Molecular mechanism of tumor necrosis factor-alpha production in 1-->3-beta-glucan (zymosan)-activated macrophages
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Published on: May 31, 2018
1Engineering Control and Technology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, National Institutes of Health, Morgantown, West Virginia 26505, USA. syoung@cdc.gov
Abstract:
The molecular details of 1-->3-beta-glucans, a fungal cell wall component, induced inflammatory responses are not well understood. In the present study, we conducted a systematic analysis of the molecular events leading to tumor necrosis factor (TNF)-alpha production after glucan stimulation of macrophages. We demonstrated that activation of nuclear factor kappaB (NF-kappaB) is essential in zymosan A (a source of 1-->3-beta-glucans)-induced TNF-alpha production in macrophages (RAW264.7 cells). Zymosan A-induced TNF-alpha protein production was associated with an increase in the TNF-alpha gene promoter activity. Activation of the TNF-alpha gene promoter was dependent on activation of NF-kappaB. Time course studies indicated that DNA binding activity of NF-kappaB preceded TNF-alpha promoter activity. Inhibition of NF-kappaB activation led to a dramatic reduction in both TNF-alpha promoter activity and TNF-alpha protein production in the response to zymosan A. Mutation of a major NF-kappaB binding site (kappa3) in the gene promoter resulted in a significant decrease in the induction of the gene promoter by zymosan A, while mutation of Egr or CRE sites failed to inhibit the response to zymosan. Together, these results strongly suggest that NF-kappaB is involved in signal transduction of 1-->3-beta-glucans-induced TNF-alpha expression.
Insights
Nuclear factor kappaB (NF-kappaB) activation is crucial for fungal beta-glucans to trigger tumor necrosis factor-alpha (TNF-alpha) production in macrophages. This pathway is key to understanding fungal-induced inflammation.
Area of Science:
- Immunology
- Molecular Biology
- Mycology
Background:
- 1-->3-beta-glucans are fungal cell wall components that can trigger inflammatory responses.
- The precise molecular mechanisms underlying glucan-induced inflammation, particularly TNF-alpha production, remain incompletely understood.
Purpose of the Study:
- To systematically analyze the molecular events involved in tumor necrosis factor-alpha (TNF-alpha) production following 1-->3-beta-glucans stimulation of macrophages.
- To elucidate the role of nuclear factor kappaB (NF-kappaB) in this inflammatory signaling pathway.
Main Methods:
- Utilized RAW264.7 macrophage cell line stimulated with zymosan A (a source of 1-->3-beta-glucans).
- Assessed TNF-alpha protein production, TNF-alpha gene promoter activity, and NF-kappaB DNA binding activity.
- Employed gene promoter mutation analysis, specifically targeting NF-kappaB binding sites.
Main Results:
- Zymosan A stimulation induced TNF-alpha production and increased TNF-alpha gene promoter activity in macrophages.
- NF-kappaB activation was essential for zymosan A-induced TNF-alpha production and promoter activity.
- NF-kappaB DNA binding activity preceded TNF-alpha promoter activation, and its inhibition significantly reduced TNF-alpha expression.
- Mutation of a key NF-kappaB binding site abrogated zymosan A-induced promoter activity.
Conclusions:
- Nuclear factor kappaB (NF-kappaB) plays a critical role in the signal transduction pathway mediating 1-->3-beta-glucans-induced TNF-alpha expression in macrophages.
- This finding clarifies a key molecular event in the innate immune response to fungal components.
