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TLR8 Senses Staphylococcus aureus RNA in Human Primary Monocytes and Macrophages and Induces IFN-β Production via a TAK1 −IKK −β IRF5 Signaling Pathway This information is current as of September 24, 2021. Bjarte Bergstrøm, Marie H. Aune, Jane A. Awuh, June F. Kojen, Kjetil J. Blix, Liv Ryan, Trude H. Flo, Tom E. Mollnes, Terje Espevik and Jørgen Stenvik J Immunol published online 17 June 2015 http://www.jimmunol.org/content/early/2015/06/17/jimmun Downloaded from ol.1403176

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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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 17, 2015, doi:10.4049/jimmunol.1403176 The Journal of Immunology

TLR8 Senses Staphylococcus aureus RNA in Human Primary Monocytes and Macrophages and Induces IFN-b Production via a TAK1–IKKb–IRF5 Signaling Pathway

Bjarte Bergstrøm,* Marie H. Aune,* Jane A. Awuh,* June F. Kojen,* Kjetil J. Blix,* Liv Ryan,* Trude H. Flo,* Tom E. Mollnes,*,†,‡,x,{ Terje Espevik,*,1 and Jørgen Stenvik*,1

Staphylococcus aureus may cause serious infections and is one of the most lethal and common causes of sepsis. TLR2 has been described as the main pattern recognition receptor that senses S. aureus and elicits production of proinﬂammatory cytokines via MyD88–NF-kB signaling. S. aureus can also induce the production of IFN-b, a cytokine that requires IFN regulatory factors (IRFs) for its transcription, but the signaling mechanism for IFN-b induction by S. aureus are unclear. Surprisingly, we demon-

strate that activation of TLR2 by lipoproteins does not contribute to IFN-b production but instead can suppress the induction of Downloaded from IFN-b in human primary monocytes and monocyte-derived macrophages. The production of IFN-b was induced by TLR8- mediated sensing of S. aureus RNA, which triggered IRF5 nuclear accumulation, and this could be antagonized by concomitant TLR2 signaling. The TLR8-mediated activation of IRF5 was dependent on TAK1 and IkB kinase (IKK)b, which thus reveals a physiological role of the recently described IRF5-activating function of IKKb. TLR8–IRF5 signaling was necessary for induction of IFN-b and IL-12 by S. aureus, and it also contributed to the induction of TNF. In conclusion, our study demonstrates

a physiological role of TLR8 in the sensing of entire S. aureus in human primary phagocytes, including the induction of IFN-b and http://www.jimmunol.org/ IL-12 production via a TAK1–IKKb–IRF5 pathway that can be inhibited by TLR2 signaling. The Journal of Immunology, 2015, 195: 000–000.

taphylococcus aureus can act as a peaceful colonizer of hemolytic and leucolytic toxins and C-targeting factors that con- the skin and the nostrils or as an aggressive pathogen tribute to immune evasion (2). TLR2 is a primary pattern recog- S causing invasive diseases and sepsis. Intracellular sur- nition receptor (PRR) for sensing of S. aureus by immune cells and vival of S. aureus, abscess formation, as well as the emergence mediates resistance of mice against experimental S. aureus infec- of methicillin-resistant strains complicate the treatment of serious tion (3). S. aureus deficient in lipoprotein synthesis (Dlgt) does not infections (1). S. aureus also produces virulence factors such as activate TLR2 and elicits reduced proinflammatory responses in by guest on September 24, 2021 human cell lines (4). Children and teenagers with MyD88- or IL-1R–associated kinase 4 deficiency are at risk for infections with *Centre of Molecular Inflammation Research, Department of Cancer Research and pyogenic bacteria, in particular S. aureus, Streptococcus pneumo- Molecular Medicine, Norwegian University of Science and Technology, Trondheim N-7491, Norway; †Department of Immunology, Oslo University Hospital Rikshospi- nia, and Pseudomonas aeruginosa, whereas their resistance against talet, Oslo N-0027, Norway; ‡K.G. Jebsen Inflammation Research Center, University infection with other pathogens is normal (5, 6). This indicates of Oslo, Oslo N-0027, Norway; xResearch Laboratory, Nordland Hospital, Bodø { a particular importance of TLR2 and/or IL-1Rs for resistance N-8092, Norway; and Institute of Clinical Medicine, University of Tromsø, Tromsø N-9037, Norway against S. aureus infection in young humans. 1T.E. and J.S. are cosenior authors. Type I IFNs are classical antiviral cytokines, but they are also induced by intracellular and extracellular bacteria. The impact of ORCID: 0000-0002-1051-9258 (J.S.). the main type I IFNs (IFN-a and IFN-b) on bacterial infections is Received for publication December 22, 2014. Accepted for publication May 20, 2015. less clear and spans from enhanced innate and cell-mediated This work was supported by the Liaison Committee between the Central Norway immunity to immune suppression and dysregulation, which may Regional Health Authority and Norwegian University of Science and Technology contribute to the progression of septic shock (7). The predomi- Grants 46056622 (to J.S.) and 46056633 (to B.B.), as well as by Research Council of nant pathway of IFN-b induction by Gram-negative bacteria is by Norway Grant 223255/F50 through its Centres of Excellence funding scheme. LPS-mediated activation of endosomal TLR4 signaling via the Address correspondence and reprint requests to Dr. Jørgen Stenvik, Department of b Cancer Research and Molecular Medicine, Norwegian University of Science and Toll/IL-1R domain–containing adapter inducing IFN- –IFN reg- Technology, Olav Kyrres Gate 10, Trondheim N-7491, Norway. E-mail address: ulatory factor (IRF)3 pathway (8, 9). For Gram-positive bacte- [email protected] ria there may not be a single predominant mechanism for IFN-b The online version of this article contains supplemental material. induction, as multiple pathogen-associated molecular patterns Abbreviations used in this article: HK, heat-killed; HS, human serum; IKK, IkB (PAMPs) and cell host receptors have been implicated in different kinase; IRF, IFN regulatory factor; lgt, prolipoprotein diacylglyceryl transferase model systems. In murine phagocytes, recognition of bacterial gene; L2K, Lipofectamine 2000; MDM, monocyte-derived macrophage; NOD2, nucleotide-binding oligomerization domain–containing protein 2; PAMP, pathogen- RNA and DNA appears as central for IFN-b production (10–12), associated molecular pattern; PBS-S, PBS with 0.05% saponin; PFA, paraformalde- and mouse TLR13 was recently identified as a sensor of RNA of hyde; pL-Arg, poly-L-arginine; PRR, pattern recognition receptor; pU, polyuridylic acid; Q-PCR, quantitative real-time PCR; RIP2, receptor interacting protein 2; microbial origin (13, 14). Induction of type I IFNs by S. aureus siRNA, small interfering RNA; STING, stimulator of IFN genes; TAK1, TGF-b– has been examined in different human and murine cell types with activated kinase 1; TBK1, TANK-binding kinase 1; TRIF, Toll/IL-1R domain– different conclusions regarding the molecular mechanisms involved. containing adapter inducing IFN-b; wt, wild-type. S. aureus PAMPs suggested to be responsible for IFN-b induction Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 include staphylococcal protein A, DNA, RNA, and lipoteichoic

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1403176 2 S. AUREUS ACTIVATES A TLR8–TAK1–IKKb–IRF5 AXIS IN MONOCYTES acid, and TLR2, TLR7, TLR9, and cytosolic PRRs were impli- Medical and Health Research Ethics (REC Central, Norway, no. 2009/ cated (15–22). 2245). Human PBMCs were isolated from buffy coats using Lympho- The aim of the present study was to examine the role of TLR2 prep (Axis-Shield) as described by the manufacturer, and monocytes were b purified by adherence in culture plates and maintained in RPMI 1640 (Life and other PRRs for S. aureus–induced IFN- production in human Technologies) supplemented with 10% pooled human serum (HS). TLR2 primary monocytes and monocyte-derived macrophages (MDMs). blocking was done by 30 min pretreatment with the anti-mouse/human Unexpectedly, we found that TLR2 activation could suppress the TLR2 mAb clone T2.5 (no. HM1054, Hycult Biotech) at 5 mg/ml, and S. aureus–induced production of IFN-b. In contrast, induction of mouse IgG1 mAb (R&D Systems) served as isotype control. Poly(I:C), pU, b and crude S. aureus lysate, with or without RNAse A (Qiagen) treatment IFN- was triggered by S. aureus RNA, which activated a TLR8– (2 ml/ml, 1 h at 37˚C), was precomplexed with pL-Arg at a 1:1 ratio (w/w) IRF5 signaling axis in a TGF-b–activated kinase 1 (TAK1)– and in Opti-MEM (Invitrogen), or with 2.5 ml L2K/mg RNA in Opti-MEM for IkB kinase (IKK)b–dependent fashion. In this study, we establish transfection. For infection of monocytes and macrophages, live bacteria TLR8 as a second MyD88-dependent PRR of S. aureus in human from overnight cultures with or without the bacterial culture media were primary monocytes and MDMs and show that it is essential for the diluted in RPMI 1640 and incubated for 1 h at room temperature before b addition to the cells. Extracellular bacteria were killed after 1 h by addition induction of IFN- production by whole bacteria via a recently of gentamicin to 100 mg/ml. For quantitative real-time PCR (Q-PCR) anal- identified IKKb–IRF5 activation pathway. We also demonstrate ysis, the cells were lysed after a total infection time of 3–4 h, and cell culture a cross-regulatory function of TLR2 in TLR8–IRF5 signaling. supernatants were harvested after 5–6 h of infection. Macrophages, small interfering RNA, and Q-PCR Materials and Methods Macrophages were derived from monocytes by differentiation in RPMI Materials 1640 with 30% pooled HS for 5–6 d. Medium was replaced with RPMI 1640 containing 10% serum for infection, stimulation, or siRNA treatment. Downloaded from Concentrations of IFN-b were determined with the VeriKine-HS human A pool of four individual ON-TARGETplus siRNAs (Dharmacon) was IFN-b serum ELISA kit (PBL Assay Science, Piscataway, NJ) typically transfected using siLentFect (Bio-Rad), yielding a final concentration of with no dilution or 1:2 dilutions of the culture supernatants. BioPlex assays 5 nM siRNA. The transfection was repeated after 3 d, and the silenced were from Bio-Rad and were analyzed as per the manufacturer’s instruc- MDMs were infected with live S. aureus for 3 h. RNA was isolated with an tions, with dilutions of the supernatants ranging from none to 200, depending RNeasy kit including DNAse treatment (Qiagen), cDNA was transcribed on the cytokine to be examined. Escherichia coli bioparticles were of the with a Maxima cDNA synthesis kit (Thermo Fisher Scientific), and relative rough K12 strain (Invitrogen). The following TLR ligands were from

quantiﬁcation by Q-PCR was done with StepOnePlus using TaqMan probes http://www.jimmunol.org/ InvivoGen: LPS from the E. coli K12 strain, FSL-1, Pam3Cys, R837, CL75, (Life Technologies) and Perfecta qPCR FastMix from Quanta. Probes used poly(I:C), and polyuridylic acid (pU). Lipofectamine 2000 (L2K) was from were: IFN-b, Hs01077958_s1; TNF, Hs00174128_m1; TLR7, Hs00152971_m1; Invitrogen, and poly-L-arginine (pL-Arg) was from Sigma-Aldrich. RNAse TLR8, Hs00607866_mH; IRF5, Hs00158114_m1; stimulator of IFN genes A was from Qiagen. The IKKb inhibitor BI605906 was provided by Prof. Sir (STING), Hs00736958_m1; and TBP, Hs00427620_m1. TBP served as en- Philip Cohen (University of Dundee, Dundee, U.K.), and IKKII–VIII and the dogenous control, and relative expression in monocytes was calculated as TAK1 inhibitor 5Z-7-oxozeaenol was from Calbiochem/Merck Millipore fold induction by stimulation or infection. For siRNA-treated macrophages, (Darmstadt, Germany). The TAK1 inhibitor NG-25 was from MedChem the level of gene expression by infected cells were normalized against Express (Monmouth Junction, NJ). noninfected cells treated with each of the respective siRNAs to correct for Bacteria and bacterial lysates potential background differences.

S. aureus 113 wild-type (wt) strain, its isogenic 113 Dlgt mutant, and the Whole blood model and flow cytometry analyses by guest on September 24, 2021 pRBlgt-reconstituted 113 Dlgt strain were provided by Prof. Friedrich The whole blood experiments were performed basically as described (23). € € Goëtz (University of Tubingen, Tubingen, Germany). The Newman and Venous blood was drawn into polypropylene tubes with lepirudin (Reflu- Cowan strains were provided by Prof. Timothy Foster (Trinity College, dan) anticoagulant (50 mg/ml final concentration). The blood was quickly Dublin, Ireland), and the Wood-46 strain was from the American Type transferred to 1.8-ml round-bottom cryotubes (Nunc) containing HK Culture Collection (10832). The bacteria were grown on tryptic soy agar S. aureus and FSL-1 and then incubated on a tube roller at 37˚C. Samples for plates that were supplemented with 10 mg/ml erythromycin or kanamycin cytokine analyses were centrifuged for collection of plasma, and samples for the Dlgt mutant or the pRBlgt reconstituted strains, respectively. For for flow cytometry analyses were fixed immediately with 0.5% parafor- preparation of bacteria, colonies were picked and grown in 5 ml tryptic soy maldehyde (PFA) for 5 min. For analysis of CD11b expression, blood cells broth during vigorously shaking at 37˚C overnight (12–18 h) for use in stimulated with HK bacteria and/or FLS-1 for 15 min were stained with infection experiments. To prepare heat-killed (HK) bacteria, a preculture anti-CD11b PE and anti-CD14 FITC (BD Biosciences) and EasyLyse was diluted 1:100 to 1:200 in 50–100 ml tryptic soy broth in 500–1000 ml erythrocyte-lysing reagent (Dako). Flow cytometry was performed on culture flasks and grown in a shaking incubator to the exponential phase a BD LSR II using side scatter and CD14 to gate for neutrophils and (∼4 h), stationary phase (∼12 h), or decline phase (20–24 h), as appro- monocytes, and CD11b expression was determined by the median fluo- priate. For heat killing of the bacteria, bacteria were spun down, resus- rescence intensity (PE) of the total cell distribution of monocytes and pended in PBS, incubated at 80˚C for 30 min, and finally washed once with granulocytes. Phagocytosis of Alexa Fluor 488–labeled HK S. aureus was PBS. For quantification of bacteria by OD measurements, a standard curve analyzed with the Phagotest kit (BD Biosystems) according to the manu- was generated with serial dilutions of HK bacteria that had been quantified facturer’s instructions. In brief, full blood was incubated with bacteria with € by manual counting in a Burker chamber. Fluorescent labeling of the HK or without FSL-1 at 37˚C for 30 min, and control tube with S. aureus was bacteria was done using Alexa Fluor 488–succinimidyl ester (Invitrogen). kept on ice. Phagocytosis was stopped by placing tubes on ice and im- This reagent was dissolved in DMSO and immediately added to a solution mediately adding ice-cold quenching solution. This was followed by 3 10 m of 2 10 S. aureus/ml in NaHCO3 (167 M, pH 8.3), yielding a final washing with cold washing buffer, lysis of erythrocytes, and fixation of concentration of 1 mg/ml dye. Incubation with agitation was done for 1 h leukocytes with lysis solution, and the DNA stain was finally added to at room temperature, and the labeled bacteria were washed twice with exclude artifacts of aggregated bacteria and cells. Monocytes and gran- 500 ml PBS and counted. Preparation of crude bacterial lysate was done by ulocytes were discriminated by forward scatter/side scatter, and a phago- a previous described protocol (4) with some modifications. Glass particles cytic index was calculated as: mean fluorescence intensity (Alexa Fluor (0.1 mm, Sigma-Aldrich) were preheated at 200˚C for 4 h to eliminate 488) of cells containing bacteria 3 fraction of cells containing bacteria. potential TLR ligand contaminants. The particles were added to 2 3 1010 S. aureus 113 Dlgt (HK, exponential growth phase) in ice-cold PBS and Western blot run in four cycles on a Precellys 24 bead-beater (Bertin Technologies, m Montigny-le-Bretonneux, France) with chilling on ice between each cycle. Cells were adhered in six-well plates, treated and lysed in 150 l lysis The glass particles and intact bacteria were spun down and the crude lysate buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 137 mM NaCl, 1% supernatants were added to new tubes for storage at 280˚C. Triton X-100, 1 mM sodium deoxycholate, 10% glycerol, 1 mM Na3VO4, 50 mM NaF, and Complete protease inhibitor [Roche]). PAGE was per- Blood, monocytes, and stimulation/infection formed with the NuPAGE system (Life Technologies) per the manufacturer’s recommendations. Immunoblotting was performed with the iBlot Fresh blood, serum, and buffy coats were acquired from healthy volunteers system (Life Technologies) per the manufacturer’s recommendations. Af- under informed written consent approved by the Regional Committee for ter blotting, nitrocellulose membranes were briefly rinsed with dH20and The Journal of Immunology 3 blocked with 5% BSA dissolved in TBST for 1 h. All incubations with ficient in mature lipoprotein production and fails to activate TLR2, primary Abs were done at 4˚C overnight, and all incubations with sec- resulting in impaired immune activation by entire bacteria (4, 24). ondary HRP-linked Abs (Dako, nos. P0399 and P0447) were done at room To examine the total immunostimulatory capacity of the different temperature for 1 h. Blots were developed with SuperSignal West Femto substrate (Pierce) and imaged with a Li-Cor Odyssey Fc system. Abs used strains, we included both the bacteria and the supernatants from for Western blots in this study were: p38 (Cell Signaling Technology, no. the bacterial cultures, and the plasmid-reconstituted 113 Dlgt 9212), phospho-p38 (Cell Signaling Technology, no. 9211), p44/42 (ERK; strain (pRBlgt) served as a genotype/phenotype control (Fig. 1A). Cell Signaling Technology, no. 4695), phospho-p44/42 (ERK, Cell Sig- Unexpectedly, the Dlgt strain induced higher levels of IFN-b than naling Technology, no. 4370), JNK (Cell Signaling Technology, no. 9252), phospho-JNK (Cell Signaling Technology, no. 4668), TANK-binding ki- did the wt and pRBlgt strains, suggesting that S. aureus lipo- nase 1 (TBK1; Cell Signaling Technology, no. 3504), phospho-TBK1 (Cell proteins inhibit IFN-b induction by the bacteria. To examine the Signaling Technology, no. 5483), phospho-IKKa/b (Cell Signaling Tech- possible contribution of factors in the bacterial culture supernatant nology, no. 2697), TAB1 (Cell Signaling Technology, no. 3226), phospho- for this phenomenon, the wt and Dlgt supernatants were swapped TAB1 (Millipore, no. 06-1334), phospho-p105 (Cell Signaling Technology, b a (Fig. 1B). This resulted in increased IFN- induction by the wt no. 4806), IkB (Cell Signaling Technology, no. 4812), GAPDH (Abcam, b no. ab8245), TLR8 (Cell Signaling Technology, no. 11886), and IRF5 strain and concomitant increased IFN- induction by the mutant (Cell Signaling Technology, no. 13496). strain, thus reaching intermediate IFN-b levels compared with the condition in Fig. 1A. This indicates that the S. aureus 113 wt Immunofluorescence and Scan^R analyses strain inhibits IFN-b induction by lipoproteins that are released Immunofluorescence labeling was done as described (9) with minor into the bacterial culture media during growth, as well as by lipo- changes. PBMCs were seeded in 96-well glass bottom plates (no. P96- proteins in the bacterial cell wall. Release of lipoproteins by the 1.5H-N, In Vitro Scientific, Sunnyvale, CA) that were precoated with HS for 1–2 h. Nonadherent cells were removed by washing with 33 HBSS. 113 wt strain during growth has been demonstrated previously (4), Downloaded from For intracellular staining the cells were fixed with ice-cold 2% PFA in PBS and with a TLR2–HEK293–NF-kB reporter assay we identified for 15 min on ice and washed three times with room-tempered PBS. TLR2 ligands in the culture supernatant of the 113 wt strain, but Permeabilization was done with PEM buffer (100 mM K-Pipes [pH 6.8], not the Dlgt strain (not shown). To examine whether TLR2 ligands 5 mM EGTA, 2 mM MgCl2, 0.05% saponin) for 10 min, quenched of free of S. aureus antagonize the induction of IFN-b during infection, aldehyde groups in 50 mM NH4Cl in PBS with 0.05% saponin (PBS-S) for 5 min, and blocked with 20% HS in PBS-S for 20 min. After a single wash we used a TLR2-blocking Ab (Fig. 1C). In the presence of bac- with 1% HS in PBS-S, the cells were incubated with primary Ab (2 mg/ml) terial culture media, the TLR2-blocing Ab increased the produc- http://www.jimmunol.org/ in with 1% HS in PBS-S overnight at 4˚C. Cells were washed twice with tion of IFN-b by monocytes upon infection of all the S. aureus room-tempered PBS-S and once with 1% HS in PBS-S, and incubated with strains examined, except for the 113 Dlgt strain. In the absence of highly cross-adsorbed Alexa Fluor 488– or 647–labeled secondary Abs (Invitrogen) at 2 mg/ml for 30 min. Subsequently, the cells were washed bacterial culture media, TLR2 blocking significantly increased the with 33 PBS-S, postfixed with 4% PFA at room temperature, and washed IFN-b production by the pathogenic S. aureus isolates Newman once with PBS-S. Nuclei were stained with Hoechst 3342 (200 ng/ml) in and Cowan, with a similar tendency for the 113 wt and Wood46 PBS-S. The following Abs were used (typically 2–10 mg/ml or 1:100- to strains. Collectively, this suggests that both lipoproteins released 1:200-fold dilution): anti-human TLR8 (Novus Biologicals, no. NBP1- into the bacterial culture media and in the bacterial cell wall can 77203), rabbit IgG control (Novus Biologicals, no. NB810-56910), anti- b human IRF5 mAb (Abcam, no. 10T1), anti-human IRF5 mAb (Abcam, no. antagonize the IFN- production from monocytes via activation of ab124792), anti-human IRF5 (Santa Cruz Biotechnology, no. D10), anti- TLR2. We further examined the role of TLR2 in the induction of by guest on September 24, 2021 human IRF5 (Sigma-Aldrich, no. HPA046700), anti-human IRF3 XP mAb other cytokines using multiplex ELISA. Secretion of IL-6, IL-8, (Cell Signaling Technology, no. 11904), anti-human IRF3 mAb (D83B9 IL-1a, and IL-12-p40 by monocytes in response to S. aureus in- mAb) (Cell Signaling Technology, no. 4302), anti-human IRF3 (Santa Cruz Biotechnology, FL425, no. sc9082), anti-human p65 XP mAb fection was partially lipoprotein-dependent, whereas secretion IL- (Cell Signaling Technology, no. 8242), anti-human p65 A (Santa Cruz 1b and IL-18 was independent of lipoproteins (Supplemental Fig. Biotechnology, no. sc-109), anti-human IRF1 XP mAb (Cell Signaling 1). Lipoproteins suppressed IL-12p70 secretion to a similar degree Technology, no. 8478), anti-human IRF7 (Cell Signaling Technology, no. as IFN-b, suggesting that these two cytokines share a regulatory 4920; EPR4718, Abcam, no. ab109255; H-246, Santa Cruz Biotechnology, mechanism (Supplemental Fig. 1). no. sc-9083), anti–phospho-IRF7 (Cell Signaling Technology, no. 5184), anti-human IRF8 (Santa Cruz Biotechnology, C-19, no. sc-6058; Sigma- TLR2 ligands antagonize the induction of IFN-b by S. aureus Aldrich, no. HPA00253; Cell Signaling Technology, no. 5628), normal goat IgG (Santa Cruz Biotechnology), and rabbit IgG XP control mAb in human blood monocytes and whole blood (Cell Signaling Technology). Automated imaging was done with the 3 To simplify our model system, we examined whether the defined Scan^R system (Olympus) using a 20 objective, up to 1 s exposure time, b and ∼100 frames were captured for each well (∼5,000–15,000 cells) synthetic TLR2/6 ligand FSL-1 alone would antagonize IFN- performed in duplicates or triplicates. Automated image analysis was done induction by HK S. aureus Dlgt, E. coli, and E. coli LPS (Fig. 2A). with the Scan^R software v1.3.0.3. Confocal images were captured with TLR2 costimulation with FSL-1 suppressed IFN-b induction by a Zeiss LSM 510 META scanning unit, and a 1.4 numerical aperture 363 HK S. aureus, but not HK E. coli and E. coli LPS. This confirmed objective. that TLR2 activation blocks S. aureus–induced IFN-b production, Statistical analyses whereas it does not affect TLR4–TRIF signaling. S. aureus lipo- peptides can be both diacylated and triacylated (25), and synthetic Statistical analyses were done on data merged from independent experi- b ments, as indicated in the figures or the figure legends. For analysis, the data triacylated TLR1/2 ligand Pam3Cys also inhibited the IFN- in- were log2 transformed to generate Gaussian distributions, and analyses duction by HK S. aureus in monocytes (not shown). were performed with GraphPad Prism v5.03. Figures without statistics We further examined the impact of the timing of TLR2 ligand show a single representative experiment out of at least three independent administration relative to S. aureus and found that maximum in- experiments. hibition of IFN-b induction was achieved when FSL-1 was added before or at the same time as the bacteria, although the inhibitory Results effect was gradually reduced when FSL-1 was added 15, 30, and b S. aureus inhibits the induction of IFN- in human blood 60 min later (Fig. 2B). Thus, the TLR2 inhibitory effect is limited monocytes via TLR2 activation to an early time frame of S. aureus sensing by monocytes. To clarify the role of TLR2 ligands in S. aureus–induced IFN-b To examine whether the inhibitory effect of TLR2 also is im- production, we infected monocytes with live S. aureus 113wt portant in the more complex physiological environment of blood strain and its isogenic mutant 113 Dlgt. The mutant strain is de- monocytes, we employed a lepirudin anticoagulated human whole 4 S. AUREUS ACTIVATES A TLR8–TAK1–IKKb–IRF5 AXIS IN MONOCYTES

FIGURE 1. S. aureus–infected blood monocytes produce IFN-b, which can be antagonized by secreted lipoproteins via TLR2. Monocytes were infected with S. aureus 113 wt strain, its isogenic lgt mutant (Dlgt), or the plasmid- reconstituted Dlgt strain (pRBlgt). Extracellular bacteria were killed with gentamicin after 1 h Downloaded from and cell culture supernatant was sampled 5–6 h later. (A) Dose-dependent induction of IFN-b with S. aureus including the bacterial culture supernatant (S). (B) Effect of swapping supernatants of the Dlgt and wt bacterial cultures on the induction of IFN-b. The IFN-b levels from http://www.jimmunol.org/ triplicates were determined by ELISA (mean 6 SD). (C) The effect of the TLR2-blocking mAb (T2.5, 5 mg/ml) on the IFN-b production induced by infection with different S. aureus strains (1 3 108/ml) with or without inclusion of the bacterial culture supernatants (mean + SEM, n = 4). Differences were tested by two- way repeated-measurement ANOVA with a Bonferroni posttest. *p , 0.05, **p , 0.01,

***p , 0.001. by guest on September 24, 2021

blood model (ex vivo) that enables crosstalk between all blood cells S. aureus RNA is an endosomal PRR ligand and most of the plasma cascades, including a C system that is Because bacterial nucleic acids are implicated in type I IFN in- functionally active under physiological conditions (23). TLR2 duction in various model systems, we examined the importance of b costimulation fully suppressed S. aureus–induced IFN- produc- RNA in crude lysate of the HK S. aureus Dlgt strain. Bacteria were tion in whole blood (Fig. 2C), conﬁrming the validity of our mechanically disrupted, treated with RNAse A, which cleaves PBMC-based model. Cytokine induction by HK S. aureus is to ssRNA, and the lysate was mixed with pL-Arg for delivery of a large extent dependent on phagocytosis and bacterial degrada- nucleic acid to the monocyte endosomal compartment (28) tion (20, 26). The TLR2 effect could thus possibly be explained by (Fig. 3). RNAse treatment eliminated the induction of IFN-b,IL- inhibition of phagocytosis. However, we found that TLR2 co- 12p40, and IL-12p70, and it strongly reduced the release of IL- stimulation did not inhibit phagocytosis, but instead signiﬁcantly 1a, IL-1b, and IL-18. In contrast, the levels of IL-6 and IL-8 increased the uptake of HK S. aureus by monocytes in whole were not affected by RNAse A (Fig. 3). This demonstrates that blood (Fig. 2E). The mechanism for the enhanced phagocytosis is in lysate of HK S. aureus deprived of TLR2 ligands, ssRNA was likely TLR2-mediated activation of the CR3 (CD11b/CD18), as a dominant PAMP for the induction of IFN-b, IL-1, IL-12, and S. aureus phagocytosis in the whole blood model is strongly C- IL-18. Still, other PAMPs distinct from RNA and lipoproteins dependent (27) and the CD11b level on both monocytes and were apparently dominating for IL-6 and IL-8 induction by these granulocytes increased strongly upon TLR2 stimulation (Fig. 2F). crude lysates. The Journal of Immunology 5

FIGURE 2. Synthetic TLR2 ligand inhibits S. aureus–induced production of IFN-b in human monocytes and whole blood, but enhances phagocytosis. Monocytes were costimulated with TLR2 ligand (FSL-1) and HK bacteria or LPS for 6 h. (A) Effect of FSL-1 (1, 10, and 100 ng/ml) cotreatment on IFN-b induction with HK E. coli (K12 strain, 1 3 107/ml), E. coli LPS (K12, 100 ng/ml), and HK S. aureus Dlgt (1 3 108/ml). (B) Effect of timing of administration of FSL-1 (10 ng/ml) relative to HK 8 S. aureus Dlgt (1 3 10 /ml). The IFN-b levels Downloaded from from triplicates were determined by ELISA (mean + SD). (C) FSL-1 inhibits induction of IFN-b by HK S. aureus Dlgt (1 3 109/ml) in a human whole blood model (3 h, mean + SEM, n = 3). The effect of FSL-1 costimulation was tested by one-way repeated-measurement

ANOVA with a Dunnett multiple comparison http://www.jimmunol.org/ test. (D) Effect of FSL-1 costimulation on phagocytosis of HK S. aureus in human whole blood (30 min, mean + SEM, n = 3). The effect of FSL-1 costimulation was tested with two-way paired t test. (E)EffectofS. aureus, FSL-1, and costimulation on the CD11b/CD18 surface level on phagocytes in human whole blood (30 min, mean + SEM, n = 3). The effect of FSL-1 costimulation was tested by one-way repeated- by guest on September 24, 2021 measurement ANOVA with a Bonferroni multiple comparison test. *p , 0.05, **p , 0.01, ***p , 0.001.

TLR2 inhibits IFN-b and IL-12 induction by both S. aureus and induced by S. aureus and pU in endosomes and was also inhibited TLR8 ligands by TLR2 activation (Fig. 4B). GU- and U-rich ssRNA stimulate To clarify the mechanism of how S. aureus RNA induces IFN-b murine TLR7 and human TLR8 (30), and the induction of IL- in monocytes, we compared the response by HK S. aureus Dlgt 12p70 is a well-described characteristic of human TLR8 (28, b with the synthetic TLR8 ligand pU and the dsRNA TLR3 ligand 31). Thus, the regulation of IFN- and IL-12p70 production by poly(I:C). The RNA was delivered to the endosomal or the cyto- monocytes is similar for S. aureus and a defined TLR8 ligand, solic compartment of monocytes by complexation with pL-Arg or including its inhibition by TLR2 activation. Additional correlative transfection with L2K, respectively, as previously shown (28). evidence for the involvement of TLR8 in the sensing of S. aureus Stimulation with S. aureus, pU/pL-Arg, and poly(I:C)/L2K in- was found using the TLR8-specific imidazoquinoline agonist duced IFN-b production, whereas stimulation with poly(I:C)/pL- CL75, as CL75-mediated IFN-b induction was antagonized by Arg and pU/L2K did not (Fig. 4A). This implies that pU and poly TLR2 costimulation (Fig. 4C). In contrast, IFN-b induction by the (I:C) induce IFN-b from the endosomal and the cytosolic com- TLR7 specific ligand R837 was not antagonized by TLR2 co- partments, respectively, and is consistent with high TLR8 and stimulation (Fig. 4D), arguing against a role of TLR7 in the IFN-b low TLR3 levels in monocytes (29). Costimulation with FSL-1 induction by S. aureus. TLR8 immunofluorescence indicated that strongly suppressed IFN-b induction by HK S. aureus and pU, TLR8 was recruited to S. aureus phagosomes (Fig. 4E). Quanti- but not poly(I:C). TLR2 activation thus interferes with TLR8 fication using a high-content screening system suggested that ∼9% signaling, but not cytosolic dsRNA sensing or TLR4–TRIF sig- of the S. aureus phagosomes stained positive for TLR8 1 h after naling (Fig. 2A). Moreover, induction of IL-12p70 was solely exposure to bacteria (Fig. 4F). 6 S. AUREUS ACTIVATES A TLR8–TAK1–IKKb–IRF5 AXIS IN MONOCYTES

silencing, but it was not as strongly affected and reached sta- tistically signiﬁcance only for the TLR7 plus TLR8 combined knockdown. Collectively, this indicates an essential role of a TLR8–IRF5 pathway in the induction of IFN-b by S. aureus, which also may contribute to S. aureus–induced TNF production by monocytes.

S. aureus and TLR8 ligands induce IRF5 nuclear accumulation, which is antagonized by TLR2 activation To further examine IRF5 activation, we established a quantitative immunoﬂuorescence method of transcription factor nuclear accumulation using high-content screening (Scan^R) (Supplemental Fig. 3). Monocytes were stimulated with HK S. aureus and TLR8 ligands, and nuclear accumulation of total p65 (NF-kB/RelA), IRF3 (Supplemental Fig. 3), and IRF5 (Fig. 6) was examined. The level of nuclear IRF5 was low in resting cells (Fig. 6A) and was strongly increased following pU/pL-Arg stimulation (Fig. 6B) and phagocytosis of HK S. aureus Dlgt (Fig.6D).FSL-1

costimulation clearly reduced the nuclear accumulation of IRF5 Downloaded from induced by both stimuli (Fig. 6C, 6E), which thus correlates with suppressed IFN-b and IL-12 induction. Moreover, when S. aureus was heat inactivated during the stationary growth phase, the bacteria were markedly less potent as IFN-b inducers (not shown). The stationary phase S. aureus did not induce IRF5

nuclear accumulation (Fig. 5F), thus again demonstrating a cor- http://www.jimmunol.org/ relation of S. aureus–induced IFN-b production and IRF5 nuclear accumulation in monocytes. Around 25% of the monocytes that had phagocytosed HK S. aureus Dlgt stained positive for nuclear IRF5 (Fig. 5G). The frequency was strongly reduced by costimulation with FSL-1, as well as when HK S. aureus from the stationary growth phase was used. Moreover, cells that did not phagocytosed bacteria, that is, “S. aureus bystanders,” did FIGURE 3. S. aureus RNA induces IFN-b, IL-12, IL-1, and IL-18, but is not have increased IRF5 nuclear accumulation. Thus, IRF5 nu- redundant for induction of IL-6 and IL-8 in monocytes. Homogenate of clear accumulation was dependent on S. aureus phagocytosis and by guest on September 24, 2021 HK S. aureus Dlgt was made by mechanical disruption using glass par- not a result of paracrine signals. Correlation of IRF5 transloca- ticles and a bead-beater. The lysate was subsequently treated with or tion and phagocytosis was also seen in a whole-well overview without RNAse A for 1 h at 37˚C. The crude lysate was delivered to the with S. aureus phagocytosis and IRF5 nuclear staining being monocytes as a complex with cationic protein (pL-Arg), and supernatants strongest around the well center (not shown). pU/pL-Arg stim- after 6 h of treatment were assayed for cytokines using ELISA (IFN-b) and ulation activated IRF5 nuclear accumulation to a similar degree BioPlex (mean + SD of triplicates). as S. aureus uptake, and was also suppressed by FSL-1 costimulation, whereas FSL-1 or LPS alone did not activate IRF5 (Fig. b S. aureus induces IFN- via TLR8 and IRF5 5H). In contrast, nuclear accumulation of p65 was seen with The correlative data suggested that TLR8 was responsible for ligands for all three TLRs examined (Fig. 5I). Only LPS acti- S. aureus–induced IFN-b production. Western blot analysis demon- vated IRF3 nuclear translocation (Fig. 5J), which is consistent strated that although S. aureus and TLR2 ligands activated TBK1 with the IRF3 phosphorylation pattern (not shown). IRF1 was phosphorylation, IRF3 was not activated (not shown). Further- constitutively localized to the nuclei in monocytes, whereas IRF7 more, preliminary data suggested a possible involvement of IRF5. and IRF8 Abs gave no specific staining (not shown). We con- To clarify the mechanism, we performed gene silencing of MDMs clude that S. aureus induces IRF5 nuclear accumulation in by transient transfection with siRNA targeting TLR7, TLR8, monocytes as a consequence of TLR8 activation and is blocked IRF5, and STING. All targets were efficiently and specifically by TLR2 signaling. silenced on the mRNA level after sequential transfections (Supplemental Fig. 2A). Clear knockdown of TLR8 and IRF5 was TLR8-induced nuclear accumulation of IRF5 is dependent on b also seen at the protein level (Supplemental Fig. 2B). However, TAK1 and IKK , whereas nuclear accumulation of p65 is two TLR8 bands of ∼100 kDa were not completely eliminated TAK1 independent even several days after mRNA knockdown. These bands probably We further examined the requirement of central signaling com- correspond to N-terminal fragments of TLR8 after cleavage in ponents in the MyD88 pathway for TLR8-mediated IRF5 and p65 endosomes and can be a functional PRR as recently shown (32). activation and TLR2-induced p65 activation. We quantified IRF5/ MDMs showed similar responses as monocytes, as costimulation p65 nuclear staining by two-color immunofluorescence (Fig. 7A– with synthetic TLR2 ligand or S. aureus lipoproteins strongly C). We then used well-characterized chemical inhibitors of central antagonized IFN-b induction by S. aureus and synthetic TLR8 signaling kinases to block monocyte signaling. To minimize ligand (not shown). Infection of silenced MDMs with live S. au- possible problems of toxicity and secondary effects of the inhib- reus Dlgt demonstrated that induction of IFN-b was dependent on itors, we chose CL75 as TLR8 ligand, as CL75 elicits more rapid TLR8 and IRF5, but not on TLR7 or STING (Fig. 5). Induction of cytokine induction than do pU/pL-Arg and S. aureus and thus TNF followed a similar trend as IFN-b upon TLR8 and IRF5 limits the required incubation time with the inhibitors. Two The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. S. aureus– and TLR8 ligand–induced IFN-b and IL-12 are antagonized by TLR2 activation. Monocytes were stimulated with HK S. aureus Dlgt (108/ml), ssRNA (pU, 0.75ug/ml), and dsRNA (poly(I:C), 0.75 mg/ml) with or without FSL-1 costimulation (10 ng/ml). The synthetic RNA was delivered as a complex with pL-Arg or cationic lipid (L2K) to examine the stimulatory effect of RNA in the endosomal (pL-Arg) versus the cytosolic (L2K) compartment. After 6 h the supernatants were sampled and assayed for (A) IFN-b and (B) IL-12p70 (mean + SD of triplicates). (C and D) Monocytes were stimulated for with TLR8-specific ligand (CL75) or TLR7-specific ligand (R837) with or without FSL-1 coadministration (100 ng/ml) for 2 h (mean 6 SEM, n = 3). The differences were tested by two-way repeated-measurement ANOVAwith a Bonferroni posttest. ***p , 0.001. (E and F) Monocytes were treated for 1 h with Alexa Fluor 488–labeled HK S. aureus Δlgt (1 3 108/ml) and then fixed and stained with (D) anti-TLR8 and (E) control Abs. (F) Quantification of the percentage of TLR8+ S. aureus phagosomes using high-content screening analysis (Scan^R) (mean + SD of triplicates). Nuclei are stained with Hoechst (blue). Scale bar, 10 mm. pI:C, poly(I:C). structurally nonrelated inhibitors of TAK1 (5Z-7-oxozeaenol and pendent on both TAK1 and IKKb (Fig. 7D). In contrast, p65 nu- NG-25) and IKKb (IKKII-VIII and BI605906) were given as a 30- clear accumulation following TLR8 and TLR2 stimulation was only min pretreatment, and their effects on TLR8- and TLR2-mediated dependent on IKKb and not on TAK1 (Fig. 7E, 7F). nuclear accumulation of IRF5 and/or p65 were examined Western blot analysis of MAPKs and other signaling inter- (Fig. 7D–F). TLR8-induced IRF5 nuclear accumulation was de- mediates was performed to verify the specificity of the inhibitors and 8 S. AUREUS ACTIVATES A TLR8–TAK1–IKKb–IRF5 AXIS IN MONOCYTES Downloaded from

FIGURE 5. S. aureus induces IFN-b production via TLR8 and IRF5 in MDMs. MDMs were treated with siRNA as indicated. Infection with live Δ 3 7 S. aureus lgt (3 10 /ml) including bacterial culture medium was done http://www.jimmunol.org/ for 3 h and the fold induction of IFN-b and TNF expression by infection was determined with reverse transcription–Q-PCR (mean + SEM, n = 4). The effect of silencing was tested by one-way repeated-measurement ANOVA with a Dunnett multiple comparison test compared with control siRNA. *p , 0.05, **p , 0.01, ***p , 0.001. to dissect further details of TLR2 and TLR8 signaling (Supplemental Fig. 4). Both TAK1 inhibitors effectively blocked TLR2- and TLR8-induced JNK and p38 phosphorylation, in contrast to the by guest on September 24, 2021 IKKb inhibitors. IKKb inhibitors still blocked the phosphorylation of p105 and ERK1/2. This is in agreement with the ca- nonical model of MyD88 signaling where JNK and p38 are downstream of TAK1, whereas activation of p105 and ERK1/2 arecontrolledbyIKKb (33). The degradation of IkBa was not FIGURE 6. S. aureus and pU/pL-Arg complexes induce nuclear accu- blocked by any of these inhibitors (Supplemental Fig. 4), and mulation of IRF5, but not IRF3, in monocytes. (A–F) Monocytes were IKKa can probably phosphorylate IkBa, leading to its degra- treated for 2–4 h with pU/pL-Arg (7.5 mg/ml) and Alexa Fluor 488–labeled Δ 3 8 dation once IKKb is lost (34). Failure of TAK1 inhibitors to HK S. aureus lgt (1 10 /ml) from exponential and stationary growth rescue IkBa from degradation fits with the TAK1-independent phases, respectively, and with or without FSL-1 (10 ng/ml) costimulation. Subsequently, the cells were fixed and immunostained for IRF5, and auto- nuclear accumulation of p65 (Fig. 7E, 7F), whereas p65 activa- mated imaging and analyses of nuclear IRF5 accumulation were done with b tion may require phosphorylation by IKK in addition to deg- Scan^R. (A) IRF5 staining in monocyte without stimulation, (B) with pU/pL- radation of IkBa (33). The TAK1 inhibitor 5Z-7-oxozeaenol Arg, (C) with pU/pL-Arg plus FSL-1, (D) with HK S. aureus from expo- efficiently antagonized IKKa/b phosphorylation and IKKb ac- nential growth phase, (E)withHKS. aureus plus FSL-1, and (F)withHK tivity (Supplemental Fig. 4B), whereas NG25 was less efficient S. aureus from stationary growth phase. Scale bar, 20 mm. (G and H)The (Supplemental Fig. 4A). frequencies of cells showing positive staining for IRF5 were determined We further used gene silencing to examine the role of TLR7, from ∼5,000–15,000 cells (100 frames/well). (G) Frequencies of IRF5+ TLR8, IRF5, IKKb, STING, and TBK1 for IRF5 nuclear ac- nuclei among monocytes having phagocytosed HK S. aureus harvested cumulation in MDMs upon infection with S. aureus Dlgt during the exponential growth phase; the effect of FSL-1 costimulation; monocytes having phagocytosed HK S. aureus harvested during the sta- (Fig. 7G). Silencing shows that also in MDMs, S. aureus activates b tionary growth phase; monocytes in wells with HK S. aureus from the ex- IRF5 via TLR8 and IKK , confirming the specificity of IRF5 ponential growth phase, but which have not taken up bacteria (S. aureus staining and the inhibitor data, whereas TLR7, TBK1, STING, and bystanders). (H–J) Monocytes were stimulated with pU/pL-Arg with or with- p65 silencing did not influence IRF5 activation. In contrast, p65 out FSL-1 for 4 h, or with FSL-1 (10 ng/ml) and E. coli K12 LPS (100 ng/ml) nuclear accumulation was reduced solely with siRNA for p65, alone for 1 h. The cells were fixed and stained with Abs for (H) total IRF5, verifying the specificity of the nuclear translocation assay also for (I) total p65 (NF-kB subunit RelA), and (J) IRF3, and the frequencies of this factor (Fig. 7H). positively stained nuclei were determined (mean + SD of duplicates and We conclude that both primary monocytes and MDMs sense triplicates). Nuclei are stained with Hoechst (blue). SA, S. aureus. S. aureus via TLR8, and that TLR8 signaling includes a novel TAK1–IKKb–IRF5 pathway that is required for IFN-b induction for TLR8 and TLR2 signaling in monocytes that includes two and that is blocked by TLR2 signaling. We thus propose a model distinct pathways (Fig. 8). The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 7. TLR8-induced IRF5 nuclear accumulation is dependent on TAK1 and IKKb, whereas TLR8- and TLR2-induced p65 nuclear accumulation is IKKb-dependent but TAK1-independent. Monocytes were stimulated with CL75 (1 mg/ml) and FSL-1 (10 ng/ml) for 1 h and then fixed and stained for IRF5 and p65 by immunofluorescence. Imaging and quantification of IRF5+ and p65+ nuclei were done with Scan^R. (A) IRF5 and p65 double-stained cells without stimulation. (B) IRF5 and p65 double-stained cells after CL75 stimuli. Scale bar, 30 mm. (C) Analysis of nuclear p65 and IRF5 staining intensity (mean fluorescence intensity [MFI]) after CL75 stimulation. (D–F) Effect of pharmacological inhibitors of TAK1 and IKKb on nuclear accumulation of IRF5 and p65 following CL75 and FSL-1 stimulation. (D)FractionofIRF5+ nuclei 1 h after CL75 stimulation (mean + SEM). (E) Fraction of p65+ nuclei 1 h after CL75 stimulation. (F) Fraction of p65+ nuclei 1 h after FSL-1 stimulation. The effect of inhibitors was tested by one-way ANOVAwith a Dunnett multiple comparison test against the “no inhibitor” condition. (G and H) Effects of gene silencing of various targets in MDMs on the induction of IRF5 and p65 nuclear translocation by S. aureus.LiveS. aureus Δlgt (3 3 107/ml) including bacterial culture medium was added to the macrophages for 3 h. The fraction of positive nuclei from independent experiments is shown (mean + SEM, n = 4). The effect of targeted siRNAs was tested by one-way repeated-measurement ANOVAwith a Dunnett multiple comparison test against control siRNA. Nuclei are stained with Hoechst (blue). *p , 0.05, **p , 0.01, ***p , 0.001. SA, S. aureus. 10 S. AUREUS ACTIVATES A TLR8–TAK1–IKKb–IRF5 AXIS IN MONOCYTES

cytes or TLR8-expressing HEK293 cells (not shown). Thus, although human TLR8 and murine TLR13 may seem functionally analogous, their ligand specificities and mechanisms of activation are different. This is also in agreement with the findings of Eigenbrod et al. (35). The role of IFN-b in S. aureus pathogenesis is controversial, and two recent studies found contradictory effects of IFN-b on the outcome of experimental murine infection (19, 20). In humans, S. aureus forms abscesses with a high local bacterial load that can leak into the surrounding tissue and the bloodstream. The maximum levels of IFN-b produced in response to HK S. aureus exceeded that of HK E. coli and LPS. A more potent induction of IFN-b by HK S. aureus from the exponential phase than the stationary phase may be related to the change in cell wall thickness (38), or it could reflect changes in the amount of stimulatory RNA. Only a fraction (e.g., 20–30%) of the monocyte population stained positive for IRF5 and IRF3 nuclear accumulation after TLR8 or TLR4 activation, respectively. This fits with a stochastic model of

IFN-b production in a cell population, which is explained by Downloaded from variations in limiting components at the cellular level (39, 40). TLR8 is also a potent inducer of IL-12 in human monocytes (28, 31), whereas IRF5 regulates macrophage polarization and drives FIGURE 8. Model of TLR2 and TLR8 activation by S. aureus and IL-12 and IL-23 production and Th1–Th17 activation (41). Th17 cross-regulation of the signaling pathways in human primary monocytes cells may also be important for protection from S. aureus infec- and MDMs. S. aureus lipoproteins activate TLR2, leading to CR3 (CD11b/ tions (42). We found that lipoproteins of S. aureus, either soluble http://www.jimmunol.org/ CD18) upregulation on the monocyte surface and enhanced phagocytosis. or in the bacterial cell wall, activated TLR2 and strongly sup- Degradation of S. aureus in the phagolysosome releases bacterial ssRNA, pressed TLR8-induced production of IFN-b and IL-12 by bacteria which activates TLR8 signaling. Both TLR2 and TLR8 signal via the being degraded in phagosomes. This may represent a control adaptor molecule MyD88 in TAK1-dependent and TAK1-independent mechanism to limit IFN-b and IL-12 production induced by ex- k pathways. The TAK1-independent pathway activates NF- B p65 nuclear tracellular bacteria. It is thus possible that dysregulation of TLR2/ translocation in an IKKb-dependent fashion and is important for proin- TLR8 signaling can lead to disease progression, and it could flammatory cytokine production. Both TLRs activate MAPKs by the TAK1-dependent pathway, but only TLR8 activates IRF5 nuclear trans- represent a new immune-evasion target for bacteria such as location via a mechanism involving TAK1 and IKKb. IRF5 nuclear ac- S. aureus. cumulation via TLR8 signaling is a specific requirement for IFN-b and Human blood monocytes also sense Borrelia burgdorferi RNA by guest on September 24, 2021 IL-12 induction by S. aureus, and it also contributes to TNF production, via TLR8, resulting in IFN-b production (43, 44). Although this whereas IL-1 and IL-18 are IRF5-independent. TLR2 activation inhibits finding is in agreement with our present study on S. aureus, the TLR8–IRF5 signaling, probably at the level of TAK1/IKKb or upstream. proposed signaling mechanisms are different, as they suggested IRF7 to be the central transcription factor involved. We show that TLR8-induced IFN-b production in monocytes and MDMs is Discussion dependent on IRF5. The activation of IRF5 by S. aureus and TLR8 In this study, we provide evidence for a novel role of TLR8 in ligands was rapid and did not occur in bystander cells not infected sensing of S. aureus by human primary phagocytes. The physio- by S. aureus, excluding the possibility that IRF5 is triggered via logical role of TLR8 is demonstrated by its contribution to the a secondary mediator. Mycobacterium tuberculosis induces IFN-b cytokine response induced by the whole bacteria during infection. in mouse macrophages through a nucleotide-binding oligomeri- This is consistent with the findings by Eigenbrod et al. (35) zation domain–containing protein 2 (NOD2)–receptor interacting showing a central role of TLR8 in recognition of live Strepto- protein 2 (RIP2)–TBK1–IRF5 pathway (45). Also, a NOD2–IRF5 coccus pyogenes in human MDMs. Although TLR2 and TLR8 mechanism of S. aureus–mediated IFN-b induction in mouse display considerable redundancy in signaling and both contributed BMDCs was recently reported (46), and IRF5 was activated by to S. aureus–induced production of proinflammatory cytokines TBK1/IKKi and RIP2 in overexpression studies (47, 48). How- such as IL-1a/b, IL-18, and TNF, we show a specific role of TLR8 ever, we found no induction of IFN-b in RNA-depleted S. aureus for the induction of IFN-b and IL-12 via IRF5 activation. TLR8 lysates, which most likely contain significant amounts of pepti- and IRF5 also contributed to TNF production. It is possible that doglycan and bacterial cell wall fragments. Moreover, silencing of IFNR signaling could have influenced the TNF response, but this TBK1 in MDMs did not affect IRF5 translocation in our studies. seems less likely given the short incubation time (3 h) used. Thus, neither NOD2 nor TBK1 seems to be involved in S. aureus– The function of human TLR8 as a sensor of bacterial RNA mediated IRF5 activation, arguing against a NOD2–RIP2–TBK1 appears similar to murine TLR13 (13, 14). However, whereas pathway for S. aureus–induced IFN-b in primary human phag- TLR13 detects a short sequence of bacterial 23S RNA with high ocytes. specificity (14, 36), TLR7 and TLR8 have weak sequence speci- In model systems, TLR7 and TLR8 can activate both IRF5 and ficity and generally detect U-rich RNA (30). Recently it was found IRF7, but not IRF3, and in THP-1 monocytic cells TLR7-induced that the natural TLR8 ligands are degradation products of U-rich IFN-a is IRF5-dependent (48). In human plasmacytoid dendritic RNA in the form of uridine and short U-containing oligomers that cells, IRF5 rather than IRF7 regulates TLR9-induced IFN-b work synergistically (37). In agreement with this model, the production together with NF-kB p50 (49). It thus appears that commercial TLR13 ligand Sa19, which has a single U residue and TLR7, TLR8, and TLR9 signaling can involve IRF5 in different is stabilized by thioester backbone, did not activate human mono- cell types. IFN-type I induction by TLR7 and TLR9 in the human The Journal of Immunology 11 plasmacytoid dendritic cell line Gen2.2 is dependent on TAK1 and 4. Stoll, H., J. Dengjel, C. Nerz, and F. Go¨tz. 2005. Staphylococcus aureus deficient b in lipidation of prelipoproteins is attenuated in growth and immune activation. IKK , but independent of IRF7 (34), which is similar to our Infect. Immun. 73: 2411–2423. findings of TLR8-induced IFN-b in primary phagocytes. An es- 5. von Bernuth, H., C. Picard, Z. Jin, R. Pankla, H. Xiao, C.-L. Ku, M. Chrabieh, sential function of IRF5 for IFN-b induction by TLR7 ligands in I. B. Mustapha, P. Ghandil, Y. Camcioglu, et al. 2008. Pyogenic bacterial infections in humans with MyD88 deficiency. Science 321: 691–696. Gen2.2 cells was recently demonstrated by Cohen and colleagues 6. von Bernuth, H., C. Picard, A. Puel, and J.-L. Casanova. 2012. Experimental and (50). They found that IKKb catalyzes IRF5 phosphorylation, natural infections in MyD88- and IRAK-4-deficient mice and humans. Eur. J. Immunol. 42: 3126–3135. leading to its dimerization and nuclear translocation and resulting € b 7. Decker, T., M. Muller, and S. Stockinger. 2005. The yin and yang of type I in induction of IFN- and IL-12, but not IL-6. Our finding on interferon activity in bacterial infection. Nat. Rev. Immunol. 5: 675–687. TLR8-induced signaling in human primary monocytes and MDMs 8. Kagan, J. C., T. Su, T. Horng, A. Chow, S. Akira, and R. Medzhitov. 2008. is similar to this newly described TLR7–TAK1–IKKb–IRF5–IFN-b TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon- b. Nat. Immunol. 9: 361–368. pathway in Gen2.2 cells. The function of IKKb as a kinase acti- 9. Husebye, H., M. H. Aune, J. Stenvik, E. Samstad, F. Skjeldal, O. Halaas, vating IRF5 was confirmed by another study (51). Our study using N. J. Nilsen, H. Stenmark, E. Latz, E. 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