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* University of Michigan Medical School, Department of Pathology and Comprehensive Cancer Center, Ann Arbor, MI 48109; and
Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602
Muramyl dipeptide (MDP), the microbial activator of nucleotide-binding oligomerization domain 2 (Nod2), induces NF-
B and MAPK activation, leading to the production of multiple anti-bacterial and proinflammatory molecules. In addition, MDP has been implicated in IL-1β secretion through the regulation of caspase-1. However, the mechanisms that mediate caspase-1 activation and IL-1β secretion in response to MDP stimulation remain poorly understood. We show here that fluorescent MDP molecules are internalized in primary macrophages and accumulate in granular structures that colocalize with markers of acidified endosomal compartments. The uptake of MDP was Nod2-independent. Upon ATP stimulation, labeled MDP was rapidly released from acidified vesicles into the cytosol, a process that required functional pannexin-1. Caspase-1 activation induced by MDP and ATP required pannexin-1 and Cryopyrin but was independent of Nod2. Conversely, induction of pro-IL-1β mRNA by MDP stimulation was abolished in Nod2-deficient macrophages but unimpaired in macrophages lacking Cryopyrin. These studies demonstrate a Nod2-independent mechanism mediated through pore-forming pannexin-1 that is required for intracellular delivery of MDP to the cytosol and caspase-1 activation. Furthermore, the work provides evidence for distinct roles of Nod2 and Cryopyrin in the regulation of MDP-induced caspase-1 activation and IL-1β secretion.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grants DK61707 and AI06331 (to G. N.). L.F. was supported by a fellowship from the Arthritis Foundation and Y.-G.K. by a fellowship from the University of Michigan Comprehensive Cancer Center. MDP derived compounds synthesis was supported by Grant GM065248 (to G.-J. B.). C.M. was supported by a Career Development Award from the Crohn’s and Colitis Foundation of America.
2 Current address: Department of Pathobiology, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195.
3 Address correspondence and reprint requests to Dr. Gabriel Núñez, University of Michigan Medical School, Department of Pathology and Comprehensive Cancer Center, 1500 East Medical Center Drive, Ann Arbor, MI 48109. E-mail address: bclx{at}umich.edu
4 Abbreviations used in this paper: Nod, nucleotide-binding oligomerization domain; KO, knockout; MDP, muramyl dipeptide; MAPK, mitogen-activated protein kinase; NLR, Nod-like receptors; P2X7R, P2X7 receptor; WT, wild type; BMDM, bone marrow-derived macrophages; DMF, dimethylformamide; DAPI, 4',6-diamidino-2-phenylindole; Ipaf, ICE-protease activating factor.
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