Christian Alexander

TLR2 -promiscuous or specific? A critical re-evaluation of a receptor expressing apparent broad specificity

Of all pattern recognition receptors (PRR) in innate immunity, Toll-like receptor 2 (TLR2) recognizes the structurally broadest range of different bacterial compounds known as pathogen-associated molecular patterns (PAMPs). TLR2 agonists identified so far are lipopolysaccharides (LPSs) from different bacterial strains, lipoproteins, (synthetic) lipopeptides, lipoarabinomannans, lipomannans, glycosylphosphatidylinositol, lipoteichoic acids (LTA), various proteins including lipoproteins and glycoproteins, zymosan, and peptidoglycan (PG). Because these molecules are structurally diverse, it seems unlikely that TLR2 has the capability to react with all agonists to the same degree. The aim of this review is to identify and describe well-defined structure-function relationships for TLR2. Because of its biomedical importance and because its genetics and biochemistry are presently most completely known among all Gram-positive bacteria, we have chosen Staphylococcus aureus as a focus. Our data together with those reported by other groups reveal that only lipoproteins/lipopeptides are sensed at physiologically concentrations by TLR2 at picomolar levels. This finding implies that the activity of all other putative bacterial compounds so far reported as TLR2 agonists was most likely due to contaminating highly active natural lipoproteins and/or lipopeptides.

Lipopolysaccharide Binding Protein Binds to Triacylated and Diacylated Lipopeptides and Mediates Innate Immune Responses

LPS binding protein (LBP) is an acute-phase protein synthesized predominantly in the liver of the mammalian host. It was first described to bind LPS of Gram-negative bacteria and transfer it via a CD14-enhanced mechanism to a receptor complex including TLR-4 and MD-2, initiating a signal transduction cascade leading to the release of proinflammatory cytokines. In recent studies, we found that LBP also mediates cytokine induction caused by compounds derived from Gram-positive bacteria, including lipoteichoic acid and peptidoglycan fragments. Lipoproteins and lipopeptides have repeatedly been shown to act as potent cytokine inducers, interacting with TLR-2, in synergy with TLR-1 or -6. In this study, we show that these compounds also interact with LBP and CD14. We used triacylated lipopeptides, corresponding to lipoproteins of Borrelia burgdorferi, mycobacteria, and Escherichia coli, as well as diacylated lipopeptides, corresponding to, e.g., 2-kDa macrophage activating lipopeptide of Mycoplasma spp. Activation of Chinese hamster ovary cells transfected with TLR-2 by both lipopeptides was enhanced by cotransfection of CD14. Responsiveness of human mononuclear cells to these compounds was greatly enhanced in the presence of human LBP. Binding of lipopeptides to LBP as well as competitive inhibition of this interaction by LPS was demonstrated in a microplate assay. Furthermore, we were able to show that LBP transfers lipopeptides to CD14 on human monocytes using FACS analysis. These results support that LBP is a pattern recognition receptor transferring a variety of bacterial ligands including the two major types of lipopeptides to CD14 present in different receptor complexes.

Recognition of pneumococcal peptidoglycan: an expanded, pivotal role for LPS binding protein.

Lipopolysaccharide binding protein (LBP) has a well-established role in LPS-induced immune responses. Here, we report that LBP also plays an essential role in the innate immune response to Gram-positive pneumococci, specifically to their major inflammatory component, pneumococcal cell wall (PCW). LBP was present in the CSF of patients with meningitis, and LBP-deficient mice failed to develop meningeal inflammation. LBP enhanced PCW-induced cell signaling and TNF-alpha release. LBP bound specifically to PCW multimers, indicating novel lipid-independent binding capability for LBP. We propose the iterative anionic groups along the glycan backbone of the cell wall are a crucial structure for recognition by LBP. Such a function for LBP expands its role to Gram-positive infections.

Acute-Phase Concentrations of Lipopolysaccharide (LPS)-Binding Protein Inhibit Innate Immune Cell Activation by Different LPS Chemotypes via Different Mechanisms

ABSTRACT The chain length of bacterial lipopolysaccharide (LPS) is a crucial factor for host-pathogen interaction during bacterial infection. While rough (R)-type and smooth (S)-type LPSs have been shown to differ in their ability to interact with the bactericidal/permeability-increasing protein, little is known about the differential mode of interaction with the acute-phase reactant LPS-binding protein (LBP). At lower concentrations, LBP catalyzes the binding of LPS to CD14 and enhances LPS-induced cellular activation via Toll-like receptor 4. In humans, however, concentrations of LBP in serum increase during an acute-phase response, and these LBP concentrations exhibit inhibitory effects in terms of cellular activation. The mechanisms of inhibition of LPS effects by LBP are not completely understood. Here, we report that human high-dose LBP (hd-LBP) suppresses binding of both R-type and S-type LPS to CD14 and inhibits LPS-induced nuclear translocation of NF-κB, although cellular uptake of R-type LPS was found to be increased by hd-LBP. In contrast, we found that hd-LBP enhanced the binding and uptake of S-type LPS only under serum-free conditions, whereas in the presence of serum, hd-LBP inhibited cellular binding and uptake. This inhibitory effect of serum could be mimicked by the addition of purified high-density lipoprotein (HDL) to serum-free medium, indicating an LBP-mediated transfer of preferentially S-type LPS to plasma lipoproteins such as HDL. A complete understanding of the hosts mechanisms to modulate the proinflammatory effects of LPS will most likely help in the understanding of inflammation and infection and may lead to novel therapeutic intervention strategies.

Proteomics Out of the Archive: Two-dimensional Electrophoresis and Mass Spectrometry Using HOPE-fixed, Paraffin-embedded Tissues

Proteome analyses provide diagnostic information which can be essential for therapeutic predictions. The application of such techniques for analyzing paraffin-embedded tissue samples is widely hampered by the use of formalin fixation requiring antigen retrieval procedures in molecular pathology. In prior studies, the HEPES-glutamic acid buffer-mediated organic solvent protection effect (HOPE) technique of tissue fixation has been shown to provide a broad array of biochemical investigations with excellent preservation of morphological structures, DNA, RNA, and proteins, thus supporting the multimethod analysis of archived specimens. Here we show that HOPE fixation is also useful in proteomic investigations by allowing two-dimensional electrophoresis (2DE) and mass spectrometry, using lung cancer tissues. Two-dimensional gels of two-protein extraction protocols derived from HOPE-fixed material displayed characteristic spot patterns with high reproducibility. For comparison, 2DE analysis of ethanol-fixed, formalin-fixed, and frozen samples from the same tissues was performed. Western blotting confirmed immunoreactivity of 2DE-separated proteins from HOPE-fixed tissue samples. Additionally, distinct spots were excised from HOPE-derived 2D gels and successfully subjected to peptide mass fingerprinting. In conclusion, paraffin archives containing HOPE-fixed tissues are applicable to a wide spectrum of molecular investigations including common biochemical methods for proteome analyses and therefore represent a unique source for molecular investigations in the rapidly growing field of molecular pathology. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Lipoproteins/peptides are sepsis-inducing toxins from bacteria that can be neutralized by synthetic anti-endotoxin peptides

Sepsis, a life-threatening syndrome with increasing incidence worldwide, is triggered by an overwhelming inflammation induced by microbial toxins released into the bloodstream during infection. A well-known sepsis-inducing factor is the membrane constituent of Gram-negative bacteria, lipopolysaccharide (LPS), signalling via Toll-like receptor-4. Although sepsis is caused in more than 50% cases by Gram-positive and mycoplasma cells, the causative compounds are still poorly described. In contradicting investigations lipoproteins/-peptides (LP), lipoteichoic acids (LTA), and peptidoglycans (PGN), were made responsible for eliciting this pathology. Here, we used human mononuclear cells from healthy donors to determine the cytokine-inducing activity of various LPs from different bacterial origin, synthetic and natural, and compared their activity with that of natural LTA and PGN. We demonstrate that LP are the most potent non-LPS pro-inflammatory toxins of the bacterial cell walls, signalling via Toll-like receptor-2, not only in vitro, but also when inoculated into mice: A synthetic LP caused sepsis-related pathological symptoms in a dose-response manner. Additionally, these mice produced pro-inflammatory cytokines characteristic of a septic reaction. Importantly, the recently designed polypeptide Aspidasept® which has been proven to efficiently neutralize LPS in vivo, inhibited cytokines induced by the various non-LPS compounds protecting animals from the pro-inflammatory activity of synthetic LP.

Biophysical characterization of the interaction of endotoxins with hemoglobins.

Bacterial endotoxin (lipopolysaccharide, LPS) is the major component of the outer leaflet of the outer membrane in gram-negative bacteria. During severe infections, bacteria may reach the blood circuit of humans, and endotoxins may be released from the bacteria due to cell division or cell death. In particular enterobacterial forms of LPS represent extremely strong activator molecules of the human immune system causing a rapid induction of cytokine production in monocytes and macrophages. Various mammalian blood proteins have been documented to display LPS binding activities mediating normally decreasing effects in the biological activity of LPS. In more recent studies, the essential systemic oxygen transportation protein hemoglobin (Hb) has been shown to amplify LPS-induced cytokine production on immune cells. The mechanism responsible for this effect is poorly understood. Here, we characterize the interaction of hemoglobin with LPS by using biophysical methods. The data presented, revealing the changes of the type and size of supramolecular aggregates of LPS in the presence of Hb, allow a better understanding of the hemoglobin-induced increase in bioactivity of LPS.

Reviewing and identifying amino acids of human, murine, canine and equine TLR4 / MD-2 receptor complexes conferring endotoxic innate immunity activation by LPS/lipid A, or antagonistic effects by Eritoran, in contrast to species-dependent modulation by lipid IVa.

There is literature evidence gathered throughout the last two decades reflecting unexpected species differences concerning the immune response to lipid IVa which provides the opportunity to gain more detailed insight by the molecular modeling approach described in this study. Lipid IVa is a tetra-acylated precursor of lipid A in the biosynthesis of lipopolysaccharide (LPS) in Gram-negative bacteria. Lipid A of the prototypic E. coli-type is a hexa-acylated structure that acts as an agonist in all tested mammalian species by innate immunorecognition via the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) receptor complex. In contrast, lipid IVa is proinflammatory in mouse cells (agonism) but it remains inactive to human macrophages and even antagonizes the action of potent agonists like E. coli-type lipid A. This particular ambivalent activity profile of lipid IVa has been confirmed in other mammalian species: in equine cells Lipid IVa also acts in a weak agonistic manner, whereas being inactive and antagonizing the lipid A-induced activation of canine TLR4/MD-2. Intriguingly, the respective TLR4 amino acid sequences of the latter species are more identical to the human (67%, 68%) than to the murine (62%, 58%) ortholog. In order to address the unpaired activity-sequence dualism for human, murine, canine and equine species regarding the activity of lipid IVa as compared to LPS and lipid A and, we review the literature and computationally pinpoint the differential biological effects of lipid IVa versus LPS and lipid A to specific amino acid residues. In contrast to lipid IVa the structurally related synthetic compound Eritoran (E5564) acts consistently in an antagonistic manner in these mammalian species and serves as a reference ligand for molecular modeling in this study. The combined evaluation of data sets provided by prior studies and in silico homology mapping of differential residues of TLR4/MD-2 complexes lends detailed insight into the driving forces of the characteristic binding modes of the lipid A domain in LPS and the precursor structure lipid IVa to the receptor complex in individual mammalian species.

Mechanism of Hbγ-35-induced an increase in the activation of the human immune system by endotoxins:

Endotoxins (LPS) are highly potent immune stimulatory molecules and are mainly known for triggering Gram-negative sepsis. However, besides their toxic effects, this stimulatory function may be advantageous, for example when used as an adjuvant during vaccination. Thus, there is always a narrow range between the useful wake-up of the immune system and its overwhelming reaction, which can lead to diseases like sepsis. This raises the question of which conformational properties are responsible for making the LPS aggregates more or less potent. As described previously, the size, type and form of LPS aggregates play a major role in their immune stimulatory activity. In this study we investigate the role of these parameters. On the one hand, we use a peptide (Pep19-2.5; Aspidasept) that causes a change of the LPS aggregate structure into a less toxic state; on the other hand, we use a potent immune stimulating peptide (Hbγ-35), leading to higher toxicity. We have found opposing effects on LPS aggregate conformations allowing a better understanding of the processes of immune stimulation.

Three-dimensional mapping of differential amino acids of human, murine, canine and equine TLR4/MD-2 receptor complexes conferring endotoxic activation by lipid A, antagonism by Eritoran and species-dependent activities of Lipid IVA in the mammalian LPS sensor system.

A literature review concerning the unexpected species differences of the vertebrate innate immune response to lipid IVA was published in CSBJ prior to the present computational study to address the unpaired activity-sequence correlation of prototypic E. coli -type lipid A and its precursor lipid IVA regarding human, murine, equine and canine species. To this end, their sequences and structures of hitherto known Toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (MD-2) complexes were aligned and their differential side chain patterns studied. If required due to the lack of the corresponding X-ray crystallographic data, three-dimensional models of TLR4/MD-2/ligand complexes were generated using mono and dimeric crystal structures as templates and in silico docking of the prototypic ligands lipid A, lipid IVA and Eritoran. All differential amino acids were mapped to pinpoint species dependency on an atomic scale, i.e. the possible concert of mechanistically relevant side chains. In its most abstract and general form the three-dimensional (3D-) models devise a triangular interface or “wedge” where molecular interactions between TLR4, MD-2 and ligand itself take place. This study identifies two areas in the wedge related to either agonism or antagonism reflecting why ligands like lipid IVA can possess a species dependent dual activity. Lipid IVA represents an imperfect (underacylated and backbone-flipped), low affinity ligand of mammalian TLR4/MD-2 complexes. Its specific but weak antagonistic activity in the human system is in particular due to the loss of phosphate attraction in the wedge-shaped region conferred by nonhomologous residue changes when compared to crystal and modeled structures of the corresponding murine and equine TLR4/MD-2 complexes. The counter-TLR4/MD-2 unit was also taken into account since agonist-mediated dimerization in a defined m-shaped complex composed of two TLR4/MD-2/agonist subunits triggers intracellular signaling during the innate immune response to bacterial endotoxin exposure.