[Construction and optimization of two real-time macrophage polarization reporter systems based on Arg1/Nos2...

To enable non-destructive, repeatable, and quantitative monitoring of macrophage polarization direction and intensity under live-cell conditions, we constructed and optimized two reporter systems centered on the Arg1 (M2-associated) and Nos2 (M1-associated) promoters, respectively. Using a secreted NanoLuc (secNluc) reporter system as the detection tool, we applied combined stimulation with lipopolysaccharide (LPS) and interferon-γ (IFN-γ), and conditioned medium from irradiated tumor cells as M1-like and tumor-associated macrophage-like (M2-skewed) inducers, respectively. In RAW264.7 cells, systematic screening was performed for truncated Arg1 promoter fragments (P_{Arg1-0/-1/-2/-3}) and Nos2 promoter fragments (P_{Nos2-0/-1/-2/-3/-4}). The relative positions of enhancers and promoters were compared, followed by the establishment and validation of stable cell lines. The results showed that P_Arg1-2 and P_Nos2-1 achieved the best balance between basal activity and inducible responsiveness. The positional effect of enhancers was gene-dependent: in the Arg1 system, downstream enhancer placement was more effective than upstream placement (7.8-fold vs. 2.0-fold), whereas in the Nos2 system, upstream enhancer placement was more effective than downstream placement (20.7-fold vs. 2.4-fold). Upon conditioned-medium or LPS/IFN-γ stimulation, Nluc activity in the stable cell lines increased by approximately 10.0-fold and 8.5-fold, respectively. Monoclonal RAW264.7-PNos2 and RAW264.7-PArg1 cell lines were further obtained by limiting dilution, and the system was then used to verify the differential regulation of Nos2 and Arg1 expression by transforming growth factor-β1 and its time-dependent pattern. In summary, the established reporter systems enable continuous, quantitative monitoring of macrophage polarization in live cells under in vitro conditions. They are suitable for evaluating polarization trends in tumor-mimicking in vitro microenvironments and hold promise for plate-based multi-condition comparison, dose-response evaluation, and high-throughput screening, thereby providing a practical tool for mechanistic studies of macrophage polarization and for screening modulatory factors.