Role of extracellular vesicles released after oxidative stress in activation of adaptive response

Zunajcelični vezikli (EV) so membranski delci, manjši od 1 μm, ki jih najdemo v krvi in drugih telesnih tekočinah. Vse celice pri normalnih fizioloških pogojih izločajo vezikle, vendar se njihova količina močno poveča pod stresnimi pogoji, kot so hipoksija, oksidativni stres in apoptoza. Na ta način EV prenašajo molekularne motive, povezane s poškodbami (DAMP) na druge celice in tako vplivajo na njihovo delovanje. V raziskovalnem delu smo preučevali učinek EV, ki se sprostijo po oksidativnem stresu (sEV), na polarizacijo naivnih makrofagov. Na podlagi rezultatov smo ugotovili, da sEV sprožijo polarizacijo makrofagov v tip M1 in ne v tip Mox, kot smo predpostavili. Zanimala nas je tudi potencialna vloga sEV pri aktivaciji adaptivnega odgovora v kardiomiocitih, ki celice lahko zaščiti pred nadaljnjimi poškodbami zaradi stresa. Rezultati so pokazali, da sEV v kardiomiocitih povečajo izražanje hem oksigenaze 1 (HO 1), ki celicam s svojimi antioksidativnimi učinki nudi zaščito pred oksidativnimi poškodbami. Še več, predinkubacija kardiomiocitov s sEV je zmanjšala sproščanje laktat dehidrogenaze po hipoksiji/reoksigenaciji, torej je celicam povečala možnost preživetja. To nakazuje, da sEV s svojim delovanjem lahko sprožijo adaptivni odgovor, in najverjetneje predstavljajo enega izmed mehanizmov oddaljenega ishemičnega kondicioniranja.Extracellular vesicles (EVs) are submicrometer-sized particles, composed of phospholipid bilayer, and can be found in blood and other bodily fluids. Cells release EVs under normal physiological conditions, but their amount increases under stress conditions such as hypoxia, oxidative stress and apoptosis. EVs serve as transmitters of damage-associated molecular patterns (DAMPs), which can trigger changes in function of target cells. We investigated the effects of EVs, released after oxidative stress (sEVs), on naïve macrophages. Based on the results, we concluded that sEVs trigger polarization of macrophages to M1 phenotype and not Mox phenotype, as hypothesised. We were also interested in the role sEVs might play by triggering adaptive response in cardiomyocytes, which can protect the cells from the forthcoming tissue injury resulting from oxidative stress. Results show that sEVs induce expression of heme oxygenase 1 (HO-1), which has anti-oxidative properties and can protect the cells from aforementioned oxidative injury. Moreover, preincubation of cardiomyocytes with sEVs decreased release of lactate dehydrogenase after hypoxia/reoxygenation leading to increased cell survival. This suggests that sEVs can trigger adaptive immune response and most likely represent one of the mechanisms present in remote ischemic conditioning

Calcium Ionophore-Induced Extracellular Vesicles Mediate Cytoprotection against Simulated Ischemia/Reperfusion Injury in Cardiomyocyte-Derived Cell Lines by Inducing Heme Oxygenase 1

Cardioprotection against ischemia/reperfusion injury is still an unmet clinical need. The 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 the isolation of EVs from blood takes considerable effort, the aim of our study was to establish a cellular model from which cardioprotective EVs can be isolated in a well-reproducible manner. EV release was induced in HEK293 cells with calcium ionophore A23187. EVs were characterized and cytoprotection was assessed in H9c2 and AC16 cell lines. Cardioprotection afforded by EVs and its mechanism were investigated after 16 h simulated ischemia and 2 h reperfusion. The induction of HEK293 cells by calcium ionophore resulted in the release of heterogenous populations of EVs. In H9c2 and AC16 cells, stressEVs induced the 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 cells, which was independent of TLR4 induction, but not that of HO-1. Calcium ionophore-induced EVs exert cytoprotection by inducing HO-1 in a TLR4-independent manner

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and β-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small β-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The β-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the β-annulus peptide for vaccine design