Differential Control of Mincle-Dependent Cord Factor Recognition and Macrophage Responses by the Transcription Factors C/EBPb and HIF1a

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 tran-scriptional response to TDB/TDM has been defined to require FcRg-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/EBPb 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/EBPb dependence was also observed after stimulation with the Dectin-1 ligand Curdlan. Inducible expression of hypoxia-inducible factor 1a (HIF1a) also required C/EBPb. In turn, HIF1a was not required for Mincle expression, kinase activation, and Egr1 or Csf3 expression, but critically contributed to NO production. Taken together, we identify C/EBPb as central hub in Mincle expression and inflam-matory gene induction, whereas HIF1a controls Nos2 expression. C/EBPb also connects TLR signals to cord factor responsiveness through MYD88-dependent upregulation of Mincle. T he intracellular pathogen Mycobacterium tuberculosis survives and multiplies in the phagosome of macro-phages, which become activated and are recruited along with other cells to the site of infection to form typical granulomas. Mycobacteria are a rich source of pathogen-associated molecular patterns that include mycobacterial DNA triggering TLR9, the 19-kDa lipopeptide that activates TLR2, and several cell-wall lipids and glycolipids (1). The mycobacterial cord factor, Tre-halose-6,6-dimycolate (TDM), is abundant in the mycobacterial cell and has long been known to be a major virulence factor of mycobacteria and a potent inducer of inflammation in animal models (2). Geisel et al. (3) demonstrated that TDM is the major cell-wall glycolipid inducing inflammatory responses by macro-phages in vitro. Importantly, TDM per se is sufficient to induce formation of granulomas when injected in oil droplets (4). TDM (5) and …