Pathogen infected macrophages release exosomes associating w

Pathogen-infected CCK Octapeptide, non-sulfated release exosomes associating with pathogen-associated molecular patterns (PAMPs) such as LPS and lipoprotein derived from microorganisms [29,30]. Exosomes released by bacterially infected, but not uninfected, macrophages stimulate macrophages to release inflammatory mediators via MyD88-dependent signaling pathway. Thus, exosomes derived from infected macrophages play important roles in innate and early immune responses [31]. However, it is still elusive whether components of exosomes are altered during stimulation to activate macrophages. In this study, we further examined the roles of secreted ERAP1, and demonstrate that ERAP1 secreted from LPS/IFN-γ-stimulated macrophages are associated with exosomes derived from activated macrophages. The ERAP1-associated exosomes enhanced the phagocytic and NO synthetic activities of macrophages. Four proteins (i.e. ERAP1, tumor necrosis factor (TNF)-α, IFN-γ, and C-C motif chemokine ligand (CCL) 3) were associated with exosomes after stimulation and found to be involved in the activation of macrophages. Our data provides new insights into the roles of exosomes derived from activated macrophages in immune and/or inflammatory responses.
Materials and methods
Results
Discussion In this study, we determined that exosomes released from macrophages in response to inflammatory stimuli enhanced phagocytosis and NO synthesis of macrophages. A small but significant subset of secreted ERAP1 derived from LPS/IFN-γ-stimulated macrophages was associated with exosomes and played important roles in macrophage activation. To our knowledge, this is the first study to demonstrate the important roles of the exosome-associated form of ERAP1. Although macrophages secreted exosomes constitutively, the association of several proteins including ERAP1 and cytokines/chemokines were observed only after treatment with LPS/IFN-γ. Therefore, it is conceivable that inflammatory stimuli mediated the changes in composition, physicochemical properties, and pathophysiological functions of exosomes released by macrophages. These results suggest that the exosome-associated form of ERAP1, as well as several cytokines/monokines plays important roles in the innate immune system, and modifies inflammatory reactions. It was reported that ERAP1/ARTS-1 was a membrane bound enzyme acting as a sheddase of several cytokine receptors [[38], [39], [40]]. In addition, exosome-like vesicular localization of secreted ERAP1 together with TNFR1 was also reported [41]. These results suggest that ERAP1 can modify inflammatory process via binding to biological membranes. ERAP1 but not ΔEx10ERAP1 effectively bound to AP(+)-exosomes, suggesting the presence of specific binding sites on the exosomes. The exon 10 sequence is known to be important for ER-retention of the enzyme [21]. Several reports described the binding proteins of ERAP1 [23,[38], [39], [40],42], and it was recently shown that ERp44 that is associated with ER membrane via KDEL receptor [43] might act as an ER-retention machinery of the enzyme [23]. In addition, EpCAM that is an integral membrane protein [44] was shown to bind to ERAP2, and act as a possible linker between the enzyme and the plasma membrane [45]. These results implicate that ERAP1 is associated with biological membranes including ER via interaction with specific proteins such as cytokine receptors and/or ERp44. Considering these interactions, it is most plausible that ERAP1 binds to exosomes via interaction with specific protein(s). Because exosomes released from various sources were often heterogeneous in terms of their compositions and pathophysiological functions, it is possible that a distinct subpopulation having unique ERAP1 binding capacity (sites) might be selected during the stimulation of macrophages. It is interesting to identify the putative binding protein on AP(+)-exosomes. At present, no evidence is available whether ERAP2 is secreted in response to inflammatory stimuli. Because ERAP2 has an exon 10 sequence that reveals ~40% homology with that of ERAP1, it is interesting to examine whether ERAP2 is secreted by the stimuli and then bind to exosomes.