Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, and its synthetic analog Trehalose-6,6-dibehenate (TDB) bind to the C-type lectin receptors macrophage-inducible C-type lectin (Mincle) and Mcl to activate macrophages. Genetically, the transcriptional response to TDB/TDM has been defined to require FcRγ-Syk-Card9 signaling. However, TDB/TDM-triggered kinase activation has not been studied well, and it is largely unknown which transcriptional regulators bring about inflammatory gene expression. In this article, we report that TDB/TDM caused only weak Syk-phosphorylation in resting macrophages, consistent with low basal Mincle expression. However, LPS-priming caused MYD88-dependent upregulation of Mincle, resulting in enhanced TDB/TDM-induced kinase activation and more rapid inflammatory gene expression. TLR-induced Mincle expression partially circumvented the requirement for Mcl in the response to TDB/TDM. To dissect transcriptional responses to TDB/TDM, we mined microarray data and identified early growth response (Egr) family transcription factors as direct Mincle target genes, whereas upregulation of Cebpb and Hif1a required new protein synthesis. Macrophages and dendritic cells lacking C/EBPβ showed nearly complete abrogation of TDB/TDM responsiveness, but also failed to upregulate Mincle. Retroviral rescue of Mincle expression in Cebpb-deficient cells restored induction of Egr1, but not of G-CSF. This pattern of C/EBPβ dependence was also observed after stimulation with the Dectin-1 ligand Curdlan. Inducible expression of hypoxia-inducible factor 1α (HIF1α) also required C/EBPβ. In turn, HIF1α was not required for Mincle expression, kinase activation, and Egr1 or Csf3 expression, but critically contributed to NO production. Taken together, we identify C/EBPβ as central hub in Mincle expression and inflammatory gene induction, whereas HIF1α controls Nos2 expression. C/EBPβ also connects TLR signals to cord factor responsiveness through MYD88-dependent upregulation of Mincle.
The T-cell adjuvanticity of mycobacterial cord factor trehalose 6,6'-dimycolate (TDM) is well established. The identification of the C-type lectin Mincle on innate immune cells as the receptor for TDM and its synthetic analogue trehalose 6,6'-dibehenate (TDB) has raised interest in development of synthetic Mincle ligands as novel adjuvants. Trehalose mono- (TMXs) and diesters (TDXs) with symmetrically shortened acyl chains [denoted by X: arachidate (A), stearate (S), palmitate (P), and myristate (M)] were tested. Upon stimulation of murine macrophages, G-CSF secretion and NO production were strongly augmented by all TDXs tested, in a wide concentration range. In contrast, the TMXs triggered macrophage activation only at high concentrations. Macrophage activation by all TDXs required Mincle, but was independent of MyD88. The superior capacity of TDXs for activating macrophages was paralleled by direct binding of TDXs, but not of TMXs, to a Mincle-Fc fusion protein. Insertion of a short polyethylene glycol between the sugar and acyl chain in TDS reduced Mincle-binding and macrophage activation. Immunization of mice with cationic liposomes containing the analogues demonstrated the superior adjuvant activity of trehalose diesters. Overall, immune activation in vitro and in vivo by trehalose esters of simple fatty acids requires two acyl chains of length and involves Mincle.
This information is current as GBP1 Antigen Presentation and Expression of Enhanced Inflammation yet Impaired towards γ Macrophage Response to IFN-Mycobacterial Cord Factor Reprograms the Lang
Excretory/secretory (ES) products of Ostertagia (Teladorsagia) circumcincta and Haemonchus contortus have been implicated in the inhibition of gastric acid secretion and vacuolation, and the loss of parietal cells associated with abomasal parasitism. Vacuolation of epithelial (HeLa) cells caused by adult O. circumcincta or L3 O. circumcincta or H. contortus ES products have been examined by differential interference contrast microscopy and by the neutral red uptake assay. ES products caused visible vacuolation of HeLa cells, and this effect was enhanced by 8 mM NH4Cl. Some parasite ES products caused a marked detachment of cells from the coverslip. At lower concentrations of ES products, neutral red uptake was usually increased above the control, but at higher concentrations of ES products, uptake was often decreased, probably because of cell detachment. Although generally consistent with direct observations of HeLa cell vacuolation by parasite chemicals, neutral red uptake was not a satisfactory quantitative assay.
Excretory/secretory (ES) products of the gastric nematode, Haemonchus contortus, have been implicated in the inhibition of gastric acid secretion which follows infection. Parietal cell vacuolation has been observed in abomasal sections from parasitised sheep, and ES prepared in vitro has been reported to cause vacuolation and to increase neutral red (NR) uptake in epithelial cell cultures. We have used the latter approach to examine, at the cellular level, the effects of ES prepared from L3 and adult nematodes. Both NR uptake and cellular vacuolation were increased following exposure to larval or adult ES products. ES preparations from adult worms induced more extensive vacuolation then those from L3 worms and, as with VacA treatment, adherent cells remained viable despite high levels of vacuolation. Whereas VacA-induced vacuolation resulted in NR uptake predominantly localised in vacuoles, this appeared not to be the case with ES-induced vacuolation, suggesting that different mechanisms might be involved. Both ES and VacA exposure was associated with an increased rate of cell detachment.
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