Post-transcriptional regulation of IFN-signaling
The innate immune system is the primary defence mechanism of a host cell which acts against a variety of pathogens upon an inflammatory stimulus. The response needs to be tightly regulated to fight infections but avoid toxicity at the same time. The Hertzog lab recently performed HITS-CLIP and RNA-Seq experiments and reported more than 100 miRNAs, a large amount of which are novel, that are induced or repressed upon IFN-β-treatment in bone marrow derived macrophages (BMMs) and target major components of innate immune response, like TLR4, IFNAR1, STAT1, JAK1 or P2rx7. Additionally, their data revealed a change in the 3’UTR-length of many IFN-modulated transcripts, which leads to loss or gain of miRNA- and protein- binding sites. Differential binding of IFN-regulated miRNAs or RNA-binding-proteins can lead to amplification or reduction of the translation level and, as recent discoveries have shown, differential binding of 3’-UTRs by scaffold proteins like HuR can change function and location of the resulting protein.
We want to validate the IFN-induced changes in 3’UTRs of different transcripts by RTPCR and characterise the mechanism of change by examining RNA binding proteins. In addition, we will characterise interactions of these novel miRNAs with their proposed targets using luciferase reporters and their implications on cell responses. We will investigate the effects of differential miRNA binding on components involved in IFN-signaling and inflammasome activation at mRNA- and protein-level and examine the biological effects. These findings will be put in context of global changes in the transcriptome induced by type-1 IFNs.
Systems analysis of paracrine effects after inflammasome activation
Myeloid cells like macrophages (MP) use several families of germline-encoded signaling receptors to recognize microbial pathogens and cell damage inflicted by pathogens. Among them, the inflammasomes are unique, as they are not triggering a transcriptional response, but a proteolytic cascade culminating in the activation of caspase-1, which cleaves precursor cytokines of the IL-1β family. However, inflammasome activation also induces the release of cellular proteins involved in inflammation and tissue repair. Additionally, inflammasome-activated MP undergo pyroptotic cell death and release their activated inflammasomes into the extracellular environment. While the activation mechanisms of inflammasomes and the activities of IL-1β family members have been extensively studied, little is known about how inflammasome- mediated unconventional release of proteins, pyroptotic cell death or the extracellularly released inflammasomes influences bystander cells, such as MP, endothelial cells or adaptive immune cells. Since inflammasome activation is important in the pathogenesis of multiple acute and chronic inflammatory disease, we here propose studying cell-to-cell communication between inflammasome-activated cells and surrounding innate immune cells, stromal cells and adaptive immune cells using a combination of systems biology, biochemical and cellular immunological approaches. The Latz and Hertzog groups have jointly generated inflammasome reporter mice critical for this and other projects of this IRTG, which allows visualizing local inflammasome activation in tissues using fluorescent microscopy. We will activate inflammasomes in vivo and analyse its influence in various tissues on cellular reprogramming and on adjacent stromal, myeloid and T cells. These in vivo activation studies will be complemented by supernatant transfer experiments from inflammasome-activated MP to different types of recipient cells. We anticipate that we will uncover and characterise in detail novel functions of inflammasomes and provide a better understanding of how acute inflammatory reactions are turned off, how tissue repair can be induced by inflammatory triggers and how chronic inflammation may cause fibrotic tissue responses. Students will have the opportunity to learn various techniques from different disciplines such as imaging, cellular immunology, genomics and bioinformatics. The project will benefit from the complementary expertise in Melbourne (genomics, bioinformatics) and Bonn (imaging, inflammasomes).