The Rapid and Facile Synthesis of Oxyamine Linkers for the Preparation of Hydrolytically Stable Glycoconjugates

The synthesis of a number of <i>N</i>-glycosyl-<i>N</i>-alkyl-methoxyamine bifunctional linkers is described. The linkers contain an <i>N</i>-methoxyamine functional group for conjugation to carbohydrates and a terminal group, such as an amine, azide, thiol, or carboxylic acid, for conjugation to the probe of choice. The strategy for the linker synthesis is rapid (3–4 steps) and efficient (51–96% overall yield), and many of the linkers can be synthesized using a three-step one-pot strategy. Moreover, the linkers can be conjugated to glycans in excellent yield and they show excellent stability toward hydrolytic cleavage

Synthesis of Branched Trehalose Glycolipids and Their Mincle Agonist Activity

The macrophage inducible C-type lectin (Mincle) is a pattern recognition receptor that recognizes trehalose dimycolate (TDM), and trehalose dibehenate (TDB) and related trehalose diesters, and thus represents a promising target for the development of vaccine adjuvants based on the trehalose glycolipid scaffold. To this end, we report on the synthesis of a series of long-chain α-branched, β-modified trehalose monoesters and diesters to explore how glycolipid structure affects signaling through Mincle. Key steps in our synthetic strategy include a Fráter-Seebach α-alkylation to install the C₂₀ aliphatic lipid on a malic acid derivative, and the formation of a β,γ-epoxide as an intermediate from which modifications to the β-position of the lipid can be made. Biological evaluation of the derivatives using nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cell lines expressing mMincle or hMincle revealed that the hMincle agonist activity of all diesters was superior to that of the current lead trehalose glycolipid adjuvant TDB, while the activity of several monoesters was similar to that of their diester counterparts for mMincle, but all showed reduced hMincle agonist activity. Taken together, diesters <b>2d</b>–<b>g</b> are thus potent Mincle agonists and promising vaccine adjuvants

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 <b>9a</b> has an important interaction with Mincle Arg183. In vivo assessment of <b>9a</b> 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 <b>9a</b> has superior in vivo adjuvant activity compared to TDB

Discovery of Lipids from B. longum subsp. <i>infantis</i> 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. <i>infantis</i>. 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

Lung anti-pneumococcal immunity is restored in chimeric Mincle KO mice reconstituted with the hematopoietic system of WT mice.

<p>Mincle KO mice received whole body irradiation (8 Gy), followed by transplantation with bone marrow cells (10⁷ cells/mouse i.v.) from either WT mice (WT onto KO, white bars), or Mincle KO mice (KO onto KO, transplantation controls, black bars). Seven weeks later, mice were infected orotracheally with 10⁷ CFU/mouse of type 19F <i>S</i>. <i>pneumoniae</i>. (A-C) Determination of BAL fluid cellular constituents (A, alveolar macrophages; B, alveolar exudate macrophages; C, alveolar recruited neutrophils) under baseline conditions (CL), or in response to infection, as indicated. (D) Analysis of Mincle expression on the cell surface of alveolar recruited neutrophils in bronchoalveolar lavage from <i>S</i>. <i>pneumoniae</i>-infected (24 h) Mincle KO mice reconstituted with WT (solid lines) or Mincle KO bone marrow cells (dashed lines). Grey histogram, isotype-stained negative control. (E,F) Determination of CFU counts in BAL fluids (E) or lung tissue (F), as indicated. (G-K) BAL fluid levels of proinflammatory cytokines TNFα (G), KC (H), IL-1β (I), or anti-inflammatory cytokines IL1ra (J), and IL-10 (K) in WT onto KO mice, or KO onto KO mice, as indicated. The data are shown as mean ± SD of n = 5 mice (A-F) or n = 4 mice (G-K) per time point and treatment group, and are representative of two experiments (Mann-Whitney U test).</p

Characterization of glycolipid Glc-DAG purified from <i>S</i>. <i>pneumoniae</i>.

<p>(A) NFAT-GFP reporter cells (4 x 10⁴ cells/well) were incubated with live <i>S</i>. <i>pneumoniae</i> at MOI 0.2, 2, 20 and 200 for 18 h followed by determination of the percentage of GFP-expressing reporter cells by flow cytometry. (B) FcRγ or Mincle+FcRγ expressing NFAT-GFP reporter cells (4 x 10⁴ cells/well) were incubated with aqueous or C:M fraction of <i>S</i>. <i>pneumoniae</i> lysates for 18 h, followed by determination of GFP-expressing reporter cells by flow cytometry. (C) HPTLC result of HPLC fractionated C:M portion of pneumococcal lysates with putative ligand indicated by an arrow head (copper acetate stain). (D) FcRγ (white bars) or Mincle + FcRγ (black bars) NFAT-GFP reporter cell assay of HPLC fractions obtained from scratched and purified HPTLC bands of <i>S</i>. <i>pneumoniae</i> lysates shown in (C). Experiments were repeated three times with similar results. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006038#ppat.1006038.s002" target="_blank">S2 Fig</a>. (E-H) Effect of TDM (E), or <i>S</i>. <i>pneumoniae</i>-derived Glc-DAG (F), or synthetic Glc-DAG (C14:0/C14:0, (G), or C18:0/C18:0, (H)) to trigger GFP reporter activation in Mincle/FcRγ (black dots), or FcRγ only (white dots) expressing NFAT-GFP reporter cells.</p

Expression of Mincle in the lungs of mice after infection with <i>S</i>. <i>pneumoniae</i>.

<p>(A) WT mice were infected with <i>S</i>. <i>pneumoniae</i> (1 x 10⁷ CFU/mouse) or were mock-infected (PBS), and lungs were harvested at the given time points and Mincle mRNA levels were analyzed. Values are shown as mean ± SD (n = 3 mice per time point and treatment group). CL (control): Mock-infection relative to untreated control, all other values in (A), relative to mock-infection. * p<0.05, relative to mock-infection. (B) Flow sorted alveolar macrophages and neutrophils purified from lung tissue of mock- versus <i>S</i>. <i>pneumoniae</i>-infected WT mice were subjected to Mincle mRNA analysis by real-time RT-PCR. Data are shown as mean ± SD (n = 4–6 mice for mock-infected and n = 5 mice for <i>S</i>. <i>pneumoniae</i>-infected mice). * p<0.05 relative to neutrophils. (C) Mincle mRNA levels in neutrophils collected by bronchoalveolar lavage from the lungs of patients with pneumococcal pneumonia, relative to peripheral blood neutrophils collected from the same patients. Data are shown as mean ± SD (n = 3 patients). * p<0.05 relative to PB neutrophils. (D-J) WT mice were left untreated (D, and CL in H-J), or were infected with <i>S</i>. <i>pneumoniae</i> (10⁶ CFU/mouse). At 0 h, 24 h, 48 h, and 72 h post-infection, expression of Mincle was analyzed on BAL AM (D (0 h), E (24 h), and H), and BAL ExMacs (F (48 h), and I), and BAL neutrophils (G (48 h), and J) (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006038#ppat.1006038.s001" target="_blank">S1 Fig</a>). Note that CL in (I,J) represents Mincle expression on resident alveolar macrophages (I) and neutrophils (J) purified from lung tissue of mock-infected WT mice. Horizontal bars: median values (n = 5–8 mice). Experiments were repeated two times with similar results. ** p<0.01, *** p<0.001, relative to mock-infection ((Mann-Whitney U test). ExMacs, exudate macrophages.</p

Impact of Mincle deficiency on lung proinflammatory cytokine release and macrophage necrosis in mice infected with <i>S</i>. <i>pneumoniae</i>.

<p>WT and Mincle KO mice were mock-infected or infected with <i>S</i>. <i>pneumoniae</i> (10⁷ CFU/mouse), and at indicated time points post-infection, BAL fluid TNF-α (A), KC (B), IL-1β (C), IL-10 (D), and IL-1ra (E) cytokine levels were measured. Data are shown as mean ± SD of n = 5–8 mice per time point and treatment group (12 h, n = 4 mice). Data are representative of two independent experiments with similar results. (F) WT and Mincle KO mice were mock-infected or low-dose infected with <i>S</i>. <i>pneumoniae</i> (5 x 10⁵ CFU/mouse). At the indicated time points, the percentage of necrotic macrophages (propidium iodide^pos/annexin V^neg) in BAL fluid was determined by flow cytometry. Values are shown as mean ± SD with n = 3 (0 h values) or 8 mice (12 and 24 h values) per time point and treatment group and are representative of two experiments. *p<0.05, **p<0.01, ***p<0.001 relative to WT mice. (Mann-Whitney U test).</p

Mincle is an essential receptor for Glc-DAG purified from <i>S</i>. <i>pneumoniae</i>.

<p>(A,B) AM were collected from the lungs of untreated WT and Mincle KO mice, or were collected by bronchoalveolar lavage from healthy volunteers (C,D). After adherence purification, murine or human AM were seeded into vehicle (isopropanol), or Glc-DAG coated (5 μg/well), or TDM coated (1 μg/well) 96-well plates (7.5 x 10⁴ cells/well), or were stimulated with LTA (dissolved in sterile water) at 1 μg/ml, as indicated. After 24 and 48 h (A,B), or after 24 h (C-F), murine (A,B, E,F) or human (C,D) TNF-α or IL-1ra release was determined in cell culture supernatants by ELISA. Data are shown as mean ± SD of 4 determinations per time point and treatment condition and the data are representative of two independent experiments. In (C,D), cytokine values are depicted as mean ± SD of quadruplicate determinations per time point and treatment condition collected from human AM of one healthy individual, with similar data obtained from two other healthy individuals. *p<0.05, **p<0.01 compared to vehicle treated AM and +p<0.05, ++p<0.01 compared to AM from WT mice (unpaired t-test).</p