An Essential Role for Perforin-2 in Type I IFN Signaling Ryan Mccormack, Richard Hunte, Eckhard R

An Essential Role for Perforin-2 in Type I IFN Signaling Ryan McCormack, Richard Hunte, Eckhard R. Podack, Gregory V. Plano and Noula Shembade This information is current as of September 26, 2021. J Immunol published online 11 March 2020 http://www.jimmunol.org/content/early/2020/03/10/jimmun ol.1901013 Downloaded from

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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 © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 11, 2020, doi:10.4049/jimmunol.1901013 The Journal of Immunology

An Essential Role for Perforin-2 in Type I IFN Signaling

Ryan McCormack,*,1 Richard Hunte,*,1 Eckhard R. Podack,*,†,2 Gregory V. Plano,* and Noula Shembade*,†

Type I IFNs play a complex role in determining the fate of microbial pathogens and may also be deleterious to the host during bacterial and viral infections. Upon ligand binding, a receptor proximal complex consisting of IFN-a and -b receptors 1 and 2 (IFNAR1, IFNAR2, respectively), tyrosine kinase 2 (Tyk2), Jak1, and STAT2 are assembled and promote the phosphorylation of STAT1 and STAT2. However, how the IFNARs proximal complex is assembled upon binding to IFN is poorly understood. In this study, we show that the membrane-associated pore-forming protein Perforin-2 (P2) is critical for LPS-induced endotoxic shock in wild-type mice. Type I IFN–mediated JAK–STAT signaling is severely impaired, and activation of MAPKs and PI3K signaling pathways are delayed in P2-deﬁcient mouse bone marrow–derived macrophages, mouse embryonic ﬁbroblasts (MEFs), and human HeLa cells upon IFN stimulation. The P2 N-glycosylated extracellular membrane attack complex/perforin domain and the P2 domain independently associate with the extracellular regions of IFNAR1 and IFNAR2, respectively, in resting MEFs. In Downloaded from addition, the P2 cytoplasmic tail domain mediated the constitutive interaction between STAT2 and IFNAR2 in resting MEFs, an interaction that is dependent on the association of the extracellular regions of P2 and IFNAR2. Finally, the constitutive association of P2 with both receptors and STAT2 is critical for the receptor proximal complex assembly and reciprocal transphosphorylation of Jak1 and Tyk2 as well as the phosphorylation and activation of STAT1 and STAT2 upon IFN-b stimulation. The Journal of Immunology, 2020, 204: 000–000. http://www.jimmunol.org/

ype I IFNs are potent cytokines induced by most cell types In humans, type I IFNs consist of a large subgroup, including in the body in response to bacterial, viral, or fungal in- IFN-a (which can be further subdivided into 13 different sub- T fections to limit the spread of these pathogenic microbes types), IFN-b,IFN-d,IFN-ε,IFN-k,IFN-t,andIFN-v1–3, and promote host survival (1, 2). The effect of type I IFN is which act in autocrine and paracrine manners. Studies have not limited to antimicrobial functions but also promotes cyto- shown that all type I IFNs exclusively bind to and signal through toxic effects, tissue damage by apoptosis induction, or suppression ubiquitously expressed heterodimeric transmembrane (TM) re- of proliferation in tissue cells (3, 4). Improper regulation of ceptors known as IFN-a and -b receptor (IFNAR) 1 and type I IFN responses can lead to the development of infectious and IFNAR2 (9). IFN-a and IFN-b are the most studied type I IFNs. by guest on September 26, 2021 inflammation-related diseases, such as septic shock, autoimmune Studies have shown a constitutive association of IFNAR1 with diseases, and inflammatory syndromes (5–8). The mechanisms of Tyk2 and IFNAR2 with Jak1 and STAT2 under normal physi- type I IFN signaling activation are complex and not well understood. ological conditions (10, 11). Upon binding of IFN to the receptor subunits IFNAR1 and IFNAR2, the receptor proximal complex is assembled and the tyrosine kinase 2 (TYK2) and *Department of Microbiology and Immunology, Miller School of Medicine, Univer- JAK1 are activated by reciprocal transphosphorylation. Acti- sity of Miami, Miami, FL 33136 †Sylvester Comprehensive Cancer Center, Univer- sity of Miami, Miami, FL 33136 vated TYK2 and JAK1 phosphorylate and activate STAT2. 1R.M. and R.H. are co-first authors. Phosphorylation of STAT2 is a critical step in STAT1 activation 2 in response to IFN stimulation (12, 13). Activated STAT1 and E.R.P. deceased. STAT2 heterodimers associate with IFN-regulatory factor 9 ORCIDs: 0000-0002-6107-1385 (R.H.); 0000-0001-7321-9589 (N.S.). (IRF9) in the cytoplasm to form a heterotrimeric transcription Received for publication August 21, 2019. Accepted for publication February 12, 2020. complex termed IFN-stimulated gene factor 3 (ISGF3) (14, 15). ISGF3 binds to upstream sequence elements (IFN-stimulated re- This work was supported by National Institutes of Health Grants CA039201, CA109094, AI0073234, and AI096396 (to E.R.P.); the Lois Pope Life Foundation sponse elements) and activates the transcription of IFN-inducible Development Fellowship (to R.M.); and American Cancer Society Grant RSG-16- genes. Type I IFN–activated STAT1 can form homodimers and 254-01-MPC (to N.S.). bind to g-activated sequences to induce proinflammatory genes R.M., R.H., E.R.P., and N.S. designed the research; R.M., R.H., and N.S. performed (6). Type I IFNs also signal through other STATs such as the experiments; R.M., R.H., E.R.P., G.V.P., and N.S. analyzed the results; N.S. wrote the paper; and R.M., R.H., and G.V.P. edited the manuscript. STAT3, STAT4, STAT5A and STAT5B, but such activation is Address correspondence and reprint requests to Dr. Noula Shembade, Department limited to specific cell types such as endothelial cells or cells of of Microbiology and Immunology, Miller School of Medicine, University of Miami, 1600 lymphoid origin (6, 12, 16, 17). Although the IFNAR1–IFNAR2 NW 10th Avenue, Miami, FL 33136. E-mail address: [email protected] interaction and the IFN-induced receptor proximal complex The online version of this article contains supplemental material. formation are critical to promote STAT1 and STAT2 activa- Abbreviations used in this article: BMDM, bone marrow–derived macrophage; tion, it remains unclear precisely how the IFNARs proximal Co-IP, coimmunoprecipitation; CT, cytoplasmic tail; IFNAR, IFN-a and -b receptor; ISRE, IFN-sensitive response element; MACPF, membrane attack complex/perforin; complex is assembled upon stimulation with IFNs. Previous MEF, mouse embryonic fibroblast; P2, Perforin-2; PBST, PBS with 0.05% Tween 20; studies have shown that the glycosylation of membrane re- Rap1, Ras-related protein 1; shRNA, short hairpin RNA; TM, transmembrane; ceptor proteins play an important role in receptor interaction TYK2, tyrosine kinase 2; VSV-GFP, vesicular stomatitis virus–encoding GFP. and subsequent activation of signal transduction pathways (18, 19). Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 The extracellular regions of both IFNAR1 and IFNAR2 possess

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901013 2 ROLE OF PERFORIN-2 IN TYPE I IFN–MEDIATED JAK–STAT SIGNALING glycosylation sites (20, 21). However, the mechanistic roles of Materials and Methods glycosylation of IFNARs in JAK–STAT activation are poorly Mice understood. 2 2 2 Eight- to ten-week-old P2+/+, P2 / , and P2+/ mice (26) (30 mice in Studies have shown that Jak1/Tyk2 activated downstream of type each group) were injected i.p. with 30 mg of LPS (from Sigma-Aldrich) I IFN also phosphorylates and activates insulin receptor substrate 1 per kilogram of body weight. All animal studies were performed according to (IRS1) and 2 (IRS2), which subsequently activate the PI3K/AKT National Institutes of Health guidelines and were approved by the University of and p38/ERK MAPK pathways (12). Given the important roles of Miami Institutional Animal Care and Use Committee. constitutive association of IFNAR1 and IFNAR2 with Tyk2 and Cells and biological reagents Jak1, respectively, it is not clear whether activated Jak1/Tyk2 is +/+ 2/2 essential for the phosphorylation of AKT because AKT phos- P2 and P2 mouse embryonic fibroblasts (MEFs) were prepared as phorylation is not impaired in Ifnar22⁄2 cells after stimulation described previously (33). E12.5 embryos were dissected free of sur- rounding tissues, washed in PBS, and the heads and livers were removed. with IFN-b (22). This diversity of type I IFN signaling may The tissue was placed in trypsin/EDTA and disrupted by forcing through a explain the broader effects of IFN-a/b in the induction of genes 6-ml syringe followed by vigorous pipetting. IFNAR1+/+ and IFNAR12/2 involved in antimicrobial responses and proapoptotic effects. MEFs were gifts from Dr. S. Balachandran (Fox Chase Cancer Center, Studies have demonstrated that IFNAR1 knockout animals are Philadelphia, PA). Bone marrow–derived macrophages (BMDMs) were isolated using a standard protocol (34). Bone marrow cells were isolated resistant to lethal endotoxemia, sepsis, and Gram-positive and from the femurs of P2+/+ and P22/2 mice. For BMDMs, bone marrow Gram-negative intracellular bacteria. However, the mechanisms cells were cultured in L929 cell–conditioned media for 7 d prior to IFN-b of promoting lethal endotoxemia and sepsis by IFNAR1 in re- stimulation. Cells were stained using CD11b and subjected to flow sponse to LPS and infections with Gram-negative bacteria are cytometry to confirm the purity of macrophages (.98%). Mouse Downloaded from poorly understood (23–25). peritoneal macrophages were extracted by flushing the peritoneal cavity with ice-cold, sterile PBS and cultured for 24 h before stimulation with Perforin-2 (P2) (also known as macrophage-expressed gene 1 IFN-b. MEFs and HeLa cells were cultured in complete DMEM (MPEG1) is a ubiquitously expressed membrane attack complex/ (Mediatech) containing 10% FBS (which was heat inactivated and perforin (MACPF) TM protein, which functions as a major sterile-filtered [Sigma-Aldrich]), L-glutamine, and 13 penicillin– antibacterial effector protein that forms pores in infected bac- streptomycin (Invitrogen/Life Technologies). The following Abs were used in this study: anti–p-STAT1 (catalog no. 7649), anti–p-STAT2 terial membranes (26, 27). It is localized in select intracellular (4441), anti-STAT2 (4597), anti–p-Tyk2 (9321), anti–p-Jak1 (3332), http://www.jimmunol.org/ membranes and at the cell surface (26). P2 limits the prolif- anti-Jak1 (3331), anti–p-AKT (9271), anti-AKT (9272), anti–p-p38 eration and spread of Gram-positive, Gram-negative, and (4511), anti-p38 (8690) anti-ISG15 (2743), anti-SOCS1 (3950) from Cell acid-fast bacteria (26, 28, 29). P2 is highly conserved Signaling, anti-STAT1 (sc-464), anti-IFNAR2 (sc-137209), anti-Mx1 throughout evolution from sea sponges to humans (30). Some (sc-166412), anti-GFP (sc-9996), anti-STAT2 (sc-476) from Santa Cruz Biotechnology, anti–p-Tyk2 (PA5-34497), anti-Tyk2 (PA5-37762), studies have also suggested that P2 may have an antimicrobial anti-SOCS1 (PA1-41009), anti–P2 (PA5-31958 and PA1-29037) from role in invertebrates, including clams, mussels and snails, as Thermo Fisher Scientific, anti–P2 (ab25146), anti–b-actin (AC-15) well as in fish (26). Unlike other MACPF family members, P2 Abcam, anti-IFNAR1 (I-401), anti–p-STAT2 (07-224) from Millipore, is a type I TM protein composed of an MACPF domain at its N and anti-FLAG (F3165) from Sigma-Aldrich. Recombinant mouse IFN-a

and IFN-b were purchased from PBL Assay Science, Piscataway, NJ. by guest on September 26, 2021 terminus, a unique P2 domain, a single TM domain, and a Recombinant human IFN-a was purchased from Peprotech. EndoH and short cytoplasmic tail (CT) at the C terminus (26, 30). Evo- PNGase F were purchased from New England Biolabs (New England lutionary studies of P2 have demonstrated that it is one of the Biolabs, Ipswich, MA), and anti-Flag M2-agarose beads (Sigma-Aldrich). oldest eukaryotic MACPF domain members, present in early Lentiviral particle production and virus infection metazoan phyla, including Porifera (sponges), and is highly conserved throughout evolution (30). The expression of P2 is To overexpress P2 in BMDMs, pCMV-P2 was used as a template for PCR- not limited to macrophages and other professional phagocytes mediated cloning into the pDUET-GFP-hygromycin and pCDH-Cuo-MCS- EFI-GFP-T2A-puro lentiviral vector. P2-specific short hairpin RNA but is ubiquitously expressed in many cells including epi- (shRNA) lentiviral vectors were purchased from Horizon Discovery/ thelial cells and fibroblasts (26, 28–31). Previous studies have Dharmacon. Lentiviral particles carrying shRNAs, P2, or control (GFP) 2⁄2 shown that P2 mice are highly susceptible to bacterial empty vector were made by transfection of HEK 293–T cells with 1 mgof infections (26). Interestingly, P2 expression is also induced control empty vector, shRNA vector, or P2 vector with 2 mg of packaging several fold after stimulation with type I IFNs (32). Although plasmids (OriGene Technologies or Horizon Discovery/Dharmacon) containing a puromycin selection marker using FuGENE 6 (Promega). the role of P2 as an antibacterial molecule has been established, Seventy-two hours posttransfection, the supernatants were collected and its complete role in innate immunity remains unknown. concentrated by ultracentrifugation, and the pellets were resuspended in Our findings demonstrate that P22⁄2 mice, like IFNAR12/2 ice-cold PBS. BMDMs were infected with lentiviral particles for 48 h prior mice, are protected from the lethal effects of i.p. LPS adminis- to stimulation with IFNs. Vesicular stomatitis virus–encoding GFP (VSV- GFP) were provided by Drs. S. Balachandran and G. N. Barber (35). For tration observed in wild-type mice. Interestingly, type I IFN– VSV-GFP infection, cells were serum starved for 1 h, inoculated with mediated JAK–STAT signaling is severely impaired and activation VSV-GFP in serum-free DMEM for 1 h, and further incubated in complete of MAPKs and PI3K-signaling are delayed in P2-deficient cells DMEM. upon IFN stimulation. We determined that the extracellular MACPF and P2 domains were glycosylated, and that this gly- Far-Western blotting cosylation was essential for their interaction with the extracel- P2 binding with IFNAR1 and IFNAR2 was assessed by far-western blotting lular regions of IFNAR1 and IFNAR2, respectively. In addition, as described (36, 37). Briefly, 60 mg of whole-cell lysates prepared from the P2 CT mediated the constitutive interaction between STAT2 HeLa cells in RIPA buffer was mixed with SDS-PAGE sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes. and IFNAR2. Furthermore, the reciprocal transphosphorylation Membranes were blocked with 5% milk in PBS with 0.05% Tween 20 of Jak1 and Tyk2, and the phosphorylation and activation of (PBST) for 2 h at room temperature after the denaturation and renaturation STAT1andSTAT2uponIFN-b stimulation, was exclusively process. P2 (bait) binding with IFNAR1 and IFNAR2 (prey) was per- dependent on P2’s association with the extracellular region of formed by incubating 5 mg of Flag-tagged recombinant P2 at 4˚C for 18 h. Membranes were washed three times in PBST and incubated with primary IFNAR2. Together, our data implicate P2 as a key regulator of (anti-Flag [bait protein]) and secondary (horseradish-conjugated) Abs in IFNAR interaction and receptor proximal complex assembly in PBST, and then detected with Western Lightning ECL reagent (Perkin type I IFN signaling. Elmer, Boston, MA). The Journal of Immunology 3

Flow cytometry ELISA IFNAR1 surface staining was conducted as described previously (22). In Cytokine concentrations in serum and supernatants from mice and brief, nonspecific Ab interactions were blocked using anti-CD16/CD32 BMDMs treated with LPS or IFN-b, respectively, were determined blocking Ab before staining with the specific Abs described below. using commercially available ELISA kits, according to the manufac- BMDMs derived from P2+/+, P22/2, and P2+/2 mice were collected and turer’s instructions. The following ELISA kits were used: mouse TNF-a stimulated with IFN-b (1000 IU/ml) for 60 min, blocked, and stained with (DY410; R&D Systems), mouse IL-12 p70 (DY419; R&D Systems), either biotinylated anti-IFNAR1 or biotinylated isotype control (IgG1) Ab mouse MCP 1 (DY479; R&D Systems), mouse IL-10 (DY417; R&D and a PE-conjugated streptavidin-labeled secondary Ab for detection. Systems), and mouse CXCL10 (DY466; R&D Systems). All samples were acquired on a BD Fortessa Flow Cytometer running FACS DIVA software. Analysis was performed using FlowJo X software Statistical analysis (TreeStar, Woodburn, OR). Student t test, one-way ANOVA with Bonferroni multiple-comparisons test, or Kruskal–Wallis nonparametric test with Dunn multiple-comparison test Coimmunoprecipitation assays and Western blotting was used for comparisons (GraphPad Prism Version 6.0b and SPSS 21.0 Coimmunoprecipitation (Co-IP) assays were performed as described were used for statistical analysis). previously (34, 38). Whole-cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose membranes, blocked in 5% milk, cut into different sizes, incubated with specific primary and secondary Abs, and Results then detected with Western Lightning ECL reagent (Perkin Elmer). P2 knockout mice are resistant to LPS-induced septic shock P2, a pore-forming protein, kills bacteria by forming pores in their Purification of recombinant P2 and in vitro protein–protein surface-exposed membranes. Recent genetic data suggest that P2 is

interaction assays Downloaded from critical for the prevention of intracellular replication and prolif- Protein purification and in vitro protein–protein interaction assays were eration of bacterial pathogens. P2 knockout mice infected with performed as described previously (39–42). Briefly, FLAG-P2 (25–655) Gram-positive or Gram-negative bacteria, by either epicutaneous or was expressed in HEK 293–T cells. Lysates were incubated with anti- FLAG M2 agarose to pull down the FLAG-P2 (25–655). The beads were orogastric routes, are unable to control systemic dissemination of washed five times with buffer prior to the elution of FLAG-P2 (25–655), bacteria and succumb to bacterial infection (26, 28, 29). However, as described previously (39). IFNAR1-Fc (Met1–Lys436) and IFNAR2-Fc whether P2 plays additional roles in innate immunity to bacterial 1 243 (Met –Lys ) expressed in HEK 293–T cells were purchased from Sino infection is unknown. The stimulation of TLR-4 by the major http://www.jimmunol.org/ Biological, Wayne, PA. Purified FLAG-P2 (25–655) was incubated with IFNAR1-Fc and IFNAR2-Fc in pull-down buffer (1 mM DTT, 5 mM outer surface membrane molecule of Gram-negative bacteria, MgCl2, 2.7 mM KCl, 130 mM NaCl, 10 mM sodium phosphate [pH 7.4]) LPS, initiates production of type I IFN (45). LPS-induced type I containing EDTA-free protease inhibitors mixture (Roche) for 1 h at IFNs (e.g., IFN-b) play critical roles in LPS-induced toxic 24˚C. The reactions were subjected to immunoprecipitation using anti-Fc Ab. shock (46). Thus, to determine whether P2 plays additional roles The protein A-agarose beads were washed three times with reaction buffer in innate immunity to bacterial infection, we used age-matched and boiled for 5 min after adding 23 Laemmli Sample Buffer (LSB) buffer and analyzed by immunoblotting. P2-deficient mice to examine the LPS-induced pathogenesis of sepsis in vivo. P2+/+, P2+/2,andP22⁄2 (30miceineachgroup) Quantitative PCR were injected i.p. with 30 mg of LPS per kilogram of body

2⁄2 by guest on September 26, 2021 Quantitative PCR was done as described previously (26). Total RNA was weight. Surprisingly, P2 mice were protected from the lethal obtained from cells by using an RNeasy Kit (Qiagen, Valencia, CA) and effects of LPS observed in P2+/+ mice(Fig.1A).Thisfinding converted to cDNA by using a First-Strand cDNA Synthesis Kit (Roche). was previously replicated with mice deficient in IFNAR1 and The following sets of primers were used to amplify gene products for PCR: viperin forward, 59-TTGGGCAAGCTTGTGAGATTC-39 and reverse, 59- Tyk2, key effectors of type I IFN signaling (22, 25, 47–49). Because TGAACCATCTCTCCTGGATAAGG-39; IFIT1 forward, 59-TCGCGTA- the toxic effects of LPS are mediated in part by proinflammatory GACAAAGCTCTTCATC-39 and reverse, 59-AGCAGAGCCCTTTTTG- and acute-phase cytokines (50), we next quantified the serum levels ATAATGTAA-39; MxA forward, 59-TGCCTGGCAGAGAGACTGACT-39 of TNF-a, IL-12p70, MCP1, and IL-10 in control P2+/+, P2+/2, and reverse, 59-GCTTGCACTCTGATGACTGCTATT-39;andRPLPO/ and P22⁄2 mice by ELISA at different time points following LPS 36B4 forward, 59-AGATGCAGCAGATCCGCAT-39 and reverse, 59- GGATGGCCTTGCGCA-39. challenge. As expected, the P2 knockout mice exhibit a significantly delayed and reduced production of inflammatory cyto- Glycosidase assays kines, including TNF-a, IL-12p70, and MCP1 (Fig. 1B–D). Cell lysates were treated with EndoH or PNGase F according to the Surprisingly, the P2 knockout animals also demonstrated an manufacturer’s instructions. Briefly, around 40 mg of lysates were dena- increased production of IL-10 (Fig. 1E). These results suggest tured and then treated with 500 U of glycosidase at 37˚C for 1 h. that deficiency of P2 alters the cellular response pathways and Isolation of cell-surface proteins likewise renders P2 knockout animals highly resistant to LPS endotoxemia. Cell-surface proteins were isolated using the Pierce Cell Surface Protein Isolation Kit (Thermo Fisher Scientific) according to the manufacturer’s Type I IFN signaling is defective in P2-deficient cells protocol. Briefly, MEFs and BMDMs were incubated with sulfo-NHS- SS-biotin to biotinylate cell-surface proteins. Cells were rinsed twice Previous studies have reported that type I IFNR knockout mice 2 2 2 2 with ice-cold PBS and unbound sulfo-NHS-SS-biotin was quenched with (Ifnar1 / ) and tyrosine kinase Tyk2 knockout mice (Tyk2 / ) 100 mM glycine for 20 min at 4˚C, and then rinsed twice with PBS. Cells are resistant to high-dose LPS treatment. Type I IFN–mediated were harvested in 1% Triton X-100–containing RIPA buffer with JAK–STAT activation is defective in IFNAR1- and Tyk2-deficient protease inhibitor mixture. The lysates were clarified by centrifugation and incubated with streptavidin–agarose resin beads. Beads were washed cells. Therefore, we next determined if P2 also plays a role in type three times in RIPA buffer, and bound proteins were eluted from the beads I IFN–mediated JAK–STAT signaling. Accordingly, MEFs lacking by boiling the samples in 23 Laemmli Sample Buffer and separated by endogenous P2 or Ifnar1 were used to determine the role of P2 in SDS-PAGE followed by immunoblotting. type I IFN–mediated JAK–STAT signaling. P2+/+ and Ifnar12/2 EMSA MEFs were used as a control in this assay owing to their previously described defective type I IFN signaling (51), P22⁄2 MEFs Nuclear extracts were prepared from stimulated and unstimulated cells as 2⁄2 described previously (43, 44). The IFN-sensitive response element (ISRE) and P2 MEFs reconstituted with wild-type Flag-tagged mouse and Oct-1 EMSA were performed using the ISRE EMSA Kit (Signosis) P2 were stimulated with 1000 IU/ml IFN-b at different time according to the manufacturer’s instructions. points. We analyzed the kinetics of type I IFN signaling and 4 ROLE OF PERFORIN-2 IN TYPE I IFN–MEDIATED JAK–STAT SIGNALING Downloaded from http://www.jimmunol.org/

FIGURE 1. P2-deficient mice are resistant to LPS-induced endotoxic shock and have defective type I IFN signaling. (A) Kaplan–Meier plot showing the percentage survival over time of age-matched P2+⁄+, P2+⁄2, and P22⁄2 mice after i.p. challenge with a lethal dose of LPS (30 mg/kg). Log-rank (Mantel– Cox) test was performed with a statistical significance of p , 0.0001. Serum levels of TNF-a (B), IL-12p70 (C), MCP1 (D), and IL-10 (E) determined by ELISA in P2+/+ and P22⁄2 mice after IP challenge with LPS (30 mg/kg). Data are representing three independent experiments with n = 5 biological replicates. (F) Activation of type I IFN signaling in control, IFNAR1- and P2-deficient MEFs, and P2-deficient MEFs reconstituted with either empty vector (EV) or FLAG-tagged wild-type P2. P2+⁄+ (WT) MEFs, IFNAR12⁄2 MEFs, P22⁄2 MEFs, and P22⁄2 MEFs reconstituted with P2 were stimulated with IFN-b for the indicated time points. Lysates were subjected to immunoblotting with Abs to anti–p-STAT1, anti-STAT1, anti–p-Tyk2, anti-Tyk2, anti–p- by guest on September 26, 2021 Jak1, anti-Jak1, anti-FLAG (P2), and anti-IFNAR2. P2 and IFNAR1 expression were examined by immunoprecipitation and immunoblotting with the same Abs. Statistical analysis by one-way ANOVA with Tukey post hoc test. *p , 0.05. examined the expression and activation of nonreceptor tyrosine with wild-type P2 BMDMs after stimulation with IFN-b but not in kinase JAK (Jak1), Tyk2, STAT1, and STAT2, which are rapidly P22⁄2 BMDMs (Supplemental Fig. 2A). These results strongly phosphorylated upon stimulation with type I IFNs (13). Sur- suggest that P2 is required for the internalization of IFNAR1 and prisingly, the phosphorylation of Jak1, Tyk2, STAT1, and activation of type I IFN–mediated JAK–STAT signaling. STAT2 after IFN stimulation was impaired in P22⁄2 MEFs, similar to Ifnar12/2 MEFs (Fig. 1F), but not in P2+/+ MEFs or P22⁄2 Type I IFN–mediated STAT1 DNA-binding activity and MEFs reconstituted with Flag-P2. Next, to determine whether P2 antiviral gene induction is defective in P2-deficient cells modulates a dose-dependent activation of JAK–STAT signaling by To investigate the effect of P2 deficiency on the downstream type I IFNs, mouse BMDMs and MEFs derived from P2+/+ and functional consequences of type I IFN signaling after IFN stim- P22⁄2 mice were stimulated with different concentrations of ulation, P2+/+, P22⁄2, and P22⁄2 BMDMs reconstituted with either IFN-a or IFN-b for 15 min. The phosphorylation of Jak1, wild-type P2 were stimulated with 1000 IU/ml IFN-b at different Tyk2, STAT1, and STAT2 was impaired in both P22⁄2 MEFs time points, and cytoplasmic and nuclear extracts were prepared. and BMDMs after IFN stimulation (Fig. 2). To determine As expected, P2 deficiency was associated with the loss of IFN-b– whether P2 also plays a role in type I IFN signaling activation in induced STAT1 and STAT2 phosphorylation, loss of STAT1 DNA human cells, endogenous P2 expression was stably suppressed binding as shown by ISRE EMSA, and induction of ISGs, in- with lentiviral-delivered shRNA in HeLa cells followed by cluding ISG15 and SOCS1 (Fig. 3A). The rapid induction of ISGs stimulation with IFN-a. As expected, IFN-a–induced phosphor- ISG15 and SOCS1 observed in primary P2+/+ BMDMs is con- ylation of STAT1 and STAT2 was impaired in HeLa cells after sistent with published studies (53, 54). Because type I IFN also knockdown of P2 (Supplemental Fig. 1A, 1B). These results activates PI3K/AKT and p38/ERK MAPK pathways (12, 55), we suggest that P2 is critical for the activation of type I IFN signaling evaluated the phosphorylation of AKT and p38 by Western blot- in both mouse and human cells. Because IFNAR1 readily un- ting using phospho-specific Abs. Although PI3K/AKT and dergoes endocytosis and degradation upon stimulation with type p38/ERK were activated in P22⁄2 BMDMs, there was a pronounced I IFNs (52), we next determined IFNAR1 internalization in delay in their activation (Fig. 3A). The induction of ISGs BMDMs. P2+/+, P22⁄2,andP22⁄2 BMDMs reconstituted with (viperin, IFIT1, and MxA) mRNA by IFN-b were significantly wild-type P2 were stimulated with IFN-b and subjected to flow reduced in P22⁄2 MEFs compared with P2+/+ MEFs (Fig. 3B). cytometry to assess IFNAR1 internalization. IFNAR1 internaliza- The IFN-induced secretion of IL-10 and CXCL10 were also sig- tion was observed in the P2+/+ and P22⁄2 BMDMs reconstituted nificantly reduced in P22⁄2 BMDMs compared with P2+/+ The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Defective type I IFN signaling in P2-deﬁcient BMDMs and MEFs. P2+/+ and P22⁄2 (A) BMDMs and (C) MEFs were stimulated with different concentrations of IFN-a as indicated. Lysates were subjected to immunoblotting with Abs to p-STAT1, STAT1, p-STAT2, STAT2, p-Tyk2, Tyk2, p-Jak1, Jak1, IFNAR2, and immunoprecipitation and immunoblotting with anti-IFNAR1. P2+/+ and P22⁄2 (B) BMDMs and (D) MEFs were stimulated with different concentrations of IFN-b as indicated. Lysates were subjected to immunoblotting with Abs to p-STAT1, STAT1, p-STAT2, STAT2, p-Tyk2, Tyk2, p-Jak1, Jak1, IFNAR2, and immunoprecipitation and immunoblotting with anti-IFNAR1. All immunoblot data shown are representative of at least three independent experiments.

BMDMs (Supplemental Fig. 2B, 2C). These results suggest an reconstituted with wild-type P2 with 1000 IU/ml IFN-b for essential role of P2 in type I IFN signaling. various times, and protein–protein interactions were examined by Co-IP assays. Immunoprecipitation with anti-IFNAR1 (Fig. 4B) P2 forms distinct ligand-independent and ligand-dependent pulled down Tyk2, which is known to be constitutively associated complexes with IFNAR1, IFNAR2, and STAT2 with IFNAR1, in all BMDMs tested. Surprisingly, IFNAR1 To further examine the mechanisms of P2 function in type I IFN was constitutively associated with P2 in unstimulated cells signaling, we ﬁrst determined whether P2 is present on the cell (Fig. 4B). Interestingly, the IFNAR1–Tyk2 complex formation surface and/or in the cytoplasm. Unstimulated BMDMs derived with IFNAR2–Jak1–STAT2 after stimulation with IFN-b was from wild-type and P22⁄2 mice were surface biotinylated and impaired in P22⁄2 BMDMs but not in P2+/+ BMDMs and P22⁄2 subsequently puriﬁed and immunoblotted with anti–P2 Ab. In- BMDMs reconstituted with wild-type P2 after stimulation with terestingly, examination of these cell-surface biotinylation ex- IFN-b (Fig. 4B). As expected, the IFN-induced phosphorylation periments revealed that P2, IFNAR1, and IFNAR2 were mostly of Jak1, Tyk2, STAT1, and STAT2 were impaired in P22⁄2 localized on the cell surface (Fig. 4A, 4D). Because both BMDMs but not in P2+/+ BMDMs and P22⁄2 BMDMs recon- IFNAR1 and IFNAR2 are localized on the cell surface (56, 57), stituted with wild-type P2 (Fig. 4B). These results strongly sug- we hypothesized that P2 may interact with either IFNAR1 and/or gest that the constitutive interaction of P2 with IFNAR1 in resting IFNAR2 and promote the assembly of IFNARs proximal com- BMDMs is critical for the IFNAR1–IFNAR2 interaction after plexes, which facilitates downstream signaling upon IFN stim- stimulation with IFN-b. We next determined whether P2 interacts ulation. To examine whether P2 interacts with IFNARs, we with IFNAR2 in BMDMs. P2+/+ BMDMs, P22⁄2 BMDMs, and stimulated P2+/+ BMDMs, P22⁄2 BMDMs, and P22⁄2 BMDMs P22⁄2 BMDMs reconstituted with wild-type P2 were stimulated 6 ROLE OF PERFORIN-2 IN TYPE I IFN–MEDIATED JAK–STAT SIGNALING

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B by guest on September 26, 2021

FIGURE 3. P2 is required for STAT1 phosphorylation, STAT1 DNA binding, and ISG induction in type I IFN signaling. (A) Kinetics of type I IFN– induced genes and PI3K/AKT and p38/ERK MAPK pathways activation in wild-type control, P22⁄2 BMDMs, and P22⁄2 BMDMs reconstituted with FLAG-tagged wild-type P2. P2+/+ BMDMs, P22⁄2 BMDMs, and P22⁄2 BMDMs reconstituted with empty vector (EV) or wild-type P2 were stimulated with IFN-b for the indicated time points. Cytosolic extracts were immunoblotted with anti–p-STAT1, anti-STAT1, anti–p-STAT2, anti-STAT2, anti–p-AKT, anti-AKT, anti–p-p38, anti-p38, anti–b-actin, anti-P2, anti-ISG15, and SOCS1. Nuclear extracts were used for ISRE and Oct-1 EMSAs. (B) Quantitative PCR analysis of ISGs (Viperin, IFIT1, and MxA) in P2+/+ and P22⁄2 MEFs after stimulation with IFN-b for different time points as indicated. *p , 0.05. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. P2 is required for the assembly of type I IFNARs proximal complexes. (A) BMDMs and (D) MEFs immunoblot analysis of cell-surface P2. Cell-surface proteins were labeled with sulfo-NHS-SS-biotin, and the biotinylated proteins were recovered with streptavidin beads. Whole-cell lysates or surface P2 and IFNAR1 were used for immunoprecipitation with anti–P2 or anti-IFNAR1 Abs and immunoblotted with anti–P2 or anti-IFNAR1 Abs. Whole-cell lysates and surface lysates were also used for immunoblots with anti–b-actin and anti-IFNAR2 Abs. (B) P2 is required for the assembly of type I IFNARs proximal complexes. P2+/+ BMDMs, P22⁄2 BMDMs, and P22⁄2 BMDMs reconstituted with empty vector (EV) or FLAG-tagged wild-type P2 were stimulated with IFN-b for the indicated times, and lysates were immunoprecipitated with IFNAR1 (B)orP2(C) Abs and detected by immunoblotting with Abs to Tyk2, IFNAR1, Jak1, STAT2, IFNAR2, and P2. Lysates were subjected to phosphorylated forms of STAT1, STAT2, Tyk2, and Jak1 and total STAT1, STAT2, Tyk2, Jak1, IFNAR2, and P2. (D) P2+/+ (WT) MEFs, IFNAR12⁄2 MEFs, P22⁄2 MEFs, and P22⁄2 MEFs (Figure legend continues) 8 ROLE OF PERFORIN-2 IN TYPE I IFN–MEDIATED JAK–STAT SIGNALING with or without 1000 IU/ml IFN-b for various times. Lysates Jak1, Tyk2, STAT1, and STAT2 phosphorylation (Fig. 6D, 6E). were coimmunoprecipitated with anti–P2 Ab and immunoblotted Furthermore, we examined interactions between IFNAR1, with anti-IFNAR1, anti-IFNAR2, anti-Jak1, anti-Tyk2, and anti- IFNAR2, and P2 in P22⁄2 MEFs reconstituted with P2 deletion STAT2 Abs. Surprisingly, P2 also constitutively interacted with mutants after IFN-b stimulation. Immunoprecipitations with anti- IFNAR2, similar to IFNAR1, in unstimulated P2+/+ BMDMs and IFNAR1 Ab under stringent conditions and immunoblotting with 2⁄2 P2 BMDMs reconstituted with wild-type P2 (Fig. 4C). As anti-IFNAR2, anti-Jak1, anti-Tyk2, anti-STAT2, and anti-Flag expected, the phosphorylation of Jak1, Tyk2, STAT1, and STAT2 Abs. As expected, the constitutive interactions between IFNAR1 2⁄2 was impaired in P2 BMDMs, but not in P2+/+ BMDMs and and Tyk2 remained intact (Fig. 6C). However, the IFNAR1 in- 2⁄2 P2 BMDMs reconstituted with wild-type P2 after stimulation teraction with IFNAR2 was severely impaired in the absence of with IFN-b (Fig.4C).Interestingly,IFNAR1andIFNAR2were MACPF, P2, EX, and TM domains of P2 after stimulation with 2⁄2 unable to interact with each other in P2 BMDMs after IFN-b (Fig. 6C). Interestingly, the IFNAR1 interaction with stimulation with IFN-b (Fig. 4B). These results suggest that the IFNAR2 was unaffected in the absence of the CT domain of P2 constitutive interaction of P2 with IFNAR2 is essential for the after stimulation with IFN-b (Fig. 6C), but the interaction between IFNAR1–IFNAR2 interaction and phosphorylation and activa- IFNAR2 and STAT2 was impaired in the absence of the CT do- tion of Jak1, Tyk2, STAT1, and STAT2 after stimulation with main of P2 after stimulation with IFN-b (Fig. 6C). Next, immu- 2⁄2 IFN-b. Similar results were obtained with P2+/+ MEFs, P2 noprecipitations with anti-Flag (P2) Ab revealed that the MACPF 2⁄2 MEFs, and P2 MEFs reconstituted with wild-type P2 after domain and P2 domain of P2 are critical for the constitutive in- stimulation with IFN-b (Fig. 4E, 4F). These results strongly teractions with IFNAR1 and IFNAR2, respectively (Fig. 6D). suggest that the constitutive associated P2 with IFNAR1 and Interestingly, the STAT2 association with IFNAR2 was impaired Downloaded from IFNAR2 may mediate IFNAR1 and IFNAR2 interactions after in the absence of P2 or CT domains of P2 (Fig. 6D). As expected, stimulation with IFN-b. we have also observed an impaired IFNAR2 interaction with Direct interactions between P2, IFNAR1, and IFNAR2 STAT2 in absence of EX and TM domains of P2 (Fig. 6D). We next repeated the above experiment, and lysates were used for Our results indicate that P2 interacts independently and constitu- immunoprecipitation with anti-IFNAR2. As expected, IFNAR2 tively with IFNAR1 and IFNAR2 in resting cells. We next ex- and IFNAR1 interacted in cells expressing wild-type P2 after http://www.jimmunol.org/ amined if P2 and IFNAR1 and IFNAR2 were directly interacting stimulation with IFN-b; however, the constitutive interaction of with each other in vitro using an in vitro protein–protein interaction IFNAR2 with P2 was lost in the absence of the P2 domain of P2 method. Flag-tagged human recombinant P2 (25–655) was (Fig. 6E). Also, the interaction between IFNAR2 and STAT2 was expressed in HEK 293–T cells and subjected to affinity pull-down impaired in the absence of the P2 domain of P2. Again, the using anti-FLAG M2 agarose beads, as described previously (39, IFNAR2 interaction with STAT2 is impaired in the absence of EX 40). Purified Flag-P2 (25–655 aa) was incubated in the presence and TM domains of P2. These results suggest that the CT domain and absence of rIFNAR1-Fc (1–436 aa) or IFNAR2-Fc (1–243 of P2 may regulate the constitutive interactions between aa). Indeed, immunoprecipitation of the receptor–Fc fusion with IFNAR2 and STAT2 only when P2 is associated with IFNAR2 protein A-agarose and immunoblotting with anti-FLAG revealed by guest on September 26, 2021 via the P2 domain (Fig 6). The IFN-b–induced phosphoryla- that P2 directly interacted with both IFNAR1 and IFNAR2 tion of Jak1, Tyk2, STAT1, and STAT2 was completely abro- (Fig. 5A, 5B). Next, we applied far-western gel blotting assay gated in cells expressing P2 deleted for MACPF, P2, EX, TM, using total lysates from HeLa cells and purified Flag-tagged or CT domains (Fig. 6). The loss of IFN-b–induced Jak1 and recombinant P2 (25–655 aa) to further confirm the validity of Tyk2 phosphorylation upon deletion of the CT domain of P2 P2 interactions with IFNAR1 and IFNAR2. Striking interactions suggests that the CT domain of P2 may regulate Jak1 and Tyk2 between P2 and protein bands corresponding in size to IFNAR1/2 interactions, which are critical for Jak1 and Tyk2 reciprocal were observed in the membrane incubated with Flag-tagged transphosphorylation and STATs activation upon IFN-b stim- recombinant P2 (Fig. 5C). These results suggest that P2 is di- ulation (Fig. 6). Together, these results strongly suggest that rectly associated with IFNAR1 and IFNAR2 proteins. the MACPF, P2, and CT domains of P2 are critical for type I The extracellular MACPF and P2 domains are associated with IFN–mediated JAK–STAT signaling. IFNAR1 and IFNAR2, respectively Given the importance of the P2 CT domain in type I IFN To address the critical functional domains of P2 in type I IFN signaling, P2-deficient MEFs were next reconstituted with a signaling, we constructed deletion mutants of P2 lacking the Flag-tagged P2 triple mutant (K678R, K680R, and K681R), MACPF, P2, EX, TM, and CT domains (Fig. 6A). To begin to whichisimpairedinP2bactericidal activity (28), followed by address the localization of P2 deletion mutants, P2-deficient MEFs stimulation with IFN-b. Surprisingly, the phosphorylation of were reconstituted with the Flag-tagged P2 deletion mutants and STAT1 and STAT2 upon stimulation with IFN-b was unaffected 2⁄2 labeled with biotin to determine if the P2 mutants are expressed at in P2 MEFs expressing the mouse P2 triple (K678R, K680R, the cell surface. Wild-type P2, DMACPF, DP2, or DCT, but not and K681R) mutant (Supplemental Fig. 3). These results sug- DEX and DTM, were present on the cell surface (Fig. 6B). Next, gest that the role of P2 in Type I IFN signaling is distinct from P2-deficient MEFs reconstituted with Flag-tagged P2 deletion its bactericidal activity. We also examined the alternative splice mutants were stimulated with 1000 IU/ml IFN-b for 20 min to variant of P2, which does not express TM and CT domains of identify the domain(s) important for the activation of type I IFN wild-type P2 (58). As expected, the phosphorylation of STAT1 signaling. Surprisingly, deletion of either MACPF, P2, EX, TM, or andSTAT2wasimpairedinIFN-b–stimulated MEFs expressing CT domains of P2 led to completely abrogated IFN-b–mediated the alternative splice variant of P2 (Supplemental Fig. 4).

reconstituted with EV or Flag-tagged wild-type P2 were stimulated with IFN-b for the indicated times, and lysates were immunoprecipitated with IFNAR1 (E)orP2(F) Abs and detected by immunoblotting with Abs to Tyk2, Jak1, STAT2, IFNAR2, IFNAR1, and P2. Lysates were subjected to phosphorylated forms of STAT1, STAT2, Tyk2, and Jak1 and total STAT1, STAT2, Tyk2, Jak1, IFNAR2, and P2. The Journal of Immunology 9

FIGURE 5. Direct interactions between P2 and IFNAR1–IFNAR2. (A and B) The indicated puriﬁed recombinant proteins were incubated together for 1 h at 24˚C. The reactions were subjected to immunoprecipitation of Fc-tagged IFNAR1 or IFNAR2 proteins, and complexes were analyzed by immunoblotting with anti-Flag Ab for P2. (C) Far-western gel blot analysis of P2 interactions with IFNAR1 and IFNAR2. Total cell extracts from HeLa cells (lane 1) and negative control BSA (lane 2) membranes were incubated with puriﬁed Flag-tagged recombinant P2 and immunoblotted with anti-Flag Ab. CBB, Coomassie Brilliant Blue. Downloaded from

N-linked glycosylation of P2 is critical for the association with glycosylation-defective P2 mutants (N185A, N269A, or N375A) IFNARs and type I IFN signaling (Fig. 7D, 7E). Next, we performed Co-IP assays to determine if glycosylation-defective P2 (N185A, N269A, or N375A) mutants Glycosylation of extracellular proteins can play critical regulatory http://www.jimmunol.org/ roles in protein–protein interactions, receptor activation, and interacted with IFNARs. As expected, wild-type P2 interacted signal transduction (19, 59); therefore, we examined if P2 was with both IFNAR1 and IFNAR2, and we also detected active re- glycosylated. Lysates from P22⁄2 MEFs reconstituted with Flag- ceptor proximal complex formation in wild-type P2–expressing tagged wild-type P2 were treated with the glycosidases EndoH or cells after stimulation with IFN-b (Fig. 7D, 7E). The P2 mutants PNGase F, which specifically hydrolyses asparagine-linked high- (N185A and N269A), which are localized in the MACPF domain, mannose oligosaccharides, and immunoblotted with anti-Flag Ab. were unable to interact with IFNAR1 (Fig. 7D, 7E). Similarly, the P2 was sensitive to EndoH and PNGase F treatment as determined interaction between P2 and IFNAR2 was impaired in the N375A by faster electrophoretic migration (Fig. 7A). We next performed a mutant, which is localized in the P2 domain of P2 (Fig. 7E). Also, glycosylation consensus sequence (Asn-X–Ser/Thr) motif search the constitutive interaction between STAT2 and IFNAR2 was by guest on September 26, 2021 (60) and confirmed these results using the Uniprot protein se- abolished with the N375A mutant of P2 (Fig. 7E). These results quence database, which both yielded three N-glycosylation sites suggest that the N-glycosylation of the MACPF domain either at (N185, N269, and N375) in mouse P2. The putative glycosylation N185 or N269 and the P2 domain at N375 of P2 are critical for the sites N185 and N269 are found in the MACPF domain, and N375 constitutive associations with IFNAR1 and IFNAR2, respectively, is found in the P2 domain of P2. To determine the functional role as well as activation of type I IFN signaling. of P2 glycosylation in type I IFN signaling, we generated P2 P2 is critical for type I-IFN–mediated inhibition of glycosylation-defective single mutants (N185A, N269A, or viral replication N375A) using site-directed mutagenesis. We first determined if Because type I IFN plays a critical role in restricting virus repli- glycosylation-defective P2 mutants are present on the cell surface cation, we next examined the role of P2 in type I IFN–mediated or in the cytoplasm. P2-deficient MEFs reconstituted with wild- inhibition of virus replication. P2-deficient MEFs or HeLa cells type P2 or P2 mutants (N185A, N269A, or N375A) were surface reconstituted with wild-type P2 or the P2 (N185A) mutant were biotinylated, purified using streptavidin beads, and immunoblotted stimulated overnight with either mouse IFN-b or human IFN-b, with anti-Flag Ab. As expected, all the glycosylation-defective P2 respectively, followed by infection with the RNA virus VSV-GFP mutants were mostly localized on the cell surface, similar to at a multiplicity of infection of 1.0 as described previously (35). IFNAR1, and IFNAR2 (Fig. 7B). We also observed faster mi- As expected, type I IFN prevented VSV-GFP replication in wild- gration of P2 N185A, N269A, or N375A mutants compared with type MEFs and HeLa cells treated with type I IFN (Fig. 8). wild-type P2 (Fig. 7B). Also, as expected, IFN-b–induced phos- However, type I IFN was unable to inhibit VSV-GFP as assessed phorylation of STAT1 was severely affected in MEFs treated with by fluorescence microscopy and anti-GFP immunoblotting in PNGase F (250 U/ml) for an hour prior to stimulation with IFN-b P2-deficient MEFs and HeLa cells, as well as P2-deficient MEFs (Fig. 7C). These results suggest that P2 glycosylation is critical for and HeLa cells reconstituted with the P2 (N185) mutant (Fig. 8). type I IFN signaling. Next, to determine the functional relevance of these P2 glycosylation sites in type I IFN signaling, we reconstituted P2-deficient MEFs with either wild-type P2 or P2 Discussion mutants (N185A, N269A, or N375A). As expected, P22⁄2 We have identified an unanticipated function for P2 in the type I MEFs showed defective Jak1, Tyk2, STAT1, and STAT2 IFN signaling pathway. Septic shock caused by the type I IFN phosphorylation after stimulation with IFN-b (Fig. 7D, 7E). response induced after challenge with LPS is absent in P2-deficient Reconstitution of P22⁄2 MEFs with wild-type P2 restored the mice. The reciprocal transphosphorylation of tyrosine kinases Jak1 phosphorylation of Jak1, Tyk2, STAT1, and STAT2 (Fig. 7D, and Tyk2, which are constitutively associated with IFNAR1 and 7E). Interestingly, phosphorylation of Jak1, Tyk2, STAT1, and IFNAR2, respectively, is impaired in the absence of P2 after STAT2 was impaired in IFN-b–stimulated cells expressing the stimulation with type I IFN. In addition, the phosphorylation and 10 ROLE OF PERFORIN-2 IN TYPE I IFN–MEDIATED JAK–STAT SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. Identiﬁcation of P2 domains required for IFNAR1, IFNAR2, and STAT2 interactions in type I IFN signaling. Cell-surface expression of wild- type and mutants of P2. (A) Schematic of truncation mutants of P2. (B) P22⁄2 MEFs were transiently transfected with the indicated mutants. After 36 h, cell-surface proteins were labeled with sulfo-NHS-SS-biotin, and the biotinylated proteins were recovered with streptavidin beads. Surface P2 wild-type and mutants or whole-cell lysates were used for immunoprecipitation with anti-Flag Ab. Requirement of the MACPF, P2, and CT domains of P2 for the activation of type I IFN signaling. P22⁄2 MEFs were transiently transfected with the indicated mutants. After 36 h, cells were stimulated with IFN-b for the indicated times. Lysates were subjected to immunoblotting with Abs to phosphorylated forms of STAT1, STAT2, Tyk2, and Jak1 and total STAT1, STAT2, Tyk2, Jak1, IFNAR2, and Flag. Lysates were immunoprecipitated with either anti-IFNAR1 (C), anti-Flag (D), or IFNAR2 (E) Abs and detected by immunoblotting with Abs to IFNAR1, IFNAR2, STAT2, Tyk2, Jak1, or Flag. activation of transcription factors STAT1 and STAT2 by the kinases and PI3K signaling pathways by type I IFN was delayed in the Jak1 and Tyk2, which are critical players in the type I IFN signaling absence of P2. Although high levels of P2 protein expression in pathways, were also impaired in the absence of P2 in response to cells such as macrophages is essential to eliminate bacterial type I IFN. Furthermore, activation of the nonclassical p38 MAPK pathogens by forming pores (26), our current ﬁndings suggest that The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. N-linked glycosylation of P2 is critical in type I IFN signaling. Deglycosylation and cell-surface biotinylation of wild-type and point mutants of P2. (A) Protein lysates from P22⁄2 MEFs reconstituted with empty vector (EV) or Flag-tagged wild-type P2 were denatured and treated with either EndoH or PNGase F at 37˚C for 1 h. (B) P2 glycosylation mutants are able to translocate to the surface. Immunoblot analysis of cell-surface Flag-tagged wild-type P2, Flag-tagged P2 mutants (N185A, N269A, and N375A), IFNAR1, and IFNAR2. Cell-surface proteins were labeled with sulfo-NHS-SS-biotin, and the biotinylated proteins were recovered with streptavidin beads. Surface lysates were used for immunoblotting with anti-Flag, anti-IFNAR2, and immunoprecipitation with anti-IFNAR1 Ab and immunoblot with anti-IFNAR1 Ab. Whole-cell lysates were also used for immunoblotting with anti-Flag and anti–b-actin Abs. (C) Receptor glycosylation is critical for type I IFN signaling. Lysates from P2+/+ MEFs pretreated with PNGase for 60 min followed by stimulation with IFN-b for 20 min were subjected to immunoblotting to detect phosphorylated and total STAT1. (D and E) Requirement of P2 N-linked glycosylation is critical for the activation of type I IFN signaling. Lysates from P22⁄2 MEFs transiently transfected with the indicated mutants for 36 h and stimulated with IFN-b for the indicated times were subjected to immunoblotting with Abs to phosphorylated forms of STAT1, (Figure legend continues) 12 ROLE OF PERFORIN-2 IN TYPE I IFN–MEDIATED JAK–STAT SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 8. P2 is critical for type I IFN–mediated inhibition of VSV replication. P2-deficient MEFs (A) or HeLa cells (C) reconstituted with empty vector (EV) or wild-type Flag-P2 or Flag-P2 (N185A) mutant were stimulated overnight with either mouse IFN-b (50 IU/ml) or human IFN-a (50 IU/ml), respectively, followed by infection with VSV-GFP at an multiplicity of infection (MOI) of 1.0. Twenty hours postinfection, the VSV-GFP signal was detected by fluorescence microscopy (original magnification 320). P2-deficient MEFs (B) or HeLa cells (D) reconstituted with EV or wild-type Flag-P2 or Flag-P2 (N185A) mutant were stimulated with IFN-b (50 IU/ml) or human IFN-a (50 IU/ml), respectively, followed by infection with VSV-GFP, as indicated in (A and C). Twenty hours postinfection, whole-cell extracts were analyzed by immunoblotting using the indicated Abs. the amount of P2 expressed in MEFs is sufficient to regulate type I It is possible that the interactions mediated via the MACPF and P2 IFN signaling. We found that P2 is expressed at the cell surface domains may play a role in preventing the intimate interaction of and is constitutively associated with IFNAR1 and IFNAR2 IFNAR1 and IFNAR2 in resting cells; thus, no interaction of these through interactions with its MACPF and P2 domains, respec- receptors was observed in the absence of P2. Alternatively, P2 tively, in resting cells. Although P2 is preassociated with IFNAR1 may be required to keep the receptors in the proper conformation and IFNAR2 in resting cells, we were unable to pull down to recognize ligand and to promote proximal complex assembly. It IFNAR1 with IFNAR2 in the absence of ligand, indicating that is also possible that the P2-mediated ligand-independent complex IFNAR1 and IFNAR2 are not associated with the same P2 protein. I with IFNAR1 and complex II with IFNAR2 and STAT2,

STAT2, Tyk2, and Jak1 and total STAT1, STAT2, Tyk2, Jak1, IFNAR2, and Flag. Lysates were immunoprecipitated with either anti-IFNAR1 or anti-Flag (C) or IFNAR2 (D) Abs and detected by immunoblotting with Abs to IFNAR1, IFNAR2, STAT2, Tyk2, Jak1, or Flag. The Journal of Immunology 13 respectively, in resting cells may play different functional roles in constitutive interactions of P2 with either IFNAR1 or IFNAR2 in cell homeostasis. The scenario in the presence of ligand is dif- absence of glycosylation resulted in impairment of type I IFN ferent, as there may be additional modifications or conformational signaling. Thus, it will be important in future studies to determine changes either in P2, IFNAR1, or IFNAR2, which may allow them the exact mechanistic roles of IFNARs and P2 glycosylation in to form a larger trimeric (or tetrameric) complex III in type I IFN type I IFN signaling. Also, generation of a P2 glycosylation mu- signaling. tant knock-in mouse model will be needed to elucidate physio- Our study has also demonstrated that IFNAR1 and IFNAR2 are logical roles of P2 glycosylation in different disease settings such constitutively associated with the MACPF and P2 domains, re- as bacterial infection. spectively, in unstimulated cells. The constitutive interaction of Our data also suggest that P2-mediated complexes I and II IFNAR2 with STAT2 is also impaired in the absence-bound P2. are critical for the ligand-dependent formation of complex III Although the constitutive interaction between IFNAR2 and STAT2 (Figs. 4, 5, 7). It will be essential to determine exactly which seems dependent on the CT domain of P2, the CT domain of P2 is domains of IFNAR1 and IFNAR2 are interacting with the MACPF not sufficient to mediate the constitutive interaction between and P2 domains in both unstimulated and IFN-stimulated cells. IFNAR2 and STAT2. Therefore, we speculate that the function of We have observed loss of IFN-induced Jak1 and Tyk2 reciprocal the CT domain of P2 may be dependent on the constitutive in- transphosphorylation in cells expressing the P2 mutant with a teraction between IFNAR2 and P2 in type I IFN signaling (Fig. 6). CT domain deletion (Fig. 6). Thus, it will be interesting to de- Importantly, reintroduction of full-length P2 in cells derived from termine the mechanistic role of the CT domain in regulating Jak1 P2 knockout mice (MEFs or BMDMs) was sufficient to restore the and Tyk2 reciprocal transphosphorylation. Another possibility normal function of type I IFN signaling. Thus, our study has is that the constitutive interaction between P2 with IFNARs Downloaded from demonstrated for the first time, to our knowledge, that P2 is an may limit the cytotoxic effect of type I IFNs. A recent study essential component of type I IFN signaling. supports this hypothesis because IFNAR12/2 mice, but not P22/2 IFNAR1 plays critical roles in type I IFN–mediated autoimmune mice, are protected after S. enterica serovar typhimurium infection and inflammatory related diseases (61, 62). We found that P2 (15, 26, 63). knockout mice are resistant to LPS-induced endotoxicity, similar The defect in the phosphorylation and activation of STAT1 and 2/2 2/2 to IFNAR1 mice (22), suggesting that P2 may play a role in STAT2 we have observed in P2 cells is strikingly similar to that http://www.jimmunol.org/ type I IFN–mediated autoimmune and inflammatory diseases. It observed with Tyk22/2cells (25). In the current study, we have will be interesting to determine whether P2 regulates proin- demonstrated that Jak1 and Tyk2 are not phosphorylated in the flammatory signals that promote cancer cell proliferation in solid absence of P2 after stimulation with type I IFN. Previous studies tumors or in leukemias and lymphomas. Future studies will ex- have demonstrated that PI3K/AKT and MAPK p38 pathways are amine the roles of P2 and type I IFN in host defense against rapidly activated by type I IFN (12, 65). It has been previously pathogenic microbial infections. In addition, because type I IFN reported that a small GTPase, Ras-related protein 1 (Rap1), which plays both beneficial and detrimental roles in pathogen infec- regulates both cellular proliferation and suppression, is known to tions (2, 3), it will be interesting to determine if P2 plays bene- be an activator of PI3K/AKT and MAPK p38 in type I IFN sig- ficial or detrimental roles in different tissues in vivo upon type I naling (35). However, the mechanism of PI3K and MAPK acti- by guest on September 26, 2021 IFN administration or pathogenic infection. Although P2 and vation by Rap1 in type I IFN signaling is poorly understood. In the IFNAR12/2 mice are similarly protected upon challenge with a current study, we have demonstrated that phosphorylation of AKT high dose of LPS and mediate STAT1 and STAT2 activation upon and p38 is delayed in P22/2 cells after IFN-b stimulation, sug- type I IFN stimulation, the functional phenotypes of P2 and gesting that P2 may be functionally linked with Rap1. Because P2 IFNAR1 toward bacteria killing are distinct. P2 kills Gram- directly interacts with both IFNAR1 and IFNAR2, it is plausible positive, Gram-negative, and acid-fast bacteria by forming pores that P2 is involved upstream of Rap1 in the activation of PI3K on the surface of bacteria. P22/2 mice succumb to Gram-negative and MAPK pathways early on in type I IFN signaling. It is Salmonella enterica serovar typhimurium bacterial infection possible that P2 serves as a critical scaffold molecule for the (26), whereas IFNAR12/2 mice are protected against S. enterica activation of Rap1 and other adaptor proteins to activate PI3K serovar typhimurium infection (15, 63). This distinct functional and MAPK in type I IFN signaling. In future studies, we will phenotype between P2 and IFNAR1 knockout mice suggest that determine the mechanism by which P2 cooperates with IFNAR1 P2 likely plays additional roles in protection against bacterial and Rap1 to mediate PI3K and MAPK activation in type I IFN infections (for example, direct pore formation). Thus, it will be signaling. interesting to determine if IFNAR1 plays any detrimental roles in Together, our findings have revealed, to our knowledge, that P2 P22/2 mice after bacterial and viral infection. Future studies will functions as a novel component of the IFNARs and receptor further determine the mechanisms of IFNAR1 regulation by P2 in proximal complex formation of type I IFN signaling and is a key viral and bacterial infections. regulator of IFNAR1-mediated internalization and signaling. Previous studies have shown that glycosylation of receptors Constitutive binding of P2 to IFNAR1 and IFNAR2 may regulate plays key roles in ligand-dependent signaling pathways (19, 59). the dynamic interactions between IFNAR1 and IFNAR2 after Although both IFNAR1 and IFNAR2 are heavily glycosylated (20, stimulation with IFNs. A more-refined understanding of how P2 64), the mechanistic roles of IFNARs glycosylation in protein– interacts with and regulates IFNARs and limits IFNAR1- protein interactions (and specifically, type I IFN signaling) is mediated inflammation and tissue damage should be deter- poorly understood. It is possible that the glycosylation of IFNARs mined in future studies by solving the crystal structure of P2 may also be involved in P2, IFNAR1, and IFNAR2 complex together with IFNAR1. formation and reciprocal transphosphorylation of Jak1 and Tyk2 in type I IFN signaling. Our data clearly indicate that the P2 Acknowledgments N-glycosylation in the extracellular MACPF and P2 domains. Our We thank Dr. Edward Harhaj for discussion and a thorough review of this data also suggest that elimination of N-glycosylation of P2 either manuscript. We thank Dr. Siddharth Balachandran (Fox Chase Cancer Cen- in the MACPF or P2 domain impairs the constitutive interaction of ter, Philadelphia, PA) and Dr. Glen N. Barber (Department of Cell Biology, P2 with IFNAR1 and IFNAR2, respectively (Fig. 7). This loss in University of Miami, FL) for providing reagents and facilities. We thank 14 ROLE OF PERFORIN-2 IN TYPE I IFN–MEDIATED JAK–STAT SIGNALING

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