have been shown to be particularly sensitiveto infection by Gram-positive bacteria, beyond what can beexplained by known TLRs. It has previously been shown thatcombined activation of TLR7 and TLR4 (18) or of TLR2 andTLR9 (19) can synergistically enhance activation of the im-mune system. Triggering of RNA-sensing receptor TLR13 canprovide a signal similar to that of TLR7 and TLR9, which, inconcert with other innate signaling pathways, could shape theimmune defense against bacterial infection. TLR13 constitutesa difference between how human and murine innate immuneresponses are activated by bacteria.

AcknowledgmentsWe thank Julia Pollmann for cloning TLR13 and TLR8, Oliver Heil

(Genomics and Proteomics Core Facility at the Deutsches Krebsforschungs-

zentrum, Heidelberg, Germany) for microarray data extraction and mathemat-

ical processing, and Gerald McInerney (Karolinska Institutet, Stockholm,

Sweden) for discussions.

DisclosuresThe authors have no financial conflicts of interest.

References1. Poltorak, A., X. He, I. Smirnova, M. Y. Liu, C. Van Huffel, X. Du, D. Birdwell,

E. Alejos, M. Silva, C. Galanos, et al 1998. Defective LPS signaling in C3H/HeJand C57BL/10ScCr Mice: mutations in Tlr4 gene. Science 282: 2085–2088.

2. Ip, W. K. E., A. Sokolovska, G. M. Charriere, L. Boyer, S. Dejardin,M. P. Cappillino, L. M. Yantosca, K. Takahashi, K. J. Moore, A. Lacy-Hulbert, andL. M. Stuart. 2010. Phagocytosis and phagosome acidification are required forpathogen processing and MyD88-dependent responses to Staphylococcus aureus.J. Immunol. 184: 7071–7081.

3. Inoue, R., T. Nagino, G. Hoshino, and K. Ushida. 2011. Nucleic acids of En-terococcus faecalis strain EC-12 are potent Toll-like receptor 7 and 9 ligands inducinginterleukin-12 production from murine splenocytes and murine macrophage cellline J774.1. FEMS Immunol. Med. Microbiol. 61: 94–102.

4. Gratz, N., M. Siller, B. Schaljo, Z. A. Pirzada, I. Gattermeier, I. Vojtek,C. J. Kirschning, H. Wagner, S. Akira, E. Charpentier, and P. Kovarik. 2008.Group A streptococcus activates type I interferon production and MyD88-dependentsignaling without involvement of TLR2, TLR4, and TLR9. J. Biol. Chem. 283:19879–19887.

5. Eberle, F., M. Sirin, M. Binder, and A. H. Dalpke. 2009. Bacterial RNA isrecognized by different sets of immunoreceptors. Eur. J. Immunol. 39: 2537–2547.

6. Gratz, N., H. Hartweger, U. Matt, F. Kratochvill, M. Janos, S. Sigel, B. Drobits,X.-D. Li, S. Knapp, and P. Kovarik. 2011. Type I interferon production induced byStreptococcus pyogenes-derived nucleic acids is required for host protection. PLoSPathog. 7: e1001345.

7. Kanneganti, T.-D., M. Body-Malapel, A. Amer, J.-H. Park, J. Whitfield,L. Franchi, Z. F. Taraporewala, D. Miller, J. T. Patton, N. Inohara, and G. Nunez.2006. Critical role for Cryopyrin/Nalp3 in activation of caspase-1 in response toviral infection and double-stranded RNA. J. Biol. Chem. 281: 36560–36568.

8. Mancuso, G., M. Gambuzza, A. Midiri, C. Biondo, S. Papasergi, S. Akira, G. Teti,and C. Beninati. 2009. Bacterial recognition by TLR7 in the lysosomes of con-ventional dendritic cells. Nat. Immunol. 10: 587–594.

9. Heil, F., H. Hemmi, H. Hochrein, F. Ampenberger, C. Kirschning, S. Akira,G. Lipford, H. Wagner, and S. Bauer. 2004. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303: 1526–1529.

10. Deshmukh, S. D., B. Kremer, M. Freudenberg, S. Bauer, D. T. Golenbock, andP. Henneke. 2011. Macrophages recognize streptococci through bacterial single-stranded RNA. EMBO Rep. 12: 71–76.

11. Tabeta, K., K. Hoebe, E. M. Janssen, X. Du, P. Georgel, K. Crozat, S. Mudd,N. Mann, S. Sovath, J. Goode, et al. 2006. The Unc93b1 mutation 3d disruptsexogenous antigen presentation and signaling via Toll-like receptors 3, 7 and 9. Nat.Immunol. 7: 156–164.

12. Brinkmann, M. M., E. Spooner, K. Hoebe, B. Beutler, H. L. Ploegh, and Y.-M. Kim. 2007. The interaction between the ER membrane protein UNC93B andTLR3, 7, and 9 is crucial for TLR signaling. J. Cell Biol. 177: 265–275.

13. Shi, Z., Z. Cai, A. Sanchez, T. Zhang, S. Wen, J. Wang, J. Yang, S. Fu, andD. Zhang. 2011. A novel Toll-like receptor that recognizes vesicular stomatitis virus.J. Biol. Chem. 286: 4517–4524.

14. Lutz, M. B., N. Kukutsch, A. L. Ogilvie, S. Rossner, F. Koch, N. Romani, andG. Schuler. 1999. An advanced culture method for generating large quantities ofhighly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223:77–92.

15. Heine, H., C. J. Kirschning, E. Lien, B. G. Monks, M. Rothe, andD. T. Golenbock. 1999. Cutting edge: cells that carry a null allele for toll-likereceptor 2 are capable of responding to endotoxin. J. Immunol. 162: 6971–6975.

16. Roach, J. C., G. Glusman, L. Rowen, A. Kaur, M. K. Purcell, K. D. Smith,L. E. Hood, and A. Aderem. 2005. The evolution of vertebrate Toll-like receptors.Proc. Natl. Acad. Sci. USA 102: 9577–9582.

17. Tabeta, K., P. Georgel, E. Janssen, X. Du, K. Hoebe, K. Crozat, S. Mudd,L. Shamel, S. Sovath, J. Goode, et al. 2004. Toll-like receptors 9 and 3 as essentialcomponents of innate immune defense against mouse cytomegalovirus infection.Proc. Natl. Acad. Sci. USA 101: 3516–3521.

18. Kasturi, S. P., I. Skountzou, R. A. Albrecht, D. Koutsonanos, T. Hua, H. I. Nakaya,R. Ravindran, S. Stewart, M. Alam, M. Kwissa, et al. 2011. Programming themagnitude and persistence of antibody responses with innate immunity. Nature470: 543–547.

19. Duggan, J. M., D. You, J. O. Cleaver, D. T. Larson, R. J. Garza, F. A. GuzmanPruneda, M. J. Tuvim, J. Zhang, B. F. Dickey, and S. E. Evans. 2011. Synergisticinteractions of TLR2/6 and TLR9 induce a high level of resistance to lung infectionin mice. J. Immunol. 186: 5916–5926.

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