In analysing Toll like Receptor (TLR) signaling induced by bacterial and viral ligands and by providing continuing access to standardized microarray technology, this project is one of the “bridge-building projects” within the Network Infection & Inflammation. Scientifically, the project aims at analysing genetic determinants of TLR initiated signaling used by various innate immune cells to discriminate “self” from pathogen (bacteria, virus) derived “non-self”. Technologically, the integrated Microarray and Bioinformatics Core Unit provides the projects of the Network I&E with state of the art transcriptional profiling and data analysis services. In addition, we make genetic tools for the study of TLR pathways in the murine and human system available to partners in the Network. In NGFN-1, we have used gene expression profiling to identify common (MyD88 dependent) and unique (TRIF dependent) gene activation programs of innate immune cells after stimulation with a variety of defined TLR-ligands. Furthermore, we learned that in addition to bacterial ligands, TLRs are also key in recognising virus derived ligands. Of note, when TLRs are triggered together with other surface receptors (bearing either “immune activating” or “inhibiting” cytoplasmatic motifs) signaling may be modulated in a positive or negative manner, implying that the integration of multiple signals determines the molecular and biological phenotype in vivo. In NGFN-2 we propose to tackle this complexity by dissecting the contribution of these additional cellular factors to the overall innate immune response. It is expected that the findings provide a basis for new strategies of disease control including vaccine development.
Overview of results from NGFN-1
Toll-like receptors are innate immunity’s sensors for detection of a wide range of infectious danger signals. Triggering of TLR-signaling during pathogen encounter by innate immune cells initiates the host response (infection resistance), leading to the release of inflammatory cytokines (causing inflammation, directing T cell polarization) and chemokines (directing cell migration).
We have addressed the question which common and unique gene expression programs are induced in murine macrophages and dendritic cells by diverse TLR ligands. Gene expression in response to the ligands for TLR1/2, TLR2/6, TLR7 and TLR9 (utilizing Myd88) was remarkably similar. In contrast, ligands for TLR3 and TLR4 uniquely up-regulated Interferon pathway genes, consistent with utilization of the TRIF adapter molecule. In primary mouse DCs, however, CpG ODN effectively induced the interferon pathway similar to LPS although the former is MyD88 dependent and the latter not. Thus, down-stream of MyD88 there must exist yet unknown signal pathway(s) that cause phosphorylation of the transcription factor IRF-3. Cell-type specificity in transcriptional responses to CpG and LPS was also revealed by profiling of B cells, that displayed only partially overlapping patterns of gene activation when compared to macrophages and DCs.
In collaboration with S. Bauer and H. Hochrein (Munich) we identified single stranded RNA motifs as physiological virus derived ligand for the Orphan receptor TLR7 and CpG-DNA motifs within the Herpes simplex DNA virus family as physiological TLR9 ligand. Finally we realised that during bacterial and viral infection, multiple TLRs are being triggered together with other receptor bearing “stimulatory” and “inhibitory” cytoplasmatic domains, an example being members of the Fc receptor family. It follows that integration of multiple signals will determine the molecular and biological phenotype in vivo.
The Microarray and Bioinformatics Core Unit Munich was established as part of the Network in NGFN-1 and is fully operational. During NGFN I, several hundred RNA-samples, provided by groups of the Network, of other NGFN-Networks, or by academic groups unrelated to the NGFN, were processed and analysed on Affymetrix GeneChips. Previously existing protocols for amplification of minute amounts of mRNA (e.g. from needle biopsies or sorted cells) have been optimized to the specific needs of the Network. The spectrum of methods for inter-array normalisation, statistical analysis, visualisation, and annotation of data now includes all major public-domain and commercially available algorithms. This enables us to tailor the analysis strategy to the specific experimental situation under investigation, in a process that is guided by rigorous quality control steps. A comparative analysis of the transcriptional signatures induced in the various models of host-pathogen interactions has been started, and genes regulated in response to most pathogens as well as such genes that are differentially expressed by the host cell in a pathogen-specific way, have been identified. Ultimately, these gene lists will help to define common and pathogen-specific signal transduction pathways operating in host cells.
Project status in NGFN-2
Modulation of TLR-induced cellular activation by cell surface receptors and intracellular signaling proteins
The use of synthetic ligands has yielded important insights into common and TLR-specific signaling pathways. However, during encounter of innate immune cells with pathogens there will be multiple signals by microbial ligands or cytokines. The impact of regulatory mechanisms on the functional response to TLR ligands in macrophages and dendritic cells is studied with a focus on:
• direct components of the TLR signaling machinery, including the adapter molecules Myd88 and TRIF, to complement the results obtained with Myd88 deficient dendritic cells and clarify whether adapter protein usage is differentially required in a cell type specific manner.
• ITAM / ITIM containing cell surface receptors, e.g. Fc receptor family, DC-SIGN and paired immunoglobulin like receptors. These receptors are triggered either by pathogen-derived ligands or ligands on other immune cells.
• intracellular proteins identified in NGFN-1 as being specifically regulated by TLR-ligands and expected to have an impact on the quality or quantitiy of the cellular response, including a group of dual specificity phosphatases and the SOCS family of proteins.
The reagents used to dissect the integration of TLR-induced signaling are primarily macrophages and dendritic cells from transgenic mice deficient in these molecules or overexpressing them. Modulating surface receptors are stimulated or blocked by using appropriate antibodies. Pilot experiments reading out simple parameters like cytokine production or NO release are used to establish optimal stimulation conditions. Then, we employ gene expression profiling to determine the impact of the activation or inhibition / deletion of individual factors on the signature response to TLR ligands.
Novel ligands for TLRs: single-stranded RNA motifs and CpG motifs within DNA viruses
Work by S. Bauer’s lab at the TU Munich has shown that single stranded RNA motifs represent the long searched physiological ligand for TLR7, that is generated e.g. in the course of Influenza virus infection. In collaboration with U. Koszinowski it was found that DNA viruses such as HSV family members trigger innate immune responses via TLR9, while it was shown that viral glycoproteins can activate TLR2 (HCMV, MCMV) and TLR4 (RSV). These observations suggest a central role of TLR family members in antiviral immune response. We are in a strong position to unravel in depth the molecular basis of TLR driven antiviral immune responses, since the molecular tools (gene-deficient mice, constructs for overexpression) are available. In collaboration with the projects of WP3 of this Network, we are planning both in vitro and in vivo experiments designed to inform us on the similarities and differences in signaling pathways governing antiviral versus antibacterial responses at the cellular and systemic level. To this, we will first use transcriptional profiling of innate immune cells stimulated with viral ligands (ssRNA, glycoproteins) or whole viruses, and secondly the appropriate gene-deficient mice (TLR2,4,7,9) for analysis of in vivo responses. It is also planned to perform reconstitution of irradiated wildtype mice with KO bone marrow cells and, vice versa, to address the contribution of TLR signaling in hemopoietic versus epithileal cells to protective and pathological responses in vivo.
Provision of gene expression analyses by the Microarray and Bioinformatics Core Unit
In NGFN-2, functional genomics using Affymetrix GeneChip technology will continue to play a major role in many projects of the KG Infection and Inflammation and in other KG or SMPs (see Networking table under I). Since there is considerable need for this type of microarray analyses, the goal of this part of the project is to provide continuing access to the infrastructure and expertise of the Microarray and Bioinformatics Core Unit which was established in NGFN-1. The platform is fully operational, including a new generation Affymetrix scanner, enabling us to perform state of the art experiments in a cost effective manner. Genome-wide transcriptome analyses will form the core of the service provided to the projects in the Network. As a new feature, genomic mapping and hegh-density SNP analyses are feasible with the existing resources. The experience gained during NGFN-1 at all levels of global gene expression expression experimentation, and the standardized procedures we established for RNA processing and data normalization / analysis procedures, are essential for optimal perfomance of microarray experiments. The set-up of provision of gene expression analyses is straightforward and is working well with numerous collaborators in the Network Infection and Inflammation: experimental RNA samples from Network projects are sent on dry ice to the Core Unit for quality control, labeling and hybridisation to Affymetrix GeneChips. If only minute amounts of RNA can be obtained, established and validated RNA amplification methods are performed. The most important aspect of the work of the Core Unit has turned out to be the tailoring of experimental planning and data analysis project-specific needs. Therefore, we have found that communication by phone and e-mail, as well as meetings between scientists from the projects and the service platform are essential for the successful use of functional genomics in the study of innate immunity to infection.
Outlook
A great deal has been learned in the last years about the recognition of infectious non-self by innate immunity’s receptors, most notably the TLR family. In elucidating the signaling pathways triggered by TLR ligands, both genetic mouse models with defects in the molecules involved and the tools of functional genomics were instrumental. Ongoing work in this project is revealing how the functional outcome of innate immune cell activation is determined by the cell type involved and signals emanating from other cell surface receptors, as well as the action of regulatory factors.
Lit.: 1. Schmitz F, Mages J, Heit A, Lang R, Wagner H. 2004. Transcriptional activation induced in macrophages by TLR ligands: from expression profiling to a model of TLR signaling. Eur J Immunol 34: 2863. 2. Ludwig H, Mages J, Staib C, Lang R, Sutter G. 2005. Role of viral factor E3L in vaccina virus MVA infection of human cells – regulation of the virus life cycle and identification of differentially expressed host genes. J Virol 79: 2584 3. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, Lipford GB, Wagner H, Bauer S. 2004. Species-specific recognition of single-stranded RNA via TLR7 and 8. Science 303: 5663 4. Lang R, Pauleau AL, Parganas E, Takahashi Y, Mages J, Ihle JN, Rutschman RL, Murray PJ. 2003. SOCS3 regulates the plasticity of gp130 signaling. Nat Immunol 4: 546.
