Bianco Nicole R, Kim Seon-Hee, Morelli Adrian E, Robbins Paul D
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Methods Mol Biol. 2007;380:443-55. doi: 10.1007/978-1-59745-395-0_28.
Initial studies in our laboratory were focused on the use of dendritic cells (DC) genetically modified to express Th2-derived cytokines (i.e., interleukin [IL]-4 and IL-10) or apoptotic proteins (i.e., Fas Ligand [FasL]) to reduce inflammation in a mouse model of experimentally induced arthritis. Exosomes are nano-sized vesicles (40-100 nm diameter) released by different cell types, including DC, that contain many of the proteins thought to be involved in regulating the immune response. We have demonstrated that exosomes derived from immature DC treated with immunomodulatory cytokines (i.e., IL-10, IL-4) are able to inhibit inflammation in a murine footpad model of delayed-type hypersensitivity (DTH) and reduce the severity of established collagen-induced arthritis (CIA). In fact, the exosomes were as therapeutic as the parental DC. Because purified DC-derived exosomes are very stable vesicles, they may be a better approach for future treatment of arthritis and other autoimmune disorders than the more unstable DC. In this chapter we detail a protocol for preparing the exosomes produced by murine bone marrow-derived DC. We also review methods to assess the purity and concentration of purified exosomes, by using electron microscopy, Western blot analysis, and flow cytometry. Finally, we describe methods to assess the function of exosomes in vitro, using the mixed leukocytes reaction, and in vivo by means of DTH and an experimental model of CIA.
