S1PR1-Mediated IFNAR1 Degradation Modulates Plasmacytoid Dendritic Cell Interferon-Α Autoamplification

S1PR1-mediated IFNAR1 degradation modulates plasmacytoid dendritic cell interferon-α autoamplification

John R. Teijaroa,b,1,2, Sean Studerb,1, Nora Leafb, William B. Kiossesc, Nhan Nguyenb, Kosuke Matsukid, Hideo Negishid, Tadatsugu Taniguchid, Michael B. A. Oldstonea,2, and Hugh Rosenb,2

aDepartment of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037; bDepartment of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037; cCore Microscopy Facility, The Scripps Research Institute, La Jolla, CA 92037; and dDepartment of Molecular Immunology, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan

Contributed by Michael B. A. Oldstone, December 23, 2015 (sent for review November 16, 2015; reviewed by Arturo Casadevall and Herbert W. Virgin) Blunting immunopathology without abolishing host defense is the curb resulting immune pathology during both infectious and foundation for safe and effective modulation of infectious and autoimmune disease states. autoimmune diseases. Sphingosine 1-phosphate receptor 1 (S1PR1) The goal of the current study was to generate a detailed mech- agonists are effective in treating infectious and multiple autoimmune anistic understanding of how S1PR1 selectively suppresses type I pathologies; however, mechanisms underlying their clinical efficacy IFN and cytokine amplification. Within this study, we demonstrate are yet to be fully elucidated. Here, we uncover an unexpected that S1PR1 signaling limits the IFN-α autoamplification loop in mechanism of convergence between S1PR1 and interferon alpha pDCs. S1PR1 agonist suppression is pertussis toxin (PT)-resistant, receptor 1 (IFNAR1) signaling pathways. Activation of S1PR1 signaling but it is inhibited by an S1PR1 C-terminal–derived transactivating by pharmacological tools or endogenous ligand sphingosine-1 phos- transcriptional activator (Tat)-fusion peptide that blocks receptor phate (S1P) inhibits type 1 IFN responses that exacerbate numerous internalization. S1PR1 agonist treatment accelerates turnover of pathogenic conditions. Mechanistically, S1PR1 selectively suppresses interferon alpha receptor 1 (IFNAR1), suppresses signal transducer the type I IFN autoamplification loop in plasmacytoid dendritic cells and activator of transcription 1 (STAT1) phosphorylation, and down- (pDCs), a specialized DC subset, for robust type I IFN release. S1PR1 modulates total STAT1 levels, thereby inactivating the autoamplifi- agonist suppression is pertussis toxin-resistant, but inhibited by an cation loop. Inhibition of S1P-S1PR1 signaling in vivo using the S1PR1 C-terminal–derived transactivating transcriptional activator selective antagonist Ex26 significantly elevates IFN-α production in (Tat)-fusion peptide that blocks receptor internalization. S1PR1 agonist response to CpG-A. Thus, multiple lines of evidence demonstrate treatment accelerates turnover of IFNAR1, suppresses signal transducer that S1PR1 signaling sets the sensitivity of pDC amplification of IFN and activator of transcription 1 (STAT1) phosphorylation, and down- responses, thereby blunting pathogenic immune responses. modulates total STAT1 levels, thereby inactivating the autoamplification loop. Inhibition of S1P-S1PR1 signaling in vivo using the selective Results antagonist Ex26 significantly elevates IFN-α production in response to To understand how S1PR1 signaling regulates IFN-α and cyto- CpG-A. Thus, multiple lines of evidence demonstrate that S1PR1 signal- kine amplification, we assessed the pulmonary cell subsets that ing sets the sensitivity of pDC amplification of IFN responses, thereby produce IFN-α and cytokines/chemokines following influenza blunting pathogenic immune responses. These data illustrate a lipid virus challenge. Although many cell types produce IFN-α G-protein coupled receptor (GPCR)-IFNAR1 regulatory loop that bal- following virus infection, two major pulmonary cell populations ances effective and detrimental immune responses and elevated endogenous S1PR1 signaling. This mechanism will likely be advantageous Significance in individuals subject to a range of inflammatory conditions. The sphingosine 1-phosphate receptor (S1PR1) is known to act by sphingosine 1-phosphate | S1PR1 | plasmacytoid dendritic cell | multiple mechanisms: limiting lymphocyte egress from secondary interferon-α | IFNAR1 lymphoid organs, suppressing proinflammatory endothelial cell function, and acting directly on neurons and astrocytes. Here, we lasmacytoid dendritic cells (pDCs) are a rare innate immune report that sphingosine 1-phosphate (S1P)-S1PR1 signaling in Pcell population in mice known for their ability to produce plasmacytoid dendritic cells (pDCs) directly inhibits IFN-α auto- large quantities of type 1 IFN (IFN-I) following stimulation with amplification by induced degradation of the interferon alpha re- viral or cellular nucleic acids. Moreover, IFN-α signaling promotes ceptor 1 (IFNAR1) receptor and suppression of signal transducer autoimmune (1), viral (2–5), and bacterial disease pathogenesis and activator of transcription 1 (STAT1) signaling. An endosomal (6). Suppression of IFN-α signaling has demonstrated efficacy in regulatory interaction of a lipid G-protein coupled receptor (GPCR) multiple autoimmune mouse models (7–9) and during influenza and IFNAR1 balances effective and detrimental components of viral infection (4, 10); however, the mechanism by which sphin- immune responses and provides a previously unidentified path- gosine 1-phosphate receptor 1 (S1PR1) signaling prevents IFN-α way that contributes to significant and unexpected efficacy in clinical trials in multiple sclerosis, ulcerative colitis, psoriasis, and amplification during these disease states is currently unknown. likely other diseases with aberrant IFN-α signatures. Recently, we found direct evidence that IFN-I induction and

the concomitant cytokine storm were chemically tractable using Author contributions: J.R.T., S.S., T.T., M.B.A.O., and H.R. designed research; J.R.T., S.S., sphingosine 1-phosphate receptor 1 (S1PR1) selective agonists. N.L., W.B.K., N.N., K.M., and H.N. performed research; J.R.T., S.S., T.T., M.B.A.O., and H.R. S1PR1 agonist therapy suppressed innate immune cell recruit- analyzed data; and J.R.T., S.S., M.B.A.O., and H.R. wrote the paper. ment and cytokine-chemokine production and improved survival Reviewers: A.C., Johns Hopkins Bloomberg School of Public Health; and H.W.V., Washington University. without postponing viral clearance, indicating that cytokine The authors declare no conflict of interest. storm was causative of disease pathogenesis and that S1P agonist 1J.R.T. and S.S. contributed equally to this work. therapy could suppress detrimental innate immune responses 2To whom correspondence may be addressed. Email: [email protected], mbaobo@ without hindering virus control (10, 11). The identification that scripps.edu, or [email protected].

S1PR1 agonists suppress detrimental innate immune responses This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. INFLAMMATION IMMUNOLOGY AND indicates that S1PR1 probes may serve as viable drug leads to 1073/pnas.1525356113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1525356113 PNAS | February 2, 2016 | vol. 113 | no. 5 | 1351–1356 Downloaded by guest on October 2, 2021 make significant quantities of IFN-α in vivo following respiratory through TLR7 signaling, we asked if CYM-5442 could also virus infection, alveolar macrophages and pDCs (11); thus, we modulate TLR9 signaling in pDCs. Two classes of immunosti- initially focused on these populations. We depleted pDCs from mulatory oligonucleotides were demonstrated to activate pDCs mice before influenza virus challenge. Depletion of pDCs before differentially through TLR9 stimulation (14, 15). Treatment of influenza virus challenge significantly reduced IFN-α levels fol- pDC with CpG-A or influenza virus resulted in elevated IFN-α lowing influenza virus infection, as well as CCL2, CCL5, and IL-6 secretion similar to previous reports, and treatment with CYM- levels (Fig. S1). We asked whether treatment of fluorescence- 5442 inhibited IFN-α induction upon both CpG-A and influenza activated cell sorter (FACS)–purified pDCs (>90%) from the virus stimulation (Fig. 2A). Surprisingly, treatment with CYM- spleen or lung with the selective S1PR1 agonist CYM-5442 5442 had no effect following CpG-B stimulation (Fig. 2A), sug- (Table 1) could inhibit the production of IFN-α following stim- gesting that S1PR1 agonist targets suppression of TLR9 signal- ulation with influenza virus in vitro. Infection of pDCs with in- ing in the early endosome. Further, CYM-5442 treatment of fluenza virus followed by CYM-5442 treatment resulted in the pDCs isolated from the peripheral blood of human donors dis- α A μ inhibition of IFN- production (Fig. 1 )atanIC50 of 1.4 M, as played similar suppressive capacity following CpG-A stimulation well as an IC50 of 500 nM using the highly potent S1PR1 agonist and no effect following CpG-B stimulation (Fig. 2B). Because RP-001 (11), demonstrating direct inhibition of IFN-α pro- CpG-A and CpG-B are known to localize to different induction from pDCs by S1PR1 agonists. Interestingly, the IC50 of tracellular compartments (14), we asked whether S1PR1 agonist- S1PR1 agonist CYM-5442 required to inhibit IFN-α amplifica- mediated suppression was specific to endosome signaling in tion exceeds the IC50 required to activate Gi/Go signaling, sug- pDCs. To alter CpG-B trafficking, the cationic lipid N-[1-(2,3- – gesting a non Gi/Go-mediated mechanism for the suppression. Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate The expression of S1PR1 has not been reported in pDCs; thus, (DOTAP) was used to encapsulate CpG-B; this approach results we assessed whether S1PR1 could be detected on pDCs purified in redistribution of CpG-B to early endosomes, resulting in an from lung and spleen. Using the S1PR1-EGFP mice described increase in IFN-α production to levels similar to CpG-A stimu- previously (11), we determined that FACS-purified pDCs int + + + lation (14). Redistributing CpG-B to the endosome resulted in (CD11c , B220 , PDCA-1 , and Siglec H ) expressed signifi- both IFN-α secretion (Fig. 2C) and CYM-5442–induced sup- cant levels of S1PR1 as determined by detection of S1PR1- pression of IFN-α production as observed following CpG-A EGFP by flow cytometry and immunoblotting (Fig. S2). These stimulation in both murine (Fig. 2C) and human (Fig. 2D)pDCs. data demonstrate that CYM-5442 suppression of cytokine The S1PR1 is a heptahelical receptor that interacts with aux- amplification during influenza virus challenge correlates with α iliary proteins through the third intracellular loop (Gi/Go) or the S1PR1 expression in pDCs. We next asked whether pDC IFN- C-terminal tail. Canonical signaling through the third intracel- production requires S1PR1 agonism or if it can be replicated lular loop involves interaction with the Gi/Go protein, whereas with a neutral antagonist of S1PR1 signaling (12). To generate signaling through the C-terminal tail involves transient interac- sufficient quantities of functional pDCs, we used i.v. hydrody- tions with ubiquitin ligase, GRK2, and β-Arrestin (16). To de- namic injection of a plasmid encoding the human Flt3 ligand termine if Gi/Go signaling is involved in the suppression of gene (hFlt3L) into a mouse. This method allowed for the gen- IFN-α, PT was used to prevent S1PR1 signaling through Gi/Go. eration of large numbers of quiescent pDCs in vivo, which Treatment of pDCs with PT alone following influenza virus (Fig. express pDC cell surface markers and produce significant 3A) or CpG-A (Fig. 3B) stimulation resulted in modest but sig- quantities of IFN-α following stimulation with influenza virus or nificant inhibition of IFN-α production. However, treatment of CpG-A DNA (Fig. S3). Treatment of purified mouse pDCs with pDC with CYM-5442, in the presence or absence of PT, sup- the S1PR1 antagonist W146 (Table 1) increased IFN-α pro- pressed IFN-α secretion following both influenza virus (Fig. 3A) duction from pDCs (Fig. 1B), indicating that suppression of pDC α and CpG-A stimulation (Fig. 3B), suggesting that S1PR1-medi- IFN- production requires S1PR1 signaling. Moreover, CYM- α 5442 treatment of pDCs following influenza virus stimulation ated suppression of IFN- production does not require Gi/Go prevented IFN-α protein expression as measured by intracellular signaling. Moreover, these data are in agreement with our sup- C D pression data with CYM-5442 (which has an IC50 for Gi/Go cytokine staining (Fig. 1 and ). We also confirmed pharma- – cological inhibition of IFN-α production in human pDCs fol- signaling of 1.2 nM) and support a non Gi/Go-mediated mech- lowing CYM-5442 treatment (Fig. 1 E–G). The inability of a anism of suppression. We next investigated the involvement of neutral S1PR1 antagonist to inhibit IFN-α amplification was the S1PR1 C terminus using an inhibitory peptide mimic of the confirmed in human pDCs using the potent and selective Ex26 S1PR1 C terminus consisting of an HIV Tat sequence coupled to S1PR1 antagonist (13) (Fig. 1E and Table 1). amino acids that correspond to the C terminus of S1PR1 (17). α Because S1PR1 signaling suppressed IFN-α production from The IFN- response to CpG-A was similar in pDCs treated with pDCs following influenza virus stimulation, which likely occurs either CYM-5442 alone or with CYM-5442 and a control scrambled C-terminal peptide (control peptide) (Fig. 3C). Con- versely, inhibition of CpG-A–induced IFN-α production by Table 1. Receptor specificity of chemical probes CYM-5442 was lost in pDCs in the presence of the C-terminal Tat-coupled peptide, with IFN-α detected at levels similar to EC or 50 vehicle treatment (Fig. 3C). Compounds IC , nM S1PR1 S1PR2 S1PR3 S1PR4 S1PR5 Ref(s). 50 The C-terminal S1PR1-signaling requirement to suppress IFN-α Agonist amplification in pDCs caused us to hypothesize that S1PR1 ag- S1P 0.5–1 +++++(18, 27) onist treatment redirects pDC signaling components for degra- CYM-5442 + −−−− dation. Agonist signaling on S1PR1 is well known to induce Antagonist S1PR1 internalization, culminating in S1PR1 sorting to lyso- W146 36 + −−−−(13, 28) somes (18). Although TLR7 and TLR9 both appear to be within Ex26 0.93 + −−−− 40 nm of S1PR1 based upon proximity ligation assay (PLA) data (Fig. S4), a constitutive PLA signal is seen in the presence of CYM-5442, R-3-amino-(3-diethoxyphenyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro- 1H-inden-1-yl amino)ethanol; Ex26, [1-(5′-((1-(4-chloro-3-methylphenyl)ethyl)ami- W146, and only a modest change in PLA signal is observed with no)-2′-fluoro-3,5-dimethyl-[1,1′-biphenyl]-4-ylcarboxamido)cyclopropanecar- CYM-5442 stimulation (Fig. S4). No significant degradation of boxylic acid]; W146, R-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic either TLR7 or TLR9 was observed by Western blot analysis in acid (ML-056). the presence of either W146 or CYM-5442 (Fig. S5), and no

1352 | www.pnas.org/cgi/doi/10.1073/pnas.1525356113 Teijaro et al. Downloaded by guest on October 2, 2021 A B Unstimulated Uninfected Vehicle Vehicle 800 CYM5442 500 CYM5442 450 600 W146 400 400 350 300 200 250 50 50 40 pg/mL 18 hrs 18 pg/mL pg/mL 18 hrs 18 pg/mL  40    30 30 20 20 IFN- IFN- 10 10 *** *** *** 0 post-infection in supernatant 0 post-infection in supernatant spleen Lung Unstimulated Vehicle C Influenza virus Stimulated D CYM-5442 Unstimulated Vehicle CYM5442 2.5   2.0 0.319 2.5 0.670

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Fig. 1. IFN-I induction in pDCs is directly inhibited by S1PR1 agonist stimulation. (A) FACS-purified pDCs isolated from the spleen and lung were cultured at 1 × 105 and 1 × 104 cells per well, respectively; stimulated with 1 multiplicity of infection (MOI) of WSN influenza virus in the presence of either vehicle (water) or CYM5442 (5 μM); and incubated for 18 h. An ELISA was used to quantify IFN-α concentration, and the data are the mean and SEM of more than six independent experiments. (B) FACS-purified pDCs were stimulated with influenza as in A and treated with vehicle, CYM5442 (5 μM), or W146 (10 μM) for 18 h. IFN-α concentrations were quantified by ELISA, and the data are the mean and SEM of two independent experiments. (C) Inhibition of IFN-α production in + + + purified pDCs following CYM5442 treatment. Purified pDCs were gated on B220 PDCA1 Siglec H cells, and IFN-α expression was detected by intracellular cytokine staining after 6 h of stimulation with 1 MOI of influenza virus. (D) Percentage of pDCs expressing IFN-α following stimulation compiled from four replicates, and data are shown as the mean and SEM. (E) pDCs purified from human peripheral blood mononuclear cells were cultured at 5 × 104 cells per well and stimulated with 1 MOI of WSN influenza virus in the presence of vehicle (water), CYM5442 (5 μM), or S1PR1 antagonist Ex26 (10 μM) and incubated for 18 h. IFN-α concentration was quantified by ELISA, and the data are the mean and SEM of three independent experiments. (F) Purified human pDCs were gated on CD123+ cells, and IFN-α expression was detected by intracellular cytokine staining after 6 h of stimulation with 1 MOI of influenza virus. (G) Per- centage of human pDCs expressing IFN-α following stimulation. Data shown are from one individual experiment and are representative of four independent experiments. Data are shown as mean ± SEM. ***P < 0.005.

− − redistribution of TLR9 was detected by confocal fluorescence effect on IFN-α production in IFNAR1 / pDCs (Fig. 4A), microscopy (Fig. S6). suggesting that S1PR1 signaling inhibits an IFN-α autocrine Given that S1PR1 agonist suppression of cytokine amplifica- feedback loop in pDCs. We next asked if IFNAR1 expression or tion in vivo requires IFN-α signaling coupled to the requirement signaling was differentially modulated following S1PR1 agonist of an IFNAR1 autocrine feedback loop for IFN-α amplification or antagonist treatment. IFNAR1 protein levels were signifi- (19), we hypothesized that S1PR1 signaling in pDCs modifies the cantly reduced and smaller IFNAR1 species were observed in IFN-α amplification loop. To test the ability of S1PR1 signaling the presence of CYM-5442 compared with W146 (Fig. 4B), to modulate IFN-α amplification, we isolated pDCs from WT or suggesting that IFNAR1 is being degraded following S1PR1 IFNAR1-KO mice and stimulated them with CpG-A in the pres- agonist treatment. Additionally, direct stimulation of pDCs with ence or absence of CYM-5442. The absence of IFNAR1 in pDCs exogenous IFN-α in the presence of CYM-5442, but not W146, α resulted in the suppression of IFN- amplification following resulted in diminished STAT1 levels and phosphorylation (Fig. INFLAMMATION CpG-A stimulation (Fig. 4A). Moreover, CYM-5442 had no 4C). Down-modulation of total STAT1 is likely a consequence of IMMUNOLOGY AND

Teijaro et al. PNAS | February 2, 2016 | vol. 113 | no. 5 | 1353 Downloaded by guest on October 2, 2021 A Mouse B Human Fig. 2. S1PR1 agonist inhibits IFN-α production Vehicle through an early endosome-specific signaling pathway. 3000 Unstimulated 10000 (A) FACS-purified pDCs isolated from the spleens of Vehicle CYM-5442 *** 8000 human Flt3 ligand gene (hFLT3L)-injected mice (SI CYM-5442 6000 Materials and Methods and Fig. S2) were cultured 2000 *** 4000 at 1 × 105 cells per well; stimulated with CpG-A pg/mL pg/mL 2000   (ODN2216), CpG-B (ODN1826), or 1 MOI of influenza *** 1000 μ 800 virus in the presence of either vehicle (water, 1 L) or IFN- IFN- 1000 μ N.S. 600 CYM5442 (5 M); and incubated for 18 h. An ELISA was 400 used to quantify IFN-α, and the data are the mean and

18 hrs post-stimulation hrs 18 200 SEM of two independent experiments. (B)pDCsiso- 18 hrs post-stimulation hrs 18 0 0 lated from peripheral blood mononuclear cells were CpG-A CpG-B CpG-A CpG-B Flu cultured at 5 × 104 cells per well and stimulated with Stimulation Stimulation either CpG-A (ODN2216) or CpG-B (ODN1826) in the presence of either vehicle (water) or CYM5442 (5 μM) and incubated for 18 h. An ELISA was used to quantify CDMouse Human IFN-α concentration, and the data are the mean and Unstimulated SEM of two independent experiments. (C)FACS-puri- Unstimulated fied pDCs isolated from the spleens of hFLT3L-injected Vehicle 80000 2000 Vehicle mice (SI Materials and Methods) were cultured at 1 × CYM-5442 CYM5442 105 cells per well and left either unstimulated or stim- 1500 60000 ulated with CpG-A (ODN2216), CpG-B (ODN1826), or CpG-B (1 μM)complexedtoDOTAP.TheIFN-α con- pg/mL pg/mL centration in the supernatants was measured by ELISA

 40000   1000 18 h poststimulation, and the data are the SEM of IFN- IFN- three independent experiments. (D) pDCs isolated from 500 20000 peripheral blood mononuclear cells were cultured at 5 × *** 4 18 hrs post-stimulation hrs 18 *** 10 cells per well and stimulated with CpG-B com- *** post-stimulation hrs 18 0 0 plexed to DOTAP as done in C. The IFN-α concen- CpG-B DOTAP tration in the supernatants was measured by ELISA Stimulation 18 h poststimulation. Data shown are from one in- CpG-B CpG-A dividual experiment and are representative of three CpG-B CpG-B (DOTAP) independent experiments. Data are shown as the Unstimulated Stimulation mean ± SEM. ***P < 0.005.

IFNAR1 turnover, because diminished STAT1 levels are seen in plasma membrane (Fig. 4D), whereas CYM-5442–treated cells − − pDC from IFNAR1 / mice (Fig. S7). CYM-5442 treatment did showed S1PR1 and IFNAR1 internalization, with significant + not alter IFNAR2 expression in pDCs following stimulation with colocalization of S1PR1 and IFNAR1 in LAMP1 terminal lyso- exogenous IFN-I (Fig. S8). somes (Fig. 4 D and E). This subcellular reorganization indicates To examine IFNAR1 subcellular localization, pDCs were im- that S1PR1 agonist-induced IFNAR1 internalization directs both aged using immunofluorescence confocal microscopy. Cells were receptors toward a degradation pathway, preventing IFN-α signaling treated with either W-146 or CYM-5442 and labeled with Abs for and cytokine autoamplification (Fig. 4 A–C). The minimal re- S1P1-GFP (green), IFNAR1 (red), and LAMP1 (orange), and the quirement for CYM-5442–induced degradation of IFNAR1 ap- nucleus was stained with Hoechst (blue) (Fig. 4D). Treatment with pears to be coexpression of the receptors, because the internalization/ W-146 showed S1PR1 and IFNAR1 receptors colocalized to the degradation paradigm can be reconstituted in Jump-In HEK 293

1200 Unstimulated A BCUnstimulated t n.s. n Vehicle t Vehicle *** n 400 *** 4000 Vehicle ***

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Fig. 3. Suppression of IFN-α amplification is dependent on signaling through the S1PR1 C terminus. (A) IFN-α secretion was measured in the supernatant of influenza-stimulated (1 MOI) pDCs at 18 h poststimulation. pDCs were treated with PT, CYM-5442 (5 μM), or both. An ELISA was used to quantify IFN-α concentration. The data are the mean and SEM of three independent experiments. (B) IFN-α secretion was measured in the supernatant of CpG-A–stimulated pDCs after 18 h of stimulation. pDCs were treated with PT, CYM-5442, or both. An ELISA was used to quantify IFN-α concentration, and the data are the mean and SEM of three independent experiments. (C) IFN-α levels were measured in the supernatant of CpG-A–stimulated (1.5 μM) pDCs after 18 h. Cells were treated as noted in the figure. An ELISA was used to quantify IFN-α concentration. Data shown are from one individual experiment and are representative of three independent experiments. Data are shown as the mean ± SEM. ***P < 0.005. n.s., not significant.

1354 | www.pnas.org/cgi/doi/10.1073/pnas.1525356113 Teijaro et al. Downloaded by guest on October 2, 2021 ABC Unstimulated CYM-5442 - + - + 2000 Vehicle IFNAR1+/+ W146 + - + - 1800 *** CYM5442 IFNAR1+/+ M.W. W146 - - + + 1600 CYM5442 IFN Vehicle IFNAR1-/- 1400 93 IFNAR1 STAT1-p 1200 CYM5442 IFNAR1-/- 70 1000 STAT1 100 41  Actin

 80 n.s. Beta-Actin ection in supernatant 60 IFN- pg/mL 18hrs 40 20 post-inf 0

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in supernatant 0.75 6Hrs post-treatment  100 10000 0.50 pg/mL 18Hrs post-stimulation IFN- pg/mL 0

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Relative DensityRelative 6 0 2 IFN- 0.00 x E e ed l M) M) Vehicle -A t ic   G a 3 1 -A p ( ( C Veh P P imul 1 S1 S CpG Unst

+ + Fig. 4. S1PR1-induced IFNAR1 destabilization limits IFN-α amplification in pDCs. (A) IFN-α secretion was measured in the supernatant of IFNAR1 / pDCs or − − IFNAR1 / pDCs 18 h after CpG-A (1 μM) stimulation. Cells were stimulated in the presence or absence of CYM-5442. An ELISA was used to quantify IFN-α levels, and the data are the mean and SEM of three independent experiments. (B) Representative immunoblot of cultured pDCs following 16 h of treatment with W-146 (20 μM) or CYM-5442 (6.0 μM). Blots were probed for IFNAR1 and β-Actin, and are representative of three independent experiments. M.W., molecular weight. (C) Representative immunoblot of cultured pDCs following 16 h of treatment with W-146 (20 μM) or CYM-5442 (6.0 μM). The indicated lanes were stimulated with 1,000 U/mL IFN-α for 15 min and blotted for STAT1, p-STAT1, and β-Actin. (D) Representative immunofluorescence confocal microscopy image of pDCs treated for 16 h with W-146 (20 μM) or CYM-5442 (6.0 μM). Cells were stained with Abs against S1PR1-GFP (488 nm, green), LAMP1 (647 nm, orange), and IFNAR1 (555 nm, red), and the nucleus was stained with Hoechst. (E) Quantification of the immunofluorescence microscopy images is represented as the percentage of colocalization of the noted proteins. The data represent the mean of four independent experiments, and the error bars represent the SD. **P < 0.01; ***P < 0.005 (determined using a Student t test). (F) pDCs isolated from peripheral blood mononuclear cells were cultured at 5 × 104 cells per well and stimulated with 2 μM CpG-A (ODN2216) in the presence of either vehicle or S1P at concentrations of 3 μM and 1 μM for 18 h. An ELISA was used to quantify IFN-α concentration, and the data are the mean and SEM of two independent experiments. (G) p-STAT1 immunoblot (Top) and relative densities of p-STAT1 (Bottom) from cultured pDCs stimulated with CpG-A following 16 h of treatment with vehicle, CYM-5442, or S1P (3 μM and 1 μM). (H) IFN-α levels measured in

the serum 6 h following CpG-DOTAP administration and treatment with either vehicle or Ex26 (30 mg/kg). Data were compiled from four independent INFLAMMATION experiments. **P < 0.01; ***P < 0.005. IMMUNOLOGY AND

Teijaro et al. PNAS | February 2, 2016 | vol. 113 | no. 5 | 1355 Downloaded by guest on October 2, 2021 cells. Reconstitution is achieved by transfecting S1PR1-EGFP, modulatory effects is important for extending treatment in addi- into HEK cells, resulting in S1PR1 ligand-dependent internalization tional autoimmune and infectious pathologies. We report here that and degradation of endogenously expressed IFNAR1 (Fig. S9). the key innate cell subset, the pDC, is a direct target of S1PR1 To determine whether the S1PR1 natural ligand, S1P, suppresses drugs. Moreover, we show that S1PR1 signaling promotes IFNAR1 α IFN- amplification, human pDCs were treated with physiologically internalization and degradation, resulting in attenuation of relevant concentrations of S1P. Treatment with S1P resulted in IFNAR1-mediated IFN-α and cytokine amplification. Amplifi- significant suppression of IFN-α production (Fig. 4F)and cation of IFN-α in vivo by blocking S1PR1 function using the inhibited STAT1 phosphorylation (Fig. 4G)inCpG-A–stimulated neutral antagonist Ex26 further suggests that endogenous S1P- pDCs. We also asked whether blocking of S1PR1 signaling in vivo would alter IFN-α amplification following CpG-A administration. S1PR1 signaling can dampen IFN-I responses physiologically. Administering CpG-A–DOTAP in the presence of the S1PR1 We further postulate that other G protein-coupled receptors antagonist Ex26 significantly increased IFN-α protein in the se- may use similar mechanisms to regulate IFN-I amplification and rum compared with vehicle-treated mice (Fig. 4H). possibly other diverse biological responses. Because pDCs are causal effectors in the pathogenesis of autoimmune disorders, Discussion including lupus and psoriasis (24), and have been linked to dis- S1PR1 is not highly expressed on all leukocyte populations, with ease progression in animal models of multiple sclerosis and ul- very low expression on myeloid DCs and alveolar macrophages cerative colitis (25, 26), our study indicates that the efficacy of (4). S1PR1 expression in pDCs and its functional coupling to S1PR1 therapies in these disease states may manifest through turnover of IFNAR1 and STAT1 down-modulation may reflect an attenuation of an IFNAR1 cytokine amplification loop. The role evolutionary pathway suited to therapeutic exploitation. We have of this pathway in therapeutic efficacy and as a biomarker for shown that S1PR1 agonism protects from influenza and mouse patient subsets that might benefit from S1PR1 agonist therapies pulmonary virus immunopathology while allowing full develop- warrants exploration. ment of sterilizing immunity, neutralizing Abs, and quantitatively normal immunological memory (10, 20–23). Thus, blunting, but not Materials and Methods abolishing, IFN-I amplification by the S1P/S1PR1 signaling axis allows host defense from pathogens. S1PR1 signaling is therefore a All methods are located in the Supporting Information. All animal experiments potential selective advantage in the face of acute pathogens, such as were approved by the Scripps Research Institute Animal Care and Use Committee (IACUC). influenza, by limiting excessive amplification of the IFN-α response, and the resulting detrimental collateral tissue damage, while ACKNOWLEDGMENTS. This article is Publication 29095 from the Department allowing pathogen sterilization through adaptive immunity. of Immunology and Microbial Science, Department of Chemical Physiology S1PR1 agonists are effective in treating multiple autoimmune and The Scripps Research Institute Molecular Screening Center, The Scripps conditions, including multiple sclerosis and ulcerative colitis, where Research Institute. This work was supported, in part, by Grant MH084512 (to H.R.), Grant U54AI057160 to the Midwest Regional Center of Excellence for clinical efficacy is only partially explained by sequestration of cir- Biodefense and Emerging Infectious Diseases Research, NIH Grants AI009484 culating lymphocytes. Thus, a more complete understanding of and AI099699 (to M.B.A.O.), and the Donald E. and Delia B. Baxter Foundation the molecular mechanisms responsible for the sustained immune Faculty Scholar Grant (to J.R.T.).

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