And IRF7 Through a Mechanism Involving IRF1, IRF3, in Dendritic

MyD88 Drives the IFN-β Response to Lactobacillus acidophilus in Dendritic Cells through a Mechanism Involving IRF1, IRF3, and IRF7 This information is current as of September 26, 2021. Gudrun Weiss, Kristina Maaetoft-Udsen, Sebastian A. Stifter, Paul Hertzog, Stanislas Goriely, Allan R. Thomsen, Søren R. Paludan and Hanne Frøkiær J Immunol published online 15 August 2012 http://www.jimmunol.org/content/early/2012/08/15/jimmun Downloaded from ol.1103491

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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. Published August 15, 2012, doi:10.4049/jimmunol.1103491 The Journal of Immunology

MyD88 Drives the IFN-b Response to Lactobacillus acidophilus in Dendritic Cells through a Mechanism Involving IRF1, IRF3, and IRF7

Gudrun Weiss,*,† Kristina Maaetoft-Udsen,* Sebastian A. Stifter,† Paul Hertzog,† Stanislas Goriely,‡ Allan R. Thomsen,x Søren R. Paludan,{ and Hanne Frøkiær*

Type I IFNs are induced by pathogens to protect the host from infection and boost the immune response. We have recently demonstrated that this IFN response is not restricted to pathogens, as the Gram-positive bacterium Lactobacillus acidophilus, a natural inhabitant of the intestine, induces high levels of IFN-b in dendritic cells. In the current study, we investigate the intracellular pathways involved in IFN-b upon stimulation of dendritic cells with L. acidophilus and reveal that this IFN-b induction requires

phagosomal uptake and processing but bypasses the endosomal receptors TLR7 and TLR9. The IFN-b production is fully Downloaded from dependent on the TIR adapter molecule MyD88, partly dependent on IFN regulatory factor (IRF)1, but independent of the TIR domain-containing adapter inducing IFN-b MyD88 adapter-like, IRF and IRF7. However, our results suggest that IRF3 and IRF7 have complementary roles in IFN-b signaling. The IFN-b production is strongly impaired by inhibitors of spleen tyrosine kinase (Syk) and PI3K. Our results indicate that L. acidophilus induces IFN-b independently of the receptors typically used by bacteria, as it requires MyD88, Syk, and PI3K signaling and phagosomal processing to activate IRF1 and IRF3/IRF7 and thereby

the release of IFN-b. The Journal of Immunology, 2012, 189: 000–000. http://www.jimmunol.org/

ype I IFNs are encoded by a multigene family comprising nounced resistance to virus replication, as animals defective in multiple a subtypes, IFN-b, and others which protect IFN production are highly sensitive to virus infection (4). Type I T cells from viral infections and regulate both innate and IFNs have been demonstrated to contribute to the induction of a adaptive immune responses (1, 2). Secreted type I IFN signals strong Th1 response via upregulation of IL-12 (5), activation of through a ubiquitously expressed two-chain receptor (IFNAR), NK cells and various chemokines including the Th1 cell recruiting thereby initiating a signaling cascade that triggers the expression chemokine CXCL10 (6). IFN-b has also been recognized for its of numerous anti-viral, anti-bacterial, and immunoregulatory IFN- anti-inflammatory activity, as it inhibits the production of IL-1b by guest on September 26, 2021 stimulated genes (ISGs) involved in the innate host response (3). It (7), TNF-a, IFN-g (8), and the neutrophil and leukocyte recruiting is well established that cells exposed to type I IFNs display pro- chemokine CXCL8 (9, 10). However, IFN-b canalsobedetri- mental, as in models of sepsis LPS-induced IFN-b contributes to lethal toxicity (2). *Department of Basic Sciences and Environment, Molecular Immunology, Faculty of Recognition of microbes by immune cells is mediated by pattern † Life Sciences, University of Copenhagen, 1871 Frederiksberg, Denmark; Centre for recognition receptors (PRRs), including TLRs and C-type lectins Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria 3168, Australia; ‡Institute of Medical Immunol- expressed on the surface of innate immune cells like monocytes, ogy, Free University of Brussels, 6041 Gosselies, Belgium; xDepartment of Interna- macrophages, neutrophils, and dendritic cells (DCs). PRRs rec- tional Health, Immunology and Microbiology, University of Copenhagen, 2200 { ognize microbe-specific pathogen-associated molecular patterns Copenhagen, Denmark; and Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark and thereby activate several intracellular signaling pathways or- Received for publication December 5, 2011. Accepted for publication July 9, 2012. chestrating both a cell type-specific and a pathogen-specific im- This work was supported by the Danish Agency for Science, Technology and Inno- mune response in the host (11). In mice, 12 TLRs have been vation and the Novo Nordisk Foundation. G.W. was supported by a postdoctoral identified (12). TLR3, TLR7, TLR8, and TLR9 are localized in fellowship and a travel grant to Australia from the University of Copenhagen, the the endosomal compartments, whereas the other TLRs are asso- Augustinus Foundation, the A.P. Møllers and Wife Chastine Mc-Kinney Møllers Foundation for Medical Science Promotion, the Christian and Ottilia Brorsons ciated with cell membranes. MyD88, an essential signaling compo- Travel Foundation for Young Scientists, the Sophus Jacobsen and Wife Astrid Jacob- nent of all TLRs except TLR3, which utilizes TIR domain-containing sens Foundation, the Oticon Foundation, and the Torben and Alice Frimodts Foun- adapter inducing IFN-b (TRIF), is required for activation of dation. MAPKs and NF-kB leading to the production of proinflammatory Address correspondence and reprint requests to Gudrun Weiss at the current address: Leukocyte Biology Section, National Heart and Lung Institute, Imperial College cytokines such as IL-12, TNF-a, and IL-6 (13). London, Exhibition Road, London SW7 2AZ, U.K. E-mail address: gudrun.weiss@ In recent years, evidence for bacterial induction of type I IFNs, imperial.ac.uk in particular IFN-b, has emerged (14). Most of the bacteria tested The online version of this article contains supplemental material. belong to the Gram-negative genera, but a number of Gram- Abbreviations used in this article: DC, dendritic cell; IRF, IFN regulatory factor; ISG, positive pathogenic bacteria are also able to activate IFN-b pro- IFN-stimulated gene; LTA, lipoteichoic acid; Mal, MyD88 adapter-like; MFI, mean fluorescence intensity; MOI, multiplicity of infection; Poly I:C, polyriboinosinic duction in immune cells [reviewed by Monroe et al. (15) and polyribocytidylic acid; PRR, pattern recognition receptor; Syk, spleen tyrosine ki- Trinchieri (16)]. Gram-negative bacteria are primarily recognized nase; TRAF3, TNFR-associated factor 3; TRIF, TIR domain-containing adapter in- by TLR4. Upon activation, TLR4 is endocytosed and delivered to ducing IFN-b; WT, wild-type. intracellular vesicles where it forms complexes with TRAM and Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 TRIF. Subsequently, TNFR-associated factor 3 (TRAF3) and the

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103491 2 L. ACIDOPHILUS-INDUCED IFN-b SIGNALING protein kinases TBK1 and IKKi are recruited, and the IFN regu- (Sigma-Aldrich, Glostrup, Denmark) were added in final concentrations of latory factor (IRF)3 is phosphorylated leading to the expression 1 mg/ml. Polyriboinosinic polyribocytidylic acid (Poly I:C) (InvivoGen), of type I IFN (17). Both TRIF and TRAM function in LPS–TLR4 a synthetic analogue of dsRNA, was added in a final concentration of 10 mg/ml. The cell cultures were incubated at 37˚C in 5% CO2. signaling independently of MyD88 (18). In the case of Gram- In the inhibitor experiments, DCs were preincubated for 30 min with BAY positive bacteria, the majority of work has been conducted on the 61-3606 (Sigma-Aldrich), a highly selective inhibitor of the specific spleen pathogenic bacteria Listeria monocytogenes and Streptococcus tyrosine kinase (Syk), in final concentrations of 5 mM and 25 mM; 30 min pyogenes. In macrophages, IFN-b induction by Group B Strep- with the Syk inhibitor piceatannol (Sigma-Aldrich) in final concentrations of 5 mM and 25 mM; and 60 min with the PI3K inhibitor LY294002 tococcus as well as L. monocytogenes was completely abrogated in (Sigma-Aldrich) in a final concentration of 50 mM. In the endosomal the absence of IRF3 but was unaffected in the absence of TLR3, inhibitor experiments, DCs were prestimulated with chloroquine (InvivoGen) TLR4, TLR7, TLR9, TRIF, TRAM, MyD88 adapter-like (Mal), in a final concentration of 10 mM; bafilomycin A1 (InvivoGen) in a final MyD88, IRF1, or IRF7. By contrast, in DCs Group B Streptococcus- concentration of 25 mM; and Dynasore (Sigma-Aldrich) in a final con- induced release of IFN-b was mainly dependent on TLR7, phag- centration of 40 mM. Viability was verified by trypan blue staining. osomal degradation, MyD88, and IRF1 (19). In a recent study, Immunostaining and flow cytometry S. pyogenes-induced IFN-b in DCs was dependent on MyD88, DCs were harvested and resuspended in PBS supplemented with 1% (v/v) IRF3, and IRF5, whereas macrophages required IRF3 and par- FBS and 0.15% (w/v) sodium azide containing anti-mouse FcgRII/III tially MyD88 (20). Thus, bacteria can activate various cell type- (3 mg/ml; BD Biosciences, San Jose, CA) to block nonspecific binding specific pathways leading to the release of IFN-b. Notably, IFN-b of Ab reagents. Allophycocyanin-conjugated anti-mouse CD86, FITC- induced by L. monocytogenes and S. pyogenes was reported to be conjugated anti-mouse CD11c (BD Pharmingen, San Diego, CA), and PE-conjugated anti-mouse CD40 (eBioscience, San Diego, CA) were used Downloaded from dependent on IRF3 but independent of TLR2 (20-23). for staining. Nonspecific binding was evaluated by matched isotype con- We have previously shown that Lactobacillus acidophilus strains trols for all Abs. DCs were analyzed using a BD FACSCanto II flow induce high amounts of IFN-b in DCs, which in turn leads to the cytometer (BD Biosciences) based on counting of 10,000 cells. Data anal- expression of hundreds of ISGs and the activation of an innate ysis was performed using the software program FlowJo (Tree Star, Ashland, immune response (24, 25). The capacity to induce IFN-b was OR). The level of expression was measured as the geometric mean fluorescence intensity (MFI). markedly reduced in TLR22/2 DCs, dependent on clathrin-mediated

endocytosis (24) and abrogated after blocking of the MAP JNK Cytokine quantification by ELISA http://www.jimmunol.org/ pathway (26). In this study, we show that L. acidophilus induces Cytokine production of was analyzed using the following commercially astrongIFN-b response in DCs, which subsequently drives an available ELISA kits: IL-12 (p70), IL-10, TNF-a, and IL-6 (R&D Systems, IFNAR1-dependent activation of the classical ISG Viperin. The Minneapolis, MN), and IFN-b (PBL Interferon Source, Piscataway, NJ). L. acidophilus-triggered IFN-b production requires dynamin- RNA extraction dependent phagocytosis and maturation of phagolysosomes but is independent of the endosomal receptors TLR7 and TLR9. The Murine bone marrow-derived DCs were harvested and total RNA was IFN-b production is dependent on the TIR-containing adapter extracted using the MagMAX sample separation system (Applied Bio- systems, Foster City, CA), including a DNAse treatment step for genomic MyD88 but not TRIF or Mal and partially dependent on IRF1. DNA removal. RNA quality was verified by Bioanalyzer (Agilent, Santa Furthermore, our findings indicate that IRF3 or IRF7 are com- Clara, CA), and the concentration was determined by Nanodrop (Thermo, by guest on September 26, 2021 plementary in the induction of IFN-b. These characteristics of an Wilmington, DE). IFN-b activation pathway have not been described previously. Quantitative real-time PCR analysis Materials and Methods Five hundred nanograms of total RNA was reverse transcribed by the Bacterial strains TaqMan Reverse Transcription Reagent kit (Applied Biosystems) using random hexamer primers according to the manufacturer’s instructions. The Gram-positive bacterium L. acidophilus NCFM (Danisco, Copenhagen, The obtained cDNA was stored in aliquots at 280˚C. The expression of the Denmark) was grown anaerobically overnight at 37˚C in de Man Rogosa genes encoding IFN-b, IL-12p40, and b-actin was detected using primers Sharp broth (Merck, Darmstadt, Germany). The Gram-negative bacterium and probes as previously described (25). HPLC-purified forward and re- Escherichia coli Nissle 1917 O6:K5:H1 (Statens Serum Institut, Copenhagen, verse primers were manufactured by DNA Technology (Aarhus, Denmark) Denmark) was grown aerobically overnight at 37˚C in Luria-Bertani broth and FAM-labeled probes by Applied Biosystems. A TaqMan Gene Ex- (Merck). The bacteria were subcultured twice, harvested by centrifugation pression Assay was purchased for the detection of Viperin (Rsad2) (Assay at 1250 3 g for 10 min, and washed twice in sterile PBS buffer. ID Mm00491265_m1; Applied Biosystems). The amplifications were carried out in a total volume of 10 ml containing 13 TaqMan Universal Generation of DCs PCR Master Mix (Applied Biosystems) using the ABI Prism 7500 (Ap- plied Biosystems) as previously described (25). The amplifications were Bone marrow-derived DCs were prepared from 6- to 10-wk-old mice. Cells normalized to the expression of the b-actin encoding gene. The samples were cultivated in RPMI 1640 with 10% heat-inactivated FCS in the generated in the Mal2/2 experiments were analyzed using the 7900HT presence of GM-CSF as described previously (27). Wild-type (WT) mice Fast Real-Time PCR System (Applied Biosystems), the TaqMan Gene were purchased from Taconic (Lille Skensved, Denmark), TRIF2/2,IRF12/2, 2/2 Expression Assay for IFN-b (Assay ID Mm00439546_s1; Applied Bio- and TLR7 mice were purchased from The Jackson Laboratory (Bar systems), and were normalized to 18S (Assay ID 4319413E; Applied Harbor, ME). MyD882/2 and TLR92/2 were kindly provided by S. Akira 2 2 Biosystems). Amplification reactions were performed in triplicate, and (Osaka University, Osaka, Japan). Mal / were housed at Monash Uni- 2/2 DNA contamination controls were included. Relative transcript levels versity (Clayton, Australia). IRF3 were obtained from T. Taniguchi 2 2/2 were calculated applying the 2( DDC(T)) method described by Livak (University of Tokyo, Tokyo, Japan). IRF3/7 were bred at the Institute and Schmittgen (28). of Medical Immunology (Gosselies, Belgium), and IRF72/2 and IFNAR12/2 were housed at the Medical University of Copenhagen. All mice used were Type I IFN bioassay on a C57BL/6 background. Antiviral activity of IFNs was determined by inhibition of cytopathic effect Stimulation of DCs with bacteria and treatments of Semliki Forest Virus on L929 cells compared with a reference standard (GU-02-901-511) as described previously (29). Primary innate DCs (2 3 106 cells/ml), tested .90% CD11c positive, were resuspended in fresh medium and seeded in 48-well tissue culture plates SDS-PAGE and Western blotting (500 ml/well) (Nunc, Roskilde, Denmark). Bacterial pellets were resuspended in antibiotic-free RPMI 1640 and added (100 ml/well) to the DCs DCs stimulated with L. acidophilus or E. coli were harvested by centri- at a multiplicity of infection (MOI) of 2:1. The TLR2 ligand lipoteichoic fugation and lysed in RIPA buffer (Thermo Fisher, Roskilde, Denmark) acid (LTA; InvivoGen, San Diego, CA) and the TLR4 ligand LPS (E. coli) supplemented with protease and phosphatase inhibitor cocktails (P8340 The Journal of Immunology 3 and P5726; Sigma-Aldrich) for 30 min on ice. After centrifugation for sion was lower compared to DCs (data not shown). L. acidophilus 20 min (4˚C, 14,000 3 g), the supernatant was stored at 220˚C. For Western likewise triggered a strong IL-12 response in DCs (Fig. 1C) with blotting, the samples were mixed with sample buffer (Invitrogen, Naerum, a gene expression profile of Il-12 similar to Ifn-b (Fig. 1D). Denmark), boiled for 5 min, and resolved on 7% Tris acetate gels (Invi- trogen) in Tris acetate SDS running buffer (Invitrogen) and transferred to MyD88 is indispensable for the induction of IFN-b and IL-12 Immun-Blot PVDF Membranes (Invitrogen) at 300 mA for 1 h. After blocking by L. acidophilus for 1 h in TBS containing 5% nonfat milk, membranes were washed three times in TBS and probed for 20 h with anti-Syk and anti–p-Syk (Tyr 525/ TLR signaling involves either MyD88, which acts as an adapter 526) (Cell Signaling) diluted in TBS. After extensive washing in TBS, molecule for TLR2, TLR4, TLR7, TLR8, and TLR9, or TRIF, which membranes were incubated for 45 min with a secondary HRP-conjugated Ab (Dako, Glostrup, Denmark), and finally washed three times with TBS. is the sole adapter molecule for TLR3 but also contributes to the The bound Abs were detected using ECL reagents according to the man- IFN-b induction through TLR4 and IRF3 (11, 30). To investigate ufacturer’s instructions (GE Healthcare). the involvement of the adapter proteins in the IFN-b response to Statistical analysis L. acidophilus, we stimulated DCs derived from mice deficient for MyD88, TRIF, and IRF3 for 10 h. In contrast to the high levels Statistical calculations were performed using the software program GraphPad of IFN-b in the supernatants of WT DCs, the IFN-b production , Prism 5. One-way ANOVA and Bonferroni tests were applied (p 0.01). was completely abolished in MyD882/2 DCs, whereas in TRIF2/2 and IRF32/2 DCs, the IFN-b release was similar to the WT (Fig. Results 2A). In comparison, the induction of IFN-b in DCs stimulated with b 2 2 L. acidophilus induces high levels of IFN- and IL-12 in DCs E. coli was reduced by 66% in the MyD88 / DCs and completely 2/2 2/2 Bone marrow-derived DCs were stimulated with L. acidophilus,the absent in TRIF and IRF3 DCs. In DCs stimulated with LPS, Downloaded from 2 2 2 2 2 2 dsRNA ligand Poly I:C, the TLR2 ligand LTA, the Gram-negative IFN-b was undetectable in MyD88 / ,TRIF / ,andIRF3 / bacterium E. coli, and the TLR4 ligand LPS. Protein levels of IFN-b DCs. IFN-b release by Poly I:C was slightly reduced in the 2 2 2 2 and IL-12 were measured in the supernatants after 10 h (Fig. 1A). MyD88 / DCs (22%) and completely absent in TRIF / and 2 2 L. acidophilus induced high amounts of IFN-b in DCs, as a protein IRF3 / DCs. We also determined the expression of the Th1 expression of 2350 pg/ml was measured. For the RT-PCR analysis, skewing cytokine IL-12 as it is induced by IFN-b in an autocrine

cells were harvested after 2, 4, 6, 8, 10, and 16 h, RNA was manner (5). Notably, the expression of IL-12 was completely absent http://www.jimmunol.org/ 2 2 extracted, and the gene expression was determined (Fig. 1B). in MyD88 / cells upon L. acidophilus stimulation but increased 2 2 2 2 E. coli,LPS,andPolyI:CinducedIfn-b ∼50-fold at 2 h, which by 35% in TRIF / and 86% in IRF3 / DCs compared with dropped at 4 h and remained very low. In contrast, Ifn-b was not WT cells (Fig. 2B). The induction of IL-12 upon addition of E. coli detected at 2 h upon stimulation with L. acidophilus but was and LPS was signiﬁcantly reduced in all three knockout mice 2 2 strongly induced at 4 h (420-fold), peaked at 6 h (510-fold), and was tested, with no or very little IL-12 production in MyD88 / and 2 2 2 2 still expressed at high levels at 8 h and 10 h (380-fold and 290-fold, TRIF / DCs and an ∼50%reductioninIRF3 / DCs, which respectively). L. acidophilus also activated the IFN-b expression is in agreement with previous observations (31). Poly I:C did not in bone marrow-derived macrophages; however, the protein expres- trigger the expression of IL-12 in any of the cells tested. by guest on September 26, 2021

FIGURE 1. L. acidophilus induces high levels of IFN-b and IL-12 in DCs. Bone marrow-derived DCs were stimulated with L. acidophilus (MOI 2:1), Poly I:C (10 mg/ml), LTA (1 mg/ml), E. coli (MOI 2:1), and LPS (1 mg/ml). (A and C) Production of IFN-b and IL-12 was measured by ELISA in supernatants after 10 h of stimulation. Data represent mean of measurements from triplicate cultures 6 SD. *p , 0.01 (versus nonstimulated cells). (B and D) Expression of Ifn-b and Il-12 analyzed by RT-PCR and normalized to b-actin after 2, 4, 5, 8, 10, and 16 h of stimulation. Data presented are from one experiment representative of at least three independent experiments. 4 L. ACIDOPHILUS-INDUCED IFN-b SIGNALING Downloaded from

FIGURE 2. MyD88 is indispensable for the induction of IFN-b and IL-12 by L. acidophilus. Bone marrow-derived DCs from WT, MyD882/2, TRIF2/2, and IRF32/2 were stimulated with L. acidophilus (MOI 2:1), Poly I:C (10 mg/ml), E. coli (MOI 2:1), and LPS (1 mg/ml). Production of IFN-b (A) and IL-12 (B) was measured by ELISA in supernatants after 10 h of stimulation. Data represent mean of measurements from triplicate cultures 6 SD from three independent experiments. *p , 0.01 (versus WT).

MyD88 is essential for the upregulation of the surface markers higher level than upon incubation with E. coli and Poly I:C. The http://www.jimmunol.org/ CD40 and CD86 in DCs stimulated with L. acidophilus induction of CD40 and CD86 upon stimulation with L. acidophilus 2/2 To assess whether molecules required for costimulation of T cells was markedly impaired in the MyD88 cells, although not are involved, we analyzed the upregulation of the maturation abrogated, whereas the induction by E. coli or Poly I:C was not 2 2 2 2 markers CD40 and CD86 by ﬂow cytometry (Fig. 3). In WT DCs affected. In the TRIF / and IRF3 / DCs, L. acidophilus up- stimulated with L. acidophilus, CD40 and CD86 was expressed at a regulated CD40, which was absent upon Poly I:C stimulation. The by guest on September 26, 2021

FIGURE 3. MyD88 is essential for the upregulation of the surface markers CD40 and CD86 in DCs stimulated with L. acidophilus. Bone marrow-derived DCs from WT, MyD882/2, TRIF2/2, and IRF32/2 were stimulated with L. acidophilus (MOI 2:1), Poly I:C (10 mg/ml), and E. coli (MOI 2:1). The expression of the surface markers CD40 (top panels) and CD86 (bottom panels) was measured by flow cytometry upon 16 h of stimulation. Numbers indicate the geometric MFI of stimulated cells, respectively. Data presented are from one experiment representative of three independent experiments. The Journal of Immunology 5 upregulation of CD86 was likewise markedly reduced in MyD882/2 lomycin A1 and chloroquine, which are both inhibitors of endosomal DCs incubated with L. acidophilus but not with E. coli or Poly I:C, acidification. To assess the importance of endosome maturation we andintheTRIF2/2 and IRF32/2 DCs only a slight upregulation included Dynasore, a selective noncompetitive GTPase inhibitor was detected upon E. coli or Poly I:C stimulation. Thus, our data that blocks dynamin-dependent endocytosis and thereby the scis- demonstrate that the adapter molecule MyD88 is indispensable for sion of endocytic vesicles. Pretreatment of DCs with chloroquine, the L. acidophilus mediated upregulation of the surface maturation bafilomycin A1, or Dynasore completely abolished the expression markers CD40 and CD86. of Ifn-b upon addition of L. acidophilus to the cells (Fig. 5A). As these results indicate that endosomal acidification is essential for b Induction of IFN- by L. acidophilus in DCs does not require Mal the induction of IFN-b, we investigated the involvement of the Mal acts to bridge MyD88 to TLR2 and TLR4 specifically via its endosomal receptors TLR7 and TLR9. We stimulated DCs from TIR domain in response to bacterial infection (30, 32), but recent WT, TLR72/2,andTLR92/2 mice with L. acidophilus and mea- reports also describe a Mal-independent MyD88 pathway in the sured the protein expression of IFN-b in the supernatants. Neither induction of the proinflammatory cytokines TNF-a andIL-6(33,34). TLR7 nor TLR9 is required for IFN-b signaling, as the IFN-b We used DCs deficient for Mal to investigate whether this adapter levels expressed were in alignment with the WT (Fig. 5B). protein is required for the induction of IFN-b by L. acidophilus. We have previously shown that the MAP JNK pathway is Surprisingly, coincubation of Mal2/2 DC with L. acidophilus in- mandatory for the expression of IFN-b in DCs stimulated with duced IFN-b protein levels more than three times compared with L. acidophilus (25, 26). We therefore sought to identify intermediate WT (Fig. 4A). This result was also confirmed by RT-PCR, as the molecules upstream of the JNK pathway. As JNK phosphorylation

Ifn-b expression was signiﬁcantly higher after 6 and 8 h compared is dependent on Syk signaling (35) and Syk collaborates with TLR Downloaded from with WT (Fig. 4B). As our next step, we used a bioassay to test receptors (36), we speculated that blockage of Syk might impair the antiviral property of the IFN released upon stimulation of the IFN-b response to L. acidophilus. Indeed, pretreatment of WT and Mal2/2 DCs with L. acidophilus (Fig. 4C). The protection DCs with the Syk inhibitors BAY 61-3606 and piceatannol prior measured was highest after 6 h in the WT and twice as pronounced to stimulation with L. acidophilus reduced the protein expression in the supernatants of Mal2/2 DCs. These results demonstrate of IFN-b (Fig. 5C). By contrast, when E. coli was added to DCs

that the adapter molecule Mal is not required for the induction of preincubated with BAY 61-3606 and piceatannol, the expression http://www.jimmunol.org/ IFN-b by L. acidophilus in DCs. of IFN-b only significantly decreased in the DCs pretreated with BAY 61-3606 but not with piceatannol. To verify the inhibitory b L. acidophilus-induced IFN- release requires phagosomal potency of piceannatol, we measured the expression of IL-12 in processing and Syk kinase signaling but not TLR7 and TLR9 the supernatants. Both Bay 61-3606 and piceatannol blocked the To rework the rationale that IFN-b induction via MyD88-dependent induction of IL-12 in DCs stimulated with L. acidophilus as well and Mal- and IRF3-independent mechanisms implies endosomal as E. coli (Supplemental Fig 1). We also pretreated DCs with TLRs, we tested whether phagosomal uptake, processing of LY294002, an inhibitor specific for PI3K. PI3K is a direct binding L. acidophilus, and the endosomal receptors TLR7 and TLR9 are partner of Syk that participates in Syk-mediated downstream essential for the release of IFN-b. We prestimulated DCs with bafi- signaling and is also involved in phagocytosis (35). As anticipated, by guest on September 26, 2021

FIGURE 4. Induction of IFN-b by L. acidophilus in DCs does not require Mal. Bone marrow-derived DCs from WT and Mal2/2 were stimulated with L. acidophilus (MOI 2:1). (A) Protein levels of IFN-b measured in the supernatants of WT and Mal2/2 DCs by ELISA after 10 h. (B) Gene expression of IFN-b determined by RT-PCR over time in WT and Mal2/2 DCs. (C) Biological activity of IFN measured via bioassay in the supernatants of WT and Mal2/2 DCs over time. Data represent mean of measurements from triplicate cultures 6 SD from three independent experiments. *p , 0.01 (versus WT). 6 L. ACIDOPHILUS-INDUCED IFN-b SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. L. acidophilus-induced IFN-b release requires phagosomal processing and Syk signaling but not TLR7 and TLR9. Bone marrow-derived DCs from WT mice were pretreated with either chloroquine (10 mM), bafilomycin A1 (25 mM), Dynasore (40 mM), or vehicle (DMSO) for 30 min prior to stimulation with L. acidophilus (MOI 2:1) for 4 h or untreated. (A) Gene expression of IFN-b was determined by RT-PCR. (B) Protein levels of IFN-b measured in the supernatants of DCs from WT, TLR72/2, and TLR92/2 mice stimulated with L. acidophilus (MOI 2:1) for 10 h. (C) Protein expression of IFN-b in the supernatants of DCs preincubated with the Syk inhibitors BAY 61-3606 (final concentrations 5 mM and 25 mM) and piceatannol (final concentrations 5 mM and 25 mM) or vehicle (DMSO) for 30 min prior to stimulation with L. acidophilus or E. coli (MOI 2:1) for 10 h. (D) Gene expression of Ifn-b in DCs preincubated with the PI3K inhibitor LY294002 (final concentration 50 mM) or vehicle (DMSO) for 60 min prior to a 4-h stimulation with L. acidophilus (MOI 2:1). (E) Phosphorylated Syk (P-Syk) detected by immunoblotting in protein extracts of DCs stimulated with L. acidophilus and E. coli (MOI 2:1) for 15 min and 30 min (upper panel). Syk Ab was used as equal loading control (lower panel). Data presented are from one experiment representative of three independent experiments. Data represent mean of measurements from triplicate cultures 6 SD. *p , 0.01 (versus L. acidophilus- or E. coli-stimulated DCs without inhibitor pretreatment or WT control). the induction of Ifn-b was downregulated by 93% when PI3K was were able to show that IRF3 is not essential for the production blocked via addition of LY294002 (Fig. 5D). To confirm our data, of IFN-b, we sought to investigate the role of IRF7, a main we analyzed the phosphorylation of Syk by Western blotting (Fig. regulator of the type I IFN induction (38). DCs from IRF12/2, 5E). Both L. acidophilus and E. coli activated Syk in DCs, but IRF32/2,IRF72/2,andIRF3/72/2 mice were stimulated with whereas E. coli-induced phosphorylation of Syk peaked at 15 min, L. acidophilus for 10 h (Fig. 6A). The protein levels of IFN-b stimulation with L. acidophilus resulted in a more pronounced were not affected by the absence of IRF3 or IRF7 but significantly expression that was first detected at 30 min of incubation. Thus, reduced in double-deficient DCs suggesting complementary roles our data indicate that Syk signaling and phagosomal maturation of IRF3 and IRF7 in regulating the gene expression of IFN-b. of L. acidophilus is required for the induction of IFN-b. In IRF1-deficient DCs, the expression of IFN-b was significantly decreased (50%) compared with the WT. Autocrine IFN-b stim- b L. acidophilus induced IFN- signals independently of IRF3 ulation was confirmed, as the classical type I IFN-induced gene and IRF7 but is reduced in IRF3/7- and IRF1-deficient DCs Viperin (Rsad2) (39) was not induced in IFNAR2/2 DCs (Fig. 6B). To further investigate the involvement of IRFs downstream of the Our findings suggest that L. acidophilus activates several path- MyD88 pathway, we used DCs deficient for IRF1, which is a pos- ways in DCs leading to the induction of IFN-b involving the itive regulator of the transcription of type I IFN genes (37). As we transcription factors IRF1, IRF3, and IRF7. The Journal of Immunology 7

FIGURE 6. L. acidophilus induced IFN-b signals independently of IRF3 and IRF7 but is reduced in IRF3/7- and IRF1-deﬁcient DCs. Bone marrow- derived DCs from IRF-32/2, IRF72/2, IRF3/72/2, IRF12/2,andIFNAR12/2 mice were stimulated with L. acidophilus (MOI 2:1). (A) Protein levels of IFN-b measured after 10-h incubation. (B) Gene expression of Viperin analyzed by RT-PCR in WT and IFNAR12/2 DCs after 10-h incubation with L. acidophilus (MOI 2:1). Data represent mean of measurements from triplicate cultures 6 SD of three independent experiments. *p , 0.01 (versus WT).

Discussion indicate that a different mechanismisinvolvedastheadapter The production of IFN-b by DCs and macrophages stimulated protein TRIF is not essential, the Ifn-b gene expression proﬁle is with pathogenic bacteria is well established. However, the levels more sustained, and the amount of protein released is signiﬁcantly induced are both bacteria- and cell-type dependent as differential higher. responses have been observed (19, 20), which might be due to A direct interaction between the adapter molecule MyD88 and Downloaded from activation of diverging cellular signaling pathways. Importantly, the transcription factor IRF1 has been demonstrated (46, 47). The the expression of IFN-b induced by L. acidophilus in DCs is association of MyD88 with IRF1 seems to be required for trans- among the highest reported for bacteria able to induce a type I location of IRF1 to the nucleus in DCs (47). IRF1 is essential for IFN response. In the current study, we aimed to identify receptors the induction of several TLR-dependent genes; however, it remains andadaptermoleculesinvolvedintheIFN-b induction of this unclear how MyD88 activates IRF1. IRF7 resides in the cytoplasm

non-pathogenic Gram-positive bacterium. Our results clearly show in an inactive form that has to be activated through PRR ligation http://www.jimmunol.org/ that MyD88 is a prerequisite for the release of IFN-b,whereas to induce type I IFN expression (48). Although the expression of absence of TRIF has no effect. Although it is well established that both genes was upregulated upon stimulation with L. acidophilus the Poly I:C pathway of IFN-b induction is MyD88 independent, in our recent study (24), the proteins may be present in the cell we also observed a reduction of IFN-b in the MyD882/2 cells prior to stimulation and thus play a role in the induction of IFN-b. stimulated with this TLR3 ligand. However, in comparison with Our observation is supported by Dietrich et al. (49), who dem- L. acidophilus, this effect is only subtle and may result from a onstrated that in macrophages uptake and trafficking of TLR2 to general defect caused by knocking down the MyD88 gene. No- the endosomal compartments induced type I IFNs via MyD88- tably, in the absence of Mal, a coadapter protein of MyD88 critical dependent IRF1 and IRF7 pathways. Whether TLR2, which we for TLR2 and TLR4 signaling (40, 41), the induction of IFN-b previously reported partly essential for the induction of IFN-b by guest on September 26, 2021 is significantly increased. Our data indicate that Mal not only is in L. acidophilus-stimulated DCs (24), translocates to the endo- dispensable in MyD88 signaling but also might act as a repres- somal compartments and thereby induces the release of IFN-b is sor for L. acidophilus-induced IFN-b. MyD88-independent TLR2 currently under investigation. signaling of Mal has been reported by Kenny et al. (33). They All TLRs, with the exception of TLR3, signal through MyD88 showed that the induction of IL-6 in macrophages and DCs is (32). As L. acidophilus requires MyD88, endocytosis, and acid- dependent on MyD88 but independent of Mal. Furthermore, they ification of the phagosome to induce IFN-b, we stimulated DCs detected a higher JNK activation upon TLR3 stimulation in the deficient for the two endosomal nucleic acid sensors TLR7 and absence of Mal. As we previously established that JNK is essential TLR9. Several bacteria are reported to activate IFN-b via endo- for the IFN-b induction (25, 26) and in the current study detected somal receptors (19, 20, 50). However, conflicting data exist whether an increase of IFN-b in Mal2/2 DCs, our findings are in accor- the Gram-positive Streptococcus induces IFN-b in DCs via TLR7 dance with these observations. Kenny and colleagues (33) con- (19, 20). Our results reveal that TLR7 and TLR9 are not essential cluded that Mal was dispensable but only at high TLR2 ligand for the induction of IFN-b by L. acidophilus in DCs; however, we concentrations. In this study, we demonstrate that the induction cannot exclude simultaneous activation of both receptors leading of IFN-b by L. acidophilus is dependent on Syk activation. We to redundancy. The PRR responsible for triggering the MyD88– therefore suggest that receptor clustering caused by a local high IRF pathway therefore remains to be determined. Stimulation with concentration of high-avidity ligands might lead to simultaneous heat-killed but not UV-killed L. acidophilus induced a signifi- activation/phosphorylation of many closely associated Syk mole- cantly lower induction of IFN-b compared with live bacteria (data cules, for example the MAPK and PI3K (42). In contrast to E. coli, not shown), thus indicating that an intact bacterium with native L. acidophilus phosphorylated Syk at a later time point, which molecules is a prerequisite for a strong expression of IFN-b. indicates that a different mechanism is involved in cellular sig- The Gram-positive bacterium L. monocytogenes is primarily naling and processing. The intracellular Syk pathway can be acti- detected by cytosolic sensing pathways due to its ability to escape vated by integrin signal transduction (43) or phagocytic processing from phagosomes. L. monocytogenes is able to disrupt the phag- (44). Our data emphasize the pivotal importance of phagocytosis osomal membrane via the pore-forming toxin listeriolysin O (51). and thereby compartmentalization of receptor–ligand interactions Listeriolysin O-deficient bacteria are trapped and unable to induce leading to the induction of IFN-b by L. acidophilus (17). Recently, type I IFN, whereas WT strains get released into the cytosol and it was shown that endocytosis of LPS from E. coli in DCs is de- induce IFN-b dependent on IRF3 (23, 52, 53). Many bacteria are pendent on Syk and CD14, which coincided with the induction of reported to induce IFN-b through IRF3-dependent mechanisms IFN-b (45). Moreover, IRF3 dimerization was induced. Although (20–22, 54). It has been proposed that the IRF3-dependent path- one or more PPRs and IRF3 or IRF3/IRF7 dimerization are likely way is activated by an unidentified cytosolic receptor after rup- to be required for the L. acidophilus-induced IFN-b, our data ture of the phagosomal membrane (22, 54), injection of bacterial 8 L. ACIDOPHILUS-INDUCED IFN-b SIGNALING products into the cytosol (54), or translocation of digested bacterial 11. Kawai, T., and S. Akira. 2010. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. 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