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SLC19A1 Transports Immunoreactive Cyclic Dinucleotides Rutger D

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SLC19A1 Transports Immunoreactive Cyclic Dinucleotides Rutger D LETTER https://doi.org/10.1038/s41586-019-1553-0 SLC19A1 transports immunoreactive cyclic dinucleotides Rutger D. Luteijn1,7, Shivam A. Zaver2,7, Benjamin G. Gowen3,4, Stacia K. Wyman3, Nick E. Garelis1, Liberty Onia1, Sarah M. McWhirter5, George E. Katibah5, Jacob E. Corn3,4,6, Joshua J. Woodward2 & David H. Raulet1* The accumulation of DNA in the cytosol serves as a key Data Figs. 1b, 2, Supplementary Tables 1, 2). The two screens yielded immunostimulatory signal associated with infections, cancer and many common hits but there were differences, such as numerous hits genomic damage1,2. Cytosolic DNA triggers immune responses in the 2′3′-cGAMP screen—including STAT2, IRF9, IFNAR1 and by activating the cyclic GMP–AMP synthase (cGAS)–stimulator IFNAR2 (Supplementary Table 2). Hence, the 2′3′-RR CDA screen may of interferon genes (STING) pathway3. The binding of DNA to have mostly been dependent on intrinsic STING signalling, whereas cGAS activates its enzymatic activity, leading to the synthesis of a second messenger, cyclic guanosine monophosphate–adenosine monophosphate (2′3′-cGAMP)4–7. This cyclic dinucleotide a 5× ISRE mmIFN-β tdTomato (CDN) activates STING8, which in turn activates the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor b 2′3′-RR CDA 2′3′-cGAMP IFN-β Control, no stimulation 100 100 100 Control, stimulation κ-light-chain-enhancer of activated B cells (NF-κB), promoting 80 80 80 STING KD, stimulation the transcription of genes encoding type I interferons and other 60 60 60 40 40 40 cytokines and mediators that stimulate a broader immune Per cent 20 20 9 of maximum 20 response. Exogenous 2′3′-cGAMP produced by malignant cells 0 0 0 10–3 0103 104 105 10–3 0103 104 105 10–3 0103 104 105 and other CDNs, including those produced by bacteria10–12 and 13,14 tdTomato expression (AU) synthetic CDNs used in cancer immunotherapy , must traverse c the cell membrane to activate STING in target cells. How these gRNA + dCas9-KRAB + Stimulated with charged CDNs pass through the lipid bilayer is unknown. Here THP-1 cells CDNs Top 25% reporter we used a genome-wide CRISPR-interference screen to identify expressing cells the reduced folate carrier SLC19A1, a folate–organic phosphate Sort Deep sequencing for antiporter, as the major transporter of CDNs. Depleting SLC19A1 gRNA enrichment in human cells inhibits CDN uptake and functional responses, and Bottom 25% reporter overexpressing SLC19A1 increases both uptake and functional expressing cells responses. In human cell lines and primary cells ex vivo, CDN de2′3′-RR CDA 2′3′-cGAMP –14 –17 10 IRF3 10 IRF3 uptake is inhibited by folates as well as two medications approved SLC19A1 CNOT4 SUPT5H CAD CAD CNOT1 for treatment of inflammatory diseases, sulfasalazine and the e e MED9 PRPF40A –8 antifolate methotrexate. The identification of SLC19A1 as the major 10 MED19 10–9 STAT2 POLR2H SMARCC1 transporter of CDNs into cells has implications for the immuno- ACBD3 SLC19A1 RRA scor CNOT3 RRA scor CNOT3 13 MEN1 RBM4 therapeutic treatment of cancer , host responsiveness to 10–2 CDN-producing pathogenic microorganisms11 and—potentially— 10–1 for some inflammatory diseases. 0 5,000 10,000 15,000 0 5,000 10,000 15,000 Genes Genes To systematically identify genes involved in intracellular transport of CDNs, we performed a genome-wide CRISPR interference (CRISPRi) Fig. 1 | Genome-wide CRISPRi screen for host factors necessary forward genetic screen in the monocytic THP-1 cell line. To visualize for stimulation by CDNs. a, Schematic overview of the tdTomato reporter construct. tdTomato expression is driven by interferon- STING activation, THP-1 cells were transduced with a CDN-inducible stimulatory response elements (ISRE) followed by a mouse minimal reporter construct (Fig. 1a, b). Consistent with previous results, the interferon-β (mmIFN-β) promoter. b, Control THP-1 cells and STING- synthetic CDN 2′3′ RR-S2 cyclic di-AMP (2′3′-RR CDA, Extended depleted (STING KD) THP-1 cells were incubated with 2′3′-RR CDA 13 Data Fig. 1a) induced a more potent response than 2′3′-cGAMP , and (1.67 μg ml−1), 2′3′-cGAMP (10 μg ml−1) or human IFN-β (100 ng ml−1). the response to both CDNs was severalfold higher than the response to After 20 h, tdTomato reporter expression was analysed by flow cytometry. human interferon-β (IFN-β). The response to CDNs was completely Data are representative of three independent experiments with similar dependent on STING expression (Fig. 1b), implying that the reporter results. c, Schematic overview of the genome-wide CRISPRi screen. primarily reported cell-intrinsic STING activity. For the screen, THP-1 A genome-wide library of CRISPRi gRNA-expressing THP-1 cells was stimulated with CDNs. Twenty hours after stimulation, cells were sorted cells expressing a catalytically inactive Cas9 (dCas9) fusion with blue low high fluorescent protein (BFP) and the transcriptional repression domain into a tdTomato group (lowest 25% of cells) and a tdTomato group (highest 25% of cells). DNA from the sorted cells was deep-sequenced to KRAB (dCas9–BFP–KRAB), and transduced with a genome-wide reveal gRNA enrichment in the two groups. d, e, Distribution of the robust guide-RNA (gRNA) library, were stimulated separately with 2′3′-RR rank aggregation (RRA) score in the comparison of hits enriched in the CDA or 2′3′-cGAMP, and the highest and lowest quartiles of reporter- tdTomatolow versus tdTomatohigh groups of THP-1 cells stimulated with expressing cells were sorted by flow cytometry before deep-sequencing 2′3′-RR CDA (d) or 2′3′-cGAMP (e). Each panel represents combined to identify gRNAs enriched in each population (Fig. 1c, d, Extended results of two independent screens. AU, arbitrary units. 1Department of Molecular and Cell Biology, and Cancer Research Laboratory, Division of Immunology and Pathogenesis, University of California, Berkeley, CA, USA. 2Department of Microbiology, University of Washington, Seattle, WA, USA. 3Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. 4Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA. 5Aduro Biotech, Berkeley, CA, USA. 6Present address: Institute of Molecular Health Sciences, Department of Biology, ETH Zurich, Zurich, Switzerland. 7These authors contributed equally: Rutger D. Luteijn, Shivam A. Zaver. *e-mail: [email protected] 434 | NATURE | VOL 573 | 19 SEPTEMBER 2019 LETTER RESEARCH a 2′3′-RR CDA2′3′-cGAMP b 2′3′-RR CDA 2′3′-cGAMP 3′3′-CDA 250 250 gRNA: 100 100 ) 200 200 100 3 + 80 80 10 150 150 80 b × control ( 60 **** c d 100 100 60 **** 60 a 40 40 50 76.7% 50 54.3% **** **** 40 0 0 tdTomato 20 **** **** **** (% of control) **** 20 20 10–3 0103 104 105 10–3 0103 104 105 0 0 0 250 250 1 200 200 IRF3-1IRF3-2 IRF3-1IRF3-2 IRF3-1IRF3-2 150 150 Control Control Control IRF3-1 SLC19A1-1SLC19A1-2 SLC19A1-1SLC19A1-2 SLC19A1-SLC19A1-2 FSC-A 100 100 50 12.0% 50 3.4% d 2′3′-RR CDA 2′3′-cGAMP IFN-β 0 0 P = 0.0075 –3 3 4 5 **** **** NS NS 10 010 10 10 10–3 0103 104 105 160 160 NS 140 140 120 250 250 + 120 120 100 200 200 100 100 80 150 150 SLC19A1-1 80 80 **** 60 60 60 100 100 40 tdTomato 40 40 50 50 (% of control) 5.4% 22.1% 20 20 20 0 0 –3 3 4 5 –3 3 4 5 0 0 0 10 010 10 10 10 010 10 10 SLC19A1 – + – + – + – + – + – + tdTomato expression (AU) Control SLC19A1-1 Control SLC19A1-1 Control SLC19A1-1 gRNA gRNA gRNA gRNA gRNA gRNA c 1.0 f **** 2′3′-RR CDA 2′3′-cGAMP 100 Vector 100 **** ion) 0.8 Vector SLC19A1 SLC19A1 ess ) 80 80 **** pr 0.6 **** (% P = ex + . **** 60 60 P = 0.0001 el 0.4 (r P = 0.0004 1 **** **** 40 P = 0.0067 40 0.2 **** **** **** 0.0018 dTomato IFNB t 20 20 0.0 2′3′-RR CDA: –+–+–+–+–+–+ 0 0 gRNA no.: Ctrl 12112 T W T W IRF3 SLC19A1 STING C1R C1R THP1 K562 293 MDA RA THP1 K562 293 MDA RA e g +STING +STING 150 150 2′3′-RR CDA 2′3′-cGAMP IFN-β 100 80 125 125 ) 80 IFN-β (% 60 100 100 P = P = + 2′3′-cGAMP 60 1.3 × 10–11 1.2 × 10–6 75 75 (% of DMSO) (% of DMSO) 2′3′-cGAMP 40 + + 40 50 50 dTomato 20 t 20 25 2′3′-RR CDA 25 dTomato dTomato t 0 0 0 t 0 2′3′-RR CDA l –/– –/– 1416 64 256 1,024 1416 64 256 1,024 o 1 1 Methotrexate (μM) 5-me-THF (μM) Contr Control SLC19A SLC19A Fig. 2 | SLC19A1 is required for CDN-induced reporter expression. and SLC19A1−/− cells (n = 9 independent clonal lines) were exposed to a, dCas9–KRAB-expressing THP-1 cells transduced with non-targeting 2′3′-RR CDA (2.22 μg ml−1) or 2′3′-cGAMP (10 μg ml−1) and reporter gRNA (control), IRF3-1 gRNA or SLC19A1-1 gRNA were exposed to tdTomato expression was analysed as in a. Data are mean ± s.e.m. 2′3′-RR CDA (1.67 μg ml−1) or 2′3′-cGAMP (15 μg ml−1). Twenty f, Various cell lines expressing a control vector or an SLC19A1 expression hours later, tdTomato expression was analysed by flow cytometry. vector were stimulated and analysed as in b. g, THP-1 cells were incubated Representative dot plots of three independent experiments are shown. with increasing concentrations of the competitive inhibitors methotrexate, b, THP-1 cells expressing the indicated CRISPRi gRNAs or non-targeting 5-me-THF or DMSO as vehicle control, before stimulation with 2′3′-RR gRNA (control), were stimulated with 2′3′-RR CDA (1.67 μg ml−1), 2′3′- CDA (1.25 μg ml−1), 2′3′-cGAMP (15 μg ml−1) or IFN-β (100 ng ml−1).
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