Related Experiment Video
Updated: Dec 10, 2025

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages
Published on: June 23, 2013
Modulation of Macrophage Function by Lactobacillus-Conditioned Medium
Yashaswini Seenappanahalli Nanjundaiah1, David A Wright1, Anwar R Baydoun2
1School of Health and Life Sciences, Teesside University, Middlesbrough, United Kingdom.
Abstract:
Probiotics are used as microbial food supplements for health and well-being. They are thought to have immunomodulatory effects although their exact physiological mechanism of action is not clear. This study investigated the influence of probiotic Lactobacillus rhamnosus GG conditioned media (LGG-CM) on macrophage phagocytosis of non-pathogenic Escherichia coli HfrC. The gentamicin protection assay was used to study the bacterial killing phases of phagocytosis. Macrophages co-incubated with E. coli for an hour allowed them to ingest bacteria and then the rate of E. coli killing was monitored for up to 300 min to determine the killing or digestion of the bacteria by recovering them from the macrophage lysate. We found that the LGG-CM significantly increased the bacterial killing by approximately 6-fold when compared with that of controls. By contrast, this killing process was found to be associated with enhanced free radical production via the activation of NADPH oxidase, stimulated by the LGG conditioned medium. We also found that the conditioned medium had small effect on nitric oxide (NO) generation, albeit to a lesser extent. This work suggests that LGG-CM may play an important role in suppressing the total microbial load within the macrophages and hence, the extent to which pro-inflammatory molecules such as free radicals and NO are generated. The modulation of inflammation-promoting signals by LGG-CM may be beneficial as it modulates bacterial killing, and thereby prevents any collateral damage to host.
Insights
Probiotic Lactobacillus rhamnosus GG conditioned media enhanced macrophage bacterial killing by 6-fold. This process involved increased free radical production, suggesting a mechanism for modulating host inflammation.
Area of Science:
- Immunology
- Microbiology
- Gastroenterology
Background:
- Probiotics are microbial supplements with potential immunomodulatory effects.
- The precise mechanisms behind probiotic-mediated immune responses remain unclear.
- Investigating probiotic effects on macrophage function is crucial for understanding their health benefits.
Purpose of the Study:
- To investigate the impact of Lactobacillus rhamnosus GG conditioned media (LGG-CM) on macrophage phagocytosis and killing of Escherichia coli.
- To elucidate the role of free radicals and nitric oxide (NO) in LGG-CM-mediated bacterial clearance.
Main Methods:
- Utilized the gentamicin protection assay to quantify bacterial killing by macrophages.
- Co-incubated macrophages with Escherichia coli and monitored bacterial survival over time.
- Measured free radical production via NADPH oxidase activation and nitric oxide generation.
Main Results:
- LGG-CM significantly enhanced the killing of Escherichia coli by macrophages approximately 6-fold compared to controls.
- Enhanced bacterial killing was associated with increased free radical production, primarily through NADPH oxidase activation.
- Nitric oxide generation was minimally affected by LGG-CM.
Conclusions:
- LGG-CM enhances macrophage-mediated bacterial clearance, potentially by increasing oxidative stress.
- This probiotic-derived factor may help control microbial load within macrophages.
- Modulation of inflammatory signals by LGG-CM could offer protective benefits against host tissue damage.

