AEC-derived secretomes play a crucial role in intercellular communication, influencing key pro-regenerative processes such as immunomodulation. The present study focused on the impact of the enriched microvesicle (MV) fraction of secretomes released from ovine AECs during expansion, characterized by their lipidic and mitochondrial cargo. To this end, a mechanical injury was induced in the caudal fin of 72-hour post fertilization (hpf)-zebrafish larvae to model acute inflammation and subsequent tissue response. The transgenic lines Tg (mpx:GFP) and Tg (lyz:DsRed), which detect neutrophils with green fluorescence and neutrophils/macrophages with red fluorescence, respectively, were employed for anatomical orientation, blood vessels identification and, tracking of immune cell migration. To demonstrate the interspecies transfer of MVs’ cargo from ovine AEC to zebrafish, mitochondrial heteroplasmy was used as a marker. Confocal microscopy analyses confirmed the transfer of active ovine mitochondria in Tg (mpx:GFP) zebrafish larvae. Internalized mitochondria were recorded at 6-hour post damage (hpd) in both damaged tissues and throughout the caudal area. Migration of MitoTracker red-stained mitochondria was detected within incorporating cells located in the wounded area. Moreover, quantification of ovine mitochondrial DNA copies by qPCR revealed a progressive degradation of ovine mitochondria from 24 to 48 hpd. Time-lapse imaging of Tg (lyz:DsRed) larvae demonstrated that the internalization of AEC-derived MVs significantly accelerated the early phase (from 0.5 hpd to 1 hpd) of immune cells recruitment at the wound site. Finally, to assess the pro-regenerative long-term effect of AEC MVs on the injured caudal fin, larvae from 72 hpf to 120 hpf, were imaged daily. Interestingly, AEC-derived MVs facilitated faster and more complete fin regeneration (11 out of 20 MV-treated larvae vs 4 out of 10 in CTR). Notably, fins of MV-treated larvae, cut at the beginning of circulation., started to recover the anatomical microarchitecture, including fin rays and pigments by 48 hpd. These findings underscore the crucial role of AEC-derived secretomes’ MV cargo in modulating immune response and tissue regeneration, paving the way for new controlled and targeted therapeutic cell-free approaches.
IN VIVO EMPLOYMENT OF AEC FOR THE SETUP OF IMMUNOMODULATORY AND REGENERATIVE CELL FREE PROTOCOLS
Ludovica Sulcanese;Giuseppe Prencipe;Monia Perugini;Annamaria Iannetta;Umberto Tosi;Giulia Capacchietti;Valentina Russo;Barbara Barboni
2024-01-01
Abstract
AEC-derived secretomes play a crucial role in intercellular communication, influencing key pro-regenerative processes such as immunomodulation. The present study focused on the impact of the enriched microvesicle (MV) fraction of secretomes released from ovine AECs during expansion, characterized by their lipidic and mitochondrial cargo. To this end, a mechanical injury was induced in the caudal fin of 72-hour post fertilization (hpf)-zebrafish larvae to model acute inflammation and subsequent tissue response. The transgenic lines Tg (mpx:GFP) and Tg (lyz:DsRed), which detect neutrophils with green fluorescence and neutrophils/macrophages with red fluorescence, respectively, were employed for anatomical orientation, blood vessels identification and, tracking of immune cell migration. To demonstrate the interspecies transfer of MVs’ cargo from ovine AEC to zebrafish, mitochondrial heteroplasmy was used as a marker. Confocal microscopy analyses confirmed the transfer of active ovine mitochondria in Tg (mpx:GFP) zebrafish larvae. Internalized mitochondria were recorded at 6-hour post damage (hpd) in both damaged tissues and throughout the caudal area. Migration of MitoTracker red-stained mitochondria was detected within incorporating cells located in the wounded area. Moreover, quantification of ovine mitochondrial DNA copies by qPCR revealed a progressive degradation of ovine mitochondria from 24 to 48 hpd. Time-lapse imaging of Tg (lyz:DsRed) larvae demonstrated that the internalization of AEC-derived MVs significantly accelerated the early phase (from 0.5 hpd to 1 hpd) of immune cells recruitment at the wound site. Finally, to assess the pro-regenerative long-term effect of AEC MVs on the injured caudal fin, larvae from 72 hpf to 120 hpf, were imaged daily. Interestingly, AEC-derived MVs facilitated faster and more complete fin regeneration (11 out of 20 MV-treated larvae vs 4 out of 10 in CTR). Notably, fins of MV-treated larvae, cut at the beginning of circulation., started to recover the anatomical microarchitecture, including fin rays and pigments by 48 hpd. These findings underscore the crucial role of AEC-derived secretomes’ MV cargo in modulating immune response and tissue regeneration, paving the way for new controlled and targeted therapeutic cell-free approaches.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
