Periodic Reporting for period 3 - POST-IT (Pathogen Oriented SNARE Trafficking for Immune Tailoring)
Período documentado: 2023-05-01 hasta 2024-10-31
Immune clearance of infectious diseases requires correct activation of an immune cell type called T cell. T cells are activated by another immune cell type called macrophage that shows fragments of pathogenic microbes to the T cells. However, macrophages can also show fragment of ‘healthy’ self-material. This is a potential problem, because activation of T cells with this self-material might cause autoimmune disease. The goal of POST-IT is to understand the cellular mechanisms how macrophages can selectively present fragments of ingested microbes, but not self-material, to T cells.
In the future, an understanding of these mechanisms could aid the development of new diagnostics or medicine for autoimmune diseases and infections. To accomplish this, the general objectives of POST-IT are:
-Objective 1: Understanding how macrophages distinguish microbial pathogens from self-material, with the hypothesis that this is due to differential intracellular sorting of organelles.
-Objective 2: Studying the role of immune signaling in this process, with the hypothesis that this alters the intracellular sorting of organelles (via phosphorylation).
-Objective 3 is to determine the functional consequences on T cell activation, using a novel and highly sensitive chemical approach.
In the future, an understanding of these mechanisms could aid the development of new diagnostics or medicine for autoimmune diseases and infections. To accomplish this, the general objectives of POST-IT are:
-Objective 1: Understanding how macrophages distinguish microbial pathogens from self-material, with the hypothesis that this is due to differential intracellular sorting of organelles.
-Objective 2: Studying the role of immune signaling in this process, with the hypothesis that this alters the intracellular sorting of organelles (via phosphorylation).
-Objective 3 is to determine the functional consequences on T cell activation, using a novel and highly sensitive chemical approach.
We wrote several reviews related to the topic of POST-IT during the lockdown of the Covid-19 pandemic. We also performed a bioinformatics analysis of the fragments that are presented to T cells by macrophages. We developed the microscopy technique for objective 1 (understanding how macrophages sort microbial pathogens), and already used this technique to show the intracellular sorting of organelles. We also established and phenotyped all the cellular models for macrophages and T cells from human blood. We also made good progress with the second objective of POST-IT (determine how sorting is affected by phosphorylation). Using a screening technique, we gained a complete overview of all phosphorylated proteins, and identified key proteins that might be responsible for the differential sorting of ingested microbes. Finally, we developed the new T cell activation assay for Objective 3.
POST-IT has led to the following technological developments and state of the art knowledge. First, the development of a new technique for measuring the intracellular sorting of organelles. We used this technique to characterize the cellular phenotype of a novel genetic disease caused by a mutation in an alternative starting codon in the gene coding for an organelle trafficking protein. Second, we developed a new approach for measuring activation of T cells, which is more sensitive than other assays. Third, we generated a comprehensive set of plasmids for measurement of pH in organelles. Given the large importance of pH regulation in both cell biological and (bio)chemical processes, this will be valuable to the broad cell biology and biochemistry communities. Using this toolset, we discovered a new cell biological mechanism: that organelles can be heterogenous is composition. This finding provides an important new insight in the intracellular processing of different pathogens by immune cells. Finally, using a screening approach, we discovered a key protein that becomes phosphorylated upon recognition of bacteria, but not upon healthy self-material. Using the approaches above, we are currently following up on this finding as this might explain how macrophages can selectively present fragments of ingested microbes, but not harmless self-material, to T cells.