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A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces
Published on: January 7, 2020
A microbial TLR2 agonist imparts macrophage-activating ability to apolipoprotein A-1
Akira Hasebe1, Nathan D Pennock, Hong-Hua Mu
1Division of Rheumatology, Department of Internal Medicine, University of Utah School of Medicine, 30 North 1900 East, Salt Lake City, UT 84132, USA.
Abstract:
There is increasing epidemiologic evidence implying a role for chronic infection in atherosclerosis and that microbial TLR agonists may contribute to this disease. Mycoplasma arthritidis is an agent of acute and chronic inflammatory disease in rodents, and has been used extensively as a model for defining the mechanisms involved in arthritis and other inflammatory diseases. We have purified a 28-kDa, apolipoprotein A-1 (apoA-1)-like TLR2-dependent macrophage-activating moiety from a culture of a virulent strain of M. arthritidis. ApoA-1 similarly isolated from uninoculated mycoplasma medium was without bioactivity. The activity of the mycoplasma-derived molecule was resistant to heat and to digestion with proteinase K, but was susceptible to alkaline hydrolysis and H(2)O(2) oxidation. Infrared profiles of normal apoA-1 and that derived from mycoplasma were distinct. Unlike the activity of other mycoplasmal TLR2 agonists such as macrophage-activating lipopeptide-2, activity of the M. arthritidis-derived 28-kDa component was dependent upon CD14, a coreceptor for LPS. Finally, we showed that bioactive lipopeptides prepared from M. arthritidis grown in serum-free medium and also from a 41-kDa known bioactive lipoprotein of M. arthritidis, avidly bound to purified apoA-1 that separated out by SDS-PAGE, induced TNF-alpha and IL-12p40 both in vitro and in vivo. ApoA-1 is a key functional component of the high-density lipoprotein cholesterol complex by scavenging and removing unwanted lipids. Our finding that this molecule can acquire macrophage-activating properties from microbial TLR2-dependent agonists suggests a novel mechanism whereby some microbial agents might reverse the protective role of apoA-1, thus contributing to the genesis of atherosclerosis.
Insights
A novel molecule from Mycoplasma arthritidis mimics apolipoprotein A-1 (apoA-1), activating macrophages via TLR2. This finding suggests a mechanism where microbial infections may disrupt apoA-1
Area of Science:
- Immunology
- Microbiology
- Cardiovascular Research
Background:
- Chronic infections are increasingly linked to atherosclerosis.
- Microbial Toll-like receptor (TLR) agonists may play a role in disease development.
- Mycoplasma arthritidis is a model organism for studying inflammatory diseases.
Purpose of the Study:
- To investigate the role of Mycoplasma arthritidis in atherosclerosis.
- To identify and characterize macrophage-activating moieties from M. arthritidis.
- To explore the interaction between microbial components and apolipoprotein A-1 (apoA-1).
Main Methods:
- Purification of a 28-kDa moiety from M. arthritidis.
- Biochemical characterization including heat resistance, proteinase K digestion, alkaline hydrolysis, and H2O2 oxidation.
- Infrared spectroscopy to compare mycoplasma-derived apoA-1 with normal apoA-1.
- Assays for TLR2-dependent macrophage activation, including CD14 dependence.
- In vitro and in vivo studies using M. arthritidis lipopeptides and apoA-1.
Main Results:
- A 28-kDa apoA-1-like molecule was purified from M. arthritidis, exhibiting TLR2-dependent macrophage-activating properties.
- The mycoplasma-derived molecule's activity was resistant to heat and proteinase K but sensitive to alkaline hydrolysis and H2O2 oxidation.
- Infrared profiles distinguished mycoplasma-derived apoA-1 from normal apoA-1.
- The M. arthritidis-derived moiety required CD14 for activity, unlike other mycoplasma TLR2 agonists.
- Bioactive lipopeptides from M. arthritidis bound to apoA-1 and induced TNF-alpha and IL-12p40 production.
Conclusions:
- Mycoplasma arthritidis produces a molecule that mimics apoA-1 and activates macrophages via TLR2.
- This microbial component can alter apoA-1's function, potentially contributing to atherosclerosis.
- Microbial TLR2 agonists may reverse the atheroprotective role of apoA-1, offering a novel disease mechanism.
