Infusion of Induced Regulatory T Cells Alleviates Atherosclerosis by Reducing Pathological Macrophage-Like Vascular Smooth Muscle Cells

Abstract

Atherosclerosis remains the primary driver of cardiovascular and cerebrovascular morbidity and mortality. A pivotal event in its pathogenesis is the phenotypic conversion of vascular smooth muscle cells (VSMCs), particularly the transition from a contractile to a macrophage-like state. Using a murine model of atherosclerosis, we demonstrate that this process is orchestrated by a progressively disrupted perivascular immune milieu characterized by an expansion of CD44⁺ memory CD4⁺ T cells at the expense of CD44⁻ naive CD4⁺ T cells. Within this niche, CD44⁺ natural regulatory T cells (nTregs) actively promote VSMCs macrophage-like reprogramming, whereas their CD44⁻ counterparts exert an opposing, protective effect. Reciprocally, macrophage-like VSMCs foster the trans-differentiation of nTregs into pathogenic Th17 cells, amplifying vascular inflammation. In contrast, induced Treg cells (iTregs) display phenotypic stability and potently inhibit VSMCs macrophage-like switching, restrict pathological VSMCs migration, and curtail VSMCs survival. Systemic infusion of iTregs selectively remodels the perivascular immune microenvironment toward an antiatherogenic profile. Adoptive transfer of iTregs at early disease stages decreased the abundance of macrophage-like VSMCs, attenuated plaque burden, and these benefits were partially mediated by transforming growth factor β signaling. Collectively, iTreg-based cellular therapy represents a promising strategy to intercept VSMCs macrophage-like transformation and limit atherosclerotic progression.

Keywords

atherosclerosis / iTregs / phenotypic switching / VSMCs

References

[1] L. Nedkoff, T. Briffa, D. Zemedikun, S. Herrington, and F. L. Wright, “Global Trends in Atherosclerotic Cardiovascular Disease,” Clinical Therapeutics 45, no. 11 (2023): 1087-1091.

[2] H. U. H. Ansari, E. Mahboob, M. A. Samad, et al., “Temporal Trends and Disparities in Atherosclerosis-associated Cerebrovascular Disease Mortality in the United States, 1999-2020,” Current Problems in Cardiology 50, no. 2 (2025): 102935.

[3] A. C. Aprotosoaie, A. D. Costache, and I. I. Costache, “Therapeutic Strategies and Chemoprevention of Atherosclerosis: What Do We Know and Where Do We Go?,” Pharmaceutics 14, no. 4 (2022): 722.

[4] A. Ajoolabady, D. Pratico, L. Lin, et al., “Inflammation in Atherosclerosis: Pathophysiology and Mechanisms,” Cell Death & Disease 15, no. 11 (2024): 817.

[5] C. Madaudo, G. Coppola, A. L. M. Parlati, and E. Corrado, “Discovering Inflammation in Atherosclerosis: Insights From Pathogenic Pathways to Clinical Practice,” International Journal of Molecular Sciences 25, no. 11 (2024): 6016.

[6] P. Kong, Z. Y. Cui, X. F. Huang, D. D. Zhang, R. J. Guo, and M. Han, “Inflammation and Atherosclerosis: Signaling Pathways and Therapeutic Intervention,” Signal Transduct Target Ther 7, no. 1 (2022): 131.

[7] H. Pan, C. Xue, B. J. Auerbach, et al., “Single-Cell Genomics Reveals a Novel Cell State during Smooth Muscle Cell Phenotypic Switching and Potential Therapeutic Targets for Atherosclerosis in Mouse and Human,” Circulation 142, no. 21 (2020): 2060-2075.

[8] M. O. J. Grootaert and M. R. Bennett, “Vascular Smooth Muscle Cells in Atherosclerosis: Time for a Re-assessment,” Cardiovascular Research 117, no. 11 (2021): 2326-2339.

[9] Y. Yu, Y. Cai, F. Yang, et al., “Vascular Smooth Muscle Cell Phenotypic Switching in Atherosclerosis,” Heliyon 10, no. 18 (2024): e37727.

[10] L. Zanoli, M. Briet, J. P. Empana, et al., “Vascular Consequences of Inflammation: A Position Statement From the ESH Working Group on Vascular Structure and Function and the ARTERY Society,” Journal of Hypertension 38, no. 9 (2020): 1682-1698.

[11] M. Liu and D. Gomez, “Smooth Muscle Cell Phenotypic Diversity,” Arteriosclerosis, Thrombosis, and Vascular Biology 39, no. 9 (2019): 1715-1723.

[12] A. Lin, J. M. Miano, E. A. Fisher, and A. Misra, “Chronic Inflammation and Vascular Cell Plasticity in Atherosclerosis,” Nat Cardiovasc Res 3, no. 12 (2024): 1408-1423.

[13] M. T. Patterson, M. M. Firulyova, Y. Xu, et al., “Trem2 promotes Foamy Macrophage Lipid Uptake and Survival in Atherosclerosis,” Nat Cardiovasc Res 2, no. 11 (2023): 1015-1031.

[14] Y. X. Liu, P. Z. Yuan, J. H. Wu, and B. Hu, “Lipid Accumulation and Novel Insight Into Vascular Smooth Muscle Cells in Atherosclerosis,” J Mol Med (Berl) 99, no. 11 (2021): 1511-1526.

[15] F. Bonacina, A. Di Costanzo, V. Genkel, et al., “The Heterogeneous Cellular Landscape of Atherosclerosis: Implications for Future Research and Therapies. A Collaborative Review From the EAS Young Fellows,” Atherosclerosis 372 (2023): 48-56.

[16] I. Tabas and A. H. Lichtman, “Monocyte-Macrophages and T Cells in Atherosclerosis,” Immunity 47, no. 4 (2017): 621-634.

[17] I. Tabas and K. E. Bornfeldt, “Macrophage Phenotype and Function in Different Stages of Atherosclerosis,” Circulation Research 118, no. 4 (2016): 653-667.

[18] Y. Wang, J. A. Dubland, S. Allahverdian, et al., “Smooth Muscle Cells Contribute the Majority of Foam Cells in ApoE (Apolipoprotein E)-Deficient Mouse Atherosclerosis,” Arteriosclerosis, Thrombosis, and Vascular Biology 39, no. 5 (2019): 876-887.

[19] S. Allahverdian, A. C. Chehroudi, B. M. McManus, T. Abraham, and G. A. Francis, “Contribution of Intimal Smooth Muscle Cells to Cholesterol Accumulation and Macrophage-Like Cells in human Atherosclerosis,” Circulation 129, no. 15 (2014): 1551-1559.

[20] P. Hou, J. Fang, Z. Liu, Y. Shi, and M. Agostini, “Macrophage Polarization and Metabolism in Atherosclerosis,” Cell death & disease 14, no. 10 (2023): 691.

[21] H. Winkels, E. Ehinger, M. Vassallo, et al., “Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA-Sequencing and Mass Cytometry,” Circulation Research 122, no. 12 (2018): 1675-1688.

[22] M. A. C. Depuydt, K. H. M. Prange, L. Slenders, et al., “Microanatomy of the Human Atherosclerotic Plaque by Single-Cell Transcriptomics,” Circulation Research 127, no. 11 (2020): 1437-1455.

[23] Y. Du, Q. Fang, and S. G. Zheng, “Regulatory T Cells: Concept, Classification, Phenotype, and Biological Characteristics,” Advances in Experimental Medicine and Biology 1278 (2021): 1-31.

[24] X. Zhang, N. Olsen, and S. G. Zheng, “The Progress and Prospect of Regulatory T Cells in Autoimmune Diseases,” Journal of Autoimmunity 111 (2020): 102461.

[25] J. D. Proto, A. C. Doran, G. Gusarova, et al., “Regulatory T Cells Promote Macrophage Efferocytosis During Inflammation Resolution,” Immunity 49, no. 4 (2018): 666-677.e666.

[26] X. Ouyang and Z. Liu, “Regulatory T Cells and Macrophages in Atherosclerosis: From Mechanisms to Clinical Significance,” Frontiers in immunology 15 (2024): 1435021.

[27] E. Maganto-García, M. L. Tarrio, N. Grabie, D. X. Bu, and A. H. Lichtman, “Dynamic Changes in Regulatory T Cells Are Linked to Levels of Diet-induced Hypercholesterolemia,” Circulation 124, no. 2 (2011): 185-195.

[28] A. J. Ali, J. Makings, and K. Ley, “Regulatory T Cell Stability and Plasticity in Atherosclerosis,” Cells 9, no. 12 (2020): 2665.

[29] M. J. Butcher, A. R. Filipowicz, T. C. Waseem, et al., “Atherosclerosis-Driven Treg Plasticity Results in Formation of a Dysfunctional Subset of Plastic IFNγ+ Th1/Tregs,” Circulation Research 119, no. 11 (2016): 1190-1203.

[30] N. R. Blatner, M. F. Mulcahy, K. L. Dennis, et al., “Expression of RORγt Marks a Pathogenic Regulatory T Cell Subset in human Colon Cancer,” Science Translational Medicine 4, no. 164 (2012): 164ra159.

[31] M. J. Butcher, B. N. Gjurich, T. Phillips, and E. V. Galkina, “The IL-17A/IL-17RA Axis Plays a Proatherogenic Role via the Regulation of Aortic Myeloid Cell Recruitment,” Circulation Research 110, no. 5 (2012): 675-687.

[32] L. Kastner, D. Dwyer, and F. X. Qin, “Synergistic Effect of IL-6 and IL-4 in Driving Fate Revision of Natural Foxp3+ Regulatory T Cells,” Journal of Immunology (Baltimore, Md: 1950) 185, no. 10 (2010): 5778-5786.

[33] H. Ait-Oufella, B. L. Salomon, S. Potteaux, et al., “Natural Regulatory T Cells Control the Development of Atherosclerosis in Mice,” Nature Medicine 12, no. 2 (2006): 178-180.

[34] H. X. Ou, B. B. Guo, Q. Liu, et al., “Regulatory T Cells as a New Therapeutic Target for Atherosclerosis,” Acta Pharmacologica Sinica 39, no. 8 (2018): 1249-1258.

[35] E. M. Shevach and A. M. Thornton, “tTregs, pTregs, and iTregs: Similarities and Differences,” Immunological Reviews 259, no. 1 (2014): 88-102.

[36] X. Zhou, N. Kong, J. Wang, et al., “Cutting Edge: All-trans Retinoic Acid Sustains the Stability and Function of Natural Regulatory T Cells in an Inflammatory Milieu,” Journal of Immunology 185, no. 5 (2010): 2675-2679.

[37] N. Kong, Q. Lan, M. Chen, et al., “Antigen-specific Transforming Growth Factor β-induced Treg Cells, but Not Natural Treg Cells, Ameliorate Autoimmune Arthritis in Mice by Shifting the Th17/Treg Cell Balance From Th17 Predominance to Treg Cell Predominance,” Arthritis and Rheumatism 64, no. 8 (2012): 2548-2558.

[38] S. Yang, X. Zhang, J. Chen, et al., “Induced, but Not Natural, Regulatory T Cells Retain Phenotype and Function Following Exposure to Inflamed Synovial Fibroblasts,” Science Advances 6, no. 44 (2020): eabb0606.

[39] M. Sharma, M. P. Schlegel, M. S. Afonso, et al., “Regulatory T Cells License Macrophage Pro-Resolving Functions during Atherosclerosis Regression,” Circulation Research 127, no. 3 (2020): 335-353.

[40] X. He and B. Liang, “Th17/Treg Imbalance and Atherosclerosis,” Disease Markers 2020 (2020): 8821029.

[41] Y. Vengrenyuk, H. Nishi, X. Long, et al., “Cholesterol Loading Reprograms the microRNA-143/145-myocardin Axis to Convert Aortic Smooth Muscle Cells to a Dysfunctional Macrophage-Like Phenotype,” Arteriosclerosis, Thrombosis, and Vascular Biology 35, no. 3 (2015): 535-546.

[42] Z. Zhang, J. Huang, Y. Wang, and W. Shen, “Transcriptome Analysis Revealed a Two-step Transformation of Vascular Smooth Muscle Cells to Macrophage-Like Cells,” Atherosclerosis 346 (2022): 26-35.

[43] C. Mazière, V. Salle, C. Gomila, and J. C. Mazière, “Oxidized Low Density Lipoprotein Increases RANKL Level in human Vascular Cells. Involvement of Oxidative Stress,” Biochemical and Biophysical Research Communications 440, no. 2 (2013): 295-299.

[44] J. M. Moreau, M. Velegraki, C. Bolyard, M. D. Rosenblum, and Z. Li, “Transforming Growth Factor-β1 in Regulatory T Cell Biology,” Science Immunology 7, no. 69 (2022): eabi4613.

[45] K. Tzavlaki and A. Moustakas, “TGF-β Signaling,” Biomolecules 10, no. 3 (2020): 487.

[46] W. Su, H. Fan, M. Chen, et al., “Induced CD4+ Forkhead Box Protein-positive T Cells Inhibit Mast Cell Function and Established Contact Hypersensitivity Through TGF-β1,” Journal of Allergy and Clinical Immunology 130, no. 2 (2012): 444-452. e447.

[47] P. Y. Chen, L. Qin, G. Li, et al., “Smooth Muscle Cell Reprogramming in Aortic Aneurysms,” Cell Stem Cell 26, no. 4 (2020): 542-557. e511.

[48] C. Pagiatakis, D. Sun, S. W. Tobin, T. Miyake, and J. C. McDermott, “TGFβ-TAZ/SRF Signalling Regulates Vascular Smooth Muscle Cell Differentiation,” The FEBS Journal 284, no. 11 (2017): 1644-1656.

[49] L. Calvier, P. Chouvarine, E. Legchenko, et al., “PPARγ Links BMP2 and TGFβ1 Pathways in Vascular Smooth Muscle Cells, Regulating Cell Proliferation and Glucose Metabolism,” Cell Metabolism 25, no. 5 (2017): 1118-1134.e1117.

[50] P. Lacolley, V. Regnault, and A. P. Avolio, “Smooth Muscle Cell and Arterial Aging: Basic and Clinical Aspects,” Cardiovascular Research 114, no. 4 (2018): 513-528.

[51] V. Sorokin, K. Vickneson, T. Kofidis, et al., “Role of Vascular Smooth Muscle Cell Plasticity and Interactions in Vessel Wall Inflammation,” Frontiers in Immunology 11 (2020): 599415.

[52] M. G. Ionita, P. van den Borne, L. M. Catanzariti, et al., “High Neutrophil Numbers in human Carotid Atherosclerotic Plaques Are Associated With Characteristics of Rupture-prone Lesions,” Arteriosclerosis, Thrombosis, and Vascular Biology 30, no. 9 (2010): 1842-1848.

[53] J. W. Han, K. Shimada, W. Ma-Krupa, et al., “Vessel Wall-embedded Dendritic Cells Induce T-cell Autoreactivity and Initiate Vascular Inflammation,” Circulation Research 102, no. 5 (2008): 546-553.

[54] S. Pryshchep, K. Sato, J. J. Goronzy, and C. M. Weyand, “T Cell Recognition and Killing of Vascular Smooth Muscle Cells in Acute Coronary Syndrome,” Circulation Research 98, no. 9 (2006): 1168-1176.

[55] G. Miao, X. Zhao, S. L. Chan, et al., “Vascular Smooth Muscle Cell c-Fos Is Critical for Foam Cell Formation and Atherosclerosis,” Metabolism 132 (2022): 155213.

[56] J. Thazhathveettil, A. K. Kumawat, I. Demirel, A. Sirsjö, and G. V. Paramel, “Vascular Smooth Muscle Cells in Response to Cholesterol Crystals Modulates Inflammatory Cytokines Release and Promotes Neutrophil Extracellular Trap Formation,” Molecular Medicine 30, no. 1 (2024): 42.

[57] G. Cao, X. Xuan, J. Hu, R. Zhang, H. Jin, and H. Dong, “How Vascular Smooth Muscle Cell Phenotype Switching Contributes to Vascular Disease,” Cell Communication and Signaling 20, no. 1 (2022): 180.

[58] J. Pan, Y. Cai, M. Liu, and Z. Li, “Role of Vascular Smooth Muscle Cell Phenotypic Switching in Plaque Progression: A Hybrid Modeling Study,” Journal of Theoretical Biology 526 (2021): 110794.

[59] Y. Chen, Z. Xu, R. Liang, et al., “CD4(+)CD126(low/-) Foxp3(+) Cell Population Represents a Superior Subset of Regulatory T Cells in Treating Autoimmune Diseases,” Molecular Therapy 28, no. 11 (2020): 2406-2416.

[60] Y. Luo and S. G. Zheng, “Hall of Fame Among Pro-inflammatory Cytokines: Interleukin-6 Gene and Its Transcriptional Regulation Mechanisms,” Frontiers in immunology 7 (2016): 604.

[61] L. Lu, Q. Lan, Z. Li, et al., “Critical Role of all-trans Retinoic Acid in Stabilizing human Natural Regulatory T Cells Under Inflammatory Conditions,” PNAS 111, no. 33 (2014): E3432-3440.

[62] P. Das, R. Bose, M. Paul, D. Nandy, T. Basak, and R. Ain, “IL1β-NFκβ-Myocardin Signaling Axis Governs Trophoblast-directed Plasticity of Vascular Smooth Muscle Cells,” Faseb Journal 38, no. 9 (2024): e23637.

[63] V. Rai, H. Singh, and D. K. Agrawal, “Targeting the Crosstalk of Immune Response and Vascular Smooth Muscle Cells Phenotype Switch for Arteriovenous Fistula Maturation,” International Journal of Molecular Sciences 23, no. 19 (2022): 12012.

[64] L. Xu, A. Kitani, I. Fuss, and W. Strober, “Cutting Edge: Regulatory T Cells Induce CD4+CD25-Foxp3- T Cells or Are Self-induced to Become Th17 Cells in the Absence of Exogenous TGF-beta,” Journal of Immunology 178, no. 11 (2007): 6725-6729.

[65] S. G. Zheng, J. Wang, and D. A. Horwitz, “Cutting Edge: Foxp3+CD4+CD25+ Regulatory T Cells Induced by IL-2 and TGF-beta Are Resistant to Th17 Conversion by IL-6,” Journal of Immunology 180, no. 11 (2008): 7112-7116.

[66] Y. Luo, Y. Xue, J. Wang, et al., “Negligible Effect of Sodium Chloride on the Development and Function of TGF-β-Induced CD4(+) Foxp3(+) Regulatory T Cells,” Cell reports 26, no. 7 (2019): 1869-1879. e1863.

[67] M. Kanamori, H. Nakatsukasa, M. Okada, Q. Lu, and A. Yoshimura, “Induced Regulatory T Cells: Their Development, Stability, and Applications,” Trends in Immunology 37, no. 11 (2016): 803-811.

[68] Z. Zhang and X. Zhou, “Foxp3 Instability Helps tTregs Distinguish Self and Non-self,” Frontiers in Immunology 10 (2019): 2226.

[69] B. H. Yang, S. Hagemann, P. Mamareli, et al., “Foxp3(+) T Cells Expressing RORγt Represent a Stable Regulatory T-cell Effector Lineage With Enhanced Suppressive Capacity During Intestinal Inflammation,” Mucosal Immunology 9, no. 2 (2016): 444-457.

[70] S. Taleb, A. Tedgui, and Z. Mallat, “IL-17 and Th17 Cells in Atherosclerosis: Subtle and Contextual Roles,” Arteriosclerosis, Thrombosis, and Vascular Biology 35, no. 2 (2015): 258-264.

[71] K. Danzaki, Y. Matsui, M. Ikesue, et al., “Interleukin-17A Deficiency Accelerates Unstable Atherosclerotic Plaque Formation in Apolipoprotein E-deficient Mice,” Arteriosclerosis, Thrombosis, and Vascular Biology 32, no. 2 (2012): 273-280.

[72] M. S. Madhur, S. A. Funt, L. Li, et al., “Role of Interleukin 17 in Inflammation, Atherosclerosis, and Vascular Function in Apolipoprotein E-deficient Mice,” Arteriosclerosis, Thrombosis, and Vascular Biology 31, no. 7 (2011): 1565-1572.

[73] W. Chen, W. Jin, and N. Hardegen, “Conversion of Peripheral CD4+CD25- naive T Cells to CD4+CD25+ Regulatory T Cells by TGF-beta Induction of Transcription Factor Foxp3,” Journal of Experimental Medicine 198, no. 12 (2003): 1875-1886.

[74] J. Wang, X. Zhao, and Y. Y. Wan, “Intricacies of TGF-β Signaling in Treg and Th17 Cell Biology,” Cell Mol Immunol 20, no. 9 (2023): 1002-1022.

[75] M. R. Bennett, S. Sinha, and G. K. Owens, “Vascular Smooth Muscle Cells in Atherosclerosis,” Circulation Research 118, no. 4 (2016): 692-702.

[76] H. Iwata, I. Manabe, K. Fujiu, et al., “Bone Marrow-derived Cells Contribute to Vascular Inflammation but Do Not Differentiate Into Smooth Muscle Cell Lineages,” Circulation 122, no. 20 (2010): 2048-2057.

[77] L. S. Shankman, D. Gomez, O. A. Cherepanova, et al., “KLF4-dependent Phenotypic Modulation of Smooth Muscle Cells Has a Key Role in Atherosclerotic Plaque Pathogenesis,” Nature Medicine 21, no. 6 (2015): 628-637.

[78] N. M. Caplice, T. J. Bunch, P. G. Stalboerger, et al., “Smooth Muscle Cells in human Coronary Atherosclerosis Can Originate From Cells Administered at Marrow Transplantation,” Proceedings of the National Academy of Sciences of the United States of America 100, no. 8 (2003): 4754-4759.

[79] H. Yu, V. Stoneman, M. Clarke, et al., “Bone Marrow-derived Smooth Muscle-Like Cells Are Infrequent in Advanced Primary Atherosclerotic Plaques but Promote Atherosclerosis,” Arteriosclerosis, Thrombosis, and Vascular Biology 31, no. 6 (2011): 1291-1299.

[80] S. G. Zheng, J. Wang, P. Wang, J. D. Gray, and D. A. Horwitz, “IL-2 Is Essential for TGF-beta to Convert Naive CD4+CD25- cells to CD25+Foxp3+ Regulatory T Cells and for Expansion of these Cells,” Journal of Immunology (Baltimore, Md: 1950) 178, no. 4 (2007): 2018-2027.

[81] X. Chen, W. Su, T. Wan, et al., “Sodium Butyrate Regulates Th17/Treg Cell Balance to Ameliorate Uveitis via the Nrf2/HO-1 Pathway,” Biochemical Pharmacology 142 (2017): 111-119.

[82] T. J. Yun, J. S. Lee, D. Shim, J. H. Choi, and C. Cheong, “Isolation and Characterization of Aortic Dendritic Cells and Lymphocytes in Atherosclerosis,” Methods in Molecular Biology (Clifton, NJ) 1559 (2017): 419-437.

[83] J. J. Patel, S. Srivastava, and R. C. Siow, “Isolation, Culture, and Characterization of Vascular Smooth Muscle Cells,” Methods in Molecular Biology (Clifton, NJ) 1430 (2016): 91-105.

[84] S. G. Zheng, J. H. Wang, M. N. Koss, F. Quismorio,, J. D. Gray, and D. A. Horwitz, “CD4+ and CD8+ Regulatory T Cells Generated Ex Vivo With IL-2 and TGF-beta Suppress a Stimulatory Graft-versus-host Disease With a Lupus-Like Syndrome,” Journal of Immunology (Baltimore, Md: 1950) 172, no. 3 (2004): 1531-1539.

[85] J. X. Rong, M. Shapiro, E. Trogan, and E. A. Fisher, “Transdifferentiation of Mouse Aortic Smooth Muscle Cells to a Macrophage-Like state After Cholesterol Loading,” Proceedings of the National Academy of Sciences of the United States of America 100, no. 23 (2003): 13531-13536.

[86] J. Zhu, B. Liu, Z. Wang, et al., “Exosomes From Nicotine-stimulated Macrophages Accelerate Atherosclerosis Through miR-21-3p/PTEN-mediated VSMC Migration and Proliferation,” Theranostics 9, no. 23 (2019): 6901-6919.

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