Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021 is online at: average * The Journal of Immunology , 17 of which you can access for free at: 2012; 188:2905-2913; Prepublished online 6 from submission to initial decision 4 weeks from acceptance to publication February 2012; doi: 10.4049/jimmunol.1102595 http://www.jimmunol.org/content/188/6/2905 Spatiotemporal Inhibition of Innate Immunity Signaling by the Tbc1d23 RAB-GAP Lesly De Arras, Ivana V. Yang, Brad Lackford, David W. H. Riches, Rytis Prekeris, Jonathan H. Freedman, David A. Schwartz and Scott Alper J Immunol cites 58 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription http://www.jimmunol.org/content/suppl/2012/02/06/jimmunol.110259 5.DC1 This article http://www.jimmunol.org/content/188/6/2905.full#ref-list-1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material References Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 27, 2021. The Journal of Immunology Spatiotemporal Inhibition of Innate Immunity Signaling by the Tbc1d23 RAB-GAP Lesly De Arras,*,† Ivana V. Yang,†,‡ Brad Lackford,x David W. H. Riches,*,‡,{ Rytis Prekeris,‖ Jonathan H. Freedman,# David A. Schwartz,†,‡ and Scott Alper*,† We previously identified Tbc1d23 as a candidate novel regulator of innate immunity using comparative genomics RNA interfer- ence screens in Caenorhabditis elegans and mouse macrophages. Using Tbc1d23 knockout mice and macrophages engineered to overexpress Tbc1d23, we now show that Tbc1d23 is a general inhibitor of innate immunity signaling, strongly inhibiting multiple TLR and dectin-signaling pathways. Tbc1d23 likely acts downstream of the TLR-signaling adaptors MyD88 and Trif and upstream of the transcription factor XBP1. Importantly, like XBP1, Tbc1d23 affects the maintenance, but not the initiation, of inflammatory cytokine production induced by LPS. Tbc1d23 acts as a RAB-GAP to regulate innate immunity signaling. Thus, Tbc1d23 exerts its inhibitory effect on innate immunity signaling in a spatiotemporal fashion. The identification of a novel Downloaded from spatiotemporal regulator of innate immunity signaling validates the comparative genomics approach for innate immunity gene discovery. The Journal of Immunology, 2012, 188: 2905–2913. he innate immune response plays a critical role in fighting including LPS. In contrast, overexpression of Tbc1d23 in mac- infection (1). However, if this response is activated too rophages inhibits the response to numerous PAMPs. Tbc1d23 is T strongly or becomes chronic, this can cause or contribute a general inhibitor of innate immunity signaling, affecting TLR http://www.jimmunol.org/ to a variety of diseases, including sepsis, Crohn’s disease, ath- and dectin signaling. The response to LPS is initiated by TLR4, erosclerosis, and cancer (2–5). We previously used comparative which signals through MyD88 and Trif to activate NF-kB, IRF3, genomics RNA interference screens in Caenorhabditis elegans and the MAPKs p38, ERK, and JNK (11–13). This has a variety of and mouse macrophages to identify novel regulators of innate effects, including production of inflammatory cytokines. We show immunity (6). Such regulators are potential targets for the devel- that Tbc1d23 likely acts downstream of MyD88 and Trif and opment of novel therapeutic and diagnostic options (3, 7–10). In upstream of the XBP1 transcription factor to inhibit TLR signal- these comparative genomics screens, we identified Tbc1d23 as ing. a novel conserved regulator of innate immunity. Nematodes har- Importantly, we find that Tbc1d23 inhibits the innate immune boring a mutation in tbc-1 (the C. elegans Tbc1d23 ortholog) have response in a spatiotemporal fashion. In contrast to the substantial by guest on September 27, 2021 altered antimicrobial gene expression and diminished survival in body of work investigating the initiation of inflammation, much the presence of pathogenic, but not nonpathogenic, bacteria (6). less is known about the maintenance of this response (14–16). We have now generated a murine knockout of Tbc1d23. In this Tbc1d23 does not alter initial activation events but instead affects article, we show that Tbc1d232/2 mice and macrophages exhibit maintenance of inflammatory gene expression several hours after increased inflammatory cytokine production when challenged LPS challenge. There has been a substantial effort to identify the with different pathogen-associated molecular patterns (PAMPs), signaling pathways that regulate innate immunity (11, 13). In contrast, the study of how the subcellular transport machinery affects innate immunity signaling is a younger field that has re- *Integrated Department of Immunology, National Jewish Health and University of Colorado, Denver, CO 80206; †Center for Genes, Environment and Health, National cently gained much attention (17). Tbc1d23 contains two con- Jewish Health, Denver, CO 80206; ‡Department of Medicine, University of Colorado, x served domains: the Tbc domain, which functions as a RAB Aurora, CO 80045; Laboratory of Molecular Carcinogenesis, National Institute of GTPase-activating protein (RAB-GAP) domain (18–20), and a Environmental Health Sciences, National Institutes of Health, Durham, NC 27709; {Program in Cell Biology, Department of Pediatrics, National Jewish Health, Denver, rhodanese superfamily domain (21, 22). We show that Tbc1d23 ‖ CO 80206; Department of Cell and Developmental Biology, University of Colorado, functions as a RAB-GAP to inhibit innate immunity signaling. Aurora, CO 80045; and #Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, NC 27709 Materials and Methods Received for publication September 9, 2011. Accepted for publication January 13, Generation and phenotyping of Tbc1d23 knockout mice 2012. Mouse experiments were approved by the National Jewish Health Insti- This work was supported by R21ES019256 from the National Institute of Environ- tutional Animal Care and Use Committee (protocol no. AS2801-06-12). mental Health Sciences and the Intramural Research Programs of the National Heart Lung and Blood Institute and the National Institute of Environmental Health Sciences Every effort was made to ensure that discomfort, distress, or pained in- (Z01ES102045 and Z01ES101946). jury to the animals was limited to that which was unavoidable in the conduct of scientifically sound research. Tbc1d23 knockout mice were generated by The array data presented in this article have been deposited in the Gene Expression injecting mouse embryonic stem cell line BA0556 (International Gene Trap Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE30840. Consortium) into blastocysts from C57BL/6 mice. The resulting Tbc1d23 gene-trapped mice were outcrossed six times to C57BL/6 (JAX). The Address correspondence and reprint requests to Dr. Scott Alper, National Jewish BA0556 gene trap was inserted in the first intron of Tbc1d23. By sequencing Health, 1400 Jackson Street, Denver, CO 80206. E-mail address: [email protected] PCR products within this intron, we determined that the gene trap was 2893 The online version of this article contains supplemental material. bp past the first base pair in exon 1 of Tbc1d23. Mice were genotyped by Abbreviations used in this article: BMDM, bone marrow-derived macrophage; CAT, PCR amplification of genomic DNA using a mix of three primers: oSA512, chloramphenicol acetyltransferase; ER, endoplasmic reticulum; PAMP, pathogen- oSA513, and oSA516 (Supplemental Table I). This generated a 313-bp associated molecular pattern; qPCR, quantitative PCR. product for the wild-type chromosome and a 221-bp product for the mutant. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102595 2906 Tbc1d23 INHIBITS INNATE IMMUNITY SIGNALING Tbc1d232/2 mice produced little or no Tbc1d23 RNA, as evidenced by implementation in Partek (St. Louis, MO), and differentially expressed quantitative PCR (qPCR) using three primer sets (Supplemental Table I) genes were identified using two-factor ANOVA in MultiExperiment Viewer that span the Tbc1d23 gene: primers that span exons 1 and 2 (2.5% of (http://www.tm4.org) (30). Differentially expressed genes in LPS-treated wild-type in spleen, 5.7% of wild-type in bone marrow-derived macro- cells compared with untreated cells in the CMV–myc–Tbc1d23 line com- phages [BMDMs]), primers that span exons 2 and 3 (2.8% spleen, 0.9% pared with the control CMV-CAT cell line were identified as interaction BMDMs), and primers that span exons 13 and 14 (3.9% spleen, 5.3% genes from the two-factor ANOVA analysis. Genes with Bonferroni- BMDMs). All experiments compared Tbc1d232/2 male mice with their corrected p values , 0.05 (corresponding to raw p , 1 3 1026) were age-matched (6–10-wk-old) wild-type littermates generated from hetero- considered statistically significant. The analysis was done separately for zygous 3 heterozygous matings. different time points. Pathway analysis of significant genes was