PI is the first author and shares corresponding authorship. The link between inflammation and cancer is particularly strong in Waldenström’s macroglobulinaemia (WM), a diffuse large B-cell lymphoma, wherein the majority of patients harbor a constitutively active mutation in the innate immune signaling adaptor MyD88. Here we report detection and a functional role of MyD88 in the extracellular vesicles (EVs) shed from WM cells. MyD88L265P was transferred via EVs into the cytoplasm of the recipient mast cells and macrophages, recruiting the endogenous MyD88 that triggered the activation of proinflammatory signaling in the absence of receptor activation. Additionally, internalization of EVs containing MyD88L265P was observed in mice with an effect on the bone marrow microenvironment. MyD88-loaded EVs were detected in the bone marrow aspirates of WM patients thus establishing the physiological role of EVs for MyD88L265P transmission and shaping of the proinflammatory microenvironment. Results establish the mechanism of transmission of signaling complexes via EVs to propagate inflammation as a new mechanism of intercellular communication.
COBISS.SI-ID: 6319130
PI is the first author. We report the enhancement of the lipopolysaccharide-induced immune response by adamantane containing peptidoglycan fragments in vitro. The immune stimulation was detected by Il-6 and RANTES chemokine expression using cell assays on immortalized macrophages. The most active compound was a alpha-D-mannosyl derivative of an adamantylated tripeptide with L-chirality at the adamantyl group attachment, whereby the mannose moiety assumed to target mannose receptors expressed on macrophage cell surfaces. The immune co stimulatory effect was also influenced by the configuration of the adamantyl center, revealing the importance of specific molecular recognition event taking place with its receptor. The immunostimulating activities of these compounds were further enhanced upon their incorporation into lipid bilayers, which is likely related to the presence of the adamantyl group that helps anchor the peptidoglycan fragment into lipid nanoparticles. We concluded that the proposed adamantane containing peptidoglycan fragments act as co-stimulatory agents and are also suitable for the preparation of lipid nanoparticle-based delivery of peptidoglycan fragments.
COBISS.SI-ID: 26490883
PI shares corresponding authorship. PhD students share the first authorship. Cardioprotection against ischemia/reperfusion injury is still an unmet clinical need. Transient activation of Toll-like receptors (TLRs) has been implicated in cardioprotection, which may be achieved by treatment with blood-derived extracellular vesicles (EVs). However, since isolation of EVs from blood is a considerable effort, our aim was to establish a cellular model from which TLR-inducing, cardioprotective EVs can be isolated in a well-reproducible manner. Induction of HEK293 cells by calcium ionophore resulted in release of heterogenous populations of EVs. In H9c2 and AC16 cells stressEVs induced downstream signaling of TLR4 and heme oxygenase 1 (HO-1) expression in H9c2 cells. StressEVs decreased necrosis due to simulated ischemia/reperfusion injury in H9c2 and AC16 cardiomyocytes, which was independent of TLR4 induction but not that of HO-1.
COBISS.SI-ID: 33505539
The CRISPR/Cas system has been developed as a potent tool for genome engineering and transcription regulation. However, the efficiency of the delivery of the system into cells, particularly for therapeutic in vivo applications, remains a major bottleneck. Extracellular vesicles (EVs), released by eukaryotic cells, can mediate the transfer of various molecules, including nucleic acids and proteins. We show the packaging and delivery of the CRISPR/Cas system via EVs to the target cells, combining the advantages of both technological platforms. A genome editing with designed extracellular vesicles (GEDEX) system generated by the producer cells can transfer the designed transcriptional regulator dCas9-VPR complexed with appropriate targeting gRNAs enabling activation of gene transcription. We show functional delivery in mammalian cells as well in the animals. The therapeutic efficiency of in vivo delivery of dCas9-VPR/sgRNA GEDEX was demonstrated in a mouse model of liver damage counteracted by upregulation of the endogenous hepatocyte growth factor, demonstrating the potential for therapeutic applications and thus presenting EVs as a alternative therapeutic delivery route for CRISPR/Cas system.
COBISS.SI-ID: 39877893
PI is the corresponding author. The first author was her PhD student. Damage-associated endogenous molecules induce innate immune response, thus making sterile inflammation medically relevant. Stress-derived EVs (stressEVs) released during oxidative stress conditions were previously found to activate Toll-like receptor 4 (TLR4) resulting in expression of a different pattern of immune response proteins in comparison to lipopolysaccharide (LPS), underlying the differences between pathogen-induced and sterile inflammation. Here we report that synergistic activities of 15-lipoxygenase (15-LO) and secreted phospholipase A2 (sPLA2) are needed for the formation of TLR4 agonists, which were identified as lysophospholipids (lysoPLs) with oxidized unsaturated acyl chain as new endogenous TLR4 agonists. Hydroxy, hydroperoxy and keto products of 2-arachidonoyl-lysoPI oxidation by 15-LO were identified by mass spectrometry and they activated the same gene pattern as stressEVs. Exreacellular PLA2 activity was detected in the synovial fluid from rheumatoid arthritis and gout patients. Furthermore, injection of sPLA2 promoted K/BxN serum induced arthritis in mice, whereby ankle swelling was partially TLR4-dependent. Results confirm the role of oxidized lysoPL of stressEVs in sterile inflammation that promotes chronic diseases. Both 15-LO and sPLA2 enzymes are induced during inflammation, which opens the opportunity for therapy without compromising innate immunity against pathogens.
COBISS.SI-ID: 30457091