Gary D. Ainge

Natural killer T cells recognize diacylglycerol antigens from pathogenic bacteria.

Natural killer T (NKT) cells recognize glycosphingolipids presented by CD1d molecules and have been linked to defense against microbial infections. Previously defined foreign glycosphingolipids recognized by NKT cells are uniquely found in nonpathogenic sphingomonas bacteria. Here we show that mouse and human NKT cells also recognized glycolipids, specifically a diacylglycerol, from Borrelia burgdorferi, which causes Lyme disease. The B. burgdorferi–derived, glycolipid-induced NKT cell proliferation and cytokine production and the antigenic potency of this glycolipid was dependent on acyl chain length and saturation. These data indicate that NKT cells recognize categories of glycolipids beyond those in sphingomonas and suggest that NKT cell responses driven by T cell receptor–mediated glycolipid recognition may provide protection against diverse pathogens.

Soluble CD36 ectodomain binds negatively charged diacylglycerol ligands and acts as a co-receptor for TLR2.

Background Cluster of differentiation 36 (CD36) is a transmembrane glycoprotein involved in many biological processes, such as platelet biology, angiogenesis and in the aetiopathology of atherosclerosis and cardiovascular diseases. Toll-like receptors (TLRs) are one of the most important receptors of the innate immune system. Their main function is the recognition of conserved structure of microorganisms. This recognition triggers signaling pathways that activate transcription of cytokines and co-stimulatory molecules which participate in the generation of an immune response against microbes. In particular, TLR2 has been shown to recognize a broad range of ligands. Recently, we showed that CD36 serves as a co-receptor for TLR2 and enhances recognition of specific diacylglycerides derived from bacteria. Methodology/ Principal Findings Here, we investigate the mechanism by which CD36 contributes to ligand recognition and activation of TLR2 signaling pathway. We show that the ectodomain of murine CD36 (mCD36ED) directly interacts with negatively charged diacylglycerol ligands, which explains the specificity and selectivity of CD36 as a TLR2 co-receptor. We also show that mCD36ED amplifies the pro-inflammatory response to lipoteichoic acid in macrophages of wild-type mice and restores the pro-inflammatory response of macrophages from mice deficient in CD36 (oblivious), but not from mice deficient in cluster of differentiation 14 (CD14) (heedless). Conclusion/ Significance These data indicate that the CD36 ectodomain is the only relevant domain for activation of TLR2 signaling pathway and that CD36 and CD14 have a non-redundant role for loading ligands onto TLR2 in the plasma-membrane. The pro-inflammatory role of soluble CD36 can be relevant in the activation of the immune response against pathogens, as well as in the progression of chronic diseases. Therefore, an increased level of soluble forms of CD36, which has been reported to be increased in type II diabetic patients, could accelerate atherosclerosis by increasing the pro-inflammatory response to diacylglycerol ligands.

Structural characterization of mycobacterial phosphatidylinositol mannoside binding to mouse CD1d.

Mycobacterial phosphatidylinositol tetramannosides (PIM4) are agonists for a distinct population of invariant human (Vα24) and mouse (Vα14) NKT cells, when presented by CD1d. We determined the crystal structure at 2.6-Å resolution of mouse CD1d bound to a synthetic dipalmitoyl-PIM2. Natural PIM2, which differs in its fatty acid composition is a biosynthetic precursor of PIM4, PIM6, lipomannan, and lipoarabinomannan. The PIM2 headgroup (inositol-dimannoside) is the most complex to date among all the crystallized CD1d ligands and is remarkably ordered in the CD1d binding groove. A specific hydrogen-bonding network between PIM2 and CD1d orients the headgroup in the center of the binding groove and above the A′ pocket. A central cluster of hydrophilic CD1d residues (Asp153, Thr156, Ser76, Arg79) interacts with the phosphate, inositol, and α1–α6-linked mannose of the headgroup, whereas additional specificity for the α1- and α2-linked mannose is conferred by Thr159. The additional two mannoses in PIM4, relative to PIM2, are located at the distal 6′ carbon of the α1-α6-linked mannose and would project away from the CD1d binding groove for interaction with the TCR. Compared with other CD1d-sphingolipid structures, PIM2 has an increased number of polar interactions between its headgroup and CD1, but reduced specificity for the diacylglycerol backbone. Thus, novel NKT cell agonists can be designed that focus on substitutions of the headgroup rather than on reducing lipid chain length, as in OCH and PBS-25, two potent variants of the highly stimulatory invariant NKT cell agonist α-galactosylceramide.

Role of phosphatidylinositol mannosides in the interaction between mycobacteria and DC-SIGN.

ABSTRACT The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is the major receptor on DCs for mycobacteria of the Mycobacterium tuberculosis complex. Recently, we have shown that although the mannose caps of the mycobacterial surface glycolipid lipoarabinomannan (ManLAM) are essential for the binding to DC-SIGN, genetic removal of these caps did not diminish the interaction of whole mycobacteria with DC-SIGN and DCs. Here we investigated the role of the structurally related glycolipids phosphatidylinositol mannosides (PIMs) as possible ligands for DC-SIGN. In a binding assay with both synthetic and natural PIMs, DC-SIGN exhibited a high affinity for hexamannosylated PIM6, which contains terminal α(1→2)-linked mannosyl residues identical to the mannose cap on ManLAM, but not for di- and tetramannosylated PIM2 and PIM4, respectively. To determine the role of PIM6 in the binding of whole mycobacteria to DC-SIGN, a mutant strain of M. bovis bacillus Calmette-Guérin deficient in the production of PIM6 (ΔpimE) was created, as well as a double knockout deficient in the production of both PIM6 and the mannose caps on LAM (ΔpimE ΔcapA). Compared to the wild-type strain, both mutant strains bound similarly well to DC-SIGN and DCs. Furthermore, the wild-type and mutant strains induced comparable levels of interleukin-10 and interleukin-12p40 when used to stimulate DCs. Hence, we conclude that, like ManLAM, PIM6 represents a bona fide DC-SIGN ligand but that other, as-yet-unknown, ligands dominate in the interaction between mycobacteria and DCs.

Soluble human TLR2 ectodomain binds diacylglycerol from microbial lipopeptides and glycolipids.

TLRs are key innate immune receptors that recognize conserved features of biological molecules that are found in microbes. In particular, TLR2 has been reported to be activated by different kinds of microbial ligands. To advance our understanding of the interaction of TLR2 with its ligands, the recombinant human TLR2 ectodomain (hTLR2ED) was expressed using a baculovirus/insect cell expression system and its biochemical, as well as ligand binding, properties were investigated. The hTLR2ED binds synthetic bacterial and mycoplasmal lipopeptides, lipoteichoic acid from Staphylococcus aureus, and synthetic lipoarabinomannan precursors from Mycobacterium at extracellular physiological conditions, in the absence of its co-receptors TLR1 and TLR6. We also determined that lipopeptides and glycolipids cannot bind simultaneously to hTLR2ED and that the phosphatidyl inositol mannoside 2 (Pim2) is the minimal lipoarabinomannan structure for binding to hTLR2ED. Binding of hTLR2ED to Pim4, which contains a diacylglycerol group with one of its acyl chains containing 19 carbon atoms, indicates that hTLR2ED can bind ligands with acyl chains longer than 16 carbon atoms. In summary, our data indicate that diacylglycerol is the ligand moiety of microbial glycolipids and lipoproteins that bind to hTLR2ED and that both types of ligands bind to the same binding site of hTLR2ED.

Phosphatidylinositol Mannosides are Efficient Mucosal Adjuvants

The development of defined sub-unit vaccines requires the inclusion in the vaccine of an immunological adjuvant. The most important property of adjuvants for vaccines aimed at inducing optimal protection against intracellular bacteria such as Mycobacterium tuberculosis or M. bovis is the ability to enhance cell-mediated immunity, specifically Th1 responses. In this paper, we describe a system where transgenic mice expressing a high proportion of T cells specific for an ovalbumin (OVA) peptide are used to assess the ability of a novel class of adjuvants to positively modulate cell-mediated immune responses. Defined fractions containing purified native or synthetic phosphatidylinositol mannosides (PIMs) from mycobacteria were assessed for their adjuvant activities in response to the model antigen (OVA). Purified PIM preparations given to mice with OVA by the subcutaneous route were shown to elicit an enhanced release of interferon-gamma (IFN-γ) in cellular responses to OVA peptide in vitro. Very little interleukin-4 (IL-4) was released by cells from mice immunized with PIMs and OVA, whereas cells from animals immunized with complete Freunds adjuvant (CFA) and OVA released IL-4 as well as IFN-γ. Synthetic preparations of PIM2 and PIM4 also acted as adjuvants in the mouse model studied. In addition, PIM preparations were shown to generate an efficient cell-mediated immune response to OVA, when the antigen/adjuvant preparations were administered via the oral route or intranasal route. PIM preparations elicited substantial release of interleukin-12 (IL-12) from dendritic cells (DCs). These data suggest that purified or synthetic PIMs act as adjuvants when administered at mucosal surfaces and represent a new class of adjuvants for mucosal immunization against intracellular pathogens.

Chemical Synthesis and Immunosuppressive Activity of Dipalmitoyl Phosphatidylinositol Hexamannoside

Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of phosphoglycolipids with significant immune-modulating properties. We present here the synthesis of dipalmitoyl phosphatidylinositol hexamannoside (PIM(6)) 1 and the first reported functional biology of a synthetic PIM(6). Key steps in the synthetic protocol included the selective glycosylation of an inositol 2,6-diol with a suitably protected mannosyl donor and construction of the glycan core utilizing a [3 + 4] thio-glycosylation strategy. The target 1 was purified by reverse phase chromatography and characterized by standard spectroscopic methods, HPLC, and chemical modification by deacylation to dPIM(6). The (1)H NMR spectrum of synthetic dPIM(6) obtained from 1 matched that of dPIM(6) obtained from nature. PIM(6) (1) exhibited dendritic cell-dependent suppression of CD8(+) T cell expansion in a human mixed lymphocyte reaction consistent with the well established immunosuppressive activity of whole mycobacteria.

Synthesis and Toll-like receptor 4 (TLR4) activity of phosphatidylinositol dimannoside analogues.

A series of five PIM(2) analogues were synthesized and tested for their ability to activate primary macrophages and modulate LPS signaling. Structural changes included replacement of the fatty acid esters of the phosphatidyl moiety of PIM(2) with the corresponding ether or amide. An AcPIM(2) analogue possessing an ether linkage was also prepared. The synthetic methodology utilized an orthogonally protected chiral myo-inositol starting material that was conveniently prepared from myo-inositol in just two steps. Important steps in the synthetic protocols included the regio- and α-selective glycosylation of inositol O-6 and introduction of the phosphodiester utilizing phosphoramidite chemistry. Replacement of the inositol core with a glycerol moiety gave compounds described as phosphatidylglycerol dimannosides (PGM(2)). Biological testing of these PIM compounds indicated that the agonist activity was TLR4 dependent. An ether linkage increased agonist activity. Removal of the inositol ring enhanced antagonist activity, and the presence of an additional lipid chain enhanced LPS-induced cytokine production in primary macrophages. Furthermore, the interruption of the LPS-induced 2:2 TLR4/MD-2 signaling complex formation by PIM(2) represents a previously unidentified mechanism involved in the bioactivity of PIM molecules.

A synthetic analogue of phosphatidylinositol mannoside is an efficient adjuvant.

We recently described the synthesis of an ether linked analogue of phosphatidylinositol dimannoside (PIM2ME). In the current study, PIM2ME was found to significantly enhance the release of the key Th1 cytokine interleukin-12 (IL-12) by dendritic cells (DCs) of naïve mice in vitro, but not interleukin-10 (IL-10). Based on this result, it was hypothesized that PIM2ME would be an effective adjuvant for cell-mediated immune responses. Injections of PIM2ME alone did not lead to weight loss and did not have toxic side effects, based on biomarkers of toxicity in serum,demonstrating that the compound induced no apparent adverse side effects. Mice were vaccinated with the core antigens of the hepatitis C virus by itself or with three different adjuvants, namely PIM2ME, a commercial preparation of monophosphoryl lipid A (MPL) or a preparation of aluminium hydroxide gel (alum). A control group of animals received the antigen only with no adjuvants. Immune responses to the Hepatitis C viral antigens were monitored by measuring antigen-specific production of interferon-gamma (IFN-γ), the p40 subunit of interleukin-12 (IL-12) and interleukin-10 (IL-10) to assess cell-mediated immune responses. Vaccination of mice with Hepatitis C viral antigens with the adjuvant PIM2ME led to a significant increase in cell-mediated immune responses (IFN-γ and IL-12). Injection of Hepatitis C viral antigens in alum led to no enhancement of the cell-mediated immune response. We conclude that PIM2ME is an efficacious adjuvant for enhancing cell-mediated immunity, and induces no observable adverse effects.