SFB796 - Sub project B6


Reprogramming of macrophages by the mycobacterial cord factor


Project summary

The glycolipid trehalose-dimycolate (TDM, also known as the cord factor) is a major constituent of the mycobacterial cell wall. TDM has been known for many years as a virulence factor of pathogenic mycobacteria, but also as a trigger for innate immune cells and potent adjuvant. In our previous work, we have identified the C-type lectin receptor Mincle as the pattern recognition receptor for TDM. Mincle-triggered FcRg-Syk-Card9 signalling activates antigen-presenting cells to produce inflammatory cytokines, which in turn direct Th1/Th17 responses. These findings have provided a molecular explanation for the well-known inflammatory effects of the cord factor and revealed the mechanism of the adjuvanticity of TDM and its synthetic analog TDB. We have also observed that the cytokine response of macrophages to M. bovis BCG is at least partially dependent on the presence of the TDM receptor Mincle, suggesting that the TDM-Mincle interaction may be important for sensing mycobacterial infection. On the other hand, TDM is a glycolipid effector molecule enabling mycobacteria to evade destruction in the macrophage. TDM can by itself inhibit phagosome maturation and thereby increase intracellular survival of mycobacteria. In addition, there is evidence that TDM and other mycobacterial cell wall and capsule components impede the inflammatory response of macrophages. It is unknown by which signalling pathways TDM impairs phagosome maturation and whether binding to Mincle is required for the inhibitory effects of TDM on macrophages.

In this project, we will investigate the reprogramming of macrophages by TDM. Our central hypothesis states that the glycolipid effector molecule TDM impairs key functions of the macrophage (inflammatory gene expression, phagolysosomal maturation and killing, and antigen presentation) by exploiting the Mincle-Syk-Card9 pathway. The role of this pathway in inhibition of phagosome maturation will be studied using macrophages from the respective knockout mice. In addition, to identify signalling proteins involved in the reprogramming by TDM, the recruitment of such proteins to the phagosome and their phosphorylation will be investigated. In addition to macrophage-autonomous phagosomal killing mechanisms, anti-mycobacterial defence requires the initiation of inflammation by the infected macrophage and the capacitating effects of IFN-γ. Therefore, in parts two and three of the project, TDM-triggered negative regulation of macrophage activation through TLR- and IFN-γ-signalling will be studied. Transcriptome profiling will be used to obtain a global perspective and to identify new candidate mediators of inhibition. The interference of TDM-triggered signalling with IFN-γ-induced macrophage gene expression will be studied with a focus on MHC class II expression and antigen presentation. The function of TDM-induced signalling modulators and transcriptional regulators identified in our previous work in antagonizing TLR- and/or IFN-γ-induced macrophage activation will be determined to provide mechanistic insight into how TDM reprograms the mycobacterial host cell.

Shown here: hierachical cluster analysis of genes regulated in macrophages after stimulation with different microbial ligands. The activation program induced by bacterial CpG-rich DNA is strikingly different from that induced by the Dectin-1 ligand Curdlan and the glyocolipid TDB. Card9-deficient macrophages do not respond to stimulation with Curdlan or TDB, showing the essential role of this pathway in TDB-ionduced innate immune activation.


Intracellular survival of mycobacteria in macrophages.
A section of the liver of a mouse infected with Mycobacterium bovis BCG. The bacteria are stained in red, cell nuclei appear in blue. Killing of intracellular mycobacteria requires activation of infected macrophages by IFNg that is produced by Th1 lymphocytes.


Project relevant publications

  • Heitmann L, Schoenen H, Ehlers S, Lang R, Holscher C.   (2012).   Mincle is not essential for controlling Mycobacterium tuberculosis infection.   Immunobiology 2012;   [in press].

  • Wenzel J, Held C, Palmisano R, Teufel S, David JP, Wittenberg T, Lang R.   (2011).   Measurement of TLR-Induced Macrophage Spreading by Automated Image Analysis: Differential Role of Myd88 and MAPK in Early and Late Responses.   Front Physiol 2011;2:71.

  • Lang R, Schoenen H, Desel C.   (2011).   Targeting Syk-Card9-activating C-type lectin receptors by vaccine adjuvants: findings, implications and open questions.   Immunobiology 2011;216:1184-91.

  • Held C, Wenzel J, Webel R, Marschall M, Lang R, Palmisano R, Wittenberg T.   (2011).   Using multimodal information for the segmentation of fluorescent micrographs with application to virology and microbiology.   Conf Proc IEEE Eng Med Biol Soc 2011:6487-90.

  • Schoenen H, Bodendorfer B, Hitchens K, Manzanero S, Werninghaus K, Nimmerjahn F, Agger EM, Stenger S, Andersen P, Ruland J, Brown GD, Wells C, Lang R.   (2010).   Cutting edge: mincle is essential for recognition and adjuvanticity of the mycobacterial cord factor and its synthetic analog trehalose-dibehenate.   J Immunol 2010;184:2756-60.

  • Andersen, C. A. S., Rosenkrands, I., Olsen, A.W., Nordly, P., Christensen, D., Lang, R., Kirschning, C.J., Bhowruth, V., Miinikin, D.E., Besra, G.S., Follman, F., Andersen, P. and Agger, E.M.   (2009).   Novel generation mycobacterial adjuvant based on liposome-encapsulated monomycoloyl glycerol from Mycobacterium bovis bacillus Calmette-Guerin.   J Immunol 2009;183:2294-302.

  • Schreiber, T., Ehlers, S., Heitmann, L., Rausch, A., Mages, J., Murray, P.J., Lang, R. and Holscher, C.   (2009).   Autocrine IL-10 Induces Hallmarks of Alternative Activation in Macrophages and Suppresses Antituberculosis Effector Mechanisms without Compromising T Cell Immunity.   J Immunol 2009;183:1301-12.

  • Werninghaus, K., Babiak, A., Gross, O., Holscher, C., Dietrich, H., Agger, E. M., Mages, J., Mocsai, A., Schoenen, H., Finger, K., Nimmerjahn, F., Brown, G. D., Kirschning, C., Heit, A., Andersen, P., Wagner, H., Ruland, J., and Lang, R.   (2009).   Adjuvanticity of a synthetic cord factor analogue for subunit Mycobacterium tuberculosis vaccination requires FcRgamma-Syk-Card9-dependent innate immune activation.   J Exp Med 206, 89-97.

  • Aly, S., Mages, J., Reiling, N., Kalinke, U., Decker, T., Lang, R. and Ehlers, S.   (2009).   Mycobacteria-induced granuloma necrosis depends on IRF-1.   J Cell Mol Med 2009;13:2069-82.

  • Agger, E. M., Rosenkrands I., Hansen, J., Brahimi, K., Vandahl, B. S., Aagaard, C., Werninghaus, K., Kirschning, C., Lang, R., Christensen, D., Theisen, M., Follmann, F. and Andersen, P.   (2008).   Cationic liposomes formulated with synthetic mycobacterial cordfactor (CAF01): a versatile adjuvant for vaccines with different immunological requirements.   PLoS ONE 3:e3116.

  • Aly, S., Laskay, T., Mages, J., Malzan, A., Lang, R. and Ehlers, S.   (2007).   Interferon-gamma-dependent mechanisms of mycobacteria-induced pulmonary immunopathology: the role of angiostasis and CXCR3-targeted chemokines for granuloma necrosis.   J Pathol 2007;212:295-305.

  • Lang, R, Rutschman RL, Greaves DR, Murray PJ.   (2002).   Autocrine deactivation of macrophages in transgenic mice constitutively overexpressing IL-10 under control of the human CD68 promoter.   J Immunol 2002;168:3402-11.