Senescence-associated β-galactosidase activity in human effector and regulatory T cells
AbstractIntroduction. The accumulation of senescent cells with age is considered to be one of the causes of dysfunction of various organs and tissues during physiological aging of the organism. The immune system participates in the elimination of senescent cells, but it is also subject to aging itself. Cell aging is usually assessed by the specific activity of the enzyme associated with senescence β-galactosidase (SA-β-Gal). However, the activity of SA-β-Gal in the lymphocyte population of donors of different ages is insufficiently studied. Therefore, studying the aging processes in the immune system and the potential use of SA-β-Gal as a marker for senescent lymphocytes is of interest.
The aim of the study – assessment of SA-β-Gal activity in peripheral blood T lymphocyte populations that differ in surface activation markers in donors of different ages.
Material and methods. The study included 20 apparently healthy donors aged 21–32 and 50–61 years. Mononuclear cells isolated from peripheral blood were phenotyped with antibodies against surface markers, incubated with a substrate to determine SA-β-Gal activity, and analyzed using flow cytometry.
Results. It was found that T lymphocyte subpopulations positive for activation markers CD25 and HLA-DR have higher SA-β-Gal activity. The regulatory T lymphocyte population with the phenotype CD3+CD4+CD25highCD127low/– (CD4+-Treg) demonstrated significantly lower SA-β-Gal activity compared to the overall pool of CD4+-T cells. Significant differences in SA-β-Gal activity among donors of the two age groups were not observed, except for subpopulations with the phenotypes CD3+CD8+HLA-DR+ and CD3+CD8+HLA-DR+CD127– (CD4+-Treg), where SA-β-Gal activity decreases with age.
Conclusion. Activated CD4+ and CD8+ effector T cells exhibit increased expression of SA-β-Gal compared to non-activated populations and decreased expression in CD4+-Treg lymphocytes, indicating differences in metabolic activity in these cell populations. We did not find a significant increase in the percent of SA-β-Galhigh-lymphocytes or an increase in its activity with age, which limits the use of this method for assessing age-related changes in the immune system. However, the activity of SA-β-Gal can be used as an additional indicator of T lymphocyte activation along with surface activation markers in donors of the studied age groups.
Keywords:SA-β-Gal; senescence; T lymphocytes; Treg
For citation: Matveeva K.S., Shevyrev D.V., Rybtsov S.A. Senescence-associated β-galactosidase activity in human effector and regulatory T cells. Immunologiya. 2024; 45 (3): 290–9. DOI: https://doi.org/10.33029/1816-2134-2024-45-3-290-299 (in Russian)
Funding. The work of Matveeva K.S. on SA-β-Gal activity detection, flow cytometry, analysis of the results and writing the chapters «Introduction», «Material and methods», «Results» was funded by the Ministry of Science and Higher Education of the Russian Federation (agreement No. 075-10-2021-093; project IMB-2102). The work of Shevyrev D.V., Rybtsov S.A on the development of research protocols, performing flow cytometry and analyzing the results, writing the «Discussion» chapter, final editing of the text was supported by the Russian Science Foundation grant No. 23-15-00443 (https http://rscf.ru/project/23-15-00443/).
Conflict of interests. The authors declare no conflict of interests.
Authors’ contribution. Concept and study design – Shevyrev D.V., Rybtsov S.A.; material collection and processing – Matveeva K.S., Shevyrev D.V.; formal analysis – Matveeva K.S., Shevyrev D.V.; text writing – Matveeva K.S. Shevyrev D.V.; editing – Shevyrev D.V., Rybtsov S.A.; approval of the final version of the article – Rybtsov S.A.; responsibility for the integrity of all article parts – Shevyrev D.V.
References
1. Masyutina A.M., Pashenkov M.V., Pinegin B.V. Cellular senescence: mechanisms and clinical implications. Immunologiya. 2024; 45 (2): 221–34. DOI: https://doi.org/10.33029/1816-2134-2024-45-2-221-234 (in Russian)
2. Valieva Y., Ivanova E., Fayzullin A., Kurkov A., Igrunkova A. Senescence-Associated β-Galactosidase Detection in Pathology. Diagnostics. 2022; 12 (10): 2309. DOI: https://doi.org/10.3390/diagnostics12102309
3. Rybtsova N., Berezina T., Kagansky A., Rybtsov S. Blood-Circulating Factors Unveil and Delay Your Biological Aging? Biomedicines. 2020; 8 (12): 615. DOI: https://doi.org/10.3390/biomedicines8120615
4. Martyshkina Y.S., Tereshchenko V.P., Bogdanova D.A., Rybtsov S.A. Reliable Hallmarks and Biomarkers of Senescent Lymphocytes. Int J Mol Sci. 2023; 24 (21): 15653. DOI: https://doi.org/10.3390/ijms242115653
5. Burmakina V.V., Gankovskaya L.V., Nasaeva E.D., Khasanova E.M., Grechenko V.V., Strazhesko I.D., Gorodishchenskaya S.V. Link between the inflammasome complex and cytokines IL-1β and IL-10 and pathological phenotype of aging in centenarians Immunologiya. 2023; 44 (6): 754–63. DOI: https://doi.org/10.33029/1816-2134-2023-44-6-754-763 (in Russian)
6. Da Silva-Álvarez S., Collado M. Cellular Senescence. In: Encyclopedia of Cell Biology. Bradshaw R.A., Stahl P. D., eds. Waltham: Academic Press. 2016; 511–7. DOI: https://doi.org/10.1016/B978-0-12-394447-4.30066-9
7. Campisi J., d’Adda di Fagagna F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol. 2007; 8: 729–40. DOI: https://doi.org/10.1038/nrm2233
8. Muñoz-Espín D., Serrano M. Cellular senescence: from physiology to pathology. Nat Rev Mol Cell Biol. 2014; 15: 482–96. DOI: https://doi.org/10.1038/nrm3823
9. Kirkland J.L., Tchkonia T. Clinical strategies and animal models for developing senolytic agents. Exp Gerontol. 2015; 68: 19–25. DOI: https://doi.org/10.1016/j.exger.2014.10.012
10. Kennedy B.K., Berger S.L., Brunet A., Campisi J., Cuervo A.M., Epel E.S., Franceschi C., Lithgow G.J., Morimoto R.I., Pessin J.E., Rando T.A., Richardson A., Schadt E.E., Wyss-Coray T., Sierra F. Geroscience: linking aging to chronic disease. Cell. 2014; 159: 709–13. DOI: https://doi.org/10.1016/j.cell.2014.10.039
11. Gankovskaya L.V., Artemyeva O.V., Grechenko V.V., Nasaeva E.D., Khasanova E.M. Age-associated diseases: the role of the inflammasome complex. Immunology. 2023; 44 (5): 640–52. DOI: https://doi.org/10.33029/1816-2134-2023-44-5-640-652 (in Russian)
12. Artemyeva O.V., Gankovskaya L.V. Inflammaging as the basis of age-associated diseases. Medical Immunology (Russia). 2020; 22 (3): 419–32. DOI: https://doi.org/10.15789/1563-0625-IAT-1938 (in Russian)
13. Garcia H.G., Kondev J., Orme N., Theriot J.A., Phillips R. Chapter Two – Thermodynamics of Biological Processes. In: Methods in Enzymology. Johnson M. L., Holt J. M., Ackers G. K., eds. Academic Press. 2011; 492: 27–59. DOI: https://doi.org/10.1016/B978-0-12-381268-1.00014-8
14. Borgoni S., Kudryashova K.S., Burka K., de Magalhães J.P. Targeting immune dysfunction in aging. Ageing Res Rev. 2021; 70: 101410. DOI: https://doi.org/10.1016/j.arr.2021.101410
15. Krishna D.R., Sperker B., Fritz P., and Klotz U. Does pH 6 beta-galactosidase activity indicate cell senescence? Mech Ageing Dev. 1999; 109 (2): 113–23. DOI: https://doi.org/10.1016/s0047-6374(99)00031-7
16. Dimri G.P., Lee X., Basile G., Acosta M., Scott G., Roskelley C., Medrano E.E., Linskens M., Rubelj I., Pereira-Smith O. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A. 1995; 92 (20): 9363–7. DOI: https://doi.org/10.1073/pnas.92.20.9363
17. 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 beta-galactosidase is lysosomal beta-galactosidase. Aging Cell. 2006; 5 (2): 187–95. DOI: https://doi.org/10.1111/j.1474-9726.2006.00199.x
18. Tan J.X., Finkel T. Lysosomes in senescence and aging. EMBO Rep. 2023; 24 (11): e57265. DOI: https://doi.org/10.15252/embr.202357265
19. Carmona-Gutierrez D., Hughes A.L., Madeo F., Ruckenstuhl C. The crucial impact of lysosomes in aging and longevity. Ageing Res Rev. 2016; 32: 2–12. DOI: https://doi.org/10.1016/j.arr.2016.04.009
20. Abe A., Shayman J.A. Sphingolipid Catabolism. In: Encyclopedia of Biological Chemistry (Second Edition). Lennarz W. J., Lane M. D., eds. Waltham: Academic Press. 2013; 287–92. DOI: https://doi.org/10.1016/B978-0-12-378630-2.00462-X
21. Bursuker I., Rhodes J.M., Goldman R. Beta-galactosidase – an indicator of the maturational stage of mouse and human mononuclear phagocytes. J Cell Physiol. 1982; 112: 385–90. DOI: https://doi.org/10.1002/jcp.1041120312
22. Calcinotto A., Kohli J., Zagato E., Pellegrini L., Demaria M., Alimonti A. Cellular Senescence: Aging, Cancer, and Injury. Physiol Rev. 2019; 99 (2): 1047–78. DOI: https://doi.org/10.1152/physrev.00020.2018
23. Tuttle C.S.L., Waaijer M.E.C., Slee-Valentijn M.S., Stijnen T., Westendorp R., Maier A.B. Cellular senescence and chronological age in various human tissues: A systematic review and meta-analysis. Aging Cell. 2020; 19 (2): e13083. DOI: https://doi.org/10.1111/acel.13083
24. Gao P., Zou X., Sun X., Zhang C. Cellular Senescence in Metabolic-Associated Kidney Disease: An Update. Cells. 2022; 11 (21): 3443. DOI: https://doi.org/10.3390/cells11213443
25. Bulbiankova D., Díaz-Puertas R., Álvarez-Martínez F.J., Herranz-López M., Barrajón-Catalán E., Micol V. Hallmarks and Biomarkers of Skin Senescence: An Updated Review of Skin Senotherapeutics. Antioxid Basel Switz. 2023; 12 (2): 444. DOI: https://doi.org/10.3390/antiox12020444
26. Lozano-Torres B., García-Fernández A., Domínguez M., Sancenón F., Blandez J.F., Martínez-Máñez R. β-Galactosidase-Activatable Nile Blue-Based NIR Senoprobe for the Real-Time Detection of Cellular Senescence. Anal Chem. 2023; 95 (2): 1643–51. DOI: https://doi.org/10.1021/acs.analchem.2c04766
27. Sikora E., Bielak-Zmijewska A., Dudkowska M., Krzystyniak A., Mosieniak G., Wesierska M., Wlodarczyk J. Cellular Senescence in Brain Aging. Front Aging Neurosci. 2021; 13: 646924. DOI: https://doi.org/10.3389/fnagi.2021.646924
28. Jurk D., Wang, C., Miwa S., Maddick M., Korolchuk V., Tsolou A., Gonos E.S., Thrasivoulou C., Saffrey M.J., Cameron K., von Zglinicki T. Postmitotic neurons develop a p21-dependent senescence-like phenotype driven by a DNA damage response. Aging Cell. 2021; 11 (6): 996–1004. DOI: https://doi.org/10.1111/j.1474-9726.2012.00870.x
29. Dungan C.M., Wells J.M., Murach K.A. The life and times of cellular senescence in skeletal muscle: friend or foe for homeostasis and adaptation? Am J Physiol Cell Physiol. 2023; 325 (1): C324–31. DOI: https://doi.org/10.1152/ajpcell.00553.2022
30. Wang L., Lankhorst L., Bernards R. Exploiting senescence for the treatment of cancer. Nat Rev Cancer. 2022; 22: 340–55. DOI: https://doi.org/10.1038/s41568-022-00450-9
31. Severino J., Allen R.G., Balin S., Balin A., Cristofalo V.J. Is beta-galactosidase staining a marker of senescence in vitro and in vivo? Exp Cell Res. 2000; 257 (1): 162–71. DOI: https://doi.org/10.1006/excr.2000.4875
32. Yegorov Y.E., Akimov S.S., Hass R., Zelenin A.V., Prudovsky I.A. Endogenous β-Galactosidase Activity in Continuously Nonproliferating Cells. Exp Cell Res. 1998; 243 (1): 207–11. DOI: https://doi.org/10.1006/excr.1998.4169
33. Yang N.-C., Hu M.-L. The limitations and validities of senescence associated-beta-galactosidase activity as an aging marker for human foreskin fibroblast Hs68 cells. Exp Gerontol. 2005; 40 (10): 813–9. DOI: https://doi.org/10.1016/j.exger.2005.07.011
34. Debacq-Chainiaux F., Erusalimsky J.D., Campisi, J., Toussaint, O. Protocols to detect senescence-associated beta-galactosidase (SA-βgal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc. 2009; 4: 1798–806. DOI: https://doi.org/10.1038/nprot.2009.191
35. de Mera-Rodríguez J.A., Álvarez-Hernán G., Gañán Y., Martín-Partido G., Rodríguez-León J., Francisco-Morcillo J. Senescence-associated β-galactosidase activity in the developing avian retina. Dev Dyn Off Publ Am Assoc Anat. 2019; 248 (9): 850–65. DOI: https://doi.org/10.1002/dvdy.74
36. de Mera-Rodríguez J.A., Álvarez-Hernán G., Gañán Y., Martín-Partido G., Rodríguez-León J., Francisco-Morcillo J. Is Senescence-Associated β-Galactosidase a Reliable in vivo Marker of Cellular Senescence During Embryonic Development? Front Cell Dev Biol. 2021; 9: 623175. DOI: https://doi.org/10.3389/fcell.2021.623175
37. Young A.R., and Narita M. Connecting autophagy to senescence in pathophysiology. Curr Opin Cell Biol. 2010; 22 (2): 234–40. DOI: https://doi.org/10.1016/j.ceb.2009.12.005
38. Kopp H.-G., Hooper A.T., Shmelkov S.V., Rafii S. Beta-galactosidase staining on bone marrow. The osteoclast pitfall. Histol Histopathol. 2007; 22: 971–6. DOI: https://doi.org/10.14670/HH-22.971
39. Martínez-Zamudio R.I., Dewald H.K., Vasilopoulos T., Gittens-Williams L., Fitzgerald-Bocarsly P., Herbig U. Senescence-associated β-galactosidase reveals the abundance of senescent CD8+ T cells in aging humans. Aging Cell. 2021; 20: e13344. DOI: https://doi.org/10.1111/acel.13344
40. Chikhirzhina E.V., Polyanichko A.M., Starkova T.Yu. Extranuclear functions of nonhistone protein HMGB1. Tsitologiya. 2020; 62 (10): 716–25. DOI: https://doi.org/10.31857/S0041377120100016 (in Russian)