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 24, 2021. Gudrun Weiss, Kristina Maaetoft-Udsen, Sebastian A. Stifter, Paul Hertzog, Stanislas Goriely, Allan R. Thomsen, Søren R. Paludan and Hanne Frøkiær J Immunol 2012; 189:2860-2868; Prepublished online 15 August 2012; Downloaded from doi: 10.4049/jimmunol.1103491 http://www.jimmunol.org/content/189/6/2860 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2012/08/15/jimmunol.110349 Material 1.DC1 References This article cites 58 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/189/6/2860.full#ref-list-1 Why The JI? Submit online. by guest on September 24, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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. 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 dem- onstrated 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: 2860–2868. 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 24, 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 The Journal of Immunology 2861 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).