key: cord-0761763-igiueg3g authors: Noack, J; Jordi, M; Zauner, L; Alessi, D; Burch, A; Tinguely, M; Hersberger, M; Bernasconi, M; Nadal, D title: TLR9 agonists induced cell death in Burkitt's lymphoma cells is variable and influenced by TLR9 polymorphism date: 2012-06-21 journal: Cell Death Dis DOI: 10.1038/cddis.2012.60 sha: 05df6a79e61e69b9113ffa6a667c3fb9b2680e70 doc_id: 761763 cord_uid: igiueg3g Toll-like receptor 9 (TLR9) triggering is a promising novel strategy to combat cancer as it induces innate and adaptive immunity responses. B-cell lymphoma is unique in this context as tumor cells express TLR9 and may harbor latent Epstein-Barr virus (EBV), a gamma-herpesvirus with remarkable oncogenic potential when latent. Latent EBV may be promoted by TLR9 triggering via suppression of lytic EBV. Here, we elaborated an initial assessment of the impact of TLR9 triggering on EBV-positive and EBV-negative B-cell lymphoma using Burkitt's lymphoma (BL) cell lines as an in vitro model. We show that, independent of the presence of EBV, the TLR9 ligand oligodeoxynucleotide (ODN) CpG-2006 may or may not induce caspase-dependent cell death in BL cells. Moreover, ODN CpG-2006-induced cell death responses of BL cells were associated with TLR9 single-nucleotide polymorphisms (SNPs) rs5743836 or rs352140, which we detected in primary BL tumors and in peripheral blood from healthy individuals at similar frequencies. Thus, our findings suggest that the effect of TLR9 agonists on BL cells should be tested in vitro before installment of therapy and TLR9 SNPs in BL patients should be determined as potential biological markers for the therapeutic response to treatment targeting innate immunity. Toll-like receptors (TLRs) are important players of the innate immune system, 1 and expression of the 10 TLRs known in humans 2,3 depends on the cell subset and differentiation status. [4] [5] [6] TLR9 is preferentially expressed by B cells and plasmacytoid dendritic cells (reviewed in reference Iwasaki and Medzhitov 1 ). Synthetic TLR9 ligands, short oligodeoxynucleotides (ODN) with unmethylated CpG motifs, 7 activate TLR9 that recruits the adapter protein myeloid differentiation factor 88 (MyD88) and induces a cascade leading to the nuclear translocation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). 2 The immune response following TLR9 engagement is highly desirable for cancer treatment. 8 Synthetic TLR9 agonists are regarded as potential anti-cancer agents. Clinical trials have shown promising results for the treatment of various cancer types with CpG ODNs. 9 Their effects can be indirect by enhancing the anti-tumor immune response or direct by inducing apoptosis in the malignant cells. Direct effects would be expected for B-cell malignancies as they express TLR9. B-cell activation can eventually lead to activation-induced cell death of cancer cells and therefore support anti-cancer treatment. 10 Nevertheless, the TLR9 agonist effects vary strongly between B-cell cancer types, and the responses of B-cell lymphomas to ODN CpG-2006 show high variability. 11 Thus, the effects of CpG ODNs on B-cell malignancies are not predictable. Moreover, CpG ODN treatment has tumor-promoting effects on benign B cells and strongly enhances their proliferation and differentiation. [12] [13] [14] Stimulation of TLR9 with ODN CpG-2006 suppresses lytic reactivation of Epstein-Barr virus (EBV), a B-cell tropic gamma-herpesvirus, and can thereby promote latent EBV. 15, 16 The latter is associated with several types of B-cell lymphomas including Burkitt's lymphoma (BL). 17 EBV is present in nearly all cases of the high-incidence form of BL ('endemic BL'), in up to 85% of the intermediate-incidence cases, and in 15% of the low-incidence cases ('sporadic' BL). Up to 40% of BL in human immunodeficiency virus carriers harbor EBV. 17 As latent EBV exhibits unique growth transformation potential on B cells in vitro 18 and TLR9 triggering enhances EBV-induced B-cell transformation 19 and promotes latent EBV in vitro, 15, 16 TLR9 stimulation in the treatment of EBV-positive BL could provoke detrimental rather than beneficial outcomes. Single-nucleotide polymorphisms (SNPs) of TLR9 are associated with increased risk for certain B-cell lymphomas 20 and have the potential to exhibit deregulation of signaling thereby promoting tumorigenesis. The responses of TLR9 SNPs in B-cell tumors to CpG ODN treatment are not reported. Here, we used BL-cell lines to model direct effects of TLR9 stimulation on malignant cells, investigate the influence of EBV, and assess the impact of TLR9 SNPs, which we found in primary BL samples or in healthy primary cells. TLR9 triggering alters gene expression and activates Akata cells in a MyD88-dependent manner. CpG ODNs activate B cells by impacting on gene expression. 21 We asked how the gene expression pattern of BL cells is affected by TLR9 triggering using CpG ODN. We performed a microarray analysis comparing ODN CpG-2006-treated versus untreated Akata cells. Most of the Z2-fold changes in gene expression were upregulation and only few downregulation. Among upregulated genes, we were mainly interested in those involved in cytokine (human interleukin 10; hIL-10) and chemokine expression (CXCL10), B-cell activation (CD40), transcription (NF-kB) and apoptosis (FAS) as these genes are those that preferentially determine cell proliferation and survival (Supplementary Table 1) . Thirteen genes, including two regulating cell cycle, one transcription factor, one involved in B-cell differentiation and apoptosis, four of diverse functions and four of unknown function, showed downregulation following ODN CpG-2006 treatment (Supplementary Table 2 ). We earlier confirmed that TLR9 triggering of Akata cells with ODN CpG-2006 increases hIL-10 expression and leads to the translocation of NF-kB to the nucleus. 16 To validate a marker for B-cell activation, in this context, we measured CD40 expression by flow cytometry. CD40 expression was increased upon ODN CpG-2006 treatment (Figure 1a ). After 48 h, CD40 expression on ODN CpG-2006-treated cells was increased 2.7-fold compared with untreated cells (Figures 1a and b) . Surprisingly, the control ODN GpC-2006, which is similar to ODN CpG-2006 but lacks the CpG motifs, also led to a 2.4-fold increase in CD40 expression (Figures 1a and b) . This might reflect a TLR9-independent mechanism or the ability of the control ODN to stimulate TLR9 in BL cells. To clarify this point, we used Akata cells overexpressing a dominant-negative mutant of the adaptor protein MyD88 (DN-MyD88). 16 Table 1 ) by quantitative real-time polymerase chain reaction (qRT-PCR) and confirmed activation of STAT3 by western blot (data not shown). Collectively, we validated our microarray data showing that triggering with ODN CpG-2006 activates Akata cells to induce CD40 and STAT3 expression. Moreover, our results indicated that both ODNs used here act in a MyD88dependent manner and that the ODN lacking CpG motifs activates Akata cells to a similar extent as the ODN containing CpG motifs. To explore the mechanism of ODN-induced cell death of Akata cells we set out to investigate whether caspases were involved. Thus, the activity of caspase-3 and -7 in ODN-treated Akata cells was detected using a luminogenic substrate, which gives rise to a luminescent signal proportional to caspase-3/7 activity. Triggering with TLR9 ligands for 48 h increased the luminescent signal about twofold as compared with no triggering. This increase was abolished by treatment with the caspase inhibitor z-VAD-FMK (Figure 3a ), indicating that TLR9 triggering resulted in increased caspase activity in Akata cells and that this activity was not increased if cells were concomitantly treated with a caspase inhibitor. These findings were corroborated using PI staining, which indicate that Akata cell death induced by treatment with ODNs can be abrogated by the caspase inhibitor z-VAD-FMK ( Figure 3b ); and by western blotting showing that PARP cleavage is increased by ODN treatment and can be reduced by the inhibitor (Figure 3c ). Thus, ODN-induced cell death of Akata cells was found to be caspase-3/7 dependent and therefore due to apoptosis. CpG ODNs differs considerably, independently of the presence of EBV. ODN CpG-2006 suppresses EBV lytic gene expression in Akata cells and primary B cells promoting latent EBV that may provide cell survival signals. 15, 16 As we observed Akata cell death upon TLR9 triggering, we asked whether the absence of EBV would result in even more pronounced cell death following TLR9 triggering. Hence, we Exogenous hIL-10 does not prevent ODN CpG-2006induced cell death. hIL-10 has an important role in B-cell lymphoma biology as it seems to act as autocrine growth factor. 22, [24] [25] [26] We previously reported 16 and corroborate here, a strong hIL-10 induction in Akata cells following TLR9 triggering with ODN CpG-2006 (Supplementary Table 1 ). Nevertheless, the very same treatment resulted in drastic Akata cell death (Figure 2) , suggesting that the latter could not be prevented by the induction of hIL-10 expression. (Figure 6b) . Thus, TLR9 triggering of distinct BL cells resulted in a broad range of hIL-10 mRNA and hIL-10 protein expression levels, and the magnitude of the level seemed independent of EBV status. Next, we determined TLR9 mRNA expression levels in BL cells by qRT-PCR. Although distinct BL cells showed up to sixfold different relative TLR9 mRNA expression levels (Figure 6c) , ODN CpG-2006 treatment-induced hIL-10 mRNA peak expression levels did not correlate with the relative TLR9 mRNA expression levels ( Figure 6d) . Collectively, TLR9 triggering in distinct BL cells resulted in distinct hIL-10 mRNA expression, not linked to presence or absence of EBV, and not correlated to TLR9 mRNA expression levels. TLR9 polymorphisms of BL cells might correlate with distinct responses to TLR9 triggering. TLR9 polymorphisms in patients are linked to different outcomes of inflammatory diseases and the development of cancer. 27, 28 We hypothesized that TLR9 polymorphisms could be a possible explanation for our observations. Thus, we isolated the genomic DNA from BL cell lines and analyzed it for the presence of TLR9 polymorphisms. We found specific SNPs for each cell line, allowing segregation of the BL cell lines into three groups (Table 1) (Figure 4c) , suggesting that the distinct BL-cell responses to TLR 9 triggering may depend on the SNPs present. Next, we analyzed TLR9 SNPs in genomic DNA from primary BLs or blood from healthy individuals. Indeed, we found the TLR9 SNPs detected in our BL cell lines in primary BL samples and blood of healthy individuals (Table 2) . Importantly, the TLR9 SNP frequencies were statistically not significantly different between BL cell lines, primary BLs and blood from healthy individuals, respectively. This suggested that if the direct response to TLR9 agonists depends on the TLR9 SNP, this could be an important factor regarding the treatment of BL patients by TLR9 triggering. We analyzed the effects of TLR9 agonists on BL cell lines as an in vitro model for B-cell tumor. We found that treatment with TLR9 ligands induces distinct cytokine expression and cell death responses in distinct BL cells. Cell death was (i) dependent on TLR9-MyD88 signaling, (ii) occurred concomitantly with activation and could be suppressed by pan caspase inhibitors, (iii) was not dependent on the presence or absence of EBV in the tumor cells and (iv) was associated with SNPs in the TLR9 gene. Our results suggest that individualized in vitro pretesting of BL responses to CpG ODNs could help to predict the outcome of therapeutic TLR9 triggering and tailor adjuvant molecular treatment of BL. Our observation that BL cells of different origin show distinct cell survival following TLR9 triggering is novel. We previously demonstrated that TLR9 triggering counteracts lytic EBV reactivation in BL cells and promotes latent EBV that is associated with B-cell lymphoproliferation. 16 Here, we observed CpG type B ODN-induced cell death by apoptosis in EBV-positive Akata and EBV-negative Akata31 cells, but not or to a much lower extent in the other EBV-positive or EBV-negative BL cell lines tested. Thus, the EBV status of the tumor cells does not rule the responses of BL cells to TLR9 triggering. TLR9-triggered BL cells consistently upregulated hIL-10 expression. hIL-10 influences the development and growth of B cells 29 and acts as an autocrine growth factor for different B-cell lymphomas. 25, 26, 30 Importantly, here, rhIL-10 did neither prevent nor enhance cell death induced by TLR9 triggering. hIL-10, as an anti-inflammatory cytokine, inhibits the Th1 immune response 31 and this effect would be detrimental in cancer therapy that is based on intact or enhanced immune responses, 9 as an unintended proliferation of the malignant cells could be provoked. Thus, the function of CpG type B ODN-induced hIL-10 expression in BL cells seems to considerably differ from that in chronic lymphocytic leukemia B cells that were reported to undergo hIL-10mediated apoptosis. 10 Surprisingly, ODN GpC-2006 induced cell death to the same extent as ODN CpG-2006. ODN GpC-2006 binds to TLR9, but in contrast to CpG-2006 it does not lead to TLR9 signaling in HEK293 cells or to changes in the secondary structure of the ectodomain. 32 Therefore, we examined whether downstream signaling of TLR9 via MyD88 is involved in the induction of cell death. Indeed, experiments using Akata cells overexpressing a MyD88 dominant-negative mutant indicated that both ODNs induce cell death of BL cells in a MyD88-dependent manner. Others demonstrated that ODNs induce cell death independently of the CpG motif, of TLR9 33 or even of the whole-ODN sequence. 34 Another pathway that is triggered by CpG ODNs, but is TLR9-independent, was described in monocytes and involves the activation of the Src family kinases Lyn and Hck. 35 In our experiments, BL cell death induced by TLR9 triggering correlated with increased caspase activity and was caspase-dependent, thus most likely due to apoptosis. The BL cell lines tested differed in their TLR9 mRNA expression level and TLR9 SNPs. mRNA expression did not correlate with cell death responses to TLR9 triggering, but the distinct TLR9 SNPs did. The role of TLR9 SNPs in BL is unknown. To start to investigate this we analyzed the frequencies of given SNPs in BL patients and healthy individuals and found them to be similar. A recent report suggests that the rs5743836 polymorphism, which was also detected here, confers an increased risk for non-Hodgkin lymphoma in people from Portugal and Italy but not from the United States. 20 The association of the C allele rs5743836 with lack of cell death upon TLR9 triggering observed here in the Mutu-I and BJAB BL cell lines is striking. It does not allow establishing a causal link, but justifies the hypothesis that the distinct cell death responses upon CpG ODN treatment in BL cells may be linked to TLR9 SNPs. Notably, the C allele of rs5743836 exhibits greater NF-kB-binding affinity because of an additional NF-kB transcriptional binding site that may lead to increased production of proinflammatory cytokines. 36 Hence, the presence of the C allele seems to result in enhanced NF-kB activation following TLR9 triggering. This may lead to protection of ODN CpG treatmentinduced apoptosis that can be observed in BL cells without the C allele. In conclusion, therapeutic TLR9 triggering appears to be a double-edge sword that may induce apoptosis, or enhance lymphoproliferation. Our findings suggest that the effect of TLR9 agonists on BL cells should be tested in vitro before installment of therapy and that TLR9 SNPs in BL patients should be evaluated as potential biological markers for the response to treatment targeting innate immunity. Cell lines. The EBV-positive BL cell lines Akata and Mutu-I, and the EBV-negative BL cell lines Akata 31, BJAB and Ramos were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, streptomycin (100 mg/ml), penicillin (100 U/ml) and L-glutamine (2 mM). EBV-positive BL Akata cells expressing a dominant-negative MyD88 (DN-MyD88 Akata) 16 were grown in the same medium supplemented with 0.4 mg/ml G418 (Promega, Mannheim, Germany). Primary BL tissue samples and peripheral blood from healthy individuals. BLs were retrieved from the database of the Institute of Surgical Pathology, University Hospital of Zurich (PathoPro Software, Institute of Medical Software, Saarbrücken, Germany). The BL diagnosis was performed according to the WHO classification of tumors of hematopoietic and lymphoid tissues. 37 DNA was extracted from paraffin-embedded whole-tissue sections, according to standard procedures. This study was in accordance with Swiss laws and approved by the official authorities of the ethical committee of the Canton of Zurich (StV2-2007). Peripheral blood was collected from 402 healthy blood donors (aged 19-70 years) who were randomly selected according to the criteria of the Swiss Red Cross. 38 Single-nucleotide polymorphism analysis. Whole-genomic DNA was extracted with the DNeasy Blood and Tissue Kit (Qiagen, Hombrechtikon, Switzerland) according to the manufacturer's instructions. Genotyping of the TLR9 polymorphisms -1237 T/C (rs5743836) and 2848 G/A (rs352140) was done by tetra-primer assays as previously described. 39 Treatment with TLR9 agonists. Cells were split to a density of 0.5 Â Microarray analysis. Akata cells were mock treated or treated with CpG-2006, and harvested 6 h later. Total RNA was isolated with RNeasy mini kit (Qiagen) according to the manufacturer's instructions. Microarray analysis was performed at the Functional Genomic Center Zurich (University of Zurich, Zurich, Switzerland). Human Exon 1.0 ST Array chips (Affymetrix, Santa Clara, CA, USA) and Genespring GX software (Agilent Technologies, Basel, Switzerland) were used for the analysis. An at least twofold change in gene expression (upregulation or downregulation) in the treated versus the mock-treated sample was regarded as significant. qRT-PCR (Taqman). Total RNA was extracted from 10 6 cells with the RNeasy Kit (Qiagen) and DNA was removed with DNA-free (Ambion Europe, Huntingdon, Cambridgeshire, UK). 1 mg of the purified RNA was used to generate cDNA with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Quantitative real-time PCR was performed with specific Taqman primers and probes on ABI 7200 (Applied Biosystems). Data were analyzed with the software SDS2.2 (Applied Biosystems). Delta cycle threshold (Ct) values for the respective genes were normalized to HMBS PE-Cy5 mouse anti-human IgG (Clone G18-145), PE mouse anti-human IgD (Clone IA6-2) or the respective isotype controls, FITC IgG1k isotype control (Clone MOPC-21), PE-Cy5 mouse IgG1k isotype control (Clone MOPC-21) Switzerland) for 30 min at 4 1C in the dark 10 5 cells were harvested, washed with phosphate-buffered saline (PBS) and resuspended in 100 ml Annexin V binding buffer (140 mM NaCl, 2.5 mM CaCl 2 , 10 mM HEPES, pH 7.4) and incubated with 5 ml Annexin V and 5 ml 7-AAD (BD Biosciences) for 15 min at room temperature in the dark. After addition of 200 ml Annexin V binding buffer ) for 15 min at room temperature and then analyzed with the FACSCanto II flow cytometer Total protein lysates were obtained after lysing the cells in RIPA complete buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA SDS, 1 Â EDTA-free protease inhibitor cocktail according to the manufacturer's instructions. To analyze protein expression by western blot, protein (20 mg/well) was loaded into a NuPAGE 4-12% Bis-Tris Gel (life technologies, Zug, Switzerland) and subjected to SDS-PAGE electrophoresis, transferred electrophoretically to a nitrocellulose membrane (GE Healthcare, Glattbrugg, Switzerland), incubated with rabbit anti-PARP antibody or rabbit anti-b-Actin antibody, and subsequently with anti-rabbit IgG, HRP-linked antibody (all diluted 1:1000 and from Cells, dispensed at 0.5 Â 10 6 cells/ml/well in a 24-well plate were treated with TLR9 ligands and incubated at 371C for 48 h. At the end of the incubation period, 100 ml of Caspase-Glo 3/7 reagent was added to 100 ml cell suspension in a white-walled 96-well plate and incubated at room temperature for 1 h One day before the experiment, the cells were split to a density of 0.5 Â 10 6 cells/ml. 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IARC: Lyon The Asp298 but not the C-786 genotype of the endothelial nitric oxide synthase is reduced with age in healthy Swiss men Tolllike receptor 4 gene polymorphism modulates phenotypic expression in patients with hereditary hemochromatosis This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this license Acknowledgements. This work was funded by the Swiss National Foundation (310040-114118), Oncosuisse and the Cancer League of the Canton of Zurich. The authors declare no conflict of interest.