Cellular Signalling 26 (2014) 1204–1212

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Toxoplasma gondii inhibits apoptosis via a novel STAT3-miR-17–92-Bim pathway in macrophages Yihong Cai a,b,c, He Chen a,d, Xuwei Mo a, Yuanyuan Tang a, Xiucai Xu a,e, Aimei Zhang a,e, Zhaorong Lun f,g, Fangli Lu f,g, Yong Wang h, Jilong Shen a,⁎ a

Anhui Provincial Laboratories of Pathogen Biology and Zoonoses, Anhui Medical University, Hefei, China Department of Health Inspection and Quarantine, School of Public Health, Anhui Medical University, Hefei, China Department of Immunology, Anhui Medical University, Hefei, China d Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China e The Central Laboratory of Affiliated Provincial Hospital, Anhui Medical University, Hefei, China f State Key Laboratory of Biocontrol, School of Life Sciences, and Key Laboratory of Tropical Diseases Control, The Ministry of Education, Zhongshan Medical College, China g Department of Pathogen Biology, Sun Yat-Sen University, Guangzhou, China h Department of Pathogen Biology, Nanjing Medical University, Nanjing, China b c

a r t i c l e

i n f o

Article history: Received 8 February 2014 Accepted 21 February 2014 Available online 28 February 2014 Keywords: MicroRNA Apoptosis STAT3 Bim Macrophage

a b s t r a c t In order to accomplish their life cycles, intracellular pathogens, including the apicomplexan Toxoplasma gondii, subvert the innate apoptotic response of infected host cells. However, the precise mechanisms of parasite interference with the apoptotic pathway remain unclear. MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level. Using T. gondii strain TgCtwh3, which was isolated from felids and possesses the predominant genotype China 1 (ToxoDB#9) in China, we analyzed the miRNA expression profile of human macrophages challenged with TgCtwh3. The results showed that miR-17–92 miRNA expression was significantly increased and Bim was decreased in TgCtwh3-infected cells. Database analysis of miR-17–92 miRNAs revealed the potential binding sites in the 3′UTR of Bim, one of the crucial effectors of pro-apoptosis. Furthermore, we demonstrated that the promoter of the miR-17–92 gene cluster which encodes miRNAs was transactivated through the promoter binding of the STAT3 following TgCtwh3 infection. Taken together, we describe a novel STAT3-miR17–92-Bim pathway, thus providing a mechanistic explanation for inhibition of apoptosis of host cells following Toxoplasma infection. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Microbial infection might provide a stress signal that triggers the intrinsic apoptotic pathway [1]. However, if the infected cell is induced to undergo apoptosis, further development, multiplication and dissemination of the invader might be disturbed. As a typical intracellular protozoan parasite of warm-blooded vertebrates, Toxoplasma gondii has elaborate mechanisms to counteract host-cell apoptosis in order to maintain survival and breed in the host cells [2–5]. Upon invasion of T. gondii, the parasite uses a specialized set of secretory organelles to inject parasite-derived effector molecules into its host cell [6]. These effector molecules can specifically modulate host cell gene expression to improve their ability to infect and proliferate, via inhibition of host immune responses, and change their gene expression to escape immunologic surveillance [7]. Quantitative analysis of the host mRNAs and proteome during Toxoplasma infection showed that upwards of 15% of ⁎ Corresponding author at: Department of Microbiology and Parasitology, Anhui Medical University, Hefei, Anhui 230032, China. Tel./fax: +86 551 5113863. E-mail address: [email protected] (J. Shen).

http://dx.doi.org/10.1016/j.cellsig.2014.02.013 0898-6568/© 2014 Elsevier Inc. All rights reserved.

mRNAs and upregulation of 213 protein spots display altered abundance relative to uninfected cells, including crucial mRNAs and proteins that were associated with the apoptotic pathway [8,9]. Bim, a BH3-only member of the Bcl-2 family, senses pro-apoptotic upstream signals and its upregulation or post-transcriptional activation precedes permeabilization of the outer mitochondrial membrane (MOMP). Bim initiates or promotes pore formation in the outer membrane of mitochondria to release cytochrome C and other already-soluble apoptogenic factors into the cytosol [10,11] Deficiency of Bim results in delayed apoptosis in leukemia and breast cancer [12,13]. MicroRNAs (miRNAs) are endogenously small (19–24 nt long) noncoding RNAs that regulate gene expression in a sequence specific manner. This is primarily accomplished through binding to 3′UTR of target mRNAs, either targeting the transcripts for degradation or blocking their translation [14]. However, molecular mechanisms underlying miRNA gene transcriptional regulation are largely unclear. Recent studies on expression of miRNA genes have revealed potential transcriptional regulation by transcription factors, such as p53, NF-κB and MAPK [7,15,16]. Recently, the signal transducer and activator of transcription (STAT) signaling pathway has emerged as a major target of exploitation

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by Toxoplasma. Infection of mouse bone marrow-derived macrophages induces rapid and sustained activation of STAT3 [17,18]. Although a number of miRNAs have been shown to be important components of the STAT3 in various cell types [19–21], the potential interaction between miRNAs and STAT3 in human macrophages, in which the activation of STAT3 by infection with Toxoplasma may result in distinct biological consequences, has not yet been established. We herein provide evidence that Toxoplasma–host cell interactions counteract the death of parasite-infected cells following interference with the apoptotic pathway. We report here that STAT3 mediates a prosurvival pathway by upregulating the miR-17–92 miRNAs that in turn targets Bim, leading to survival of host cells with Toxoplasma infection. Moreover, we show that macrophages with T. gondii express elevated levels of miR-17–92 miRNAs that is closely associated with decreased Bim. Thus, the STAT3-miR-17–92-Bim pathway is postulated to be an important apparatus in Toxoplasma biology.

macrophages were harvested using TRIzol (Invitrogen) and miRNeasy min kit (Qiagen) according to manual instructions. After having passed RNA quantity measurement using the NanoDrop 1000, the samples were labeled using the miRCURYTM Hy3TM/Hy5TM Power labeling kit and hybridized on the miRCURYTM LNA Array (v.16.0). Following the washing steps the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA). Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Replicated miRNAs were averaged and miRNAs that are having intensities ≥50 in all samples were chosen for calculating normalization factor. Expressed data were normalized using the Median normalization. After normalization, differentially expressed miRNAs were identified through Volcano Plot filtering. Hierarchical clustering was performed using MEV software (v4.6, TIGR).

2. Materials and methods

Macrophages were exposed to TgCtwh3 or LPS (1 μg/ml) as control for different duration (6, 12 and 24 h). For qPCR analysis of mature miRNAs, total RNA was reverse-transcribed from 0.05 μg total RNAs and determined with All-in-One™ miRNA qRT-PCR detection kit (GeneCopoeia) by Applied Biosystems 7500 real-time PCR System (Applied Biosystems). The specific primers used in this reaction were as follows: miR-19b; miR-17-5p; miR-18a; miR-19a; miR-20a; miR-92a; miR-33b; and miR-141 (all from GeneCopoeia). All reactions were run in triplicate. The Ct values were analyzed using the comparative Ct (ΔΔCt) method. Normalization was performed by using the small housekeeping U6 relative to the control (non-treated cells).

2.1. Ethics statement Ethical permission was obtained from the Institutional Review Board (IRB) of the Institute of Biomedicine at Anhui Medical University (Permit Number: AMU26-08610), which records and regulates all research activities in the school. The IRB of the Anhui Medical University Institute of Biomedicine approved both animal and human protocols. The study follows the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All subjects gave written informed consent.

2.6. qRT-PCR analysis

2.2. Parasites 2.7. Protein extraction and Western blotting The T. gondii strain, named TgCtwh3 with atypical genotype China 1 and high virulence to mice as previously identified [22,23],was kept in the laboratory by mouse passage. Tachyzoites were maintained by twice weekly passage on HF (human fibroblast cells) in culture medium (DMEM with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, Gibco). 2.3. Human macrophage separation, culture and determination Human PBMCs for in vitro parasite infection assays were isolated from whole blood of 6 healthy individuals with written informed consent (1 female and 5 males, mean age 22 ± 3 years). PBMCs were used as host macrophages after induction to differentiate with 1000 U/ml recombinant human GM-CSF (Preprotech) for 48 h. Macrophages were stained with FITC-conjugated anti-CD14 to determine the purity of CD14+ cells. Control and infected cells were stained with Wright– Giemsa for 8 min, rinsed with distilled water and air dried. Cell morphology was observed by light microscopy [24]. THP-1 cells (Cell bank, CAS) were cultured in DMEM medium supplemented with 10% FCS, and treated with 20 nmol of phorbol 12-myristate 13-acetate (PMA, Sigma) to induce THP-1 cell differentiation into macrophagelike THP-1 cells [25]. 2.4. Parasite infection and apoptosis induction TgCtwh3 tachyzoites were separated from HF cells by differential centrifugation. Human macrophages were infected at parasite-to-host cell ratios of 1:1 and kept for 6, 12 or 24 h. Infected cells were treated with 1 μM staurosporine at 0, 3, 9 or 21 h postinfection for additional 3 h. 2.5. MiRCURYTM LNA array analysis of miRNAs The Exiqon (Vedbaek, Denmark) mercury LNA microRNA arrays and service were used to process the samples. Briefly, human macrophages were exposed to TgCtwh3 for 6 h, 12 h or 24 h. Total RNAs from

To obtain total cellular lysates, TgCtwh3-infected cells and uninfected controls were lysed in ice-cold cell lysis buffer [20 nM tris, 250 mM NaCl, 2 mM EDTA, 0.1% Triton X-100, 0.01 mg/ml aprotinin, 0.003 mg/ ml leupetin, 0.4 mM phenylmethylsulfonyl fluoride, and 4 mM NaVO4] (Sigma). The nuclear proteins were isolated from macrophages using the NucBuster Protein Extraction Kit (Novagen). The protein concentration was determined with the Bradford protein assay kit (BESTBIO) using a c-globulin standard curve. Proteins were separated by standard SDS-PAGE and transferred onto nitrocellulose membranes (Beyotime). Nonspecific binding sites were blocked using 5% dry skimmed milk, 0.2% Tween-20 in PBS (pH 7.4) for overnight at 4 °C and then incubated with primary antibodies to STAT3 (Proteintech), phosphor-STAT3 (Tyr705) (Cell Signaling), Bim (Enzo), and GAPDH (Proteintech). 2.8. Plasmid construction and transcriptional reporter assays For construction of a constitutively active form of human STAT3, named STAT3-C, the amino acid residues at A661 and N663 were mutated to cysteine, the sequences were chemi cally synthetized and cloned into pEZ; M02 (GeneCopeia). (200 ng), pEZ-M02-STAT3-C (300 ng) or empty vector was individually cotransfected into THP-1 cells, together with appropriate miR-17–92 gene cluster promoter reporter plasmids (200 ng) by using Effectene Transfection Reagent (Qiagen). In each transfection, cells were also cotransfected with pRLCMV (Renilla luciferase reporter plasmid, Promega). Firefly and Renilla luciferase activity was assayed using a Dual-Luciferase Reporter Assay System (Promega). The magnitude of activation values was measured relatively to the levels of Firefly luciferase activity in THP-1 cells transfected with empty vectors and normalized by Renilla luciferase activity. A mutation was introduced into the STAT3 binding site (STAT3-BR-1) using the following ; antiprimers: sense (alterations sense

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underlined). The correct sequence of each insert was confirmed by sequencing. Bim-3′UTR-1, Bim-3′UTR-2, Bim-3′UTR-3 and Bim-3′UTR-3 mutant were constructed into pSI-CHECK2-report plasmid (Promega). Plasmids and pEZ-M02-miR-17–92 (250 ng), miR-17-5p inhibitor or miR-20a inhibitor were cotransfected into THP-1 cells (2 × 104) by using the Effectene Transfection Reagent (Qiagen). The magnitude of activation values was measured relatively to the levels of Renilla luciferase activity in THP-1 cells transfected with negative control oligonucleotide and normalized by Firefly luciferase activities. For inhibition of miRNAs, a similar protocol was applied with anti-miR-17-5p and antimiR-20a (all from GeneCopoeia, 100 nM/well) instead of the miR-17– 92 encoding vector. 2.9. SiRNA THP-1 cells were transiently transfected with STAT3 siRNA or siRNAcontrol (40 nM, Santa Cruz) for 24 h using SuperFectin II in vitro siRNA Transfection Reagent (PuFei Biotech) according to the manufacturer's protocols. 2.10. Flow cytometry Cell death following induction of apoptosis and infection with T. gondii was quantified by flow cytometry. Annexin-V/propidium iodide (PI) double assay was performed using the Annexin V-EGFP Apoptosis Detection Kit (BESTBIO). Infected or non-infected macrophages were washed twice with PBS. 1 × 106 cells were resuspended in 400 μl binding buffer and stained with 5 μl Annexin V-EGFP according to the manual's recommendations. 10 μl PI was added and allowed to incubate

with cells for 5 min at 4 °C in the dark. Then cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences). 3. Results 3.1. T. gondii leads to anti-apoptosis in macrophages Human macrophages were separated from whole blood and cultured after 48 hs. Cells were stained with FITC-conjugated CD14 antibody and subjected to flow cytometry to determine the purity of macrophages. The percentages of CD14+ cells were 94.7 ± 0.60% after induction with GM-CSF in PBMCs (data not shown). Cell death was quantified by staining with Annexin V and PI to determine whether the activation of apoptosis would be inhibited by T. gondii infection in macrophages and lack of activation of apoptosis would induce necrotic death of tachyzoite-infected cells. After treatment with staurosporine, the percentage of macrophages that were positive for Annexin V and negative for PI (Annexin V+/PI−, early apoptotic) and to a lesser extent positive for Annexin V and PI (Annexin V +/PI +, late apoptotic and/or necrotic) increased considerably. Prior infection with T. gondii dramatically decreased the number of Annexin V+/PI− macrophages at 24 h (P b 0.05). However, the parasite-mediated percentage of Annexin V+/PI+ macrophages did not increase after treatment with staurosporine (P N 0.05). Consequently, the number of Annexin V−/PI−macrophages (viable cells) increased in stauroporine-treated macrophages by prior infection with parasites (P b 0.05; Fig. 1A, B). Through observation using optical microscope, we noted that triggering of the apoptotic pathway did not affect the number of parasite-infected macrophages (Fig. 1C). These results established

Fig. 1. Triggering of the apoptotic pathway does not induce cell death of Toxoplasma-infected host cells. (A, B) Macrophages were infected with TgCtwh3 at different time points postinfection (0, 6, 12 and 24 h) or control cultivation. TgCtwh3-infected cells were treated with staurosporine (Stau+) at 0, 3, 9 or 21 h postinfection for an additional 3 h or untreated with stauroporine (Stau−). The apoptosis of macrophages was analyzed by flow cytometry following staining with Annexin V and PI. (A) Dot plots depict the amounts of macrophages stained with Annexin V and/or PI; numbers indicate percentages of cells in each quadrant from a representative experiment. (B) Percentage of cells with the indicated phenotypes after treatment with staurosporine (gray bars), untreatment with staurosporine (black bars) and control cultivation (white bars). (C) Percentages of parasite-positive macrophages (Wright-Giemsa stain as shown in the figure) after treatment with staurosporine (black bars) or untreatment with staurosporine (white bars). Values are presented as mean ± SD (n = 3 in A–C).

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that activation of the apoptotic pathway was counteracted by T. gondii, thus increasing the viability of parasite-infected host cells. 3.2. Database analysis of upregulated miRNAs in macrophages following T. gondii infection Alterations in the mature miRNA expression profile of T. gondiiinfected macrophages suggest that host miRNA gene expression is finely controlled in response to the parasite infection. We profiled the levels of miRNAs extracted at 6 h, 12 h and 24 h from uninfected and tachyzoite-infected human macrophages using miRCURYTM LNA Array (v.16.0). The 6th generation of miRCURYTM LNA Array (v.16.0) contains more than 1891 capture probes, covering all human, mouse and rat miRNAs annotated in miRBase 16.0, as well as all virus miRNAs related to these species. In addition, the array contains capture probes for 66 new miRPlus™ human miRNAs. Of the miRNAs expressed, miR19b, miR-20a and miR-18a (the members from the miR-17–92 gene cluster) expression marginally increased at 6 h postinfection and remarkably increased at 12 h and 24 h postinfection (P b 0.05; Fig. 2A). To validate the microarray data and to specifically measure the effects of Toxoplasma infection on miRNAs encoded by miR-17–92 gene cluster, qRT-PCR analysis using primers for mature miRNAs was performed to assess the kinetics of miR-17–92 miRNAs in macrophages following TgCtwh3 infection. Increased expression of miR-17-5p, miR-19a, miR-19b, miR-20a, miR18a and miR-92a was noted in macrophages at 6 h and 12 h postinfection, the abundance of these miRNAs significantly increased by ~23.5-fold at 24 h postinfection. The qRT-PCR analysis of mature miR-17–92 was also performed on macrophages treated with

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LPS (1 μg/ml for 6 h, 12 h and 24 h). The results showed that LPStreated cells did not significantly alter the levels of mature miRNAs of miR-17–92 at any time-point in the assay. Especially, increased expression of mature miRNAs of miR-17–92 was identified in Toxoplasmainfected cells but not in cells exposed to LPS at 24 h (P b 0.05; Fig. 2B). No LPS contamination in the TgCtwh3 preparation was detected using the Limulus Amebocyte Lysate (LAL) test kit (Bio-Whittaker) (data not shown). 3.3. T. gondii infection attenuates Bim via miR-17–92 miRNAs We searched the MicroCosm and miRBase databases for the potential targets of miR-17–92 miRNAs. Among the candidates identified, the 3′UTR of Bim contains several putative regions that matched perfectly to the miR-17–92 miRNA “seed” region (Fig. 3A, Table 1). Because Bim is known to facilitate the apoptotic pathway [26,27], we examined if Bim is indeed down-regulated by T. gondii infection, which may be involved in up-regulation of miR-17–92 miRNAs in macrophages with the parasite infection. To investigate this, we determined the level of Bim protein down-regulation by Western blotting at 6 h (BimEL: 0.57 ± 0.18-fold, BimL: 0.55 ± 0.17-fold, BimS: 0.74 ± 0.1-fold compared with 0 h) postinfection and the result showed that dramatical decrease of Bim expression was noted at 12 h (BimEL: 0.43 ± 0.17-fold, BimL: 0.39 ± 0.16-fold, BimS: 0.59 ± 0.13-fold) and 24 h (BimEL: 0.24 ± 0.07-fold, BimL: 0.29 ± 0.12fold, BimS: 0-fold) postinfection (P b 0.05; Fig. 3B). Next, we sought to test whether the miR-17–92 miRNA overexpression would decrease Bim expression, a gene which may be involved in Toxoplasma infection. The overexpression of miR-17–92 miRNAs in THP-

Fig. 2. Expression profiling of mature miRNAs in macrophages following Toxoplasma infection. Macrophages were infected with TgCtwh3 at different time points postinfection (6, 12 and 24 h). RNA sample were taken at the indicated time points and analyzed on mature miRNAs content by miRNA array and qRT-PCR. (A) miRNA expression profile in macrophages following TgCtwh3 infection. The left panel shows a heat-map of selected miRNAs indicating altering in expression in macrophages following TgCtwh3 infection. The filtered miRNA array data were subjected to unsupervised hierarchical clustering analysis. The metric was set as the Euclidean distance. The right panel shows expression miRNAs in macrophages following TgCtwh3 infection. The fold change contains the ratio of normalized intensities between TgCtwh3-infected macrophages (Group T) and non-infected macrophages (Group C). The P values are from the t test. (B) Quantitative RT-PCR (qRT-PCR) analysis of miR-17–92 miRNAs in macrophages following TgCtwh3 infection or LPS stimulation at different time points (6, 12 and 24 h). Data are representative of three independent experiments.

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1 cells after transfection with pEZ-miR-17–92 resulted in a significant decrease of Bim (BimEL: 0.29 ± 0.12-fold, BimL: 0.63 ± 0.22-fold, BimS: 0fold compared to transfection with pEZ-vector) at the protein level as analyzed by Western blotting (P b 0.05). Similarly, Bim protein expression was found to be lower with Toxoplasma infection than with uninfected cells after 24 h (BimEL: 0.30 ± 0.11-fold, BimL: 0.58 ± 0.19-fold, BimS: 0-fold) (P b 0.05). This reduction, however, was blocked by pretreatment with miR-17-5p inhibitor (BimEL: 2.48 ± 0.19-fold, BimL: 2.32 ± 0.21fold, BimS: 0.85 ± 0.32-fold) (P b 0.05) or miR-20a inhibitor (BimEL:

1.19 ± 0.23-fold, BimL: 1.90 ± 0.11-fold, BimS: 0.94 ± 0.15-fold) (P b 0.05). Furthermore, the Bim protein expression was significantly increased with cotransfected miR-17-5p inhibitor and miR-20a inhibitor (BimEL: 2.09 ± 0.28-fold, BimL: 3.03 ± 0.13-fold, BimS: 1.59 ± 0.39fold) as compared with untreated cells (P b 0.05) (Fig. 3C). Additionally, we explored whether Bim is indeed directly downregulated by the miR-17–92 miRNAs. To this end, we introduced pSICHECK2 luciferase reporter plasmids of the Bim1–3M (pSI-Bim-3′ UTR-1, pSI-Bim-3′UTR-2, pSI-Bim-3′UTR-3 and pSI-Bim-3′UTR-3M),

Fig. 3. The miR-17–92 miRNAs target Bim and are controlled by TgCtwh3 infection. (A) The Bim 3′UTR contains multiple predicted binding sites for the miR-17–92 miRNAs. Bim-1–Bim-3 are the fragments of the 3′UTRs cloned into the pSICHECK2 vector for reporter assays in (D) and (E). (B) Complete cellular lysates separated by SDS-PAGE and analyzed by immunoblotting using primary antibodies against Bim at different time points postinfection without treatment of staurosporine. (C) THP-1 cells were transfected separately with pEZ-miR-17–92 vector or inhibitors of miRNAs as indicated vectors, and proteins were collected 72 h later. Then, reporter gene studies on the interaction between 3′UTRs of Bim and the miR17–92 miRNAs in THP1. THP-1 cells were transfected with or without indicated plasmids for 72 h or infected with TgCtwh3 for 24 h. Whole-cell lysates were subjected to Western blot analysis. (D) Schematic illustration of pSICHECK2-based luciferase reporter constructs used for examining the effect of miR-17–92 miRNAs on the 3′UTR of Bim. 3M represents mutations introduced into these fragments. See Table 1 for details. (E) THP-1 cells were cotransfected with the indicated reporter constructs and Renilla luciferase plasmids. Twenty-four hours later, reporter activity was measured by luciferase assays. Consistently, THP-1 cells were transfected with Renilla luciferase plasmids, followed by measurement of reporter activity by luciferase assays. Values are presented as mean ± SD (n = 3 in B, C, E).

Y. Cai et al. / Cellular Signalling 26 (2014) 1204–1212 Table 1 Sites of miRNA binding to Bim 3′UTR and cloned fragments for reporter assays. miRNA site

Position of Bim 3′UTR

Sequence

Mutated sequence

miR-17-5p

2115 878 1998 4056 2694 2725 2725 2115 1998 878 4056 668 1998 1210

CACTTTG GCACTTT GCACTTT GCACTTTA GEGCAAT TTGCACA TTTGCAC CACTTTG GCACTTT GCACTTT GCACTTTA ACGCCAC GCACTTT TCAGTTC

Not mutated Not mutated Not mutated GGAGTATA GAGCATT TAGGATA TAGGATA Not mutated Not mutated Not mutated GGAGTATA Not mutated Not mutated Not mutated

miR-92 miR-19a miR-19b miR-20a

miR-18a

which are the fragments and mutants of the 3′UTR of Bim (Fig. 3A, D and Table 1). Cotransfection of the pSI-Bim-3′UTR-1–3 and pEZ-miR-17–92 yielded a lower relative luciferase activity than pSI-vector (P b 0.05). This result, however, was reversed when the 3′UTR was mutated (Fig. 3E). Collectively, these results substantiate the evidence that Bim is a target of miR-17–92 miRNAs that is modulated in macrophages with T. gondii infection. 3.4. STAT3 transcriptionally regulates promoter of the miRNA-17–92 gene cluster to decrease Bim in macrophages with Toxoplasma infection One potential mechanism for selectively altering miRNA levels is that transcription factors take part in specific intracellular signaling pathways of transcriptional regulation of miRNA genes [15]. It has been previously reported that STAT3 can regulate miRNA gene transcription in macrophages with Toxoplasma infection [24]. In order to identify whether Toxoplasma infection could induce STAT3 activation, macrophages were infected with TgCtwh3 tachyzoites and kept for 6 h, 12 h or 24 h and the proteins of total STAT3 and pSTAT3 (Tyr705 phosphorylation) were analyzed with Western blotting. As a consequence, TgCtwh3 induced strong pSTAT3 expression at 6 h and the phosphorylation of STAT3 sustained for at least 24 h (P b 0.05; Fig. 4A). Similar to the induction of miR-17–92 miRNA gene expression following TgCtwh3 infection, the pSTAT3 levels were increased in macrophages following TgCtwh3 infection, suggesting an association between the endogenous activation of STAT3 and miR-17–92 miRNAs in macrophages with the parasite infection. Additionally, we found that knock-down of STAT3 by siRNA blocked the increase of Toxoplasma-induced miR-17–92 miRNA expression, whereas constitutively active pSTAT3 significantly upregulated miR-17–92 miRNA expression (P b 0.05; Fig. 4B). To further determine the relationship between the STAT3 and miR17–92 miRNA expression in macrophages with TgCtwh3 infection, we analyzed the miRNA-17–92 gene cluster, which encodes the miR17–92 miRNAs, including miR-17-5p, miR-18a, miR-19a, miR19b, miR20a, and miR-92a. Examination of the flanking genomic DNA region identified two putative STAT3 binding sites located from 1039 to 988 bp upstream of the miRNA-17–92 gene cluster promoter. pEZSTAT3-C (a constitutively active form of STAT3) increased the transcriptional activity of the luciferase reporters (STAT3-BR-WT, and STAT3-BR2, containing the STAT3 binding region), but had no significant impact on the luciferase reporter lacking the transactivation domain (STAT3BR-1) (Fig. 4C, D). However, pEZ-STAT3-WT cotransfection failed to have any effect on those luciferase reporters even containing the STAT3-BR (Fig. 4D). In addition, knock-down of STAT3 by siRNA markedly inhibited activation of the STAT3-BR to Toxoplasma infection mediated by STAT3 (Fig. 4D). Consistently, Toxoplasma infection upregulated the endogenous miR-17–92 miRNA expression levels, strongly suggesting that the promoter of miR-17–92 gene cluster is a STAT3 transcriptional target in macrophages infected with T. gondii tachyzoites.

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To explore in depth whether the STAT3 phosphorylation might affect the protein levels of Bim through altering the expression of miR17–92 miRNAs after Toxoplasma infection, siRNA experiments were performed to knock-down the endogenous STAT3 expression in THP-1 cells (Fig. 4E). Consequently, overexpression of STAT3-C resulted in decreased expression of Bim as compared with pEZ-vector transfected cells (P b 0.05). This was further demonstrated in THP-1 cells with STAT3 knockdown by siRNA. Deficiency in STAT3 reduced the expression of Bim in response to T. gondii infection (P b 0.05; Fig. 4F). STAT3 has been shown to be differentially regulated by polymorphic effectors of Toxoplasma genotypes of I, II, and III strains. The identification of a crucial polymorphic leucine/serine residue at position 503 in ROP16 that determines the genotype difference with respect to STAT3 activation. The leucine at position 503 in ROP16 may sustain STAT3 phosphorylation [6,17]. Through sequence analysis of the Toxoplasma TgCtwh3 ROP16, we found that this isolate has the same amino acid substitution of the kinase domain of ROP16 at 503 bp (503L) as that of TGGT1 strain (503L) with archetypical type I (Fig. 4G). Therefore the identical amino acid polymorphism might account for the identical way of activation to STAT3 as type I. To link together all of the data presented so far, the STAT3-miR17–92-Bim pathway in macrophages, in combination with T. gondii infection in vitro, has a functional significance with respect to antiapoptosis. 4. Discussion More recently, several reports have shown that transcription factors could be involved in regulation of miRNA expression. Here we found that the pSTAT3 was upregulated by Toxoplasma infection in human macrophages and similar results were seen in the miR-17–92 miRNAs. Previous investigation showed that a single amino acid substitution in the kinase domain of Toxoplasma ROP16 with strain difference determined STAT3 activation between type I and type II. Significantly, we found that TgCtwh3 strain with atypical genotype China 1 (ToxoDB#9) has the same amino acid substitution of the kinase domain of ROP16 at 503 bp (503L, Fig. 4G) as that of TGGT1 strain (503L) with archetypical type I, which might account for the identical way of activation to STAT3 as type I [17]. With respect to the link of STAT3 to the miR-17–92 miRNAs, we demonstrated that the deficiency of STAT3 remarkably reduced the expression of the miR-17–92 miRNAs in response to Toxoplasma infection. Moreover, knock-down of STAT3 similarly blocked Toxoplasma-induced down-regulation of Bim expression in macrophages. In general, the expression and phosphorylation of STAT3 is finely balanced by negative feedback loops in normal cells. In order to confirm the novel miRNA regulation, we constructed an expression vector for constitutive activation of STAT3, and subsequent transfection of this vector promptly resulted in the increase of miR-17–92 miRNAs and decreased expression of Bim as compared with mock or STAT3 WT transfected cells. By applying promoter studies, we found that the promoter of miR-17–92 gene contains two putative STAT3 binding sites. This STAT3-BR is transcriptionally responsive to Toxoplasma infection, which increased the transcriptional activity of the luciferase reporters (STAT3-BR-WT and STAT3-BR-2), but had no influence on those lacking the region. When STAT3-C was transfected, the transcriptional activity of the STAT-BR was enhanced, whereas it was limited when mock or STAT-WT was used. Additionally, knock-down of STAT3 markedly inhibited activation of the STAT3-BR-containing luciferase reporters, indicating that the response of the STAT3-BR to Toxoplasma infection is mediated by STAT3. One of the important observations in the present study is that triggering of the apoptotic pathway did not induce cell death of Toxoplasma-infected macrophages. In order to exclude the possibility that activation of the intrinsic pathway of apoptosis by staurosporine could induce necrotic death of parasite-infected host cells, we quantified the parasite-infected macrophages and cell-death phenotypes of parasite-

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Fig. 4. The miR-17–92 gene is a transcriptional target of STAT3 and is involved in the biological significance of the STAT3-miR-17–92-Bim pathway in macrophages with Toxoplasma infection. Transient transfection of constitutively active STAT3 downregulates Bim in THP-1 cells after 72 h. Reporter gene assay studies identified STAT3-binding site in the promoter of miR17–92 gene. (A) Macrophages were infected with TgCtwh3 at different time points postinfection, the cells were lysed and subjected to Western blotting. Activation of STAT3 was also determined by Western blotting analysis of cell nuclear extracts using anti-phospho-STAT3 Tyr705. STAT3 and GAPDH levels are shown as loading controls. (B) Quantification of mature miR17–92 miRNAs was performed in STAT3-WT, STAT2-C and siRNA-STAT3 transfected cells with TgCtwh3 infection. (C) A schematic illustration of pGL-based reporter constructs was used in dual luciferase assays to examine the transcriptional activity of the STAT3-BR in response to TgCtwh3 infection. Dotted lines indicated deleted region. (D) THP-1 cells were cotransfected with the indicated reporter constructs and Renilla luciferase plasmids. Twenty-four hours later, transcriptional activity was determined by luciferase assays. THP-1 cells with or without STAT3 knock-down by siRNA were cotransfected with the indicated reporter constructs and Renilla luciferase plasmids. Twenty-four hours later, cells were exposed to TgCtwh3 for another 24 h, followed by measurement of reporter activity by using luciferase assays. (E) Western blot analysis showing stable knock-down of STAT3 by siRNA. (F) THP-1 with or without transfection of the indicated reporter constructs for 72 h or exposure to TgCtwh3 for 24 h. Whole-cell lysates were subjected to Western blot analysis. (G) ROP16 amino acid sequence from type I (TGGT1), type II (ME49) and typical genotype China 1 (TgCtwh3) were compared by using NCBI server (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The residues at position 503 is indicated by square frame. Values are presented as mean ± SD (n = 3 in A, B, D, E and F).

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infected macrophages. As a consequence, neither did the number of parasite-infected cells decrease after pro-apoptotic stimulation, nor did the percentage of necrotic cells specifically increase after treatment of parasite-infected cells with staurosporine. Conversely, the number of viable parasite-infected cells was augmented due to an anti-apoptotic activity. These results indicate that the parasite–host interaction might indeed facilitate the intracellular development of the parasite. Because previous findings have demonstrated the parasitemediated blockade of cytochrome C release following pro-apoptotic signaling [28–31], we could narrow down the level of parasite interference to a step between Bcl-2 family and MOMP. Bim is a pro-apoptotic member of the Bcl-2 protein family. Alternative splicing generates three Bim isoforms (BimS, BimL and BimEL), which can all neutralize the activity of pro-survival Bcl-2-like proteins through their BH3 domain [32–34]. Importantly, the total protein levels of BimEL and BimL were altered after parasitic infection. This indicated that the blockage of Bim-mediated signaling is responsible for apoptotic inhibition in T. gondii infection. The previous report, however, by Hippe et al. [5] described an alternative pathway of Bax-mediated inhibition of apoptosis in Toxoplasma infection. The role of Bim in the process was not involved since the quantification of the BimEL and BimL levels was not presented and the three isoforms vary considerably in their pro-apoptotic activity in that study. This is partly due to sequestration of BimL and BimEL, but not BimS, to cytoskeletal structure via association with dynein light chain LC8 [35]. It might also be speculated that the use of different Toxoplasma strains (type II NTE in the study by Hippe; TgCtwh3 in the present study) explains this discrepancy because both lineages have been shown to differentially modulate host-cell signaling [6]. The results presented here provide an evidence that Bim was significantly downregulated in Toxoplasma-infected macrophages. However, the results of Toxoplasma infection in macrophages treated with staurosporine suggested that inhibition of the apoptosis pathway induced by Toxoplasma infection does not seem to be restricted to Bim-induced apoptosis. Although Toxoplasma can infect any kind of nucleated cells, macrophages and related mononuclear phagocytes are its preferred target in vivo, and the parasite seems to have multiple ways of avoiding being killed. Therefore, macrophage-centric research is essential to understand the basic strategies of the parasite–host interaction [36]. MiRNA profiles in macrophages induced by Toxoplasma infection remains to be clarified although prior studies have reported that Toxoplasma (type I RH) infection specifically increases the levels of miRNA in HFF cells [37]. The probes used in the microarray analysis in this study are human-miRNA specific with minimal cross-interaction with known miRNAs from other species. The microarray profiling showed that upwards of 8.3% and down-regulation of 4.3% miRNAs display abundantly altered, relatively to uninfected cells (data not shown). The analyses of miRNAs upregulated in Toxoplasma-infected macrophages revealed that the 3′UTR of Bim contains several regions that matches to the miR-17–92 miRNA “seed” region. However, we found that miR-17–92 miRNAs only marginally increased at 6 h after infection. This is inconsistent with the finding that at that time point BimEL and BimL were already considerably decreased. Indeed, miRNAs recognize their target mRNAs mainly through a limited base-pairing interaction between the 5′ end “seed” region and the complementary sequences present in the 3′UTRs of target mRNAs. The majority of animal miRNAs imprecisely match their targets [38]. The miR-17–92 miRNAs have the same “seed” region as many other miRNAs, such as miR-106b–25 and miR-106a– 363. It is possible that the other miRNAs could bind to 3′UTR of Bim after parasite infection. So the decreases of BimL and BimEL were not parallel to the increase of miR-17–92 miRNAs in the study. The miR17–92 miRNAs consist of six members, miR-17-5p, miR-19a, miR-19b, miR-20a, miR18a and miR-92a, and all are encoded by the miR-17–92 gene cluster. The miR-17–92 miRNAs have been shown to influence the functionally intertwined pathways of apoptosis and G1/S cell cycle progression by targeting multiple components of each pathway. To investigate the role of miR-17–92 miRNAs in the regulation of Bim, we

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used a mammalian pEZ expression vector encoding the miR-17–92 gene, the result showed that constitutive activation of this cluster led to a reduction of Bim protein expression, which is similar to the result of Toxoplasma infection. Conversely, this reduction could be mostly blocked by pretreatment with the anti-miR-17–92 miRNAs. To provide further evidence for the direct interaction of miR-17–92 miRNAs with the 3′UTR of Bim, we constructed a reporter gene system containing a luciferase gene and the predicted seed matches for miR-17–92 miRNAs. This assay presented repressed luciferase activity following overexpression of miR-17–92 miRNAs. Consistent with these data, Bim is a bona fide target for the miR-17–92 miRNAs that is modulated in macrophages subjected to Toxoplasma infection. The results presented here demonstrate that Toxoplasma infection increases miR-17–92 miRNA expression in macrophages via a STAT3dependent mechanism. Additionally, Toxoplasma-induced increase of miR-17–92 miRNA expression is associated with down-regulation of Bim in macrophages. Taken together, our findings offer an emerging evidence that Toxoplasma infection confers resistance against apoptosis via down-regulation of Bim based on a phylogenetically conserved STAT3-miR-17–92 miRNAs pathway, which contributes to the survival of host cells with T. gondii infection. A better understanding of miRNA expression in macrophages following T. gondii infection will play a critical role in inhibition of apoptosis of host cells and it may provide new insights into the relationship between the intracellular parasite and its host cells. 5. Conclusion Our studies support that STAT3 mediates a prosurvival pathway by upregulating the miR-17–92 miRNAs that in turn targets Bim, leading to the survival of host cells with Toxoplasma infection. Competing interests The authors declare that they have no competing interests towards any aspect of the work described in this paper. Authors' contributions YHC and JLS conceived and designed the study. YHC, HC, XWM and YYT performed the experiments. YHC, XCX and AMZ analyzed the data. YHC, JLS, ZRL, FLL and YW drafted the manuscript. All authors read and approved the final manuscript. Acknowledgments This work was supported by Natural Basic Research Program of China (Grant No. 2010CB530001) and National Natural Science Foundation of China (Grants No. 81101272). Our thanks are given to Dr. Peter Hotez (Texas Children's Hospital, Texas, USA) for his valuable comments to the manuscript, and Prof. Geoff Hide (Center for Parasitology and Disease, University of Salford, UK) for polishing the language in the manuscript. References [1] K. Labbe, M. Saleh, Cell Death Differ. 15 (2008) 1339–1349. [2] K.J. Song, H.J. Ahn, H.W. Nam, Korean J. Parasitol. 50 (2012) 1–6. [3] E.K. Persson, A.M. Agnarson, H. Lambert, N. Hitziger, H. Yagita, B.J. Chambers, A. Barragan, A. Grandien, J. Immunol. 179 (2007) 8357–8365. [4] C.G. Luder, U. Gross, Curr. Top. Microbiol. Immunol. 289 (2005) 219–237. [5] D. Hippe, A. Weber, L. Zhou, D.C. Chang, G. Hacker, C.G. Luder, J. Cell Sci. 122 (2009) 3511–3521. [6] J.P. Saeij, S. Coller, J.P. Boyle, M.E. Jerome, M.W. White, J.C. Boothroyd, Nature 445 (2007) 324–327. [7] R. Zhou, G. Hu, J. Liu, A.Y. Gong, K.M. Drescher, X.M. Chen, PLoS Pathog. 5 (2009) e1000681. [8] M.M. Nelson, A.R. Jones, J.C. Carmen, A.P. Sinai, R. Burchmore, J.M. Wastling, Infect. Immun. 76 (2008) 828–844.

1212 [9] [10] [11] [12] [13] [14] [15]

[16] [17]

[18] [19] [20] [21]

[22] [23]

Y. Cai et al. / Cellular Signalling 26 (2014) 1204–1212 I.J. Blader, I.D. Manger, J.C. Boothroyd, J. Biol. Chem. 276 (2001) 24223–24231. D.D. Newmeyer, S. Ferguson-Miller, Cell 112 (2003) 481–490. A.C. Faber, H. Ebi, C. Costa, J.A. Engelman, Adv. Pharmacol. 65 (2012) 519–542. J. Tanizaki, I. Okamoto, S. Fumita, W. Okamoto, K. Nishio, K. Nakagawa, Oncogene 30 (2011) 4097–4106. S.T. Wilkinson, M.E. Tome, M.M. Briehl, Int. J. Mol. Sci. 13 (2012) 10212–10228. L. He, G.J. Hannon, Nat. Rev. Genet. 5 (2004) 522–531. L. Jin, W.L. Hu, C.C. Jiang, J.X. Wang, C.C. Han, P. Chu, L.J. Zhang, R.F. Thorne, J. Wilmott, R.A. Scolyer, P. Hersey, X.D. Zhang, M. Wu, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 15840–15845. X. Liu, H. Fang, H. Chen, X. Jiang, D. Fang, Y. Wang, D. Zhu, PLoS One 7 (2012) e38659. M. Yamamoto, D.M. Standley, S. Takashima, H. Saiga, M. Okuyama, H. Kayama, E. Kubo, H. Ito, M. Takaura, T. Matsuda, D. Soldati-Favre, K. Takeda, J. Exp. Med. 206 (2009) 2747–2760. B.A. Butcher, B.A. Fox, L.M. Rommereim, S.G. Kim, K.J. Maurer, F. Yarovinsky, D.R. Herbert, D.J. Bzik, E.Y. Denkers, PLoS Pathog. 7 (2011) e1002236. B. Cai, J. Li, J. Wang, X. Luo, J. Ai, Y. Liu, N. Wang, H. Liang, M. Zhang, N. Chen, G. Wang, S. Xing, X. Zhou, B. Yang, X. Wang, Y. Lu, Stem Cells 30 (2012) 1746–1755. Q. Chen, H. Wang, Y. Liu, Y. Song, L. Lai, Q. Han, X. Cao, Q. Wang, PLoS One 7 (2012) e42971. K. Sugimura, H. Miyata, K. Tanaka, R. Hamano, T. Takahashi, Y. Kurokawa, M. Yamasaki, K. Nakajima, S. Takiguchi, M. Mori, Y. Doki, Clin. Cancer Res. 18 (2012) 5144–5153. L. Wang, H. Chen, D. Liu, X. Huo, J. Gao, X. Song, X. Xu, K. Huang, W. Liu, Y. Wang, F. Lu, Z.R. Lun, Q. Luo, X. Wang, J. Shen, PLoS One 8 (2013) e53483. Z.W. Chen, J.M. Gao, X.X. Huo, L. Wang, L. Yu, F. Halm-Lai, Y.H. Xu, W.J. Song, G. Hide, J.L. Shen, Z.R. Lun, Vet. Parasitol. 183 (2011) 166–170.

[24] [25] [26] [27] [28]

[29] [30] [31] [32] [33] [34] [35] [36] [37] [38]

Y. Cai, H. Chen, L. Jin, Y. You, J. Shen, Parasit. Vectors 6 (2013) 356. P.R. Kramer, S. Wray, J. Steroid Biochem. Mol. Biol. 81 (2002) 203–216. S. Gillespie, J. Borrow, X.D. Zhang, P. Hersey, Apoptosis 11 (2006) 2251–2265. P. Bouillet, J.F. Purton, D.I. Godfrey, L.C. Zhang, L. Coultas, H. Puthalakath, M. Pellegrini, S. Cory, J.M. Adams, A. Strasser, Nature 415 (2002) 922–926. L. Morales-Sainz, A. Escobar-Ramirez, V. Cruz-Torres, A. Reyes-Prieto, M. Vazquez-Acevedo, R. Lara-Martinez, L.F. Jimenez-Garcia, D. Gonzalez-Halphen, Biochim. Biophys. Acta 1777 (2008) 202–210. P. Keller, F. Schaumburg, S.F. Fischer, G. Hacker, U. Gross, C.G. Luder, FEMS Microbiol. Lett. 258 (2006) 312–319. D.C. McFadden, S. Tomavo, E.A. Berry, J.C. Boothroyd, Mol. Biochem. Parasitol. 108 (2000) 1–12. C.M. Collazo, C. Miller, G. Yap, S. Hieny, P. Caspar, R.H. Schwartz, A. Sher, Infect. Immun. 68 (2000) 2713–2719. M.L. Colli, T.C. Nogueira, F. Allagnat, D.A. Cunha, E.N. Gurzov, A.K. Cardozo, M. Roivainen, A. Op de Beeck, D.L. Eizirik, PLoS Pathog. 7 (2011) e1002267. M. Rahmani, M.M. Aust, E. Attkisson, D.C. Williams Jr., A. Ferreira-Gonzalez, S. Grant, Blood 119 (2012) 6089–6098. K.Y. Soo, J.D. Atkin, M. Farg, A.K. Walker, M.K. Horne, P. Nagley, PLoS One 7 (2012) e35413. L.A. O'Reilly, L. Cullen, J. Visvader, G.J. Lindeman, C. Print, M.L. Bath, D.C. Huang, A. Strasser, Am. J. Pathol. 157 (2000) 449–461. P.J. Murray, Cell Host Microbe 9 (2011) 445–447. G.M. Zeiner, K.L. Norman, J.M. Thomson, S.M. Hammond, J.C. Boothroyd, PLoS One 5 (2010) e8742. F. Fazi, C. Nervi, Cardiovasc. Res. 79 (2008) 553–561.