Cutting Edge: TLR13 Is a Receptor for Bacterial RNA Asa Hidmark, Antonia von Saint Paul and Alexander H. Dalpke This information is current as of September 24, 2021. J Immunol 2012; 189:2717-2721; Prepublished online 15 August 2012; doi: 10.4049/jimmunol.1200898 http://www.jimmunol.org/content/189/6/2717 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cutting Edge: TLR13 Is a Receptor for Bacterial RNA Asa Hidmark, Antonia von Saint Paul, and Alexander H. Dalpke Bacterial RNA (bRNA) can induce cytokine production RNA of Streptococcus pyogenes induces IFN-b in myeloid DCs in macrophages and dendritic cells (DCs) through a pre- in an MyD88-dependent but TLR7-independent manner (6). viously unidentified receptor. expression analysis In addition, RNA-dependent induction of TNF-a by group B of murine DCs showed that bRNA induced gene regu- Streptococcus in macrophages has been shown to be indepen- lation similar to that induced by stimulation of TLR7 dent of nucleic acid-sensing TLR3, 7, and 9, although re- with R848. Although TLR7 was dispensable for cyto- quiring MyD88 and UNC93B (10), needed for the function of kine induction by bRNA, TLR-associated endosomal TLRs (11). Thus, the receptor for bRNA, which MyD88 and UNC93B were required. TLR13 is an may play an important role in recognizing Gram-positive bacteria, has not yet been identified.

endosomal murine TLR that has been described to Downloaded from interact with UNC93B with, so far, no characterized We found that murine TLR13, an UNC93B-interacting ligand. Small interfering RNA against TLR13 reduced (12, 13), could mediate the recognition of bRNA and whole Gram-positive bacteria when expressed in cytokine induction by bRNA in DCs. Moreover, Chi- Chinese hamster ovary (CHO) cells. When TLR13 expression nese hamster ovary cells transfected with TLR13, but k was targeted in DCs, using small interfering RNA (siRNA), not with TLR7 or 8, could activate NF- B in response cytokine induction by bRNA was reduced. Our results indi- to bRNA or Streptococcus pyogenes in an RNA-specific cate that TLR13 is required and sufficient for the induction of http://www.jimmunol.org/ manner. TLR7 antagonist IRS661 could, in addition, cytokines in response to bRNA. inhibit TLR13 signaling and reduced recognition of whole Gram-positive bacteria by DCs, also in the ab- Materials and Methods sence of TLR7. The results identify TLR13 as a recep- Mice tor for bRNA. The Journal of Immunology, 2012, Wild-type (wt) C57BL/6 mice were purchased from Charles River Labora- 2 2 189: 2717–2721. tories. MyD88 / mice were provided by H. Wagner (Munich, Germany). 2 2 TLR7 / mice were provided by S. Bauer (Marburg, Germany). UNC93B mice harboring H412R missense mutation (3D) (11) were supplied by by guest on September 24, 2021 eceptors of the innate immune system detect mo- M. Freudenberg (Freiburg, Germany). Animal experiments were approved by lecular patterns during the life cycles of infecting local authorities. R pathogens, depending on the localization and avail- Cells ability of these patterns. TLRs recognize a wide range of Bone marrow-derived DCs were generated as previously described (14). GM- microbial components and are important for raising innate CSF was provided to the culture by adding supernatant of 363-Ag8 cells, immune defenses against bacterial infections. Whereas LPS supplied by M. Lutz (Wu¨rzburg, Germany). DCs were harvested at day 7 and 5 from Gram-negative bacteria commonly activates the innate seeded in 24-well plates (8 3 10 DCs per well). CHO cells (DSMZ, #ACC110) were grown in Ham’s F12 medium. Media were supplemented immune system via TLR4 (1), it is less well understood how with 10% heat-inactivated FCS, penicillin G, and streptomycin sulfate Gram-positive bacteria are recognized. Although TLR adaptor (100 IU/ml) (Life Technologies). molecule MyD88 is important for activation of the innate immune system by Staphylococcus aureus (2) and strains of Bacteria and stimulations Enterococcus and Lactobacillus (3), activation by group A Staphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 6051), Listeria Streptococcus can occur independently of TLR2, 4, and 9 (4). monocytogenes (ATCC 15313), Enterococcus faecalis (ATCC 29212), Lacto- bacillus sp. (patient isolate), and Streptococcus pyogenes (ATCC 12344) were Several studies have shown that bacterial RNA (bRNA) acti- grown to midlog phase. S. pyogenes was heat killed for 45 min at 70˚C. RNA vates the innate immune system (5–8). RNA receptor TLR7, was purified from lysozyme-treated bacteria or eukaryotic cells (THP-1 or which recognizes single-stranded viral RNA (9), has been RAW 264.7), using TRIzol (Invitrogen) followed by High Pure RNA b Isolation Kit (Roche) with DNase I digest. RNase A (Invitrogen) digestion suggested to induce IFN- (8) or IL-12 (3) production by of bRNA was performed at room temperature. bRNA (3 mg/ml from myeloid dendritic cells (DCs) in response to bRNA. However, L. monocytogenes unless otherwise stated) was packaged with 2.5 ml DOTAP

Department of Infectious Diseases, Medical Microbiology and Hygiene, University of Neuenheimer Feld 324, 69120 Heidelberg, Germany. E-mail address: asa.hidmark@med. Heidelberg, 69120 Heidelberg, Germany uni-heidelberg.de Received for publication March 22, 2012. Accepted for publication July 16, 2012. The online version of this article contains supplemental material. This work was supported by a grant from the Medical Faculty of the University of Abbreviations used in this article: bRNA, bacterial RNA; CHO, Chinese hamster ovary; Heidelberg (to A.H.). DC, dendritic cell; pI:C, polyinosinic–polycytidylic acid; siRNA, small interfering RNA; wt, wild-type. The microarray data presented in this article have been submitted to the ArrayExpress database (www.ebi.ac.uk/arrayexpress/experiments) under accession number E-MTAB-1139. Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 Address correspondence and reprint requests to Dr. Asa Hidmark, Department of In- fectious Diseases, Medical Microbiology and Hygiene, University of Heidelberg, Im www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200898 2718 CUTTING EDGE: TLR13 IS A RECEPTOR FOR BACTERIAL RNA per microgram of RNA, as previously described (5). Ultrapure LPS from TLR expression vectors Salmonella minnesota (100 ng/ml) was provided by U. Seydel (Borstel, Germany). Other agents used were R848 (InvivoGen), polyinosinic–poly- Murine TLR13 from (Mammalian Gene Collection; http://mgc.nci.nih.gov/) m pCR-Blunt II-TOPO-TLR13 (I.M.A.G.E. IRAMp995J1823Q) was cloned cytidylic acid (pI:C, 50 g/ml; Sigma-Aldrich), Pam3Csk4 (InvivoGen), and CpG ODN 1668 (MWG-Biotech). Phosphorothioate oligonucleo- into pcDNA3.1 (Invitrogen) using ApaI/XhoI. TLR8, from pCR4-TOPO- tide inhibitor IRS661 (59-TGCTTGCAAGCTTGCAAGCA-39;Eurofins TLR8 (I.M.A.G.E. IRCLp5011B0512D), was digested with SpeI/FspI and MWG Operon) was added 1 h before stimulation. ligated into pcDNA3.1 cut with NheI/XbaI. TLR7 in pcDNA3 was a gift from T. Espevik (Trondheim, Norway).

DNA transfection, selection, and luciferase reporter assay CHO cells were cotransfected with vectors expressing TLR and NF-kB luciferase reporter pGL4.32[luc2P/NF-kB-RE/Hygro] (Promega), using Lipofectamine 2000 (Invitrogen) in 100-mm plates and incubated overnight. Bulk transfected cells were distributed in 24-well plates and stimulated upon seeding. Clones were 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 per well 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 High Downloaded from Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and measured with ABsolute SYBR Green Rox Mix Kit (ABgene House), using ABI PRISM 7700 (Applied Biosystems). Primer sequences are available on request.

siRNA transfection

siRNA against TLR13 (target sequence: 59-CACCTATGTTCTTGTAAG- http://www.jimmunol.org/ TAA-39) or Negative Control-N3 siRNA (Riboxx) was delivered with the Nucleofector Kit VPA-1011 (Lonza) or riboxx FECT Transfection Reagent (Riboxx) according to the manufacturer’s protocols. The riboxx FECT transfection was performed upon seeding 1 3 105 DCs per 48 wells in 150 ml media. Each nucleofection was divided into 96 wells. Media were changed after 24 h, and the cells were stimulated 48 h post siRNA transfection.

ELISA

Supernatants were analyzed using BD OptEIA Mouse ELISA Set IL-12p40 or by guest on September 24, 2021 IL-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 2 2 induction in DCs from wt or MyD88 / mice treated with bRNA or bRNA RNAse; induction of (B) IL-12p40 mRNA and (C) IL-12p40 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 FIGURE 2. bRNA and R848 induce similar gene regulation in DCs. 2 2 TLR7 / mice. (F) IL-12p40 mRNA in DCs from wt and UNC93B-mu- Microarray analysis of gene regulation in DCs treated with bRNA, RNase A tated 3D mice treated with bRNA, bRNA RNase, pI:C, LPS, R848, CpG digest of bRNA (bRNA RNase), R848 (1 mg/ml), or mock (untreated); log2 m m $ A B DNA (1 M), or Pam3Csk4 (1 g/ml) (Pam3C). n 3 mice from at least of signal intensity values of ( ) bRNA RNase versus bRNA, ( ) bRNA three independent experiments. Error bars show mean 6 SD. **p # 0.01, RNase versus R848, (C) R848 versus bRNA, or (D) untreated versus bRNA ***p # 0.001. RNase. Diagonal lines indicate 2-fold changes. n = 4 mice. The Journal of Immunology 2719

FACS Statistical analysis Brefeldin A (2 mg/ml; Sigma-Aldrich) was added at 5 h post stimulation. At Two-tailed unpaired Student t tests were used. Statistical significance indi- 16 h, cells were fixed and stained using Cytofix/Cytoperm (BD Biosciences) cated: *p # 0.05, **p # 0.01, and ***p # 0.001. Error bars show mean 6 with APC-conjugated anti-mouse IL-12 (p40/p70) Ab (BD #554480) and SD. assayed using a FACSCanto (BD Biosciences).

Microarray Results and Discussion Cytokine induction by bRNA requires MyD88 and UNC93B but is RNA was purified 6 h post stimulation, as described for quantitative RT-PCR, independent of TLR7 hybridized to Mouse Microarray Ref-8 v2.0 Expression BeadChips (Illumina) at the Genomics and Proteomics Core Facility at the Deutsches Krebsfor- RNA from Gram-positive bacteria, but not from eukaryotic schungszentrum (Heidelberg, Germany) and analyzed as described in the cells, has been shown to induce cytokines in myeloid DCs supplemental information online. The microarray data from this publication have been submitted to the ArrayExpress database, www.ebi.ac.uk/arrayexpress/ when delivered to the endosome (5, 8, 10). We found that experiments/E-MTAB-1139, and assigned the identifier E-MTAB-1139. bRNA from a number of bacterial species induced IL-12p40 Downloaded from http://www.jimmunol.org/

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 k R848. Stable CHO clones transfected with NF- B lu- by guest on September 24, 2021 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) 2 2 or TLR7 / (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 m (200 nM), or Pam3Csk4 (5 g/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. 2720 CUTTING EDGE: TLR13 IS A RECEPTOR FOR BACTERIAL RNA mRNA in murine bone marrow-derived myeloid DCs (Fig. completely inhibited IL-12p40 induction by bRNA in wt and 2 2 1A). RNase A digestion of the bRNA significantly reduced IL- TLR7 / DCs (Fig 3E, 3F). The IL-12p40 production in 12p40 induction. Previous studies have been contradictory response to several Gram-positive bacteria was significantly 2 2 regarding the ability of bRNA to induce cytokines in the inhibited by IRS661 treatment of wt and TLR7 / DCs, absence of MyD88. We showed that induction of IL-12p40 compared with induction by CpG via TLR9 or Pam3Csk4 via mRNA (Fig. 1B), secreted IL-12p (Fig. 1C), and IFN-b TLR2, which were not inhibited. Thus, inhibition of TLR13 mRNA (Fig. 1D) by bRNA from Gram-positive bacteria re- reduced the innate immune induction by several Gram- quired MyD88. TLR7 has been suggested to mediate IFN-b positive bacteria independently of TLR7 (4, 6). (8) or IL-12p40 (3) production in response to bRNA in myeloid DCs, whereas other studies have shown TLR7 to be nonessential for bRNA recognition (5, 6, 10). Using knock- out cells, we found TLR7 to be dispensable for induction of IL-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 found that induction of IL-12p40 mRNA by bRNA was abolished in DCs from UNC93B defect 3D mice (11) (Fig. 1F). Downloaded from bRNA induces signaling similar to that induced by TLR7 in myeloid DCs To characterize gene regulation induced by bRNA in DCs, we performed a microarray analysis (Fig. 2, Supplemental Table I). Both bRNA and TLR7 ligand R848 induced more than 2-fold regulation of several hundred , compared http://www.jimmunol.org/ with 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 and DOTAP did not induce any gene regulation, compared with untreated control, showing that the bRNA preparations did not contain any other components, in addition to RNA, ca-

pable of DC activation (Fig. 2D). The microarray result by guest on September 24, 2021 supported the hypothesis that bRNA induced signaling via an endosomal, MyD88-dependent TLR.

TLR13 activates NF-kB in response to bRNA and mediates recognition of Gram-positive bacteria TLR13 is a murine orphan TLR expressed by myeloid DCs, B cells, and macrophages (13). To investigate whether TLR13 could mediate bRNA signaling, CHO cells were cotransfected with an NF-kB luciferase reporter and vectors expressing TLR13, 7, or 8, or empty vector, before treatment with bRNA (Fig. 3A). Treatment with bRNA induced NF-kB activation specifically in TLR13-transfected cells (Fig. 3A). A clone stably transfected with TLR13 and NF-kB luciferase reporter induced up to 6-fold NF-kB activation in a dose- dependent response to bRNA (Fig. 3B). CHO cells have a frameshift mutation rendering TLR2 defective, whereas they are capable of responding to LPS (15). Responses to bRNA were of the same magnitude as responses to stimulation by FIGURE 4. siRNA targeting TLR13 reduces cytokine production in re- LPS. The TLR13-expressing CHO cell clone induced NF-kB sponse to bRNA treatment. DCs were transfected with TLR13 or control siRNA, using riboxx FECT and treated with OptiMEM (Mock), S. aureus luciferase reporter activity in response to both live and heat- RNA (bRNA) (wedge represents 0.25 and 0.5 mg/ml), or CpG (100 nM). killed S. pyogenes, but not in response to RNase A-treated (A) Relative expression of TLR13 mRNA was assayed in mock-treated bacteria (Fig. 3C), showing that TLR13 can contribute to samples. The samples were analyzed for (B) IL-12p40 mRNA and (C) IL-6 recognition of group A streptococci. TLR7-inhibitory oligo- mRNA. (D) Supernatants (6 h) were assayed for IL-6 by ELISA. (E) DCs nucleotide IRS661 has previously been shown to inhibit were harvested at 16 h post treatment, stained with an anti–IL-12-APC Ab, bRNA signaling (3). We show that IRS661 inhibited TLR13- and assayed by FACS. IL-12 is displayed as mean fluorescence intensity k (MFI). DCs were transfected with TLR13 or control siRNA using nucleo- mediated activation of NF- B luciferase reporter in response fection and treated with OptiMEM (Mock); S. aureus RNA (bRNA), 3 mg/ml; tobRNA,butnottoLPS(Fig.3D).Weproceededtoin- or CpG. The samples were analyzed for (F)IL-6mRNAand(G) IL-12p40 vestigate if TLR7/13 antagonist IRS661 could inhibit mRNA. n = 5 mice. Error bars show mean 6 SD. *p # 0.05, **p # 0.01, bRNA-induced signaling in DCs. Pretreatment with IRS661 ***p # 0.001. The Journal of Immunology 2721

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Nakaya, ical processing, and Gerald McInerney (Karolinska Institutet, Stockholm, R. Ravindran, S. Stewart, M. Alam, M. Kwissa, et al. 2011. Programming the Sweden) for discussions. magnitude and persistence of responses with innate immunity. Nature 470: 543–547. 19. Duggan, J. M., D. You, J. O. Cleaver, D. T. Larson, R. J. Garza, F. A. Guzma´n Pruneda, M. J. Tuvim, J. Zhang, B. F. Dickey, and S. E. Evans. 2011. Synergistic Disclosures interactions of TLR2/6 and TLR9 induce a high level of resistance to lung infection The authors have no financial conflicts of interest. in mice. J. Immunol. 186: 5916–5926. Supplementary material Mx2 7.46 6.26 Acsl1 7.44 6.60 Gene R848/ bRNA/ Ms4a6d 7.36 5.78 bRNA bRNA RNase RNase Dusp2 7.31 5.77 Cxcl1 67.52 50.78 Ifi47 7.23 5.47 Il1a 66.88 40.23 Ppap2b 7.03 5.02 Cxcl2 44.65 24.67 Cxcl9 7.01 5.35 Saa3 32.04 29.49 Mx1 6.76 5.61 Cd40 31.31 23.58 Ccrl2 6.72 7.19 LOC100038882 21.31 14.05 Gbp2 6.65 4.09 Ch25h 21.14 11.53 Lad1 6.64 4.06 Nfkbiz 20.98 25.67 Igtp 6.47 5.40 Rsad2 19.79 14.11 Oasl2 6.47 4.56 Slc7a2 19.46 11.26 Clec4e 6.40 5.99 Il6 17.76 10.51 Edn1 6.21 4.16 Inhba 17.23 9.21 Nt5c3 6.19 4.96 Oasl1 16.48 9.59 Slfn1 6.13 4.20 Cxcl10 15.91 15.54 Ifi205 6.08 3.49 Ifit3 14.77 8.23 Oas1g 6.04 4.01 Ifit2 14.52 9.77 Casp4 5.92 3.90 LOC100048346 13.07 10.19 Gbp3 5.90 4.32 Ppfia3 12.95 6.58 Upp1 5.87 4.20 Socs3 12.81 12.75 Gpr109a 5.81 6.02 Gbp5 12.56 12.92 Trim21 5.67 4.09 Il1b 12.54 12.15 Irgm 5.50 5.22 Tyki 12.47 9.77 Acpp 5.47 2.68 Cd69 12.46 12.27 Ppap2a 5.42 4.44 Il12a 12.44 6.34 Ccl4 5.37 5.56 Tnf 12.02 12.18 Il27 5.35 3.99 Irf7 10.96 6.43 Ddx58 5.13 3.88 Slco3a1 10.69 6.01 Lox 5.11 2.01 Usp18 10.42 8.34 Batf2 4.95 3.93 9930022N03Rik 10.29 8.41 Stat1 4.93 3.69 Phf11 10.22 5.54 Serpina3g 4.92 3.54 LOC667370 10.14 5.79 Ptx3 4.87 3.97 Marco 9.52 8.83 Jmjd2a 4.87 3.46 Lcn2 9.39 5.66 Schip1 4.85 3.62 D14Ertd668e 9.26 6.01 Zc3h12a 4.82 3.58 Ccnd2 8.90 5.11 Spic 4.82 2.86 Gpr84 8.55 7.44 Fpr1 4.80 4.30 Cav1 8.40 5.59 BC006779 4.76 3.67 Car13 8.22 4.71 Ifi35 4.74 3.22 Cfb 8.18 5.23 Dhx58 4.70 3.78 Fabp3 7.95 3.64 Clecsf9 4.69 4.61 Parp12 4.67 3.75 Irak3 3.85 3.78 Dgat2 4.64 2.83 Atp10a 3.85 2.41 Itga5 4.62 3.41 Agrn 3.83 2.99 Ptges 4.57 3.28 Trim30 3.81 3.17 Fst 4.52 3.31 St3gal5 3.78 3.32 Ccl22 4.48 3.42 Stat2 3.78 3.22 Il18 4.44 2.78 LOC100044190 3.76 2.36 Il1rn 4.42 3.19 Gpd2 3.76 2.81 Lta 4.39 3.58 Trex1 3.76 3.38 Ifitm3 4.38 3.09 AI451557 3.76 2.92 Marcksl1 4.37 4.18 Parp14 3.69 3.69 BC094916 4.33 3.44 Cd14 3.69 3.57 Tnfaip3 4.33 3.66 Tmem2 3.68 2.47 Ube2l6 4.33 2.87 Pou2f2 3.61 3.42 Batf 4.28 2.74 Rgs1 3.60 3.04 Vnn3 4.26 2.40 Traf1 3.59 2.95 Gpr31c 4.24 2.75 A630077B13Rik 3.59 2.64 St3gal3 4.23 2.95 Dusp16 3.58 2.77 Isg20 4.23 2.66 Il10 3.56 Osbpl3 4.17 4.05 Lgals9 3.55 2.40 AI451617 4.17 3.44 9030625A04Rik 3.54 3.47 LOC233529 4.16 2.81 Mlp 3.52 3.89 Mefv 4.14 4.38 Ebi3 3.52 3.00 Casp7 4.14 2.79 Tyms 3.51 2.48 Serpinb2 4.13 3.72 Micall2 3.49 Rcl1 4.10 2.56 Il15 3.46 2.48 Pml 4.08 3.03 Slc6a12 3.45 2.84 Hdc 4.07 2.92 Map3k8 3.45 3.64 Daxx 4.05 3.73 Slc1a2 3.45 Dnajb6 4.02 2.95 Lass6 3.45 3.16 Apol7c 4.02 3.57 Stard5 3.44 2.66 Gadd45b 4.01 3.59 Trem1 3.43 2.54 Ube1l 4.01 2.30 Btla 3.42 2.45 Oas3 3.98 2.24 Bst2 3.39 2.94 Tnfrsf1b 3.96 2.94 Sp100 3.38 2.52 Dusp4 3.95 C330023M02Rik 3.37 2.51 Vnn3 3.95 2.35 Palld 3.35 2.52 Siat9 3.95 3.69 Cd200 3.34 2.13 Eif2ak2 3.93 3.42 Trem3 3.34 2.81 Iigp2 3.88 4.19 Tpst1 3.34 2.95 Tlr1 3.87 3.61 Gvin1 3.33 2.95 Gpr18 3.86 3.71 Lrp11 3.33 2.32 Xkr8 3.86 2.97 Enpp4 3.33 2.63 Ccl5 3.86 3.48 Wnt6 3.32 3.03 Dtx2 3.31 2.22 Vcam1 2.90 2.57 Scarf1 3.31 2.66 Lgals9 2.90 2.31 Gfi1 3.31 2.98 Jak2 2.88 2.35 1200009I06Rik 3.30 2.94 Malt1 2.88 2.42 Car4 3.30 3.13 Rgs1 2.87 2.66 Tnfsf15 3.29 3.83 Gca 2.86 2.10 Sp140 3.28 2.73 Tlr6 2.86 2.40 Ifi204 3.25 2.59 Cav2 2.85 2.28 Mlkl 3.22 2.03 Apbb2 2.85 2.23 Nos2 3.21 Aoah 2.85 2.41 Tap1 3.21 2.94 Arhgef3 2.84 3.24 Ccl3 3.20 3.12 Adra2a 2.83 2.43 Mmp13 3.20 Tap2 2.83 2.10 Tyms-ps 3.18 2.44 Trim56 2.82 2.35 Ppfibp2 3.18 2.56 Smpdl3b 2.82 2.36 Pik3ap1 3.16 2.62 Csf3 2.81 Src 3.15 2.12 A430084P05Rik 2.81 2.07 Slamf7 3.14 2.46 Mov10 2.81 Ogfr 3.14 2.39 Nod1 2.80 2.42 St3gal1 3.14 2.66 Hspa1a 2.80 2.25 Prdx5 3.13 2.81 1700047I17Rik1 2.80 2.56 Oas2 3.13 2.51 2210009G21Rik 2.79 2.11 Tmem178 3.12 2.71 Fnbp1l 2.78 2.94 Appl1 3.12 2.29 Tpbg 2.78 2.29 LOC100045567 3.10 2.85 Six1 2.78 2.86 Tagap 3.07 2.77 LOC100045343 2.77 2.30 Snn 3.06 2.53 Sirpa 2.76 2.11 Zfp313 3.06 2.63 Hmgn3 2.76 Hp 3.06 2.81 Plek 2.75 3.34 Tor3a 3.05 2.62 EG665378 2.73 Rasgrp1 3.03 3.11 Pilrb1 2.73 3.07 Tapbp 3.00 2.10 Sap30 2.72 2.93 Plagl2 2.99 2.43 Cd274 2.71 2.43 Slpi 2.99 2.32 Cd86 2.71 Cdk5r1 2.98 2.03 Phlda1 2.71 2.26 LOC100046781 2.97 3.29 Pdzk1ip1 2.71 Fscn1 2.97 2.35 Kal1 2.70 2.07 Slc7a11 2.97 3.00 Epsti1 2.70 Mina 2.96 2.26 Fpr2 2.70 3.04 D16Ertd472e 2.96 2.80 Ms4a7 2.70 2.06 Ehd1 2.95 2.97 Cdkn1a 2.69 2.94 Blnk 2.95 2.42 5730528L13Rik 2.69 2.19 Orm1 2.93 Slc25a37 2.69 2.10 Tgm2 2.93 2.36 Frag1 2.69 2.40 Slfn2 2.69 2.82 Ccdc86 2.47 2.42 Oas1a 2.68 2.09 Emr1 2.46 2.16 BC046404 2.67 2.18 Ly6c1 2.46 2.96 Kctd14 2.67 2810439F02Rik 2.46 LOC100044702 2.66 2.12 Ms4a6c 2.45 2.32 Ier3 2.66 2.12 Tmem39a 2.45 2.04 Bcl2a1d 2.66 2.59 Adar 2.44 LOC100047963 2.66 2.64 1300018I05Rik 2.44 Chi3l1 2.66 2.24 Treml4 2.44 C130032J12Rik 2.66 2.10 Gbp6 2.44 2.07 Irf1 2.65 2.79 Itpr2 2.44 Xylt2 2.65 2.65 Mmp2 2.43 Tor1aip1 2.64 2.38 F10 2.43 2.23 Trafd1 2.64 2.41 Met 2.43 2.14 Il20rb 2.64 Ikbkg 2.42 1100001G20Rik 2.63 2.32 LOC621823 2.42 2.01 Has1 2.62 Plk3 2.42 2.20 Ppp1r15b 2.62 2.24 Mtdh 2.42 2.25 Denr 2.61 2.34 Zmynd15 2.41 Tnfaip8l3 2.61 3.39 Tcf4 2.41 2.23 Clec4a1 2.60 3.51 Tmco3 2.41 Ptprj 2.60 2.42 Rasgef1b 2.41 2.96 Zbp1 2.60 Ets2 2.40 2.35 Chpt1 2.59 Nampt 2.40 2.21 Cp 2.57 2.04 Sipa1l1 2.40 2.16 A030007L17Rik 2.56 2.19 Kremen 2.39 AI504432 2.56 2.59 Pvrl2 2.39 2.12 Rbpms 2.56 2.44 Tdrd7 2.38 Sbds 2.54 2.30 Artn 2.38 2.90 Pcgf5 2.54 2.38 Dusp1 2.38 Nfkb1 2.54 2.48 Plac8 2.37 2.36 Pim3 2.53 2.18 Ppbp 2.37 3.98 Sdc4 2.53 2.11 Dnmt3l 2.35 Pols 2.52 2.57 Ppp1cb 2.35 2.07 Thbs1 2.51 3.25 H2-M2 2.35 2.24 Gja1 2.51 Slc31a1 2.34 2.43 Kif3c 2.51 2.13 Cxcl4 2.34 2.94 2610103J23Rik 2.51 2.37 Slc11a1 2.34 2.16 2610208M17Rik 2.50 Slc25a25 2.33 2.12 Cd70 2.50 Rel 2.33 2.15 AA960436 2.49 2.16 Nfkbib 2.33 2010106G01Rik 2.48 2.07 Hmox1 2.32 Fyb 2.48 2.25 Slc15a3 2.32 6330409N04Rik 2.48 2.07 Rtp4 2.31 LOC100048556 2.31 2.07 Aldh1b1 2.15 Fcgr2b 2.31 2.08 Stap1 2.15 2.26 Areg 2.31 Srxn1 2.14 Cxcl16 2.30 2.09 Ptpn1 2.14 2.07 Nlrp3 2.30 2.25 Jmjd3 2.13 2.13 Stat5a 2.29 Procr 2.13 Lrrk2 2.29 Psmd10 2.13 Psmb8 2.29 Asb13 2.13 Cxcl3 2.28 2700055A20Rik 2.12 2.13 Mdm2 2.28 Sod2 2.12 2.14 Ube2e2 2.28 2.10 Lgals3bp 2.12 Pilra 2.27 2.42 Spsb1 2.12 Tnip1 2.26 2.30 Pdlim7 2.12 Leng9 2.26 Fbxw17 2.12 Ece1 2.25 Lcp2 2.11 2.03 Zhx2 2.25 2.19 Cias1 2.11 2.15 Serpina3f 2.24 Axud1 2.10 2.18 Bcl2a1c 2.24 2.11 Psmb10 2.10 B230339H12Rik 2.24 Camkk2 2.10 Peli1 2.23 Fgl2 2.10 Il4i1 2.23 Tlk2 2.10 Srgn 2.23 Coq10b 2.09 Pstpip2 2.23 2.29 Igf2bp2 2.09 Ripk2 2.23 2.31 Tank 2.09 Mmp14 2.22 2.28 Whdc1 2.08 Slc31a2 2.22 2.27 Nr1h3 2.08 Myd116 2.21 2.39 H2-T24 2.08 Cox18 2.21 Gpc1 2.07 Cbwd1 2.20 Rassf4 2.07 2.01 Slc16a10 2.20 2.78 Akt3 2.06 LOC100044475 2.20 Magohb 2.06 2.08 Pvr 2.20 2.03 Ccdc50 2.06 2.18 Mtpn 2.19 Elk3 2.06 2.39 Psmb9 2.18 Mfap3l 2.06 Hsd17b11 2.18 Rab10 2.05 Mfsd7 2.18 2.23 Zfp238 2.05 2.10 Clcn7 2.18 Samhd1 2.05 LOC435565 2.17 Rhbdf2 2.05 2.03 Pla2g4f 2.17 Rffl 2.04 Ddx24 2.17 2.00 Vti1a 2.04 Ak3l1 2.17 2.37 Trim26 2.04 Zfhx2 2.16 Slc7a8 2.04 Irak2 2.16 2.11 Gm885 2.04 Zeb1 2.15 Tapbpl 2.04 Gpr141 2.03 2.07 P2ry1 0.49 0.50 Il15ra 2.03 Zfhx3 0.49 Susd2 2.02 Nfatc1 0.49 0.50 LOC100048858 2.02 Zdhhc14 0.49 Ncoa5 2.02 Pacs2 0.49 Stk39 2.01 Bdh1 0.49 Tnfsf4 2.01 Plec1 0.49 Irf8 2.01 Casp2 0.49 Arid5b 2.00 2.14 Tex261 0.49 Bcl6 2.00 Cyhr1 0.49 Nrap 2.10 LOC381629 0.49 Grhl1 2.01 Rab40c 0.49 Lmo4 2.04 Tgfbr1 0.49 Tsc22d1 2.34 Mthfd1 0.49 P2ry14 2.11 Nme3 0.49 Cited2 2.26 Lage3 0.49 Tnfaip2 2.01 Rnu6 0.49 Klrb1b 2.34 Tmem205 0.49 P2ry6 0.49 Nqo2 0.49 Ctgf 0.49 Iap 0.49 Slc40a1 0.34 Gng2 0.48 Mertk 0.48 Hhex 0.48 Pdgfa 0.49 Dolpp1 0.48 Dyrk1b 0.47 Cyb561 0.48 Acpl2 0.50 Vat1 0.48 Ivd 0.50 Centb5 0.48 Exosc7 0.50 Msrb2 0.48 1110002B05Rik 0.50 2310079N02Rik 0.48 2310047M10Rik 0.50 Syngr1 0.48 Cyp27a1 0.50 0.47 Rgl2 0.48 Ndufa6 0.50 Mvd 0.48 Cnnm2 0.50 AA407659 0.48 0.50 Mpv17 0.50 BC028528 0.48 Ppm1m 0.50 Tec 0.48 0.46 Zdhhc9 0.50 Rab31 0.48 Tmem147 0.50 Mic2l1 0.48 Pdlim2 0.50 Cd300a 0.48 C1qc 0.50 Exosc5 0.48 1500032D16Rik 0.50 Zfp704 0.48 Cugbp2 0.49 Rreb1 0.48 Mcm4 0.49 Gpnmb 0.48 Aldoc 0.49 D9Ertd392e 0.48 0.48 2410002F23Rik 0.49 0.50 Clec4b1 0.48 Trappc1 0.49 Cd151 0.48 Pld4 0.48 Fes 0.46 Depdc6 0.48 Pop5 0.46 Mical1 0.48 Fgd2 0.46 0.46 3110001D03Rik 0.48 Ptplad2 0.46 Parp1 0.48 Pstpip1 0.46 C1qa 0.48 Tnfaip8l2 0.46 Idh2 0.48 Cuta 0.46 Reep5 0.48 Fkbp4 0.46 Lman2 0.48 Nfam1 0.46 Tgfbi 0.48 Clecsf12 0.45 Gng10 0.48 Nrm 0.45 Ctns 0.48 Ly86 0.45 0.41 Rell1 0.47 Metrn 0.45 Ivns1abp 0.47 Efcab4a 0.45 0.45 Cyb5r1 0.47 Casp9 0.45 Txnip 0.47 Sh3glb2 0.45 Cdk2 0.47 Arrb1 0.45 1500031L02Rik 0.47 0.45 Amz1 0.45 Asph 0.47 Ppap2c 0.45 Klk1b27 0.47 D15Wsu169e 0.45 4732429D16Rik 0.47 Lmo2 0.45 Fbxl10 0.47 Tns4 0.45 0.47 Stard4 0.47 Nuak1 0.45 LOC333331 0.47 Elovl6 0.45 Epb4.1l1 0.47 LOC100048461 0.44 Aes 0.47 Rpa1 0.44 Ard1 0.47 Socs6 0.44 Nudt19 0.47 Gpr146 0.44 0.39 Nqo2 0.47 Rab3il1 0.44 AI467606 0.47 0.47 B3gnt1 0.44 Mrpl28 0.47 2610039C10Rik 0.44 Qdpr 0.47 Deb1 0.44 Dnase2a 0.47 Tnfrsf22 0.44 LOC100044204 0.47 0.48 Dnajc15 0.44 Bbc3 0.47 0.45 Dscr1l2 0.44 Asgr2 0.47 0.49 Rps6ka1 0.44 Pqlc1 0.47 Lat2 0.44 Slc39a11 0.47 Cxcr4 0.44 Myo7a 0.46 Cryl1 0.44 Capn2 0.46 Fcgr3 0.44 Gpr171 0.46 Gpr65 0.44 Tmem14c 0.46 Itgb7 0.44 0.46 Nqo2 0.46 1110032A13Rik 0.44 Coro1a 0.46 Rassf5 0.44 D10Bwg1364e 0.43 0.50 Pnpla7 0.41 0.48 Ankrd47 0.43 Pqlc3 0.41 Pigx 0.43 Mbnl1 0.41 Mylc2b 0.43 Dusp7 0.41 0.45 Vav3 0.43 Ccdc85b 0.41 0.48 Lass5 0.43 Lpxn 0.41 0.41 Pitpna 0.43 Phlda3 0.41 0.48 Adk 0.43 Plp2 0.41 Paqr7 0.43 Ap1b1 0.40 Sidt2 0.43 Bnip3l 0.40 0610007C21Rik 0.43 Msr2 0.40 0.44 Ccng1 0.43 Klf9 0.40 0.47 Mgll 0.43 0.42 Tmem41a 0.40 0.45 Cd300lg 0.43 0.43 Erp29 0.40 0.49 Gusb 0.43 Uap1l1 0.40 Acaa2 0.43 Pira3 0.40 Itgb5 0.43 0.42 Acadm 0.40 Tmem141 0.43 Card11 0.40 0.47 Bcas1 0.42 Mgl1 0.40 0.41 Cdc7 0.42 Arhgef6 0.40 0.44 Rnf166 0.42 Ech1 0.40 Slc35c2 0.42 LOC100045877 0.40 Rnase4 0.42 0.45 Glt25d1 0.40 Tmem53 0.42 4930570C03Rik 0.40 Eif4ebp2 0.42 Them2 0.40 1810015A11Rik 0.42 Bin1 0.39 Ethe1 0.42 0.47 Ptpn18 0.39 0.42 Arhgap18 0.42 0.50 Alox5ap 0.39 Nav1 0.42 0.47 2700094K13Rik 0.39 0.49 Timm8a1 0.42 Uck2 0.39 Dab2 0.42 0.43 Ptprs 0.39 Angptl4 0.42 Hsd17b10 0.39 Scd2 0.41 0.49 Xpr1 0.39 0.48 Siva1 0.41 0.48 Dgkg 0.39 0.44 Klf2 0.41 Tubb2b 0.39 0.44 Cyp4f16 0.41 0.48 Scamp5 0.39 0.47 Pcyox1 0.41 Diras2 0.39 0.40 Syn1 0.41 Acox3 0.39 0.44 Ptpro 0.41 D9Ertd280e 0.39 0.47 Slc27a3 0.41 0.41 Hmha1 0.38 0.40 Urod 0.41 Tlr4 0.38 0.42 Suv420h2 0.41 0.47 Inpp5d 0.38 0.46 Ctnnbip1 0.41 Ebpl 0.38 Cenpa 0.41 Zmat3 0.38 Paox 0.38 0.40 2310005P05Rik 0.34 0.36 Hfe 0.38 0.43 Oxct1 0.34 0.45 Aacs 0.38 0.43 Ifngr1 0.34 0.43 Mknk2 0.38 0.38 BC031353 0.34 0.36 LOC100038908 0.38 0.50 Adrb2 0.34 0.45 Gcnt1 0.38 0.43 Klf4 0.34 0.35 Nup210 0.38 0.49 Bach1 0.34 0.46 9830130M13Rik 0.38 0.38 Emb 0.33 0.45 9130211I03Rik 0.38 Trp53inp1 0.33 0.38 Tbxas1 0.38 0.50 Elovl5 0.33 0.46 Coro1c 0.37 0.49 Sh2d1b1 0.33 0.48 Rcan3 0.37 0.46 Idh1 0.33 0.46 Pygl 0.37 Ccdc80 0.33 0.39 Tm6sf1 0.37 Abhd14b 0.32 0.39 Ephx1 0.37 0.39 1190002A17Rik 0.32 0.40 Bzw2 0.37 2410004L22Rik 0.32 0.42 Tiam1 0.37 0.46 Tmem86a 0.32 0.31 Arhgap9 0.37 0.45 Pycard 0.32 0.41 Gmpr 0.37 0.43 D10Ertd322e 0.32 0.45 Appl2 0.37 0.43 0910001L09Rik 0.32 Emilin2 0.37 0.50 Hebp1 0.31 0.49 Tspan14 0.37 0.47 Pros1 0.31 0.41 Acss1 0.36 0.38 Fos 0.31 Scd1 0.36 0.47 Rnase6 0.31 0.34 Fcgrt 0.36 0.37 Gadd45g 0.31 0.33 Arhgap25 0.36 0.40 Akr7a5 0.31 0.40 Cbfa2t3h 0.36 0.38 Kcnab2 0.31 0.41 9330186A19Rik 0.36 0.41 Prcp 0.31 0.43 Abcd1 0.36 F2rl2 0.30 B3gnt8 0.36 0.38 1700025G04Rik 0.30 0.41 Sort1 0.36 P2ry5 0.30 0.31 Xrcc6 0.36 0.45 Plcb2 0.30 0.38 Cd97 0.36 0.35 Rab11fip5 0.29 0.35 Zfp608 0.35 0.48 H2-DMa 0.29 0.36 Ptpn22 0.35 0.42 Efcab4a 0.29 0.34 6430548M08Rik 0.35 0.45 Elmo2 0.29 0.37 Ffar2 0.35 A130092J06Rik 0.29 0.38 Plekhg3 0.35 0.36 Nfe2 0.29 0.33 Hip1 0.35 Smarca2 0.28 0.36 Acot1 0.35 0.38 Sbk 0.28 0.34 Dhrs7 0.35 0.44 Snx24 0.28 0.37 Pparg 0.35 Lrmp 0.27 0.37 Lpin1 0.35 0.38 Adi1 0.27 0.34 Sesn1 0.35 0.41 Ckb 0.27 0.30 Kcnk13 0.27 0.33 1110032E23Rik 0.16 0.17 Tsc22d3 0.27 0.31 Lbh 0.15 0.23 Fblim1 0.27 0.32 Tmem154 0.14 0.18 Hr 0.26 E330036I19Rik 0.13 0.27 Kctd12 0.26 0.50 St6gal1 0.09 0.16 Add3 0.26 0.38 Trem2 0.26 0.33 Camk1 0.25 0.41

Galnt9 0.25 0.32

Arl11 0.25 0.31 LOC676420 0.25 0.30 Aatk 0.25 0.34 Olfm1 0.24 0.31 Fads1 0.24 0.36 Tgfbr2 0.24 0.36 Adssl1 0.24 0.34

Lypla3 0.24 0.28 Cd93 0.24 0.29 Tmem51 0.24 0.35 Chn2 0.24 0.32 Sept9 0.23 0.34 Gnpda1 0.23 0.34 Slc46a3 0.23 0.30

Hdac5 0.23 0.31 Eps8 0.22 0.25 Ank 0.22 0.30 1200013B08Rik 0.22 0.33 Manba 0.22 0.33 Lmna 0.22 0.33 Ppargc1b 0.22 0.29

Mrc1 0.21 0.28 Emp1 0.21 0.25 X99384 0.20 0.38 Klc4 0.20 0.28 Timp2 0.19 0.28 Ypel3 0.19 0.27 Tspan32 0.19 0.29

Tpcn1 0.19 0.27 E2f2 0.19 0.24 Tbc1d2 0.18 0.23 Cnr2 0.17 0.22 Ramp1 0.17 0.22 Aadacl1 0.17 0.23 Ltb4r1 0.17 0.23 Supplementary Table 1. Genes regulated >2-fold as compared to Mock

Middle column shows fold regulation of genes by R848 as compared to an RNase A digest of RNA from L. monocytogenes (bRNA RNase). Right column shows fold regulation of the gene by RNA from L. monocytogenes (bRNA) as compared to bRNA RNase. RNA from BM-DCs from 4 individual mice were purified at 6 h post stimulation as described for quantitative RT-PCR and hybridized to Illumina Mouse

Microarray Ref-8 v2.0 Expression BeadChips following the manufacturer’s recommendations (Illumina) at the Genomics and Proteomics Core Facility at the

DKFZ (Heidelberg). Microarray scanning was done using a Bead station array scanner. Analysis was performed on the bead-level data using the programming language R. Briefly, outliers were removed on bead level by taking only beads with an expression value >20 and subsequently filtering values which had a median absolute deviation (MAD) <2.5. Remaining data points were used for the calculation of the mean average signal for a given probe and standard deviations were calculated for each probe. Two-tailed t-tests were performed over all beads of all samples of a group in original scale. Benjamini-Hochberg correction was applied over all p-values of the differential expression analysis.

Data were subsequently filtered according to a p-value <0.01 and analyzed with

MicrosoftExcel. Fold changes 2.0 or 0.5 were regarded as significant.