Application of an intermediate concentration of cyclophosphamide does not specifically deplete regulatory T cells in a mouse experimental model
DOI:
https://doi.org/10.2298/ABS230715032RKeywords:
Cyclophosphamide, immunosuppression, lymphocytes, mouse model system, regulatory T cells (Tregs)Abstract
Paper description:
- Cyclophosphamide (100 mg/kg), applied once intraperitoneally to C57BL/6 mice, does not alter proportions or absolute counts of CD4+ cells or CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) in the blood.
- Cyclophosphamide (100 mg/kg), applied two times 5 days apart, decreased to the same extent the absolute numbers of CD4+ and Tregs.
- Decreased Treg counts in peripheral blood after cyclophosphamide treatment mirrored the diminished splenic Treg number.
- Two-dose application of cyclophosphamide does not specifically affect the Treg population in peripheral blood or the spleen and cannot be used as a model for in vivo Treg depletion.
Abstract: Cyclophosphamide (CP) is a cytostatic, widely used to treat different carcinomas and autoimmune diseases. It is commonly used in experimental designs modeling immunosuppression in laboratory animals, with different approaches for CP treatment but without a consensus on the dose, timing, and route of administration. We aimed to establish if treatment with CP in C57BL/6 mice depletes regulatory T cells (Tregs). Tregs are a crucial component of the immune system that helps maintain immune tolerance and prevent excessive immune reactions. They are significant in autoimmune diseases, allergies, and immune-related therapies. CP was applied intraperitoneally (i.p.) twice in a 5-day interval in doses of 100 mg/kg. Monitoring of Treg prevalence in peripheral blood after each treatment and in the spleen after the second treatment with CP revealed a drop in the number of Tregs after two doses of CP because of the decreased number of total lymphocytes but not as a specific response of the Tregs. The prevalence of Tregs in peripheral blood after CP treatment mirrored the change in Treg number in the spleen. CP treatment induced a decrease in the number of CD3+ cells in the spleen while increasing their proportion, indicating that CP affected the B lymphocyte population rather than T cells. Our results suggest that CP treatment cannot be used as a specific Treg-depleting agent in the C57BL/6 animal model.
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Farago AF, Drapkin BJ, de Ramos J, Galmarini CM, Nunez R, Kahatt C, Paz-Arez L. ATLANTIS: a Phase III study of lurbinectedin/doxorubicin versus topotecan or cyclophosphamide/doxorubicin/ vincristine in patients with small-cell lung cancer who have failed one prior platinum-containing line. Future Oncol. 2019;15(3):231-9. https://doi.org/10.2217/fon-2018-0597
Lee CK, Scott C, Lindeman GJ, Hamilton A, Lieschke E, Gibbs E, Asher R, Badger H, Paterson R, Macnab L, Kwan EM, Francis PA, Boyle F, Friedlander M. Phase 1 trial of olaparib and oral cyclophosphamide in BRCA breast cancer, recurrent BRCA ovarian cancer, non-BRCA triple-negative breast cancer, and non-BRCA ovarian cancer. Br J Cancer. 2019;120(3):279-85. https://doi.org/10.1038/s41416-018-0349-6
Xu Y, Wang H, Zhou S, Yu M, Wang X, Fu K, Qian Z, Zhang H, Qiu L, Liu X, Wang P. Risk of second malignant neoplasms after cyclophosphamide-based chemotherapy with or without radiotherapy for non-Hodgkin lymphoma. Leuk Lymphoma. 2013;54(7):1396-404. https://doi.org/10.3109/10428194.2012.743657
Lee Ching C, Kenyon L, Berk M, Park C. Rheumatoid meningitis sine arthritis. J Neuroimmunol. 2019;328:73-5. https://doi.org/10.1016/j.jneuroim.2018.12.001
Cooray S, Zhang H, Breen R, Carr-White G, Howard R, Cuadrado M, D'Cruz D, Sanna G. Cerebral tuberculosis in a patient with systemic lupus erythematosus following cyclophosphamide treatment: a case report. Lupus. 2018;27(4):670-5. https://doi.org/10.1177/0961203317722849
Yu Q, Nie SP, Wang JQ, Huang DF, Li WJ, Xie MY. Molecular mechanism underlying chemoprotective effects of Ganoderma atrum polysaccharide in cyclophosphamide-induced immunosuppressed mice. J Funct Foods. 2015;15:52-60. https://doi.org/10.1016/j.jff.2015.03.015
Becker JC, Schrama D. The Dark Side of Cyclophosphamide: Cyclophosphamide-Mediated Ablation of Regulatory T Cells. J Invest Dermatol. 2013;133:1462-5. https://doi.org/10.1038/jid.2013.67
Ge Y, Domschke C, Stoiber N, Schott S, Heil J, Rom J, Blumenstein M, Thum J, Sohn C, Schneeweiss A, Beckhove P, Schuetz F. Metronomic cyclophosphamide treatment in metastasized breast cancer patients: immunological effects and clinical outcome. Cancer Immunol Immunother. 2012;61(3):353-62. https://doi.org/10.1007/s00262-011-1106-3
Wlodarczyk M, Ograczyk E, Kowalewicz-Kulbat M, Druszczyńska M, Rudnicka W, Fol1 M. Effect of Cyclophosphamide Treatment on Central and Effector Memory T Cells in Mice. Int J Toxicol. 2018;37(5):373-82. https://doi.org/10.1177/1091581818780128
Huyan X, Lin Y, Gao T, Chen RY, Fan YM. Immunosuppressive effect of cyclophosphamide on white blood cells and lymphocyte subpopulations from peripheral blood of Balb/c mice. Int Immunopharmacol. 2011;11(9):1293-7. https://doi.org/10.1016/j.intimp.2011.04.011
Wójcik R, Dabkowska A. The effect of cyclophosphamide on the selected parameters of immunity in rats. Cent Eur J Immunol. 2010;35(1):1-9.
Ghiringhelli F, Larmonier N, Schmitt E, Parcellier A, Cathelin D, Garrido C, Chauffert B, Solary E, Bonnotte B, Martin F. CD4+CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative. Eur J Immunol. 2004;34:336-44. https://doi.org/10.1002/eji.200324181
Tanaka H, Tanaka J, Kjaergaard J, Shu S. Depletion of CD4+ CD25+ regulatory cells augments the generation of specific immune T cells in tumor-draining lymph nodes. J Immunother. 2002;25:207-17. https://doi.org/10.1097/00002371-200205000-00003
Motoyoshi Y, Kaminoda K, Saitoh O, Hamasaki K, Nakao K, Ishii N, Nagayama Y, Eguchi K. Different mechanisms for anti-tumor effects of low-and high-dose cyclophosphamide. Oncol Rep. 2006;16(1):141-6. https://doi.org/10.3892/or.16.1.141
Zhao J, Cao Y, Lei Z, Yang Z, Zhang B, Huang B. Selective depletion of CD4+CD25+Foxp3+ regulatory T cells by low-dose cyclophosphamide is explained by reduced intracellular ATP levels. Cancer Res. 2010;70(12):4850-8. https://doi.org/10.1158/0008-5472.CAN-10-0283
Zhang WN, Gong LL, Liu Y, Zhou ZB, Wan CX, Xu JJ, Wu QX, Chen L, Lu YM, Chen Y. Immunoenhancement effect of crude polysaccharides of Helvella leucopus on cyclophosphamide-induced immunosuppressive mice. J Funct Foods. 2020;69:103942. https://doi.org/10.1016/j.jff.2020.103942
Kim HI, Kim DS, Jung Y, Sung NY, Kim M, Han IJ, Nho EY, Hong JH, Lee JK, Boo M, Kim HL, Baik S, Jung KO, Lee S, Kim CS, Park J. Immune-Enhancing Effect of Sargassum horneri on Cyclophosphamide-Induced Immunosuppression in BALB/c Mice and Primary Cultured Splenocytes. Molecules. 2022;27:8253. https://doi.org/10.3390/molecules27238253
Zdravkovic N, Shahin A, Arsenijevic N, Lukic ML, Mensah-Brown EP. Regulatory T cells and ST2 signaling control diabetes induction with multiple low doses of streptozotocin. Mol Immunol. 2009;47(1):28-36. https://doi.org/10.1016/j.molimm.2008.12.023
Brode S, Raine T, Zaccone P, Cooke A. Cyclophosphamide-induced type-1 diabetes in the NOD mouse is associated with a reduction of CD4+CD25+Foxp3+ regulatory T cells. J Immunol. 2006;177(10):6603-12. https://doi.org/10.4049/jimmunol.177.10.6603
Kiesel U, Greulich B, Moumé CM, Kolb H. Induction of experimental autoimmune diabetes by low-dose streptozotocin treatment in genetically resistant mice. Immunol Lett. 1981;3(4):227-30. https://doi.org/10.1016/0165-2478(81)90079-1
Kaur S, Tan WL, Soo C, Cheung CC, Stewart J, Reddy S. An immunohistochemical study on the distribution and frequency of T regulatory cells in pancreatic islets of NOD mice during various stages of spontaneous and cyclophosphamide-accelerated diabetes. Pancreas. 2010;39(7):1024-33. https://doi.org/10.1097/MPA.0b013e3181da9037
Heylmann D, Bauer M, Becker H, van Gool S, Bacher N, Steinbrink K, Kaina B. Human CD4+CD25+ regulatory T cells are sensitive to low dose cyclophosphamide: implications for the immune response. PLoS One. 2013;8(12):e83384. https://doi.org/10.1371/journal.pone.0083384
Man S, Bocci G, Francia G, Green SK, Jothy S, Hanahan D, Bohlen P, Hicklin DJ, Bergers G, Kerbel RS. Antitumor effects in mice of low-dose (metronomic) cyclophosphamide administered continuously through the drinking water. Cancer Res. 2002;62(10): 2731-5.
Feng L, Huang Q, Huang Z, Li H, Qi X, Wang Y, Liu Z, Liu X, Lu L. Optimized animal model of cyclophosphamide-induced bone marrow suppression. Basic Clin Pharmacol Toxicol. 2016;119(5):428-35. https://doi.org/10.1111/bcpt.12600
Hou F, Yang H, Yu T, Chen W. The immunosuppressive effects of 10 mg/kg cyclophosphamide in Wistar rats. Environ Toxicol Pharmacol. 2007;24(1):30-6. https://doi.org/10.1016/j.etap.2007.01.004
Gonzalez EJ, Peterson A, Malley S, Daniel M, Lambert D, Kosofsky M, Vizzard MA. The effects of tempol on cyclophosphamide-induced oxidative stress in rat micturition reflexes. Sci World J. 2015;2015:545048. https://doi.org/10.1155/2015/545048
Li P, Chen F, Zheng J, Yang Y, Li Y, Wang Y, Chen X. Cyclophosphamide abrogates the expansion of CD4+Foxp3+ regulatory T cells and enhances the efficacy of bleomycin in the treatment of mouse B16-F10 melanomas. Cancer Biol Med. 2021;18(4):1010-20. https://doi.org/10.20892/j.issn.2095-3941.2021.0027
Zhong H, Lai Y, Zhang R, Daoud A, Feng Q, Zhou J, Shang J. Low Dose Cyclophosphamide Modulates Tumor Microenvironment by TGF-β Signaling Pathway. Int J Mol Sci. 2020;21(3):957. https://doi.org/10.3390/ijms21030957
Noh EM, Kim JM, Lee HY, Song HK, Joung SO, Yang HJ, Kim MJ, Kim KS, Lee YR. Immuno-enhancement effects of Platycodon grandiflorum extracts in splenocytes and a cyclophosphamide-induced immunosuppressed rat model. BMC Complement Altern Med. 2019;19(1):322. https://doi.org/10.1186/s12906-019-2724-0
Salem ML, Al-Khami AA, El-Nagaar SA, Zidan AA, Al-Sharkawi IM, Marcela Díaz-Montero C, Cole DJ. Kinetics of rebounding of lymphoid and myeloid cells in mouse peripheral blood, spleen and bone marrow after treatment with cyclophosphamide. Cell Immunol. 2012;276(1-2):67-74. https://doi.org/10.1016/j.cellimm.2012.03.010
Huang R, Zhang J, Liu Y, Hao Y, Yang C, Wu K, Cao S, Wu C. Immunomodulatory effects of polysaccharopeptide in immunosuppressed mice induced by cyclophosphamide. Mol Med Rep. 2013;8(2):669-75. https://doi.org/10.3892/mmr.2013.1542
Zhang Z, Pan T, Liu C, Shan X, Xu Z, Hong H, Lin H, Chen J, Sun H. Cyclophosphamide induced physiological and biochemical changes in mice with an emphasis on sensitivity analysis. Ecotoxicol Environ Saf. 2021;211:111889. https://doi.org/10.1016/j.ecoenv.2020.111889
Yang H, Choi K, Kim KJ, Park SY, Jeon JY, Kim BG, Kim JY. Immunoenhancing Effects of Euglena gracilis on a Cyclophosphamide-Induced Immunosuppressive Mouse Model. J Microbiol Biotechnol. 2022;32(2):228-37. https://doi.org/10.4014/jmb.2112.12035
Ikezawa Y, Nakazawa M, Tamura C, Takahashi K, Minami M, Ikezawa Z. Cyclophosphamide decreases the number, percentage and the function of CD25+ CD4+ regulatory T cells, which suppress induction of contact hypersensitivity. J Dermatol Sci. 2005;39(2):105-12. https://doi.org/10.1016/j.jdermsci.2005.02.002
Salem ML, Kadima AN, El-Naggar SA, Rubinstein MP, Chen Y, Gillanders WE, Cole DJ. Defining the ability of cyclophosphamide preconditioning to enhance the antigen-specific CD8+ T-cell response to peptide vaccination: creation of a beneficial host microenvironment involving type I IFNs and myeloid cells. J Immunother. 2007;30(1):40-53. https://doi.org/10.1097/01.cji.0000211311.28739.e3
Shurlygina AV, Mel'nikova EV, Trufakin VA. Chronodependent effect of interleukin-2 on mouse spleen cells in the model of cyclophosphamide immunosuppression. Bull Exp Biol Med. 2015;158(4):471-4. https://doi.org/10.1007/s10517-015-2787-y
Zdravkovic N, Pavlovic S, Zdravkovic V, Pejnovic N, Arsenijevic N, Lukic ML. ST2 gene-deletion reveals a role of Foxp3+ regulatory T cells in diabetes modulation in BALB/c mice. Transl Res. 2013;161(2):118-29. https://doi.org/10.1016/j.trsl.2012.10.005
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