BRIEF RESEARCH REPORT article
Front. Immunol.
Sec. Inflammation
Volume 16 - 2025 | doi: 10.3389/fimmu.2025.1661497
A Long-Term Culture Model for Investigating Senescence-Associated Dysregulation in Macrophages
Provisionally accepted
- 1National Institute of Respiratory Diseases-Mexico (INER), Mexico City, Mexico
- 2Universidad Nacional Autonoma de Mexico Facultad de Ciencias, Mexico City, Mexico
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Macrophage senescence is increasingly recognized as a key contributor to immune dysfunction during aging and chronic inflammation. Here, we developed a reproducible long-term in vitro culture model to investigate the senescence's phenotypic and functional consequences in human macrophages. Monocyte-derived macrophages (MDMs) were cultured for 7, 14, or 21 days and assessed for canonical senescence markers and immune function. While macrophages at day 7 exhibited minimal expression of β-galactosidase, H2AXpS139, and CDKN2A (p16INK4a), these markers were significantly upregulated at days 14 and 21, indicating progressive acquisition of a senescent phenotype. Cell viability remained above 95% across all time points, confirming the system's stability. High-dimensional flow cytometry and unsupervised clustering revealed marked phenotypic remodeling over time, shifting from anti-inflammatory CD163⁺/CD206⁺ profiles to proinflammatory CD14⁺/CD64⁺/TLR2⁺ populations. Five distinct macrophage subpopulations were identified, each with dynamic temporal distribution and unique marker expression, highlighting a loss of plasticity and emergence of senescence-associated states. Functionally, day 7 macrophages produced a diverse cytokine panel responding to lipopolysaccharide (LPS), including IL-2, IL-12p70, IL-4, IL-5, TNF-α, GM-CSF, IFN-γ, and IL-10. In contrast, macrophages at days 14 and 21 displayed a markedly restricted cytokine profile, retaining the secretion of cytokines but significantly downregulating the secretion of Th1-type cytokines. This long-term culture model constitutes a robust, physiologically relevant tool to study immune aging, senescence-driven immune reprogramming, and inflammation in macrophages with the potential for the preclinical evaluation of therapeutic strategies to ameliorate inflammaging and restore immune competence.
Keywords: senescence, Macrophages, Immune dysregulation, Inflammation, M1 polarization, Flow Cytometry
Received: 07 Jul 2025; Accepted: 02 Sep 2025.
Copyright: © 2025 Ruiz, Lucero-Gil, Torres and Juárez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Esmeralda Juárez, National Institute of Respiratory Diseases-Mexico (INER), Mexico City, Mexico
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