Related Experiment Video
Updated: Aug 5, 2025
Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages
Published on: December 10, 2013
[Effect of Trichomonas vaginalis macrophage migration inhibitory factor on THP-1 macrophages]
1Department of Laboratory Medicine, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, China.
Objective:
To investigate the effect of Trichomonas vaginalis macrophage migration inhibitory factor (TvMIF) on THP-1 macrophages.
Methods:
Recombinant TvMIF protein was prokaryotic expressed and purified, and endotoxin was removed after identification. Following exposure to TvMIF at concentrations of 0, 1, 5, 10, 50 and 100 ng/mL, the cytotoxicity of the recombinant TvMIF protein to THP-1 macrophages was tested using cell counting kit (CCK)-8 assay, and the apoptosis of THP-1 macrophages and reactive oxygen species (ROS) were detected using flow cytometry. The relative expression of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), caspase-1, interleukin-1β (IL-1β) and IL-18 genes was quantified using real-time fluorescent quantitative PCR (qPCR) assay, and the expression of caspase-1, NLRP3, gasdermin D (GSDMD), gasdermin D N-terminal (GSDMD-NT) and pro-IL-1β proteins were determined using Western blotting assay.
Results:
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) displayed successful expression and purification of the recombinant TvMIF protein with a molecular weight of 15.5 kDa, and the endotoxin activity assay showed the successful removal of endotoxin in the recombinant TvMIF protein (endotoxin concentration < 0.1 EU/mL), which was feasible for the subsequent studies on protein functions. Flow cytometry revealed that the recombinant TvMIF protein at a concentration of 10 ng/mL and less promoted the apoptosis of THP-1 macrophages, and the highest apoptotic rate of THP-1 macrophages was seen following exposure to the recombinant TvMIF protein at a concentration of 5 ng/mL, while the recombinant TvMIF protein at concentrations of 50 and100 ng/mL inhibited the apoptosis of THP-1 macrophages. Exposure to the recombinant TvMIF protein at a concentration 1 ng/mL resulted in increased ROS levels in THP-1 macrophages. qPCR assay quantified significantly elevated caspase-1, NLRP3, IL-18 and IL-1β expression in THP-1 macrophages 8 hours post-treatment with the recombinant TvMIF protein at a concentration 1 ng/mL, and Western blotting determined increased caspase-1, NLRP3, pro-IL-1β, GSDMD and GSDMD-NT protein expression in THP-1 macrophages following exposure to the recombinant TvMIF protein at a concentration 1 ng/mL. Pretreatment with MCC950 significantly reduced GSDMD and GSDMD-NT protein expression.
Conclusions:
High-concentration recombinant TvMIF protein inhibits macrophage apoptosis, while low-concentration recombinant TvMIF protein activates NLRP3 inflammasome and promotes macrophage pyroptosis.
Insights
Low-concentration Trichomonas vaginalis macrophage migration inhibitory factor (TvMIF) activates the NLRP3 inflammasome and promotes pyroptosis in THP-1 macrophages. High concentrations of TvMIF inhibit apoptosis, revealing a dual role in macrophage response.
Area of Science:
- Immunology
- Cell Biology
- Parasitology
Context:
- Trichomonas vaginalis is a protozoan parasite that infects the urogenital tract.
- Macrophage migration inhibitory factor (MIF) plays a crucial role in immune responses.
- The specific role of TvMIF in modulating host macrophage functions remains largely unexplored.
Purpose:
- To investigate the effect of recombinant TvMIF on THP-1 macrophage apoptosis, reactive oxygen species (ROS) production, and NLRP3 inflammasome activation.
- To elucidate the concentration-dependent effects of TvMIF on macrophage behavior.
Summary:
- Recombinant TvMIF was expressed, purified, and tested on THP-1 macrophages at various concentrations.
- Low concentrations (≤10 ng/mL) of TvMIF promoted THP-1 macrophage apoptosis and increased ROS production.
- TvMIF at 1 ng/mL significantly upregulated NLRP3 inflammasome components (NLRP3, caspase-1, IL-1β, IL-18) and pyroptosis markers (GSDMD, GSDMD-NT).
- High concentrations (50 and 100 ng/mL) of TvMIF inhibited macrophage apoptosis.
Impact:
- This study reveals a dual role for TvMIF in regulating macrophage apoptosis and pyroptosis.
- Low concentrations of TvMIF activate the NLRP3 inflammasome, suggesting a mechanism for parasite-induced inflammation.
- Understanding TvMIF's immunomodulatory functions can inform therapeutic strategies against trichomoniasis.
More Related Videos
07:21Author Spotlight: Unraveling the Interplay Between Trichoderma stromaticum and the Mammalian Immune System
Published on: October 20, 2023
06:38Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line
Published on: August 2, 2021