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Endotoxin{middle dot}albumin complexes transfer endotoxin monomers to MD-2 resulting in activation of TLR4
Gregory A Esparza1, Athmane Teghanemt, DeSheng Zhang
1Immunology Program, University of Iowa Graduate College, Iowa City, Iowa, USA.
Abstract:
Response to Gram-negative bacteria (GNB) is partially mediated by the recognition of GNB-derived endotoxin by host cells. Potent host response to endotoxin depends on the sequential interaction of endotoxin with lipopolysaccharide binding protein (LBP), CD14, MD-2 and TLR4. While CD14 facilitates the efficient transfer of endotoxin monomers to MD-2 and MD-2·TLR4, activation of MD-2·TLR4 can occur in the absence of CD14 through an unknown mechanism. Here, we show that incubation of purified endotoxin (E) aggregates (E(agg), M ( r ) ≥ 20 million) in PBS with ≥ 0.1% albumin in the absence of divalent cations Ca(2+) and Mg(2+), yields E·albumin complexes (M ( r ) ∼70,000). E·albumin transfers E monomers to sMD-2 or sMD-2·TLR4 ectodomain (TLR4(ecd)) with a 'K (d)' of ∼4 nM and induces MD-2·TLR4-dependent, CD14-independent cell activation with a potency only 10-fold less than that of monomeric E·CD14 complexes. Our findings demonstrate, for the first time, a mechanistic basis for delivery of endotoxin monomers to MD-2 and for activation of TLR4 that is independent of CD14.
Insights
This study reveals a novel mechanism for activating host immune responses to Gram-negative bacteria. Albumin facilitates endotoxin transfer to MD-2 and TLR4, bypassing CD14 for potent cell activation.
Area of Science:
- Immunology
- Microbiology
- Biochemistry
Background:
- Host immune responses to Gram-negative bacteria involve endotoxin recognition.
- Endotoxin recognition relies on lipopolysaccharide binding protein (LBP), CD14, MD-2, and TLR4.
- CD14 facilitates endotoxin transfer to MD-2 and TLR4, but CD14-independent activation mechanisms remain unclear.
Purpose of the Study:
- To elucidate the CD14-independent mechanism of MD-2·TLR4 activation by endotoxin.
- To characterize the formation and function of endotoxin-albumin complexes in immune activation.
Main Methods:
- Incubation of purified endotoxin aggregates with albumin in the absence of divalent cations.
- Characterization of endotoxin-albumin complexes using molecular weight determination.
- Assessing the transfer of endotoxin monomers to soluble MD-2 and TLR4 ectodomain.
- Measuring cell activation potency in a CD14-independent manner.
Main Results:
- Endotoxin aggregates incubated with albumin form E·albumin complexes.
- E·albumin complexes efficiently transfer endotoxin monomers to sMD-2 or TLR4 ectodomain.
- These complexes induce potent MD-2·TLR4-dependent, CD14-independent cell activation.
Conclusions:
- A novel mechanistic basis for CD14-independent endotoxin delivery to MD-2 and TLR4 activation is demonstrated.
- Albumin acts as a crucial mediator in this alternative pathway of immune activation.
- Findings provide new insights into host-pathogen interactions and immune signaling.
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