Viktória Szeifert1, Ákos M. Lőrincz2, András Ács3, László Drahos4, Ágnes Kittel5, Erzsébet Ligeti6
1PhD student, Department of Physiology, Semmelweis University, Budapest
2Research fellow, Department of Physiology, Semmelweis University, Budapest
3Assistant research fellow, MS Proteomic Research Group, Hungarian Academy of Sciences, Budapest
4Senior research fellow, MS Proteomic Research Group, Hungarian Academy of Sciences, Budapest
5Scientific advisor, Experimental Research Institute of Hungarian Academy of Sciences, Budapest
6Professor, Department of Physiology, Semmelweis University, Budapest
Introduction: Our group previously described three distinct extracellular vesicle (EV) populations released from human neutrophilic granulocytes (PMN): EVs formed spontaneously (sEVs), upon activation with different opsonized particles (aEVs) and during apoptosis (apoEVs).
Aims: We aimed to examine the effect of an inflammatory cytokine, TNF- α pre-treatment on the production and functional properties of these populations of PMN EVs.
Method: We isolated EVs by a two-step centrifugation and filtration from PMNs isolated from the peripheral blood of healthy volunteers. We examined the morphology and size of the EVs by transmission electron microscopy. We evaluated the EV production by flow cytometry, the EV’s protein content by Bradford assay and we also examined their protein cargo by LC-MS/MS. We performed functional analysis of the EVs by the measurement of the bacterial survival of S. aureus by optical density-based measurement and in parallel, by our previously published flow cytometry based method.
Results: TNF-α pre-treatment did not cause change in the morphology of the EVs, but resulted a shift to smaller EV size. This pre-treatment itself increased the sEV release, but the protein-content did not change. Meanwhile, the TNF-α could not increase further the aEV formation, therefore the previously observed aEV/sEV ratio decreased. TNF-α was not able to substitute the opsonization in case of the non-opsonized particles induced aEV formation. The TNF-α pre-treatment slightly increased the ratio of the granule proteins in sEVs, and increased the granule protein content even more in aEVs. The TNF-α pre-treatment itself did not result in EVs with antibacterial effect and this pre-treatment could not potentiate further antibacterial effect of the aEVs.
Conclusion: Our findings emphasize that the effect of TNF-α on the EV release of human neutrophilic granulocytes is highly dependent on the secondary activator such as opsonins.
Funding: NKFIH K119236, Hungary
Doctoral School: Molecular Medicine
Program: Cellular and Molecular Physiology
Supervisor: Ákos M. Lőrincz
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