Macrophages are at the heart of mechanobiology research. The physical properties of macrophages, namely stiffness, adhesion, and ECM (extracellular matrix) sensing, are closely related to phenotypic polarization and immune function. Pro-inflammatory M1 macrophages typically exhibit higher cell stiffness, whereas anti-inflammatory tissue-repairing M2 macrophages are mechanically more flexible, and these mechanical properties determine how the cell responds to physical cues in the microenvironment. For decades, penstreptomycin has been used at a standard 1% v/v concentration in cell cultures to prevent bacterial contamination, but the effects of penstreptomycin on the mechanophenotype of macrophages, a mechanical property that defines cellular function, had never been systematically investigated.
Penstrep promotes time-dependent macrophage stiffening and rewires ECM mechanosensing
The researchers quantified mechanical changes in RAW264.7 macrophages treated with penstreptomycin over a five-day period using typical mechanobiology measurement techniques: atomic force microscopy (AFM) for cell stiffness and single cell force spectroscopy (SCFS) for adhesion strength. The results demonstrated that macrophage stiffness increased steadily in a time-dependent manner. The cell elastic modulus increased significantly within 24 h, peaking at approximately 2.5 kPa at day 5, more than twice the baseline stiffness of untreated macrophages. Adhesion strength showed only a temporary decrease (complete recovery by day 3), confirming that penstrept exerts a selective effect on cellular machinery rather than overall adhesion capacity.
Additionally, mechanobiology assays revealed that PenStrep induces substrate-specific rewiring of macrophage ECM mechanosensing, a core process by which cells detect and respond to physical and biochemical cues in their microenvironment. When cultured on common mechanobiology research substrates, penstrept-treated macrophages:
- Increased spreading (decreased circularity) on PDMS rubber, collagen I, laminin, polyamino acid, and polyRGD peptide substrates, which are widely used to study cell-matrix mechanical interactions.
- Reduced spreading on type IV collagen, a basement membrane ECM component important for tissue-specific mechanosensing.
- No significant morphological changes were observed on glass, highlighting context-dependent modulation of mechanical signal perception.
Molecular mechanobiology analysis identified the transcriptional drivers of these changes. Pen-streptomycin syndrome YAP-1 and TAZ, master regulators of the Hippo mechanotransduction pathway that control cell stiffness and cytoskeletal remodeling, were upregulated, and β1 integrin, a key “molecular clutch” mediating ECM mechanical signal sensing and focal adhesion formation, was downregulated. Remarkably, other core adhesion proteins (paxillin, vinculin) remained unchanged, explaining the transient nature of the adhesion defect and corroborating the targeted effects of penstrept on mechanotransduction pathways.
Mechanophenotypic changes lead to macrophage immune dysfunction
A core tenet of mechanobiology is that cellular mechanical properties directly govern biological function, and this study confirms that penstrept-induced mechanophenotypic changes in macrophages lead to severe impairment of key innate immune functions.
- Decreased phagocytic capacity: The ability of macrophages to engulf and destroy pathogens and cell debris, an immune function closely related to the mechanical flexibility of the cytoskeleton, was significantly reduced in penstrept-treated cells.
- Non-canonical phenotypic polarization: pro-inflammatory M1 mechanophenotype ( T.N.F., Cxcl9 gene expression) was broadly downregulated, whereas M2-associated genes showed a heterogeneous response.argument 1 and anti-inflammatory effect Il10 Upregulated and M2 marker Mrc1 (CD206) It was downregulated, creating a non-classical polarized state that was uncoupled from the canonical mechanophenotypic link.
- Elevated intracellular reactive oxygen species (ROS): ROS levels, which mediate antibacterial activity and are regulated by cytoskeletal mechanical signaling, were significantly increased and induced oxidative stress.
- Moderate migration impairment: Directional migration of macrophages, which is essential for recruitment to sites of infection/injury and relies on mechanical plasticity, was slightly reduced, likely due to increased cell stiffness that impairs cytoskeletal dynamics.
Notably, penstrept had no effect on macrophage proliferation (as measured by Ki67 staining), confirming that its effects were selective for mechanophenotypic and functional traits rather than general cell viability.
Paradigm shift in mechanobiology research and laboratory practice
Macrophages are a model cell type in mechanobiology, and the study of their mechanical properties informs research in all areas of inflammation, cancer, tissue engineering, and regenerative medicine, where penstreptomycin is used ubiquitously. The results of this study revealed that this “routine” cell culture reagent introduces hidden mechanobiological variables that can alter experimental results, compromising the reproducibility and translational relevance of in vitro mechanobiology studies.
“Mechanobiology research aims to reveal how physical forces shape cellular function, but we have unknowingly used reagents that actively modulate the very physical properties of important immune cells,” said corresponding author Dr. Yang Song, from the Institute of Biomedical Engineering at Sichuan University. “This finding means that countless mechanobiology studies on macrophages may have incorrectly captured mechanophenotypes altered by penstrept changes, rather than the native cellular mechanical responses we are trying to understand. This is a call to action for the field to reevaluate common cell culture reagents through a mechanobiology lens.”
The findings raise broader questions for clinical practice beyond basic research. Penstreptomycin Streptococcus is widely used to treat bacterial infections in humans and animals, and its ability to modulate macrophage mechanotransduction and immune function may result in off-target in vivo effects such as altering inflammatory responses, tissue repair, or pathogen clearance in situations where cellular mechanical function is important.
Future direction of mechanobiology research
The research team plans to extend this finding with two important studies focused on mechanobiology. First, we will validate our findings in primary human macrophages (we used a mouse cell line in this study) and identify the precise molecular mechanisms by which penstreptomycin modulates YAP/TAZ and β1 integrin-mediated mechanotransduction. Second, we assess whether penstrept exerts similar mechanophenotypic effects on other cell types that are central to mechanobiology research (e.g., fibroblasts, endothelial cells, stem cells). The team also aims to screen alternative antimicrobial agents for mechanobiology research to identify options that do not alter the mechanical properties of cells.
For the mechanobiology community, this study highlights an important principle. Common cell culture reagents cannot be assumed to be mechanobiologically inert, and their potential effects on the mechanical properties of cells must be considered in experimental design and data interpretation.
sauce:
Shanghai Jiao Tong University
Reference magazines:
Hu, S. Others. (2025). Penicillin-streptomycin affects the mechanical properties of macrophages and the mechanosensation of the microenvironment. Mechanobiology in medicine. DOI: 10.1016/j.mbm.2025.100173. https://www.sciencedirect.com/science/article/pii/S2949907025000610?via%3Dihub
