Athmane Teghanemt

Isolation of an endotoxin–MD-2 complex that produces Toll-like receptor 4-dependent cell activation at picomolar concentrations

Host proinflammatory responses to minute amounts of endotoxins derived from many Gram-negative bacteria require the interaction of lipopolysaccharide-binding protein (LBP), CD14, Toll-like receptor 4 (TLR4) and MD-2. Optimal sensitivity to endotoxin requires an ordered series of endotoxin–protein and protein–protein interactions. At substoichiometric concentrations, LBP facilitates delivery of endotoxin aggregates to soluble CD14 (sCD14) to form monomeric endotoxin–sCD14 complexes. Subsequent interactions of endotoxin–sCD14 with TLR4 and/or MD-2 have not been specifically defined. This study reports the purification of a stable, monomeric, bioactive endotoxin–MD-2 complex generated by treatment of endotoxin–sCD14 with recombinant MD-2. Efficient generation of this complex occurred at picomolar concentrations of endotoxin and nanogram per milliliter doses of MD-2 and required presentation of endotoxin to MD-2 as a monomeric endotoxin–CD14 complex. TLR4-dependent delivery of endotoxin to human embryonic kidney (HEK) cells and cell activation at picomolar concentrations of endotoxin occurred with the purified endotoxin–MD-2 complex, but not with purified endotoxin aggregates with or without LBP and/or sCD14. The presence of excess MD-2 inhibited delivery of endotoxin–MD-2 to HEK/TLR4 cells and cell activation. These findings demonstrate that TLR4-dependent activation of host cells by picomolar concentrations of endotoxin occurs by sequential interaction and transfer of endotoxin to LBP, CD14, and MD-2 and simultaneous engagement of endotoxin and TLR4 by MD-2.

Biochemical and Functional Characterization of Membrane Blebs Purified from Neisseria meningitidis Serogroup B

Studies with purified aggregates of endotoxin have revealed the importance of lipopolysaccharide-binding protein (LBP)-dependent extraction and transfer of individual endotoxin molecules to CD14 in Toll-like receptor 4 (TLR4)-dependent cell activation. Endotoxin is normally embedded in the outer membrane of intact Gram-negative bacteria and shed membrane vesicles (“blebs”). However, the ability of LBP and CD14 to efficiently promote TLR4-dependent cell activation by membrane-associated endotoxin has not been studied extensively. In this study, we used an acetate auxotroph of Neisseria meningitidis serogroup B to facilitate metabolic labeling of bacterial endotoxin and compared interactions of purified endotoxin aggregates and of membrane-associated endotoxin with LBP, CD14, and endotoxin-responsive cells. The endotoxin, phospholipid, and protein composition of the recovered blebs indicate that the blebs derive from the bacterial outer membrane. Proteomic analysis revealed an unusual enrichment in highly cationic (pI > 9) proteins. Both purified endotoxin aggregates and blebs activate monocytes and endothelial cells in a LBP-, CD14-, and TLR4/MD-2-dependent fashion, but the blebs were 3-10-fold less potent when normalized for the amount of endotoxin added. Differences in potency correlated with differences in efficiency of LBP-dependent delivery to and extraction of endotoxin by CD14. Both membrane phospholipids and endotoxin are extracted by LBP/soluble CD14 (sCD14) treatment, but only endotoxin·sCD14 reacts with MD-2 and activates cells. These findings indicate that the proinflammatory potency of endotoxin may be regulated not only by the intrinsic structural properties of endotoxin but also by its association with neighboring molecules in the outer membrane.

Molecular Basis of Reduced Potency of Underacylated Endotoxins

Potent TLR4-dependent cell activation by Gram-negative bacterial endotoxins depends on sequential endotoxin-protein and protein-protein interactions with LPS-binding protein, CD14, myeloid differentiation protein 2 (MD-2), and TLR4. Previous studies have suggested that reduced agonist potency of underacylated endotoxins (i.e., tetra- or penta- vs hexa-acylated) is determined by post-CD14 interactions. To better define the molecular basis of the differences in agonist potency of endotoxins differing in fatty acid acylation, we compared endotoxins (lipooligosaccharides (LOS)) from hexa-acylated wild-type (wt), penta-acylated mutant msbB meningococcal strains as well as tetra-acylated LOS generated by treatment of wt LOS with the deacylating enzyme, acyloxyacylhydrolase. To facilitate assay of endotoxin:protein and endotoxin:cell interactions, the endotoxins were purified after metabolic labeling with [3H]- or [14C]acetate. All LOS species tested formed monomeric complexes with MD-2 in an LPS-binding protein- and CD14-dependent manner with similar efficiency. However, msbB LOS:MD-2 and acyloxyacylhydrolase-treated LOS:MD-2 were at least 10-fold less potent in inducing TLR4-dependent cell activation than wt LOS:MD-2 and partially antagonized the action of wt LOS:MD-2. These findings suggest that underacylated endotoxins produce decreased TLR4-dependent cell activation by altering the interaction of the endotoxin:MD-2 complex with TLR4 in a way that reduces receptor activation. Differences in potency among these endotoxin species is determined not by different aggregate properties, but by different properties of monomeric endotoxin:MD-2 complexes.

Specific High Affinity Interactions of Monomeric Endotoxin·Protein Complexes with Toll-like Receptor 4 Ectodomain

Potent Toll-like receptor 4 (TLR4) activation by endotoxin has been intensely studied, but the molecular requirements for endotoxin interaction with TLR4 are still incompletely defined. Ligand-receptor interactions involving endotoxin and TLR4 were characterized using monomeric endotoxin·protein complexes of high specific radioactivity. The binding of endotoxin·MD-2 to the TLR4 ectodomain (TLR4ECD) and transfer of endotoxin from CD14 to MD-2/TLR4ECD were demonstrated using HEK293T-conditioned medium containing TLR4ECD ± MD-2. These interactions are specific, of high affinity (KD < 300 pm), and consistent with the molecular requirements for potent cell activation by endotoxin. Both reactions result in the formation of a Mr ∼ 190,000 complex composed of endotoxin, MD-2, and TLR4ECD. CD14 facilitates transfer of endotoxin to MD-2 (TLR4) but is not a stable component of the endotoxin·MD-2/TLR4 complex. The ability to assay specific high affinity interactions of monomeric endotoxin·protein complexes with TLR4ECD should allow better definition of the structural requirements for endotoxin-induced TLR4 activation.

Monomeric endotoxin:protein complexes are essential for TLR4-dependent cell activation

Potent TLR4-dependent cell activation by Gram-negative bacterial endotoxin depends on sequential endotoxin—protein and protein—protein interactions with LBP, CD14, MD-2 and TLR4. LBP and CD14 combine, in an albumin-dependent fashion, to extract single endotoxin molecules from purified endotoxin aggregates (Eagg) or the bacterial outer membrane and form monomeric endotoxin:CD14 complexes that are the preferred presentation of endotoxin for transfer to MD-2. Endotoxin in endotoxin:CD14 is readily transferred to MD-2, again in an albumin-dependent manner, to form monomeric endotoxin:MD-2 complex. This monomeric endotoxin:protein complex (endotoxin:MD-2) activates TLR4 at picomolar concentrations, independently of albumin, and is, therefore, the apparent ligand in endotoxin-dependent TLR4 activation. Tetra-, penta-, and hexa-acylated forms of meningococcal endotoxin (LOS) react similarly with LBP, CD14, and MD-2 to form endotoxin:MD-2 complexes. However, tetra- and penta-acylated LOS:MD-2 complexes are less potent TLR4 agonists than hexa-acylated LOS:MD-2. This is mirrored in the reduced activity of tetra-, penta- versus hexa-acylated LOS aggregates (LOS agg) + LBP toward cells containing mCD14, MD-2, and TLR4. Therefore, changes in agonist potency of under-acylated meninigococcal LOS are determined by differences in properties of monomeric endotoxin:MD-2.

Regulation of interactions of endotoxin with host cells

Potent Toll-like receptor 4 (TLR4)-dependent cell activation by endotoxin requires lipopolysaccharide-binding protein (LBP) and CD14-dependent delivery of endotoxin to cells containing MD-2 and TLR4. We have used metabolically labeled [14C] meningococcal lipooligosaccharide (LOS), purified recombinant endotoxin-binding proteins, and cultured endothelial cells to better define protein: endotoxin intermediates key in cell activation in the absence of functional membrane (m) CD14. Protein:endotoxin complexes or aggregates (agg) were purified by gel sieving and characterized by immunocapture and bio-assays. Cell activation closely correlated with LBP, albumin and soluble (s) CD14-dependent conversion of endotoxin agg (Mr ≥ 20 × 106) to monomeric (M ~55 × 10 3) endotoxin:sCD14 complexes. Ordered interaction of LBP (+ albumin) and sCD14 with rLOSagg was required for the efficient formation of a bioactive endotoxin:sCD14 complex and potent cell activation. Increasing the ratio of LBP/sCD14 or addition of bactericidal/permeability-increasing protein (BPI) reduced accumulation of endotoxin:sCD14 complexes and instead yielded aggregates of endotoxin (Mr ~1—20 × 106) containing LBP or BPI that were taken up by cells in a CD14- and TLR4-independent manner without inducing pro-inflammatory responses. These findings strongly suggest that host machinery linked to TLR4-dependent cellular activation or TLR4-independent cellular clearance of endotoxin selectively recognizes different protein:endotoxin complexes. At the outset of infection, the low concentrations of LBP present and absence of extracellular BPI favor formation of pro-inflammatory endotoxin:CD14 complexes. The mobilization of LBP and BPI that is triggered by inflammation directs endotoxin for clearance and hence resolution of endotoxin-triggered inflammation.

Novel roles in human MD-2 of phenylalanines 121 and 126 and tyrosine 131 in activation of Toll-like receptor 4 by endotoxin.

Potent mammalian cell activation by Gram-negative bacterial endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). TLR4 activation requires simultaneous binding of MD-2 to endotoxin (E) and the ectodomain of TLR4. We now describe mutants of recombinant human MD-2 that bind TLR4 and react with E·CD14 but do not support cellular responsiveness to endotoxin. The mutants F121A/K122A MD-2 and Y131A/K132A MD-2 react with E·CD14 only when co-expressed with TLR4. Single mutants K122A and K132A each react with E·CD14 ± TLR4 and promote TLR4-dependent cell activation by endotoxin suggesting that Phe121 and Tyr131 are needed for TLR4-independent transfer of endotoxin from CD14 to MD-2 and also needed for TLR4 activation by bound E·MD-2. The mutant F126A MD-2 reacts as well as wild-type MD-2 with E·CD14 ± TLR4. E·MD-2F126A binds TLR4 with high affinity (Kd ∼ 200 pm) but does not activate TLR4 and instead acts as a potent TLR4 antagonist, inhibiting activation of HEK/TLR4 cells by wild-type E·MD-2. These findings reveal roles of Phe121 and Tyr131 in TLR4-independent interactions of human MD-2 with E·CD14 and, together with Phe126, in activation of TLR4 by bound E·MD-2. These findings strongly suggest that the structural properties of E·MD-2, not E alone, determine agonist or antagonist effects on TLR4.

Evidence of a specific interaction between new synthetic antisepsis agents and CD14

Synthetic molecules derived from natural sugars with a positively charged amino group or ammonium salt and two lipophilic chains have been shown to inhibit TLR4 activation in vitro and in vivo. To characterize the mechanism of action of this class of molecules, we investigated possible interactions with the extracellular components that bind and shuttle endotoxin [lipopolysaccharide (LPS)] to TLR4, namely, LBP, CD14, and MD-2. Molecules that inhibited TLR4 activation inhibited LBP.CD14-dependent transfer of endotoxin monomers derived from aggregates of tritiated lipooligosaccharide ([(3)H]LOS) from Neisseria meninigitidis to MD-2.TLR4, resulting in a reduced level of formation of a ([(3)H]LOS.MD-2.TLR4(ECD))(2) (M(r) approximately 190000) complex. This effect was due to inhibition of the transfer of [(3)H]LOS from aggregates in solution to sCD14 with little or no effect on [(3)H]LOS shuttling from [(3)H]LOS.sCD14 to MD-2. These compounds also inhibited transfer of the [(3)H]LOS monomer from full-length CD14 to a truncated, polyhistidine-tagged CD14. Dose-dependent inhibition of the transfer of [(3)H]LOS between the two forms of CD14 was observed with each of three different synthetic compounds that inhibited TLR4 activation but not by another structurally related analogue that lacked TLR4 antagonistic activity. Saturation transfer difference (STD) NMR data showed direct binding to CD14 by the synthetic TLR4 antagonist mediated principally through the lipid chains of the synthetic compound. Taken together, our findings strongly suggest that these compounds inhibit TLR4 activation by endotoxin by competitively occupying CD14 and thereby reducing the level of delivery of activating endotoxin to MD-2.TLR4.

Endotoxin-binding Proteins Modulate the Susceptibility of Bacterial Endotoxin to Deacylation by Acyloxyacyl Hydrolase

Acyloxyacyl hydrolase (AOAH) is an eukaryotic lipase that partially deacylates and detoxifies Gram-negative bacterial lipopolysaccharides and lipooligosaccharides (LPSs or LOSs, endotoxin) within intact cells and inflammatory fluids. In cell lysates or as purified enzyme, in contrast, detergent is required for AOAH to act on LPS or LOS (Erwin, A. L., and Munford, R. S. (1990) J. Biol. Chem. 265, 16444–16449 and Katz, S. S., Weinrauch, Y., Munford, R. S., Elsbach, P., and Weiss, J. (1999) J. Biol. Chem. 274, 36579–36584). We speculated that the sequential interactions of endotoxin (E) with endotoxin-binding proteins (lipopolysaccharide-binding protein (LBP), CD14, and MD-2) might produce changes in endotoxin presentation that would allow AOAH greater access to its substrate, lipid A. To test this hypothesis, we measured the activity of purified AOAH against isolated, metabolically labeled meningococcal LOS and Escherichia coli LPS that were presented either as aggregates (LOSagg or LPSagg) ± LBP or as monomeric protein (sCD14 or MD-2)-endotoxin complexes. Up to 100-fold differences in the efficiency of endotoxin deacylation by AOAH were observed, with the following rank order of susceptibility to AOAH: E:sCD14 ≥endotoxin aggregates (Eagg):LBP (molar ratio of E/LBP 100:1) ≫Eagg, Eagg:LBP (E/LBP ∼1, mol/mol), or E:MD-2. AOAH treatment of LOS-sCD14 produced partially deacylated LOS still complexed with sCD14. The underacylated LOS complexed to sCD14 transferred to MD-2 and thus formed a complex capable of preventing TLR4 activation. These findings strongly suggest that LBP- and CD14-dependent extraction and transfer of endotoxin monomers are accompanied by increased exposure of fatty acyl chains within lipid A and that the acyl chains are then sequestered when LOS binds MD-2. The susceptibility of the monomeric endotoxin-CD14 complex to AOAH may help constrain endotoxin-induced TLR4 activation when endotoxin and membrane CD14 are present in excess of MD-2/TLR-4.

Isolation of Monomeric and Dimeric Secreted MD-2: ENDOTOXIN·sCD14 AND TOLL-LIKE RECEPTOR 4 ECTODOMAIN SELECTIVELY REACT WITH THE MONOMERIC FORM OF SECRETED MD-2*

Potent cell activation by endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). MD-2 plays an essential role by bridging endotoxin (E) recognition initiated by lipopolysaccharide-binding protein and CD14 to TLR4 activation by presenting endotoxin as a monomeric E·MD-2 complex that directly and potently activates TLR4. Secreted MD-2 (sMD-2) exists as a mixture of monomers and multimers. Published data suggest that only MD-2 monomer can interact with endotoxin and TLR4 and support cell activation, but the apparent instability of MD-2 has thwarted efforts to more fully separate and characterize the individual species of sMD-2. We have taken advantage of the much greater stability of sMD-2 in insect culture medium to fully separate sMD-2 monomer from dimer by gel sieving chromatography. At low nanomolar concentrations, the sMD-2 monomer, but not dimer, reacted with a monomeric complex of E·sCD14 to form monomeric E·MD-2 and activate HEK293/TLR4 cells. The monomer, but not dimer, also reacted with the ectodomain of TLR4 with an affinity comparable with the picomolar affinity of E·MD-2. These findings demonstrate directly that the monomeric form of sMD-2 is the active species both for reaction with E·CD14 and TLR4, as needed for potent endotoxin-induced TLR4 activation.