ABSTRACT: Toll like receptors (TLRs) sense microbial products and initiate adaptive immune responses by activating dendritic cells (DCs). Since pathogens may contain several agonists we asked whether different TLRs may synergize in DC activation. We report that in human and mouse DC TLR3 or TLR4 potently synergize with TLR7, TLR8 or TLR9 in the induction of selected cytokine genes. Upon synergistic stimulation, IL-12, IL-23 and Delta-4 are induced at levels 50-100 fold higher than those induced by optimal concentrations of single agonists, leading to enhanced and sustained TH1 polarizing capacity. Using microarray analysis we show that only 1.5% of the transcripts induced by single TLR agonists are synergistically regulated by combinations of TLR4 and TLR8 agonists.. These results identify a combinatorial code by which DCs discriminate pathogens and provide (suggest) a rationale to design adjuvants for TH1 responses. Series_overall_design: 3 untreated, 3 treated with LPS at 2h, 3 treated with LPS at 8h, 3 treated with R848 at 2h, 3 treated with R848 at 8h, 3 treated with LPS + R848 at 2h, 3 treated with LPS + R848 at 8h Keywords: other
INSTRUMENT(S): [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array
Project description:Toll like receptors (TLRs) sense microbial products and initiate adaptive immune responses by activating dendritic cells (DCs). Since pathogens may contain several agonists we asked whether different TLRs may synergize in DC activation. We report that in human and mouse DC TLR3 or TLR4 potently synergize with TLR7, TLR8 or TLR9 in the induction of selected cytokine genes. Upon synergistic stimulation, IL-12, IL-23 and Delta-4 are induced at levels 50-100 fold higher than those induced by optimal concentrations of single agonists, leading to enhanced and sustained TH1 polarizing capacity. Using microarray analysis we show that only 1.5% of the transcripts induced by single TLR agonists are synergistically regulated by combinations of TLR4 and TLR8 agonists.. These results identify a combinatorial code by which DCs discriminate pathogens and provide (suggest) a rationale to design adjuvants for TH1 responses. Series_overall_design: 3 untreated, 3 treated with LPS at 2h, 3 treated with LPS at 8h, 3 treated with R848 at 2h, 3 treated with R848 at 8h, 3 treated with LPS + R848 at 2h, 3 treated with LPS + R848 at 8h
Project description:Immunosuppressive microenvironments block the activity of tumoricidal T and NK cells, allowing cancers to avoid immune elimination. Monocytic myeloid-derived suppressor cells (mMDSC) are an important component of these immunosuppressive milieus. Human mMDSC respond to stimulation via their Toll-like receptors by differentiating into macrophage. Agonists targeting TLRs 1/2 (such as PAM3) induce mMDSC to mature into immumosuppressive M2-like macrophage through a process that involves TNF and IL6. Agonists targeting TLRs 7/8 (such as R848) cause the same precursors to mature into tumoricidal M1-like macrophages, a process in which IL12 plays a central role. The immune suppression mediated by mMDSC is reversed by exposure to TLR 7/8 agonists which thus might be useful adjuncts for tumor immunotherapy. In this study we looked at several time points. hMDSC from four different donors were sorted from PBMC then cultured in the presence of R848, Pam 3 or left unstimulated for 30, 75 and 225 minutes. Late time points included samples from seven donors after 3 days in culture with the same TLR ligands. For microarray experiments total RNA was extracted, amplified into aRNA and coupled to Cy5. Similarly amplified RNA from universal RNA was coupled to Cy3 and used as a reference for each array.
Project description:The innate immune system is the organism’s first line of defense against pathogens. Pattern recognition receptors (PRRs) are responsible for sensing the presence of pathogen-associated molecules. The prototypic PRRs, the membrane-bound receptors of the Toll-like receptor (TLR) family, recognize pathogen-associated molecular patterns (PAMPs) and initiate an innate immune response through signaling pathways that depend on the adaptor molecules MyD88 and TRIF. Deciphering the differences in the complex signaling events that lead to pathogen recognition and initiation of the correct response remains challenging. Here we report the discovery of temporal changes in the protein signaling components involved in innate immunity. Using an integrated strategy combining unbiased proteomics, transcriptomics and macrophage stimulations with three different PAMPs, we identified differences in signaling between individual TLRs and revealed specifics of pathway regulation at the protein level. Overall design: RAW264.7 cells were treated with individual TLR ligands (100 ng/mL LPS, 1 μM P3C, and 1 μM R848 for stimulation of TLR4, TLR2, and TLR7, respectively) for either 1, 2 or 4 h. Two replicates for each condition.
Project description:Toll-like receptor (TLR) signalling activation by pathogens is critical to the induction of immune responses, and demands tight regulation. Chemokine ligand 2 (CCL2) secretion triggered by TLR4 or TLR8 engagement is strongly inhibited upon simultaneous activation of both TLRs in human monocyte-derived dendritic cells (MD-DC). Impaired CCL2 secretion occurs concomitantly to IL-12 up-regulation, being part of a complex regulatory circuit ensuring optimal Th type 1 polarization. Interestingly, triggering selected TLRs or their combinations differently affects nuclear factor-kB p65 activation and microRNA expression. To investigate in details such different modulation we performed a microarray profiling of MD-DCs stimulated by different TLRs agonist or their combination in three different donors. We found that CCL2 supplies an important immunomodulatory role to DCs, and may contribute to dictate the cytokine profile in Th type 1 responses induced by DCs.
Project description:Goal was to detect differences in response to TLR7 versus TLR8 agonists in human monocytes from healthy donors 3 deidentified donors from the Red Cross, monocytes from each donor incubated overnight with either vehicle, TLR7 agonist or TLR8 agonist
Project description:Oxidative stress is a hallmark of inflammation in infection or sterile tissue injury. We show that partially oxidized phospholipids of microvesicles (MVs) from plasma of patients with rheumatoid arthritis or cells exposed to oxidative stress induce activation of TLR4. MVs from healthy donors or reconstituted synthetic MVs can be converted to TLR4 agonists by limited oxidation, while prolonged oxidation abrogates the activity. Activation by MVs mimics the mechanism of TLR4 activation by LPS. However, LPS and MVs induce significantly different transcriptional response profile in mouse BMDMs with a strong inflammation-resolving component induced by the endogenous signals. MVs thus represent a ubiquitous endogenous danger signal released under the oxidative stress, which underlies the pervasive role of TLR4 signaling in inflammation. Overall design: BMDMs (bone marrow derived macrophages) from C57BL/6J strain were stimulated with LPS (100ng/ml), oxidative stress-derived MVs (5000 MV/ml) or MVs-FR (5000 MV/ml). RNA was extracted 4 hours after stimulation. 3 biological replicates (BMDM cells isolated from three different mice) per group.
Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. Here we define the role of TLR4 and MD-2 in LPS signaling by using genome wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin, and are both absolutely required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation. The gene expression profile of macrophages from C57BL/6 and MD-2-deficient mice following either 10 ng LPS /mL, 100 ng lipid A/mL or 10 nM Pam2 stimulation for 2 hours were compared to PBS-stimulated control cells . In vitro differentiated macrophages from two individual WT and MD-2-deficient mice were cultured and stimulated with agonists separately, comparing the gene expression to PBS-stimulated control cells from the same mouse. Comparisons of PBS-stimulated WT cells to PBS-stimulated MD-2-deficient cells were performed to directly compare basal gene expression in the two genotypes.
Project description:Macrophages play a crucial role in HIV-1 pathogenesis. Toll-like receptors (TLRs) are fundamental for innate and adaptive immune responses, but their role in HIV-1 infection is still incompletely understood. The TLR3 and TLR4 ligands poly(I:C) and LPS are known to modulate HIV-1 infection of and replication in monocyte-derived macrophages (MDMs), but the mechanism is incompletely understood. We found that MDMs stimulation with poly(I:C) or LPS abrogated infection by CCR5-using, macrophage-tropic HIV-1, or by VSV-G-pseudotyped HIV-1 virions, while TLR7 and TLR9 agonists Imiquimod and CpG only reduced infection to varying extent. Suppression of infection, or lack thereof, did not correlate with differential effects on CD4 or CCR5 expression, type I interferon induction, or production of pro-inflammatory cytokines. Furthermore, integrated pro-viruses were readily detected in unstimulated, TLR7- and TLR9-stimulated cells, but not in TLR3- or TLR4-stimulated MDMs, suggesting the alteration of post-entry, pre-integration event(s). MicroRNA (miRNA) microarray and real time PCR demonstrated increased miR-155 levels in MDMs upon TLR3/4, but not TLR7, stimulation, and a miR-155 inhibitor partially restored infectivity in poly(I:C)-stimulated MDMs. Finally, miR-155 over-expression in MDMs and cell lines remarkably diminished HIV-1 infection, inducing an accumulation of late reverse transcription products, concurrently with a decrease in mRNA levels of several HIV-1 dependency factors involved in nuclear import of pre-integration complexes. Our results suggest that miR-155 may target mRNA(s) for host cell protein(s) that either participate in or facilitate post-entry, pre-integration events, resulting in severely diminished HIV-1 infection. miRNA profiles were investigated in total RNA isolated from unstimulated and TLR3-, TLR4- and TLR7-stimulated human MDMs from a single normal donor
Project description:The inflammatory response initiated by microbial products signaling through Toll-like receptors (TLRs) of the innate immune system is essential for host defense against infection. Because inflammation can be harmful to host tissues, the innate response is highly regulated. Negative regulation of TLR4, the receptor for bacterial lipopolysaccharide (LPS), results in LPS tolerance, defined as hyporesponsiveness to repeated stimulation with LPS. LPS tolerance is thought to protect the host from excessive inflammation by turning off TLR4 signal, which then shuts down TLR-induced genes. However, TLR signaling induces hundreds of genes with very different functions. We reasoned that genes with different functions should have different requirements for regulation. Specifically, genes encoding proinflammatory mediators should be transiently inactivated to limit tissue damage, while genes encoding antimicrobial effectors, which directly target pathogens, should remain inducible in tolerant cells to protect the host from infection. Using an in vitro system of LPS tolerance in macrophages, here we show that TLR-induced genes may indeed be divided into two distinct categories based on their functions and regulatory requirements. Further, we show these distinct groups are regulated by gene-specific, and not signal-specific mechanisms. Keywords: Treatment Comparison Overall design: We examined gene expression using affymetrix genechips in 3 groups of murine bone-marrow derived macrophages: Naive (untreated), Naive stimulated with LPS, and Tolerant stimulated with LPS. Two biological replicates were performed for each group.
Project description:Rationale: Previous work demonstrated that pre-exposure to ozone primes innate immunity and increases TLR4-mediated response to subsequent stimulation with lipopolysaccharide (LPS). To further explore the pulmonary innate immune response to ozone exposure, we investigated the effect of ozone in combination with Pam3CYS, a synthetic TLR2/TLR1 agonist. Methods: Bronchoalveolar lavage (BAL) and lungs were harvested from C57Bl/6 mice after exposure to ozone or filtered air followed by saline or Pam3CYS 24 hours later. Cells and cytokines in the BAL, surface expression of TLRs on macrophages, and lung RNA genomic expression profiles were examined. Results: We demonstrate an increased BAL cell influx, increased IL-6 and KC, and decreased MIP-1α and TNF-α in response to Pam3CYS as a result of ozone pre-exposure. We also observed increased cell surface expression of TLR4, TLR2 and TLR1 on macrophages as a result of ozone alone or in combination with Pam3CYS. Gene expression analysis of lung tissue revealed a significant increase in expression of genes related to injury repair and cell cycle as a result of ozone exposure. When comparing Pam3CYS treated animals to saline treated animals with or without ozone, genes associated with inflammation were significantly increased. Potentially novel ozone exposure candidate genes (CCK, RELM-α and β) were identified. Conclusion: Our results extend previous findings with ozone/LPS to other TLRs PAMPs and suggest that ozone priming of innate immunity is a general mechanism. Gene expression profiling of lung tissue identified transcriptional networks and genes that contribute to the priming of innate immunity at the molecular level. Overall design: Mice were exposed to either 2ppm ozone or filtered air (FA) for 3 hours. 24 hours following ozone exposure, mice from either the ozone or FA group were treated intratracheally with either 100ug of Pam3CYS in saline or saline alone. Animals were euthanized 4 or 24 hours post-Pam3CYS exposure. RNA from whole lung tissue from 4 animals per group was profiled.