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Published on: June 23, 2013
Differential Modulation of Macrophage Polarization and NF-κB Signaling by Distinct Antibiotics in an in vitro Acute
Chunmin Chen1, Jingdi Diao1, Yuan Chen1
1Emergency Department, Lianyungang Hospital of Jiangsu Provincial Hospital of Traditional Chinese Medicine, Lianyungang, Jiangsu, 222004, People's Republic of China.
Objective:
This study aimed to compare the regulatory effects of cefotaxime (CTX), levofloxacin (LVFX), and azithromycin (AZM) on macrophage polarization and NF-κB signaling in an in vitro LPS-induced acute inflammation model.
Methods:
RAW264.7 murine macrophages were pretreated with CTX (25 μg/mL), LVFX (25 μg/mL), or AZM (10 μg/mL) for 1 h and subsequently stimulated with LPS (100 ng/mL). Cell viability was assessed using CCK-8 assays. TNF-α, IL-1β, and IL-10 levels were quantified by ELISA. M1-associated markers (iNOS and CD86) and M2-associated markers (Arg-1 and CD206) were evaluated using qRT-PCR and immunofluorescence. NF-κB pathway activation was examined by Western blot analysis of p65 nuclear translocation, IκBα expression, and IKKα/β and p65 phosphorylation.
Results:
All three antibiotics attenuated LPS-induced proinflammatory responses, as shown by reduced TNF-α and IL-1β levels and increased IL-10 secretion. CTX, LVFX, and particularly AZM suppressed M1-associated marker expression while increasing M2-associated marker expression. Western blot analysis showed that antibiotic pretreatment inhibited NF-κB activation by reducing IKKα/β and p65 phosphorylation, limiting p65 nuclear translocation, and restoring IκBα expression, with AZM exerting the strongest effect among the three antibiotics.
Conclusion:
CTX, LVFX, and AZM exerted differential immunomodulatory effects in LPS-stimulated RAW264.7 macrophages. AZM showed the most pronounced ability to attenuate inflammatory activation, promote an M2-like macrophage phenotype, and suppress NF-κB signaling. These findings provide preliminary in vitro evidence that antibiotics may differ in their immunomodulatory properties, although further validation in primary macrophages, pharmacologically relevant exposure ranges, and in vivo inflammatory models is required.
Insights
This study shows that cefotaxime, levofloxacin, and azithromycin modulate macrophage polarization and NF-κB signaling in inflammation. Azithromycin demonstrated the strongest anti-inflammatory and immunomodulatory effects in this in vitro model.
Area of Science:
- Immunology
- Pharmacology
- Cell Biology
Background:
- Macrophage polarization is crucial in regulating inflammatory responses.
- The NF-κB signaling pathway plays a central role in inflammation.
- Antibiotics may possess immunomodulatory properties beyond their antimicrobial effects.
Purpose of the Study:
- To compare the immunomodulatory effects of cefotaxime (CTX), levofloxacin (LVFX), and azithromycin (AZM) on macrophage polarization.
- To investigate the impact of these antibiotics on NF-κB signaling in an in vitro LPS-induced inflammation model.
Main Methods:
- RAW264.7 murine macrophages were stimulated with lipopolysaccharide (LPS) and pretreated with CTX, LVFX, or AZM.
- Macrophage polarization markers (M1/M2), cytokine levels (TNF-α, IL-1β, IL-10), and NF-κB pathway activation were assessed.
- Techniques included CCK-8 assays, ELISA, qRT-PCR, immunofluorescence, and Western blot analysis.
Main Results:
- All tested antibiotics reduced pro-inflammatory cytokines (TNF-α, IL-1β) and increased IL-10 secretion.
- CTX, LVFX, and AZM suppressed M1 markers and promoted M2 markers, with AZM showing the most significant effect.
- Antibiotic pretreatment inhibited NF-κB activation by reducing p65 phosphorylation and nuclear translocation, and restoring IκBα expression.
Conclusions:
- Cefotaxime, levofloxacin, and azithromycin exhibit distinct immunomodulatory effects on LPS-stimulated macrophages.
- Azithromycin displayed the most potent anti-inflammatory activity, promoting an M2-like phenotype and suppressing NF-κB signaling.
- These findings suggest differential immunomodulatory properties among antibiotics, warranting further in vivo and primary cell studies.
