Problems of Radiation Medicine and Radiobiology

L. M. Zvarych, D. A. Bazyka

State Institution «National Research Center for Radiation Medicine, Hematology and Oncology of the
National Academy of Medical Sciences of Ukraine», 53 Yuriia Illienka Str., Kyiv, 04050, Ukraine

DYSREGULATION OF AUTOPHAGY IN PERIPHERAL BLOOD LEUCOCYTES IS A FACTOR IN THE DEVELOPMENT OF INFLAMMAGING IN IMMUNOCOMPROMISED PERSONS ON THE EXAMPLE OF THE SERVICEMEN OF THE DEFENSE FORCES OF UKRAINE AND CLEAN-UP WORKERS OF THE CHORNOBYL ACCIDENT

Objective. To assess the functional state and age-related characteristics of autophagy in peripheral blood leukocytes as a risk factor for the development of inflammaging using the example of the servicemen of the Defense Forces of Ukraine and clean-up workers of the Chornobyl accident.
Materials and methods. A total of 103 male patients aged 28–77 (56,48 ± 9,05) years were examined. They included: the main group – 23 servicemen of the Defense Forces of Ukraine aged 44–59 (50,21 ± 5,13) years; the comparison group – 57 clean-up workers of the Chornobyl accident aged 56–63 (60,31 ± 1,78) years; and the control group – 23 civilians aged 28–77 (53,26 ± 15,98) years. The individuals in the main and control groups were divided according to age into subgroups under 50 years and over 50 years. Clean-up workers were divided into 3 subgroups depending on the radiation dose: І – D < 100 mSv, ІІ – 100 < D < 500 mSv and ІІІ – D ≥ 500 mSv. Analysis of autophagy parameters in peripheral blood leukocytes (PB) was performed using flow cytometry and polymerase chain reaction.
Results. In patients of the main group, the autophagy activity factor (AAF) of granulocytes and the expression of the SQSTM1 gene in PB leukocytes decreased. A decrease in chloroquine-induced accumulation of LC3B protein in leukocytes, AAF in PB monocytes and the expression of the MTOR, RB1CC1 and MAP1LC3B genes was revealed in servicemen of the Defense Forces of Ukraine under 50 years of age. The spontaneous levels of LC3B protein and AAF in monocytes and the expression level of PIK3C3, ULK1 and MAP1LC3B genes in PB leukocytes were increased in servicemen of the Defense Forces of Ukraine over 50 years of age. The clean-up workers of different dose groups showed a decrease in the AAF in lymphocytes and granulocytes, the LC3B level in monocytes after incubation with chloroquine, the expression of the MTOR, RB1CC1, SQSTM1, ULK1, MAP1LC3B, BECN1 and PIK3C3 genes in PB leukocytes, and the AAF of monocytes was higher. Similar changes were revealed in the indices of chloroquine-induced LC3B accumulation in lymphocytes and monocytes of the clean-up workers and servicemen of both age groups, as well as the spontaneous LC3B protein level in PB monocytes of the clean-up workers irradiated at doses above 100 mSv and civilians over 50 years old.
Conclusions. Unidirectional dysregulation of autophagy was established in the servicemen of the Defense Forces of Ukraine and the clean-up workers of the Chornobyl accident. The existing changes in autophagy parameters can lead to disruption of the functioning of the autophagic apparatus of leukocytes at the level of mRNA and protein, as well as signaling pathways, and be associated with age-related changes at both the cellular and organismal levels. The emergence of new and persistent earlier stress factors as a result of the war creates an additional load on the mechanisms of maintaining homeostasis, which is observed in individuals exposed to ionizing radiation more than 30 years later. The found intergroup differences and similarities can activate the same or similar mechanisms of pathological processes, which will ultimately increase the risks of developing age-associated chronic somatic pathology in younger age groups.
Key words: autophagy; LC3B; leukocytes; inflammaging; immune system; stress; servicemen; war; ionizing radiation; Chornobyl accident.

Problems of Radiation Medicine and Radiobiology.
2024;29:327-358. doi: 10.33145/2304-8336-2024-29-327-358

full text

1. Salminen A, Kaarniranta K, Kauppinen A. Inflammaging: disturbed interplay between autophagy and inflammasomes. Aging (Albany NY). 2012;4(3):166-175.
https://doi.org/10.18632/aging.100444  

2. Tabibzadeh S. Cell-centric hypotheses of aging. Front Biosci. (Landmark Ed). 2021;26(1):1-49.
https://doi.org/10.2741/4888  

3. Barnes PJ. Mechanisms of development of multimorbidity in the elderly. Eur Respir J. 2015;45(3):790-806.
https://doi.org/10.1183/09031936.00229714  

4. Rezus E, Cardoneanu A, Burlui A, Luca A, Codreanu C, Tamba BI, et al. The link between inflammaging and degenerative joint diseases. Int J Mol Sci. 2019;20(3):614.
https://doi.org/10.3390/ijms20030614  

5. Antuna E, Cachan-Vega C, Bermejo-Millo JC, Potes Y, Caballero B, Vega-Naredo I, et al. Inflammaging: Implications in sarcopenia. Int J Mol Sci. 2022;23(23):15039.
https://doi.org/10.3390/ijms232315039  

6. Rea IM, Gibson DS, McGilligan V, McNerlan SE, Alexander HD, Ross OA. Age and age-related diseases: Role of inflammation triggers and cytokines. Front Immunol. 2018;9:586.
https://doi.org/10.3389/fimmu.2018.00586  

7. Cuervo AM, Macian F. Autophagy and the immune function in aging. Curr Opin Immunol. 2014;29:97-104.
https://doi.org/10.1016/j.coi.2014.05.006  

8. Shi C-S, Shenderov K, Huang N-N, Kabat J, Abu-Asab M, Fitzgerald KA, et al. Activation of autophagy by inflammatory signals limits IL-1? production by targeting ubiquitinated inflammasomes for destruction. Nat Immunol. 2012;13(3):255-63.
https://doi.org/10.1038/ni.2215  

9. Bharath LP, Nikolajczyk BS. Next steps in mechanisms of inflammaging. Autophagy. 2020;16(12):2285-6.
https://doi.org/10.1080/15548627.2020.1822089  

10. Popat A, Patel AA, Warnes G. A flow cytometric study of ER stress and autophagy: Flow cytometric study. Cytometry A. 2019;95(6):672-82.
https://doi.org/10.1002/cyto.a.23665  

11. Zhu Z, Yang C, Iyaswamy A, Krishnamoorthi S, Sreenivasmurthy SG, Liu J, et al. Balancing mTOR signaling and autophagy in the treatment of Parkinson's disease. Int J Mol Sci. 2019;20(3):728.
https://doi.org/10.3390/ijms20030728  

12. Li Y, Chen Y. AMPK and autophagy. Adv Exp Med Biol. 2019;1206:85-108.
https://doi.org/10.1007/978-981-15-0602-4_4  

13. Rong Z, Zheng K, Chen J, Jin X. Function and regulation of ULK1: From physiology to pathology. Gene. 2022;840(146772):146772.
https://doi.org/10.1016/j.gene.2022.146772  

14. Chen S, Wang C, Yeo S, Liang C-C, Okamoto T, Sun S, et al. Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model. Genes Dev. 2016;30(7):856-69.
https://doi.org/10.1101/gad.276428.115  

15. Kang R, Zeh HJ, Lotze MT, Tang D. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ. 2011;18(4):571-80.
https://doi.org/10.1038/cdd.2010.191  

16. Dikic I, Elazar Z. Mechanism and medical implications of mammalian autophagy. Nat Rev Mol Cell Biol. 2018;19(6):349-64.
https://doi.org/10.1038/s41580-018-0003-4  

17. Fraiberg M, Elazar Z. Genetic defects of autophagy linked to disease. Prog Mol Biol Transl Sci. 2020;172:293-323.
https://doi.org/10.1016/bs.pmbts.2020.04.001  

18. Nakatogawa H. Mechanisms governing autophagosome biogenesis. Nat Rev Mol Cell Biol. 2020;21(8):439-58.
https://doi.org/10.1038/s41580-020-0241-0  

19. Kumar AV, Mills J, Lapierre LR. Selective autophagy receptor p62/SQSTM1, a pivotal player in stress and aging. Front Cell Dev Biol. 2022;10:793328.
https://doi.org/10.3389/fcell.2022.793328  

20. Xian H, Yang Q, Xiao L, Shen H-M, Liou Y-C. STX17 dynamically regulated by Fis1 induces mitophagy via hierarchical macroautophagic mechanism. Nat Commun. 2019;10(1):2059.
https://doi.org/10.1038/s41467-019-10096-1  

21. Levine B, Mizushima N, Virgin HW. Autophagy in immunity and inflammation. Nature. 2011;469(7330):323-35.
https://doi.org/10.1038/nature09782  

22. Choi AJS, Ryter SW. Autophagy in inflammatory diseases. Int J Cell Biol. 2011;2011:732798.
https://doi.org/10.1155/2011/732798  

23. Harris J. Autophagy and cytokines. Cytokine. 2011;56(2):140-4.
https://doi.org/10.1016/j.cyto.2011.08.022  

24. Djavaheri-Mergny M, Amelotti M, Mathieu J, Besancon F, Bauvy C, Souquere S, et al. NF-kappaB activation represses tumor necrosis factor-alpha-induced autophagy. J Biol Chem. 2006;281(41):30373-82.
https://doi.org/10.1074/jbc.M602097200  

25. Saitoh T, Fujita N, Jang MH, Uematsu S, Yang B-G, Satoh T, et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production. Nature. 2008;456(7219):264-8.
https://doi.org/10.1038/nature07383  

26. Bharath LP, Agrawal M, McCambridge G, Nicholas DA, Hasturk H, Liu J, et al. Metformin enhances autophagy and normalizes mitochondrial function to alleviate aging-associated inflammation. Cell Metab. 2020;32(1):44-55.e6.
https://doi.org/10.1016/j.cmet.2020.04.015  

27. Yang Z, Goronzy JJ, Weyand CM. Autophagy in autoimmune disease. J Mol Med. 2015;93(7):707-17.
https://doi.org/10.1007/s00109-015-1297-8  

28. Harrison DE, Strong R, Sharp ZD, Nelson JF, Astle CM, Flurkey K, et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature. 2009;460(7253):392-5..
https://doi.org/10.1038/nature08221  

29. Dong X, Chu D, Wang Z. Leukocyte-mediated delivery of nanotherapeutics in inflammatory and tumor sites. Theranostics. 2017;7(3):751-63.
https://doi.org/10.7150/thno.18069  

30. Mihalache CC, Yousefi S, Conus S, Villiger PM, Schneider EM, Simon H-U. Inflammation-associated autophagy-related programmed necrotic death of human neutrophils characterized by organelle fusion events. J Immunol. 2011;186(11):6532-42.
https://doi.org/10.4049/jimmunol.1004055  

31. Wu M-Y, Lu J-H. Autophagy and macrophage functions: Inflammatory response and phagocytosis. Cells. 2019;9(1):70.
https://doi.org/10.3390/cells9010070  

32. van Beek AA, Van den Bossche J, Mastroberardino PG, de Winther MPJ, Leenen PJM. Metabolic alterations in aging macrophages: Ingredients for inflammaging? Trends Immunol. 2019;40(2):113-27.
https://doi.org/10.1016/j.it.2018.12.007  

33. Reynolds LM, Ding J, Taylor JR, Lohman K, Soranzo N, de la Fuente A, et al. Transcriptomic profiles of aging in purified human immune cells. BMC Genomics. 2015;16(1):333.

https://doi.org/10.1186/s12864-015-1522-4  

34. Zhang H, Alsaleh G, Feltham J, Sun Y, Napolitano G, Riffelmacher T, et al. Polyamines control eIF5A hypusination, TFEB translation, and autophagy to reverse B cell senescence. Mol Cell. 2019;76(1):110-25.e9.

https://doi.org/10.1016/j.molcel.2019.08.005  

35. Phadwal K, Alegre-Abarrategui J, Watson AS, Pike L, Anbalagan S, Hammond EM, et al. A novel method for autophagy detection in primary cells: impaired levels of macroautophagy in immunosenescent T cells. Autophagy. 2012;8(4):677-89.

https://doi.org/10.4161/auto.18935  

36. Bektas A, Schurman SH, Gonzalez-Freire M, Dunn CA, Singh AK, Macian F, et al. Age-associated changes in human CD4+ T cells point to mitochondrial dysfunction consequent to impaired autophagy. Aging (Albany NY). 2019;11(21):9234-63.

https://doi.org/10.18632/aging.102438  

37. Arnold CR, Pritz T, Brunner S, Knabb C, Salvenmoser W, Holzwarth B, et al. T cell receptor-mediated activation is a potent inducer of macroautophagy in human CD8(+)CD28(+) T cells but not in CD8(+)CD28(-) T cells. Exp Gerontol. 2014;54:75-83.

https://doi.org/10.1016/j.exger.2014.01.018  

38. Raz Y, Guerrero-Ros I, Maier A, Slagboom PE, Atzmon G, Barzilai N, et al. Activation-induced autophagy is preserved in CD4+ T-cells in familial longevity. J Gerontol A Biol Sci Med Sci. 2017;72(9):1201-6.
https://doi.org/10.1093/gerona/glx020  

39. Monick MM, Powers LS, Walters K, Lovan N, Zhang M, Gerke A, et al. Identification of an autophagy defect in smokers' alveolar macrophages. J Immunol. 2010;185(9):5425-35.
https://doi.org/10.4049/jimmunol.1001603  

40. Fujii S, Hara H, Araya J, Takasaka N, Kojima J, Ito S, et al. Insufficient autophagy promotes bronchial epithelial cell senescence in chronic obstructive pulmonary disease. Oncoimmunology. 2012;1(5):630-41.
https://doi.org/10.4161/onci.20297  

41. Qian S, Han Y, Shi Y, Xu W, Zhu Y, Jiang S, et al. Benzene induces haematotoxicity by promoting deacetylation and autophagy. J Cell Mol Med. 2019;23(2):1022-33.
https://doi.org/10.1111/jcmm.14003  

42. Srivastava RK, Traylor AM, Li C, Feng W, Guo L, Antony VB, et al. Cutaneous exposure to lewisite causes acute kidney injury by invoking DNA damage and autophagic response. Am J Physiol Renal Physiol. 2018;314(6):F1166-76.
https://doi.org/10.1152/ajprenal.00277.2017  

43. So K-Y, Lee B-H, Oh S-H. The critical role of autophagy in cadmium-induced immunosuppression regulated by endoplasmic reticulum stress-mediated calpain activation in RAW264.7 mouse monocytes. Toxicology. 2018;393:15-25.
https://doi.org/10.1016/j.tox.2017.10.016  

44. Gu J, Wang Y, Liu Y, Shi M, Yin L, Hou Y, et al. Inhibition of autophagy alleviates cadmium-induced mouse spleen and human B cells apoptosis. Toxicol Sci. 2019;170(1):109-22.
https://doi.org/10.1093/toxsci/kfz089  

45. Eghtedardoost M, Ghazanfari T, Sadeghipour A, Hassan ZM, Ghanei M, Ghavami S. Delayed effects of sulfur mustard on autophagy suppression in chemically-injured lung tissue. Int Immunopharmacol. 2020;80(105896):105896.

https://doi.org/10.1016/j.intimp.2019.105896  

46. Colasanti T, Fiorito S, Alessandri C, Serafino A, Andreola F, Barbati C, et al. Diesel exhaust particles induce autophagy and citrullination in normal human bronchial epithelial cells. Cell Death Dis. 2018;9(11):1073.
https://doi.org/10.1038/s41419-018-1111-y  

47. King KE, McCormick JJ, McManus MK, Janetos K-MT, Goulet N, Kenny GP. Impaired autophagy following ex vivo cooling of simulated hypothermic temperatures in peripheral blood mononuclear cells from young and older adults. J Therm Biol. 2024;121(103831):103831.
https://doi.org/10.1016/j.jtherbio.2024.103831  

48. Qu T-T, Deng J-X, Li R-L, Cui Z-J, Wang X-Q, Wang L, et al. Stress injuries and autophagy in mouse hippocampus after chronic cold exposure. Neural Regen Res. 2017;12(3):440-6.
https://doi.org/10.4103/1673-5374.202932  

49. Kostrzewa-Nowak D, Trzeciak-Ryczek A, Wityk P, Cembrowska-Lech D, Nowak R. Post-effort changes in autophagy- and inflammation-related gene expression in white blood cells of healthy young men. Cells. 2021;10(6):1406.
https://doi.org/10.3390/cells10061406  

50. Weng T-P, Huang S-C, Chuang Y-F, Wang J-S. Effects of interval and continuous exercise training on CD4 lymphocyte apoptotic and autophagic responses to hypoxic stress in sedentary men. PLoS One. 2013;8(11):e80248.
https://doi.org/10.1371/journal.pone.0080248  

51. McCormick JJ, Cote MD, King KE, McManus MK, Goulet N, Dokladny K, et al. Autophagic response to exercise in peripheral blood mononuclear cells from young men is intensity-dependent and is altered by exposure to environmental heat. Am J Physiol Regul Integr Comp Physiol. 2022;323(4):R467-82.
https://doi.org/10.1152/ajpregu.00110.2022  

52. McCormick JJ, McManus MK, King KE, Goulet N, Kenny GP. The intensity-dependent effects of exercise and superimposing environmental heat stress on autophagy in peripheral blood mononuclear cells from older men. Am J Physiol Regul Integr Comp Physiol. 2024;326(1):R29-42.
https://doi.org/10.1152/ajpregu.00163.2023  

53. Moberg M, Hendo G, Jakobsson M, Mattsson CM, Ekblom-Bak E, Flockhart M, et al. Increased autophagy signaling but not proteasome activity in human skeletal muscle after prolonged low-intensity exercise with negative energy balance. Physiol Rep. 2017;5(23).
https://doi.org/10.14814/phy2.13518  

54. Carneiro A, Macedo-da-Silva J, Santiago VF, de Oliveira GS, Guimaraes T, Mendonca CF, et al. Urine proteomics as a non-invasive approach to monitor exertional rhabdomyolysis during military training. J Proteomics. 2022;258(104498):104498.
https://doi.org/10.1016/j.jprot.2022.104498  

55. Carneiro A, Viana-Gomes D, Macedo-da-Silva J, Lima GHO, Mitri S, Alves SR, et al. Risk factors and future directions for preventing and diagnosing exertional rhabdomyolysis. Neuromuscul Disord. 2021;31(7):583-95.
https://doi.org/10.1016/j.nmd.2021.04.007  

56. Wang J, Lu K, Liang F, Li X, Wang L, Yang C, et al. Decreased autophagy contributes to myocardial dysfunction in rats subjected to nonlethal mechanical trauma. PLoS One. 2013;8(8):e71400.
https://doi.org/10.1371/journal.pone.0071400  

57. Mufson EJ, Perez SE, Nadeem M, Mahady L, Kanaan NM, Abrahamson EE, et al. Progression of tau pathology within cholinergic nucleus basalis neurons in chronic traumatic encephalopathy: A chronic effects of neurotrauma consortium study. Brain Inj. 2016;30(12):1399-413.
https://doi.org/10.1080/02699052.2016.1219058  

58. Goldstein LE, Fisher AM, Tagge CA, Zhang X-L, Velisek L, Sullivan JA, et al. Chronic traumatic encephalopathy in blast-exposed military veterans and a blast neurotrauma mouse model. Sci Transl Med. 2012;4(134):134ra60.
https://doi.org/10.1126/scitranslmed.3004862  

59. Zhou Y, Song Y, Zhu L. Activation of autophagy after blast-induced traumatic brain injury in mice. Neuroreport. 2023;34(15):759-66.
https://doi.org/10.1097/WNR.0000000000001951  

60. Saykally JN, Ratliff WA, Keeley KL, Pick CG, Mervis RF, Citron BA. Repetitive mild closed head injury alters protein expression and dendritic complexity in a mouse model. J Neurotrauma. 2018;35(1):139-48.
https://doi.org/10.1089/neu.2017.5070  

61. Scaini G, Mason BL, Diaz AP, Jha MK, Soares JC, Trivedi MH, et al. Dysregulation of mitochondrial dynamics, mitophagy and apoptosis in major depressive disorder: Does inflammation play a role? Mol Psychiatry. 2022;27(2):1095-102.
https://doi.org/10.1038/s41380-021-01312-w  

62. Raju RP, Terry AV. Dysregulation of cellular energetics in Gulf War Illness. Toxicology. 2021;461(152894):152894.
https://doi.org/10.1016/j.tox.2021.152894  

63. Du C-H, Wu Y-D, Yang K, Liao W-N, Ran L, Liu C-N, et al. Apoptosis-resistant megakaryocytes produce large and hyperreactive platelets in response to radiation injury. Mil Med Res. 2023;10(1):66.
https://doi.org/10.1186/s40779-023-00499-z  

64. Chaurasia M, Gupta S, Das A, Dwarakanath BS, Simonsen A, Sharma K. Radiation induces EIF2AK3/PERK and ERN1/IRE1 mediated pro-survival autophagy. Autophagy. 2019;15(8):1391-406.
https://doi.org/10.1080/15548627.2019.1582973  

65. Lin W, Yuan N, Wang Z, Cao Y, Fang Y, Li X, et al. Autophagy confers DNA damage repair pathways to protect the hematopoietic system from nuclear radiation injury. Sci Rep. 2015;5:12362. doi: 10.1038/srep12362 Erratum in: Sci Rep. 2016;6:30095.
https://doi.org/10.1038/srep12362  

66. Hou J, Han Z-P, Jing Y-Y, Yang X, Zhang S-S, Sun K, et al. Autophagy prevents irradiation injury and maintains stemness through decreasing ROS generation in mesenchymal stem cells. Cell Death Dis. 2013;4(10):e844.
https://doi.org/10.1038/cddis.2013.338  

67. Rodrigues-Moreira S, Moreno SG, Ghinatti G, Lewandowski D, Hoffschir F, Ferri F, et al. Low-dose irradiation promotes persistent oxidative stress and decreases self-renewal in hematopoietic stem cells. Cell Rep. 2017;20(13):3199-211.
https://doi.org/10.1016/j.celrep.2017.09.013  

68. Xu F, Fang Y, Yan L, Xu L, Zhang S, Cao Y, et al. Nuclear localization of Beclin 1 promotes radiation-induced DNA damage repair independent of autophagy. Sci Rep. 2017;7:45385.
https://doi.org/10.1038/srep45385  

69. Zhuang X, Lin X, Xu R, Zhang Z, Zhou B, Deng F. ATG7-mediated autophagy protects human gingival myofibroblasts from irradiation-induced apoptosis. J Oral Pathol Med. 2023;52(10):996-1003.
https://doi.org/10.1111/jop.13490  

70. Chen X, Qin W, Wang L, Jin Y, Tu J, Yuan X. Autophagy gene Atg7 regulates the development of radiation-induced skin injury and fibrosis of skin. Skin Res Technol. 2023;29(6):e13337.
https://doi.org/10.1111/srt.13337  

71. Kalamida D, Karagounis IV, Giatromanolaki A, Koukourakis MI. Important role of autophagy in endothelial cell response to ionizing radiation. PLoS One. 2014;9(7):e102408.
https://doi.org/10.1371/journal.pone.0102408