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Reprograming Model of Human Monocyte-derived Macrophages for In-vitro Assays
Published on: April 18, 2025
Characterization and partial purification of a macrophage-stimulating factor from Mycoplasma hominis
Morgan R Peltier1, Angela J Freeman, Hong H Mu
1Department of Obstetrics, Gynecology and Reproductive Sciences, University of Medicine and Dentistry-Robert Wood Johnson Medical School, New Brunswick, NJ 08901, USA. peltiemr@umdnj.edu
Problem:
Mycoplasma hominis is one of the most common pathogens of the genital tract and is associated with increased production of proinflammatory cytokines in reproductive tissues during preterm labor. The mechanism by which M. hominis, an organism lacking cell walls, increases the production of proinflammatory cytokines is unknown.
Method Of Study:
We characterized and purified a macrophage-activating factor from this organism.
Results:
Extraction of whole organisms with Triton-X-114 demonstrated that the activity was primarily associated with the detergent phase. Macrophage-stimulating activity (MSA) of detergent extracts of M. hominis was not inhibited by polymyxin B or heating but was completely abrogated by alkaline hydrolysis and partially reduced by proteinase K digestion. Further experiments that utilized Toll-like receptor (TLR)-2- and TLR-4-transfected cells, revealed that the detergent extracts activate TLR-2 but not TLR-4 signal transduction. Purification of the activity using preparative SDS-PAGE and reverse phase chromatography experiments led to the isolation of a 29-kDa protein.
Conclusions:
These experiments suggest that the MSA of M. hominis is due to a lipophillic factor that interacts with TLR-2 rather than TLR-4 (as does lipopolysaccharide), to increase tumor necrosis factor (TNF)-alpha by macrophages. It is known that TNF-alpha can cause preterm labor and intrauterine fetal death and that it is upregulated in amniotic fluid samples infected with M. hominis.
Insights
Mycoplasma hominis infection stimulates macrophages via a lipophilic factor activating Toll-like receptor-2 (TLR-2), leading to increased tumor necrosis factor-alpha, a key contributor to preterm labor.
Area of Science:
- Reproductive immunology
- Microbial pathogenesis
Background:
- Mycoplasma hominis is a common genital tract pathogen linked to preterm labor.
- The mechanism by which M. hominis induces pro-inflammatory cytokines is unclear.
Purpose of the Study:
- To identify and characterize the macrophage-activating factor from M. hominis responsible for cytokine induction.
Main Methods:
- Macrophage-stimulating activity (MSA) was extracted and purified from M. hominis.
- Toll-like receptor (TLR)-2 and TLR-4 transfected cells were used to assess activation pathways.
- Protein purification involved SDS-PAGE and reverse-phase chromatography.
Main Results:
- The MSA was a lipophilic factor, resistant to polymyxin B and heat, but sensitive to alkaline hydrolysis.
- M. hominis extracts activated TLR-2 but not TLR-4 signal transduction.
- A 29-kDa protein was isolated as the active component.
Conclusions:
- The MSA of M. hominis is a lipophilic factor that activates TLR-2, not TLR-4.
- This activation leads to increased macrophage production of tumor necrosis factor-alpha (TNF-alpha).
- Elevated TNF-alpha is implicated in preterm labor and intrauterine fetal death.
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