Alsteens, DavidPoncin, MéganeVan Meerbeeck, PierrePierreVan Meerbeeck2025-05-142025-05-142025-05-142019https://hdl.handle.net/2078.2/12481Stroke is one of the major public health concerns worldwide. While part of mortality is related to the lack of blood supply to the brain, another part is caused by the triggering of a detrimental inflammatory process due to the restoration of the blood supply, known as post-ischemic reperfusion inflammation. This causes the immune system, which is normally dedicated to protecting our body, to further damage brain cells. Recent evidence suggests that this inflammation is initiated by the activation of Toll-like receptors (TLRs) by a family of proteins called peroxiredoxins (PRDXs) that are released from damaged cells. Among others, peroxiredoxin 5 (PRDX5) plays a central role by binding TLR4. However, this interaction needed to be more characterized, as it remained unclear whether (i) PRDX5 redox state could influence the interaction with TLR4, and whether (ii) accessory molecules, such as cluster of differentiation 14 (CD14) or myeloid differentiation factor 2 (MD-2), were implicated in the intracellular signalization. We first investigated the interaction of multiple PRDX5 redox forms (reduced, oxidized and hyperoxidized) with TLR4 using force-distance curve-based atomic force microscopy (AFM). This method allows imaging samples at high resolution, mapping biophysical properties and quantifying biomolecular interactions at the same time. By probing the interactions between single PRDX5 redox forms grafted on the AFM tip and TLR4s immobilized on model surfaces, we observed specific interactions between PRDX5 and TLR4. Moreover, the interaction properties seem independent on the redox state. Then, using AFM combined with fluorescence microscopy, we followed the mechanical properties of the human embryotic kidney 293 cells (HEK293) upon binding with PRDX5. We observed an increase of the cellular stiffness only on HEK293 transfected with CD14, MD-2 and TLR4 (HEK293 MD2-CD14 TLR4). Next, we probed the influence of both accessory molecules, MD-2 and CD14, in the immunogenicity of PRDX5. To this end, we exposed HEK293 MD2-CD14 TLR4 to PRDX5, and we measured the release of the cytokine interleukin-8 (IL-8) with an enzyme-linked immunosorbent assay (ELISA). The results seem to show that MD-2 and/or CD14 mediate(s) the activation of a TLR4-independent signaling pathway leading to the secretion of IL-8. In addition, a sufficient level of TLR4 expression is required to allow cytokine secretion. However, these experiments were preliminary, and further controls are required. As perspectives, AFM combined with fluorescence could be used to put in evidence the in situ activation of the NF-κB pathway leading to cytokine secretion after PRDX5 binding.Atomic Force MicroscopeDamage-associated molecular patternPeroxiredoxin5Toll-like receptor 4Sterile inflammationtext::thesis::master thesisthesis:19644