Different STAT Transcription Complexes Drive Early and Delayed Responses to Type I IFNs

This information is current as Ali A. Abdul-Sater, Andrea Majoros, Courtney R. Plumlee, of September 27, 2021. Stuart Perry, Ai-Di Gu, Carolyn Lee, Sujan Shresta, Thomas Decker and Christian Schindler J Immunol 2015; 195:210-216; Prepublished online 27 May 2015;

doi: 10.4049/jimmunol.1401139 Downloaded from http://www.jimmunol.org/content/195/1/210

Supplementary http://www.jimmunol.org/content/suppl/2015/05/22/jimmunol.140113 Material 9.DCSupplemental http://www.jimmunol.org/ References This article cites 44 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/195/1/210.full#ref-list-1

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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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Different STAT Transcription Complexes Drive Early and Delayed Responses to Type I IFNs

Ali A. Abdul-Sater,*,1,2 Andrea Majoros,†,1 Courtney R. Plumlee,*,3 Stuart Perry,‡ Ai-Di Gu,* Carolyn Lee,* Sujan Shresta,‡ Thomas Decker,† and Christian Schindler*,x

IFNs, which transduce pivotal signals through Stat1 and Stat2, effectively suppress the replication of Legionella pneumophila in primary murine macrophages. Although the ability of IFN-g to impede L. pneumophila growth is fully dependent on Stat1, IFN- ab unexpectedly suppresses L. pneumophila growth in both Stat1- and Stat2-deficient macrophages. New studies demonstrating that the robust response to IFN-ab is lost in Stat1-Stat2 double-knockout macrophages suggest that Stat1 and Stat2 are func- tionally redundant in their ability to direct an innate response toward L. pneumophila. Because the ability of IFN-ab to signal through Stat1-dependent complexes (i.e., Stat1-Stat1 and Stat1-Stat2 dimers) has been well characterized, the current studies focus on how Stat2 is able to direct a potent response to IFN-ab in the absence of Stat1. These studies reveal that IFN-ab is able to Downloaded from drive the formation of a Stat2 and IFN regulatory factor 9 complex that drives the expression of a subset of IFN-stimulated , but with substantially delayed kinetics. These observations raise the possibility that this pathway evolved in response to microbes that have devised strategies to subvert Stat1-dependent responses. The Journal of Immunology, 2015, 195: 210–216.

egionella pneumophila, the causative agent of Legion- IFNs, initially identified for their potent antiviral activity, me-

naires’ disease, continues to account for 4–20% of U.S. diate their biological responses through the STAT family of http://www.jimmunol.org/ L cases of community-acquired pneumonia (1). Its ability to transcription factors. They direct the rapid and robust expression of subvert the host’s immune response is dependent on the Icm/Dot a large family of IFN-stimulated genes (ISGs; reviewed in Refs. 5–9). type IV secretion system, which serves to inject numerous effector Type I IFNs (IFN-Is; IFN-ab) mediate their response through the into host cells (reviewed in Refs. 2–4). Even though IFN-a and two associated JAKs, Jak1 and Tyk2, where L. pneumophila is a human pathogen, it can infect murine mac- the activity of Jak1 is dominant (10). This culminates in the re- rophages. This model has been exploited to identify several cruitment and subsequent JAK-dependent phosphorylation of L. pneumophila pathogen-associated molecular patterns, as well as Stat1 and Stat2. Once activated, these STATs form either Stat1- their corresponding pattern recognition receptors (3). These pat- Stat1 or Stat1-Stat2 dimers. The Stat1 homodimers directly by guest on September 27, 2021 tern recognition receptors then direct the production of potent bind to IFN-g activation site (GAS) enhancers to drive a rapid inflammatory mediators, including TNF-a, IL-1b, and type I IFNs expression of target genes (e.g., IFN regulatory factor [IRF]-1, (IFN-Is). LMP2, and Stat1) (11, 12), whereas the Stat1-Stat2 hetero- dimers associate with IRF-9 to form ISG factor (ISGF) 3. This binds to the IFN-ab–stimulated responsive *Department of Microbiology and Immunology, Columbia University, New York, NY 10032; †Department of Microbiology, Immunology, and Genetics, University of element (ISRE) to direct the rapid expression of a distinct set of Vienna, Vienna A-1030, Austria; ‡Division of Vaccine Discovery, La Jolla Institute genes, which include Mx-1, Oas, inducible NO synthase (iNOS), x for Allergy and Immunology, La Jolla, CA 92037; and Department of Medicine, ISG15, Ifit2, Ifit3, Ddx58 (a.k.a., RIG-I), Dusp1, Dusp2, and Bst2, as Columbia University, New York, NY 10032 well as many other genes (6–8). Of note, this group of genes also 1A.A.A.-S. and A.M. contributed equally to this work. includes the suppressor of signaling (Socs) 1, responsible for 2Current address: Department of Immunology, University of Toronto, Toronto, ON, Canada. rapidly downregulating JAK activity (12, 13). Intriguingly, there is 3Current address: Department of Immunology, University of Connecticut, Farming- also compelling evidence that basally secreted IFN-Is play an im- ton, CT. portant role in host homeostasis, including directing basal Stat1 and ORCID: 0000-0003-1598-1529 (C.S.). Stat2 expression (11, 12, 14). In contrast, type II IFN (IFN-g)ex- Received for publication May 8, 2014. Accepted for publication April 24, 2015. pression is more restricted and mediates its response through a sig- This work was supported by National Institutes of Health Grants AI 058211 (to naling cascade, consisting of the IFN-g receptor, Jak1, Jak2, and Stat1 A.A.A.-S., C.R.P., C.L., and C.S.), AI096088 (to A.A.A.-S., C.L., and C.S.), 5T32 (5, 8, 15). Analogous to IFN-Is, IFN-g–activated Stat1 homodimers AI07525 (to C.R.P.), 5T32 DK007328 (to C.R.P.), and AI082185 (to S.P. and S.S.) and Fonds zur Fo¨rderung der Wissenschaftlichen Forschung Grants SFB-28 and induce the expression of GAS-driven genes. W1220-B09 (to A.M. and T.D.). Even though type I and II IFNs both effectively suppress Address correspondence and reprint requests to Dr. Christian Schindler, Columbia L. pneumophila replication in macrophages, their responses are University, HHSC 1208, 701 West 168th Street, New York, NY 10032. E-mail address: mediated by distinct signaling pathways (16–18). The ability of [email protected] IFN-g, but not IFN-I, to suppress L. pneumophila growth is ab- The online version of this article contains supplemental material. rogated in Stat12/2 macrophages. IFN-Is also retain their ability to Abbreviations used in this article: BMM, bone marrow–derived murine macrophage; effectively suppress bacterial growth in Stat22/2 macrophages (17). ChIP, chromatin immunoprecipitation; GAS, IFN-g activation site; iBMM, immor- talized BMM; IFN-I, type I IFN; iNOS, inducible NO synthase; IRF, IFN regulatory Similar observations have been reported for measles, lymphocytic factor; ISG, IFN-stimulated ; ISGF, ISG factor; ISRE, IFN-ab–stimulated responsive choriomeningitis, and Dengue viruses (19, 20). To explore these element; P6, pyridone 6; Socs, suppressor of cytokine signaling; WT, wild-type. observations, Stat1-Stat2 double-knockout macrophages were gener- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 ated. Unexpectedly, IFN-Is lost their ability to suppress L. pneumophila www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401139 The Journal of Immunology 211 growth in these macrophages, suggesting that Stat1 and Stat2 function Invitrogen, Carlsbad, CA). A quantity amounting to 200 ng (2 mg in Fig. 4) redundantly in their ability to mediate this response. Although the of total RNA was reverse transcribed (Moloney murine leukemia virus; mechanism by which Stat1 independently signals is well understood Invitrogen) (17, 34). cDNA was PCR amplified by either standard or quantitative approaches with SYBR Green master mix (Promega, Madison, (i.e., Stat1 homodimers) (5), the mechanism by which Stat2 signals WI; or Applied Biosystems, Foster City, CA) on a real-time thermocycler independently of Stat1 has not been fully characterized (19–25). The (Stratagene MX3005p or Eppendorf Mastercycler EP Realplex) with gene- current study exploits primary Stat12/2 macrophages to explore this specific primers (see Supplemental Table I). Expression was normalized to Stat2-only pathway. Genetic and biochemical studies reveal that acti- either a GAPDH or b-actin control. vated Stat2 associates with IRF-9, whereupon it binds to the ISRE Chromatin immunoprecipitation element to drive the of target genes. The kinetics of this Chromatin immunoprecipitation (ChIP) assays were performed essentially response is both delayed and dependent on persistent stimula- as described (19). Briefly, BMMs were fixed in 1% formaldehyde and lysed tion. These observations raise the possibility that this pathway poten- in 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 1% SDS, and sonicated tially evolved to function as a backup response in the setting of either an and diluted with 9 parts 50 mM Tris-HCl (pH 8.0), 167 mM NaCl, 1.1% inherited or acquired loss of Stat1 (26–28). Triton X-100, and 0.11% sodium deoxycholate. A total of 10 mg sonicate was then immunoprecipitated ( G–agarose beads; Pierce Thermo Scientific, Rockford, IL) with a murine-specific Stat2 Ab and eluted at Materials and Methods 65˚C. Eluted DNA was treated with RNase A and proteinase K, and then Legionella pneumophila The L. pneumophila JR32 (restriction-defective Philadelphia-1, streptomycin- resistant) strain was grown in AYE broth or on CYE plates, as previously described (17). C57BL/6J bone marrow–derived murine macrophages Downloaded from (BMMs) were infected with a L. pneumophila strain that was also Fla2 (Flagellin deficient) (17). Mice The 129 and C57BL/6J mice were purchased from Jackson ImmunoResearch Laboratories and bred in a specific pathogen-free facility. Homozygous Stat12/2, Stat22/2, IRF-92/2, and Stat12/2/Stat22/2 double-knockout http://www.jimmunol.org/ mice were either from 129 (Fig. 1) or C57BL/6J backgrounds, as previ- ously reported (11, 29–31). The Columbia University Institutional Animal Care and Use Committee approved all animal studies in New York, and the institutional ethics committee at the University of Vienna determined that all studies carried out in Vienna were in accordance with Austrian law (permit GZ 680 205/67-BrGt/2003). Cell culture Primary murine macrophages were prepared by culturing bone marrow cells in RPMI 1640 or DMEM (Invitrogen-Life Technologies, Grand Island, by guest on September 27, 2021 NY), 10% FCS (Hyclone, Logan, UT), Penn/Strep (Life Technologies), and 20% L929 conditioned media for day 7–10 cultures, as previously reported (17, 32). Cells were stimulated with murine IFN-aA/D (1000 U/ml; PBL, Piscataway, NJ), which is active on human and murine cells; murine IFN-b (250 U/ml; PBL); or murine IFN-g (50 U/ml; PBL). In some IFN-I–treated cells, the JAK inhibitor tetracyclic pyridone 6 (P6; 2 mM; Calbiochem, La Jolla, CA) was added 1 or 4 h prior to harvest. Some macrophages were immortalized with a v-/v-raf–expressing retrovirus (33). Growth curves In vivo bacterial growth was evaluated by infecting (multiplicity of infec- tion = 0.25) day 6 BMMs (2.5 3 105 BMMs per well of 24-well plate) with postexponential phase L. pneumophila, as reported (17). All infections were carried out in triplicate and verified through at least three independent studies. Biochemical studies

Whole-cell extracts were prepared from IFN-aA/D– or IFN-b–treated cells and evaluated by immunoblotting with Abs specific for Stat1 (Santa Cruz Biotechnology, Dallas, TX) (11, 12), phospho-Stat1 (Cell Signaling, Beverly, MA), Stat2 (11, 12), phospho-Stat2 (UBI/EMD-Millipore, Temecula, CA), phospho-Stat1 (Cell Signaling), Jak1 and phospho-Jak1 (UBI/EMD-Millipore), and tubulin (Sigma-Aldrich, St. Louis, MO), as previously reported (11, 12, 34). For EMSA, whole-cell or nuclear extracts were prepared and evaluated, FIGURE 1. L. pneumophila growth in Stat12/2 and Stat22/2 BMMs. as previously reported (11, 35). Briefly, extracts (1–4 ml) were incubated L. pneumophila (JR-32; Lp: multiplicity of infection = 0.25) growth was with a [32P]dATP–labeled ([32P]-g-dATP; 6000 Ci/mMol; PerkinElmer, Waltham, MA) dsOAS oligonucleotide probe with binding buffer. In some evaluated by a colony-forming assay (24, 48, or 72 h postinfection) in 129 A 2/2 B 2/2 C 2/2 studies, extracts were preincubated (30 min, 4˚C) with 1–2 ml Abs (11, 12, (WT) control, ( ) Stat1 ,( ) Stat2 , and ( ) Stat1/Stat2 double- 5 21) or competed with a 7-fold excess of cold dsOAS oligonucleotide si- knockout BMMs in the 129 background (2.5 3 10 /well of 24-well plate), multaneous to the addition of radiolabeled probe. as previously described (17). Some BMMs were pretreated with a single dose of IFN-aA/D (1000 U/ml) or IFN-g (50 U/ml) 2 h prior to infection. RT-PCR Please note that (A) and (B) were previously published (17) and are in- cluded solely for comparison’s sake. Studies are representative of more Total RNA was prepared from day 6 BMMs before or after IFN-aA/D treatment with the Nucleospin RNA (Macherey-Nagel, Dueren, Ger- than three independent experiments. Similar results were obtained in the many), according to manufacturer’s instructions, or by TRIzol (Fig. 4; C57BL/6J background. 212 Stat2–IRF-9 SIGNALING purified with QIAquick PCR Purification kit (Qiagen, Valencia, CA) prior significant roles for other IFN-I–stimulated mediators, like p38 to SYBR Green PCR base Q-PCR on the Light-Cycler 480 PCR System and phosphoinositide 3 kinases in the suppression of L. pneumo- (Roche, Indianapolis, IN) for 45 cycles (see Supplemental Table I for phila growth (17, 38) (see also Supplemental Fig. 1A). These primers). The percentage of input DNA was determined by comparing cycle threshold value obtained with immunoprecipitated DNA and cycle observations were consistent with a number of other reports (19, threshold value obtained from input DNA. 20, 23) and raised the intriguing possibility that Stat1 and Stat2 are redundant in their ability to suppress L. pneumophila in response Generation of lentiviral particles and knockdown of IRF-9 in to IFN-Is. Because IFN-I–dependent Stat1 homodimer formation immortalized BMMs and transcriptional activity have been well documented (11, 12), pGIPZ lentiviral short hairpin RNAvectors specific for murine IRF-9 (clone subsequent studies focused on how Stat2 may signal indepen- V2LMM_167172; see Supplemental Table I) and a proprietary control dently of Stat1 (i.e., in Stat12/2 macrophages). (RHS4346) were obtained from Open Biosystems (Huntsville, AL). Fresh, filtered (0.45-mm) lentivirus, prepared as previously reported (36), was 2/2 used to infect immortalized wild-type (WT) and Stat12/2 BMMs. Posi- Stat2 is able to direct ISRE-driven gene expression in Stat1 tive populations were selected and maintained on puromycin (5 mg/ml; cells Thermo-Fisher). Studies exploring the mechanism by which IFN-Is suppress L. pneumophila had excluded an important role for iNOS, an IFN Results target gene (17, 34). However, they also surprisingly revealed that IFN-Is direct a Stat2-dependent suppression of L. pneumophila IFN-I–dependent iNOS expression was equivalent in WT and growth Stat12/2 BMMs at 24 h of stimulation (17). This observation Classic studies identified a Stat1-Stat2 heterodimer as critical in suggested that Stat2 could direct iNOS expression independently Downloaded from transducing the biological response to IFN-Is (5, 11, 37). Unex- of Stat1, consistent with other recent in vitro studies on IFN-I– pectedly, however, recent studies revealed that IFN-I (IFN-ab) dependent ISG expression (i.e., Apobec3g, Adar1, Nox2, and Oas) was able to robustly suppress L. pneumophila growth in both (20, 22–25). Moreover, our contemporaneous studies on Dengue Stat12/2 and Stat22/2 macrophages, whereas the ability of IFN-g virus–infected WT and Stat12/2 BMMs revealed overlapping to suppress growth was completely dependent on Stat1 (17). To patterns of ISG expression (19). more rigorously explore this unexpected finding, Stat1-Stat2 To further investigate these findings, expression of two well- http://www.jimmunol.org/ double-knockout (Stat12/2/Stat22/2) mice were generated. As characterized ISRE-driven ISGs, ISG-15 and Mx-1, was carefully previously reported (17), IFN-a effectively suppressed L. pneu- evaluated by quantitative PCR in WT, Stat12/2, Stat22/2,andIRF-92/2 mophila growth in WT, Stat12/2, and Stat22/2 macrophages in BMMs (7, 8, 11, 35). As anticipated, both genes were rapidly induced both the 129 (Fig. 1A, 1B) and C57BL/6J backgrounds (data not by IFN-I in WT BMMs (Fig. 2A). Consistent with previous studies, shown). Intriguingly, this response was lost in Stat12/2/Stat22/2 both genes were difficult to detect in Stat12/2 BMMs early after IFN-I macrophages, in both the 129 (Fig. 1C) and C57BL/6J back- stimulation (i.e., by 4 or 8 h), and they were essentially undetectable grounds (data not shown). The robust IFN-a–dependent activation in Stat22/2 and IRF-92/2 BMMs(Fig.2A)(11,29,39).Remark- of Stat3 in Stat12/2/Stat22/2 macrophages excluded the possi- ably, however, at later time points both genes were robustly induced bility that Stat3 might play an important role in the suppression of by IFN-I in Stat12/2 BMMs. Similar observations were made with by guest on September 27, 2021 L. pneumophila growth (Supplemental Fig. 1A). Likewise, pre- other well-known ISRE-driven ISGs (i.e., Ddx58, Ifit2, Ifit3, Dusp1, vious studies in Stat12/2 and Stat22/2 macrophages had excluded Dusp2, ADAR, Bst2, and Oas2; see Supplemental Fig. 1B).

FIGURE 2. Delayed kinetics of IFN-a–stim- ulated gene expression and Stat2 activation. (A)

The kinetics of IFN-aA/D (1000 U/ml)–dependent Mx-1 and ISG-15 expression was evaluated by quantitative PCR in C57BL/6J (WT), Stat12/2, Stat22/2, and IRF-92/2 BMMs. Similar results were obtained with IFN-b treatment and BMMs from the 129 background. (B) Whole-cell extracts (WCEs) were prepared from day 6 (d6) C57BL/6J (WT) (top), Stat12/2 (middle), and Stat22/2 (bottom) BMMs after stimulation with

IFN-aA/D (1000 U/ml), as indicated. Extracts were fractionated and immunoblotted with Abs specific for phospho-Stat1 (pSt1; Cell Signal- ing), phospho-Stat2 (pSt2; UBI), and tubulin (Sigma-Aldrich). The same extracts were refrac- tionated by SDS-PAGE and immunoblotted for total-Stat1 (tStat1; Santa Cruz) and total-Stat2 (tStat2) (11). Studies are representative of more than three independent experiments. Similar results were obtained with IFN-b treatment and in the 129 background. The Journal of Immunology 213

To determine whether this delayed expression correlated with the correlation between the delayed kinetics of Stat2 activation IFN-I–dependent STAT activation, extracts from WT, Stat12/2, and ISG expression in IFN-I–stimulated Stat12/2 BMMs. 2/2 and Stat2 BMMs were evaluated by immunoblotting with Abs 2/2 specific for the activated (i.e., tyrosine-phosphorylated) isoforms Stat2 directs the formation of ISRE-binding complex in Stat1 of Stat1 and Stat2. As expected, IFN-I stimulated the rapid, robust, cells and transient activation of Stat1 and Stat2 in WT BMMs (Fig. 2B, To explore the possibility that Stat2 independently directs the top panel) (12, 37). Yet, in Stat12/2 BMMs, significant quantities expression of ISRE-driven genes in Stat12/2 BMMs, EMSAs and of phospho-Stat2 did not begin to accumulate until after prolonged ChIP studies were carried out. As previously reported for WT IFN-I stimulation (i.e., 12, 18, and 24 h; Fig. 2B, middle panel). BMMs, IFN-I stimulated robust activation of ISGF3 (Stat1:Stat2: Moreover, this delayed activation correlated with the belated, IFN- IRF-9) DNA-binding activity (see Fig. 3A) (11, 12, 37). Consis- I–dependent nuclear accumulation of Stat2 in the Stat12/2 BMMs tent with the preceding phospho-immunoblotting results, ISGF3 (see Supplemental Fig. 2). Consistent with previous results (11, activity peaked early (0.5–2 h; data not shown) in WT cells (12), 12), IFN-I–treated Stat22/2 BMMs exhibited a rapid (i.e., 0.5 h; after which it rapidly decayed. In contrast, the ISRE DNA-binding see Fig. 2B, bottom panel), albeit modest Stat1 activation that activity observed in IFN-I–treated Stat12/2 BMMs was negligible corresponded closely with the expression of GAS-driven target at early time points (e.g., 0.5 h), but became pronounced by genes (e.g., IRF-1 and Stat1; Supplemental Fig. 1B) (11, 12). 16 h (Fig. 3A), reflecting the pattern of Stat2 phosphorylation Intriguingly, there was also a second peak of phospho-Stat1, cor- (Fig. 2B). Moreover, this complex exhibited a distinct and slower relating with the delayed Stat2 activation observed in Stat12/2 mobility that did not appear to correlate with the faster IRF-1

BMMs, as well as a prolonged pattern of GAS-driven gene ex- ISRE-binding complex observed in IFN-I–treated cells (40). Downloaded from pression (see Supplemental Fig. 1B). These observations highlight Further excluding IRF-1 was its limited expression in Stat12/2 http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. IFN-Is activate a novel ISRE- binding complex in Stat12/2 BMMs. (A) Nuclear extracts from IFN-aA/D–treated (1000 U/ml; 0.5 and 16 h), day 6 C57BL/6J (WT), and Stat12/2 BMMs were evaluated with a radiolabeled ISRE probe. In some samples, a 7-fold excess of cold ISRE probe (comp) or Abs specific for Stat2 (a-St2) or IRF-9 (a-IRF-9) were added. Migra- tion of ISGF3 and the novel complex are indi- cated. Studies are representative of more than three independent experiments. Similar results were obtained in the 129 background. (B) For ChIP studies, IFN-a–treated C57BL/6J (WT), Stat12/2, and Stat12/2Stat22/2 BMMs, as above, were cross-linked, sonicated, and immunoprecipi- tated with a Stat2-specific Ab and interrogated by quantitative PCR. Error bars represent the SEM, and asterisks denote statistically significant differ- ences (*p , 0.05, **p , 0.01, ***p , 0.0005). 214 Stat2–IRF-9 SIGNALING

BMMs (see Supplemental Fig. 1B). Of note, the slower migrating DNA-binding complex observed in the Stat12/2 BMMs was remi- niscent of one identified when Stat2 and IRF-9 were overexpressed in U3A cells (21). Moreover, this slower migrating complex was supershifted by Abs specific for murine Stat2 and IRF-9, as was the case for bona fide ISGF3 in WT BMMs (Fig. 3A) (11, 12, 41). Next, ChIP studies were employed to determine whether com- ponents of this novel complex were recruited to the promoters of bona fide ISRE-driven genes, as we recently reported for Dengue virus–infected cells (19). Although limitations with the IRF-9 Ab precluded directly evaluating recruitment of this component, the Stat2 Ab highlighted effective Stat2 recruitment to Mx-1 and ISG15 promoters at both 0.5 and 16 h of IFN-I stimulation in WT BMMs. In contrast, in Stat12/2 BMMs Stat2 was only recruited to these two promoters after 16 h of IFN-I stimulation. These observations are not only consistent with prior reports suggesting a Stat2–IRF-9 complex may drive the expression of some genes (21–23, 25), but also demonstrated that this more slowly migrating

ISRE-binding complex is recruited to the promoters of bona fide Downloaded from ISGs. To confirm that IRF-9 was required for the delayed expression of ISGs observed in Stat12/2 BMMs, IRF-9 was knocked down in immortalized BMMs (iBMMs) through lentiviral-mediated short hairpin IRF-9 RNA (Fig. 4A). Consistent with studies on IRF-92/2

BMMs (Fig. 2A), Mx-1 failed to be expressed in WT iBMMs in http://www.jimmunol.org/ which IRF-9 had been knocked down (Fig. 4A). More importantly, IRF-9 was required for the IFN-I–dependent expression of both Mx-1 and ISG-15 in Stat12/2 iBMMs (Fig. 4A). An additional analysis in Stat1/Stat2 double-knockout BMMs revealed that Stat2 is absolutely required for IFN-I–dependent gene expression in Stat1-deficient macrophages (Fig. 4B). These data provide addi- tional support for the model that, absent Stat1, a Stat2–IRF-9 complex directs the expression of ISRE-driven genes, albeit with substantially delayed kinetics. FIGURE 4. IRF-9 is required for IFN-I–stimulated ISG expression in by guest on September 27, 2021 Stat12/2 BMMs. (A) Efficiency of IRF-9 knockdown was evaluated by 2/2 Activation of the Stat2–IRF-9 complex requires continuous quantitative PCR in C57BL/6J (WT) and Stat1 iBMMs (top left). IFN- IFN-I–dependent signaling aA/D–dependent Mx-1 expression was determined by quantitative PCR in C57BL/6J (WT) iBMMs stably knocked down with either short hairpin

Next, studies were undertaken to explore why Stat2 was activated IRF-9 or a short hairpin control (Open Biosystems; top right). IFN-aA/D– 2/2 with such delayed kinetics in Stat1 BMMs. Because the basal dependent Mx-1 and ISG-15 expression was similarly evaluated in Stat12/2 2 2 level of Stat2 was reduced in Stat1 / BMMs (Fig. 2), the first set iBMMs stably knocked down with either short hairpin IRF-9 or a short B of studies probed Stat2 t1/2 through cycloheximide-dependent hairpin control. ( ) IFN-aA/D–dependent Mx-1 and ISG-15 expression was similarly evaluated in C57BL/6J (WT) or Stat1/Stat2 double-knockout turnover (Supplemental Fig. 3). These studies revealed that 2 2 2/2 BMMs (Stat1/2 / ). Data represent three independent replicates. Stat2 protein had a considerably shorter t1/2 in Stat1 than WT BMMs. However, Stat2 levels increased steadily upon IFN-I treatment in both WT and Stat12/2 BMMs, although the re- these cells were treated with a potent JAK inhibitor, P6. As sponse was delayed in Stat12/2 BMMs (e.g., Fig. 5B). Consistent anticipated,theadditionofP61hpriortoharvestafterashort with this, the level of Stat2 transcripts increased more rapidly in IFN-I treatment (i.e., 0.5 or 1 h) of WT BMMs led to a sub- WT than Stat12/2 BMMs upon IFN-I treatment (Supplemental stantial reduction in STAT phosphorylation, demonstrating this Fig. 1B), suggesting that resting Stat12/2 cells may not express drug effectively blocked JAK-dependent activation (Fig. 5B, top sufficient, stable levels of Stat2 to enable a rapid and robust sig- panels). Likewise, a 4-h pulse of P6 prior to harvesting IFN-I– naling response. treated WT cells at 18 or 22 h had little effect because the signal A second set of studies explored whether the ligand-dependent had already decayed. However, in Stat12/2 BMMs, the 4-h (data not shown) and delayed Stat2 activation observed in Stat12/2 P6 pulse significantly impaired the delayed activation of Stat2 BMMs was associated with a prolonged activation of Jak1, the (Fig. 5B, bottom panels). Consistent with this, the addition of P6 dominant JAK in IFN-I response (10). As previously reported, in the final 4 h of IFN-a stimulation led to a significant reduction Jak1 activity, interrogated through phospho-immunoblotting, was in ISG-15 and Mx-1 gene expression in Stat12/2 BMMs, as well rapidly induced in IFN-I–stimulated WT BMMs. But this activa- as WT BMMs (Fig. 5C). These studies confirmed that prolonged tion was quite transient, a response that has been attributed to the Stat2 activation in Stat12/2 BMMs was dependent on prolonged IFN-I–dependent expression of an important negative regulator, and ligand-dependent Jak1 activity. Socs1 (12, 13, 42). In contrast, Jak1 phosphorylation was more A final set of studies explored whether a defect in Socs1 ex- robust and prolonged in the Stat12/2 BMMs, indicating an ex- pression might account for prolonged Jak1 activity in IFN-I–treated tended duration of activity (Fig. 5A). Stat12/2 BMMs. In contrast to the rapid and robust expression To determine whether this enhanced Jak1 activity was im- profile in WT BMMs, Socs1 expression was substantially reduced portant for the delayed activation of Stat2 in Stat12/2 BMMs, in IFN-I–stimulated Stat12/2 BMMs, but less dramatically in the The Journal of Immunology 215 Downloaded from

2/2 FIGURE 5. Jak1 is activated with delayed kinetics in IFN-I–stimulated Stat1 BMMs. (A) Jak1 was immunoprecipitated from IFN-aA/D C57BL/6J 2/2 (WT) and Stat1 BMM WCEs and immunoblotted with an Ab specific for activated Jak1, as in Fig. 2. (B) IFN-aA/D–stimulated WCEs were prepared from C57BL/6J (WT) and Stat12/2 BMMs and immunoblotted as in Fig. 2 (left panels). P6 inhibitor (2 mM) was added for either 1 or 4 h before IFN-I– stimulated extracts were harvested, as indicated. Samples were then immunoblotted, as described in Fig. 2. (C) Mx1 and ISG15 expression was evaluated by B D 2/2 2/2 quantitative PCR in samples from ( ), as detailed in Fig. 2. ( ) Socs1 expression was evaluated by quantitative PCR in C57BL/6J (WT), Stat1 -, Stat2 -, http://www.jimmunol.org/ and IRF-92/2–treated BMMs, as described in Fig. 2. Data represent three independent replicates.

Stat22/2 and IRF-92/2 BMMs (Fig. 5D). This was consistent with BMMs failed to robustly induce Socs1 expression, an important prior studies that highlighted an important role for ISGF3 in Socs1 negative regulator of IFN-I–stimulated JAK activation (12, 13). expression and IFN-I signal decay (12, 13, 42). In summary, these Third, a dose-response study revealed that the amounts of IFN-I observations suggest that, in the absence of ISGF3, Stat12/2 required to achieve equivalent levels of phospho-Stat2 in WT and 2 2 BMMs exhibit reduced basal levels of Stat2 and defective Socs1 Stat1 / BMMs led to equivalent levels of target gene expression expression. This culminates in a prolonged and IFN-I–dependent at early time points in WT BMMs versus later time points in by guest on September 27, 2021 2 2 Jak1 activation (and likely Tyk2) (5, 42), as well as the accu- Stat1 / BMMs, respectively (Supplemental Fig. 3B). These ob- mulation of Stat2 protein. After 12–16 h of IFN-I stimulation, servations are consistent with a model, in which over time, ac- sufficient levels of Stat2 accumulate to drive significant ISG cumulating Stat2 protein and prolonged JAK activity reach the expression. threshold of phospho-Stat2 required for IFN-I–dependent signal- ing. However, it is certainly possible that rate-limiting concen- trations of IRF-9, the second component of the signaling complex, Discussion may also contribute to this delay. Additional models could be The ability of IFN-Is to mediate their potent biological responses considered, including a specific restriction in nuclear translocation through canonical Stat1-Stat1 and Stat1-Stat2 (plus IRF-9) dimers (44), elevated phosphatase activity (45), or an enhanced capacity has been well documented (5, 8, 14). However, the capacity of for phospho-Stat2 degradation in Stat12/2 BMMs. 2/2 IFN-Is to effectively suppress L. pneumophila growth in Stat1 The ability of a Stat2–IRF-9 complex to drive the expression 2/2 and Stat2 macrophages, as well as the more important role of a subset of ISGs was presaged by several cell line–based, Stat2 plays in the innate response to Dengue virus, suggested the overexpression studies. The first such study demonstrated that involvement of an additional pathway (17, 43). Likewise, the with ectopic overexpression in HEK-293 cells, activated Stat2 observation that IFN-I– dependent L. pneumophila suppression could dimerize and, with the addition of ectopic IRF-9, form 2/2 2/2 was lost in Stat1 /Stat2 macrophages raised the intriguing a more slowly migrating ISRE-binding complex (21). Additional possibility that Stat1 and Stat2 may function redundantly. To ex- overexpression studies in Stat1-deficient U3A cells revealed plore how Stat2 signals independently of Stat1, biochemical a physical interaction between IRF-9 and activated Stat2 (22). 2 2 studies were carried out in primary Stat1 / BMMs. They de- Moreover, this complex, along with the unusually prolonged termined that, absent Stat1, IFN-Is direct a delayed activation of Stat2 activation kinetics observed in NB4 cells, was ascribed Stat2, which then associates with IRF-9 to bind ISRE sites and aroleinIFN-I–stimulatedRIG-G expression. Likewise, Stat1 2 2 drive the late expression of a subset of ISGs. Stat1 / BMMs and Stat2 knockdown studies in Hep3B cells highlighted a more exhibited a number of features likely to contribute to the delayed important role for Stat2 (versus Stat1) in the IFN-I–dependent kinetics of target gene expression. First, basal Stat2 expression induction of several ISGs, including APOBEC3G (23); but IRF- was reduced, likely because of increased basal turnover and re- 9 knockdown was associated with only a partial block of ISG duced basal transcription (11, 14), raising the possibility that this expression. Remarkably, a parallel knockdown of Stat1 in HEK- basal Stat2 level was below the threshold required for effective 293 cells revealed an essential role for this STAT in the IFN-I– signaling. Consistent with this model, IFN-I treatment increased dependent expression of the same ISGs (23). Similarly, two both Stat2 transcription and protein stability, culminating in an studies in primary cells, including our own with Dengue virus, increased Stat2 protein level. Second, absent ISGF3, Stat12/2 revealed a more important role for Stat2 than Stat1 in the innate 216 Stat2–IRF-9 SIGNALING response to several viruses, but did not elucidate the signaling 20. Hahm, B., M. J. Trifilo, E. I. Zuniga, and M. B. Oldstone. 2005. Viruses evade the immune system through type I -mediated STAT2-dependent, but pathway responsible for this effect (19, 20). Finally, a more re- STAT1-independent, signaling. Immunity 22: 247–257. cent study has ascribed a more important role for Stat2 in the 21. Bluyssen, H. A., and D. E. Levy. 1997. Stat2 is a transcriptional activator that ability of IFN-b and TNF-a to synergistically stimulate the ex- requires sequence-specific contacts provided by and p48 for stable inter- action with DNA. J. Biol. Chem. 272: 4600–4605. pression of DUOX2 NADPH oxidase in two epithelial lines (25). 22. Lou, Y. J., X. R. Pan, P. M. Jia, D. Li, S. Xiao, Z. L. Zhang, S. J. Chen, Z. Chen, Confirming several earlier overexpression studies, our results and J. H. Tong. 2009. IRF-9/STAT2 [corrected] functional interaction drives demonstrate that, in primary Stat12/2 cells, Stat2 can associate retinoic acid-induced gene G expression independently of STAT1. Cancer Res. 69: 3673–3680. with IRF-9 to drive the delayed expression of a subset of ISGs 23. Sarkis, P. T., S. Ying, R. Xu, and X. F. Yu. 2006. STAT1-independent cell type- important in the innate response to L. pneumophila, Dengue virus, specific regulation of antiviral APOBEC3G by IFN-alpha. J. Immunol. 177: as well as potentially other viruses. Although we find little evi- 4530–4540. 24. George, C. X., S. Das, and C. E. Samuel. 2008. Organization of the mouse RNA- dence of a significant quantity of activated Stat2–IRF-9 complex specific adenosine deaminase Adar1 gene 59-region and demonstration of in WT BMMs, it is intriguing to speculate that this pathway STAT1-independent, STAT2-dependent transcriptional activation by interferon. Virology 380: 338–343. evolved as a backup response to defend against pathogens that 25. Fink, K., L. Martin, E. Mukawera, S. Chartier, X. De Deken, E. Brochiero, impede Stat1 activity (e.g., Paramyxovirus) (27). It is also possible F. Miot, and N. Grandvaux. 2013. IFNb/TNFa synergism induces a non- that, during severe immune stress, Stat1 may become functionally canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH oxidase-mediated airway antiviral response. Cell Res. 23: 673–690. deficient, providing an additional setting in which the Stat2–IRF-9 26. Zhang, S. Y., S. Boisson-Dupuis, A. Chapgier, K. Yang, J. Bustamante, A. Puel, pathway could provide a backup response. In future studies, it will C. Picard, L. Abel, E. Jouanguy, and J. L. Casanova. 2008. Inborn errors of be interesting to explore whether this delayed pathway serves to interferon (IFN)-mediated immunity in humans: insights into the respective roles of IFN-alpha/beta, IFN-gamma, and IFN-lambda in host defense. Immunol. Rev. afford a more durable IFN-I response during chronic microbial 226: 29–40. Downloaded from challenges, or potentially to integrate responses between IFN-I 27. Ramachandran, A., and C. M. Horvath. 2009. Paramyxovirus disruption of interferon and TNF-a (25). signal transduction: STATus report. J. Interferon Cytokine Res. 29: 531–537. 28. Versteeg, G. A., and A. Garcı´a-Sastre. 2010. Viral tricks to grid-lock the type I interferon system. Curr. Opin. Microbiol. 13: 508–516. Disclosures 29. Meraz, M. A., J. M. White, K. C. Sheehan, E. A. Bach, S. J. Rodig, A. S. Dighe, D. H. Kaplan, J. K. Riley, A. C. Greenlund, D. Campbell, et al. 1996. Targeted The authors have no financial conflicts of interest. disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in

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