Pen-strep treatment rewires mechanical sensing in immune cells

Macrophages are central to mechanobiology research: their physical characteristics-stiffness, adhesion, and ECM (extracellular matrix) sensing-are inextricably linked to their phenotypic polarization and immune function. Pro-inflammatory M1 macrophages typically exhibit higher cellular stiffness, while anti-inflammatory, tissue-repair M2 macrophages are more mechanically flexible, and these mechanical traits dictate how the cells respond to physical cues in their microenvironment. For decades, pen-strep has been used at a standard 1% v/v concentration in cell culture to prevent bacterial contamination, but its impact on macrophage mechano-phenotypes, the mechanical characteristics that define cellular function, had never been systematically investigated.

Pen-strep drives time-dependent macrophage stiffening, rewires ECM mechanosensing

Using gold-standard mechanobiology measurement techniques-atomic force microscopy (AFM) for cellular stiffness and single-cell force spectroscopy (SCFS) for adhesion strength-the research team quantified mechanical changes in RAW264.7 macrophages treated with pen-strep over five days. The results demonstrated a robust, time-dependent increase in macrophage stiffness: the cells' elastic modulus rose significantly within 24 hours and peaked at ~2.5 kPa on day 5, more than doubling the baseline stiffness of untreated macrophages. Adhesion strength showed only a transient reduction (recovering fully by day 3), confirming pen-strep exerts a selective effect on cellular mechanics rather than global adhesion capacity.

Further mechanobiology assays revealed pen-strep induces substrate-specific rewiring of macrophage ECM mechanosensing-a core process where cells detect and respond to physical and biochemical cues in their microenvironment. When cultured on common mechanobiology research substrates, pen-strep-treated macrophages:

• Increased spreading (reduced roundness) on PDMS rubber, collagen I, laminin, poly-amino acids, and poly-RGD peptides-substrates widely used to study cell-matrix mechanical interactions;
• Decreased spreading on type IV collagen, a key basement membrane ECM component critical for tissue-specific mechanosensing;
• Showed no significant morphological change on glass, highlighting a context-dependent modulation of mechanical signal perception.

Molecular mechanobiology analyses identified the transcriptional drivers of these changes: pen-strep upregulated YAP-1 and TAZ-master regulators of the Hippo mechanotransduction pathway that control cellular stiffness and cytoskeletal remodeling-and downregulated β1 integrin, a critical "molecular clutch" that mediates ECM mechanical signal sensing and focal adhesion formation. Notably, other core adhesion proteins (paxillin, vinculin) remained unaltered, explaining the transient nature of the adhesion defect and confirming pen-strep's targeted impact on mechanotransduction pathways.

Mechanophenotypic Shifts Translate to Impaired Macrophage Immune Function

A core tenet of mechanobiology is that cellular mechanical traits directly govern biological function-and the study confirmed pen-strep-induced mechanophenotypic changes in macrophages lead to profound impairments in key innate immune functions:

• Diminished phagocytic capacity: The ability of macrophages to engulf and destroy pathogens/cellular debris-an immune function tightly linked to cytoskeletal mechanical flexibility-was significantly reduced in pen-strep-treated cells;
• Non-canonical phenotypic polarization: Pro-inflammatory M1 mechanophenotypes (marked by Tnf, Cxcl9 gene expression) were broadly downregulated, while M2-associated genes showed a heterogeneous response-Arg1 and anti-inflammatory Il10 were upregulated, and M2 marker Mrc1 (CD206) was downregulated-creating a non-classical polarization state uncoupled from standard mechanical-phenotypic links;
• Elevated intracellular reactive oxygen species (ROS): ROS levels, which mediate antimicrobial activity and are regulated by cytoskeletal mechanical signaling, were significantly increased, inducing oxidative stress;
• Modest migration impairment: Directional macrophage migration-essential for recruiting to sites of infection/injury and dependent on mechanical plasticity-was slightly reduced, likely due to increased cellular stiffness compromising cytoskeletal dynamics.

Notably, pen-strep had no effect on macrophage proliferation (measured via Ki67 staining), confirming its effects are selective for mechanophenotypic and functional traits, not general cell viability.

A paradigm shift for mechanobiology research and lab practice

Macrophages are a model cell type in mechanobiology, with studies of their mechanical properties informing research into inflammation, cancer, tissue engineering, and regenerative medicine-all fields where pen-strep is used ubiquitously. The study's findings reveal that this "routine" cell culture reagent introduces a hidden mechanobiological variable that can alter experimental outcomes, potentially compromising the reproducibility and translational relevance of in vitro mechanobiology research.

"Mechanobiology research aims to uncover how physical forces shape cellular function, yet we've been using a reagent that actively modulates those very physical traits in key immune cells without realizing it," said Dr. Yang Song, corresponding author from the Institute of Biomedical Engineering at Sichuan University. "This discovery means countless mechanobiology studies on macrophages may have inadvertently captured pen-strep-altered mechanophenotypes, not the native cellular mechanical responses we aim to understand. It's a call to action for the field to re-evaluate common cell culture reagents through a mechanobiological lens."

Beyond basic research, the findings raise broader questions for clinical practice: pen-strep is widely used to treat bacterial infections in humans and animals, and its ability to modulate macrophage mechanotransduction and immune function could have off-target in vivo effects-altering inflammatory responses, tissue repair, or pathogen clearance in contexts where cellular mechanical function is critical.

Future mechanobiology research directions

The research team plans to expand this discovery with two key mechanobiology-focused investigations: first, to validate the findings in primary human macrophages (the current study used a murine cell line) and identify the precise molecular mechanisms by which pen-strep modulates YAP/TAZ and β1 integrin-mediated mechanotransduction; second, to assess whether pen-strep exerts similar mechanophenotypic effects on other cell types 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 cellular mechanical traits.

For the mechanobiology community, the study underscores a critical principle: common cell culture reagents cannot be assumed to be mechanobiologically inert, and their potential impact on cellular mechanical properties must be considered in experimental design and data interpretation.