Preconditioned Mesenchymal Stromal Cell-Derived Extracellular Vesicles (EVs) Counteract Inflammaging
Abstract
:1. Introduction
2. Materials and Methods
2.1. Mice
2.2. Isolation and Culture of Human Adipose Tissue-Derived Mesenchymal Stromal Cells (hMSCs)
2.3. Isolation and Culture of Mouse Bone Marrow-MSCs (mBMSCs)
2.4. Collection of hMSC and mBMSC Conditioned Medium (CM)
2.5. Immunohistochemistry
2.6. Extracellular Vesicle (EV) Separation and Characterization
2.7. Macrophage Isolation and Culture
2.8. Macrophage Polarization and Treatment with EVs
2.9. RNA Extraction and Quantitative Real Time-PCR
2.10. Proteome Profiler Array
2.11. Evaluation of Oxygen Consumption Rate
2.12. FoF1 ATP-Synthase Activity Assay
2.13. P/O Ratio
2.14. Cell Homogenate Preparation
2.15. ATP and AMP Intracellular Content Evaluation
2.16. Lactate Dehydrogenase Activity Assay
2.17. Malondialdehyde Evaluation
2.18. Statistical Analysis
3. Results
3.1. The Bone Marrow (BM) of Aged Mice Contains a Higher Number of Pro-Inflammatory Macrophages Compared to the Young Counterpart
3.2. The Aged Microenvironment Induces an Accumulation of CD38POS BM-Macrophages
3.3. Characterization of HypINF-hMSC-Derived EVs
3.4. EVs Released by HypINF-hMSCs Exert an Anti-Inflammatory Role on Aged Macrophages
3.5. HypINF hMSCs-Derived EVs Counteract Age-Associated Mitochondrial Dysfunction on Macrophages
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Khan, S.S.; Singer, B.D.; Vaughan, D.E. Molecular and physiological manifestations and measurement of aging in humans. Aging Cell 2017, 16, 624–633. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- López-Otín, C.; Blasco, M.A.; Partridge, L.; Serrano, M.; Kroemer, G. The hallmarks of aging. Cell 2013, 153, 1194. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Schmauck-Medina, T.; Molière, A.; Lautrup, S.; Zhang, J.; Chlopicki, S.; Madsen, H.B.; Cao, S.; Soendenbroe, C.; Mansell, E.; Vestergaard, M.B.; et al. New hallmarks of ageing: A 2022 Copenhagen ageing meeting summary. Aging 2022, 14, 6829–6839. [Google Scholar] [CrossRef] [PubMed]
- Ferrucci, L.; Fabbri, E. Inflammageing: Chronic inflammation in ageing, cardiovascular disease, and frailty. Nat. Rev. Cardiol. 2018, 15, 505–522. [Google Scholar] [CrossRef] [PubMed]
- Pinti, M.; Appay, V.; Campisi, J.; Frasca, D.; Fülöp, T.; Sauce, D.; Larbi, A.; Weinberger, B.; Cossarizza, A. Aging of the immune system—focus on inflammation and vaccination. Eur. J. Immunol. 2016, 46, 2286. [Google Scholar] [CrossRef] [Green Version]
- Rea, I.M.; Gibson, D.S.; McGilligan, V.; McNerlan, S.E.; Denis Alexander, H.; Ross, O.A. Age and age-related diseases: Role of inflammation triggers and cytokines. Front. Immunol. 2018, 9, 586. [Google Scholar] [CrossRef]
- Fagiolo, U.; Cossarizza, A.; Scala, E.; Fanales-Belasio, E.; Ortolani, C.; Cozzi, E.; Monti, D.; Franceschi, C.; Paganelli, R. Increased cytokine production in mononuclear cells of healthy elderly people. Eur. J. Immunol. 1993, 23, 2375–2378. [Google Scholar] [CrossRef]
- Freund, A.; Orjalo, A.V.; Desprez, P.Y.; Campisi, J. Inflammatory networks during cellular senescence: Causes and consequences. Trends Mol. Med. 2010, 16, 238–246. [Google Scholar] [CrossRef] [Green Version]
- Maggio, M.; Guralnik, J.M.; Longo, D.L.; Ferrucci, L. Interleukin-6 in Aging and Chronic Disease: A Magnificent Pathway. J. Gerontol. Ser. A 2006, 61, 575–584. [Google Scholar] [CrossRef]
- Franceschi, C.; Bonafè, M.; Valensin, S.; Olivieri, F.; De Luca, M.; Ottaviani, E.; De Benedictis, G. Inflamm-aging. An evolutionary perspective on immunosenescence. Ann. N. Y. Acad. Sci. 2000, 908, 244–254. [Google Scholar] [CrossRef]
- Van Deursen, J.M. The role of senescent cells in ageing. Nature 2014, 509, 439. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Coppé, J.P.; Patil, C.K.; Rodier, F.; Sun, Y.; Muñoz, D.P.; Goldstein, J.; Nelson, P.S.; Desprez, P.Y.; Campisi, J. Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor. PLoS Biol. 2008, 6, e301. [Google Scholar] [CrossRef] [PubMed]
- Young, A.R.J.; Narita, M. SASP reflects senescence. EMBO Rep. 2009, 10, 228–230. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Coppé, J.P.; Desprez, P.Y.; Krtolica, A.; Campisi, J. The Senescence-Associated Secretory Phenotype: The Dark Side of Tumor Suppression. Annu. Rev. Pathol. 2010, 5, 99–118. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Malaquin, N.; Martinez, A.; Rodier, F. Keeping the senescence secretome under control: Molecular reins on the senescence-associated secretory phenotype. Exp. Gerontol. 2016, 82, 39–49. [Google Scholar] [CrossRef]
- Yarbro, J.R.; Emmons, R.S.; Pence, B.D. Macrophage Immunometabolism and Inflammaging: Roles of Mitochondrial Dysfunction, Cellular Senescence, CD38, and NAD. Immunometabolism 2020, 2, e200026. [Google Scholar] [CrossRef]
- Covarrubias, A.J.; Kale, A.; Perrone, R.; Lopez-Dominguez, J.A.; Pisco, A.O.; Kasler, H.G.; Schmidt, M.S.; Heckenbach, I.; Kwok, R.; Wiley, C.D.; et al. Senescent cells promote tissue NAD+ decline during ageing via the activation of CD38+ macrophages. Nat. Metab. 2020, 2, 1265–1283. [Google Scholar] [CrossRef]
- Chini, E. CD38 as a regulator of cellular NAD: A novel potential pharmacological target for metabolic conditions. Curr. Pharm. Des. 2009, 15, 57–63. [Google Scholar] [CrossRef] [Green Version]
- Shi, Y.; Wang, Y.; Li, Q.; Liu, K.; Hou, J.; Shao, C.; Wang, Y. Immunoregulatory mechanisms of mesenchymal stem and stromal cells in inflammatory diseases. Nat. Rev. Nephrol. 2018, 14, 493–507. [Google Scholar] [CrossRef]
- Varderidou-Minasian, S.; Lorenowicz, M.J. Mesenchymal stromal/stem cell-derived extracellular vesicles in tissue repair: Challenges and opportunities. Theranostics 2020, 10, 5979–5997. [Google Scholar] [CrossRef]
- Hou, Y.; Li, J.; Guan, S.; Witte, F. The therapeutic potential of MSC-EVs as a bioactive material for wound healing. Eng. Regen. 2021, 2, 182–194. [Google Scholar] [CrossRef]
- Gorgun, C.; Ceresa, D.; Lesage, R.; Villa, F.; Reverberi, D.; Balbi, C.; Santamaria, S.; Cortese, K.; Malatesta, P.; Geris, L.; et al. Dissecting the effects of preconditioning with inflammatory cytokines and hypoxia on the angiogenic potential of mesenchymal stromal cell (MSC)-derived soluble proteins and extracellular vesicles (EVs). Biomaterials 2021, 269, 120633. [Google Scholar] [CrossRef]
- Fei, F.; Lee, K.M.; McCarry, B.E.; Bowdish, D.M.E. Age-associated metabolic dysregulation in bone marrow-derived macrophages stimulated with lipopolysaccharide. Sci. Rep. 2016, 6, 22637. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Spees, J.L.; Lee, R.H.; Gregory, C.A. Mechanisms of mesenchymal stem/stromal cell function. Stem Cell Res. Ther. 2016, 7, 125. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Panfoli, I.; Ravera, S.; Podestà, M.; Cossu, C.; Santucci, L.; Bartolucci, M.; Bruschi, M.; Calzia, D.; Sabatini, F.; Bruschettini, M.; et al. Exosomes from human mesenchymal stem cells conduct aerobic metabolism in term and preterm newborn infants. FASEB J. 2016, 30, 1416–1424. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bruschi, M.; Santucci, L.; Ravera, S.; Candiano, G.; Bartolucci, M.; Calzia, D.; Lavarello, C.; Inglese, E.; Ramenghi, L.A.; Petretto, A.; et al. Human urinary exosome proteome unveils its aerobic respiratory ability. J. Proteom. 2016, 136, 25–34. [Google Scholar] [CrossRef] [PubMed]
- Gorgun, C.; Reverberi, D.; Rotta, G.; Villa, F.; Quarto, R.; Tasso, R. Isolation and Flow Cytometry Characterization of Extracellular-Vesicle Subpopulations Derived from Human Mesenchymal Stromal Cells. Curr. Protoc. Stem Cell Biol. 2019, 48. [Google Scholar] [CrossRef] [Green Version]
- Ravera, S.; Bertola, N.; Pasquale, C.; Bruno, S.; Benedicenti, S.; Ferrando, S.; Zekiy, A.; Arany, P.; Amaroli, A. 808-nm Photobiomodulation Affects the Viability of a Head and Neck Squamous Carcinoma Cellular Model, Acting on Energy Metabolism and Oxidative Stress Production. Biomedicines 2021, 9, 1717. [Google Scholar] [CrossRef]
- Cappelli, E.; Degan, P.; Bruno, S.; Pierri, F.; Miano, M.; Raggi, F.; Farruggia, P.; Mecucci, C.; Crescenzi, B.; Naim, V.; et al. The passage from bone marrow niche to bloodstream triggers the metabolic impairment in Fanconi Anemia mononuclear cells. Redox Biol. 2020, 36, 101618. [Google Scholar] [CrossRef]
- MM, B. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [Google Scholar] [CrossRef]
- Cappelli, E.; Bertola, N.; Bruno, S.; Degan, P.; Regis, S.; Corsolini, F.; Banelli, B.; Dufour, C.; Ravera, S. A Multidrug Approach to Modulate the Mitochondrial Metabolism Impairment and Relative Oxidative Stress in Fanconi Anemia Complementation Group A. Metabolites 2021, 12, 6. [Google Scholar] [CrossRef] [PubMed]
- Cappelli, E.; Cuccarolo, P.; Stroppiana, G.; Miano, M.; Bottega, R.; Cossu, V.; Degan, P.; Ravera, S. Defects in mitochondrial energetic function compels Fanconi Anaemia cells to glycolytic metabolism. Biochim. Biophys. Acta—Mol. Basis Dis. 2017, 1863, 1214–1221. [Google Scholar] [CrossRef] [PubMed]
- Ravera, S.; Dufour, C.; Cesaro, S.; Bottega, R.; Faleschini, M.; Cuccarolo, P.; Corsolini, F.; Usai, C.; Columbaro, M.; Cipolli, M.; et al. Evaluation of energy metabolism and calcium homeostasis in cells affected by Shwachman-Diamond syndrome. Sci. Rep. 2016, 6, 25441. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yue, Z.; Nie, L.; Zhang, P.; Chen, Q.; Lv, Q.; Wang, Q. Tissue-resident macrophage inflammaging aggravates homeostasis dysregulation in age-related diseases. Cell. Immunol. 2021, 361, 104278. [Google Scholar] [CrossRef]
- Xue, Q.; Yan, Y.; Zhang, R.; Xiong, H. Regulation of iNOS on Immune Cells and Its Role in Diseases. Int. J. Mol. Sci. 2018, 19, 3805. [Google Scholar] [CrossRef] [Green Version]
- Lefèvre, L.; Iacovoni, J.S.; Martini, H.; Bellière, J.; Maggiorani, D.; Dutaur, M.; Marsal, D.J.; Decaunes, P.; Pizzinat, N.; Mialet-Perez, J.; et al. Kidney inflammaging is promoted by CCR2 + macrophages and tissue-derived micro-environmental factors. Cell. Mol. Life Sci. 2021, 78, 3485–3501. [Google Scholar] [CrossRef]
- Duong, L.; Radley, H.; Lee, B.; Dye, D.; Pixley, F.; Grounds, M.; Nelson, D.; Jackaman, C. Macrophage function in the elderly and impact on injury repair and cancer. Immun. Ageing 2021, 18, 4. [Google Scholar] [CrossRef]
- Lee, B.Y.; Han, J.A.; Im, J.S.; Morrone, A.; Johung, K.; Goodwin, E.C.; Kleijer, W.J.; DiMaio, D.; Hwang, E.S. Senescence-associated β-galactosidase is lysosomal β-galactosidase. Aging Cell 2006, 5, 187–195. [Google Scholar] [CrossRef]
- Sharpless, N.E.; Sherr, C.J. Forging a signature of in vivo senescence. Nat. Rev. Cancer 2015, 15, 397–408. [Google Scholar] [CrossRef]
- Ravera, S.; Podestà, M.; Sabatini, F.; Dagnino, M.; Cilloni, D.; Fiorini, S.; Barla, A.; Frassoni, F. Discrete Changes in Glucose Metabolism Define Aging. Sci. Rep. 2019, 9, 10347. [Google Scholar] [CrossRef]
- Hinkle, P.C. P/O ratios of mitochondrial oxidative phosphorylation. Biochim. Biophys. Acta—Bioenergetics 2005, 1706, 1–11. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Licastro, F.; Candore, G.; Lio, D.; Porcellini, E.; Colonna-Romano, G.; Franceschi, C.; Caruso, C. Innate immunity and inflammation in ageing: A key for understanding age-related diseases. Immun. Ageing 2005, 2, 8. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Chung, H.Y.; Kim, D.H.; Lee, E.K.; Chung, K.W.; Chung, S.; Lee, B.; Seo, A.Y.; Chung, J.H.; Jung, Y.S.; Im, E.; et al. Redefining Chronic Inflammation in Aging and Age-Related Diseases: Proposal of the Senoinflammation Concept. Aging Dis. 2019, 10, 367–382. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Sicco, C.L.; Reverberi, D.; Balbi, C.; Ulivi, V.; Principi, E.; Pascucci, L.; Becherini, P.; Bosco, M.C.; Varesio, L.; Franzin, C.; et al. Mesenchymal stem cell-derived extracellular vesicles as mediators of anti-inflammatory effects: Endorsement of macrophage polarization. Stem Cells Transl. Med. 2017, 6, 1018–1028. [Google Scholar] [CrossRef] [PubMed]
- Ulivi, V.; Tasso, R.; Cancedda, R.; Descalzi, F. Mesenchymal stem cell paracrine activity is modulated by platelet lysate: Induction of an inflammatory response and secretion of factors maintaining macrophages in a proinflammatory phenotype. Stem Cells Dev. 2014, 23, 1858–1869. [Google Scholar] [CrossRef] [PubMed]
- Tasso, R.; Ulivi, V.; Reverberi, D.; Lo Sicco, C.; Descalzi, F.; Cancedda, R. In vivo implanted bone marrow-derived mesenchymal stem cells trigger a cascade of cellular events leading to the formation of an ectopic bone regenerative niche. Stem Cells Dev. 2013, 22, 3178–3191. [Google Scholar] [CrossRef] [Green Version]
- Gibon, E.; Loi, F.; Córdova, L.A.; Pajarinen, J.; Lin, T.; Lu, L.; Nabeshima, A.; Yao, Z.; Goodman, S.B. Aging Affects Bone Marrow Macrophage Polarization: Relevance to Bone Healing. Regen. Eng. Transl. Med. 2016, 2, 98–104. [Google Scholar] [CrossRef] [Green Version]
- Li, W.; Li, Y.; Jin, X.; Liao, Q.; Chen, Z.; Peng, H.; Zhou, Y. CD38: A Significant Regulator of Macrophage Function. Front. Oncol. 2022, 12, 487. [Google Scholar] [CrossRef]
- Amici, S.A.; Young, N.A.; Narvaez-Miranda, J.; Jablonski, K.A.; Arcos, J.; Rosas, L.; Papenfuss, T.L.; Torrelles, J.B.; Jarjour, W.N.; Guerau-de-Arellano, M. CD38 is robustly induced in human macrophages and monocytes in inflammatory conditions. Front. Immunol. 2018, 9, 1593. [Google Scholar] [CrossRef] [Green Version]
- Petersen, K.S.; Smith, C. Ageing-Associated Oxidative Stress and Inflammation Are Alleviated by Products from Grapes. Oxid. Med. Cell. Longev. 2016, 2016. [Google Scholar] [CrossRef]
- Zuo, L.; Prather, E.R.; Stetskiv, M.; Garrison, D.E.; Meade, J.R.; Peace, T.I.; Zhou, T. Inflammaging and Oxidative Stress in Human Diseases: From Molecular Mechanisms to Novel Treatments. Int. J. Mol. Sci. 2019, 20, 4472. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Cui, H.; Kong, Y.; Zhang, H. Oxidative Stress, Mitochondrial Dysfunction, and Aging. J. Signal Transduct. 2012, 2012, 1–13. [Google Scholar] [CrossRef] [PubMed]
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Gorgun, C.; Africano, C.; Ciferri, M.C.; Bertola, N.; Reverberi, D.; Quarto, R.; Ravera, S.; Tasso, R. Preconditioned Mesenchymal Stromal Cell-Derived Extracellular Vesicles (EVs) Counteract Inflammaging. Cells 2022, 11, 3695. https://doi.org/10.3390/cells11223695
Gorgun C, Africano C, Ciferri MC, Bertola N, Reverberi D, Quarto R, Ravera S, Tasso R. Preconditioned Mesenchymal Stromal Cell-Derived Extracellular Vesicles (EVs) Counteract Inflammaging. Cells. 2022; 11(22):3695. https://doi.org/10.3390/cells11223695
Chicago/Turabian StyleGorgun, Cansu, Chiara Africano, Maria Chiara Ciferri, Nadia Bertola, Daniele Reverberi, Rodolfo Quarto, Silvia Ravera, and Roberta Tasso. 2022. "Preconditioned Mesenchymal Stromal Cell-Derived Extracellular Vesicles (EVs) Counteract Inflammaging" Cells 11, no. 22: 3695. https://doi.org/10.3390/cells11223695