The Bacterial Peptidoglycan-Sensing Molecules NOD1 and NOD2 Promote CD8 + Thymocyte Selection This information is current as Marianne M. Martinic, Irina Caminschi, Meredith O'Keeffe, of September 24, 2021. Therese C. Thinnes, Raelene Grumont, Steve Gerondakis, Dianne B. McKay, David Nemazee and Amanda L. Gavin J Immunol published online 15 February 2017 http://www.jimmunol.org/content/early/2017/02/15/jimmun ol.1601462 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/02/15/jimmunol.160146 Material 2.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 15, 2017, doi:10.4049/jimmunol.1601462 The Journal of Immunology The Bacterial Peptidoglycan-Sensing Molecules NOD1 and NOD2 Promote CD8+ Thymocyte Selection Marianne M. Martinic,*,1 Irina Caminschi,†,‡,2 Meredith O’Keeffe,†,2 Therese C. Thinnes,* Raelene Grumont,†,2 Steve Gerondakis,†,2 Dianne B. McKay,x David Nemazee,* and Amanda L. Gavin*,† Nucleotide-binding and oligomerization domain (NOD)–like receptors NOD1 and NOD2 are cytosolic innate immune receptors that recognize microbial peptidoglycans. Although studies have addressed the role of NOD proteins in innate immune responses, little attention has been given to their impact on the developing adaptive immune system. We have assessed the roles of NOD1 and NOD2 deficiency on T cell development in mice. Our results demonstrate that NOD1 and NOD2 promote the positive selection/ maturation of CD8 single-positive thymocytes in a thymocyte-intrinsic manner. TCR-mediated ERK phosphorylation is signifi- cantly reduced in the absence of NOD proteins, but receptor-interacting protein 2 is not involved in CD8 single-positive thymocyte Downloaded from selection or ERK signaling. Commensal bacteria–free animals have thymocyte maturation defects, and exogenous NOD ligands can enhance thymocyte maturation in culture. These results raise the intriguing possibility that abnormal lymphocyte responses observed in NOD-dependent inflammatory diseases are not driven solely by microbial signals in the gut, but may also involve intrinsic lymphocyte defects resulting from impaired CD8 T cell thymic development. The Journal of Immunology, 2017, 198: 000–000. http://www.jimmunol.org/ ucleotide-binding and oligomerization domain (NOD)1 peptidoglycan ligand–activated NOD–RIP2 complex promotes the and NOD2 are intracellular innate immune sensors for potent activation of NF-kB, leading to the production of inflam- N the bacterial peptidoglycan-derived molecules g-D- matory mediators and chemokines (8). glutamyl-meso-diaminopimelic acid and muramyl dipeptide NOD1 and NOD2 are important for epithelial and myeloid cell (MDP), respectively (1, 2). Their expression is normally associ- signaling in response to microbial products from a variety of ated with myeloid and nonlymphoid cell types. Upon sensing bacteria, including Mycobacterium sp., Salmonella enterica these ligands via leucine-rich repeat (LRR) domains (3, 4), di- serovar Typhimurium, Helicobacter sp., and Listeria mono- merization occurs via the oligomerization domain, leading to cytogenes (9). In addition to signaling the presence of pathogens, by guest on September 24, 2021 caspase-recruitment domain–dependent interaction with the serine the sensing of commensal microbial products via NOD molecules threonine kinase receptor-interacting protein 2 (RIP2) (5–7). The appears to be important for an increasingly diverse range of bio- logical functions. These include intestinal lymphoid tissue genesis (10), the generation or maintenance of functional Paneth cells in *Department of Immunology and Microbial Science, The Scripps Research Institute, the intestine (11, 12), and systemic neutrophil priming at sites La Jolla, CA 92037; †Centre for Immunology, Burnet Institute, Melbourne, Victoria 3004, Australia; ‡The Walter and Eliza Hall Institute of Medical Research, Parkville, distal to the gut (13). x Victoria 3052, Australia; and Division of Nephrology, Department of Medicine, Polymorphisms in NOD2 are associated with two important University of California San Diego, La Jolla, CA 92093 human inflammatory diseases, Crohn disease and Blau syndrome 1 Current address: Vaccine Immunotherapeutics, Pfizer Global Research and Devel- (14–16). Although the amino acid changes in the LRR domains of opment, San Diego, CA. NOD2 that are associated with Crohn disease reduce MDP ligand– 2Current address: Monash Biomedicine Discovery Institute, Department of Biochem- istry and Molecular Biology, Monash University, Clayton, VIC, Australia. driven signaling, Blau syndrome–associated sequence changes ORCIDs: 0000-0003-4407-3423 (M.M.M.); 0000-0002-4857-6039 (D.B.M.); 0000- appear to constitutively activate NF-kB, at least when expressed in 0002-4769-6311 (D.N.); 0000-0001-9921-4225 (A.L.G.). epithelial cell lines (5, 17). NOD1 and NOD2 can associate with Received for publication October 13, 2016. Accepted for publication January 22, the autophagy-related molecule ATG16L1 to promote autophagic 2017. double membrane restriction of invasive bacteria (18). As hypo- This work was supported National Institutes of Health Grant A1067403, National functional polymorphisms of other autophagy genes are also as- Health and Medical Research Council Project Grant 1009538, and by the Victorian sociated with Crohn disease (19, 20), it appears that inappropriate Operational Infrastructure Support Program. A.L.G. was supported by an Australian Research Council Future Fellowship, M.M.M. was supported by a Swiss National control of bacteria by autophagy and inappropriate inflammatory Science Foundation postdoctoral fellowship, and M.O. was supported by a National responses driven by NF-kB and MAPK are linked to this chronic Health and Medical Research Council Career Development Award. inflammatory disease (21). Address correspondence and reprint requests to Dr. Amanda L. Gavin, The Scripps The resulting ileal inflammation in Crohn disease consists Research Institute, Maildrop IMM29, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: [email protected] mainly of T lymphocyte infiltrates that are associated with high The online version of this article contains supplemental material. levels of IFN-g and IL-17 expression (22). During their devel- Abbreviations used in this article: 7AAD, 7-aminoactinomycin D; DN, double- opment in the thymus, all a/b TCR T cells, including a/b TCR negative; DP, double-positive; HSA, heat-stable Ag; LRR, leucine-rich repeat; MDP, intraepithelial lymphocytes, are subject to positive selection at the muramyl dipeptide; NOD, nucleotide-binding and oligomerization domain; RIP2, CD4+CD8+ (double-positive [DP]) stage of differentiation (23). receptor-interacting protein 2; SP, single-positive; Tg, transgenic; WT, wild-type. The signaling generated by newly rearranged ab TCRs interacting Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 with self-peptide–MHC complexes expressed on cortical thymic www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601462 2 NOD MOLECULES PROMOTE CD8 THYMOCYTE MATURATION epithelial cells promotes positive selection that initiates the dif- unlabeled Nod12/2Nod22/2 cells. After loading with Indo-1 AM (2 mM; ferentiation of DPs into MHC class II–restricted CD4 single- Molecular Probes), cells were washed and then stained for 20 min on ice positive (SP) or MHC class I–restricted CD8SP thymocytes with biotinylated Abs specific for CD3 (145-2C11) and CD4 (RM4.4), PerCP-Cy5.5–conjugated anti-CD4 (GK1.5), and PE-Cy7–conjugated (reviewed in Refs. 24, 25). anti-CD8 (53-6.7; all from BioLegend). Cells were prewarmed to 37˚C Mature human T lymphocytes express both NOD1 and NOD2 and streptavidin was added to cross-link biotinylated Abs. Maximum (26, 27), and a CD4 T cell–intrinsic function for mouse NOD2 has Ca2+ flux was achieved by the addition of ionomycin (500 ng/ml; Cal- been reported (28). Although both NOD1 and NOD2 transcripts biochem). The mean fluorescence ratio of Indo-1 violet to Indo-1 blue was calculated using FlowJo software (Tree Star). have been detected in mouse thymic tissue, it is unknown whether 2 2 NOD1 and NOD2 play roles in developing lymphocyte signaling OT-I TCR Tg Tap1 / stimulation assay (29, 30). In this study, we provide evidence that both NOD1 and To avoid cell signaling triggers by purification, total unpurified 5 3 106 NOD2 function intrinsically in thymocytes to promote CD8SP thymocytes from WT or Nod12/2Nod22/2 OT-I TCR Tg Tap12/2 mice thymocyte-positive selection. Furthermore, we identify a RIP2- were cultured in RPMI 1640 with 10% FCS, 100 U/ml penicillin, 0.1 mg/ml independent pathway where NOD proteins are required for effi-