Impact of exosomes derived from adipose stem cells on lymphocyte proliferation and phenotype in mouse skin grafts

Abstract

Aim: Exosomes derived from adipose-derived stem cells (ASCs) in mice have been reported to influence immune regulation. Yet, the potential immunological effects of ASCs-derived exosomes and their interaction with lymphocytes during transplant immunity remain understudied.

Methods: ASCs from BALB/c mice, along with their conditioned culture medium, were collected for the extraction, isolation, and comprehensive characterization of exosomes. Splenic cell suspensions were isolated from BALB/c mice and subsequently processed for downstream analyses. Lymphocytes were isolated via gradient centrifugation and stimulated in vitro with the purified exosomes to assess their functional responses. Lymphocyte proliferation was quantified using the CCK8 assay, and the relative frequencies of CD4+ T cells, CD8+ T cells, Treg cells, NK (natural killer) cells, macrophages, B cells, dendritic cells (DCs), and Th17 cells were determined through flow cytometric analysis. Before establishing the skin transplantation model, the mice were administered PBS, 0.5 × 10⁸ exosomes, 1 × 10⁸ exosomes, 1.5 × 10⁸ exosomes, or ASCs via intravenous injection through the tail vein. Seven days after transplantation, the spleens, drainage lymph nodes, and blood samples were harvested for lymphocyte isolation and further downstream analyses.

Results: Exosomes derived from ASCs significantly increased the CD4+/CD8+ ratio and Treg cell levels, without inducing any notable changes in Th17 cell content or CTLA-4 protein expression in CD4+ T cells. Compared to the PBS-treated group, both ASC and exosome treatment groups demonstrated an enhanced CD4+/CD8+ ratio, increased Treg cell content, and elevated CTLA-4 protein expression in spleen tissue following skin transplantation, while Th17 cell levels remained unaffected. Compared to the ASC treatment group, the exosome group exhibited a higher CD4+/CD8+ ratio and Treg cell levels, alongside a reduced proportion of PD-1+ Treg cells and lower CTLA-4 protein expression in CD3+CD4+ T cells. No significant differences were observed in the proportions of NK cells, macrophages, B cells, and DCs in the spleens across all treatment groups. In peripheral blood, an increased proportion of CD3+ T cells, macrophages, and DCs was detected, accompanied by a reduced proportion of NK cells and B cells. In the draining lymph nodes, no significant changes were observed in the proportions of CD3+ T cells and B cells, while macrophages, NK cells, and DCs showed elevated proportions. In the exosome-treated group, mouse grafts exhibited a disorganized and thinner granular layer, accompanied by focal regions of inflammatory cell infiltration. Both exosome and ASC treatments significantly extended the survival of skin grafts.

Conclusion: Exosomes derived from ASCs promote lymphocyte proliferation and modulate their phenotypic profiles in mouse skin graft models, effectively extending graft survival.

Keywords

Adipose-derived stem cells / exosome / mouse skin graft / transplant immunity

References

[1] Duneton C,Ford ML.Activation and regulation of alloreactive T cell immunity in solid organ transplantation.Nat Rev Nephrol2022;18:663-76 PMCID:PMC9968399

[2] Ochando J,Madsen JC,Mulder WJM.Trained immunity in organ transplantation.Am J Transplant2020;20:10-8 PMCID:PMC6940521

[3] Cui X,Xu Y.Allograft rejection following immune checkpoint inhibitors in solid organ transplant recipients: a safety analysis from a literature review and a pharmacovigilance system.Cancer Med2023;12:5181-94 PMCID:PMC10028127

[4] Carroll RP,Gebski V.Immune checkpoint inhibitors in kidney transplant recipients: a multicentre, single-arm, phase 1 study.Lancet Oncol2022;23:1078-86

[5] Azzi Y,Scalea J,Akalin E.COVID-19 and solid organ transplantation: a review article.Transplantation2021;105:37-55

[6] Parlakpinar H.Transplantation and immunosuppression: a review of novel transplant-related immunosuppressant drugs.Immunopharmacol Immunotoxicol2021;43:651-65

[7] González-Cubero E,Esteban-Blanco M.The therapeutic potential of adipose-derived mesenchymal stem cell secretome in osteoarthritis: a comprehensive study.Int J Mol Sci2024;25:11287 PMCID:PMC11508730

[8] Bacakova L,Travnickova M.Stem cells: their source, potency and use in regenerative therapies with focus on adipose-derived stem cells - a review.Biotechnol Adv2018;36:1111-26

[9] Mazini L,Admou B,Malka G.Hopes and limits of adipose-derived stem cells (ADSCs) and mesenchymal stem cells (MSCs) in wound healing.Int J Mol Sci2020;21:1306 PMCID:PMC7072889

[10] Al-Ghadban S.Adipose tissue-derived stem cells: immunomodulatory effects and therapeutic potential.Physiology2020;35:125-33

[11] Welsh JA, Goberdhan DCI, O’Driscoll L, et al; MISEV Consortium. Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches. J Extracell Vesicles. 2024;13:e12404. PMCID:PMC10850029

[12] An Y,Tan X.Exosomes from adipose-derived stem cells and application to skin wound healing.Cell Prolif2021;54:e12993 PMCID:PMC7941238

[13] Jiang L,Zhou X.Augmenting peripheral nerve regeneration with adipose-derived stem cells.Stem Cell Rev Rep2022;18:544-58 PMCID:PMC8858329

[14] Kalluri R.The biology, function, and biomedical applications of exosomes.Science2020;367:eaau6977 PMCID:PMC7717626

[15] Zhang Y.Adipose-derived stem cells exosome and its potential applications in autologous fat grafting.J Plast Reconstr Aesthet Surg2023;76:219-29

[16] Li C,Wang X.Exosome-based targeted RNA delivery for immune tolerance induction in skin transplantation.J Biomed Mater Res A2020;108:1493-500

[17] Qiu H,Wu K,Cao L.Prospective application of exosomes derived from adipose-derived stem cells in skin wound healing: a review.J Cosmet Dermatol2020;19:574-81

[18] Jin J,Gong J.Exosome secreted from adipose-derived stem cells attenuates diabetic nephropathy by promoting autophagy flux and inhibiting apoptosis in podocyte.Stem Cell Res Ther2019;10:95 PMCID:PMC6419838

[19] Heidari N,Namaki S.Adipose-derived mesenchymal stem cell-secreted exosome alleviates dextran sulfate sodium-induced acute colitis by Treg cell induction and inflammatory cytokine reduction.J Cell Physiol2021;236:5906-20

[20] Qu Y,Cai X.Exosomes derived from miR-181-5p-modified adipose-derived mesenchymal stem cells prevent liver fibrosis via autophagy activation.J Cell Mol Med2017;21:2491-502 PMCID:PMC5618698

[21] Wang G,Lyu Y.Chemical-induced epigenome resetting for regeneration program activation in human cells.Cell Rep2023;42:112547

[22] Li X,Wu B.Landscape of immune cells heterogeneity in liver transplantation by single-cell RNA sequencing analysis.Front Immunol2022;13:890019 PMCID:PMC9127089

[23] Re F,Manferdini C,Lisignoli G.Bone regeneration improves with mesenchymal stem cell derived extracellular vesicles (EVs) combined with scaffolds: a systematic review.Biology2021;10:579 PMCID:PMC8301056

[24] Xiong M,Hu W.Exosomes from adipose-derived stem cells: the emerging roles and applications in tissue regeneration of plastic and cosmetic surgery.Front Cell Dev Biol2020;8:574223 PMCID:PMC7511773

[25] Lyu K,Chen Y.A “cell-free treatment” for tendon injuries: adipose stem cell-derived exosomes.Eur J Med Res2022;27:75 PMCID:PMC9148514

[26] Lou G,Yang F.Exosomes derived from miR-122-modified adipose tissue-derived MSCs increase chemosensitivity of hepatocellular carcinoma.J Hematol Oncol2015;8:122 PMCID:PMC4627430

[27] Deng S,Ge Z.Exosomes from adipose-derived mesenchymal stem cells ameliorate cardiac damage after myocardial infarction by activating S1P/SK1/S1PR1 signaling and promoting macrophage M2 polarization.Int J Biochem Cell Biol2019;114:105564

[28] Cohen GS,Jayaraman S.Transplantation elicits a clonally diverse CD8⁺ T cell response that is comprised of potent CD43⁺ effectors.Cell Rep2023;42:112993 PMCID:PMC10727118