Contribution of MINCLE–SYK Signaling to Activation of Primary Human APCs by Mycobacterial Cord Factor and the Novel Adjuvant TDB

Abstract

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.

Rational design of adjuvants targeting the C-type lectin Mincle

“…Macrophages actually exhibited a bias toward TLR2 vs. Mincle signaling compared with BMDCs. In addition, human cells appeared to be surprisingly poorly responsive to Mincle stimulation compared with mouse cells, although this effect might be donor-dependent (34). This result is in contrast with data from Andersen et al (26), who recently reported that the CAF01 liposomal adjuvant system, composed of DDA and TDB, promotes long-lived M. tuberculosisspecific T-cell responses in humans, in agreement with previous data obtained in mice (27).…”

Intracellular metabolite β-glucosylceramide is an endogenous Mincle ligand possessing immunostimulatory activity

“…β-GlcCer is localized in endoplasmic reticulum/Golgi in healthy cells, whereas Mincle is expressed not only on the cell surface but also in intracellular compartments, such as endosomes or lysosomes (48). Thus, Mincle may encounter β-GlcCer intracellularly, particularly when the localization of β-GlcCer is dysregulated upon cell damage.…”

C-type lectin receptors in the control of T helper cell differentiation

“…Interestingly, the function of MINCLE seems to be partially dependent on MCL, as this CLR controls the expression of MINCLE through the formation of heterocomplexes that stabilize MINCLE at the surface 84,85 . A recent study suggests that MINCLE can act independently of MCL 49 , but its function can be modulated by MCL co-expression 86 . MCL-mediated stabilization increases expression of MINCLE and thereby influences its signalling to different pathogens.…”