Hypotheses and aims: The immunoregulatory functions of MSCs depend on the activation of specific receptors. Activation of Toll-like receptors TLR3 and TLR4 modulate the polarization of MSCs towards an anti- and pro-inflammatory phenotype, respectively. MSC-EVs have been reported to influence the polarization of human macrophages, eliciting a shift from M1 (pro-inflammatory) to M2 (anti-inflammatory) phenotype, however, the implication of TLR activation has not been addressed in this context. We postulate that the differentiation of MSCs towards an anti- or pro-inflammatory phenotype emerges from the respective TLR receptors that are activated, and that this phenotype is passed on to secreted MSC-EVs. Regarding the effect of MSC-EVs on monocytes, we thus propose that TLR4 activation triggers a pro-inflammatory phenotype in MSC-EVs resulting in a shift towards CD16⁺ intermediate monocytes and non-classical monocytes (see PhD projects 1 and 2), while TLR3 activation leads to an anti-inflammatory response maintaining the classical CD16^- monocyte population.

To address this dependence of the immunoregulatory properties of MSC-EVs on TLR2, TLR3 and TLR4 activation, we will generate optogenetic TLR2, TLR3 and TLR4 MSC cell lines, from which EVs expressing light-inducible TLRs will be isolated. Targeting these three receptors will allow us to assess the impact of MyD88-dependent (TLR2), MyD88-independent (TLR3), and biased (TLR4) signaling. In contrast to cellular induction by chemical agents or endotoxins, which can induce non-specific activation, the use of optogenetic cell lines allows for the precise stimulation of individual receptors.

Methods: We will use a lentiviral delivery system for stable integration (knock in) of the opto-TLR constructs into MSCs isolated from the placental amnion. Light induction will allow for the activation or inactivation of signaling pathways downstream of the respective TLRs. The activation of TLR2, TLR3, and TLR4 in the optogenetic cell lines will be characterized by analysis of the phosphorylation of their downstream targets using Western blotting. The expression of TLR2, TLR3, and TLR4 on secreted MSC-EVs will be analyzed by flow cytometry. Activation of the respective signaling pathways will be determined by characterization of the downstream adaptor proteins (MyD88 and TRAF6 for TLR2, TRIF and TRAF3 for TLR3, biased signaling for TLR4) using Western blotting. MSC-EVs will be isolated by differential centrifugation and characterized by nanoparticle tracking analysis and flow cytometry as described in PhD project 2. EVs derived from MSCs after specific activation of TLR2, TLR3, or TLR4 will be co-cultured with monocytes (isolated as described in PhD project 2) either before or after monocyte activation by platelets (PhD project 1), and their potential to induce a shift towards CD16⁺ monocytes or to revert the shift will be determined by flow cytometry.

Main supervisor: C. Wiesner (IMC University of Applied Sciences Krems)

Proposed mobility: B. Giebel, Essen (characterization of optogenetic MSC cell lines and MSC-EVs)