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Published on: February 5, 2020
Detection of HBD1 peptide in peripheral blood mononuclear cell subpopulations by intracellular flow cytometry
Lisa K Ryan1, Gill Diamond, Sheela Amrute
1Department of Oral Biology, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, NJ 07103, USA. ryanlk@umdnj.edu
Abstract:
Production of human beta-defensin1 (HBD1) in response to LPS in monocytes, myeloid dendritic cells and plasmacytoid dendritic cells (PDC) was examined. Since PDC make up only 0.1-0.5% of the peripheral blood mononuclear cell population, we developed a method to determine HBD1 peptide levels using four-color flow cytometry, which can examine several cell surface or intracellular markers at once. Coupled with intracellular flow cytometry, we determined that PDC and monocytes only made significant amounts of HBD1 when exposed to >50ng/ml LPS for 2h. This response was limited to monocytes when ultrapure LPS was used, and was inhibited in PDC by chloroquine treatment.
Insights
Human beta-defensin 1 (HBD1) production by monocytes and plasmacytoid dendritic cells (PDC) was studied. A novel flow cytometry method revealed HBD1 is significantly produced by these cells upon stimulation with high lipopolysaccharide (LPS) levels.
Area of Science:
- Immunology
- Cell Biology
- Infectious Disease
Background:
- Human beta-defensin 1 (HBD1) is a crucial antimicrobial peptide.
- Understanding HBD1 production in immune cells is vital for host defense mechanisms.
- Plasmacytoid dendritic cells (PDC) are rare but important immune cells.
Purpose of the Study:
- To investigate the production of HBD1 by monocytes and dendritic cells in response to lipopolysaccharide (LPS).
- To develop and utilize a sensitive method for quantifying HBD1 in rare cell populations like PDC.
- To characterize the conditions and modulators affecting HBD1 production.
Main Methods:
- Development of a four-color flow cytometry assay for intracellular HBD1 detection.
- Analysis of HBD1 peptide levels in monocytes, myeloid dendritic cells, and PDC.
- Exposure of cells to varying concentrations of LPS and assessment of HBD1 production.
- Investigation of the effect of chloroquine on HBD1 production in PDC.
Main Results:
- A novel flow cytometry method enabled HBD1 quantification in rare PDC populations.
- Significant HBD1 production by PDC and monocytes required high LPS concentrations (>50ng/ml) for 2 hours.
- The response was primarily limited to monocytes when using ultrapure LPS.
- Chloroquine treatment inhibited HBD1 production in PDC.
Conclusions:
- PDC and monocytes exhibit a dose-dependent and time-dependent production of HBD1 in response to LPS.
- The sensitivity of PDC to LPS stimulation for HBD1 production is highlighted.
- Chloroquine acts as an inhibitor of HBD1 production in PDC, suggesting specific signaling pathways involved.
