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

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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 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 required for tran- immunity is an important component of host defense because this scription of 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 ; 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 . 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 guidelines; animal protocols were approved by centrifuged twice at 100,000 3 g for 4 h. The pellet was resuspended in the Animal Care and Use Committee at the Rocky Mountain Laboratories. buffer containing 50 mM Tris/HCl and 1 mM EDTA, then dialyzed against SchuS4 lipids and E. coli LPS were administered intranasally. Briefly, PBS using 3000 m.w. cutoff Slide-A-Lyzer cassettes (Pierce). lipids were resuspended at 25 mg/25 ml in PBS. Mice were anesthetized by concentration of MF was determined using a BCA Protein Assay Reagent i.p. injection of 100 ml 12.5 mg/ml ketamine + 3.8 mg/ml xylazine. Lipids Kit according to the manufacturer’s instructions. MF was then aliquoted, were administered in a volume of 25 ml into a single naris. Mice treated irradiated to render it sterile, and stored at 280˚C. As indicated, MF was with ethanol served as negative controls. At 18 h after exposure to lipids, heated at 56˚C for 4 h or incubated with 2 mg/ml pronase in 0.1 M Tris mice were anesthetized as described above, and 200 ng/25 ml E. coli LPS buffer, pH 7.0, at 40˚C for 3 h, followed by heating at 87˚C for 30 min to O127:B8 (Sigma-Aldrich) was delivered into a single nare. At 5 h after delivery of LPS, mice were euthanized and bronchoalveolar lavage (BAL) deactivate the pronase prior to use. Downloaded from fluid was collected for assessment of cytokines by ELISA and cellular Preparation of Francisella lipids populations by flow cytometry, as previously described (8, 20). Lipids were isolated from LVS and SchuS4, using the modified Folch Assessment of cytokines method for isolation of bacterial lipids, as previously described (16–19). Human TNF-a, IL-6, IL-10, IL-12p40, and TGF-b were assessed by Briefly, 1 3 109 bacteria were thawed and plated onto eight 150-mm petri commercially available ELISA kits according to the manufacturer’s dishes containing MMH agar. Bacteria were incubated at 37˚C/7% CO for 2 instructions (BD Biosciences). Concentrations of mouse TNF-a, IL-6, and

48 h. Bacteria were collected from the agar plates and added to 100 ml http://www.jimmunol.org/ IL-12p40 in BAL fluid were assessed by commercially available ELISA HPLC grade choloroform:methanol (2:1) (both from Sigma-Aldrich). The kits according to the manufacturer’s instructions (BD Biosciences). resulting mixture was stirred vigorously for 30 min at room temperature. Then, 20 ml endotoxin-free water was added, and the mixture was stirred Flow cytometry for an additional 10 min. The mixture was centrifuged at 4000 3 g for 10 min at room temperature to separate organic and aqueous phases. Organic Influx of inflammatory cells into mouse airways and expression of hDC cell and aqueous phases were pipetted into separate containers and dried or surface receptors were assessed by flow cytometry, as previously described concentrated under nitrogen. Dried organic samples were reconstituted in (8, 10, 11). Briefly, directly conjugated Abs for these analyses were pur- absolute ethanol (Warner–Graham) to 20 mg/ml. Average yield of lipids chased from eBiosciences (San Diego, CA). The following Abs in various from Francisella was 80 mg/4 g wet weight of bacteria, representing ∼ 2% combinations were used for flow cytometric analysis: PeCy7 anti-mouse of wet weight. Thus, 30 mg/ml lipid is 0.00075% wet weight of bacteria. CD11b (clone M1/70), PerCp-Cy5.5 anti-mouse CD11c (clone N418), PE The aqueous phase was concentrated to 1 ml. Both preparations were anti-mouse Ly6C (clone HK1.4), FITC anti-mouse Ly6G (clone 1A8), by guest on September 29, 2021 stored at 4˚C for up to 2 mo. Absence of proteins and carbohydrates present PeCy7 anti-human CD1a (clone HI149), PerCpCy5.5 anti-human HLA- in the organic phase was confirmed by analysis of preparations on silver- DR (clone LN3), FITC anti-human CD80 (clone 2D10.4), APC anti- stained SDS-PAGE gels, quantification of protein using the Bradford assay human CCR7 (clone 3D12), and Alexa Fluor 488 anti-human CD86 (clone (Sigma-Aldrich) according to the manufacturer’s instruction, periodate stain IT2.2). Staining with directly conjugated Abs was done in FACS buffer at of SDS-PAGE gels, and Western blotting for Francisella O-Ag and capsule. 4˚C. Then, the cells were washed and fixed in 1% paraformaldehyde for No evidence of proteins or carbohydrates was observed in organic phase 30 min at 4˚C. Cells were washed a final time, resuspended in FACS buffer, preparations. and stored at 4˚C until analyzed. Flow cytometry was done using an LSR II flow cytometer (BD Biosciences). Analysis gates were set on viable un- Generation and stimulation of human dendritic cells stained cells and were designed to include all viable cell populations. Ap- proximately 10,000 events were analyzed for each sample. Isotype control Primary human dendritic cells (hDCs) were differentiated from apheresed Abs were included when analyses and panels were first being performed to , as previously described (7, 10, 11). Human monocytes, en- ensure specificity of staining but were not routinely included with each ex- riched by apheresis, were obtained from peripheral blood provided by the periment. Neutrophils were defined as Ly6G+ cells. Macrophages were Department for Transfusion Medicine and the National Institutes of Health identified as CD11c+/CD11b+/2 cells. Data were analyzed using FlowJo Clinical Center at the National Institutes of Health (Bethesda, MD) under software (TreeStar, Ashland, OR). a protocol approved by the National Institutes of Health Clinical Center Institutional Review Board. Signed, informed consent was obtained from Microscopy each donor, acknowledging that his or her donation would be used for research purposes by intramural investigators throughout the National The hDCs were infected with SchuS4 or treated with PBS, ethanol (vehicle Institutes of Health. Monocytes were further enriched using Ficoll-Paque control), or Schu4 lipids for the indicated times, followed by 10 ng/ml E.coli PREMIUM (GE Healthcare) and were differentiated into hDCs following strain O127:B7 LPS (Sigma-Aldrich). The hDCs were spun onto a glass culture in RPMI 1640 supplemented with 10% heat-inactivated FCS, 0.2 slide using a Shandon Cytospin 4 and fixed in 3% paraformaldehyde for 20 mM L-glutamine, 1 mM HEPES buffer, and 0.1 mM nonessential amino min. The hDCs were incubated with 0.3% Triton X-100 in PBS + 10% acids (all from Invitrogen) [complete RPMI 1640 (cRPMI)] and 100 ng/ml horse serum for 1 h at room temperature, followed by addition of rabbit GM-CSF and 20 ng/ml IL-4 over the course of 4 d. On day 3 of culture, anti–NF-kB p65 XP Ab, anti–IFN regulatory factor (IRF)–1 XP Ab, or 100% of each per well in 1 ml cRPMI was added and cells were anti-IRF3 XP Ab (all from Cell Signaling Technology, Beverly, MA) for used on day 4 of culture. The resulting differentiated hDCs were . 97% 1 h at room temperature. Slides were washed with PBS and incubated with CD1a+/DC-SIGN+ and , 1% CD14+. The hDCs were seeded at 5 3 105 Alexa Fluor 568–conjugated goat anti-rabbit Ab (Cell Signaling Tech- cells per milliliter in cRPMI. MF or lipid isolated from SchuS4 and LVS nology) and Alexa Fluor 488–conjugated mouse anti-F. tularensis LPS Abs was added at the indicated concentrations. Cells treated with ethanol (United States Biological, Swampscott, MA) and counterstained with (EtOH) served as vehicle control samples. At 24 h after addition of MF or DAPI to label cell nuclei. Samples were visualized using a Carl Zeiss LSM lipid supernatants, cells were harvested and assessed for cytokine, as de- 710 confocal scanning laser microscope for quantitative analysis and im- scribed below. Viability of hDCs following incubation with EtOH and lipid age acquisition. Confocal images were acquired and assembled using was assessed by trypan blue exclusion assay. The percent of viable cells Adobe Photoshop CS2 software (Adobe Systems, San Jose, CA). All was calculated using the following formula: Number of trypan blue neg- confocal images were taken at 3400. Translocation was quantitated using ative cells/total cells counted 3 100. As indicated, ultrapure E. coli LPS or a Zeiss Axio Imager M1 fluorescence microscope. Starting in the middle E. coli LPS O127:B8 (10 ng/ml), Pam3CSK4 (5–10 mg/ml), Pam2CSK4 of the slide, three to four fields in succession were counted, for a total 1278 BACTERIAL MODULATION OF IRF FAMILY MEMBERS of .300 cells per sample. Data were pooled from three identical experi- contrast, and consistent with our previous observations in hDCs ments with three different donors. infected with LVS, both LVS MF and E. coli LPS readily in- Quantification of NF-kB translocation duced production of IL-12p40 from hDCs (Fig. 1A and Ref. 7). Translocation of NF-kB in hDCs was quantified using a commercially available TransAM assay, as previously described (21). Briefly, hDCs were treated with PBS, EtOH (vehicle control), SchuS4 lipids, and/or ultrapure E. coli LPS. For assessment of SchuS4 lipid-mediated translocation of NF- kB, cells were harvested 3 h after addition of stimuli or vehicle controls. For assessment of lipid-mediated inhibition of NF-kB translocation, cells were incubated with PBS, EtOH, or SchuS4 lipids for 24 h, followed by addition of ultrapure E. coli LPS. Cells were harvested 3 h after addition of LPS. Nuclear extracts were obtained using a Nuclear Extract Kit (Active Motif; Carlsbad, CA) according to the manufacturer’s directions. Nuclear protein in each sample was quantified using the Bradford assay (Pierce) andadjustedto5mg per sample prior to application in the NF-kB TransAM Assay Kit (Active Motif).

DNA affinity binding assays Determination of IRF1 and IRF8 binding to IL-12p40 sequences was performed as previously described, with minor modifications (22). The hDCs were lysed in a non-denaturing lysis buffer (20 mM Tris HCl, 150 Downloaded from mM NaCl, 10% glycerol, and 1% Triton) containing Halt Protease In- hibitor mixture (Thermo Scientific) and Phosphatase Inhibitor Cocktail (Active Motif). Total protein was quantified by BCA assay (Thermo Sci- entific). M280 Streptavidin-coated Dynabeads (Invitrogen) were prepared per the manufacturer’s instructions and ligated to 59 biotinylated dsDNA (Integrated DNA Technologies) corresponding to the 2244 bp to 2154 bp

region of the human IL-12p40 promoter containing the Ets2 sequence http://www.jimmunol.org/ or the 2101 to 211 region of the IL-12p40 promoter containing the ISRE sequence (Supplemental Fig. 1). Beads were washed in TGED buffer (10 mM Tris HCl, 10% glycerol, 0.1 mM EDTA, and 0.01% Triton 3 100) and blocked overnight at 4˚C in TGED + 0.5% BSA. Whole hDC lysate (100 mg) was added to 200 mg DNA-coated beads in a total of 500 ml TGED buffer + 0.1 M NaCl, and the mixture was incubated with constant mixing at 4˚C overnight. Beads were then washed four times with TGED buffer + 0.1 M NaCl, and protein attached to DNA sequences was eluted in 20 ml 13 sample buffer + reducing agent (Invitrogen). The presence of IRF1 and IRF8 was assessed by SDS-PAGE and Western blotting using anti-IRF1 XP and anti-IRF8 Abs (both from Cell Signaling Technology). by guest on September 29, 2021

Statistical analyses For comparison between three or more groups, analysis was done by one- way ANOVA, followed by a Tukey multiple comparisons test or with significance determined at p , 0.05. For comparison of two groups, analysis was done by unpaired Student t test, with significance determined at p , 0.05.

Results Total MF derived from virulent F. tularensis SchuS4 inhibits production of IL-12p40 An important mechanism of virulence associated with virulent subspecies of F. tularensis is its ability to evade and suppress mul- tiple inflammatory responses in human cells, including production of IL-12 (7, 9, 10, 23, 24). The absence of IL-12 and the subse- quent suppression of this cytokine among human cells are unique to virulent subspecies of F. tularensis, such as SchuS4 (7). We have previously established that heat-stable components of viru- FIGURE 1. Inhibition of IL-12p40 in hDCs is mediated by components in lent F. tularensis SchuS4 secreted or shed from the bacteria have SchuS4 membranes. (A) hDCs were incubated with the indicated concentration the capacity to inhibit host inflammatory responses (10). Thus, we of total MF isolated from SchuS4 or LVS for 18 h. Culture supernatants were postulate that the component of F. tularensis mediating inhibition assessed for IL-12p40 by ELISA. *Significantly greater than untreated of IL-12p40 is a bacterial lipid or carbohydrate. Lipids and car- or SchuS4 MF–treated hDCs (p , 0.05). (B) hDCs were incubated with the bohydrates are often incorporated and associated with membrane indicated concentration of SchuS4 MF for 18 h, followed by addition of E. coli structures in bacteria. Therefore, we tested total MF enriched from LPS (10 ng/ml). At 20 h later, supernatants were assessed for IL-12p40 by ELISA. *Significantly less than LPS only–treated control (p , 0.05). (C and D) whole-cell lysates of SchuS4 for its ability to stimulate and/or hDCs were incubated with MF, MF heat treated for 4 h at 56˚C (C), or MF inhibit cytokine production from hDCs. As a positive control for treated with pronase (D)(allat10mg/ml) for 18 h, followed by addition of LPS. stimulation of IL-12p40 by bacterial components, we also tested At 20 h later, supernatants were assessed for IL-12p40 by ELISA. *Signifi- MF obtained from attenuated F. tularensis LVS. Similar to our cantly less than LPS only–treated control (p , 0.05). In each experiment, each previous observations using intact bacteria, MF from SchuS4 condition was tested in triplicate. Error bars represent SEM. Data are repre- does not elicit IL-12p40 from hDCs (Fig. 1A and Refs 7, 10). In sentative of three experiments of similar design, using different donors. The Journal of Immunology 1279

We also assessed whether the absence of IL-12p40 was asso- ciated with induction of anti-inflammatory cytokines. However, we do not detect either IL-10 or TGF-b in supernatants of hDCs treated with SchuS4 MF (data not shown). Because SchuS4 MF fails to induce secretion of detectable IL- 12p40, we next determined if SchuS4 MF inhibits the ability of hDCs to respond to secondary stimuli, as previously observed following infection with viable SchuS4 (10). The hDCs pretreated with SchuS4 MF secrete significantly less IL-12p40 in response to LPS, compared with cells treated with LPS alone (Fig. 1B). We next determined if the “active” component of the SchuS4 MF had FIGURE 2. SchuS4 lipids inhibit secretion of IL-12p40 by hDCs. hDCs characteristics shared with lipid- and/or carbohydrate-containing were treated with the indicated concentration of lipids isolated from compounds, such as being heat stable and pronase resistant. Sim- SchuS4 or LVS for 18 h, followed by addition of LPS for an additional ilar to our previous observations, the components of SchuS4 MF 20 h. EtOH served as vehicle control. Culture supernatants were assessed for capable of inhibiting secretion of IL-12p40 by hDCs in response to IL-12p40 by ELISA. *Significantly less than EtOH + LPS–treated controls , E. coli LPS are heat stable and pronase resistant (Fig. 1C, 1D). (p 0.05). In each experiment, each condition was tested in triplicate. Error bars represent SEM. Data are representative of three experiments of similar These data strongly suggest that anti-inflammatory molecule(s) design, using different donors. associated with SchuS4 may be a lipid and/or carbohydrate. Downloaded from Lipids derived from virulent F. tularensis SchuS4 inhibit of several cell surface receptors, a process known as phenotypic inflammatory responses maturation. Therefore, we also examined changes in CD80, CD86, HLA-DR, and CCR7 on the surface of hDCs treated with lipids Data generated with the MF of SchuS4 suggest that the compo- before and after exposure to Pam3CSK4. SchuS4 lipids do not nents associated with the MF that inhibit inflammatory responses induce increased expression of any cell surface assessed are either lipids or carbohydrates. Therefore, we separated lipid (Fig. 3). Interestingly, SchuS4 lipids have no effect on the upreg- and nonlipid materials from SchuS4 and LVS, using a modified http://www.jimmunol.org/ ulation of HLA-DR and CCR7 among hDCs treated with Pam3Csk4 Folch method, and determined their ability to activate or inhibit (Fig. 3). However, in correlation with the inhibition of cytokine inflammatory responses in hDCs. Nonlipid components of the production, hDCs treated with SchuS4 lipids express significantly bacteria present in the aqueous phase of the SchuS4 or LVS pre- less CD80 and CD86 on the cell surface following addition of parations do not stimulate a measurable cytokine response in hDCs Pam3CSK4 (Fig. 3). Thus, SchuS4 lipids also affect the ability of at the concentrations tested (Supplemental Fig. 2). When examined hDCs to undergo phenotypic maturation. for suppressive activity, nonlipid products present in aqueous preparations from SchuS4 and LVS are capable of inhibiting in- SchuS4 lipids inhibit inflammatory responses in vivo flammatory responses in hDCs (Supplemental Fig. 2). However, the We have previously demonstrated that viable SchuS4 inhibits by guest on September 29, 2021 nonstimulatory yet suppressive activity of nonlipid material as- inflammatory responses in vivo, as measured by interference of sociated with LVS is inconsistent with the ability of viable LVS and recruitment of neutrophils following intranasal infection (8). Given total MF isolated from LVS to provoke inflammatory responses in the ability of SchuS4 lipids to inhibit inflammatory responses hDCs. Thus, the nonlipid products isolated from both SchuS4 and LVS do not faithfully replicate the differential stimulatory and/or inhibitory activity of SchuS4 and LVS in hDCs. Because we are pursuing identification of a heat- and pronase-resistant molecule that shares the unique nonstimulatory but inhibitory properties observed among hDCs infected with SchuS4, we tested and compared lipid material found in the organic phase of our extra- ctions of SchuS4 and LVS. Neither lipids isolated from LVS nor those from SchuS4 induce detectable concentrations of IL-12p40 from hDCs following incubation of cells with doses of lipid as high as 30 mg/ml (Fig. 2). Lipids isolated from LVS do not inhibit hDC responsiveness to LPS (Fig. 2). In contrast, SchuS4 lipids potently suppress secretion of IL-12p40 by hDCs in response to E. coli LPS (Fig. 2). Similarly to SchuS4 MF, SchuS4 lipids do not induce secretion of the anti-inflammatory cytokines IL-10 or TGF-b (data not shown). In addition, SchuS4 lipids also inhibit the hDC re- sponse to other TLR agonists, including Pam3CSK4 (TLR2/1), Pam2CSK4 (TLR2/6), LTA (TLR2), R848 (TLR8), and ssRNA (TLR8) (Supplemental Fig. 3). Viability of hDCs treated with SchuS4 lipid was 99.1% compared with 98.2% of EtOH-treated cells and 99% viability in untreated cells. Thus, suppression of cytokine production observed in hDCs treated with SchuS4 lipids FIGURE 3. SchuS4 lipids partially inhibit phenotypic maturation of hDCs. hDCs were incubated with SchuS4 lipids (30 mg/ml) for 18 h, fol- was not associated with toxicity of SchuS4 lipid to hDCs. To- lowed by Pam3CSK4 (10 mg/ml) for an additional 20 h. Changes in surface gether these data show that lipids isolated from SchuS4 interfere expression of the indicated receptors was monitored by flow cytometry. with proinflammatory responses activated following engagement *Significantly less than hDCs treated with Pam3CSK4 alone (p , 0.05). In of multiple TLRs in hDCs. each experiment, each condition was tested in triplicate. Error bars represent In addition to production of cytokines, activation of hDCs by SEM. Data are representative of three experiments of similar design, using exposure to TLR ligands is measured by the increases in expression different donors. 1280 BACTERIAL MODULATION OF IRF FAMILY MEMBERS in vitro (Fig. 2), we next determined if these bacterial components could suppress pulmonary inflammation in vivo. Mice were given 25 mg of SchuS4 lipid intranasally prior to administration of 200 ng E. coli LPS via the same route. At 5 h after inoculation with LPS, airway cells and fluid were collected and assessed for the presence of neutrophils and cytokines. Instillation of E. coli LPS fails to reproducibly induce production of IL-12p40 in the airways of mice; thus we assessed secretion of TNF-a and IL-6 as rep- resentative proinflammatory cytokines. Administration of SchuS4 lipids does not elicit recruitment of neutrophils or secretion of TNF-a or IL-6 into the airways of treated mice (data not shown). Rather, SchuS4 lipids significantly inhibit recruitment of neu- trophils and production of TNF-a and IL-6 in the airways of mice treated with LPS (Fig. 4). Thus, SchuS4 lipids are also capable of inhibiting inflammatory responses in vivo. SchuS4 lipids inhibit translocation of NF-kB to the nucleus Production of IL-12p40 is dependent on activation and translo- cation of specific transcription factors to induce expression. Downloaded from One of these factors includes NF-kB, specifically NF-kB p65 (25). In a resting cell, p65 is bound to IkBa in the host cytosol. Upon cellular activation, the kinases in the IKK family phosphorylate IkBa, targeting it for ubiquitination and degradation. This activity, in turn, results in release of the p65 subunits for activation and

translocation to the nucleus, where they participate in transcription http://www.jimmunol.org/ of proinflammatory genes. Inhibition of cytokine production by SchuS4 lipids in hDCs may be due to interference of activation and/or translocation of NF-kB transcription factors to the nucleus of the host cell. We first examined the ability of SchuS4 lipids to modulate phosphorylation of IkBa. SchuS4 lipids do not inde- pendently induce phosphorylation and/or degradation of IkBa (Fig. 5A), nor do they interfere with activation of IkBa following incubation of cells with E. coli LPS (Fig. 5B).

Data above suggest that SchuS4 lipids are not modulating ac- by guest on September 29, 2021 tivation of NF-kBp65 through canonical pathways. However, ac- tivation of p65 can occur independently of IkBa, and the absence of an effect on the activation of IkBa by SchuS4 lipids does not rule out modulation of NF-kBp65 (26, 27). Using a quantitative assay, we next determined if SchuS4 lipids had any effect on translocation of p65 to the host nucleus. SchuS4 lipids do not independently induce translocation of NF-kB family members in hDCs, but readily inhibit translocation of p65 in response to LPS (Fig. 5C, 5D). Inhibition of p65 translocation is confirmed by FIGURE 4. SchuS4 lipids inhibit inflammatory responses in vivo. Mice microscopy. In agreement with our quantitative results, ∼ 50% of (n = 5 per group) were given 25 mg/25 ml SchuS4 lipids or EtOH (vehicle hDCs pretreated with SchuS4 lipid do not translocate the NF-kB control) intranasally, followed by intranasal instillation of 200 ng/25 ml E. p65 to their nucleus in response to LPS (Fig. 5E). Overall, our data coli LPS 18 h later. At 5 h after administration of LPS, BAL cells and fluid suggest that the suppression of cytokine production in hDCs by were collected and assessed for neutrophils, TNF-a, and IL-6. *Signifi- , SchuS4 lipid is not mediated through manipulation of canonical cantly less than EtOH-treated controls (p 0.05). Error bars represent pathways upstream of NF-kB, but rather is affecting activation SEM. Data are representative of three experiments of similar design. –, Completely untreated mice; BLD, below level of detection. and translocation of NF-kB family members either directly or via noncanonical pathways. within the context of IL-12p40 transcription is examination of its SchuS4 lipids inhibit activation of IRFs ability to bind specific sequences (Ets2 and ISRE) in the promoter In addition to p65, transcription of IL-12p40 is also dependent on of the gene encoding IL-12p40 (22, 32, 33). IRF1 and IRF8 form IRFs, including IRF1 and IRF8 (28–30). IRF1 is also a critical a heterocomplex, and thus, both bind to Ets2 and ISRE promoter transcription factor for several IFN-g–regulated genes involved in regions in IL-12p40 (32). Therefore, we determined whether lipid host defense against intracellular pathogens (31). Thus, we ex- affected the ability of IRF1 and IRF8 to bind to the IL-12p40 amined the impact SchuS4 lipids had on the activation and promoter in primary hDCs. Binding of IRF1 and IRF8 is opti- translocation of IRF1 in hDCs. SchuS4 lipids do not induce mal following stimulation of hDCs with LPS + IFN-g. Before translocation of IRF1, but do inhibit translocation of IRF1 among assessing the effect SchuS4 lipids had on the ability of IRF1 and hDCs responding to LPS (Fig. 6A, 6B). In addition to IRF1, IRF8 IRF8 to bind regions in the IL-12p40 promoter, we first ascer- is also essential for transcription of the IL-12p40 gene (32). No tained whether SchuS4 lipids could inhibit production of IL-12p40 detection methods are available to view IRF8 translocation by in hDCs stimulated with LPS + IFN-g. Similar to hDCs stimulated microscopy. The accepted technique for analyzing IRF8 activity with LPS alone, exposure to SchuS4 lipids results in secretion of The Journal of Immunology 1281

significantly less IL-12p40 by hDCs treated with LPS + IFN-g (Fig. 7A). We next examined DNA binding activity of IRF1 and IRF8 in SchuS4 lipid-treated cells. SchuS4 lipids have no effect on the ability of IRF1 and IRF8 to bind to the ISRE in the IL-12p40 promoter following stimulation with LPS + IFN-g (Fig. 7B). In contrast, SchuS4 lipids inhibit binding of both IRF1 and IRF8 to the Ets2 sequence in the IL-12p40 promoter of hDCs treated with LPS + IFN-g, compared with EtOH-treated controls (Fig. 7B). To determine if the inhibitory effect of SchuS4 lipids on IRF1 and IRF8 activity is a global phenomenon having impact on multiple IRF family members, we also examined the ability of SchuS4 to affect activity of an IRF that is not required for transcription of IL- 12p40. Similar to IRF1, SchuS4 lipids do not induce translocation of IRF3 in hDCs (Fig. 6C, 6D). However, in contrast to their effect on IRF1 and IRF8, SchuS4 lipids do not inhibit translocation of IRF3 following stimulation of hDCs with LPS (Fig. 6C, 6D). To- gether, these data suggest that SchuS4 lipids inhibit production of IL-12p40 by inhibiting translocation of IRF1 and binding of IRF1

and IRF8 to the Ets2 element in the IL-12p40 promoter. Downloaded from Viable SchuS4 inhibits translocation of NF-kB and IRF1 It is well documented that virulent F. tularensis inhibits production of IL-12p40 in mouse and human cells (7–11, 34). However, the mechanism by which this occurs is not clear. Data presented above

indicate that lipids isolated from virulent SchuS4 inhibit IL-12p40 http://www.jimmunol.org/ production by interfering with activation of NF-kB and IRF1. Thus, we next sought to confirm that interference with these transcription factors also occurred among hDCs infected with viable F. tularensis SchuS4. In agreement with previous reports and activity observed with SchuS4 lipids, infection with viable SchuS4 inhibited the ability of hDCs to secrete IL-12p40 (Fig. 8A and Ref. 7). Furthermore, similar to data presented above, viable SchuS4 inhibits translocation of both NF-kB and IRF1 among

infected hDCs (Fig. 8B, 8C), but has no effect on translocation of by guest on September 29, 2021 IRF3 (Fig. 8D). Thus, consistent with suppression mediated by SchuS4 lipids, viable F. tularensis SchuS4 also inhibits translo- cation of the same transcription factors tied to production of IL- 12p40 that are affected by SchuS4 lipids.

Discussion Production of proinflammatory cytokines, including IL-12, is a critical step in the control and eradication of invading micro- organisms. Evasion and suppression of this host response are important features embodied by highly virulent pathogens. As a case in point, in contrast to more attenuated strains and sub- species, virulent F. tularensis both evades triggering inflammatory responses and subsequently inhibits the ability of the host to mount effective responses at key, early time points post infection (8–10, 35). However, the bacterial components, and specific host pathways modulated by these components, are largely undefined. Dendritic FIGURE 5. SchuS4 lipids inhibit activation of NF-kBp65. (A) hDCs cells represent a target for the intracellular replication of F. tularensis, were treated with SchuS4 lipids (30 mg/ml), EtOH (vehicle control), PBS, and pulmonary infection is typically considered the most dan- or LPS (10 ng/ml), and cell lysates were collected at the indicated time gerous manifestation of tularemia (4, 10, 20, 36). Thus, we used points. Phosphorylation and degradation of IkBa were assessed by model systems incorporating primary hDCs and intranasal instil- Western blot. (B) hDCs were treated with 30 mg/ml SchuS4 lipid, EtOH, or lation in the mouse to identify the components of F. tularensis and PBS for 18 h, followed by addition of LPS. At the indicated time points specific signaling pathways involved in immunosuppression me- after LPS addition, cell lysates were collected and assessed for phos- diated by this organism. phorylation and degradation of IkBa by Western blot. (C) hDCs were treated with SchuS4 lipid, EtOH, PBS, or LPS, and translocation of NF- kBp65 (p65) to the cell nucleus was assessed 3 h later, using quantitative TransAM assay. *Significantly greater than all other samples (p , 0.05). (D and E) hDCs were treated with SchuS4 lipid, EtOH, PBS (2) for 18 h, samples (p , 0.05). In each experiment, each condition was tested in followed by LPS. At 3 and 1 h after addition of LPS, translocation of NF- triplicate. Error bars represent SEM. Graphed data are pooled from three kBp65 to the cell nucleus was assessed using quantitative TransAM assay experiments using different donors. Microscopy images are representative (D) or by microscopy (E), respectively. *Significantly less than all other of three experiments of similar design, using different donors. 1282 BACTERIAL MODULATION OF IRF FAMILY MEMBERS Downloaded from

FIGURE 6. SchuS4 lipids inhibit translocation of IRF1 in hDCs. hDCs were treated with SchuS4 lipid (30 mg/ml) or EtOH (vehicle control) for 18 h, followed by LPS (10 ng/ml). At 90 min after addition of LPS, translocation of IRF1 (A, B) and IRF3 (C, D) to the cell nucleus was assessed by microscopy. Translocation of IRF1 and IRF3 was quantitated http://www.jimmunol.org/ by scoring . 500 cells for the presence of nuclear IRF1 or IRF3. *Sig- nificantly less than all other samples (p , 0.05). In each experiment each condition was tested in duplicate. Error bars represent SEM. Graphed data are pooled from three experiments using different donors. Microscopy FIGURE 7. SchuS4 lipids inhibit binding of IRF1 and IRF8 to the hu- images are representative of three experiments of similar design, using man IL-12p40 promoter. hDCs were treated with SchuS4 lipids or EtOH different donors. (vehicle control), followed by LPS + IFN-g.(A) Supernatants were assessed for IL-12p40 by ELISA. SchuS4 lipids significantly inhibited the ability of hDCs to secrete IL-12p40 in response to LPS and IFN-g.*p , 0.05, compared with EtOH-treated and untreated controls. Error bars by guest on September 29, 2021 In this article, we provide evidence that lipids isolated from represent SEM. Data are pooled from four separate experiments using four virulent, but not attenuated, F. tularensis are an important bacterial different donors. (B) SchuS4 lipids inhibit binding of IRF1 and IRF8 to constituent for the suppression of human and murine innate in- the Ets sequence, but not the ISRE sequence, in the IL-12p40 promoter. flammatory responses. We demonstrate that, although not pro- Whole-cell extracts from hDCs treated with EtOH, SchuS4 lipids, and/or voking an inflammatory response, lipids isolated from virulent LPS + IFN-g, as indicated, were incubated with a biotinylated DNA probe F. tularensis SchuS4 readily inhibited upregulation of select cell corresponding to the Ets or ISRE sequence conjugated to streptavidin surface receptors on hDCs associated with hDC activation and beads. Beads were washed, and bound materials eluted for assessment of also suppressed production of IL-12p40 by primary hDCs in re- IRF1 and IRF8 by SDS-PAGE, followed by Western blotting. Data are sponse to a variety of secondary stimuli. Using a mouse model of representative of two experiments using two different donors. pulmonary inflammation, we also found that SchuS4 lipids po- tently inhibited inflammation in vivo. Our data also suggest that respond to secondary stimuli. In this study, we have found that the suppressive lipids associated with SchuS4 are not associated following biochemical extraction, both nonlipid (present in the with LPS—that is, lipid A—because the lipid products described aqueous phase) and lipids (present in the organic phase) isolated in this article are fully capable of suppressing responses in hDCs, from SchuS4 are able to modulate the ability of hDCs to respond whereas SchuS4 LPS does not inhibit hDC function in any de- to various secondary stimuli. However, material isolated from the tectable manner (10). We extended our studies to decipher two aqueous phase of both attenuated LVS and SchuS4 was capable of signaling pathways required for IL-12 transcription that are ma- inhibiting cytokine secretion in hDCs. LVS was derived via serial nipulated by SchuS4 lipids. SchuS4 lipids inhibit translocation of passage from a virulent Type B subspecies and is capable of causing NF-kBp65 in a manner independent of IkBa degradation. SchuS4 overt disease in some humans (37). LVS also retains a route- and lipids also selectively target IRF family members by inhibiting dose-dependent virulence in mice (5, 38). Furthermore, the com- nuclear localization of IRF1, but not IRF3, in LPS-stimulated position of the O-Ag polysaccharide that represents the primary hDCs. Finally, our data point toward a physiological role for nonlipid species in membranes of Francisella is very similar among SchuS4 lipids in mediating the suppression observed in hDCs LVS and Type A isolates (39). Thus, LVS may retain features in its infected with viable SchuS4 by demonstrating that both SchuS4 carbohydrate profile that are associated with pathogenicity that may lipids and viable SchuS4 inhibit NF-kB and IRF1 activation fol- be more prominently featured in fully virulent isolates. Neverthe- lowing infection of hDCs. less, intact LVS and MF isolated from LVS provoke production of This work is an extension of our original observation that heat- proinflammatory cytokines from hDCs, whereas intact SchuS4 and stable products either secreted or shed from virulent F. tularensis MF derived from SchuS4 do not (Fig. 1 and Ref. 10). Thus, the SchuS4 were able to interfere with hDC responsiveness to E. coli nonstimulatory, but suppressive, activity of nonlipid components LPS (10). These data strongly suggest that a nonprotein compo- present in LVS is not consistent with the activity of the intact or- nent derived from SchuS4 contributes to the inability of hDCs to ganism with human cells. The Journal of Immunology 1283 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 8. Viable F. tularensis SchuS4 inhibits translocation of NF-kBp65 and IRF1 in hDCs. The hDCs were infected with a multiplicity of infection = 50 viable SchuS4. As indicated, at 24 h post infection, cells were treated with either Pam3CSK4 (5 mg/ml) or LPS (10 ng/ml). At 18 h later, culture supernatants were assessed for IL-12p40 by ELISA (A), and 90 min after addition of Pam3CSK4, cells were assessed for translocation of NF-kBp65 by microscopy(B). At 90 min after addition of LPS, cells were assessed for translocation of IRF1 and IRF3 by microscopy (C, D). Translocation of NF-kBp65, IRF1, and IRF3 was quantitated by scoring . 500 cells for the presence of nuclear NF-kBp65, IRF1, or IRF3. *Significantly less than uninfected cells (p , 0.05). In each experiment, each condition was tested in duplicate. Error bars represent SEM. Graphed data are pooled from three experiments using different donors. Microscopy images are representative of three experiments of similar design, using different donors.

In contrast to nonlipid material present in the aqueous phase of ious TLR ligands (7, 10). This finding suggests that SchuS4 our extraction, lipid products present in the organic phase of the contains immunosuppressive lipid species that are either unique or SchuS4 extractions are unique in their ability to interfere with more abundant than those found in LVS. inflammatory response in hDCs. Neither LVS nor SchuS4 lipids To date, information on the lipid composition of virulent provoke cytokine production from hDCs. However, only SchuS4 Francisella is sparse. A handful of reports characterize the lipid lipids are able to inhibit the ability of hDCs to secrete IL-12 in species associated with attenuated and virulent strains of F. response to multiple TLR agonists, which is consistent with the tularensis. Virulent F. tularensis has a lipid profile and content that ability of viable SchuS4 to evade induction of proinflammatory are unusual and unique among Gram-negative bacteria. Whereas cytokines while potently suppressing hDC responsiveness to var- the membranes from most Gram-negative bacteria contain ∼ 10–20% 1284 BACTERIAL MODULATION OF IRF FAMILY MEMBERS lipid, F. tularensis membranes are composed of nearly 70% lipid host cell, specifically via the inhibition of phosphorylation of (40). Some have suggested that a large portion of this lipid is free IkBa subunits that (in their nonphosphorylated state) retain p65 in lipid A unassociated with O-Ag, which would partition to the the cytosol (45). Our data demonstrate that SchuS4 is capable of organic phase of our extraction (41), although this phenomenon preventing p65 translocation independently of activation of IkBa. has been documented only for F. novicida and not the other sub- Although it is widely accepted that phosphorylation of IkBa is species of F. tularensis. Lipid A is the lipid portion of the large a critical step in the nuclear translocation of p65, it is not sufficient LPS present in the cell wall of all Gram-negative bacteria. The for the activation of NF-kB subunits. For example, it has been lipid A of LPS is the portion recognized by TLR4 on host cells. A shown that several kinases are important for phosphorylation of direct association exists between the number of acyl groups present p65 and that interference with their activity can result in poor on the lipid A and the stimulatory activity of this molecule (42). translocation and DNA binding of p65 (46, 47). Thus, SchuS4- Specifically, lipid A species with four or fewer acyl groups do not mediated inhibition of p65 translocation may lie in the interfer- provoke inflammatory responses from host cells (42). Among at- ence with kinases required for phosphorylation of p65 downstream tenuated strains—LVS and F. novicida—lipid A is tetraacylated of IkBa. and thus is a poor stimulator for cytokine responses (43, 44). Given In addition to disrupting the activation of p65, we also show that the presence of such unique lipid A in Francisella subspecies, one SchuS4 selectively inhibits activation of members of the IRF family could postulate that perhaps this is the molecule mediating the of transcription factors, specifically IRF1 and IRF8. The IRF family suppressive effect in our preparations. However, several factors consists of nine different proteins originally characterized as argue against this hypothesis. transcription factors that regulated from Type I

First, although the presence of tetraacylated lipid A has not been IFN and IFN-inducible genes (48). It is now appreciated that IRFs Downloaded from confirmed among Type A strains such as SchuS4, purified LPS also play critical roles in cell survival, oncogenesis, and mediation from SchuS4 also lacks the stimulatory properties observed in lipid of signals through pattern recognition receptors for cytokine A and LPS isolated from LVS and F. novicida (10). Moreover, in production, including IL-12 (48). Initially, our finding that SchuS4 addition to the weak ability of SchuS4 LPS to stimulate responses and SchuS4 lipids modulated activation of IRF1 was surprising in host cells, it does not inhibit the ability of cells to respond to because we had previously shown SchuS4 induced rapid, transient

secondary stimuli (10). Second, new evidence suggests that rec- production of IFN-b and, as described above, IRFs are intimately http://www.jimmunol.org/ ognition of altered lipid A/LPS molecules is not the same among involved in transcription of Type I IFN (7). However, depending mice and humans. For example, pentaacylated LPS isolated from on the receptors engaged, transcription of IFN-b requires specific Neisseria meningitidis stimulates weak responses from human IRFs. For example, expression of IFN-b stemming from engage- cells but retains its ability to induce cytokine production from ment of pattern recognition receptors is dependent on IRF3 (49, mouse cells (as reviewed in Ref. 42). Thus, assuming lipid A is 50). Thus, owing to its ability to induce IFN-b, SchuS4 may not involved, one hypothesis may be that the lack of suppressive ac- influence IRF3 activation. Indeed, no significant difference can be tivity of LVS lipids in hDCs, compared with SchuS4 lipids, is noted in the translocation of IRF3 among cells infected with attributable to the fact that we were examining human cells. SchuS4 or treated with SchuS4 lipid. This finding supports our However, we also observed that SchuS4 lipids readily inhibit in- previous work that SchuS4 is not negatively affecting Type I IFN by guest on September 29, 2021 flammatory responses in the mouse (Fig. 4 and D. Crane, R. Ire- pathways. land, J. Alinger, P. Small, and C. Bosio, submitted for publication). In contrast to IFN-b, transcription of IL-12p40 requires IRF1 Therefore, the ability of SchuS4 lipids to modulate inflammatory and IRF8 (33). Others have shown that microbial toxins, such as responses is not dependent on the species of the host, thus arguing cholera toxin, inhibits binding of IRF8, but not IRF1, to specific against a role for lipid A. Finally, in addition to the major lipid A sites in the promoter region of the IL-12p40 gene, to suppress species found in Francisella, low abundant forms of unique lipid A transcription of this cytokine (22). We found that SchuS4 lipids species have also been described (44). However, the presence inhibit binding of both IRF1 and IRF8 at the Ets2 site within the and relative abundance of these minor lipid A species were not IL-12p40 promoter. Thus, the inhibition of IRF1 and IRF8 by different among subspecies of F. tularensis tested—that is, viru- SchuS4 as a mechanism to interfere with production of IL-12p40 lent Type B F. tu la re ns is, F. no vi cid a,andF. philomargia. Together, is unique. IRF1 also plays integral roles in the transcription of these findings suggest it is unlikely that a unique lipid A species several genes encoding elements required for control of F. tularensis, found in SchuS4 is responsible for suppressing inflammatory including inducible NO synthase and caspase-1 (13, 31, 51, 52). responses in human cells. Rather, we propose that other lipids Thus, the targeted interference of IRF1 activation by SchuS4 found in SchuS4 act to inhibit proinflammatory responses in hDCs. represents a mechanism by which the bacterium broadly incapa- In support of this hypothesis, preliminary data from our laboratory citates host innate immune responses. suggest that fatty acids, which we would propose to be present as Regulation of IRF1 activity and its interaction with IRF8 are not free fatty acids rather than those found attached to the lipid A well defined. To date, casein kinase II is the only kinase shown to backbone, are the classes of SchuS4 lipids that mediate anti- phosphorylate IRF1 (53). This interaction has not been confirmed inflammatory responses observed in hDCs (R. Ireland and in primary cells. IRF1 activity can also be regulated by two other C. Bosio, unpublished observations). IRFs. IRF2 can act as a repressor of IRF1 via its ability to compete In addition to identifying the class of molecules found in SchuS4 for binding at the same DNA binding sites (54). However, we that inhibit inflammatory responses, we also identify three host observe an inhibition of IRF1 translocation to the host nucleus. signaling molecules required for transcription of genes encoding Further, it is not clear if IRF2 is capable of binding the Ets2 region proinflammatory cytokines that are modulated by both SchuS4 of the human IL-12p40 promoter. Considered together, this in- lipids and viable SchuS4. NF-kBp65 is critical for the transcription formation suggests that IRF1 competition with IRF2 is not the of multiple inflammatory cytokines, including IL-12 (25). Others mechanism for SchuS4-mediated inhibition of IL-12p40. We also have suggested that F. tularensis may inhibit translocation of this found that binding of IRF8 to the Ets2 site was inhibited by host protein as a mechanism of virulence. However, in these SchuS4 lipid. Heterocomplexes of IRF1 and IRF8 have been studies it was suggested that interference with p65 activation shown to be required for transcription of IL-12p40 (32). Thus, the occurs through modulation of canonical signaling pathways in the impact of SchuS4 lipid on IRF1 and IRF8 binding to the Ets2 site The Journal of Immunology 1285 may be due to poor formation of IRF1–IRF8 heterocomplexes. It 13. Ireland, R., N. Olivares-Zavaleta, J. M. Warawa, F. C. Gherardini, C. Jarrett, B. J. Hinnebusch, J. T. Belisle, J. Fairman, and C. M. Bosio. 2010. Effective, is not clear if the heterocomplex of IRF1 and IRF8 that binds to broad spectrum control of virulent bacterial infections using cationic DNA li- the IL-12p40 promoter must form prior to its entry to the nucleus. posome complexes combined with bacterial antigens. PLoS Pathog. 6: It is possible that the formation of this complex is inhibited by e1000921. 14. Sutherland, M. D., A. W. Goodyear, R. M. Troyer, J. C. Chandler, S. W. Dow, SchuS4 lipids in the cytosol. Alternatively, IRF1 has a short half- and J. T. Belisle. 2012. Post-exposure immunization against Francisella tular- life and is targeted for ubiquitination and degradation by the host ensis membrane proteins augments protective efficacy of gentamicin in a mouse cell (55). It is possible that SchuS4 accelerates the degradation of model of pneumonic tularemia. Vaccine 30: 4977–4982. 15. Huntley, J. F., G. T. Robertson, and M. V. Norgard. 2010. 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Supplemental Figure 1. DNA probes containing Ets2 and ISRE sequences utilized for immunoprecipitation. Schematic of the -244 to -154 sequence of the IL-12p40 promoter with the Ets2 region underlined and in bold. Schematic of the -101 to -11 sequence of the IL-12p40 promoter with the ISRE region underlined and in bold.

Supplemental Figure 2. Non-lipid molecules from Francisella inhibit secretion of IL-12p40 by hDC. hDC were treated with the non-lipid molecules present in the aqueous phase of preparations from LVS and SchuS4 diluted 1:100, 1:500 and 1:1000 in cRPMI for 18 hours followed by addition of LPS for an additional 20 hours. Culture supernatants were assessed for

IL-12p40 by ELISA. ns= not significantly different. * = significantly less than LPS alone treated controls (p<0.05). Triangles represent increasing dilutions of aqueous phase. In each experiment each condition was tested in triplicate. Error bars represent SEM. Data is representative of three experiments of similar design using different donors.

Supplemental Figure 3. SchuS4 lipids inhibit secretion of IL-12p40 by hDC following addition of multiple TLR agonists. hDC were treated with 30 μg/ml SchuS4 lipids for 18 hours followed by addition of Pam3CSK4 (10 μg/ml), Pam2CSK4 (10 μg/ml), LTA (100 μg/ml), R848 (2 ng/ml) or ssRNA40/LysoVec (ssRNA40) (0.25 μg/ml) for an additional 20 hours. EtOH served as vehicle control. Culture supernatants were assessed for IL-12p40 by ELISA. ns= not significantly different. * = significantly less than EtOH+TLR agonist treated controls (p<0.05). In each experiment each condition was tested in triplicate. Error bars represent SEM. Data is representative of three experiments of similar design using different donors.