Toxoplasma Gondii Dysregulates IFN-␥-Inducible Gene Expression in Human Fibroblasts: Insights from a Genome-Wide Transcriptional Proﬁling1

The Journal of Immunology

Toxoplasma gondii Dysregulates IFN-␥-Inducible Gene Expression in Human Fibroblasts: Insights from a Genome-Wide Transcriptional Proﬁling1

Seon-Kyeong Kim, Ashley E. Fouts, and John C. Boothroyd2

Toxoplasma gondii is an obligate intracellular parasite that persists for the life of a mammalian host. The parasite’s ability to block the potent IFN-␥ response may be one of the key mechanisms that allow Toxoplasma to persist. Using a genome-wide microarray analysis, we show here a complete dysregulation of IFN-␥-inducible gene expression in human fibroblasts infected with Toxo- plasma. Notably, 46 of the 127 IFN-␥-responsive genes were induced and 19 were suppressed in infected cells before they were exposed to IFN-␥, indicating that other stimuli produced during infection may also regulate these genes. Following IFN-␥ treatment, none of the 127 IFN-␥-responsive genes could be significantly induced in infected cells. Immunofluorescence assays showed at single-cell levels that infected cells, regardless of which Toxoplasma strain was used, could not be activated by IFN-␥ to up-regulate the expression of IFN regulatory factor 1, a transcription factor that is under the direct control of STAT1, whereas uninfected cells in the same culture expressed IFN regulatory factor 1 normally in response to IFN-␥. STAT1 trafficked to the nucleus normally and indistinguishably in all uninfected and infected cells treated with IFN-␥, indicating that the inhibitory effects of Toxoplasma infection likely occur via blocking STAT1 transcriptional activity in the nucleus. In contrast, a closely related apicomplexan, Neospora caninum, was unable to inhibit IFN-␥-induced gene expression. A differential ability to interfere with the IFN-␥ response may, in part, account for the differences in the pathogenesis seen among Toxoplasma and Neospora parasite strains. The Journal of Immunology, 2007, 178: 5154–5165.

oxoplasma gondii is an obligate intracellular parasite that distant tissues such as the lungs, muscles, and brain within a few induces a strong IFN-␥-driven cell-mediated immune re- days (14). It is plausible that the parasite reaches these tissues T sponse in its mammalian hosts. This response is critical for before the arrival of IFN-␥-secreting NK and T cells and, given the resolution of acute infection (1) and control of a chronic, latent time to establish an infection and co-opt/subvert host cell pro- infection in the CNS (2). Various cell types are activated by IFN-␥ cesses, Toxoplasma can effectively inhibit the IFN-␥ signaling to acquire potent toxoplasmacidal mechanisms, including induc- pathway and enhance its intracellular survival and persistence. ible NO synthase and IFN-␥-induced GTPases expression (3–6). Indeed, recent studies have shown that several IFN-␥-responsive In addition, IFN-␥ plays a role in driving the conversion of genes could not be induced by IFN-␥ in Toxoplasma-infected cells tachyzoites (the acute phase form) to bradyzoites (the chronic (11–13). IFN-␥ exerts its effects via the transcriptional activation phase form) (7) and suppresses the reactivation to tachyzoites (8). of numerous genes involved in antimicrobial activity, Ag process- IFN-␥ is produced at high levels in acutely infected mice (100– ing and presentation, lymphocyte trafficking, cell growth, and ap- 300 ng/ml serum at 7 days postinfection) (9) and remains at 1–6 optosis (15). IFN-␥ binds a ubiquitously expressed cell surface ng/ml serum even at 3 wk postinfection (10). Clearly, IFN-␥ is a IFN-␥ receptor, which is associated with JAK1 and JAK2 that potent immune effector and, yet, Toxoplasma is not cleared but phosphorylate STAT1 at Tyr701 (16). Phosphorylated STAT1 persists in immunocompetent hosts. For optimal control of Toxo- forms homodimers that then translocate to the nucleus and initiate plasma replication in vitro, host cells should be activated with the transcription of IFN-␥ target genes. Additional phosphoryla- IFN-␥ before infection (3–6); if the cells are infected first, subse- tion at Ser727 is thought to be necessary for a maximal transcrip- quent exposure to IFN-␥ is unable to control infection and the tional activity (17, 18). Toxoplasma appears to use various mech- parasite grows normally (11–13). Toxoplasma disseminates rap- anisms to interfere with the STAT1-dependent IFN-␥ signaling idly following oral infection of a mammalian host and reaches pathway: in heavily infected murine macrophages, a type I Toxo- plasma strain (BK) was reported to induce a high-level SOCS1 mRNA expression and degradation of STAT1 protein (13). In con- Department of Microbiology and Immunology, Stanford University School of Med- trast, a type II strain (NTE) inhibited IFN-␥-inducible gene ex- icine, Stanford, CA 94305 pression without altering STAT1 protein stability (11, 12). As dis- Received for publication September 14, 2006. Accepted for publication February cussed in Ref. 11, STAT1 trafficking was found to be intact in 7, 2007. infected NIH3T3 cells overexpressing STAT1-GFP fusion protein. The costs of publication of this article were defrayed in part by the payment of page In contrast, the same group reported in (12) that nuclear trafficking charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. of endogenous STAT1 appeared to be partially inhibited by infec- 1 This work was supported by National Institutes of Health Grants AI41014 and tion in heavily infected macrophages. These previous studies ex- AI21423. amined the expression of only several IFN-␥-responsive genes 2 Address correspondence and reprint requests to Dr. John C. Boothroyd, Department mostly by performing RT-PCR using a population of heavily in- of Microbiology and Immunology, Stanford University School of Medicine, 299 fected macrophages. The extent to which Toxoplasma blocks IFN- Campus Drive, Stanford, CA 94305-5124. E-mail address: [email protected] ␥-inducible gene expression, the effects of infection in cell types Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 other than macrophages (e.g., in nonphagocytic cells in which the www.jimmunol.org The Journal of Immunology 5155 parasite persists at low numbers), and the exact mechanisms of or lacking recombinant human IFN-␥ (100 U/ml) was added. After 2, 4, and8hofIFN-␥ treatment, total RNA was extracted using TRIzol reagent parasite interference remain unknown. ϩ In this study, we examined how Toxoplasma-infected human (Invitrogen Life Technologies) and poly(A) mRNA was isolated using ␥ the Oligotex kit (Qiagen). As a reference, mRNA was isolated from com- fibroblasts respond to IFN- . Fibroblasts in the smooth muscle mercially available total RNA pooled from 10 human cell lines may be important cell types for infection and persistence (19, 20). (Stratagene). Fibroblasts are now known to play an important role in shaping the cDNA preparation and hybridization were essentially conducted as de- course of an immune response in tissues by regulating the switch scribed previously (30). Briefly, reference and sample cDNAs were directly labeled with Cy3-dUTP and Cy5-dUTPs (Amersham), respectively, from acute inflammation to adaptive immunity and tissue repair through random nonamer priming using Klenow enzyme. Following quan- (21). Nonphagocytic, nonprofessional APCs, such as fibroblasts, titation and purification, reference (250 ng) and sample (300 ng) cDNA critically depend on IFN-␥ to develop antimicrobial properties and were simultaneously hybridized to human cDNA microarrays spotted on an ability to interact with T cells effectively (15). It would be to the Corning Ultragap slides (Stanford Functional Genomics Facility, Stanford parasite’s advantage to infect these cells and block the expression University, Stanford, CA; 40,996 spots representing 23,228 unique puta- ␥ tive genes). Duplicate cultures were analyzed for each time point. of IFN- target genes to persist. We compared whether and by Microarrays were scanned using Axon Genepix 4000A and gridded us- what mechanisms three major clonal types (I, II, and III) of Tox- ing Genepix 5.1 (Molecular Devices). Data were entered into Stanford oplasma (22) and its closely related apicomplexan species Neos- Microarray Database and two-dimensional spatial local estimation was pora caninum (23, 24) inhibit the IFN-␥-inducible gene expres- used to normalize the spots. Data were filtered to remove poor quality spots (regression correlation Ͻ0.6 and Cy3 channel intensity Ͻ3-fold back- sion, because the host range, virulence, and ability to persist vary ground) and retrieved as Log2 of Cy5/Cy3 normalized ratio (median). Two- greatly among these parasites (24–26), although IL-12 and IFN-␥ class SAM (statistical analysis of microarrays) (31) was performed using are key to controlling infection in all cases (1, 27, 28). MeV 3.0 (www.tigr.org) to identify the genes showing significantly dif- We show in this study that Toxoplasma, but not Neospora, suc- ferent expression levels in uninfected cells before and after IFN-␥ cessfully blocks the effects of IFN-␥. Microarray analysis reveals treatment. Biological network analyses were performed using the software avail- that by some means (probably an early proinflammatory cytokine able from the Ingenuity Systems (www.ingenuity.com) with the IFN-␥- response) Toxoplasma infection causes a differential expression of responsive genes showing differential expression levels in uninfected and ϳ50% of the 127 IFN-␥-inducible genes in human foreskin fibro- infected cells before IFN-␥ treatment. 3 ␥ blasts (HFFs) before infected cells are exposed to IFN- , but that Cytokine Ab arrays the other half shows no such change. Addition of IFN-␥ to infected cells had no significant effect on any of the 127 IFN-␥-inducible For qualitative determination of the kinds of cytokines and chemokines secreted by infected HFFs, cytokine Ab arrays were performed with culture genes, indicating a highly efficient means by which Toxoplasma supernatant. Confluent HFF monolayers were left uninfected or infected defends itself against this potent immune mediator. with Pru strain parasites at a nominal MOI of 3 as described in human cDNA microarray analysis. Culture supernatant (containing 10% FCS) was Materials and Methods harvested after 18 h and used in human cytokine Ab arrays according to the manufacturer’s instructions (RayBiotech). Briefly, supernatant was incu- Parasites and cell culture bated with an array membrane on which 42 different human cytokine Abs The following T. gondii strains representing the three major clonal types were bound. The membrane was then incubated with a mixture of biotin- were used in this study: RH (type I) (10) and CL14 (type III) (provided by ylated anti-cytokine Abs followed by HRP-conjugated streptavidin. Detec- Dr. J. Saeij, Stanford University, Stanford, CA) expressing GFP under the tion was by chemiluminescence reagents provided by the manufacturer. GRA1 promoter, and Pru (type II) expressing GFP under the GRA2 pro- Immunofluorescence microscopy moter. The minimal GRA2 promoter region (29) was PCR-cloned from the Pru strain genomic DNA using the following primers: 5Ј-GACCAAGCT HFFs grown on coverslips were infected with indicated parasite strains for TCCCGTCGCACGGTGATACTG-3Ј and 5Ј-CGGCATGCATTGTGAG 18 h at a MOI of 0.5 and treated with IFN-␥ (100 U/ml) for the indicated GCGATATGTGGAGAA-3Ј. The 317-bp PCR product was digested with amount of time. Cells were fixed in 3.7% formaldehyde (20 min at room HindIII and NsiI and replaced the GRA1 promoter in the pGRA1-GFP/ temperature), blocked in PBS containing 3% FCS (30 min at room tem- pDHFR-HPT plasmid as described in Ref. 10. Transformation of the Pru perature), and permeabilized in 0.2% Triton X-100 (1 h at room temper- strain and selection for hypoxanthine-xanthine-guanine phosphoribosyl- ature or overnight at 4°C). Cells were incubated with the following mouse transferase activity was performed as described previously (10). Toxo- mAbs in PBS containing 3% FCS (1 h at room temperature): anti-IFN plasma strains and N. caninum (NC-1) were maintained by passage in HFF regulatory factor (IRF)-1 (BD Biosciences; clone 20); anti-total STAT1 monolayers cultured in DMEM supplemented with 10% FCS, penicillin (BD Biosciences; clone 42; recognizes a C-terminal epitope shared by (100 U/ml), streptomycin (100 ␮g/ml), and L-glutamine (2 mM) (Invitro- STAT1␣ and STAT1␤); anti-pTyr701 STAT1 (BD Biosciences; clone 4a); gen Life Technologies) in a humidified, 5% CO2 incubator. and anti-HLA-DR/DP/DQ (BD Biosciences; clone TU¨ 39). Detection Ab ␥ was goat anti-mouse IgG coupled with Alexa Fluor 594 (Molecular Probe). IFN- treatment of infected HFFs Coverslips were mounted on a glass slide for fluorescence microscopy as Parasites were released from intracellular vacuoles by syringe lysis as de- described previously (10). scribed previously (10) and washed three times in complete medium before Western blots inoculation of HFFs grown to confluency in flasks or on coverslips. Cells were infected or left uninfected for 18 h at a nominal multiplicity of in- Western blots were performed as previously described (10) with total cel- fection (MOI) of 3 for microarray and Western blot experiments and at a lular proteins prepared from HFFs grown in 25-cm2 flasks (ϳ106 cells per MOI of 0.5 for immunofluorescence microscopy (IFM). For IFN-␥ treat- flask) infected or not with the indicated parasite strains at a MOI of 3. At ment, the cells were given fresh medium containing recombinant human 18 h postinfection, Ͼ70% of the cells were infected with 1–3 vacuoles IFN-␥ at 100 U/ml (ϳ2 ng/ml) (R&D Systems) and incubated further for containing 2–8 parasites. Cells were washed three times in ice-cold PBS the indicated amount of time. and scraped into ice-cold lysis buffer (400 ␮l per flask). Lysates were boiled in the presence of 0.1 M DTT, and 20 ␮l was loaded per lane in a Human cDNA microarray analysis 12% denaturing SDS-PAGE (8% gel for JAK1 and JAK2 immunoblots). Proteins were transferred to nitrocellulose membrane and probed with the Type II Toxoplasma strain, Pru, was used to infect confluent monolayers of 701 2 following Abs: mouse monoclonal IgG against total STAT1; pTyr - HFFs in 175-cm flasks. At 18 h postinfection, culture medium was re- STAT1 and IRF1 that were also used in IFM; rabbit polyclonal anti- moved from all uninfected or infected flasks and fresh medium containing pSer727-STAT1, anti-pTyr1022/1023-JAK1, and anti-pTyr1007/1008-JAK2 IgG (Cell Signaling Technology); rabbit polyclonal anti-suppressor of cytokine 3 Abbreviations used in this paper: HFFs, human foreskin fibroblasts; MOI, multi- signaling (SOCS)1 IgG (Santa Cruz Biotechnology; clone H-93); and plicity of infection; IFM, immunofluorescence microscopy; SAM, statistical analysis mouse monoclonal anti-GAPDH IgG (Calbiochem; clone 6C5). Mem- of microarrays; IRF, IFN regulatory factor; SOCS1, suppressor of cytokine signaling branes were incubated overnight at 4°C with primary Abs diluted in 2% 1; FDR, false discovery rate; PIAS, protein inhibitor of activated STAT. nonfat dry milk in Tris-buffered saline (140 mM NaCl, 2.7 mM KCl, 25 5156 INHIBITION OF IFN-␥-INDUCIBLE GENE EXPRESSION BY Toxoplasma

mM Tris base (pH 7.4)). HRP-conjugated anti-mouse and anti-rabbit IgG were used as secondary reagents (Bio-Rad). Detection was by SuperSignal chemiluminescence reagents (Pierce Biotechnology). The manufacturer of anti-pTyr1022/1023-JAK1 and anti-pTyr1007/1008-JAK2 IgG noted a possible mutual cross-reactivity. Results A genome-wide microarray analysis reveals a complete dysregulation of IFN-␥-inducible gene expression in Toxoplasma-infected human fibroblasts To understand the extent of Toxoplasma inhibition of IFN-␥-inducible gene expression in human fibroblasts, we performed mRNA expression profiling using human cDNA microarrays hav- ing 40,996 spots representing 23,228 unique putative genes. Be- cause the effects of IFN-␥ are mediated largely by the transcriptional activation of target genes, the microarray experiments were performed with a population of HFFs in which Ͼ90% of the cells were infected to detect parasite inhibition of IFN-␥-inducible gene expression. At 18 h postinfection with a type II strain (Pru) at a MOI of 3, infected cells had 1–3 vacuoles containing 2–8 parasites per vacuole. At this time, cells were treated with IFN-␥ (100 U/ml) for 0, 2, 4, and 8 h (named P0, P2, P4, and P8, respectively). Uninfected HFFs treated with IFN-␥ for equal amounts of time were used as controls (named N0, N2, N4, and N8, respectively). Duplicate cultures were analyzed for each condition. Among the 17,004 good quality spots represented in at least 9 of the total 16 arrays, 150 spots representing 127 unique putative genes were identified as being significantly up-regulated in uninfected cells following IFN-␥ treatment (2-class SAM of N0 vs N2, N4, and N8 at 10% false discovery rate (FDR)) (Fig. 1 and Sup- plemental Table I) (the on-line version of this article contains supplemental material). No genes were identified as being significantly suppressed by IFN-␥ in uninfected cells. Remarkably, none of the 127 IFN-␥-responsive genes were found to be significantly induced by IFN-␥ in infected HFFs by 2-class SAM of P0 vs P2, P0 vs P4, or P0 vs P8 at 10% FDR (Fig. 1). Notably, 65 of 127 IFN-␥-responsive genes showed differential expression in Pru-infected (P0) vs uninfected cells (N0) before IFN-␥ treatment (Table I) (see Supplemental Table I for infection- induced fold change values for all IFN-␥-responsive genes): with the P0:N0 ratio of 2 as a cutoff value for a significant difference in expression levels, 46 genes showed increased expression in infected cells (“P0 Ͼ N0” group) and 19 genes showed decreased expression in infected cells (P0 Ͻ N0 group), whereas the remaining 64 genes were expressed at similar levels (P0 ϭ N0 group). Although type I IFNs and IFN-␥ share numerous common target genes (32), type I IFNs is an unlikely cause for the P0 Ͼ N0 and P0 Ͻ N0 groups because Toxoplasma-infected HFFs produce lit- tle, if any, IFN-␣ and -␤ (M. Lodoen, unpublished data). Addi- tionally, infected HFFs may be in an unresponsive state to IFN-␣ and -␤, because the expression of IRF9 and STAT2 that form a

FIGURE 1. IFN-␥-inducible gene expression in human ﬁbroblasts uninfected or infected with Toxoplasma. Human cDNA microarrays were performed with HFFs uninfected (N) or infected with type II Pru strain for 18 h (P) and then treated with IFN-␥ (100 U/ml) for the given amount of time. For example, N2 indicates an uninfected culture 2 h after IFN-␥ treatment. Data from duplicate arrays per time point were retrieved as

log2(sample/reference) and are shown as a color scheme. Shown are data for all 150 spots (representing 127 unique putative genes) identified as being significantly induced by IFN-␥ in uninfected HFFs (2-class SAM of N0 vs N2, N4, and N8 at 10% FDR). Gene symbols and GenBank iden- tifications are shown. Gray rectangles indicate no data. See Supplemental Table I for gene names and fold change values. The Journal of Immunology 5157

Table I. Basal expression levels of IFN-␥-inducible genes in uninfected and Toxoplasma-infected HFFs before IFN-␥ treatmenta

Gene Symbol GenBank Identification Gene Symbol GenBank Identification Gene Symbol GenBank Identification

P0 Ͼ N0 group (46 genes) R91258 FLJ13273 N32085 PBEF1 AA489629 AA487750 FLJ34922 AA465168 PHLDA1 AA258396 AA281932 FLJ34922 H80525 PIK3AP1 R62339 AA991624 FLJ39370 N38839 PMAIP1 AA458838 H57105 FST AA701860 PRKAA1 R33152 C18orf54 AA281744 HPS3 AA918982 RIPK2 AA913804 C1GALT1 N73031 ICAM1 N92072 SCML1 AI537061 C21orf91 AA400378 IRF7 AA477347 SLC15A2 AA425352 C6orf192 R67222 KLF6 AA156946 SLCO1B1 H62893 C8orf1 H25041 LOC25391 AA400157 SOD2 T60269 CBR3 AI352345 LOC90355 R52538 SOD2 W78148 CCL2 T77816 MSX1 R33154 STAMBP1 AI017607 CCL2 AA425102 MT1G AI745626 VAV2 AA682337 CCNB2 AI932735 MYCBP AA424831 VRK2 AA282291 DDX46 H89698 NFKBIZ H70961 ZNF9 AA625995 DIP2C AA873341 NT5E R60343 FAS AA293570 NUDCD1 N32587

P0 Ͻ N0 group (19 genes) H11453 IFITM1 AA419251 SAMD9L AA490264 AA971543 IGF1 AA456321 SAMD9L AA988857 GBP2 W72748 ISGF3G AA291389 SAMD9L AA996042 GBP2 W01896 MX1 AA405416 SOCS1 AA280136 IFIT1 AI953299 MX2 AA286908 TRAFD1 N21170 IFIT1 AA074989 OAS1 AA146772 TRIB2 AA053865 IFIT1 AA489640 OAS2 R72243 UBE2L6 AW071596 IFIT2 AA143609 RARRES3 W47350 UBE2L6 AA292031 IFIT2 N63988 RNF36 AA133281

P0 ϭ N0 group (64 genes) T55558 GBP1 AA486849 PELI1 W86504 AA149783 GBP3 R78509 PLSCR1 AA058514 AA433993 GBP4 AI268082 PLSCR1 N25945 T72621 HERC6 AA487462 PSMB8 AI983836 AA402719 HLA-E R94660 RALA H97948 AI275489 HSPB8 H57493 SAMHD1 AA421603 AA973917 HSPB8 AA010110 SFRS1 AA455164 AFF3 AI004671 IFI16 AA287732 SLMAP N49107 APOL6 R68682 IFI35 AA827287 SP110 AA504832 ATXN7 R63241 IFIH1 AA911194 STAT1 AA486367 BCL6 R99749 INDO AA478279 TAP1 AA487429 BCL6 AA521434 ING1 N47308 TAP1 AI346384 C12orf11 AA479106 IRF2 AA416883 TMPRSS13 AA290867 CCDC75 AA781508 IRF7 AA877255 TRIM25 N73575 CFHL3 T74567 JUN AA293362 TRIM25 AA464251 CMAH N29639 JUN W96134 TRIM25 AA281936 CSF1 AA878257 KCTD3 AA448160 USP42 R71889 CSTF3 AA465143 KIAA1033 R53810 WARS AA857888 DMN AA877815 KLF4 AW008766 WARS AA664040 EPSTI1 AI286247 LAP3 R69306 XPR1 AA453474 ETV1 T60063 MX1 AA456886 XRN1 AA504116 FLJ20160 AA259115 NCOA3 AI302669 ZKSCAN1 W84769 G1P2 AA120862 PAPD5 AA699802 ZNF313 R38967 G1P3 AA432030 PAPD5 AA029273 ZNF313 AA504825 G1P3 AA075725 PARP14 T64956 ZNFX1 AA099652

a Expression levels were compared in HFFs uninfected (N0) and infected with Pru strain for 18 h (P0) that were not treated with IFN-␥. Two-fold increase or decrease in expression levels was used as cutoff value to classify genes into the P0 Ͼ N0, P0 Ͻ N0, or P0 ϭ N0 group. Data for 150 spots representing 127 unique genes are shown. Two spots for MX1 and IRF7 are represented in two different groups, causing the sum of all genes in three groups to be 129. trimeric complex with STAT1 in response to IFN-␣ and -␤ (16) and IL-1␤ pathways that lead to the activation of downstream tran- appear to be suppressed in infected cells (P0:N0 ratio ϭ 0.24 and scription factor NF-␬B (Fig. 2A) (36, 37). Indeed, many of the 0.12, respectively; see Supplemental Table II for information on IFN-␥-inducible genes in the P0 Ͼ N0 group have been shown to all 3,245 genes differentially expressed in N0 and P0). be NF-␬B target genes (e.g., CCL2, FAS, FST, ICAM1, IRF7, Although IFN-␥ signaling is mainly transduced via the JAK/ MSX1, NFKBIZ, and SOD2) (http://people.bu.edu/gilmore/nf-kb/ STAT1 pathway, other cytokines, such as IL-1␤ (33, 34) and target/index.html). In addition, IL-1␤ is one of the most highly TNF-␣ (35), are also known to activate some IFN-responsive induced genes by infection (140-fold increase) and likely ex- genes (16). A biological network analysis of the IFN-␥-inducible pressed as a functional protein, because its known target gene genes in the P0 Ͼ N0 group identiﬁed one major network that PTGS2/COX2 is also up-regulated 174-fold during infection (also showed an association of the genes in our data set with the TNF-␣ see Fig. 3 below). TNF-␣ was induced 1.7-fold in infection and its 5158 INHIBITION OF IFN-␥-INDUCIBLE GENE EXPRESSION BY Toxoplasma

FIGURE 2. Biological networks relevant to the IFN-␥-inducible genes differentially expressed in Toxoplasma-infected cells before IFN-␥ exposure. Three groups of IFN-␥-responsive genes (Table I) were uploaded onto the Ingenuity Pathway Analysis (www.ingenuity.com) to identify molecular networks in which these genes are known to function. The analyses show direct (e.g., binding, modification; indicated as thin lines) and indirect (e.g., regulation of gene expression; indicated as dotted lines) relationships between genes that are known in the literature. The analyses found only one major network for each of the following three groups of IFN-␥-responsive genes. The software chose a certain number of IFN-␥-inducible genes in each of the three groups as focus genes to build molecular networks (indicated by underlining and bolding); genes shown in plain face are interacting genes that the software identified but that did not emerge as IFN-␥-inducible genes in our microarray analysis. Gene names are positioned in the figures based on their known subcellular localization. Such information is not available for the four genes shown in the “Unknown” box in B and C. A, Analysis for the IFN-␥-inducible genes in the P0 Ͼ N0 group (i.e., expression levels increased Ն2-fold during infection). The software used 13 of 46 genes from this group as focus genes in creating networks. B, Analysis for the IFN-␥-inducible genes in the P0 Ͻ N0 group (i.e., expression levels decreased Ն2-fold during infection). Twelve of 19 genes from this group were used as focus genes. C, Analysis of the IFN-␥-inducible genes in the P0 ϭ N0 group (i.e., infection-induced changes in expression levels were Ͻ2-fold). Eighteen of 64 genes from this group were used as focus genes. target genes, TNFAIP3 and TNFAIP6 (TNF-␣-induced proteins), posure to IFN-␥. This result is consistent with a recent report that were up-regulated up to 30-fold. FAS, encoding a member of the NF-␬B p50/p65 dimers were basally bound to the promoters of TNF receptor superfamily known to activate NF-␬B, is induced some IFN-responsive genes and that the stimulation of these genes 3.66-fold. Both TNF-␣ and IL-1␤ are known to be potent inducers by IFN was higher in NF-␬B-deficient mouse fibroblasts than in of NF-␬B-dependent CCL2 production (38), and CCL2 was found wild-type cells (39). to be induced 25-fold during infection in our microarray experi- Nineteen IFN-␥-responsive genes in the P0 Ͻ N0 group (Table ments. The expression of NF-␬B1 itself was found to be induced I) are also associated with the TNF-␣ pathway (Fig. 2B). Among 5-fold during infection. Taken together, the above data suggest that these genes, IFIT1, IFIT2, IFITM1, MX1, MX2, and OAS1 have early proinflammatory cytokines produced by type II Toxoplasma- been shown to be up-regulated in reovirus-infected HeLa cells in infected HFFs may also regulate a large number of IFN-␥-respon- a NF-␬B-dependent manner (40). Thus, it seems that the IFN-␥- sive genes via the recruitment of NF-␬B and independently of responsive genes in the P0 Ͻ N0 group may also be regulated by STAT1, and that these genes cannot be further induced upon ex- NF-␬BinToxoplasma-infected HFFs, although mechanisms by

FIGURE 3. Human cytokine arrays with Toxoplasma-infected HFF culture supernatant. Culture supernatant from uninfected HFFs (A) and HFFs infected with Pru parasites for 18 h at MOI of 3 (B) were analyzed in human cytokine arrays as described in Ma- terials and Methods. Both uninfected and infected cells were cultured in medium containing 10% FCS. The table identiﬁes the position of a given cytokine analyzed in the arrays. Cytokines abundantly produced by infected cells are indicated in bold. Aster- isks indicate the cytokines also produced by uninfected cells at high levels. The Journal of Immunology 5159

FIGURE 4. IFN-␥-inducible IRF1 expression in Toxoplasma- and Neospora-infected HFFs. A, Top panels are IFM images showing IRF1 expression (anti- IRF1 in red) in uninfected cells after IFN-␥ treatment (100 U/ml) for the indicated amount of time. Same exposure time was used for all IRF1 images. Correspond- ing Hoechst nuclear staining is shown in middle panels. Bottom panels for 0- and 0.5-h time points are longer exposure images for the same cells shown at top and demonstrate the presence of IRF1 in the host cell nucleus. B, IFN-␥-induced IRF1 expression in HFFs infected with GFP-expressing Toxoplasma strains RH, Pru, and CL14 representing types I, II, and III, respectively. HFFs were infected for 18 h and then treated with IFN-␥ for2or6h.Top panels in each time point show parasite GFP (green) and anti-IRF1 (red); lower panels in each vertical pair are images after merging Hoechst nuclear staining (blue). C, IFN-␥-induced IRF-1 expression (red) in HFFs infected with N. caninum for 18 h. The NC-1 strain used lacks GFP expression and infected cells can be identiﬁed in the phase images. D, Western blots showing basal expression levels of STAT1 (91 kDa), IRF1 (48 kDa), and SOCS1 (24 kDa), in HFFs uninfected or infected with indicated parasite strains for 18 h at a MOI of 3. Approximately 70–80% of the cells were infected with one to three vacuoles per cell containing two to eight parasites per vacuole at the time of lysate preparation. GAPDH (36 kDa) was used as a loading control. E, Western blots showing SOCS1 expression levels in HFFs uninfected or infected with RH or Pru strains as in D following IFN-␥ stimulation for 2 and 4 h. Data shown are rep- resentative of three experiments performed.

which these genes are suppressed in Toxoplasma infection are un- increased the repertoire of secreted factors, and infected cells clear. It is possible that Toxoplasma is able to directly suppress a produced IL-1␤, GRO-␣ (ϭ CXCL1), MCP-2 (ϭ CCL8), specific subset of IFN-␥-responsive genes. MCP-3 (ϭ CCL7), and RANTES (ϭ CCL5) in addition to The IFN-␥-responsive genes in the P0 ϭ N0 group (Table I) MCP-1, IL-6, and IL-8. These cytokines and chemokines were largely associated with the IFN-␥/STAT1 signaling pathway produced by infected HFFs are known to be target genes of (Fig. 2C). This group of genes may be regulated mainly by IFN-␥ NF-␬B (http://people.bu.edu/gilmore/nf-kb/target/index.html). in a STAT1-dependent manner, and not by other cytokines such as Thus, data indicate the activation of NF-␬B in infected HFFs, IL-1␤ and TNF-␣. Included in this group are STAT1 itself and the which may regulate the expression of a subset of IFN-␥-respon- genes that function in the MHC Ag processing and presentation sive genes. pathway (i.e., PSMP8, LAP3, TAP1, and HLA-E). In summary, our microarray experiments demonstrated that We then performed a human cytokine array analysis to examine none of the IFN-␥-responsive genes could be significantly up-reg- the kind of cytokines and chemokines produced by infected HFFs ulated in Toxoplasma-infected HFFs upon IFN-␥ treatment, indi- that may regulate the expression of a subset of IFN-␥-responsive cating an efficient means by which the parasite blocks the effects of genes in the absence of IFN-␥. Interestingly, we found that unin- IFN-␥. Moreover, we have shown that numerous IFN-␥-respon- fected HFFs secreted several cytokines, chemokines, and growth sive genes can also be regulated by other stimuli produced during factors into the culture medium, including high levels of GRO (ϭ infection. CXC), MCP-1 (ϭ CCL2), IL-6, and IL-8 (Fig. 3A). This finding was not simply due to the presence of 10% FCS in the culture All three clonal types of Toxoplasma, but not N. caninum, block ␥ medium, because uninfected cells cultured for 24 h without FCS the IFN- -inducible expression of the transcription factor IRF1 still produced the same cytokines but at somewhat reduced As mentioned above, various cytokines and chemokines produced amounts (data not shown). Infection with Pru strain (type II) at high levels in Toxoplasma-infected cultures may modulate the 5160 INHIBITION OF IFN-␥-INDUCIBLE GENE EXPRESSION BY Toxoplasma expression of numerous IFN-␥-inducible genes. Such immune me- Thus, parasite induction of inhibitory regulators such as SOCS1 diators may have played a role in the inability to detect IFN-␥- and protein inhibitor of activated STAT (PIAS) 1 is unlikely to be inducible gene expression in macrophages heavily infected with responsible for the lack of IRF1 induction in Toxoplasma-infected Toxoplasma (11–13), given that macrophages are potent producers HFFs. It is more likely that the block in IRF1 expression is due to of proinflammatory cytokines and chemokines, and the milieu pro- a defect in STAT1 activation and/or its transcriptional activity in duced by infected macrophages may be far more complex than that the nucleus. produced by HFFs. To minimize the effects of other cytokines produced during infection and examine specific interactions be- ␥ tween Toxoplasma and the STAT1-dependent IFN-␥ signaling IFN- -induced STAT1 nuclear translocation is normal in pathway, we performed IFM using HFFs infected at a low MOI of Toxoplasma-infected cells but does not result in autoinduction 0.5 with parasites that had been thoroughly washed. of STAT1 or MHC class II expression STAT1 transcription factor is the main signal transducer of the IFN-␥ binding to its cell surface receptor triggers the phosphory- ␥ IFN- response and is required for resistance to Toxoplasma (41). lation of cytoplasmic STAT1 at Tyr701, a prerequisite for STAT1 To see whether Toxoplasma specifically interferes with STAT1 in dimerization and nuclear translocation (48). We examined whether ␥ HFFs, we examined by IFM the IFN- -inducible expression of STAT1 activation and nuclear translocation is intact in Toxoplas- IRF1, a transcription factor that is under the direct control of ma-infected HFFs in response to IFN-␥. ␥ Ϫ/Ϫ STAT1 and cannot be induced by IFN- in STAT1 cells (42). In uninfected HFFs not treated with IFN-␥, IFM for total ϳ IRF1 protein turns over rapidly (half life 30 min) and is consti- STAT1 (i.e., irrespective of phosphorylation state) showed low tutively expressed at low levels to drive the basal expression of levels of STAT1 broadly distributed in the cytoplasm and nucleus MHC class I and other genes involved in Ag presentation, anti- of all cells (Fig. 5A; 0 h). This finding is consistent with the fact Ϫ/Ϫ microbial activity, and apoptosis (43). IRF1 mice are ex- that STAT1 is found in the nuclei of unstimulated cells (49, 50) tremely susceptible to Toxoplasma infection (44), consistent with and functions as a constitutive transcription factor for some genes ␥ a crucial role for this downstream mediator of the IFN- response. without needing ligand-mediated tyrosine phosphorylation and ho- HFFs constitutively expressed low levels of IRF1 that were pre- modimer formation (51). At 0.5 h of IFN-␥ treatment, cytoplasmic dominantly localized in the nucleus (Fig. 4A; 0 h). As expected, STAT1 intensity decreased and nuclear STAT1 intensity increased ␥ IFN- treatment rapidly induced IRF1 expression in HFFs and sharply in all HFFs (Fig. 5A), indicating mobilization of cytoplas- resulted in a strong nuclear staining with anti-IRF1 after2hof mic STAT1 to the nucleus. At2hofIFN-␥ treatment, some ␥ IFN- treatment (Fig. 4A). Uninfected cells within Toxoplasma- STAT1 reappeared in the cytoplasm, likely indicating redistribu- infected cultures also showed normal levels of IRF1 induction af- tion of nuclear STAT1. Cytoplasmic STAT1 staining increased ter stimulation with IFN-␥ (Fig. 4B). However, all cells infected noticeably at 6 h and was very bright at 12 h of IFN-␥ treatment with Toxoplasma, regardless of which strain was used, failed to (Fig. 5A), indicating new STAT1 protein synthesis. up-regulate IRF1 expression within2hofIFN-␥ treatment (Fig. In Toxoplasma-infected cultures, all cells, infected or not and 4B). IFN-␥ stimulation for 6 h (Fig. 4B) and 24 h (data not shown) regardless of infecting strains, showed total STAT1 expression did not result in IRF1 induction in Toxoplasma-infected cells. levels and localization patterns similar to that in uninfected cul- Even increasing the IFN-␥ concentration up to 500 U/ml did not tures at 0, 0.5, and2hofIFN-␥ treatment (Fig. 5B for Pru) (data induce IRF1 in Toxoplasma-infected cells (data not shown). Often, not shown for RH and CL14). Thus, early events of STAT1 acti- we observed a lack of IFN-␥-induced IRF-1 expression in cells vation in the cytoplasm and nuclear trafficking proceeded normally containing a vacuole of only one or two parasites (Fig. 4B; Pru at in infected cells. At6hofIFN-␥ treatment, uninfected cells within 2 h and RH at 6 h), suggesting that impairment of the STAT1- the infected culture began to accumulate STAT1 in the cytoplasm, dependent IFN-␥ signaling pathway is not dependent on parasite but Pru-infected cells tend to lack such increase in cytoplasmic replication. In contrast, IFN-␥ induced normal levels of IRF1 ex- STAT1 despite the persistent presence of STAT1 in the nucleus pression in cells infected with N. caninum (Fig. 4C). (Fig. 5B). At 12 h of IFN-␥ treatment, regardless of which Toxo- Previously, it was reported that infection with type I Toxo- plasma strain was used, 20–40% of infected cells lacked cyto- plasma caused STAT1 degradation and induced high levels of plasmic STAT1 staining despite positive nuclear staining (Fig. 5B, SOCS1 mRNA in murine macrophages (MOI of 10 for 4 h) (13). 12 h (a)), and the remaining 60–80% of infected cells lacked both In our microarray experiments, STAT1 and IRF1 mRNA levels cytoplasmic and nuclear STAT1 staining (Fig. 5B, 12 h (b)). The were similar in uninfected and infected HFFs (MOI of 3 for 18 h), STAT1 levels at 6 and 12 h of IFN-␥ treatment may be the balance and infection did not change basal STAT1 and IRF1 protein levels of normal turnover of STAT1 molecules that were already present (Fig. 4D). Thus, under the conditions used here, the effects of in the cell before IFN-␥ treatment and the lack of new protein Toxoplasma infection do not seem to be through degradation of synthesis due to parasite inhibition of IFN-␥ signaling. Although STAT1 and IRF1 mRNA or their protein products. Also, in our the half-life of STAT1 is known to be 16–24 h in unstimulated microarray experiments, mRNA levels of SOCS1 and PIAS1, neg- cells (52, 53), it may change depending on culture conditions. The ative regulators of STAT1-dependent IFN-␥ signaling pathway effects of Toxoplasma infection and IFN-␥ stimulation on the (45, 46), were not induced by infection. Consistent with this result, STAT1 half-life in HFFs are not known. neither RH (type I) nor Pru (type II) altered SOCS1 protein levels Western blot results were consistent with the IFM data: total in infected cells (Fig. 4D). In uninfected HFFs, SOCS1 protein STAT1 protein levels were similar in uninfected and Pru-infected levels increased ϳ1.5-fold after 2 and4hofIFN-␥ treatment (Fig. cultures (MOI of 3 for 18 h) (Fig. 5C). Stimulation with IFN-␥ for 4E), consistent with a previous report that SOCS1 mRNA levels 24 h substantially increased STAT1 protein levels in uninfected significantly increase in hepatic cells during 3–6 h of IFN-␥ treat- cultures. Infected cultures also showed an increase in STAT1 lev- ment (47). However, IFN-␥-induced increase in SOCS1 protein els due to the presence of uninfected cells in the cultures but this levels was not observed with RH- or Pru-infected HFFs (Fig. 4E), increase was much less than that in uninfected cultures, indicating as expected from our microarray data showing that none of the an inhibition in STAT1 expression due to infection. Similar results IFN-␥-responsive genes could be induced in infected cells. were obtained with RH-infected cultures (data not shown). The Journal of Immunology 5161

FIGURE 5. STAT1 nuclear trafﬁcking and autoinduction in Toxoplasma- and Neospora-infected HFFs following exposure to IFN-␥. A and B, IFM images of anti-total STAT1 staining using a mAb that recognizes STAT1 irrespective of its phosphorylation state. Unin- fected HFFs (A) and HFFs infected with a type II Tox- oplasma Pru for 18 h (MOI of 0.5) (B) are compared. Times indicate the duration of IFN-␥ treatment. Phase images in B allow for the delineation of infected cells. White triangles indicate parasite vacuoles. C, Western blots with uninfected HFFs or HFFs infected with Pru strain for 18 h (MOI of 3) and then treated with IFN-␥ for 24 h or left untreated. D, IFM for total STAT1 in HFFs infected with N. caninum for 18 h (MOI of 0.5). Triangles indicate parasite vacuoles.

Thus, as with IRF1, all three types of Toxoplasma had inhibitory IL-1, IL-6, MCP-3, RANTES, and other cytokines by HFFs that effects on the IFN-␥-inducible STAT1 expression, and the inhibi- activate the PI3K, protein kinase C, and MAPK pathways (16, 54). tion was entirely conﬁned to infected cells. In contrast, cells in- In Western blots, the extent of IFN-␥-induced phosphorylation fected with N. caninum up-regulated STAT1 expression normally of STAT1 at Tyr701 appeared to be reduced by infection. Before when stimulated with IFN-␥: at 12 h of IFN-␥ treatment, infected IFN-␥ treatment, infected cultures showed low levels of pTyr701- cells showed equally high levels of STAT1 expression both in the STAT1 that were undetectable in uninfected cultures (Fig. 7A). cytoplasm and nucleus to those seen in uninfected cells (Fig. 5D). IFN-␥ caused a substantial increase in pTyr701-STAT1 in both One of the critical effects of IFN-␥ on nonprofessional APCs is the up-regulation of MHC class II and costimulatory molecules to enable the cells to interact with CD4ϩ T cells. Nonprofessional APCs, such as HFFs, do not normally express MHC class II molecules (Fig. 6A). After 24 h of IFN-␥ treatment, ϳ50% of the HFFs in the uninfected culture could be induced to express MHC class II molecules (Fig. 6B). All cells in Pru-infected cultures lacked MHC class II expression before stimulation with IFN-␥ (Fig. 6C). When exposed to IFN-␥, ϳ50% of the uninfected cells in the infected culture expressed MHC class II but none of the infected cells did (Fig. 6D). Cells infected with Toxoplasma types I and III strains were also unable to express MHC class II upon exposure to IFN-␥, whereas N. caninum-infected cells did (data not shown).

Toxoplasma modulates the Tyr701 phosphorylation state of STAT1 in the nucleus of some infected host cells Immunoﬂuorescence assays with anti-total STAT1 Ab suggested that early events of STAT1 activation in the cytoplasm and subsequent nuclear trafﬁcking were intact in Toxoplasma-infected cells but did not lead to the downstream gene expression. Indeed, Western blot analysis showed that the extent and kinetics of tyrosine phosphorylation of JAK1/2 in Pru-infected cultures was similar to that in uninfected cultures following IFN-␥ treatment FIGURE 6. Lack of MHC class II expression by Toxoplasma-infected ␥ 727 (Fig. 7A). IFN- -induced phosphorylation of STAT1 at Ser also HFFs after IFN-␥ treatment. HFF monolayers uninfected (A and B)or appeared unchanged by infection (Fig. 7A). Under the culture con- infected with Pru (type II) strain for 18 h (MOI of 0.5) (C and D) were ditions used, both uninfected and Pru-infected cultures showed low treated with IFN-␥ (100 U/ml) for 24 h (B and D). Parasite GFP is shown 727 but detectable levels of pSer -STAT1 before IFN-␥ treatment in green, staining with mouse monoclonal anti-HLA-DR/DP/DQ Ab in (Fig. 7A; 0 min). This effect may be caused by the production of red, and Hoechst nuclear staining in blue. 5162 INHIBITION OF IFN-␥-INDUCIBLE GENE EXPRESSION BY Toxoplasma

FIGURE 7. STAT1 phosphorylation at the Tyr701 residue in Toxoplasma- and Neospora-infected cells. A, Western blots performed with HFFs uninfected or infected with Pru strain for 18 h at a MOI of 3 before IFN-␥ treatment for the indicated amount of time. Abs used are rabbit polyclonal Abs to pTyr1007/1008-JAK2 (may cross-react with pTyr1022/1023-JAK1) and pSer727- STAT1, and mouse mAb to pTyr701-STAT1. B–F, HFFs were uninfected or infected with indicated parasite strains for 18 h at a MOI of 0.5. Cells were subse- quently treated, or not, with IFN-␥ (100 U/ml) for 45 min before IFM. For Toxoplasma-infected cultures, parasite GFP (green) and anti-pTyr701-STAT1 (red) are shown in top panels at each time point; Hoechst nuclear staining (blue) is merged onto the corresponding bottom panels. For N. caninum that lacks GFP, anti-pTyr701- STAT1 (red) are shown in top panels; infected cells can be identiﬁed in corresponding phase images shown in bottom panels.

infected and uninfected cultures, but the increase was less for in- 5D), N. caninum did not alter the phosphorylation state of STAT1: fected cultures, indicating that infection may interfere with the anti-pTyr701-STAT1 staining was predominantly nuclear in all in- tyrosine phosphorylation state of STAT1. fected cells after 45 min of IFN-␥ treatment and indistinguishable We then performed IFM to examine STAT1 phosphorylation at from that seen in uninfected cells (Fig. 7F). Tyr701 at single-cell levels. Before IFN-␥ exposure, IFM with anti- pTyr701-STAT1 revealed a predominantly cytoplasmic or perinu- Discussion clear staining in uninfected cells (Fig. 7B; 0 min). Toxoplasma- The ability of Toxoplasma to modify host cell processes and infected cells, regardless of which strain was used, showed similar render the cell unresponsive to IFN-␥ may be one of the key cytoplasmic or perinuclear localization of pTyr701-STAT1 before mechanisms that allow the parasite to persist in the face of a IFN-␥ exposure (Fig. 7, C–E; 0 min). What causes this basal level robust IFN-␥-mediated cellular immunity elicited in immune- of STAT1 phosphorylation at Tyr701 and why this does not induce competent hosts. Using HFFs as a model, we investigated how nuclear translocation of STAT1 are not clear. Nonetheless, data Toxoplasma infection of nonphagocytic cells in tissues affects indicate that Toxoplasma does not appear to cause dephosphory- the cell’s responsiveness to IFN-␥, which is critical for devel- lation of STAT1 at Tyr701 in the cytoplasm. oping antimicrobial properties and maturing into a fully func- Forty-five minutes after IFN-␥ treatment, anti-pTyr701-STAT1 tional APC. We compared three major clonal types of Toxo- staining was strongly nuclear in uninfected cultures (Fig. 7B) and plasma and its closely related species, N. caninum, to examine in uninfected cells within the Toxoplasma-infected cultures (Fig. 7, whether their ability or inability to inhibit the IFN-␥ response C–E). In contrast, ϳ60% of type I RH-infected cells lacked such would correlate with the differences in host range, virulence, nuclear staining (Fig. 7C). With type II Pru and type III CL14, the and persistence. effect was less dramatic with only 20–30% of infected cells lack- Over 200 genes are known to be regulated by IFN-␥ in various ing the nuclear staining and the remainder showing nuclear stain- cell types, and functional significance is yet to be attributed to ing similar to that seen in uninfected cells (Fig. 7, D and E). Be- many of them (15, 42). Our microarray experiments with human cause Tyr701 phosphorylation is required for STAT1 nuclear fibroblasts identified 127 unique genes that can be induced by translocation, and because total STAT1 staining is nuclear in all IFN-␥, and no genes were found to be suppressed by IFN-␥ (in infected cells soon after exposure to IFN-␥ (Fig. 5B), our data mouse macrophages, ϳ10% of the IFN-␥-responsive genes appear suggest that Toxoplasma does not interfere with the pTyr701- to be suppressed by IFN-␥ (42)). Remarkably, we have found that STAT1 formation in the cytoplasm but may cause dephosphory- none of the 127 IFN-␥-responsive genes could be up-regulated in lation of pTyr701-STAT1 in some cells, not in the cytoplasm, but Toxoplasma (type II Pru)-infected HFFs following IFN-␥ treat- after it reaches the nucleus. Indeed, infected cells lacking nuclear ment. This unresponsiveness can be accounted for by two different pTyr701-STAT1 staining still showed cytoplasmic staining at 45 mechanisms: first, infection causes the activation of a large number min of IFN-␥ treatment (Fig. 7C). of IFN-␥-responsive genes, presumably through the activation of In contrast to Toxoplasma, and as expected from the normal NF-␬B, and these genes cannot be further induced by subsequent IFN-␥-induced expression of IRF1 (Fig. 4C) and STAT1 (Fig. exposure to IFN-␥. Secondly, Toxoplasma seems to specifically The Journal of Immunology 5163 target and disrupt the STAT1 transcriptional activity in the host increased expression of SOCS1 in HFFs. Thirty minutes after cell nucleus, thus blocking the expression of STAT1-depedent, IFN-␥ treatment, STAT1 was found in the nucleus in all infected IFN-␥-responsive genes. and uninfected cells (according to IFM with anti-total STAT1 Ab), We found that ϳ50% of the 127 IFN-␥-responsive genes were indicating that early events of STAT1 activation (phosphorylation already induced or suppressed during infection before IFN-␥ treat- at Tyr701 by JAK1/2 in the cytoplasm and subsequent nuclear ment, and, according to biological network analyses, these genes translocation of STAT1 dimers) are intact in infected HFFs. How- were associated with the NF-␬B pathway regulated by proinflam- ever, we found that not all STAT1 in the nucleus was phosphor- matory cytokines, such as TNF-␣ and IL-1␤. Although several ylated at the Tyr701 residue, suggesting that infection appeared to reports have suggested that TNF-␣ and IL-1␤ could activate IFN- cause dephosphorylation of STAT1 in some cells after it reaches responsive genes, only a limited number of genes have been ex- the nucleus. This could negatively regulate STAT1 transcriptional amined in their studies (33–35). Our microarray experiments sug- activity because tyrosine phosphorylation is essential for the tight gest that a remarkable number of IFN-␥-responsive genes may be binding of STAT1 dimers to DNA. However, such dephosphory- transcriptionally regulated by these and other cytokines produced lation occurred at varying degrees, depending on parasite strains during infection. It has been shown that the promoter regions of used, and cannot fully account for the lack of IFN-␥-inducible some IFN-inducible genes already occupied by NF-␬B are not IRF1 and STAT1 expression seen in every infected cell. readily accessible to STAT1 dimers (39). This may be one of the In contrast to the parasite modulation of STAT1 phosphoryla- reasons why the genes belonging to the P0 Ͼ N0 and P0 Ͻ N0 tion at Tyr701 that was clearly detectable in Western blots, pSer727- groups could not be further up-regulated by IFN-␥ in STAT1 formation did not appear to be altered by infection. How- Toxoplasma-infected HFFs. ever, it is possible that the parasite causes dephosphorylation of The other half of the 127 IFN-␥-responsive genes showed sim- STAT1 at the Ser727 residue in the nucleus, which is thought to be ilar expression levels in infected and uninfected cells (P0 ϭ N0 necessary for a maximal transcriptional activity of STAT1. We group) and was closely associated with the IFN-␥/STAT1 network. have been unable to address this possibility conclusively because This result suggests that promoter regions of these genes may re- anti-pSer727-STAT1 Abs acquired from two independent commer- main free of other transcription factors during infection and may cial sources were contaminated with reactivities toward Toxo- be regulated primarily by STAT1. Investigation of the promoter plasma in addition to host cell nuclei in IFN-␥-treated cells (data regions of the genes in the P0 Ͼ N0, P0 Ͻ N0, and P0 ϭ N0 not shown). Because we know that some secreted Toxoplasma groups may lead to new information as to the mechanisms by proteins are targeted to the host cell nucleus (see below), we can- which a given set of genes is regulated by several distinct path- not rule out that the nuclear staining we observed may be due to ways and why a second signal cannot override the existing con- reactivities to parasite Ags. ditions of the promoter regions to further activate the target genes. Another possible mechanism of STAT1 inhibition is an auto- Moreover, the genes that are regulated by IFN-␥ but not by other crine effect within Toxoplasma-infected cells. It has been shown cytokines (i.e., the P0 ϭ N0 group) may hold an answer to why the that TGF-␤1 production restricted to type I Toxoplasma (RH)- immune system has evolved to produce IFN-␥ in addition to many infected murine microglial cells could inhibit the induction of in- other cytokines and type I IFNs, which apparently accomplish ducible NO synthase by IFN-␥ (55). Recently, it has been reported many redundant functions. In this regard, it is noteworthy that that TGF-␤1 may block the IFN-␥-inducible NO production in STAT1 itself and such genes as PSMP8, LAP3, TAP1, and HLA-E dendritic cells and macrophages by inhibiting tyrosine phosphor- that function in the MHC Ag processing and presentation path- ylation of STAT1, presumably, as a result of a physical associ- way belong to the P0 ϭ N0 group, indicating a uniquely im- ation between TFG-␤1 receptor and IFN-␥ receptor (56). This portant role of IFN-␥ in facilitating the interaction between autocrine effect of TFG-␤1 is an unlikely explanation for the APCs and T cells (15). impaired STAT1 activity we observed in Toxoplasma-infected We have demonstrated by IFM that all three types of Toxo- HFFs because STAT1 nuclear translocation that requires phos- plasma, but not N. caninum, block the IFN-␥-inducible expression phorylation at Tyr701 in the cytoplasm is intact in all infected of IRF1 and STAT1. The inhibition was confined to infected cells, cells. and neighboring, uninfected cells expressed IRF1 and STAT1 nor- Based on the fact that STAT1 activation and nuclear trans- mally in response to IFN-␥. This strongly argues against the pos- location occurs normally in infected cells despite the lack of its sibility that soluble factors secreted by infected cells into the cul- downstream effects on gene expression, we propose that se- ture medium exert an inhibitory paracrine effect. For IRF1 and creted Toxoplasma proteins present in the host cell nucleus (i.e., STAT1, as well as for other IFN-␥-responsive genes of the P0 ϭ Tyr-phosphatase or PIAS1-like inhibitory factors) may interfere N0 group, the recruitment of NF-␬B to their promoter regions with the STAT1 transcriptional activity. Many secreted Toxo- alone cannot explain the lack of their expression in Toxoplasma- plasma molecules are now known to be targeted to specific infected cells following IFN-␥ treatment; consistent with our mi- destinations within a host cell (57). Notably, among the secre- croarray analysis results with a type II Pru strain suggesting the tome are rhoptry proteins that include at least one protein phos- activation of NF-␬B, Toxoplasma type II strains have been shown phatase (PP2C-hn) (58) and one protein kinase (Rop16) that are to cause a persistent presence of NF-␬B in the host cell nucleus but targeted to the host cell nucleus (59). For Rop16, it has been types I and III strains do not (J. Saeij, unpublished results). N. shown that the presence of this highly polymorphic kinase is caninum also recruits NF-␬B to the host cell nucleus (G. Alvarez, associated with strain-specific activation of STAT3 and STAT6 unpublished results). Because IRF1 expression cannot be induced in infected cells, such that at 18 h postinfection, cells infected by IFN-␥ in STAT1Ϫ/Ϫ cells (42) and is blocked by all three types with type I or III strain, but not type II, contained phosphory- of Toxoplasma but not by N. caninum, our data indicate that all lated STAT3 and STAT6 in the host cell nuclei (59). The fact Toxoplasma strains have evolved a common ability to directly tar- that infection itself does not cause STAT1 activation regardless get and disturb STAT1 transcriptional activity, which has probably of which strain was used (see Fig. 5 above) and that the IFN- been key to their intracellular survival in a wide variety of hosts. ␥-inducible gene expression can be blocked by all three Toxo- Unlike a previous report (13), we have observed that neither plasma strains indicates that a mechanism shared by all three type I nor type II Toxoplasma causes the degradation of STAT1 or strains and independent of STAT3/6 regulation is responsible 5164 INHIBITION OF IFN-␥-INDUCIBLE GENE EXPRESSION BY Toxoplasma for the disruption of the IFN-␥ signaling pathway. The results 11. Lang, C., M. Algner, N. Beinert, U. Gross, and C. G. Luder. 2006. Diverse ␥ with Rop16 suggest that the as-yet-unidentified parasite mole- mechanisms employed by Toxoplasma gondii to inhibit IFN- -induced major histocompatibility complex class II gene expression. Microbes. Infect. 8: cules might be targeted to the host cell nucleus to specifically 1994–2005. interfere with STAT1 activity. 12. Luder, C. G., W. Walter, B. Beuerle, M. J. Maeurer, and U. Gross. 2001. Tox- oplasma gondii down-regulates MHC class II gene expression and antigen pre- In striking contrast to Toxoplasma, its closely related apicom- sentation by murine macrophages via interference with nuclear translocation of plexan cousin N. caninum lacked the ability to block the IFN-␥- STAT1␣. Eur. J. Immunol. 31: 1475–1484. inducible expression of IRF1, STAT1, and MHC class II. Al- 13. Zimmermann, S., P. J. Murray, K. Heeg, and A. H. Dalpke. 2006. 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