Long‐Chain Lipids Are Required for the Innate Immune Recognition of Trehalose Diesters by Macrophages

Abstract

Going to any length? Trehalose diesters of various chain lengths have been synthesised in order to determine the effect of lipid length on innate immune recognition, as determined by NO and cytokine production by macrophages. In this work, we show that longer lipids (C(20) -C(26)) are required for macrophage activation, with C(22) giving optimal activity.

Influence of trehalose 6,6′-diester (TDX) chain length on the physicochemical and immunopotentiating properties of DDA/TDX liposomes

“…The Mincle receptor has been extensively studied in terms of immune activation mechanisms and structural characteristics of ligands [13,14,18,40,41]. When TDX is incorporated into DDA liposomes, the TDX molecules are arranged in the lipid bilayer with their hydrophobic tails inserted into the DDA bilayer and the hydrophilic headgroups facing the surrounding aqueous environment.…”

Rational design of adjuvants targeting the C-type lectin Mincle

“…Given our findings that GlcMM is at least as active as TDM in stimulating Mincle reporter cell lines, we used glucose derivatives monoacylated at position 6 to investigate the role of the fatty acid structure in ligand recognition by Mincle. Chain length was previously shown to be critical for macrophage activation by trehalose derivatives diacylated by saturated linear fatty acids, with optimal activity achieved for fatty acids containing 22 carbon atoms (behenic acid), such as TDB (21). Surprisingly, glucose monobehenate (GlcMB/GlcC22), in contrast to TDB, did not show any activity on reporter cell lines ( Fig.…”

The Uptake of Trehalose Glycolipids by Macrophages Is Independent of Mincle

“…The ability of the F-TGLs to activate macrophages was then determined by comparing the level of NO production by GM-CSF-differentiated bone-marrow-derived macrophages (BMMs) obtained from C57BL/6 and Mincle À/À mice in response to stimulation with F-TGLs. [23] BMMs were cultured with each of the four F-TGLs 3 a-d, the lipidated azide intermediates 10 and 16, and the nonlipidated iodo-and azide-functionalised trehalose sugars 13 and 14, so as to garner further information about the requirements of the trehalose-glycolipid binding site in Mincle. [16,17,24] In all experiments TDB (2 a) and the potent TLR-4 ligand LPS were used as positive controls, [11,24] and medium only was used as a negative control.…”

“…[21,22] Our target F-TGLs 3 a-d were chosen to mimic the linear glycolipids TDB 2 a and trehalose diester (TDE) 2 b, which activate macrophages in a Mincle-dependent manner. [23,24] As only one long-chain ester group on trehalose is sufficient to activate macrophages in a Mincle-dependent manner, [24] probes containing a fluorophore with and without a linker (i.e., 3 a and 3 b, and 3 c and 3 d, respectively) were deemed suitable tools for the study of glycolipid uptake. We also envisioned the use of fluorescent beads coated with TDB (2 a) to determine whether trehalose glycolipids increase the uptake of larger particles in a Mincle-dependent manner.…”

“…Time-course experiments also demonstrated that NO production by BMMs in response to trehalose glycolipids 3 a-b, 10 and 16 reached a plateau between 72 to 96 h (data not shown), as has been previously reported for a number of linear trehalose diesters. [23] Having demonstrated the ability of F-TGLs 3 a-d to activate BMMs in a Mincle-dependent manner, we then sought to study the uptake of the probes by BMMs by confocal microscopy. Unfortunately, initial experiments indicated that the dansyl probes were too prone to photobleaching to give a re- an increase in glycolipid uptake with increasing glycolipid concentration.…”