The advances in subunit vaccines development have intensified the search for potent adjuvants, particularly adjuvants inducing cellmediated immune responses. Identification of the C-type lectin Mincle as one of the receptors underlying the remarkable immunogenicity of the mycobacterial cell wall, via recognition of trehalose-6,6′-dimycolate (TDM), has opened avenues for the rational design of such molecules. Using a combination of chemical synthesis, biological evaluation, molecular dynamics simulations, and protein mutagenesis, we gained insight into the molecular bases of glycolipid recognition by Mincle. Unexpectedly, the fine structure of the fatty acids was found to play a key role in the binding of a glycolipid to the carbohydrate recognition domain of the lectin. Glucose and mannose esterified at O-6 by a synthetic α-ramified 32-carbon fatty acid showed agonist activity similar to that of TDM, despite their much simpler structure. Moreover, they were seen to stimulate proinflammatory cytokine production in primary human and murine cells in a Mincle-dependent fashion. Finally, they were found to induce strong Th1 and Th17 immune responses in vivo in immunization experiments in mice and conferred protection in a murine model of Mycobacterium tuberculosis infection. Here we describe the rational development of new molecules with powerful adjuvant properties. mycobacteria | glycolipid | innate immunity

Mycobacterium tuberculosis mannose-capped lipoarabinomannan inhibits the release of proinflammatory cytokines by LPS-stimulated human dendritic cells (DCs) via targeting the C-type lectin receptor DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN). With the aim of mimicking the bioactive supramolecular structure of mannose-capped lipoarabinomannan, we designed and synthesized a set of poly(phosphorhydrazone) dendrimers grafted with mannose units, called mannodendrimers, that differed by size and the number and length of their (α1→2)-oligommanoside caps. A third-generation dendrimer bearing 48 trimannoside caps (3T) and a fourth-generation dendrimer bearing 96 dimannosides (4D) displayed the highest binding avidity for DC-SIGN. Moreover, these dendrimers inhibited proinflammatory cytokines, including TNF-α, production by LPS-stimulated DCs in a DC-SIGN-dependent fashion. Finally, in a model of acute lung inflammation in which mice were exposed to aerosolized LPS, per os administration of 3T mannodendrimer was found to significantly reduce neutrophil influx via targeting the DC-SIGN murine homolog SIGN-related 1. The 3T mannodendrimer therefore represents an innovative fully synthetic compound for the treatment of lung inflammatory diseases. T o secure their colonization and survival, some bacterial intracellular pathogens have evolved tactics to undermine host innate immune responses, including inflammation. Mycobacterium tuberculosis, the causative agent of human tuberculosis, uses multiple mechanisms to survive within its host cellular niches of alveolar macrophages and dendritic cells (DCs). In particular, M. tuberculosis exposes surface lipoglycans at its cell envelope, namely mannose-capped lipoarabinomannans (ManLAMs), which inhibit the production of proinflammatory cytokines IL-12 and TNF-α by LPS-stimulated human DCs (1-3) via binding to the C-type lectin DC-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN) (4, 5). DC-SIGN reportedly modulates immune responses to several other pathogens, supporting its important role as an immunomodulatory receptor (6). ManLAMs are complex amphipathic macromolecules with an average molecular weight of 17 kDa that are composed of three domains: (i) a mannosyl-phosphatidyl-myo-inositol (MPI) anchor; (ii) a heteropolysaccharidic core composed of D-mannan and D-arabinan; and (iii) mannose caps consisting of mono, (α1→2)-di-, and (α1→2)-trimannosides (7). MPI anchor fatty acyl appendages induce a supramolecular organization of ManLAMs in aqueous solution, resulting in the formation of a 30-nm spherical structure of ∼450 molecules with the mannose caps exposed at the surface (8). This multivalent supramolecular structure allows multipoint attachment of ManLAMs, via mannose caps, to multimeric DC-SIGN receptors (9, 10) expressed at the surface of DCs, thereby ensuring high-affinity binding to the receptor (8-10) and induction of antiinflammatory activity (1, 2, 7).The strategy used by M. tuberculosis to down-regulate the...