Spatiotemporal Inhibition of Innate Immunity Signaling by the Tbc1d23 RAB-GAP

This information is current as Lesly De Arras, Ivana V. Yang, Brad Lackford, David W. H. of September 28, 2021. Riches, Rytis Prekeris, Jonathan H. Freedman, David A. Schwartz and Scott Alper J Immunol 2012; 188:2905-2913; Prepublished online 6 February 2012;

doi: 10.4049/jimmunol.1102595 Downloaded from http://www.jimmunol.org/content/188/6/2905

Supplementary http://www.jimmunol.org/content/suppl/2012/02/06/jimmunol.110259 Material 5.DC1 http://www.jimmunol.org/ References This article cites 58 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/188/6/2905.full#ref-list-1

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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 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 28, 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 , 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 (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 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 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 performed Endotoxic shock was induced in mice using a high dose of LPS with no in GATHER (31). D-galactosamine sensitization (23, 24). In brief, mice were injected i.p. with 20 mg/kg body weight Escherichia coli 0111:B4 LPS (Sigma). Six ELISA and qPCR studies hours after injection, mice were euthanized, serum was collected via car- PAMP exposures for ELISA were performed in 96-well format using diac bleed, and serum cytokine levels were measured by ELISA (24). In 100,000 cells/well. ELISAs were performed on mouse serum (24) or cell the same mice, inflammatory cell infiltration was monitored by peritoneal culture supernatants. PAMP exposures for qPCR were performed in 24- lavage (25). To determine the composition of these cells, cytospin slides well format using 250,000 cells/well. RNA was purified using the QIA- were prepared, and differential cell counts were determined (26). GEN RNeasy Mini kit. qPCR was performed using an ABI 7900 Real Time thermocycler and the QuantiTect SYBR Green RT-PCR Assay Kit Generation and phenotyping of BMDMs (Invitrogen), according to the manufacturer’s instructions. Relative ex- BMDMs were generated using standard techniques (24). Briefly, marrow pression levels were normalized using primers specific for b-actin. Primer was harvested from the femurs and tibias, filtered, and plated in DMEM sequences used for qPCR are shown in Supplemental Table I. Actinomycin D was from Santa Cruz Biotech. Tunicamycin was from Sigma.

(Invitrogen) supplemented with 10% FBS (Invitrogen), penicillin and Downloaded from streptomycin (Fisher), and 20 ng/ml recombinant mouse M-CSF (R&D XBP1 mRNA splicing was also monitored by subjecting RNA to RT-PCR Systems). Six days later, the cells were washed, and adherent cells were and subsequently monitoring the amplified product using 3% agarose gel collected by trypsinization and used in further experiments. The extent of electrophoresis with primers and techniques as described (32). bone marrow stem cells differentiating into macrophages was similar be- tween Tbc1d232/2 mice and their wild-type siblings, as determined by F4/ Trafficking assays + 80 staining (wild-type BMDMs: 95.4 6 3.3% F4/80 , 40.8 6 7.1 mean Uptake of transferrin-Alexa Fluor 488 (Invitrogen) was analyzed, as de- 2/2 + fluorescence intensity; Tbc1d23 BMDMs: 94.9 6 1.9% F4/80 , 41.4 6 scribed (33, 34). Briefly, cells were incubated in the presence of labeled 3.4 mean fluorescence intensity, n =5,p = 0.95). PAMP exposures were transferrin for 40 min at 37˚C, washed twice, resuspended in PBS + 1% http://www.jimmunol.org/ performed for 6 h, except in the time-course experiments. PAMPs used for paraformaldehyde, and analyzed by flow cytometry. Uptake and cleavage BMDM and cell line exposures were from InvivoGen, except for E. coli of DQ-Green-BSA (Invitrogen) was analyzed, as described (34). In brief, O111:B4 LPS, which was from List Biological Laboratories. We con- cells were incubated in the presence of DQ-Green-BSA for 1 h at 37˚C, firmed that PAMP exposures did not alter cell viability using fluorescein washed, and fixed in the presence of PBS + 1% paraformaldehyde. diacetate, as described (6, 27). Phagocytosis of FITC-labeled E. coli particles was assayed using the Vybrant Phagocytosis Assay Kit (Invitrogen), according to the manu- Generation and phenotyping of macrophage lines facturer’s instructions. overexpressing Tbc1d23 Western blots The Tbc1d23 cDNA clone (no. 4194266) was obtained from MRC Gene- service. Tbc1d23 was cloned downstream of the CMV promoter by Western blots were performed on SDS-polyacrylamide gels using primary by guest on September 28, 2021 digesting the Tbc1d23 cDNA clone with EcoRI and NotI and ligating the antisera that recognize phospho-JNK Thr183/Tyr185, phospho-ERK cDNA fragment into pCDNA3.1 (Invitrogen) that had been digested with Thr202/Tyr204, or phospho-p38 Thr180/Try182 (Cell Signaling Technol- EcoRI and NotI. We also generated a Tbc1d23 overexpression clone that ogy), IkBa (Santa Cruz Biotech), and actin (Millipore) and visualized was tagged with the 10-aa Myc epitope at its N terminus by PCR amplifying using HRP-conjugated secondary Abs (GE Healthcare) and the ECL the Tbc1d23 cDNA with primers oSA571 and oSA572 (Supplemental Western blot detection kit (Pierce). Table I), digesting the amplified product with EcoRI and BamHI, and li- gating this fragment into pCMVtag3A (Stratagene) that had been digested Statistical analyses with EcoRI and BamHI. A control plasmid that overexpresses chloram- All data are from a minimum of three biological replicates; statistical phenicol acetyltransferase (CAT) (pCDNA3.1CAT; Invitrogen) was also analyses were performed in GraphPad Prism 5 using unpaired t tests. used. These plasmids were transfected into the RAW264.7 mouse mac- rophage cell line using the Amaxa nucleofector shuttle, according to the manufacturer’s instructions, and stable lines were generated by selection Results with 400 mg/ml G418 (Invitrogen). Cells were grown in DMEM supple- Tbc1d23 inhibits LPS-induced inflammation mented with 10% FBS, penicillin, and streptomycin. We generated a mouse Tbc1d23 knockout using an embryonic We used the PCR fusion technique (28) to generate cell lines over- 2/2 expressing myc-Tbc1d23–R50A. First, the 59 half of the Tbc1d23 gene stem cell gene trap (35); Tbc1d23 mice produced little or no was amplified by PCR using primers CMV-f and oSA582 (Supplemental Tbc1d23 mRNA (Materials and Methods). To monitor the effect Table I), and the 39 half of the Tbc1d23 gene was amplified using primers of LPS in these mice, the mice were injected i.p. with a high dose oSA581 and CMV-r (amplified from the myc-Tbc1d23 plasmid). These of LPS with no D-galactosamine, a model of endotoxic shock that PCR products were then mixed and reamplified with primers oSA595 and oSA596 to generate the full-length Tbc1d23 clone with the R50A muta- mimics some features of sepsis (23). Six hours after injection, tion. This PCR product was digested with EcoRI and SapI and ligated into mice were euthanized, and serum cytokine production and re- pCDNA3.1(+) (Invitrogen) that had been digested with EcoRI and SapI. cruitment of inflammatory cells into the peritoneum were moni- The EcoRI-ApaI fragment of this clone was then cloned into pCDNA3.1 tored. Tbc1d232/2 mice produced higher quantities of several 2 ( ) (Invitrogen) that had been digested with EcoRI and ApaI to generate proinflammatory cytokines in serum than did their wild-type sib- the mutant Tbc1d23-R50A construct downstream of the CMV promoter. 2/2 All clones were verified by DNA sequencing. lings (Fig. 1A–C). Tbc1d23 mice also recruited more inflam- matory cells to the peritoneum (Fig. 1D), although the com- Microarray analysis position of these cells was not altered by Tbc1d23. Total RNA was labeled with Cy3 using the QuickAmp labeling kit and To examine the effect of Tbc1d23 at the cellular level, we hybridized on mouse whole genome 4 3 44 arrays using Agilent protocols. generated BMDMs and found that Tbc1d232/2 BMDMs produced Arrays were scanned on an Agilent microarray scanner, and intensities were more IL-6 and TNF-a when stimulated with LPS than did extracted from array images using Agilent Feature Extraction Software. BMDMs from their wild-type siblings (Fig. 1E, 1F). In the ab- Array data have been deposited in the Gene Expression Omnibus database 2/2 (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE30840. sence of stimulation, Tbc1d23 BMDMs and wild-type Data were normalized and scaled using robust multiarray analysis (29) BMDMs produced little or no IL-6 or TNF-a (data not shown). The Journal of Immunology 2907

FIGURE 1. Tbc1d232/2 mice exhibit increased PAMP-induced cytokine production. Serum cytokine production (A–C; n = 10) and inflammatory cells in the peritoneum (D; n = 5) following i.p. LPS injection. Cytokine production from BMDMs exposed for 6 h to either the indicated concentrations of LPS (E and F; solid black lines = wild-type, dashed gray lines = Tbc1d232/2)or50mg/ml zymosan (H). (G) IL-6 RNA levels at the indicated times in BMDMs treated with 100 ng/ml LPS; IL-6 RNA levels are normalized so that 1 = wild-type BMDMs in the absence of stimulation. (n = 13, E–H)*p , 0.07, **p ,

0.05, ***p , 0.0001. w.t., Wild-type. Downloaded from

The effect of Tbc1d23 likely occurred at the level of signaling, ing Tbc1d23 did not accumulate cytokines (IL-6: 40 6 15 pg/ml because IL-6 RNA levels also increased in Tbc1d232/2 BMDMs control versus 0 pg/ml Tbc1d23 overexpresser; TNF-a: 597 6 following LPS challenge (Fig. 1G). 136 pg/ml control versus 139 6 11 pg/ml Tbc1d23 overexpresser, We also generated stable macrophage cell lines that overexpressed 200 ng/ml LPS, 6-h exposures), arguing against this possibility.

Tbc1d23. We cloned two variants of Tbc1d23 downstream of the http://www.jimmunol.org/ CMV promoter: wild-type full-length Tbc1d23 cDNA (CMV- Tbc1d23 inhibits the response to many TLR agonists Tbc1d23) and Tbc1d23 cDNA with a 10-aa myc epitope at its N In addition to inhibiting the production of cytokines induced by terminus (CMV–myc–Tbc1d23). Stable lines were generated in the TLR4 agonist LPS (14–16), Tbc1d23 overexpression inhibited the RAW264.7 mouse macrophage cell line. As negative controls, cytokine production induced by the TLR2/1 agonist PAM3CSK4 we generated stable lines overexpressing CAT (CMV-CAT), which (36) and the TLR3 agonists poly(I:C) and poly(A:U) (37, 38) (Fig. was not expected to alter innate immunity, and also used the 2C–E). Tbc1d23 overexpression also inhibited the response to RAW264.7 cell line as a control. Overexpression of Tbc1d23 zymosan (Fig. 2F), which signals through both TLR2/6 and greatly reduced LPS-induced production of IL-6 and TNF-a (Fig. dectin-1 (39). The response to depleted zymosan (zymosan treated 2A, 2B) compared with control cells, a phenotype opposite to that with hot alkali), which signals only through dectin-1 (40), was by guest on September 28, 2021 induced by Tbc1d23 deletion in BMDMs. Because Tbc1d23 en- also inhibited by Tbc1d23 (Fig. 2G). This suggests that Tbc1d23 codes a presumed trafficking regulator, it was possible that cytokines can inhibit multiple innate immunity signaling pathways and were produced but not secreted when Tbc1d23 was overexpressed. not only TLR pathways. However, the effect of Tbc1d23 was However, ELISAs on cell lysates showed that cells overexpress- not universal to all stimuli, because Tbc1d23 overexpression had

FIGURE 2. Tbc1d23 overexpression inhibits PAMP- induced cytokine production (A–H). Production of the indicated cytokines in response to the indicated PAMP challenges. Cells overexpressing Tbc1d23 (red), myc- Tbc1d23 (blue), or CAT (gray) and the RAW264.7 cell line (black) are depicted. Exposures were 6 h. TNF-a levels were typically ,10 pg/ml in the absence of stimulation. 2908 Tbc1d23 INHIBITS INNATE IMMUNITY SIGNALING a more modest effect on the response of the NF-kB activator PMA (41) (Fig. 2H). In confirmation of the general effect of Tbc1d23 on TLR signaling, we found that, in Tbc1d232/2 BMDMs, zymosan increased production of inflammatory cytokines (Fig. 1H). Tbc1d23 regulates maintenance, but not initiation, of inflammatory cytokine production We performed time-course experiments to monitor the production of inflammatory cytokines following LPS stimulation in cells that overexpressed Tbc1d23. In one experiment, we monitored cytokine production by taking small aliquots of media at each time point FIGURE 4. Tbc1d23 overexpression does not decrease TNF-a RNA to gauge total cytokine accumulation (Fig. 3A, 3B). In a second stability. Either RAW264.7 cells (solid black line) or cells overexpressing Tbc1d23 (dashed gray line) were stimulated with 50 ng/ml LPS for 2 h, experiment, we collected and replaced the medium at each time actinomycin D was subsequently added to inhibit further transcription, and cells were lysed in RLT buffer (QIAGEN) at the indicated times after stimulation. TNF-a mRNA levels were monitored by qPCR and normal- ized relative to the RAW264.7 cells at time 0.

point with fresh medium containing LPS to monitor differential cytokine production over time (Fig. 3C). TNF-a production starts Downloaded from soon after LPS exposure (16, 42, 43). In both experiments, TNF-a production in the first hour after LPS exposure was similar in cells that overexpress Tbc1d23 and control macrophages (Fig. 3A–C). However, at later times, significantly less TNF-a was produced and accumulated (Fig. 3A, 3C). This temporal effect was also

observed when cells that overexpressed myc-Tbc1d23 were http://www.jimmunol.org/ compared with control cells that overexpress CAT (Fig. 3D). This effect likely occurred at the level of cytokine RNA regulation, because a similar pattern was observed using qPCR to monitor TNF-a mRNA levels. TNF-a mRNA levels increased during the first hour after LPS exposure at similar rates in control macro- phages and macrophages overexpressing Tbc1d23 (Fig. 3E). However, TNF-a mRNA levels returned to baseline levels (16, 42, 43) more rapidly in macrophages that overexpressed Tbc1d23 than in control cells (Fig. 3E). IL-6 production starts at a later time by guest on September 28, 2021 than does TNF-a production following LPS exposure (16, 42, 43). Consistent with the differential temporal effects of Tbc1d23, little or no IL-6 protein or mRNA was induced by LPS when Tbc1d23 was overexpressed (Fig. 3F, 3G). In confirmation of the temporal effect of Tbc1d23, we found that TNF-a levels increased in Tbc1d232/2 BMDMS late, but not early, following LPS exposure (Fig. 3H). Cytokine levels are regulated both at the level of transcription and posttranscriptionally at the level of RNA stability. To determine whether Tbc1d23 affected TNF-a mRNA stability, we exposed cells that overexpress Tbc1d23 to LPS for 2 h and then inhibited transcription with actinomycin D and monitored TNF-a mRNA decay. We found that Tbc1d23 overexpression did not decrease

FIGURE 3. Tbc1d23 inhibits late, but not early, LPS-induced cytokine Table I. LPS-induced NF-kB p65 nuclear translocation production. Time courses showing cytokine protein or RNA production A C F G in response to 20 ng/ml LPS stimulation. ( - , , and ) Cells over- Time (min) Strain Nuclear (%) n expressing Tbc1d23 (dashed gray lines) or RAW264.7 cells (solid black lines). (D) Cells overexpressing myc-Tbc1d23 (dashed gray line) or CAT 0 RAW 3.3 154 (solid black line). (E) CAT-overexpressing cells (black bars) and Tbc1d23- 0 CMV-CAT 3.1 191 overexpressing cells (open bars). In (A) and (F), a small aliquot of su- 0 CMV-Tbc 4.3 94 0 CMV–myc–Tbc 5.2 136 pernatant was removed at each time point. (B) is an expansion of the first 30 RAW 94.8 211 A C few hours of ( ). In ( ), all media were removed at each time point, and 30 CMV-CAT 94.7 225 fresh media with LPS were added back to the wells. Thus, all panels depict 30 CMV-Tbc 97.2 146 total cytokine accumulation, with the exception of (C), which depicts 30 CMV–myc–Tbc 97.6 86 differential cytokine production over time. (H) Accumulation of cytokines 60 RAW 53.2 158 in LPS-stimulated Tbc1d232/2 (dashed gray line) or wild-type (solid black 60 CMV-CAT 54.9 248 line) BMDMs (n = 13). RNA in (E) and (G) are normalized so that cytokine 60 CMV-Tbc 79.7 123 RNA in control cells in the absence of stimulation = 1. 60 CMV–myc–Tbc 91.8 98 The Journal of Immunology 2909

The effect of Tbc1d23 is largely specific to innate immunity regulation Because Tbc1d23 is a presumed intracellular trafficking regulator, we examined macrophages that overexpressed Tbc1d23 (compared with control macrophages) and Tbc1d232/2 BMDMs (compared with wild-type BMDMs) for gross morphological defects using immunofluorescence microscopy and antiserum that labels dif- ferent organelles. We did not observe a gross difference in the structure of the cis-Golgi (GM130), trans-Golgi network (golgin- 97), nucleus (DAPI), or recycling endosomes (labeled transferrin uptake) in macrophages with decreased or increased Tbc1d23 expression (n . 50 cells, all conditions). We also monitored several subcellular trafficking events in these cells and found ei- ther modest or no defects in endocytosis (uptake of transferrin- Alexa Fluor 488), phagocytosis (uptake of FITC-E. coli particles), or protein trafficking to lysosomes (DQ-Green-BSA, which is cleaved and becomes fluorescent upon entry into the lysosome) in cells in which Tbc1d23 was overexpressed or mutated (Supple- mental Fig. 2). Although not comprehensive, these data suggested Downloaded from FIGURE 5. Early IkBa destruction and MAPK phosphorylation in- that intracellular trafficking processes were generally unaffected duced by LPS stimulation is not inhibited by Tbc1d23 overexpression. by Tbc1d23. Cells that overexpress either CAT (left side)ormyc-Tbc1d23(right side) To determine what effect Tbc1d23 had on innate immunity were stimulated with LPS for the indicated times (in minutes), the cells specifically and other cellular processes more generally, we per- were lysed, and protein was prepared and subjected to Western blot formed DNA microarray analysis to investigate global gene ex- analysis. Blots were probed with anti-IkBa antiserum (A), anti–phospho- http://www.jimmunol.org/ p38 (C), anti–phospho-ERK (D), and anti–phospho-JNK (E). (B and F) pression changes induced by overexpression of Tbc1d23. We The blots were also probed with anti-actin antiserum to control for gel exposed macrophages overexpressing myc-Tbc1d23 and control loading. macrophages overexpressing CAT to LPS for 0, 1, 3, or 5 h and then collected RNA. We analyzed induction of LPS-induced genes at each time point and determined the effect of Tbc1d23 overex- TNF-a mRNA stability (Fig. 4), suggesting that Tbc1d23 inhibi- pression. Tbc1d23 overexpression inhibited expression of many ted cytokine production at the transcriptional level. cytokines and chemokines, including IL-6, TNF-a, IL-1a, IL-1b, We also monitored several early signaling events induced by RANTES, IP-10, and CXCL11. This is consistent with a general LPS, including activation of NF-kB and the MAPKs p38, JNK, effect on innate immunity and consistent with our qPCR and by guest on September 28, 2021 and ERK. We monitored LPS-induced nuclear translocation of ELISA data (full list of genes with altered LPS induction is pro- NF-kB p65 by immunofluorescence microscopy (Table I, Sup- vided in Supplemental Table II). plemental Fig. 1), destruction of the NF-kB inhibitory protein To determine which signaling pathways were significantly dif- IkBa by Western blot (Fig. 5A, 5B), and activation of the MAPKs ferent when Tbc1d23 was overexpressed compared with control by Western blot using Abs that recognize phosphorylated forms macrophages, we used the GATHER utility (31) (Table II). In the of the (Fig. 5C–F). Despite the strong inhibitory effect of absence of LPS stimulation, Tbc1d23 overexpression had a sig- Tbc1d23 on cytokine production, these early LPS-induced sig- nificant effect on only a few pathways, most notably neuroactive naling events were not inhibited by Tbc1d23 overexpression. ligand–receptor interactions (Table II). At 1 h post-LPS exposure,

Table II. Pathways altered by Tbc1d23 overexpression

Pathway p Value No LPS (basal comparison) path:mmu04080: neuroactive ligand–receptor interaction 1.57E205 path:mmu04910: insulin-signaling pathway 0.0021 path:mmu00193: ATP synthesis 0.0023 path:mmu04070: phosphatidylinositol-signaling system 0.0032 1 h post-LPS path:mmu04620: TLR-signaling pathway 8.61E205 3 h post-LPS path:mmu04010: MAPK-signaling pathway 5.81E204 path:mmu04620: TLR-signaling pathway 0.0024 path:mmu04060: cytokine–cytokine receptor interaction 0.0024 path:mmu04080: neuroactive ligand–receptor interaction 0.0042 path:mmu00910: nitrogen metabolism 0.01 5 h post-LPS path:mmu04620: TLR-signaling pathway 1.57E205 path:mmu04060: cytokine–cytokine receptor interaction 5.81E204 path:mmu04080: neuroactive ligand–receptor interaction 7.46E204 path:mmu04010: MAPK-signaling pathway 0.0018 path:mmu00240: pyrimidine metabolism 0.0028 2910 Tbc1d23 INHIBITS INNATE IMMUNITY SIGNALING only the TLR-signaling pathway was differentially regulated. Five modulate inflammatory gene expression. In particular, we moni- pathways were significantly differentially regulated at the 3- and tored the XBP1 transcription factor, which is activated by both TLR 5-h post-LPS time points. In each case, three of these five path- signaling and endoplasmic reticulum (ER) stress (32, 44–46) and ways were innate immunity signaling pathways: TLR signaling, which, like Tbc1d23, affects late, but not early, inflammatory gene MAPK signaling, and cytokine-cytokine receptor signaling. These expression (32). XBP1 is transcribed as an inactive precursor data indicate that Tbc1d23 overexpression strongly affects innate mRNA (XBP-1 U = unspliced). TLR signaling or ER stress immunity signaling but has a limited effect on other pathways, induces IRE1 to splice 26 bp out of the primary XBP1 transcript to demonstrating the specificity of Tbc1d23. produce spliced, active XBP1 mRNA (XBP-1 S = spliced) (32, 47, 48). We used qPCR to monitor production of active and inactive Tbc1d23 inhibits XBP1 activation XBP1 mRNA using primers that could distinguish between the To determine how Tbc1d23 regulates TLR signaling, we used two mRNA splice forms and found that Tbc1d23 overexpression qPCR to survey expression of transcription factors known to inhibited LPS-induced activation of XBP1 (Fig. 6A–D). XBP-1(S) Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. Tbc1d23 inhibits LPS-induced, but not ER stress-induced, activation of XBP1. Relative amounts of active spliced XBP1 mRNA (A, C) and the inactive unspliced precursor mRNA (B, D). RNA levels were determined by qPCR and normalized so that XBP1-S and XBP1-U were 1 in the absence of treatment in control cells. (A–L) Cells overexpressing Tbc1d23 (dashed gray lines) or the RAW264.7 cell line (solid black lines). (M and N) Photographs of agarose gels in which RT-PCR was used to analyze XBP1 spliced and unspliced mRNA using the indicated strains and treatments (no treatment, 100 ng/ ml LPS, or 0.5 mg/ml tunicamycin). Exposures were 5 h (except for the time courses). (O) BMDMs from Tbc1d232/2 mice and their wild-type siblings [100 ng/ml LPS, n =7,p = 0.0015 XBP1 (S), p = 0.36 XBP1 (U)]. (P) RAW264.7 cells (increasing shades of solid gray solid lines, 0, 0.25, or 0.5 mg/ml tunicamycin) and cells overexpressing Tbc1d23 (increasing shades of dashed gray lines, 0, 0.25, or 0.5 mg/ml tunicamycin). Tunicamycin increases LPS- induced cytokine production in wild-type cells but not when Tbc1d23 is overexpressed. The Journal of Immunology 2911 levels were lower in the absence of LPS stimulation when gene discovery (6). Using a combination of knockout and over- Tbc1d23 was overexpressed and remained lower over a range of expression approaches, we find that Tbc1d23 is a general inhibitor LPS doses (Fig. 6E, 6F) and over a range of times following LPS of innate immunity signaling. Our previous RNA interference data exposure (Fig. 6I, 6J). In contrast, the ER stress inducer tunica- with Tbc1d23 in macrophage cell lines (6) suggested that mycin could still activate XBP1 splicing when Tbc123 was Tbc1d23 stimulated cytokine production. In other cases investi- overexpressed (Fig. 6G, 6H, 6K, 6L). We confirmed that Tbc1d23 gating our candidate innate immune genes, we found that mouse inhibited LPS-mediated, but not ER stress-mediated, activation of knockouts confirmed our small interfering RNA data (24). It is XBP1 by subjecting RNA from exposed cells to RT-PCR and possible that Tbc1d23 small interfering RNA could be inhibiting subsequent agarose gel electrophoresis (Fig. 6M, 6N). Consistent one of the .50 other related Tbc family members. Consistent with with these data, Tbc1d232/2 BMDMs exhibited increased levels this possibility, we identified several other Tbc family members of XBP1(S) (Fig. 6O). To determine whether the only effect of that regulate innate immunity (data not shown). The strong con- Tbc1d23 on TLR signaling was the inhibition of XBP1 activation, cordance between the Tbc1d23 knockout and overexpression data we exposed cells overexpressing Tbc1d23 to both LPS and tuni- definitively shows that Tbc1d23 is a general innate immunity camycin. Although tunicamycin rescued the XBP1 splicing defect signaling inhibitor. caused by Tbc1d23 overexpression (data not shown), it did not Tbc1d23 inhibits the response to TLRs acting at the cell surface rescue the Tbc1d23-mediated inhibition of cytokine production (TLR2) and in the endosome (TLR3) and that signal through (Fig. 6P). MyD88 (TLR2) or Trif (TLR3) (11, 13, 17). Thus, Tbc1d23 likely is acting downstream of these signaling adaptors to inhibit TLR The RAB-GAP activity of Tbc1d23 is required to inhibit innate signaling. Our microarray analysis is consistent with this, because Downloaded from immunity Tbc1d23 overexpression specifically affected TLR and other in- Tbc1d23 has two conserved domains: a Tbc RAB-GAP domain nate immunity-signaling pathways. We also found that Tbc1d23 (18–20) and a rhodanese superfamily domain (21, 22). To deter- inhibited mRNA splicing of the XBP1 transcription factor. This mine whether the RAB-GAP domain is required for the innate effect was specific, because Tbc1d23 affected the TLR-mediated, immune regulatory function of Tbc1d23, we expressed a mutant but not ER stress-mediated, activation of XBP1. XBP1 splicing

Tbc1d23 variant in macrophages to determine whether that variant is regulated downstream of Traf6 but upstream of Nemo (32); http://www.jimmunol.org/ altered the ability of Tbc1d23 overexpression to inhibit the LPS therefore, it acts in parallel to NF-kB and MAPK activation in response. To knock out the RAB-GAP domain, we mutated the TLR-signaling pathways. We found that using tunicamycin to highly conserved R50 (49) to A; the crystal structure and func- bypass the defect in XBP1 splicing caused by Tbc1d23 over- tional studies with other RAB-GAPs indicate that this arginine is expression did not restore cytokine production in the presence of in the RAB-GAP catalytic site (50). Moreover, mutation of the LPS. This suggests that Tbc1d23 is still affecting another aspect of analogous R→A in the related family member Tbc1d20 abolished TLR signaling and likely acts upstream of the fork between XBP1 RAB-GAP activity on the cognate RAB for Tbc1d20, Rab1 (51). activation and NF-kB signaling. Consistent with this more general Thus, our myc–Tbc1d23–R50A variant very likely will have lost effect of Tbc1d23 than XBP1 is the observation that Tbc1d23 also its RAB-GAP activity. Overexpression of myc-Tbc1d23–R50A affects the response to TLR3 agonists [XBP1 does not (32)], and by guest on September 28, 2021 failed to inhibit LPS-induced cytokine production (Fig. 7). myc- the stronger phenotype induced by Tbc1d23 overexpression than Tbc1d23–R50A was expressed at levels higher than wild-type XBP1 mutation. Thus, we conclude that Tbc1d23 inhibits TLR myc-Tbc1d23, and its subcellular localization (monitored with signaling upstream of the fork that leads to Ire1-XBP1 activation anti-myc antiserum) was the same as the wild-type (data not [upstream of Nemo (32)]. shown). In contrast to the substantial focus on the initiation of innate immunity signaling, the maintenance of innate immunity has Discussion been much less studied. Tbc1d23, like XBP1, regulates main- Innate immunity is a conserved defense mechanism present in tenance, but not initiation, of inflammatory cytokine production organisms as diverse as humans and nematodes. We previously induced by LPS; further understanding of this interaction should demonstrated that the C. elegans Tbc1d23 ortholog regulates shed light on how XBP1 is regulated and inflammation is main- nematode innate immunity (6). We now show that the mouse gene tained. also regulates innate immunity signaling, demonstrating the val- How does Tbc1d23 regulate TLR signaling? Tbc1d23 contains idity of a comparative genomics approach for innate immunity two conserved domains: a Tbc RAB-GAP domain and a rhodanese superfamily domain. Our data demonstrate that the Tbc RAB-GAP domain is required for Tbc1d23’s innate immune regulatory function. We speculate that Tbc1d23 could control the movement and activity of one of the many negative regulators of TLR sig- naling induced by LPS treatment (12, 52, 53). It is rather re- markable that a RAB-GAP has such a specific effect on innate immunity. Three RABs are known to affect TLR signaling (54– 56), although none of them is likely the cognate RAB for Tbc1d23. Spatial regulators of innate immunity have been im- plicated in several diseases, including Griscelli syndrome (57), Crohn’s disease (58), HSV encephalitis (59), and systemic lupus erythematosus (60). Thus, the identification of genes that transport FIGURE 7. The Tbc1d23 RAB-GAP domain is necessary for Tbc1d23 to inhibit LPS-induced cytokine production. Stable lines were generated important immune mediators offers another level of regulation that overexpress either wild-type myc-Tbc1d23 or myc-Tbc1d23–R50A. that could be used to identify critical immune modulators and These cell lines were exposed to LPS, and cytokine production was human disease genes, and our current and future studies with monitored. myc-Tbc1d23, but not myc-Tbc1d23–R50A, inhibited LPS- Tbc1d23 should help us to better understand this additional level induced cytokine production. of immune regulation. 2912 Tbc1d23 INHIBITS INNATE IMMUNITY SIGNALING

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