Abstract
Nod1 and Nod2 are pattern recognition receptors of the mammalian innate immune system. They respond to bacterial peptidoglycan fragments and are implicated in host defense against a variety of different bacterial pathogens. Recent studies furthermore support additional functions of these proteins in the control of adaptive immune responses and intestinal homeostasis. Activation of Nod1 and Nod2 by their cognate elicitors triggers inflammatory responses driven by the activation of NF-κB and MAPK pathways. In this chapter, we describe a quick and reliable cell-based assay using a luciferase reporter to measure Nod1- and Nod2-mediated NF-κB activation. The described protocol was successfully applied to analyze the influences of overexpressed proteins and siRNA-mediated knock-down to provide new insights into the regulation of Nod1/2-specific signaling pathways. Furthermore, this method is well suited for downscaling to high-throughput screening applications.
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References
Akira, S., Uematsu, S., and Takeuchi, O. (2006) Pathogen recognition and innate immunity. Cell 124, 783–801.
Fritz, J. H., Ferrero, R. L., Philpott, D. J., and Girardin, S. E. (2006) Nod-like proteins in immunity, inflammation and disease. Nat Immunol 7, 1250–7.
Chamaillard, M., Hashimoto, M., Horie, Y., Masumoto, J., Qiu, S., Saab, L., Ogura, Y., Kawasaki, A., Fukase, K., Kusumoto, S., Valvano, M. A., Foster, S. J., Mak, T. W., Nunez, G., and Inohara, N. (2003) An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat Immunol 4, 702–7.
Girardin, S. E., Boneca, I. G., Carneiro, L. A., Antignac, A., Jehanno, M., Viala, J., Tedin, K., Taha, M. K., Labigne, A., Zahringer, U., Coyle, A. J., DiStefano, P. S., Bertin, J., Sansonetti, P. J., and Philpott, D. J. (2003) Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 300, 1584–7.
Girardin, S. E., Boneca, I. G., Viala, J., Chamaillard, M., Labigne, A., Thomas, G., Philpott, D. J., and Sansonetti, P. J. (2003) Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem 278, 8869–72.
Inohara, N., Ogura, Y., Fontalba, A., Gutierrez, O., Pons, F., Crespo, J., Fukase, K., Inamura, S., Kusumoto, S., Hashimoto, M., Foster, S. J., Moran, A. P., Fernandez-Luna, J. L., and Nunez, G. (2003) Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn’s disease. J Biol Chem 278, 5509–12.
Inohara, Chamaillard, McDonald, C., and Nunez, G. (2005) NOD-LRR proteins: role in host-microbial interactions and inflammatory disease. Annu Rev Biochem 74, 355–83.
Lukasik, E., and Takken, F. L. (2009) STANDing strong, resistance proteins instigators of plant defence. Curr Opin Plant Biol 12, 427–36.
Inohara, N., Koseki, T., del Peso, L., Hu, Y., Yee, C., Chen, S., Carrio, R., Merino, J., Liu, D., Ni, J., and Nunez, G. (1999) Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB. J Biol Chem 274, 14560–7.
Tanabe, T., Chamaillard, M., Ogura, Y., Zhu, L., Qiu, S., Masumoto, J., Ghosh, P., Moran, A., Predergast, M. M., Tromp, G., Williams, C. J., Inohara, N., and Nunez, G. (2004) Regulatory regions and critical residues of NOD2 involved in muramyl dipeptide recognition. Embo J 23, 1587–97.
Kufer, T. A. (2008) Signal transduction pathways used by NLR-type innate immune receptors. Mol Biosyst 4, 380–6.
Girardin, S. E., Tournebize, R., Mavris, M., Page, A. L., Li, X., Stark, G. R., Bertin, J., DiStefano, P. S., Yaniv, M., Sansonetti, P. J., and Philpott, D. J. (2001) CARD4/Nod1 mediates NF-kappaB and JNK activation by invasive Shigella flexneri. EMBO Rep 2, 736–42.
Opitz, B., Puschel, A., Beermann, W., Hocke, A. C., Forster, S., Schmeck, B., van Laak, V., Chakraborty, T., Suttorp, N., and Hippenstiel, S. (2006) Listeria monocytogenes activated p38 MAPK and induced IL-8 secretion in a nucleotide-binding oligomerization domain 1-dependent manner in endothelial cells. J Immunol 176, 484–90.
Hsu, Y. M., Zhang, Y., You, Y., Wang, D., Li, H., Duramad, O., Qin, X. F., Dong, C., and Lin, X. (2007) The adaptor protein CARD9 is required for innate immune responses to intracellular pathogens. Nat Immunol 8, 198–205.
Fritz, J. H., Le Bourhis, L., Sellge, G., Magalhaes, J. G., Fsihi, H., Kufer, T. A., Collins, C., Viala, J., Ferrero, R. L., Girardin, S. E., and Philpott, D. J. (2007) Nod1-mediated innate immune recognition of peptidoglycan contributes to the onset of adaptive immunity. Immunity 26, 445–59.
Magalhaes, J. G., Fritz, J. H., Le Bourhis, L., Sellge, G., Travassos, L. H., Selvanantham, T., Girardin, S. E., Gommerman, J. L., and Philpott, D. J. (2008) Nod2-dependent Th2 polarization of antigen-specific immunity. J Immunol 181, 7925–35.
Bouskra, D., Brezillon, C., Berard, M., Werts, C., Varona, R., Boneca, I. G., and Eberl, G. (2008) Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis. Nature 456, 507–10.
Kain, S. R., and Ganguly, S. (2001) Overview of genetic reporter systems. Curr Protoc Mol Biol Chapter 9, Unit9 6.
White, E. H., Rapaport, E., Seliger, H. H., and Hopkins, T. A. (1971) The Chemi- and Bioluminescence of Firefly Luciferin: An Efficient Chemical Production of Electronically Excited States. Bioorganic Chemistry 1, 92–122.
Miller, J. H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Kurt-Jones, E. A., Sandor, F., Ortiz, Y., Bowen, G. N., Counter, S. L., Wang, T. C., and Finberg, R. W. (2004) Use of murine embryonic fibroblasts to define Toll-like receptor activation and specificity. J Endotoxin Res 10, 419–24.
Chen, G., and Goeddel, D. V. (2002) TNF-R1 signaling: a beautiful pathway. Science 296, 1634–5.
Kawai, T., and Akira, S. (2007) Signaling to NF-kappaB by Toll-like receptors. Trends Mol Med 13, 460–9.
McDonald, C., Chen, F. F., Ollendorff, V., Ogura, Y., Marchetto, S., Lecine, P., Borg, J. P., and Nunez, G. (2005) A role for Erbin in the regulation of Nod2-dependent NF-kappaB signaling. J Biol Chem 280, 40301–9.
Barnich, N., Aguirre, J. E., Reinecker, H. C., Xavier, R., and Podolsky, D. K. (2005) Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor-{kappa}B activation in muramyl dipeptide recognition. J Cell Biol 170, 21–6.
Kufer, T. A., Kremmer, E., Adam, A. C., Philpott, D. J., and Sansonetti, P. J. (2008) The pattern-recognition molecule Nod1 is localized at the plasma membrane at sites of bacterial interaction. Cell Microbiol 10, 477–86.
Huang, S., Zhao, L., Kim, K., Lee, D. S., and Hwang, D. H. (2008) Inhibition of Nod2 signaling and target gene expression by curcumin. Mol Pharmacol 74, 274–81.
Barnich, N., Hisamatsu, T., Aguirre, J. E., Xavier, R., Reinecker, H. C., and Podolsky, D. K. (2005) GRIM-19 interacts with nucleotide oligomerization domain 2 and serves as downstream effector of anti-bacterial function in intestinal epithelial cells. J Biol Chem 280, 19021–6.
Kufer, T. A., Kremmer, E., Banks, D. J., and Philpott, D. J. (2006) Role for erbin in bacterial activation of Nod2. Infect Immun 74, 3115–24.
Yamamoto-Furusho, J. K., Barnich, N., Xavier, R., Hisamatsu, T., and Podolsky, D. K. (2006) Centaurin beta1 down-regulates nucleotide-binding oligomerization domains 1- and 2-dependent NF-kappaB activation. J Biol Chem 281, 36060–70.
Bielig, H., Zurek, B., Kutsch, A., Menning, M., Philpott, D. J., Sansonetti, P. J., and Kufer, T. A. (2009) A function for AAMP in Nod2-mediated NF-kappaB activation. Mol Immunol 46, 2647–54.
Munoz, E., Courtois, G., Veschambre, P., Jalinot, P., and Israel, A. (1994) Tax induces nuclear translocation of NF-kappa B through dissociation of cytoplasmic complexes containing p105 or p100 but does not induce degradation of I kappa B alpha/MAD3. J Virol 68, 8035–44.
Werts, C., le Bourhis, L., Liu, J., Magalhaes, J. G., Carneiro, L. A., Fritz, J. H., Stockinger, S., Balloy, V., Chignard, M., Decker, T., Philpott, D. J., Ma, X., and Girardin, S. E. (2007) Nod1 and Nod2 induce CCL5/RANTES through the NF-kappaB pathway. Eur J Immunol 37, 2499–508.
Zurek et al. (2011) Innate Immun, PMID:21310790.
Bielig et al. (2010) ChemMedChem 5(12), 2065–71. PMID:20973121.
Acknowledgments
We thank Dr. Katja Lautz and Andreas Neerincx for critical reading of the manuscript. The authors acknowledge support by the Deutsche Forschungsgemeinschaft (DFG) grant SFB670-NG01.
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Zurek, B., Bielig, H., Kufer, T.A. (2011). Cell-Based Reporter Assay to Analyze Activation of Nod1 and Nod2. In: Rast, J., Booth, J. (eds) Immune Receptors. Methods in Molecular Biology, vol 748. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-139-0_7
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DOI: https://doi.org/10.1007/978-1-61779-139-0_7
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