Gram-positive bacterial lipoglycans based on a glycosylated diacylglycerol lipid anchor are microbe-associated molecular patterns recognized by TLR2.

Innate immune recognition is the first line of host defense against invading microorganisms. It is a based on the detection, by pattern recognition receptors (PRRs), of invariant molecular signatures that are unique to microorganisms. TLR2 is a PRR that plays a major role in the detection of Gram-positive bacteria by recognizing cell envelope lipid-linked polymers, also called macroamphiphiles, such as lipoproteins, lipoteichoic acids and mycobacterial lipoglycans. These microbe-associated molecular patterns (MAMPs) display a structure based on a lipid anchor, being either an acylated cysteine, a glycosylated diacylglycerol or a mannosyl-phosphatidylinositol respectively, and having in common a diacylglyceryl moiety. A fourth class of macroamphiphile, namely lipoglycans, whose lipid anchor is made, as for lipoteichoic acids, of a glycosylated diacylglycerol unit rather than a mannosyl-phosphatidylinositol, is found in Gram-positive bacteria and produced by certain Actinobacteria, including Micrococcus luteus, Stomatococcus mucilaginosus and Corynebacterium glutamicum. We report here that these alternative lipoglycans are also recognized by TLR2 and that they stimulate TLR2-dependant cytokine production, including IL-8, TNF-α and IL-6, and cell surface co-stimulatory molecule CD40 expression by a human macrophage cell line. However, they differ by their co-receptor requirement and the magnitude of the innate immune response they elicit. M. luteus and S. mucilaginosus lipoglycans require TLR1 for recognition by TLR2 and induce stronger responses than C. glutamicum lipoglycan, sensing of which by TLR2 is dependent on TLR6. These results expand the repertoire of MAMPs recognized by TLR2 to lipoglycans based on a glycosylated diacylglycerol lipid anchor and reinforce the paradigm that macroamphiphiles based on such an anchor, including lipoteichoic acids and alternative lipoglycans, induce TLR2-dependant innate immune responses.

LNA-based oligonucleotide electrotransfer for miRNA inhibition.

Micro-RNAs (miRNAs) are small regulatory RNAs that play an important role in disease development and progression and therefore represent a potential new class of therapeutic targets. However, an effective and safe clinical approach for miRNA inhibition remains elusive, primarily due to the lack of effective delivery methods. We proposed to inhibit miRNA by electrotransferring an antisense DNA oligomer containing locked nucleic acids (LNAs) (LNA/DNA oligomer). We observed that electropulsation (EP) led to a strong cellular uptake of LNA/DNA oligomer. The LNA/DNA oligomer electrotransfer mechanism and intracellular localization were visually investigated in real time at the single-cell level. Cyanine 5-labeled oligonucleotide entered exclusively during pulse application on the side of the permeabilized cell membrane facing the cathode, driven by electrophoretic forces. Minutes after the electrotransfer, the LNA/DNA oligomer diffused into the nucleus. EP provided the anti-miRNA oligomer with immediate and direct access to its cytoplasmic mature miRNA target and/or its nuclear precursor miRNA target. We then demonstrated using a LNA/DNA oligomer anti-miR34a that LNA/DNA oligomer electrotransfer decreased the level of the miR34a target and induced its functional inhibition. Our findings show that using the electrotransfer technique for LNA-based oligonucleotide delivery is a promising therapeutic strategy to silence deleterious miRNAs overexpressed in diseases.

Lipoglycans contribute to innate immune detection of mycobacteria.

Innate immune recognition is based on the detection, by pattern recognition receptors (PRRs), of molecular structures that are unique to microorganisms. Lipoglycans are macromolecules specific to the cell envelope of mycobacteria and related genera. They have been described to be ligands, as purified molecules, of several PRRs, including the C-type lectins Mannose Receptor and DC-SIGN, as well as TLR2. However, whether they are really sensed by these receptors in the context of a bacterium infection remains unclear. To address this question, we used the model organism Mycobacterium smegmatis to generate mutants altered for the production of lipoglycans. Since their biosynthesis cannot be fully abrogated, we manipulated the biosynthesis pathway of GDP-Mannose to obtain some strains with either augmented (∼1.7 fold) or reduced (∼2 fold) production of lipoglycans. Interestingly, infection experiments demonstrated a direct correlation between the amount of lipoglycans in the bacterial cell envelope on one hand and the magnitude of innate immune signaling in TLR2 reporter cells, monocyte/macrophage THP-1 cell line and human dendritic cells, as revealed by NF-κB activation and IL-8 production, on the other hand. These data establish that lipoglycans are bona fide Microbe-Associated Molecular Patterns contributing to innate immune detection of mycobacteria, via TLR2 among other PRRs.