The Effects of M2 Macrophages-Derived Exosomes on Urethral Fibrosis and Stricture in Scar Formation

Xiang Ren,1,* Zhixian Wang,2,3,* Jing Wang,1 Xing Li,1 Huizhi Wei,4 Chang Liu,5 Shiliang Liu,6 Yunpeng Zhu,7 Chunxiang Feng,8 Yisheng Yin,1 Yiqun Tian,1 Minglong Wu,1 Xiaoyong Zeng1 1Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China; 2Department of Urology, Traditional Chinese and Western Medicine Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China; 3Department of Urology, Wuhan No. 1 Hospital, Wuhan, People’s Republic of China; 4School of Pharmacy, Shanxi Medical University, Taiyuan, People’s Republic of China; 5Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China; 6Department of Ultrasonography, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China; 7Department of Thoracic, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China; 8Department of Urology, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Minglong Wu, Email [email protected] Xiaoyong Zeng, Email [email protected]: Macrophages are highly plastic cells, and macrophage-derived exosomes (M-Exos) have been implicated in inflammation-related pathophysiologies, such as tissue injury and fibrosis repair. This study aimed to investigate the possible effects of M-Exos on the initiation and development of urethral fibrosis and stricture after injury, and to elucidate the underlying mechanisms.Methods: In this study, we used time-tracking immunofluorescence staining for M1 and M2 macrophage markers to characterize sequential properties in the site of injured urethra. Next, we harvested these exosomes from different macrophages to co-culture with fibroblasts to further confirm the role of exosome-mediated M2 macrophage-fibroblast communication. Then, high-throughput micro-RNA (miRNA) sequencing was performed to detect the candidate exosomal miRNA and its target gene. Furthermore, fibroblasts were transfected with mRFP-GFP-LC3 plasmid to detect the autophagy role of SIRT1 in the urethral fibroblasts fibrogenesis.Results: Here we found that M2-polarized macrophages triggered and dominated the fibrotic scene, purified exosomes from M2 macrophages exacerbated urethral fibroblast fibrogenesis, and the inhibition of exosome secretion abolished fibroblast fibrogenesis. Moreover, miR-34a-5p, which is highly enriched and packaged within M2-Exos, can be transferred from M2 macrophages into urethral fibroblasts, resulting in deterioration of proliferation and fibrogenesis. Mechanistically, M2-Exos miR-34a-5p could directly interact with the 3ʹ-UTR of SIRT1, thereby suppressing SIRT1 expression in fibroblasts, leading to the blockage of autophagosome-lysosome fusion to impair urethral fibroblast autophagy flux and further exacerbate fibrogenesis. More importantly, repression of miR-34a-5p in M2-Exos mitigated-urethral strictures in rats with damaged urethra.Conclusion: M2 macrophage-derived exosomes miR-34a-5p could aggravate the development of urethral fibrosis and stricture by blocking autophagosome-lysosome fusion in urethral fibroblasts and further accelerating fibrogenesis by directly targeting SIRT1, suggesting that M2-Exo miR-34a-5p and SIRT1 could serve as promising therapeutic targets for urethral stricture.Keywords: exosomes, urethral stricture, macrophage, autophagy, scar formation