Differential Control of Mincle-Dependent Cord Factor Recognition and Macrophage Responses by the Transcription Factors C/EBPβ and HIF1α

Abstract

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.

Toll-Like Receptor 2 and Mincle Cooperatively Sense Corynebacterial Cell Wall Glycolipids

“…7). First, we observed that the TLR2-driven induction of Mincle surface expression is operative and consistent with data from previous reports on TLR4 action (37,40), MyD88-dependent upregulation in response to Mycobacterium bovis BCG (42), and TLR2 activity (43). This C/EBP␤-dependent feed-forward loop enhancing the response to Mincle ligands (37) most likely accounts for the TLR2-driven G-CSF production in response to cell wall glycolipid challenge, although we acknowledge that formal proof of this mechanism would require the demonstration that the overexpression of Mincle, perhaps in association with other CLRs like Mcl, circumvents TLR2 dependence.…”

C-Type Lectin Receptor MCL Facilitates Mincle Expression and Signaling through Complex Formation

“…We and other groups have demonstrated that TDM-induced Mincle expression is severely impaired in MCL-deficient mice (6,10,11). Mincle was also induced by TLR-dependent pathogen-associated molecular patterns, such as LPS or zymosan, in wild-type (WT) BMDCs and bone marrowderived macrophages, whereas its inducible expression on the cell surface was compromised in MCL-deficient mice (Fig.…”

“…MCL has been proposed to be an activating receptor (9). We and other groups have reported that MCL also plays a crucial role in TDM responses (6,10,11). MCL deficiency results in impaired TDMinduced Mincle expression and, consequently, MCL-deficient mice show a severe defect in TDM responses.…”

“…Mincle expression is also induced by other pathogen-associated molecular patterns and cytokines (10,12,13). Mycobacteria contain a wide variety of immunostimulatory components other than Mincle ligand (14).…”

“…Mycobacteria contain a wide variety of immunostimulatory components other than Mincle ligand (14). Mincle induction upon M. tuberculosis infection or LPS stimulation was impaired in MCL-deficient mice (6,10,12,13). It is therefore possible that MCL may also have additional functions on Mincle beyond transcriptional regulation.…”