Hypotheses and aims: Natural IgM antibodies bind OSE-carrying EVs, mediating their clearance and uptake by target cells, including monocytes, macrophages (MФs), and endothelial cells (ECs). OSE-carrying EVs are predominantly generated during inflammatory processes associated with oxidative stress but may also accumulate in medical blood products upon storage (PhD project 4).
We hypothesize that the presence of OSEs on EVs serves as signal for their differential uptake by target cells and that the uptake, clearance and potential effects in the target cells are influenced by specific natural IgM antibodies and other OSE-binding plasma proteins, such as C-reactive protein. The main objectives of PhD project 5 will be to study the cellular uptake of OSE-carrying EVs in vitro and their clearance in vivo. We will particularly focus on the fate of platelet-derived EVs carrying OSEs and their uptake by monocytes, MФs, and ECs. We propose that binding of IgM will facilitate the uptake and clearance of OSE-carrying EVs by the respective target cells.
Methods: EVs will be isolated from septic plasma as well as from platelet concentrates or red blood cell units stored as described in PhD projects 1 and 4. EVs will be characterized by flow cytometry for the expression of malondialdehyde (MDA). OSE-positive and OSE-negative EVs will be fluorescently labeled and their uptake by the target cells (monocytes, MФs, and ECs) will be studied by flow cytometry and confocal microscopy. Monocytes will be isolated from whole blood and matured to monocyte‐derived macrophages with macrophage colony‐stimulating factor (M-CSF) and granulocyte-macrophage colony‐stimulating factor (GM-CSF) for 5 days. ECs will be isolated from the umbilical vein. The expression of TLR2, TLR4, and the scavenger receptor CD36 on the target cells will be analyzed. Cellular responses initiated by the uptake of OSE-carrying EVs will be assessed by RNA-Seq profiling, cytokine arrays of supernatants, and functional assays (endothelial barrier function, phagocytosis, differentiation potential). Experiments will be performed in presence and absence of OSE-specific IgM. The in vivo clearance of fluorescently labeled OSE-carrying EVs will be studied following injection into mice (wildtype, Rag-1 ko, Siglec-G ko) with and without OSE-specific IgM antibodies. Clearance rates as well as tissue localization will be assessed by histological analysis and flow cytometry of tissues and peripheral blood. In addition, effects on inflammatory gene expression will be studied by RT-qPCR of liver tissues and aortas of injected mice. Chemokine and cytokine levels in plasma will be assessed by multiplex bead assays.
Main supervisor: C.J. Binder (Medical University of Vienna)
Proposed mobility: E. Boilard, Québec (flow cytometry and intravital imaging)