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    • Treatment of RAW cells with AP

    2023-01-16

    Treatment of RAW264.7 4μ8C with AP(+)-exosomes caused an increase in their phagocytic activity. In the presence of amastatin, phagocytic activity was not completely suppressed, suggesting that at least two components were responsible for the activity; one of which is aminopeptidase(s). When the activity of AP(+)-exosomes was compared to AP(−)-exosomes, the difference between them was comparable with that measured in the presence or absence of amastatin, suggesting that amastatin-sensitive phagocytic activity was almost exclusively attributable to ERAP1. Therefore, our data strongly supports the notion that ERAP1 in the AP(+)-exosomes was one of the key players in the phagocytosis-enhancing activity of the exosomes. In our previous work, we found that enzymatically active but not inactive ERAP1 enhanced the phagocytic activity. It is plausible that secreted and exosome-associated ERAP1 produce phagocytosis-enhancing peptide(s) via cleavage of substrate(s) in the extracellular space [18]. With regards to amastatin-insensitivity, we found that CCL3 was critical to the phagocytosis-enhancing activity of AP(+)-exosomes. In the presence of J113863, while the phagocytosis-enhancing activity of AP(+)-exosomes was maintained, that of AP(−)-exosomes decreased to around the basal level. Therefore, we conclude that almost all the phagocytosis-enhancing activity of AP(+)-exosomes was attributable to the combination of ERAP1 and CCL3. However, it is noteworthy that in AP(+)-exosomes, only a small amount of ERAP1 was required for the maximal enhancement of phagocytosis. Moreover, AP(+)-exosomes still retained a significant level of phagocytic activity even in the presence of J113863 (Fig. 5C). To the same level, a large amount of ERAP1 was required when treated alone (Fig. 5D). Thus it is plausible that in addition to CCL3, another unidentified protein is also included in the exosome-mediated phagocytosis-enhancement in a synergistic manner. We also determined that AP(+)-exosomes, but not N-exosomes, drastically enhanced NO synthesis in RAW264.7 cells. In contrast to the observed phagocytosis-enhancing activity, the NO synthesis-enhancing activities of AP(+)- and AP(−)-exosomes were comparable. Thus, the role of ERAP1 in NO synthesis was unclear in conditioned medium containing adequate free Arg. However, when the activity was measured in Arg-free medium, ERAP1-mediated enhancement was observed. We found that a significant portion of the NO synthesis-enhancing activity was dependent on a substrate of ERAP1 with N-terminal Arg, in the absence of free Arg. Moreover, in the presence of TNF-α antagonist and/or IFNGR1, a partial but significant suppression of the activity was observed, indicating that TNF-α and IFN-γ were involved in the NO synthesis-enhancing activity of AP(+)-exosomes. While iNOS was induced by AP(+)-exosomes in the presence of IFN-β, TNF-α antagonist and IFNGR1 suppressed its expression, indicating that TNF-α and IFN-γ acted as iNOS inducers in the exosomes. In summary, cytokines (i.e. IFN-β, IFN-γ and TNF-α) and ERAP1 enhance NO synthesis in RAW264.7 cells. The cytokines function as iNOS inducers, and ERAP1 (if any) as a supplier of free Arg. ERAP1 cleaves N-terminal Arg of its substrate peptide and thus maintains the free Arg levels in the blood vessel. However, other components must be included in the NO synthesis-enhancing activity of AP(+)-exosomes since considerable activity still remained. It should be stressed that we obtained the same results by employing murine peritoneal macrophages, which further supports the pathophysiological relevance of ERAP1-involved-macrophage activation mediated by exosomes. It is now evident that exosomes are involved in intracellular communication by interacting with target cells and delivering their contents [46]. Using proteomic and transcriptomic analyses, many proteins and mRNAs were identified in exosomes [[47], [48], [49], [50], [51], [52]], supporting the notion that exosomes can participate in pathophysiological processes such as immune defense. In addition, it was reported that cells released distinct exosome subpopulations with unique compositions that might elicit differential effects on recipient cells [53]. It was also reported that the protein and mRNA compositions of breast cancer exosomes differed from exosomes derived from salivary glands [54]. In this study, we demonstrate that exosomes mediated the phagocytosis- and NO synthesis-enhancing activities of LPS/IFN-γ-stimulated macrophages after their composition changed from N- to AP(+)-exosomes. Since the protein concentration of exosomes in the blood vessels was reported to be 30 to 500 μg/mL [55,56], which was within the range employed in this study, the data presented should have some pathophysiological relevance. Given that exosomes are consistently released from cells, continuous and specific targeting of the exosomes to recipient cells might enhance the local concentrations and prevent diffusion and dilution of effector molecules in the blood vessels and/or body fluids. Packaging and condensation of several effector molecules into AP(+)-exosomes together might be advantageous to facilitate their efficient actions on macrophages.