Aryl-functionalised α,α'-Trehalose 6,6'-Glycolipid Induces Mincle-independent Pyroptotic Cell Death.

α,α'-Trehalose 6,6'-glycolipids have long been known for their immunostimulatory properties. The adjuvanticity of α,α'-trehalose 6,6'-glycolipids is mediated by signalling through the macrophage inducible C-type lectin (Mincle) and the induction of an inflammatory response. Herein, we present an aryl-functionalised trehalose glycolipid, AF-2, that leads to the release of cytokines and chemokines, including IL-6, MIP-2 and TNF-α, in a Mincle-dependent manner. Furthermore, plate-coated AF-2 also leads to the Mincle-independent production of IL-1ß, which is unprecedented for this class of glycolipid. Upon investigation into the mode of action of plate-coated AF-2, it was observed that the treatment of WT and Mincle-/- bone marrow derived macrophages (BMDM), murine RAW264.7 cells, and human monocytes with AF-2 led to lytic cell death, as evidenced using Sytox Green and lactate dehydrogenase assays, and confocal and scanning electron microscopy. The requirement for functional Gasdermin D and Caspase-1 for IL-1ß production and cell death by AF-2 confirmed pyroptosis as the mode of action of AF-2. The inhibition of NLRP3 and K+ efflux reduced AF-2 mediated IL-1ß production and cell death, and allowed us to conclude that AF-2 leads to Capase-1 dependent NLRP3 inflammasome-mediated cell death. The unique mode of action of plate-coated AF-2 was surprising and highlights how the physical presentation of Mincle ligands can lead to dramatically different immunological outcomes.

Trehalose diamide glycolipids augment antigen-specific antibody responses in a Mincle-dependent manner.

Many studies have investigated how trehalose glycolipid structures can be modified to improve their Macrophage inducible C-type lectin (Mincle)-mediated adjuvanticity. However, in all instances, the ester-linkage of α,ά-trehalose to the lipid of choice remained. We investigated how changing this ester-linkage to an amide influences Mincle signalling and agonist activity and demonstrated that Mincle tolerates this functional group change. In in vivo vaccination studies in murine and ovine model systems, using OVA or Mannheimia haemolytica and Mycoplasma ovipneumoniae as vaccine antigens, respectively, it was demonstrated that a representative trehalose diamide glycolipid was able to enhance antibody-specific immune responses. Notably, IgG titres against M. ovipneumoniae were significantly greater when using trehalose dibehenamide (A-TDB) compared to trehalose dibehenate (TDB). This is particularly important as infection with M. ovipneumoniae predisposes sheep to pneumonia.

Cholesteryl glucosides signal through the carbohydrate recognition domain of the macrophage inducible C-type lectin (mincle).

Cholesteryl α-d-glucosides (αGCs) are unique metabolic products of the cancer-causing human pathogen Helicobacter pylori. Via signalling through the Macrophage inducible C-type lectin (Mincle) and the induction of a pro-inflammatory response, they are thought to play a role in the development of gastric atrophy. Herein, we prepared the first library of steryl d-glucosides and determined that they preferentially signal through the carbohydrate recognition domain of human Mincle, rather than the amino acid consensus motif. Lipidated steryl d-glucosides exhibited enhanced Mincle agonist activity, with C18 cholesteryl 6-O-acyl-α-d-glucoside (2c) being the most potent activator of human monocytes. Despite exhibiting strong Mincle signalling, sito- (5b) and stigmasterol glycosides (6b) led to a poor inflammatory response in primary cells, suggesting that Mincle is a potential therapeutic target for preventing H. pylori-mediated inflammation and cancer.

ortho-Substituted lipidated Brartemicin derivative shows promising Mincle-mediated adjuvant activity.

The macrophage inducible C-type lectin (Mincle) is a pathogen recognition receptor (PRR) that is a promising target for the development of Th1-polarising vaccine adjuvants. We recently reported on the synthesis and evaluation of lipidated Brartemicin analogues that showed Mincle agonist activity, with our lead agonist exhibiting potent Th1 adjuvant activity that was greater than that of trehalose dibehenate (TDB). Herein, we report on the efficient synthesis and subsequent biological evaluation of additional lipidated Brartemicin analogues that were designed to determine the structural requirements for optimal Mincle signalling. While all the Brartemicin analogues retained their ability to signal through Mincle and induce a functional response, the o-substituted and m,m-disubstituted derivatives (5a and 5d, respectively) induced a potent inflammatory response when using cells of both murine and human origin, with this response being the greatest observed thus far. As the inflammatory response elicited by 5a was slightly better than that induced by 5d, our findings point to o-substituted Brartemicin analogues as the preferred scaffold for further adjuvant development.

The effects of trehalose glycolipid presentation on cytokine production by GM-CSF macrophages.

Trehalose glycolipids (TGLs) are promising vaccine adjuvants, however effects of glycolipid presentation in the in vitro evaluation, and ultimate selection, of lead vaccine adjuvants are often overlooked. To this end, we synthesised a variety of TGLs and determined how the physicochemical presentation of these lipids influenced the cytokine response by bone marrow derived macrophages (BMMs). The TGLs were presented to wild-type and Mincle-/- BMMs as micellar solutions, coated on plates, coated on beads or surfactant solubilised. Medium to long-chain TGLs, either coated on plates or surfactant solubilised, resulted in the highest BMM activation. Stimulation of BMMs with TGLs coated on beads led to a decreased cytokine response, as compared to TGLs alone. All the TGL responses were Mincle dependent, however the mode of presentation did not have the same effect for each individual TGL. This was most apparent for the C22 trehalose monoester, which showed reduced activity compared to its diester counterpart when presented on a plate, but similar activity to the diester when presented as micelles or on beads. Taken together, our findings support the use of several in vitro assays for selecting lead vaccine adjuvants, particularly if structural differences between the adjuvants are pronounced. Graphical abstract The mode of glycolipid presentation, such as micellar solutions, coated on plates, coated on beads or surfactant solubilised, influences the immune response to trehalose glycolipids.

Lipidated Brartemicin Analogues Are Potent Th1-Stimulating Vaccine Adjuvants.

Effective Th1-stimulating vaccine adjuvants typically activate antigen presenting cells (APCs) through pattern recognition receptors (PRRs). Macrophage inducible C-type lectin (Mincle) is a PRR expressed on APCs and has been identified as a target for Th1-stimulating adjuvants. Herein, we report on the synthesis and adjuvanticity of rationally designed brartemicin analogues containing long-chain lipids and demonstrate that they are potent Mincle agonists that activate APCs to produce inflammatory cytokines in a Mincle-dependent fashion. Mincle binding, however, does not directly correlate to a functional immune response. Mutation studies indicated that the aromatic residue of lead compound 9a has an important interaction with Mincle Arg183. In vivo assessment of 9a highlighted the capability of this analogue to augment the Th1 response to a model vaccine antigen. Taken together, our results show that lipophilic brartemicin analogues are potent Mincle agonists and that 9a has superior in vivo adjuvant activity compared to TDB.

Discovery of lipids from B. longum subsp. infantis using whole cell MALDI analysis.

Bifidobacteria are dominant members of the microbial community in the intestinal tract of infants, and studies have shown that glycolipids extracted from the cell surface of these bacteria elicit beneficial immune responses. Accordingly, the identification and structural characterization of glycolipids from the cell wall of bifidobacteria is the first step in correlating glycolipid structure with biological activity. Using whole cell MALDI as a screening tool, we herein present for the first time the identification and structural elucidation of the major polar lipids from Bifidobacterium longum subs. infantis. The lipids identified include an unprecedented plasmenyl cyclophosphatidic acid and a mixed acetal glycolipid, with the latter subsequently being isolated and found to suppress the innate immune response.

Synthesis and biological activity of the lipoteichoic acid anchor from Streptococcus sp. DSM 8747.

In this study, the role of lipoteichoic acid (LTA) anchors in the activation of the innate immune response was investigated through the chemical synthesis of a series of LTA derivatives and the determination of their ability to induce NO production in bone marrow-derived macrophages (BMM). To this end, an efficient synthesis of the sn-3-O-(α-D-galactofuranosyl)-1,2-di-O-acylglycerol LTA core was developed, which was then used as a key structure to produce both phosphate and glycerylphosphate-funtionalised LTA anchors, as well as galactofuranosyldiglycerides with different fatty acid chain lengths. With a series of LTA anchors in hand, we then determined the effect of these glycolipids on the innate immune response by exploring their capacity to activate macrophages. Here, we report that several of the LTA-derivatives were able to induce NO production by BMMs. In general, the unnatural (sn-1) core glycolipid anchors showed lower levels of activity than the corresponding natural (sn-3) analogues, and the activity of the glycolipids also appears to be dependent on the length of lipid present, with an optimum lipid length of C20 for the sn-3 derivatives. Interestingly, a triacylated anchor and the 6-O-phosphorylated anchor, showed only modest activity, while the 6-O-glycerophosphorylated derivative was unable to induce NO production. Taken as a whole, our results highlight the subtle effects that glycolipid length can have on the ability to activate BMMs.