• Characterization of Schu S4 delta aroD as a putative vaccine candidate against pulmonary tularemia

      Cunningham, Aimee L.; Santiago, Araceli E.; Mann, Barbara J., Ph.D.; Qin, Aiping; Grassel, Christen; Vogel, Stefanie N.; Barry, Eileen M. (2015-03-09)
      Francisella tularensis is the etiological agent of the human disease tularemia and a Tier 1 Select Agent. There is a need for an efficacious vaccine against this pathogen due to its low infectious dose, high mortality rate, and ability to be spread by aerosol. To this end, we have engineered a defined live attenuated strain derived from the highly human virulent F. tularensis WT strain Schu S4, designated Schu S4ΔaroD; this strain lacks aroD, the 3rd enzyme in the chorismate biosynthesis pathway used for synthesis of aromatic amino acids. Schu S4ΔaroD is attenuated for growth in broth cultures and in both J774 and primary murine peritoneal macrophages, with a pulmonary LD50 > 105 CFU in C57BL/6 mice (compared to WT Schu S4 LD50 < 10 CFU). Intranasal immunization with Schu S4ΔaroD protects against high-dose WT pulmonary challenge in C57BL/6 mice. A single 50 CFU dose of Schu S4ΔaroD generated 80% protection against 100 CFU challenge, and addition of a 105 CFU booster to the initial 50 CFU vaccination is 80% and 40% protective against 500 or 1000 CFU challenge, respectively. Further studies revealed that a 103 or 105 CFU priming dose followed by a 108 CFU boost is 100% protective against 1000 CFU WT Schu S4 pulmonary challenge. This level of protection has not previously been demonstrated by any tularemia vaccine candidate. Bacterial burden following i.n. vaccination found that regardless of initial dose, immunized mice show identical bacterial loads (107 CFU/g tissue in the lung and 105 CFU/g tissue in the liver) at the peak of infection. However, time to peak burden was dose-dependent, as animals receiving a higher initial inoculum (at least 5600 CFU) reach this peak at day 3, as opposed to day 7 in lower initial inoculum groups. Peak bacterial burden correlates with maximal associated histopathology in the liver and peak pro-inflammatory cytokine and chemokine production in lungs, liver, and spleen (TNF-α, IL-1β, Cox-2, KC, iNOS, MCP-1). Our study shows Schu S4ΔaroD serves as an efficacious defined live attenuated vaccine against pulmonary tularemia. We have revealed a dose-dependent response to Schu S4ΔaroD, which may help determine the protective capacity of this vaccine and assist in development of vaccine-associated correlates of protection in the mouse model.
    • Identification of novel mechanisms that regulate pattern-recognition receptor expression and signaling

      Nhu, Quan Minh; Vogel, Stefanie N. (2009)
      Pattern-recognition receptors (PRRs) of the innate immune system detect infection and tissue injury by binding pathogen-associated molecular patterns (PAMPs) and endogenous "danger" ligands, respectively. We proposed the classification of PRRs into classical PRRs that recognize conserved microbial molecules as ligands and nonclassical PRRs that recognize a proteolytic function. This project focused on the identification of novel mechanisms that regulate the expression and signaling of two transmembrane PRRs: classical PRRs, i.e., Toll-like receptors (TLRs), and a nonclassical PRR, i.e., proteinase-activated receptor 2 (PAR<sub>2</sub>). Analysis of basal and lipopolysaccharide (LPS)-induced TLR gene expression in murine macrophages revealed that TLRs 3-6 and 9 were transcriptionally regulated by two key counteracting transcription factors, interferon regulatory factor-1 (IRF-1) and IRF-2. An unexpected role for IRF-2 as a transcriptional activator was identified, in contrast to its well characterized role as a transcriptional repressor, for murine TLR3 gene expression. An examination of signaling interactions between TLRs and PAR<sub>2</sub> identified a novel paradigm for receptor cooperativity between PAR<sub>2</sub> and TLR4 that centers on the shared utilization of TLR adapters. In HEK293T transfectants, PAR<sub>2</sub>-induced activation of NF-κB and IRF-3 reporters was TRIF/TRAM-dependent. Although PAR<sub>2</sub> agonist did not directly activate TLR4, co-expression of PAR<sub>2</sub> with TLR4 resulted in MyD88-dependent synergistic enhancement of PAR<sub>2</sub>-driven NF-κB activation, whereas PAR<sub>2</sub>-induced IRF-3 activation was diminished. We demonstrated a physical interaction between PAR<sub>2</sub> and TLR4, and between PAR<sub>2</sub> and TRIF. Differential, cell-type specific responses were found to occur when PAR<sub>2</sub> and TLRs were simultaneously activated. In murine macrophages, concurrent activation of TLR4 and PAR<sub>2</sub> led to a "T<sub>h</sub>2"-like pattern of cytokine expression, findings that were consistent with PAR<sub>2</sub>-mediated synergistic enhancement of IL-4-induced alternative activation of macrophages. In epithelial cell lines, PAR<sub>2</sub> activation augmented TLR3-induced NF-κB activation while down-regulating TLR3-induced activation of IRF-3 antiviral pathway. In vivo, PAR<sub>2</sub><sup>-/-</sup> and TLR4<sup>-/-</sup> mice were both resistant to PAR<sub>2</sub> agonist peptide-induced footpad edema and to mouse-adapted H1N1 influenza virus-induced lethality. These results provide compelling support for the hypothesis that regulating the extracellular proteinase/anti-proteinase balance might represent an effective therapeutic approach to controlling inflammation and suggest that targeting PAR<sub>2</sub> and/or TLR4 receptors and signaling pathways may offer protection against influenza.
    • Inhibition of TLR2 Signaling by Small Molecule Inhibitors Targeting a Pocket Within the TLR2 TIR Domain

      Mistry, Pragnesh Dilipkumar; Vogel, Stefanie N. (2015)
      Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll-Interleukin-1 receptor resistance (TIR) domains. For all TLRs, except TLR3, recruitment of the adapter, MyD88, to the TIR domains results in downstream signaling that culminates in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate diseases caused by TLR-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein-protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a "pocket" adjacent to the highly conserved P681 and G682 residues of the BB loop. Using "Computer-Aided Drug Design" (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this "BB loop pocket" and, potentially, disrupt TLR2 signaling. In silico screening identified 149 lead compounds and 20 FDA-approved drugs based on their predicted ability to bind in the BB loop pocket. These were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. In this screen, C16H15NO4 ("C29") was identified as a potential TLR2 inhibitor. C29, and a derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages induced by synthetic and bacterial TLR2 agonists. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6 signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle for this novel CADD approach for the identification of inhibitors of TLR signaling.
    • “Interest-STING”: Inhibition of Innate Immune Signaling by Prostaglandin E2

      Mathena, Reilley; Vogel, Stefanie N.; 0000-0002-2049-8139 (2020)
      The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway senses cytosolic double-stranded DNA from microbial or host cells. cGAS-STING activation elicits an inflammatory response, including production of type I interferons, through activation of TBK1 kinase and the transcription factor IRF3. Therefore, negative regulation of cGAS-STING activity would be predicted to prevent microbial-induced or autoimmune inflammatory damage. Based on prior work, we tested the hypothesis that the inflammatory product prostaglandin E2 (PGE2) acts on immune cells to control inflammation induced by cGAS-STING. STING pathway activating agents, 5,6 dimethylxanthenone-4-acetic acid (DMXAA) and cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), were used to initiate STING signaling. Exogenous PGE2 suppressed DMXAA- and cGAMP-induced STING signaling in murine embryonic fibroblasts and primary murine macrophages. Cells treated with PGE2, followed by DMXAA or cGAMP, exhibited decreased activation of TBK1 and IRF3, and decreased inflammatory gene expression, arguing that PGE2 signaling may be a mechanism restricting cGAS-STING activation.
    • TLR4/MyD88/PI3K Interactions Regulate TLR4 Signaling

      Laird, Michelle Helen Weber; Vogel, Stefanie N. (2009)
      Toll-like receptors (TLRs) activate immune responses by sensing microbial structures such as bacterial lipopolysaccharides (LPS), viral RNA, and endogenous "danger" molecules released by damaged host cells. Myeloid differentiation primary response gene 88 (MyD88) is an adapter protein that mediates signal transduction for most TLRs and the IL-1 receptor family and leads to activation of NF-kappaB, MAP kinases, and production of proinflammatory cytokines. TLR4-mediated signaling also leads to rapid activation of phosphoinositide 3'-kinase (PI3K), one of a family of kinases involved in regulation of cell growth, apoptosis, and motility. LPS stimulates phosphorylation of Akt, a downstream target of PI3K, in wild-type (WT) mouse macrophages. LPS-induced phosphorylation of Akt serine 473 was blunted in MyD88-/- macrophages and completely TLR4-dependent. MyD88 and p85 were previously shown to co-immunoprecipitate and an YXXM motif within the Toll-IL-1-Resistance (TIR) domain of MyD88 was suggested to be important for this interaction. To test this hypothesis, we compared expressed MyD88 variants with mutations within the YXXM motif, or lacking the TIR domain or death domain (DD), and measured their capacities to bind PI3K p85, MyD88, and TLR4 by co-immunoprecipitation analyses and their ability to activate LPS-induced NF-kappaB. Additionally, we tested cell-permeable decoy peptides that correspond to the YXXM motif of MyD88 as an alternative way to analyze its role in TLR4 signaling. The cell permeable decoy peptides are presumed to interact with the target protein, thus precluding the target protein's interaction with the protein from which the peptide sequence was derived. The YXXM -> YXXA mutant MyD88 bound more strongly to p85, TLR4, and WT MyD88 than the other variants, yet was significantly less active than WT MyD88, suggesting that these three proteins (TLR4, MyD88, PI3K) interact simultaneously in the signaling platform and that sustained interaction of MyD88/PI3K with the TLR4 intracellular signaling platform negatively regulates signaling. In addition, the YXXM cell permeable peptides had a broad inhibitory effect on TLR4 signaling suggesting the importance of the MyD88 YXXM motif in formation of a fully functional TLR4 signaling platform. We propose a hypothetical model in which sustained PI3K activity at the membrane limits the availability of PI3K substrate, thereby negatively regulating signaling.