Hypotheses and aims: Oxidation-specific epitopes (OSEs) are present on (lipo)proteins, apoptotic cells, cell debris and modified proteins that accumulate in response to inflammatory processes. They represent an important class of damage-associated molecular patterns and are recognized by membrane-bound pattern recognition receptors (PRRs), including TLR2, TLR3, and TLR4, as well as by soluble PRRs, including natural IgM antibodies, complement factor H, and C-reactive protein.
We have previously identified a subset of EVs exposing OSEs on their surface that accumulate at sites of inflammation and have shown that OSE-specific natural IgM antibodies bind to these EVs and have the capacity to block their pro-inflammatory and pro-coagulant potential. The putative role of OSE-carrying EVs in inducing a shift towards pro-inflammatory CD16+ monocytes have not been addressed so far, and it remains to be assessed whether the interaction of OSE-carrying EVs with monocytes is associated with enhanced TF expression.
We postulate that OSE-carrying EVs can promote a shift from classical monocytes towards CD16+ intermediate and non-classical monocytes, whereas binding of natural IgM antibodies to OSE-carrying EVs can block or reduce the pro-inflammatory effect of OSE-carrying EVs. We will therefore determine the effect of OSE-carrying EVs on the relative abundance of monocyte subsets and assess the impact of IgM binding on the interaction of OSE-carrying EVs with monocytes.
OSE-carrying EVs accumulate not only during inflammation but may also be enriched in blood products upon prolonged storage (PhD project 1). The characterization of pEVs and rbcEVs obtained in PhD project 1 will thus be extended to the presence of oxidation-specific epitopes. The function of IgM binding in initiating the uptake of OSE-carrying EVs by target cells, such as endothelial cells, monocytes, and macrophages will be addressed in PhD project 5.
Methods: EVs will be isolated from septic plasma as well as from platelet concentrates or packed red blood cells stored as described in PhD project 1 (fresh vs. 5d storage for platelet concentrates, fresh vs. 28d storage and normoxic vs. hypoxic storage for red blood cells). They will be characterized for the presence of OSEs and TF using flow cytometry. OSE-carrying EVs will be characterized by malondialdehyde (MDA) expression and will be isolated by cell sorting. Their interaction with monocytes and the effect on monocyte subset distribution will be determined using the experimental set-up from PhD project 1. Experiments will be performed in the presence and absence of IgM to define the shielding effect of natural IgM antibodies. Monocytic cells with light-inducible TLR2, TLR4, and with light-inducible CD36 will be developed using the approach described in PhD project 3 and will be used to define the impact of individual TLRs on monocyte interaction with OSE-carrying EVs.
Main supervisor: C. Wiesner (IMC University for Applied Sciences Krems)
Proposed mobility: M. Radisic, Toronto (signal transduction, organs-on-a-chip for inflammation research)