Rapid Biodistribution of Fluorescent Outer-Membrane Vesicles from the Intestine to Distant Organs via the Blood in Mice.

A cell's ability to secrete extracellular vesicles (EVs) for communication is present in all three domains of life. Notably, Gram-negative bacteria produce a specific type of EVs called outer membrane vesicles (OMVs). We previously observed the presence of OMVs in human blood, which could represent a means of communication from the microbiota to the host. Here, in order to investigate the possible translocation of OMVs from the intestine to other organs, the mouse was used as an animal model after OMVs administration. To achieve this, we first optimized the signal of OMVs containing the fluorescent protein miRFP713 associated with the outer membrane anchoring peptide OmpA by adding biliverdin, a fluorescence cofactor, to the cultures. The miRFP713-expressing OMVs produced in E. coli REL606 strain were then characterized according to their diameter and protein composition. Native- and miRFP713-expressing OMVs were found to produce homogenous populations of vesicles. Finally, in vivo and ex vivo fluorescence imaging was used to monitor the distribution of miRFP713-OMVs in mice in various organs whether by intravenous injection or oral gavage. The relative stability of the fluorescence signals up to 3 days post-injection/gavage paves the way to future studies investigating the OMV-based communication established between the different microbiotas and their host.

Real-time visualization of dextran extravasation in intermittent hypoxia mice using noninvasive SWIR imaging.

Imaging tools are crucial for studying the vascular network and its barrier function in various physiopathological conditions. Shortwave infrared (SWIR) window optical imaging allows noninvasive, in-depth exploration. We applied SWIR imaging, combined with vessel segmentation and deep learning analyses, to study real-time dextran probe extravasation in mice experiencing intermittent hypoxia (IH)-a characteristic of obstructive sleep apnea associated with potential cardiovascular alterations due to early vascular permeability. Evidence for permeability in this context is limited, making our investigation significant. C57Bl/6 mice were exposed to normoxia or intermittent hypoxia for 14 days. Then SWIR imaging between 1,250 and 1,700 nm was performed on the saphenous artery and vein and on the surrounding tissue after intravenous injection of labeled dextrans of two different sizes (10 or 70 kDa). Postprocessing and segmentation of the SWIR images were conducted using deep learning treatment. We monitored high-resolution signals, distinguishing arteries, veins, and surrounding tissues. In the saphenous artery and vein, after 70-kD dextran injection, tissue/vessel ratio was higher after intermittent hypoxia (IH) than normoxia (N) over 500 seconds (P < 0.05). However, the ratio was similar in N and IH after 10-kD dextran injection. The SWIR imaging technique allows noninvasive, real-time monitoring of dextran extravasation in vivo. Dextran 70 extravasation is increased after exposure to IH, suggesting an increased vessel permeability in this mice model of obstructive sleep apnea.NEW & NOTEWORTHY We demonstrate that SWIR imaging technique is a useful tool to monitor real-time dextran extravasation from vessels in vivo, with a high resolution. We report for the first time an increased real-time dextran (70 kD) extravasation in mice exposed to intermittent hypoxia for 14 days compared with normoxic controls.

3D-Printed Osteoinductive Polymeric Scaffolds with Optimized Architecture to Repair a Sheep Metatarsal Critical-Size Bone Defect.

The reconstruction of critical-size bone defects in long bones remains a challenge for clinicians. A new osteoinductive medical device is developed here for long bone repair by combining a 3D-printed architectured cylindrical scaffold made of clinical-grade polylactic acid (PLA) with a polyelectrolyte film coating delivering the osteogenic bone morphogenetic protein 2 (BMP-2). This film-coated scaffold is used to repair a sheep metatarsal 25-mm long critical-size bone defect. In vitro and in vivo biocompatibility of the film-coated PLA material is proved according to ISO standards. Scaffold geometry is found to influence BMP-2 incorporation. Bone regeneration is followed using X-ray scans, µCT scans, and histology. It is shown that scaffold internal geometry, notably pore shape, influenced bone regeneration, which is homogenous longitudinally. Scaffolds with cubic pores of ≈870 µm and a low BMP-2 dose of ≈120 µg cm-3 induce the best bone regeneration without any adverse effects. The visual score given by clinicians during animal follow-up is found to be an easy way to predict bone regeneration. This work opens perspectives for a clinical application in personalized bone regeneration.

Lipoprotein interactions with water-soluble NIR-II emitting aza-BODIPYs boost the fluorescence signal and favor selective tumor targeting.

Following intravenous administration, the interaction of fluorescent exogenous molecules with circulating endogenous transporters can influence their photophysical properties as well as their fate and distribution, and possibly their recognition by different cell types. This type of interaction can be used to optimize the drug delivery but also the imaging properties of a compound of interest. In this study, we investigated the behavior of SWIR-WAZABY-01 fluorophore, a water-soluble aza-BODIPY dye emitting in the NIR-II region, both in vitro and in vivo. While the fluorescence emission of SWIR-WAZABY-01 was weak in aqueous solutions, it was intensely magnified in plasma (∼ ×30). Further analyses using lipoprotein gel electrophoresis and ultracentrifugation revealed interactions between SWIR-WAZABY-01 and plasma lipoproteins in vitro and ex vivo, in particular with LDL. The tumor uptake mechanism of SWIR-WAZABY-01 was investigated based on the presence of low-density lipoprotein (LDL) receptors and passive tumor uptake. Overall, we found that SWIR-WAZABY-01 interacts with lipoproteins enhancing their NIR-II fluorescence emission, and driving the tumor accumulation with regards to the expression of lipoprotein receptors (LDLR, SR-BI). Moreover, SWIR-WAZABY-01, by exploiting endogenous lipoproteins, arises as a new, potent and relevant tool to efficiently label LDL involved in pathologies.

Chronic intermittent hypoxia, a hallmark of obstructive sleep apnea, promotes 4T1 breast cancer development through endothelin-1 receptors.

The association between obstructive sleep apnea (OSA) and cancer is still debated and data are scarce regarding the link between OSA and breast cancer progression. Since conclusive epidemiological studies require large sample sizes and sufficient duration of exposure before incident cancer occurrence, basic science studies represent the most promising approach to appropriately address the topic. Here we assessed the impact of intermittent hypoxia (IH), the major hallmark of OSA, on the development of breast cancer and explored the specific involvement of the endothelin signaling pathway. Original in vitro and in vivo models were used where 3D-spheroids or cultures of murine 4T1 breast cancer cells were submitted to IH cycles, and nude NMRI mice, orthotopically implanted with 4T1 cells, were submitted to chronic IH exposure before and after implantation. The role of the endothelin-1 in promoting cancer cell development was investigated using the dual endothelin receptor antagonist, macitentan. In vitro exposure to IH significantly increased 4T1 cell proliferation and migration. Meta-analysis of 4 independent in vivo experiments showed that chronic IH exposure promoted tumor growth, assessed by caliper measurement (overall standardized mean difference: 1.00 [0.45-1.55], p < 0.001), bioluminescence imaging (1.65 [0.59-2.71]; p < 0.01) and tumor weight (0.86 [0.31-1.41], p < 0.01), and enhanced metastatic pulmonary expansion (0.77 [0.12-1.42]; p = 0.01). Both in vitro and in vivo tumor-promoting effects of IH were reversed by macitentan. Overall, these findings demonstrate that chronic intermittent hypoxia exposure promotes breast cancer growth and malignancy and that dual endothelin receptor blockade prevents intermittent hypoxia-induced tumor development.

Tumor-Specific Imaging with Angiostamp800 or Bevacizumab-IRDye 800CW Improves Fluorescence-Guided Surgery over Indocyanine Green in Peritoneal Carcinomatosis.

Complete surgical removal of lesions improves survival of peritoneal carcinomatosis and can be enhanced by intraoperative near-infrared fluorescence imaging. Indocyanine green (ICG) is the only near-infrared fluorescent dye approved for clinical use, but it lacks specificity for tumor cells, highlighting the need for tumor-selective targeting agents. We compared the tumor-specific near-infrared fluorescent probes Bevacizumab-IRDye 800CW and Angiostamp800, which target tumor angiogenesis and cancer cells, to ICG for fluorescence-guided surgery in peritoneal carcinomatosis of ovarian origin. The probes were administered to mice with orthotopic peritoneal carcinomatosis prior to conventional and fluorescence-guided surgery. The influence of neoadjuvant chemotherapy was also assessed. Conventional surgery removed 88.0 ± 1.2% of the total tumor load in mice. Fluorescence-guided surgery allowed the resection of additional nodules, enhancing the total tumor burden resection by 9.8 ± 0.7%, 8.5 ± 0.8%, and 3.9 ± 1.2% with Angiostamp800, Bevacizumab-IRDye 800CW and ICG, respectively. Interestingly, among the resected nodules, 15% were false-positive with ICG, compared to only 1.4% with Angiostamp800 and 3.5% with Bevacizumab-IRDye 800CW. Furthermore, conventional surgery removed only 69.0 ± 3.9% of the total tumor burden after neoadjuvant chemotherapy. Fluorescence-guided surgery with Angiostamp800 and Bevacizumab-IRDye 800CW increased the total tumor burden resection to 88.7 ± 4.3%, whereas ICG did not improve surgery at all. Bevacizumab-IRDye 800CW and Angiostamp800 better detect ovarian tumors and metastases than the clinically used fluorescent tracer ICG, and can help surgeons completely remove tumors, especially after surgery neoadjuvant chemotherapy.

An Orthotopic Murine Model of Peritoneal Carcinomatosis of Ovarian Origin for Intraoperative PDT.

Advanced ovarian cancer is the most serious among gynecological malignancies and is associated with 35% five-year overall survival. Surgery is the first therapeutic indication, and the absence of remaining macroscopic lesions is the most important prognostic factor. However, tumor dissemination over the whole abdominal cavity largely contributes to the difficulty of complete surgical resection. Consequently, any therapeutic approach that may complete surgical resection should improve patient survival. Considering that some sites are not suitable for surgery because of their close location to vital organs, intraoperative photodynamic therapy (ioPDT) appears to be a complementary therapeutic approach to surgery to obtain the lowest residual disease.Relevant in vivo cancer models that closely resemble human ovarian cancer are essential for preclinical research of alternative antitumor therapeutic strategies. Thus, we propose a comprehensive protocol to set up an orthotopic ovarian xenograft in mice leading to peritoneal carcinomatosis that could be harnessed for antitumor therapeutic application and evaluation.

FGF-2 promotes angiogenesis through a SRSF1/SRSF3/SRPK1-dependent axis that controls VEGFR1 splicing in endothelial cells.

BACKGROUND: Angiogenesis is the process by which new blood vessels arise from pre-existing ones. Fibroblast growth factor-2 (FGF-2), a leading member of the FGF family of heparin-binding growth factors, contributes to normal as well as pathological angiogenesis. Pre-mRNA alternative splicing plays a key role in the regulation of cellular and tissular homeostasis and is highly controlled by splicing factors, including SRSFs. SRSFs belong to the SR protein family and are regulated by serine/threonine kinases such as SRPK1. Up to now, the role of SR proteins and their regulators in the biology of endothelial cells remains elusive, in particular upstream signals that control their expression. RESULTS: By combining 2D endothelial cells cultures, 3D collagen sprouting assay, a model of angiogenesis in cellulose sponges in mice and a model of angiogenesis in zebrafish, we collectively show that FGF-2 promotes proliferation, survival, and sprouting of endothelial cells by activating a SRSF1/SRSF3/SRPK1-dependent axis. In vitro, we further demonstrate that this FGF-2-dependent signaling pathway controls VEGFR1 pre-mRNA splicing and leads to the generation of soluble VEGFR1 splice variants, in particular a sVEGFR1-ex12 which retains an alternative last exon, that contribute to FGF-2-mediated angiogenic functions. Finally, we show that sVEGFR1-ex12 mRNA level correlates with that of FGF-2/FGFR1 in squamous lung carcinoma patients and that sVEGFR1-ex12 is a poor prognosis marker in these patients. CONCLUSIONS: We demonstrate that FGF-2 promotes angiogenesis by activating a SRSF1/SRSF3/SRPK1 network that regulates VEGFR1 alternative splicing in endothelial cells, a process that could also contribute to lung tumor progression.

Evaluation of the human type 3 adenoviral dodecahedron as a vector to target acute myeloid leukemia.

Intensive systemic chemotherapy is the gold standard of acute myeloid leukemia (AML) treatment and is associated with considerable off-target toxicities. Safer and targeted delivery systems are thus urgently needed. In this study, we evaluated a virus-like particle derived from the human type 3 adenovirus, called the adenoviral dodecahedron (Dd) to target AML cells. The vectorization of leukemic cells was proved very effective at nanomolar concentrations in a time- and dose-dependent manner, without vector toxicity. The internalization involved clathrin-mediated energy-dependent endocytosis and strongly correlated with the expression of αVß3 integrin. The treatment of healthy donor peripheral blood mononuclear cells showed a preferential targeting of monocytes compared to lymphocytes and granulocytes. Similarly, monocytes but also AML blasts were the best-vectorized populations in patients while acute lymphoid leukemia blasts were less efficiently targeted. Importantly, AML leukemic stem cells (LSCs) could be addressed. Finally, Dd reached peripheral monocytes and bone marrow hematopoietic stem and progenitor cells following intravenous injection in mice, without excessive spreading in other organs. These findings reveal Dd as a promising myeloid vector especially for therapeutic purposes in AML blasts, LSCs, and progenitor cells.

Nanobiosensor Reports on CDK1 Kinase Activity in Tumor Xenografts in Mice.

Probing the dynamics and quantifying the activities of intracellular protein kinases that coordinate cell growth and division and constitute biomarkers and pharmacological targets in hyperproliferative and pathological disorders remain a challenging task. Here engineering and characterization of a nanobiosensor of the mitotic kinase CDK1, through multifunctionalization of carbon nanotubes with a CDK1-specific fluorescent peptide reporter, are described. This original reporter of CDK1 activity combines the sensitivity of a fluorescent biosensor with the unique physico-chemical and biological properties of nanotubes for multifunctionalization and efficient intracellular penetration. The functional versatility of this nanobiosensor enables implementation to quantify CDK1 activity in a sensitive and dose-dependent fashion in complex biological environments in vitro, to monitor endogenous kinase in living cells and directly within tumor xenografts in mice by fluorescence imaging, thanks to a ratiometric quantification strategy accounting for response relative to concentration in space and in time.

Optimization of spatial resolution and scattering effects for biomedical fluorescence imaging by using sub-regions of the shortwave infrared spectrum.

We evaluated the impact of light-scattering effects on spatial resolution in different shortwave infrared (SWIR) sub-regions by analyzing two SWIR emissive phantoms made of polydimethylsiloxane (PDMS)-gold nanoclusters (Au NCs) composite covered with mice skin, or capillary tubes filled with Au NCs or IRDye 800CW at different depth in intralipids and finally, after administration of the Au NCs intravenously in mice. Our findings highlighted the benefit of working at the highest tested spectral range of the SWIR region with a 50% enhancement of spatial resolution measured in artificial model when moving from NIR-II (1000-1300 nm) to NIR-IIa (1300-1450 nm) region, and a 25% reduction of the scattering from the skin determined by point spread function analysis from the NIR-II to NIR-IIb region (1500-1700 nm). We also confirmed that a series of Monte Carlo restoration of images significantly improved the spatial resolution in vivo in mice in deep tissues both in the NIR-II and NIR-IIa spectral windows.

Two Antagonistic Microtubule Targeting Drugs Act Synergistically to Kill Cancer Cells.

Paclitaxel is a microtubule stabilizing agent and a successful drug for cancer chemotherapy inducing, however, adverse effects. To reduce the effective dose of paclitaxel, we searched for pharmaceutics which could potentiate its therapeutic effect. We screened a chemical library and selected Carba1, a carbazole, which exerts synergistic cytotoxic effects on tumor cells grown in vitro, when co-administrated with a low dose of paclitaxel. Carba1 targets the colchicine binding-site of tubulin and is a microtubule-destabilizing agent. Catastrophe induction by Carba1 promotes paclitaxel binding to microtubule ends, providing a mechanistic explanation of the observed synergy. The synergistic effect of Carba1 with paclitaxel on tumor cell viability was also observed in vivo in xenografted mice. Thus, a new mechanism favoring paclitaxel binding to dynamic microtubules can be transposed to in vivo mouse cancer treatments, paving the way for new therapeutic strategies combining low doses of microtubule targeting agents with opposite mechanisms of action.

Noninvasive monitoring of liver metastasis development via combined multispectral photoacoustic imaging and fluorescence diffuse optical tomography.

Rationale:In vivo molecular imaging in preclinical animal models is a tool of choice for understanding the pathophysiological mechanisms involved in cancer development and for conducting drug development research. Moreover, combining several imaging modalities can provide multifaceted, complementary and cross-validated information. Photoacoustic imaging (PAI) is a promising imaging modality that can reflect blood vasculature and tissue oxygenation as well as detect exogenous molecules, but one shortcoming of PAI is a lack of organic photoacoustic contrast agents capable of providing tumor contrast. Methods: In the present study, we designed an animal model of liver metastases from colon cancer and monitored metastasis development by in vivo bioluminescence and X-ray microcomputed tomography. Contrast-agent-free PAI was used to detect the respective amounts of oxy- and deoxyhemoglobin and, thus, liver tissue oxygenation. two contrast agents, Angiostamp800 and indocyanin green (ICG), respectively with and without tumor targeting specificity, were then evaluated for their dual fluorescence and photoacoustic detectability and were then used for combined PAI and fluorescence diffuse optical tomography (fDOT) at various disease development stages. Findings: Contrast-agent-free PAI reflected tumor angiogenesis and gradual hypoxia during metastasis development. Multispectral PAI enabled noninvasive real-time monitoring of ICG blood pharmacokinetics, which demonstrated tumor-related liver dysfunction. Both PAI and fluorescence ICG signals were clearly modified in metastasis-bearing livers but did not allow for differentiation between different disease stages. In contrast, there was a significant improvement achieved by using the tumor-specific marker Angiostamp800, which provided gradually increasing PAI and fDOT signals during metastasis development. Conclusion: We demonstrated for the first time the value of using Angiostamp800 as a bimodal tumor-targeting contrast agent for combined PAI and fluorescence imaging of liver metastasis progression in vivo.

High-Resolution Shortwave Infrared Imaging of Vascular Disorders Using Gold Nanoclusters.

We synthesized a generation of water-soluble, atomically precise gold nanoclusters (Au NCs) with anisotropic surface containing a short dithiol pegylated chain (AuMHA/TDT). The AuMHA/TDT exhibit a high brightness (QY ∼ 6%) in the shortwave infrared (SWIR) spectrum with a detection above 1250 nm. Furthermore, they show an extended half-life in blood (t1/2ß = 19.54 ± 0.05 h) and a very weak accumulation in organs. We also developed a non-invasive, whole-body vascular imaging system in the SWIR window with high-resolution, benefiting from a series of Monte Carlo image processing. The imaging process enabled to improve contrast by 1 order of magnitude and enhance the spatial resolution by 59%. After systemic administration of these nanoprobes in mice, we can quantify vessel complexity in depth (>4 mm), allowing to detect very subtle vascular disorders non-invasively in bone morphogenetic protein 9 (Bmp9)-deficient mice. The combination of these anisotropic surface charged Au NCs plus an improved SWIR imaging device allows a precise mapping at high-resolution and an in depth understanding of the organization of the vascular network in live animals.

Stapled peptide targeting the CDK4/Cyclin D interface combined with Abemaciclib inhibits KRAS mutant lung cancer growth.

CDK4/cyclin D kinase constitutes an attractive pharmacological target for development of anticancer therapeutics, in particular in KRAS-mutant lung cancer patients, who have a poor prognosis and no targeted therapy available yet. Although several ATP-competitive inhibitors of CDK4 have been developed for anticancer therapeutics, they suffer from limited specificity and efficacy. Methods: As an alternative to ATP-competitive inhibitors we have designed a stapled peptide to target the main interface between CDK4 and cyclin D, and have characterized its physico-chemical properties and affinity to bind cyclin D1. Results: We have validated a positive correlation between CDK4/cyclin D level and KRAS mutation in lung cancer patients. The stapled peptide enters cells rapidly and efficiently, and inhibits CDK4 kinase activity and proliferation in lung cancer cells. Its intrapulmonary administration in mice enables its retention in orthotopic lung tumours and complete inhibition of their growth when co-administered with Abemaciclib. Conclusion: The stapled peptide targeting the main interface between CDK4 and cyclin D provides promising therapeutic perspectives for patients with lung cancer.

Verteporfin-Loaded Lipid Nanoparticles Improve Ovarian Cancer Photodynamic Therapy In Vitro and In Vivo.

Advanced ovarian cancer is the most lethal gynecological cancer, with a high rate of chemoresistance and relapse. Photodynamic therapy offers new prospects for ovarian cancer treatment, but current photosensitizers lack tumor specificity, resulting in low efficacy and significant side-effects. In the present work, the clinically approved photosensitizer verteporfin was encapsulated within nanostructured lipid carriers (NLC) for targeted photodynamic therapy of ovarian cancer. Cellular uptake and phototoxicity of free verteporfin and NLC-verteporfin were studied in vitro in human ovarian cancer cell lines cultured in 2D and 3D-spheroids, and biodistribution and photodynamic therapy were evaluated in vivo in mice. Both molecules were internalized in ovarian cancer cells and strongly inhibited tumor cells viability when exposed to laser light only. In vivo biodistribution and pharmacokinetic studies evidenced a long circulation time of NLC associated with efficient tumor uptake. Administration of 2 mg.kg-1 free verteporfin induced severe phototoxic adverse effects leading to the death of 5 out of 8 mice. In contrast, laser light exposure of tumors after intravenous administration of NLC-verteporfin (8 mg.kg-1) significantly inhibited tumor growth without visible toxicity. NLC-verteporfin thus led to efficient verteporfin vectorization to the tumor site and protection from side-effects, providing promising therapeutic prospects for photodynamic therapy of cancer.

Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display.

Self-assembling virus-like particles represent highly attractive tools for developing next-generation vaccines and protein therapeutics. We created ADDomer, an adenovirus-derived multimeric protein-based self-assembling nanoparticle scaffold engineered to facilitate plug-and-play display of multiple immunogenic epitopes from pathogens. We used cryo-electron microscopy at near-atomic resolution and implemented novel, cost-effective, high-performance cloud computing to reveal architectural features in unprecedented detail. We analyzed ADDomer interaction with components of the immune system and developed a promising first-in-kind ADDomer-based vaccine candidate to combat emerging Chikungunya infectious disease, exemplifying the potential of our approach.

The Bone Morphogenetic Protein Signaling Inhibitor LDN-193189 Enhances Metastasis Development in Mice.

Breast cancer with bone metastasis is essentially incurable with current anticancer therapies. The bone morphogenetic protein (BMP) pathway is an attractive therapeutic candidate, as it is involved in the bone turnover and in cancer cell formation and their colonization of distant organs such as the bone. We previously reported that in breast cancer cells, the ZNF217 oncogene drives BMP pathway activation, increases the metastatic growth rate in the bone, and accelerates the development of severe osteolytic lesions in mice. In the present study, we aimed at investigating the impact of the LDN-193189 compound, a potent inhibitor of the BMP type I receptor, on metastasis development in vivo. ZNF217-revLuc cells were injected into the left ventricle of nude mice (n = 16) while control mice (n = 13) were inoculated with control pcDNA6-revLuc cells. Mice from each group were treated or not with LDN-193189 for 35 days. We found that systemic LDN-193189 treatment of mice significantly enhanced metastasis development, by increasing both the number and the size of metastases. In pcDNA6-revLuc-injected mice, LDN-193189 also affected the kinetics of metastasis emergence. Altogether, these data suggest that in vivo, LDN-193189 might affect the interaction between breast cancer cells and the bone environment, favoring the emergence and development of multiple metastases. Hence, our report highlights the importance of the choice of drugs and therapeutic strategies used in the management of bone metastases.

The Toxoplasma effector TEEGR promotes parasite persistence by modulating NF-κB signalling via EZH2.

The protozoan parasite Toxoplasma gondii has co-evolved with its homeothermic hosts (humans included) strategies that drive its quasi-asymptomatic persistence in hosts, hence optimizing the chance of transmission to new hosts. Persistence, which starts with a small subset of parasites that escape host immune killing and colonize the so-called immune privileged tissues where they differentiate into a low replicating stage, is driven by the interleukin 12 (IL-12)-interferon-γ (IFN-γ) axis. Recent characterization of a family of Toxoplasma effectors that are delivered into the host cell, in which they rewire the host cell gene expression, has allowed the identification of regulators of the IL-12-IFN-γ axis, including repressors. We now report on the dense granule-resident effector, called TEEGR (Toxoplasma E2F4-associated EZH2-inducing gene regulator) that counteracts the nuclear factor-κB (NF-κB) signalling pathway. Once exported into the host cell, TEEGR ends up in the nucleus where it not only complexes with the E2F3 and E2F4 host transcription factors to induce gene expression, but also promotes shaping of a non-permissive chromatin through its capacity to switch on EZH2. Remarkably, EZH2 fosters the epigenetic silencing of a subset of NF-κB-regulated cytokines, thereby strongly contributing to the host immune equilibrium that influences the host immune response and promotes parasite persistence in mice.