conducted on CLR assigned to the so-called Dectin-1 or Dectin-2 clusters, localized within 51 the NK cell gene cluster on human chromosome 12 or mouse chromosome 6 (1-3). Several 52 excellent reviews on the function of these CLR in anti-microbial defense and homeostasis are 53 available (4, 5). In this review we summarize the current knowledge about signaling 54 downstream of the activating CLR Dectin-1 (Clec7a), Dectin-2 (human Clec6a, mouse 55 Clec4n), Mincle (Clec4e) and Mcl (Clec4d) that is largely dependent on the kinase spleen 56 tyrosine kinase (Syk). Table I provides an overview of defined ligands and microorganisms 57 bound by this group of PRR. In addition to microbial carbohydrate and glycolipid structures 58 acting as PAMP, several CLR bind endogenous ligands such as SAP130 released by dying 59 cells or cholesterol crystals. Thus, these CLR are involved in homeostatic responses and 60 inflammatory conditions (6-9), in addition to host response to pathogens and commensals (10-61 (Fig. 1). 103 Syk-Card9 coupled CLR is observed across cell-types and species, promoting the 111 differentiation of IL-17-producing CD4+ T cells (23, 24, 26, 27). Remarkably, to date there is 112 only very limited information about the effects of combined stimulation of CLR and TLR 113 pathways on global gene expression. 114

Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, is an abundant cell wall glycolipid and major virulence factor of Mycobacterium tuberculosis. Its synthetic analog trehalose-6,6-dibehenate (TDB) is a new adjuvant currently in phase I clinical trials. In rodents, the C-type lectin receptors Mincle and Mcl bind TDB/TDM and activate macrophages and dendritic cells (DC) through the Syk-Card9 pathway. However, it is unknown whether these glycolipids activate human innate immune cells through the same mechanism. We performed in vitro analysis of TDB/TDM-stimulated primary human monocytes, macrophages, and DC; determined C-type lectin receptor expression; and tested the contribution of SYK, MINCLE, and MCL by small interfering RNA knockdown and genetic complementation. We observed a robust chemokine and cytokine release in response to TDB or TDM. MCSF-driven macrophages secreted higher levels of IL-8, IL-6, CCL3, CCL4, and CCL2 after stimulation with TDM, whereas DC responded more strongly to TDB and GM-CSF-driven macrophages were equally responsive to TDB and TDM. SYK kinase and the adaptor protein CARD9 were essential for glycolipid-induced IL-8 production. mRNA expression of MINCLE and MCL was high in monocytes and macrophages, with MINCLE and MCL proteins localized intracellularly under resting conditions. Small interfering RNA-mediated MINCLE or MCL knockdown caused on average reduced TDB- or TDM-induced IL-8 production. Conversely, retroviral expression in murine Mincle-deficient DC revealed that human MINCLE, but not MCL, was sufficient to confer responsiveness to TDB/TDM. Our study demonstrates that SYK-CARD9 signaling plays a key role in TDB/TDM-induced activation of innate immune cells in man as in mouse, likely by engagement of MINCLE.

Membrane microparticles (MMP) released from apoptotic cells deliver signals that secure the anti-inflammatory response beyond the nearest proximity of the apoptotic cell. Plasmacytoid dendritic cells (pDC) are sentinels prepared to detect cellular processes that endanger the organism. They play a key role in the regulation of both pro- and anti-inflammatory immune responses. Based on the assumption that pDC could participate in the initiation of the anti-inflammatory response to apoptotic cells, we investigated the effects of apoptotic cell-derived MMP on human pDC. The results obtained in our experiments confirmed that MMP released from apoptotic cells trigger IFN-α secretion from human pDC. They further suggest that pDC activation results from sensing of DNA contained in MMP. MMP-DNA displays a particularly strong stimulatory activity compared with MMP-RNA and other sources of DNA. Inhibition of MMP-induced IFN-α secretion by cytochalasin D, chloroquine, and an inhibitory G-rich oligodeoxynucleotide identify TLR9 as the receptor for MMP-DNA. In marked contrast to the pDC response in autoimmune patients, in healthy subjects MMP-mediated stimulation of pDC-derived IFN-α was found to be independent of FcγRIIA (CD32A). Based on our findings, we conclude that induction of pDC-derived IFN-α by MMP is a physiological event; future investigations are necessary to elucidate whether pDC activation promotes inflammation or propagates tolerance in the context of apoptotic cell clearance.