Characterization of Inflammation-induced changes in exosomes and evaluation of their diagnostic potential

• Applicant: Dr. Mitja Leonard Heinemann
• Subject Area: Public Health, Healthcare Research, Social and Occupational Medicine
• Term: from 2017 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 391125364

Exosomes are small (d=40-100 nm), bilipid-layer enclosed vesicles, which are actively shed by different cell types and contain a variety of proteins, lipids, and nucleic acids. Depending on the originating cell type, the contents of an exosome will vary. Exosomes are currently believed to play an important role in intercellular communication. Depending on their contents, exosomes may induce various cellular reactions. Current work on Exosomes lays a primary focus on exosomal nucleic acids (e.g. miRNA, mRNA) and surface proteins within the exosomal membrane (e.g. CD63). Little is known on the effect of exosomal bioactive lipids, which are to be investigated in this project. For this purpose, previous work has been dedicated to the development of a method for the analysis of exosomal lipids, such as eicosanoids and polyunsaturated fatty acids (PUFA). The objective of this project is to examine whether the lipid profile of monocyte exosomes changes in inflammatory conditions. Additionally, the functional effects of exosomes derived from monocytes exposed to inflammatory conditions will be investigated. To explore these objectives, exosomes of a monocyte cell line will be isolated by means of an isolation method that has been developed in previous works. The yielded exosomes will be analyzed with respect to known exosomal characteristics (size, surface proteins), and the reproducibility of the exosome isolation will be assessed. A monocyte cell system will be used for the investigation of inflammation-induced changes within the exosomes: Monocytes will first be differentiated into macrophages. Then, both monocytes and macrophages will be treated with pro-inflammatory cytokines. As a control, some monocytes and macrophages will remain untreated. Exosomes will then be isolated from the four different cellular conditions. In addition to the assessment of exosomal characteristics (size, surface markers), lipid profiles of the exosomes will be analyzed and compared with regards to changes induced by inflammation. Following the assessment of exosomal composition, functional assays will be performed in order to investigate whether exosomes may induce an inflammatory response in target cells. Exosomes from the monocytes and macrophages, some of which were treated with pro-inflammatory cytokines, will then be added to adipocytes. After incubation with exosomes, the inflammatory response of the adipocytes will be assessed by analysis of NFKB activation and Inflammasome activation. Subsequent to this project, a follow-up application is planned in order to translate the findings into an in-vivo model.

• DFG Programme: Research Grants