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Updated: Aug 15, 2026
Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy
Published on: July 9, 2014
Macrophage internalization of fungal beta-glucans is not necessary for initiation of related inflammatory responses
Frances McCann1, Eva Carmona, Vishwajeet Puri
1Thoracic Diseases Research Unit, Division of Pulmonary, Critical Care and Internal Medicine, Department of Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
Abstract:
Cell wall beta-glucans are highly conserved structural components of fungi that potently trigger inflammatory responses in an infected host. Identification of molecular mechanisms responsible for internalization and signaling of fungal beta-glucans should enhance our understanding of innate immune responses to fungi. In this study, we demonstrated that internalization of fungal beta-glucan particles requires actin polymerization but not participation of components of caveolar uptake mechanisms. Using fluorescence microscopy, we observed that uptake of 5-([4,6-dichlorotriazin-2-yl] amino)-fluorescein hydrochloride-Celite complex-labeled Saccharomyces cerevisiae beta-glucan by RAW macrophages was substantially reduced in the presence of cytochalasin D, which antagonizes actin-mediated internalization pathways, but not by treatment with nystatin, which blocks caveolar uptake. Interestingly, beta-glucan-induced NF-kappaB translocation, which is necessary for inflammatory activation, and tumor necrosis factor alpha production were both normal in the presence of cytochalasin D, despite defective internalization of beta-glucan particles following actin disruption. Dectin-1, a major beta-glucan receptor on macrophages, colocalized to phagocytic cups on macrophages and exhibited tyrosine phosphorylation after challenge with beta-glucan particles. Dectin-1 localization and other membrane markers were not affected by treatment with cytochalasin D. Furthermore, dectin-1 receptors rather than Toll-like receptor 2 receptors were shown to be necessary for both efficient internalization of beta-glucan particles and cytokine release in response to the fungal cell wall component.
Insights
Fungal beta-glucans trigger inflammation by requiring actin polymerization for internalization, not caveolar pathways. Dectin-1 receptor is crucial for uptake and cytokine release, independent of NF-kappaB translocation.
Area of Science:
- Immunology
- Cell Biology
- Microbiology
Background:
- Fungal beta-glucans are key triggers of host inflammatory responses.
- Understanding fungal beta-glucan internalization and signaling is vital for innate immunity research.
- The roles of specific cellular pathways and receptors in fungal recognition are not fully elucidated.
Purpose of the Study:
- To elucidate the molecular mechanisms of fungal beta-glucan particle internalization and signaling in macrophages.
- To investigate the involvement of actin polymerization and caveolar pathways in beta-glucan uptake.
- To determine the role of Dectin-1 and Toll-like receptor 2 in beta-glucan recognition and immune response.
Main Methods:
- Fluorescence microscopy was used to track the uptake of labeled Saccharomyces cerevisiae beta-glucan by RAW macrophages.
- Pharmacological inhibitors (cytochalasin D and nystatin) were employed to disrupt actin polymerization and caveolar pathways, respectively.
- NF-kappaB translocation, tumor necrosis factor alpha production, and Dectin-1 localization were assessed following beta-glucan challenge.
Main Results:
- Beta-glucan particle internalization by macrophages was dependent on actin polymerization but independent of caveolar uptake mechanisms.
- Disruption of actin polymerization impaired beta-glucan uptake but did not affect NF-kappaB translocation or TNF-alpha production.
- Dectin-1, a key beta-glucan receptor, localized to phagocytic cups and was tyrosine phosphorylated upon stimulation.
- Dectin-1, not TLR2, was essential for efficient beta-glucan internalization and subsequent cytokine release.
Conclusions:
- Macrophage internalization of fungal beta-glucans relies on actin polymerization, not caveolae.
- Dectin-1 is the primary receptor mediating beta-glucan uptake and initiating inflammatory cytokine release.
- These findings clarify innate immune recognition pathways for fungal components.
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