MARCO, TLR2, and CD14 Are Required for Macrophage Cytokine Responses to Mycobacterial Trehalose Dimycolate and Mycobacterium tuberculosis

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Abstract
Virtually all of the elements of Mycobacterium tuberculosis (Mtb) pathogenesis, including pro-inflammatory cytokine production, granuloma formation, cachexia, and mortality, can be induced by its predominant cell wall glycolipid, trehalose 6,6′-dimycolate (TDM/cord factor). TDM mediates these potent inflammatory responses via interactions with macrophages both in vitro and in vivo in a myeloid differentiation factor 88 (MyD88)-dependent manner via phosphorylation of the mitogen activated protein kinases (MAPKs), implying involvement of toll-like receptors (TLRs). However, specific TLRs or binding receptors for TDM have yet to be identified. Herein, we demonstrate that the macrophage receptor with collagenous structure (MARCO), a class A scavenger receptor, is utilized preferentially to “tether” TDM to the macrophage and to activate the TLR2 signaling pathway. TDM-induced signaling, as measured by a nuclear factor-kappa B (NF-κB)-luciferase reporter assay, required MARCO in addition to TLR2 and CD14. MARCO was used preferentially over the highly homologous scavenger receptor class A (SRA), which required TLR2 and TLR4, as well as their respective accessory molecules, in order for a slight increase in NF-κB signaling to occur. Consistent with these observations, macrophages from MARCO−/− or MARCO−/−SRA−/− mice are defective in activation of extracellular signal-related kinase 1/2 (ERK1/2) and subsequent pro-inflammatory cytokine production in response to TDM. These results show that MARCO-expressing macrophages secrete pro-inflammatory cytokines in response to TDM by cooperation between MARCO and TLR2/CD14, whereas other macrophage subtypes (e.g. bone marrow–derived) may rely somewhat less effectively on SRA, TLR2/CD14, and TLR4/MD2. Macrophages from MARCO−/− mice also produce markedly lower levels of pro-inflammatory cytokines in response to infection with virulent Mtb. These observations identify the scavenger receptors as essential binding receptors for TDM, explain the differential response to TDM of various macrophage populations, which differ in their expression of the scavenger receptors, and identify MARCO as a novel component required for TLR signaling. The causative agent of tuberculosis, Mycobacterium tuberculosis, has a lipid-rich cell wall that contains a high percentage of mycolic acids. These mycolic acids contribute to both the impermeable nature of the cell wall and to the immunostimulatory properties of the bacterium. Indeed, it has been known for over 50 years that trehalose 6,6′-dimycolate (TDM/cord factor) is the major immunogenic lipid of M. tuberculosis, which induces potent pro-inflammatory responses from macrophages, although the receptor has not been identified. We have demonstrated that the toll-like receptor (TLR) pathway is required for pro-inflammatory cytokine production in response to TDM; however, the TLRs alone, or in conjunction with known co-receptors, are not sufficient to induce a response. We demonstrate that the macrophage receptor MARCO, a scavenger receptor, is utilized preferentially to “tether” TDM to the macrophage and activate the TLR2 signaling pathway, and is used preferentially over the related SRA. Macrophages from MARCO−/− mice are defective in activation of TDM-induced signaling and subsequent pro-inflammatory cytokine production in response to both TDM-coated beads and virulent M. tuberculosis. By identifying the macrophage receptors involved in initial recognition we can now explain variable responses to TDM between different macrophage populations (which differ in scavenger receptor expression), and have identified a novel co-receptor that may be involved in lipid presentation to TLRs.