Project description:Infectious pneumonias exact an unacceptable mortality burden worldwide. Efforts to protect populations from pneumonia have historically focused on antibiotic development and vaccine-enhanced adaptive immunity. However, we have recently reported that the lungs’ innate defenses can be therapeutically induced by inhalation of a combination of synthetic TLR ligands that synergize to protect mice against otherwise lethal pneumonia. Simultaneous treatment with ligands for TLR2/6 and TLR9 conferred robust, synergistic protection against virulent Gram-positive and Gram-negative pathogens, as well as viruses. Protection is associated with rapid pathogen killing in the lungs, and pathogen killing can be induced from lung epithelial cells in isolation. Here we explore the mechanisms underlying this dramatic phenomenon by performing microarray gene expression analysis of mouse lungs treated by aerosol with PBS (sham treatment), Pam2CSK4 (TLR 2/6 ligand), ODN2395 (TLR9 ligand), or both TLR ligands.
Project description:Infectious pneumonias exact an unacceptable mortality burden worldwide. Efforts to protect populations from pneumonia have historically focused on antibiotic development and vaccine-enhanced adaptive immunity. However, we have recently reported that the lungs’ innate defenses can be therapeutically induced by inhalation of a bacterial lysate that protects mice against otherwise lethal pneumonia. Here, we tested in mice the hypothesis that Toll-like receptors (TLRs) are required for this antimicrobial phenomenon, and found that resistance could not be induced in the absence of the TLR signaling adaptor protein MyD88. We then attempted to recapitulate the protection afforded by the bacterial lysate by stimulating the lung epithelium with aerosolized synthetic TLR ligands. While most single or combination treatments yielded no protection, simultaneous treatment with ligands for TLR2/6 and TLR9 conferred robust, synergistic protection against virulent Gram-positive and Gram-negative pathogens. Protection was associated with rapid pathogen killing in the lungs, and pathogen killing could be induced from lung epithelial cells in isolation. Taken together, these data demonstrate the requirement for TLRs in inducible resistance against pneumonia, reveal a remarkable, unanticipated synergistic interaction of TLR2/6 and TLR9, reinforce the emerging evidence supporting the antimicrobial capacity of the lung epithelium, and may provide the basis for a novel clinical therapeutic that can protect patients against pneumonia during periods of peak vulnerability. MLE-15 cells were treated with 20 ul volumes of PBS (sham treatment), ODN2395 (0.4 ug), Pam2CSK4 (0.2 ug) or ODN2395+Pam2CSK4. At least 5 unique samples per group. Treated for 2 hours. Hybridized to Illumina Sentrix MouseRef-8 v2 Beadhips.
Project description:Infectious pneumonias exact an unacceptable mortality burden worldwide. Efforts to protect populations from pneumonia have historically focused on antibiotic development and vaccine-enhanced adaptive immunity. However, we have recently reported that the lungs’ innate defenses can be therapeutically induced by inhalation of a combination of synthetic TLR ligands that synergize to protect mice against otherwise lethal pneumonia. Simultaneous treatment with ligands for TLR2/6 and TLR9 conferred robust, synergistic protection against virulent Gram-positive and Gram-negative pathogens, as well as viruses. Protection is associated with rapid pathogen killing in the lungs, and pathogen killing can be induced from lung epithelial cells in isolation. Here we explore the mechanisms underlying this dramatic phenomenon by performing microarray gene expression analysis of mouse lungs treated by aerosol with PBS (sham treatment), Pam2CSK4 (TLR 2/6 ligand), ODN2395 (TLR9 ligand), or both TLR ligands. C57BL/6J mice were placed, unrestrained, in a aerosolization chamber and inhalationally exposed to 20 minute treatment with an 8 ml volume of PBS (sham), Pam2CSK4 10 ug/ml, ODN 2395 20 ug/ml, or the combination. 4 h after treatment, the mice were deeply anesthetized, their lungs were harvested, homogenized, and total RNA was extracted. Amplified cRNA was hybridized to Illumina Sentrix MouseRef-8 v2 Beadhips, labeled with Cy3, and scanned on an Illumina iScan. At least 8 unique samples were obtained per condition.
Project description:Among both healthy and immunocompromised patient populations, pneumonia is a leading cause of death worldwide. Yet, despite structural vulnerability resulting in recurrent exposure to pathogens, the lungs’ mucosal immunity successfully suppresses most infections. We recently reported that these innate defenses can be substantially augmented by inhalational exposure to a crude bacterial lysate, protecting broadly against respiratory pathogens, including lethal pneumonia caused by bacteria, fungi or viruses. The phenomenon of inducible resistance is associated with rapid pathogen killing in the lungs and persists in the absence of the typical leukocytes of innate immunity. Rather, the respiratory epithelium appears to be the predominant effector. Toll-like receptors (TLRs) are highly conserved pattern recognition receptors crucial to host defense through the sensing of pathogen associated molecular patterns. Given the importance of TLRs to mucosal immunity, the presence of numerous pathogen associated molecular patterns in the bacterial lysate, and the induction of many TLR-dependent genes following lysate treatment, we hypothesized that induced resistance follows simultaneous stimulation of multiple TLRs. To test this, we challenged mice deficient in TLR/IL1R adaptor proteins and found that resistance could not be induced in mice lacking MyD88. Having identified this phenomenon to be MyD88-dependent, we sought to determine whether the protective phenomenon could be recapitulated by treatment with synthetic TLR agonists. Mice were treated with aerosolized TLR ligands, alone and in combination, prior to infection with virulent pathogens. While limited protection against pneumonia was afforded by the individual TLR ligands, we discovered that the synergistic combination of diacylated lipopetide TLR2/6 agonist Pam2CSK4 and CpG oligodeoxynucleotide TLR9 agonist ODN2395 induced profound resistance against all tested pathogens. This combination also induced greater than additive pathogen killing in the lungs of challenged mice, and we found that the combination could effectively induce pathogen killing by respiratory epithelial cells in vitro. In order to better understand the mechanisms underlying the inducible pathogen killing by this unique combination of TLR agonists, we performed microarray analysis of murine MLE-15 respiratory epithelial cells following 4 h treatment with PBS (sham treatment), Pam2CSK4 alone, ODN2395 alone, or the combination of both agonists, using Illumina Sentrix MouseRef-8 v2 BeadChips. This allows for assessment of differential gene expression, not only between treated and untreated, but between single and combination treated. The intent of the experiment is to gain insight into the transcriptionally-regulated means by which TLR2/6 and TLR9 signaling pathways synergistically interact. Keywords: Differential expression, epithelium, in vitro
Project description:Infectious pneumonias exact an unacceptable mortality burden worldwide. Efforts to protect populations from pneumonia have historically focused on antibiotic development and vaccine-enhanced adaptive immunity. However, we have recently reported that the lungs’ innate defenses can be therapeutically induced by inhalation of a bacterial lysate that protects mice against otherwise lethal pneumonia. Here, we tested in mice the hypothesis that Toll-like receptors (TLRs) are required for this antimicrobial phenomenon, and found that resistance could not be induced in the absence of the TLR signaling adaptor protein MyD88. We then attempted to recapitulate the protection afforded by the bacterial lysate by stimulating the lung epithelium with aerosolized synthetic TLR ligands. While most single or combination treatments yielded no protection, simultaneous treatment with ligands for TLR2/6 and TLR9 conferred robust, synergistic protection against virulent Gram-positive and Gram-negative pathogens. Protection was associated with rapid pathogen killing in the lungs, and pathogen killing could be induced from lung epithelial cells in isolation. Taken together, these data demonstrate the requirement for TLRs in inducible resistance against pneumonia, reveal a remarkable, unanticipated synergistic interaction of TLR2/6 and TLR9, reinforce the emerging evidence supporting the antimicrobial capacity of the lung epithelium, and may provide the basis for a novel clinical therapeutic that can protect patients against pneumonia during periods of peak vulnerability.
Project description:Epithelial malignancies are effectively treated by antiangiogenics; however, acquired resistance is a major problem in cancer therapeutics. Epithelial tumors commonly have mutations in the MAPK/Pi3K-AKT pathways, which leads to high-rate aerobic glycolysis. Here, we show how novel multikinase inhibitor antiangiogenics (TKIs) induce hypoxia correction in spontaneous breast and lung tumor models. When this happens, the tumors down-regulate glycolysis and switch to long-term reliance on mitochondrial respiration. A transcriptomic, metabolomic and phosphoproteomic study revealed that this metabolic switch is mediated by down-regulation of HIF1α and AKT and up-regulation of AMPK, allowing uptake and degradation of fatty acids and ketone bodies. The switch renders mitochondrial respiration necessary for tumor survival. Agents like phenformin or ME344 induce synergistic tumor control when combined with TKIs, especially in a sequential schedule, leading to metabolic synthetic lethality. Our study uncovers new mechanistic insights in the process of tumor resistance to TKIs, and may have clinical applicability
Project description:Immunological memory is generally thought to be mediated exclusively by lymphocytes such as memory T and B cells. However, enhanced innate immune responses caused by a previous infection increase protection against reinfection suggesting the presence of innate immunological memory. Here, we describe expression profile of peritoneal macrophages from wild-type mice pre-administrated with TLR ligands or from ATF7 knockout mice. ATF7 suppresses a group of innate-immunity genes in macrophage by recruiting H3K9 dimethyltransferase G9a. TLR ligands induce ATF7 phosphorylation, leading to release of ATF7 from chromatin and reduction in H3K9me2 level. Partially disrupted chromatin structure and increased basal expression on target genes are maintained for a long period, increasing resistance pathogens. Therefore we speculate ATF7 is important factor in controlling innate immunological memory. This series contains seven sets of exression array data. For all sample, we use four CEL files generated by four biological-independent experiments.
Project description:Many successful vaccines induce persistent antibody responses that can last a lifetime. The mechanisms by which they do so remain unclear, but emerging evidence suggests that activate dendritic cells (DCs) via Toll-like receptors (TLRs). For example, the yellow fever vaccine YF-17D, one of the most successful empiric vaccines ever developed, activates DCs via multiple TLRs to stimulate pro-inflammatory cytokines. Triggering specific combinations of TLRs in DCs can induce synergistic production of cytokines, which results in enhanced T cell responses, but its impact on antibody responses remain unknown. Learning the critical parameters of innate immunity that programs such antibody responses remains a major challenge in vaccinology. We demonstrated that immunization of mice with synthetic nanoparticles containing antigens plus Toll-like receptor (TLR) ligands 4 (MPL) + 7 (R837) induces synergistic increases in antigen-specific, neutralizing antibodies compared to immunization with a single TLR ligand. To determine whether there was any early programming of B cells, we isolated isotype switched, TCRbeta-CD11b-CD19+IgD-IgG+ B cells by FACS at 7 days post immunization with nanoparticles containing various adjuvants plus OVA, and performed microarray analyses to assess their molecular signatures. Two independent sets of samples at day 7 post-treatment were used in our analyses. Each set is comprised by B-cells from mice treated with MPL + R837 or from those treated with either individual MPL or R837 alone.