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IRF8 Dendritic Cells by Inhibition of IRF1 and Lipids Inhibit IL-12P40 In Francisella tularensis SchuS4 and SchuS4 Lipids Inhibit IL-12p40 in Primary Human Dendritic Cells by Inhibition of IRF1 and IRF8 This information is current as of September 29, 2021. Robin Ireland, Rong Wang, Joshua B. Alinger, Pamela Small and Catharine M. Bosio J Immunol 2013; 191:1276-1286; Prepublished online 1 July 2013; doi: 10.4049/jimmunol.1300867 Downloaded from http://www.jimmunol.org/content/191/3/1276 Supplementary http://www.jimmunol.org/content/suppl/2013/07/01/jimmunol.130086 http://www.jimmunol.org/ Material 7.DC1 References This article cites 55 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/191/3/1276.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 29, 2021 • 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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Francisella tularensis SchuS4 and SchuS4 Lipids Inhibit IL-12p40 in Primary Human Dendritic Cells by Inhibition of IRF1 and IRF8 Robin Ireland,* Rong Wang,*,1 Joshua B. Alinger,* Pamela Small,† and Catharine M. Bosio* Induction of innate immunity is essential for host survival of infection. Evasion and inhibition of innate immunity constitute a strat- egy used by pathogens, such as the highly virulent bacterium Francisella tularensis, to ensure their replication and transmission. The mechanism and bacterial components responsible for this suppression of innate immunity by F. tularensis are not defined. In this article, we demonstrate that lipids enriched from virulent F. tularensis strain SchuS4, but not attenuated live vaccine strain, inhibit inflammatory responses in vitro and in vivo. Suppression of inflammatory responses is associated with IkBa-independent inhibition of NF-kBp65 activation and selective inhibition of activation of IFN regulatory factors. Interference with NF-kBp65 Downloaded from and IFN regulatory factors is also observed following infection with viable SchuS4. Together these data provide novel insight into how highly virulent bacteria selectively modulate the host to interfere with innate immune responses required for survival of infection. The Journal of Immunology, 2013, 191: 1276–1286. irulent Francisella tularensis ssp. tularensis is a Gram- allowing the adaptive response time to develop. Unlike attenuated negative bacterium and the causative agent of the fatal subspecies and strains, virulent F. tularensis is not sensed by host http://www.jimmunol.org/ V disease coined tularemia. Francisella has four major receptors or other detection machinery (7–10). In addition to evading subspecies (1). F. tularensis ssp. novicida and ssp. mediasiatica are detection by the host, virulent F. tularensis also suppresses the ability generally considered attenuated for humans. F. tularensis ssp. of host cells to mount inflammatory responses (7, 8, 11). The holarctica (Type B; F. holarctica) causes serious disease in humans ability of the bacterium to both evade and suppress innate immune but is not typically fatal. F. tularensis ssp. tularensis (Type A; F. responses is a primary mechanism of virulence. tularensis) is highly infectious and can cause a lethal infection Generation of novel vaccines and therapeutics for treatment of following inhalation of as few as 10 organisms in both humans and tularemia has been hampered by our lack of understanding of the rodent models (2, 3). F. tularensis ssp. tularensis was previously host pathways associated with innate immunity that are modulated used as a biological weapon and thus presents a viable concern as a by virulent F. tularensis. Similarly, we have not identified those by guest on September 29, 2021 potential bioweapon today (4). Although a vaccine generated from components of the bacterium that facilitate suppression of innate serial passage of F. ho larc ti ca—that is, live vaccine strain (LVS)— inflammatory responses in the host. Identification of both the host was produced in the 1960s, it is no longer licensed as a vaccine for signaling pathways and components uniquely associated with viru- use against tularemia (5). Further, treatment of individuals infected lent F. tularensis that mediate inhibition of inflammation will greatly with F. tularensis with antibiotic (when delivered in a timely man- facilitate development of new therapeutics and vaccines. ner) does not always result in complete clearance of the bacterium In this article, we demonstrate that lipids isolated from fully virulent (6). Thus, a need has arisen for development of both novel vaccines F. tularensis strain SchuS4, but not attenuated LVS, inhibit innate and therapeutics to combat this highly virulent pathogen. immune responses in primary human cells in vitro and in the mouse The effectiveness of vaccines and therapeutics is tied to their lung in vivo. Inhibition of inflammatory responses by SchuS4 lipids ability to trigger innate immune responses. Induction of innate was a result of targeting specific host proteins required for tran- immunity is an important component of host defense because this scription of genes necessary for innate immunity. Importantly, these response slows replication and dissemination of microorganisms, findings were recapitulated following infection with viable SchuS4. Materials and Methods *Immunity to Pulmonary Pathogens Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Reagents † National Institutes of Health, Hamilton, MT 59840; and Department of Microbiol- Ultrapure E. coli strain K12 LPS, Pam3CSK4, Pam2CSK4, lipoteichoic ogy, University of Tennessee, Knoxville, TN 37996 acid (LTA), ssRNA40/LyoVec, and R848 (imidazoquinolone compound) 1 Current address: U.S. Meat Animal Research Center, Clay Center, NE. were purchased from Invivogen (San Diego, CA). E. coli strain O127:B7 Received for publication April 1, 2013. Accepted for publication May 31, 2013. LPS was purchased from Sigma-Aldrich (St. Louis, MO). Recombinant GM-CSF and IL-4 were purchased from PeproTech (Rocky Hill, NJ). This work was supported by the Intramural Research Program of the National In- stitutes of Health, National Institute of Allergy and Infectious Diseases. Pronase was obtained from Roche Diagnostics (Indianapolis, IN). Address correspondence and reprint requests to Dr. Catharine M. Bosio, Immunity to Bacteria Pulmonary Pathogens Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories/NIAID/NIH, Hamilton, MT 59840. E-mail address: bosioc@niaid. Virulent F. tularensis ssp. tularensis strain SchuS4 was kindly provided by nih.gov Jeannine Peterson, Ph.D. (Centers for Disease Control, Fort Collins, CO). The online version of this article contains supplemental material. Attenuated F. tularensis ssp. holarctica LVS was originally obtained from Abbreviations used in this article: BAL, bronchoalveolar lavage; cRPMI, complete Dr. Jean Celli (Rocky Mountain Laboratories, Hamilton, MT). Stock vials RPMI 1640; EtOH, ethanol; hDC, human dendritic cell; IRF, IFN regulatory factor; of SchuS4 and LVS in broth were generated as previously described LTA, lipoteichoic acid; LVS, live vaccine strain; MF, membrane fraction. (10, 12). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300867 The Journal of Immunology 1277 Isolation of total membrane fraction (10 mg/ml), LTA (100 mg/ml), R848 (5 mg/ml), or LPS (10 ng/ml) + 100 U/ml IFN-g (PeproTech) was added 18 h after MF or lipid treatment, and Total membrane fraction (MF) from LVS and SchuS4 were isolated as cells were cultured for an additional 20 h before supernatants were col- previously described (13–15). Briefly, SchuS4 was grown in modified lected for assessment of cytokines. Mueller–Hinton broth as described earlier (10, 12, 13). Following over- night culture, bacteria were pelleted by centrifugation for 15 min at 8000 3 g. Mice and in vivo assessment of inflammation The resulting pellet was resuspended in the following buffer: 50 mM Tris/ HCl, 0.6 mg/ml DNase, 0.6 mg/ml RNase, 1 mM EDTA (all from Sigma- Specific pathogen–free, 6- to 8-wk-old male C57BL/6J mice (wild type) Aldrich), and 1 complete EDTA-Free Protease Inhibitor Cocktail Tablet (n = 5 per group) were purchased from The Jackson Laboratory (Bar Harbor, (Roche), followed by centrifugation and resuspension in the buffer de- ME). Mice were housed in sterile microisolator cages in the ABSL-2 fa- scribed above. Bacteria were lysed via processing in FastPrep Lysing cility at the National Institute of Allergy and Infectious Diseases/Rocky Matrix B tubes using a FastPrep-24 (MPBio) for 10 cycles of 45 s with 2- Mountain Laboratories. All mice were provided sterile water and food ad min rest periods on ice between each cycle. The resulting slurry was then libitum, and all research involving animals was conducted in accordance centrifuged at 10,000 rpm for 10 min. The supernatant was collected and with Animal Care and Use
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