PHOSPHORYLATION DYNAMICS OF TOLL-LIKE RECEPTOR SIGNALING

Innate immune system is the first important barricade of microbial threats such as infection by pathogens such as viruses and bacteria. In addition, they also sense for danger signals arising from dead cells that needs to be cleared without triggering any unwanted inflammation. Our immune system performs this surveillance by multiple cell types of which macrophages are one of the most central is executing these specialized tasks. These cells contain a number of sensor proteins that constantly police our body for pathogens. Once they see a pathogen or a danger signal, the sensor alarms the system to prepare the body for attacking the pathogen leading to various symptoms of inflammation that ultimately leads to clearance of the pathogen. At the same time, it is crucial to turn off this response so that the body comes to a normal state. Any failure in this information flow can be harmful for the body and can lead to several inflammatory diseases. One of the key components in this information flow are reversible chemical modifications of the proteins in these immune cells. Addition of phosphate group to proteins - called phosphorylation (one of the major chemical modifications), is critical for several steps in this information flow. In this study, we are applying cutting-edge approaches to decode how specific phosphorylation events dictate the type and outcome of the immune cell upon sensing a danger signal. So far, our experiments has verified our hypothesis that there is indeed a type-specific outcome and we are continuing this on a larger scale. We have also identified a new gene that is necessary for the resolution of the inflammation. We are currently performing some focused studies to understand the mechanism behind the function of this gene.

Through this grant, we have established several novel technologies and workflows, especially the use of multiOmics technologies and CRISPR on identification of several novel signaling pathways and host factors essential for mediating inflammation and host-response to infection. One of the seminal publications is Pinto et al., in the journal iScience where we performed the first multiOmics analysis of host-response to a SARS-CoV-2 isolate from Norway resulting in the identification of several novel signaling pathways that could be targeted. Additionally, this study also serves as a model for identifying host-directed therapeutics that is crucial for future pandemic prepardeness studies. Through our work, now it is almost a common methodolgy to use multiOmics technologies and CRISPR in our department at NTNU.

This proposal aims to investigate the role of phosphorylation in the first line defense - the innate immune response. Innate immune signaling can be activated under threat from microbial organisms or upon tissue damage. Toll-like receptors are the largest family of molecules that sense these danger or pathogen-associated signals and trigger inflammatory response resulting in clearance of the pathogen and restoration of cellular homeostasis. Post-translational modifications such as phosphorylation, are crucial for these cellular processes and has not been systematically studied yet. Ours and published data suggests that the family of kinases and phosphorylation-based signaling are crucial in a range of inflammatory processes. In addition, the data also points to potential existence of a phosphorylation code for specific Toll-like receptor signaling pathways. This proposal aims to systematically investigate the role of highly druggable kinases and phosphorylation-based signaling in Toll-like receptor signaling pathways. Successful implementation of this proposal will yield new insights into innate immune signaling and will have implications for development of therapeutic strategies in inflammatory diseases and beyond.